A synthetic compound, 4-acetyl-3-methyl-6-(3,4,5-trimethoxyphenyl)pyrano[3, 4-c]pyran-1,8-dione, ameliorates ovalbumin-induced asthma

Article abstract of DOI:10.1016/j.bmc.2013.08.045

Eosinophilia is one of the characteristic signs of allergic inflammation. Massive migration of eosinophils to the airways can cause epithelial tissue injury, contraction of airway smooth muscle and increased bronchial responsiveness. Previously, we discov

Full text of DOI:10.1016/j.bmc.2013.08.045

Bioorganic & Medicinal Chemistry 21 (2013) 6359–6365
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A synthetic compound, 4-acetyl-3-methyl-6-(3,4,5-trimethoxy-
phenyl)pyrano[3,4-c]pyran-1,8-dione, ameliorates
ovalbumin-induced asthma
Hwan-Suck Chung a, Youngeun Kim a, Sei Joong Oh a, Hankyum Kim a,b, Seung Ill Choi c, Yujuan Zhang c,
Jin-Hyun Jeong c, Hyunsu Bae a,
⇑
a Department of Physiology, College of Korean Medicine, Kyung Hee University, #1 Hoeki-Dong, Dongdaemoon-gu, Seoul 130-701, Republic of Korea
b Department of Oriental Rehabilitation Medicine, Jaseng Hospital of Oriental Medicine, Seoul 135-896, Republic of Korea
c College of Pharmacy, Yonsei University, 162-1 Songdo-dong Yonsooku, Inchon 406-840, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2013
Revised 19 August 2013
Accepted 24 August 2013
Available online 3 September 2013
Eosinophilia is one of the characteristic signs of allergic inflammation. Massive migration of eosinophils
to the airways can cause epithelial tissue injury, contraction of airway smooth muscle and increased
bronchial responsiveness. Previously, we discovered a new compound, 1H,8H-pyrano[3,4-c]pyran-1,8-
dione (PPY), derived from the fruit of Vitex rotundifolia L. and evaluated its anti-inflammatory and anti-
asthmatic properties. In this study, we synthesized a new modified compound, 4-acetyl-3-methyl-6-
(3,4,5-trimethoxyphenyl) pyrano[3,4-c]pyran-1,8-dione (PPY-345), which was based on the PPY skeleton,
and we evaluated its anti-asthmatic effects. To evaluate the anti-asthmatic effect of PPY-345 in vitro,
A549 lung epithelial cells were stimulated with TNF-alpha, IL-4 and IL-1-beta to induce the expression
of CCL11 (Eotaxin), a chemokine involved in eosinophil chemotaxis. To characterize the anti-asthmatic
properties of PPY-345 in vivo, we examined the influence of PPY-345 in an ovalbumin (OVA)-induced
asthma model. PPY-345 treatments significantly reduced CCL11 secretion. PPY-345 treatment did not
Keywords:
4-Acetyl-3-methyl-6-(3,4,5-
trimethoxyphenyl)pyrano[3,4-c]pyran-1,8-
dione
Asthma
CCL11
Ovalbumin
Eosinophil
inhibit the translocation of NF-jB into the nucleus but suppressed the phosphorylation of signal trans-
ducers and activators of transcription 6 (STAT6). PPY-345 treatment significantly reduced airway hyper-
reactivity as measured by whole-body plethysmography. PPY-345 further reduced total cells, including
eosinophil, macrophage and lymphocytes, in the BAL fluid, goblet cell hyperplasia and myosin light chain
2 positive smooth muscle cell area in the lung tissue. Additionally, PPY-345 significantly suppressed the
levels of OVA–IgE present in the serum. These results suggested that PPY-345 could improve asthma
symptoms in OVA-sensitized mice.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
always present in excess in the airways of asthma patients, but
their accumulation in the lungs decreases as asthmatic symptoms
Asthma is a chronic inflammatory disease characterized by air-
way inflammation, increased mucus production, intermittent air-
way obstruction and hyperresponsiveness.1,2 Airway remodeling
is specifically characterized by structural and morphometric
changes to the airway, including subepithelial fibrosis, epithelial
