Antiviral activity of diarylheptanoid stereoisomers against respiratory syncytial virus in vitro and in vivo

Article abstract of DOI:10.1007/s11418-013-0743-6

We previously showed that (5S)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenylhept- 3-one (AO-0011) and (5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) isolated from Alpinia officinarum exhibited stronger anti-influenza virus activity and anti-respiratory syncytia

Full text of DOI:10.1007/s11418-013-0743-6

J Nat Med
DOI 10.1007/s11418-013-0743-6
ORIGINAL PAPER
Antiviral activity of diarylheptanoid stereoisomers against
respiratory syncytial virus in vitro and in vivo
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Katsuhiko Konno Motofumi Miura Masaharu Toriyama Shigeyasu Motohashi Rie Sawamura
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Wataru Watanabe Hiroki Yoshida Masahiko Kato Ryuichi Yamamoto Ken Yasukawa Masahiko Kurokawa
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•
Received: 7 November 2012 / Accepted: 6 January 2013
Ó The Japanese Society of Pharmacognosy and Springer Japan 2013
Abstract We previously showed that (5S)-5-hydroxy-7-
(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011) and
(5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) iso-
lated from Alpinia officinarum exhibited stronger anti-
influenza virus activity and anti-respiratory syncytial virus
(RSV) activity, respectively, than the other isolated
diarylheptanoids. In this study, we synthesized an enantio-
mer (AO-0503) and racemate (AO-0504) of AO-0011 and
an enantiomer (AO-0514) of AO-0016. The anti-RSV
activities of the three stereoisomers (AO-0503, AO-0504,
and AO-0514) and AO-0011 were examined in vitro and
in vivo to evaluate the stereoisomeric effect on anti-RSV
activity. In a plaque reduction assay using human epidermoid
carcinoma cells, all four diarylheptanoids significantly
exhibited anti-RSV activity, and AO-0514 and AO-0016
exhibited stronger anti-RSV activity than AO-0503, AO-
0504, and AO-0011. In a murine RSV infection model, all
four diarylheptanoids with anti-RSV activity in vitro were
also significantly effective in reducing virus titers in the lungs
of RSV-infected mice. In the histopathological analysis of
RSV-infected lungs, the oral administration of even AO-
0514, which showed the lowest reduction of virus titers in the
lungs, was significantly effective in reducing the infiltration
of lymphocytes and in reducing the interferon-c level, which
is a marker of severity of pneumonia due to RSV infection, in
bronchoalveolar lavage fluids prepared from RSV-infected
mice. Although the stereoisomeric effects of diarylhepta-
noids on anti-RSV activity varied moderately, all four di-
arylheptanoids examined were suggested to ameliorate
pneumonia and have a potential anti-RSV activity in vivo.
They are possibly mother compounds for the development of
an anti-RSV drug in the future.
Keywords Alpinia officinarum ꢀ Diarylheptanoid ꢀ RSV ꢀ
Antiviral ꢀ Stereoisomer ꢀ Synthesis of diarylheptanoids
Introduction
Respiratory syncytialvirus(RSV) is a commonand worldwide
pathogen, and RSV infection is a highly significant cause of
bronchiolitis and pneumonia [1–3]. The infection becomes a
serious cause of deadly disease not only in infants but also in
K. Konno
W. Watanabe
Department of Microbiology, Graduate School of Clinical
Pharmacy, Kyushu University of Health and Welfare,
1714-1 Yoshino, Nobeoka, Miyazaki 882-8508, Japan
Department of Clinical Veterinary Medicine, School
of Pharmaceutical Sciences, Kyushu University
of Health and Welfare, 1714-1 Yoshino, Nobeoka,
Miyazaki 882-8508, Japan
M. Kato
Department of Applied Veterinary Medicine, School
of Pharmaceutical Sciences, Kyushu University
of Health and Welfare, 1714-1 Yoshino, Nobeoka,
Miyazaki 882-8508, Japan
M. Miura ꢀ M. Toriyama ꢀ S. Motohashi ꢀ K. Yasukawa
School of Pharmacy, Nihon University, 7-7-1 Narashinodai,
Funabashi-shi, Chiba 274-8555, Japan
R. Sawamura ꢀ H. Yoshida ꢀ M. Kurokawa (&)
Department of Biochemistry, Graduate School of Clinical
Pharmacy, Kyushu University of Health and Welfare,
1714-1 Yoshino, Nobeoka, Miyazaki 882-8508, Japan
e-mail: b2mk@phoenix.ac.jp
R. Yamamoto
First Department of Pharmacology, Graduate School of Clinical
Pharmacy, Kyushu University of Health and Welfare,
1714-1 Yoshino, Nobeoka, Miyazaki 882-8508, Japan
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the elderly and high-risk patients such as immunocompro-
mised or transplanted patients [4–6]. Ribavirin has been used
for the treatment of RSV infection, and palivizumab and
motavizumab have been used prophylactically [7–11].
