Synthesis of sialic acid derivatives as ligands for the myelin-associated glycoprotein (MAG)

Article abstract of DOI:10.1016/j.bmc.2007.04.038

The trisaccharide substructure 13 of the ganglioside GQ1bα shows a remarkable affinity for the myelin-associated glycoprotein (MAG). In the search for structurally simplified and pharmacokinetically improved mimics of 13, sialosides with modifications at

Full text of DOI:10.1016/j.bmc.2007.04.038

Bioorganic & Medicinal Chemistry 15 (2007) 4951–4965
Synthesis of sialic acid derivatives as ligands for the
myelin-associated glycoprotein (MAG)
Sachin V. Shelke,a Gan-Pan Gao,a Stefanie Mesch,a Heiko Ga¨thje,b Soerge Kelm,b
Oliver Schwardta and Beat Ernsta,*
aInstitute of Molecular Pharmacy, Pharmacenter, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland
bDepartment of Physiological Biochemistry, University of Bremen, 28334 Bremen, Germany
Received 7 November 2006; revised 11 April 2007; accepted 20 April 2007
Available online 25 April 2007
Abstract—The trisaccharide substructure 13 of the ganglioside GQ1ba shows a remarkable affinity for the myelin-associated glyco-
protein (MAG). In the search for structurally simplified and pharmacokinetically improved mimics of 13, sialosides with modifica-
tions at the reducing and non-reducing end were synthesized. The biological evaluation of mimics 12a–o was performed in a
competitive target-based assay. It was found that the relative inhibitory potency (rIP) of antagonist 12h was enhanced by more than
1000-fold in comparison to the reference trisaccharide 13, despite the former having a much simpler structure. In addition, the sialic
acid derivatives, for example, 12h, have clearly improved pharmacokinetic properties due to the presence of aromatic moieties, a
lower molecular weight, and a reduced number of polar hydroxy functions compared to the reference compound 13.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
MAG has been identified as a sialic acid-binding immu-
noglobulin-like lectin (Siglec-4). Its role11 as one of sev-
eral myelin components inhibiting axonal regrowth after
injury has drawn a lot of attention.12 Although the
mechanism is still unclear, it is believed that blocking
this inhibitory activity of MAG could support the regen-
eration after injury to the CNS. Schnaar13 reported that
GQ1ba, GD1a, and GT1b, known to be expressed on
myelinated neurons in vivo, are functional ligands of
MAG. These gangliosides have been synthesized in pre-
parative amounts,14 and were therefore used to establish
a structure affinity relationship (SAR). Thereafter, the
SAR profile was refined by numerous synthetic contri-
butions based on neuraminic acid derivatives and gan-
glioside fragments.15 The recently reported ability to
reverse MAG inhibition with monovalent glycosides16
encourages further exploration of glycans and glycan
mimics as inhibitors of MAG-mediated axonal out-
growth inhibition.
The adult mammalian central nervous system (CNS)
inherently lacks the capacity for regeneration.1 How-
ever, it could be shown that neurite outgrowth is princi-
pally possible,2 but actively inhibited by inhibitor
proteins expressed by myelinating glia cells, oligoden-
drocytes, and Schwann cells.3 Three major inhibitor
proteins have been identified so far: Nogo A,4 oligoden-
drocyte myelin glycoprotein (OMpg),5 and myelin-asso-
ciated glycoprotein (MAG).6 All three inhibitor proteins
bind to the Nogo receptor (NgR)7 located on the neuron
surface. The NgR then forms a complex with the co-
receptor p75NTR to transduce the inhibitory signal to
the cytosol of the neuron.8 There, it activates the
RhoA-ROCK9 cascade, which finally leads to growth
cone collapse. This same cascade is probably also trig-
gered by MAG binding to gangliosides. Again, it is be-
lieved that the interaction with the coreceptor p75NTR
10
initiates the inhibitory cascade.
The binding properties of all siglecs studied so far rely
on their interactions with sialylated glycans, strongly
suggesting that these Sia-dependent interactions are also
important for their biological function in vivo. Several
potential sialylated binding partners for MAG have
been identified,15 however, the full biological role of its
sialic acid-binding activity has remained unclear. A
Keywords: Sialic acid derivatives; Myelin-associated glycoprotein
(MAG); Topliss operational scheme.
*
Corresponding author. Tel.: +41 267 15 51; fax: +41 267 15 52;
e-mail: beat.ernst@unibas.ch
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.04.038

4952
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
potent and selective inhibitor with drug-like properties
would provide a tool required to further investigate
both, the role of sialic acid-dependent interactions of
MAG and the therapeutic potential of MAG
antagonists.
In order to obtain MAG antagonists with further en-
hanced affinity, we combined the various modifications
reported to date in one molecule (!12a, Scheme 1). This
new lead structure was then the starting point for a lead
optimization program.
In this report, we describe the synthesis and biological
evaluation of the sialic acid derivative 12a obtained by
combining the reported beneficial modifications in the
2- and 9-position of one molecule.15,17 This new lead
12a was then further optimized applying a Topliss
approach.18
Starting from neuraminic acid (1), the sialyl donor 3 was
synthesized according to a reported procedure.20 The
reaction of 3 with benzyl alcohol in the presence of
NIS/TfOH yielded an anomeric mixture of the benzyl
sialosides 4a (41%) and 4b (18%), which were chromato-
graphically separated.21 Subsequent saponification of 4a
and 4b gave the test compounds 5a and 5b (Table 1, en-
tries b and c).
2. Results and discussion
For the extension of the non-reducing end of 4a, the ace-
´
Kelm et al. reported that Neu5AcaBn has a 10-fold
higher affinity for MAG than the corresponding methyl
sialoside.15a In addition, substitution of the hydroxy
group in the 9-position of sialic acid by an amino group
also enhanced binding, while substitution by halogens
abolished binding, suggesting the important role of a
hydrogen donor at this position.15a Finally, acylation
of the amino group in the 9-position further increased
the affinity for MAG significantly.15f,19
tate protection was removed under Zemplen conditions
(!6), the 9-OH selectively tosylated (!7),22 and then
transformed into the corresponding azide 8 using so-
dium azide and 18-crown-6 in DMF.23 Subsequent
saponification afforded the test compound 9 (Table 1,
entry d). Alternatively, acetylation of 8 using acetic
anhydride and pyridine gave compound 10, which was
used for amidation under modified Staudinger condi-
tions24 with a wide range of aromatic and aliphatic acyl
OAc
OAc
OAc
OAc
OH
OH
CO2Me
SMe
CO2Me
OAc
AcO
CO2H
OH
AcO
HO
ii
i
O
O
O
AcHN
AcHN
AcHN
[20]
[20]
AcO
AcO
HO
2
1
3
OR1
OH
OH
CO2R2
R1O
OR1
CO2Me
O
RO
iii
v
O
1
O
O
AcHN
AcHN
R1O
HO
2
4α/β (R = Ac, R = Me)
6 (R = H)
7 (R = Ts)
iv
vi
1
2
5α/β (R = H, R = Na)
OR1
OAc
OAc
CO2R2
OR1
H
N
R
N3
CO2Me
vii
ix
O
O
O
O
O
AcHN
AcHN
R1O
1
AcO
2
11a-o
8 (R = H, R = Me)
iv
1
2
viii
9 (R = H, R = Na)
1
2
10 (R = Ac, R = Me)
OH
H
N
R
CO2Na
OH
iv
O
O
O
AcHN
HO
12a-o
˚
Scheme 1. Reagents: (i) a—MeOH, Amberlyst 15, b—Ac2O, DMAP, pyr, 81%; (ii) TMSSMe, TMSOTf, MS 3 A, DCE 82%; (iii) PhCH2OH, NIS/
TfOH, MeCN, 41% a, 18% b; (iv) a—10% NaOH, MeOH, b—Dowex 50 · 8 (Na+), 70–95%; (v) NaOMe, MeOH, 2 h, 82%; (vi) pTsCl, pyr, 56%;
(vii) NaN3, DMF, 18-C-6, 75%; (viii) Ac2O, DMAP, pyr, 73%; (ix) RCOCl, PPh3, DCM, 54–70%.

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
Table 1. Relative inhibitory potencies (rIPs) of sialosides
4953
OH
OH
2
CO2Na
R
1
O
R
AcHN
HO
Entry
Compound
R1
R2
rIP
a
b
c
13
5a
5b
9
Gal-b(1–3)-GalNAc-OSE
HO–
HO–
HO–
N3–
1
aOBn
bOBn
aOBn
0.62
n.a.
0.05
d
H
N
e
f
12a
12b
12c
aOBn
aOBn
aOBn
690
191
75
O
H
N
O
H
N
g
O
O
HN
O
h
12d
aOBn
6.7
H
N
N
H
O
H
H C
3
N
i
j
12e
12f
aOBn
aOBn
8.6
12
O
H
N
H C
3
O
H
H C
3
N
k
l
12g
16
aOBn
aOBn
12
O
2.8
O
Cl
H
m
n
12h
12i
aOBn
aOBn
1074
N
Cl
O
Cl
690
H
N
O
F3C
H
N
o
p
12j
aOBn
aOBn
207
35
O
Cl
Cl
H
N
12k
O
(continued on next page)

4954
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
Table 1 (continued)
Entry
Compound
R1
R2
rIP
414
O2N
H
q
r
s
t
12l
aOBn
N
O
MeO
H
12m
12n
12o
aOBn
aOBn
aOBn
290
806
483
N
O
Me
H
N
O
Cl
H
N
O
The rIP of each sialoside was calculated by dividing the IC50 of the reference compound 13 by the IC50 of the compound of interest. This results in
rIPs above 1.0 for derivatives binding better than 13 and rIPs below 1.0 for compounds with a lower affinity than 13; n.a., not applicable, less than
50% inhibition at the highest concentration tested (20 mM).
chlorides. 9-amido-substituted sialosides 11a–g were ob-
tained in 54–70% yield. Finally, deprotection yielded the
amides 12a–g ready for biological investigation in a hap-
ten inhibition assay (Table 1, entries e–k).
ganglioside analogue a(2–3)/a(2–6)disialyllactotetraosyl
2-(trimethylsilyl)ethyl.28 Only in the case of the a-ano-
mer the vital salt bridge between the carboxylate of
the ligand and the guanidinium group of Arg97 can be
formed.
For the inhibition assays, a recombinant protein consist-
ing of the three N-terminal domains of MAG and the Fc
part of human IgG (Fc-MAGd1–3) was produced by
expression in CHO cells and affinity purification on pro-
tein A-agarose.25 The relative inhibitory potencies (rIP)
of the benzyl sialosides 5a, 5b, 9, and 12a–g were deter-
Similar to the finding of Kelm et al. with methyl a-sialo-
sides,15f,19 a benzamide in the 9-position of benzyl a-
sialoside (12a) improves binding affinity by almost three
orders of magnitude as well. The sterically more
demanding biphenyls 12b and 12c (entries 6 and 7) led
to decreased activity. However, aromatic amides (12a–
c) generally turned out to be significantly more active
than their aliphatic counterparts (12e–g). Finally, a
derivative with a heteroaromatic substituent in the 9-po-
sition also exhibited reduced affinity (12d).
mined in microtiter plates coated with covalently at-
26
tached sialic acids as binding target for Fc-MAGd1–3
.
By complexing the Fc-part with alkaline phosphatase-
labeled anti-Fc antibodies and measuring the initial
velocity of fluorescein release from fluorescein diphos-
phate, the amount of bound MAG could be determined.
At least three independent titrations were performed for
each compound. To ensure comparability with earlier
reported results,15 the affinities were measured relative
to the reference compound 13 (Table 1, entry a),27 which
has an IC50 of 265 lM and rIP of 1. Compound 13 was
selected as the reference compound because it represents
the minimal carbohydrate epitope required for binding
to MAG as identified by Schnaar et al. in their studies
on gangliosides.13 High rIPs represent compounds with
high affinity, whereas low rIPs indicate low affinity
compounds.
Unlike the other test compounds in Table 1, the 9-azido
derivative 10 is lacking the hydrogen donor properties at
the non-reducing end. This might be the reason for the
20-fold reduction in affinity compared to the reference
compound 13, similar to the reduced affinity reported
for 9-halogeno derivatives.15a In order to confirm the
importance of the hydrogen bond formation initiated
by the hydroxy group in the 9-position, the correspond-
ing benzyl ether 16 was synthesized (Scheme 2). In con-
trast to the benzamide, the benzyl ether has the
advantage of higher flexibility facilitating the formation
of possible hydrophobic interactions. On the other
hand, it is missing the hydrogen bond donor potential.
Starting from 6, the 8,9-diol was regioselectively pro-
tected as benzylidene acetal to afford 14 as a diastereo-
meric mixture29 which was then regioselectively
transformed into the 9-O-Bn derivative 15 using
BH3ÆNMe3, AlCl3 and traces of H2O in THF. After
hydrolysis with LiOH in dioxane-H2O and ion exchange
chromatography the sodium salt 16 was obtained in a
good overall yield.
As reported earlier,15a only a-sialosides fulfill the geo-
metrical requirements for binding to MAG. It was
found that the a-sialoside 5a showed an affinity slightly
below the one of the trisaccharide 13, which was used as
a reference compound. For the corresponding b-ano-
mer, no activity could be observed at concentrations
up to 20 mM. These results correspond entirely with
the binding mode deduced from the crystal structure
of siglec-7 in complex with a sialylated ligand, the

