Synthesis and biological evaluation of E-selectin antagonists that present different carbohydrate ligands in a multivalent format

Article abstract of DOI:10.1055/s-2005-865299

The synthesis of the first polylysine conjugates that present different carbohydrate ligands in a multivalent format is reported. Glycopolymers 3c and 3d have been prepared with a highly predictable composition as indicated by ¹H NMR. They func

Full text of DOI:10.1055/s-2005-865299

PAPER
1491
Synthesis and Biological Evaluation of E-Selectin Antagonists that Present
Different Carbohydrate Ligands in a Multivalent Format
M
ultivalent E-Sele
e
ctin
A
ntago
b
nists hard Thoma,* Franz Schwarzenbach
Novartis Institutes for BioMedical Research, Lichtstrasse 35, WSJ-507.4.12, 4056 Basel, Switzerland
Fax +41(61)3246735; E-mail: gebhard.thoma@pharma.novartis.com
Received 12 January 2005
Dedicated to Prof. Bernd Giese on the occasion of his 65th birthday
Abstract: The synthesis of the first polylysine conjugates that
present different carbohydrate ligands in a multivalent format is re-
ported. Glycopolymers 3c and 3d have been prepared with a highly
predictable composition as indicated by 1H NMR. They function as
multivalent E-selectin inhibitors but their potencies are not superior
compared to previously-described related compounds.
Key words: sialyl Lewis X, slectins, glycopolymers, multivalency,
carbohydrate chemistry
The interactions of E-selectin, a vascular endothelial cell
surface protein, with its physiological glycoprotein ligand
mediates leukocyte rolling on the blood vessel wall.1 Inhi-
bition of this early stage of the inflammatory response
Figure 1 E-Selectin antagonists and carbohydrate ligands incorpo-
rated in polylysine conjugates. gal: D-galactose; glcNAc: N-acetyl-D-
glucosamine; sia: sialic acid; fuc: L-fucose; THP: tetrahydropyran
prevents excessive leukocyte recruitment and thus is a po-
tential therapy for acute and chronic inflammatory disor-
ders such as reperfusion injuries, psoriasis, rheumatoid
arthritis, or respiratory diseases.2 The tetrasaccharide sia-
lyl Lewis X (1, sLex; Figure 1) is the minimum epitope
recognized by E-selectin.3 The concentration of sLex to
achieve 50% inhibition (IC50) was determined to be 1000–
2000 mM in a static, cell-free E-selectin binding assay4
which is in line with the observation that monovalent car-
bohydrate-protein interactions are generally very weak
(KD = 10–3–10–4 M–1).5 While modifying sLex we discov-
ered the simplified analogue 2 (Figure 1) which showed
30-fold improved potency (IC50 = 36 mM) compared with
sLex.6,7
ysine conjugates with different carbohydrate ligands
(Scheme 1).
The essential pharmacophores of sLex and 2 required to
bind to E-selectin are the carboxylic acid function, all
three OH-groups of the L-fucose as well as the 4-OH and
6-OH groups of the galactose.14 We have shown that the
tetrahydropyran of antagonist 2 mainly functions as spac-
er to keep the pharmacophores in the appropriate positions
with respect to each other.15 To study if random expres-
sion of all important pharmacophores in a multivalent for-
mat would lead to a potent antagonist we designed
glycoconjugate 3c containing multiple copies of galactose
derivative R4-S- and fucose derivative R5-S-, which are
fragments of antagonist 2.
