Ligand interactions with E-selectin. Identification of a new binding site for recognition of N-acyl aromatic glucosamine substituents of sialyl Lewis X

Article abstract of DOI:10.1021/jm9600611

Several N-acylglucosamine derivatives of sialyl Lewis X (1-3) were prepared using a combined chemical enzymatic approach and evaluated as an inhibitor of E-selectin-mediated cellular adhesion. Compounds with aromatic functionality, 1 and 2, were found to

Full text of DOI:10.1021/jm9600611

J . Med. Chem. 1996, 39, 1357-1360
1357
Liga n d In ter a ction s w ith E-Selectin . Id en tifica tion of a New Bin d in g Site for
Recogn ition of N-Acyl Ar om a tic Glu cosa m in e Su bstitu en ts of Sia lyl Lew is X
J ohn Y. Ramphal,† Mariann Hiroshige, Boliang Lou, J ohn J . Gaudino,† Masaji Hayashi,‡ Shiow Meei Chen,§
Lin C. Chiang,§ Federico C. A. Gaeta, and Shawn A. DeFrees*
Cytel Corporation, 3525 J ohn Hopkins Court, San Diego, California 92121, Sumitomo Pharmaceuticals,
1-98, Kasugade Naka 3-chrome, Konohana-ku, Osaka, J apan, and NuMega Resonance Labs, Inc.,
11855 Sorrento Valley Road, Suite J , San Diego, California 92121
Received J anuary 19, 1996X
Several N-acylglucosamine derivatives of sialyl Lewis X (1-3) were prepared using a combined
chemical enzymatic approach and evaluated as an inhibitor of E-selectin-mediated cellular
adhesion. Compounds with aromatic functionality, 1 and 2, were found to be 3-10 times more
potent than the N-acetyl derivative (14) in an ELISA E-selectin cell adhesion assay.
Conformational analysis with NMR indicated that the sialyl Lewis x domain of 1 retained the
conformation of the N-acetyl derivative (14) despite the presence of the N-naphthamido group.
The dramatic order of magnitude increase in potency of these monovalent structures can be
utilized to design more potent selectin-based cell adhesion inhibitors.
One process of leukocyte trafficking during an inflam-
matory response begins with the carbohydrate mediated
cell adhesion of leukocytes to endothelial cells involving
E-, P-, and L-selectin.1 These oligosaccharides have
been shown to be composed of the 3-O-sialylated and
sulfated structures of Lewis x2 and Lewis a2d as well as
6-O-sulfate sialyl Lewis x3 for L-selectin. Many groups
have investigated derivatizing these structures in at-
tempts to increase the potency of the native oligosac-
charide and include such sialyl Lewis x (SLex) analogs
as lactose (NANAR2-3Galâ1-4(FucR1-3)Glc)4a and glu-
cosamine N-acyl alkyl4b structures. With the exception
of multivalent SLex constructs,5 however, analogs of
monovalent SLex have thus far failed to significantly
F igu r e 1. Inhibition of HL-60 cell binding to rsE-selectin by
SLex analogs. Each data point is the average of duplicates.
Binding is expressed as the percentage of rsE-selectin binding
in the absence of inhibitor. The SLex analogs: (4) 1; (9) 2;
(O) 3; (2) 14.
increase the inhibitory potency of this class of com-
pounds over the natural structures.
During our studies of the structure-activity relation-
ships of SLex and its interaction with E-selectin, we
have found that aromatic N-acyl substitutions on the
glucosamine of sialyl Lewis x (1 and 2) increased the
inhibitory potency of this class of oligosaccharide when
tested as inhibitors of E-selectin-mediated cell adhe-
sion.6 Compound 1 was found to be 10-fold more potent
than N-acetyl SLex (14)7 (1, IC50 ) 0.08 mM verses 14,
IC50 ) 1.0 mM), and 2 was found to be ∼3-fold more
potent (IC50 ) 0.3 mM) (Figure 1).
One possible explanation for the increased inhibitory
potencies of 1 and 2 may be the result of topostructural
changes in the orientation of the neighboring Gal’s and
Fuc due to steric interactions with the aryl substituent.
