Characteristic of neuraminidase inhibitory xanthones from Cudrania tricuspidata

Article abstract of DOI:10.1016/j.bmc.2009.02.042

Natural polyphenolic compounds generally transpire to show relatively low inhibition against glycosidase including neuraminidase. In addition the inhibition modes of such compounds are rarely competitive. In this manuscript, a series of xanthone derivativ

Full text of DOI:10.1016/j.bmc.2009.02.042

Bioorganic & Medicinal Chemistry 17 (2009) 2744–2750
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Characteristic of neuraminidase inhibitory xanthones
from Cudrania tricuspidata
a
c
a
a
a
a
Young Bae Ryu , Marcus J. Curtis-Long , Ji Won Lee , Jin Hyo Kim , Jun Young Kim , Kyu Young Kang ,
b,
a,
*
*
Woo Song Lee , Ki Hun Park
a
Division of Applied Life Science (BK21 program), EB-NCRC, Institute of Agriculture and Life Science, Graduate School of Gyeongsang National University,
Jinju 660-701, Republic of Korea
Bioindustry Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 580-185, Republic of Korea
b
c
1
2 New Road, Nafferton, East Yorkshire YO25 4JP, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural polyphenolic compounds generally transpire to show relatively low inhibition against glycosi-
dase including neuraminidase. In addition the inhibition modes of such compounds are rarely competi-
tive. In this manuscript, a series of xanthone derivatives from Cudrania tricuspidata are shown to display
nanomolar inhibitor activity against neuraminidase (EC 3.2.1.18) as well as competitive inhibition modes.
Received 14 January 2009
Revised 17 February 2009
Accepted 19 February 2009
Available online 26 February 2009
Compound
IC50 = 0.08 ± 0.01
derived neuraminidase inhibitor, mangiferin (IC50 = 16.2 ± 4.2
a crucial role for inhibitory activity because compound 4, which has one of these hydroxyl groups preny-
lated was inactive (33% at 200 M), whereas other compounds (1–3 and 6–8) showed nanomolar activity
0.08–0.27 M) and competitive inhibition modes. Interestingly all inhibitors manifested enzyme isom-
8
bearing vicinal dihydroxy group on the A-ring displays nanomolar activity
M), a 200-fold increase in activity relative to that of the first reported xanthone-
M). The 6,7-vicinal dihydroxy group plays
(
l
Keywords:
Cudrania tricuspidata
Xanthone
Neuraminidase
Time-dependent
l
l
(
l
2
vk6
erization inhibition against neuraminidase. The most potent inhibitor, compound 8 showed similar inter-
action with a transition-state analogue of neuraminic acid in active site.
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Neuraminidase is a processing hydrolase involved in removing
general mobility of the virus in the respiratory tract. Development
of inhibitors of neuraminidase provides a new therapeutic ap-
proach for the treatment of the influenza virus and other diseases
1
,2
11
of terminal sialic acids from a variety of glycoconjugates. It is
known to play an important role in pathogenesis, bacterial nutri-
tion, and cellular interaction. This enzyme specifically cleaves N-
acetylneuraminic acid (NANA) from cell-surface glycoprotein
when the sialic acid is joined to galactose via an
linkage.
the terminal position of the carbohydrate groups of glycolipids
and glycoproteins. They have been proposed to play important
roles in various biological processes by influencing the conforma-
tion of glycoproteins, recognizing and masking the biological sites
of the molecules and their binding sites to cells.6 Neuraminidase
is especially believed to play at least two critical roles in influenza
that arise from hydrolysis of sialic acid. With the ever-present
threat of a pandemic derived from the influenza virus, the impor-
1
2
tance of neuraminidase inhibitors escalates.
There is considerable interest in the development of neuramin-
idase inhibitors from edible natural plants as these can be readily
applied to neutraceutics for the prevention of virus infection: cat-
echin derivatives are known to be strong inhibitors of the replica-
a
2?3 of
a2?6
3
–5
Neuraminic acids (sialic acid) are generally found in
1
3
14
tion processes of both HIV and the influenza virus, in addition
isoscutellarin-8-O-glucuronide from the leaf of Scutellaria baicalen-
sis also possess potent mouse liver sialidase inhibition properties
–8
15
(IC50 = 40 lM). When these and similar results are combined
with data from cell-culture, it has been reported that flavonoids
9
–11
viral life cycle:
the facilitation of virion progeny release; and
have a great potential to function as neuraminidase inhibitors.
1
5–17
Polyphenolic compounds are the most popular targets for
neuraminidase, but so far their potency has been limited as IC50
values have been at best in the M range.
Abbreviations: I, intensity at ex: 365 nm, em: 450 nm; IC50, the inhibitor
app
concentration leading to 50% activity loss; K
obs, apparent first-order rate constant for the transition from
velocity; , steady-state rate.
Corresponding author. Tel.: +82 63 570 5170 (W.S.L.); tel.: +82 55 751 5472;
i
, inhibition constant; Ki , apparent K
i
;
l
k
v
i
to , initial
v
s
;
v
i
Cudrania tricuspidata belongs to the Moraceae family and is
widely distributed in Korea, China, and Japan. The roots of C. tricu-
spidata have been used for the treatment of gonorrhea, rheuma-
tism, amenorrhea, bruising, and dysmenorrheal. Prenylated
v
s
*
fax: +82 55 757 0178 (K.H.P.).
E-mail addresses: wslee@kribb.re.kr (W.S. Lee), khpark@gsnu.ac.kr (K.H. Park).