Although new candidates for anti-RSV drugs such as a small
interference RNA drug (ALN-RSV01), fusion inhibitor (TMC
35311), and RNA polymerase inhibitor (RSV604) [12–14]
are in development and clinical trials, there are few specific
anti-RSV drugs that are effective clinically and practically.
Alpinia officinarum (A. officinarum), family Zingibera-
ceae, has been widely cultivated in Asia from ancient times
and is well known as lesser galangal. Its rhizome is used in
various Asian cuisines and as an important traditional med-
icine in Asia. Diarylheptanoids isolated from A. officinarum
[15–20] have been shown to exhibit various bioactivities in
inhibiting tumor promotion [15], inducing apoptosis [16],
anti-inflammatory, anti-nociceptive, and anti-psychiatric
effects [17], melanogenesis [20], and promoting an anti-
oxidant effect [18, 19]. Recently, we showed the first
evidence demonstrating the anti-influenza virus activity of
eleven diarylheptanoids isolated from A. officinarum in vitro.
Among them, influenza virus was more susceptible to (5S)-5-
hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-
0011) than to the others [21]. Influenza virus and RSV are
members of the Orthomyxoviridae and Paramyxoviridae,
respectively, and have similar characteristics, such as an
envelope and negative-sense single-stranded RNA. Thus,
AO-0011 may be also effective against RSV. On the other
hand, we demonstrated that (5S)-5-methoxy-1,7-diphe-
nylhept-3-one (AO-0016) showed anti-RSV activity
in vitro and its therapeutic index ([6.1) was the highest of
nine diarylheptanoids isolated from A. officinarum [22].
Thus, AO-0011 and AO-0016 are possible candidates for
mother compounds to develop anti-RSV compounds.
In this study, we synthesized the stereoisomers of AO-
0011 and AO-0016 to evaluate the stereoisomeric effect of
diarylheptanoids on anti-RSV activity and examined their
anti-RSV activity in vitro and in vivo. We found that all
synthesized stereoisomers of diarylheptanoids, including
AO-0011, significantly exhibited potent anti-RSV activity
in vitro and in vivo, although the anti-RSV activity of the
stereoisomers varied moderately. They are suggested to be
mother compounds for the development of anti-RSV drugs.
The relationships between the structure and anti-RSV
activity of the stereoisomers are discussed.
AO-0011 and AO-0016 were isolated from the rhizome of
A. officinarum as described previously [21, 22], and their
chemical structures are given in Fig. 1. The purities of AO-
0011 and AO-0016 were validated at more than 95 % by
1H-NMR.
Synthesis of diarylheptanoid stereoisomers
(5R)-5-Hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one
(AO-0503) and (5RS)-5-hydroxy-7-(4-hydroxyphenyl)-1-
phenylhept-3-one (AO-0504) were synthesized as an
enantiomer and racemate, respectively, of AO-0011
(Fig. 1). (5R)-5-Methoxy-1,7-diphenylhept-3-one (AO-
0514) was synthesized as an enantiomer of AO-0016
(Fig. 1).