In some cases high affinity inhibitors can be obtained by
multivalent presentation of low affinity ligands.8 Poly-
mers, liposomes and protein conjugates containing sLex or
similar carbohydrates have been described.9 We have pre-
pared multivalent sLex–polyaspartic acid conjugates10 and
poly-L-lysine–sLex conjugates11 but did not observe im-
proved potencies. However, multivalent polylysine conju-
gates of antagonist 2, such as 3a and 3b (Scheme 1,
Table 1) were highly active E-selectin inhibitors with IC50
values as low as 0.04 mM in the binding assay. Further-
more, these glycopolymers reduced neutrophil rolling on
activated endothelial cells both in vitro and in vivo.12,13
We here report on the synthesis, characterization and bio-
logical evaluation of compounds 3c and 3d, the first polyl-
SYNTHESIS 2005, No. 9, pp 1491–1495
x
x
.
x
x
.2
0
0
5
Advanced online publication: 07.04.2005
DOI: 10.1055/s-2005-865299; Art ID: C00205SS
© Georg Thieme Verlag Stuttgart · New York
Scheme 1 Synthesis of carbohydrate-polylysine conjugates

1492
G. Thoma, F. Schwarzenbach
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R4-SH. Likewise, fucose R5-SH was prepared from 1119
which was glycosylated (→ 12), deprotected and subse-
quently treated with g-thiobutyrolactone (Scheme 3). The
glycopolymers 3c and 3b were prepared from chloro-
acetylated polylysine 13.11 Conjugate 3c was obtained by
reacting 13 with 30 mol% of R4-SH and 30 mol% of R5-
SH in the presence of DBU followed by the addition of an
excess of R6-SH. Reaction of 13 with 20 mol% of R2-SH
and subsequent treatment with a small excess of R4-SH
furnished glycopolymer 3d. As we have shown that the
degree of polymerization of the L-polyslysine starting ma-
terial (n ca. 1200) is not altered under the reaction condi-
tions applied11 the molecular weight (Mn) of 3c and 3b can
be estimated to ca 400–500 kD. Both composition and in-
tegrity of the novel glycopolymers were confirmed by
Table 1 E-Selectin Inhibitiona
Com
Ri
Rii
Riii
(% cont.)
IC50 bind.b
[mM]
pound
(% cont.)
(% cont.)
1
monovalent
monovalent
R2 (35)
1000–2000
36
2
3a
3b
3c
3d
–
R6 (65)
R6 (80)
R6 (40)
R4 (80)
0.04
R2 (20)
–
0.18
R4 (30)
R5 (30)
–
1900
0.40
R2 (20)
a For R groups, see Figure 1.
b For multivalent compounds concentrations refer to carbohydrate
ligand concentrations but not to macromolecule concentration.
1
means of H NMR spectroscopy (Figure 2). Within the
experimental error carbohydrate incorporation occurred
quantitatively.
The glycoconjugates were evaluated in the binding assay.
Compound 3c was found to inhibit E-selectin with an IC50
of 1900 mM. Thus, random expression of all important
pharmacophores in a multivalent format resulted in a in-
hibitory potential similar to monovalent sLex (Table 1,
Figure 1). Conjugate 3d was highly active showing an
IC50 value comparable to 3b also containing 20% of R2-S-
Scheme 2 Reagents and conditions: (a) NIS, F3CSO3H, CH2Cl2, –10
°C; (b) 1. Pd/C, H2, dioxane–H2O 5:1, 20 °C; 2. NaOH, CH3OH, 20
°C; (c) g-thiobutyrolactone, Et3N, CH3OH, 64 °C; NIS: N-iodosuc-
cinimide; Z: benzyl oxycarbonyl; Bn: benzyl; Bz: benzoyl
The structure of the physiological E-selectin ligand has
not been fully elucidated16 but there is evidence that addi-
tional fucose residues in the proximity of sLex are re-
quired for high affinity binding.17 Glycopolymer 3d with
fucose derivative R5-S- in addition to the potent sLex
mimic R2-S- was designed to study the role of additional
fucose residues in close proximity to the potent sLex-like
E-selectin inhibitor 2 in a multivalent format.
The E-selectin ligand R2-SH was prepared as reported
earlier.11 The modified galactose R4-SH was obtained
from 718 (Scheme 2). Glycosylation with propanolamine 8
furnished compound 9, which was deprotected to give 10.
