Rational design of novel, potent small molecule pan-selectin antagonists

Article abstract of DOI:10.1021/jm060536g

This report describes the first results of a rational hit-finding strategy to design novel small molecule antiinflammatory drugs targeting selectins, a family of three cellular adhesion molecules. Based on recent progress in understanding of molecular interaction between selectins and their natural ligands as well as progress in clinical development of synthetic antagonists like 1 (bimosiamose, TBC1269), this study was initiated to discover small molecule selectin antagonists with improved pharmacological properties. Considering 1 as template structure, a ligand-based approach followed by focused chemical synthesis has been applied to yield novel synthetic small molecules (MW_r < 500) with a trihydroxybenzene motif, bearing neither peptidic nor glycosidic components, with nanomolar in vitro activity. Biological evaluation involves two kinds of in vitro assays, a static molecular binding assay, and a dynamic HL-60 cell attachment assay. As compared to controls, the novel compounds showed improved biological in vitro activity both under static and dynamic conditions.

Full text of DOI:10.1021/jm060536g

J. Med. Chem. 2007, 50, 1101-1115
1101
Rational Design of Novel, Potent Small Molecule Pan-Selectin Antagonists
Remo Kranich, Anke S. Busemann, Daniel Bock, Sabine Schroeter-Maas, Diana Beyer, Bo Heinemann, Michael Meyer,
Katrin Schierhorn, Rainer Zahlten, Gerhard Wolff, and Ewald M. Aydt*
ReVotar Biopharmaceuticals AG, Neuendorfstrasse 24a, 16761 Hennigsdorf, Germany
ReceiVed May 9, 2006
This report describes the first results of a rational hit-finding strategy to design novel small molecule
antiinflammatory drugs targeting selectins, a family of three cellular adhesion molecules. Based on recent
progress in understanding of molecular interaction between selectins and their natural ligands as well as
progress in clinical development of synthetic antagonists like 1 (bimosiamose, TBC1269), this study was
initiated to discover small molecule selectin antagonists with improved pharmacological properties.
Considering 1 as template structure, a ligand-based approach followed by focused chemical synthesis has
been applied to yield novel synthetic small molecules (MWr < 500) with a trihydroxybenzene motif, bearing
neither peptidic nor glycosidic components, with nanomolar in Vitro activity. Biological evaluation involves
two kinds of in Vitro assays, a static molecular binding assay, and a dynamic HL-60 cell attachment assay.
As compared to controls, the novel compounds showed improved biological in Vitro activity both under
static and dynamic conditions.
Introduction
in the inflammatory response and thus constitutes an attractive
target for therapeutic intervention.
Transmigration of leukocytes (e.g., monocytes, lymphocytes,
eosinophils, neutrophils, and basophils) from the circulation into
the surrounding tissue is of vital importance to maintain innate
and adaptive immune defense mechanisms under physiological
conditions and pathological stages like injury or inflammatory
response. This multistep process is governed by a coordinated
interplay involving a broad spectrum of adhesion and signaling
molecules.1 Different from this highly regulated cascade,
dysregulation of leukocyte transmigration results in uncontrolled
excessive and pathological infiltration of leukocytes into organs.
Finally, it leads to acute and chronic inflammatory diseases such
as acute lung injury (ALI), acute respiratory distress syndrome
(ARDS), asthma, chronic obstructive pulmonary diseases (COPD),
psoriasis, and rheumatoid arthritis. Therefore, adhesion and
signaling molecules are an attractive target to develop novel
drugs for the treatment of acute and chronic inflammatory
diseases.
The first step in transmigration is mediated by the selectin
family of cell adhesion molecules, which consists of three
structurally related calcium-dependent carbohydrate binding
proteins, E-, P-, and L-selectin.2 P-selectin is a rapidly inducible
selectin found only on activated platelets and vascular endot-
helium. E-selectin is an intermediate inducible selectin found
primarily on activated vascular endothelial cells. L-selectin is
constitutively expressed on the surface of several leukocyte
subtypes, including neutrophils, monocytes, the majority of
circulating B- and T-cells, and on a subset of natural killer cells.
Physiologically, selectins mediate the initial rolling or tethering
of leukocytes on the vascular endothelium to allow them to
adhere to the vascular endothelium and, consequently, to initiate
transmigration from the vascular circulation into the surrounding
tissue. Under pathological conditions, however, e.g., local or
systemic inflammation and/or injury of the vascular system, this
fundamental process is up-regulated, at least in part, due to an
increase in the expression of E- and P-selectin. The process of
leukocyte rolling is generally considered to be a primary event
For the interaction between selectins and their natural ligands,
the tetrasaccharide sialyl LewisX (sLeX, [Neu5AcR2-3Galâ1-
4(FucR1-3)GlcNAc], 2, Figure 1), and related oligosaccharides
have been identified as key functional groups.3 SLeX which
binds with low affinity to E-, P-, and L-selectin was found to
be a common epitope in many natural selectin ligands, such as
P-selectin glycoprotein ligand 1 (PSGL-1), glycosylated cell
adhesion molecule 1 (GlyCAM-1), CD34, mucosal addressin
cell adhesion molecule 1 (MAdCAM-1), and E-selectin ligand
1 (ESL-1).4 The identification of this epitope and its importance
has guided the design of selectin inhibitors in the past. Thus,
efforts to develop novel selectin antagonists have been mainly
focused on the design of sLeX glycomimetics, glycosylated
peptides, or peptides.5 Since many natural high affinity ligands
provide multivalent arrangement of binding epitopes to selectins,
one approach to overcome the low affinity of monomeric sLeX-
mimetics is multivalent assembly of sLeX-mimetics.6 However,
these compounds may suffer from several problems regarding
development as pharmaceutical agents. As compared to the large
number of reports on glycosidic and peptidic sLeX-mimetics,
only few reports on nonglycosylated and nonpeptidic selectin
antagonists are published.7-9 In this regard, several excellent
reviews were published summarizing the history and current
activities in the field of selectin antagonists.10-11
To date, almost all efforts to design novel therapeutically
effective sLeX-mimicking selectin antagonists failed, which is
supposed to be caused by different reasons.12 First, many selectin
antagonists effectively inhibit only one or two of the three
selectins. However, there is functional redundancy between
selectins, e.g., between E- and P-selectin on the vascular
endothelial surface, allowing to bypass inhibition of single-type
selectin family members.13 Second, there is only a weak
interaction between selectins and sLeX implying KDs in the mM
range for P- and L-selectin and in the higher µM range for
E-selectin.14 Thus, novel compounds designed on the basis of
sLeX only are likely to be a selectin antagonists with high KD
values. Third, sLeX and structurally related low affinity ligands
failed to inhibit P- and L-selectin mediated rolling in ViVo.
* To whom correspondence should be addressed. E-mail: e.aydt@
revotar-ag.de. Tel: +49 (0)3302 202 5069. Fax: +49 (0)3302 202 5030.
10.1021/jm060536g CCC: $37.00 © 2007 American Chemical Society
Published on Web 02/16/2007

1102 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Kranich et al.
Figure 1. Structure of 1 (bimosiamose, TBC1269), a dimer-like (R-D-Mannopyranosyloxy)biphenyl-substituted carboxylic acid. Sialyl LexisX (2)
is itself a natural low affinity ligand of selectins as well as a crucial binding moiety of many natural ligands of selectins, such as the high affinity
P-selectin glycoprotein ligand 1 (PSGL-1). Structure of 3 (TBC265), a substructure of 1, and first generation lead structure in the development of
1. Stereochemically equivalent hydroxyls at C-2, C-3, and C-4 of mannose in 1 or 3 and C-4, C-3, and C-2 of fucose in sLeX are highlighted. The
boxes around the carboxylic acids in compound 1 and 3 indicate potentially crucial groups for competing with tyrosine-sulfate-51 in PSGL-1. Note
that in compound 1 only one of two carboxylic acids is marked. SAR studies showed that only one carboxylic acid in compound 1 is required for
activity.23
E-selectin-mediated in ViVo rolling is only influenced at high
doses.15 High affinity of natural selectin ligands is supposed to
be, at least in part, due to multivalent arrangement of binding
epitopes as well as extensive surface contacts between selectins
and their ligands.16 Recently, for instance, the nature of the high
affinity binding of a glycoprotein-type ligand and P-selectin has
been solved in atomic detail. P-selectin glycoprotein ligand 1
(PSGL-1) binds with nanomolar in Vitro activity to P-selectin,
but it also binds to E- and L-selectin. It was shown by
biochemical and structural investigations that sulfated tyrosine
residues are in addition to the occurrence of sLeX mandatory
for high affinity binding to P-selectin.17 Even though the
interaction between PSGL-1 and P-selectin comprises a large
surface area, the close proximity between essential binding
features offers the opportunity for the design of selectin
antagonists that compete with PSGL-1 and PSGL-1-like ligands
for selectin binding.
Using PSGL-1 as major high affinity template structure, a
rational drug design approach to discover novel potent synthetic
nonglycosidic and nonpeptidic small molecule pan-selectin
antagonists is presented. Based on 1 (bimosiamose, TBC1269,
Figure 1), a ligand-based design strategy followed by focused
chemical synthesis was applied. In silico considerations led to
synthetic polyphenolic small molecules with MWr < 500 and
nanomolar in Vitro activity in the static molecular binding assay.
In this assay synthetic antagonists compete with a PSGL-1-
like ligand for selectin binding. In addition, the compounds
inhibit the in Vitro attachment of HL-60 cells to E- and P-selectin
matrices. To our knowledge, this is the first time that synthetic
small molecules MWr < 500 and yet with nanomolar in Vitro
activity are reported.
the selectins belong to a class of molecules which are not readily
amenable to receptor- or structure-based drug design for virtual
screening even if the 3-D structure is available. This is due to
structural characteristics of the ligand-binding site, which is an
almost flat binding surface. Most docking algorithms work well
for receptors or enzymes with more or less deep cavities
providing spatial restraints to small molecule ligands, but due
to the large number of degrees of freedom, shallow surfaces
still represent a challenge for many docking algorithms. The
applicability of an automated docking procedure to search for
novel scaffolds was investigated in this study. The results of
this analysis indicate that applying automated docking, as
implemented in MOE,20 is not applicable to quickly identify
novel chemical scaffolds in the hit-finding process.
Therefore, a ligand-based approach has been employed being
based on 1, a selectin antagonist that was tested in Phase I and
Phase IIa trials for the treatment of asthma and psoriasis.21-22
Presently, further preclinical and clinical studies are ongoing.
The molecule was identified as the most advanced pharmaceuti-
cal compound in the field of selectin antagonists,10 and it appears
to be an appropriate template for a ligand-based drug design.
This approach resulted in a concept for a possible pharmacoph-
ore, followed by focused chemical synthesis.
The pan-selectin antagonist 1 has been rationally designed
by Kogan et al. as sLeX (2) mimetic.23 It is a dimer type (R-
D-mannopyranosyloxy)biphenyl-substituted carboxylic acid with
MWr ) 862.9. As compared to the natural ligand sLeX, 1
showed improved in Vitro potency. Due to the fact, except for
compound 1, that none of the former reported drug discovery
programs for the development of novel small molecule sLeX
mimetics succeeded in providing novel chemical entities with
clinical efficacy, it was suggested that 1 might bind, at least in
part, differently to selectins as compared to sLeX. Molecular
alignments of compound 3 (TBC265, Figure 1), a substructure
of 1, with sLeX as well as comprehensive retrospective docking
investigations of compound 1 on P-selectin revealed that 1 may
mimic both sLeX-specific binding features and a crucial mo-
lecular binding feature being specific for the N-terminal peptidic
Rational Drug Design
The structure of E- and P-selectin has been solved in atomic
detail.18,19 In addition, cocrystals of P-selectin with sLeX and
PSGL-1 as well as cocrystals of E- selectin with sLeX have been
solved.19 A structure-based approach in order to search for new
selectin antagonists seems therefore to be appropriate. However,

