Studies on selectin blocker. 1. Structure-activity relationships of sialyl Lewis X analogs

Article abstract of DOI:10.1021/jm9506478

As a part of our studies of selectin blockers, we prepared 1-deoxy-3'-O- sulfo Le(x) analogs (1-3), 1-deoxy-3'-O-phosphono Le(x) analogs (4), and 1- deoxy sLe(x) analogs (5-7), and examined their inhibitory activities against natural ligand (sLe(x)) bindi

Full text of DOI:10.1021/jm9506478

J . Med. Chem. 1996, 39, 1339-1343
1339
Stu d ies on Selectin Block er . 1. Str u ctu r e-Activity Rela tion sh ip s of Sia lyl
Lew is X An a logs
Hiroshi Ohmoto,† Kenji Nakamura,† Tomomi Inoue,† Noriko Kondo,† Yoshimasa Inoue,‡ Kohichiro Yoshino,† and
Hirosato Kondo*,‡
Department of Biology, The Institute of Cancer Research Laboratories, Department of Medicinal Chemistry, New Drug
Research Laboratories, Kanebo Ltd., 5-90 Tomobuchi-Cho, Miyakojima-Ku, Osaka 534, J apan
Hideharu Ishida,§ Makoto Kiso,§ and Akira Hasegawa*,§
Department of Applied Bioorganic Chemistry, Gifu University, Gifu 501-11, J apan
Received August 30, 1995X
As a part of our studies of selectin blockers, we prepared 1-deoxy-3′-O-sulfo Lex analogs (1-3),
1-deoxy-3′-O-phosphono Lex analogs (4), and 1-deoxy sLex analogs (5-7), and examined their
inhibitory activities against natural ligand (sLex) binding to E-selectin, P-selectin, and L-selectin.
The 1-deoxy sLex 5 was up to 20 times more potent an inhibitor than the sLex tetrasaccharide
toward P- and L-selectin binding. This indicates that the modification of the 1 or 2 position of
sLex is useful in the design of a more potent selectin blocker.
Cell adhesion molecules (CAMs) mediate the regula-
tion of inflammation.1 The selectins are a family of
CAMs that plays an important role in the initial
interactions of leukocyte homing, platelet binding, and
neutrophil extravasation.2
Lex analogs (4), and 1-deoxy sLex analogs (5-7) (see
Chart 1) and examined their inhibitory activities toward
the natural ligand (sLex) binding to each of the selectins.
Resu lts a n d Discu ssion
Recent studies have indicated the involvement of
selectin-oligosaccharide interactions in various inflam-
matory diseases.3 It is known that E-selectin (ELAM-
1), P-selectin (GMP-140), and L-selectin (LECAM-1)
play important roles in the migration of inflammatory
cells from the blood stream to inflammatory sites. The
selectin family is expressed on a variety of cell surfaces.
For example, ELAM-1 is an adhesion molecule that is
expressed on vascular endothelial cells during inflam-
mation.4 GMP-140 is an adhesion molecule that is
expressed on platelets and vascular endothelial cells,5
and LECAM-1 is an adhesion molecule expressed on
leukocytes.6 These selectins are believed to be involved
in the progression of the clinical manifestations of
complicated diseases, such as chronic inflammation.7
Attempts have been made, therefore, to find a selectin
blocker that effectively inhibits their cell-adhesion
activities at an early stage of inflammation.8 To this
end, it would be desirable for the blocker to exert its
cell adhesion-inhibitory effects on all members of the
selectin family.
Phillips et al. reported that the native ligand for
ELAM-1 is the tetrasaccharide sialyl Lewis X (sLex).9
Although the natural ligand for each selectin has not
been completely characterized, it was reported recently
that both P-selectin and L-selectin can also recognize
the sLex structure.10 Since then, a number of its
derivatives have been reported.11 These derivatives
have activities similar to that of sLex against the natural
ligand (sLex)-E-selectin binding. However, there are
few reports regarding their inhibitory activities toward
either sLex-P-selectin or -L-selectin binding. As a part
of our studies of selectin blockers, we prepared 1-deoxy-
3′-O-sulfo Lex analogs (1-3), 1-deoxy-3′-O-phosphono
Ch em istr y. Compounds 1-7 were synthesized ac-
cording to published procedures.12 For the synthesis of
compound 1, the key intermediate 8 was treated with
a sulfur trioxide pyridine complex in DMF for 1 h at
room temperature to afford the sulfated Lex analog 9,
and this was transformed quantitatively by the removal
of the protecting groups into the desired compound 1
(Scheme 1). Compounds 2 and 3 were synthesized from
the corresponding intermediates 10 and 12 by a similar
method.
For the synthesis of compound 4, the key intermediate
12 was treated with diphenyl chlorophosphate in pyri-
dine for 12 h at room temperature to give the 3′-O-
diphenylphosphono derivative 14, which upon hydro-
genolysis of the phenyl groups in the presence of a
prereduced Adams platinum catalyst, and subsequent
treatment with sodium methoxide in methanol gave the
3′-O-phosphono Lex analog 4 in good yield.
For the synthesis of compound 5, hydrogenolysis of
the benzyl groups in 15 and subsequent acetylation gave
the protected sLex analog 16 in an 85% yield (Scheme
2). Compound 16 was deacylated with a sodium meth-
oxide in methanol, and hydrolysis of the methyl group
quantitatively yielded the desired sLex analog 5. Com-
pounds 6 and 7 were synthesized from the correspond-
ing intermediates 17 and 19, respectively.