hypertrophy, goblet cell hyperplasia and smooth muscle hypertro-
phy.3,4 The initial symptoms of asthma include airway inflamma-
tion, in which eosinophils play a crucial role.5 Eosinophils are
decrease. CCL11 (Eotaxin) is a small protein that is produced in the
lungs of asthmatic patients and is a potent eosinophil chemoattrac-
tant. CCL11, a CC chemokine, stimulates the migration of eosino-
phils from small blood vessels to lung tissue by acting on the CC
chemokine receptor CCR3, located on the leukocyte cell surface.6
Asthmatic patients commonly take two types of medication: pre-
ventative and relief agents.7 Oral or inhaled steroids are common
drugs for the treatment or prevention of asthma, but steroids have
many unpleasant side effects. Thus, research is currently underway
to find asthma treatment alternatives.8
We previously isolated a novel natural compound, 1H,8H-
Pyrano[3,4-c]pyran-1,8-dione (PPY), from the fruit of Vitex
rotundifolia L. and evaluated its anti-inflammatory and anti-
asthmatic effects. We reported that PPY treatment significantly
reduced the expression of eotaxin, IL-8, IL-16, and vascular cell
adhesion molecule-1 (VCAM-1) mRNA in A549 lung epithelial cells
Abbreviations: PPY-345, 4-acetyl-3-methyl-6-(3,4,5-trimethoxyphenyl)pyr-
ano[3,4-c]pyran-1,8-dione; BALF, bronchoalveolar lavage fluid; VCAM-1, vascular
cell adhesion molecule-1; STAT6, signal transducers and activators of transcription
6; Dexa, dexamethasone; OVA, ovalbumin; AHR, airway hyperresponsiveness; H&E,
hematoxylin and eosin; PAS, periodic acid Schiff; MLC2, myosin light chain 2; HRP,
streptavidin-horseradish peroxidase; Penh, enhanced pause.
⇑
Corresponding author. Tel./fax: +82 2 962 9316.
E-mail address: hbae@khu.ac.kr (H. Bae).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.045

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H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
that had been stimulated with cytokines. Additionally, in an oval-
bumin-challenged mouse model, PPY profoundly inhibited eosino-
phil accumulation in the airways and reduced the levels of IL-4,
IL-5, and IL-13 in the bronchoalveolar lavage fluid (BALF).9 As a
and powdered KOH (12 mmol) in dry DMF(50 mL) was stirred at
room temperature for 10–14 h. After completion of reaction, the
mixture was poured into iced water with constant stirring. The
precipitate thus obtained was filtered and purified on a silica-gel
column with chloroform as eluent in 45% yield: 1H NMR
(400 MHz, CDCl3, ppm) 7.05 (s, 2H), 6.59 (s, 1H), 3.94 (s, 6H),
2.73 (s, 3H).
follow-up study, we have synthesized
a new compound,
4-acetyl-3-methyl-6-(3,4,5-trimethoxyphenyl)pyrano[3,4-c]pyran-
1,8-dione (PPY-345) based on the PPY skeleton and evaluated its
anti-asthmatic effects in vivo and in vitro.
2.2.2. Synthesis of PPY-345
A
mixture of compound 3 (1 mmol), pentane-2,4-dione
2. Materials and methods
2.1. Cells
(1 mmol) and KOH (1.5 mmol) in dry DMF (10 ml) was stirred for
24 h at room temperature. The reaction mixture was poured onto
crushed ice with vigorous stirring and then neutralized with 10%
HCl. The precipitate obtained was filtered, washed with water
and finally purified on a silica-gel column using 0.5% and 5% ethyl
acetate in hexane as eluent for 1H,8H-pyrano[3,4-c]pyran-1,8-
dione and 6-(2-bromophenyl)-4-(methylthio)-2-oxo-2H-pyran-3-
carbonitrile, respectively, in 40% yield: 1H NMR (400 MHz, CDCl3,
ppm) 7.55 (s, 1H), 7.28 (s, 2H), 3.88 (s, 9H), 2.17 (s, 6H). 4-Acet-
yl-3-methyl-6-(3,4,5-trimethoxyphenyl)pyrano[3,4-c]pyran-1,8-
dione (PPY-345) was dissolved in 50% ethanol. The chemical struc-
tures of PPY and PPY-345 and synthesis procedure of PPY-345 are
shown in Figure 1.