If this were to occur, the steric energies resulting from
acyl substitution must exceed the structural stabilizing
exo-anomeric effects8 of Gal and Fuc which normally fix
the glycosidic torsion angles into a specific topographic
orientation. Alternatively, the increase in potency
observed with 1 and 2 may be the result of a new
complementary binding site for SLex on E-selectin which
is not accounted for in current models of oligosaccharide
binding. Conformational analysis of compound 1 using
NMR9 has indicated that the topographic orientation
is essentially identical to those described for other
N-acetamido SLex’s (Table 1).5 These findings suggest
that the increase in potency of 1 and 2 results from the
direct interaction of the aromatic acyl group with
E-selectin presumably through a hydrophobic mecha-
nism.
† Current address: Amgen Inc., 4765 Walnut Street, Boulder, CO
80301.
‡ Sumitomo Pharmaceuticals.
§ NuMega Resonance Labs, Inc.
Current address: Geron, 200 Constitution Drive, Menlo Park, CA
94025.
X Abstract published in Advance ACS Abstracts, March 15, 1996.
0022-2623/96/1839-1357$12.00/0 © 1996 American Chemical Society

1358 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Ta ble 1. 1H and 13C Chemical Shift Assignments (ppm) of 1
Ramphal et al.
Neu5Ac
Gal
Fuc
GlcN
Gal
carbon
H
C
H
C
H
C
H
C
H
C
C1
C2
C3
C4
C5
C6
C7
C8
177.6
103.4
43.5
72.0
55.3
78.9
71.4
75.6
66.3
25.6
4.57
3.53
4.09
3.92
3.59
3.68
105.3
73.0
79.4
71.0
71.6
65.1
5.19
3.60
3.90
3.75
4.84
1.15
102.2
78.6
73.0
75.6
70.4
18.8
4.96
105.8
60.4
78.4
76.8
76.6
63.2
4.32
106.1
73.3
86.1
72.0
78.5
64.6
4.26
4.11
4.04
3.63
3.48
3.72
4.17
3.63
3.72
1.78, 2.72
3.67
3.82
3.66
3.57
3.89
3.63, 3.87
2.05
3.93, 4.02
C9
CH3
CH2
CdO
1.13
3.59, 3.86
17.5
69.8
178.7
Sch em e 1a
The importance of the aromatic character of the
N-acyl GlcN substituent of 1 and 2 for selectin inhibition
is exemplified by the inhibition results of 3, which
contains a cyclohexane ring in place of the benzene ring
of 2. Compound 3 (IC50 ) 2.9 mM) was found to be less
potent than the N-acetyl SLex derivative (14) (IC50 ) 1
mM). Recent reports have also shown that fatty acyl
GlcN substituents of SLex with varying alkyl lengths
were no more potent than N-acetyl SLex as E-selectin
adhesion inhibitors.4b,10 These findings suggest that the
observed increased inhibitory potencies of 1 and 2
resulted from strong (potent) E-selectin interactions
with the aromatic substituents on the GlcN nitrogen
and may be due to π-π interactions. When 1 and 2
were incorporated into several proposed SLex-E-selec-
tin binding models,11,12 specific interactions could not
be identified although these models are speculative and
SLex or these analogs could well bind at different sites
or in different ways.