Biologica l Activities. The method using selectin-
IgG chimeras reported by Foxall et al. was followed.10c
First, the sLex tetrasaccharide was found to inhibit
the binding of the ligand (sLex pentasaccharide ceram-
ide) to purified human E-, P-, and L-selectin-Ig, with
IC50 values of 0.6, > 1.0, and > 1.0 mM, respectively.
As shown in Table 1, compounds 1-3, the 1-deoxy-
3′-O-sulfo Lex analogs, were all less potent (IC50 > 1.0
mM) than sLex (IC50 0.6 mM) in the ligand-E-selectin
competitive binding assay. In contrast, compounds 2
† The Institute of Cancer Research Laboratories.
‡ Kanebo New Drug Research Laboratories.
§ Gifu University.
X Abstract published in Advance ACS Abstracts, February 15, 1996.
0022-2623/96/1839-1339$12.00/0 © 1996 American Chemical Society

1340 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Ohmoto et al.
Ch a r t 1
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) 10% Pd-C, EtOH, AcOH, Ac2O-
pyridine (16, 85%; 18, 73%); (b) NaOMe, MeOH, Amberlite IR-
120(H+) resin (5, 100%; 6, 100%); (c) 10% Pd-C, EtOH, AcOH,
NaOMe, MeOH, KOH, Amberlite IR-120(H+) resin (7, 100%).
a
Reagents and conditions: (a) pyr‚SO3, DMF (9, 93%; 11, 85%;
sLex. These results indicate that the binding mode of
the 1-deoxy sLex analogs (5-7) to each selectin is
different from that of the 1-deoxy-3′-O-sulfo Lex analogs
(1-3).
13, 100%); (b) NaOMe, MeOH (1, 100%; 2, 98%; 3, 100%); (c)
ClPO(OPh)2, pyridine MeOH, (14, 100%); (d) PtO2, EtOH, NaMe
(4, 89%).
and 3 were more potent (IC50 0.56 mM for 2, 0.67 mM
for 3) than sLex (IC50 >1.0 mM) against the P-selectin
in the competitive binding assay, which demonstrated
that the substitution of the 2-position of 1-deoxy-3′-O-
sulfo Lex was not essential for binding to P-selectin.
As shown in Table 1, compounds 5-7, the 1-deoxy
sLex analogs, were examined for their inhibitory activi-
ties toward ligand binding to each of the selectins.
Compounds 6 and 7 inhibited the ligand-E-selectin
binding at IC50 values of 0.36 and 0.24 mM, respectively.
Interestingly, compound 5, which lacks the 1-hydroxyl
group from sLex, very strongly inhibited P-selectin and
L-selectin-ligand binding, with IC50 values of 0.044 and
0.035 mM, respectively. In contrast, the inhibitory
effect of 5 for E-selectin was less potent than that of
The solution conformations of sLex have been reported
by several groups.13 It was further proposed that the
fucose and galactose moieties, as well as the negatively
charged sialic acid residues of sLex, would be critical
for binding to E-selectin.13b The carboxylate group,
therefore, serves to mimic the sialic acid in sLex as a
type of negative charge, such as that within a sulfonate
or phosphonate group. Our results of the blocking
activities of compounds 3 (sulfonate), 4 (phosphonate),
and 7 (sialic acid), shown in Table 1, indicate that the
sialic acid residue was most favorable for the recognition
by E-selectin; however, it was not necessarily optimal
for the recognition by P-selectin and L-selectin. It was
found that 1-deoxy-3′-O-sulfo Lex 2, 3, and 1-deoxy sLex
5 inhibited the P-selectin-ligand binding more potently

Studies on Selectin Blocker
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1341
Ta ble 1. Blocking Activity of Compounds 1-7 and sLex
F igu r e 1. Schematic diagram of ELISA inhibition assay using
selectin-Ig chimera. For details, see the Experimetal Section.
cDNA in pCIneo vector (Promega) by the lipofectamine (Gibco
BRL). Selectin-Igs were affinity-purified from culture media
using protein A silica gel (Nihon Gaishi Co.).
IC50, mM
In h ibition Assa y of E-, P -, a n d L-selectin -sLex Bin d -
in g. A solution of sLex-pentaceramide in a 1:1 mixture of
methanol and distilled water was pipetted into microtiter plate
wells (96 wells, Falcon PRO-BIND) at 100 pmol/50 µL per well
and adsorbed by evaporating the solvent. The wells were
washed twice with distilled water, blocked with 5% BSA
(bovine serum albumin)-PBS (phosphate buffered saline) for
1 h at room temperature, and washed with PBS three times.
