Design, synthesis, and structure-activity relationships of 2-benzylidene-1-indanone derivatives as anti-inflammatory agents for treatment of acute lung injury

Article abstract of DOI:10.2147/DDDT.S160314

Purpose: The purpose of this study was to design and synthesize novel 2-benzylidene-1-indanone derivatives for treatment of acute lung injury. Methods: A series of 39 novel 2-benzylidene-indanone structural derivatives were synthesized and evaluated for anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated murine primary macrophages. Results: Most of the obtained compounds effectively inhibited the LPS-induced expression of IL-6 and TNF-α. The most active compound, 8f, was found to significantly reduce LPS-induced pulmonary inflammation, as reflected by reductions in the concentration of total protein, inflammatory cell count, as well as the lung wet/dry ratio in bronchoalveolar lavage (BAL) fluid. Furthermore, 8f effectively inhibited mRNA expression of several inflammatory cytokines after LPS challenge in vitro and in vivo. Administration of 8f also blocked LPS-induced activation of the proinflammatory NF-κB/MAPK signaling pathway. Conclusion: The simple synthetic preparation and biological properties of these derivatives make these 2-benzylidene-indanone scaffolds promising new entities for the development of anti-inflammatory therapeutics for the treatment of acute lung injury.

Full text of DOI:10.2147/DDDT.S160314

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Open access FullText article
O r i g i n a l r e s e a r c h
Design, synthesis, and structure–activity
relationships of 2-benzylidene-1-indanone
derivatives as anti-inflammatory agents for
treatment of acute lung injury
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
siyang Xiao1,*
Wenxin Zhang1,*
hongjin chen1
Bo Fang1
Purpose: The purpose of this study was to design and synthesize novel 2-benzylidene-1-
indanone derivatives for treatment of acute lung injury.
Methods: A series of 39 novel 2-benzylidene-indanone structural derivatives were synthesized
and evaluated for anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated murine
primary macrophages.
Yinda Qiu2
Results: Most of the obtained compounds effectively inhibited the LPS-induced expression
of IL-6 and TNF-α. The most active compound, 8f, was found to significantly reduce LPS-
induced pulmonary inflammation, as reflected by reductions in the concentration of total protein,
inflammatory cell count, as well as the lung wet/dry ratio in bronchoalveolar lavage (BAL) fluid.
Furthermore, 8f effectively inhibited mRNA expression of several inflammatory cytokines after
LPS challenge in vitro and in vivo. Administration of 8f also blocked LPS-induced activation
of the proinflammatory NF-κB/MAPK signaling pathway.
Xianxin chen1
lingfeng chen1
sheng shu1
Yali Zhang1
Yunjie Zhao1
Zhiguo liu1
guang liang1
Conclusion: The simple synthetic preparation and biological properties of these derivatives
make these 2-benzylidene-indanone scaffolds promising new entities for the development of
anti-inflammatory therapeutics for the treatment of acute lung injury.
1chemical Biology research center,
school of Pharmaceutical sciences,
Wenzhou Medical University,
Wenzhou, Zhejiang, china; 2college
of life and environmental science,
Wenzhou University,Wenzhou,
Zhejiang, china
Keywords: indanone, acute lung injury, drug design, anti-inflammation, synthesis
Introduction
Acute lung injury (ALI) is characterized by neutrophil infiltration into the lung
interstitium and airways, and the development of several disorders including pneu-
monia, aspiration syndromes, sepsis, and major trauma and shock.1,2 Despite extensive
research, the mortality rate from ALI remains high, at ~40%, which is similar to that of
breast cancer. Effective methods of treating acute inflammatory disease are extremely
limited at present.3 Increasing evidence indicates that dysregulation of cytokines is
an important step in mediating, amplifying, and perpetuating ALI processes. These
cytokines mainly include tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β,
IL-12, and IL-8.4,5 Observations strongly suggest that the inflammatory processes
occur in association with lung injury, and effective treatment should be given early
in the ALI development process. Therefore, the development of anti-inflammatory
candidates to inhibit the excessive production of cytokines, including IL-6 and TNF-α,
has been a promising strategy for the prevention and treatment of ALI. This research
has also provided a new direction for anti-inflammatory drug development.6,7
*These authors contributed equally
to this work
correspondence: Yunjie Zhao;
Zhiguo liu
chemical Biology research center,
school of Pharmaceutical sciences,
Wenzhou Medical University,
1210 University Town, Wenzhou,
Zhejiang 325035, china
Tel +86 577 8669 9892
Fax +86 577 8669 9892
email aabye1100@aliyun.com;
lzgcnu@163.com
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http://dx.doi.org/10.2147/DDDT.S160314

Xiao et al
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Xanthohumol (XAN) is a major chalcone derivative IL-6 (% inhibition=69.28%) in RAW 264.7 macrophages.
isolated from hops (Humulus lupulus L.), which contains To improve the inhibition of IL-6, we further performed a lead
the bitter flavors commonly used in brewing and medicine.8 optimization program to modify the A ring of the 4d to yield
XAN displays a wide range of biological activities, including 19 new 2-benzylidene-1-indanone derivatives. Preliminary
anticancer,9,10 anti-oxidation,11 and properties that prevent structure–activity relationship (SAR) analyses were performed
and treat diabetes.12,13 Moreover, XAN has been reported to outline the relationships between structures and their
to have additional anti-inflammatory effects via the down- pharmacological activity. The active compound 8f showed
regulation of NF-κB activation in lipopolysaccharide improved anti-inflammatory activities in vitro and significant
(LPS)-stimulated RAW264.7 macrophages.14 Unfortunately, therapeutic effects in a mouse model, suggesting the potential
XAN has an acute problem due to poor solubility in an of 2-benzylidene-1-indanone derivatives for the development
aqueous solution and poor bioavailability (,1%), which of new anti-inflammatory agent for the treatment of ALI.
