Thiosialoside clusters using carbosilane dendrimer core scaffolds as a new class of influenza neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.10.085

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with α-thioglycoside of sialic acid was accomplished. The results of a preliminary study on biological responses against influenza virus sialidases using thiosialoside clusters showed that some of the glycodendrimers have inhibitory potencies against the sialidases.

Full text of DOI:10.1016/j.bmcl.2006.10.085

Bioorganic & Medicinal Chemistry Letters 17 (2007) 717–721
Thiosialoside clusters using carbosilane dendrimer core scaffolds as
a new class of influenza neuraminidase inhibitorsq
Jun-Ichi Sakamoto,a Tetsuo Koyama,a Daisei Miyamoto,b,c Sangchai Yingsakmongkon,d
Kazuya I. P. J. Hidari,b,c Wipawee Jampangern,e Takashi Suzuki,b,c Yasuo Suzuki,c,f
Yasuaki Esumi,g Ken Hatano,a Daiyo Terunumaa and Koji Matsuokaa,*
aDivision of Materials Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan
bDepartment of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences, and COE program in the 21st century,
Suruga, Shizuoka 422-8526, Japan
cCore Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency,
Kawaguchi, Saitama 332-0012, Japan
dDepartment of Microbiology and Immunology, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand
eDepartment of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
fDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Aichi 487-8501, Japan
gThe Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-0198, Japan
Received 13 September 2006; revised 23 October 2006; accepted 26 October 2006
Available online 1 November 2006
Abstract—An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with a-thioglycoside of sialic acid was
accomplished. The results of a preliminary study on biological responses against influenza virus sialidases using thiosialoside clusters
showed that some of the glycodendrimers have inhibitory potencies against the sialidases.
Ó 2006 Elsevier Ltd. All rights reserved.
Influenza viruses have different types of carbohydrate-
related proteins on their surfaces.1 One of the proteins
is hemagglutinin (HA), which is a kind of lectin and
binds to sialyl oligosaccharides as specific receptors on
host cells.2 The other one is glycosidase, which is re-
ferred to as either sialidase or neuraminidase (NA)
and cleaves sialic acid residues from sialoglycoproteins
as well as gangliosides on the surfaces of the host cells.3
Therefore, the virus is significantly unique and the pro-
teins on the surfaces of influenza viruses have completely
different roles, such as adhesion to the host cell and
secession from the host cell.1 Neuraminidase inhibitors,
such as zanamivir4 and oseltamivir,5 have been synthe-
sized and widely used as therapeutic agents in the clini-
cal treatment of influenza A and B viruses.6 These drugs
have extremely high inhibitory potencies to the release
of influenza virions from infected cells; however, NA
inhibitor-resistant viruses have already been generated.7
Although the specific ligand of NA of influenza viruses
A and B is natural sialic acid, the inhibitors are designed
as transition-state analogues after cleavage of sialic acid
residues by a sialidase.4,5 Therefore, we examined the
use of thioglycoside of sialic acid as a natural epitope
for an NA inhibitor because thioglycosidic linkage is
usually not hydrolyzed by glycosidases, such as NA.8
However, since monomeric sialoside does not have
inhibitory potency for NA, we planned to use the su-
gar-clustering effect.9 In addition, NAs on the virus sur-
face display tetrameric structures10 and a glycocluster is
expected to be a promising multivalent-type therapeutic
agent.11
As to glycoclusters, we have reported syntheses of vari-
ous carbosilane dendrimers having bioactive carbohy-
drate determinants on the terminal ends12 and
biological activities of the compounds.11 In this paper,
we describe the synthetic assembly of thiosialoside moi-
eties using a series of carbosilane dendrimers as the core
Keywords: Thioglycosides; Enzyme inhibitors; Sialic acid; Influenza
virus; Sialidases; Glycoclusters.
q
Glyco-Silicon Functional Materials. Part 9. For Part 8, see Ref. 12c.
