Potent small molecule mouse CD22-inhibitors: Exploring the interaction of the residue at C-2 of sialic acid scaffold

Article abstract of DOI:10.1016/j.bmcl.2009.08.044

Our previous study revealed that compound 1 (9-(4′-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5Gcα2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and

Full text of DOI:10.1016/j.bmcl.2009.08.044

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5573–5575
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Potent small molecule mouse CD22-inhibitors: Exploring the interaction
of the residue at C-2 of sialic acid scaffold
Hajjaj H. M. Abdu-Allah a, , Kozo Watanabe b,c,d, Koji Hayashizaki b,c, Chiaki Takaku b,c, Taichi Tamanaka b,c
,
*
Hiromu Takematsu e, Yasunori Kozutsumi e, Takeshi Tsubata b,c,d, Hideharu Ishida a, Makoto Kiso a,f,
*
a Department of Applied Bio-organic Chemistry, The United Graduate School of Agricultural Sciences, Gifu University, Gifu 501-1193, Japan
b Laboratory of Immunology, Graduate School of Biomedical Sciences, Tokyo Medical and Dental University, Japan
c Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, Japan
d CREST, Science and Technology Agency (JST), Saitama, Japan
e Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Japan
f Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, Japan
a r t i c l e i n f o
a b s t r a c t
1
(9-(40-hydroxy-4-biphenyl)acetamido-9-deoxy-
Article history:
Our previous study revealed that compound
Received 13 January 2009
Revised 8 August 2009
Accepted 12 August 2009
Available online 15 August 2009
Neu5Gca2-6GalOMP) has the most promising affinity for mCD22. Replacing the subterminal galactose
residue of 1 with benzyl or biphenylmethyl as aglycone led to 38- and 20-fold higher potency, respec-
tively. This discovery represents a new direction in inhibitor design suitable for pharmaceutical
development.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
CD22
Sialoside
Hanessian reaction
Inhibitor
ELISA
CD22 is a B cell-specific sialic acid binding immunoglobulin-like
lectin (Siglec) whose function as a regulator of B cell signaling ap-
pears to be modulated by its interaction with glycan ligands bear-
selectivity.7–14 CD22 showed high specificity for Neu5Ac/Gc
6Gal. The axial position of the carboxyl group in -sialosides is
a2-
a
essential for the binding. In contrast to most members of siglec
family, human (hCD22) and mouse (mCD22) show high affinity
for Neu5Gc.7 Moreover, 9-biphenylcarboxamido and 9-bipheny-
lacetamido derivatives of Neu5Ac/Gc showed high affinity and
selectivity for hCD22 and mCD22, respectively.8,9 These key fea-
tures of the CD22-sialic acid binding, provide the basis for sialoside
design aimed at developing compounds with enhanced affinity and
selectivity for CD22.
ing the sequence Neu5Ac/Gca
2-6Gal.1 It exhibits low intrinsic
affinity for its in vivo ligands (Kd = 0.2 mM), and their abundance
provides cis and trans interactions that effectively mask the ligand
binding site.2 To overcome the threshold set by these interactions,
high affinity ligand probes are required. Interestingly, high affinity
ligand conjugated with toxin was shown to bind CD22 and eventu-
ally endocytosed, inducing B cell death. This finding represents a
promising approach for B cell targeting.3
CD22 binding, similar to other siglecs, is almost entirely medi-
ated through interactions with the Neu5Ac/Gc template, while
the subterminal galactose residue contribute to a lesser degree
and its hydroxyl groups are not involved in binding.10
In addition to treatment of hematologic malignancies,4 B cell-
directed therapies have an important role in the treatment of auto-
immune disorders.5 It has been suggested that CD22-inhibitors
could shorten the time required for antibody production, thereby
augmenting the host defense against acute infectious diseases
regardless of pathogens; as a ‘universal vaccination’.6
A number of ligand specificity studies for CD22 have been car-
ried out which have shown the importance of C-2, C-5 and C-9 sub-
stituents on sialic acids in modulating binding affinity and
Recently,14 we have reported the dramatic improvement of
binding affinity for CD22 achieved by the modifications at 9-posi-
tion of Neu5Gc
a2-6GalOMP core as exemplified by compound 1,
Figure 1, (9-(40-hydroxy-4-biphenyl)acetamido-9-deoxy-Neu5G-
ca2-6GalOMP) which exhibited the highest potency for mCD22.
Our predicted binding model for compound 1,14 revealed that the
interactions of GalOMP residue with its binding site is mainly
hydrophobic in nature.
* Corresponding authors. Tel./fax: +81 (0) 58 293 2918/2840 (M.K.).
E-mail addresses: moazhajjaj@yahoo.com (H.H.M. Abdu-Allah), kiso@gifu-u.ac.jp
(M. Kiso).
In this respect, Neu5Ac
aBn was 8–10-fold more potent than
Nue5Ac
a
Me for MAG (siglec-4) and sialoadhesin (Sn).7 Since the
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.044