Separately, a 1:1 volumetric mixture of a 1:500 dilution in
1% BSA-PBS of biotin-anti-human IgG (Fc) (BioSource
International Inc., Lot 1201)/streptavidin-alkaline phos-
phatase (Zymed Lab Inc., Lot 50424702) and a E-selectin-
immunoglobulin fusion protein (E-selectin-Ig) was incubated
at room temperature for 30 min to form a complex. The test
compounds were dissolved in distilled water at 1.0 mM and
finally diluted to final concentrations of 100, 25, 6.25, and 1.56
µM, respectively. Reactant solutions were prepared by incu-
bating 30 µL of this solution at each concentration with 30
µL of the above complex solution for 30 min at room temper-
ature. This reactant solution was then added to the above
microtiter wells at 50 µL/well and incubated at 37 °C for 45
min. The wells were washed three times with PBS and
distilled water, and the mixture was added to the solution of
p-nitrophenyl phosphate (1 mg/mL) and 0.01% of MgCl2 in 1
M diethanolamine (pH 9.8) at 50 µL/well. The reactant
mixture was developed for 120 min at 23 °C, and the absor-
bance at 405 nm was measured. Percent binding was calcu-
lated by the following equation:
compd
R1
R2
E-selectin P-selectin L-selectin
1
2
3
4
5
NHAc SO3Na
OH
H
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
0.67
0.56
>1.0
0.044
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
SO3Na
SO3Na
PO3Na
H
0.42
NHAc NeuAca
OH
H
0.035
0.22
>1.0
6
NeuAc
NeuAc
0.24
7
0.36
0.60
sLex
>1.0
a
NeuAc, sialic acid.
than sLex. In addition, 1-deoxy-3′-O-phosphono Lex 4
and 1-deoxy sLex 5 and 6 were more effective inhibitors
of the L-selectin-ligand binding than sLex. These
results suggest that the combination of the negative
charge of the 3′-O position and the substituent at the 2
position of 1-deoxy Lex skeleton is very important for
the recognition of the P- and L-selectins by the ligand.
Especially, 1-deoxy sLex 5 was up to the 20 times more
potent than sLex tetrasaccharide toward P- and L-
selectin binding.
This study supports the proposition that the interac-
tion between the ligand and each selectin becomes
favorable by the removal of the 1-hydroxyl group in sLex,
and the hydrophobicity of the 1-position of GlcNAc may
be important for tight binding to each selectin. Our
data indicate that the modification of the 1- or 2-position
of sLex could be useful for the design of more potent
selectin blockers, which could be specific to each selectin
or generally used toward all selectins.
% binding ) (X - C/A - C) × 100
where X is the absorbance of wells containing the test
compounds at each concentration, C is the absorbance of wells
not containing the selectin-Ig and test compounds, and A is
the absorbance of control wells not containing the test com-
pounds. Inhibition of P- or L-selectin-sLex binding was
repeated except that P-selectin-Ig or L-selectin-Ig was
replaced for E-selectin-Ig. The results of inhibitory activities
are presented in Table 1 as IC50 values, the concentrations of
blockers necessary to cause 50% inhibition of the ligand
binding to each of the selectins by the compounds 1-7. The
number of replicates is two. For a schematic representation
of the ELISA blocking assay, see Figure 1.
O-(2,4,6-Tr i-O-ben zoyl-3-O-su lfo-â-D-galactopyr an osyl)-
(1-4)-O-[(2,3,4-tr i-O-a cetyl-r-L-fu cop yr a n osyl)-(1-3)]-2-
a ceta m id o-1,5-a n h yd r o-6-O-ben zoyl-D-glu citol P yr id in e
Sa lt (9). To a solution of 812a (33 mg, 0.031 mM) in DMF (0.3
mL) was added sulfur trioxide pyridine complex (25 mg), and
the mixture was stirred for 1 h. MeOH (0.1 mL) was added
to the mixture and concentrated at 25 °C. Column chroma-
tography (15:1 CH2Cl2-MeOH) of the residue on silica gel (10
g) gave 9 (35 mg, 93%) as an amorphous mass: [R]D -5.5° (c
1.2, CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.01 (d, J ) 6.2 Hz,
3H, H-6b), 1.88-2.08 (4s, 12H, 3AcO, AcN), 7.10-8.10 (m, 25H,
4Ph, pyridine). Anal. Calcd (C59H62N2O24S) C, H, N.
Exp er im en ta l Section
Con str u ction , Exp r ession , a n d P u r ifica tion of Selec-
tin -Im m u n oglobu lin F u sion P r otein . The construction of
selectin-immunoglobulin was carried out according to the
previous paper.10c
Selectin-immunogloblin fusion proteins (selectin-Ig) used
in ELISA assays are recombinant chimeric molecules contain-
ing the lectin domain, epidermal growth factor domain, and
two L-selectin-Ig, two P-selectin-Ig, or two E-selectin-Ig
complement reguratory repeats coupled to the hinge, CH2, and
CH3 regions of human IgG1. The corresponding E-selectin
cDNA and P-selectin cDNA domain was amplified from HU-
VEC mRNA by RT-PCR. L-selectin domain was amplified
from J urkat cells mRNA by RT-PCR. Selectin cDNAs were
fused to the hinge and Fc region of human IgG1 heavy chain.
Selectin-Ig was expressed in COS7 (American Type Culture
Collection CRL 1651) cells by transient transfection with
selectin-Ig cDNA in pCDM8 vector (Funakoshi Co.) or ex-
pressed in CHO cells by stable transfection with selectin-Ig

1342 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6
Ohmoto et al.