Corresponding author. Tel./fax: +81 48 858 3099; e-mail:
koji@fms.saitama-u.ac.jp
*
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.085

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J.-I. Sakamoto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 717–721
OH
COOH
OH
O
Neu5AcS :
HO
AcHN
Neu5AcS
S S
2
Neu5AcS
Neu5AcS
S
OH
1
SNeu5Ac
SNeu5Ac
SNeu5Ac
SNeu5Ac
O
S
S
Ph Si
S
O
Ph Si
S
S
S
SNeu5Ac
O
SNeu5Ac
3
6
Neu5AcS
Neu5AcS
SNeu5Ac
SNeu5Ac
SNeu5Ac
SNeu5Ac
Neu5AcS
Neu5AcS
O
O
O
O
S
S
S
S
S
S
S
Si
4
Si
7
S
SNeu5Ac
Neu5AcS
SNeu5Ac
SNeu5Ac
Neu5AcS
O
O
Me
Me
Me
Me
S
S
S
S
S
S
Si
O
SNeu5Ac
SNeu5Ac
Si
S
Neu5AcS
O
S
Si
Si
Si
5
Neu5AcS
Neu5AcS
S
Si
S
S
S
O
SNeu5Ac
O
8
Neu5AcS
Figure 1. A series of multivalent-type synthetic substrates having thioglycosidic linkages of sialic acids.
frames and the preliminary biological evaluations using
influenza viruses.
Scheme 2. Known tribromide 1212b was condensed with
6 equivalent molar of thioacetate 11, which means the
ratio of Br/SAc is 1:2, in the presence of sodium methox-
ide in methanol—DMF followed by re-acetylation to
Schematic structures of target dendrimers are shown in
Figure 1, where monomeric substrate 1, dimeric sub-
strate 2, trimeric substrates 3 and 6, which have fan
shapes, tetrameric substrates 4 and 7, which have ball
shapes, and hexameric substrates 5 and 8, which have
dumbbell shapes, are illustrated.
afford fully protected 13 in 80.2% yield accompanied
33
D
by a disulfide 14, 13, ½aꢀ +26° (c 1.10, CHCl3), integral
ratio of the H atoms by 1H NMR/SiCH2/CH2SCH2/Ph/
H-8 = 6:12:5:3, HRMS (ESI+) calcd for [M+H]+:
28
D
2056.71535; found m/z: 2056.72008, and 14, ½aꢀ +28°
(c 1.13, CHCl3), FABMS calcd for [M+H]+: 1217.4;
found m/z: 1218.0. Deprotection of 13 was accomplished
The synthetic scheme for preparation of a common thi-
oacetate 11 of sialic acid having thioglycosidic linkage is
shown in Scheme 1. Known chloride was converted into
a-thioacetate 9 by the previously reported procedures,13
and 9 was allowed to react with 5-bromo-pentene in the
presence of potassium carbonate in methanol to give thio-
´
by a combination of Zemplen’s manner and alkali
hydrolysis to provide water-soluble 3 in 87.4% yield
after chromatographic purification by using Sephadex
29
G-25, ½aꢀ +28° (c 1.03, H2O), HRMS (ESI+) calcd for
D
[M+H]+: 1510.54162; found m/z: 1510.53494. The same
reaction was applied for known tetrabromide 1512c and
the coupling reaction proceeded smoothly to yield 16
(75.2%), in which ester functions were removed by the
glycoside 10 in 84.2% yield after re-acetylation followed
27
by chromatographic purification, ½aꢀ +29° (c 1.13,
D
CHCl3), J7,8 = 8.2 Hz, Dd jH-9a-H9bj = 0.20 ppm.14
Transesterification and subsequent saponification to 10
gave water-soluble sialoside 1 as a monomeric substrate
for the biological assay in quantitative yield after lyophi-
lization. Quantitative transformation of the terminal dou-
ble bond into corresponding primary thioacetate was
method described for 3 to give white powdery 4 in
24
D
99.5% yield, 16, ½aꢀ +27° (c 1.19, CHCl3), integral ratio
of the
H
atoms by 1H NMR/SiCH2/CH2SCH2/
H-8 = 8:16:4, HRMS (ESI+) calcd for [M+Na]+:
31
D
2651.88866; found m/z: 2651.87993, and 4, ½aꢀ +23°
accomplished by a radical addition of thioacetic acid in
(c 1.06, H2O), HRMS (ESI+) calcd for [M+H]+:
1901.67508; found m/z: 1901.66668. The same reaction
was also carried out for 1712c and the condensed product
18 having hexa-sialic acid moieties in the molecule was
obtained in 53.5% yield, which was then treated in same
28
D
the presence of AIBN15 to afford 11, ½aꢀ +25° (c 1.11,
CHCl3).