Each reaction with air- and moisture-sensitive compo-
nents was performed under an argon atmosphere in a
flame-dried reaction flask. Reaction solvents used were
anhydrous grade (using solvent supply systems) tetrahy-
drofuran (THF) and dichloromethane (CH2Cl2) purchased
from Kanto Chemical Co. Inc. Silica gel (F254) was used
for all analytical thin layer chromatography. Flash column
chromatography was carried out using Kanto silica gel
60 N (spherical, neutral, 40–50 lm), containing 0.5 %
1
fluorescence reagent 254 and a quartz column. H-NMR
spectra were recorded at 600 MHz and 13C-NMR spectra
were recorded at 150 MHz, as solutions in deuteriochlo-
roform (CDCl3), on a JEOL ECA-600. Chemical shifts are
reported in ppm downfield from TMS. Mass spectra were
obtained on a JEOL JMS-GCMATE.
Materials and methods
Chemicals
Fig. 1 Chemical structures of diarylheptanoids. Chemical structures
of AO-0011 and AO-0016 are cited from reference [21] and [22],
respectively
Dimethyl sulfoxide (DMSO) and ribavirin were purchased
from Wako Pure Chemical Industries, Ltd., Osaka, Japan.
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Cells and viruses
by a Trypan blue exclusion test. The 50 % cytotoxic con-
centration (CC50) was determined as the concentration that
reduced cell destruction by 50 % [24].
Human epidermoid carcinoma (HEp-2) cells (CCL-23,
American Type Culture Collection, Rockville, MD, USA)
were purchased from Dainippon Pharmaceutical (Osaka,
Japan) and grown and maintained in Eagle’s minimum
essential medium (EMEM; Nissui Pharmaceutical Co. Ltd.,
Tokyo, Japan) supplemented with heat-inactivated 10 %
and 2 % fetal calf serum. The A2 strain of RSV was
obtained from American Type Culture Collection and
grown in HEp-2 cell cultures.
Experimental murine RSV infection
Experimental RSV infection was performed as reported
previously [25]. Briefly, 6-week-old female mice were
infected intranasally with 1 9 106 PFU per 0.1 ml of A2
strain of RSV under anesthesia. Diarylheptanoids (AO-
0011, AO-0503, AO-0504, and AO-0514) dissolved in
1 % DMSO were administered orally to mice by gavage at
30 mg/kg of body weight once at 4 h prior to, once at 1 h
after, and once again at 6 h after virus infection on day 0,
and three times daily from day 1 to day 3 after infection. A
1 % DMSO solution was used as a control. The mice were
weighed daily from day 0 to day 4 through the experiment.
On day 4 after infection, the lungs were removed for virus
titration, immediately frozen in liquid N2, and stored at
-80 °C. Frozen lungs were homogenized with cold quartz
sand in a homogenizer, and viral titers in the supernatants
of the homogenates were measured by a plaque assay. For
the measurement of interferon (IFN)-c levels in broncho-
alveolar lavage fluid (BALF) of mice, BALF was obtained
from the mice under anesthesia by instilling 1.0 ml of cold
phosphate-buffered saline into the lungs and aspirating it
from the trachea using a tracheal cannula [26]. Ice-cold
BALF was centrifuged at 100g at 4 °C for 10 min. The
supernatant was stored at -80 °C until use.
Animals
Female BALB/c mice (6 weeks old, 19–21 g) were pur-
chased from Kyudo Animal Laboratory (Kumamoto,
Japan). The mice were housed in groups of five per cage
with food and water ad libitum under a 12-h light/dark
diurnal cycle (light at 7:30 a.m.). The temperature in the
room was kept at 23 2 °C. The mice were acclimated for
at least 5 days before starting experimental procedures.
Animal studies followed the animal experimentation
guidelines of Kyushu University of Health and Welfare
(Nobeoka, Japan) and were carried out in an approved
biosafety level facility there.