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1103
Table 1. Biological in Vitro Data
IC50 [µM] or % inhibition at 100 µMa
% inhibition HL-60 attachmente
compound
E-selectin
P-selectin
L-selectin
E-selectin
P-selectin
1 (bimosiamose)
>500b
134.2
>500b
8.3%
>500b
19.5%
31.7
95.0
>4000d
>500b
1.7%
19.1
-16.1%
31.8
11.8
>500b
>4000d
>500b
13.5%
27.6
1.3%
38.5
15.2
1.1%
12.2%
51.7%
4.3%
83.6%
1.5
28
32
34
47
50
2 (sLeX)c
3 (TBC265)
4
n.s.f
n.s.f
24
5
6
7
8
9
10
11
12
13
14
29a
29b
34
35a
35b
36a
36b
38a
38b
39a
39b
40a
40b
41a
41b
42a
42b
44
46
48
50
52
54
56
58
60
n.s.f
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
16
n.s.f
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
n.a.g
35
>500b
6.0%
25.6%
37.6%
15.0%
3.1%
>500b
4.9
-3.0%
-1.0%
52.0%
-5.3%
77.0%
0.87
4.2
43.2
57.2
1.1
19.6
2.1
2.3
2.4
10.2
2.8
3.4
14.8
8.7
18
20
29
22
22
85.4
64.9
154.4
1.4
46.6
>500b
2.3
>500b
0.8
n.s.f
n.s.f
n.s.f
n.s.f
n.s.f
11
n.s.f
n.s.f
27
18.7
1.0
2.3
3.3
41.9
3.0
3.1
8.7
19
35
35
3.1
13.6
3.3
11
n.s.f
n.s.f
16
15
5.7
n.s.f
20
26.2
30.8
64.5
4.2
8.4
34.9
42.8
2.9
21
21
24
17
>500b
1.3
22
n.s.f
19
n.a.g
>500b
21.7
7.6
>500b
109
n.s.f
n.s.f
n.s.f
n.s.f
47
8.4
>500b
52.4
n.s.f
26
14.6
105.1
41.7
85.8
21.3
4.3
53.9
24.8
28.6
19.5
3.4
n.s.f
26
>500b
>500b
48.6
n.s.f
28
n.s.f
23
57.2
n.s.f
61
56
55
45
25
132.0
>500b
113.7
61.4
5.8
9.8
21.8
212.1
1.2
37.6
244.0
1.6
32
n.s.f
53
61
98
62
63
9.7
48.0
0.57
1.1
0.79
1.6
47
33
83
58
64
65
15.7
0.74
3.6
1.1
9.7
58
93
58
>500b
n.s.f
a IC50 [µM] values are given for the static molecular binding assay for E-, P-, and L-selectin. Maximal tested concentration is 500 µM. If IC50s are not
available, %-inhibition at an inhibitor concentration of 100 µM will be given. b No IC50 value determinable up to a concentration of 500 µM. c SLeX has
been tested up to a concentration of 4000 µM. d No IC50 value determinable up to a concentration of 4000 µM. e %-inhibition of HL-60 cell attachment is
given. Inhibitor concentration was 109 µM. f Not significant. g Not available.
and/or hydrogen bonding to P-selectin. Considering the equiva-
lent stereochemical arrangement of hydroxyl groups at C-2, C-3,
and C-4 of mannose in compound 1 or 3 and at C-4, C-3, and
C-2 of fucose in sLeX, it can be assumed that the mannosyl
unit binds to the calcium ion on the surface of selectins in a
similar manner as the fucosyl unit of sLeX does (Figure 1). The
stereochemical pattern of hydroxyl groups is also reflected by
other selectin antagonists taking advantage of arabinose instead
of fucose, further supporting the meaning of a common
pharmacophore.25 In addition, the docking investigations indicate
Figure 2. Early pharmacophoric model used for 3-D database searches.
The pharmacophore represents an abstraction of three hydroxyl groups
that one of the carboxylic acid groups of 1 is likely to mimic
the function of tyrosine sulfate-51 (Tys 51) of PSGL-1, another
which are considered to be important for binding to selectins and can
hot spot of the high affinity ligand PSGL-1, which in turn binds
to Arg-85 of P-selectin.19 In conclusion, hot spots mandatory
for high affinity binding of PSGL-1 to P-selectin can also be
found in 1. Therefore, compound 1 can be considered as PSGL-
1-glycopeptido-mimetic rather than sLeX glyco-mimetic, whereby
one of the mannosyl groups of 1 is supposed to bind to calcium
and one of the carboxylic acids to Arg-85 of P-selectin. This
be found in the calcium binding sugars mannose, fucose, and arabinose.
Each sphere represents a hydrogen bond acceptor and donator. Distances
between the centers are 2.64 Å, 2.85 Å, and 4.30 Å. The tolerance
radius was set to 0.8 Å.
fraction of PSGL-1.24 According to the cocrystals of sLeX and
sLeX-bearing PSGL-1 with P-selectin, the hydroxyl groups of
fucose at C-4, C-3, and C-2 are involved in calcium binding

1104 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Kranich et al.
Figure 3. Selected hits of the in silico search procedure.
hypothesis is in agreement with biological in Vitro experiments,
where 1 potently competes with a PSGL-1-like ligand for
P-selectin binding (Table 1).
was employed. The root-mean-square distance (rmsd) of 0.098
Å clearly demonstrates the structural equivalence of the hydroxyl
groups and confirms validity of the energy minimization of
compound 3 in a protein-free state. Accordingly, a 3-D database
search against vendor databases was performed in order to
identify molecular fragments appropriate for the replacement
of mannose, which resulted in a large number of in silico hits.
The hit set was then reduced by manual inspection whereby
main criteria for exclusion were (1) molecular weight of the
molecule, (2) no potential for chemical modifications, and (3)
a peptidic and glycosidic nature of the molecular fragments.
The resulting compounds were then acquired and tested in our
in Vitro assays. Examples from this exercise are shown in Figure
3. Both compounds 4 (pyrogallol) and 5 (gallic acid) showed
in Vitro inhibitory activity for P- and L-selectin in the static
molecular binding assay with IC50 values in the lower µM range.
Compound 4, but not 5, also showed activity in the dynamic
HL-60 cell attachment assay. Compound 8, a regioisomer of 5,
showed IC50 values comparable to that of 5. Removing one of
the hydroxyls of 5 or 8 resulted in compounds 6 and 9,
respectively, which were devoid of inhibitory effect for P- and
L-selectin. However, inhibition of E-selectin was increased by
both 6 and 9. Calcium binding and hydrogen bonding of the
hydroxyls were assumed, but electronic effects (e.g., π-bonding),
or a combination of both might be possible as well. Further
structure-activity relationship (SAR) studies are required and
are part of ongoing research activities.
Other in silico hits, such as 10 (shikimic acid), which is
known to bind to C-type lectin MBP-A,27 as well as salicylates
(not shown here), have also been investigated for their inhibitory
action but with moderate activity in the static molecular binding
assay. Interestingly, Kaila et al. recently reported on novel
selectin antagonists. The authors describe quinic acid-derived
compounds, a biochemical precursor of compound 10, which
was developed through structure-based design.9 The authors
applied a fragment-based design approach to identify molecular
entities with moderate activity. Selected synthetic quinic acid
derivatives showed promising biological activity both in Vitro
and in ViVo. Two further in silico hit compounds, 11 (Baicalein)
and 12 (Chrysin), also showed moderate biological in Vitro
activity. Compound 11 (5,6,7-trihydroxyflavone) is one of the
three major flavonoids of Scutellaria baicalensis Georgi, a
traditional Chinese herb with known antiinflammatory activity.28
On the basis of these findings, compound 1 is regarded as
the template structure for a ligand-based design of novel
nonpeptidic and nonglycosidic small molecule selectin antago-
nists with improved pharmacodynamic and pharmacokinetic
properties. Due to its large number of rotatable bonds, compound
1 is, however, not the best starting point for a ligand-based
design approach. Interestingly, Kogan et al. showed that only
one of the two carboxylic groups in compound 1 is mandatory
for selectin binding, indicating that both structurally identical
subunits of 1 may contribute differently to selectin binding.23
Therefore, compound 3, a substructure of 1 and historically the
first generation lead structure in the development of 1 was taken
as a starting structure.26 This substructure is basically half of 1
and, like 1, is supposed to interfere with not only the sLeX
binding feature but also a peptidic binding feature of PSGL-1.
Although 3 did not show inhibitory activity in the PSGL-1-like
setting (static molecular binding assay) at given concentrations
(Table 1), the compound showed activity in another in Vitro
selectin assay at higher concentration and can therefore be
considered as a weak binder.26 As already mentioned above,
the hydroxyl groups at C-2, C-3, and C-4 of mannose in 3, which
are likely to reflect the function of the hydroxyl groups at C-4,
C-3, and C-2 of fucose in sLeX, are supposed to be crucial for
selectin binding. In the case of sLeX, two of the fucosyl
hydroxyls bind to calcium on the shallow surface of the C-type
lectin domain of selectins.19 All three hydroxyl groups partici-
pate in hydrogen bonding to selectins. Furthermore, the car-
boxylic acid group of 3, which is supposed to mimic Tys 51 of
PSGL-1, is also considered to be crucial for selectin binding.
Taking compound 3 as the new template, initial efforts were
focused on the replacement of the mannose moiety. The three-
dimensional structure of 3 was minimized by employing a
stochastic search algorithm and the MMFF94x force field. From
the position of hydroxyls at C-2, C-3, and C-4 of mannose, a
pharmacophore was derived (Figure 2). In order to analyze
conformational differences between the positions of the hydroxyl
groups in the minimized mannose and the corresponding
hydroxyl groups of the fucosyl unit in sLeX as solved by
crystallography,19 a rigid superposition of the three hydroxyl
groups of the minimized mannose of compound 3 with fucose

