A novel class of potent nonglycosidic and nonpeptidic pan-selectin inhibitors

Article abstract of DOI:10.1021/jm060468y

An early step of the inflammatory response, the rolling of leukocytes on activated endothelial cells, is mediated by selectin/carbohydrate interactions. The tetrasaccharide sialy Lewis^X is a ligand for E-, P-, and L-selectin and therefore serve

Full text of DOI:10.1021/jm060468y

5988
J. Med. Chem. 2006, 49, 5988-5999
A Novel Class of Potent Nonglycosidic and Nonpeptidic Pan-Selectin Inhibitors§
Holger K. Ulbrich,*,†,‡ Andreas Luxenburger,†,‡ Philip Prech,† Einar E. Eriksson,# Oliver Soehnlein,# Pierre Rotzius,#
Lennart Lindbom,# and Gerd Dannhardt†
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Johannes Gutenberg UniVersity, Staudingerweg 5,
DE-55099 Mainz, Germany, and Department of Physiology and Pharmacology, Karolinska Institute, Von Eulers Va¨g 8,
SE-171 77 Stockholm, Sweden
ReceiVed April 20, 2006
An early step of the inflammatory response, the rolling of leukocytes on activated endothelial cells, is mediated
by selectin/carbohydrate interactions. The tetrasaccharide sialy Lewisx is a ligand for E-, P-, and L-selectin
and therefore serves as a lead structure for the development of analogues. A combination of synthesis and
structure-based design allowed rapid optimization. The current lead 2a was evaluated in our E-selectin cell
flow chamber assay where it proved to inhibit rolling and adhesion with an IC50 of 28 ( 7 µM. The assays
used are predictive for the in vivo efficacy of test compounds as shown for 2a in a proteose peptone induced
peritonitis model of acute inflammation in mice.
Introduction
Molecular Modeling
A number of published studies have established key structural
features of sLex necessary for binding to selectin proteins. The
latest and most significant crystallographic experiments by
Somers et al. involve solving the structure of sLex bound to
E-selectin as well as P-selectin and the structure of the PSGL-1
N-terminus bound to P-selectin.8 The 3- and 4-hydroxy groups
of the fucose moiety coordinate the calcium ion and interact
additionally with further protein side chains. The 4-hydroxy
functionality of galactose undergoes hydrogen bonding to the
side chain of Tyr94, while the 6-OH group binds to the Glu92
side chain. The sialic acid interacts with the side chains of Arg97
and Tyr48 of E-selectin. This cocomplex of sLex and E-selectin
is shown in Figure 2. The majority of the formed bonds within
the binding pocket are of electrostatic nature. The buried surface
area is relatively small with 549 Å2.17 Previous molecular
dynamics simulations18 of ligand/E-selectin complexes also
suggested the existence of three essential binding sites of ligands
for E-selectin as follows: (1) hydroxy groups coordinate to the
calcium ion; (2) branched alkyl chains interfere with two
hydrophobic regions on the surface of E-selectin; (3) a
negatively charged group of the ligand interacts with the basic
residues of E-selectin. These results gave rise to the synthesis
of a variety of nonglycosidic and nonpeptidic low-molecular-
weight selectin inhibitors in which the sugars in sLex have been
substituted. The general outline that was previously published
by Kondo and co-workers is shown in Figure 3a.18 On the basis
of this pharmacophore model, our model consists of the
following features (Figure 3b): (1) one of the carboxylic acids
would coordinate to calcium; (2) the other at a distance of 8-9
Å is to form bonds by electrostatic interactions with Arg97 (E-
selectin) or Lys99 in the case of P-selectin; (3) a long alkyl
chain would interact with the shallow hydrophobic region
consisting of Lys114, Ala9, Tyr49, etc. on E-selectin.8 The
following results indicate that our pharmacophore model could
have potential to find further selectin antagonists.
Recruitment of circulating leukocytes from blood to tissue
is an essential process in response to infection or tissue injury.