Antiviral and cytotoxic assays
The anti-RSV activity of diarylheptanoids was examined
by a plaque reduction assay using HEp-2 cells [23]. Briefly,
HEp-2 cells grown in 24-well plates were infected with 100
plaque-forming units (PFU)/0.2 ml of RSV at 37 °C for
1 h. The cells were overlaid with 1 ml of maintenance
EMEM containing 0.8 % methylcellulose and various
concentrations of diarylheptanoids or ribavirin, and main-
tained in a humidified atmosphere containing 5 % CO2 at
37 °C for 4–5 days. The infected cells were fixed and
stained, and the number of plaques was counted. All
diarylheptanoids were dissolved in DMSO and diluted with
culture medium to make the various final concentrations.
The concentration of DMSO in each medium was less than
1 %. Ribavirin was dissolved in DMSO and used as a
control. The 50 % effective antiviral concentration (EC50)
was the concentration that reduced virus-induced cell
destruction by 50 %, as described previously [23].
Histopathological examination
For histopathological examination of the infected lungs,
RSV-infected mice were killed on day 4 after infection,
and the lungs were removed and placed in buffered for-
malin for a minimum of 24 h. The tissue was then
embedded in low-melting-point paraffin, sectioned at a
thickness of 5 lm, and stained with hematoxylin and eosin
(HE).
Pathological changes (hemorrhage, infiltration of neu-
trophils, hyperplasia of macrophages, infiltration of lym-
phocytes, edema, degeneration of tracheal epithelium,
formation of hyaline membrane, and bacterial flora) in
tissue sections were scored as 0, negative; 1, minimal; 2,
mild; 3, moderate; 4, severe. The means of each patho-
logical change between AO-0514-administered mice and
control mice were compared statistically.
The cytotoxicity of diarylheptanoids was assessed by a
Trypan blue exclusion test using mock-infected HEp-2
cells. Cells were seeded at a concentration of 5 9 104
cells/ml in 24-well plates and cultured at 37 °C for 24 h.
The culture medium was then replaced with fresh medium
containing diarylheptanoids at various concentrations, and
the cells were further incubated for 48 h. The cells were
trypsinized, and the number of viable cells was determined
ELISA
IFN-c levels in BALF were measured using a specific
ELISA kit (Ready-set-go, eBioscience Inc., San Diego,
CA, USA) according to the manufacturer’s instructions.
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This product was tested and found to conform to all
eBioscience Inc. quality control release specifications. The
lower limit of detection sensitivity in the kit is 4 pg/ml for
IFN-c. The intra- and inter-assay coefficients of variation
for the ELISA were less than 10 %.
Reduction of 3 by Red-Al afforded the desired allylic
alcohol 4a.
4b and AO-0504 were synthesized from b-hydroxyke-
tone 7 as a common starting material, which was prepared
by aldol reaction between 4-phenyl-2-butanone 5 and
arylpropioaldehyde 6 (R=OTBDPS). TBAF-mediated
deprotection of 7 gave racemic b-hydroketo AO-0504.
Alternatively, dehydration of 7 with TsOH furnished the
enone product 8, which was subjected to Luche reduction
to give the allylic alcohol 4b [27].
Statistical analysis
Statistical significances of differences between the EC50
and CC50 values, and in virus titers, scores of pathological
changes, and IFN-c levels were evaluated using Student’s
t test. A P value of 0.05 or less was considered to be
statistically significant.
Optically active b-methoxyketo AO-0514 and b-hydroxy-
keto AO-0503 were synthesized from b-hydroxyketones 4a
(R=H) and 4b (R=OTBDPS) according to the following pro-
cedures. The Sharpless asymmetric epoxidation of 4a or 4b
gave enantio-enriched anti-epoxy alcohols 9 through kinetic
resolution [28], followed by Dess–Martin oxidation to afford
epoxy ketones 10. Treatment of 10 with diphenyldiselenide
under reductive conditions gave the enantio-enriched b-
hydroxy products 11. Finally, b-methoxyketo 11a was
obtained by treating AO-0514 (R=H) with MeOTf and 2,6-
DTBP; deprotection of 11b (R=OTBDPS) by TBAF yielded b-
hydroxyketo AO-0503. Detailed spectroscopic data of the
synthesized compounds can be obtained from us by request.