O-(3-O-Su lfo-â-D-galactopyr an osyl)-(1-4)-O-[r-L-fu copy-
r a n osyl-(1-3)]-2-a cet a m id o-1,5-a n h yd r o-D-glu cit ol So-
d iu m Sa lt (1). To a solution of 912a (50 mg, 0.041 mM) in
MeOH (1 mL) was added NaOMe (5 mg), and the mixture was
stirred overnight at room temperature then concentrated at
30 °C. Column chromatography (4:1 MeOH-H2O) of the
residue on Sephadex LH-20 (30 g) gave 1 (25.5 mg, 100%) as
an amorphous mass: [R]D -24.0° (c 0.6, 1:1 MeOH-H2O); 1H
NMR (270 MHz, D2O) δ 1.20 (d, J ) 6.6 Hz, 3H, H-6b), 2.25
(s, 3H, AcN), 3.33 (t, J ) 11.2 Hz, 1H, H-1a), 4.58 (d, J ) 7.9
Hz, 1H, H-1c). The mass spectrum of 1 (negative ion mode)
showed the base peak at m/z 592.5 (M - H)-.
mass spectrum of 4 (negative ion mode) showed the base peak
at m/z 534.9 (M - 2H)2-.
O-(Meth yl 5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-3,5-d id e-
oxy-D-glycer o-r-D-ga la cto-2-n on u lop yr a n oson a te)-(2-3)-
O-(2,4,6-t r i-O-b e n zoyl-â-D-ga la ct op yr a n osyl)-(1-4)-O-
[(2,3,4-tr i-O-acetyl-r-L-fu copyr an osyl)-(1-3)]-2-acetam ido-
1,5-an h ydr o-6-O-ben zoyl-2-deoxy-D-glu citol (16). A solution
of 1512a (158 mg, 0.095 mM) in EtOH (20 mL) and AcOH (5
mL) was stirred with 10% Pd-C (160 mg) overnight at room
temperature under hydrogen, then filtered, and concentrated.
The residue was treated with Ac2O (1 mL) and pyridine (2 mL)
overnight at room temperature and concentrated. Column
chromatography (50:1 CH2Cl2-MeOH) of the product on silica
gel (30 g) gave 16 (120 mg, 85%) as an amorphous mass: [R]D
O-(4-O-Acet yl-2,6-d i-O-b en zoyl-3-O-su lfo-â-D-ga la ct o-
pyr an osyl)-(1-4)-O-[(2,3,4-tr i-O-acetyl-r-L-fu copyr an osyl)-
(1-3)]-1,5-a n h yd r o-2,6-d i-O-ben zoyl-D-glu citol P yr id in e
Sa lt (11). To a solution of 1012b(126 mg, 0.12 mM) in DMF (1
mL) was added sulfur trioxide pyridine complex (95 mg), and
the mixture was stirred for 1 h. MeOH (0.2 mL) was added
to the mixture and concentrated at 25 °C. Column chroma-
tography (20:1 CH2Cl2-MeOH) of the residue on silica gel (60
g) gave 11 (121 mg, 85%) as an amorphous mass: [R]D -2.0°
1
+0.5° (c 1.5, CHCl3); H NMR (270 MHz, CDCl3) δ 1.12 (d, J
) 6.4 Hz, 3H, H-6b), 1.49-2.06 (9s, 27H, 7AcO, 2AcN), 2.40
(dd, J ) 12.5 Hz, 4.5 Hz), 3.78 (s, 3H, MeO), 4.77 (m, 1H, H-4d),
5.35 (d, J ) 3.8 Hz,1H, H-1b), 5.48 (dd, J ) 8.1, 10.1 Hz, 1H,
H-2c), 5.65 (m, 1H, H-8d), 7.26-8.15 (m, 20H, 4Ph). Anal.
Calcd C74H83N2O32) C, H, N.
O-(5-Acet a m id o-3,5-d id eoxy-D-glycer o-r-D-ga la cto-2-
n on u lop yr a n osylon ic a cid )-(2-3)-O-â-D-ga la ctop yr a n o-
syl-(1-4)-O-[R-L-fu cop yr a n osyl-(1-3)]-2-a cet a m id o-1,5-
a n h yd r o-2-d eoxy-D-glu citol (5). To a solution of 16 (80 mg,
0.054 mM) in MeOH (2 mL) was added NaOMe (6 mg), and
the mixture was stirred overnight at room temperature.
Water (1 mL) was added to the mixture, and this was stirred
for 5 h at room temperature, neutralized with Amberlite IR-
120 (H+) resin, and filtered, the resin was washed with MeOH,
and the combined filtrate and washings were concentrated.
Column chromatography (MeOH) of the residue on Sephadex
LH-20 (30 g) gave 5 (42 mg, 100%) as an amorphous mass:
1
(c 0.9, CHCl3); H NMR (270 MHz, CDCl3) δ 1.29 (d, J ) 6.4
Hz, 3H, H-6b), 1.85-2.15 (4s, 12H, 4AcO), 3.24 (t, J ) 10.6
Hz, 1H, H-1a), 5.14 (dd, J ) 11.1 Hz, 4.0 Hz, 1H, H-3b), 5.45
(d, J ) 3.4 Hz, 1H, H-4c), 7.13-8.06 (m, 25H, 4Ph, pyridine).
Anal. Calcd (C59H61NO25S) C, H, N.