a
Key; (a) (i) Pd(Ph3P)4, polymethylsiloxane, THF (68%); (ii)
AcOH, MeOH, H2O, 55 °C (72%); (b) NaHCO3, CH2Cl2, 2-naphthoyl
chloride (77%); (c) (i) tri-O-benzyl-R/â-L-fucopyranosyl fluoride,
AgClO4, SnCl2, TMU, dichloromethane, 4 Å sieves (57%); (d) (i)
cyclohexene, 5% Pd/BaSO4, ethanol, 80 °C (75%); (ii) NaOMe,
MeOH, H2O (95%); (e) NaHCO3, CH2Cl2, acyl chloride (8, benzoyl
chloride, 80%; 10, Cbz chloride, 65%); (f) tri-O-benzyl-R-L-fucopy-
ranosyl bromide, Et4NBr, DMF, dichloromethane, 4 Å sieves (62%);
(g) (i) NaOMe, MeOH, water (89%); (ii) Pearlman’s catalyst,
hydrogen, water, ethanol (97%); (h) tri-O-benzyl-R/â-L-fucopyra-
nosyl fluoride, AgClO4, SnCl2, TMU, dichloromethane, 4 Å sieves
(73%); (i) 10% Pd/C, ethanol, NH4CO3, reflux (96%); (j) (i) cyclo-
hexanecarbonyl chloride, NaHCO3, CH2Cl2, (ii) NaOMe, MeOH,
water (93%).
The synthesis of 1-3 was accomplished with a
combined chemical and enzymatic approach in which
the starting tetrasaccharide (4) was prepared by enzy-
matic sialylation and galactosylation which was then
acetylated as reported previously (Scheme 1).7 The
GlcN hydroxyl to be fucosylated was easily and selec-
tively deprotected using the 2-amino group of GlcN to
direct the hydrolysis.13 Acylation with the appropriate
acyl chlorides provided 6, 8, or 10 which were subse-
quently fucosylated using tri-O-benzyl fucosyl halide7
(Br or F) to provide the protected pentasaccharides (7,
9, and 11). Compounds 7 and 9 were then deprotected
using hydrogenation14 and deacetylation to provide 1
and 2. The Cbz-protected pentasaccharide (1) was
hydrogenated and then acylated with cyclohexanecar-
bonyl chloride to provide 12. Compound 3 was obtained
after hydrolysis of the acetyl and ester groups.
In summary, this study suggests that the interaction
of these SLex analogs with E-selectin encompasses both
the proposed topostructure of SLex containing the
carboxylate, Gal, and Fuc functionalities as well as an
additional complementary hydrophobic binding site on
E-selectin which is adjacent to the glucosamine nitrogen
as seen in 15. Although the exact nature of this ad-
ditional binding site and its role in ligand adhesion to
E-selectin has not been fully characterized, further
studies with additional N-acylglucosamine substituents
of SLex should clarify the mechanism of this E-selectin
interaction and suggest new routes for the design of
more potent antiadhesion molecules.
Exp er im en ta l Section
All reactions were monitored by thin layer chromatography
carried out on 0.25 mm Whatman silica gel plates (60F-254)
using UV light and anisaldehyde reagent1 as developing agent.
E. Merck silica gel (60, particle size 0.040-0.063 mm) and
Bakerbond octadecyl silica gel (C18, particle size 40 µm) were
used for flash chromatography.
All reactions were carried out under an argon atmosphere
with anhydrous solvents from Aldrich unless otherwise noted.
Yields refer to chromatographically and spectroscopically (1H
NMR) homogeneous materials unless otherwise stated. NMR
spectra were recorded on a 300 MHz General Electric QE-300
NMR and a Bruker AM-500 NMR spectrometer. The FAB

Ligand Interactions with E-Selectin
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7 1359
mass spectra and exact mass calculations were acquired on a
VG Fisons ZAB 2SE mass spectrometer.