Subsequent reaction with g-thiobutyrolactone gave thiol
Figure 2 1H NMR (500 MHz) spectra of glycopolymers 3c and 3d
in D2O at 60 °C. Several baseline-separated signals can be assigned to
the corresponding R groups. Integration allows the determination of
the composition within NMR accuracy.
Scheme 3 Reagents and conditions: (a) 1. Br2, CH2Cl2, 0 °C, 2.
Et4NBr, CH2Cl2–DMF 3:2; (b) 1. Pd(OH)2/C, H2, dioxane/H2O 4:1,
20 °C; 2. g-thiobutyrolactone, Et3N, DMF, 90 °C
Synthesis 2005, No. 9, 1491–1495 © Thieme Stuttgart · New York

PAPER
Multivalent E-Selectin Antagonists
1493
but with R6-S- instead of additional fucose (Table 1,
Figure 1). Thus, incorporation of fucose in a multivalent
format does not lead to improved potency.
OCH2CH2CH2NH), 3.36 (dd, J = 9.5, 3.0 Hz, 1 H, H3), 3.54 (dd, J =
9.5, 8.0 Hz, 1 H, H2), 3.63 (m, 1 H, OCHaHbCH2), 3.66–3.77 (m,
3 H, H5, H6, H6¢), 3.87 (br d, J = 3.0 Hz, 1 H, H4), 3.93 (m, 2 H,
OCHaHbCH2, OCHCO2H), 4.35 (d, J = 8.0 Hz, 1 H, H1).
13C NMR (D2O): d = 22.6, 25.3, 25.5, 25.7, 27.8, 29.0, 31.3, 32.8,
33.1, 33.9, 35.9, 40.7, 60.6, 65.7, 67.3, 69.5, 74.0, 78.8, 82.4, 102.2,
175.6, 182.2.
In conclusion we have prepared the first polylysine conju-
gates, which present different carbohydrate ligands in a
multivalent format with a highly predictable composition.
These compounds inhibit E-selectin but are not superior
compared to previously-described related analogues.
HRMS: m/z [M + Na]+ calcd for C22H39NO9S: 516.2238; found:
516.2240.
Compound R5-SH
All reactions were carried out under an atmosphere of anhyd Ar.
Commercially available absolute solvents were used. The NMR
spectra were recorded on a Bruker Avance DPX 400 spectrometer.
Chemical shifts of the 1H NMR signals of water-soluble compounds
are reported relative to the shift of the DHO peak (4.75 ppm). The
signal assignments are based on 2D 1H–1H-correlation (COSY) and
1H–13C-correlation spectroscopy (HSQC). The MS spectra were ob-
tained on a Finnigan MAT 90 mass spectrometer, the HRMS spec-
A mixture of 12 (1000 mg, 1.60 mmol), Pd(OH)2 (500 mg, 10% on
charcoal), dioxane (16 mL) and H2O (4 mL) was hydrogenated by
means of a balloon at 20 °C for 16 h. Following filtration the solvent
was removed to give a colorless oil which was dissolved in de-
gassed DMF (20 mL). g-Thiobutyrolactone (1630 mg, 16.0 mmol)
and Et3N (1620 mg, 16.0 mmol) were added and the mixture was
stirred for 16 h at 90 °C. The solvent and volatile side products were
removed in vacuo and the residue subjected to chromatography on
silica gel (CHCl3–MeOH–H2O, 3:1:0 → 3:1:0.3). The product
R5–SH was isolated as a colorless oil (383 mg, 74%); [a]D20 –112.2
(c = 0.76, CH3OH).
tra on
a Bruker Daltonics 9.4T APEX-III FT-MS mass
spectrometer. Ultrafiltrations were performed using Amicon stirred
cells 8010 (volume: 10 mL; diameter: 25 mm) and Amicon disc
membranes YM3 (molecular weight cut-off: 3000).