The synthesized AO-0503, AO-0504, and AO-0514 were
Results
Synthesis of diarylheptanoid stereoisomers
As shown in Fig. 2, racemic syntheses of allylic alcohols
4a and 4b, as well as b-hydroxyketo AO-0504 were carried
as follows. Preparation of 4a was initiated by lithiation of
4-phenyl-1-butyne 1 by n-BuLi, followed by dropwise
addition of 3-phenylpropanal 2 to form alkynyl alcohol 3.
Fig. 2 Synthesis ofdiarylheptanoid analogues. DIPT diisopropyltartrate,
DMP Dess–Martin periodinane, 2,6-DTBP 2,6-di-tert-butylpyridine,
TBAF tert-butylammonium fluoride, TBDPS tert-butyldiphenylsilyl,
TBHP tert-butylhydroperoxide. The enantiomeric excess values of AO-
0503 and AO-0514 were determined by HPLC equipped with chiral
phase supported column (OD-3)
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identified as follows. The purity of AO-0503, AO-0504, and
AO-0514 were validated as more than 99 % by 1H-NMR.
35.7, 31.4, 29.5 ppm; HRMS (EI) m/z: 296.17803 (calcd
for C20H24O2: 296.17762).
Anti-RSV activity of diarylheptanoids in vitro
(5R)-5-Hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one
(AO-0503)
Four diarylheptanoids (AO-0011, AO-0503, AO-0504, and
AO-0514, Fig. 1) were examined for their anti-RSV
activity and cytotoxicity in vitro. AO-0011 has been
reported to have anti-influenza virus activity [21] but its
anti-RSV activity was unknown. As shown in Table 1, the
EC50 values of AO-0011, AO-0503, AO-0504, and AO-
0514 were 40.7 3.5, 44.7 1.5, 24.3 0.6, and
7.0 1.4 lg/ml, respectively, and the EC50 values were
significantly lower than their CC50 values (85.5 26.2,
56.8 6.4, 82.5 28.6, and [100 lg/ml, respectively).
All diarylheptanoids examined exhibited anti-RSV activity
in vitro. The diarylheptanoids (AO-0011, AO-0503, and
AO-0504) are stereoisomers and their CC50/EC50 values
were similar (2.1, 1.3, and 3.4, respectively). However, the
CC50/EC50 values of AO-0016, which have been reported
by Konno et al. [22] and are given in Table 1, and AO-
0514 (6.1 and 14.3, respectively) were higher than those of
AO-0011, AO-0503, and AO-0504. In particular, the EC50
value of AO-0514 was much lower than that of AO-0016,
and the CC50/EC50 value ([14.3) of AO-0514 was more
than double that of AO-0016. DMSO at 1 %, which was
used as a maximum concentration to dissolve diarylhep-
tanoids in the culture medium, was not cytotoxic. In this
assay, the EC50 value of ribavirin, used as a positive con-
trol, was similar to the results reported previously [29, 30].
AO-0011, AO-0503, AO-0504, and AO-0514 all exhibited
anti-RSV activity in vitro, and, of them, AO-0514 seemed
to be most effective.
[a]D = -0.34° (c = 2.30, acetone); 1H-NMR (CDCl3,
600 MHz) d: 7.29–7.26 (m, 2 H), 7.20–7.16 (m, 3 H), 7.03
(d, 2 H, J = 8.6 Hz), 6.74 (d, 2 H, J = 8.6 Hz), 5.04 (s, 1
H), 4.05–4.02 (m, 1 H), 3.10 (m, 1 H), 2.90–2.87 (m, 2 H),
2.76–2.68 (m, 3 H), 2.62–2.51 (m, 3 H), 1.80–1.74 (m, 1
H), 1.64–1.62 ppm (m, 2 H); 13C-NMR (CDCl3, 150 MHz)
d: 211.3, 153.8, 140.6, 133.8, 129.5, 128.6, 128.3, 126.2,
115.2, 66.9, 49.2, 45.0, 38.2, 30.8, 29.5 ppm; HRMS (EI)
m/z: 298.15672 (calcd for C19H22O3 : 298.15688).