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.042

Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
2745
O
O
OH
O
O
OH
O
O
OH
O
OH
HO
HO
HO
HO
HO
O
HO
HO
7
A
5
C
O
B
4
OH
OH
OH
OH
1
2
3
4
O
OH
O
O
OH
O
O
OH
HO
HO
HO
HO
O
OH
HO
HO
O
O
HO
HO
O
OCH
3
O
OH
5
6
7
8
Figure 1. Chemical structures of isolated compounds 1–8 from the Cudrania tricuspidata.
xanthones and flavonoids are the major compound components of
strong inhibitory activity against neuraminidase. More detailed
bioassays of the isolated compounds (1–8) were subsequently con-
ducted. As shown in Table 1, all 6,7-vicinal dihydroxy substituted
xanthones (1–8) examined exhibited a dose-dependent inhibitory
effect on the hydrolysis activity of neuraminidase (IC50 0.08–
C. tricuspidata. Previous biological studies have shown that the con-
18
stituents of C. tricuspidata exhibit anticancer and antioxidant
properties as well as monoamine oxidase19 (MAO) inhibitory
activity. In our recent publications, we have begun to elucidate
the vast biological potential of these extracts through various bio-
logical studies. For instance, xanthones possessing a vicinal dihy-
droxy group on one phenyl ring were shown to behave as
excellent radical scavengers.18 Xanthone-derived species were la-
ter proven to strongly suppress LDL oxidation.20 We associated this
with the anti-atherosclerotic and anti-inflammatory properties of
C. tricuspidata. Our research has also brought to light that natural
18
1.27
lM) (Fig. 2A). These derivatives showed a 20-fold higher
activity than quercetin and were 200 times more activate than
1
7,22
the positive control, mangiferin (Table 1).
bears a mono alkylated 6,7-dihydroxy motif was shown to have a
much higher IC50 value (around 300 M) (Table 1).
Compound 4 which
l
Raising the concentrations of the inhibitors drastically lowered
residual enzyme activity (Fig. 2B). All inhibitors manifested a sim-
ilar relationship between enzyme activity and enzyme concentra-
tion. The relevant data for compound 1 is illustrated in Figure 2B
as an example. Plots of residual enzyme activity versus enzyme
concentration at different concentrations of compound 1 gave a
family of straight lines with a y-axis intercept of 0, indicating that
1 is a reversible inhibitor. Augmenting the concentration of com-
pound 1 resulted in a reduction of the slopes of the lines.
xanthones can inhibit
level).
a-glucosidase potently (at the low lM
2
1
In this manuscript, we found that xanthones from C. tricuspidata
inhibited neuraminidase at the nanomolar level. Moreover, these
strongly potent inhibitors exhibited competitive inhibition, which
is very rare in polyphenolic compounds. We also wish to report a
detailed enzyme mechanism study and molecular docking
experiments.
2
.3. Time-dependent inhibitory effects of xanthones on
neuraminidase
2
2
. Results and discussion
To further investigate the inhibition mechanism, the time
dependence of the inhibition of the hydrolysis of neuraminic acid
by these inhibitors was subsequently probed. This was achieved
.1. Isolation and identification of xanthones derivatives
The EtOAc extract of the root of C. tricuspidata was selected for
purification through activity (against neuraminidase)-guided frac-
tionation. Repeated column chromatography of this extract fur-
nished eight xanthones (1ꢀ8) (Fig. 1). The spectroscopic data of
Table 1
Inhibitory effects of isolated compounds 1ꢀ8 on neuraminidase activities
1
8,20,21
compounds (1ꢀ8) agree with those previously published
Entry
IC50a
(lM)
i
Inhibition type (K , lM)
for Cudratricusxanthone (1), Macluraxanthone (2), Cudraxanthone
L (3), 1,3,7-trihydroxy-4-(1,1-dimethyl-2-propenyl)-5,6-(2,2-dim-
ethylchromeno) xanthone (4), Cudratricusxanthone F (5), Cudrax-
anthone D (6), Cudraxanthone M (7), and 1,3,6,7-tetrahydroxy-2-
1
2
3
4
5
6
7
8
0.245 ± 0.03
0.186 ± 0.02
0.228 ± 0.01
33% at 200
1.271 ± 0.21
0.278 ± 0.08
0.186 ± 0.04
0.080 ± 0.01
2.7 ± 0.5
Competitive (0.136 ± 0.01)
Competitive (0.103 ± 0.02)
Competitive (0.138 ± 0.02)
Not tested
Competitive (0.950 ± 0.02)
Competitive (0.143 ± 0.02)
Competitive (0.098 ± 0.01)
Competitive (0.058 ± 0.01)
Not tested
lM
(
3-methylbut-2-enyl)-8-(2-methylbut-3-en-2-yl)-9H-xanthen-9-
one (8).
2
.2. Effects of isolated xanthones on the activity of
b
Quercetin
neuraminidase
Apigeninb
17.4 ± 0.7
16.2 ± 4.24
Not tested
Not tested
Mangiferinb
In a preliminary screening using neuraminidase, we observed
that EtOAc extracts of C. tricuspidata roots showed significant inhi-
bition of the hydrolysis of neuraminic acid. In fractionation, guided
by neuraminidase inhibitory activity, EtOAc fractions showed
a
All compounds were examined in a set of experiments repeated three times;
IC50 values of compounds represent the concentration that caused 50% enzyme
activity loss.
b
These compounds were used as a positive control.

2
746
Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
7
A 100
B
6
5
4
3
2
1
0
8
6
4
2
0
0
0
0
0
0
.01
0.1
1
10
100
0
1
2
3
4
5
6
[
I] (µM)
[Neuraminidase] (mU/ml)
Figure 2. (A) Effects of compounds on the activity of neuraminidase for hydrolysis of neuraminic acid. (compound 1, d; compound 2, s; compound 3, .; compound 5,
, respectively). (B) The hydrolytic activity of neuraminidase as function of enzyme
; 80 nM, .; 160 nM, s; 320 nM, d, respectively).