1H NMR (D2O): d = 1.15 (d, J = 6.5 Hz, 3 H, H6), 1.79 (m, 2 H,
OCH2CH2CH2NH), 1.83 (quint, J = 7.5 Hz, 2 H, COCH2CH2-
CH2SH), 2.31 (t, J = 7.5 Hz, 2 H, COCH2CH2CH2SH), 2.49 (t, J =
Compound 3c
DBU (91 mg, 0.60 mmol) was added at 20 °C to a solution of 13 (50
mg, 0.25 mmol) and R2-SH (37 mg; 0.05 mmol) in a mixture of
DMF (4 mL) and water (0.1 mL). The mixture was stirred for 15
min. Then, R5-SH (120 mg, 0.37 mmol) and DBU (73 mg, 0.49
mmol) were added and stirring continued for 2 h. The solution was
added dropwise to a mixture of EtOH–Et2O (30 mL, 1:1). The
formed precipitate was filtered off and washed with EtOH. The
crude product was dissolved in water and further purified by means
of ultrafiltration (5 × 10 down to 2 mL, with the volume being made
up with distilled water on each occasion). Following lyophilization,
the product 3c was isolated as a colorless powder (128 mg, 91%).
7.5 Hz,
2 H, COCH2CH2CH2SH), 3.17–3.33 (m, 2 H,
OCH2CH2CH2NH), 3.45 (m, 1 H, OCHaHbCH2), 3.68 (m, 1 H,
OCHaHbCH2), 3.71 (dd, J = 10.5, 4.0 Hz, 1 H, H2), 3.74 (br d, J =
3.5 Hz, 1 H, H4), 3.81 (dd, J = 10.5, 3.5 Hz, 1 H, H3), 3.98 (br q,
J = 6.5 Hz, 1 H, H5), 4.81 (d, J = 4.0 Hz, 1 H, H1).
13C NMR (CDCl3): d = 15.8, 23.7, 28.6, 29.1, 34.3, 36.7, 65.6, 65.8,
68.8, 70.8, 71.4, 98.1, 172.1.
HRMS: m/z [M + Na]+ calcd for C13H25NO6S: 346.1295; found:
346.1295.
1H NMR: see Figure 2.
Compound 9
Under rigorous exclusion of moisture CF3SO3H (70 mL) was added
at –10 °C to a solution of 7 (1070 mg, 5.13 mmol), 8 (2000 mg, 2.56
mmol) and NIS (866 mg, 3.85 mmol) in CH2Cl2 and the mixture
stirred for 30 min. EtOAc was added and the mixture extracted with
Na2S2O3, NaHCO3 and brine. The solvent was removed and the res-
idue subjected to flash chromatography on silica gel (hexane–
EtOAc, 3:1 → 1:1). Compound 9 was isolated as a colorless oil
(2070 mg, 87%); [a]D20 +17.3 (c = 0.74, CH3OH).
1H NMR (CDCl3): d = 0.44–1.79 (m, 15 H, CH2c-C6H11,
OCH2CH2CH2NH), 3.14 (m, 2 H, OCH2CH2CH2NH), 3.55 (m, 1 H,
OCHaHbCH2), 3.90 (dd, J = 9.5, 3.0 Hz, 1 H, H3), 3.94 (m, 1 H,
OCHaHbCH2), 3.97 (br t, J = 6.0 Hz, 1 H, H5), 4.19 (dd, J = 8.0,
4.5 Hz, 1 H, OCHCO2H), 4.43 (dd, J = 11.5, 5.0 Hz, 1 H, H6), 4.49
(dd, J = 11.5, 7.0 Hz, 1 H, H6¢), 4.55 (d, J = 8.0 Hz, 1 H, Hvv1), 4.99
(br t, J = 6.0 Hz, 1 H, NH), 5.03 (m, 2 H, OBn), 5.08 (d, J = 12 Hz,
1 H, CO2Bn), 5.19 (d, J = 12 Hz, 1 H, CO2Bn), 5.63 (dd, J = 10.0,
8.0 Hz, 1 H, H2), 5.94 (br d, J = 3.0 Hz, 1 H, H4), 7.29–8.16 (m, 25
H, ArH).