However, excessive influx of leukocytes may result in adverse
reactions that can be manifested in reperfusion injury, autoim-
mune diseases, and other acute or chronic disorders.1-4 The
initial step in the inflammatory response is capture of leukocytes
and their rolling along the endothelium in postcapillary venules.5
It has been shown that a set of inducible adhesion molecules,
the so-called selectins, are critical in this process. They constitute
a family of three structurally related calcium-dependent cell
adhesion molecules that are expressed on the surface of activated
vascular endothelial cells (E- and P-selectin), activated platelets
(P-selectin), and leukocytes (L-selectin).6 To date, a number of
natural selectin ligands have been identified.7 The common
motifs recognized by all three selectins are the sialylated and
fucosylated tetrasaccharide sialyl Lewisx (sLex) (Figure 1) and
sialyl Lewisa (sLea), respectively.8,9 Thus, the ability to inhibit
the recruitment of leukocytes by regulating interactions between
the selectins and sLex or other ligands could have potential utility
in a number of clinical diseases. Indeed, research on the design
and synthesis of sLex mimetics has led to the development of
a number of sugar- or nonsugar-based selectin inhibitors as well
as related structures consisting of glycosylated peptides10-15
(Figure 1). Most of the inhibitors suffer from a lack of potency
or poor pharmacokinetic properties and have often been difficult
to synthesize. Nonetheless, the pan-selectin inhibitor bimosia-
mose (Revotar Biopharmaceuticals) has been effective in
asthma, representing a proof of principle for selectin blockade
in the treatment of inflammatory disease.16 Here, we describe
the development of a novel benzoic acid type compound 1a
(Figure 1) with potent E-, P-, and L-selectin-blocking properties.
This compound and closely related structures (Chart 1) represent
new opportunities for the inhibition of selectin function in
inflammation.
Ligand-E-selectin complexes were generated using the
flexible docking procedure of the ICM (version 3.0) by Molsoft.
Structural coordinates for the “receptor” were derived from the
cocrystal structure of sLex and E-selectin from which the ligand
was removed. Receptor grid maps were calculated with a grid
spacing of 0.5 Å. The grid was defined in such a way that it
§ Dedicated to Prof. Dr. Dr. Dres. h.c. E. Mutschler on the occasion of
his 75th birthday.
* To whom correspondence should be addressed. Phone: +49 6131 39-
24339. Fax: +49 6131 39-23062. E-mail: ulbrich@uni-mainz.de.
† Johannes Gutenberg University.
‡ These authors contributed equally.
# Karolinska Institute.
10.1021/jm060468y CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/08/2006

Pan-Selectin Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5989
Figure 1. Representive chemical structures of some known selectin blockers.
included the ligand and key residues plus 3-5 Å in each of the
x, y, z directions. For the ligands, 2D to 3D conversion is
followed by energy minimization using the MMFF option as a
force field. The docking algorithm implemented in ICM (version
3.0) optimizes the entire ligand in the receptor field, using a
multistart Monte Carlo minimization procedure in internal
coordinate space.
the corresponding imines 10a,b, which were then treated with
NaBH4 in methanol at room temperature to give the desired
amines 7d-e in good yields.23
Subsequent deprotonation of the amines 7a-e with sodium
hydride in n-Bu2O and further reaction with 6-bromohexanoyl
chloride gave rise to compounds 11a-e (Scheme 1b).24 To
synthesize the phenolic coupling partners 15 and 18, we
etherified phloroglucin 14 in 1-decanol in the presence of HCl
gas to produce 3,5-didecyloxyphenol 15,25 and resorcine 17 was
converted to 3-decyloxyphenol 1826 by treatment of 17 with
potassium hydroxide in the presence of 1-bromodecane (Scheme
2). Compounds 11a-e were then refluxed with 15 or 18 in the
presence of potassium carbonate and a catalytic amount of
potassium iodide or TBAI in cyclohexanone to achieve the
desired coupling products 13a-g. Saponification by stirring
13a-g with a 0.5 M aqueous lithium hydroxide solution in THF
accomplished the dicarboxylic acids 1a-g (parts a and b of
Scheme 1).
Chemistry
According to our molecular modeling considerations, we
focused our synthesis on connecting the envisaged dicarboxylic
acid functionalities with different types of spacer scaffolds.
Our approach toward the dicarboxylic acids 1a-c (Figure
4) started from compounds 6a-c, which were initially prepared
by a Goldberg coupling reaction following the procedure
described in ref 19 (Scheme 1a). Subsequent cleavage of the
acetyl group combined with transesterification by refluxing
6a-c in the presence of sodium methoxide in methanol provided
the required diphenylamine building blocks 7a-c. As the
coupling products 6a-c and thus the desired compounds 7a-c
as well were only obtained in moderate yields, we also became
interested in looking into several other methods reported in the
literature.20-22 When the copper-catalyzed methodology reported
by Buchwald and co-workers22 was employed, the overall yields
for the preparation of 7a and 7b improved significantly from
40-50% to 76-78% for the two steps.
