Inhibition of human neuraminidase 3 (NEU3) by C9-triazole derivatives of 2,3-didehydro-N-acetyl-neuraminic acid

Article abstract of DOI:10.1016/j.bmcl.2010.09.111

We report the synthesis of a series of C9 and N5Ac modified analogs of 2,3-didehydro-N-acetyl-neuraminic acid (DANA) and their inhibitory potency for the human neuraminidase 3 (NEU3) enzyme. We were able to generate a small library of compounds through the synthesis of azide derivatives of DANA, followed by Cu-catalyzed azide-alkyne cycloaddition (CuAAC) to generate triazole-containing inhibitors. Our results suggest that NEU3 can tolerate large hydrophobic groups at the C9 position; however, none of the derivatives made at the N5Ac side-chain were active. We identify three new inhibitors that have comparable potency to the best reported inhibitors of the enzyme.

Full text of DOI:10.1016/j.bmcl.2010.09.111

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7529–7533
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Inhibition of human neuraminidase 3 (NEU3) by C9-triazole derivatives
of 2,3-didehydro-N-acetyl-neuraminic acid
⇑
Yao Zou, Amgad Albohy, Mahendra Sandbhor, Christopher W. Cairo
Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 July 2010
Revised 19 September 2010
Accepted 22 September 2010
Available online 29 September 2010
We report the synthesis of a series of C9 and N5Ac modified analogs of 2,3-didehydro-N-acetyl-neuram-
inic acid (DANA) and their inhibitory potency for the human neuraminidase 3 (NEU3) enzyme. We were
able to generate a small library of compounds through the synthesis of azide derivatives of DANA, fol-
lowed by Cu-catalyzed azide–alkyne cycloaddition (CuAAC) to generate triazole-containing inhibitors.
Our results suggest that NEU3 can tolerate large hydrophobic groups at the C9 position; however, none
of the derivatives made at the N5Ac side-chain were active. We identify three new inhibitors that have
comparable potency to the best reported inhibitors of the enzyme.
Keywords:
Neuraminidase
Glycosidase
Inhibitor
Ó 2010 Elsevier Ltd. All rights reserved.
CuAAC
Triazole
Sialic acid
The mammalian sialidase enzymes are a family of glycosyl
hydrolases which cleave the terminal sialic acid residue from gly-
coproteins and glycolipids.1 Dysfunction of these enzymes can re-
were first proposed by Magesh et al. in 2006; however, many of the
specific features of the models remain to be experimentally
tested.21 For example, the predicted catalytic residues have only
been confirmed in NEU219 and NEU3.22 There are few reports of
inhibitors tested against the family of human NEU enzymes. A pa-
nel of 9-amino-9-deoxy-2,3-didehydro-N-acetyl-neuraminic acid
derivatives were tested against all four enzymes; the most potent
of these inhibitors showed only low micromolar activity against
NEU1.23 A library of benzoic acid derivatives have also been tested
against all four isozymes.24 The viral neuraminidase inhibitors
zanamivir and oseltamivir have limited potency against all of the
sult in sialic acid storage disorders, such as type
I and II
sialidosis.2,3 Mammalian sialidase enzymes also play essential
roles in cellular function, with reported effects on diverse pro-
cesses including neurite outgrowth,4 immune cell activation,5 cel-
lular communication,6–8 signaling,5,9–11 adhesion,12 as well as the
apoptosis13,14 and metastasis of malignant cells.15,16 Selective,
tight-binding inhibitors of sialidase isoforms could provide an
essential tool for discerning the role of individual isozymes in cell
biology. However, while nanomolar inhibitors are known for path-
ogenic neuraminidase enzymes, most notably the influenza viral
neuraminidase, no inhibitors of similar potency are known for
the human enzymes.17
human sialidases.25 However, tight-binding (<1
lM) and selective
inhibitors for individual isozymes remain to be identified.