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1105
Figure 5. On the left-hand side molecule 4 (pyrogallol; carbon atoms
in green) is aligned with a minimized structure of 3 (TBC265; carbon
atoms in blue). The spatial correspondence of oxygens (red) is evident.
On the right-hand side the best scoring superposition of 14 (carbon
atoms in green) and 3 (carbon atoms in blue) is depicted. A flexible
alignment procedure was applied with equally weighted similarity terms
for hydrogen-bond donators and acceptors, aromaticity, acid/base, and
volume, as implemented in MOE.20 The structure of mannose in 3 was
kept fixed in a minimized conformation. All other structural elements
in compound 3 and 14 were flexible. Hydrogen atoms are omitted from
the structures for clearance.
Figure 4. Hit compound 14 represents the gallic acid analogue of 3.
Presumably crucial binding motifs are highlighted. Motif 15 represents
a generic structure for the compounds described in the following.
Compound 12 (5,7-dihydroxyflavone) belongs to the class of
isoflavones with various biological activities.29 Both compounds
showed activity in the static molecular binding assay in the
intermediate µM range. Actually, flavonoids are well-known
as compounds with antiinflammatory effect30 and have been
described as selectin antagonists.31 Finally compound 13 showed
inhibitory activity against P- and L-selectin.
Esterification of 3-bromophenylacetic acid with benzyl
bromide and NaHCO3 delivered the first building block (17).
THPa1 protection of phenol with 3,4-DHP and PPTS followed
by selective ortho-boration with n-BuLi and tri-isopropyl borate
provided the second building block (18). Suzuki coupling of
17 and 18 with Pd(PPh3)4 to generate related orthogonally
protected biphenyl 19 and release of phenolic hydroxyl under
acidic conditions (p-TsOH, MeOH) led to 20. Subsequent
reaction with 5-(chlorocarbonyl)benzene-1,2,3-triyl triacetate
(21) and final cleavage of acetyl groups of 22 with aqueous
ammonia followed by Pd/C-catalyzed hydrogenation gave 14.
Initially, molecule 14 was modified to investigate the effect
of -CH2- and -CH2CH2- spacer between the pyrogallol
moiety and the carboxylic acid part of the molecule. Addition-
ally, the ester function between the biphenyl and the gallic acid
was replaced with an amide bond to facilitate chemical
accessibility of the target molecules and to enhance the
compatibility with a variety of reaction conditions (as well as
with in Vitro/in ViVo conditions for later assays) leading to
derivatives outlined in Scheme 2. Suzuki reaction of 3-bromo-
phenylacetic acid methyl ester (24) with 2-aminophenylboronic
acid hydrochloride in the presence of Pd(PPh3)4 under basic
aqueous conditions provided desired biphenyl 25. Amides 27
were then obtained by EDC coupling of compound 25 and 3,4,5-
trimethoxyphenyl acetic (26a) or propionic acid (26b). Car-
boxylic acids 29 were subsequently prepared from 27 either by
direct demethylation with BBr3 (29a) or by analogue demethyl-
ation of the saponified intermediate 28 (LiOH in MeOH) leading
to 29b.
Taking synthetic aspects and the screening results of Table
1 into account, compound 5 seem to be the most appropriate
replacement for mannose in 3. Compound 10 has also been
further investigated as a substitute for mannose but without
success (data not shown). Substitution of mannose in 3 with 5
led to compound 14 (Figure 4), a gallic acid analogue of 3.
This compound proved to have IC50 values for P- and L-selectin
in the high nM range and low µM range (see Table 1),
respectively. However, compound 14 did not show inhibitory
activity for E-selectin up to a concentration of 500 µM.
Compound 14 has also been investigated in the dynamic HL-
60 cell attachment assay and inhibited cell attachment to E-
(16%) and P-selectin (35%), with inhibition rates below the
values of the reference compound 1 (E: 28%; P: 50%).
Furthermore, compound 14 can easily be aligned with compound
3 (Figure 5). After having found a new chemical scaffold by
applying a ligand-based approach, a focused diversity approach
followed. Here, a series of compounds with the general structure
15 (Figure 4) were synthesized and assayed for their biological
activity. The intention of this procedure was to discover selectin
antagonists with improved in Vitro activity belonging to the
structural space of 15.
Chemistry
The first objective of this approach was to synthesize
compound 14, a gallic acid analogue of 3. Thus a synthetic route
analogous to the preparation of 3 was pursued (Scheme 1),
whereby a compatible orthogonal protecting group strategy had
to be applied to obtain 14. The first challenge at this stage was
to avoid the cleavage of the ester bond between the biphenyl
and the gallic acid moiety throughout the reaction sequence.
Due to the sensitivity of this ester bond against strong basic
and acidic reaction conditions, the carboxylic acid function on
the biphenyl moiety was protected as benzyl ester which could
be cleaved afterward by hydrogenation. The hydroxyls of the
gallic acid subunit were acetylated before the coupling step and
could be cleaved later on under mild basic conditions (aqueous
ammonia).
Next, γ-aminobutyric acid (GABA) was chosen as a spacer
to further expand the distance between the carboxylic acid and
pyrogallol portion of the molecule. As shown in Scheme 3,
γ-amino butyric acid ethyl ester was treated with 3,4,5-
trimethoxybenzoic acid chloride followed by ester hydrolysis
with aqueous LiOH resulting in building block 31. Following
an analogous sequence as described above, EDC coupling of
a Abbreviations: THP, tetrahydropyranyl; DHP, dihydro-2H-pyran;
PPTS, pyridinium p-toluenesulfonate; EDC, 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide; DMAP, 4-N,N′-dimethylaminopyridine; DIC, diiso-
propylcarbodiimide; Pd/C, palladium on carbon (10% w/w); rt, room
temperature; aq, aqueous, tR, retention time; MWr, relative molecular weight.

1106 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Scheme 1. Synthesis of TBC265 (3)-Analogous Compound 14
Kranich et al.
Scheme 2. Synthesis of Homologous Compounds 29 via Biphenyl 25a
a For a, n ) 1 and b n ) 2.
Scheme 3. Synthetic Route to 34
31 with biphenyl 25, hydrolysis with LiOH, and release of the
phenolic hydroxyls with BBr3 led to carboxylic acid 34.
We then focused our efforts on a series of compounds
containing a carboxylic acid-substituted head part and a pyro-
gallol type tail part connected by a -CH2- and -CH2CH2-
spacer (Scheme 4). The general synthetic route to these novel
pyrogallol type compounds is analogous to the one in Scheme
2, whereby 3,4,5-trimethoxyphenyl acetic (26a) and propionic
acid (26b) were employed as starting materials, as well as the
appropriately substituted primary amines or anilines. The
aminophenyl-thiopheneacetic acid building block 37 was syn-
thesized in two steps starting from 2-thiopheneacetic acid methyl

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1107
Scheme 4. Synthetic Routes to 3,4,5-Trimethoxyphenyl Acetic and Propionic Acid Amides 35, 36, and 38-42a
a For a, n ) 1 and b n ) 2; (i) EDC, DMAP, Et3N, CH2Cl2; (ii) LiOH, MeOH; (iii) BBr3, CH2Cl2.
Scheme 5. Synthetic Routes to Further 38-Related Homologous Compounds 44, 46, 48, and 50a
a (i) Pyridine, CH2Cl2; (ii) EDC, DMAP, Et3N, CH2Cl2; (iii) LiOH, MeOH; (iv) BBr3, CH2Cl2.
ester which was brominated selectively in the 5-position with
NBS in glacial acetic acid and CHCl3 followed by a Suzuki
type coupling with 2-aminophenylboronic acid in the presence
of Pd(PPh3)4.
Striving toward a more rigid molecular geometry, the number
of rotatable bonds was reduced by inserting a double bond or
phenyl spacer according to Scheme 6. Again, using 37 as the
key building block, the compounds 52, 54, 56, 58, and 60 were
obtained. The synthetic route remained analogous to the one
previously described (Scheme 5). Starting materials were either
generated from the appropriate trimethoxycinnamic acids (in
terms of 51 and 53) or by Suzuki coupling of the appropriate
trimethoxyphenylboronic acids and methyl bromobenzoates
followed by subsequent ester hydrolysis with aqueous LiOH
solution, and formation of the acid chlorides 55, 57, and 59
with oxalyl chloride.
Homologous and isomeric analogues of 56 were then
synthesized as in Scheme 7. Synthetic routes to compounds 61-
65 were adapted from the sequence leading to 56 beginning
with the appropriate starting materials. For the generation of
the initial trimethoxybiphenyls leading to 63 and 64, an alternate
Because of the promising assay data on 38b (Table 1), the
phenylthiopheneacetic acid motif was used as the backbone for
further homologous and isomeric derivatives outlined in Scheme
5 (44, 46, 48, and 50), where spacers and substitution pattern
at the pyrogallol subunit (2,3,4- vs 3,4,5-trihydroxy) were
initially exchanged. Depending on the nature of the carboxylic
acid, either EDC coupling or amidation via the related acid
chloride were employed. For carboxylic acid building blocks
having any alkyl spacer between the carboxylic acid function
and the aromatic ring usually EDC coupling succeeded best.
However, in case of 46 and 48 the amidation using the
corresponding acid chlorides 45 and 47, respectively, provided
better yields.