D;
compound 6, j; compound 7, h; compound 8, ꢀ; quercetin, }; apigenin, N; mangiferin,
r
concentrations at different concentrations of compound 1 (0,
D
Table 2
2.4. Determination of the inhibition type of xanthones on
neuraminidase
Kinetic parameters for time-dependent inhibitor of neuraminidase by compounds
1ꢀ3 and 5ꢀ8)
(
K ai pp (nM)a
k
5
(s
ꢀ1
)
ꢀ1
The inhibition mechanisms displayed by isolated xanthones
Entry
6
k (s )
(
1ꢀ3 and 5ꢀ8) were then studied. This began with analysis of
1
2
3
5
6
7
8
213.5 ± 37.2
477.2 ± 98.6
567.6 ± 41.8
2230 ± 300
435.9 ± 11.2
510.1 ± 27.2
127.4 ± 10.7
0.0093 ± 0.001
0.0066 ± 0.002
0.0092 ± 0.001
0.0173 ± 0.004
0.0112 ± 0.005
0.0091 ± 0.001
0.0065 ± 0.001
0.00015 ± 0.0001
0.00029 ± 0.0001
0.00044 ± 0.0002
0.00002 ± 0.0000
0.00068 ± 0.0007
0.00047 ± 0.0002
0.00015 ± 0.0001
the mode of inhibition using both Lineweaver–Burk (Fig. 4A–F)
and Dixon plot which is useful in determining the K value for
i
inhibitor (Fig. 4G and H). As illustrated in Figure 4, the kinetic plots
show that compounds (1ꢀ3 and 5ꢀ8) have respective inhibition
constants, K
i
of 0.136, 0.103, 0.138, 0.950, 0.143, 0.098, and
0
.058 lM (Table 1). This analysis also delineates that the inhibitors
a
All compounds were examined in a set of experiments repeated three times.
are competitive because increasing concentration of substrate re-
sulted in a family lines which declined with a common intercept
on the x-axis (ꢀK
by measuring initial velocities of substrate hydrolysis as a function
of preincubation time of the enzyme with the inhibitor. As a de-
crease in residual activity was seen as a function of preincubation
time, compound 8 emerged to be a slow-binding inhibitor at low
concentrations (Fig. 3A). Increasing preincubations time of com-
pounds (1ꢀ6 and 8) led to a decrease in both the initial velocity
i
).
The predominant inhibition mode shown by naturally occurring
neuraminidase inhibitors is noncompetitive. Interestingly, the
inhibition of other enzymes by xanthone derives from C. tricuspida-
ta is also rarely competitive. However, the xanthones in this man-
uscript showed time-dependent competitive inhibition of
neuraminidase (enzyme isomerization inhibition). Thus these
inhibitors may be correlated to TamifluÒ which inhibits neuramin-
idase from influenza virus A (Tokyo/3/67, N2) is a similar man-
(
3
v
i
s
) and the steady-state rate (v ) (Fig. 3B). Representatively, Figure
C showed typical progress curves for slow binding behavior, when
the hydrolysis of substrate by neuraminidase in the presence of
different compound 8 concentrations (0, 0.09, 0.19, 0.39, 0.78,
.56, and 3.12 lM). As shown in Figure 3C and D, the progress
curves obtained using the differing concentrations of the inhibitors
were fitted to Eq. 3 to determine kobs. The plot presents the rela-
tionship between kobs and concentrations of inhibitors [I]. The y
intercept of the curve provides an estimate of the rate constant
6
k , while the maximum value of kobs expected at infinite inhibitor
concentration. The kinetic parameters k
2
4
ner. This suggests that further research on these important lead
compounds may possibly show them to be alternative new anti-
influenza treatments.
1
The qualitative SAR of these compounds can be described as fol-
lows. A vicinal dihydroxy motif on the A ring is mandatory for high
activity, as compound 4, in which one of the alcohol functions
within this unit is blocked, showed at least 500 times less activity
than 1ꢀ3 and 5ꢀ8 all of which have a free vicinal dihydroxy group
in the ring A. Interestingly, blocking of one of the hydroxyl units of
the 1,3-diol in the B ring appears to exert a variable but not insig-
nificant effect on activity. Thus compounds 5, 6, and 7 all of which
are alkylated at C3OH (on the B ring) have IC50 values 1.271
0.278 M, and 0.186 M, respectively, making them some of the
least active species tested. When these are compared to the corre-
sponding compounds bearing a free OH at C3, (1, 2, and 3) the IC50
app
5 6
, k , and Ki , were derived
from the plots by fitting the results (Table 2). This, analysis
unearthed the following values: k
and Ki = 127.4 nM. The kinetic model can be written as:
ꢀ
1
ꢀ1
5
= 0.0065 s , k
6
= 0.00015 s ,
app
23
k1
kcat
lM,
E + S
E S
E + P
k2
l
l
+
I
(A)
s
k4 k3
E I
of the latter are generally (but not in the case of 3 vs 7) lower. This
outlines a moderately importantly role for the 1,3-diol unit in the B
ring, which is presumably modified by other factors. The affects of
alkyl substitution around the ring seem to be suitable and within
experimental error are difficult to interpret.
k5
k6
E' I
The plot showed a hyperbolic dependence on the concentration
of the compound 8, so the inhibition of neuraminidase by com-
pound 8 is believed to followed mechanism A. The results indicated
that 8 inhibits neuraminidase by rapid formation of an enzyme
substrate complex (EꢁI) which slowly isomerizes to form a modi-
2.5. In silico molecular docking simulation of xanthones
The excellent results outlined above spurred us on to investi-
gate the principal interactions between the inhibitor and enzyme
0
fied enzyme complex (E ꢁI).

Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
2747
A
1000
B
1.0
0.8
0.6
0.4
8
6
4
2
00
00
00
00
0
v
v
1
2
3
4
5
6
8
control
0
1
2
3
min
0
.2
0 min
0 min
0 min
0.0
0
50 100 150 200 250 300
Time (sec)
0
5
10 15 20 25 30
Preincubation time (min)
D 0.006
C 1000
0
0
0
.005
.004
.003
8
6
4
2
00
00
00
00
0
control
90 nM
k
190 nM
390 nM
780 nM
1560 nM
3120 nM
0.002
0
0
.001
.000
0
50 100 150 200 250 300
Time (sec)
0
100 200 300 400 500
I] (nM)
[
Figure 3. (A) Time-dependent inhibition of neuraminidase in the presence of 8 (at 80 nM). (B) Inhibition as a function of preincubation time for active compounds (1ꢀ6 and
) at IC50. (C) Slow-binding inhibition of compound 8 at time course. The kobs values at each inhibition concentration were determined by fitting the data to Eq. 2. (D)
Dependence of the values for kobs on the concentration of compound 8. The kobs values, determined in panel A, were fitted to Eq. 3.
8
A
0
1
B
0
C 2.5
0
D
1.6
.4
1.2
0
1
1
1
0
0
0
0
.4
.2
.0
.8
.6
.4
.2
1.4
1.2
1.0
0.8
0.6
0.4
0.2
20 nM
90 nM
180 nM
360 nM
100 nM
200 nM
400 nM
600 nM
1200 nM
2000 nM
1
240 nM
60 nM
2.0
1.5
1.0
0.5
3
1
0
0
0
0
.0
.8
.6
.4
.2
-
100 -50
0
1
50 100 150 200
/[S] (mM)
-100 -50
0
50 100 150 200
-50
0
50 100 150 200
1/[S] (mM)
-50
0
50 100 150 200
1/[S] (mM)
1/[S] (mM)
1
1
1
1
1
0
0
0
0
.8
1.4
.2
1.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
E
F
0
G
H
0.00625 mM
0.0125 mM
0.025 mM
0
1
.2
.6
.4
.2
.0
.8
.6
.4
.2
100 nM
2
3
60 nM
80 nM
160 nM
1
00 nM
60 nM
1.0
0
0
0
0
.8
.6
.4
.2
0.8
0.6
0.4
0.2
0
.00625 mM
0.0125 mM
.025 mM
0
-
100 -50
0
1
50 100 150 200
/[S] (mM)
-0.10 -0.05 0.00 0.05 0.10 0.15
1/[S] ( M)
-400 -200
0
200 400 600
-200 -100
0
100
200
µ
[I] (nM)
[I], nM
Figure 4. Graphical determination of the type of inhibition for compounds (1–3, 5, 7, and 8). (A–F) Lineweaver–Burk plot for inhibition of compounds (1–3, 5, 7, and 8) on
neuraminidase for the hydrolysis of substrate. (G and H) Dixon plot for inhibition of compounds 1 and 8 on neuraminiase for the hydrolysis of substrate.

2
748
Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
more thoroughly. The most potent inhibitor of neuraminidase,
compound 8, was analyzed via molecular docking analysis for its
interactions with protein residues in the original ligand site. The
aminidase. Our detailed kinetic analysis of these species has un-
veiled that they are all competitive, slow binding inhibitors. Such
characteristics are highly unusual for xanthones obtained from
natural sources. We proceeded to investigate the interaction of
these compounds with the enzyme by molecular modeling, which
disclosed important information about the principal interactions
bringing about activity in this series. Most importantly, the most
active inhibitor, 8, appeared to interact with the enzyme in a sim-
ilar fashion to a known transition-state analogue. We strongly be-
lieve that all this information implicates xanthones 1ꢀ8 as
potentially fruitful areas for antiviral drugs of the future.
2
5
X-ray structure of neuraminidase determined by Newstead was
obtained from the protein data bank (PDB Code 2vk6). Molecular
2
6
docking studies were carried our using CDOCKER methodology
because of our kinetic study showing competitive inhibition mode.
Figure 5A shows an overlay of the active site of the ligand free
structure and compound 8. Our modeling calculation unveiled H-
bond interaction in the original ligand site between 8 and:
Arg1266, Arg1285, Arg1555, and Asp1328 (Fig 5B). Interestingly,
the binding pocket matches compound 8 very well. Furthermore
this interaction appears to be similar to the interaction between
the enzyme and 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid
4
4
. Materials and methods
(
Neu5Ac2en) (Fig 5D inset)25 which is a transition-state analogue
.1. General apparatus and chemicals
of neuraminic acid as shown in Figure 5C and D. By comparing
the interactions of these two ligands with Asp1328 and Arg1266,
it seems likely that the 6,7-dihydroxy group of A-ring may bind
to the protein in a similar fashion to the 4-OH and 5-NHAc of
Neu5Ac2en, while the 7-OH of the xanthone assumes the role of
All the reagent grade chemicals were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). Chromatographic separations
were carried out by Thin–layer Chromatography (TLC) (E. Merck
Co., Darmastdt, Germany), using commercially available glass plate
pre–coated with silica gel and visualized under UV at 254 and
66 nm sprayed with p-anisaldehyde staining reagent. Column
chromatography was carried out using 230–400 mesh Silica Gel
kieselgel 60, Merck, Germany). Silica gel (230–400 mesh, Merck),
RP-18 (ODS-A, 12 nm, S-150 mM, YMC), and Sephadex LH-20
Amersham Biosciences) were used for column chromatography.
5
8
-NHAc on Neu5Ac2en. In addition the carbonyl group of xanthone
may serve as a similar donor to the carboxylic acid moiety of
3
Neu5Ac2en, both of which were found to interact with Arg1555.
This result furnishes some additional support to our affirmation
that xanthone 8 is a new lead competitive inhibitor for neuramin-
idase. Although this preliminary molecular modeling study cannot
constitute a definitive proof for our argument, it is a positive sign
that our inhibitor resides in the active site, as a competitive inhib-
itor should. It is also encouraging that taken as a whole the conclu-
sion from modeling and SAR are in agreement.