poses a severe limitation for pharmacological application.15
Materials and methods
1-indanone, with a benzocyclopentanone skeleton, is a natu-
rally occurring component found in various edible natural chemical experimental procedures
plants and is a useful synthons for synthesis of various The syntheses of the 2-benzylidene-1-indanone deriva-
carbocyclic and heterocyclic molecules. Thus, it may be used tives (4a-t and 8a-s) are summarized in Schemes 1 and 2.
in the preparation of key intermediates for several drugs and As depicted in Scheme 1, tetrahydropyranyl (THP) protection
natural products.16,17 Studies have been performed showing of commercially available 6-hydroxy-1-indanone (1) obtained
that compounds possessing a 1-indanone moiety are of 6-[(tetrahydro-2H-pyran-2-yl)oxy]-1-indanone (2), which
great pharmacological importance, as they exhibit various was subjected to a key Claisen Schmidt condensation with
beneficial biological activities, including anti-inflammatory, various benzaldehydes to give THP protected 2-benzylidene-
anticancer, antioxidant, and antimicrobial properties.18,19 1-indanone intermediates (3a-b, 3e-t) in the presence of
In addition, several important drugs contain 1-indanone as a ethanolic sodium hydroxide. Deprotection of the above inter-
core moiety, such as Donepezil, which in 1996 was approved mediates with hydrochloric acid in ethanol gave the desired
by the US Food and Drug Administration (FDA) for the treat- 2-benzylidene-1-indanone derivatives 4a-b and 4e-t. Alterna-
ment of moderate-to-severe Alzheimer’s disease.20
tively, Aldol condensation between 6-hydroxy-1-indanone (1)
Considering the structural similarity that exists between and hydroxyl-benzaldehydes using hydrochloric acid gas as a
chalcone and 1-indanone, and since the hydroxyl group catalyst gave the products 4c and 4d. Similarly, as described
contributes to improving the pharmacological profile of in Scheme 1, a condensation reaction catalyzed by acid or
bioactive compounds, we designed and synthesized 20 base was utilized to synthesize the desired 2-benzylidene-1-
novel 2-benzylidene-1-indanone derivatives by substituting indanone derivatives 8a-s with 4-hydroxy-3-methoxybenz-
a hydroxyl group at the C-6 position of indanone (Figure 1). aldehyde and substituted 1-indanones as the start materials
In vitro profiling indicated that 4d showed the promising (Scheme 2). All of the target compounds were characterized
1
anti-inflammatory activity against LPS-induced TNF-α by H-NMR, 13C-NMR, and ESI-MS. The analytical and
expression (% inhibition=83.73%), but lacked potency in spectral data of the final compounds are in full agreement
HO
OH
OH
OH
OMe
R1
OMe O
B
Structural
modification
Xanthohumol
Drug design
A
R2
HO
O
O
6-hydroxy-2-benzylidene-
1-indanone derivatives
2-benzylidene-1-indanone derivatives
O
1-indanone
Figure 1 Design strategy based on 1-indanone and anti-inflammatory natural product XAN.
Abbreviation: Xan, xanthohumol.
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2-benzylidene-1-indanone derivatives for acute lung injury
R1
R1
O
O
O
O
HO
HO
THPO
THPO
a
b
c
1
2
3a-b, 3e-t
4a-t
d
Synthesis of compounds 4c and 4d
Scheme 1 general synthetic method for 6-hydroxyl-2-benzylidene-1-indanone derivatives 4a-t. reagents and conditions: (a) 3,4-dihydro-2H-pyran, PPTs, 4 h, ch2cl2, 40°c;
(b) various benzaldehydes, etOh, 20% (w/v) naOh, room temperature, overnight; (c) 1.0 mol/l hcl, etOh, room temperature, 5 h; (d) hcl (gas), etOh, room temperature,
overnight.
with the proposed structures. The detailed synthesis and (San Diego, CA, USA). C57BL/6 mice were intraperitoneally
spectral characterization of new or unreported compounds injected with 6% thioglycollate solution (0.3 g beef extract,
are described in the Supplementary materials.
1 g tryptone, 0.5 g NaCl dissolved in 100 mL ddH2O, and
filtrated through 0.22 µm filter membrane, 3 mL per mouse)
and kept in pathogen-free conditions for 3 days before the
isolation of murine peritoneal macrophages (MPMs). Total
MPMs were harvested by washing the peritoneal cavity
with RPMI-1640 medium (Gibco/BRL life Technologies,
Eggenstein, Germany), centrifuged, and suspended in RPMI-
1640 medium (Gibco/BRL life Technologies), with 10%
fetal bovine serum (Hyclone, Logan, UT, USA), 100 U/mL
penicillin, and 100 mg/mL streptomycin. Nonadherent cells
were removed by washing with medium 4 h after seeding.
Before treatment, MPMs were cultured in 35 mm plates and
incubated overnight at 37°C in 5% CO2-humidified air.
animals
The animal protocol was approved by Wenzhou Medical
University Animal Policy and Welfare Committee (Approval
documents: 2013/APWC/0361). Male C57BL/6 mice
weighing 18–22 g were purchased from the Animal Center
of Wenzhou Medical University (Wenzhou, China). Animals
involved in this experiment were treated in accordance
with the Guide for Care and Use of Laboratory Animals of
National Institutes of Health. Animals were maintained in
sterile microisolator cages and fed autoclaved-pelleted diet
and water ad libitum.
Anti-inflammatory evaluation of synthetic
cells and reagents
LPS were purchased from Sigma (St Louis, MO, USA). compounds
Saline was prepared as 0.9% NaCl solution. The mouse IL-6 The inhibition effects of IL-6 and TNF-α release of novel
enzyme-linked immunosorbent assay (ELISA) kit and mouse 2-benzylidene-1-indanone derivatives were evaluated in
TNF-α ELISA kit were purchased from eBioscience, Inc. LPS-stimulated MPMs, as previously described.21 After
OH
O
O
OMe
OH
a
+
HO
HO
OHC
OMe
5
6: 7-OH
7: 5-OH
8a: 7-OH
8b: 5-OH
b
OTHP
OH
O
O
OMe
OMe
OTHP
OMe
d
c
R2
R2
OHC
9
10c-s
8c-s
Scheme 2 general synthetic method for 2-benzylidene-1-indanone derivatives 8a-s; reagents and conditions: (a) hcl (gas), etOh, room temperature , overnight; (b) 3,4-dihydro-
2H-pyran, PPTs, 4 h, ch2cl2, 40°c; (c) various benzaldehydes, etOh, 20% (w/v) naOh, room temperature, overnight; (d) 1.0 mol/l hcl, etOh, room temperature, 5 h.