1108 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Kranich et al.
Scheme 6. Syntheses of 38-Related More Rigid Compounds 52, 54, 56, 58 and 60a
a (i) Pyridine, CH2Cl2; (ii) LiOH, MeOH; (iii) LiOH, MeCN; (iv) BBr3, CH2Cl2.
Scheme 7. Peripheric Compounds of 56
approach using umpoled building blocks (1-bromo-2,3,4-tri-
methoxybenzene and appropriate borated carboxylic acid esters)
had to be applied.
The commonly known substrate-dependency of Suzuki
couplings in terms of steric effects and electron density forced
us to vary the reaction conditions for the individual coupling
steps mentioned above, with respect to solvent, base, or reaction
time.
ing. Most candidates were also screened in the dynamic HL-60
cell attachment assay, which measures attachment of HL-60 cells
under flow conditions. HL-60 cells provide a complex pattern
of selectin ligands on their surface.32 Discrepancies between
results from static molecular binding assay and cell-based assays
might be due to hydrodynamic differences and different
presentations of selectin ligands in both systems. Overall,
compounds with best screening results in both settings are
considered to be most promising. Table 1 summarizes the in
Vitro screening data. Since polyphenolic compounds are known
for their pH-dependent antioxidative effects,33 all samples
screened in the assays were freshly prepared. As outlined above,
a number of catechol and pyrogallol type compounds such as 5
and 4 were screened. Accordingly compound 14, an analogue
of 3 (TBC265), was synthesized (Scheme 1) and tested. The
Results and Discussion
The compounds presented here were biologically evaluated
employing two in Vitro assays. A static molecular binding assay
was used to determine IC50 values or %-inhibition at 100 µM.
Here, potential selectin antagonists compete with a polymeric
PSGL-1-mimicking ligand (PAA-sLeX-TYS) for selectin bind-

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1109
compound showed IC50 values for P-selectin in the nM range
and in the lower µM range for L-selectin. However, E-selectin
was not inhibited by 14 in the static molecular binding assay.
Yet, HL-60 cell attachment for E- and P-selectin was inhibited
by 14. Subsequently, the length of the alkyl spacer between
the pyrogallol subunit and the biphenyl moiety was varied. In
parallel we switched to an amide connection between the two
molecule parts to accelerate and to shorten the chemical access
to the target molecules. As compared to 14, compounds 29a
and 29b in Scheme 2 have one and two additional methylene
groups, respectively, between the amide and pyrogallol motif.
In compound 34, a GABA spacer was introduced between the
gallic acid and the biphenyl moiety to further expand the
distance (Scheme 3). Out of this set compounds 29a and 29b
showed the best in Vitro activity profiles with IC50s for E-, P-,
and L-selectin in the lower µM range and comparable inhibition
for E- and P-selectin in the cell-based assay. Next, the effect of
the biphenyl acetic acid moiety of 29a and 29b on selectin
binding was investigated. Thus, a library of 14 compounds was
synthesized where this moiety was substituted by more simple
aromatic carboxylic acids and also by a bioisosteric building
block (26a,b, Scheme 4). Compound 35a showed a remarkable
IC50 value for E-selectin in the static molecular binding assay.
However, this compound was not active in the dynamic HL-60
cell attachment assay for either E- or P-selectin. Out of this
focused library, other compounds displayed low IC50 values,
whereby compounds 38a, 40a, and 40b gave the best overall
in Vitro activity profile. Due to its overall good activity profile,
phenylthiopheneacetic acid analogues 38a and 38b were chosen
for further optimization. Scheme 5 depicts the synthesis of
additional phenylthiopheneacetic acid derivatives. Variation of
the spacer length and substitution pattern of pyrogallol (2,3,4-
and 3,4,5-trihydroxy) did not result in improved activity, and
none of these compounds showed a balanced profile in the static
molecular binding and the dynamic HL-60 cell attachment assay.
However, compound 48 performed almost as well as 1 (bimo-
siamose) in the dynamic HL-60 cell attachment assay but was
not active in the static molecular binding assay for E-selectin.
It was decided to proceed with the general pattern containing
the phenylthiopheneacetic acid and pyrogallol subunits and
introduce more rigid spacers as the next step (Scheme 6). This
strategy led to compounds with a very promising overall profile.
The introduction of a double bond gave the regioisomers 52
and 54. The 2,3,4-trihydroxy-substituted compound 54 demon-
strated, as compared to compound 1, improved inhibition of
cell attachment for P-selectin. Inhibition of cell attachment to
E-selectin was not significant. Changing the substitution pattern
to 3,4,5-trihydroxy, as in compound 52, resulted in a loss of
inhibition for P-selectin attachment but gave moderate inhibition
of E-selectin attachment. IC50s for both 52 and 54 were in the
lower µM range. The most promising compound of this group
was 56. As compared to 1, compound 56 showed improved in
Vitro activities in both assays. IC50s were in the lower µM range,
and inhibition of HL-60 cell attachment to E- and P-selectin
was 61% and 55%, respectively. In 56 the alkyl- or alkene-
spacer has been replaced with a 1,3-phenyl group. Substitution
pattern of the pyrogallol motif is 2,3,4-trihydroxy. Interestingly,
switching the pyrogallol substitution pattern from 2,3,4- (56)
to 3,4,5-trihydroxy (58) diminishes activity. Switching from the
1,3-linked isomer 58 to the 1,2-phenyl-linked compound 60
resulted in poor activity data as well. HL-60 cell attachment to
E-selectin was not significant and IC50s particularly for P- and
L-selectin were worse than for 58. This might indicate the
capability of the phenyl-spacer to build additional hydrophobic
interactions with the selectins. Specific orientation of these
groups seems to be crucial for activity. As outlined in Scheme
7, the further influence of small variations of 56 (homologous
and isomeric derivatives) was investigated. Hereby the focus
was directed to the 2,3,4-trihydroxy substitution of the pyrogallol
substructure. From this library, the best activity data were
observed for compound 64. In 64, the thiophene moiety was
connected to the meta position of the amide, rather than the
ortho position, as in 56. The compound showed IC50s in the
lower µM range for E- and L-selectin and a nanomolar IC50 for
P-selectin in the static molecular binding assay. Inhibition of
HL-60 cell attachment to E- and P-selectin was 58% and 93%,
respectively. Similar activity data were achieved with 61 which
is a lower homologue of 56 missing the methylene group
between the carboxylic acid and the thiophene moiety. Nano-
molar IC50s for P- and L-selectin in the static molecular binding
assay were observed for compound 62 wherein the thiophene
moiety is connected para to the amide, rather than meta, as in
compound 64. Also for compound 62, inhibition of HL-60 cell
attachment for E- and P-selectin was improved, as compared
to molecule 1. For compound 63, the 2,3,4-trihydroxyphenyl
subunit was connected para to the amide linker. Activities of
63 were also improved as compared to 1; however, the HL-60
cell attachment data were worse when compared to 56, 61, 62,
or 64. Introduction of a methylene group between the amide
group and the pyrogalloylphenyl unit in 56 resulted in the
homologous compound 65. This modification resulted in a loss
of activity for E-selectin in both the static molecular binding
and the dynamic HL-60 cell attachment assay, whereas IC50s
for P- and L-selectin in the static molecular binding assay as
well as inhibition of HL-60 cell attachment for P-selectin were
noteworthy.
The compounds presented here are the result of a rational
hit-finding procedure with focused diversity. While initial hits
of a ligand-based approach such as gallic acid derivatives
showed moderate biological activity, the homologous com-
pounds such as 3,4,5- or 2,3,4-trihydroxyphenylacetic acid
amides showed higher potency. Further modifications led us to
compound 56 and related derivatives, e.g., 61-64. These
compounds already showed improved in Vitro activity, as
compared to 1, with IC50s up to the upper nanomolar range and
high inhibition values in the dynamic HL-60 cell attachment
assay. Having relative molecular weights below 500, these
compounds can be considered as small molecules. At this point
it seems that hydrophobic interactions might be important for
selectin binding. It remains to be analyzed what the functions
of the pyrogallol part and of the carboxylic acid are. Compound
64, for example, seems to be a promising starting point for a
more detailed SAR study including comprehensive docking
studies in the course of hit-to-lead and lead optimization
programs and is the subject of ongoing research activities.
However, it should be mentioned that obtaining a meaningful
SAR for compounds which bind to selectins is a particular
challenge. Beside other proposed binding regions, the surface
of the C-type lectin domain of selectins has been identified as
important binding site for natural, sLeX-bearing ligands. Indeed,
the selectin antagonists presented herein are supposed to address
this binding site, which is also reflected by the type of molecular
binding assay described here. In contrast to the shape of binding
sites of many other drug targets, selectins provide a shallow
surface with highly flexible amino acids to their interaction
partners, rather than a buried cavity with rotationally more
restricted amino acids. This, of course, impacts drug discovery
of synthetic small molecule selectin antagonists. Since even