(
(
Melting points were measured on a Thomas Scientific Capillary
Melting Point Apparatus and are uncorrected. IR spectra were re-
corded on a Bruker IFS66 infrared Fourier transform spectropho-
1
13
tometer (KBr). H and C NMR along with 2D-NMR data were
1
13
obtained on a Bruker AM 500 ( H NMR at 500 MHz, C NMR at
125 MHz) spectrometer in CDCl acetone-d , DMSO-d , and CD OD,
3
. Conclusion
3
6
6
3
EIMS and HREIMS data were collected on Jeol JMS-700 spectrome-
ter. Optical rotations were measured on Perkin–Elmer 343
polarimeter.
Eight polyhydroxylated xanthones from C. tricuspidiata were
isolated and shown to be particularly effective inhibitors of neur-
Figure 5. (A) Overlay of the active site of the ligand free structure and compound 8. (B) Hydrogen bond interactions between compound 8 and neuraminidase. (C and D)
Compound 8 binding pocket matches with Neu5Ac2en on the structure at active site of neuraminidase (green color, Neu5Ac2en).

Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
2749
4
.2. Isolation of neuraminidase inhibitors from C. tricuspidata
The root bark of C tricuspidata (Carr.) Bureau was collected in
(2H, d, J = 7.1 Hz, H-16), 5.23 (1H, m, H-17), 1.65 (3H, s, H-19),
1
3
1
3
.85 (3H, s, H-20). C NMR (125 MHz, CD OD) d 163.7 (C-1),
116.3 (C-2), 165.7 (C-3), 95.5 (C-4), 157.6 (C-4a), 151.3 (C-4b),
116.3 (C-5), 152.9 (C-6), 144.2 (C-7), 106.7 (C-8), 114.0 (C-8a),
182.1 (C-9), 103.7 (C-9a), 42.5 (C-11), 29.9 (C-12), 29.9 (C-13),
152.0 (C-14), 108.5 (C-15), 23.7 (C-16), 123.3 (C-17), 133.1 (C-
Hapchun (Korea) in August, 2002, and identified by Professor Jae-
Hong Pak of Kyungpook National University. A voucher specimen
(
Park, K. H. 110) of this raw material is deposited at the Herbarium
1
8,20,21
of Kyungpook National University (KNU). The root bark of C. tricu-
spidata (2 kg) were air-dried, pulverized, and extracted with EtOAc
for a week at room temperature. The combined extract was evap-
orated to dryness under reduced pressure at a temperature below
18), 18.6 (C-19), 26.4 (C-20).
Compound obtained as
(500 MHz, CDCl ) d 6.15 (1H, s, H-2), 6.77 (1H, s, H-8), 1.43 (3H,
4
a
yellowish powder; 1H NMR
3
s, H-12), 1.43 (3H, s, H-13), 6.39 (1H, dd, J = 17.8, 10.5 Hz, H-14),
5.41 (1H, d, J = 10.5 Hz, H-15), 5.39 (1H, d, J = 17.8 Hz, H-15), 7.93
(2H, d, J = 10.2 Hz, H-16), 5.75 (1H, d, J = 10.2 Hz, H-17), 1.62 (3H,
3
5 °C to afford EtOAc-soluble extracts (61 g). The EtOAc extract
was subjected to column chromatography on silica gel
1
3
(
(
10 ꢂ 30 cm, 230–400 mesh, 600 g) using hexane/EtOAc [40:1
3
s, H-19), 1.63 (3H, s, H-20). C NMR (125 MHz, CDCl ) d 162.2
1.5 L), 20:1 (1.5 L), 10:1 (1.5 L), 5:1 (1.5 L), 3:1 (1.5 L), 1:1 (3 L)
(C-1), 100.6 (C-2), 162.4 (C-3), 109.4 (C-4), 155.6 (C-4a), 137.4
(C-4b), 120.0 (C-5), 151.4 (C-6), 153.0 (C-7), 102.3 (C-8), 108.5
(C-8a), 183.2 (C-9), 105.0 (C-9a), 41.3 (C-11), 28.4 (C-12), 28.4
(C-13), 149.7 (C-14), 113.7 (C-15), 121.3 (C-16), 132.8 (C-17),
and only EtOAc (3 L)] mixtures to give fractions F1–F5. Fraction
F3 (18 g) was purified by column chromatography using a glass
column packed with silica gel in hexane/EtOAc. The column was
then eluted using hexane/acetone (40:1?1:1) mixtures of increas-
ing polarity. Altogether, 80 fractions of 100 mL each were collected
and combined to give seven major subfractions (F3.1–F3.7), based
on the comparison of TLC profile using hexane/acetone (3:1) as
developing solvent. Subfraction F3.1 (1.8 g) was rechromato-
graphed using the same solvent gradient, to yields compound 3
1
8,20,21
77.5 (C-18), 27.7 (C-19), 27.7 (C-20).