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4955
Ph
OH
OH
OH
O
O
HO
BnO
BnO
CO2Me
OBn
CO2Me
OBn
CO2Na
O
OBn
CO2Me
OH
OH
OH
OH
ii
i
iii
O
O
O
OBn
AcHN
AcHN
AcHN
AcHN
HO
HO
HO
HO
16
6
14
15
Scheme 2. Reagents and conditions: (i) a,a-Dimethoxytoluene, pTsOHÆH2O, CH3CN, rt, 1.5 h; (ii) BH3ÆNMe3, AlCl3, THF, cat. H2O, rt, 4 h
(6 ! 15, 70%); (iii) a—LiOH, H2O-dioxane, rt, 1.5 h, b—Dowex 50 · 8 (Na+), 87%.
The replacement of the benzamide in 12a by a benzyl ether
(16) led to a reduction in the affinity by more than two or-
ders of magnitude (Table 1, entry l). The nonexistence of a
hydrogen bridge donor at the 9-position in 16 might there-
fore be the reason for the dramatic drop in activity.
2,4-Substitution (12k) was found to be detrimental to
the affinity. Steric limitations within the binding site or
a change in the torsional angle between the phenyl
group and the carbonyl carbon may explain this result.
For substituents in the 3,4-position (12i), the electronic
and steric effects seem to compensate each other. Final-
ly, 4-substitution with CF3, having a strongly increased
p- and r-effect also led to a decrease in affinity.
A very common problem in drug design is to find the
optimal substitution on an aromatic ring in an active
lead compound (e.g. 12a) in order to maximize its po-
tency. Since there are many possible substitutions and
several different ring positions, the number of potential
compounds to be considered is very large. With the
development of the Hansch method for treatment of
structure activity correlations,30 a more rational ap-
proach to this problem became available. Thus, a lim-
ited group of substituents, which will give good
discrimination between p (hydrophobic effects), r (elec-
tronic effects), and ES (steric effects) can be selected.
Compared to the trisaccharide 13, which was used as
reference compound, the new mono-sialoside 12h shows
an improvement of affinity toward MAG of more than a
factor 1000. In addition, it is supposed to have improved
pharmacokinetic properties because the number of
hydrogen donors and acceptors is dramatically reduced
compared to the reference compound, the molecular
weight is close to 500, and the Clog P (1.47) is in the
range of oral availability.31 A further important issue
to be addressed is the metabolic stability of the pre-
sented sialosides. In general, the substrate specificity of
sialidases from mammalian tissues is determined by
the type of bond at the terminal sialic acid residue (2–
3, 2–6 or 2–8) and does not depend on the structure of
the glycoconjugate chain.32 Therefore, it cannot be ex-
cluded that the presented mimics are metabolically
cleaved by sialidases.
The Topliss operational scheme is a manual, non-math-
ematical application of the Hansch analysis to drug de-
sign, developed to guide towards the most active
analogue of a lead compound with the least synthetic
investment.18 The Topliss scheme starts with the unsub-
stituted benzamide 12a (Scheme 1). The first derivative
to be synthesized is the p-Cl-benzamide 12h. It turned
out to have a higher affinity than the parent compound
12a (rIP: 690 vs 1074), which most probably can be
attributed to a +p-effect, a +r-effect or to a combination
of both. m,p-Dichloro-benzamide 12i was synthesized
next, because of the increase in both p- and r-values
when summed for the two substituents. Since the po-
tency of 12i was lower than that of the precursor 12h
(rIP: 1074 vs 690), 12j–l were the next synthetic targets.
All three compounds showed decreased affinities, which
might be ascribed to either an unfavorable steric effect of
2,4-substitution in 12k or to the exceedance of the opti-
mal lipophilicity or the electron withdrawing capacity of
the substituents. To cross check, the derivatives 12m,
12n, and 12o were also synthesized. As expected they
turned out to have lower affinity than the best com-
pound identified so far, the p-chloro-benzamide 12h.
4. Experimental
4.1. Chemistry
NMR spectra were recorded on a Bruker Avance DMX-
C
500 (500 MHz) spectrometer. Assignment of 1H and 13
NMR spectra was achieved using 2D methods (COSY,
HSQC, TOCSY). Chemical shifts are expressed in
ppm using residual CHCl3, CHD2OD, and HDO as ref-
erences. Optical rotations were measured using Perkin-
Elmer Polarimeters 241 and 341. ESI-MS analyses were
carried out using a Waters Micromass ZQ Detector sys-
tem. The spectra were recorded in positive ESI mode.
HR-MS were measured on a Bruker micrOTOF detec-
tor in positive mode. Reactions were monitored by
TLC using glass plates coated with silica gel 60 F254
(Merck) and visualized by using UV light and/or by
charring with a molybdate solution (a 0.02 M solution
of ammonium cerium sulfate dihydrate and ammonium
molybdate tetrahydrate in aqueous 10% H2SO4). Col-
umn chromatography was performed on silica gel (Flu-
ka, 40–60 mesh). Methanol (MeOH) was dried by
refluxing with sodium methoxide and distilled immedi-
ately before use. Pyridine was freshly distilled under
3. Conclusions
Although the Topliss approach can be highly useful in
lead optimization, it did not furnish a significantly more
potent compound in the presented case. The increase in
potency from 12a (rIP 690) with an unsubstituted phenyl
group to the most potent compound 12h (rIP 1074) is
less than one order of magnitude in difference.

4956
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
argon over CaH2. Dichloromethane (DCM), dichloro-
ethane (DCE), acetonitrile (MeCN), toluene, and ben-
zene were dried by filtration over Al2O3 (Fluka, type
5016 A basic). Molecular sieves (3 A) were activated in
vacuo at 500 ꢁC for 2 h immediately before use.
under argon at 50 ꢁC for 5 h and then for 16 h at rt After
dilution with DCM (25 mL) the reaction mixture was
subsequently washed with satd aqueous NaHCO3
(100 mL) and H2O (100 mL), dried (Na2SO4), filtered,
and concentrated. Purification by column chromatogra-
phy on silica gel (DCM/MeOH 40:1) yielded 3 (5.75 g,
82%) as a colorless foam, which was directly used in
the next step.
˚
4.2. General procedure A for the synthesis of 9-substituted
amides 11a–o
To a solution of 10 (1 equiv) and acid chloride (2–
4 equiv) in toluene, DCM or DCE was added a solution
of triphenyl phosphine (1.2–2.2 equiv) in DCM, DCE or
toluene. After stirring for 1–3 h at room temperature,
the solvent was evaporated. Purification by column
chromatography on silica gel (DCM/MeOH 100:1 to
20:1) yielded 11a–11o as solids.
4.6. Methyl (benzyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-D-glycero-a- and b-D-galacto-2-nonulopyr-
anosid)onate (4a and 4b)
A suspension of 3 (5.20 g, 10.0 mmol), benzyl alcohol
˚
(1.62 g, 15.0 mmol), and molecular sieves (3 A, 5.0 g)
in MeCN (100 mL) was cooled to ꢀ40 ꢁC under argon.
NIS (2.68 g, 12.0 mmol) and TfOH (600 mg, 4.00 mmol)
were added successively at ꢀ40 ꢁC. After stirring for
30 min, the reaction mixture was warmed to ꢀ30 ꢁC
and stirring was continued for additional 14 h. The mix-
ture was diluted with DCM and filtered through a pad
of Celite. After washing with 20% aqueous Na2S2O3
(200 mL) and satd aqueous NaHCO3 (200 mL), the or-
ganic layer was dried (Na2SO4), filtered, and concen-
4.3. General method B for deprotection
To a solution of the protected compound (25–50 mg) in
MeOH (2 mL) was added 10% aq NaOH (0.2 mL). The
mixture was stirred at room temperature for 3 h. The
solution was concentrated and the residue was purified
by reversed phase chromatography (RP-18 column, 5%
gradient MeOH in water), Dowex 50 · 8 (Na+ type)
ion-exchange chromatography, and Sephadex G-15 size
exclusion chromatography to afford the target molecule
as colorless solid after a final lyophilization from water.
trated to give
a syrup. Purification by column
chromatography on silica gel (petrol ether/DCM/i-PrOH
8:4:1) afforded 4a (2.40 g, 41%) and 4b (1.04 g, 18%).
1
Compound 4a: H NMR (500 MHz, CDCl3): d 1.90 (s,
4.4. Methyl (5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-
dideoxy-D-glycero-D-galacto-2-nonulopyranosid)onate (2)20
3H, NHCOCH3). 2.03 (s, 3H, OCOCH3), 2.03 (m, 4H,
H-3a, OCOCH3), 2.05, 2.14, 2.17 (3s, 9H, 3 OCOCH3),
2.66 (dd, J = 4.6, 12.9 Hz, 1H, H-3e), 3.67 (s, 3H,
OCH3), 4.09–4.15 (m, 3H, H-5, H-6, H-9a), 4.33 (dd,
J = 2.7, 12.4 Hz, 1H, H-9b), 4.43, 4.82 (A, B of AB,
J = 12.0 Hz, 2H, CH2Ph), 4.87 (m, 1H, H-4), 5.21 (m,
1H, NH), 5.35 (dd, J = 2.1, 8.5 Hz, 1H, H-7), 5.47 (m,
1H, H-8), 7.25–7.37 (s, 5H, C6H5); ESI-MS Calcd for
C27H35NNaO13 [M+Na+]: 604.2; Found: 604.2.
N-Acetyl neuraminic acid (1) (6.18 g, 20.0 mmol) and ion-
exchange resin Amberlyst 15 (5.00 g) were stirred at rt in
MeOH (300 mL) for 16 h. The reaction mixture was fil-
tered and the residual solid thoroughly washed with
MeOH. After evaporation of the solvent, the methyl ester
(5.50 g) was obtained and taken as such for the acetyla-
tion step. The above product was dissolved in pyridine
(68 mL) and stirred at 0 ꢁC for 15 min under argon before
DMAP (323 mg, 2.70 mmol) and Ac2O (76.6 mL,
715 mmol) were added at 0 ꢁC. The mixture was stirred
at rt for 14 h and then concentrated in vacuo. Chromatog-
raphy of the residue on silica gel (DCM/MeOH 20:1)
yielded 2 (7.25 g, 81%) as a white foam.
Compound 4b: 1H NMR (500 MHz, CDCl3): 1.87 (s, 3H,
NHCOCH3), 1.96 (m, 4H, H-3a, OCOCH3), 2.01, 2.03,
2.16 (3s, 9H, 3 OCOCH3), 2.56 (m, 1H, H-3e), 3.73 (s,
3H, OCH3), 3.99 (dd, J = 2.2, 10.5 Hz, 1H, H-9a),
4.10–4.15 (m, 2H, H-5, H-9b), 4.52 (A, B of AB,
J = 11.9 Hz, 2H, CH2Ph), 4.84 (dd, J = 2.5, 12.5 Hz,
1H, H-6), 5.28–5.41 (m, 4H, H-4, H-7, H-8, NH),
7.28–7.36 (m, 5H, C6H5); ESI-MS Calcd for
C27H35NNaO13 [M+Na+]: 604.2; Found: 604.3.
1H NMR (500 MHz, CDCl3): d 1.90 (s, 3H, NHC-
OCH3), 2.04 (m, 10H, H-3a, 3 OCOCH3), 2.14, 2.15
(2s, 6H, 2 OCOCH3), 2.66 (dd, J = 5.0, 13.5 Hz, 1H,
H-3e), 3.79 (s, 3H, OCH3), 4.12 (m, 3H, H-5, H-6, H-
9a), 4.49 (dd, J = 2.5, 12.4 Hz, 1H, H-9b), 5.08 (m,
1H, H-8), 5.26 (m, 1H, H-4), 5.38 (m, 2H, H-7, NH);
ESI-MS Calcd for C22H31NNaO14 [M+Na+]: 556.2;
Found: 556.1.
4.7. Sodium (benzyl 5-acetamido-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosid)onate (5a)
According to General procedure B, compound 4a
(50.0 mg, 86.0 lmol) was treated with 10% aqueous
NaOH (0.2 mL). Compound 5a (33.9 mg, 94%) was ob-
tained as a colorless solid after purification.
4.5. Methyl (methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-2-thio-D-glycero-D-galacto-2-nonulopyrano-
sid)onate (3)20
25
D
1
½aꢁ ꢀ 4:3 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.60 (t, J = 11.9 Hz, 1H, H-3a), 1.89 (s, 3H, NHC-
OCH3), 2.68 (dd, J = 3.5, 12.0 Hz, 1H, H-3e), 3.49–
3.77 (m, 7H, H-4, H-5, H-6, H-7, H-8, H-9), 4.42, 4.65
(A, B of AB, J = 11.0 Hz, 2H, CH2Ph), 7.30 (m, 5H,
To a stirred solution of 2 (7.20 g, 13.5 mmol) in DCE
˚
(100 mL) containing activated molecular sieves (3 A,
5.0 g), TMSSMe (2.26 g, 18.9 mmol) and TMSOTf
(2.24 g, 10.1 mmol) were added. The mixture was stirred
C6H5); 13C NMR (125 MHz, D2O):
d
22.4