1110 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Kranich et al.
{2′-[(3,4,5-Trihydroxybenzoyl)oxy]-1,1′-biphenyl-3-yl}-ace-
tic Acid (14). Step 1. (3-Bromo-phenyl)-acetic acid (16, 2.15 g,
10 mmol) was dissolved in anhydrous DMF (12.5 mL) at rt, and
sodium bicarbonate (2.1 g, 25 mmol) was added, followed by slow
addition of benzyl bromide (2.99 mL, 25 mmol). The reaction was
stirred at 100 °C overnight and then mixed with water and extracted
with EtOAc (three times). The combined extracts were washed with
brine, dried with Na2SO4, and concentrated. The crude product was
purified by preparative radial chromatography (CyH/EtOAc 3:1)
to obtain benzyl (3-bromophenyl)-acetate (17, 3.05 g, quant.) as a
colorless oil. 1H NMR (400 MHz, CDCl3): 3.62 (s, 2 H); 5.12 (s,
2 H); 7.14-7.22 (m, 2 H); 7.26-7.41 (m, 6 H) 7.43 (s, 1 H).
Step 2. Phenol (941 mg, 10.0 mmol) was dissolved in dichloro-
methane (80 mL), PPTS (251 mg, 1.0 mmol) and then 3,4-dihydro-
2H-pyran (2.75 mL, 30.0 mmol) were added, and the mixture was
stirred at rt under nitrogen overnight. The reaction was mixed with
water and extracted with EtOAc (three times). The combined
extracts were washed with half-saturated brine, dried with Na2SO4,
and then concentrated. The crude 2-phenoxytetrahydro-2H-pyran
(1.78 g, quant.) was obtained as a white solid. 1H NMR (400 MHz,
CDCl3): 1.51-1.73 (m, 3 H); 1.81-1.88 (m, 2 H); 1.94-2.06 (m,
1 H); 3.55-3.62 (m, 1 H); 3.90 (Ψtd, 1 H, J1 ) 9.0 Hz, J2 ) 3.0
Hz); 5.40 (Ψt, 1 H, J ) 3.0 Hz); 6.96 (t, 1 H, J ) 7.3 Hz); 7.03
(d, 2 H, J ) 8.0 Hz); 7.26 (t, 2 H, J ) 7.6 Hz).
2-Phenoxytetrahydro-2H-pyran (1.00 g, 5.6 mmol) was dissolved
in anhydrous THF (20 mL) under a N2 atmosphere, and then the
solution was cooled to -78 °C. A solution of n-BuLi in hexane
(5.25 mL of a 1.6 M solution, 8.40 mmol) was added slowly, and
the reaction mixture was stirred for 15 min at -78 °C and then an
additional 30 min at rt. The mixture was cooled again to -78 °C
before triisopropyl borate (3.25 mL, 14 mmol) was added. The
reaction mixture was slowly warmed up to rt and stirred for
additional 2 h. The reaction was mixed with water and then
extracted with EtOAc (three times). The combined extracts were
washed with brine, dried with Na2SO4, and concentrated to obtain
2-(tetrahydro-2H-pyran-2-yloxy)phenyl]boronic acid (18, 1.35 g,
79%) as an adhesive off-white solid, which was used without further
purification.
Step 3. [2-(Tetrahydro-2H-pyran-2-yloxy)phenyl]boronic acid
(18, 153 mg, 0.69 mmol) was dissolved in toluene (10.0 mL) and
ethanol (1.5 mL), and the mixture was treated with tetrakis-
(triphenylphosphine)-palladium(0) (50 mg, 0.04 mmol) and aq
Na2CO3 (2.0 mL of a 2.5 M solution, 5.0 mmol) followed by
addition of benzyl (3-bromophenyl)-acetate (17, 200 mg, 0.65
mmol) in toluene (10.0 mL). The reaction mixture was degassed
under nitrogen (five times), flooded with N2 again and stirred
vigorously for 2 h at 100 °C. The resulting mixture was then filtered
through silica gel, which was washed with EtOAc. The combined
filtrates were concentrated, and the crude product was purified by
preparative radial chromatography (CyH/EtOAc 1:1) to obtain an
inseparable mixture of benzyl [2′-(tetrahydro-2H-pyran-2-yloxy)-
1,1′-biphenyl-3-yl]-acetate (19) and starting material 17 (403 mg,
about 1:1) as a colorless oil.
Step 4. Benzyl [2′-(tetrahydro-2H-pyran-2-yloxy)-1,1′-biphenyl-
3-yl]-acetate (19, 156 mg, 0.7 mmol) was dissolved in MeOH (20
mL) at rt. pTsOH·H2O (4.0 mg, 0.02 mmol) was added, and the
reaction mixture was stirred for 2 h at 40 °C. A mixture of EtOAc
(10 mL) and brine (10 mL) was added to the cooled reaction and
mixed well. The aqueous phase was extracted with EtOAc, and
the combined organic phases were washed with brine, dried with
Na2SO4, and concentrated under reduced pressure. The crude
product was purified by preparative radial chromatography (CyH/
EtOAc 3:1) to obtain benzyl (2′-hydroxy-1,1′-biphenyl-3-yl)-acetate
(20, 88 mg, 76%) as a colorless oil. 1H NMR (400 MHz, CDCl3):
3.72 (s, 2 H); 5.14 (s, 2 H); 5.15 (br. s, 1 H); 6.92-7.00 (m, 2 H);
7.16-7.23 (m, 2 H) 7.28-7.47 (m, 9 H).
structurally similar ligands show an increased probability to bind
to selectins with different binding modes, clear structure-
activity-relationship may be difficult to obtain.
The novel class of synthetic selectin antagonists presented
herein are polyphenolic compounds tethered to carboxylic acid
through a variety of structural units. Phenolic and polyphenolic
compounds are common in many natural products, food
constituents, and marketed drugs. Members of this compound
class can be found in green tea, red wine, grapes, apples, and
chocolate, for instance, and they are believed to be responsible
for protective antioxidative, anticancer, antiinflammatory, and
cardiovascular protecting effects, commonly known as the
French paradox.8,34 Consequently, pharmacological action of
natural polyphenolics is the subject of intensive research. The
inhibitory activity of short peptides against P-selectin, found
by phage display, could be improved to nanomolar IC50 values
by the N-terminal attachment of gallic acid.35 Accordingly, the
authors investigated the inhibitory effect of 5 on P-selectin.8 It
could be shown, that molecule 5 binds to P-selectin in a relative
potent manner. However, the authors did not observe binding
to E-selectin and binding to L-selectin was moderate.
In conclusion, novel, rationally designed synthetic small
molecules with nanomolar in Vitro activity against E-, P-, and
L-selectin and with a relative molecular weight below 500 have
been presented. Current research activities are directed toward
further medicinal chemistry including comprehensive compu-
tational investigations in order to optimize pharmacodynamic
and pharmacokinetic properties of this novel class of com-
pounds, and results from these studies will be disclosed in due
course.
Experimental Section
Computational Chemistry. Molecular simulations such as
stochastic conformational searches, setup of 3D databases of vendor
compound collections, pharmacophore searches, and molecular
alignment have been performed with MOE of the Chemical
Computing Group Inc.20 The MMFF94x force field has been applied
in all energy-based calculation. The flexible alignment procedure
was applied with equally weighted similarity terms for hydrogen-
bond donors and acceptors, aromaticity, acid/base, and volume.
Chemistry. Solvents and reagents were used as received from
commercial distributors without further purification. Anhydrous
reactions were conducted under a nitrogen atmosphere. Proton NMR
spectra were recorded at a 400 MHz Bruker Avance 400. Chemical
shifts δ are reported in ppm units. Molecular mass was determined
by liquid chromatography mass spectrometry (LC/MS) using an
LCQ Advantage ion trap mass spectrometer from Thermo Finnigan
equipped with an electrospray interface and connected to a Surveyor
HPLC (Thermo Finnigan). Positive and negative ion mass spectra
were recorded (mass range m/z 150-1000) in normal scan mode.
Preparative reversed phase (RP) HPLC was run on a Shimadzu
LC8A instrument using a Phenomenex Synergi 10 µ Hydro RP
80A (15 mm i.d. × 250 mm) C-18 column. The flow rate was 5
mL/min, the mobile phase A was water and the mobile phase B
acetonitrile, containing 0.05% TFA each as modifier. Purity was
determined using a Shimadzu 10ADvp instrument with C18 RP
column. Two methods were applied: method A using a gradient
of water and acetonitrile (from 5% B to 95% B in 8.5 min) and
method B using a gradient of water and methanol (from 5% B to
95% B in 4.5 min followed by 5 min at 95% B). Each solvent
contained 0.05% TFA, flow rate was 1.5 mL/min and UV detection
was performed at 254 nm. Pure fractions were pooled and
lyophilized. For purifications with preparative radial thin layer
chromatography a Harrison Research Chromatotron No. 7924 T
was employed using glass disks coated with silica gel 60PF254
(containing gypsum from Merck, Germany; 1 mm, 2 mm, or 4 mm
layer thickness) as stationary phase. The tetrasaccharide sLeX has
been acquired from Carbohydrate Synthesis Ltd.
Step 5. Benzyl (2′-hydroxy-1,1′-biphenyl-3-yl)-acetate (20, 46
mg, 0.14 mmol) was dissolved in anhydrous dichloromethane (1.0
mL) and Et3N (29 µL, 0.21 mmol) was added. The solution was
stirred for 5 min at rt, and then 5-(chlorocarbonyl) benzene-1,2,3-
triyl triacetate (21, 50 mg, 0.16 mmol) was added. The reaction