O-(3-O-Su lfo-â-D-galactopyr an osyl)-(1-4)-O-[r-L-fu copy-
r a n osyl-(1-3)]-1,5-a n h yd r o-D-glu citol Sod iu m Sa lt (2). To
a solution of 11 (120 mg, 0.1 mM) in MeOH (2 mL) was added
NaOMe (20 mg), and the mixture was stirred overnight at
room temperature and then concentrated at 25 °C. Column
chromatography (4:1 MeOH-H2O) of the residue on Sephadex
LH-20 (60 g) gave 2 (56 mg, 98%) as an amorphous mass: [R]D
-9.7° (c 1.1, 1:1 MeOH-H2O); 1H NMR (270 MHz, D2O) δ 1.53
(d, J ) 6.6 Hz, 3H, H-6b), 3.63 (t, J ) 10.6 Hz, 1H, H-1a). The
mass spectrum of 2 (negative ion mode) showed the base peak
at m/z 550.5 (M - H)-.
1
[R]D -25.0° (c 0.7, MeOH); H NMR (270 MHz, DMSO-d6) δ
1.12 (d, J ) 6.2 Hz, 3H, H-6b), 1.89, 1.98 (2s, 6H, 2AcN), 3.19
(t, 1H, H-1a), 4.42 (d, J ) 7.6 Hz, 1H, H-1c), 5.02 (d, J ) 2.8
Hz, 1H, H-1b). Anal. Calcd (C31H52N2O22) C, H, N.
O-(Meth yl 5-a ceta m id o-4,7,8,9-tetr a -O-a cetyl-3,5-d id e-
oxy-D-glycer o-r-D-ga la cto-2-n on u lop yr a n oson a te)-(2-3)-
O-(4-O-a cetyl-2,6-d i-O-ben zoyl-â-D-ga la ctop yr a n osyl)-(1-
4)-O-[(2,3,4-t r i-O-a cet yl-R-L-fu cop yr a n osyl)-(1-3)]-1,5-
a n h yd r o-2,6-d i-O-ben zoyl-D-glu citol (18). A solution of
1712b (98 mg, 0.06 mM) in EtOH (10 mL) and AcOH (2 mL)
was stirred with 10% Pd-C (100 mg) for 2 days at 40 °C under
hydrogen. The precipitate was collected, and the solution was
concentrated. Treatment of the residue with Ac2O (1 mL) and
pyridine (2 mL) in the presence of 4-dimethylaminopyridine
(5 mg) occurred overnight at 45 °C. MeOH was added to the
mixture, and the mixture was concentrated. Column chro-
matography (200:1 CH2Cl2-MeOH) of the residue on silica gel
gave 18 (66 mg, 73%) as an amorphous mass: [R]D +14.5° (c
1.0, CHCl3); 1H NMR (270 MHz, CDCl3) δ 1.44 (d, J ) 6.6 Hz,
3H, H-6b), 1.45-2.13 (9s, 27H, 8AcO, AcN), 2.52 (dd, J ) 12.4
Hz, 4.4 Hz), 3.19 (t, J ) 10.6 Hz, 1H, H-1a), 3.68 (s, 3H, MeO),
7.27-8.17 (m, 20H, 4Ph). Anal. Calcd (C74H83NO34) C, H, N.
O-(5-Acet a m id o-3,5-d id eoxy-D-glycer o-r-D-ga la cto-2-
n on u lop yr a n osylon ic a cid )-(2-3)-O-â-D-ga la ctop yr a n o-
syl-(1-4)-O-[r-L-fu cop yr a n osyl-(1-3)]-1,5-a n h yd r o-D-glu -
citol (6). To a solution of 18 (53 mg, 0.033 mM) in MeOH (2
mL) was added NaOMe (10 mg), and the mixture was stirred
overnight at room temperature. Water (0.5 mL) was added
to the mixture, and this was stirred for a further 12 h at room
temperature, neutralized with Amberlite IR-120 (H+) resin,
and filtered, the resin was washed with MeOH, and the
combined filtrate and washings were concentrated. Column
chromatography (MeOH) of the residue on Sephadex LH-20
(30 g) gave 6 (26 mg, 100%) as an amorphous mass: [R]D
O-(4-O-Acet yl-2,6-d i-O-b en zoyl-3-O-su lfo-â-D-ga la ct o-
pyr an osyl)-(1-4)-O-[(2,3,4-tr i-O-acetyl-r-L-fu copyr an osyl)-
(1-3)]-1,5-a n h yd r o-6-O-ben zoyl-2-d eoxy-D-a r a bin o-h ex-
itol P yr id in e Sa lt (13). To a solution of 1212b (150 mg, 0.16
mM) in DMF (0.3 mL) was added sulfur trioxide pyridine
complex (127 mg), and the mixture was stirred for 1 h. MeOH
(0.2 mL) was added to the mixture and concentrated at 25 °C.
Column chromatography (20:1 CH2Cl2-MeOH) of the residue
on silica gel (60 g) gave 13 (173 mg, 100%) as an amorphous
mass: [R]D -23.0° (c 1.0, CHCl3); 1H NMR (270 MHz, CDCl3)
δ 1.29 (d, J ) 6.4 Hz, 3H, H-6b), 1.85-2.15 (4s, 12H, 4AcO),
7.23-8.57 (m, 20H, 3Ph, pyridine). Anal. Calcd (C52H57NO23S)
C, H, N.