(5RS)-5-Hydroxy-7-(4-hydroxyphenyl)-1-phenylhapt-3-one
(AO-0504)
1H-NMR (CDCl3, 600 MHz) d: 7.29–7.26 (m, 2 H),
7.20–7.16 (m, 3 H), 7.03 (d, 2 H, J = 8.6 Hz), 6.74 (d, 2 H,
J = 8.6 Hz), 5.04 (s, 1 H), 4.05–4.02 (m, 1 H), 3.10 (m, 1
H), 2.90–2.87 (m, 2 H), 2.76–2.68 (m, 3 H), 2.62–2.51 (m,
3 H), 1.80–1.74 (m, 1 H), 1.64–1.62 ppm (m, 2 H); 13C-
NMR (CDCl3, 150 MHz) d: 211.3, 153.8, 140.6, 133.8,
129.5, 128.6, 128.3, 126.2, 115.2, 66.9, 49.2, 45.0, 38.2,
30.8, 29.5 ppm; HRMS (EI) m/z: 298.15672 (calcd for
C19H22O3: 298.15688).
(5R)-5-Methoxy-1,7-diphenylhept-3-one (AO-0514)
[a]D = -10.2° (c = 0.33; CHCl3); 1H-NMR (CDCl3,
600 MHz) d: 7.28–7.26 (m, 4 H), 7.19–7.16 (m, 6 H),
3.73–3.68 (m, 1 H), 3.30 (s, 3 H), 2.90–2.88 (m, 2 H),
2.80–2.60 (m, 5 H), 2.47–2.43 (m, 1 H), 1.82–1.74 ppm
(m, 2 H); 13C-NMR (CDCl3, 150 MHz) d: 208.5, 141.9,
141.0, 128.5, 128.4, 126.1, 125.9, 76.7, 57.0, 47.4, 45.4,
Anti-RSV activity of diarylheptanoids in mice
To assess the potential anti-RSV activity of the diaryl-
heptanoids in vivo, virus titers in the lungs of infected mice
Table 1 Anti-RSV activity and cytotoxicity of diarylheptanoids
Compounds
ECa50 (lg/ml)
CCa50 (lg/ml)
CC50/EC50
(5S)-5-Hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011)d
(5R)-5-Hydroxy-7-(400-hydroxyphenyl) -1-phenylhept-3-one (AO-0503)
(5RS)-5-Hydroxy-7-(400-hydroxyphenyl) -1-phenylhept-3-one (AO-0504)
(5S)-5-Methoxy-1,7-diphenylhept-3-one (AO-0016)d
(5R)-5-Methoxy-1,7-diphenylhept-3-one (AO-0514)
Ribavirin
40.7 3.5b
44.7 1.5b
24.3 0.6b
16.3 3.5b,e
7.0 1.4b
85.5 26.2
56.8 6.4
82.5 28.6
[100e
2.1
1.3
3.4
6.1e
14.3
NDc
[100
NDc
0.67 0.08
a
Mean SD of four independent experiments
b
P \ 0.05 versus CC50 by Student’s t test
Not done
c
d
AO-0011 and AO-0016 are reported by reference [21] and [22], respectively
e
EC50, CC50, and CC50/EC50 values are cited from reference [22]
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Table 2 RSV titers of lungs of infected mice
Compounds
Titer (log10PFU/ml)a SD
% of control
Experiment 1
Controlc
5.31 0.15
3.97 0.33d
4.45 0.30d
4.81 0.20d
100
4.6
AO-0011b
AO-0503b
AO-0504b
Experiment 2
Controlc
13.8
31.6
4.02 0.08
3.87 0.13d
100
AO-0514b
69.6
Experiments 1 and 2 were performed independently
a
Mean SD for five mice
b
Diarylheptanoids were administered daily at 30 mg/kg/mouse
1 % DMSO solution was administered daily as the control
P \ 0.05 versus control by Student’s t test
c
d
administered diarylheptanoids at 30 mg/kg and 1 %
DMSO as a control were compared on day 4 after infection.