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4957
(NHCOCH3), 40.9 (C-3), 52.2 (C-5), 62.9 (C-9), 67.5
(CH2Ph), 68.5 (C-7), 68.7 (C-4), 72.0 (C-8), 73.1 (C-6),
101.3 (C-2), 128.7, 129.0, 129.1, 137.4 (6C, C6H5),
173.9, 175.4 (2 CO); HR-MS Calcd for C18H24NNa2O9
[M+Na+]: 444.1246; Found: 444.1245.
CH3), 2.74 (dd, J = 4.6, 12.8 Hz, 1H, H-3e), 3.52 (dd,
J = 1.5, 8.5 Hz, 1H, H-7), 3.65 (dd, J = 1.5, 10.4 Hz,
1H, H-6), 3.69 (m, 1H, H-4), 3.77 (m, 1H, H-5), 3.79
(s, 3H, OCH3), 4.05 (ddd, J = 2.1, 6.4, 9.4 Hz, 1H, H-
8), 4.13 (dd, J = 6.4, 10.1 Hz, 1H, H-9a), 4.40 (dd,
J = 2.2, 10.1 Hz, 1H, H-9b), 4.49, 4.77 (A, B of AB,
J = 11.6 Hz, 2H, CH2Ph), 7.33, 7.45, 7.83 (m, 9H,
C6H4, C6H5); ESI-MS Calcd for C26H33NNaO11S
[M+Na+]: 590.2; Found: 590.2.
4.8. Sodium (benzyl 5-acetamido-3,5-dideoxy-D-glycero-
b-D-galacto-2-nonulopyranosid)onate (5b)
According to General procedure B, compound 4b
(50.0 mg, 86.0 lmol) was treated with 10% aqueous
NaOH (0.2 mL). Compound 5b (34.0 mg, 94%) was ob-
tained as a colorless solid after purification.
4.11. Methyl (benzyl 5-acetamido-9-azido-3,5,9-trideoxy-
D-glycero-a-D-galacto-2-nonulopyranosid)onate (8)
A suspension of 7 (910 mg, 1.60 mmol), crown ether 18-
C-6 (169 mg, 0.64 mmol), and NaN3 (520 mg,
8.00 mmol) was stirred in DMF (30 mL) at 60 ꢁC for
24 h. The mixture was filtered through a pad of Celite
and the filtrate was evaporated to dryness. Purification
by column chromatography on silica gel (DCM/acetone
7:3) gave 8 (525 mg, 75%) as a white solid.
25
D
1
½aꢁ ꢀ 1:3 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.68 (t, J = 12.1 Hz, 1H, H-3a), 1.90 (s, 3H, NHC-
OCH3), 2.38 (dd, J = 4.7, 13.1 Hz, 1H, H-3e), 3.57 (d,
J = 9.6 Hz, 1H, H-7), 3.67 (dd, J = 6.5, 12.0 Hz, 1H,
H-9a), 3.83–3.97 (m, 4H, H-5, H-6, H-8, H-9b), 4.02
(m, 1H, H-4), 4.27, 4.60 (A, B of AB, J = 10.1 Hz, 2H,
CH2Ph), 7.39–7.47 (m, 5H, C6H5); 13C NMR
(125 MHz, D2O): d 23.6 (NHCOCH3), 40.3 (C-3), 52.4
(C-5), 63.9 (C-9), 65.3 (CH2Ph), 67.4 (C-4), 68.6 (C-7),
70.3 (C-8), 70.6 (C-6), 100.5 (C-2), 128.7, 129.1, 129.2,
137.5 (6C, C6H5), 175.1, 181.9 (2 CO); HR-MS Calcd
for C18H24NNa2O9 [M+Na+]: 444.1246; Found: 444.1248.
1H NMR (500 MHz, CD3OD): d 1.84 (t, J = 12.6 Hz,
1H, H-3a), 2.06 (s, 3H, NHCOCH3), 2.77 (dd, J = 4.6,
12.8 Hz, 1H, H-3e), 3.43 (dd, J = 6.2, 12.9 Hz, 1H, H-
9a), 3.54 (dd, J = 1.4, 8.9 Hz, 1H, H-7), 3.60 (dd,
J = 2.5, 12.8 Hz, 1H, H-9b), 3.75 (m, 2H, H-4, H-6),
3.81 (s, 3H, OCH3), 3.83 (m, 1H, H-5), 4.06 (m, 1H,
H-8), 4.57, 4.85 (A, B of AB, J = 11.6 Hz, 2H, CH2Ph),
7.34–7.38 (m, 5H, C6H5); ESI-MS Calcd for
C19H26N4NaO8 [M+Na+]: 461.2; Found: 461.1.
4.9. Methyl (benzyl 5-acetamido-3,5-dideoxy-D-glycero-
a-D-galacto-2-nonulopyranosid)onate (6)
A solution of 4a (2.38 g, 3.95 mmol) in MeOH (90 mL)
was treated with 1 M NaOMe/MeOH (10.5 mL) at rt for
2 h. The reaction mixture was neutralized with Amber-
lyst 15 ion-exchange resin and filtered through a pad
of Celite. The Celite was washed thoroughly with meth-
anol (3 · 5 mL), and the combined filtrates were evapo-
rated to dryness to give 6 as colorless foam (1.40 g, 82%).
4.12. Sodium (benzyl 5-acetamido-9-azido-3,5,9-trideoxy-
D-glycero-a-D-galacto-2-nonulopyranosid)onate (9)
According to General procedure B, compound 8
(25.0 mg, 45.0 lmol) was treated with 10% aqueous
NaOH. Compound 9 (17.0 mg, 85%) was obtained as
a colorless solid after purification.
1H NMR (500 MHz, CD3OD): d 1.85 (t, J = 12.6 Hz,
1H, H-3a), 2.05 (s, 3H, NHCOCH3), 2.78 (dd, J = 4.6,
12.8 Hz, 1H, H-3e), 3.58 (dd, J = 1.6, 11.0 Hz, 1H, H-
9a), 3.67–3.74 (m, 3H, H-4, H-6, H-9b), 3.81 (s, 3H,
OCH3), 3.83–3.95 (m, 4H, H-5, H-7, H-8, NH), 4.56,
4.86 (A, B of AB, J = 11.6 Hz, 2H, CH2Ph), 7.28–7.33
(m, 5H, C6H5); ESI-MS Calcd for C19H27NNaO9
[M+Na+]: 436.2; Found: 436.1.
25
D
1
½aꢁ ꢀ 3:6 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.67 (t, J = 12.2 Hz, 1H, H-3a), 2.03 (s, 3H, NHC-
OCH3), 2.76 (dd, J = 4.6, 12.4 Hz, 1H, H-3e), 3.46
(dd, J = 6.1, 13.2 Hz, 1H, H-9a), 3.57 (dd, J = 1.8,
9.1 Hz, 1H, H-7), 3.63 (dd, J = 2.6, 13.2 Hz, 1H, H-
9b), 3.68 (ddd, J = 4.1, 4.6, 11.8 Hz, 1H, H-4), 3.74 (d,
J = 10.4 Hz, 1H, H-6), 3.79–3.83 (m, 2H, H-5, H-8),
4.53, 4.72 (A, B of AB, J = 11.0 Hz, 2H, CH2Ph),
7.39–7.43 (m, 5H, C6H5); 13C NMR (125 MHz, D2O):
d 20.5 (NHCOCH3), 39.1 (C-3), 50.4 (C-5), 51.5 (C-9),
65.8 (CH2Ph), 66.8 (C-4), 67.4 (C-7), 68.8 (C-8), 71.0
(C-6), 99.6 (C-2), 126.8, 127.2, 127.2, 135.7 (6C,
C6H5), 172.0, 173.6 (2 CO); HR-MS Calcd for
C18H24N4NaO8 [M+H+]: 447.1492; Found: 447.1490.
4.10. Methyl [benzyl 5-acetamido-3,5-dideoxy-9-O-(4-
toluenesulfonyl)-D-glycero-a-D-galacto-2-nonulopyrano-
sid]onate (7)
To a solution of 6 (1.20 g, 2.90 mmol) in pyridine was
added p-TsCl (608 mg, 3.20 mmol) at 0 ꢁC. After 2 h,
an additional portion of p-TsCl (220 mg, 1.16 mmol)
was added and stirring continued for 16 h at 5 ꢁC. The
reaction mixture was warmed to rt, diluted with MeOH
(40 mL), and stirring continued for 30 min. After re-
moval of the solvents the remaining syrup was purified
by chromatography on silica gel (DCM/MeOH 19:1)
to yield 7 (929 mg, 56%) as a colorless foam.
4.13. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
azido-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulopyr-
anosid)onate (10)
Compound 8 (480 mg, 1.09 mmol) was dissolved in pyri-
dine (4.38 mL, 54.3 mmol) and stirred at 0 ꢁC for 15 min
under argon before DMAP (21.4 mg, 0.175 mmol) and
Ac2O (3.27 mL, 46.0 mmol) were added at 0 ꢁC. The mix-
ture was stirred at rt for 14 h and then concentrated in va-
1H NMR (500 MHz, CD3OD): d 1.80 (t, J = 12.7 Hz,
1H, H-3a), 2.04 (s, 3H, NHCOCH3), 2.45 (s, 3H,