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1111
mixture was stirred another 30 min at rt, then quenched with sat.
aqueous NH4Cl solution and extracted with dichloromethane. The
extracts were combined and washed with brine, dried with Na2-
SO4, and then concentrated under reduced pressure. The crude
product was purified by preparative radial chromatography (CyH/
EtOAc 3:1) to obtain 3′-[2-(benzyloxy)-2-oxoethyl]-1,1′-biphenyl-
2-yl 3,4,5-tris(acetyloxy) benzoate (22, 55 mg, 64% yield) as an
adhesive colorless solid. H NMR (400 MHz, CDCl3): 2.24 (s, 6
H); 2.27 (s, 3 H); 3.61 (s, 2 H); 5.06 (s, 2 H); 7.16-7.41 (m, 13
H); 7.74 (s, 2 H).
was dissolved in MeOH (2.5 mL) at rt, and aq LiOH (385 µL of a
1 M solution, 385 µmol) was added. The reaction mixture was
stirred for 17 h at rt and then quenched with 1 M aq HCl (pH ∼
3), with cooling as necessary. The mixture was then extracted with
EtOAc (three times), and the extracts were combined, washed with
water and then brine, dried with Na2SO4, and then concentrated,
to obtain {2′-[3-(3,4,5-trimethoxy-phenyl)-propionylamino]-biphe-
nyl-3-yl}-acetic acid (28, 36 mg, quant.) as an adhesive white solid
1
1
that was used without further purification. H NMR (400 MHz,
CDCl3): 2.46 (t, 2 H, J ) 7.2 Hz); 2.86 (t, 2 H, J ) 7.3 Hz); 3.65
(s, 2 H); 3.74 (s, 6 H); 3.77 (s, 3 H); 6.36 (s, 2 H); 7.09-7.22 (m,
5 H); 7.25 (d, 1 H, J ) 7.6 Hz); 7.30-7.40 (m, 2 H); 8.28 (br. d,
1 H, J ) 8.1 Hz).
Step 6. 3′-[2-(Benzyloxy)-2-oxoethyl]-1,1′-biphenyl-2-yl 3,4,5-
tris(acetyloxy) benzoate (22, 55 mg, 0.09 mmol) was dissolved in
acetone (0.5 mL) and then treated with an aqueous NH3 solution
(58 µL, 1.0 mmol). The reaction mixture was stirred for 15 min at
rt and then quenched with a 1 N aq HCl solution (pH ∼ 3). The aq
phase was extracted with EtOAc, and the combined organic phases
were dried with Na2SO4 and then concentrated under reduced
pressure. The crude 3′-[2-(benzyloxy)-2-oxoethyl]-1,1′-biphenyl-
2-yl 3,4,5-trihydroxybenzoate (23) was dissolved in EtOAc (1.0
mL), and Pd/C (10 mg) was added. The resulting mixture was
carefully evacuated, flooded with hydrogen three times, and then
stirred under a hydrogen atmosphere at rt overnight. The reaction
system was evacuated again and flushed with N2. The reaction
mixture was filtered and then concentrated under reduced pressure.
The crude product was purified by preparative RP HPLC to obtain
{2′-[(3,4,5-trihydroxybenzoyl)oxy]-1,1′-biphenyl-3-yl} acetic acid
Step 4. {2′-[3-(3,4,5-Trimethoxy-phenyl)-propionylamino]-bi-
phenyl-3-yl}-acetic acid (28, 33 mg, 0.073 mmol) was dissolved
in anhydrous dichloromethane (2.5 mL) under nitrogen, and then
the solution was cooled to -78 °C. A solution of BBr3 in
dichloromethane (440 µL of a 1 M solution, 0.44 mmol) was added
dropwise. The reaction mixture was stirred for 30 min at -78 °C,
warmed slowly to rt, and stirred an additional 90 min at rt. The
reaction was quenched by dropwise addition of ice-water, the
layers were separated, and the aq phase was extracted with EtOAc
(three times). The combined organic phases were washed with water
and then brine, dried with Na2SO4, and then concentrated. The crude
product was purified by preparative RP HPLC to obtain {2′-[3-
(3,4,5-trihydroxy-phenyl)-propionylamino]-biphenyl-3-yl}-acetic acid
(29b, 21 mg, 71%). 1H NMR (400 MHz, CD3OD): 2.48 (t, 2 H, J
) 7.6 Hz); 2.71 (t, 2 H, J ) 7.6 Hz); 3.69 (s, 2 H); 6.24 (s, 2 H);
7.17 (dt, 1 H, J1 ) 7.6 Hz, J2 ) 1.3 Hz); 7.28-7.42 (m, 6 H); 7.60
(d, 1 H, J ) 7.6 Hz). MS (ESI) m/z 408.1 ([M + H]+).
1
(14, 9 mg, 14%). H NMR (400 MHz, CD3CN): 3.59 (s, 2 H);
7.05 (s, 2 H); 7.21 (d, 1 H, J ) 7.6 Hz); 7.27 (d, 1 H, J ) 7.8 Hz);
7.32 (t, 1 H, J ) 7.6 Hz); 7.34-7.49 (m, 5 H).
{2′-[3-(3,4,5-Trihydroxy-phenyl)-propionylamino]-biphenyl-
3-yl}-acetic Acid (29b). Step 1. Tetrakis-(triphenylphosphine)-
palladium(0) (30 mg, 0.025 mmol) and Na2CO3 decahydrate (944
mg, 3.30 mmol dissolved in 1.2 mL of H2O) were added to ethanol
(0.8 mL), followed by a solution of 2-amino-benzeneboronic acid
(201 mg, 1.30 mmol) in toluene (6.0 mL). The reaction mixture
was carefully degassed under nitrogen five times, and then flooded
with N2 again. A solution of (3-bromo-phenyl)-acetic acid methyl
ester (24, 270 mg, 1.18 mmol) in toluene (6.0 mL) was then added,
and the reaction mixture was degassed again (five times) and then
stirred overnight at 100 °C. The reaction mixture was then
partitioned between EtOAc and brine (1:1), and then the aqueous
phase was extracted with EtOAc (three times). The combined
organic phases were washed with brine, dried with Na2SO4, and
concentrated. The crude product was purified by preparative radial
chromatography (CyH/EtOAc 3:1) to obtain (2′-amino-biphenyl-
3-yl)-acetic acid methyl ester (25, 304 mg, 81%) as an orange oil.
1H NMR (400 MHz, CDCl3): 3.66 (s, 2 H); 3.69 (s, 3 H); 3.62-
3.86 (br. s, 2 H); 6.75 (d, 1 H, J ) 8.1 Hz); 6.80 (t, 1 H, J ) 7.3
Hz); 7.11 (d, 1 H, J ) 7.3 Hz); 7.15 (d, 1 H, J ) 8.1 Hz); 7.22-
7.26 (br. m, 1 H); 7.32-7.42 (m, 3 H).
Step 2. EDC hydrochloride (61 mg, 0.32 mmol) and Et3N (44
µL, 0.32 mmol) were mixed with anhydrous dichloromethane (1.0
mL), and the resulting mixture was stirred under nitrogen for 5
min at rt. 3-(3,4,5-Trimethoxy-phenyl)-propionic acid (26b, 55 mg,
0.23 mmol) and DMAP (2 mg, 0.02 mmol) were added, and the
resulting solution was stirred for 15 min. (2′-Amino-biphenyl-3-
yl)-acetic acid methyl ester (25, 50 mg, 0.21 mmol) was then added,
and the reaction mixture was stirred overnight at rt and then
quenched with water. The aq phase was extracted with dichlo-
romethane (three times), and the combined organic phases were
washed with brine, dried with Na2SO4, and concentrated. The crude
product was purified by preparative radial chromatography (EtOAc/
CyH 1:1) to obtain {2′-[3-(3,4,5-trimethoxy-phenyl)-propiony-
lamino]-biphenyl-3-yl}-acetic acid methyl ester (27b, 46 mg, 48%)
as a yellow oil. 1H NMR (400 MHz, CDCl3): 2.50 (t, 2 H, J ) 7.6
Hz); 2.90 (t, 2 H, J ) 7.7 Hz); 3.64 (s, 2 H); 3.65 (s, 3 H); 3.77 (s,
6 H); 3.78 (s, 3 H); 6.38 (s, 2 H); 7.09-7.18 (m, 3 H); 7.19-7.28
(m, 3 H); 7.34 (d, 1 H, J ) 8.1 Hz); 7.38 (d, 1 H, J ) 7.8 Hz);
8.31 (br. d, 1 H, J ) 7.8 Hz).
{2′-[4-(3,4,5-Trihydroxy-benzoylamino)-butyrylamino]-biphen-
yl-3-yl}-acetic Acid (34). Step 1. 4-Amino-butyric acid ethyl ester
hydrochloride (3.98 g, 23.74 mmol) was dissolved in anhydrous
dichloromethane (120 mL) at rt under nitrogen, and triethylamine
(8.30 mL, 59.55 mmol) was added. The reaction mixture was stirred
for 15 min at rt, 3,4,5-trimethoxy-benzoyl chloride (6.58 g, 28.53
mmol) was added in portions, and the mixture was stirred for
additional 24 h at rt. The reaction mixture was quenched with
methanol, filtered through a short pad of silica gel with EtOAc/
CyH (3:1), and concentrated. The crude product was purified by
flash chromatography (EtOAc/CyH 2:1 and then 3:1) to obtain
4-(3,4,5-trimethoxy-benzoylamino)-butyric acid ethyl ester (30, 4.48
1
g, 58%) as a white solid. H NMR (400 MHz, CDCl3): 1.22 (t, 3
H, J ) 7.1 Hz); 1.95 (Ψquint, 2 H, J ) 6.5 Hz); 2.44 (t, 2 H, J )
6.5 Hz); 3.48 (td, 2 H, J1 ) 6.5 Hz, J2 ) 5.8 Hz); 3.85 (s, 3 H);
3.89 (s, 6 H); 4.10 (q, 2 H, J ) 7.1 Hz); 6.72 (br. s, 1 H); 7.02 (s,
2 H).
Step 2. 4-(3,4,5-Trimethoxy-benzoylamino)-butyric acid ethyl
ester (30, 4.48 g, 13.79 mmol) was dissolved in acetonitrile (100
mL) at rt, and a solution of aq LiOH (41.4 mL of a 1 M solution,
41.4 mmol) was added. The reaction mixture was stirred for 18 h
at rt and then quenched by addition of 1 M aq HCl (50 mL), with
cooling as necessary. The mixture was then extracted with
chloroform (three times), and the combined organic phases were
washed with brine, dried with Na2SO4, and concentrated, to obtain
4-(3,4,5-trimethoxy-benzoylamino)-butyric acid (31, 4.07 g, 99%)
as a white solid. 1H NMR (400 MHz, CDCl3): 1.94 (Ψquint, 2 H,
J ) 6.7 Hz); 2.46 (t, 2 H, J ) 6.7 Hz); 3.50 (br. td, 2 H); 3.85 (s,
3 H); 3.88 (s, 6 H); 6.61 (br. s, 1 H); 7.00 (s, 2 H).
Step 3. EDC hydrochloride (55 mg, 0.29 mmol) and Et3N (40
µL, 0.29 mmol) were mixed with anhydrous dichloromethane (1.0
mL) under nitrogen, and the resulting solution was stirred for 5
min at rt. 4-(3,4,5-Trimethoxy-benzoylamino)-butyric acid (31, 63
mg, 0.21 mmol) was added and then DMAP (2 mg, 0.02 mmol),
and the resulting solution was stirred for 10 min. (2′-Amino-
biphenyl-3-yl)-acetic acid methyl ester (25, 46 mg, 0.19 mmol) was
added, and the reaction was stirred for 15 h at rt. The reaction was
then quenched by addition of sat. aq NH4Cl and water. The layers
were separated, and the aq phase was extracted with dichlo-
romethane (three times). The combined organic phases were washed
with water and then brine, dried with Na2SO4, and concentrated.
Step 3. {2′-[3-(3,4,5-Trimethoxy-phenyl)-propionylamino]-bi-
phenyl-3-yl}-acetic acid methyl ester (27b, 35 mg, 0.075 mmol)

