Synthesis and biological evaluation of a sialyl Lewis X mimic with significantly improved E-selectin inhibition

Article abstract of DOI:10.1016/S0960-894X(01)00092-0

The synthesis of the highly potent E-selectin inhibitor 5 is described. Sialyl Lewis X mimic 5 was rationally designed by combining two previously disclosed beneficial sLe^x modifications in a single molecule. The compound was found to be 30-fold more potent than sLe^x in a static, cell-free equilibrium assay. Furthermore, compound 5 was highly active (IC₅₀ = 10 μM) in a dynamic non-equilibrium assay in which sLe^x did not inhibit neutrophil rolling at up to 1000 μM.

Full text of DOI:10.1016/S0960-894X(01)00092-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 923–925
Synthesis and Biological Evaluation of a Sialyl Lewis X Mimic
with Significantly Improved E-selectin Inhibition
Gebhard Thoma,a,* John L. Magnanib and John T. Pattonb
aNovartis Pharma AG, PO Box, CH-4002 Basel, Switzerland
bGlycoTech Corporation, 14915 Broschart Road, Rockville, MD 10850, USA
Received 18 December 2000; accepted 7February 2001
Abstract—The synthesis of the highly potent E-selectin inhibitor 5 is described. Sialyl Lewis X mimic 5 was rationally designed by
combining two previously disclosed beneficial sLex modifications in a single molecule. The compound was found to be 30-fold more
potent than sLex in a static, cell-free equilibrium assay. Furthermore, compound 5 was highly active (IC50=10 mM) in a dynamic
non-equilibrium assay in which sLex did not inhibit neutrophil rolling at up to 1000 mM. # 2001 Elsevier Science Ltd. All rights
reserved.
Introduction
inhibitor 5 (Fig. 1) which combines both the glucosa-
mine modification and the sialic acid replacement. Com-
pound 5 was found to be 30-fold more potent than sialyl
Lewis X. Furthermore, under in vitro flow conditions
compound 5 inhibited rolling of polymorphonuclear
leukocytes (PMNs) on stimulated human umbilical vein
endothelial cells (HUVECs) with an IC50 value of 10 mM.
The recruitment of leukocytes to sites of inflammation is
a multistep process.1 The selectins are cell-surface gly-
coproteins which promote both the initial attachment of
leukocytes (tethering) and their movement (rolling) over
the blood vessel wall.2 Integrins mediate the subsequent
firm adhesion preceding extravasation into the under-
lying tissue. L-Selectin is expressed constitutively on
leukocytes whereas E- and P-selectin are induced on the
surface of vascular endothelium in response to inflam-
matory stimuli. Excessive infiltration of leukocytes can
cause acute or chronic reactions as observed in reperfu-
sion injuries, stroke, psoriasis, rheumatoid arthritis, or
respiratory diseases. Thus, the adverse effects could be
prevented by selectin blockade.3 The tetrasaccharide
sialyl Lewis X (sLex 1, Fig. 1)4 is a physiologically rele-
vant recognition component for all three members of
the selectin family.5 It is a weak inhibitor of E-selectin
(KDꢀ1000 mM) and became a prominent lead stucture
for the design of more potent mimics which could be of
therapeutic interest.6 Thus, the introduction of N-acyl
aromatic substituents into sLex led to up to 10-fold
more potent analogues.7 We have found the 3,4-di-
methoxybenzoate 2 (Fig. 1) to be 6-fold more potent
than sLex.8 Previously, we have reported on potent sLex
mimetics, such as 3 and 4 (Fig. 1), containing S-cyclo-
hexyllactic acid as a replacement for sialic acid.9 Here
we describe the synthesis of the novel, potent E-selectin
Synthesis
Compound 5 was prepared starting from suitably pro-
tected glucosamine derivative 610 (Scheme 1). Treatment
of a mixture of 6 and alcohol 7 (1.3 equiv) with TMS
triflate (2.5 equiv) at ꢁ35 ꢂC gave selectively the b-gly-
coside 8 (85%) which was subsequently deacetylated
applying catalytic amounts of NaOMe in methanol. The
crude triol intermediate was reacted with benzaldehyde
dimethylacetal in acetonitrile in the presence of catalytic
amounts of PTSA to selectively block the 4- and 6-
hydroxyl functions as a benzylidene acetal leaving the 3-
hydroxyl group unprotected. Compound 9 was isolated
in 88% yield over two steps. Fucose was introduced
using fucosyl donor 1011 which was transformed into
the corresponding glycosylbromide by slow addition of
a small excess of bromine at 0 ꢂC to a solution of 10 in
methylene chloride. Unreacted bromine was quenched
with cyclohexene. This solution was added to com-
pound 9 dissolved in methylene chloride/DMF (3:2).
The mixture was kept at ambient temperature for 16 h.
Disaccharide 1112 was isolated by flash chromatography
in 89% yield. The benzylidene acetal was opened by
*Corresponding author. Tel.:+41-61-3243342; fax: +41-61-3246735;
e-mail: gebhard.thoma@pharma.novartis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00092-0