Compound 3d
DBU (55 mg, 0.37 mmol) was added at 20 °C to a solution of 13 (50
mg, 0.25 mmol), R4-SH (39 mg, 0.08 mmol) and R5-SH (26 mg;
0.08 mmol) in a mixture of DMF (4 mL) and water (0.2 mL). The
mixture was stirred for 15 min. Then, R6-SH (79 mg, 0.73 mmol)
and Et3N (0.2 mL) were added and stirring continued for 2 h. The
solution was added dropwise to a mixture of EtOH–Et2O (30 mL,
1:1). The formed precipitate was filtered off and washed with
EtOH. The crude product was dissolved in water and further puri-
fied by means of ultrafiltration (5 × 10 down to 2 mL, with the vol-
ume being made up with distilled water on each occasion).
Following lyophilization, the product 3d was isolated as a colorless
powder (107 mg, 100%).
1H NMR: see Figure 2.
Compound R4-SH
Under rigorous exclusion of oxygen a solution of 10 (1000 mg, 2.56
mmol), g-thiobutyrolactone (2600 mg, 25.6 mmol) and Et3N (2600
mg, 25.6 mmol) in degassed CH3OH (30 mL) was heated under re-
flux for 16 h. The solvent and volatile side products were removed
in vacuo and the residue subjected to chromatography on silica gel
13C NMR (CDCl3): d = 16.2, 28.6, 39.7, 66.0, 66.1, 67.6, 72.9, 73.0,
74.3, 75.7, 77.3, 79.1, 97.8, 127.0–138.4 (signals of aromatic C-at-
oms), 158.2.
HRMS: m/z [M + Na]+ calcd for C54H57NO13: 950.3722; found:
950.3724.
(EtOAc–i-PrOH–water, 1:1:0.25). Lyophilization afforded the
product R4-SH as a colorless powder (956 mg, 76%); [a]D
33.5 (c = 0.76, CH3OH).
–
20
1H NMR (D2O): d = 0.90–1.75 (m, 13 H, CH2-cC6H11), 1.79 (quint,
J = 6.5 Hz, 2 H, OCH2CH2CH2NH), 1.83 (quint, J = 7.5 Hz, 2 H,
COCH2CH2CH2SH), 2.30 (t, J = 7.5 Hz, 2 H, COCH2CH2CH2SH),
2.49 (t, J = 7.5 Hz, 2 H, COCH2CH2CH2SH), 3.25 (m, 2 H,
Compound 10
A mixture of 9 (2.07 g, 2.23 mmol), Pd(OH)2 (1.00 g, 10% on char-
coal), dioxane (40 mL), H2O (10 mL) and HOAc (0.34 mL) was hy-
drogenated by means of a balloon at 20 °C for 16 h. Following
Synthesis 2005, No. 9, 1491–1495 © Thieme Stuttgart · New York

1494
G. Thoma, F. Schwarzenbach
PAPER
filtration the solvent was removed and the residue dissolved in a
mixture of MeOH (50 mL) and NaOH (2 N, 11 mL). The solution
was stirred at 60 °C for 4 h. The solvents were removed and the res-
idue subjected to flash chromatography on silica gel (EtOAc–i-
PrOH–H2O, 1:1:0.5). Compound 10 was isolated as a colorless oil
(0.855 g, 98%).