A549 cells, a human type II-like epithelial lung cell line, were
obtained from the Korean Cell Line Bank (Cancer Research Insti-
tute, Seoul, Korea). The cells were cultured in 100 mm tissue cul-
ture plates (Corning, Corning, NY, USA) in RPMI 1640 medium
(Welgene, Daegu, Korea) supplemented with 10% heat-inactivated
fetal bovine serum (Hyclone, Logan, UT, USA) and 100 U mlÀ1 pen-
icillin–streptomycin (Invitrogen, Rockville, MD, USA). The plates
were incubated at 37 °C at 100% humidity and 5% CO2. The cells
were sub-cultured every 3–4 days.
2.2. Synthesis of 4-acetyl-3-methyl-6-(3,4,5-
trimethoxyphenyl)pyrano[3,4-c]pyran-1,8-dione (PPY-345)
2.3. Cell viability assay
The synthetic pyranopyrandione was obtained10 from the reac-
tion of 3-methoxyphenol and diethyl ethoxymethylenemalonate,
albeit in poor yield. The only structural commonality found in
compounds is the benzopyran moiety, but they differ in their site
of fusion with the other pyranone ring. The chemistry of pyr-
ano[3,4-c]pyran-4,5-diones is largely unexplored. Except for MO
calculations, that is, correlation of delocalization energy, p-bond
order and p-charge density of 20 different theoretical pyranopy-
randiones including pyrano[3,4-c]pyran-4,5-dione III, no other
additional information is available in the literature. This has in-
spired us to develop an innovative route for the construction of this
class of compounds in order to explore their therapeutic potential.
PPY-345 was synthesized by the following procedure.10 But PPY
was not synthesized. At this point, PPY is available from extract.
Furthermore, structure–activity relationship is under study.
The effect of PPY-345 on cell viability was examined by a 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulf-
ophenyl)-2H-tetrazolium (MTS) assay. Briefly, A549 cells were
seeded into 96-well plates at a density of 2 Â 104 cells wellÀ1 and
incubated at 37 °C for 24 h. The cells were then treated with 0, 5,
10, or 50 lM of PPY-345 for 24 h. After the addition of 20 l
l wellÀ1
of CellTiter 96Ò Aqueous One Solution Reagent (Promega, Madison,
WI), the cells were cultured for an additional 4 h and the absor-
bance was recorded at 490 nm using a microplate reader (Sunrise,
Tecan, Mannedorf, Switzerland). All experiments were performed
in triplicate.
2.4. Eotaxin inhibition assay
Cells were plated in 24-well flat-bottomed plates (Corning,
Corning, NY, USA). Upon reaching confluence, the medium was
changed to serum free RPMI and the samples were then incubated
2.2.1. Synthesis of 6-(3,4,5-trimethoxyphenyl)-4-(methylthio)-
2-oxo-2H-pyran-3-carbonitrile (3)
A mixture of methyl 2-cyano-3,3-bis(methylthio)acrylate (1)
(10 mmol), 1-(3,4,5-trimethoxyphenyl)ethanone (2) (11 mmol),
for an additional 24 h. Concentrations of 0, 5, 10, or 50 lM of
PPY-345 were added to the medium for 1 h, and the cells were
stimulated with a combination of IL-1b (10 ng mlÀ1) (Biosource,
Figure 1. Chemical structures of (A) PPY and PPY-345; (B) and the synthesis procedure of PPY-345.

H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
6361
Camarillo, CA, USA), TNF-
a
(100 ng mlÀ1) (Biosource), and IL-4
had free access to food and water during the experiments. The
study was conducted according to the Rules for Animal Care and
the Guiding Principles for Animal Experiments Using animals of
the University of Kyung Hee Animal Care and Use Committee
and in accordance with the recommendations of the Weatherall re-
port ‘The use of non-human primates in research.’ The experimen-
tal procedure protocols were approved by the Animal Welfare and
Animal Care Committee of the University of Kyung Hee Animal
Care and Use Committee.