2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osid e (7). A solution of
compound 6 (135 mg, 0.093 mmol), tri-O-benzyl-R/â-L-fucosyl
fluoride2 (283 mg, 0.65 mmol), 4 Å sieves (100 mg), tetra-
methylurea (122 µL, 1.02 mmol), and dichloroethane (10 mL)
was stirred for 4 h. Silver perchlorate (67 mg, 0.33 mmol)
followed by SnCl2 (61 mg, 0.33 mmol) was then added, and
the reaction mixture was stirred for 2 days. Ethyl acetate (300
mL) was then added and the suspension filtered. The filtrate
was washed with water (100 mL) and dried (MgSO4). Con-
centration and chromatography (silica, 8% acetone/ethyl ac-
etate) afforded 99 mg (57%) of white solid: Rf ) 0.39 (8%
acetone/ethyl acetate); 1H NMR (300 MHz, CDCl3) δ 8.03 (s, 1
H, H-1 naphthalene), 7.74 (d, J ) 7 Hz, 1 H, aromatic), 7.68
(d, J ) 7 Hz, 1 H, aromatic), 7.47 (m, 2 H, aromatic), 7.37-
7.11 (m, 16 H, aromatic), 6.98 (d, J ) 7 Hz, 1 H, aromatic),
6.35 (d, J ) 6.4 Hz, 1 H, NH), 5.57-5.35 (m, 3 H), 5.12-5.03
(m, 3 H), 4.97-4.45 (m, 8 H), 4.32-4.25 (m, 2 H), 4.14-3.72
(m, 22 H), 3.81 (s, 3 H, OMe), 3.71-3.60 (m, 2 H), 3.48-3.43
(m, 1 H), 3.25 (m, 1 H), 2.55 (dd, J ) 4.5, 12.4 Hz, 1 H, H-3eq
SA), 2.24 (s, 3 H, OAc), 2.22 (s, 3 H, OAc), 2.16 (s, 3 H, OAc),
2.13 (s, 3 H, OAc), 2.11 (s, 3 H, OAc), 2.09 (s, 3 H, OAc), 2.08
(s, 3 H, OAc), 2.06 (s, 3 H, OAc), 2.05 (s, 3 H, OAc), 2.02 (s, 3
H, OAc), 2.00 (s, 3 H, OAc), 1.85 (s, 3 H, NHAc), 1.68 (dd, J )
12.4, 12.4 Hz, 1 H, H-3ax SA), 1.18 (d, J ) 6.5 Hz, 3 H, H-6
Fuc), 1.08 (t, 3 H, Me). Anal. (C92H112N2O39‚2H2O) Calcd: C,
57.97; H, 6.12; N, 1.47. Found: C, 57.89; H, 5.98; N, 1.37.
Eth yl (Sod iu m 5-a ceta m id o-3,5-d id eoxy-r-D-glycer o-D-
ga la cto-2-n on u lop yr on osylon a t e)-(2,3)-O-(â-D-ga la ct o-
p yr a n osyl)-(1,4)-O-(r-L-fu cop yr a n osyl)-(1,3)-O-[2-(2-n a p h -
t h a m i d o )-2-d e o x y -â-D-g lu c o p y r a n o s y l]-(1,3)-O -â-D-
ga la ctop yr a n osid e (1). Compound 7 (14 mg, 7.49 µmol) was
dissolved in ethanol (5 mL) and degassed under vacuum.
Cyclohexene (50 µL) followed by 5% Pd/BaSO4 (30 mg) was
then added and suspension heated at 80 °C for 18 h. The
reaction mixture was filtered, concentrated, and chromato-
graphed (silica, hexane/ethyl acetate/ethanol, 2/2/1) to afford
9 mg (75%) of a white solid: Rf ) 0.28 (silica, hexane/ethyl
Eth yl (Meth yl 5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -O-
a cet yl-r-D-glycer o-D-ga la cto-2-n on u lop yr on osylon a t e)-
(2,3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1,4)-O-
(6-O-a cetyl-2-a m in o-2-d eoxy-â-D-glu cop yr a n osyl)-(1,3)-O-
2,4,6-t r i-O-a cet yl-â-D-ga la ct op yr a n osid e (5). Tetrakis-