924
G. Thoma et al. / Bioorg. Med. Chem. Lett. 11 (2001) 923–925
Figure 1. Structures of selected E-selectin inhibitors.
Scheme 1. Synthesis of E-selectin antagonist 5.
slowly adding a freshly prepared saturated solution of
HCl in ether to a mixture of 11, molecular sieves (3 A)
and NaCNBH3 (10 equiv) in THF to afford compound
12. Complete conversion of 11 gave only low yields of 12
due to cleavage of the acid labile a-glycosidic linkage.
Thus, the reaction had to be monitored carefully (TLC)
and stopped at 70–80% conversion. Compound 12 was
isolated in 60% yield along with 20% of unreacted 11.

G. Thoma et al. / Bioorg. Med. Chem. Lett. 11 (2001) 923–925
925
It is worthwhile to mention that this rather simple pro-
References and Notes
tecting group manipulation turned out to be the most
critical step in the whole synthesis. Next, the amine
protecting group was removed using catalytic quantities
of Pd(PPh3)4 (0.1 equiv) in THF in the presence of
morpholine (30 equiv). The corresponding amine was
isolated in 96% yield. Treatment with active ester 13
(2.5 equiv) in a mixture of DMF/2,6-lutidine (5:1) at
80 ꢂC for 16 h gave benzamide 14 in 93% yield. Galac-
tose was introduced activating a mixture of 14, selec-
tively protected thiogalactoside 1511 (1.5 equiv) and
molecular sieves (3 A) in CH2Cl2 with dimethyl-
(methylthio)sulfonium triflate (DMTST). Trisaccharide
1612 was obtained in 85% yield. The benzoates were
removed applying catalytic amounts of NaOMe in
methanol (quant.), the triol heated in methanol for 16 h
in the presence of dibutyltin oxide13 (1.75 equiv), the
solvent removed and the residue dried. This material
and triflate 179c (3.0 equiv) were dissolved in DME, CsF
(2.5 equiv) was added and the mixture kept at ambient
temperature for 4 h. Compound 18 was isolated in 60%
yield. Hydrogenation of 18 in a mixture of dioxane/
water/acetic acid (0.7:0.25:0.05) using Pd(OH)2 (20%)
on charcoal as a catalyst followed by transformation of
the carboxylic acid into the sodium salt afforded the
potent E-selectin inhibitor 512 in 99% yield.
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Biological Evaluation
Compound 5 was tested in a static, cell-free ligand
binding assay which measures E-selectin inhibition
under equilibrium conditions.14 To compare the data
for different compounds obtained on different test plates
sLex 1 (IC50=1000–1500 mM) was assayed on each plate
as a reference. This allows the determination of IC50
values relative to sLex which are defined as relative
IC50=IC50(test compound)/IC50(sLex). The relative
IC50 for 5 was determined in three independent
measurements to be 0.030ꢃ0.015. In addition, 5 was