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treatment of cells with indicated compounds and LPS, the
TNF-α and IL-6 levels in medium were determined by an
ELISA kit (eBioScience, San Diego, CA, USA) according to
the manufacturer’s instructions. Briefly, cells were seeded into
six-well plates at a density of 5×105 cells per well in Dulbecco’s
Modified Eagle’s Medium and incubated at 37°C in 5% CO2 for
24 h. Cells were pretreated with 10 µM compounds for 30 min,
then treated with LPS (0.5 µg/mL) for 24 h. After treatment, the
culture media and cells were collected separately. The levels
of TNF-α and IL-6 in the media were determined by ELISA.
The total protein in cultural plates was collected, and the
concentrations were determined using Bio-Rad protein assay
reagents (Bio-Rad Laboratories; Hercules, CA, USA). The
total amount of the inflammatory factor in the media was nor-
malized to the total protein amount of the viable cell pellets.
IL-6 sense: 5′-GAGGATACCACTCCCAACA
GACC-3′;
antisense: 5′-AAGTGCATCATCGTTGTTCAT
ACA-3′.
IL-1β sense: 5′-ACTCCTTAGTCCTCGGCCA-3′
antisense: 5′-CCATCAGAGGCAAGGAGGAA-3′
β-actin sense: 5′-TGGAATCCTGTGGCATCCATG
AAAC-3′;
antisense: 5′-TAAAACGCAGCTCAGTAACAG
TCCG-3′.
VCAM-1 sense primer: 5′-TGCCGAGCTAAATTAC
ACATTG-3′;
antisense primer: 5′-CCTTGTGGAGGGATGTAC
AGA-3′.
ICAM-1 sense primer: 5′-GCCTTGGTAGAGGTG
ACTGAG-3′;
antisense primer: 5′-GACCGGAGCTGAAAA
GTTGTA-3′.
assessment of cytotoxicity of synthetic
compounds
To assess the safety of the 2-benzylidene-1-indanone deriva-
The amount of each gene was determined and normalized
tives, compounds were tested for their toxicity in the MPMs by the amount of β-actin.
by MTT at a concentration of 10 µM. MPMs were cultured
in 96-well plates at a density of 5,000 cells per well in 1640 lPs-induced ali in mice
medium, supplemented with 10% heat inactivated serum, The 32 male C57BL/6 mice were randomly divided into four
100 U/mL penicillin, and 100 mg/mL streptomycin. The groups, on average: the control group, LPS group, 8f+LPS
cells were incubated at 37°C in 5% CO2-humidified air. group, and XAN+LPS group. The mice in the 8f+LPS and
Compounds were dissolved in dimethyl sulfoxide (DMSO) XAN+LPS groups were oral pretreated with 8f (dissolved in
and diluted with 1640 medium to the final concentrations 0.5% CMC-Na) and XAN (dissolved in 0.5% CMC-Na) at a
of 10 µM and incubated for 24 h before the MTT assay.22 dosage of 20 mg/kg/day, respectively, for three consecutive
The solution of MTT (5 mg/mL) dissolved in NaCl solution days. The control-Vehicle and LPS-Vehicle groups were
(0.9%) was added to each well of the 96-well plate and incu- given the same volume of 0.5% CMC-Na. After being anes-
bated for 4 h. After treatment with DMSO (150 µL/well), the thetized by diethyl ether, the mice from the LPS, 8f+LPS,
plate was analyzed in a multi-well-plate reader at 490 nm.
and XAN+LPS groups were intratracheally instilled with
LPS (5 mg/kg) to induce lung injury. Mice were killed under
ether anesthesia at 6 h after LPS instillation. Bronchoalveo-
real-time quantitative Pcr
CellswerehomogenizedinTRIZOLkit(Invitrogen, Carlsbad, lar lavage fluid, serum, and lung tissues were collected for
CA) for extraction of RNA according to each manufacturer’s further tests.
protocol. Both reverse transcription and quantitative PCR
were carried out using a two-step M-MLV Platinum SYBR histological analysis
Green qPCR SuperMix-UDG kit (Invitrogen). Eppendorf Lung tissues were fixed in 4% paraformaldehyde solution,
Mastercycler ep realplex detection system (Eppendorf, embedded in paraffin, and ectioned at 5 µm. After dehy-
Hamburg, Germany) was used for quantitative PCR analysis. dration, tissues were stained with hematoxylin and eosin
The primers of genes including TNF-α, IL-6, IL-1β, (H&E) and observed with a Nikon Eclipse E800 microscope
VCAM-1, ICAM-1, and β-actin were synthesized by Invit- (200× amplification; Nikon). The severity of microscopic
rogen. PCR primers were designed using Primer Premier injury was graded from 0 (normal) to 4 (severe), based on
Version 5.0 software (Premier Biosoft, Palo Alto, CA, USA), the following categories: neutrophil infiltration, interstitial
and sequences were as follows (Invitrogen):
TNF-α sense: 5′-TGGAACTGGCAGAAGAGG-3′;
antisense: 5′-AGACAGAAGAGCGTGGTG-3′.
edema, hemorrhage, and hyalinemembrane. The sum of
all scores was combined to calculate a composite score, as
described previously.23
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2-benzylidene-1-indanone derivatives for acute lung injury
immunohistochemistry analysis
antibodies(1:3,000)for1hatroomtemperature.Blotswerethen
Lung tissues (5 µm thickness) were prepared, deparaffinized visualizedusingenhancedchemiluminescencereagent(Bio-Rad
in xylene, and hydrated using an ethanol gradient. A pressure Laboratories). The density of the immunoreactive bands was
cooker was used for heat-induced antigen retrieval (10 mM analyzed using ImageJ software (NIH, Bethesda, MD, USA).
sodium citrate buffer, pH 6.5). After treatment with 30% of
statistical analysis
hydrogen peroxide, all sections were blocked in 5% bovine
Data are expressed as the mean ± standard error of the mean
(SEM). Student’s t-test was employed to analyze the differ-
ences between sets of data. Statistics were performed using
GraphPad Pro (GraphPad, San Diego, CA, USA). P-values
less than 0.05 (P,0.05) were considered indicative of signifi-
cance. All experiments were repeated at least three times.
serum albumin and incubated with primary anti-F4/80 and
anti-LY-6G antibody overnight at 4°C. The slides were then
incubated with HRP-labeled secondary antibody for 1 h.