(triphenylphosphine)palladium (1.29 g, 1.12 mmol) was added
to a solution of polymethylsiloxane (2.98 mL, 8.95 mmol), ethyl
(methyl 5-acetamido-3,5-dideoxy-4,7,8,9-tetra-O-acetyl-R-D-
glycero-D-galacto-2-nonulopyronosylonate)-(2,3)-O-(2,4,6-tri-O-
acetyl-â-D-galactopyranosyl)-(1,4)-O-(3,6-di-O-acetyl-2-(allyl-
oxycarbamoyl)-2-deoxy-â-D-glucopyranosyl-(1,3)-O-2,4,6-tri-O-
acetyl-â-D-galactopyranoside7 (4) (30 g, 22.37 mmol), and THF
(250 mL). The solution was stirred overnight and diluted with
ethyl acetate (1.2 L). The solution was washed with water
(400 mL) and brine (400 mL), and the combined aqueous layers
were extracted again with ethyl acetate (2 × 250 mL). The
combined organic layers were dried (Na2SO4), concentrated,
and chromatographed (silica, 20% acetone/ethyl acetate) to
yield 19.3 g (68%) of a yellow solid: Rf ) 0.31 (silica, 20%
1
acetone/ethyl acetate); H NMR (300 MHz, CDCl3) δ 5.47 (m,
1 H), 5.42 (d, J ) 2.6 Hz, 1 H), 5.38 (d, J ) 3.0 Hz, 1 H), 5.06-
5.01 (m, 2 H), 4.95-4.84 (m, 3 H), 4.66 (d, J ) 8.1 Hz, 1 H,
â-anomer), 4.63 (d, J ) 12.4 Hz, 1 H), 4.50 (dd, J ) 3.3, 10.1
Hz, 1 H), 4.41-4.34 (m, 3 H), 4.15-3.93 (m, 7 H), 3.90-3.80
(m, 4 H), 3.83 (s, 3 H, OMe), 3.77-3.51 (m, 5 H), 2.56 (dd, J )
4.4, 12.7 Hz, 1 H, H-3eq SA), 2.21 (s, 3 H, OAc), 2.14 (s, 3 H,
OAc), 2.11 (s, 3 H, OAc), 2.07 (s, 6 H, OAc), 2.05 (s, 9 H, OAc),
2.03 (s, 3 H, OAc), 1.99 (s, 6 H, OAc), 1.84 (s, 3 H, NHAc),
1.67 (dd, J ) 12.4, 12.4 Hz, 1 H, H-3ax SA), 1.17 (t, J ) 7.0 Hz,
3 H, Me).
A solution of the above compound (19.2 g, 14.33 mmol),
acetic acid (847 µL, 14.79 mmol), methanol (1.26 L), and water
(312 mL) was heated to 55 °C for 24 h. The mixture was
concentrated and chromatographed (silica, ethyl acetate/ether/
acetone, 6/1/3) to afford 13.54 g (72%) of a yellow solid: Rf )
1
1
acetate/ethanol, 2/2/1); H NMR (300 MHz, CDCl3) δ 8.35 (s,
0.30 (silica, 20% acetone/ethyl acetate); H NMR (300 MHz,
1 H, H-1 naphthalene), 7.94 (d, J ) 7.1 Hz, 1 H, aromatic),
7.86 (s, 2 H, aromatic), 7.84 (d, J ) 10.2 Hz, 1 H, aromatic),
7.54 (m, 2 H, aromatic), 6.94 (bd, J ) 7.2 Hz, 1 H, NH), 5.55-
5.51 (m, 1 H), 5.45-5.41 (m, 2 H), 5.17-5.07 (m, 4 H), 4.96-
4.85 (m, 3 H), 4.74 (m, 2 H), 4.57 (m, 2 H), 4.38-4.33 (m, 3
H), 4.30-3.99 (m, 6 H), 3.95-3.71 (m, 9 H), 3.84 (s, 3 H, OMe),
3.66-3.59 (m, 4 H), 3.50 (m, 1 H), 2.58 (dd, J ) 4.4, 12.4 Hz,
1 H, H-3eq SA), 2.24 (s, 3 H, OAc), 2.17 (s, 3 H, OAc), 2.13 (s,
3 H, OAc), 2.11 (s, 3 H, OAc), 2.08 (s, 3 H, OAc), 2.07 (s, 3 H,
OAc), 2.04 (s, 6 H, OAc), 2.03 (s, 6 H, OAc), 2.00 (s, 3 H, OAc),
1.86 (s, 3 H, NHAc), 1.70 (dd, J ) 12.4, 12.4 Hz, 1 H, H-3ax
SA), 1.29 (d, J ) 6.6 Hz, 3 H, H-6 Fuc), 1.12 (t, 3 H, Me). Anal.