Construction of carbosilane dendrimers 3–5 functional-
ized with thioglycosides of sialic acid is summarized in
manner as that described for 3 to give quantitatively 5 as
24
a white powder after lyophilization, 18, ½aꢀ +24° (c
D
0.664, CHCl3), integral ratio of the H atoms by 1H
NMR/SiCH3/SiCH2/CH2SCH2/H-8 = 6:20:24:6, HRMS
(ESI+) calcd for [M+2H]2+/2: 2072.71705; found m/z:
All new compounds with specific rotation data gave satisfactory
results of elemental analyses or high-resolution mass spectra.

J.-I. Sakamoto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 717–721
719
OR1
OR1
OAc
OAc
COOR2
S
OAc
Cl
COOMe
SAc
OAc
O
R1O
AcHN
ii)
i)
AcO
AcHN
AcO
AcHN
O
O
OR1
COOMe
OAc
OAc
10 : R1 = Ac, R2 = Me
1 : R1 = R2 = H
9
iii)
OAc
COOMe
S
OAc
iv)
AcO
AcHN
O
SAc
OAc
11
Scheme 1. Reagents and conditions: (i) Ref. 13; (ii) 5-Bromo-1-pentene, K2CO3, MeOH—DMF, 0°C ! rt, overnight, then Ac2O—Pyr, rt, overnight;
(iii) NaOMe, MeOH, rt, 0.5 h, then 0.5 M aqueous NaOH, rt, overnight; (iv) AIBN, HSAc, 1,4-dioxane, 50 °C ! 80°C, 3 h.
OR1
OR1
R1O
AcHN
COOR2
S
O
OR1
OR1
R1O
COOR2
S
i)
S
S
Ph Si
Br
R1O
AcHN
COOR2
O
OR1
+
3
S
Si Ph
OR1
OR1
3
12
S
O
R1O
AcHN
13 : R1 = Ac, R2 = Me
3 : R1 = R2 = H
ii)
R1O
14 : R1 = Ac, R2 = Me
2 : R1 = R2 = H
ii)
OR1
OR1
COOR2
S
i)
Si
Br
R1O
AcHN
O
OR1
4
14
S
Si
+
4
15
16 : R1 = Ac, R2 = Me
4 : R1 = R2 = H
ii)
OR1
COOR2
S
Me
Me
OR1
i)
Me
Me
Si
Si
Br
R1O
AcHN
O
3
S
Si
Si
14
2
+
OR1
3
17
2
18 : R1 = Ac, R2 = Me
5 : R1 = R2 = H
ii)
Scheme 2. Reagents and conditions: (i) 11 (2 molar excess vs SAc), NaOMe, MeOH—DMF, 0°C ! rt, overnight, then Ac2O—Pyr, rt, overnight;
(ii) NaOMe, MeOH, rt, 3 h, then 0.5 M aqueous NaOH, rt, overnight.
27
D
2072.71684, and 5, ½aꢀ +23° (c 0.99, H2O), HRMS
the dendritic core and sugars, because the binding sites
of sialic acid residue in tetrameric sialidases in influenza
virus are located around in 40–50 distances.8 Therefore,
an elongation of each spacer-arm in the known dendri-
mer scaffolds was first needed, and the synthetic route
for the preparation of dendrimer scaffolds and the con-
struction of glycodendrimers using elongated core
frames are shown in Scheme 3. The starting trihydroxy
compound 1916 was subjected to Williamson’s ether
synthesis with allyl bromide giving triallyl derivative
20, which underwent hydroboration to give 21. The
(ESI+) calcd for [M+2H]2+/2: 1504.5614; found m/z:
1504.5558. Removal of protective groups in dimeric
sialoside 14 was also carried out in a similar manner
as that described for 3 to give quantitatively 2 as a white
30
D
powder after lyophilization, ½aꢀ +25° (c 1.03, H2O),
FABMS calcd for [M+H]+: 853.3; found m/z: 853.0.