5574
H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5573–5575
Table 1
HO
Inhibitory potencies of representative Neu5Ac/Gc
a
2-6Gal derivatives for mCD22
Y
COOH
O
OH
O
HO
RHN
NH
O
O
COOH
O
OH
O
HO
HO
HO
HO
HO
HOCH2COHN
OX
HO
HO
OH
O
O
a
Compd
Neu5Ac
Neu5Gc
GSC-715
GSC-633
Y
R
X
IC50
(l
M) SD
HO
a
a
2-GalbSE
2-GalbSE
OH
OH
OH
Ac
Gc
Gc
Gc
SE
N. D.
OMe
SE
MP
MP
544.8 23.5
305.2 20.4
82.9 4.5
Figure 1. Compound 1.14
NH2
a
Sialoside concentration which leads to 50% inhibition of binding. The values are
the mean SD of triplicates. MP, p-methoxyphenyl; SE, 2-(trimethylsilyl)ethyl. N.
D.; IC50 >1000 lM.
binding sites of Sn and CD22 possess a high degree of sequence
similarity;12 we expected that hydrophobic substituent at the C-2
of sialic acid scaffold could improve the affinity for CD22 in analogy
to Sn.
Table 2
To explore the importance of a2-6 linkage, contribution of gal-
Inhibitory Potencies of compounds 1, 8 and 9 for mCD22
actose residue to the binding and the nature of interaction at C-2 of
sialic acid; mimetics 8 and 9 were synthesized by replacing Ga-
lOMP residue of 1 with benzyl or biphenylmethyl, respectively,
as reducing end substituents.
rIPb
a
Compd
IC50
(
l
M) SD
1
8
9
3.84 0.32
0.10 0.00
0.19 0.00
1
38.4
20.2
The synthesis of the target compounds is depicted in Scheme 1.
Compound 2 was synthesized in seven steps starting from neuram-
inic acid.14 Selective debenzylation of 2 with Hanessian reaction
method15 followed by acetylation afforded compound 3 in 87%.
Glycosylation of 3 with benzyl alcohol or biphenylmethanol un-
der NIS/TfOH condition at ꢀ40 °C in acetonitrile gave the corre-
a
Footnote of Table 1.
The rIP of each sialoside was calculated by dividing the IC50 of the reference
b
compound 1 by the IC50 of the compound of interest. This results in rIPs above 1.0
for derivatives binding better than 1 and rIPs below 1.0 for compounds with a lower
affinity than 1.
sponding sialoside
4
or
5
in about 76%
(a/b = 66/10).
ment of the affinity of GSC-633 upon amidation of 9-amino to give
compound 1.14
Saponification of the
a-sialosides followed by chemoselective
reduction of azide with Me3P gave 9-amino derivative 6 and 7.
Acylation of the 9-amine with N-hydroxysuccinimide ester of 40-
hydroxybiphenyl-4-acetic acid14 yielded the target compounds 8
and 9.
A competition enzyme-linked immunosorbent assay (ELISA)
based on a biotinylated form of 1 has been developed.16Therefore
the binding affinity of various synthetic sialosides was accurately
and reliably determined. Inhibitory potencies of some previously
reported compounds (Table 1), 1, 8 and 9 (Table 2) were deter-
mined (see Supplementary data). The results are shown in Tables
1 and 2.
Interestingly, replacing the subterminal galactose residue of
compound 1 with benzyl or biphenyl as aglycone enhanced the po-
tency. Benzyl sialoside (8) exhibited an IC50 value 38-fold more po-
tent than 1. The sterically more demanding biphenyl of 9 led also