Compound obtained as
yellowish needle; 1H NMR
(500 MHz, CDCl ) d 6.32 (1H, s, H-2), 6.80 (1H, s, H-5), 1.62 (3H,
5
a
3
s, H-12), 1.62 (3H, s, H-13), 6.25 (1H, dd, J = 17.4, 10.6 Hz, H-14),
4.84 (1H, d, J = 10.6 Hz, H-15), 4.85 (1H, d, J = 17.4 Hz, H-15), 4.30
(2H, d, J = 6.8 Hz, H-16), 5.30 (1H, m, H-17), 1.77 (3H, s, H-19),
1
3
(
12 mg) and compound 4 (33 mg). Subfraction F3.3 (1.2 g) were
3 3
1.88 (3H, s, H-20), 3.82 (OCH ). C NMR (125 MHz, CDCl ) d
purified using sephadex LH-20 column chromatography, eluting
with 95% MeOH to afford compound 1 (30 mg), compound 2
162.4 (C-1), 95.6 (C-2), 165.1 (C-3), 112.7 (C-4), 154.7 (C-4a),
153.8 (C-4b), 101.3 (C-5), 151.4 (C-6), 140.0 (C-7), 127.6 (C-8),
111.3 (C-8a), 183.6 (C-9), 104.5 (C-9a), 41.5 (C-11), 29.8 (C-12),
30.1 (C-13), 151.2 (C-14), 107.3 (C-15), 26.4 (C-16), 121.9 (C-17),
(
22 mg), and compound 5 (14 mg). Furthermore, compounds 1ꢀ3
were purified by recrystallized from hexane/acetone. Subfraction
1
8,20,21
F3.6 (2.2 g) was subjected to silica gel column (3 ꢂ 50 cm, 230–
135.9 (C-18), 26.2 (C-19), 18.5 (C-20), 55.9 (OCH
3
).
Compound 6 obtained as a pale yellow; H NMR (500 MHz, ace-
tone-d ) d 6.84 (1H, s, H-5), 7.41 (1H, s, H-8), 1.35 (3H, s, H-12),
1
4
00 mesh, 250 g) chromatography with hexane/EtOAc
(
40:1?1:1) and then purified by reversed-phase column chroma-
tography. Thus, this subfraction was loaded onto a glass column
packed with RP-18 (ODS-A, 12 nm, S-150
M, 40 g). The column
was then eluted using MeOH/H O (4:1) to afford compound 8
8 mg). Fraction 4 (10 g) was applied to silica gel column [eluent:
6
1.11 (3H, s, H-13), 4.39 (1H, q, J = 13.1, 6.6 Hz, H-14), 1.26 (3H, d,
J = 6.6 Hz, H-15), 3.26 (2H, d, J = 7.2 Hz, H-16), 5.27 (1H, br t,
J = 7.2 Hz, H-17), 1.72 (3H, s, H-19), 1.52 (3H, s, H-20). 13C NMR
l
2
(
6
(125 MHz, acetone-d ) d 157.7 (C-1), 117.0 (C-2), 164.6 (C-3),
hexane/EtOAc (40:1?1:1)] to afford 12 fractions (F4.1–F4.12).
Subfraction F4.7 (750 mg) was resubjected to silica gel chromatog-
raphy with hexane/acetone (30:1?1:1) to yield compound 6
103.2 (C-4), 156.1 (C-4a), 153.0 (C-4b), 103.9 (C-5), 154.5 (C-6),
144.5 (C-7), 109.6 (C-8), 114.0 (C-8a), 181.6 (C-9), 104.6 (C-9a),
44.8 (C-11), 21.4 (C-12), 26.0 (C-13), 91.7 (C-14), 15.1 (C-15),
23.0 (C-16), 123.3 (C-17), 132.5 (C-18), 18.4 (C-19), 26.3 (C-
(
11 mg) and 7 (9 mg). All of isolated compounds were identified
1
8,20,21
on the basis of the following spectroscopic data.
20).
Compound obtained as
3
(500 MHz, CDCl ) d 6.33 (1H, s, H-4), 7.50 (1H, s, H-8), 1.26 (3H,
1
a
yellowish powder; 1H NMR
Compound 1 obtained as a yellowish solid; H NMR (500 MHz,
7
CDCl
3
) d 6.22 (1H, s, H-2), 6.79 (1H, s, H-5), 1.68 (3H, s, H-12), 1.68
(
3H, s, H-13), 6.45 (1H, dd, J = 17.8, 10.5 Hz, H-14), 5.35 (1H, d,
s, H-12), 1.51 (3H, s, H-13), 4.54 (1H, q, J = 6.6 Hz, H-14), 1.42
(3H, d, J = 6.6 Hz, H-15), 3.25 (2H, m, H-16), 5.14 (1H, m, H-17),
1.81 (3H, s, H-19), 1.65 (3H, s, H-20). C NMR (125 MHz, CDCl )
3
J = 10.5 Hz, H-15), 5.44 (1H, d, J = 17.8 Hz, H-15), 4.27 (2H, d,
J = 6.7 Hz, H-16), 5.30 (1H, m, H-17), 1.75 (3H, s, H-19), 1.87 (3H,
s, H-20). C NMR (125 MHz, CDCl
1
3
1
3
3
) d 161.7 (C-1), 100.0 (C-2),
d 158.1 (C-1), 116.7 (C-2), 165.8 (C-3), 89.3 (C-4), 158.0 (C-4a),
150.0 (C-4b), 115.1 (C-5), 149.9 (C-6), 141.6 (C-7), 105.1 (C-8),
112.4 (C-8a), 180.3 (C-9), 103.4 (C-9a), 43.4 (C-11), 20.7 (C-12),
25.2 (C-13), 91.1 (C-14), 14.4 (C-15), 22.2 (C-16), 121.0 (C-17),
1
1
1
1
2
61.8 (C-3), 108.9 (C-4), 155.0 (C-4a), 152.9 (C-4b), 100.6 (C-5),
50.9 (C-6), 140.0 (C-7), 127.4 (C-8), 111.1 (C-8a), 183.0 (C-9),
04.6 (C-9a), 40.8 (C-11), 28.1 (C-12), 28.1 (C-13), 149.4 (C-14),
13.1 (C-15), 26.0 (C-16), 121.6 (C-17), 135.0 (C-18), 18.1 (C-19),
1
8,20,21
132.8 (C-18), 17.9 (C-19), 25.7 (C-20).
Compound 8 obtained as a yellowish solid; H NMR (500 MHz,
CDCl ) d 7.43 (1H, s, H-4), 6.29 (1H, s, H-5), 1.59 (3H, s, H-12), 1.59
1
8,20,21
1
5.9 (C-20).