4958
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
cuo. Chromatography of the residue on silica gel (DCM/
MeOH 20:1) yielded 10 (470 mg, 73%).
ride (72.5 mg, 336 lmol) and PPh3 (48.4 mg, 184 lmol)
in DCM (2 mL) for 1 h. After purification 11c
(40.0 mg, 66%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.89 (s, 3H, NHC-
OCH3), 2.03 (s, 3H, OCOCH3), 2.04 (t, J = 12.6 Hz,
1H, H-3a), 2.17, 2.19 (2s, 6H, 2 OCOCH3), 2.67 (dd,
J = 4.6, 12.9 Hz, 1H, H-3e), 3.28 (dd, J = 5.9, 13.5 Hz,
1H, H-9a), 3.59 (dd, J = 3.0, 13.5 Hz, 1H, H-9b), 3.70
(s, 3H, OCH3), 4.12 (m, 1H, H-5), 4.44, 4.80 (A, B of
AB, J = 11.9 Hz, 2H, CH2Ph), 4.87 (m, 1H, H-4), 5.19
(m, 1H, NH), 5.36 (m, 3H, H-6, H-7, H-8), 7.28–7.34
(m, 5H, Ar-H); ESI-MS Calcd for C25H32N4NaO11
[M+Na+]: 587.2; Found: 587.4.
1H NMR (500 MHz, CDCl3): d 2.02 (m, 4H, H-3a, NHC-
OCH3), 2.08, 2.15, 2.17 (3s, 9H, OCOCH3), 2.63–2.70 (m,
2H, H-3e, H-9a), 3.69 (s, 3H, OCH3), 3.94 (dd, J = 2.3,
10.7 Hz, 1H, H-6), 4.06–4.15 (m, 2H, H-5, H-9b), 4.39,
4.79 (A, B of AB, J = 12.0 Hz, 2H, CH2Ph), 4.67 (dd,
J = 2.2, 9.4 Hz, 1H, H-7), 4.83 (m, 1H, H-4), 4.99 (m,
1H, NH-5), 5.12 (m, 1H, H-8), 5.99 (m, 1H, NH-9),
7.31–7.59 (m, 14H, C6H4, 2 C6H5); ESI-MS Calcd for
C38H42N2NaO12 [M+Na+]: 741.3; Found: 741.3.
4.17. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-orotinoylamido-D-glycero-a-D-galacto-
2-nonulopyranosid)onate (11d)
4.14. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
benzamido-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonu-
lopyranosid)onate (11a)
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with orotinoyl chloride
(63.8 mg, 336 lmol) and PPh3 (48.4 mg, 184 lmol) in
DCM (2 mL) for 1 h. After purification 11d (40.0 mg,
70%) was obtained as a colorless solid.
According to General procedure A, 10 (42.0 mg,
74.4 lmol) was reacted with benzoyl chloride (20.0 mg,
142 lmol) and PPh3 (24.0 mg, 91.5 lmol) in toluene
(2 mL) for 1 h. After purification 11a (25.0 mg, 54%)
was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.92 (s, 3H, NHCOCH3),
2.05 (m, 4H, H-3a, OCOCH3), 2.13, 2.26 (2s, 6H,
OCOCH3), 2.67 (dd, J = 4.6, 12.7 Hz, 1H, H-3e), 2.93
(m, 1H, H-9a), 3.99 (s, 3H, OCH3), 4.03 (dd, J = 2.0,
10.7 Hz, 1H, H-6), 4.21 (m, 1H, H-5), 4.27 (m, 1H, H-
9b), 4.41, 4.79 (A, B of AB, J = 12.0 Hz, 2H, CH2Ph),
4.84 (m, 1H, H-4), 5.04 (dd, J = 2.0, 9.9 Hz, 1H, H-7),
5.32 (m, 2H, NH-5, H-8), 6.08 (s, CH= C), 6.40 (s,
C= CH) 7.30–7.34 (s, 5H, C6H5), 7.49 (m, 1H, NH-9),
8.65 (m, 1H, allylic NH), 8.82 (s, 1H, NH); ESI-MS Calcd
for C30H37N4NaO14 [M+H+]: 677.2; Found: 677.2.
1H NMR (500 MHz, CDCl3): d 1.99 (s, 3H, NHCOCH3),
2.05 (m, 4H, H-3a, OCOCH3), 2.15, 2.27 (2s, 6H, 2
OCOCH3), 2.67 (dd, J = 4.6, 12.8 Hz, 1H, H-3e), 2.96
(dd, J = 3.3, 11.5 Hz, 1H, H-9a), 3.65 (s, 3H, OCH3),
4.06 (d, J = 10.7 Hz, 1H, H-6), 4.21 (q, J = 10.7 Hz, 1H,
H-5), 4.39 (m, 1H, H-9b), 4.44, 4.83 (A, B of AB,
J = 12.0 Hz, 2H, CH2Ph), 4.85 (m, 1H, H-4), 5.16–5.18
(m, 2H, NH-5, H-7), 5.33 (m, 1H, H-8), 7.09 (m, 1H,
NH-9), 7.33–7.44, 7.84 (m, 10H, 2 C6H5); ESI-MS Calcd
for C32H38N2NaO12 [M+Na+]: 665.2; Found: 665.3.
4.15. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-(4-
biphenylcarboxamido)-3,5,9-trideoxy-D-glycero-a-D-galac-
to-2-nonulopyranosid]onate (11b)
4.18. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
butyrylamido-3,5-dideoxy-D-glycero-a-D-galacto-2-nonu-
lopyranosid)onate (11e)
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with biphenyl-4-carbonyl chlo-
ride (36.3 mg, 168 lmol) and PPh3 (26.2 mg, 100 lmol)
in toluene (2 mL) for 2 h. After purification 11b
(14.0 mg, 24%) was obtained as a colorless solid.
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with butyryl chloride (35.8 mg,
336 lmol) and PPh3 (48.4 mg, 184 lmol) in DCM
(2 mL) for 3 h. After purification 11e (43.0 mg, 84%)
was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.91 (s, 3H, NHC-
OCH3), 2.06 (m, 3H, H-3a, OCOCH3), 2.16, 2.28 (2s,
6H, 2 OCOCH3), 2.67 (dd, J = 4.6, 12.7 Hz, 1H, H-
3e), 2.98 (m, 1H, H-9a), 3.65 (s, 3H, OCH3), 4.07 (dd,
J = 2.0, 10.8 Hz, 1H, H-6), 4.22 (dd, J = 10.3, 10.8 Hz,
1H, H-5), 4.41 (m, 1H, H-9b), 4.45, 4.85 (A, B of AB,
J = 12.0 Hz, 2H, CH2Ph), 4.86 (m, 1H, H-4), 5.20 (m,
2H, H-7, NH-5), 5.35 (m, 1H, H-8), 7.15 (m, 1H, NH-
9), 7.33–7.39, 7.47, 7.62–7.67, 7.91 (m, 14H, C6H4, 2
C6H5); ESI-MS Calcd for C38H42N2NaO12 [M+Na+]:
741.3; Found: 741.2.
1H NMR (500 MHz, CDCl3): d 0.96 (t, J = 8.3 Hz, 3H,
H-40), 1.63–1.68 (m, 2H, H-30), 1.91 (s, 3H, NHC-
OCH3), 2.04 (m, 4H, H-3a, OCOCH3), 2.13 (s, 3H,
OCOCH3), 2.17 (t, J = 7.4 Hz, 2H, H-20), 2.21 (s, 3H,
OCOCH3), 2.66 (dd, J = 4.5, 12.8 Hz, 1H, H-3e), 2.78
(m, 1H, H-9a), 3.66 (s, 3H, OCH3), 4.04–4.20 (m, 3H,
H-5, H-6, H-9b), 4.42, 4.82 (A, B of AB, J = 12.0 Hz,
2H, CH2Ph), 4.86 (m, 1H, H-4), 5.09 (d, J = 10.6 Hz,
1H, H-7), 5.21–5.25 (m, 2H, H-8, NH-5), 6.17 (m, 1H,
NH-9), 7.32 (m, 5H, C6H5); ESI-MS Calcd for
C29H40N2NaO12 [M+Na+]: 631.2; Found: 631.3.
4.19. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-hexanoylamido-D-glycero-a-D-galacto-
2-nonulopyranosid)onate (11f)
4.16. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-(2-
biphenylcarboxamido)-3,5,9-trideoxy-D-glycero-a-D-galac-
to-2-nonulopyranosid]onate (11c)
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with caproyl chloride (45.6 mg,
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with biphenyl-2-carbonyl chlo-

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4959
336 lmol) and PPh3 (48.4 mg, 184 lmol) in DCM
(2 mL) for 3 h. After purification 11f (25.9 mg, 49%)
was obtained as a colorless solid.
4.22. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
(3,4-dichlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (11i)
1H NMR (500 MHz, CDCl3): d 0.90 (t, J = 6.9 Hz, 3H,
H-60), 1.28–1.32 (m, 4H, H-40, H-50), 1.62 (m, 2H, H-30),
1.91 (s, 3H, NHCOCH3), 2.04 (m, 4H, H-3a, OCOCH3),
2.13 (s, 3H, OCOCH3), 2.18 (t, J = 6.9 Hz, 2H, H-20),
2.21 (s, 3H, OCOCH3), 2.56 (dd, J = 4.6, 12.8 Hz, 1H,
H-3e), 2.79 (m, 1H, H-9a), 3.66 (s, 3H, OCH3), 4.09
(m, 2H, H-6, H-9b), 4.18 (q, J = 10.4 Hz, 1H, H-5),
4.42, 4.82 (A, B of AB, J = 12.0 Hz, 2H, CH2Ph), 4.86
(ddd, J = 5.7, 6.5, 10.4 Hz, 1H, H-4), 5.01 (dd, J = 2.1,
9.7 Hz, 1H, H-7), 5.23–5.25 (m, 2H, H-8, NH-5),
6.14 (m, 1H, NH-9), 7.26–7.33 (m, 5H, C6H5); ESI-
MS Calcd for C31H44N2NaO12 [M+Na+]: 659.3; Found:
659.3.
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with 3,4-dichlorobenzoyl chlo-
ride (58.5 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol)
in DCE (2 mL) for 14 h. After purification 11i
(31.1 mg, 63%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.91 (s, 3H, NHC-
OCH3), 2.04 (m, 4H, H-3a, OCOCH3), 2.14, 2.27 (2s,
6H, 2 OCOCH3), 2.67 (dd, J = 4.6, 12.8 Hz, 1H, H-
3e), 2.95 (m, 1H, H-9a), 3.65 (s, 3H, OCH3), 4.06 (dd,
J = 2.0, 10.7 Hz, 1H, H-6), 4.22 (q, J = 10.4 Hz, 1H,
H-5), 4.35 (m, 1H, H-9b), 4.43 (A of AB, J = 12.0 Hz,
1H, CH2Ph), 4.85 (m, 2H, H-4, CH2Ph), 5.14 (dd,
J = 2.1, 9.9 Hz, 1H, H-7), 5.25 (d, J = 10.1 Hz, 1H,
NH-5), 5.33 (m, 1H, H-8), 7.12 (m, 1H, NH-9), 7.33,
7.51, 7.62, 7.94 (m, 8H, C6H3, C6H5); ESI-MS
Calcd for C32H36Cl2N2NaO12 [M+Na+]: 733.2; Found:
733.1.
4.20. Methyl (benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
decanoylamido-3,5,9-trideoxy-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (11g)
According to General procedure A, 10 (50.0 mg,
88.6 lmol) was reacted with decanoyl chloride
(65.2 mg, 336 lmol) and PPh3 (48.4 mg, 184 lmol) in
DCM (2 mL) for 3 h. After purification 11g (30.2 mg,
51%) was obtained as a colorless solid.
4.23. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-(4-trifluoromethyl-benzamido)-D-glyce-
ro-a-D-galacto-2-nonulopyranosid]onate (11j)
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with 4-trifluoromethyl-benzoyl
chloride (58.2 mg, 280 lmol) and PPh3 (40.3 mg,
154 lmol) in DCE (2 mL) for 14 h. After purification
11j (30.0 mg, 60%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 0.87 (t, J = 6.8 Hz, 3H,
H-100), 1.26–1.31 (m, 14H, H-40, H-50, H-60, H-70, H-80,
H-90), 1.60 (m, 2H, H-30), 1.91 (s, 3H, NHCOCH3), 2.04
(m, 4H, H-3a, OCOCH3), 2.13 (s, 3H, OCOCH3), 2.18
(t, J = 7.9 Hz, 2H, H-20), 2.21 (s, 3H, OCOCH3), 2.66
(dd, J = 4.5, 12.7 Hz, 1H, H-3e), 2.79 (m, 1H, H-9a),
3.65 (s, 3H, OCH3), 4.06–4.20 (m, 3H, H-5, H-6, H-
9b), 4.42, 4.82 (A, B of AB, J = 12.0 Hz, 2H, CH2Ph),
4.86 (ddd, J = 4.9, 6.2, 10.3 Hz, 1H, H-4), 5.09 (d,
J = 8.8 Hz, 1H, H-7), 5.18–5.25 (m, 2H, H-8, NH-5),
6.13 (m, 1H, NH-9), 7.27–7.33 (m, 5H, C6H5); ESI-
MS Calcd for C35H52N2NaO12 [M+Na+]: 715.3; Found:
715.4.
1H NMR (500 MHz, CDCl3): d 1.82 (s, 3H, NHC-
OCH3), 1.97 (m, 4H, H-3a, OCOCH3), 2.07, 2.20 (2s,
6H, OCOCH3), 2.60 (dd, J = 4.6, 12.8 Hz, 1H, H-3e),
2.92 (m, 1H, H-9a), 3.57 (s, 3H, OCH3), 3.99 (m, 1H,
H-6), 4.14 (q, J = 10.4 Hz, 1H, H-5), 4.29 (m, 1H, H-
9b), 4.36 (A of AB, J = 12.0 Hz, 1H, CH2Ph), 4.76 (m,
2H, H-4, CH2Ph), 5.08 (dd, J = 2.1, 9.9 Hz, 1H, H-7),
5.29 (m, 2H, H-8, NH-5), 7.13–7.26, 7.63, 7.86 (m,
10H, NH-9, C6H4, C6H5); ESI-MS Calcd for
C33H37F3N2NaO12 [M+Na+]: 733.2; Found: 733.2.
4.21. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-(4-
chlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-galacto-
2-nonulopyranosid]onate (11h)
4.24. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-
(2,4-dichlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (11k)
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with p-chlorobenzoyl chloride
(49.0 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol) in
DCE (2 mL) for 14 h. After purification 11h (32.0 mg,
68%) was obtained as a colorless solid.
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with 2,4-dichlorobenzoyl chlo-
ride (58.5 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol)
in DCE (2 mL) for 14 h. After purification 11k
(30.8 mg, 62%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.89 (s, 3H, NHC-
OCH3), 2.04 (m, 4H, H-3a, OCOCH3), 2.14, 2.26 (2s,
6H, 2 OCOCH3), 2.67 (dd, J = 4.6, 12.8 Hz, 1H, H-
3e), 2.96 (m, 1H, H-9a), 3.64 (s, 3H, OCH3), 4.06 (dd,
J = 2.0, 10.7 Hz, 1H, H-6), 4.20 (q, J = 10.4 Hz, 1H,
H-5), 4.35 (m, 1H, H-9b), 4.43 (A of AB, J = 12.0 Hz,
1H, CH2Ph), 4.83 (m, 2H, H-4, CH2Ph), 5.15 (dd,
J = 2.0, 9.9 Hz, 1H, H-7), 5.27 (d, J = 10.1 Hz, 1H,
NH-5), 5.34 (m, 1H, H-8), 7.09 (m, 1H, NH-9), 7.25–
7.43, 7.77 (m, 9H, C6H4, C6H5); ESI-MS Calcd for
C32H37ClN2NaO12 [M+Na+]: 699.2; Found: 699.2.
1H NMR (500 MHz, CDCl3): d 1.82 (s, 3H, NHC-
OCH3), 1.97 (m, 4H, H-3a, OCOCH3), 2.09, 2.15 (2s,
6H, 2 OCOCH3), 2.60 (dd, J = 4.6, 12.8 Hz, 1H, H-
3e), 2.87 (m, 1H, H-9a), 3.59 (s, 3H, OCH3), 4.01 (dd,
J = 2.0, 10.8 Hz, 1H, H-6), 4.11 (q, J = 10.4 Hz, 1H,
H-5), 4.29 (m, 1H, H-9b), 4.37, 4.76 (A, B of AB,
J = 12.0 Hz, 2H, CH2Ph), 4.80 (m, 1H, H-4), 5.11 (dd,
J = 1.9, 9.7 Hz, 1H, H-7), 5.22–5.24 (m, 2H, H-8, NH-
5), 6.84 (m, 1H, NH-9), 7.18–7.26, 7.35, 7.49 (m, 8H,