Our group has studied the substrate recognition and catalysis of
human NEU3, the plasma membrane-associated sialidase, which
modifies glycolipid substrates.22 This enzyme is particularly chal-
lenging due to its hydrophobic character and propensity for aggre-
gation. We tested the predictions of a NEU3 homology model using
site-directed mutagenesis, and validated both the catalytic resi-
dues and several residues which are essential for substrate recog-
nition. As part of this study, we performed molecular docking of a
well-known sialidase inhibitor, 2,3-didehydro-N-acetyl-neurami-
nic acid (DANA). Our molecular docking studies of DANA in the
NEU3 binding site revealed a relatively large hydrophobic pocket
adjacent to the C9 position of sialic acid. We hypothesized that this
region of the binding site could be used to design improved inhib-
itors of NEU3 based on the DANA core structure.
Currently identified members of the sialidase enzyme family in
humans consist of NEU1, NEU2, NEU3, and NEU4. All four enzymes
are classified as exo-sialidases (EC 3.2.1.18) and members of glyco-
side hydrolase family 33 in the CaZy database.18 Among the mam-
malian enzymes, only the human NEU2 structure has been studied
by crystallography, revealing that the enzyme adopts a six-bladed
b-propeller fold.19,20 Homology models of all four human enzymes
Abbreviations: CuAAC, Cu-catalyzed azide–alkyne cycloaddition; DANA, 2,3-
didehydro-N-acetyl-neuraminic acid; MBP, maltose binding protein; NEU, neur-
aminidase; 4MU-NA, 4-methylumbelliferyl
a
-
D
-N-acetylneuraminic acid.
⇑
Corresponding author. Tel.: +1 780 492 0377; fax: +1 780 492 8231.
E-mail address: ccairo@ualberta.ca (C.W. Cairo).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.111

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Y. Zou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7529–7533
Known substrates of NEU3 include
(Table 1) found in glycolipid substrates, such as GM3.26 Addition-
ally, (2,8)-linked sialosides are known to be substrates for the
a
(2,3)-linked sialosides
ated by incorporating an azide at C2 of Neu5Ac.29 We set out to
generate a small library of DANA analogs with modifications at
the C9 and N5Ac as a means to explore the binding site topology
of NEU3 and to identify new inhibitors of the enzyme.
a
enzyme, and there is evidence that the enyzme has a preference
for this linkage.13 A common strategy for the design of sialidase
inhibitors relies on the use of DANA as a transition state analog.
The inhibition of NEU3 by DANA has been reported by several
groups, and is usually found to be in the mid-micromolar range
(Table 1).22,23,25 Interestingly, zanamivir, a potent viral NEU inhib-
itor has been reported to have low micromolar activity against
NEU3, while oseltamivir is essentially inactive against the en-
zyme.25 Magesh et al. found that NEU3 could tolerate a relatively
bulky phenyl-amide group at the C9 position of DANA, although
these derivatives had relatively low potency (Table 1).23 We con-
sidered that a potential strategy for rapidly exploring the structure
activity relationships (SAR) of DANA analogs for NEU3 could re-
volve around a modular synthetic strategy which introduced mod-
The basis of our strategy relied on the generation of two
derivatives, the N5-azidoacetyl- (6) and 9-azido-9-deoxy (12) DANA
derivatives (Scheme 1). We began our synthesis from N5-acetyl-
neuraminic acid (Neu5Ac), which could be converted to the
peracetylated methyl ester, 1, following reported methods.30,31 We
obtained moderate yields of the acetyl-protected 2,3-didehydro
analog (2) using triphenylphosphine hydrobromide (see Supple-
mentary scheme SI1).32,33 To prepare compound 6 and the related
triazole derivatives, 2 was deacetylated34 and the O9 position was
selectively converted to a sulfonate ester, 4.35 The sulfonate could
then be displaced with sodium azide to give the methyl ester of 6,
compound 5.23,35 Hydrolysis of 5 under basic conditions provided
the target compound 6. Alternatively, 5 could be used to generate
triazole derivatives using the copper-catalyzed 1,3-dipolar Huisgen
addition of an alkyne, also known as a Cu-catalyzed azide–alkyne
cycloaddition (CuAAC).36 We generated a series of nine triazole
derivatives of 6 using standard conditions (compounds 6a–i, Table
2).