(C71H94N2O39) Calcd: C, 53.31; H, 5.92; N, 1.75. Found: C,
53.57; H, 6.30; N, 1.72.
This compound (1.34 g, 0.839 mmol) was then dissolved in
MeOH (30 mL), and a solution of 20% NaOMe in MeOH (1.0
mL) was added. The solution was stirred for 18 h and water
(5 mL) added. After 24 h, the solution pH was adjusted to 7.0
with acetic acid and the solution concentrated. Chromatog-
raphy (Bakerbond C-18, water then 10% MeOH in water)
afforded 0.92 g (95%) of a white solid after lyophilization: Rf
) 0.52 (1 M NH4OAc/2-propanol, 1/3); 1H NMR (500 MHz, D2O)
δ 8.38 (s, 1H, H-1 naphth), 8.10-7.99 (m, 3 H, H-4, 5 and 8
naphth), 7.81 (dd, J ) 1.6, 8.5 Hz, 1 H, H-3 naphth), 7.72-
7.63 (m, 2 H, H-6 and 7 naphth), 5.19 (d, J ) 4.0 Hz, 1 H, H-1
Fuc), 4.96 (d, J ) 8.4 Hz, 1 H, H-1 GlcN), 4.84 (m, 1 H, H-5
Fuc), 4.57 (d, J ) 7.9 Hz, 1 H, H-1 Gal), 4.32 (d, J ) 8.0 Hz,
1 H, H-1, Gal′), 4.26 (bt, 1 H, H-2 GlcN), 4.17 (d, J ) 3.3 Hz,
1 H, H-4, Gal′), 4.12-4.02 (m, 4 H, H-3 and 4 GlcN, H-3 Gal,
H-6′ GlcN), 3.96-3.84 (m, 7 H), 3.78-3.54 (m, 14 H), 3.48 (dd,
J ) 2.1, 9.2 Hz, 1 H, H-2 Gal′), 2.76 (dd, J ) 4.6, 12.4 Hz, 1 H,
H-3 SA), 2.03 (s, 3 H, NHAc SA), 1.78 (dd, J ) 12.2, 12.2 Hz,
1 H, H-3 SA), 1.17 (d, J ) 6.6 Hz, 3 H, H-6 Fuc), 1.13 (dd, J )
7.9, 7.9 Hz, 3 H, CH2CH3); 13C NMR (120 MHz, D2O) see Table
1. Anal. (C48H69N2O28Na‚6H2O) Calcd: C, 46.00; H, 6.51; N,
2.23. Found: C, 45.97; H, 6.47; N, 2.23.
CDCl3) δ 5.53-5.49 (bm, 1 H), 5.41-5.37 (m, 2 H), 5.16 (dd, J
) 8.1, 10.0 Hz, 1 H), 5.10-4.96 (m, 2 H), 4.95-4.84 (m, 2 H),
4.67-4.49 (m, 3H), 4.42-4.24 (m, 3 H), 4.21-3.42 (m, 16 H),
3.84 (s, 3 H, OMe), 2.70 (bs, 1 H, OH), 2.56 (dd, J ) 4.4, 12.4
Hz, 1 H, H-3eq SA), 2.25 (s, 3 H, OAc), 2.16 (s, 3 H, OAc), 2.10
(s, 6 H, OAc), 2.08 (s, 6 H, OAc), 2.06 (s, 6 H, OAc), 2.05 (s, 6
H, OAc), 2.00 (s, 3 H, OAc), 1.85 (s, 3 H, NHAc), 1.67 (dd, J )
12.4, 12.4 Hz, 1 H, H-3ax SA), 1.20 (t, 3 H, Me).