4960
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
C6H3, C6H5); ESI-MS Calcd for C32H36Cl2N2NaO12
[M+Na+]: 733.2; Found: 733.2.
H-4, CH2Ph), 5.17 (dd, J = 1.9, 9.8 Hz, 1H, H-7), 5.24
(d, J = 10.1 Hz, 1H, NH-5), 5.33 (m, 1H, H-8), 7.04
(m, 1H, NH-9), 7.22–7.33, 7.73 (m, 9H, C6H4, C6H5);
ESI-MS Calcd for C33H40N2NaO12 [M+Na+]: 679.2;
Found: 679.2.
4.25. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-(4-nitrobenzamido)-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (11l)
4.28. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-9-(3-
chlorobenzamido)-3,5,9-trideoxy-D-glycero-a-D-galacto-
2-nonulopyranosid]onate (11o)
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with p-nitrobenzoyl chloride
(51.8 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol) in
DCE (2 mL) for 14 h. After purification 11l (28.1 mg,
58%) was obtained as a colorless solid.
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with m-chlorobenzoyl chloride
(49.0 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol) in
DCE (2 mL) for 14 h. After purification 11o (28.0 mg,
59%) was obtained as a colorless solid.
1H NMR (500 MHz, CDCl3): d 1.82 (s, 3H, NHCOCH3),
1.99 (m, 4H, H-3a, OCOCH3), 2.08, 2.20 (2s, 6H, 2
OCOCH3), 2.60 (dd, J = 4.6, 12.7 Hz, 1H, H-3e), 2.93
(m, 1H, H-9a), 3.58 (s, 3H, OCH3), 3.99 (dd, J = 2.0,
10.7 Hz, 1H, H-6), 4.15 (q, J = 10.4 Hz, 1H, H-5), 4.30
(m, 1H, H-9b), 4.36 (A of AB, J = 11.9 Hz, 1H, CH2Ph),
4.76 (m, 2H, H-4, CH2Ph), 5.08 (dd, J = 2.0, 9.8 Hz, 1H,
H-7), 5.27 (m, 2H, H-8, NH-5), 7.18–7.30, 7.91, 8.20 (m,
10H, NH-9, C6H4, C6H5); ESI-MS Calcd for
C32H37N3NaO14 [M+Na+]: 710.2; Found: 710.2.
1H NMR (500 MHz, CDCl3): d 1.84 (s, 3H, NHC-
OCH3), 1.99 (m, 4H, H-3a, OCOCH3), 2.07, 2.19 (2s,
6H, OCOCH3), 2.60 (dd, J = 4.6, 12.9 Hz, 1H, H-3e),
2.87 (m, 1H, H-9a), 3.57 (s, 3H, OCH3), 3.99 (d,
J = 10.7 Hz, 1H, H-6), 4.14 (q, J = 10.7 Hz 1H, H-5),
4.29 (m, 1H, H-9b), 4.36 (A of AB, J = 12.0 Hz, 1H,
CH2Ph), 4.77 (m, 2H, H-4, CH2Ph), 5.09 (d,
J = 9.9 Hz, 1H, H-7), 5.22 (m, 2H, H-8, NH-5), 7.04
(m, 1H, NH-9), 7.14–7.41, 7.60, 7.76 (m, 9H, C6H4,
C6H5); ESI-MS Calcd for C32H37ClN2NaO12
[M+Na+]: 699.2; Found: 699.2.
4.26. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-(4-methoxybenzamido)-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (11m)
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with p-anisoyl chloride
(47.6 mg, 280 lmol) and PPh3 (40.3 mg, 154 lmol) in
DCE (2 mL) for 14 h. After purification 11m (25.0 mg,
53%) was obtained as a colorless solid.
4.29. Sodium (benzyl 5-acetamido-9-benzamido-3,5,9-tri-
deoxy-D-glycero-a-D-galacto-2-nonulopyranosid)onate
(12a)
According to General procedure B, compound 11a
(25.0 mg, 38.0 lmol) was treated with 10% aqueous
NaOH. Compound 12a (19.0 mg, 95%) was obtained
as a colorless solid after purification.
1H NMR (500 MHz, CDCl3): d 1.89 (s, 3H, NHC-
OCH3), 2.04 (m, 4H, H-3a, OCOCH3), 2.14, 2.25 (2s,
6H, 2 OCOCH3), 2.67 (dd, J = 4.6, 12.7 Hz, 1H, H-
3e), 2.95 (m, 1H, H-9a), 3.64, 3.84 (2s, 6H, 2 OCH3),
4.06 (dd, J = 2.1, 10.7 Hz, 1H, H-6), 4.21 (q, J = 10.4
Hz, 1H, H-5), 4.35 (m, 1H, H-9b), 4.44 (A of AB,
J = 11.9 Hz, 1H, CH2Ph), 4.84 (m, 2H, H-4, CH2Ph),
5.18 (dd, J = 2.0, 9.8 Hz, 1H, H-7), 5.26 (d, J =
10.1 Hz, 1H, NH-5), 5.33 (m, 1H, H-8), 6.92, 6.96, 7.24,
7.34, 7.79 (m, 10H, NH-9, C6H4, C6H5); ESI-MS Calcd
for C33H40N2NaO13 [M+Na+]: 695.2; Found: 695.3.
25
D
1
½aꢁ ꢀ 2:0 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.65 (t, J = 11.8 Hz, 1H, H-3a), 1.99 (s, 3H, NHCOCH3),
2.77 (dd, J = 3.5, 11.8 Hz, 1H, H-3e), 3.46–3.52 (m, 2H,
H-7, H-9a), 3.68 (m, 1H, H-4), 3.75–3.84 (m, 4H, H-5, H-
6, H-8, H-9b), 4.53, 4.70 (A, B of AB, J = 11.2 Hz, 2H,
CH2Ph), 7.27–7.39, 7.52, 7.60, 7.78 (m, 10H, 2 C6H5);
13C NMR (125 MHz, D2O): d 21.5 (NHCOCH3), 40.1
(C-3), 42.1 (C-9), 51.4 (C-5), 66.9 (CH2Ph), 67.8 (C-4),
69.4 (C-7), 69.8 (C-8), 72.2 (C-6), 101.0 (C-2), 126.6,
127.7, 128.2, 128.3, 131.7, 133.2, 136.8 (12C, 2 C6H5),
170.7, 172.5, 174.6 (3 CO); HR-MS Calcd for
C25H30N2NaO9 [M+H+]: 525.1849; Found: 525.1850.
4.27. Methyl [benzyl 5-acetamido-4,7,8-tri-O-acetyl-
3,5,9-trideoxy-9-(4-methylbenzamido)-D-glycero-a-D-
galacto-2-nonulopyranosid]onate (11n)
According to General procedure A, 10 (40.0 mg,
70.9 lmol) was reacted with p-toluyl chloride (43.1 mg,
280 lmol) and PPh3 (40.3 mg, 154 lmol) in DCE
(2 mL) for 14 h. After purification 11n (26.2 mg, 56%)
was obtained as a colorless solid.
4.30. Sodium [benzyl 5-acetamido-9-(4-biphenylcarbox-
amido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosid]onate (12b)
According to General procedure B, compound 11b
(14.0 mg, 19.0 lmol) was treated with 10% aqueous
NaOH. Compound 12b (7.1 mg, 64%) was obtained as
a colorless solid after purification.
1H NMR (500 MHz, CDCl3): d 1.89 (s, 3H, NHC-
OCH3), 2.04 (m, 4H, H-3a, OCOCH3), 2.14, 2.26 (2s,
6H, 2 OCOCH3), 2.39 (s, 3H, PhCH3), 2.67 (dd,
J = 4.6, 12.8 Hz, 1H, H-3e), 2.96 (m, 1H, H-9a), 3.64
(s, 3H, OCH3), 4.06 (dd, J = 1.9, 10.8 Hz, 1H, H-6),
4.20 (q, J = 10.4 Hz, 1H, H-5), 4.36 (m, 1H, H-9b),
4.43 (A of AB, J = 12.0 Hz, 1H, CH2Ph), 4.84 (m, 2H,
25
D
1
½aꢁ ꢀ 4:8 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.69 (t, J = 12.2 Hz 1H, H-3a), 2.01 (s, 3H, NHCOCH3),
2.77 (dd, J = 4.6, 12.2 Hz, 1H, H-3e), 3.44 (dd, J = 8.5,
14.7 Hz 1H, H-9a), 3.54 (d, J = 8.5 Hz, 1H, H-7), 3.68