Compound 2 obtained as a yellowish prism; H NMR (500 MHz,
CDCl ) d 6.41 (1H, s, H-5), 7.51 (1H, s, H-8), 1.68 (3H, s, H-12), 1.68
3H, s, H-13), 6.53 (1H, dd, J = 17.8, 10.5 Hz, H-14), 5.40 (1H, d,
1
3
3
(3H, s, H-13), 6.46 (1H, dd, J = 17.8, 10.6 Hz, H-14), 5.34 (1H, d,
J = 10.6 Hz, H-15a), 5.34 (1H, d, J = 17.8 Hz, H-15b), 3.09 (2H, d,
J = 7.0 Hz, H-16), 5.01 (1H, d, J = 7.0 Hz, H-17), 1.56 (3H, s, H-19),
(
J = 10.5 Hz, H-15), 5.30 (1H, d, J = 17.8 Hz, H-15), 3.44 (2H, d,
J = 7.0 Hz, H-16), 5.20 (1H, m, H-17), 1.70 (3H, s, H-19), 1.83 (3H,
s, H-20). C NMR (125 MHz, CDCl
1
3
3
1.72 (3H, s, H-20). C NMR (125 MHz, CDCl ) d 163.0 (C-1),
1
3
3
) d 160.4 (C-1), 113.2 (C-2),
113.6 (C-2), 163.3 (C-3), 95.8.9 (C-4), 156.4 (C-4a), 142.0 (C-4b),
105.4 (C-5), 150.7 (C-6), 150.1 (C-7), 115.6 (C-8), 112.6 (C-8a),
180.8 (C-9), 103.1 (C-9a), 41.5 (C-11), 27.5 (C-12), 27.4 (C-13),
150.2 (C-14), 113.9 (C-15), 22.3 (C-16), 121.3 (C-17), 133.2 (C-
1
1
1
1
2
61.1 (C-3), 107.2 (C-4), 153.3 (C-4a), 151.8 (C-4b), 102.1 (C-5),
52.8 (C-6), 141.9 (C-7), 107.0 (C-8), 112.1 (C-8a), 180.3 (C-9),
02.6 (C-9a), 41.5 (C-11), 27.4 (C-12), 27.4 (C-13), 149.9 (C-14),
13.4 (C-15), 22.1 (C-16), 122.3 (C-17), 131.8 (C-18), 17.9 (C-19),
2
1
18), 26.1 (C-19), 18.3 (C-20).
1
8,20,21
5.8 (C-20).
Compound
500 MHz, CD
s, H-12), 1.61 (3H, s, H-13), 6.35 (1H, dd, J = 17.8, 10.6 Hz, H-14),
.81 (1H, d, J = 10.6 Hz, H-15), 4.90 (1H, d, J = 17.5 Hz, H-15), 3.51
3
obtained as
a
yellowish needle; 1H NMR
4.3. Assay of neuraminidase activity
(
3
OD) d 6.32 (1H, s, H-4), 7.35 (1H, s, H-8), 1.61 (3H,
Neuraminidase activity was measured by a modification of the
1
7,27
4
method described by Poteir et al.
4-Methylumbelliferyl-a-D-N-

2
750
Y. B. Ryu et al. / Bioorg. Med. Chem. 17 (2009) 2744–2750
acetylneuraminic acid sodium salt hydrate (SIGMA, M8639)
.125 mM in 50 mM sodium acetate buffer (pH 5.0) was used as
Acknowledgments
0
a substrate. Neuraminidase (E.C. 3.2.1.18. from Clostridium perfrin-
gens, SIGMA, N2876) 0.1 U/mL in acetate buffer was used as the en-
zyme. The isolated compounds were dissolved in MeOH and
diluted to the corresponding concentrations in acetate buffer. For
This work was supported by a grant from Agricultural R&D Pro-
motion Center, Korea Rural Economic Institute and the MOST/KO-
SEF to the EB-NCRC (R15-2003-012-0200-0), Republic of Korea.
J.W.L. was supported by a scholarship from the BK21 Program,
the Ministry of Education and Human Resources Development,
Republic of Korea.
assay, enzyme (15
mixed with buffer (510
L of substrate at 37 °C. 4-Methylumbelliferone was immedi-
lL) was added to 15
lL of sample solution
lL) in a cuvette, and then was added
60 l
ately quantified by fluorometric determination with an LS-55 lumi-
nescence spectrometer (Perkin–Elmer Limited, Beaconsfield,
Buckinghamshire, United Kingdom). The excitation wavelength
was 365 nm, with the excitation slits set at 2.5 nm, and the emis-
sion wavelength was 450 nm, with the emission slits set at
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.02.042.
2
0 nm. For the determination of enzyme activity (by fitting exper-
imental data to the logistic curve by Eq. 1), used time-drive proto-
col with initial velocity was recorded over range of
References and notes
a
1. Schauer, R.; Kelm, S.; Reuter, G.; Roggenin, P. In Biology of the Sialic Acid;
Rosenberg, A., Ed.; Plenum Press: New York, 1995; pp 7–67.
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regression program [Sigma Plot (SPCC Inc., Chicago, IL)].
2.
3.
4.
Saito, M.; Yu, R. K. In Biology of the Sialic Acid; Rosenberg, A., Ed.; Plenum Press:
New York, 1995; pp 216–313.
Matrosovich, M. N.; Matrosovich, T. Y.; Gray, T.; Roberts, N. A.; Klenk, H. D. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 4620.
Activity ð%Þ ¼ 100½1=ð1 þ ð½Iꢃ=IC50ÞÞꢃ
ð1Þ
Shinya, K.; Ebina, M.; Yamada, S.; Ono, M.; Kasai, N.; Kawaoka, Y. Nature 2006,
4
40, 435.