Given the success of synthetic assembly of thiosialoside
using various carbosilane scaffolds, our attention was
directed toward an elongation of spacer length between

720
J.-I. Sakamoto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 717–721
i)
ii)
Ph Si
O
R
Ph Si
O
Ph Si
OH
OH
3
3
3
21 : R = OH
22 : R = OMs
23 : R = Br
iii)
iv)
20
19
v)
Si
Si
O
Br
4
4
25
24
Me
Si
Me
v)
Si
OH
Si
Si
O
Br
3
3
2
Me
Me
2
27
26
Scheme 3. Reagents and conditions: (i) Allyl bromide, NaH, THF, 0 ! 70 °C, 2 h; (ii) 1 M BH3—THF, cyclohexene, THF, 0 °C ! rt, 3 h, then 3 M
aqueous NaOH, H2O2, 60°C, overnight; (iii) MsCl, Pyr, 0°C, 3 h; (iv) NaBr, DMF, 60 °C, overnight; (v) (i)–(iv).
alcohols were all O-mesylated to afford 20 and replaced
i)
Ph Si
O
R
23
25
with bromo anions to give tribromo compound 23 in
55.4% yield (4 steps), FABMS calcd for [M+H]+:
643.1; found m/z: 642.7. The same reaction sequences
were performed for known alcohols 2416 and 26,12c
and all of the reactions proceeded smoothly to provide
tetrabromo compound 25 in 54.4% yield (4 steps) and
hexabromo compound 27 in 39.4% yield (4 steps),
respectively, 25, FABMS calcd for [M+H]+: 745.0;
found m/z: 748.5. Although the molecular ion peak of
27 was unfortunately not observed at this stage, a com-
bination of the results of elemental analysis, IR, and 1H
and 13C NMR for 27 supported its structure.
3
28 : R = A
6 : R = B
ii)
i)
Si
O
R
4
29 : R = A
7 : R = B
ii)
Me
Me
i)
Si
Si
O
R
27
3
2
Coupling of 11 with 23, 25, and 27 was performed in the
same way as that for the preparation of 13, giving fully
protected dendrimers 28 (55.9% yield), 29 (59.2% yield),
and 30 (36.1% yield), which carry three, four, and six
thiosialoside moieties, respectively, 28, HRMS (ESI+)
calcd for [M+Na]+: 2252.82289; found m/z:
2252.82345, 29, HRMS (ESI+) calcd for [M+2Na]2+/2:
1453.52295; found m/z: 1453.51809, and 30, HRMS
(ESI+) calcd for [M+3Na]3+/3: 1505.55837; found m/z:
1505.55195. Finally, transesterification and subsequent
saponification of 28, 29, and 30 gave corresponding
30 : R = A
8 : R = B
ii)
OAc
OAc
COOMe
S
A : AcO
AcHN
O
S
OAc
OH
COOH
S
OH
O
B : HO
water-soluble 6, 7, and 8 in quantitative yields, respec-
S
AcHN
24
tively, 6, ½aꢀ +25° (c 1.07, H2O), HRMS (ESI+) calcd
OH
D
for [M+Na]+: 1706.64916; found m/z: 1706.65283, 7,
25
½aꢀ +29° (c 1.12, H2O), HRMS (ESI+) calcd for
Scheme 4. Reagents and conditions: (i) 11 (2 molar excess vs SAc),
NaOMe, MeOH—DMF, 0 °C ! rt, overnight, then Ac2O—Pyr, rt,
overnight; (ii) NaOMe, MeOH, rt, 3 h, then 0.5 M aqueous NaOH, rt,
overnight.
D
[M+Na]+: 2155.82448; found m/z: 2155.81847, and 8,
25
D
½aꢀ +20° (c 1.05, H2O), HRMS (ESIꢁ) calcd for
[Mꢁ2H]2ꢁ/2: 1676.67135; found m/z: 1676.67301 (see
Scheme 4).
Preliminary results of inhibitory activity17 of a series of
thiosialoside clusters against influenza virus sialidases
are summarized in Table 1. Interestingly, dendrimers
6, 7, and 8 showed inhibitory potencies not only for
H3N2-type sialidase but also for H1N1-type sialidase,
while monomeric substrate 1 and dimeric one 2 as well
as dendrimers 3, 4, and 5 did not show any activities.
The results suggested that topological orientation of
each sialoside and distances between the sugar moieties
are important for effective binding to the binding sites in
the tetrameric sialidases on virus surfaces. Indeed, den-
drimers 6, 7, and 8 having longer spacer-arm moieties
than those of dendrimers 3, 4, and 5 show the inhibitory
activities.

J.-I. Sakamoto et al. / Bioorg. Med. Chem. Lett. 17 (2007) 717–721
721
Table 1. Preliminary results of inhibition assays for influenza virus
sialidases
D. B.; Tai, C. Y.; Laver, W. G.; Stevens, R. C. J. Am.
Chem. Soc. 1997, 119, 681.