(100 ng mlÀ1) (Biosource) and incubated for 24 h. The cell-free cul-
ture supernatants were then collected and assayed for eotaxin
using enzyme-linked immunosorbent assay (ELISA) kits (BD Biosci-
ences, San Jose, CA, USA) according to the manufacturer’s
instructions.
2.5. Preparation of nuclear extracts
Nuclear extracts were prepared to assess nuclear translocation
of NF-
jB and phosphorylation of signal transducers and activators
2.8. Induction of allergic asthma
of transcription 6 (STAT6). Briefly, dishes with cultured cells were
washed with ice-cold PBS. The dishes were then scraped and the
cells were transferred to microtubes. The cells were allowed to
swell by adding 200 ll lysis buffer (10 mM HEPES, pH 7.5,
10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM NaF,
Balb/c male mice 6 weeks of age were sensitized by intraperito-
neal (ip) injection on days 0 and 14 with 100
(OVA) (Sigma–Aldrich, St. Louis, MO, USA) precipitated with
20 mg of aluminum hydroxide in 200 l of PBS. The mice were then
challenged by administering 1% OVA in 50 l PBS or PBS directly
lg of ovalbumin
l
1 mM DTT, and 0.1 mM Na3VO4). The samples were incubated for
l
15 min on ice and gently mixed periodically. Next, 12.5
l
l of 10%
into the nostrils using a micropipette on days 15, 17, 19, 21, 23,
and 25. Negative control mice were sensitized and challenged with
PBS alone. On day 27, the mice were sacrificed and various tissues
were collected for analyses.
NP-40 was added and mixed carefully followed by a 5 min incuba-
tion. The tubes were vortexed twice for 10 s to disrupt the cell
membranes and centrifuged for 30 s at 4 °C. Cytosol containing
the supernatants was transfer to another tube. Pellets containing
crude nuclei were resuspended in 10
ll extraction buffer (25 mM
2.9. Experimental protocol and design
HEPES, pH 7.5, 500 mM NaCl, 10 mM NaF, 1 mM DTT, 10% Glycerol,
0.2% NP-40 and 5 mM MgCl2) and sonicated for 10 s three times.
The samples were centrifuged at 15,000 rpm for 10 min to obtain
the supernatant containing nuclear extracts. The extracts were
stored at À70 °C until further usage.
All mice were randomly divided into four groups (n = 6 mice per
group): (1) normal control mice (PBS group) were sensitized and
challenged with normal saline; (2) OVA control mice (OVA group)
were sensitized and challenged with OVA; (3) Positive control mice
that were OVA-sensitized and orally injected 1 mg kgÀ1 dexameth-
asone (Sigma) (Dexa group); (4) OVA-sensitized mice that were
intraperitoneally injected 10 mg kgÀ1 PPY-345 (PPY-345 group)
(Fig. 2).
2.6. Western blotting
The nuclear extracts were separated by 4-12% Tris-glycine
(KOMA Biotech, Seoul, Korea) gel electrophoresis and transferred
to a nitrocellulose membrane. The membrane was then blocked
with 5% skim milk in TBS-Tween-20 for 1 h at room temperature,
after which it was incubated with anti-p65 or p-STAT-6 or prolifer-
ating cell nuclear antigen (PCNA) antibody at 4 °C for overnight.
After washing three times in TBS-Tween-20, the blot was incu-
bated with a secondary antibody for 1 h. Antibody-specific proteins
were then visualized with an enhanced chemiluminescence detec-
tion system as recommended (Amersham Corp., Newark, NJ). Band
intensity was quantified by ImageJ analysis.
2.10. Measuring AHR to methacholine and analyzing BAL cells
Airway hyperresponsiveness (AHR) was measured 24 h after
the final allergen challenge. Mice were placed in a barometric ple-
thysmographic chamber (All Medicus, Seoul, Korea), and baseline
readings were taken for 3 min. The enhanced pause (Penh) was cal-
culated according to the manufacturer’s protocol [i.e., (expiratory
time/relaxation time À 1) Â (peak expiatory flowÁpeak inspiratory
flowÀ1)]. The results are expressed as the percentage increase in
Penh following challenge with each concentration of methacholine
(50 and 100 mg mlÀ1).