profiled in a dynamic in vitro assay which allows
monitoring of E-selectin-dependent rolling of neutro-
phils on activated endothelial cells and, hence, mimics
the non-equilibrium in vivo conditions.15 sLex (1)
showed no inhibition in this more relevant assay at up to
1000 mM. Compound 5 was tested at 200, 50 and 10 mM
and showed 97, 93 and 54% inhibition of the number of
rolling cells, respectively. Thus, the IC50 was estimated
to be ꢄ10 mM.
J. L.; Banteli, R. J. Med. Chem. 1999, 42, 4909. (d) Banteli, R.;
¨
¨
Herold, P.; Bruns, C.; Patton, J. T.; Magnani, J. L.; Thoma,
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bohydr. Res. 1990, 197, 7 5.
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186312
12. 11: MS/EI 936 (MꢁH)ꢁ; 1H NMR (400 MHz, CDCl3)
selected signals d 0.75 (3H, d, J=6.5 Hz, H-6 Fuc), 3.67(s, 3H,
CO2CH3), 4.82 (1H, d, J=8.0 Hz, H-1 Glc), 5.15 (1H, d,
J=3.5 Hz, H-1 Fuc), 5.18 (1H, dq, J=10.5, 1.5 Hz,
OCH2CH¼CHEHZ), 5.27(1H, dq,
J=17.5, 1.5 Hz,
OCH2CH¼CHEHZ), 5.48 (1H, s, Ar-CH-), 5.87(1H, ddt,
J=17.5, 1.5 Hz, OCH2CH¼CHEHZ); 16: MS/EI 1582
1
(MꢁH)ꢁ; H NMR (400 MHz, CDCl3) selected signals d 1.17
(3H, d, J=6.5 Hz, H-6 Fuc), 3.64 (s, 3H, CO2CH3), 3.74 (3H,
s, ArOCH3), 3.87(3H, s, ArOC H3), 4.35 (1H, m, H-1 Glc),
4.91 (1H, d, J=8.0 Hz, H-1 Gal), 5.47(1H, s (br), H-1 Fuc),
5.89 (1H, d, J=3.5 Hz, H-4 Gal), 6.62 (1H, d, J=8.5 Hz, Ar-
H), 6.63 (1H, d, J=8.0 Hz, NH); 5: MS/EI 974 (MꢁNa)ꢁ;
1H NMR (400 MHz, D2O) selected signals d 1.06 (3H, d,
J=6.5 Hz, H-6 Fuc), 3.54 (s, 3H, CO2CH3), 3.77 (3H, s,
ArOCH3), 3.76 (3H, s, ArOCH3), 4.38 (1H, d, J=8.0 Hz, H-1
Gal), 4.55 (1H, m, H-1 Glc), 4.70 (1H, q, J=6.5 Hz, H-5 Fuc),
5.01 (1H, s (br), H-1 Fuc), 7.00 (1H, d, J=8.5 Hz, Ar-H), 7.32
(1H, d, J=2.0 Hz, Ar-H), 7.32 (1H, dd, J=8.5, 2.0 Hz, Ar-H).
13. For regioselective manipulations of hydroxyl groups via
organotin derivatives see David, S; Hanessian, S. Tetrahedron
1985, 41, 643.
Conclusion
E-selectin antagonist 5 was rationally designed com-
bining two previously discovered beneficial structural
elements (cyclohexyllactic acid and amide modification)
in a single molecule. In a static equilibrium assay 5
(relative IC50=0.030) was found to be equally potent as
compound 4 (rel. IC50=0.031). In the dynamic flow
assay 5 (IC50=ꢄ10 mM) is even more potent than 4
(IC50=30–40 mM). Thus, compound 5 is one of the
most potent E-selectin inhibitors to date. Simplified
analogues of 5 are currently being evaluated.
14. Thoma, G.; Magnani, J. L.; Oehrlein, R.; Ernst, B.;
Schwarzenbach, F.; Duthaler, R. O. J. Am. Chem. Soc. 1997,
119, 7414.
15. (a) Thoma, G.; Patton, J. T.; Magnani, J. L.; Ernst, B.;
Oehrlein, R.; Duthaler, R. O. J. Am. Chem. Soc. 1999, 121,
5919.