The preparation of the target dicarboxylic acids 2a,b (Figure
4) began with a borane reduction of the amide function in 11a
and 11d (Scheme 3).27 The resulting amines 20a,b were
subsequently coupled with didecyloxyphenol 15, applying the
same reaction conditions as for the synthesis of compounds
13a-g so that 21a,b were isolated in good yields. Final
saponification with potassium hydroxide accomplished the
dicarboxylic acids 2a,b in good yields.
The phenylbenzylamine precursors 7d-e required for the
synthesis of the dicarboxylic acids 1d-g (Figure 4) were
obtained from the condensation of 4-formylbenzoic acid methyl
ester 8 and the aminobenzoic acid methyl esters 9a,b to give
The synthesis of the dicarboxylic acids 3a-c (Figure 4)
started from the iodide methyl esters 4b,c, which were treated
with i-PrMgCl at low temperatures (Scheme 4). In a halogen
magnesium exchange reaction28 the corresponding organomag-

5990 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Ulbrich et al.
Chart 1. Chemical Structures of the Investigated Compounds Representing Three Structural Classes: Amides (1a-g and 13a-g),
Amines (2a,b and 21a,b), and Oximes (3a-c and 25a-c)
nesium intermediates were generated and reacted further with
4-formylbenzoic acid methyl ester 8 to provide alcohols 22a,b
in good yields. PCC oxidation led to the benzophenone
derivatives 23a,b, which were converted into the desired oximes
24a,b by refluxing in ethanol in the presence of hydroxylamine
hydrochloride and sodium acetate in good yields over two steps.
24b was isolated as an inseparable mixture of Z/E isomers, and
the ratio was determined to be 1:5 from the proton NMR
spectrum. The required coupling partners 16 and 19 were
obtained by additional etherification of 15 and 18 with 1,6-
dibromohexane (Scheme 2). Compounds 24a,b were then both
coupled with 16 and 24a additionally with 19 by deprotonating
the oximes 24a,b with sodium hydride in DMF and adding the
corresponding bromide 16 or 19 together with a catalytic amount
of TBAI at room temperature. Thus, the dimethyl esters 25a-c
were isolated in good yields. Finally, 25a-c were saponified
by stirring with a 0.5 M aqueous sodium hydroxide solution in
a mixture of ethanol and THF to provide the desired dicarboxylic
acids 3a-c in good yields.
The dichloro compound 28 was prepared from commercially
available 4,4′-dichlorobezophenone 26 (Scheme 5), which was
readily converted into the corresponding oxime 27 by employing
the same reaction conditions as for the synthesis of 24a,b.
Figure 3. (a) From molecular modeling investigations by Kondo et
al.,18 three critical interactions of ligands toward E-selectin were
clarified. (b) Pharmacophore model that conserves the three essential
groups to the desirable positions for E-selectin binding.
Figure 2. Crystal structure of sLex bound to the lectin domain of
E-selectin. The bound calcium ion is represented by a green sphere,
8
and sLex is shown in a capped stick model.

Pan-Selectin Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5991
Figure 4. Synthetic targets.
Coupling of 27 with 16 furnished the dichloro compound 28 in
80% yield in two steps.
consequence of the natural ligand binding weakly to the receptor
necessitates the use of a multimeric presentation of the receptor
or the ligand in the ELISA-based assays. For example, it is
necessary to utilize strepavidin in this assay. A multimeric
presentation ensures that a sufficient interaction survives wash
steps and produces adequate signals to measure differences in
inhibition of binding. For this reason this glyco-ELISA is more
sensitive than comparable cell-free E-selectin binding assays.
Furthermore, neutralizing anti-E-selectin antibodies completely
blocked binding to E-selectin. This demonstrates the specifity
of the assay system.30 The compounds were simultaneously
evaluated for their toxicity in the same cells using different
assays (Table 2) to detect false positives.
Following the pharmacophore considerations by Kondo et
al.18 (Figure 3a), three minimum criteria are supposed to be
essential for activity: (I) a calcium ion binding structure, (II) a
negatively charged group, and (III) a hydrophobic moiety.
