Design, synthesis, and structure-affinity relationships of novel series of sialosides as CD22-specific inhibitors

Article abstract of DOI:10.1021/jm8000696

Sialosides incorporating substituted amides or amines at 9-position of sialic acid moiety have been synthesized and evaluated as CD22 inhibitors. Several derivatives exhibited inhibitory potency in sub- to low micromolar range (e. g., 80, 9d, 9g, and 9k s

Full text of DOI:10.1021/jm8000696

J. Med. Chem. 2008, 51, 6665–6681
6665
Design, Synthesis, and Structure-Affinity Relationships of Novel Series of Sialosides as
CD22-Specific Inhibitors
Hajjaj H. M. Abdu-Allah,*,† Taichi Tamanaka,‡ Jie Yu,‡,§ Lu Zhuoyuan,‡,§ Magesh Sadagopan,| Takahiro Adachi,‡
Takeshi Tsubata,‡, Soerge Kelm,# Hideharu Ishida,† and Makoto Kiso*,†,|,
Department of Applied Bio-organic Chemistry, The United Graduate School of Agricultural Sciences, Gifu UniVersity, Gifu 501-1193, Japan,
Laboratory of Immunology, School of Biomedical Science, Tokyo Medical and Dental UniVersity, Japan, Department of Immunology, School of
Basic Medical Sciences, Peking UniVersity Health Science Center, China, Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto
UniVersity, Kyoto, Japan, CREST, Science and Technology Agency (JST), Saitama, Japan, Centre for Biomolecular Interactions, Department of
Biology and Chemistry, Bremen UniVersity, Bremen, Germany
ReceiVed January 23, 2008
Sialosides incorporating substituted amides or amines at 9-position of sialic acid moiety have been synthesized
and evaluated as CD22 inhibitors. Several derivatives exhibited inhibitory potency in sub- to low micromolar
range (e. g., 8o, 9d, 9g, and 9k showed IC50 values 0.40, 0.47, 0.24, and 0.23 µM, respectively, for hCD22,
while 8p, 8q, and 9f, showed IC50 values 1.70, 2.90, and 4.10 µM, respectively, for mCD22). The most
significant result was the strongly enhanced affinity of 9g and 9k containing 9-(2′ or 4′-hydroxy-4-biphenyl)
methylamino substituents (600-fold more potent for hCD22 than the corresponding 9-hydroxy derivative;
7a). Molecular modeling study was carried out to get some insights into the molecular basis of CD22
inhibition. To the best of our knowledge, this is the first systematic structure-affinity relationship study on
inhibition of CD22.
Introduction
affinity values reported for monovalent cell surface receptor-
carbohydrate interactions.
The Sigleca (Sialic acid-binding immunoglobulin [Ig]-like
lectins) proteins are subset of the Ig superfamily of cell
recognition molecules that bind to sialic acid-containing glycans
of cell surface glycoconjugates as ligands.1,2 Each Siglec has a
N-terminal V-set sialic acid-binding Ig domain, a variable
number of additional C-set Ig domains, a transmembrane
domain, and a cytoplasmic domain that typically contains
tyrosine-based motifs. These motifs are implicated in the
regulation of cell signaling and endocytosis. Each Siglec has a
characteristic cell-type-dependent expression pattern and a
distinct binding specificity for sialylated glycoconjugates.3,4 The
recognition of Siglecs for sialylated glycoconjugates was found
to depend on the nature of sialic acid, its linkage to substituted
sugars, and the underlying neutral oligosacchrides. In general,
Siglecs show low affinity (a Kd of 0.1-3 mM) for Neu5AcR2-
3Gal and Neu5AcR2-6Gal.1 This typifies the relatively low
CD22 (Siglec-2) is a well-characterized B cell-specific
transmembrane protein of the Siglec family. It is a B-cell
inhibitory receptor that regulates multiple B-cell functions,
including the cellular activation thresholds and survival.1
Therefore, CD22 modulates B-cell dependent immune response,
prevents autoimmunity, and controls the homing of recirculating
B cells back to the bone marrow.5 Also, binding of CD22 to
glycoprotein ligands on T cells modulates T cell signaling.5
Importantly, many of these CD22 functions depend on its ligand
binding ability.1
A number of studies have established the key structural
features for binding to CD22.6-12 CD22 is unique in having a
strong preference for Neu5GcR2-6Gal and Neu5AcR2-6Gal
structures.6,7 Powell et al.8 reported that the minimal motif
required for recognition by human CD22 is a disaccharide R-D-
Neu5Ac-2,6-Hex(NAc), Hex(NAc) being Gal, GalNAc, or
GlcNAc. Recent data from NMR experiments, computational
docking model, X-ray data of a complex of R2-3-sialyllactose
and sialoadhesin (Sn)/Siglec-1 revealed that Sn mainly recog-
nizes the Neu5Ac and a small part of the Gal moiety.12 The
interaction of Siglecs with sialosides modified at the fifth
position of sialic acid moiety varies from Siglec to Siglec
significantly. While mCD22 showed strong preference for
Neu5Gc, hCD22 binds Neu5Gc and Neu5Ac equally well. In
contrast, Sn and myelin-associated glycoprotein (MAG)/Siglec-4
do not show significant binding to Neu5Gc and bind well to
cells carrying Neu5Ac.10
* To whom correspondence should be addressed. Phone/Fax: +81-(0)
58-293-2919/2840. E-mail: Kiso@gifu-u.ac.jp (M.K.); moazhajjaj@
yahoo.com (H.H.M.A.).
†
Department of Applied Bio-organic Chemistry, The United Graduate
School of Agricultural Sciences, Gifu University.
‡
Laboratory of Immunology, School of Biomedical Science, Tokyo
Medical and Dental University.
§
Department of Immunology, School of Basic Medical Sciences, Peking
University Health Science Center.
|
Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University.
CREST, Science and Technology Agency (JST).
Centre for Biomolecular Interactions, Department of Biology and
#
Chemistry, Bremen University.
CD22 binding, as for all Siglecs, requires the recognition of
not only the sialic acid negative charge but also the side chain
that comprises the C-7, C-8, and C-9 positions. It has been
shown that substitution of the hydroxyl group at the 9-position
of sialic acid by halogens or esterification abolished binding,
suggesting the important role of a hydrogen donor at this
position.10 Furthermore, substitution of this group by an amino
group enhanced binding, and acylation of this amino group by
a Abbreviations: Siglec, sialic acid-binding immunoglobulin-like lectin;
Ig, immunoglobulin; hCD22, human CD22; mCD22, murine CD22; Sn,
sialoadhesin (Siglec-1); MAG, myelin-associated glycoprotein (Siglec-4);
Neu5Gc, N-glycolylneuraminic acid; Neu5Ac, N-acetylneuraminic acid;
GalNAc, N-acetyl-ꢀ-D-galactosamine; Gal, ꢀ-D-galactose; GlcNAc, N-acetyl-
ꢀ-D-glucosamine; BPC, biphenylcarboxamido; BPA, biphenylacetamido;
LacNAc, N-acetyllactoseamine (Galꢀ1-4GlcNAc); Sia, sialic acid type
unspecified; MP, p-methoxyphenyl; SE, 2-(trimethylsilyl)ethyl; IC50, con-
centration giving 50% inhibition of binding.
10.1021/jm8000696 CCC: $40.75
2008 American Chemical Society
Published on Web 10/08/2008

6666 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
Figure 1. Chemical structures of the known 9-biphenyl derivatives of Neu5Ac or Neu5Gc and their comparative inhibitory potencies.13,16
hydrophobic moieties has yielded increased binding to CD22,
MAG, and sialoadhesin.13,14 Interestingly, 9-biphenyl-4-car-
boxamido derivative of Neu5Ac (BPC-Neu5Ac)13 showed high
affinity and selectivity for hCD22 and was found to augment B
cell receptor (BCR) signaling by inhibiting CD22-mediated
signal inhibition and in turn enhances B cell response. In
contrast, biphenylacetamido derivative (BPA-Neu5Ac)13 was
more potent and selective inhibitor for mCD22 than BPC-
Figure 2. Target compounds. Y ) OH, NH2, substituted amide, or
substituted amine.
Neu5Ac (Figure 1).
Molecular modeling studies of Zaccai et al.15 explored a
hydrophobic pocket in CD22 adjacent to the 9-position of sialic
acid. They further identified biphenyl substituents at that
position, BPC and BPA, that increased the affinity and
selectively of Neu5Ac for hCD22 and mCD22, respectively.
Moreover, the sequence alignment and the structural analysis
of members of the Siglec family showed that variations in the
amino acid residues that create this hydrophobic pocket (which
are not well conserved) might contribute to the differences in
binding affinity and specificity.15 Such differences provide
promising opportunity for the design of siglec-specific sialosides.
Recently, BPC-Neu5Ac and BPA-Neu5Gc were assembled,
chemoenzymatically, into the sequences 9-BPC-NeuAcR2-
6Galꢀ1-4GlcNAcꢀ-ethylamine (BPC-Neu5Ac-LNAcꢀR) and
9-BPA-NeuGc-R2-6Gaꢀ1-4GlcNAcꢀ-ethylamine (BPA-Neu5Gc-
LNAcꢀR) (Figure 1).16 This assembly resulted in high-affinity
sialoside ligands that could compete with cis ligands, bind to
CD22 on native B cells, and served as a ligand-based markers
of CD22 endocytosis. Conjugation of these sialoside probes to
the toxin saporin resulted in toxin uptake and toxin-mediated
killing of B lymphoma cell lines, suggesting an alternative
approach to antibody-mediated therapy for targeting CD22 for
the treatment of B cell lymphoma. This interesting finding
indicates that any therapeutic agent or radioisotope could be
targeted to B cells by attaching it to high affinity CD22 ligands.16
Thus, synthetic CD22 ligands could be useful in developing a
novel strategy to regulate the immune responses and can also
be used as delivery agents for treating B cell-related diseases
(e.g., lymphoma).13,16,17
selective CD22 inhibitors. As part of our continuing research
into exploring sialoside specificity of Siglec binding,18 the
present study was aimed at the systematic investigation of
the structure-affinity relationship and eventually developing
more potent and selective CD22 inhibitors. Herein we report
the synthesis and CD22 inhibitory potencies of a series of
synthesized compounds of the general scaffold shown in (Figure
2). This library of compounds (35 compounds) has allowed us
to carry out the first systematic structure-affinity relationship
study on inhibition of CD22.
Rational Design. Crystal structure analysis of two Siglecs,
Sn19 and Siglec 7,20 have revealed a shallow surface with highly
flexible amino acids and this feature is predicted to be present
in all siglecs.15 This means that CD22 belongs to a class of
molecules which are not readily amenable to receptor- or
structure-based drug design for virtual screening as recently
reported for selectins.21 Instead, structurally similar analogues
of natural ligand can increase the possibility of getting more
selective and higher affinity ligands. Therefore, analogue-based
design approach and chemical synthesis have been employed
based on sialic acid template. The basic binding motif in
endogenous ligands for CD22 has been identified as Neu5AcR2-
6-LacNAc.22 Further studies by Powell et al.8 suggested that
this basic binding motif for CD22 could be further stripped to
the disaccharide Neu5AcR2-6-Hex(NAc) without loss of affinity.
Moreover, compound Neu5AcR2-6GalNAcONP (ONP, o-ni-
trophenyl) displayed a 3-fold higher affinity for CD22 compared
with the reference trisaccharide structure Neu5AcR2-6LacNAc.8
The same study revealed that CD22 binding was significantly
influenced by the structure of the group at the reducing terminus
of Gal. For example a sialoside with o-nitrophenyl group as
The above-mentioned facts gave us an insight into the factors
govern the ligand binding of CD22, possible routes for inhibitor
design, and the crucial importance of gaining potent and

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6667
Scheme 1. Preparation of Gal Acceptora
aglycone, was 40-fold better than the same sialoside with a
benzyl group instead.8
Because CD22 does not recognize R2-3 sialosides, the Gal
C5-C6 face of Neu5AcR2-6Gal must be involved in CD22
binding as these positions are the key structural features which
distinguish R2-6 from R2-3 sialosides. The unfavorable effect
of C6 methylation of Gal likewise indicates that this region of
the disaccharide faces the binding site.8 An X-ray crystal-
lographic analysis of the N-terminal domain of Sn complexed
with R2-3-sialyllactose showed a network of close interactions
between the protein, the Sia, and Gal residues, while the GlcNAc
residue is bonded via water molecules.19 Furthermore, combined
NMR experiments with a molecular modeling study revealed
that Sn mainly recognizes the sialic acid moiety and a small
part of the Gal moiety.12 This binding model is probably also
representative for CD22 because the binding sites of Sn and
CD22 possess a high degree of sequence similarity.23 These
findings indicate that CD22 mainly recognizes SiaR2-6Gal motif
while GlcNAc residue is of less importance.
a Reagents and conditions: 80% AcOH, 60 °C for 24 h, 89%.
Scheme 2. Synthesis of Neu5Gc Donors 5a, ba
As already mentioned above, BPC-Neu5Ac and BPA-Neu5Ac
displayed strong affinity with high selectivity for hCD22 and
mCD22, respectively.13 Furthermore, incorporation of BPC-
Neu5Ac into BPC-Neu5Ac-LNAcꢀR sequence resulted in an
inhibitory potency for hCD22 15-fold more than BPC-Neu5Ac
alone.16 Similarly, incorporation of BPA-Neu5Ac into BPA-
Neu5Gc-LNAcꢀR sequence resulted in an inhibitory potency
for mCD22 250-fold more than BPA-Neu5Ac alone.16 Accord-
ingly, the affinity for CD22 can be enhanced by further
optimization of the structure attached to the reducing end of
the sialic acid moiety.
a Reagents and conditions: (a) MsOH, CH3OH, 65 °C, 24 h; (b)
BnOCH2COONSu, TEA, r.t., 24 h, two steps 75%; (c) Ac2O, pyr., 0 °C-rt,
94.5%; (d) 1- CBr4, Ph3P, NaN3, DMF; 2- Ac2O, pyr, 0 °C-rt, two steps
83.5%.
Scheme 3. Sialylation and Deprotection 7a
To maintain the functionality while improving binding
affinity, selectivity, and structural simplicity, we combined all
the structural requirements for CD22 receptor recognition
(Figure 2). Therefore, our lead optimization strategy was based
on the following modifications: (1) Employing SiaR2-6Gal as
the principal motif for CD22 binding. (2) N-Gc as the substituent
at the fifth position, which is recognized mainly by CD22,
Neu5GcR2-6Gal is the main source of specificity. (3) Introduc-
ing free or substituted NH2 group at 9-position of sialic acid
moiety to enhance the affinity and selectivity. The inclusion of
varying functionality at this position was designed to explore
the effects of hydrophobic, hydrophilic, and sterically demanding
groups upon their interaction with CD22. (4) Inserting a
p-methoxyphenyl moiety as the aglycon, which is not only
expected to enhance the affinity but also it is advantageous from
synthetic point of view. The phenyl group will be useful for
the detection of the oligosaccharides and serve as a marker to
check the purity of the product by NMR.
a Reagents and conditions: (a) NIS, TfOH, MS 3 A, (MeCN/DCM; 5/1),
-20 °C, 16 h, 64%; (b) LiOH, aq.EtOH, 6 h, rt; (c) H2, Pd(OH)2, EtOH,
AcOH, H2O, 40 °C, 48 h, two steps 84.5%.
(Scheme 2). The succinimidyl ester was prepared by DCC
coupling acylation in analogy to that of O-acetyl glycolic acid.26
Acetylation of 4 afforded 5a in quantitative yield. The
synthesis of 5b required the synthesis of 9-azido derivative
of 4. To achieve this purpose, 4 was reacted with p-
toluenesulfonyl chloride in pyridine to give 9-O-tosyl deriva-
tive in 55% yield. Azidation of this tosylate ester with sodium
azide in aqueous acetone provided 9-azido derivative in 90%
yield. In an alternative approach toward improving the overall
yield, we attempted to employ direct selective replacement
of primary hydroxyl group by azide.27 Accordingly, treatment
of 4 with sodium azide in the presence of triphenyphosphine
(Ph3P) and carbon tetrabromide (CBr4) followed by acety-
lation gave the desired 9-azido derivative 5b in an excellent
yield (83.5%). Direct acetylation gave the donor 5b and made
it easier to get rid of the problematic triphenylphosphine
oxide. We decided to introduce azido group at this stage
because of its stability during other manipulations. The
structure of 5b from one-pot reaction was identical to that
from azidation of tosylate ester and confirmed by IR (2100
Chemistry. The target sialosides 7a, 7b, 8a-s, and 9a-n
(Tables 1-7) were synthesized as described in Schemes 1-4.
Scheme 1 depicts the synthesis of Gal acceptor 2 by the acid
hydrolysis of the known 4-methoxyphenyl 4,6-O-benzylidene-
2,3-di-O-benzoyl-ꢀ-D- galactopyranoside 1.23 Neu5Gc donor 5
was obtained from methyl (phenyl 5-actamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-D-glycero-R- and -ꢀ-D-galacto-2-
nonulopyranoside)onate 3, which is known as one of the most
useful Neu5Ac donors, and the anomeric phenylthio group is
stable in many organic manipulations.24 Therefore, we synthe-
sized the thioglycoside of Neu5Gc 4 as a potential good donor
by exchanging the N-acetyl group of 3 with the N-glycolyl group
(Scheme 2). The thioglycoside 3 was deacetylated with meth-
anesulfonic acid (MsOH), and the resulting amine was treated
with N-hydroxysuccinimide ester of O-benzylglycolic acid25 to
give the N-glycolyl derivative 4 in 75% yield in two steps
1
cm-1, azide), 13C NMR (δ 50.0, C-9), and by H NMR in
comparison with 5a.