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4961
(m, 1H, H-4), 3.79–3.83 (m, 4H, H-5, H-6, H-8, H-9b),
4.48, 4.68 (A, B of AB, J = 11.2 Hz, 2H, CH2Ph),
7.22–7.32, 7.39, 7.54, 7.72 (m, 14H, 2 C6H5, C6H4);
13C NMR (125 MHz, D2O): d 22.3 (NHCOCH3), 40.5
(C-3), 42.5 (C-9), 52.2 (C-5), 67.5 (CH2Ph), 68.6 (C-4),
70.4 (C-7), 70.8 (C-8), 73.0 (C-6), 101.5 (C-2), 127.3,
127.9, 128.4, 128.6, 128.8, 128.9, 129.4, 132.5, 137.4,
139.6, 144.2 (18C, 2 C6H5, C6H4), 171.5, 174.6, 176.3
(3 CO); HR-MS Calcd for C31H33N2Na2O9 [M+Na+]:
623.1981; Found: 623.1981.
NaOH. Compound 12e (31.8 mg, 96%) was obtained
as a colorless solid after purification.
25
1
½aꢁ ꢀ 1:2 (c 1.00, H2O); H NMR (500 MHz, D2O): d
D
0.88 (t, J = 7.4 Hz, 3H, H-40), 1.59 (m, 2H, H-30), 1.68
(t, J = 12.2 Hz, 1H, H-3a), 2.01 (s, 3H, NHCOCH3),
2.24 (t, J = 7.3 Hz, 2H, H-20), 2.75 (dd, J = 4.7,
12.4 Hz, 1H, H-3e), 3.30 (dd, J = 7.1, 14.2 Hz, 1H, H-
9a), 3.45 (dd, J = 1.7, 9.0 Hz, 1H, H-7), 3.53 (dd,
J = 2.8, 14.1 Hz, 1H, H-9b), 3.68 (ddd, J = 2.9, 4.7,
11.8 Hz, 1H, H-4), 3.72–3.82 (m, 3H, H-5, H-6, H-8),
4.51, 4.70 (A, B of AB, J = 12.0 Hz, 2H, CH2Ph), 7.38
(m, 5H, C6H5); 13C NMR (125 MHz, D2O): d 12.1 (C-
40), 18.4 (C-30), 22.6 (NHCOCH3), 37.0 (C-20), 39.8
(C-3), 41.0 (C-9), 51.2 (C-5), 66.5 (CH2Ph), 67.6 (C-4),
68.9 (C-7), 69.3 (C-8), 71.9 (C-6), 100.4 (C-2), 127.6,
128.0, 128.0, 136.5 (6C, C6H5), 174.3, 176.7, 180.8 (3
CO); HR-MS Calcd for C22H31N2Na2O9 [M+Na+]:
513.1825; Found: 513.1825.
4.31. Sodium [benzyl 5-acetamido-9-(2-biphenylcarbox-
amido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosid]onate (12c)
According to General procedure B, compound 11c
(40.0 mg, 55.1 lmol) was treated with 10% aqueous
NaOH. Compound 12c (31.9 mg, 97%) was obtained
as a colorless solid after purification.
25
D
1
½aꢁ ꢀ 2:7 (c 1.00, H2O); H NMR (500 MHz, D2O): d
4.34. Sodium (benzyl 5-acetamido-3,5,9-trideoxy-9-hexa-
noylamido-D-glycero-a-D-galacto-2-nonulopyrano-
sid)onate (12f)
1.51 (t, J = 11.4 Hz, 1H, H-3a), 1.92 (s, 3H, NHCOCH3),
2.63 (dd, J = 4.3, 10.5 Hz, 1H, H-3e), 2.97 (dd, J = 9.0,
13.9 Hz, 1H, H-9a), 3.27 (d, J = 9.1 Hz, 1H, H-7), 3.47
(m, 1H, H-8), 3.55–3.59 (m, 4H, H-4, H-5, H-6, H-9b),
4.36, 4.58 (A, B of AB, J = 10.7 Hz, 2H, CH2Ph), 6.93,
7.02 7.07, 7.18–7.34 (m, 14H, 2 C6H5, C6H4); 13C NMR
(125 MHz, D2O): d 22.4 (NHCOCH3), 40.7 (C-3), 43.5
(C-9), 52.2 (C-5), 67.5 (CH2Ph), 68.6 (C-4), 70.2 (C-8),
70.4 (C-7), 72.8 (C-6), 101.2 (C-2), 127.9, 128.0, 128.1,
128.7, 128.9, 129.0, 129.3, 130.5, 130.9, 135.1, 137.2,
129.9, 140.1 (18C, 2 C6H5, C6H4), 173.5, 173.8, 175.4 (3
CO); HR-MS Calcd for C31H33N2Na2O9 [M+Na+]:
623.1981; Found: 623.1984.
According to General procedure B, compound 11f
(26.1 mg, 40.1 lmol) was treated with 10% aqueous
NaOH. Compound 12f (20.2 mg, 96%) was obtained
as a colorless solid after purification.
25
½aꢁ ꢀ 1:15 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
D
0.81 (t, J = 7.0 Hz, 3H, H-60), 1.23–1.27 (m, 4H, H-40,
H-50), 1.57 (m, 2H, H-30), 1.66 (t, J = 12.2 Hz, 1H, H-
3a), 2.02 (s, 3H, NHCOCH3), 2.25 (t, J = 7.4 Hz, 2H,
H-20), 2.75 (dd, J = 4.7, 12.5 Hz, 1H, H-3e), 3.22 (dd,
J = 7.7, 14.2 Hz, 1H, H-9a), 3.46 (dd, J = 1.7, 9.0 Hz,
1H, H-7), 3.58 (dd, J = 7.7, 14.1 Hz, 1H, H-9b), 3.68
(ddd, J = 6.6, 9.8, 12.8 Hz, 1H, H-4), 3.72–3.80 (m, 3H,
H-5, H-6, H-8), 4.50, 4.70 (A, B of AB, J = 11.0 Hz, 2H,
CH2Ph), 7.40 (s, 5H, C6H5); 13C NMR (125 MHz,
D2O): d 20.1 (C-60), 20.6 (C-50), 21.8 (NHCOCH3), 23.7
(C-40), 28.9 (C-30), 34.2 (C-20), 39.0 (C-3), 40.3 (C-9),
50.4 (C 5), 65.8 (CH2Ph), 66.8 (C-4), 68.2 (C-7), 68.7 (C-
8), 71.1 (C-6), 99.6 (C-2), 126.8, 127.2, 127.3, 135.6 (6C,
C6H5), 173.6, 176.1, 180.1 (3 CO); HR-MS Calcd for
C24H35N2Na2O9 [M+Na+]: 541.2138; Found: 541.2137.
4.32. Sodium (benzyl 5-acetamido-3,5,9-trideoxy-9-oroti-
noylamido-D-glycero-a-D-galacto-2-nonulopyrano-
sid)onate (12d)
According to General procedure B, compound 11d
(40.0 mg, 58.9 lmol) was treated with 10% aqueous
NaOH. Compound 12d (32.1 mg, 96%) was obtained
as a colorless solid after purification.
25
D
1
½aꢁ ꢀ 3:7 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.69 (t, J = 12.1 Hz, 1H, H-3a), 2.01 (s, 3H, NHC-
OCH3), 2.76 (dd, J = 4.5, 12.4 Hz, 1H, H-3e), 2.97 (m,
1H, H-9a), 3.44–3.52 (m, 2H, H-7, H-9b), 3.66–3.84
(m, 4H, H-4, H-5, H-6, H-8), 4.54, 4.69 (A, B of AB,
J = 11.3 Hz, 2H, CH2Ph), 6.07, 6.22 (2s, 2H, oroti-
noyl-H), 7.31–7.41 (m, 5H, C6H5); 13C NMR
(125 MHz, D2O): d 23.6 (NHCOCH3), 42.1 (C-3), 44.9
(C-9), 52.2 (C-5), 67.7 (CH2Ph), 68.6 (C-4), 70.1 (C-7),
70.4 (C-8), 72.9 (C-6), 98.2 (HN–C@C–), 100.8
(HN-C= C-), 128.5, 128.9, 129.1, 137.7 (6C, C6H5), 161.4,
166.7, 174.0, 176.0, 182.0 (5 CO); HR-MS Calcd for
C23H27N4Na2O11 [M+Na+]: 581.1472; Found: 581.1460.
4.35. Sodium (benzyl 5-acetamido-9-decanoylamido-
3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulopyrano-
sid)onate (12g)
According to General procedure B, compound 11g
(30.0 mg, 43.0 lmol) was treated with 10% aqueous
NaOH. Compound 12g (24.0 mg, 97%) was obtained
as a colorless solid after purification.
25
1
½aꢁ ꢀ 4:1 (c 1.00, H2O); H NMR (500 MHz, D2O): d
D
0.56 (t, J = 6.8 Hz, 3H, H-100), 0.92–1.00 (m, 12H, H-
40, H-50, H-60, H-70, H-80, H-90), 1.32 (m, 2H, H-30),
1.46 (t, J = 12.2 Hz, 1H, H-3a), 1.81 (s, 3H, NHC-
OCH3), 2.01 (t, J = 7.2 Hz, 2H, H-20), 2.55 (dd,
J = 4.5, 12.2 Hz, 1H, H-3e), 2.94 (dd, J = 8.3, 14.1 Hz,
1H, H-9a), 3.25 (d, J = 11.1 Hz, 1H, H-7), 3.42–3.50
(m, 2H, H-4, H-9b), 3.54–3.63 (m, 3H, H-5, H-6, H-8),
4.33. Sodium (benzyl 5-acetamido-9-butyrylamido-3,5,9-
trideoxy-D-glycero-a-D-galacto-2-nonulopyranosid)onate
(12e)
According to General procedure B, compound 11e
(42.0 mg, 69.0 lmol) was treated with 10% aqueous

4962
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4.28, 4.49 (A, B of AB, J = 10.9 Hz, 2H, CH2Ph), 7.10–
7.17 (m, 5H, C6H5); 13C NMR (125 MHz, D2O): d 13.9
(C-100), 22.4 (C-90), 22.5 (NHCOCH3), 25.9 (C-80), 28.7
(C-70), 29.0 (2C, C-50, C-60), 29.2 (C-40), 31.6 (C-30), 36.2
(C-20), 41.0 (C-3), 42.4 (C-9), 52.2 (C-5), 68.0 (CH2Ph),
68.7 (C-4), 70.3 (C-7), 70.7 (C-8), 72.9 (C-6), 101.2 (C-2),
128.6, 129.0, 129.2, 137.4 (6C, C6H5), 173.6, 175.3, 177.3
(3 CO); HR-MS Calcd for C28H43N2Na2O9 [M+Na+]:
597.2764; Found: 597.2768.
NaOH. Compound 12j (21.0 mg, 70%) was obtained
as a colorless solid after purification.
25
D
½aꢁ ꢀ 2:37 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
1.68 (t, J = 12.2 Hz, 1H, H-3a), 2.00 (s, 3H,
NHCOCH3), 2.76 (dd, J = 4.7, 12.5 Hz, 1H, H-3e),
3.44–3.52 (m, 2H, H-7, H-9a), 3.68 (m, 1H, H-4),
3.75–3.84 (m, 4H, H-5, H-6, H-8, H-9b), 4.49, 4.68 (A,
B of AB, J = 11.2 Hz, 2H, CH2Ph), 7.24–7.35, 7.75,
7.84 (m, 9H, C6H5, C6H4); 13C NMR (125 MHz,
D2O): d 22.4 (NHCOCH3), 40.9 (C-3), 43.1 (C-9), 52.3
(C-5), 67.6 (CH2Ph), 68.6 (C-4), 70.3 (C-7), 70.6 (C-8),
73.0 (C-6), 101.6 (C-2), 126.0, (CF3), 128.0, 128.5,
128.9, 129.0, 129.5, 129.6, 132.5, 137.5 (12C, C6H5,
C6H4), 170.0, 173.8, 175.4 (3 CO); HR-MS Calcd for
C26H29F3N2NaO9 [M+H+]: 593.1723; Found: 593.1722.
4.36. Sodium [benzyl 5-acetamido-9-(4-chlorobenzamido)-
3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulopyrano-
sid]onate (12h)
According to General procedure B, compound 11h
(32.0 mg, 47.1 lmol) was treated with 10% aqueous
NaOH. Compound 12h (21.0 mg, 75%) was obtained
as a colorless solid after purification.
4.39. Sodium [benzyl 5-acetamido-9-(2,4-dichorobenzam-
ido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulopyr-
anosid]onate (12k)
25
D
½aꢁ ꢀ 1:95 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
1.68 (t, J = 12.2 Hz, 1H, H-3a), 1.99 (s, 3H, NHC-
OCH3), 2.76 (dd, J = 4.6, 12.4 Hz, 1H, H-3e), 3.45
(dd, J = 8.0, 14.4 Hz, 1H, H-9a), 3.51 (d, J = 8.9 Hz,
1H, H-7), 3.64–3.84 (m, 5H, H-4, H-5, H-6, H-8, H-
9b) 4.51, 4.69 (A, B of AB, J = 11.2 Hz, 2H, CH2Ph),
7.26–7.36, 7.46, 7.69 (m, 9H, C6H5, C6H4); 13C NMR
(125 MHz, D2O): d 22.3 (NHCOCH3), 40.9 (C-3), 43.0
(C-9), 52.2 (C-5), 67.7 (CH2Ph), 68.6 (C-4), 70.3 (C-7),
70.6 (C-8), 73.0 (C-6), 101.6 (C-2), 128.5, 128.9, 129.0,
129.0, 129.1, 132.4, 137.6, 137.9 (12C, C6H5, C6H4),
170.4, 173.5, 176.5 (3 CO); HR-MS Calcd for
C25H28ClN2Na2O9 [M+Na+]: 581.1279; Found: 581.1278.
According to General procedure B, compound 11k
(31.0 mg, 42.9 lmol) was treated with 10% aqueous
NaOH. Compound 12k (18.0 mg, 72%) was obtained
as a colorless solid after purification.
25
D
½aꢁ ꢀ 1:17 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
1.66 (t, J = 12.1 Hz, 1H, H-3a), 2.01 (s, 3H,
NHCOCH3), 2.75 (dd, J = 4.7, 12.3 Hz, 1H, H-3e),
3.53–3.83 (m, 6H, H-4, H-5, H-6, H-7, H-9), 3.89 (m,
1H, H-8), 4.49, 4.71 (A, B of AB, J = 11.0 Hz, 2H,
CH2Ph), 7.33–7.41, 7.55 (m, 8H, C6H5, C6H3); 13C
NMR (125 MHz, D2O): d 20.0 (NHCOCH3), 38.5 (C-
3), 40.1 (C-9), 49.9 (C-5), 65.1 (CH2Ph), 66.1 (C-4),
67.4 (C-7), 67.9 (C-8), 70.5 (C-6), 98.9 (C-2), 125.4,
126.2, 126.6, 126.7, 127.1, 127.4, 127.6, 129.0, 131.3,
134.1, 135.0 (12C, C6H5, C6H3), 167.6, 171.4, 173.0 (3
CO); HR-MS Calcd for C25H28Cl2N2NaO9 [M+H+]:
593.1070; Found: 593.1070.
4.37. Sodium [benzyl 5-acetamido-9-(3,4-dichloroben-
zamido)-3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulo-
pyranosid]onate (12i)
According to General procedure B, compound 11i
(31.0 mg, 42.8 lmol) was treated with 10% aqueous
NaOH. Compound 12i (20.1 mg, 75%) was obtained
as a colorless solid after purification.
4.40. Benzyl 5-acetamido-3,5,9-trideoxy-9-(4-nitroben-
zamido)-D-glycero-a-D-galacto-2-nonulopyranosidic acid
(12l)
25
D
½aꢁ ꢀ 2:45 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
1.68 (t, J = 12.1 Hz, 1H, H-3a), 2.01 (s, 3H, NHC-
OCH3), 2.76 (dd, J = 4.6, 12.4 Hz, 1H, H-3e), 3.39
(dd, J = 14.4, 8.5 Hz, 1H, H-9a), 3.52 (m, 1H, H-7),
3.64–3.85 (m, 5H, H-4, H-5, H-6, H-8, H-9b), 4.47,
4.69 (A, B of AB, J = 11.1 Hz, 2H, CH2Ph), 7.21–7.32,
7.43, 7.53, 7.75 (m, 8H, C6H5, C6H3); 13C NMR
(125 MHz, D2O): d 22.4 (NHCOCH3), 41.0 (C-3), 43.2
(C-9), 52.2 (C-5), 67.6 (CH2Ph), 68.6 (C-4), 70.4 (C-7),
70.7 (C-8), 73.0 (C-6), 101.5 (C-2), 126.9, 128.5, 128.9,
129.4, 130.9, 132.5, 133.7, 135.8, 137.4 (12C, C6H5,
C6H3), 168.5, 173.7, 175.4 (3 CO); HR-MS Calcd for
According to General procedure B, compound 11l
(28.0 mg, 40.0 lmol) was treated with 10% aqueous
NaOH. After workup the crude material was purified
by reversed-phase chromatography (RP-18 column, 5%
gradient MeOH in water), and Sephadex G-15 size
exclusion chromatography to afford 12l (21.0 mg, 95%)
as colorless solid after a final lyophilization from water.
25
D
½aꢁ ꢀ 2:45 (c 1.00, H2O); 1H NMR (500 MHz, D2O): d
1.67 (t, J = 12.2 Hz, 1H, H-3a), 1.99 (s, 3H, NHC-
OCH3), 2.74 (dd, J = 4.7, 12.4 Hz, 1H, H-3e), 3.46
(dd, J = 7.7, 14.0 Hz, 1H, H-9a), 3.52 (dd, J = 1.7,
8.8 Hz, 1H, H-7), 3.67 (m, 1H, H-4), 3.74–3.84 (m,
4H, H-5, H-6, H-8, H-9b), 4.48, 4.67 (A, B of AB,
J = 11.2 Hz, 2H, CH2Ph), 7.23–7.34, 7.88, 8.25 (m,
9H, C6H4, C6H5); 13C NMR (125 MHz, D2O): d 20.4
(NHCOCH3), 39.0 (C-3), 41.3 (C-9), 50.3 (C-5), 65.7
(CH2Ph), 66.6 (C-4), 68.4 (C-7), 68.6 (C-8), 71.1 (C-6),
99.6 (C-2), 122.3, 126.6, 126.8, 127.0, 127.0, 135.6,
C25H28Cl2N2NaO9
593.1066.
[M+H+]:
593.1070;
Found:
4.38. Sodium [benzyl 5-acetamido-3,5,9-trideoxy-9-(4-
trifluoromethyl-benzamido)-D-glycero-a-D-galacto-2-non-
ulopyranosid]onate (12j)
According to General procedure B, compound 11j
(36.0 mg, 50.0 lmol) was treated with 10% aqueous