Therefore, several different groups were chosen as linker X to
establish the carboxylic acid groups R1-R2 (Figure 3b) in the
envisaged positions. Our original lead compound 1a (Figure 1)
proved to be potent in the static assay (100%) in our cell-based
flow assay with an IC50 of 35 ( 26 µM (n ) 10) and showed
inhibition of 58% in the P-selectin-ELISA assay at 500 µM.
But the major drawback of 1a was a notable toxicity against
MM6 cells (Table 2). We found that compounds containing a
diphenylamide (1a) or -amino (2a), a phenylbenzylamide (1f)
or -amino (2b), or a benzophenone oxime ether (3a) core
structure, which act as spacers between the calcium binding
substituent and the negatively charged residue at R1-R2, gave
equivalent inhibitory activities in the static assay (100%, 107%,
93%, 100%, and 90%) (Table 1). A diphenylamide (1a), a
phenylbenzylamide (1e), or amino scaffold (2b) as a bridge
between the two carboxy groups led to analogues with potent
adhesion blocking but significant cytotoxic profiles in the static
assay. All tested methyl ester precursors, e.g., 21b and 13a,
turned out to be less active or inactive in the static assay
compared to the corresponding free acid derivatives 2b and 1a
(61% and 10% vs 100% and 100%). The only exception was
the phthalic acid dimethyl ester 13c, which was as potent as its
free acid derivative 1c (115% vs 93%). Replacement of the
carboxylic acid residues in 3a by chloro atoms resulted in the
Results and Discussion
As a primary screen for the antiadhesive activities of our
synthesized compounds, we used a cell-based, computerized in
vitro assay to quantify leukocyte adhesion to endothelial cells
under static conditions.37 In this assay compounds at 100 µM
were evaluated for inhibition of adhesion of MM6 cells to BAEC
and sLex was chosen as reference. To compare the results for
compounds that have been tested on different plates, the number
of adherent MM6 cells on TNF-R-stimulated endothelial cells
is set to 100% (Table 1). Advantages of this static cell-based
assay are its reproducibility and the option for high-throughput
screening. On the other hand, it has to be noted that rolling of
leukocytes on activated endothelium mediated by selectin/
oligosaccharide interactions is a nonequilibrium process that
occurs under hydrodynamic flow. Therefore, static in vitro
assays that measure inhibition under equilibrium conditions
might generate data that are not predictive for in vivo activity
of test compounds under flow conditions. To overcome this,
potent compounds were further tested in our second assay. In a
cell-based flow assay the ability of compounds to inhibit the
adhesion of MM6 cells to recombinant selectin proteins was
measured (see Experimental Section), whereas a cell-free,
competitive ligand binding assay (glyco-ELISA) was applied
to characterize selectin/ligand interactions.29 The test principle
in the cell-free assay involves immobilization of the used selectin
onto an ELISA microtiter plate to which the substances and
the ligand (sialyl Lewisx tyrosinesulfate bound to biotinylated
polyacrylamide) are added. After the incubation period the
bound complex is detected by the streptavidin peroxidase
reaction measuring the optical density at 414 nm in a microplate
reader. The absorption of the empty plate is subtracted from all
measured values. As control, we used wells only coated with
sLex, whose values were set to 0%, and EDTA, which prevents
the Ca2+-dependent selectin ligand binding completely ()100%).
This demonstrates the specificity of the assay system. The results
are indicated as proportional inhibition of the sLex binding. This
assay takes full advantages of the fact that the interactions
between the selectins and its carbohydrate ligands are low. The

5992 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Ulbrich et al.
Scheme 1a
a Reagents and conditions for part a: (a) method A (preparation of 7a-c), Cu, CuI, K2CO3, n-Bu2O, 4 days, reflux;19 method B (preparation of 7d,e), CuI
(1 mol %), K3PO4, trans-1,2-cyclohexanediamine (10 mol %), n-Bu2O, 5 h, reflux; (b) NaOMe, MeOH, 1.5 h, reflux; (c) MeOH, 1 h, reflux; (d) NaBH4,
MeOH, 24 h, room temp. Reagents and conditions for part b: (a) NaH, n-Bu2O, 2 h, room temp; 6-bromohexanoyl chloride, 4 h, reflux; (b) 0.5 M LiOH,
THF, 8 h, 5 °C, then 16 h, 15 °C; (c) 15 or 18, K2CO3, KI (or TBAI), cyclohexanone, 5 h, reflux.
quiet weak selectin blocker 28 (90% vs 24%), providing
evidence that the presence of the carboxylic acid groups is
essential for activity. In the course of our research we also
investigated the influence of the carboxylic acid functions in
different positions.