6668 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Scheme 4. Amidation or Reductive Alkylation 8a-s and 9a-na
Abdu-Allah et al.
a Reagents and conditions: (a) appropriate succinimidyl ester, NaHCO3 (MeCN/H2O; 5/1), rt, 24 h, 70-85%; (b) 4,4′-biphenyl dicarboxylic acid, DMF,
N-methylmorpholine, isobutyl choroformate, 12 h, rt, 65%; (c) appropriate aldehyde, NaBH3CN either in phosphate buffer pH 7, rt, 48 h, and then AcOH,
3 h (compds 9a-c), or in MeOH/AcOH (compds 9d-n), 70-80%.
Scheme 5. Synthesis of 3′ and 4′-Hydroxybiphenyl-4-acetic Acid-NHS estersa
a Reagents and conditions: (a) TsOH, EtOH, 96%; (b) 3- or 4-hydroxyphenylboronic acid, Pd(PPh3)4, Na2CO3, benzene, reflux, dark condition, 85%; (c)
LiOH, EtOH, H2O, 6 h; (d) N-hydroxysuccinimide, DCC, AcOEt, 0 °C, 24 h, two steps 80%.
Table 1. Influence of the Substituents at 5,9 Positions of Sialic Acid
Moiety and at the Reducing End of Gal on Affinity (compds 7a, 7b, 10,
11): Building the Main Scaffold
Scheme 6. Synthesis of
3′-Methoxycarbonyl-biphenyl-4-carboxylic Acida
a Reagents and conditions: (a) MeOH, SOCl2, 90%; (b) NaClO2,
H2NSO3H, 70%.
IC50 (µM)a ( SD
Scheme 7. Synthesis of 4-Biphenylacetaldehydea
compd no.
Y
R
X
hCD22
mCD22
NDb
11
10
7a
7b
OH
OH
OH
NH2
Ac
Gc
Gc
Gc
SE
SE
MP
MP
683.33 ( 28.87
550.00 ( 45.09
148.33 ( 59.65
85.00 ( 43.30
833.33 ( 57.74
496.67 ( 28.87
93.10 ( 54.1
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations. b ND means
that 50% inhibition was not observed until 1000 µM “IC50 > 1000 µM”.
a Reagents and conditions: (a) (MeO)3CH, TsOH, MeOH, reflux, 4 h,
99%; (b) DIBAL, toluene, -78°C, 2 h, 96%.
Sialylation of 2 with 5 was promoted by N-iodosuccinimide/
triflic acid (NIS/TfOH)28 in CH3CN-CH2Cl2 at -20 °C,
furnishing the R-sialoside 6 in 64% and ꢀ-form in 14% yield
(Scheme 3). The anomeric configuration of the resulting
sialosides were determined by analysis of 1H NMR spectra based
on the chemical shift values of the H3beq and H4b signals, the
coupling constant J7b,8b, and the value of ∆δ (H9b′′-H9b′); for
R-sialoside these values are 2.66 ppm, 4.91 ppm, 7.4 Hz, and
0.38 ppm, respectively, while for the ꢀ-form, the values are
2.55 ppm, 5.28 ppm, 2.0 Hz, and 0.88 ppm, respectively. These
values are in accordance with the empirical rules for defining
the anomeric configuration of sialosides.29
To minimize the number of steps required for the synthesis
of substituted amides at 9-position of 6b, we decided to use
reductive acylation under modified Staudinger conditions as
commonly used for preparation of sugar amides.30 Unfortu-
nately, attempts at forming 9-N-Bz derivative by treatment of
6b under different conditions failed to furnish any of the desired
1
product. H NMR and mass data referred to the occurrence of
O to N acetyl migration. So we concluded that reduction of
azide to amine followed by acylation is the best route to get
our target compounds. Disappointingly, Zemplen deacetylation,
followed by catalytic hydrogenation and acylation with acid
chloride or active ester using standard acylation conditions,

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6669
Table 2. Inhibitory Potencies of Aryl, Heteroraryl, Arylalkyl, Alicyclic,
and Aliphatic Carboxylic Acid Derivatives (Compds 8a-i)
Table 3. Inhibitory Potencies of Dicyclic Aromatic Carboxylic Acid
Derivatives (compds 8j-n)
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations.
Table 4. Inhibitory Potencies of 9-Biphenylcarboxamido Derivatives
(compds 8l, 8o, 8r, and 8s)
a Sialoside concentration which leads to 50% inhibition of binding.
The values are means of at least three independent determinations.
failed to afford any of the desired compounds instead resulted
in a complex reaction mixture. We expected that the methyl
ester of the sialic acid moiety may undergo lactamization during
reduction and under acylation condition. This expectation is
confirmed by the usual use of free sialic acid to prepare 9-N-
acylated derivatives.31 Also, lactamization of methyl ester during
azide reduction was used as a one-pot reaction for the synthesis
of GM3-Lactam, while for azide reduction in presence of free
carboxylic group, no lactamization occurred.32 Accordingly, de-
esterifiction with lithium hydroxide (LiOH) in aq ethanol.
followed by catalytic hydrogenation in weakly acidic medium.
afforded the 7 in 84.5% yield (Scheme 3). For amidation, at
first we tried nitrophenyl active ester; thereby, nitrophenyl-4-
biphenylcarboxylate14 was used to prepare amide 8l. The main
problem was the laborious purification of the target from
p-nitrophenol. To overcome this problem, we tried succinimidyl
ester, which afforded the corresponding amides (8a-r) in good
to excellent yields (Scheme 4). N-Hydroxysuccinimidyl (NHS)
esters required as acylating agents were prepared via DCC-
mediated coupling of NHS with the corresponding carboxylic
acids essentially as reported.33 Synthesis of compounds 8p and
8q required the synthesis of 3′- and 4′-hydroxybiphenyl-4-acetic
acid, respectively (Scheme 5), which were synthesized from
phenylacetic acid by esterification followed by Suzuki coupling
with 3- or 4-hydroxyphenylboronic acid in analogy to reported
procedures.34 On the other hand, compound 8r was obtained
IC50 (µM)a ( SD
compd
X
hCD22
mCD22
8l
H
1.10 ( 0.22
0.40 ( 0.06
7.13 ( 2.00
73.25 ( 5.44
52.93 ( 9.60
8.63 ( 3.50
16.60 ( 6.40
23.43 ( 5.70
8o
8r
8s
4-OH
3-COOH
4-COOH
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations.
by amidation of 7b with succinimidyl ester of 3′-methoxycar-
bonyl-biphenyl-4-carboxylic acid, followed by de-esterification
with LiOH. This succinimidyl ester was synthesized from 3-(p-
formylphenyl)benzoic acid by esterification, aldehydic oxidation,
and then NHS esterification (Scheme 6) as reported for the
preparation of monomethyl ester of terephthalic acid.35 Acyla-
tion of 7b with 4,4′-biphenyl dicarboxylic acid under mixed
anhydride condition afforded 8s (Scheme 4) in analogy to
acylation of a uracil derivative with 2,6-naphthalene dicarboxylic
acid.36
To study the effect of replacing the amidic carbonyl with
methylene group on affinity, compounds 9a-n (Scheme 4) were

6670 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
Table 5. Inhibitory Potencies of 9-Biphenylacetamido Derivatives
(compds 8m, 8p, and 8q)
Table 7. Effect of Varying Outer Ring Substituent on Inhibitory
Potency (compds 9d and 9g-n)
IC50 (µM)a ( SD
compd
X
hCD22
mCD22
9d
9g
9h
9i
9j
9k
9l
H
4-OH
4-COOH
3-COOH
3-OH
2-OH
4-OMe
4-Me
0.47 ( 0.20
0.24 ( 0.02
43.00 ( 24.00
7.48 ( 3.50
1.73 ( 0.21
0.23 ( 0.02
1.76 ( 0.68
9.83 ( 3.75
45.00 ( 8.89
16.33 ( 3.33
6.43 ( 1.74
10.00 ( 3.00
32.7 ( 12.1
16.50 ( 3.91
12.50 ( 1.80
30.00 ( 3.61
27.00 ( 1.00
48.00 ( 4.36
IC50 (µM)a ( SD
compd
X
hCD22
mCD22
8m
8p
8q
H
3.70 ( 1.41
0.90 ( 0.01
4.30 ( 1.20
6.20 ( 0.50
1.70 ( 0.30
2.90 ( 0.40
4-OH
3-OH
9m
9n
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations.
4-CF3
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations.
Table 6. Inhibitory Potencies of 9-Amino Derivatives (compds 9a-f)
4-carbaldehydes were prepared by Suzuki coupling of p-
bromobenzaldehyde and the appropriate phenylboronic acid by
adaptation of published procedures.40 Compounds 10 (Neu5GcR2-
6GalꢀSE, 2-(trimethylsilyl)ethyl (3,5-dideoxy-5- glycolamido-
D-glycero-R-D-galacto-2-nonulopyranosylonic acid)-(2-6)-ꢀ-D-
galactopyranoside, and 11 (Neu5AcR2-6GalꢀSE, 2-(trimeth-
ylsilyl)ethyl (5-acetamido-3,5-dideoxy-D-glycero-R-D-galacto-
2-nonulopyranosylonic acid)-(2,6)-ꢀ-D-galactopyranoside) were
already synthesized by our group and used for exploring the
biological roles of CD22.17
Results and Discussion
Biological Evaluation. The inhibitory potencies (IC50) of the
synthesized compounds were determined by measuring the
binding inhibition of IgG fusion protein of mCD22 and hCD22
(mCD22-Fc and hCD22-Fc, respectively) to their cellular ligands
expressed on the mouse myeloma transfectants J558LST6.17 The
parent line J558L do not express R2,6-sialic acid due to lack of
the expression of ST6GalI, a sialyl transferase required for the
synthesis of R2,6-sialic acid-containing glycans, whereas its
ST6GalI transfectants (J558LST6) restore expression of R2,6-
sialic acid. By using these cells, specific binding of mCD22-Fc
and hCD22-Fc to R2,6-sialic acid can be measured. Thus when
J558LST6 cells incubated with CD22-Fc in the presence of
various concentrations of synthetic R2,6-sialosides, binding of
CD22-Fc to J558LST6 cells was reduced in a dose-dependent
manner. Results are shown in Tables 1-7 for different classes
of compounds.
Structure-Affinity Relationships. IC50 of the target com-
pounds 7a,7b, 8a-s, and 9a-n are reported in Tables 1-7.
Compounds 10 and 1117 were included in the present study for
the evaluation of the influence of the substituents at the reducing
end of Gal and at C-9 of sialic acid moiety and in turn a more
complete SAR investigation.
a Sialoside concentration which leads to 50% inhibition of binding. The
values are means of at least three independent determinations.
As shown in Table 1, mCD22 has strong specificity for N-Gc
(10) over N-Ac (11) while hCD22 can bind both. Compound
7a showed higher potency than 10. This result displays the
contribution of the substituent at the reducing end of Gal to the
binding affinity and the favorable effect of MP group over SE.
Interestingly, replacement of the 9-hydroxy of 7a with amino
group (7b) resulted in a further improvement in affinity. Thus
prepared by reductive alkylation of 7b with the appropriate
aldehyde, following the procedures described in ref 37. The
aldehydes, when not commercially available, were synthesized
as shown for 4-biphenylacetaldehyde (Scheme 7), which was
synthesized in two steps from biphenylacetic acid by DIBAL
reduction38 of its methyl ester.39 The other substituted biphenyl-

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6671
7b exhibited 5-fold higher potency than 7a for mCD22. This
result is in accordance with the reported data.10 On the basis of
these findings, compound 7b was taken as a valuable structural
basis for optimization of the affinity. To study the influence of
9-amino derivatives of 7b on affinity, divergent substituents with
different requirements in space and interaction possibilities were
synthesized and evaluated for their inhibition potencies. Our
design is primary based on molecular modeling studies and
molecular features previously reported to enhance the affinity
for CD22.13,15
As can be seen in Table 2, substitution of the amino group
of 7b with benzoyl 8a, salicyloyl 8b, or chlorosalicyloyl 8c
resulted in little change in affinity. While introduction of
nicotinoyl 8d seems to improve the affinity for mCD22 (3-fold
more potent), the cyclohexanecarbonyl (8e) and hexanoyl (8f)
showed, to some extent, better affinity for hCD22 (6- and 2-fold,
respectively). These results could suggest that aliphatic chain
can bind hCD22 with higher affinity than mCD22. Accordingly,
inserting alkyl side chain between the amidic carbonyl and the
aromatic ring improved the affinity for hCD22; 8g and 8h were
10-fold more potent than 7b. However, these compounds did
not show improved affinity for mCD22 (2-fold loss of potency).
Unexpectedly, increasing the bulkiness of the aromatic moiety
did not improve the affinity for CD22 (8i vs 8g).
Next we switched to study the effect of dicyclic aromatic
substituents based on the presence of a relatively large pocket
about 9-position of sialic acid moiety.15 The results shown in
Table 3 show that this class of compound has much higher
affinity for hCD22 and mCD22. Of special note is that
biphenylcarbonyl (8l) exhibited the most promising affinity for
hCD22 (IC50 ) 1.10 µM; 77-fold more potent than 7b).
Importantly, this compound was less active for mCD22 (IC50
) 52.93 µM).
Because of the promising assay data on compound 8l, the
biphenyl motif was considered as the backbone for further
homologous and isomeric derivatives where spacers and biphe-
nyl substitution pattern were changed. Therefore, inserting
methylene (CH2) between the biphenyl ring and amidic carbonyl
group of compound 8l gave compound 8m, which showed lower
affinity than 8l for hCD22 but showed strong promising affinity
for mCD22 (IC50 ) 6.20 µM, 8-fold more potent than 8l). The
improved affinity of 8m may be due to better flexibility and
favorable fitting to the binding site. Similarly, introducing CH2
between the naphthyl ring and the amidic carbonyl group of
compound 8j gave 8n with promising affinity for hCD22 (IC50
) 1.00 µM, 9-fold more potent), but it was not so effective for
mCD22 (IC50 ) 25.00 µM, 3.6-fold more potent). It can be
concluded from the above-mentioned data that 8l (9-biphenyl-
carbonyl) is the lead compound for hCD22 inhibition, while
8m (9-biphenylacetyl) is the lead compound for mCD22
inhibition.
On the other hand, the 4′-hydroxyl group at the biphenyl ring
of 8m gave 8p with better affinity for hCD22 and mCD22 ((IC50
) 0.90 and 1.70 µM, respectively). Accordingly, compound 8p
showed the highest potency of this series for mCD22 (290-fold
more potent than 7a). Compound 8q, a regioisomer of 8p,
showed little change in potency.
Zaccai et al.15 clearly showed that the amidic NH at 9-position
is involved in a key hydrogen bond between the ligand and the
main chain carbonyl of amino acid residue at the binding side,
but the amidic carbonyl group does not seem to form a hydrogen
bond. These features may imply that the amide moiety is
exposed for recognition, but it is not clear if it has the structural
role of maintaining the substituent in a particular geometry.
Therefore, we envisaged that the replacement of the amidic
carbonyl (CO) with CH2 would be very interesting. It may alter
the position of the substituent in the binding groove as a result
of the different geometry, electronic, and steric characters. As
a consequence, the affinity and selectivity could be modulated.
Table 6 shows that replacing the amidic carbonyl with CH2 was
found to improve the affinity for mCD22 with comparable
affinity for hCD22 (9a and 9c vs 8a and 8e). Thereby, we
decided to replace the amidic carbonyl in 8l with CH2, which
gave 9d. Interestingly, 9d showed better affinity for hCD22 (IC50
) 0.47 µM) and mCD22 (16.33 µM; 3-fold more potent than
8l). This result may be due to increased rotational flexibility of
the biphenyl ring, which can make it easier to adopt the
appropriate conformation to enter the pocket. Furthermore, 9d
was modified to investigate the effect of increasing the chain
length of the spacer between the biphenyl ring and the amino
group on affinity. The results suggest that increasing the chain
length decreases the affinity for hCD22 (9d vs 9e and 9f), while
it is tolerated by mCD22.
At this point, we decided to study the additive effect of
combining 4′-hydroxylation of biphenyl ring and replacement
of amidic carbonyl with the CH2 group on inhibition of CD22.
Accordingly, compound 9g with 4′-hydroxyl group at biphenyl
ring of 9d revealed the highest potency for hCD22 (IC50 ) 0.24
µM) and better affinity for mCD22. The favorable effect of
introducing 4′-OH (8o, 8p, and 9g) may be due to its ability to
form additional hydrogen bond interactions.
The promising affinity of 9d encouraged us to investigate
the steric and electronic effects of structural modifications of
the biphenyl ring (Table 7). Therefore, substitution by o-
hydroxyl (9k) exhibited promising affinity similar to 9g. In
contrast, m-hydroxyl derivative 9j displayed lower potency (7-
fold loss of potency). This variation in affinity may be due to
correct spatial orientations of the dipole originated by the
hydroxyl groups in the para and ortho position of 9g and 9k,
which may allow the formation of a hydrogen bond with the
corresponding receptor site. Moreover, substitution by electron
donating groups, 4′-OMe (9l) or 4′-Me (9m), diminished the
affinity. In a similar manner, replacement of the p-hydroxyl (9g)
by an electron withdrawing substituent; COOH (9h) or CF3 (9n),
diminished the inhibitory affinity dramatically probably due to
unfavorable stereochemical interactions. The results discussed
above suggest that the binding site for our compounds is not so
deep as to accept a large substituent at the para position and
could suggest that small polar group at this site could be better.
As a consequence, the influences of some ring substituents
of 8l and 8m on affinity were studied and the results are shown
in Tables 4 and 5, respectively. Interestingly, introducing 4′-
OH to the biphenyl ring of 8l gave 8o with improved affinity
for hCD22 (IC50 ) 0.40 µM) and was 6-fold more potent for
mCD22 (IC50 ) 8.63 vs 52.90 µM). However, introducing a
carboxyl group at 3′-position (8r) decreased the affinity for
hCD22, while at 4′-position (8s) led to dramatic drop in affinity
(IC50 ) 73.25 µM). This decrease in affinity may be due to an
unfavorable stereochemical interaction inside the ligand binding
site. In contrast, the carboxyl derivatives of 8l showed slight
improvement in affinity for mCD22 (8r and 8s vs 8l).
Molecular Modeling. To rationalize the above-described
results, the structural basis of observed affinity was investigated
by means of combined approach of homology modeling and
receptor-ligand docking. The homology models of hCD22 and
mCD22 were constructed using the crystal structure of Sn in
complex with BPC-Neu5Ac.15 These ligand bound models were