As shown in Table 2, AO-0011, AO-0503, AO-0504, and
AO-0514 were significantly effective in reducing the virus
titers of lungs of infected mice compared with the controls
(P \ 0.05). There were no significant differences between
the mean SE of body weights of mice administered AO-
0011, AO-0503, and AO-0504 (20.2 0.4, 19.5 0.3,
19.4 0.4 g, respectively, in experiment 1) and the con-
trol (18.2 0.9 g in experiment 1) on day 4 after infection,
and AO-0514 (20.1 0.4 g in experiment 2) and the
control (20.4 0.4 g in experiment 2). All four diaryl-
heptanoids exhibited potential anti-RSV activity in mice.
Fig. 3 Lungs of RSV-infected mice at 4 days after infection. Five
mice per group were used. Representative HE-stained images (9140)
are shown. The arrowhead indicates infiltration of lymphocytes and
hemorrhage. a Control mouse with RSV infection. b AO-0514-
administered mouse with RSV infection
Histopathological analysis of lungs of infected mice
The virus titers in lungs of RSV-infected mice were
reduced to 4.6, 13.8, 31.6, and 69.6 % of the controls by
the oral administration of AO-0011, AO-0503, AO-0504,
and AO-0514, respectively (Table 2). AO-0514 showed
the weakest antiviral activity in the reduction of virus titers
of lungs. To evaluate whether the oral administration of
AO-0514 reflected the alleviation of histopathological
damage of lungs, we histopathologically analyzed the lungs
of infected mice administered AO-0514. Figure 3 shows
representative HE-stained images of lungs of mice
administered AO-0514 or 1 % DMSO solution as a con-
trol. AO-0514 administration markedly reduced infiltration
of immune cells. As shown in Table 3, the hemorrhage,
inflammation of neutrophils, hyperplasia of macrophages,
infiltration of lymphocytes, and edema scores in control
infected mice were higher than those of mice administered
AO-0514. Administration of AO-0514 was especially
significantly effective in reducing infiltration of lympho-
cytes. Pathological changes such as edema, degeneration of
Table 3 Histopathological change scores
Histopathological changes
Scorea SD
Controlc
AO-0514b
Hemorrhage
1.6 1.8
1.6 0.9
1.6 0.9
1.4 0.5
0.4 0.9
0
0.8 0.8
Infiltration of neutrophils
Hyperplasia of macrophages
Infiltration of lymphocytes
Edema
1.2 0.4
0.8 0.8
0.6 0.5d
0
0
0
0
Degeneration of tracheal epithelium
Formation of hyaline membrane
Bacterial flora
0
0
a
Mean SD for five mice
b
Diarylheptanoids were administered daily at 30 mg/kg/mouse
1 % DMSO solution was administered daily as the control
P \ 0.05 versus control by Student’s t test
c
d
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enantiomer of AO-0016 to evaluate stereoisomeric effects
on anti-RSV activity. The synthesized diarylheptanoids
(AO-0503, AO-0504, and AO-0514) including AO-0011
were examined for their anti-RSV activity in vitro and
in vivo. All four diarylheptanoids examined exhibited
significant anti-RSV activity in vitro and in vivo, although
the anti-RSV activity of the four diarylheptanoids varied
moderately. Thus, these diarylheptanoids may be candi-
dates for mother compounds for the development of anti-
RSV drugs.
Diarylheptanoids AO-0011 and AO-0503 are enantio-
mers of the hydroxyl group and AO-0504 is the racemate.
In Table 1, based on stoichiometric composition of enan-
tiomers in a racemate, the EC50 value (24.3 0.6 lg/ml)
of AO-0504 did not reflect the additive value of enantio-
mers AO-0011 (40.7 3.5 lg/ml) and AO-0503 (44.7
1.5 lg/ml). The CC50 value (82.5 28.6 lg/ml) of
AO-0504 did not reflect the additive value of enantiomers
AO-0011 (85.5 26.2 lg/ml) and AO-0503 (56.8
6.4 lg/ml), either. The biological activity including anti-
viral activity or cytotoxicity of racemate has been reported
to not always reflect the additive activity of the enantio-
mers [32–34]. It would be interesting to analyze their dif-
ferences to obtain the better anti-RSV diarylheptanoids.