After the sections were incubated with 3,3-diaminobenzidine
tetrahydrochloride for color development and counterstained
with hematoxylin, the slides were evaluated under a micro-
scope (200× amplification; Nikon). The percentage of F4/80
anti-LY-6G-positive inflammatory cells was calculated in
five randomly chosen fields (200× amplification; Nikon)
per section.
Results and discussion
Evaluation of anti-inflammatory and
cytotoxic activities
LPS is the most potent innate immune-activating stimuli
in the outer membrane of Gram-negative bacteria, which
Wet/dry ratio24
The lung wet-to-dry (W/D) ratio was calculated to observe induces the activation of monocytes and macrophages,
the pulmonary edema. At the end of the experiment, animals resulting in the increased production of proinflammatory
were sacrificed at 6 h after LPS challenge. After the middle cytokines including TNF-α, IL-6, and IL-12.25,26 Evi-
lobe of the right lung was collected, the wet weight was dence from experimental and clinical studies shows that
recorded. Then the lung was heated in a thermostatic oven these proinflammatory cytokines play a significant role in
at 65°C for 48 h and weighed to determine the baseline lung accelerated inflammation-mediated ALI pathogenesis.27
dry mass levels.
All the synthesized 2-benzylidene-1-indanone derivatives
were evaluated for their in vitro anti-inflammatory activity
toward TNF-α and IL-6 release in LPS-stimulated MPMs,
and XAN was used as a positive control. The macrophages
Protein concentration in bronchoalveolar
lavage fluid (BALF)
BALF was obtained at the end of the experimental by were preincubated for 30 min with 10 µM test compounds,
irrigating the left lung with saline (4 × 200 µL). This fluid XAN, and DMSO, which was used as the control medium.
was centrifuged at 1,000 rpm for 10 min, and the protein Subsequently, the cells were incubated with LPS (0.5 µg/mL)
concentration in the supernatant was determined using a for 24 h at 37°C. The ability (% inhibition) of the tested
BCA protein assay (Pierce, Rockford, IL, USA).
compounds to reduce proinflammatory cytokines IL-6 and
TNF-α by ELISA is summarized in Table 1. The majority
of these derivatives showed moderate potency against both
Western blot analysis
Macrophages were pretreated with vehicle (DMSO) or the LPS-induced expression of IL-6 and TNF-α, and had a pro-
new compounds of interest at 10 µM for 30 min, followed by nounced increase on IL-6 expression. Notably, compound
incubation with LPS (0.5 µg/mL) for 30 min. Then, cells were 4d, with an installment of 4-hydroxy and 3-methoxy groups
lysed and total protein was collected. Bradford assay (Bio-Rad at R1, exhibited the most potent effect on LPS-induced IL-6
Laboratories)wasusedtodetecttheproteinconcentration.After and TNF-α expression, and therefore was chosen to be the
balancing and transgender, protein samples were separated lead molecule for the next structural optimization.
by 10% SDS-PAGE and transferred onto a PVDF membrane
SAR of benzene ring B: identification of
(Bio-Rad Laboratories). Membranes were blocked with 5%
milk in Tris-buffered saline containing 0.05% Tween-20 for lead 4d
1.5hatroomtemperature. Primaryantibodieswereappliedand To the best our knowledge, data are very limited about the
membranes incubated overnight at 4°C. The membranes were cytokine-inhibitory effects of 2-benzylidene-1-indanone
then washed in Tris-buffered Saline with Tween 20 (TBST) derivatives and their SAR. Compounds 4a, with a 4′-methoxy
and reacted with secondary horseradish peroxidase-conjugated group in the benzene ring B, had an equivalent potency to
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Table 1 2-benzylidene-1-indanone derivatives 4a-t inhibited lPs-induced il-6 and TnF-α secretion in MPM
5′
R1
6″
4
3
1′
2
2′
HO
1
6
O
4a-t
Compound
R1
(%) Inhibition (10 µM)*
Compound
R1
(%) Inhibition (10 µM)*
IL-6
TNF-α
IL-6
TNF-α
4a
4b
4c
4d
4e
4f
4′-OMe
4′-Oet
3′,4′-Oh
3′-OMe, 4′-Oh
2′,3′-OMe
2′,4′-OMe
3′,4′,5′-OMe
2′,3′,4′-OMe
2′,4′,6′-OMe
2′,4′,5′-OMe
2′-F
61.61±6.21
35.54±1.21
70.32±3.27
69.28±1.58
41.35±1.70
52.29±2.09
55.69±1.63
55.33±9.97
54.21±1.02
54.78±2.28
41.28±1.98
48.26±1.14
14.42±6.14
45.59±3.59
83.73±4.05
34.13±3.19
44.52±3.61
67.94±1.21
67.92±3.85
44.16±3.58
28.40±1.82
15.68±3.64
4l
2′-cl
2′-Br
3′-Br
4′-Br
45.77±1.79
63.04±2.13
47.37±6.65
53.71±3.29
59.99±1.32
52.24±5.33
17.39±1.51
54.08±1.13
47.37±3.16
62.45±1.83
26.36±4.99
na
4m
4n
4o
4p
4q
4r
17.87±1.89
29.40±0.61
41.99±1.44
36.07±2.03
18.93±1.19
33.89±5.37
36.51±2.91
53.27±1.65
4′-F
4′-cF3
3′,4′-F
3′,4′-cl
3′-nO2, 4′-cl
4g
4h
4i
4s
4t
4j
Xan
4k
Notes: Macrophages were plated at a density of 5.0×105/plate at 37°c and 5% cO2 overnight. cells were pretreated with vehicle and compounds (10 µM) for 30 min, and
subsequently treated with lPs (0.5 µg/mL) for 24 h. The culture media were collected and the inflammatory cytokines, TNF-α and il-6, in media were detected by elisa
and normalized by total protein concentration. *Values are the mean of at least n=3 independent experiments±seM.