6672 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
Figure 4. Binding mode of compound 8m in the binding site of
mCD22. The amino acids residues which interact with the C9
substituent are given in yellow color. Green line represents the hydrogen
bonding.
Figure 3. Binding mode of compound 10 in hCD22. Green line
represents the hydrogen bonding.
built according to the reported sequence alignment of N-terminal
domain, which contains the groups responsible for ligand
recognition and binding.15 A series of antagonists (10, 8l, 8m,
9d, 9g, 9h, and 9i) were manually docked into homology models
in a proposed binding mode. The resulting complexes were
energetically refined to maximize the interactions and also to
reduce the steric clashes.
To look into the key modification of present study, i.e.,
p-methoxyphenyl substitution at the reducing end of the SiaR2-
6Gal, the binding pose of the compound 7a in both of the models
was examined. In the hCD22 model, the p-methoxyphenyl group
may interact with the hydrophobic patch formed by the residues
Phe37, Lys44, Arg98, and Lys105 at the vicinity of the binding
site via steric and/or noncovalent interactions (Figure 3). This
hydrophobic patch also conserved in mCD22, where only
Lys105 is replaced with equivalent substitution Arg107. This
observation suggests that these interactions of ligand with the
hydrophobic patch of the receptor could increase the binding
affinity as compared to the control compound 10.
The docked pose of 8l revealed that the biphenyl group
sterically fits into the hydrophobic channel in hCD22 and it
appears to be more favorable than mCD22, where side chain
rigid Pro111 in mCD22 is replaced by flexible Arg109 in
hCD22. This finding is in line with a previous study, which
suggested that the biphenyl substituent could be sandwiched
between the side chain residues of hCD22 hydrophobic pocket.15
This fact could be a reason for the increased affinity of 8l to
hCD22 over mCD22. Conversely, the side chain residues of
mCD22 at the ligand binding site preclude this highly favorable
packing mode.
Interestingly, the binding pose of 8m in mCD22 seems as
favorable as hCD22, as the tail phenyl group of biphenyl can
form more stable stacking interactions with Tyr42 because of
its increased flexibility fetched in by the CH2 group between
the aromatic ring and the amidic carbonyl group (Figure 4).
Another interesting observation is that the binding mode of
9d in both models looks favorable, probably differentiated shape
of the binding sites and the rotational flexibility at C9 of 9d
could be responsible for the slight differences in the binding
affinities and the same is applicable for 9g. It is worth noting
that three out of the six amino acid residues that accommodate
C9 substituents are different although they offer comparable
hydrophobic face.
Figure 5. Binding mode of compound 9h in the binding site of hCD22.
The amino acids residues which interact with the C9 substituent are
given in yellow color. Green and pink lines represent the hydrogen
bonding and steric clash, respectively.
binding site which could be accounted for by its slight
decrease in the binding affinity and these steric clashes are
apparently not observed in mCD22. Conversely, the docked
pose of 9i in hCD22 looks more favorable than mCD22,
where biphenyl ring orientation with the mCD22 Tyr42 is
slightly disturbed and that is reasonable to apprehend the
decreased binding affinity (Figure 6). Another possibility not
to negate is that different amino acid substitutions at identical
positions (Gln94 (hCD22) and Asn96 (mCD22)) can create
differential steric volume, which also could be speculated
for the differential binding of 9h and 9i. The lower activities
exhibited by 9l-n can be attributed to the presence of
relatively bulky substituents (methoxy, methyl, and triflurom-
ethyl, respectively) in proximity to the polar amino acid
residues, Gln94 (hCD22) and Asn96 (mCD22), which can
potentially form unfavorable interactions.
Conclusions
In summary, we have documented the development and
structural optimization of a new series of sialosides as CD22-
specific inhibitors. The steric space around 9-position of sialic
Additionally, the docked pose of 9h in hCD22 (Figure 5)
shows some possible steric clashes with the distal end of the

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6673
Wako Pure Chemical Industries, Ltd. HPLC grade water and
methanol (WAKO) were used for separation of the final compounds.
Solvent systems in chromatography were specified in v/v. All
evaporations and concentrations were carried out in vacuo.
p-Methoxyphenyl 2,3-di-O-benzoyl-ꢀ-D-galactopyranoside (2). A
solution of compound 1 (3.07 g, 5 mmol) in AcOH/H2O (20 mL,
8:2, v/v) was heated at 60 °C and monitored by TLC. The excess
solvent was removed under reduced pressure and the residue
chromatographed on silica gel (2% methanol/chloroform) to afford
compound 2 (1.97 g, 89%) as a colorless solid: Rf 0.49 (1:9
26
1
methanol/chloroform); [R]D +106.3° (c 4.8, CH3OH). H NMR
(CDCl3): δ 8.01s7.96 (m, 4 H, Ar-H), 7.54s7.48 (m, 2 H, Ar-H),
7.40-7.37 (m, 4 H, Ar-H), 6.94 (d, 2 H, Ar-H), 6.76 (d, 2 H,
Ar-H), 5.99 (dd, J ) 8.0, 10.3 Hz, 1 H, H2), 5.26 (dd, J ) 2.8,
10.3 Hz, 1 H, H3), 5.15 (d, J ) 8.0 Hz, 1 H, H1), 4.97 (d, J ) 5.1
Hz, 1 H, C4-OH), 4.44 (m, 1 H, H4), 4.37 (t, J ) 6.0, 5.7 Hz, 1 H,
H5), 3.91 (m, J ) 5.7 Hz, 2 H, H6a, H6b), 3.83 (t, J ) 5.7 Hz, 1 H,
6-OH), 3.74 (s, 3 H, OCH3). 13C NMR (CDCl3): δ 165.8, 151.1,
133.5, 133.2, 129.7, 129.4, 128.4, 101.2, 74.4, 74.2, 69.4, 68.3,
62.6, 55.6. MALDI-TOF MS calcd for C27H26O9Na (M + Na)+,
517.14; found, 517.21.
Figure 6. Binding mode of compound 9i in the binding site of mCD22.
The amino acids residues which interact with the C9 substituent are
given in yellow color. Green line represents the hydrogen bonding.
Methyl (phenyl 5-benzyloxyacetamido-3,5-dideoxy-2-thio-D-glyc-
ero-ꢀ-D-galacto-2- nonulopyranosid)onate (4). To a stirred solution
of 3 (11 g, 18.83 mmol) in dry methanol (125 mL) was added
methanesulfoic acid (3 mL, 46.25 mmol). The mixture was stirred
for 24 h at 60 °C, then neutralized with triethylamine (6.2 mL)
and concentrated in vacuo. To a solution of the residue in
CH3CN-H2O (15:1, 200 mL) were added succinimidyl ester of
O-benzylglycolic acid (6.25 g, 23.78 mmol) and triethylamine (4
mL). After being stirred for 24 h at room temperature, the mixture
was concentrated in vacuo.
acid moiety has been explored using relatively small and large
bulky substituents. As concluded from the preliminary SAR
studies, the presence of biphenyl ring at 9-position of sialic acid
moiety is the key structural element for the affinity. 2′- and 4′-
Hydroxyl derivative of biphenyl ring and the replacement of
amidic carbonyl group with methylene yielded the most potent
compounds in this series (9g and 9k). Replacement of hydroxyl
group of 9g with electron donating or electron withdrawing
substituents decreased the inhibitory activities. The effect of
these modifications was more pronounced for mCD22 than for
hCD22, while the later responded better for alkyl chain
modifications. The best results of our lead optimization efforts
are compounds 8o, 9d, 9g, and 9k (IC50 values 0.40, 0.47, 0.24,
0.23 µM, respectively) for hCD22 and 8p, 8q, and 9f (IC50
values 1.70, 2.90, and 4.10 µM, respectively) for mCD22.
Docking studies were carried out to visualize the possible
interactions at the molecular level and therefore help in
extending our understanding of the nature of interactions with
the proposed binding site.
Column chromatography (2% methanol/chloroform) of the
residue on silica gel followed by crystallization from ethanol-water
gave 4 (6.94 g, 75%) as a colorless solid: Rf 0.64 (1:5 methanol/
23
1
chloroform); [R]D -60.8 (c 10.2, CH3OH). H NMR (CD3OD):
δ 7.60s7.58 (m, 2 H, Ar-H), 7.42s7.30 (m, 8 H, Ar-H),
4.64-4.62 (m, 3 H, PhCH2, H6), 4.22 (td, J ) 4.6, 12.0 Hz, 1 H,
H4), 4.03 (s, 2 H, CH2CO), 3.97 (t, J ) 10.3 Hz, 1 H, H5), 3.82
(dd, J ) 3.4, 14.3 Hz, 1 H, H9a), 3.79 (dt, J ) 3.4, 5.1 Hz, 1 H,
H8), 3.67 (dd, J ) 5.1, 14.3 Hz, 1 H, H9b), 3.57 (br-d, J ) 10.3
Hz, 1 H, H7), 3.49 (s, 3 H, OCH3), 2.70 (dd, J ) 4.6, 13.7 Hz, 1
H, H3eq), 1.96 (t, J ) 12.0, 13.7 Hz, 1 H, H3ax). 13C NMR (CD3OD):
δ 173.8, 170.7, 138.6, 137.5, 137.3, 131.5, 130.5, 129.9, 129.8,
129.7, 129.5, 129.3, 129.0, 74.5, 74.4, 73.2, 71.3, 70.6, 70.1, 67.7,
65.1, 53.9, 52.9, 42.4, 36.6. MALDI-TOF MS calcd for
C25H31NO9SNa (M + Na)+, 544.16; found, 544.24.
These CD22 specific inhibitors may be useful tools to study
the biology of B-cells, modulate B-cell dependent immune
response, and deliver therapeutic agents or toxins to B-cells,
including cancerous B-cells (B-cell targeting).
Methyl (phenyl 4,7,8,9-tetra-O-acetyl-5-benzyloxyacetamido
3,5-dideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonulopyranosid)-
onate (5a). Compound 4 (1.2 g, 2.3 mmol) was treated with acetic
anhydride (4 mL) and pyridine (4 mL) for 6 h at room temperature.
The solution concentrated in vacuo. The residue was subjected for
column chromatography (4:5 ethyl acetate/hexane) to yield 5a (1.5
g, 94.5%) as a colorless solid: Rf 0.34 (1:1 ethyl acetate/hexane);
[R]D22 -131.4 (c 10.2, CH3OH).1H NMR (CDCl3): 7.47s7.32 (m,
10 H, Ar-H), 6.66 (d, J ) 10.8 Hz, 1 H, NH), 5.50 (dd, J ) 2.3,
4.6 Hz, 1 H, H7), 5.43 (td, J ) 6.5, 10.5 Hz, 1 H, H4), 4.70 (dd, J
) 2.3, 10.5 Hz, 1 H, H8), 4.63 (d, J ) 11.4 Hz, 1 H, PhCH), 4.54
(d, J ) 11.4 Hz, 1 H, PhCH), 4.48 (dd, J ) 2.3, 10.3 Hz, 1 H, H6),
4.19 (q, J ) 10.8 Hz, 1 H, H5), 4.02 (dd, J ) 8.6, 12.0 Hz, 1 H,
H9a), 3.87 (d, J ) 9.7 Hz, 2 H, glycolyl CH2CO), 3.60 (s, 3 H,
OCH3), 3.35 (dd, J ) 9.1, 13.1 Hz, 1 H, H9b), 2.71 (dd, J ) 4.6,
13.7 Hz, 1 H, H3eq), 2.13-2.00 (m, 13 H, 4 OAc, H3ax). 13C NMR
(CDCl3): δ 170.7, 170.4, 170.3, 170.2, 170.1, 168.1, 136.7, 136.1,
129.7, 129.0, 128.8, 128.6, 128.3, 128.2, 128.0, 73.4, 72.9, 68.9,
68.7, 68.6, 68.0, 67.05, 62,5, 52.6, 52.1, 51.3, 48.5, 37.4, 21.0, 20.8,
20.7, 20.6. MALDI-TOF MS calcd for C33H39NO13SNa (M + Na)+,
712.20; found, 712.21.
Experimental Section
Chemistry. General. Specific rotations were recorded in a 1
dm cell with a JASCO DIP-360 polarimeter. Elemental analyses
were performed by using Micro Corder (MT-6) of J Science Lob.
at the Life Science Research Centre, Gifu University. H and 13C
1
NMR spectra were recorded on JEOL JNM-EX-400 (400 MHz)
or JEOL JNM-ECA-500 (500 MHz). Chemical shifts are expressed
in ppm (δ) relative to the signal of Me4Si. Gal protons assigned a,
while those of sialic acid assigned b. In some spectra, H1a
overlapped with the water signal from D2O (8r, 9h, and 9i).
MALDI-TOF MS spectra were recorded in positive ion mode on a
Bruker Autoflex with the use of R-cyano-4-hydroxycinnamic acid
(CHCA) as a matrix. Some spectra were recorded in negative ion
mode. Solvents and reagents were used as received from com-
mercial distributors without further purification. Anhydrous reac-
tions were conducted under an argon atmosphere. TLC analysis
was carried out on Merck TLC (silica gel 60F254 on glass plate),
and compounds were visualized by exposure to UV light and/or
by charring with 10% H2SO4 in ethanol. Silica gel (80 mesh and
300 mesh) manufactured by Fuji Silysia Co. was used for flash
column chromatography. The quantity of silica gel was usually
estimated as 100- to 150-fold weight of the sample to be charged.
Reversed phase silica gel (Wakogel 50C18) was purchased from
Methyl (phenyl 4,7,8-tri-O-acetyl-9-azido-5-benzyloxyacetamido
3,5,9-trideoxy-2-thio-D-glycero-ꢀ-D-galacto-2-nonulopyranosid)-
onate (5b). To a mixture of compound 4 (6.4 g, 12.30 mmol),
triphenylphosphine (3.6 g, 13.66 mmol) and sodium azide (4.01 g,