to improved affinity but to a lower extent; 20-fold more potent
than 1. The differences in binding of these compounds must arise
from the differences in the interactions made by the substituents
at the C-2 of sialic acid moiety. These substituents are either benzyl
or biphenylmethyl and the interaction may therefore be expected
to be primarily hydrophobic in nature. However, dimerization of
CD22 molecules, mediated through the phenyl (8) or biphenyl (9)
of the ligand may be occurred as already observed in the crystal
The results in Table 1 are included to show the higher affinity of
mCD22 for N-Gc at C-5 of sialic acid and the dramatic improve-
structure of Neu5Ac
be concluded that the
a
Bn with Sn.11 Based on these results it could
2-6 linkage and the subterminal galactose
a
residue are not essential for CD22 binding and could be replaced
with non-carbohydrate moiety with even improved affinity and
drug like properties.
N3
N3
Ar
SPh
OAc
O
OAc
SPh
OAc
O
i
HO
COOMe
AcO
AcO
COOMe
BnOCH2COHN
AcOCH2COHN
ii
OAc
Further investigation is under way to clarify in more detail the
role of subterminal glycan in CD22 binding affinity and selectivity.
It is worth noting that benzyl and biphenyl groups were success-
fully employed to mimic Galb(1-4)GlcNAc core in selectin antago-
nists and the Galb(1-3)GalNAc core in MAG antagonists.17a–d
Biphenyl moiety was identified as a feasible replacement for the
core disaccharide Galb(1-3)GalNAc according to saturation transfer
difference (STD) NMR and molecular modeling investigations.17c
In summary, replacing the galactose residue of compound 1
with benzyl or biphenylmethyl resulted in compounds with sim-
plified structure and improved potency. Benefits also include easier
synthetic access and possibly enhanced bioavailability.
2
3
H2N
N3
COOH
O
OH
O
OH
COOMe
O
OAc
O
OAc
iv
HO
iii
AcO
HOCH2COHN
AcOCH2COHN
Ar
Ar
6 Ar = phenyl
4 Ar = phenyl
7 Ar = biphenyl
5 Ar = biphenyl
HO
NHS ester
O
O
O
O
N
NH
HO
COOH
O
OH
O
OH
O
HOCH2COHN
Ar
8 Ar = phenyl
9 Ar = biphenyl
HO
Acknowledgments
Scheme 1. Reagents and conditions: (i) (a) 10 equiv PhSSiMe3, 5 equiv ZnI2,
1.5 equiv Bu4NI, DCE, 60 °C, 8 h; (b) Ac2O/pyridine, two steps 87%; (ii) NIS, TfOH,
MeCN, ꢀ40 °C; 36 h, 76%; (iii) (a) LiOH, EtOH/H2O; (b) Me3P, MeOH/H2O, rt, 24 h,
two steps 84%; (iv) NHS ester, NaHCO3, MeCN/H2O, 75%.
H.H.M.A. is grateful to the Egyptian Government for Ph.D. fund-
ing scholarship. This work was partly supported by the ministry of
Education, Culture, Sports, Science, and Technology (MEXT) of

H. H. M. Abdu-Allah et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5573–5575
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Supplementary data
Supplementary data (the synthetic procedures, characterization
of the compounds, and inhibition assay) associated with this article
can be found, in the online version, at doi:10.1016/
j.bmcl.2009.08.044.
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