Abbreviations: il-6, interleukin-6; lPs, lipopolysaccharide; TnF-α, tumor necrosis factor-α; MPM, murine primary macrophage; elisa, enzyme-linked immunosorbent
assay; na, no activity; Xan, xanthohumol; seM, standard error of the mean.
XAN in either IL-6 or TNF-α. The increasing size of the C-4′ particular, compound 4r (3′,4′-fluoro) expressed the weakest
substituents from the methoxy to the ethyl group (4b) signifi- anti-inflammatory capacity (17.39% and 18.93% inhibitory
cantly decreased both IL-6 and TNF-α inhibitory properties rates for IL-6 and TNF-α, respectively). This suggests that
from 61.61% (for IL-6), 48.6% (for TNF-α) for compound mono- or di-substitution of electron-withdrawing functional
4a to 35.54% (for IL-6), and 14.42% (for TNF-α), indicating groups on benzene ring B is not tolerated.
that a larger alkoxy group at this position was not well-
Optimization at the benzene ring a of
tolerated in the anti-inflammatory activity. Incorporation of
3′, 4′-hydroxy groups in the benzene ring B yields compound 1-indanone to obtain 8f
4c, which exhibited a significant gain in the IL-6 inhibitory Tofurtherimprovetheanti-inflammatoryactivityand,inpartic-
profile (% Inhibition=69.28%) compared to compound 4a. ular, inhibition on IL-6, we next directed our optimization
Methylation of the 3′-hydroxy group of compounds 4c led to effort to modify the benzene ring A of 4d by introducing
compound 4d, which was equipotent in IL-6 inhibition with different types of groups. These results are shown in Table 2.
4c, and resulted in an approximate 1.8-fold increase in TNF-α Incorporation of hydroxyl group at the 7- and 5-position of
activity. This indicates that a methoxy group at this position benzene ring A led to compounds 8a and 8b, which, respec-
is preferred for the activity. Disappointedly, introduction of tively, showed either markedly reduced or no activity. Interest-
di- or trimethoxy groups on ring B yielded compounds 4e ingly, methoxylated compounds 8c-8e displayed almost equal
(2′, 3′-methoxy), 4f (2′, 4′-methoxy), 4g (3′, 4′, 5′-methoxy), IL-6 and TNF-α inhibitory activity to that of 8a. However,
4h (2′, 3′, 4′-methoxy), 4i (2′, 4′, 6′-methoxy), and 4j (2′, to our surprise, compound 8f, with a 5-, 6-dimethoxyl sub-
4′, 5′-methoxy), which exhibited a dramatic decrease in stitution at ring A displayed the most promising effect on
their IL-6 or TNF-α inhibitory activity, with inhibitory rate LPS-induced IL-6 and TNF-α production, with its inhibitory
values ranging from 26.47% to 67.94%. Comparatively, rates reaching 81.41% and 79.29%, respectively. Meanwhile,
electron-withdrawing substituted compounds exhibited very etherification at the 4-, 5-, 6-, or 7-hydroxyl group with
weak or no activity. For example, about one- and three-fold O-propyl or O-isopropyl moieties produced compounds
lower IL-6 and TNF-α inhibitory profiles were observed for 8g-8n. All of the compounds, except for 8h, 8m, and 8n, had
4k−4t, respectively, compared to the control XAN, and, in marked improvement in blocking TNF-α production, with
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2-benzylidene-1-indanone derivatives for acute lung injury
Table 2 2-benzylidene-1-indanone derivatives 8a-s inhibited lPs-induced il-6 and TnF-α secretion in MPM
OH
OMe
R2
O
8a-s
Compound R2
(%) Inhibition (10 µM)* HL7702 cell
IL-6 TNF-α Survival rate (%)
Compound R2
(%) Inhibition (10 µM)* HL7702 cell
IL-6 TNF-α Survival rate (%)
4d
8a
8b
8c
8d
8e
8f
6-Oh
69.12±4.29 80.80±3.33 77.43±3.69
40.00±1.65 44.65±1.31 84.92±4.16
8j
4-OPr
69.13±1.76 41.63±1.38 93.96±7.95
83.17±0.60 62.85±1.05 70.03±7.54
78.45±1.46 56.85±7.76 89.64±6.97
62.53±4.05 16.67±4.75 97.62±1.87
49.99±1.77 35.92±2.38 75.61±5.71
50.05±4.75 29.12±2.48 85.90±6.81
7-Oh
8k
8l
7-O(i-Pr)
6-O(i-Pr)
5-O(i-Pr)
4-O(i-Pr)
6-n(et)2
5-Oh
17.15±2.77 na
82.68±5.95
6-OMe
5-OMe
4-OMe
50.07±4.14 37.71±1.65 96.36±3.84
40.04±1.60 48.27±2.33 98.31±1.92
56.36±2.05 60.27±2.04 82.75±4.23
8m
8n
8o
8p
8q
8r
8s
5,6-OMe 81.41±3.68 79.29±2.55 91.09±1.12
6-n(-ch2-)4 74.11±3.61 68.93±0.99 96.47±3.13
6-n(-ch2-)5 69.71±2.32 39.14±3.27 99.01±2.03
6-n(-ch2-)6 62.50±4.48 56.37±4.22 98.58±8.14
6-nh-cOMe 18.60±2.88 24.78±1.66 85.85±1.65
8g
8h
8i
7-OPr
6-OPr
5-OPr
84.92±1.10 71.99±0.88 74.06±4.28
57.74±5.84 55.36±6.48 97.00±1.69
76.62±2.67 na
92.83±6.16
Notes: Macrophages were plated at a density of 5.0×105/plate at 37°c and 5% cO2 overnight. cells were pretreated with vehicle and compounds (10 µM) for 30 min, and
subsequently treated with lPs (0.5 µg/mL) for 24 h. The culture media were collected and the inflammatory cytokines, TNF-α and il-6, in media were detected by elisa
and normalized by total protein concentration. *Values are the mean of at least n=3 independent experiments±seM.