6674 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
61.54 mmol) in dry DMF (50 mL) was added carbon tetrabromide
(4.53 g, 13.66 mmol) at room temperature. The mixture was stirred
at room temperature for 24 h,and then methanol was added until
the mixture turned into a clear solution. Stirring was continued for
another 3 h. The solution concentrated in vacuo, and the residue
was treated with acetic anhydride (20 mL) and pyridine (20 mL)
for 6 h at room temperature. The solution concentrated in vacuo.
The residue was subjected for column chromatography (1:1 ethyl
acetate/hexane) to yield 5b (6.88 g, 83.5%) as a colorless solid: Rf
0.3 (5:4 ethyl acetate/hexane); [R]D26 -151.4 (c 10.8, CH3OH).IR
(KBr) 3361, 3030, 2950, 2360, 2100, 1746, 1687, 1525, 1237, 750,
696 cm-1.1H NMR (CDCl3): δ 7.46s7.35 (m, 10 H, Ar-H), 6.51
(d, J ) 10.8 Hz, 1 H, NH), 5.46 (dd, J ) 2.3, 4.6 Hz, 1 H, H7),
5.39 (m, 1 H, H4), 4.79 (m, 1 H, H8), 4.60 (d, J ) 11.4 Hz, 2 H,
PhCH, H6), 4.54 (d, 1 H, PhCH), 4.17 (q, J ) 10.8 Hz, 1 H, H5),
3.87-3.83 (2 d, 2 H, glycolyl CH2CO), 3.67-3.64 (m, 4 H, OCH3,
H9a), 3.32 (dd, J ) 9.1, 13.1 Hz, 1 H, H9b), 2.73 (dd, J ) 4.5, 13.7
Hz, 1 H, H3eq), 2.13-2.00 (m, 10 H, 3 OAc, H3ax). 13C NMR
(CDCl3): δ 170.6, 170.3, 170.2, 170.1, 168.1, 135.9, 130.1, 129.3,
128.6, 128.4, 128.2, 128.0, 88.6, 76.6, 74.2, 73.4, 72.9, 68.9, 68.8,
68.7, 52.6, 49.8, 48.5, 37.5, 20.9, 20.8, 20.7. MALDI-TOF MS
calcd for C31H36N4O11SNa (M + Na)+, 695.20; found, 695.36.
General Procedure for Sialylation. To a solution of 2 (0.644 g,
1.26 mmol, 1.0 equiv and 5 (1.041 g, 1.51 mmol, 1.2 equiv) in dry
acetonitrile-dichloromethane (5:1, 48 mL) was added molecular
(m, 2 H, H5b, H6b), 4.02-3.96 (m, 2 H, H5a, H6a′), 3.92-3.81 (m,
6 H, H6a′′, glycolyl CH2CO, COOCH3), 3.75 (s, 3 H, OCH3), 3.64
(dd, J ) 6.0, 13.7 Hz, 1 H, H9b′), 3.26 (dd, J ) 6.0, 13.7 Hz, 1 H,
H9b′′), 2.82 (br d, J ) 5.5, 1 H, 4a-OH), 2.66 (dd, J ) 4.5, 12.5
Hz, 1 H, H3beq), 2.17, 2.03, 2.00 (3s, 9 H, 3 OAc), 1.99 (t, J )
12.6 Hz, 1 H, H3bax).13C NMR (CDCl3): δ 170.3, 170.2, 170.1,
167.8, 165.9, 165.3, 155.6, 136.7, 133.4, 133.1, 129.8, 129.7, 129.5,
129.1, 128.6, 128.4, 128.3, 128.2, 128.0, 119.1, 114.4, 101.4, 99.0,
73.4, 72.9, 72.8, 69.9, 69.5, 69.0, 68.6, 67.9, 67.0, 63.2, 50.9, 48.4,
21.0, 20.7, 20.6. MALDI-TOF MS calcd for C52H56N4O20Na (M
+ Na)+, 1079.34; found, 1079.43.
General Procedure for Deprotection and Catalytic Hydrogena-
tion. To a solution of compound 6 (0.85 g, 0.8 mmol) in ethanol
(20 mL) was added solution of LiOH ·H2O (0.2 g, 4.8 mmol, 6
equiv) in water (5 mL). After stirring at room temperature for
6 h, the reaction mixture was treated with acidic (Dowex 50, H+)
and the suspension was filtered. The filterate was concentrated in
vacuo. The residue was dissolved in ethanol/water (1:1 v/v; 20 mL).
Palladium hydroxide on carbon 20% (0.9 g) and acetic acid (80
µL) were added to the solution. The reaction mixture was
hydrogenolyzed under H2 gas atmosphere with stirring at 50 °C
for 24 h. The mixture was filtered through a plug of celite. The
filterate was concentrated in vacuo, reconstituted into water, and
applied onto a silica reversed-phase column. The compound was
eluted with a gradient of methanol-water (0:1 to 10:90 methanol/
water) to afford compound 7 (0.4 g, 84.5%) as a white fluffy solid
after a final lyophilization from water. The solvent system (ethyl
acetate/methanol/water/acetic acid 10:3:3:1) was used for following
up the reaction and determining Rf values.
´
sieves 3 Å (8.0 g), and the mixture was stirred for 6 h at room
temperature under argon atmosphere then cooled to -20 °C.
N-Iodosuccinimide (NIS, 1.024 g, 4.54 mmol, 3.0 equiv) was added
to the cooled mixture while stirring. After 20 min,trifluoromethane-
sulfonic acid (TfOH, 45.5 µL, 0.45 mmol) was added dropwise to
the reaction mixture. After 16 h at -20 °C, the precipitate was
filtered off and thoroughly washed with chloroform. The filterate
and washings were combined and the solution was successively
washed with 1 M Na2CO3, 1 M Na2S2O3,and brine, dried
(Na2SO4),and concentrated. The residue was applied to a silica gel
column (0:1 to 1:99 methanol-chloroform) to furnish compound 6
(0.85 g, 64%) as a colorless solid.
p-Methoxyphenyl (3,5-dideoxy-5-glycolamido-D-glycero-r-D-ga-
lacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-galactopyranoside (7a).
According to general procedure without employing acetic acid
23
during hydrogenation. Rf 0.1; [R]D -44.2 (c 2.6, MeOH). 1H
NMR (CD3OD): δ 7.03 (d, J ) 9.16 Hz, 2 H, Ar-H), 6.83 (d, J
) 9.16 Hz, 2 H, Ar-H), 4.69 (d, J ) 7.44 Hz, 1 H, H1a), 4.03 (d,
J ) 2.30 Hz, 2 H, glycolyl CH2CO), 3.95-3.93 (m, 2 H, H6a′
H8b), 3.85 (m, 1 H, H4b), 3.83-3.67 (m, 10 H, H2a, H3a, H5a, H6a′′
,
,
p-Methoxyphenyl (methyl 4,7,8,9-tetra-O-acetyl-5-benzyloxyac-
etamido 3,5-dideoxy-D-glycero-r-D-galacto-2-nonulopyranosylonate)-
(2f6)-2,3-di-O-benzoyl-ꢀ-D-galactopyranoside (6a). Rf 0.38 (1%
H4b, H5b, H6b, OCH3), 3.60 (dd, J ) 5.7, 9.6 Hz, 1 H, H9a),
3.57-3.54 (m, 1 H, H7b), 3.50 (d, J ) 9.6 Hz, 1 H, H9b′), 2.86 (dd,
J ) 4.6, 12.6 Hz, 1 H, H3beq), 1.62 (t, J ) 12.6 Hz, 1 H, H3bax).
13C NMR (CD3OD): δ 177.8, 177.3, 156.5, 153.2, 119.3, 115.5,
103.8, 75.2, 74.5, 73.8, 72.3, 72.2, 69.9, 69.0, 64.1, 62.5, 56.2, 53.6,
43.8, 42.4. MALDI-TOF MS calcd for C24H35NO16Na (M + Na)+,
616.18; found, 616.17. Anal. (C24H35NO16 ·3H2O) N, H. C: calcd,
44.51; found, 44.00.
23
1
methanol/chloroform); [R]D +29.9 (c 11.2, CH3OH). H NMR
(CDCl3): δ 8.01 (dd, J ) 7.2, 8.3 Hz, 4 H, Ar-H), 7.50-7.48 (m,
2 H, Ar-H), 7.39-7.26 (m, 9 H, Ar-H), 7.02 (d, J ) 9.2 Hz, 2
H, Ar-H), 6.77 (d, J ) 9.2 Hz, 2 H, Ar-H), 6.37 (d, J ) 9.7 Hz,
1 H, NH), 6.00 (dd, J ) 8.0, 10.5 Hz, 1 H, H2a), 5.43 (m, 1 H,
H8b), 5.37 (dd, J ) 3.4, 10.3 Hz, 1 H, H3a), 5.32 (d, J ) 7.4 Hz, 1
H, H7b), 5.15 (d, J ) 8.0 Hz, 1 H, H1a), 4.92 (m, 1 H, H4b), 4.56
(q, J ) 12.0, 25.2 Hz, 2 H, PhCH2), 4.42-4.39 (m, 2 H, H4a, H9b′),
4.16-4.10 (m, 2 H, H5b, H6b), 4.06(dd, J ) 6.3, 12.6 Hz, 1 H,
p-Methoxyphenyl (9-amino-3,5,9-trideoxy-5-glycolamido-D-glyc-
ero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-galacto-
25
1
pyranoside (7b). Rf 0.25; [R]D -49.5 (c 10.2, H2O). H NMR
(D2O with a few drops of CD3OD): δ 7.07 (d, J ) 9.2 Hz, 2 H,
Ar-H), 6.85 (d, J ) 9.2 Hz, 2 H, Ar-H), 4.73 (d, J ) 7.8 Hz, 1
H, H1a), 4.07-4.02 (m, 3 H, glycolyl CH2CO, H8b), 3.94-3.93 (m,
2 H, H4a, H6a′), 3.85 (m, 1 H, H4b), 3.80-3.72 (m, 8 H, H2a, H5a,
H6a′′, H5b, H6b, OCH3), 3.59 (dd, J ) 3.2, 9.6 Hz, 1 H, H3a), 3.43(d,
J ) 8.5 Hz, 1 H, H7b), 3.27 (dd, 1 H, H9b′), 2.95 (dd, J ) 12.0 Hz,
1 H, H9b′′), 2.86 (dd, J ) 5.0, 12.0 Hz, 1 H, H3beq), 1.65 (t, J )
12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.3, 156.5, 153.2,
119.3, 115.5, 103.8, 75.2, 74.5, 73.8, 72.3, 72.2, 69.9, 69.0, 64.1,
62.5, 56.2, 53.6, 43.8, 42.4. MALDI-TOF MS calcd for
C24H38N2O15 (M + H)+, 593.22; found, 593.30. Anal. (C24H36-
N2O15 ·2H2O) H, C. N: calcd, 4.46; found, 4.03.
H9b′′), 3.99-3.95 (m, 2 H, H5a, H6a′), 3.92-3.83 (m, 6 H, H6a′′
,
glycolyl CH2CO, COOCH3), 3.74 (s, 3 H, OCH3), 3.15 (br. s, 1 H,
4a-OH), 2.65 (dd, J ) 4.6, 12.6 Hz, 1 H, H3beq), 2.14, 2.12, 2.08
(3s, 9 H, 3 OAc), 2.04-1.97 (m, 4 H, OAc, H3bax).13C NMR
(CDCl3): δ 170.9, 170.3, 170.2, 170.1, 170.0, 167.8, 165.8, 165.3,
155.5, 151.4, 136.7, 133.2, 133.0, 129.8, 129.7, 129.5, 129.2, 128.5,
128.3, 128.2, 128.1, 128.0, 118.9, 114.3, 101.2, 98.9, 74.3, 73.4,
72.9, 72.7, 69.6, 69.0, 68.7, 68.6, 67.2, 66.7, 62.8, 62.6, 55.5, 53.0,
48.4, 21.0, 20.7, 20.6, 20.5. MALDI-TOF MS calcd for
C54H59NO22Na (M + Na)+, 1096.34; found, 1096.45.
p-Methoxyphenyl (methyl 4,7,8-tri-O-acetyl-9-azido-5-benzy-
loxyacetamido 3,5,9-trideoxy-D-glycero-r-D-galacto-2-nonulopyra-
nosylonate)-(2f6)-2,3-di-O-benzoyl-ꢀ-D-galactopyranoside (6b). Rf
General Procedure for Amidation. p-Methoxyphenyl (3,5,9-
trideoxy-5-glycolamido-9-substituted carboxamido-D-glycero-r-D-
galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-galactopyranoside
(8a-r). To compound 7 (40 mg, 0.067 mmol) in water (1 mL)
was added solution of the appropriate N-hydroxysuccinimidyl ester
(0.08 mmol) in acetonitrile (5 mL) while maintaining the pH
between 8.0-9.0 with saturated sodium hydrogen carbonate. The
mixture was stirred at room temperature for 48 h. The solvent was
evaporated, and the residue was reconstituted into water and applied
onto a silica reversed phase column pre-equilibrated in water. The
compound was eluted with a gradient of methanol-water (0:1 to
31
0.30 (1% methanol/chloroform); [R]D +45.4 (c 12.0, CH3OH).
IR (KBr) 3300, 3030, 2950, 2100, 1717, 1507, 1217, 712 cm-1.1H
NMR (CDCl3): δ 8.02-7.97 (dd, 4 H, Ar-H), 7.52-7.48 (m, 2
H, Ar-H), 7.40-7.33 (m, 9 H, Ar-H), 7.02 (d, J ) 9.2 Hz, 2 H,
Ar-H), 6.78 (d, J ) 9.2 Hz, 2 H, Ar-H), 6.34 (d, J ) 9.2 Hz, 1H,
NH), 5.97 (dd, J ) 8.0, 10.5 Hz, 1 H, H2a), 5.37 (m, 1 H, H8b),
5.34 (dd, J ) 3.4, 10.3 Hz, 1 H, H3a), 5.30 (d, J ) 7.4 Hz, 1 H,
H7b), 5.14 (d, J ) 8.0 Hz, 1 H, H1a), 4.91 (m, 1 H, H4b), 4.56 (q,
J ) 11.4, 32.5 Hz, 2 H, PhCH2), 4.41 (br. d, 1 H, H4a), 4.16-4.13