The CC50/EC50 values of AO-0011, AO-0503, and AO-
0504 (2.1, 1.3, and 3.4, respectively) were lower than those
(6.1 [22] and 14.3) of AO-0016 and AO-0514, which are
enantiomers of the methoxy group (Fig. 1). A methoxy
group at position 5 of diarylheptanoids may be necessary to
produce stronger anti-RSV activity than a hydroxyl group.
In the former enantiomers and racemate (AO-0011, AO-
0503, and AO-0504), anti-RSV activity in vitro was not
greatly influenced by the stereoisomeric differences of the
hydroxyl group. However, in the latter enantiomers (AO-
0016 and AO-0514), the R form (AO-0514) showed
markedly stronger anti-RSV activity than the S form (AO-
0016). Thus, the stereoisometric effect of a methoxy group
at position 5 on anti-RSV activity may be larger than that
of a hydroxyl group. The CC50 values ([100 lg/ml) of
AO-0016 and AO-514 were higher than those (56.8–85.5
lg/ml) of AO-0011, AO-0503, and AO-0504. In our
previous report [22], the cytotoxicity of (5S)-
5-methoxy-7-(400-hydroxyphenyl)-1-phenyl-3-heptanone,
which has a hydroxyl group at position 400, was shown to be
higher than that of AO-0016, without that group. The
existence of a hydroxyl group in diarylheptanoids may
reflect cytotoxicity rather than anti-RSV activity. The
methoxy group at position 5 of diarylheptanoids is possibly
indispensable to provide effective anti-RSV activity
in vitro.
Fig. 4 Concentration of IFN-c in BALF of RSV-infected mice.
BALF was obtained from 5 mice per group at 4 days after infection.
AO-0514 at 30 mg/kg and 1 % DMSO solution as the control were
administered orally to RSV-infected mice. The IFN-c levels were
expressed as the mean SD in each group represented. *P \ 0.05
versus control by Student’s t test
tracheal epithelium, formation of hyaline membrane, and
bacterial flora were hardly observed in both groups.
It has been reported that the IFN-c level in BALF of
RSV-infected mice increases greatly and is a marker of
severity of pneumonia due to RSV infection [31]. We
compared the IFN-c levels in BALF of RSV-infected mice
administered AO-0514 and 1 % DMSO solution as a
control. As shown in Fig. 4, the oral administration of AO-
0514 significantly reduced the IFN-c levels in BALF of
RSV-infected mice (P \ 0.05). Thus, although AO-0514
was not very effective in reducing virus titers in the lungs
of infected mice as compared with the other three diaryl-
heptanoids, AO-0514 administration was found to ame-
liorate the histopathological damage to lungs by RSV
infection.
Discussion
We have previously reported that two diarylheptanoids
(AO-0011 and AO-0016) isolated from A. officinarum
exhibited effective anti-influenza virus and anti-RSV
activity, respectively, in vitro [21, 22]. In this study, we
synthesized AO-0503 and AO-0504 as the enantiomer and
racemate, respectively, of AO-0011, and AO-0514 as the
In the murine RSV infection model, all four diarylhep-
tanoids examined significantly exhibited anti-RSV activ-
ity in mice. Among them, diarylheptanoids (AO-0011,
123

J Nat Med
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inconsistent with the in-vitro results that diarylheptanoids
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Acknowledgments We thank Dr. Tomomi Takeshita, Ms. Akane
Hino, and Ms. Yukiko Shimoda for their excellent technical assis-
tance and Ms. Katherine Ono for her editorial assistance. We would
also like to thank Dr. Kouichi Metori (Analytical Center, School of
Pharmacy, Nihon University) for performing mass spectroscopy. This
study was partly supported by a Grant-in-Aid for Scientific Research
(No. 24790182) from the Japan Society for the Promotion of Science
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