Abbreviations: il-6, interleukin-6; lPs, lipopolysaccharide; TnF-α, tumor necrosis factor-α; MPM, murine primary macrophage; elisa, enzyme-linked immunosorbent
assay; na, no activity; seM, standard error of the mean.
inhibitory rates ranging from 69.13% (for 8j) to 84.92% (for group on the benzene ring, these compounds exhibited a sig-
8g), but most of these compounds (except for 8g) exhibited nificantly decreased anti-inflammatory ability (18.60% and
dramatically reduced 1.3–5-fold IL-6 inhibition compared 24.78%, for IL-6 and TNF-α, respectively). All other deriva-
with that of 4d. This result indicates that the three-carbon tives, including those with different size of diethylamino
chain alkoxy groups at the C-7 position (for 8g and 8k) were (8o), butylamine (8p), pentenylamino (8q), and hexylamino
beneficial to the activity. Further screening of substituents (8r) groups, displayed comparable IL-6 but diminished
around 4d revealed that incorporation of various substituted TNF-α inhibitory activities compared to 4d. Illustration of
amino groups at the o-position on the phenyl ring provided the structure–activity relationship of these 2-benzylidene-1-
compounds 8o-8s. Except for 8s, which has an acetylamino indanone derivatives is presented in Figure 2.
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Figure 2 Structure–activity relationships of 2-benzylidene-1-indanone derivatives. Anti-inflammatory activity was measured as inhibition on LPS-induced IL-6 and TNF-α
production.
Abbreviations: il-6, interleukin-6; lPs, lipopolysaccharide; TnF-α, tumor necrosis factor-α.
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Before further bioactivity evaluation, derivatives 8a-8s promising compounds 4d, 8f, and 8g were selected to assess
and 4d were tested for their cytotoxicity and safety in the dose-dependent inhibitory effects against LPS-induced
human normal hepatic cell line, HL-7702, by MTT after TNF-α and IL-6 release. MPMs were pretreated with
treatment of the cells for 24 h with compounds at a concentra- compounds in a series of concentrations (1.25, 2.5, 5, and
tion of 10 µM. As shown in Table 2, all of these compounds 10 µM) for 30 min and were subsequently incubated with
showed no significant toxicity in hepatic cells, indicating that LPS (0.5 µg/mL) for 24 h. The release of IL-6 and TNF-α
they are relatively safe.
was determined by ELISA. As shown in Figure 3A and B,
compounds 4d, 8f, 8g, and XAN exhibited dose-dependent
inhibition on both TNF-α and IL-6 release stimulated by
LPS. Compound 8f showed the strongest inhibition against
the expression of both IL-6 and TNF-α. These results
active compounds inhibit lPs-induced
cytokines release in a dose-dependent
manner
To further confirm the anti-inflammatory effect of synthetic further support the potential of these compounds as anti-
2-benzylidene-1-indanone derivatives in vitro, the most inflammatory agents.
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Figure 3 compounds 8f, 8g, 4d, and Xan inhibited lPs-induced il-6 release in a dose-dependent manner. MPMs were plated at a density of 5.0×105/plate overnight in
37°c and 5% cO2. cells were pretreated with compounds in a series concentration of 1.25, 2.5, 5, and 10 µM for 30 min and subsequently incubated with or without lPs
(0.5 µg/ml) for 24 h. (A) TnF-α and (B) il-6 levels in the culture medium were measured by elisa and were normalized by the total protein. The results were presented
as the percentage of lPs control. each bar represents the mean ± SEM of the three independent experiments. Statistical significance relative to the LPS group was indicated,
*P,0.05; **P,0.01, ***P,0.001.
Abbreviations: Xan, xanthohumol; lPs, lipopolysaccharide; TnF-α, tumor necrosis factor-α; MPMs, murine primary macrophages; elisa, enzyme-linked immunosorbent
assay; seM, standard error of the mean; cOn, control group.
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2-benzylidene-1-indanone derivatives for acute lung injury
active compounds suppressed mrna
expressions in lPs-stimulated
macrophages
effects of 8f on lPs-induced lung
pathophysiologic changes in ali mice
LPS has been used extensively to induce animal models of
ALI, which closely resembles human ALI. LPS-induced
ALI is characterized by rapid alveolar injury, vascular
leakage, lung inflammation, neutrophil accumulation, and
induced cytokines production, leading to lung edema.28 To
investigate the possible mechanism underlying the protective
effect of 2-benzylidene-1-indanone derivatives, we further
determined the concentration of total protein, inflammatory
cell count, inflammatory mediator count, lung W/D ratio,
and neutrophil count, to evaluate whether the representative
compounds were able to relieve lung injury in the BALF of
LPS-induced ALI mice. Compound 8f, which demonstrated
the highest activities and low cytotoxicity, was selected for
this study. The details of animal experiments were described
in the experimental section.
To determine whether the decrease in LPS-induced mac-
rophages might be associated with transcriptional inhibition,
messenger RNA (mRNA) levels of TNF-α, IL-6, IL-1β,
and intercellular adhesion molecule 1 (ICAM-1) were mea-
sured by a real-time quantitative PCR (RT-qPCR) method.