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6675
40:100) to afford the title compounds 8a-r in (70-85%) yields
as a white fluffy solid after a final lyophilization from water. The
solvent system (ethyl acetate/methanol/water/acetic acid 10:3:3:1)
was used for following up the reaction and determining Rf values.
p-Methoxyphenyl (9-cyclohexanecarboxamido-3,5,9-trideoxy-
5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (8e). The yield was 80%; Rf 0.30;
27
1
[R]D -34.3 (c 10.2, H2O). H NMR (CD3OD): δ 6.93 (d, J )
9.0 Hz, 2 H, Ar-H), 6.73 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.59 (d, J
) 7.4 Hz, 1 H, H1a), 3.94 (s, 2 H, glycolyl CH2CO), 3.86 (d, J )
3.4 Hz, 1 H, H4a), 3.85-3.75 (m, 2 H, H8b, H6a′), 3.73-3.6 (m, 9
H, H2a, H5a, H6a′′, H4b, H5b, H6b, OCH3), 3.50-3.46 (m, 2 H, H7b,
H3a), 3.23 (d, J ) 12.0 Hz, 1 H, H9b′), 3.11 (dd, J ) 8, 12 Hz, 1 H,
H9b′′), 2.75 (dd, J ) 5.0, 13.0 Hz, 1 H, H3beq), 2.10-2.00 (m, 1 H,
cyclohexane H), 1.61 (m, 6 H, cyclohexane 5 H, H3bax), 1.35-1.02
(m, 5 H, cyclohexane H). 13C NMR (CD3OD): δ 179.4, 177.0,
174.6, 156.4, 153.2, 119.1, 115.4, 103.8, 102.1, 75.0, 74.6, 74.0,
72.4, 72.0, 71.4, 69.6, 69.3, 63.6, 62.6, 56.0, 53.7, 49.5, 48.4, 46.3,
43.5, 42.6, 30.7, 30.6, 26.8, 26.77, 26.7. MALDI-TOF mass for
C31H46N2O16Na (M + Na)+, 725.27; found, 724.95. Negative ion
mode calcd for C31H45N2O16 (M - H)-, 701.27; found, 701.44.
Anal. (C31H46N2O16 ·4H2O) C, N. H: calcd, 7.03; found, 6.52.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-hexanamido-
D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-gal-
actopyranoside (8f). The yield was 77%; Rf 0.54; [R]D26 -30.2 (c
10.4, H2O). 1HNMR (CD3OD): δ 6.93 (d, J ) 9.5 Hz, 2 H, Ar-H),
6.73 (d, J ) 9.5 Hz, 2 H, Ar-H), 4.59 (d, J ) 8.0 Hz, 1 H, H1a),
3.94 (s, 2 H, glycolyl CH2CO), 3.86 (d, J ) 3.5 Hz, 1 H, H4a),
3.82-3.80 (m, 2 H, H8b, H6a′), 3.69-3.60 (m, 9 H, H4b, H5b, H6b,
H2a, H5a, H6b, OCH3), 3.54 (dd, J ) 11.0 Hz, 1 H, H7b), 3.47 (dd,
J ) 3.5, 10.0 Hz, 1 H, H3a), 3.23 (d, J ) 13.5 Hz, 1 H, H9b′), 3.07
(dd, J ) 8.0, 13.5 Hz, 1 H, H9b′′), 2.75 (dd, J ) 4.5, 12.5 Hz, 1 H,
p-Methoxyphenyl (9-benzamido-3,5,9-trideoxy-5-glycolamido-
D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-ga-
26
lactopyranoside (8a). The yield was 85%; Rf 0.35; [R]D -17.1
(c 6.7, H2O). 1H NMR (CD3OD): δ 7.68 (d, 2 H, Ar-H), 7.38-7.30
(m, 3 H, Ar-H), δ 6.89 (d, J ) 8.6 Hz, 2 H, Ar-H), 6.67 (d, J )
8.6 Hz, 2 H, Ar-H), 4.59 (d, J ) 7.4 Hz, 1 H, H1a), 3.94-3.59
(m, 15 H, glycolyl CH2CO, H8b, H4b, H6b, H5b, H2, H4a, H5a, H6a′
,
H6a′′, OCH3, H3a), 3.59-3.25 (m, 3 H, H7b, H9b′, H9b′′), 2.75 (br. s,
1 H, H3beq), 1.56 (br. s, 1 H, H3bax). 13C NMR (CD3OD): δ 156.5,
153.2, 135.5, 132.6, 129.5, 128.3, 119.1, 115.4, 103.8, 75.0, 74.6,
74.0, 72.4, 69.8, 69.7, 63.7, 62.5, 56.0, 53.8, 44.4, 42.5. MALDI-
TOF MS calcd for C31H40N2O16Na (M + Na)+, 719.22; found,
719.30. Negative ion mode calcd for C31H39N2O16 (M - H)-,
695.64; found, 695.41. Anal. (C31H40N2O16 ·3H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(2-hydroxy-
benzamido)-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (8b). The yield was 75%; Rf 0.40;
27
1
[R]D -15.1° (c 10.9, H2O). H NMR (CD3OD): δ 7.76 (m, 1 H,
Ar-H), 7.33 (m, 1 H, Ar-H), 7.01 (d, J ) 8.6 Hz, 2 H, Ar-H),
6.88-6.86 (m, 2 H, Ar-H), 6.77 (d, J ) 8.6 Hz, 2 H, Ar-H), 4.69
(d, J ) 7.4 Hz, 1 H, H1a), 4.03-3.69 (m, 15 H, H2a, H3a, H4a, H5a,
H6a′, H6a′′, H4b, H5b, H6b, H8b, glycolyl CH2CO, OCH3), 3.59-3.25
(m, 3 H, H7b, H9b′, H9b′′), 2.79 (br-d, 1 H, H3beq), 1.77 (br-d, 1 H, H3bax).
13C NMR (CD3OD): δ 156.5, 153.2, 134.6, 129.5, 120.2, 119.2, 119.1,
118.2, 115.4, 103.9, 75.0, 74.7, 74.3, 72.3, 69.8, 63.8, 56.0, 53.6, 49.8,
49.6, 49.5, 49.4, 49.3, 48.5, 44.4. MALDI-TOF MS calcd for
C31H39ClN2O17Na (M + Na)+, 769.183; found, 769.240. Negative ion
mode calcd for C31H38ClN2O17 (M - H)-, 745.186; found, 745.460.
Anal. (C31H40N2O17 ·3H2O) C, H, N.
H3beq), 2.04 (t, J ) 7.5 Hz, 2 H, hexane CH2), 1.52 (t, J ) 12.5
Hz, 1 H, H3bax), 1.46 (m, 2 H, hexane CH2), 1.19 (m, 4 H, hexane
CH2CH2), 0.78 (t, J ) 6.5, 7.5 Hz, 3 H, hexane CH3). 13C NMR
(CD3OD): δ 177.1, 176.5, 174.6, 156.5, 153.3, 119.1, 115.4, 103.9,
102.1, 75.0, 74.6, 74.0, 72.4, 72.1, 71.3, 69.6, 69.3, 63.6, 62.6, 56.0,
53.7, 49.5, 48.4, 43.8, 42.6, 37.0, 32.5, 26.7, 23.3, 14.2. MALDI-
TOF MS calcd for C30H46N2O16Na (M + Na)+, 713.27; found,
713.18. Negative ion mode calcd for C30H45N2O16 (M - H)-,
689.28; found, 689.38. Anal. (C30H52N2O19 · 3H2O) N. C: calcd,
48.38; found, 47.93. H: calcd, 7.04; found, 6.57.
p-Methoxyphenyl
(9-(5-chloro-2-hydroxybenzamido)-3,5,9-
trideoxy-5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosy-
lonic acid)-(2f6)-ꢀ-D-galactopyranoside (8c). The yield was 70%;
27
1
Rf 0.38, [R]D -15.1 (c 10.9, H2O). H NMR (CD3OD): δ 7.82
(d, J ) 2.5 Hz, 1 H, Ar-H), 7.29 (dd, J ) 2.5, 9.0 Hz, 1 H, Ar-H),
7.00-6.98 (m, 2 H, Ar-H), 6.85 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.7
(d, J ) 9.0 Hz, 2 H, Ar-H), 4.68 (d, J ) 7.5 Hz, 1 H, H1a),
4.05-4.00 (m, 3 H, glycolyl CH2CO, H8b), 3.96-3.80 (m, 2 H,
H4a, H6a′), 3.83-3.69 (m, 11 H, H2a, H5a, H6a′′, H4b, H5b, H6b, H7b,
OCH3), 3.57 (dd, J ) 5.0, 9.0 Hz, 1 H, H3a), 3.46-3.39 (m, 2 H,
H9b′, H9b′′), 2.85 (dd, J ) 4.0, 12.0 Hz, 1 H, H3beq), 1.63 (t, J )
12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 156.4, 153.2,
134.0, 129.4, 124.8, 119.9, 119.1, 118.2, 115.3, 103.9, 75.0, 74.6,
74.0, 72.4, 72.2, 71.3, 69.7, 69.3, 63.7, 62.5, 56.0, 53.7, 49.8, 49.5,
49.2, 48.4, 43.9, 42.6. MALDI-TOF MS calcd for C31H39ClN2O17Na
(M + Na)+, 769.18; found, 769.24. Negative ion mode calcd for
C31H38ClN2O17 (M - H)-, 745.18; found, 745.46. Anal. (C31H39-
ClN2O17 ·3.5H2O) C, N. H: calcd, 5.72; found, 5.18.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(3-phenylpro-
pionamido)-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (8g). The yield was 76%; Rf 0.57;
[R]D27 -32.9 (c. 4.1, H2O). 1H NMR (CD3OD): δ 7.14-7.04 (m,
5 H, Ar-H), 6.92 (d, J ) 9 Hz, 2 H, Ar-H), 6.70 (d, J ) 9 Hz,
2 H, Ar-H), 4.6 (d, J ) 7.5 Hz, 1 H, H1a), 3.93 (s, 2 H, glycolyl
CH2CO), 3.86 (d, J ) 3.0 Hz, 1 H, H4a), 3.82-3.78 (m, 2 H, H8b,
H6a′), 3.68-3.59 (m, 9 H, H2a, H5a, H6a′′, H4b, H5b, H6b, OCH3),
3.55 (dd, J ) 11.0 Hz, 1 H, H7b), 3.47 (dd, J ) 3.0, 10.0 Hz, 1 H,
H3a), 3.24 (d, J ) 11.5 Hz, 1 H, H9b′), 3.03 (dd, J ) 8.5, 11.5 Hz,
1 H, H9b′′), 2.77-2.73 (m, 3 H, propionyl CH2, H3beq), 2.34 (t, J )
9.0, 7.5 Hz, 2 H, propionyl CH2), 1.51 (t, J ) 12.0 Hz, 1 H, H3bax).
13C NMR (CD3OD): δ 177.1, 175.4, 174.6, 162.1, 156.5, 153.3,
142.3, 129.4, 129.3, 127.1, 119.1, 115.4, 103.9, 102.1, 75.0, 74.7,
74.0, 72.4, 72.2, 71.3, 69.6, 69.3, 63.6, 62.6, 56.0, 53.7, 49.6, 49.5,
48.4, 43.8, 42.7, 38.9, 32.9. MALDI-TOF MS calcd for
C33H44N2O16Na (M + Na)+, 747.26; found, 747.44. Negative ion
mode calcd for C33H43N2O16 (M - H)-, 723.26; found, 723.630.
Anal. (C33H44N2O16 ·3.5H2O) C, N. H: calcd, 6.53; found, 6.10.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(3-(p-hydrox-
yphenyl) propionamido)-D-glycero-r-D-galacto-2-nonulopyranosyl-
onic acid)-(2f6)-ꢀ-D-galactopyranoside (8h). The yield was 70%;
Rf 0.56; [R]D27 -30.9 (c 10, H2O). 1H NMR (CD3OD): δ 6.92 (d,
J ) 9.0 Hz, 2 H, Ar-H), 6.88 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.70
(d, J ) 9.0 Hz, 2 H, Ar-H), 6.57 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.6
(d, J ) 7.5 Hz, 1 H, H1a), 3.94 (s, 2 H, glycolyl CH2CO), 3.86 (d,
J ) 3.0 Hz, 1 H, H4a), 3.82-3.78 (m, 2 H, H8b, H6a′), 3.68-3.59
(m, 9 H, H2a, H5a, H6a′′, H4b, H5b, H6b, OCH3), 3.55 (dd, J ) 11.0
Hz, 1 H, H7b), 3.47 (dd, J ) 3.5, 10.0 Hz, 1 H, H3a), 3.22 (br-d, 1
H, H9b′), 3.03 (dd, J ) 8.0, 13.0 Hz, 1 H, H9b′′), 2.74 (dd, J ) 8.0,
12.0 Hz, 1 H, H3beq), 2.66 (t, J ) 7.5 Hz, 2 H, propionyl CH2),
2.28 (t, J ) 8.0, 7.5 Hz, 2 H, propionyl CH2), 1.53 (t, J ) 12.0 Hz,
1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 175.7, 174.6, 156.6,
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-nicotinoyl-
amino-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-
27
ꢀ-D-galactopyranoside (8d). The yield was 78%; Rf 0.38; [R]D
1
-3.12 (c 11.2, H2O). H NMR (CD3OD): δ 8.93 (d, J ) 2.3 Hz,
1 H, Ar-H), 8.60 (dd, J ) 1.7, 5.1 Hz, 1 H, Ar-H), 8.18 (d, J )
8.6 Hz, 1 H, Ar-H), 7.45 (t, J ) 4.0, 8.0 Hz, 1 H, Ar-H), 6.97
(d, J ) 9.0 Hz, 2 H, Ar-H), 6.75 (d, J ) 9.0 Hz, 2 H, Ar-H),
4.68 (d, J ) 7.4 Hz, 1 H, H1a), 4.02 (m, 3 H, glycolyl CH2CO,
H8b), 3.95 (d, J ) 3.5 Hz, 1 H, H4a), 3.92 (dd, J ) 5.0, 9.0 Hz 1
H, H6a′), 3.89-3.70 (m, 11 H, H4b, H5b, H6b, H7b, H2a, H5a, H6a′′
,
OCH3), 3.58 (dd, J ) 3.5, 10.0 Hz, 1 H, H3a), 3.47-3.38 (m, 2 H,
H9b′, H9b′′), 2.84 (dd, J ) 4.0, 12.0 Hz, 1 H, H3beq), 1.63 (t, J )
12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 168.0, 156.4,
153.2, 152.4, 149.2, 137.0, 132.0, 125.0, 119.0, 119.1, 115.4, 103.8,
102.1, 75.0, 74.7, 74.1, 72.4, 71.4, 69.7, 69.3, 63.7, 62.6, 56.0, 53.7,
49.7, 49.5, 48.4, 44.6, 42.6. MALDI-TOF MS calcd for
C30H39N3O16Na (M + Na)+,720.22; found, 720.30. Negative ion
mode calcd for C30H38N3O16 (M - H)-, 696.22; found, 696.46.
Anal. (C30H39N3O16 ·4H2O) C, N. H: calcd, 6.15; found, 5.64.