Our data suggest that in four of the most active selectin
inhibiting compounds in the static assay (1a, 100%; 2b, 100%;
13c, 115%; 2a, 107%) the carboxy group is located in the para
position except for the phenylbenzylamide derivative 1e, which
carries one carboxylic acid in the meta position of the phenyl
ring and possessed good inhibitory activity compared to its para,
para substituted analogue 1d (82% vs 56%).
varying concentrations of compound 2a.30 As a result, the critical
micelle concentration (cmc) could not be seen at IC50 of 28
µM. These findings demonstrate that the alkyl chains with a
moderate carbon length would be necessary for the strong
bindings to all selectins. To verify to what extend the decyl
chains are responsible for the selectin blocking activity or
cytotoxicity, we were also seeking to replace the lipophilic side
chains. When a phloroglucin ether group with two decyl chains
(3a) was substituted for a resorcin ether group carrying only
one decyl chain (3c), good activity was maintained in vitro (90%
vs 67%) in the static cell based assay (Table 1). But the viability
of MM6 cells was more potently diminished with 3c as observed
for 3a (12% vs 84%). In contrast, the resorcin ether derivative
1f combined potent selectin inhibitory activity (93%) with no
loss in viability of BAEC (100%) whereas its phloroglucin
analogue 1d was less active (56%) by an augmented decrease
To clarify the importance of the alkyl chains of the com-
pounds toward the inhibitory activity, we have studied the active
form of 2a, like micellar species, in solution. Namely, we have
measured the surface tension by the capillary rise method with

Pan-Selectin Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5993
Scheme 2a
a Reagents and conditions: (a) 1-decanol, HCl-gas, 3 h, 70 °C; (b) 1,6-dibromohexane, K2CO3, cyclohexanone, reflux; (c) 1-bromodecane, EtOH, reflux,
KOH.
Scheme 3a
a Reagents and conditions: (a) BH3‚THF, 1 h, reflux; (b) 15 or 18, K2CO3, KI (or TBAI), cyclohexanone, 5 h, reflux; (c) 0.5 M KOH, THF, room temp.
Scheme 4a
a Reagents and conditions: (a) i-PrMgCl, THF, 40 min, -30 f -20 °C; (b) 4-formylbenzoic acid methyl ester 8, THF, -20 °C, room temp, overnight;
(c) PCC, CH2Cl2, 24 h, room temp; (d) HCl‚H2NOH, EtOH, NaOAc, 24 h, reflux; (e) 16 or 19, NaH, DMF, TBAI, 2 h, room temp; (f) 0.5 M NaOH,
EtOH/THF, 24 h, room temp.
in viability (36%). Taken together, no clear conclusion can be
drawn in regard to the relationship of the activity, cytotoxicity,
and the function of the decyl chains. For the purpose of
determining the role of the hydrophobic tail in general, we also
became interested in subjecting the synthetic precursors 12b
and 12a, in which the lipophilic didecyloxyphenoxy residue was
not yet installed, to our test assays. As a result, the activity was
completely lost when no hydrophobic tail was present (12b and
12a, 2% vs 4%). To optimize the length of the side chains,
groups with similar log P values will be evaluated in the future.

5994 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Ulbrich et al.
Scheme 5a
a Reagents and conditions: (a) HCl‚H2NOH, EtOH, NaOAc, 24 h, reflux; (b) 16, NaH, DMF, TBAI, 2 h, room temp.