1H NMR (D2O): d = 0.83–1.73 (m, 13 H, CH2c-C6H11), 1.98 (m, 2 H,
OCH2CH2CH2NH), 3.14 (t, J = 7.0 Hz, 2 H, OCH2CH2CH2NH),
3.39 (dd, J = 10.0, 3.0 Hz, 1 H, H3), 3.58 (br t, J = 9.0 Hz, 1 H, H2),
3.68 (m, 1 H, H5), 3.74 (m, 2 H, H6, H6¢), 3.79 (m, 1 H, OCHaHb),
3.89 (d, J = 3.0 Hz, 1 H, H4), 3.94 (dd, J = 8.0, 3.0 Hz, 1 H,
OCHCO2H), 4.03 (m, 1 H, OCHaHbv), 4.39 (d, J = 8.0 Hz, 1 H,
H1).
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MS: m/z = 392 [M + H]+.
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Compound 12
A solution of Br2 (2.21 g, 13.8 mmol) in CH2Cl2 (20 mL) was added
dropwise at 0 °C to a solution of 11 (6.0 g, 12.6 mmol) in CH2Cl2
(20 mL). After stirring for 30 min at 0 °C cyclohexene (2.5 mL) was
added to consume excessive Br2. The solution was added within 10
min to a mixture of 8 (5.25 g, 25.1 mmol) and Et4NBr (6.30 g, 30.1
mmol; dried for 2 h at 200 °C) in DMF–CH2Cl2 (100 mL, 1:1). The
mixture was stirred for 90 h at 20 °C, diluted with EtOAc, washed
with NaHCO3, H2O, HCl (0.5 M) and brine and dried with Na2SO4.
The solvent was removed and the residue subjected to flash–chro-
matography on silica gel (hexane–acetone, 6:1). Compound 12 was
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Chem. Int. Ed. 1998, 37, 2754. (b) Lee, R. T.; Lee, Y. C.
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Drug Discovery, Vol. 2; Wong, C. H., Ed.; Wiley-VCH:
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Mortell, K. H. In Glycoscience – Chemistry and Chemical
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Eds.; Springer: New York, 2001, 1817.
(9) (a) Manning, D. D.; Hu, X.; Beck, P.; Kiessling, L. L. J. Am.
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J.; Oren, D.; Beck, P. J.; Ronen, A.; Kiessling, L. L. J. Biol.
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14869.
20
isolated as a colorless oil (7.0 g, 89%); [a]D –74.5 (c = 1.2,
CH3OH).
1H NMR (CDCl3): d = 1.09 (d, J = 6.5 Hz, 3 H, H6), 1.80 (quint, J =
6.0 Hz, 2 H, OCH2CH2CH2NH), 3.25 (m, 1 H, OCH2CH2-
CHaHbNH), 3.39–3.53 (m, 2 H, OCHaHbCH2CHaHbNH), 3.59
(m, 1 H, H4), 3.78–3.86 (m, 2 H, H5, OCHaHb), 3.88 (dd, J = 10.0,
2.0 Hz, 1 H, H3), 4.01 (dd, J = 10.0, 4.0 Hz, 1 H, H2), 4.65 (m, 3 H,
OBn), 4.71 (d, J = 4.0 Hz, 1 H, H1), 4.78 (d, J = 11.5 Hz, 1 H, OBn),
4.80 (d, J = 11.5 Hz, 1 H, OBn), 4.96 (d, J = 11.5 Hz, 1 H, OBn),
5.03 (d, J = 12.0 Hz, 1 H, OBn), 5.10 (d, J = 12.0 Hz, 1 H, OBn),
5.90 (m, 1 H, NH), 7.24–7.36 (m, 20 H, ArH).
13C NMR (CDCl3): d = 25.1, 25.4, 25.7, 29.1, 32.2, 32.9, 33.0, 37.6,
40.0, 62.4, 65.9, 66.2, 67.0, 69.4, 71.5, 72.2, 76.7, 77.8, 101.1,
127.5–135.1 (signals of aromatic C-atoms), 156.2, 164.8, 165.5,
165.9, 172.1.
HRMS: m/z [M + Na]+ calcd for C38H43NO7: 648.2932; found:
648.2931.
Acknowledgment
We thank Mr. François Nuninger for technical assistance.
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