6676 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
156.5, 153.2, 133.0, 130.2, 119.1, 116.2, 115.4, 103.9, 102.1, 75.0,
74.6, 74.0, 72.4, 72.1, 71.3, 69.6, 69.3, 63.7, 62.6, 56.0, 53.7, 49.6,
49.5, 48.4, 43.8, 42.7, 39.9, 32.2. MALDI-TOF MS calcd for
C33H44N2O17Na (M + Na)+, 763.25; found, 763.02. Negative ion
mode calcd for C33H43N2O17 (M - H)-, 739.25; found, 739.43.
Anal. (C33H44N2O17 ·3.5H2O) C, H, N.
129.9, 128.9, 128.1, 127.9, 119.0, 115.3, 103.8, 75.0, 74.6, 74.1,
72.4, 69.7, 69.2, 62.6, 55.9, 53.8, 49.5, 48.4, 44.5, 42.6. MALDI-
TOF MS calcd for C37H44N2O16Na (M + Na)+, 795.26; found,
795.31. Negative ion mode calcd for C37H43N2O16 (M - H)-,
771.26; found, 771.36. Anal. (C37H44N2O16 ·4.5H2O) C, H, N.
p-Methoxyphenyl (9-(4-biphenyl)acetamido-3,5,9-trideoxy-5-gly-
colamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4(1-pyre-
nyl)butanoylamino)-D-glycero-r-D-galacto-2-nonulopyranosylon-
ic acid)-(2f6)-ꢀ-D-galactopyranoside (8i). The yield was 70%; Rf
0.63; [R]D27 -18.4 (c 10.3, H2O). 1H NMR (CD3OD): δ 8.22-7.98
(m, 8 H, Ar-H), 7.82 (d, J ) 8.0 Hz, 1 H, Ar-H), 6.94 (d, J ) 9.0
Hz, 2 H, Ar-H), 6.71 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.66 (d, J ) 8.0
Hz, 1 H, H1a), 4.10 (s, 2 H, glycolyl CH2CO), 3.98 (d, J ) 3.5 Hz, 1
H, H4a), 3.97 (dd, J ) 8.0 Hz, 1 H, H6a′), 3.91 (dd, J ) 6.0, 9.5 Hz,
1 H, H8b), 3.87-3.78 (m, 3 H, H5a, H6a′′, H4b), 3.74-3.65 (m, 4 H,
H2a, H5b, H6b, H7b), 3.61 (d, J ) 3.5, 10.5 Hz, 1 H, H3a), 3.59 (s, 1 H,
3 H, OCH3), 3.47 (d, J ) 9.5 Hz, 1 H, H9b′), 3.27-3.21 (m, 3 H, H9′′,
CH2CH2CO), 2.85 (dd, J ) 4.0, 12.5, Hz, H3beq), 2.42 (dd, J ) 7.5,
15.0 Hz, 2 H, CH2CH2CH2CO), 2.16-1.98 (m, 2 H, CH2CH2CH2CO),
1.70 (t, J ) 12.5 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 175.6, 175.3,
154.5, 151.3, 131.0, 130.6, 127.0, 126.9, 126.1, 125.6, 124.5, 124.4,
124.3, 122.9, 117.6, 114.1, 101.97, 100.58, 73.2, 72.7, 72.3, 70.6, 70.3,
70.1, 67.9, 67.7, 62.1, 60.9, 54.9, 51.9, 42.3, 40.7, 35.3, 32, 27.4.
MALDI-TOF MS calcd for C33H44N2O16Na (M + Na)+, 747.26;
found, 747.44. Negative ion mode calcd for C33H43N2O16 (M - H)-,
723.26; found, 723.630. Anal. (C44H50N2O16 ·4H2O) C, N. H: calcd,
6.25; found, 5.75.
29
ꢀ-D-galactopyranoside (8m). The yield was 76%; Rf 0.59; [R]D
-35.5 (c 10.0, H2O). 1H NMR (CD3OD): δ 7.47 (d, J ) 7.5 Hz, 2 H,
Ar-H), 7.42 (d, J ) 9.0 Hz, 2 H, Ar-H), 7.32-7.28 (m, 2 H, Ar-H),
7.22-7.20 (m, 3 H, Ar-H), 6.94 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.73
(d, J ) 9.0 Hz, 2 H, Ar-H), 4.61 (d, J ) 8.0 Hz, 1 H, H1a), 3.93 (s,
2 H, glycolyl CH2CO), 3.86-3.78 (m, 3 H, H4a, H6a′, H8b), 3.72-3.60
(m, 1 H, H2a, H6a′′, H4b, H5b, H6b, acetyl CH2, OCH3), 3.50-3.39 (m,
3 H, H3, H5a, H7b), 3.21-3.20 (m, 1 H, H9b′), 3.09 (dd, J ) 8.0, 13.0
Hz, H9b′′), 2.75 (dd, J ) 4.5, 12.0 Hz,1 H, H3beq), 1.53 (t, J ) 12.0
Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 174.5, 174.1, 156.5,
153.3, 142.0, 141.0, 136.0, 130.7, 129.8, 128.2, 128.1, 127.9, 119.2,
115.5, 103.9, 102.2, 75.0, 74.7, 74.0, 72.4, 72.2, 71.3, 69.6, 69.3, 63.7,
62.6, 56.0, 53.7, 44.0, 43.3, 42.7. MALDI-TOF MS calcd for
C38H46N2O16Na (M + Na)+, 809.27; found, 809.28. Negative ion mode
calcd for C38H45N2O16 (M - H)-, 785.27; found, 785.53. Anal.
(C38H46N2O16 ·3.5H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(2-naphtyl)-
acetamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (8n). The yield was 75%; Rf 0.58;
27
1
[R]D -32.9 (c 10.5, H2O). H NMR (CD3OD): δ 7.67 (m, 3 H,
Ar-H), 7.61 (s, 1 H, Ar-H), 7.33-7.27 (m, 3 H, Ar-H), 6.94 (d,
J ) 8.5 Hz, 2 H, Ar-H), 6.72 (d, J ) 8.5 Hz, 2 H, Ar-H), 4.60
(d, J ) 8.0 Hz, 1 H, H1a), 3.90 (s, 2 H, glycolyl CH2CO), 3.87-3.78
(m, 3 H, H4, H6a′, H8b), 3.75-3.52 (m, 12 H, H2a, H5a, H6a′′, H4b,
H5b, H6b, H7b, acetyl CH2, OCH3), 3.46 (dd, J ) 3.5, 10.0 Hz, 1 H,
H3a), 3.21-3.20 (m, 1 H, H9b′), 3.01 (dd, J ) 10.0, 12.0 Hz, H9b′′),
2.75 (dd, J ) 4.5, 12.0 Hz, 1 H, H3beq), 1.53 (t, J ) 12.0 Hz, 1 H,
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(2-naphtamido)-
D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-ga-
26
lactopyranoside (8j). The yield was 74%; Rf 0.38; [R]D -17.9 (c
1
10.3, H2O). H NMR (CD3OD): δ 8.34 (s, 1 H, Ar-H), 7.92-7.85
(m, 4 H, Ar-H), 7.55-7.52 (m, 2 H, Ar-H), 6.95 (d, J ) 9.0 Hz, 2
H, Ar-H), 6.69 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.68 (d, J ) 8.0 Hz, 1
H, H1a), 4.12-3.95 (m, 4 H, H6a′, H8b, glycolyl CH2CO), 3.98-3.70
(m, 7 H, H2a, H4a, H5a, H6a′′, H4b, H5b, H6b, H7b), 3.61-3.58 (m, 4 H,
H3a, OCH3), 3.52 (m, 2 H, H9b′, H9b′′), 2.85 (br-d, 1 H, H3beq), 1.65 (t,
1 H, H3bx). 13C NMR (CD3OD): δ 177.1, 156.4, 153.1, 136.2, 134.0,
132.9, 130.1, 129.2, 128.8, 128.7, 127.7, 124.9, 119.0, 115.3, 103.8,
75.0, 74.6, 74.1, 72.5, 72.4, 71.6, 69.6, 69.3, 63.7, 62.6, 55.9, 53.7,
49.8, 44.6, 42.7. MALDI-TOF MS calcd for C35H42N2O16Na (M +
Na)+, 769.24; found, 769.25. Negative ion mode calcd for C35H41N2O16
(M - H)-, 745.24; found, 745.46. Anal. (C35H42N2O16 ·4.5H2O) C,
N. H: calcd, 6.21; found, 5.76.
H
3bax). 13C NMR (CD3OD): δ 177.1, 174.5, 174.1, 156.5, 153.3,
135.0, 134.4 133.8, 129.1, 128.8, 128.7, 128.6, 128.3, 127.1, 126.6,
119.2, 115.5, 103.9, 102.2, 75.0, 74.7, 74.0, 72.4, 72.2, 71.3, 69.6,
69.3, 63.7, 62.6, 56.0, 53.7, 49.8, 44.0, 43.8, 42.6. MALDI-TOF
MS calcd for C36H44N2O16Na (M + Na)+, 783.26; found, 783.26.
Negative ion mode calcd for C36H43N2O16 (M - H)-, 759.26;
found, 759.50. Anal. (C36H44N2O16 ·2.5H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4′-hydroxy-
4-biphenyl-carboxamido)-D-glycero-r-D-galacto-2-nonulopyrano-
sylonic acid)-(2f6)-ꢀ-D-galactopyranoside (8o). The yield was
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-diphenylac-
etamido-D-glycero-D-r-galacto-2-nonulopyranosylonic acid)-(2f6)-
27
70%; Rf 0.60; [R]D -12.6 (c 10.3, H2O). 1H NMR (CD3OD):
26
ꢀ-D-galactopyranoside (8k). The yield was 76%; Rf 0.52; [R]D
δ 7.72 (d, J ) 9.0 Hz, 2 H, Ar-H), 7.48 (d, J ) 9.0 Hz, 2 H, Ar-H),
7.39 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.88 (d, J ) 9.0 Hz 2 H, Ar-H),
6.77 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.65 (d, J ) 9.0 Hz, 2 H, Ar-H),
4.59 (d, J ) 7.5 Hz, 1 H, H1a), 3.98-3.92 (m, 3 H, glycolyl CH2CO,
H8b), 3.88 (d, J ) 3.0 Hz, 1 H, H4a), 3.83 (dd, J ) 6.0 Hz, 1 H, H6a′),
3.78-3.68 (m, 5 H, H5a, H6a′′, H4b, H5b, H6b), 3.65-3.60 (m, 2 H, H2,
H7b), 3.54 (s, 3 H, OCH3), 3.49 (dd, J ) 3.5, 9.5 Hz, 1 H, H3a),
3.37-3.30 (m, 2 H, H9b′, H9b′′), 2.76 (dd, J ) 4.5, 12.0 Hz, 1 H, H3beq),
1.55 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 174.6,
174.1, 170.2, 159.0, 156.4, 153.2, 145.0, 133.3, 132.4, 129.2, 128.8,
127.2, 119.0, 116.8, 115.4, 103.8, 102.2, 75.0, 74.7, 74.1, 72.5, 72.4,
71.5, 69.6, 69.3, 63.7, 62.6, 56.0, 53.7, 44.5, 43.3, 42.7. MALDI-TOF
MS calcd for C37H44N2O17Na (M + Na)+, 811.25, found, 811.56.
Negative ion mode calcd for C37H43N2O17 (M - H)-, 787.25; found,
787.44. Anal. (C37H44N2O17 ·4.5H2O) C, H, N.
1
-17.9 (c 11.2, H2O). H NMR (CD3OD): δ 7.26-7.18 (m, 10 H,
Ar-H), 7.03 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.80 (d, J ) 9.0 Hz, 2
H, Ar-H), 4.99 (s, 1 H, acetyl CH), 4.70 (d, J ) 7.5 Hz, 1 H,
H1a), 3.98 (s, 2 H, glycolyl CH2CO), 3.94-3.90 (m, 2 H, H6a′, H8b),
3.82-3.60 (m, 11 H, H2a, H4a, H5a, H6a′′, H4b, H5b, H6b, H7b, OCH3),
3.55 (td, J ) 6.5 Hz, H3a), 3.34-3.25 (m, 2 H, H9b′, H9b′′), 2.8
(br-d, 1 H, H3beq), 1.6 (t, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1,
174.9, 156.5, 153.3, 141.2, 130.0, 129.9, 129.4, 128, 119.2, 115.5,
103.9, 75.0, 74.6, 74.0, 72.4, 72.1, 71.2, 69.7, 69.3, 63.6, 62.6, 59.1,
55.9, 53.7, 44.0, 42.6. MALDI-TOF MS calcd for C38H46N2O16Na
(M + Na)+, 809.27; found, 809.49. Negative ion mode calcd
for C38H45N2O16 (M - H)-, 785.27; found, 785.63. Anal.
(C38H46N2O16 ·3.5H2O) C, H, N.
p-Methoxyphenyl (9-(4-biphenylcarboxamido)-3,5,9-trideoxy-
5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (8l). The yield was 80%; Rf 0.40;
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4′-hydroxy-
4-biphenyl)-acetamido-D-glycero-r-D-galacto-2-nonulopyranosy-
lonic acid)-(2f6)-ꢀ-D-galactopyranoside (8p). The yield was 72%;
24
1
[R]D -41.4 (c 10.5, H2O). H NMR (CD3OD): δ 7.87 (d, J )
8.5 Hz, 2 H, Ar-H), 7.76-7.63 (m, 4 H, Ar-H), 7.45 (m, 2 H,
Ar-H), 7.36 (m, 1 H, Ar-H), 7.01 (d, J ) 7.0 Hz, 2 H, Ar-H),
6.77 (d, J ) 7.0 Hz, 2 H, Ar-H), 4.71 (d, J ) 8.0 Hz, 1 H, H1a),
4.1-3.95 (m, 4 H, H6a′, H8b, glycolyl CH2CO), 3.94-3.78 (m, 7
H, H4b, H6b, H5b, H2a, H4a, H5a, H6a′′), 3.74 (dd, J ) 8.0, 9.5 Hz, 1
H, H7b), 3.66 (s, 3 H, OCH3), 3.51 (dd, J ) 7.5, 14.0 Hz, 1 H,
H9b′), 3.46 (d, J ) 7.5 Hz, 1 H, H9b′′), 2.73 (br-d, 1 H, H3beq), 1.80
(t, 1 H, H3bax). 13C NMR (CD3OD): δ 156.4, 153.1, 145.5, 141.3,
32
Rf 0.61; [R]D -31.7 (c 10.1, H2O). 1H NMR (CD3OD): δ
7.45-7.40 (m, 4 H, Ar-H), 7.26 (d, J ) 9.0 Hz, 2 H, Ar-H),
7.03 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.84-6.81 (m, 4 H, Ar-H),
4.70 (d, J ) 7.2 Hz, 1 H, H1a), 4.02 (s, 2 H, glycolyl CH2CO),
3.95-3.91 (m, 3 H, H4a, H6a′, H8b), 3.85-3.63 (m, 9 H, H2a, H5a,
H6a′′, H4b, H5b, H6b, OCH3), 3.60-3.52 (m, 2 H, H3a, H7b), 3.50 (s,
2 H, acetyl CH2), 3.35-3.30 (m, 1 H, H9b′), 3.20 (dd, J ) 8.0,
12.0 Hz, 1 H, H9b′′) 2.84 (dd, J ) 4.5, 12.0 Hz,1 H, H3beq), 1.63 (t,

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6677
J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 174.5,
174.3, 158.2, 156.5, 153.3, 140.9, 134.9, 133.3, 130.6, 128.9, 128.1,
127.5, 119.1, 116.6, 115.5, 103.9, 102.2, 75.0, 74.7, 74.1, 72.4,
72.1, 71.4, 69.7, 69.3, 63.7, 62.6, 56.0, 53.7, 44.0, 43.3, 42.7.
MALDI-TOF MS calcd for C38H46N2O17Na (M + Na)+, 825.27,
found, 825.28. Anal. (C38H46N2O17 ·5H2O) C, N. H: calcd, 6.32;
found, 5.84.
General Procedures for Reductive Alkylation. p-Methoxyphe-
nyl (3,5,9-trideoxy-5-glycolamido-9-substituted amino-D-glycero-
r-D-galacto-2-nonulopyranosylonic Acid)-(2f6)-ꢀ-D-galactopyra-
noside (9a-n). For compounds 9a-c: To a mixture of the amine
7b (40 mg, 0.067 mmol) and appropriate aldehyde (0.043 mmol)
in 0.2 M phosphate buffer pH ) 7 (1 mL) was added sodium
cyanoborohydride; NaBH3CN (5.8 mg, 0.093 mmol). The reaction
mixture was stirred at room temperature for 48 h then acetic acid
(11 µL) was added and stirring was continued for further 3 h. For
compounds 9d-n: To a mixture of the amine 7 (50 mg, 0.084
mmol) and appropriate aldehyde (0.043 mmol) in dry MeOH (3
mL) was added acetic acid (0.1 mL), followed by NaBH3CN (1.0
M in THF, 60 µL, 0.060 mmol). A total of 24 h later, a TLC
indicated a complete disappearance of the aldehyde. The reaction
mixture was concentrated, reconstituted into water, and loaded on
a silica reversed-phase column pre-equilibrated in water. The
compounds were eluted with a gradient of methanol-water (0:1
to 60:100) to afford the title compounds 9a-n in (70-80%) as a
a white fluffy solid after a final lyophilization from water. The
solvent system (ethyl acetate/methanol/water/acetic acid 10:3:3:1)
was used for following up the reaction and determining Rf values.
p-Methoxyphenyl (9-benzylamino-3,5,9-trideoxy-5-glycolamido-
D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-ga-
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(3′-hydroxy-
4-biphenyl)-acetamido-D-glycero-r-D-galacto-2-nonulopyranosy-
lonic acid)-(2f6)-ꢀ-D-galactopyranoside (8q). The yield was 75%;
32
1
Rf 0.61; [R]D -31.5 (c 10.8, H2O). H NMR (CD3OD): δ 7.48
(m, 2 H, Ar-H), 7.29 (d, J ) 8.5 Hz, 2 H, Ar-H), 7.21 (t, J ) 8.5
Hz, 1 H, Ar-H), 7.03 (d, J ) 9.0 Hz, 4 H, Ar-H), 6.82 (d, J )
9.0 Hz, 2 H, Ar-H), 6.75 (dd, J ) 2.5, 6.0 Hz, 1 H, Ar-H), 4.70
(d, J ) 7.5 Hz, 1 H, H1a), 4.04 (s, 2 H, glycolyl CH2CO), 3.95-3.92
(m, 3 H, H4a, H6a′, H8b), 3.79-3.66 (m, 10 H, H2a, H5a, H6a′′, H4b,
H5b, H6b, H7b, OCH3), 3.58 (dd, J ) 3.5, 9.5 Hz, 1 H, H3a), 3.52 (s,
2 H, acetyl CH2), 3.35 (br-d, J ) 8.0 Hz,, H9b′), 3.20 (dd, J ) 8.0
Hz, 1 H, H9b′′), 2.85 (dd, J ) 4.5, 12.0 Hz,1 H, H3beq), 1.64 (t, J )
12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.1, 174.6, 174.2,
158.8, 156.5, 153.2, 143.5, 141.0, 136.0, 130.8, 130.6, 128.0, 119.2,
119.1, 115.5, 115.2, 114.7, 103.8, 102.2, 75.0, 74.6, 74.0, 72.4,
72.1, 71.5, 69.7, 69.3, 63.7, 62.6, 56.0, 53.7, 44.0, 43.3, 42.6.
MALDI-TOF MS calcd for C38H46N2NaO17Na (M + Na)+, 825.27;
found, 825.26. Anal. (C38H46N2O17 ·4.5H2O) C, H, N.
26
lactopyranoside (9a). The yield was 73%; Rf 0.61; [R]D -41.9
(c 7.5, H2O). 1H NMR (D2O with a few drops of CD3OD): δ
7.35-7.31 (m, 5 H, Ar-H), 6.95 (d, J ) 9.0 Hz, 2 H, Ar-H),
6.70 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.60 (d, J ) 8.0 Hz, 1 H, H1a),
4.45 (brs, 1 H, NH), 4.10 (s, 2 H, glycolyl CH2CO), 4.02 (td, J )
8.5 Hz, 1 H, H8b), 3.94 (s, 2 H, benzylic CH2), 3.83-3.81 (m, 2 H,
p-Methoxyphenyl (9-(3′-carboxy-4-biphenylcarboxamido)-3,5,9-
trideoxy-5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosy-
lonic acid)-(2f6)-ꢀ-D-galactopyranoside (8r). Obtained after LiOH
29
hydrolysis of methyl ester. The yield was 70%; Rf 0.59; [R]D
H4a, H6a′), 3.75 (m, 1 H, H4b), 3.65-3.55 (m, 8 H, H2, H5a, H6a′′
,
1
-8.3 (c 10.8, H2O). H NMR (CD3OD): δ 8.00 (s, 1 H, Ar-H),
H5b, H6b, OCH3), 3.46 (dd, J ) 3.5, 9.5 Hz, 1 H, H3a), 3.28 (d, J
) 8.5 Hz, 1 H, H7b), 3.25 (dd, 1 H, H9b′), 2.86 (dd, J ) 10.0, 12.0
Hz, 1 H, H9b′′), 2.76 (dd, J ) 4.5, 12.0 Hz, 1 H, H3beq), 1.53 (t, J
) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.4, 174.6, 156.6,
153.3, 132.6, 131.0, 130.5, 130.2, 119.3, 115.4, 103.9, 102.0, 75.3,
74.7, 74.0, 72.7, 72.4, 69.9, 69.1, 68.4, 64.1, 62.6, 56.0, 53.7, 52.3,
51.3, 42.6. MALDI-TOF MS calcd for C31H43N2O15 (M + H)+,
683.26; found, 683.25. Anal. (C31H42N2O15 ·3H2O) C, N. H: calcd,
6.57; found, 6.12.
7.91 (d, 1 H, Ar-H), 7.67-7.66 (m, 3 H, Ar-H), 7.44-7.41 (m,
3 H, Ar-H), 6.90 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.22 (d, J ) 9.0
Hz, 2 H, Ar-H), 4.76 (br-d, 1 H, H1a), 4.17-4.08 (m, 4 H, H4a,
glycolyl CH2CO, H8b), 4.05-3.88 (m, 5 H, H2a, H6a′, H4b, H5b, H6b),
3.85-3.75 (m, 3 H, H5a, H6a′′, H7b), 3.61 (d, J ) 9.0 Hz, 1 H, H3a)
3.60 (s, 3 H, OCH3), 3.45-3.32 (m, 2 H, H9b′, H9b′′), 2.77 (br-d, 1
H, H3beq), 1.85 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD):
δ 169.3, 154.1, 150.9, 142.3, 139.3, 132.1, 131.5, 130.0, 129.0,
128.8, 127.5, 127.4, 126.5, 117.2, 114.3, 101.5, 73.1, 72.5, 70.5,
70.3, 70.1, 67.8, 67.3, 62.3, 60.9, 55.0, 51.5. MALDI-TOF MS
calcd for C38H44N2O18Na (M + Na)+, 839.25; found, 839.39. Anal.
(C38H44N2O18 ·5H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(3,5-dimethox-
ybenzyl)amino-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (9b). The yield was 75%; Rf 0.54;
26
1
[R]D -104.4 (c 10.2, H2O). H NMR (D2O with a few drops of
CD3OD): δ 7.03 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.80 (d, J ) 9.0 Hz,
2 H, Ar-H), 6.61 (d, J ) 2.5 Hz, 2 H, Ar-H), 6.50 (s, 1 H, Ar-H),
4.70 (d, J ) 7.5 Hz, 1 H, H1a), 4.16-4.08 (m, 3 H, H8b, glycolyl
CH2CO), 4.04 (s, 2 H, benzylic CH2), 3.94-3.91 (m, 2 H, H4a,
H6a′), 3.83 (m, 1 H, H4b), 3.75-3.65 (m, 14 H, H2a, H5a, H6a′′, H5b,
H6b, 3 OCH3), 3.55 (dd, J ) 3.5, 10.0 Hz, 1 H, H3a), 3.38 (d, J )
8.5 Hz, 1 H, H7b), 3.31 (dd, 1 H, H9b′), 2.96 (dd, J ) 12.0 Hz, 1 H,
H9b′′), 2.86 (dd, J ) 4.5, 12.0 Hz, 1 H, H3beq), 1.63 (t, J ) 12.0 Hz,
1 H, H3bax). 13C NMR (CD3OD): δ 177.4, 174.6, 162.8, 156.6,
153.2, 134.6, 119.3, 115.4, 108.7, 103.9, 102.1, 102.0, 75.2, 74.7,
74.0, 72.7, 72.4, 69.9, 69.1, 68.4, 64.0, 62.6, 56.0, 53.7, 52.3, 51.3,
42.5. MALDI-TOF MS calcd for C33H47N2O17 (M + H)+, 743.28;
found 743.41. Anal. (C33H46N2O17 ·3H2O) N. C: calcd, 49.75; found,
49.29. H: calcd, 6.58; found, 6.11.
Mixed Anhydride Method. p-Methoxyphenyl (9-(4′-carboxy-4-
biphenylcarboxamido)-3,5,9-trideoxy-5-glycolamido-D-glycero-r-
D-galacto-2-nonulopyranosylonic acid)-(2f6)-ꢀ-D-galactopyrano-
side (8s). To a suspension of 4,4′-biphenyl dicarboxylic acid (16.2
mg, 0.067 mmol) in DMF (2 mL), N-methylmorpholine (7.3 µL,
0.067 mmol) and isobutyl chloroformate (9 µL, 0.067 mmol) were
added under cooling on an ice bath. To the obtained mixture
compound 7 (48 mg, 0.08 mmol) was added portionwise, and the
mixture was stirred at room temperature for 12 h. DMF was
removed under vacuum and the residue was reconstituted into water
and applied onto a silica reversed-phase column pre-equilibrated
in water. The compound was eluted with a gradient of methanol/
water (0:1 to 20:100) to afford compound 8s (65%) as a colorless
solid: Rf 0.58 (ethyl acetate/methanol/water/acetic acid 10:3:3:1);
31
1
[R]D -14.44 (c 10.4, H2O). H NMR (CD3OD): δ 7.83 (d, J )
8.0 Hz, 2 H, Ar-H), 7.59-6.49 (m, 6 2 H, Ar-H), 6.73 (d, J )
9.0 Hz, 2 H, Ar-H), 6.54 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.78 (d, J
) 7.5 Hz, 1 H, H1a), 3.97-3.90 (m, 4 H, H4a, glycolyl CH2CO,
H8b), 3.82-3.38 (dd, J ) 6.0 Hz, 1 H,), 3.78-3.68 (m, 8 H, H2a,
H5a, H6a′, H6a′′, H4b, H5b, H6b, H7b), 3.60 (s, 3 H, OCH3), 3.49 (d, J
) 9.0 Hz, 1 H, H3a), 3.47-3.38 (m, 2 H, H9b′, Hb′′), 2.77 (d, J )
12.0 Hz, 1 H, H3beq), 1.84 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR
(CD3OD): δ 169.3, 154.1, 150.9, 142.3, 139.3, 132.1, 131.5, 130.0,
129.0, 128.8, 127.5, 127.4, 126.5, 117.2, 114.3, 101.5, 73.1, 72.5,
70.5, 70.3, 70.1, 67.8, 67.3, 62.3, 60.9, 55.0, 51.5. MALDI-TOF
MS calcd for C38H44N2O18 (M + H)+, 839.25; found, 839.29. Anal.
(C38H44N2O18 ·5H2O) N. C: calcd, 50.33; found, 49.86. H: calcd,
6.00; found, 5.45.
p-Methoxyphenyl (9-cyclohexylmethylamino-3,5,9-trideoxy-5-
glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (9c). The yield was 80%; Rf 0.55;
25
1
[R]D -45.2 (c 10.5, H2O). H NMR (D2O with a few drops of
CD3OD): δ 7.05 (d, J ) 9.0 Hz, 2 H, Ar-H), 6.83 (d, J ) 9.0 Hz,
2 H, Ar-H), 4.71 (d, J ) 8.0 Hz, 1 H, H1a), 4.08-4.05 (m, 3 H,
glycolyl CH2CO, H8b), 3.95-3.87 (m, 2 H, H4a, H6a′), 3.83 (m, 1
H, H4b), 3.74-3.70 (m, 8 H, H2a, H5a, H6a′′, H5b, H6b, OCH3), 3.56
(dd, J ) 3.5, 9.5 Hz, 1 H, H3a), 3.38 (d, J ) 8.5 Hz, 1 H, H7b),
3.18 (dd, J ) 12.5 Hz, 1 H, H9b′), 2.87 (m, 2 H, H9b′′, H3beq), 2.71
(d, J ) 7.0 Hz, 2 H, CH2NH), 1.74-1.61 (m, 7 H, cyclohexane
CH2CH2CH2, H3bax), 1.28-1.18 (m, 3 H, cyclohexane CH2CH),
0.95-0.92 (m, 2 H, cyclohexane CH2). 13C NMR (CD3OD): δ
177.3, 174.6, 156.5, 153.3, 119.2, 115.4, 103.9, 102.0, 75.2, 74.7,