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4963
138.1, 147.8 (12C, C6H4, C6H5), 172.4, 173.5, 176.5 (3
CO); HR-MS Calcd for C25H28N3Na2O11 [M+Na+]:
592.1519; Found: 592.1512.
9a), 3.63–3.83 (m, 5H, H-4, H-5, H-6, H-8, H-9), 4.53, 4.71
(A, B of AB, J = 11.2 Hz, 2H, CH2Ph), 7.29–7.39, 7.44,
7.53, 7.62 (m, 9H, C6H5, C6H4); 13C NMR (125 MHz,
D2O): d 21.9 (NHCOCH3), 40.5 (C-3), 42.6 (C-9), 51.8
(C-5), 67.2 (CH2Ph), 68.2 (C-4), 69.8 (C-7), 70.1 (C-8),
72.6 (C-6), 101.2 (C-2), 125.3, 127.1, 128.1, 128.5, 128.5,
130.2, 131.8, 134.0, 135.3, 137.2 (12C, C6H5, C6H4),
169.5, 173.4, 175.0 (3 CO); HR-MS Calcd for
C25H28ClN2Na2O9 [M+Na+]: 581.1279; Found:
581.1276.
4.41. Sodium [benzyl 5-acetamido-3,5,9-trideoxy-9-(4-
methoxybenzamido)-D-glycero-a-D-galacto-2-nonulopyr-
anosid]onate (12m)
According to General procedure B, compound 11m
(25.0 mg, 36.9 lmol) was treated with 10% aqueous
NaOH. Compound 12m (16.0 mg, 80%) was obtained
as a colorless solid after purification.
4.44. Methyl (benzyl 5-acetamido-8,9-O-benzylidene-3,5-
dideoxy-D-glycero-a-D-galacto-2-nonulopyranosid)onate
(14)
25
D
1
½aꢁ ꢀ 3:22 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.68 (t, J = 12.2 Hz, 1H, H-3a), 1.98 (s, 3H, NHC-
OCH3), 2.76 (dd, J = 4.7, 12.4 Hz, 1H, H-3e), 3.44
(dd, J = 8.2, 14.6 Hz, 1H, H-9a), 3.51 (dd, J = 1.7,
8.7 Hz, 1H, H-7), 3.64–3.84 (m, 8H, H-4, H-5, H-6, H-
8, H-9b, OCH3), 4.51, 4.68 (A, B of AB, J = 11.2 Hz,
2H, CH2Ph), 7.01, 7.24–7.36, 7.74 (m, 9H, C6H5,
To a stirred solution of 6 (36.0 mg, 87.1 lmol) and a,a-
dimethoxytoluene (26.0 lL, 174 lmol) in MeCN (2 mL)
at 0 ꢁC was added p-TsOHÆH2O (1.30 mg, 6.70 lmol).
The mixture was stirred at rt for 1.5 h and then
quenched by adding a few drops of NEt3. After concen-
tration under reduced pressure, the residue (ꢂ50 mg)
was used directly in the next step. In accordance with,29a
C6H4); 13C NMR (125 MHz, D2O):
d
22.4
(NHCOCH3), 40.9 (C-3), 42.9 (C-9), 52.2 (C-5), 55.8
(OCH3), 67.7 (CH2Ph), 68.6 (C-4), 70.3 (C-7), 70.7 (C-
8), 73.0 (C-6), 101.6 (C-2), 114.3, 126.3, 128.5, 128.9,
129.0, 129.5, 137.6, 162.3 (12C, C6H5, C6H4), 170.6,
1
the H NMR data of 14 showed a 1:1 mixture of exo/
endo isomers.
173.8,
175.4
(3
CO);
HR-MS
Calcd
for
4.45. Methyl (benzyl 5-acetamido-9-O-benzyl-3,5-dide-
oxy-D-glycero-a-D-galacto-2-nonulopyranosid)onate (15)
C26H31N2Na2O10 [M+Na+]: 577.1774; Found: 577.1773.
4.42. Sodium [benzyl 5-acetamido-3,5,9-trideoxy-9-(4-
methylbenzamido)-D-glycero-a-D-galacto-2-nonulopyr-
anosid]onate (12n)
Compound 14 (87.1 lmol), BH3ÆNMe3 (25.4 mg,
348 lmol), and AlCl3 (69.6 mg, 523 lmol) were dis-
solved in THF (2 mL) under argon. After 5 min, H2O
(2.60 lL, 131 lmol) was added and the mixture was stir-
red at rt for 4 h. The reaction was quenched by adding
H2O (1 mL) and 0.1 N HCl (1 mL). The solution was di-
luted with DCM (10 mL) and washed with 5% aq NaH-
CO3 (2 · 5 mL) and H2O (5 mL). The organic layer was
dried (Na2SO4), filtered, and concentrated in vacuo. The
residue was purified by silica gel chromatography (1%
gradient MeOH in DCM) to afford 15 (34.8 mg, 70%
6 ! 15) as white foam.
According to General procedure B, compound 11n
(26.0 mg, 39.0 lmol) was treated with 10% aqueous
NaOH. Compound 12n (20.2 mg, 87%) was obtained
as a colorless solid after purification.
25
D
1
½aꢁ ꢀ 2:07 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.68 (t, J = 12.2 Hz, 1H, H-3a), 1.98 (s, 3H, NHCOCH3),
2.38 (s, 3H, PhCH3), 2.76 (dd, J = 4.6, 12.4 Hz, 1H, H-3e),
3.33–3.51 (m, 2H, H-7, H-9a), 3.67 (m, 1H, H-4), 3.72–
3.83 (m, 4H, H-5, H-6, H-8, H-9b), 4.53, 4.69 (A, B of
AB, J = 11.3 Hz, 2H, CH2Ph), 7.26, 7.36, 7.69 (m, 9H,
C6H5, C6H4); 13C NMR (125 MHz, D2O): d 20.9
(PhCH3), 22.3 (NHCOCH3), 40.9 (C-3), 42.9 (C-9), 52.2
(C-5), 67.7 (CH2Ph), 68.6 (C-4), 70.2 (C-7), 70.6 (C-8),
73.0 (C-6), 101.7 (C-2), 127.5, 128.5, 128.9, 129.0, 129.7,
131.0, 137.6, 143.6 (12C, C6H5, C6H4), 171.3, 173.8,
175.4 (3 CO); HR-MS Calcd for C26H31N2Na2O9
[M+Na+]: 561.1825; Found: 561.1821.
25
½aꢁ þ 4:8 (c 0.51, MeOH); 1H NMR (500 MHz,
D
CD3OD): d 1.80 (t, J = 12.5 Hz, 1H, H-3a), 2.00 (s,
3H, NHCOCH3), 2.74 (dd, J = 4.6, 12.8 Hz, 1H, H-
3e), 3.60 (dd, J = 1.5, 8.9 Hz, 1H, H-7), 3.63–3.69 (m,
3H, H-4, H-6, H-9a), 3.76 (s, 3H, OCH3), 3.78–3.83
(m, 2H, H-5, H-9b), 4.02 (ddd, J = 2.3, 5.8, 8.6 Hz,
1H, H-8), 4.50, 4.80 (A, B of AB, J = 11.7 Hz, 2H,
CH2Ph), 4.57, 4.59 (A, B of AB, 2H, CH2Ph), 7.24–
7.37 (m, 10H, 2 C6H5); 13C NMR (125 MHz, CD3OD):
d 22.6 (NHCOCH3), 41.7 (C-3), 53.2 (OCH3), 53.8 (C-
5), 67.2 (CH2Ph), 68.4 (C-4), 70.1 (C-7), 71.3 (C-8),
72.6 (C-9), 74.4 (CH2Ph), 74.8 (C-6), 100.1 (C-2),
128.5, 128.6, 128.8, 128.8, 129.2, 129.2, 138.8 (12C, 2
C6H5), 170.9, 175.1 (2 CO); Anal. Calcd for
C26H33NO9+1/2 H2O: C, 60.93; H, 6.69; N, 2.73.
Found: C, 61.14; H, 6.62; N, 2.65.
4.43. Sodium [benzyl 5-acetamido-9-(3-chlorobenzamido)-
3,5,9-trideoxy-D-glycero-a-D-galacto-2-nonulopyrano-
sid]onate (12o)
According to General procedure B, compound 11o
(28.0 mg, 41.1 lmol) was treated with 10% aqueous
NaOH. Compound 12o (16.0 mg, 69%) was obtained
as a colorless solid after purification.
4.46. Sodium (benzyl 5-acetamido-9-O-benzyl-3,5-dide-
oxy-D-glycero-a-D-galacto-2-nonulopyranosid)onate (16)
25
D
1
½aꢁ ꢀ 1:77 (c 1.00, H2O); H NMR (500 MHz, D2O): d
1.69 (t, J = 11.8 Hz, 1H, H-3a), 2.00 (s, 3H, NHCOCH3),
2.77 (dd, J = 4.6, 12.4 Hz, 1H, H-3e), 3.49 (m, 2H, H-7, H-
To a solution of 15 (35.0 mg, 69.5 lmol) in H2O/dioxane
(1:1, 4 mL) under argon was added LiOH (16.8 mg,