Table 1. In Vitro Potencies of the Compounds
flow chamber assayb,d
adherent MM6,
E-selectin (100 µM)
control (0%)
static assaya
% inhibition
at 100 µM
glyco-ELISA
% inhibition
glyco-ELISA
% inhibition
glyco-ELISA
% inhibition
compd
E-selectin (100 µM)c,d
P-selectin (500 µM)c,d
L-selectin (500 µM)c,d
1a
1b
1c
1d
1e
1f
1g
2a
2b
3a
3b
3c
100 ( 4
60 ( 9
44 ( 3%, IC50 ) 35 ( 20 µM
nt
nt
62
nt
43 ( 5
nt
42
94
nt
nt
nt
nt
5 ( 3
nt
5 ( 12 (500 µM)
73 (500 µM)
nt
65
18
nt
2
58
20
102
49
95
nt
103
53
14
102
50
92
nt
103
50 ( 5%
93 ( 16
56 ( 12
82 ( 19
93 ( 18
70 ( 11
107 ( 7
100 ( 18
90 ( 4
nt
nt
nt
nt
nt
IC50 ) 28 ( 7 µM
nt
29 ( 10%, IC50 ) 20 ( 14 µM
68 ( 4%
81 (35% at 100 µM)
nt
2
34
nt
12
19
16
90
27
78
79
nt
82 (41% at 100 µM)
nt
6
50
nt
6
100 ( 15
67 ( 8
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
12a
12b
13a
13c
13d
13f
13g
21b
28
4 ( 8
2 ( 6
5
10 ( 20
115 ( 7
26 ( 13
26 ( 22
45 ( 16
61 ( 10
24 ( 8
16%
95
26
75
86
nt
5
2
nt
sLex
41 ( 7
nt
nt
a In this assay compounds at 100 µM were evaluated for inhibition of adhesion of MM6 cells to BAEC, and sLex was chosen as reference. To compare
the results for compounds that have been tested on different plates, the number of adherent MM6 cells on TNF-R-stimulated endothelial cells is set to 100%.
b In a cell-based flow assay the ability of compounds to inhibit the adhesion of MM6 cells to recombinant E-selectin protein was measured. Cells were
counted as adhesive cells when they stuck at least 30 s to the plates. c The test principle in the cell-free assay involves immobilization of the used selectin
onto an ELISA microtiter plate to which the substances and the ligand (sialyl Lewisx tyrosinesulfate bound to biotinylated polyacrylamide) are added. After
the incubation period the bound complex is detected by the streptavidin-peroxidase reaction, measuring the optical density at 414 nm in a microplate reader.
The absorption of the empty plate is subtracted from all measured values. As control, we used wells only coated with sLex, whose values were set to 0%,
and EDTA, which prevents the Ca2+-dependent selectin ligand binding completely ()100%). The results are indicated as proportional inhibition of the sLex
binding. d nt ) not tested.
Our findings are in line with previous conclusions by Slee et
al.31 who investigated conceptually similar imidazole type
compounds and observed that a hydrophobic side chain was
required for potency in general. Slee also reported that the most
potent derivatives in the cell-based assay systems contain a C16
alkyl chain and removal of the hydrophobic tail structure resulted
in significant loss of activity. More recently, this was also
supported by Girard et al.32 and Kaila et al.33 who investigated
quinic acid derivatives as sLex mimetics. All tested analogues
missing such lipophilicity enhancing structural features have
shown only moderate affinity. In both studies inhibitions ranged
from 46% at 50 mM in a cell selectin assay32 to an IC50 of 225
µM in a cell flow assay, and it could be demonstrated that
presence of hydrophobic substituents resulted in a moderate
increase in activity. By evaluation of the compounds in the
glyco-ELISA, it was confirmed that inhibition of adhesion in
the cell-based static assay was due to the inhibition of the
specific ligand/receptor interactions. A good correlation was
observed between the inhibition of adhesion in the static assay
and the decrease in the binding of sLex toward E-, P-, and
L-selectin. Among six compounds that showed inhibition of at
least 80% in the cell adhesion assay, four compounds tested in
the P- and L-selectin glyco-ELISA (2a, 1c, 1e and 13c)
Table 2. In Vitro Cellular Toxicities of the Compounds
% viability
% viability (XTT)a,b
control (100%),
MM6 (100 µM)
(XTT)a,b
control (100%),
BAEC
% viability
(ATP-lite)a,b
control (100%)
compd
1a
1c
1d
1e
1f
1g
2a
2b
3a
3c
12b
13c
13f
13g
21b
12 ( 2
75 ( 7
nt
11 ( 4
nt
101 ( 5
112 ( 6
10 ( 2
84 ( 2
12 ( 1 (50 µM)
nt
50 ( 4
80 ( 4
98 ( 9
5 ( 1
107 ( 6
nt
nt
5
nt
0
nt
4
77
nt
nt
nt
nt
58
68
62
nt
36 ( 7
46 ( 7
100 ( 3
20 ( 4
102 ( 10
nt
110 ( 7
40 ( 1
75 ( 5
90 ( 7
nt
99 ( 10
111 ( 5
a To quantify antiproliferative and cytotoxic effects of our synthesized
compounds, we employed the Roche-XTT assay and the ATP-lite assay.