2.7. Animals
Balb/c male littermates (6 weeks of age, weighing 20–25 g)
were purchased from Orient Bio (Seoungnam, South Korea). All
mice were kept under specific pathogen-free conditions using air
conditioners and a 12 h lightÁdark cycleÀ1. In addition, all mice
2.11. Bronchoalveolar lavage (BAL)
Twenty-four hours after measuring the airway parameters, the
mice were sacrificed with pentobarbital sodium (65 mg kgÀ1, ip).
Figure 2. Schematic diagram of the experimental protocol.

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H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
BAL was collected by infusion and extraction of 1 ml of ice-cold
PBS. This was repeated three times, and the lavages were pooled
(mean volume, 2.0 ml). Recovered BAL (70–80%) was centrifuged
at 13,000 rpm for 10 min. The cell pallets were resuspended in 1 ml
PBS, and the cells were adhered to glass slides using cytocentrifu-
gation. Total viable cell counts were determined using a hemocy-
tometer with trypan blue exclusion. Differential counts of
eosinophils, neutrophils, lymphocytes, and macrophages were
determined on cytospin smears of BALF samples (5 Â 105 -
PPY-345 could inhibit CCL11 secretion in human lung epithelial
cells stimulated with TNF- , IL-4 and IL-1b. As shown in Figure 3B,
CCL11 secretion was decreased by 75% in response to treatment
with 5 M PPY-345.
a
l
3.2. PPY-345 inhibits phosphorylation of STAT6 on cytokines
stimulated lung epithelial cells
To further investigate the underlying mechanism behind the ef-
fects of PPY-345, we investigated its influence on NF-jB and
cellsÁ200
l
l cellsÀ1) from individual mice stained with Diff-Quick
staining (Life Technologies, Auckland, New Zealand) with 500 cells
counted per sample. The BAL fluid was then centrifuged and the
supernatants were kept at À70 °C. The results are expressed as to-
tal cell number  104.
STAT6, well-known transcription factors involved in allergic
inflammation. When we analyzed p65 and p-STAT6 protein level
in the nuclear fraction by Western blotting, we found that while
translocation of p65 subunits to the nucleus was not inhibited, p-
STAT6 protein levels were inhibited by PPY-345 treatment. These
data indicated that PPY-345 treatment suppressed CCL11 produc-
tion via inhibiting the STAT6 phosphorylation pathway (Fig. 4).
2.12. Determination of IgE titers using ELISA
For serum analysis, 96-well immunomicroplates (Costar, NY,
USA) were coated with monoclonal anti-mouse IgE antibodies.
The serum was diluted 1:250 with 5% FBS in PBS (assay diluent),
and IgE levels (BD Pharmingen) were measured using standardized
sandwich ELISAs according to the manufacturer’s protocol. Optical
density was measured at 450 nm in a microplate reader (SOFT max
PRO, version 3.1 software, CA, USA).
3.3. PPY-345 treatment reduced AHR
In an OVA-induced murine asthma model, AHR was signifi-
cantly reduced by PPY-345 treatment as measured by whole-
body plethysmography. AHR was evaluated by measuring
enhanced pause (Penh) in response to nebulized methacholine.
Bronchopulmonary hyperresponsiveness was significantly reduced
2.13. Histological examination
Trachea and lung tissues were removed from the mice, fixed in
4% paraformaldehyde and embedded in paraffin. After dehydra-
tion, the tissues were cut into 4 lm sections and stained with
hematoxylin and eosin (H&E) or periodic acid Schiff reagent
(PAS). For immunohistochemical detection of myosin light chain
2 (MLC2), 4 lm section of the lower trachea and lung tissue were
treated with 0.3% H2O2–methanol for 20 min to block endogenous
peroxidases, after which they were incubated at 4 °C overnight
with anti-MLC2 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA). After washing in PBS, the slide was treated with a biotin-
ylated secondary antibody for 20 min and then streptavidin-horse-
radish peroxidase (HRP) for 20 min. Immunoreactivity signals were
visualized with DAB substrate (Zymed Laboratories, South San
Francisco, CA, USA). Slides were counterstained with hematoxylin
and finally mounted using Canada balsam (Showa Chemical Co.