To prepare derivatives of compound 12, we required selective
deprotection of the N5 side-chain. To achieve this, we first pro-
tected N5 with di-tert-butyldicarbonate (Boc2O), followed by
deacetylation to generate 7 (Scheme 2).37 Subsequent O-acetyla-
tion provided compound 8, which could be selectively deprotected
to reveal the free amine at N5 (9). With the free amine in hand, we
could introduce a modified N-acyl group that contained an azide
handle. We employed an activated ester of azidoacetic acid,38,39
which gave the azide 10 in excellent yield. Removal of the O-acetyl
protecting groups provided the methyl ester 11, which could be
hydrolyzed to the target compound 12. As before, we generated a
series of triazole derivatives of 12 from 11 by CuAAC, followed
by hydrolysis of the methylester (compounds 12a–i, Table 2).
We previously reported a bacterial expression system for NEU3
which could be exploited to test the potency of inhibitors.22
Although there are previous reports of bacterial expression of this
enzyme,40 we found that expression of the protein as an N-termi-
nal fusion with the maltose binding protein (MBP) helped to stabi-
lize the protein and prevent aggregation. Additionally, producing
the protein in bacterial cells avoided background signal due to con-
taminating activity of other sialidase isoforms present in mamma-
lian cells. Using purified MBP-NEU3, we tested the inhibitory
potency of the C9 (compounds 6 and 6a–i) and N5Ac (compounds
12 and 12a–i) derivatives against NEU3 using a fluorescence assay
ifications at the C9- and N5Ac-positions from
a common
intermediate. Li et al. first reported the use of click chemistry to
generate viral NEU inhibitors from
a C4-azido-derivative of
DANA.27 Lu and Gervay-Hague expanded on this strategy by gener-
ating C4- and C7-azido DANA analogs, which could be elaborated
to triazoles and multivalent derivatives of zanamivir.28 In a related
strategy, non-hydrolyzable sialoside derivatives have been gener-
Table 1
Previously reported inhibitors of NEU3
Entry
Compound
Structure
NEU3 IC50 (lM)
O-
O
O
OH
HO
HN
1
a
-Sialoside
na
O
HO OH
OH
O
O
70 1525a
6123b
O
O-
HO
43 722b
O
2
3
4
DANA
HN
OH
HO
OH
HO
O
O-
O
325a
HN
HO
Zanamivir
Oseltamivir
7
NH
NH2
O
HN
O
O-
based on the hydrolysis of 4-methylumbelliferyl
raminic acid (4MU-NA) (Table 2).
a-D-N-acetylneu-
H2N
NH
>10,00025a
O
As expected, we observed that DANA was a good inhibitor of
O
NEU3, with an IC50 of approximately 40
l
M.22,23,25 We found that
modification of DANA at the C9 position by a triazole with a hydro-
phobic group which lacked a hydrogen bond donor lead to
improved activity. The most potent compounds were the phenyl
OH
O
O-
NH
6a (20 10
l
M), hexyl 6b (23
4 lM), and phenoxymethyl 6e
5
6
10bc
32023b
O
O
(45 M) derivatives. Importantly, the data suggest that the
3
l
HN
HO
triazole portion of the inhibitor is partly responsible for the in-
creased activity, as the azide derivative 6 has slightly reduced po-
OH
O
tency when compared to DANA (70 20
lM). Bulky triazole
OH
O
NH
O-
O
O
OH
OH
10ac
>100023b
HO
HN
N3
O
O-
O
HN
HO
O-
O
O
OH
HN
O
OH
OH
HO
HO
N3
a
b
c
12
6
Determined by inhibition of GM3 hydrolysis.
O
O
Determined by inhibition of 4MU-NA hydrolysis.
Nomenclature as used in Magesh et al.23
Scheme 1. DANA analog synthetic targets.

Y. Zou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7529–7533
7531
Table 2
Inhibition of NEU3
N
N
N
OH
R
OH
O
O
HO
HN
O
O-
O-
O
HN
HO
OH
OH
12a-i
6a-i
R
HO
N
O
O
N
N
a
Compound
R
IC50
(lM)
Compound
R
IC50 (lM)
DANA
6
na
na
48
70 20
5
DANA
12
na
na
48
21
4
8
6a
20 10
12a
>500
6b
6c
23
4
12b
12c
>500
>500
O
O
300 200
O
O
6d
6e
130 20
12d
12e
>500
>500
O
O
45
3
O
O
6f
300 400
400 600
500 200
12f
12g
12h
>500
>500
>500
OH
OH
OH
OH
6g
6h
OH
OH
OH
OH
6i
300 500
12i
>500
a
Inhibitors which did not show significant change (>50% decrease from control) were fit using the maximum inhibition value for DANA. See Supplementary data for IC50
curves.