O-(3-O-Su lfo-â-D-galactopyr an osyl)-(1-4)-O-[r-L-fu copy-
r an osyl-(1-3)]-1,5-an h ydr o-2-deoxy-D-a r a bin o-h exitol So-
d iu m Sa lt (3). To a solution of 13 (173 mg, 0.16 mM) in
MeOH (4 mL) and THF (2 mL) was added NaOMe (10 mg),
and the mixture was stirred overnight at room temperature
and then concentrated at 25 °C. Column chromatography (4:1
MeOH-H2O) of the residue on Sephadex LH-20 (60 g) gave 3
(89.7 mg, 100%) as an amorphous mass: [R]D -92.5° (c 0.8,
1
MeOH); H NMR (270 MHz, D2O) δ 1.03 (d, J ) 6.4 Hz, 3H,
H-6b), 4.41 (d, J ) 7.5 Hz, 1H, H-1c), 4.44 (m, 1H, H-5b), 4.71
(d, J ) 3.4 Hz, 1H, H-1b). The mass spectrum of 3 (negative
ion mode) showed the base peak at m/z 534.9 (M - H)-.
O-(3-O-P h osp h on o-â-D-ga la ctop yr a n osyl)-(1-4)-O-[r-L-
fu copyr an osyl-(1-3)]-1,5-an h ydr o-2-deoxy-D-a r a bin o-h ex-
itol Sod iu m Sa lt (4). A solution of 1412b (187 mg, 0.16 mM)
in EtOH (8 mL) was stirred for 6 h at room temperature in
the presence of prereduced Adams platinum catalyst (200 mg)
under hydrogen. The catalyst was collected, and the solution
was concentrated. To a solution of the residue in dry MeOH
(5 mL) was added NaOMe (20 mg), and the mixture was stirred
for 24 h at room temperature then concentrated at 25 °C.
Column chromatography (1:1 MeOH-H2O) of the residue on
Sephadex LH-20 (80 g) gave 4 (80.5 mg, 89%) as an amorphous
1
+20.5° (c 0.5, MeOH); H NMR (270 MHz, DMSO-d6) δ 0.97
(d, J ) 6.4 Hz, 3H, H-6b), 1.84 (s, 3H, AcN), 2.45 (dd, J )
13.2, 3.2 Hz, H-3d), 4.25 (d, J ) 7.7 Hz, 1H, H-1c), 5.14 (d, J
) 3.2 Hz, 1H, H-1b). Anal. Calcd (C29H49NO22) C, H, N.
O-(5-Acet a m id o-3,5-d id eoxy-D-glycer o-r-D-ga la cto-2-
n on u lop yr a n osylon ic a cid )-(2-3)-O-â-D-ga la ctop yr a n o-
syl-(1-4)-O-[r-L-fu cop yr a n osyl-(1-3)]-1,5-a n h yd r o-2-d e-
oxy-D-glu citol (7). A solution of 1912b (130 mg, 0.086 mM)
in EtOH (15 mL) and AcOH (5 mL) was hydrogenolyzed in
the presence of 10% Pd-C (130 mg) for 48 h at 40 °C, then
1
mass: [R]D -23.5° (c 1.1, MeOH-H2O); H NMR (270 MHz,
D2O) δ 1.37 (d, J ) 6.6 Hz, 3H, H-6b), 4.67 (d, J ) 7.7 Hz, 1H,
H-1c), 4.80 (m, 1H, H-5b), 5.19 (d, J ) 3.9 Hz, 1H, H-1b). The

Studies on Selectin Blocker
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 6 1343
x. Science 1990, 250, 1130-1132. (b) Walz, G.; Aruffo, A.;
Kolanus, W.; Bevilacqua, M.; Seed, B. Recognition by ELAM-1
of the Sialyl-Lex Determinant on Myeloid and Tumor Cells.
Science 1990, 250, 1132-1134.
Le
filtered and concentrated. To a solution of the residue in
MeOH (5 mL) was added NaOMe (20 mg), and the mixture
was stirred for 24 h at room temperature. Potassium hydrox-
ide (0.2 M, 3 mL) was added the mixture, and this was stirred
for 6 h at room temperature, neutralized with Amberlite IR-
120 (H+) resin, and filtered, the resin was washed with MeOH,
and the combined filtrate and washings were concentrated.
Column chromatography (MeOH) of the residue on Sephadex
LH-20 (30 g) gave 7 (64 mg, 100%) as an amorphous mass:
(10) (a) Watson, S. A.; Imai, Y.; Fennie, C.; Geoffroy, J . S.; Rosen, S.
D.; Lasky, L. A. A Homing Receptor-IgG Chimera as a Probe
for Adhesive Ligands of Lymph Mode High Endotherial Venules.
J . Cell Biol. 1990, 110, 2221-2229. (b) Aruffo, A.; Kolanus, W.;
Walz, G.; Fredman, P.; Seed, B. CD62/P-selectin Recognition of
Myeloid and Tumor Cell Sulfatides. Cell, 1991, 67, 35-44. (c)
Erbe, D. V.; Watson, S. R.; Presta, L. G.; Wolitzky, B. A.; Foxall,
C.; Brandley, B. K.; Lasky, L. A. P- and E-selectin use Common
Sites for Carbohydrate Ligand Recognition and Cell Adhesion.
J . Cell. Biol. 1993, 120 (5), 1227-1235. (d) Faxall, C.; Watson,
S. R.; Dowbenko, D.; Fennie, C.; Lasky, L. A.; Kiso, M.;
Hasegawa, A.; Brandley, B. K. The Three Members of the
Selectin Receptor Family Recognize a Common Carbohydrate
Epitope, the Sialyl Lewis x Oligosaccharide. J . Cell Biol. 1992,
117, 895-902.