1112 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6
Kranich et al.
The crude product was purified by preparative radial chromatog-
raphy (EtOAc/CyH 3:1 and then 6:1) to obtain {2′-[4-(3,4,5-
trimethoxy-benzoylamino)-butyrylamino]-biphenyl-3-yl}-acetic acid
preparative radial chromatography (CyH/EtOAc 5:1] to obtain [5-(2-
amino-phenyl)-thiophen-2-yl]-acetic acid methyl ester (37, 634 mg,
51%) as a brown oil. 1H NMR (400 MHz, CDCl3): 3.73 (s, 3 H);
3.83 (s, 2 H); 3.92-4.07 (br. s, 2 H); 6.74 (d, 1 H); 6.76 (td, 1 H,
J1 ) 7.6 Hz, J2 ) 1.3 Hz); 6.92 (d, 1 H, J ) 3.5 Hz); 7.02 (d, 1
H, J ) 3.5 Hz); 7.11 (td, 1 H, J1 ) 7.6 Hz, J2 ) 1.5 Hz); 7.23 (dd,
1 H, J1 ) 7.6 Hz, J2 ) 1.5 Hz). MS (ESI) m/z 248.1 ([M + H]+).
HPLC tR ) 8.36 min, 84% pure.
1
methyl ester (32, 36 mg, 37%) as a yellowish oil. H NMR (400
MHz, CDCl3): 1.91-2.00 (m, 2 H); 2.43 (t, 2 H, J ) 6.2 Hz);
3.41-3.50 (m, 2 H); 3.64 (s, 2 H); 3.68 (s, 3 H); 3.81 (s, 6 H);
3.83 (s, 3 H); 7.06 (s, 2 H); 7.13-7.27 (m, 5 H); 7.29 (t, 1 H, J )
7.7 Hz); 7.35-7.45 (m, 3 H); 8.11 (d, 1 H, J ) 8.1 Hz).
Step 4. {2′-[4-(3,4,5-Trimethoxy-benzoylamino)-butyrylamino]-
biphenyl-3-yl}-acetic acid methyl ester (32, 36 mg, 0.07 mmol)
was dissolved in MeOH (2.0 mL) at rt, and a solution of aq LiOH
(490 µL of a 1 M solution, 0.49 mmol) was added. The reaction
mixture was stirred for 17 h at rt, and then the reaction mixture
was partitioned between chloroform and 1 M hydrochloric acid.
The aq phase was separated and extracted with chloroform (three
times). The combined organic phases were washed with brine, dried
with Na2SO4, and concentrated, to obtain {2′-[4-(3,4,5-trimethoxy-
benzoylamino)-butyryl-amino]-biphenyl-3-yl}-acetic acid (33, 37
mg of crude product, quant.) as a brown solid. 1H NMR (400 MHz,
CDCl3): 1.97 (Ψquint, 2 H, J ) 6.9 Hz); 2.38 (t, 2 H, J ) 7.3
Hz); 3.51-3.60 (m, 2 H); 3.65 (s, 2 H); 3.83 (s, 6 H); 3.84 (s, 3
H); 6.67 (br. s, 1 H); 6.99 (s, 2 H); 7.15 (t, 1 H, J ) 7.3 Hz);
7.20-7.30 (m, 4 H); 7.32 (t, 1 H, J ) 7.8 Hz); 7.37 (s, 1 H), 7.44
(t, 1 H, J ) 7.4 Hz); 8.27 (d, 1 H, J ) 8.3 Hz).
Step 3. A solution of 2,3,4-trimethoxyphenylboronic acid (1.40
g, 6.60 mmol) in toluene (15 mL) under nitrogen was treated with
EtOH (2.0 mL), tetrakis-(triphenylphosphine)-palladium(0) (208 mg,
0.18 mmol), and then Na2CO3 decahydrate (4.81 g, 16.80 mmol)
in water (5.2 mL). The resulting mixture was carefully degassed
under nitrogen (five times), a solution of methyl-3-bromobenzoate
(1.29 g, 6.00 mmol) in toluene (9.0 mL) was added by syringe,
and the resulting mixture was again carefully degassed and stirred
overnight at 100 °C under nitrogen. The mixture was partitioned
between brine/EtOAc (1:1), and the layers were separated. The aq
phase was extracted with EtOAc (three times), and the combined
organic phases were washed with brine, dried with Na2SO4, and
concentrated. The crude product was purified by preparative radial
chromatography (EtOAc/CyH 1:5) to obtain 2′,3′,4′-trimethoxy-
biphenyl-3-carboxylic acid methyl ester (1.07 g, 58%) as an
yellowish oil. 1H NMR (400 MHz, CDCl3): 3.66 (s, 3 H); 3.89 (s,
3 H); 3.92 (s, 6 H); 6.74 (d, 1 H, J ) 8.6 Hz); 7.03 (d, 1 H, J )
8.6 Hz); 7.44 (t, 1 H, J ) 7.8 Hz); 7.70 (d, 1 H, J ) 7.6 Hz); 7.97
(d, 1 H, J ) 7.8 Hz); 8.15 (br. s, 1 H).
Step 5. {2′-[4-(3,4,5-Trimethoxy-benzoylamino)-butyryl-amino]-
biphenyl-3-yl}-acetic acid (33, 37 mg, 0.07 mmol) was dissolved
in anhydrous dichloromethane (1.0 mL) under nitrogen, the solution
was cooled to -78 °C, and then BBr3 (62 µL, 0.66 mmol) was
added dropwise. The reaction mixture was stirred for 30 min at
-78 °C, warmed slowly to rt, and stirred an additional 2 h at rt.
The reaction mixture was then cooled to 0 °C and quenched by
dropwise addition of water. The resulting mixture was partitioned
between water and EtOAc. The aq phase was extracted with EtOAc
(two times) and dichloromethane (two times), and the combined
organic phases were washed with brine, dried with Na2SO4, and
concentrated, to obtain {2′-[4-(3,4,5-trihydroxy-benzoylamino)-
butyrylamino]-biphenyl-3-yl}-acetic acid (34, 17 mg, 53%) as a
Step 4. 2′,3′,4′-Trimethoxy-biphenyl-3-carboxylic acid methyl
ester (566 mg, 1.87 mmol) was dissolved in acetonitrile (19 mL)
at rt, and aq LiOH (9.36 mL of a 1 M solution, 9.36 mmol) was
added. The reaction mixture was stirred overnight at rt and then
quenched by addition of 1 M aq HCl (pH ∼ 3), with cooling as
necessary. The mixture was extracted with EtOAc (three times),
and the combined extracts were washed with brine, dried with Na2-
SO4, and concentrated. The crude product was crystallized from
EtOAc/CyH 1:3 to obtain 2′,3′,4′-trimethoxy-biphenyl-3-carboxylic
1
acid (392 mg, 72%) as a white solid. H NMR (400 MHz, CD3-
1
white solid. H NMR (400 MHz, CD3CN): 1.72-1.82 (m, 2 H);
OD): 3.68 (s, 3 H); 3.93 (br. s, 6 H); 6.92 (d, 1 H, J ) 8.6 Hz);
7.11 (d, 1 H, J ) 8.6 Hz); 7.54 (t, 1 H, J ) 7.7 Hz); 7.75 (d, 1 H,
J ) 7.6 Hz); 8.01 (d, 1 H, J ) 7.8 Hz); 8.18 (br. s, 1 H).
Step 5. 2′,3′,4′-Trimethoxy-biphenyl-3-carboxylic acid (107 mg,
0.37 mmol) was dissolved in anhydrous dichloromethane (3.0 mL)
under nitrogen, and anhydrous DMF (3 drops, cat. amount) was
added. Oxalyl chloride (42 µL, 0.48 mmol) was then added slowly,
keeping the temperature at ca. 15 °C (water bath), and the turbid
mixture was stirred for additional 2 h at rt. The resulting solution
of crude 2′,3′,4′-trimethoxy-biphenyl-3-carbonyl chloride (55) was
added to an ice-cooled solution of [5-(2-amino-phenyl)-thiophen-
2-yl]-acetic acid methyl ester (37, 70 mg, 0.28 mmol) in anhydrous
dichloromethane (4.5 mL) and anhydrous pyridine (0.75 mL). The
cooling bath was removed, and the reaction was stirred for 3 h at
rt and then mixed with an ice-cooled solution of 1 M aq HCl. The
mixture was then extracted with dichloromethane (three times), and
the combined organic phases were washed with brine, dried with
Na2SO4, and concentrated. The crude product was purified by
preparative radial chromatography (EtOAc/CyH 1:3, later 1:2) to
obtain (5-{2-[(2′,3′,4′-trimethoxy-biphenyl-3-carbonyl)-amino]-
phenyl}-thiophen-2-yl)-acetic acid methyl ester (96 mg, 65%) as
an adhesive brownish solid. 1H NMR (400 MHz, CDCl3): 3.64 (s,
3 H); 3.71 (s, 3 H); 3.84 (s, 2 H); 3.90 (s, 3 H); 3.92 (s, 3 H); 6.75
(d, 1 H, J ) 8.8 Hz); 6.97 (d, 1 H, J ) 3.5 Hz); 7.01 (d, 1 H, J )
8.8 Hz); 7.03 (d, 1 H, J ) 3.5 Hz); 7.16 (br. t, 1 H, J ) 7.6 Hz);
7.36-7.43 (m, 2 H); 7.46 (t, 1 H, J ) 7.7 Hz); 7.67 (Ψdd, 2 H, J1
) 7.6 Hz, J2 ) 1.5 Hz); 7.91 (br. s, 1 H); 8.41 (br. s, 1 H); 8.50
(d, 1 H, J ) 8.6 Hz).
2.23 (t, 2 H, J ) 7.4 Hz); 3.27-3.35 (m, 2 H); 3.63 (s, 2 H); 6.60
(s, 1 H); 6.84 (s, 2 H); 7.11 (br. s, 1 H); 7.18-7.37 (m, 5 H); 7.41
(t, 1 H, J ) 7.6 Hz); 7.79 (s, 1 H); 7.86 (d, 1 H, J ) 8.3 Hz). MS
(ESI) m/z 465.4 ([M + H]+).
(5-{2-[(2′,3′,4′-Trihydroxy-biphenyl-3-carbonyl)-amino]-phen-
yl}-thiophen-2-yl)-acetic Acid (56). Step 1. Thiophene-2-yl-acetic
acid methyl ester (2.0 g, 12.8 mmol) was dissolved in anhydrous
chloroform (9.0 mL) and glacial acetic acid (9.0 mL) under nitrogen,
and N-bromosuccinimide (2.3 g, 13.0 mmol) was added in portions.
The mixture was stirred for 3 d at rt, and then water was added to
the reaction mixture. The layers were separated, and the aq phase
was extracted with dichloromethane. The combined organic phases
were extracted several times with a 1 M aq NaOH and then water
followed by brine, dried with Na2SO4, and concentrated. The crude
product was purified by preparative radial chromatography (CyH/
EtOAc 5:1] to obtain (5-bromo-thiophen-2-yl)-acetic acid methyl
ester as a yellow oil (2.46 g, 81%) which was used without any
1
further purification. H NMR (400 MHz, CDCl3): 3.71 (s, 3 H);
3.75 (s, 2 H); 6.67 (d, 1 H, J ) 3.8 Hz); 6.88 (d, 1 H, J ) 3.8 Hz).
Step 2. Tetrakis-(triphenylphosphine)-palladium(0) (289 mg, 0.25
mmol) and Na2CO3 decahydrate (4.0 g, 14.0 mmol) were dissolved
in water (5.2 mL) and ethanol (3.7 mL) under nitrogen, and the
mixture was treated with a solution of 2-amino-benzeneboronic acid
hydrochloride (910 mg, 5.25 mmol) in toluene (52 mL). The
reaction mixture was carefully degassed under nitrogen five times
and flooded with N2 again. A solution of (5-bromo-thiophen-2-
yl)-acetic acid methyl ester (1.17 g, 5.0 mmol) in toluene (4.5 mL)
was added, and the reaction was degassed again (five times) and
stirred for 22 h at 100 °C. The reaction mixture was then partitioned
between EtOAc and brine, and the layers were separated. The aq
phase was extracted with EtOAc (three times), and the combined
organic phases were washed with water and then brine, dried with
Na2SO4, and concentrated. The crude product was purified by
Step 6. (5-{2-[(2′,3′,4′-Trimethoxy-biphenyl-3-carbonyl)-amino]-
phenyl}-thiophen-2-yl)-acetic acid methyl ester (589 mg, 1.14
mmol) was dissolved in acetonitrile (10 mL) at rt and then treated
with aq LiOH (5.7 mL of a 1 M solution, 5.7 mmol). The reaction
mixture was stirred for 17 h at rt and then quenched by addition of
1 M aq HCl (pH ∼ 3), with cooling as necessary. The mixture was