6678 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Abdu-Allah et al.
74.0, 72.7, 72.4, 69.8, 69.2, 69.0, 63.9, 62.6, 56.0, 55.8, 53.7, 53.0,
42.6, 36.8, 31.8, 31.7, 27.2, 26.7, 26.6. MALDI-TOF MS calcd
for C31H49N2O15 (M + H)+, 689.31; found, 689.20. Anal.
(C31H48N2O15 ·2.5H2O) C, H, N.
Hz, 1 H, H1a), 4.20 (d, J ) 4.0 Hz, 2 H, glycolyl CH2CO), 4.15
(td, J ) 7.5, 15.0 Hz, 1 H, H8b), 4.04 (s, 2 H, CH2NH), 3.95-3.91
(m, 2 H, H4a, H6a′), 3.85 (m, 1 H, H4b), 3.75-3.71 (m, 5 H, H2a,
H5a, H6a′′, H5b, H6b), 3.67 (s, 3 H, OCH3), 3.55 (dd, J ) 3.5, 10.0
Hz, 1 H, H3a), 3.40 (br-dd, J ) 8.5, 1 H, H7b), 3.35 (dd, 1 H, H9b′),
2.99 (dd, J ) 12.0 Hz, 1 H, H9b′′), 2.88 (dd, J ) 4.5, 12.0 Hz,1 H,
p-Methoxyphenyl (9-(4-biphenyl)methylamino-3,5,9-trideoxy-
5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (9d). The yield was 70%; Rf 0.61;
H
3beq), 1.64 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ
25
1
177.4, 158.7, 156.6, 153.3, 143.6, 132.6, 131.6, 130.3, 129.1, 128.0,
119.3, 116.8, 115.4, 104.0, 75.3, 74.7, 74.0, 72.7, 72.4, 69.9, 69.1,
68.4, 64.1, 62.6, 56.0, 53.8, 52.0, 51.2, 42.6. MALDI-TOF MS
calcd for C37H47N2O16 (M + H)+, 775.29; found, 775.27. Anal.
(C37H46N2O16 ·3H2O) C, H, N.
[R]D -52.5 (c 8.0, H2O). H NMR (D2O with a few drops of
CD3OD): δ 7.65 (d, J ) 8 Hz, 2 H, Ar-H), 7.60 (d, J ) 9 Hz, 2
H, Ar-H), 7.52 (d, J ) 8.0 Hz, 2 H, Ar-H), 7.44 (t, J ) 7.5 Hz,
2 H, Ar-H), 7.35 (m, 1 H, Ar-H), 7.04 (d, J ) 9.0 Hz, 2 H,
Ar-H), 6.80 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.70 (d, J ) 7.5 Hz, 1
H, H1a), 4.24 (s, 2 H, glycolyl CH2CO), 4.16 (td, J ) 7.5 Hz, 1 H,
H8b), 4.04 (s, 2 H, CH2NH), 4.00-3.90 (m, 2 H, H4a, H6a′), 3.84
(m, 1 H, H4b), 3.78-3.68 (m, 8 H, H2a, H5a, H6a′′, H5b, H6b, OCH3),
3.55 (dt, J ) 9.5 Hz, 1 H, H3a), 3.41 (d, J ) 8.5 Hz, 1 H, H7b),
3.36 (dd, J ) 12.0 Hz, 1 H, H9b′), 3.00 (brt, J ) 12.0 Hz, 1 H,
H9b′′), 2.88 (dd, J ) 12.0 Hz, 1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1
H, H3bax). 13C NMR (CD3OD): δ 177.4, 174.6, 156.6, 153.3, 143.6,
141.4, 131.7, 131.5, 130.0, 128.8, 128.7, 128.0, 119.3, 115.4, 104.0,
102.0, 75.3, 74.7, 74.0, 72.7, 72.4, 69.9, 69.1, 68.4, 64.1, 62.6, 56.0,
53.8, 52.0, 51.2, 42.6. MALDI-TOF MS calcd for C37H47N2O15
(M + H)+, 759.30; found, 759.38. Anal. (C37H46N2O15 ·3H2O) C,
N. H: calcd, 6.45; found, 6.01.
p-Methoxyphenyl (9-(4′-carboxy-4-biphenyl)methylamino-3,5,9-
trideoxy-5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosyl-
onic acid)-(2f6)-ꢀ-D-galactopyranoside (9h). The yield was 75%;
25
1
Rf 0.60; [R]D -30.3 (c 10.4, H2O). H NMR (D2O with a few
drops of CD3OD): δ 8.07 (d, J ) 7.0 Hz, 2 H, Ar-H), 7.73 (m, 4
H, Ar-H), 7.58 (d, J ) 7.5 Hz, 2 H, Ar-H), 7.05 (d, J ) 9.0 Hz,
2 H, Ar-H), 6.81 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.28 (s, 2 H, glycolyl
CH2CO), 4.16 (td, 1 H, H8b), 4.06 (s, 2 H, CH2NH), 3.98-3.87
(m, 2 H, H4a, H6a′), 3.82 (m, 1 H, H4b), 3.80-3.64 (m, 8 H, H2a,
H5a, H6a′′, H5b, H6b, OCH3), 3.61 (dd, 1 H, H3a), 3.44-3.39 (m, 2
H, H7b, H9b′), 3.04 (dd, J ) 12.0 Hz, 1 H, H9b′′), 2.83 (dd, J ) 12.0
Hz, 1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR
(CD3OD): δ 174.7, 156.1, 152.8, 142.0, 131.7, 131.3, 128.8, 127.9,
119.2, 115.6, 74.9, 74.1, 72.0, 72.4, 56.3, 48.3. MALDI-TOF MS
calcd for C38H47N2O17 (M + H)+, 803.29; found, 803.34. Anal.
(C38H46N2O17 ·2H2O) C, H, N.
p-Methoxyphenyl (9-2(4-biphenyl)ethylamino-3,5, 9-trideoxy-
5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (9e). The yield was 70%; Rf 0.61;
25
1
[R]D -39.3 (c 7.5, H2O). H NMR (D2O with a few drops of
CD3OD): δ 7.58-7.55 (m, 4 H, Ar-H), 7.43-7.40 (m, 2 H,
Ar-H), 7.33-7.32 (m, 3 H, Ar-H), 7.04 (d, J ) 9.0 Hz, 2 H,
Ar-H), 6.80 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.70 (d, J ) 8.0 Hz, 1
H, H1a), 4.13 (td, J ) 3.0 Hz, 1 H, H8b), 4.05 (s, 2 H, glycolyl
CH2CO), 3.95 (dd, J ) 3.5 Hz, 1 H, H6a′), 3.92 (d, J ) 3.5 Hz, 1
p-Methoxyphenyl (9-(3′-carboxy-4-biphenyl)methylamino-3,5,9-
trideoxy-5-glycolamido-D-glycero-r-D-galacto-2-nonulopyranosyl-
onic acid)-(2f6)-ꢀ-D-galactopyranoside (9i). The yield was 70%;
26
1
Rf 0.60; [R]D -22.7 (c 11.0, H2O). H NMR (D2O with a few
drops of CD3OD): δ 8.19 (s, 1 H, Ar-H), 7.92 (d, J ) 7.6 Hz, 1
H, Ar-H), 7.72-7.71 (m, 3 H, Ar-H), 7.55-7.52 (m, 3 H, Ar-H),
7.08 (d, J ) 9.0, 2 H, Ar-H), 6.85 (d, J ) 9.0 Hz, 2 H, Ar-H),
4.27 (s, 2 H, glycolyl CH2CO), 4.18 (td, 1 H, H8b), 4.10 (s, 2 H,
CH2NH), 3.99-3.95 (m, 2 H, H4a, H6a′), 3.89-3.73 (m, 5 H, H2a,
H5a, H6a′′, H4b, H6b), 3.72-3.57 (m, 5 H, H5b, OCH3, H3a), 3.51 (d,
J ) 7.6 Hz, 1 H, H7b), 3.43 (br-d, J ) 12.8 Hz,, 1 H, H9b′), 3.09
(dd, J ) 10.0, 12.8 Hz, 1 H, H9b′′), 2.80 (br-d, J ) 12.4 Hz,1 H,
H, H4a), 3.85 (m, 1 H, H4b), 3.76-3.70 (m, 8 H, H2a, H5a, H6a′′
,
H5b, H6b, OCH3), 3.55 (dd, J ) 3.5, 10.0 Hz, 1 H, H3a), 3.44 (d, J
) 6.5, 1 H, H7b), 3.40 (dd, J ) 3.0, 12.5 Hz, 1 H, H9b′), 3.28-3.25
(m, 2 H, CH2CH2NH), 3.08 (brd, J ) 12.5 Hz, 1 H, H9b′′),
3.05-3.01 (m, 2 H, CH2CH2NH), 2.88 (dd, J ) 5.5, 12.0 Hz, 1 H,
H
3beq), 1.65 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ
177.4, 174.6, 156.6, 153.3, 142.0, 141.4, 136.9, 130.3, 129.9, 128.5,
128.4, 127.9, 119.3, 115.5, 104.0, 102.0, 75.3, 74.7, 74.0, 72.7,
72.4, 69.9, 69.1, 68.6, 64.1, 62.6, 56.0, 53.8, 52.1, 50.2, 42.6, 32.8.
MALDI-TOF MS calcd for C38H49N2O15 (M + H)+, 773.31; found,
773.39. Anal. (C38H48N2O15 ·3H2O) C, N. H: calcd, 6.58; found,
6.03.
H
3beq), 1.68 (t, J ) 12.4 Hz, 1 H, H3bax). 13C NMR (D2O): δ 176.4,
175.7, 174.0, 155.0, 151.5, 141.6, 140.0, 137.5, 131.0, 130.1, 130.0,
129.5, 128.8, 128.0, 127.9, 118.6, 115.4, 102.3, 101.1, 74.0, 73.0,
72.8, 71.1, 70.8, 68.8, 68.4, 67.8, 63.4, 61.5, 56.1, 52.1, 51.0, 49.7,
40.8. MALDI-TOF MS calcd for C38H47N2O17 (M + H)+, 803.29;
found, 803.36. Anal. (C38H46N2O17 ·5.5H2O) C, N. H: calcd, 6.37;
found, 5.95.
p-Methoxyphenyl (9-4(4-biphenyl)butylamino-3,5,9-trideoxy-5-
glycolamido-D-glycero-r-D-galacto-2-nonulopyranosylonic acid)-
(2f6)-ꢀ-D-galactopyranoside (9f). The yield was 70%; Rf 0.62;
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(3′-hydroxy-
4-biphenyl)-methylamino-D-glycero-r-D-galacto-2-nonulopyrano-
sylonic acid)-(2f6)-ꢀ-D-galactopyranoside (9j). The yield was 72%;
Rf 0.42; [R]D24 -54.4 (c 10.2, MeOH). 1H NMR (CD3OD): δ 7.61
(d, J ) 8.3 Hz, 2 H, Ar-H), 7.48 (d, J ) 8.3 Hz, 2 H, Ar-H),
7.25 (t, J ) 8.0 Hz, 1 H, Ar-H), 7.07-7.03 (m, 4 H, Ar-H),
6.79-6.77 (m, 3 H, Ar-H), 4.70 (d, J ) 8.0 Hz, 1 H, H1a), 4.20
(br. s, 2 H, glycolyl CH2CO), 4.14 (td, J ) 8.6 Hz, 1 H, H8b), 4.04
(s, 2 H, CH2NH), 3.95-3.91 (m, 2 H, H4a, H6a′), 3.83 (m, 1 H,
H4b), 3.74-3.71 (m, 5 H, H2a, H5a, H6a′′, H5b, H6b), 3.67 (s, 3 H,
OCH3), 3.55 (dd, J ) 3.4, 9.7 Hz, 1 H, H3a), 3.40 (dd, J ) 8.6, 1
H, H7b), 3.34 (dd, 1 H, H9b′), 2.97 (dd, J ) 12.6 Hz, 1 H, H9b′′),
2.87 (dd, J ) 4.6, 12.0 Hz,1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1 H,
25
1
[R]D -41.0 (c 5.0, H2O). H NMR (D2O with a few drops of
CD3OD): δ 7.58-7.55 (m, 4 H, Ar-H), 7.42-7.39 (m, 2 H,
Ar-H), 7.31-7.25 (m, 3 H, Ar-H), 7.04 (d, J ) 9.0 Hz, 2 H,
Ar-H), 6.81 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.70 (d, J ) 8.0 Hz, 1
H, H1a), 4.09 (td, 1 H, H8b), 4.05 (s, 2 H, glycolyl CH2CO),
3.93-3.90 (m, 2 H, H4a, H6a′), 3.84 (brs, 1 H, H4b), 3.77-3.65 (m,
8 H, H2a, H5a, H6a′′, H5b, H6b, OCH3), 3.54 (dd, J ) 3.5, 9.5 Hz, 1
H, H3a), 3.41 (d, J ) 8.5, 1 H, H7b), 3.31-3.30 (m, 1 H, H9b′),
3.00-3.96 (m, 3 H, H9b′′, CH2CH2CH2CH2NH), 2.88 (dd, J ) 5.5,
12.0 Hz, 1 H, H3beq), 2.67 (m, 2 H, CH2CH2CH2CH2NH), 1.68 (m,
5 H, CH2CH2CH2CH2NH, H3bax). 13C NMR (CD3OD): δ 177.4,
156.5, 153.3, 142.3, 142.0, 140.2, 130.0, 129.8, 128.1, 128.0, 127.8,
119.2, 115.5, 103.8, 102.0, 75.3, 74.7, 74.0, 72.6, 72.4, 69.9, 69.1,
68.5, 64.1, 62.6, 56.0, 53.8, 52.0, 50.2, 42.6, 35.8, 29.3, 26.6.
MALDI-TOF MS calcd for C40H53N2O15 (M + H)+, 801.34; found,
801.43. Anal. (C41H62N2O18 ·3H2O) C, H, N.
H
3bax). 13C NMR (CD3OD): δ 175.2, 153.2, 142.8, 131.5, 131.02,
128.6, 119.3, 115.7, 115.4, 75.2, 72.7, 72.4, 68.5, 56.0, 52.0, 42.6.
MALDI-TOF MS calcd for C37H47N2O16 (M + H)+, 775.29; found,
775.27. Anal. (C37H46N2O16 ·H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4′-hydroxy-
4-biphenyl)-methylamino-D-glycero-r-D-galacto-2-nonulopyrano-
sylonic acid)-(2f6)-ꢀ-D-galactopyranoside (9g). The yield was 75%;
Rf 0.40; [R]D -41.0 (c 10.0, H2O). H NMR (D2O with a few
drops of CD3OD): δ 7.58 (d, J ) 8.0 Hz, 2 H, Ar-H), 7.45 (m, 4
H, Ar-H), 7.04 (d, J ) 7.0 Hz, 2 H, Ar-H), 6.86 (d, J ) 9 Hz,
2 H, Ar-H), 6.78 (d, J ) 9.0 Hz, 2 H, Ar-H), 4.70 (d, J ) 8.0
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(2′-hydroxy-
4-biphenyl)-methylamino-D-glycero-r-D-galacto-2-nonulopyrano-
sylonic acid)-(2f6)-ꢀ-D-galactopyranoside (9k). The yield was 70%;
Rf 0.40; [R]D24 -42.4 (c 10.6, MeOH). 1H NMR (CD3OD): δ 7.60
(d, J ) 8.0 Hz, 2 H, Ar-H), 7.44 (d, J ) 8.0 Hz, 2 H, Ar-H),
7.24 (dd, J ) 8.0 Hz, 1 H, Ar-H), 7.17-7.14 (m, 1 H, Ar-H),
7.05-7.03 (m, 2 H, Ar-H), 6.90-6.88 (m, 2 H, Ar-H), 6.87-6.78
31
1