Ltd, Tokyo, Japan).
2.14. Statistical analysis
All results were expressed as the means SEM of at least three
independent experiments performed in duplicate. One-way analy-
sis of variance (ANOVA) and Tukey’s test were used to compare the
groups. Differences were considered to be significant at P <0.05.
3. Results
3.1. PPY-345 inhibits CCL11 secretion by cytokine-stimulated
lung epithelial cells
To investigate the effects of PPY-345 on cultured cells, cell via-
bility was monitored using the MTS assay in A549 lung epithelial
cells. Concentrations of PPY-345 high as 50 lM did not affect cell
Figure 3. Effect of PPY-345 on (A) cell viability and (B) eotaxin secretion levels in
A549 cells. (A) A549 cells were treated with various concentrations of PPY-345 and
cultured for 24 h. Cell viability was measured by the MTS assay. (B) A549 cells were
viability (Fig. 3A). A solution of 0.5% ethanol was used as a vehicle
control, which did not show any significant effects on A549 viabil-
ity (data not shown). It is known that CCL11 (eotaxin-1) produced
by lung epithelial cells is a potent chemoattractant for eosinophils.
Therefore, inhibiting CCL11 secretion is potential one way to pre-
vent asthma progression. In this study, we examined whether
treated with different concentrations of PPY-345 for 1 h, after which TNF-
a
(100 ng mlÀ1), IL-4 (100 ng mlÀ1), and IL-1b (10 ng mlÀ1) were added. The cells
were then incubated for 24 h, and the supernatants were used assessed by ELISA.
Inhibition was calculated as ‘Inhibition of X (%) = (cytokines mixture treated sample
value À X) Â 100/(cytokines mixture treated sample value À blank value)’. The
values shown are the mean SEM of three independent experiments. ⁄P <0.01
compared with the PPY-345 non-treated samples value. ‘B’ indicates blank.

H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
6363
Figure 5. Effects of PPY-345 on airway hyperresponsiveness to methacholine in an
OVA-induced murine asthma model. After the mice inhaled 50 or 100 mg mlÀ1 of
methacholine, AHR was measured and is shown as Penh values. Data are
represented as the mean SEM (n = 6 mice per group). ⁄P <0.05; ⁄⁄P <0.01; ⁄⁄⁄P
<0.001 versus OVA.
Figure 4. Influence of PPY-345 on the nuclear translocation of NF-
phosphorylation in A549 cells stimulated with TNF-
(100 ng mlÀ1), IL-4
(100 ng mlÀ1), and IL-1b (10 ng mlÀ1). (A) A549 cells treated with 50
M PPY-345
were incubated for 1 h with TNF- , IL-4, and IL-1b. Nuclear extracts were prepared
and analyzed for the presence of NF- B, phosphorylated STAT6 and PCNA by
western blotting, as described in Section 2. (B) Band intensities of p-STAT6/PCNA or
p65/PCNA were quantified by densitometric analysis of three experiments. ⁄⁄P
<0.01.
jB and STAT6
a
l
Figure 6. Effects of PPY-345 treatment on systemic IgE production in the serum of
OVA-sensitized asthmatic mice. Total IgE levels were measured by ELISA. The data
shown represent the means SEM. ⁄P <0.05; ⁄⁄P <0.01; ⁄⁄⁄P <0.001.
a
j
lungs.12 To evaluate the effects of PPY-345 treatment on the
recruitment of immune cells to the airways, we measured total cell
numbers as well as the numbers of eosinophils, lymphocytes and
macrophages in BAL fluid. In the OVA-immunized animals, the to-
tal number of infiltrating cells was approximately 2.5-fold higher
in the BAL fluid than in control animals. However, PPY-345 treat-
ment decreased the total number of infiltrating cells up to similar
level as that observed in the dexamethasone treatment group
(Fig. 7). Eosinophils were counted based on their morphological
characteristics and were visualized by Diff-Quick staining. Eosino-
phils numbers in the PPY-345-treated group were significantly de-
creased compared to the OVA group. The numbers of lymphocytes
and macrophages in the BAL fluid were also significantly lowered
by PPY-345 treatment (Fig. 7).
in PPY-345-treated mice when compared with the OVA group at
100 mg mlÀ1 of methacholine (Fig. 5). The dexamethasone-treated
group, used as a positive control, also exhibited significantly re-
duced Penh values (P <0.001). These data indicate that PPY-345
suppressed AHR in asthmatic mice.