OH
OAc
O
O
OMe
HO
AcO
a.
O
b.
O
OMe
H
H
N
2
N
O
O
OH
OAc
7
8
HO
AcO
O
O
OAc
OAc
O
OMe
AcO
O
AcO
c.
d.
O
OAc
O
OAc
OMe
H
N
+-H3N
CF3CO2
9
10
N3
AcO
AcO
O
OH
HO
O
OH
O
HO
O
e.
O-
f.
O
OH
H
N
OMe
H
N
OH
R
R
N
HO
11
12a-i
N3
HO
O
O
N
N
Scheme 2. Synthesis of N5Ac-azido and -triazolyl derivatives. Reagents and conditions: (a) Boc2O, N,N-dimethylaminopyridine, THF, 2 h (quant.); then NaOMe, MeOH (92%);
(b) Ac2O, pyridine, 12 h, 0 °C (96%); (c) TFA, CH2Cl2, 1.5 h; (d) N-hydroxysuccinimidyl-2-azidoacetate, triethylamine, 3 h, 0 °C (99% over two steps); (e) NaOMe, MeOH, 1 h,
(52%); (f) sodium ascorbate, CuSO4, H2O/tBuOH/DCM (1:2:1); then NaOH, Amberlite IR-120(H+) (yields ranged from 31–96%).
substituents, such as 6d, had reduced potency (130 20
lM). More
(21
8 lM), suggesting that the azide may provide some beneficial
polar groups, including ethoxy 6c, hydroxymethyl 6f, hydroxyethyl
6g, and hydroxypropyl 6h all showed significantly reduced activity
as compared to the parent DANA, or the azide 6. Assays of com-
interactions. However, our data imply that the N5Ac pocket is rel-
atively small, as all triazole derivatives of compound 12 tested
(12a–i) had potencies that were significantly reduced when com-
pound 12 found
a
slightly improved potency over DANA
pared to DANA (>500 lM).

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Y. Zou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7529–7533
In order to understand the interactions responsible for the differ-
These results support our previous study of the active site
topology of NEU3 using small molecule inhibitors based on
DANA.22 The compounds tested here suggest that the N5Ac pocket
of NEU3 is sterically limited, and cannot accommodate large
groups such as the triazole derivatives tested here (12a–i). Impor-
tantly, we observed that modifications of the C9 position are well
tolerated, and may have some specific requirements for potent
inhibitors of the enzyme. Although previous studies have identified
a tolerance for bulky groups at C9 (Table 1),23 we observed that the
addition of a triazole linker significantly improved activity (com-
pounds 6a, 6b and 6e). Based on our model of the active site, we
propose that groups which bind in the C9 pocket should contain
H-bond acceptors, to interact with Y181, and also hydrophobic
side-chains, to interact with V222 and V224 (Fig. 1). Additional
H-bond acceptors distal from C9 may take advantage of contacts
with K195 or H277. Our results suggest that potent inhibitors of
NEU3 may be developed by incorporation of a modified C9 side-
chain of DANA analogs. Importantly, we identified compounds in
this study with equivalent potency (6a) to the best reported
ences in potency among both series of compounds, we conducted
molecular docking experiments using a previously reported homol-
ogy model of NEU3.22 After docking, structures were subjected to
molecular dynamics and minimized (see Supplementary data).
Examination of the structures of DANA and compound 6a in the
NEU3 active site suggest that a hydrophobic pocket adjacent to the
glycerol-binding pocket (C7–C9) could contribute to the activity of
compounds related to 6a (Fig. 1). The hydrophobic pocket is made
up of residues V222, V224, P247, and H277. We observed two
changes to the active site residues upon binding of 6a, D50 became
reoriented to interact with HO7, and an apparent edge–face interac-
tion developed between the phenyl ring and H277 (Fig. 1).41 The
model of DANA binding is consistent with the limitations we
observed for the N5Ac pocket, which would be predicted to be
unable to accommodate the bulky triazole groups found in com-
pounds 12a–i. Additionally, the N5Ac pocket is relatively hydropho-
bic, although a carboxylate group from N88 is positioned close
enough to contact C4 H-bond donors.