4
.4. Progress curves determination and time-dependent assay
5. Couceiro, J. N.; Paulson, J. C.; Baum, L. G. Virus Res. 1993, 29, 155.
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7
.
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Achyuthan, K. E.; Achyuthan, A. M. Comp. Biochem. Physiol. B: Biochem. Mol. Biol.
Time-dependent assays and progress curves were carried out
using 0.1 U/mL units neuraminidase, and 4-methylumbelliferyl-
-N-acetylneuraminic acid sodium salt hydrate (SIGMA,
M8639) as a substrates in 0.05 M sodium acetate buffer (pH 5.0)
at 37 °C. Enzyme activities were measured by fluorimetry continu-
ously for 5 min. To determine the kinetic parameters associated
with time-dependent inhibition of neuraminidase, progress curves
for 300 s were obtained at several inhibitor concentrations using
fixed substrate concentrations. The data were analyzed using the
a nonlinear regression program [Sigma Plot (SPCC Inc., Chicago,
2001, 129, 29.
8
.
Rosenberg, A.; Schengrund, C.-L. In Biological Roles of Sialic Acid; Rosenberg, A.,
Ed.; Plenum: New York, 1976; pp 295–359.
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a-D
9
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1
1
0. Ohuchi, M.; Asaoka, N.; Sakai, T.; Ohuchi, R. Microbes Infect. 2006, 8, 1287.
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3. (a) Fassina, G.; Buffa, A.; Benelli, R.; Varnier, O. E.; Noonanm, S. K.; Li, C.; Liu, S.
T. Biochem. Biophys. Res. Commum. 2002, 16, 939; (b) Nakane, H.; Ono, K.
Biochemistry 1990, 29, 2841.
14. (a) Song, J.-M.; Lee, K.-H.; Seong, B.-L. Antiviral Res. 2005, 68, 66; (b) Song, J. M.;
Park, K. D.; Lee, K. H.; Byun, Y. H.; Park, J. H.; Kim, S. H.; Kim, J. H.; Seong, B. L.
Antiviral Res. 2007, 76, 178.
IL)] to give the individual parameters for each curve;
velocity), (steady-state velocity), kobs (apparent first-order rate
constant for the transition from to ), I (intensity at ex:
(apparent K
v
i
(initial
15. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Yamada, H. Biochem. Biophys. Res.
v
s
Commun. 1989, 163, 25.
16. Nagai, T.; Miyaichi, Y.; Tomimori, T.; Yamada, H. Chem. Pharm. Bull. 1990, 38,
v
i
v
s
app
i
1329.
3
65 nm, em: 450 nm), and K
i
) according to the fol-
17. Ryu, Y. B.; Curtis-Long, M. J.; Kim, J. H.; Jeong, S. H.; Yang, M. S.; Lee, K. W.; Lee,
2
3
lowing equations:
W. S.; Park, K. H. Bioorg. Med. Chem. Lett. 2008, 18, 6046.
1
8. (a) Lee, B. W.; Lee, J. H.; Lee, S. T.; Lee, H. S.; Lee, W. S.; Jeong, T. S.; Park, K. H.
Bioorg. Med. Chem. Lett. 2005, 15, 5548; (b) Lee, B. W.; Gal, S. W.; Park, K. M.;
Park, K. H. J. Nat. Prod. 2005, 68, 456.
I ¼
v
s
t þ ð
v
i
ꢀ
v
s
Þ½1 ꢀ expðꢀkobstÞꢃ=kobs
ð2Þ
ð3Þ
app
k
obs ¼ k
6
þ ½ðk ꢂ ½IꢃÞ=ðKi þ ½IꢃÞꢃ
5
1
2
2
2
9. Han, X. H.; Hong, S. S.; Hwang, J. S.; Jeong, S. H.; Hwang, J. H.; Lee, M. H.; Lee, M.
K.; Lee, D.; Ro, J. S.; Hwang, B. Y. Arch. Pharm. Res. 2005, 28, 1324.
0. Park, K. H.; Park, Y. D.; Han, J. M.; Im, K. R.; Lee, B. W.; Jeong, I.-Y.; Jeong, T. S.;
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S.; Park, K. H. Bioorg. Med. Chem. Lett. 2007, 17, 6421.
4
.5. Simulation of molecular docking
The neuraminidase three-dimensional structures were obtained
2. Li, X.; Ohtsuki, T.; Shindo, S.; Sato, M.; Koyano, T.; Preeprame, S.;
Kowithayakorn, T.; Ishibashi, M. Planta Med. 2007, 73, 1195.
2
5
from Protein Data Bank (PDB), accession code no. was 2vk6.
CDOCKER protocol in Discovery Studio platform was used for the
docking studies. After water molecules and inhibitor were re-
moved from 2vk6 (whole structure), the crystal structure was de-
fined as the receptor and the active site was defined as a sphere
within 8 Å reach of the inhibitor. Random ligand conformations
are generated from the initial ligand structure through high tem-
23. Morrison, J. F.; Walsh, C. T. Adv. Enzymol. 1988, 61, 201.
2
2
2
2
4. Kati, W. M.; Saldivar, A. S.; Mohamadi, F.; Sham, H. L.; Laver, W. G.;
Kohlvrenner, W. E. Biochem. Biophys. Res. Commun. 1998, 244, 408.
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Withers, S. G.; Taylor, G. L. J. Biol. Chem. 2008, 283, 9080.
6. Wu, G.; Robertson, D. H.; Brooks, C. L., III; Vieth, M. J. Comput. Chem. 2003, 24,
1549.
7. Potier, M.; Mameli, L.; Belisle, M.; Dallaire, L.; Melancon, S. B. Anal. Biochem.
1979, 94, 287.
2
6
perature molecular dynamics, followed by random rotations.