Small Molecule Pan-Selectin Antagonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 6 1113
extracted with EtOAc (three times), and the combined organic
phases were washed with water and then brine, dried with Na2-
SO4, and then concentrated, to obtain (5-{2-[(2′,3′,4′-trimethoxy-
biphenyl-3-carbonyl)-amino]-phenyl}-thiophen-2-yl)-acetic acid (586
mg of crude product, quant.) as a yellow solid. 1H NMR (400 MHz,
CDCl3): 3.62 (s, 3 H); 3.87 (s, 2 H); 3.89 (s, 3 H); 3.92 (s, 3 H);
6.73 (d, 1 H, J ) 8.6 Hz); 6.96-.99 (m, 1 H); 6.99 (d, 1 H, J ) 8.6
Hz); 7.02 (d, 1 H, J ) 3.5 Hz); 7.16 (td, 1 H, J1 ) 7.6 Hz, J2 )
1.0 Hz); 7.36-7.43 (m, 2 H); 7.45 (t, 1 H, J ) 7.6 Hz); 7.62 (d, 1
H, J ) 7.8 Hz); 7.70 (d, 1 H, J ) 7.8 Hz); 7.86 (s, 1 H); 8.37 (br.
s, 1 H); 8.48 (d, 1 H, J ) 8.0 Hz).
flow shear of 1 dyn/cm2 in the presence of 109 µM compound or
vehicle alone (DMSO) was counted from five low power fields.
Mean inhibition [% of vehicle control] was calculated from the
mean cells/field of at least two independent experiments.
Acknowledgment. We thank Mrs. Sabine Sommer, Mrs.
Petra Lindenberg, Mrs. Gabriele Dudda, and Mr. Frank Rein-
hardt for excellent technical support. In particular we would
like to thank Dr. Brian Dupre´, Encysive Pharmaceuticals Inc.,
Houston, TX, for in-depth reviewing this manuscript. This
project is supported by grants of the ministry of economics of
the State of Brandenburg and the European Union. Responsibil-
ity for the content of this publication rests upon the authors.
Step 7. (5-{2-[(2′,3′,4′-Trimethoxy-biphenyl-3-carbonyl)-amino]-
phenyl}-thiophen-2-yl)-acetic acid (300 mg, 0.59 mmol) was
dissolved in anhydrous dichloromethane (12 mL) at -78 °C under
nitrogen, treated dropwise with a solution of BBr3 in dichlo-
romethane (3.6 mL of a 1 M solution, 3.6 mmol), stirred for
additional 30 min at -78 °C, warmed slowly to rt, and stirred an
additional 2 h at rt. The mixture was cooled to 0 °C, and then water
was added dropwise, followed by dichloromethane with vigorous
stirring. The reaction mixture was partitioned between water and
EtOAc, and the aq phase was extracted with EtOAc (three times).
The combined organic phases were washed with brine, dried with
Na2SO4, and concentrated. The crude product was purified by
preparative RP HPLC to obtain (5-{2-[(2′,3′,4′-trihydroxy-biphenyl-
3-carbonyl)-amino]-phenyl}-thiophen-2-yl)-acetic acid (56, 110 mg,
Supporting Information Available: Additional synthesis pro-
cedures and analytical data. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
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1
40%) as a beige solid. H NMR (400 MHz, CD3OD): 3.86 (s, 2
H); 6.49 (d, 1 H, J ) 8.6 Hz); 6.74 (d, 1 H, J ) 8.6 Hz); 6.98 (d,
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Biology. Static Molecular Binding Assay. The binding assay
was performed with slight modifications as previously described.36
In summary, 384-well Maxisorp plates (NUNC Roskilde/Denmark)
were coated overnight in carbonate buffer pH 9.6 with goat anti
human Fc mAB (10 µg/mL, Sigma, Steinheim, Germany). After
being washed in assay buffer (25 mM HEPES, 150 mM NaCl, 1
mM CaCl2 pH 7.4) and blocked (3% BSA in assay buffer), the
plates were incubated for 2 h at 37 °C with human E-, P- or
L-selectin/Fc-chimera (0.61 nM, R&D Systems, Wiesbaden, Ger-
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carrying 15 mol % sLeX, 10 mol % tyrosine sulfate, and 5 mol %
biotin was complexed with Streptavidin-Peroxidase (Roche, Man-
nheim, Germany) in assay buffer without CaCl2. This ligand
complex (1:4500 in assay buffer), together with compounds in
varying concentrations (assay buffer, 1%DMSO), was added to the
wells precoated with E-, P-, or L-selectin/Fc-chimera. After
incubation for 2 h at 37 °C, wells were washed six times with assay
buffer incl. 0.005% TWEEN20 using a plate washer (BIO-TEK
Instruments, Germany), developed for 10-15 min with TMB/H2O2
substrate solution (Boehringer Ingelheim, Mannheim, Germany),
and stopped with 1 M H2SO4. Bound sLeX-tyrosine sulfate ligand
complex was determined by measuring optical density at 450 nm
vs 620 nm in a Fusion R-FP reader (Packard Bioscience, Dreieich,
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blocked with bovine serum albumin (Sigma, Steinheim, Germany).
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onto an inverted phase-contrast microscope (Olympus, Hamburg,
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personal computer. Employing a peristaltic pump (Ismatec, Wer-
theim-Mondfeld, Germany), flow chamber experiments were
performed in a recirculating configuration (without any visible
pulsation) with a final concentration of 2.4 million HL-60 cells/
mL (DSMZ, Braunschweig, Germany). The number of cells that
attached to the substrate 5 min after continuous flow at a calculated
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