Compounda
Influenza virus subtype
A/Menphis/1/71 (H3N2) A/PR/8/34 (H1N1)
6. Gubareva, L. V.; Kaiser, L.; Hyden, F. G. The Lancet
2000, 355, 827.
7. (a) Le, Q. M.; Kiso, M.; Someya, K.; Sakai, Y. T.;
Nguyen, T. H.; Nguyen, K. H. L.; Pham, N. D.; Ngyen,
H. H.; Yamada, S.; Muramoto, Y.; Horimoto, T.;
Takada, A.; Goto, H.; Suzuki, T.; Suzuki, Y.; Kawaoka,
Y. Nature (London) 2005, 437, 1108; (b) deJong, M. D.;
Thanh, T. T.; Khanh, T. H.; Hien, V. M.; Smith, G. J. D.;
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Peiris, J. S. M.; Hien, T. T.; Farrar, J. N. Engl. J. Med.
2005, 353, 2667.
1
2
3
4
5
6
7
8
NDb
NDb
NDb
NDb
NDb
5.00
NDb
NDb
NDb
NDb
NDb
5.00
5.00
1.25
5.00
5.00
8. Kiefel, M. J.; von Itzstein, M. Chem. Rev. 2002, 102, 471.
9. (a) Lee, Y. C. Carbohydr. Res. 1978, 67, 509; (b) Lee, Y.
C.; Townsend, R. R.; Hardy, M. R.; Lo¨nngren, J.;
Arnarp, J.; Haraldsson, M.; Lo¨nn, H. J. Biol. Chem.
1983, 258, 199.
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(London) 1983, 303, 35; (b) Colman, P. M.; Varghese, J.
N.; Laver, W. G. Nature (London) 1983, 303, 41.
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Mizuguchi, M.; Hino, K.; Miyazawa, S.; Yamasaki, C.;
Aoki, J.; Takashima, S.; Yamakawa, Y.; Nishijima, M.;
Terunuma, D.; Kuzuhara, H.; Natori, Y. Proc. Natl.
Acad. Sci. U.S.A. 2002, 99, 7669; (b) Nishikawa, K.;
Matsuoka, K.; Watanabe, M.; Igai, K.; Hino, K.; Hatano,
K.; Yamada, A.; Abe, N.; Terunuma, D.; Kuzuhara, H.;
Natori, Y. J. Infect. Dis. 2005, 191, 2097.
12. (a) Matsuoka, K.; Terabatake, M.; Esumi, Y.; Terunuma,
D.; Kuzuhara, H. Tetrahedron Lett. 1999, 40, 7839; (b)
Matsuoka, K.; Terabatake, M.; Umino, A.; Esumi, Y.;
Hatano, K.; Terunuma, D.; Kuzuhara, H. Biomacromol-
ecules 2006, 7, 2274; (c) Matsuoka, K.; Terabatake, M.;
Esumi, Y.; Hatano, K.; Terunuma, D.; Kuzuhara, H.
Biomacromolecules 2006, 7, 2284.
IC50 values are indicated in mM concentration.
a The structures are shown in Figure 1.
b ND means not determined due to weak binding potency.
In conclusion, an efficient synthesis of a series of dendri-
mers having thioglycosidic linkage of sialic acid 3–8 was
accomplished using functionalized thioglycoside of sialic
acid and brominated dendrimers. Biological responses
of these dendrimers against influenza virus sialidases
were preliminary evaluated, and the results showed that
some of the dendrimers have inhibitory potency for the
sialidase. Further manipulations of dendritic core struc-
tures and the introduction of sialoside into the scaffolds
to broaden the library are now in progress. The details
of the results presented here and further synthetic proce-
dures including other homologues and biological activi-
ties will be reported elsewhere in the near future.
Acknowledgment
13. (a) Hasegawa, A.; Nakamura, J.; Kiso, M. J. Carbohydr.
Chem. 1986, 5, 11; (b) Kanie, O.; Nakamura, J.; Itoh, Y.;
Kiso, M.; Hasegawa, A. J. Carbohydr. Chem. 1987, 6, 117;
(c) Hasegawa, A.; Morita, M.; Ito, Y.; Isida, H.; Kiso, M.
J. Carbohydr. Chem. 1990, 9, 369.
We are grateful to Snow Brand Milk Products Co., Ltd.
for providing the sialic acid used in this study.
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