Eth yl (Meth yl 5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -O-
a cet yl-r-D-glycer o-D-ga la cto-2-n on u lop yr on osylon a t e)-
(2,3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctop yr a n osyl)-(1,4)-O-
[6-O -a c e t y l-2-(2-n a p h t h a m i d o )-2-d e o x y -â-D -g lu c o -
p yr a n osyl]-(1,3)-O-2,4,6-t r i-O-a cet yl-â-D-ga la ct op yr a n o-
sid e (6). The 2-naphthoyl chloride (1.28 g, 6.72 mmol) was
added to a suspension of compound 5 (5.81 g, 4.48 mmol) and
sodium bicarbonate (3.01 g, 35.8 mmol) in CH2Cl2 (80 mL).
The mixture was stirred overnight and filtered. The filtrate
was washed with saturated NaHCO3 (10 mL) and dried (Na2-
SO4). Concentration and chromatography (silica, 35% acetone/
CH2Cl2) afforded 5.03 g (77%) of a white solid: Rf ) 0.45 (10%
acetone/ethyl acetate); 1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1
H, H-1 naphthalene), 7.88 (m, 4 H, aromatic), 7.57 (m, 2 H,
aromatic), 6.58 (d, J ) 5.3 Hz, 1 H, NH), 5.53 (m, 1 H), 5.44
(d, J ) 2.9 Hz, 1 H, H-4 Gal), 5.39-5.23 (m, 3 H), 5.17-5.01
(m, 3 H), 4.89 (d, J ) 3 Hz, 1 H), 4.68 (d, J ) 8 Hz, 1 H, H-1
Gal), 4.57 (dd, J ) 3, 7 Hz, 1 H), 4.42-3.28 (m, 19 H), 3.81 (s,
3 H, OMe), 3.25 (q, J ) 6 Hz, 1 H, CH2), 2.57 (dd, J ) 4.3,
12.5 Hz, 1 H, H-3eq SA), 2.27 (s, 3 H, OAc), 2.15 (s, 3 H, OAc),
2.12 (s, 3 H, OAc), 2.08 (s, 6 H, OAc), 2.07 (s, 6 H, OAc), 2.03
(s, 3 H, OAc), 1.99 (s, 3 H, OAc), 1.92 (s, 3 H, OAc), 1.84 (s, 3
H, NHAc), 1.68 (dd, J ) 12.4, 12.4 Hz, 1 H, H-3ax SA), 1.12 (t,
J ) 6 Hz, 3 H, Me). Anal. (C65H84N2O35‚2.5H2O) Calcd: C,
52.10; H, 5.98; N, 1.86. Found: C, 52.18; H, 5.81; N, 1.75.
Eth yl (Meth yl 5-a ceta m id o-3,5-d id eoxy-4,7,8,9-tetr a -O-
a cet yl-r-D-glycer o-D-ga la cto-2-n on u lop yr on osylon a t e)-
(2,3)-O-(2,4,6-tr i-O-a cetyl-â-D-ga la ctoyp yr a n osyl)-(1,4)-O-
(2,3,4-tr i-O-ben zyl-r-L-fu cop yr a n osyl)-(1,3)-O-[6-O-a cetyl-
2-(2-n a p h th a m id o)-2-d eoxy-â-D-glu cop yr a n osyl]-(1,3)-O-
NMR Exp er im en ts. Proton and carbon NMR experiments
were conducted in D2O at 295 K using a Bruker AM-500 NMR

1360 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 7
Ramphal et al.
(5) (a) DeFrees, S. A.; Gaeta, F. C. A.; Lin, Y.-C.; Ichikawa, Y.; Wong,
C.-H. Ligand Recognition by E-Selectin: Analysis of Conforma-
tion and Activity of Synthetic Monomeric and Bivalent Sialyl
Lewis X Analogs. J . Am. Chem. Soc. 1993, 115, 7549-7550. (b)
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 of E-Selectin: Synthesis, Inhibitory Activity, and
Conformational Analysis of Bivalent Sialyl Lewis x Analogs. J .
Am. Chem. Soc. 1995, 117, 66-79. (c) Welply, J . K.; Abbas, S.