Macrophages were treated with LPS (0.5 µg/mL) for 6 h
and examined for the expression of proinflammatory genes
with or without compounds 4d, 8f, or 8g as treatment. The
results, summarized in Figure 4A–D clearly demonstrate that
the mRNA levels of TNF-α, IL-6, IL-1β, and ICAM-1 were
all upregulated by LPS incubation. Except for the inhibitory
effects against IL-1β, pretreatment with the three compounds
effectively downregulate the expressions of TNF-α, IL-6,
IL-1β, and ICAM-1 with statistical significance. In particular,
compound 8f was the most active compound in inhibiting
mRNA expressions of those inflammatory mediators. Sub-
sequently, we used ELISA to analyze the effect of active
compounds on the production of TNF-α and IL-6 in cell
lysates after LPS challenge. As shown in Figure 4E and F,
compounds 4d, 8f, 8g, and XAN significantly reduced the
production of TNF-α and IL-6, compared with the LPS
The results in Figure 5 indicated that total cells
(Figure 5A), total protein concentration (Figure 5B), lung
W/D ratio (Figure 5C), and neutrophils (Figure 5D) sig-
nificantly increased in the BALF of ALI mice after LPS
challenge, compared to those of the control group. Pretreat-
ment with 8f or XAN at 20 mg/kg was found to significantly
decrease the relative amount of cells, neutrophils, and the
group. These data provide evidence for the anti-inflammatory total protein concentration compared with the LPS group, and
effect of 2-benzylidene-1-indanones 4d, 8f, and 8g, which also slightly inhibited the lung W/D ratio in BALF. We also
partly affect the cytokine profile at the mRNA level.
determined TNF-α and IL-6 levels in the blood and tissues
A
D
B
E
C
IL-6
TNF-α
IL-1β
150
100
50
150
100
50
150
100
50
**
**
**
***
***
0
0
0
CON LPS 8f
8g
4d
CON LPS 8f
8g
4d
CON LPS 8f
8g
4d
F
ICAM-1
TNF-α
IL-6
150
100
50
150
100
50
150
100
50
*
**
*
**
**
**
***
**
***
***
0
0
0
CON LPS 8f
8g
4d
CON LPS 8f 8g 4d XAN
CON LPS 8f 8g 4d XAN
Figure 4 Active compounds inhibited the inflammatory genes expression and intracellular inflammatory factor by LPS. The mRNA levels of the inflammatory mediators
(A) TnF-α, (B) il-6, (C) il-1β, and (D) ICAM-1 were quantified by RT-qPCR. The mRNA values for each gene were normalized to internal control β-actin mrna and were
expressed as a ratio to lPs. each bar represents mean ± SEM of three to five independent experiments. (E) The cell lysate level of the cytokine TnF-α. (F) The cell lysate
level of the cytokine IL-6. Statistical significance relative to LPS group was indicated, *P,0.05, **P,0.01, ***P,0.001.
Abbreviations: IL-6, interleukin-6; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid; TNF-α, tumor necrosis factor-α; icaM-1, intercellular adhesion molecule 1;
rT-qPcr, real-time quantitative Pcr; seM, standard error of the mean; cOn, control group; Xan, xanthohumol.
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Figure 5 8f and Xan attenuate the lPs-induced ali in mice. Mice were administrated with 8f and Xan for three consecutive days before given intratracheal
instillation of LPS, 6 h later mice were anesthetized and killed. Bronchoalveolar lavage fluid and lung tissues were collected for further tests. (A) Total amount of cells
in BalF. (B) Protein concentration in BalF. (C) W/D ratio. (D) The number of neutrophils in BalF. (E) serum level of the cytokine TnF-α. (F) serum level of the
cytokine il-6. (G) BalF level of the cytokine TnF-α. (H) BALF level of the cytokine IL-6. Statistical significance relative to the LPS group was indicated, n$5, *P,0.05,
**P,0.01.
Abbreviations: XAN, xanthohumol; LPS, lipopolysaccharide; ALI, acute lung injury; BALF, bronchoalveolar lavage fluid; W/D, wet/dry; TNF-α, tumor necrosis factor-α;
cOn, control group.
of LPS-induced ALI mice. As shown in Figure 5E and F,
compound 8f ameliorated
histopathological changes and reduced
mrna levels in lung tissues of
lPs-induced ali mice
To verify the inhibitory effect of 8f against macrophage
serum TNF-α and IL-6 levels after LPS injection were signif-
icantly elevated in comparison with the control group, while
pretreatment with 8f markedly prevented such increases.
Oppositely, XAN treatment has no significant statistical dif-
ference in inhibiting LPS-induced TNF-α and IL-6 activities.
To further evaluate the anti-inflammatory actions of 8f,
infiltration into the lung tissue, we further performed immu-
nohistochemistry analysis with F4/80 and LY-6G, which
are macrophage and neutrophils markers.28,29 As shown in
levels of cytokines TNF-α and IL-6 were measured in BAL
fluid collected from ALI mice. As illustrated in Figure 5G
Figure 6A and C, LPS alone induced a significant infiltration
and H, TNF-α and IL-6 were all elevated in BAL fluid in
of F4/80 and LY-6G-positive macrophages and neutrophils
in the lung sections. In contrast, macrophage and neutrophils
infiltration were significantly ameliorated after treatment with
8f (20 mg/kg) and the positive control (XAN, 20 mg/kg).
Subsequently, we evaluated histological changes after 8f treat-
ment in LPS-challenged mice by H&E staining (Figure 6B).
response to LPS challenge compared with native mice.
Treatment with 8f alone significantly decreased the levels of
cytokines TNF-α and IL-6, but no statistical difference was
observed in the XAN treatment group. The results indicated
that 8f has a remarkable protective effect on LPS-induced
ALI in mice.
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2-benzylidene-1-indanone derivatives for acute lung injury
A
CON
LPS
LPS+8f (20 mg/kg)
LPS+XAN (20 mg/kg)
100 µm
100 µm
100 µm
100 µm
100 µm
100 µm
100 µm
B
C
100 µm
100 µm
100 µm
100 µm
100 µm
D
TNF-α
IL-6
IL-1β
VCAM-1
150
250
200
150
100
50
200
200
150
100
50
150
100
50
100
50
0
**
**
0
0
0
CON LPS
8f
XAN
CON LPS
8f
XAN
CON LPS
8f
XAN
CON LPS
8f
XAN
Figure 6 The 8f and XAN inhibited macrophages in filtration through F4/80-staining, LY-6G-staining, ameliorated histopathological changes, and inflammatory gene expression
in lung tissue of lPs-stimulated ali mice. (A) F4/80 immunohistochemical staining. (B) h&e staining. (C) lY-6g immunohistochemical staining. (D) The mrna levels of the
inflammatory mediators TNF-α, il-6, il-1β, and VCAM-1. Statistical significance relative to the LPS group was indicated, n$5, **P,0.01.