Figure 1. Molecular docking of DANA and 6a. Inhibitors were docked and subjected to molecular dynamics using a homology model of NEU3 to determine the binding mode
of the triazole derivative 6a.22 (a) The binding site of NEU3 has a large hydrophobic pocket adjacent to the C9 position including V222, V224, P247 and H277 which can
accommodate hydrophobic groups, (b) an electrostatic potential map is shown for the docked structure of 6a, and (c) DANA. Several residues have reoriented to accommodate
6a, notably H277 is rotated to engage in an edge–face interaction with the phenyl ring of 6a,41 and D50 has become more solvent exposed.

Y. Zou et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7529–7533
7533
inhibitors of the enzyme (zanamivir).25 This feature of NEU3 recog-
nition may indicate that the enzyme can tolerate unusual sialic
acid modifications found at C9.42 Future work will examine the
specificity of these compounds for the NEU3 isoform, and test addi-
tional side-chain modifications at C9 that could increase potency.
13. Azuma, Y.; Sato, H.; Higai, K.; Matsumoto, K. Biol. Pharm. Bull. 2007, 30, 1680.
14. Kakugawa, Y.; Wada, T.; Yamaguchi, K.; Yamanami, H.; Ouchi, K.; Sato, I.;
Miyagi, T. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10718.
15. Uemura, T.; Shiozaki, K.; Yamaguchi, K.; Miyazaki, S.; Satomi, S.; Kato, K.;
Sakuraba, H.; Miyagi, T. Oncogene 2009.
16. Miyagi, T.; Wada, T.; Yamaguchi, K.; Hata, K. Glycoconjugate J. 2003, 20, 189.
17. Dyason, J. C.; von Itzstein, M. Aust. J. Chem. 2001, 54, 663.
18. Cantarel, B. L.; Coutinho, P. M.; Rancurel, C.; Bernard, T.; Lombard, V.;
Henrissat, B. Nucleic Acids Res. 2009, 37, D233.
19. Chavas, L. M. G.; Tringali, C.; Fusi, P.; Venerando, B.; Tettamanti, G.; Kato, R.;
Monti, E.; Wakatsuki, S. J. Biol. Chem. 2005, 280, 469.
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Tettamanti, G.; Monti, E.; Wakatsuki, S. J. Med. Chem. 2010, 53, 2998.
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Acknowledgments
This work was funded by a grant from the Natural Sciences and
Engineering Research Council of Canada (NSERC) and the Alberta
Ingenuity Centre for Carbohydrate Sciences (AICCS). We acknowl-
edge infrastructure support from the Canada Foundation for Inno-
vation (CFI).
22. Albohy, A.; Li, M. D.; Zheng, R. B.; Zou, C.; Cairo, C. W. Glycobiology 2010, 20,
1127.
23. Magesh, S.; Moriya, S.; Suzuki, T.; Miyagi, T.; Ishida, H.; Kiso, M. Bioorg. Med.
Chem. Lett. 2008, 18, 532.
Supplementary data
24. Magesh, S.; Savita, V.; Moriya, S.; Suzuki, T.; Miyagi, T.; Ishida, H.; Kiso, M.
Bioorg. Med. Chem. 2009, 17, 4595.
25. Hata, K.; Koseki, K.; Yamaguchi, K.; Moriya, S.; Suzuki, Y.; Yingsakmongkon, S.;
Hirai, G.; Sodeoka, M.; Von Itzstein, M.; Miyagi, T. Antimicrob. Agents Chemother.
2008, 52, 3484.
26. Valaperta, R.; Chigorno, V.; Basso, L.; Prinetti, A.; Bresciani, R.; Preti, A.; Miyagi,
T.; Sonnino, S. FASEB J. 2006, 20.
27. Li, J.; Zheng, M.; Tang, W.; He, P.-L.; Zhu, W.; Li, T.; Zuo, J.-P.; Liu, H.; Jiang, H.
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Supplementary data (characterization and synthetic methods)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2010.09.111.
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