3.4. PPY-345 decreased plasma IgE concentration
Cross-linking of allergen specific IgE antibodies on the surface of
mast cells causes them to be degranulated and subsequently leads
to AHR.11 To investigate the systemic effects of PPY-345 treatment,
IgE levels were measured in blood after 48 h of the last challenge,
as elevated IgE production is a key characteristic of systemic asth-
matic responses. Plasma levels of IgE were significantly decreased
in PPY-345-treated mice (Fig. 6).
3.6. Effect of PPY-345 on eosinophil infiltration and goblet cell
hyperplasia in lung
3.5. PPY-345-inhibited inflammatory cell influx into the BAL
fluid
H&E staining of lung sections revealed that more eosinophils
had infiltrated between the trachea and blood vessels in OVA-sen-
sitized mice than in normal control mice (Fig. 8A). In asthmatic
mice treated with PPY-345, eosinophil infiltration was significantly
One hallmark of allergic airway disease is the accumulation of
eosinophils, neutrophils, lymphocytes, and macrophages in the

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H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
Figure 7. Effects of PPY-345 on inflammatory cells accumulation in the BAL fluid. BAL cells were separated using a cytospin, after which they were stained with Diff-Quick.
Differential cell counting was performed using standard morphological criteria. The data shown represent the means SEM. ⁄P <0.05; ⁄⁄P <0.01; ⁄⁄⁄P <0.001.
decreased in the lung. PAS staining further revealed goblet cell
hyperplasia in the airways of the asthmatic mice. PPY-345 or dexa-
methasone treatment suppressed this goblet cell hyperplasia
(Fig. 8B). In addition, OVA-challenged mice expressed myosin light
chain 2 (MLC2) in the peribronchial muscle layer of the lung, and
PPY-345 treatment decreased the thickness of this smooth muscle
layer (Fig. 8C and D). These data demonstrated that PPY-345 had
significant potential to effectively inhibit airway remodeling.
the phosphorylated docking tyrosine residues on IL-4Ra and
phosphorylated by JAKs, resulting in STAT6 dimerization and
translocation to the nucleus. STAT6 is required for many IL-4-
and IL-13-mediated responses, including CCL11 expression.13,16,17
Bronchial asthma is characterized by reversible airway obstruc-
tion in response to allergens, chronic eosinophilic airway inflam-
mation, and non-specific airway hyper-responsiveness (AHR).18
In this study, we used an OVA-induced murine model of allergic
airway disease. In the OVA-immunized asthma model, AHR is clo-
sely associated with eosinophilia, as evidenced by increased num-
bers of eosinophils in the BAL fluid.19 The results of the present
study indicated that PPY-345 treatment improved methacholine-
induced AHR in OVA-immunized asthmatic mice (Fig. 5). Specific
IgE production is a hallmark of allergic diseases.20 In the present
study, PPY-345 treatment suppressed the increased antigen-spe-
cific IgE production characteristic of OVA-induced allergic asthma
in mice (Fig. 6).
The infiltration of inflammatory cells, including eosinophils,
into the BAL fluid was lowered to the levels observed in the dexa-
methasone or control groups following PPY-345 treatment (Fig. 7).
The decreased accumulation of eosinophils was also confirmed in
the PPY-345-treated lung samples (Fig. 8A). During an asthma at-
tack, in addition to tracheal contraction, goblet cells secrete more
mucus, which causes obstruction and difficulty breathing through
narrowed airways. In our study, PPY-345 treatment reduced the
MLC2 positive muscle area and the numbers of PAS-positive goblet
cells, suggesting that PPY-345 effectively inhibited airway remod-
eling in asthma (Fig. 8).