4964
S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
2. (a) Tello, F. Trab. Lab. Invest. Biol. 1911, 9, 123; (b)
Davies, S. J.; Goucher, D. R.; Doller, C.; Silver, J.
J. Neurosci. 1999, 19, 5810.
3. (a) Schwab, M. E.; Caroni, P. J. Neurosci. 1988, 8, 2381;
(b) Caroni, P.; Savio, T.; Schwab, M. E. Prog. Brain Res.
1988, 78, 363.
69.5 lmol). The mixture was stirred at rt for 1.5 h, then
neutralized with 0.5% HCl and concentrated. The residue
was purified by silica gel chromatography (10% gradient
MeOH in DCM), Dowex 50 · 8 (Na+ type) ion-exchange
chromatography, and Sephadex G-15 size exclusion
chromatography to afford 16 as a white solid (31.0 mg,
87%) after a final lyophilization from H2O.
4. (a) Caroni, P.; Schwab, M. E. Neuron 1988, 1, 85; (b)
Caroni, P.; Schwab, M. E. J. Cell Biol. 1988, 106, 128; (c)
Chen, M. S.; Huber, A. B.; van der Haar, M. E.; Frank,
M.; Schnell, L.; Spillmann, A. A.; Christ, F.; Schwab, M.
E. Nature 2000, 403, 434; (d) GrandPre, T.; Nakamura, F.;
Vartanian, T.; Strittmatter, S. M. Nature 2000, 403, 439.
5. (a) Kottis, V.; Thibault, P.; Mikol, D.; Xiao, Z. C.; Zhang,
R.; Dergham, P.; Braun, P. E. J. Neurochem. 2002, 82,
1566; (b) Wang, K. C.; Koprivica, V.; Kim, J. A.;
Sivasankaran, R.; Guo, Y.; Neve, R. L.; He, Z. Nature
2002, 417, 941.
6. (a) McKerracher, L.; David, S.; Jackson, D. L.; Kottis, V.;
Dunn, R. J.; Braun, P. E. Neuron 1994, 13, 805; (b)
Mukhopadhyay, G.; Doherty, P.; Walsh, F. S.; Crocker,
P. R.; Filbin, M. T. Neuron 1994, 13, 757; (c) Vinson, M.;
Strijbos, P. J. L. M.; Rowles, A.; Facci, L.; Moore, S. E.;
Simmons, D. L.; Walsh, F. S. J. Biol. Chem. 2001, 276,
20280.
25
D
½aꢁ ꢀ 16:1; (c 0.65, H2O); 1H NMR (500 MHz, D2O): d
1.68 (t, J = 12.2 Hz, 1H, H-3a), 2.03 (s, 3H, NHC-
OCH3), 2.77 (dd, J = 4.6, 12.4 Hz, 1H, H-3b), 3.60–
3.64 (m, 2H, H-7, H-9a), 3.67–3.73 (m, 2H, H-4, H-6),
3.78–3.82 (m, 2H, H-5, H-9b), 3.86 (ddd, J = 2.0, 6.3,
8.6 Hz, 1H, H-8), 4.49, 4.69 (A, B of AB, J = 11.0 Hz,
2H, CH2Ph), 4.58, 4.61 (A, B of AB, J = 12.0 Hz, 2H,
CH2Ph), 7.35–7.46 (m, 10H, 2 C6H5); 13C NMR
(125 MHz, D2O): d 22.0 (NHCOCH3), 40.5 (C-3), 51.9
(C-5), 67.1 (CH2Ph), 68.2, 68.4 (C-4, C-7), 70.4 (C-8),
70.8 (C-9), 72.6 (C-6), 73.0 (CH2Ph), 101.0 (C-2),
128.2, 128.3, 128.3, 128.7, 128.7, 128.7, 137.1, 137.5
(12C, 2 C6H5), 173.5, 175.1 (2 CO); HR-MS Calcd for
C25H31NNaO9 [M+H+]: 512.1897; Found: 512.1896.
7. Fournier, A. E.; GrandPre, T.; Strittmatter, S. M. Nature
2001, 409, 341.
4.47. In vitro binding assay
8. (a) Wang, K. C.; Kim, J. A.; Sivasankaran, R.; Segal, R.;
He, Z. Nature 2002, 420, 74; (b) Wong, S. T.; Henley, J.
R.; Kanning, K. C.; Huang, K. H.; Bothwell, M.; Poo, M.
M. Nat. Neurosci. 2002, 5, 1302.
9. Mi, S.; Lee, X.; Shao, Z.; Thill, G.; Ji, B.; Relton, J.;
Levesque, M.; Allaire, N.; Perrin, S.; Sands, B.; Crowell,
T.; Cate, R. L.; McCoy, J. M.; Pepinsky, R. B. Nat.
Neurosci. 2004, 7, 221.
A recombinant protein consisting of the N-terminal three
domains of MAG and the Fc part of human IgG (Fc-
MAGd1–3) was produced by expression in CHO cells
and affinity purification on protein A-agarose as de-
scribed.25 For the hapten inhibition assay a modification
of the previously described assay15a was used. Instead of
human erythrocytes as target for sialic acid-dependent
binding of MAG, commercially available microtiter
plates with immobilized Neu5Ac (Lundonia, Lund, Swe-
den) were used. In brief, to each well 10 lL of an oligosac-
charide solution was added followed by 20 lL of
Fc-MAGd1–3 which had been precomplexed with an
anti-Fc antibody labeled with alkaline phosphatase. After
an overnight incubation at 4 ꢁC unbound Fc-MAG com-
plexes were removed by washing and the amount of
bound Fc-MAG was determined via the alkaline phos-
phatase with fluorescein diphosphate as substrate. For
each oligosaccharide at least eight concentrations were
used to determine the concentration required for 50%
inhibition (IC50). In order to compare the results from dif-
ferent assays compound 13 was included in each test and
used as a reference to calculate the relative inhibitory
potencies (rIP). At least three independent titrations were
performed for each compound.
10. Yamashita, T.; Higuchi, H.; Tohyama, M. J. Cell. Biol.
2002, 157, 565.
11. (a) Kelm, S.; Pelz, A.; Schauer, R.; Filbin, M. T.; Tang, S.;
de Bellard, M. E.; Schnaar, R. L.; Mahoney, J. A.;
Hartnell, A.; Bradfield, P.; Crocker, P. R. Curr. Biol. 1994,
4, 965; (b) Crocker, P. R.; Clark, E. A.; Filbin, M. T.;
Gordon, S.; Jones, Y.; Kehrl, J. H.; Kelm, S.; Le Douarin,
N. M.; Powell, L.; Roder, J.; Schnaar, R. L.; Sgroi, D.;
Stamenkovic, I.; Schauer, R.; Schachner, M.; Tedder, T.;
van den Berg, T. K.; van der Merwe, P. A.; Watt, S. M.;
Varki, A. Glycobiology 1998, 8, Glycoforum 2 v–vi.
12. McKerracher, L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
7811.
13. Yang, L. J.-S.; Zeller, C. B.; Shaper, N. L.; Kiso, M.;
Hasegawa, A.; Shapiro, R. E.; Schnaar, R. L. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 814.
14. (a) Hotta, K.; Ishida, H.; Hasegawa, A. J. Carbohydr.
Chem. 1995, 14, 491; (b) Ito, H.; Ishida, H.; Ando, S.;
Kiso, M. Glycoconjugate J. 1999, 16, 585; (c) Ito, H.;
Ishida, H.; Kiso, M. J. Carbohydr. Chem 2001, 20, 207.
15. (a) Kelm, S.; Brossmer, R.; Isecke, R.; Gross, H.-J.;
Strenge, K.; Schauer, R. Eur. J. Biochem. 1998, 255, 663–
672; (b) Strenge, K.; Schauer, R.; Bovin, N.; Hasegawa,
A.; Ishida, H.; Kiso, M.; Kelm, S. Eur. J. Biochem. 1998,
258, 677; (c) Sawada, N.; Ishida, H.; Collins, B. E.;
Schnaar, R. L.; Kiso, M. Carbohydr. Res. 1999, 316, 1; (d)
Collins, B. E.; Ito, H.; Sawada, N.; Ishida, H.; Kiso, M.;
Schnarr, R. L. J. Biol. Chem. 1999, 274, 37637; (e)
Schwizer, D.; Ga¨thje, H.; Kelm, S.; Porro, M.; Schwardt,
O.; Ernst, B. Bioorg. Med. Chem. 2006, 14, 4944; (f) Kelm,
S.; Brossmer, R. PCT patent WO 03/000709A2.
16. Vyas, A. A.; Blixt, O.; Paulson, J. C.; Schnaar, R. L.
J. Biol. Chem. 2005, 280, 16305.
Acknowledgments
We thank the VW Foundation (center project grant con-
formational control of biomolecular functions) for
financial support. We would also like to thank Mr. Oleg
Khorev for proof-reading the manuscript.
References and notes
17. (a) van der Merve, P. A.; Crocker, P. R.; Vinson, M.;
Barclay, A. N.; Schauer, R.; Kelm, S. J. Biol. Chem. 1996,
1. Ramon y Cajal, S. Degeneration and Regeneration of the
Nervous System; Oxford Univ Press: London, 1928.

S. V. Shelke et al. / Bioorg. Med. Chem. 15 (2007) 4951–4965
4965
271, 9273; (b) Kelm, S.; Schauer, R.; Manuguerra, J. C.;
Gross, H. J.; Crocker, P. R. Glycoconj. J. 1994, 11, 576; (c)
Sjoberg, E. R.; Powell, L. D.; Klein, A.; Varki, A. J. Cell.
Biol. 1994, 126, 549; (d) Collins, B. E.; Yang, L. J.;
Mukhopadhyay, G.; Filbin, M. T.; Kiso, M.; Hasegawa,
A.; Schnaar, R. L. J. Biol. Chem. 1997, 272, 1248; (e)
Collins, B. E.; Kiso, M.; Hasegawa, A.; Tropak, M. B.;
Roder, J. C.; Crocker, P. R.; Schnaar, R. L. J. Biol. Chem.
1997, 272, 16889.
Handbook of Experimental Immunology, 5th ed.; Herzen-
berg, L. A.; Weir, D. M., Eds.; Blackwell Science: Boston,
1996; Vol. 4, p 166.1.
26. Bock, N.; Kelm, S. Binding and Inhibition Assay for
Siglecs. In Methods in Molecular Biology; Brockhausen,
I., Ed.; Humana Press Inc: Totowa N, 2006; vol. 347, p
359.
27. Schwardt, O.; Gao, G.-P.; Visekruna, T.; Rabbani, S.;
Gassmann, E.; Ernst, B. J. Carbohydr. Chem. 2004, 23, 1.
28. Attrill, H.; Takazawa, H.; Witt, S.; Kelm, S.; Isecke,
R.; Brossmer, R.; Ando, T.; Ishida, H.; Kiso, M.;
Crocker, P. R.; van Aalten, D. M. F. Biochem. J. 2006,
397, 271.
18. (a) Topliss, J. G. J. Med. Chem. 1972, 15, 1006; (b)
Topliss, J. G. J. Med. Chem. 1977, 20, 463.
19. Kelm, S.; Bock, N.; Strenge, K.; Brossmer, R. Synthetic
Sialic Acid Analogues Binding To Siglecs With High
Affinity And Selectivity. International Carbohydrate Sym-
posium: Cairns, Australia, 2002.
20. Hasegawa, A.; Ohki, H.; Nagahama, T.; Ishida, H.; Kiso,
M. Carbohydr. Res. 1991, 212, 277.
21. Ren, C.-T.; Chen, C.-S.; Wu, S.-H. J. Org. Chem. 2002, 67,
1376.
22. Fitz, W.; Rosenthal, P. B.; Wong, C. H. Bioorg. Med.
Chem. 1996, 4, 1349.
23. Hossain, N.; Zapata, A.; Wilstermann, M.; Nilsson, U. J.;
Magnusson, G. Carbohyr. Res. 2002, 337, 569.
24. Boullanger, P.; Maunier, V.; Lafont, D. Carbohydr. Res.
2000, 324, 97.
29. (a) Demchenko, A. V.; Boons, G.-J. Chem. Eur. J. 1999, 5,
1278; (b) De Meo, C.; Demchenko, A. V.; Boons, G.-J.
J. Org. Chem. 2001, 66, 5490.
30. (a) Hansch, C.; Fujita, T. J. Am. Chem. Soc. 1964, 86,
1616; (b) Hansch, C. In Drug Design; Ariens, E. J., Ed.;
Academic Press: New York, 1971; Vol. 1, p 271.
31. (a) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.;
Feeney, P. J. Adv. Drug Deliv. Rev. 1997, 23, 3; (b)
Veber, D. F.; Johnson, S. R.; Cheng, H.-Y.; Smith, B.
R.; Ward, K. W.; Kopple, K. D. J. Med. Chem. 2002,
45, 2615.
32. (a) Bieberich, E.; Liour, S. S.; Yu, R. K. Meth. Enzymol.
2000, B, 339; (b) Kopitz, J.; Sinz, K.; Brossmer, R.; Cantz,
M. Eur. J. Biochem. 1997, 248, 527.
25. Crocker, P. R.; Kelm, S. Methods for studying the cellular
binding properties of lectin-like receptors. In Weir’s