Z.; Scudder, P.; Keene, J . L.; Broschat, K.; Casnocha, S.; Gorka,
C.; Steininger, C.; Howard, S. C.; Schmuke, J . J .; Graneto, M.;
Rotsaert, J . M.; Manger, I. D.; J acob, G. S. Multivalent Sialyl-
LeX: Potent Inhibitors of E-Selectin-Mediated Cell Adhesion;
Reagent for Staining Activated Endothelial Cells. Glycobiology
1994, 4, 259-265.
(6) The ELISA assay were carried out according to the procedure
described previously.5b Human soluble recombinant E-selectin
was coated on plates, followed by addition of HL-60 cells and
carbohydrates. After incubation, the plates were rinsed and the
adhesion determined by the cell lysis and myeloperoxidase
method. IC50 was the concentration that inhibited cell adhesion
by 50%. This method gave consistent results with 10% devia-
tion.
(7) (a) Ramphal, J . Y.; Zheng, Z.-L.; Perez, C.; Walker, L. E.;
DeFrees, S. A.; Gaeta, F. C. A. Structure-Activity Relationships
of Sailyl Lewis x-Containing Oligosaccharides 1. Effect of
Modification of the Fucose Moiety. J . Med. Chem. 1994, 37,
3459-3463. (b) Compound 14, NANAR(2,3)Galâ(1,4)[FucR(1,3)]-
GlcNAcâ(1,3)-GalâOEt (Scheme 1, R1 ) R-L-fucopyranosyl; R2
) Ac; R3 ) H; R4 ) Na+).
(8) Lemieux, R. U.; Pavia, A. A.; Martin, J . C.; Watanabe, K. A.
Solvation Effects on Conformational Equilibrium. Studies Re-
lated to Related Methyl Glycopyranosides. Can. J . Chem. 1969,
47, 4427.
spectrometer equipped with an X-32 computer and an ASPECT-
3000 process controller. The sample was not spun. 1H
chemical shifts were referenced to internal HOD at 4.76 ppm,
and 13C chemical shifts were referenced to external DMSO at
39.5 ppm. All NMR data were processed and analyzed with
the Felix program run on a Sun SPARC station or a Silicon
Graphics Indigo workstation. For further experimental de-
tails, see ref 5a.
Ack n ow led gm en t. We wish to express our thanks
to Heping Zhang, Cytel Corporation, and Hiroshi Miyau-
chi and Masashi Tanaka, Sumitomo Pharmaceuticals,
for their chemistry support. We also thank William
Way for performing the E-selectin cell adhesion assay
and Dr. Timothy Kogan for his helpful discussions.
Su p p or tin g In for m a tion Ava ila ble: 1D spectra (1H and
13C NMR) of 1, Roesy of 1, procedures for the synthesis of 2
and 3, and physical data (12 pages). Ordering information is
given on any current masthead page.
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(9) Both 1D and 2D techniques were employed. The experimental
procedures used were as described.5b
(10) In a separate study, we found that C8-C12 acyl alkyl substitu-
tions on glucosamine had no effect on activity. Hiroshige, M.;
DeFrees, S. A. Unpublished results.
(11) Graves, B. J .; Crowther, R. L.; Chandran, C.; Rumberger, J . M.;
Li, S.; Huang, K.-S.; Presky, D. H.; Familletti, P. C.; Wolitzky,
B. Z.; Burns, D. K. Insight into E-selectin/ligand Interaction from
the Crystal Structure and Mutagenesis of the lec/EGF Domains.
Nature 1994, 367, 532-538.
(12) Kogan, T. P.; Revelle, B. M.; Tapp, S.; Scott, D.; Beck, P. J . A
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(13) Selective 3-O-deacetylation on GlcNH2 is successful with various
glycosylated forms. Zheng, Z.-L.; Ito, Y.; DeFrees, S.; Amore,
C.; Ratcliffe, R. M.; Gaeta, F. C. A. Unpublished results.
(14) Hydrogenation of 7 using hydrogen and palladium catalysts such
as Pd/C, Pearlman’s catalyst, and Pd/BaSO4 produced varying
amounts of an overreduced naphthamide product. The catalytic
phase transfer hydrogenation conditions were found to produce
no detectable amounts of the overreduced product.
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