Abbreviations: Xan, xanthohumol; il-6, interleukin-6; lPs, lipopolysaccharide; ali, acute lung injury; h&e, hematoxylin and eosin; VcaM-1, vascular cell adhesion
molecule 1; TnF-α, tumor necrosis factor-α; cOn, control group.
Lung tissues from the control group showed a normal structure cytokine mRNAs. Except for the inhibitory effects against
and no histopathological change under a light microscope. IL-1β and VCAM-1, 8f at 20 mg/kg was found to effectively
LPS instillation resulted in marked pathologic changes, such downregulate the expressions of TNF-α and IL-6, with sta-
as inflammatory cell infiltration, interalveolar septal thick- tistical significance. Meanwhile, injection with 8f resulted
ening, and interstitial edema. However, these pathological in more potently reduced mRNA expressions of those
changes induced by LPS were remarkably ameliorated by inflammatory mediators compared to those injected with
20 mg/kg of XAN treatment, whereas an even more obvious XAN at the same concentration. These data showed that anti-
pathological amelioration became visible in the treatment inflammatory compound 8f partly affects the cytokine profile
with 20 mg/kg 8f. These results were consistent with our at the mRNA level. Our studies reveal that 2-benzylidene-
previous F4/80 and LY-6G staining analysis, which showed 1-indanone derivatives are promising anti-inflammatory
that 8f had a remarkable protective effect on LPS-induced agents and should be further developed as candidates for the
histopathological changes in a mice model of ALI.
treatment of acute inflammatory diseases.
LPS exposure also elevated the mRNA levels of inflam-
matory cytokines in mice lung tissues. Therefore, we next
investigated the inhibitory effect of compounds 8f on mRNA
compound 8f inhibited the lPs-induced
activation of nF-κB/MaPK signaling
abundance for important proinflammatory factors, including pathway
TNF-α, IL-6, IL-1β, and VCAM-1. As shown in Figure 6D, In addition, to clarify the mechanism of anti-inflammatory
LPS stimulated a marked increase in the accumulation of effects of the most active compound 8f, we attempted to
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Xiao et al
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LPS (0.5 µg/mL)
–
–
+
–
+
+
–
+
–
–
+
–
+
+
–
+
–
–
+
–
+
+
–
+
8f (10 µM)
p-JNK
54 kDa
46 kDa
54 kDa
46 kDa
44 kDa
42 kDa
44 kDa
42 kDa
36 kDa
37 kDa
p-ERK
ERK
IκB-α
JNK
GAPDH
150
150
150
100
50
0
100
50
0
100
50
0
*
**
**
CON
LPS 8f+LPS
8f
CON
LPS 8f+LPS
8f
CON
LPS 8f+LPS
8f
Figure 7 effects of compound 8f on the lPs-induced activation of nF-κB and MaPK signaling pathway. MPMs pretreated with vehicle or compound 8f (10 µM) for 30 min
followed by incubation with lPs (0.5 µg/ml) for 20 min. The protein levels of iκB-α, p-JnK, JnK, p-erK, erK, and gaPDh were measured by immunoblot analysis. statistical
significance relative to the LPS group was indicated, n$5, *P,0.05, **P,0.01.
Abbreviations: lPs, lipopolysaccharide; MPMs, murine primary macrophages; cOn, control group.
identify the possible pathways responsible for the produc- analyses suggested that mono- or di-substitution of electron-
tion of proinflammatory cytokines that are inhibited by withdrawing functional groups on benzene ring B were not
our 8f compound. NF-κB and MAPK (mainly JNK, and tolerated, but incorporation of the three-carbon chain alkoxy
ERK) signaling pathways have been recently shown to play groups at the C-7 position of ring A was beneficial to the
important roles in regulating LPS-induced acute inflam- activity. The in vivo experiment reveals that three active com-
mation responses in vitro and in vivo.30,31 We investigated pounds, especially 8f, could effectively inhibit the mRNA
the possible mechanisms of NF-κB/MAPK signaling in levels of multiple inflammatory cytokines in LPS-induced
the anti-inflammatory action of 8f. While in an inactivated MPMs. Furthermore, pretreatment with active compound 8f
state, NF-κB is located in the cytosol complexed with the significantly attenuated LPS-induced ALI in mice via the
inhibitory protein IκB. In response to LPS stimulation, IκB reduction of inflammatory cytokine production, protein con-
kinase β phosphorylates cytoplasmic IκB-α; this phospho- centration in BALF, pathological changes, and macrophage
rylation results in the dissociation of IκB-α from NF-κB. infiltration. Moreover, 8f displayed a potential inhibitory
The activated NF-κB will translocate from the cytoplasm effect on MAPKs/NF-κB activation of LPS-stimulated
to nucleus and promotes the transcription of inflammatory MPMs. These multifunctional properties highlight 8f as a
genes. Activation of NF-κB is regulated by the degradation promising candidate for further studies aimed at the develop-
of the IκB-α; thus, we evaluated the effects of the analog on ment of novel anti-inflammatory drugs to treat ALI.
IκB-α degradation in LPS-stimulated MPMs.
As shown in Figure 7, 8f effectively reduced LPS-induced
Acknowledgments
IκB-α degradation at a concentration of 10 µM. Regarding
MAPK signaling, administration of LPS for 20 min induced
Financial support was provided by the National Natural
Science Funding of China (81773579, 21472142, 21572166)
a significant phosphorylation of ERK and JNK, respectively,
and Zhejiang Natural Science Funding (LY17B020008).
while pretreatment with compound 8f reversed LPS-induced
MAPK phosphorylation in the MPMs. These results sug-
Disclosure
The authors report no conflicts of interest in this work.
gest that the inhibition of NF-κB/MAPK activation may
mediate the anti-inflammatory effects of 8f.
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