4. Discussion
In our previous study, we screened the anti-asthmatic effects of
270 medicinal plants and we found that the fruit of Vitex rotundifolia
L. had the highest effects in our in vitro experiments. After several
rounds of fractionation and activity testing, a novel compound
named PPY was isolated.9 As a follow-up study, we synthesized a
PPY derivative based on the PPY skeleton and evaluated its activity.
We demonstrated that PPY-345 inhibited the production of CCL11
in proinflammatory cytokine-activated A549 cells. PPY-345 did not
inhibit the proinflammatory cytokine-induced nuclear transloca-
tion of NF-
We previously showed that PPY treatment suppresses the
MAPK/NF-
B pathway.9 Therefore, we hypothesized that the PPY
derivative, PPY-345, would also inhibit the MAPK/NF- B pathway.
However, PPY-345 did not inhibit the translocation of NF- B into
jB but did inhibit the phosphorylation of STAT6.
j
j
j
nucleus (Fig. 4). According to Fulkerson et al., three chemokines,
CCL11, CCL17, and CCL22, are known to be STAT6 dependent.13
Murine models of allergic airway inflammation have demonstrated
that overexpression of the cytokine products of Th2 cells, specifi-
cally IL-4 and IL-13, is sufficient for the induction of numerous lung
chemokines and the development of pulmonary eosinophilia.14,15
In conclusion, we demonstrated that PPY-345 treatment
profoundly inhibited CCL11 production by cytokine-activated lung
epithelial cells in addition to impairing the recruitment of
inflammatory cells, including eosinophils, in a murine asthma
model. These results indicated that PPY-345 reduced airway
hyperresponsiveness, OVA-specific IgE production, and airway
IL-4 and IL-13 share the IL-4 receptor
mediates the phosphorylation of JAK1 and JAK3 and, subsequently,
phosphorylation of IL-4R . STAT6 monomers are then recruited to
a chain (IL-4Ra), which
a

H.-S. Chung et al. / Bioorg. Med. Chem. 21 (2013) 6359–6365
6365
Figure 8. Effect of PPY-345 on eosinophil infiltration and goblet cell hyperplasia in the lung. (A) H&E staining of lung tissue to detect eosinophil infiltration. (B) Lung sections
were stained with PAS stain to analyze goblet cell hyperplasia. Goblet cells are indicated by the arrows. (C) MLC2 expression was analyzed by IHC staining to evaluate airway
smooth muscle thickness. (D) Graphs represent the area of MLC2-positive smooth muscle in 5 bronchi. The data shown represent the means SEM. ⁄⁄P <0.01.
7. Thomson, N. C.; Bicknell, S.; Chaudhuri, R. Curr. Opin. Allergy Clin. Immunol.
2012, 12, 241.
8. Mark, J. D. Pediatr. Clin. North Am. 2007, 54, 1007.
remodeling in a murine asthma model at least in part via inhibiting
eosinophil infiltration to the lungs.
9. Lee, H.; Han, A. R.; Kim, Y.; Choi, S. H.; Ko, E.; Lee, N. Y.; Jeong, J. H.; Kim, S. H.;
Bae, H. Int. J. Immunopathol. Pharmacol. 2009, 22, 591.
10. Singh, F. V.; Dixit, M.; Chaurasia, S.; Raghunandan, R.; Maulik, P. R.; Goel, A.
Acknowledgments
Tetrahedron Lett. 2007, 48, 8998.
This work was supported by the National Research Foundation
of Korea (NRF) grant funded by the Korea Government [MEST] (No.
2012-0005755).
11. Bradding, P.; Walls, A. F.; Holgate, S. T. J. Allergy Clin. Immunol. 2006, 117, 1277.
12. Busse, W. W.; Lemanske, R. F., Jr. N. Engl. J. Med. 2001, 344, 350.
13. Fulkerson, P. C.; Zimmermann, N.; Hassman, L. M.; Finkelman, F. D.;
Rothenberg, M. E. J. Immunol. 2004, 173, 7565.
14. Li, L.; Xia, Y.; Nguyen, A.; Feng, L.; Lo, D. J. Immunol. 1998, 161, 3128.
15. Wills-Karp, M.; Luyimbazi, J.; Xu, X.; Schofield, B.; Neben, T. Y.; Karp, C. L.;
Donaldson, D. D. Science 1998, 282, 2258.
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