Series of Sialosides as CD22-Specific Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6679
(m, 2 H, Ar-H), 4.70 (d, J ) 8.0 Hz, 1 H, H1a), 4.22 (br. s, 2 H,
glycolyl CH2CO), 4.15 (td, J ) 9.7, 8.6, 6.3 Hz, 1 H, H8b), 4.04 (s,
2 H, CH2NH), 3.97-3.91 (m, 2 H, H4a, H6a′), 3.84 (m, 1 H, H4b),
3.74-3.70 (m, 5 H, H2a, H5a, H6a′′, H5b, H6b), 3.67 (s, 3 H, OCH3),
3.54 (dd, J ) 3.4, 9.7 Hz, 1 H, H3a), 3.40 (dd, J ) 8.6, 1 H, H7b),
3.35 (br. d, 1 H, H9b′), 2.99 (dd, J ) 9.7, 12.6 Hz, 1 H, H9b′′), 2.88
(dd, J ) 4.6, 12.0 Hz,1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1 H, H3bax).
13C NMR (CD3OD): δ 177.4, 156.6, 155.5, 153.2, 141.6, 131.6,
131.1, 130.7, 130.6, 129.9, 128.8, 121.0, 119.3, 117.0, 115.5, 103.9,
75.2, 74.7, 74.0, 72.7, 72.4, 69.9, 69.1, 68.5, 64.1, 62.6, 56.0, 53.7,
52.1, 51.2, 42.6. MALDI-TOF MS calcd for C37H47N2O16 (M +
H)+, 775.29; found, 775.23. Anal. (C37H46N2O16 ·2H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4′-methoxy-
4-biphenyl)-methylamino-D-glycero-r-D-galacto-2-nonulopyrano-
sylonic acid)-(2f6)-ꢀ-D-galactopyranoside (9l). The yield was 75%;
transfectants (J558LST6), which express R2-6 linked sialic acids,
were cultured as previously described.42 The chimeric protein
containing the N-terminal three domains of murine or human CD22
and the Fc fragment of human IgG1 (mCD22-Fc and hCD22-Fc,
respectively) were prepared and biotinylated as reported.43 Cells
were incubated with 1 µg biotinylated mCD22-Fc or hCD22-Fc
alone or together with synthetic sialosides at 1.00, 0.10, 0.01, 0.001,
0.0001, and 0.00001 mM. After washing, the cells were stained
with 1% Alexa Fluor 647-streptavidin conjugate (Molecular Probes,
Eugene, Oregon) for 30 min on ice and then analyzed by flow
cytometry using a FACS Calibur (BectonsDickinson, San Jose,
CA). The percentage of binding was calculated according to the
following equation: % binding ) (A - B)/(C - B) × 100%, where
A is the mean of fluorescent intensity (MFI) of J558LST6 cells in
the presence of the synthetic sialoside (sample), B is MFI of J558L
cells (negative control), and C is MFI of J558LST6 cells (positive
control). Obviously, the lower the binding rates, the higher the
inhibitory affinity of the synthetic sialosides.
Molecular Modeling. Model construction and antagonist docking
were carried out on an Intel P4 based Microsoft Windows 2000
workstation using Discovery Studio Modeling 1.5 and 2.0 packages
(Accelrys, Inc., San Diego, CA). The X-ray structure of the Sn in
complex with a substrate-analogue inhibitor BPC-Neu5Ac (coded
1oda) obtained from the Protein Data Bank was used for modeling
analysis. The sequence comparisons between Sn and the equivalents
regions of hCD22 and mCD22 have been described in detail
elsewhere.15 Homology models of hCD22 and mCD22 in complex
with BPC-Neu5Ac were constructed using the MODELER program,
based upon the individual sequence/template alignments. The
obtained complexes were then subjected to geometry optimization
using the following parameters: CHARMm force field, conjugate
gradient minimization, distance-dependent dielectric constant 4, 10
Å nonbonded cutoff, and 2000 maximum iterations. Fitness of the
models sequence in their current 3D environment was evaluated
by Profiles-3D, and the stereochemical quality of the final structure
was analyzed using the program PROCHECK.
22
Rf 0.50; [R]D -101.8 (c 10.9, MeOH). 1H NMR (CD3OD): δ
7.60 (d, J ) 8.0 Hz, 2 H, Ar-H), 7.54 (d, J ) 1.7, 6.8 Hz, 2 H,
Ar-H), 7.05-6.9 (m, 4 H, Ar-H), 6.8 (dd, J ) 1.7, 6.8 Hz, 2 H,
Ar-H), 4.70 (d, J ) 8.0 Hz, 1 H, H1a), 4.20 (br. s, 2 H, glycolyl
CH2CO), 4.17-4.12 (m, Hz, 1 H, H8b), 4.04 (s, 2 H, CH2NH),
3.97-3.91 (m, 2 H, H4a, H6a′), 3.85-3.78 (m, 4 H, H4b, OCH3),
3.74-3.69 (m, 5 H, H2a, H5a, H6a′′, H5b, H6b), 3.67 (s, 3 H, OCH3),
3.54 (dd, J ) 3.4, 9.7 Hz, 1 H, H3a), 3.40 (dd, J ) 8.6, 1 H, H7b),
3.35 (br. d, 1 H, H9b′), 2.99 (dd, J ) 9.7, 12.6 Hz, 1 H, H9b′′), 2.88
(dd, J ) 4.6, 12.0 Hz,1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1 H, H3bax).
13C NMR (CD3OD): δ 177.4, 174.7, 161.1, 156.5, 153.3, 143.2,
133.7, 131.6, 130.6, 129.1, 128.1, 119.3, 115.4, 103.9, 102.0, 75.2,
74.6, 74.0, 72.6, 72.4, 69.9, 69.1, 68.4, 64.1, 62.5, 56.0, 55.7, 53.7,
52.0, 51.1, 42.6. MALDI-TOF MS calcd for C38H49N2O16 (M +
H)+, 789.30; found, 789.32. Anal. (C38H48N2O16 ·H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-5-glycolamido-9-(4′-methyl-4-
biphenyl)-methylamino-D-glycero-r-D-galacto-2-nonulopyranosy-
lonic acid)-(2f6)-ꢀ-D-galactopyranoside (9m). The yield was 69%;
Rf 0.58; [R]D22 -44.6 (c 10.3, MeOH). 1H NMR (CD3OD): δ 7.60
(d, J ) 8.0 Hz, 2 H, Ar-H), 7.47 (d, J ) 8.0 Hz, 2 H, Ar-H),
7.22 (m, J ) 8.6 Hz, 2 H, Ar-H), 7.03 (d, J ) 9.2 Hz, 2 H, Ar-H),
6.77 (d, J ) 9.2 Hz, 2 H, Ar-H), 4.69 (d, J ) 8.0 Hz, 1 H, H1a),
4.22-4.17 (m, 3 H, glycolyl CH2CO, H8b), 4.05 (s, 2 H, CH2NH),
3.96-3.91 (m, 2 H, H4a, H6a′), 3.85-3.78 (m, 1 H, H4b), 3.75-3.72
(m, 5 H, H2a, H5a, H6a′′, H5b, H6b), 3.65 (s, 3 H, OCH3), 3.55 (dd,
J ) 3.4, 9.7 Hz, 1 H, H3a), 3.42 (dd, J ) 8.2, 1 H, H7b), 3.34 (br.
d, 1 H, H9b′), 2.99 (t, J ) 12.6 Hz, 1 H, H9b′′), 2.89 (dd, J ) 12.0
Hz,1 H, H3beq), 1.64 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR
(CD3OD): δ 177.4, 174.7, 156.5, 153.3, 143.3, 138.8, 138.4, 131.6,
131.1, 130.6, 128.4, 127.8, 119.2, 115.5, 103.9, 102.0, 75.2, 74.6,
73.9, 72.6, 72.4, 69.9, 69.0, 68.5, 64.1, 62.6, 56.0, 51.2, 42.6.
MALDI-TOF MS calcd for C38H49N2O16 (M + H)+, 773.31; found,
773.32. Anal. (C38H48N2O15 ·0.5H2O) C, H, N.
p-Methoxyphenyl (3,5,9-trideoxy-9-(4′-trifluromethyl-4-biphe-
nyl)methylamino-5-glycolamido-D-glycero-r-D-galacto-2-nonulopy-
ranosylonic acid)-(2f6)-ꢀ-D-galactopyranoside (9n). The yield was
70%; Rf 0.52; [R]D22 -25.7 (c 9.7, MeOH). 1H NMR (CD3OD): δ
7.78 (d, J ) 8.6 Hz, 2 H, Ar-H), 7.72 (d, J ) 8.02 Hz, 2 H,
Ar-H), 7.62 (d, J ) 8.6 Hz, 2 H, Ar-H), 7.43 (d, J ) 8.02 Hz,
2 H, Ar-H), 7.03 (d, J ) 2.3, 6.3 Hz, 2 H, Ar-H), 6.78 (d, J )
2.3, 6.3 Hz, 2 H, Ar-H), 4.71 (d, J ) 8.0 Hz, 1 H, H1a), 4.08-4.04
(m, 3 H, glycolyl CH2CO, H8b), 3.96-3.94 (m, 2 H, H4a, H6a′),
3.89-3.72 (m, 8 H, CH2NH, H2a, H5a, H6a′′, H4b, H5b, H6b), 3.71 (s,
3 H, OCH3), 3.58 (dd, J ) 3.4, 9.7 Hz, 1 H, H3a), 3.35 (dd, J )
8.2, 1 H, H7b), 2.99 (dd, J ) 8.6, 12.0 Hz, 1 H, H9b′), 2.85 (dd, J
) 4.6, 12.0 Hz,1 H, H3beq), 2.99 (t, J ) 8.6, 12.0 Hz, 1 H, H9b′′),
1.63 (t, J ) 12.0 Hz, 1 H, H3bax). 13C NMR (CD3OD): δ 177.2,
174.6, 156.5, 153.3, 145.9, 140.4, 139.7, 138.4, 130.4, 128.5, 128.3,
126.7, 119.2, 115.4, 103.9, 102.0, 75.07, 74.6, 72.9, 72.7, 72.4,
70.5, 69.6, 69.3, 63.6, 62.6, 56.0, 53.7, 52.9, 51.2, 42.6. MALDI-
TOF MS calcd for C38H46F3N2O15 (M + H)+, 827.28; found,
827.32. Anal. (C38H45F3N2O15 ·2.5H2O) C, H, N.
For the manual docking of antagonists (8m, 9h, 9i, 10), their
structures were built using BPC-Neu5Ac in the model complexes
as a template. The individual antagonists were superimposed onto
the BPC-Neu5Ac in the models, confirming that interactions which
are essential for competitive binding are maintained throughout the
docking process and a special emphasis was given to core carboxylic
acid group’s salt-bridge formation with Arginine residue (hCD22
Arg98, mCD22 Arg100). The obtained complexes were then
energetically minimized with 500-1000 iterations of “in situ ligand
minimization algorithm” using the Smart Minimizer program. The
antagonist binding modes were analyzed to investigate the relative
differences between interactions to understand the selectivity
differences.
Acknowledgment. H.H.M.A. thanks the Egyptian govern-
ment for a Ph.D. scholarship. The first author thanks Masanori
Yamaguchi, Akihiro Imamura, and Hirano Takaku for valuable
discussions. This work was partly supported by the ministry of
Education, Culture, Sports, Science, and Technology (MEXT)
of Japan (Grant-in-Aid for Scientific Research to Prof M. Kiso,
no. 17101007) and CREST of JST (Japan Science and Technol-
ogy Corporation). We thank Prof. Mitsuhiro Yoshimatsu
(Department of Chemistry, Faculty of Education, Gifu Univer-
sity) for determination of elemental analyses.
Supporting Information Available: 1H NMR spectra, combus-
tion analysis data of the target compounds, and molecular modeling
coordinates. This material is available free of charge via the Internet
at http://pubs.acs.org.
Inhibition Assay. Materials and Methods. The binding assay was
performed as previously described with slight modifications.17
Mouse myeloma line J558L,41 which do not express R2-6 linked
sialic acid due to lack of ST6GalI expression, and its ST6GalI
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