1
[R]D -23.0° (c 1.5, MeOH); H NMR (270 MHz, DMSO-d6) δ
1.03 (d, J ) 6.4 Hz, 3H, H-6b), 1.87 (s, 3H, AcN), 2.57 (dd, J
) 13.2, 3.2 Hz, H-3d), 4.37 (d, J ) 7.2 Hz, 1H, H-1c), 4.80 (d,
J ) 3.2 Hz, 1H, H-1b). Anal. Calcd (C29H49NO21) C, H, N.
Ack n ow led gm en t. We wish to express our many
thanks to Dr. T. Saito for his helpful discussion.
(11) (a) Raga, J . A.; Cooper, K. Synthesis of a Galactose-Fucose
Disaccharide Mimic of Sialyl Lewis X. Bioorg. Med. Chem. Lett.
1994, 4, 2563-2566. (b) Stahl, W.; Sprengard, U.; Kretzschmar,
G.; Kunz, H. Synthesis of Deoxy Sialyl Lewis x Analogues,
Potential Selectin Antagonist. Angew. Chem., Int. Ed. Engl.
1994, 33, 2096-2098. (c) Kiso, M.; Furui, H.; Ando, K.; Ishida,
H.; Hasegawa, A. Systematic Synthesis of N-Methyl-1-Deoxy-
nojirimycin-Containing, Lex, Lea, Sialyl-Lex and Sialyl-Lea
Epitopes Recognized by Selectins. Bioorg. Med. Chem., 1994, 2,
1295-1308. (d) Ramphal, J . Y.; Zheng, Z.-L.; Perez, C.; Walker,
L. E.; DeFrees, S. A.; Gaeta, F. C. A. Structure-Activity
Relationships of Sialyl Lewis x-Containing Oligosaccharides.1.
Effect of Modifications of the Fucose Moiety. J . Med. Chem. 1994,
37, 3459-3463. (e) Sprengard, U.; Kretzschmar, G.; Bartnik, E.;
Hu¨ls, C.; Kunz, H. Synthesis of an RGD-Sialyl-Lewisx Glyco-
conjugate: A New Highly Active Ligand for P-Selectin. Angew.
Chem., Int. Ed. Engl. 1995, 34, 990-993. (f) J ain, R. K.; Vig, R.;
Rampal, R.; Chandrasekaran, E. V.; Matta, K. L. Total Synthesis
of 3′-O-Sialyl, 6′-O-Sulfo Lewisx, NeuAcR2-3(6-O-SO3Na)-
Galâ1-4(FucR1-3)-GlcNAcâ-OMe: A Major Capping Group of
GLYCAM-1. J . Am. Chem. Soc. 1994, 116, 12123-12124. (g)
Uchiyama, T.; Vassilev, V. P.; Kajimoto, T.; Wong, W.; Huang,
H.; Lin, C.-C.; Wong, C.-H. Design and Synthesis of Sialyl Lewis
X Mimetics. J . Am. Chem. Soc. 1995, 117, 5395-5396. (h)
DeFrees, S. A.; Kosch, W.; Way, W.; Paulson, J . C.; Sabesan, S.;
Halcomb, R. L.; Huang, D.-H.; Ichikawa, Y.; Wong, C.-H. ligand
Recognition by E-Selectin: Synthesis, Inhibitory Activity, and
Conformational Analysis of Bivalent Sialyl Lewis x Analogs. J .
Am. Chem. Soc. 1995, 117, 66-79. (i) Huang, H.; Wong, C.-H.
Synthesis of Biologically Active Sialyl Lewis X Mimetics. J . Org.
Chem. 1995, 60, 3100-3106. (j) Prodger, J . C.; Bamford, M. J .;
Gore, P. M.; Holmes, D. S.; Saez, V.; Ward, P. Synthesis of a
Novel Analogue of Sialyl Lewis X. Tetrahedron Lett. 1995, 36,
2339-2342.
Refer en ces
(1) Springer, T. A. Adhesion Receptors of the Immune System.
Nature 1990, 346, 425-434.
(2) (a) Hallman, R.; J utila, M. A.; Smith, C. W.; Anderson, D. C.;
Kishimoto, T. K.; Butcher, E. C. The Peripheral Lymph Node
Homing Receptor LECAM-1 is Involved in CD18 - Independent
Adhesion of Human Neutrophils to Endothelium. Biochem.
Biophys. Res. Commun. 1991, 174, 236-243. (b) Lawrence, M.
B.; Springer, T. A. Leukocytes Roll on a Selectin at Physiologic
Flow Rates: Distinction from and Prerequisite for Adhesion
through Integrins. Cell 1991, 65, 859-873. (c) Watson, S. R.;
Fennie, C.; Lasky, L. A. Neutrophil Influx into an Inflammatory
Site Inhibited by a Soluble Homing Receptor - IgG Chimera.
Nature 1991, 349, 164-167. (d) Mayades, T. N.; J ohnson, R. C.;
Rayburn, H.; Hynes, R. O.; Wagner, D. D. Leukocyte Rolling and
Extravasation are Severely Compromised in P-selectin - Defi-
cient Mice. Cell 1993, 74, 541-549.
(3) (a) Cummings, R. D.; Smith, D. F. The Selectin Family of
Carbohydrate-Binding Proteins: Structure and Importance of
Carbohydrate Ligands for Cell Adhesion. BioEssays 1992, 14,
849-856. (b) McEver, R. P. Selectins. Curr. Opin. in Immunol.
1994, 6, 75-84.
(4) Bevilacqua, M. P.; Stengelin, S.; Gimbrone, M. A. Endothelial
leukocyte Adhesion Molecule 1: An Inducible Receptor for
Neutrophils Related to Complement Regulatory Proteins and
Lectins. Science 1989, 243, 1160-1165.
(5) Isenberg, W. M.; McEver, R. P.; Shuman, M. A.; Bainton, D. F.
Topographic Distribution of a Granule Membrane Protein (GMP-
140) that is Expressed on the Platelet Surface after Activation:
an Immunogold-Surface Replica study. Blood Cells 1986, 12,
191-204.
(6) Butcher, E. C. Leukocyte-Endothelial Cell Recognition: Three
(or more) Steps to Specificity and Diversity. Cell 1991, 67, 1033-
1036.
(7) (a) Berg, E. L.; Yoshino, T.; Rott, L. S.; Robinson, M. K.; Warnock,
R. A.; Kishimoto, T. K.; Picker, L. J .; Butcher, E. C. The
Cutaneous Lymphocyte Antigen is a Skin Lymphocyte Horming
Receptor for the Vascular Lectin Endothelial Cell- leukocyte
Adhesion Molecule 1. J . Exp. Med. 1991, 174, 1461-1466. (b)
Picker, L. J .; Kishimoto, T. K.; Smith, C. W.; Warnock, R.A.;
Butcher, E. C. ELAM-1 is an Adhesion Molecule for Skin-
Homing T cells. Nature, 1991, 349, 796-799. (c) Lorant, D. E.;
Topham, M. K.; Whatley, R. E.; McEver, R. P.; Mclntyre, T. M.;
Prescott, S. M.; Zimmerman, G. A. Inflammatory Roles of
P-selectin. J . Clin. Invest. 1993, 92, 559-570. (d) Symon, F. A.;
Walsh, G. M.; Watson, S. R.; Wardlaw, A. J . Eosinophil Adhesion
to Nasal Polyp Endothelium is P-Selectin-Dependent. J . Exp.
Med. 1994, 80, 371-376. (e) Akbar, A. N.; Salmon, M.; J anossy,
G. The Synergy between Naive and Memory T Cells during
Activation. Immunol. Today 1991, 12, 184-188. (f) Duijvestijn,
A. M.; Horst, E.; Pals, S. T.; Rouse, B. N.; Steere, A. C.; Picker,
L. J .; Meijer, C. J . L. M.; Butcher, E. C. High Endothelial
Differenciation in Human Lymphoid and Inflammatory Tissue
Defined by Monoclonal Antibody HECA-452. Am. J . Pathol.
1988, 130, 147-155.
(12) (a) Maeda, H.; Ishida, H.; Kiso, M.; Hasegawa, A. Synthetic
Studies on Sialoglycoconjugates 71: Synthesis of Sulfo- and
Sialyl-Lewis X Epitope Analogs Containing the 1-Deoxy-N-
acetylglucosamine in Place of N-Acetylglucosamine Residue. J .
Carbohydr. Chem. 1995, 14 (3), 369-385. (b) Maeda, H.; Ito, K.;
Ishida, H.; Kiso, M.; Hasegawa, A. Synthetic Studies on Sia-
loglycoconjugates 72: Synthesis of Sulfo-, Phosphono- and Sialyl-
Lewis X Analogs Containing the 1-Deoxy- and 1,2-Dideoxy
Hexopyranoses in place of N-Acetylglucosamine Residue. J .
Carbohydr. Chem. 1995, 14 (3), 387-406.
(13) (a) Lin, Y.-C.; Hummel, C. W.; Huang, D.-H.; Ichikawa, Y.;
Nicolaou, K. C.; Wong, C.-H. Conformational Studies of Sialyl
Lewis X in Aqueous Solution. J . Am. Chem. Soc. 1992, 114,
5452-5454. (b) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen,
G.-J .; Garcia-J unceda, E.; Williams, M. A.; Bayer, R.; Ketcham,
C.; Walker, L. E.; Paulson, J . C.; Wong, C.-H. J . Am. Chem. Soc.,
1992, 114, 9283-9298. (c) Miller, K. E.; Mukhopadhyay, C.;
Cagas, P.; Bush, C. A. Solution structure of the Lewis
X
oligosaccharide determined by NMR spectroscopy and molecular
dynamics simulations. Biochemistry 1992, 31, 6703-6709. (d)
Rutherford, T. J .; Spackman, D. G.; Simpson, P. J .; Homans, S.
W. 5 Nanosecond Molecular Dynamics and NMR Study of
Conformational Transitions in the Sialyl Lewis X Antigen.
Glycobiology, 1994, 4, 59-68.
(8) Wein, M.; Bochner, B. S. Adhesion molecule Antagonists: Fur-
ther Therapies for Allergic Diseases? Eur. Respir. J . 1993, 6,
1239-1242.
(9) (a) Phillips, M. L.; Nudelman, E.; Gaeta, F. C. A.; Perez, M.;
Singhal, A. K.; Hakomori, S.-T.; Paulson, J . C. ELAM-1 Mediates
Cell Adhesion by Recognition of a Carbohydarate Ligand, Sialyl-
J M9506478