The assay is based on the conversion of the yellow tetrazolium salt XTT
into an orange formazan dye by metabolically active cells. To compare the
results for compounds that have been tested on different plates, the viability
of untreated control cells is set to 100%. b nt ) not tested.

Pan-Selectin Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5995
Figure 5. Effect of compound 2a on leukocyte recruitment in proteose
peptone induced mouse peritonitis. Leukocyte extravasation into the
peritoneal cavity was analyzed in mice after intraperitonical treatment
with proteose peptone for 6 h (open bars). One group of animals
received compound 2a (50 mg/kg) intraperitonically 1 h before
challenge with proteose peptone (black bars). Leukocytes harvested
from the peritoneal cavity after 6 h were stained with subgroup specific
PE-conjugated antibodies and differentiated according to forward and
side scatter parameters and fluorescence intensity. Values are the mean
( SEM of four independent experiments: (/) P < 0.05 vs control.
possessed a selectin-inhibiting potency in the same manner.
Surprisingly, 1a showed a weak potency and 3a failed to reduce
binding of sLex toward P- and L-selectin (58% vs 53% and 2%
vs 6%) despite excellent activity in our static assay (100% vs
90%). From analysis of the reverse case, with substances that
cause a small or a not notable inhibition of adhesion in the static
assay, the corresponding results in the glyco-ELISA assay could
be observed (13a, 12b, 12a, 13d, and 1d); i.e., no compound
with less activity in the cell-based static assay was potent in
the selectin binding assay.
Figure 6. (A) Docking model of a ribbon representation of 2a bound
to E-selectin (PDB-code: 1G1T). The E-selectin calcium is shown as
a green sphere. (B) Compound 2a docked into the binding site of
E-selectin. The accessible surface of the binding site of E-selectin was
calculated with ICM-Pro and is colored by lipophilicity: Hydrophobic
amino acids are yellow (Ala, Val, Phe, Met, Ile, Leu), hydrophilic amino
acids are gray (Ser, Thr, Tyr, His, Cys, Asn, Gln, Trp, Gly), positive
charged amino acids are blue (Lys, Arg), and negative charged amino
acids are red (Asp, Glu).
There were four compounds (2a, 1c, 13c, 1e) that showed
high potency in the cell-based static assay and the P- and
L-selectin glyco-ELISA binding assays, but only 2a had a
favorable toxicity profile in all cytotoxicity assays. Thus,
compound 2a was further evaluated in the E-selectin cell flow
chamber assay where it proved to inhibit rolling and adhesion
with an IC50 of 28 ( 7 µM (Table 1). In addition, 2a turned
out to be nontoxic in an additional ATP-lite toxicity assay, a
standard method used to monitor cell proliferation. Conse-
quently, 2a was chosen for further in vivo evaluation. The effect
of 2a on leukocyte recruitment in vivo was tested in a mouse
peritonitis model. Leukocyte extravasation was induced by ip
proteose peptone (3%) treatment. As seen in Figure 5, pretreat-
ment with 2a (50 mg/kg) reduced the total cell count in the
peritoneal cavity, the majority of which were PMNs (-44%)
(n ) 4, P < 0.05).34 Only few monocytes and lymphocytes
were extravasated in response to proteose peptone stimulation.
sLex was inactive in this model of acute inflammation at doses
up to 100 mg/kg.35 This indicates that our in vitro selectin assays
predicted anti-inflammatory efficacy of the test compound in
vivo.
acid group orients itself to the Ca2+ ion, filling out the
coordination sphere in a manner similar to the way the fucose
subunit is believed to bind in the natural ligand structure sLex.
The second carboxy group is directed to Arg97 (E-selectin) or
to Lys99 in the case of P-selectin, while the alkyl chains interact
almost completely in hydrophobic regions of the E-selectin
surface (Figure 6B; yellow amino acids are Ala, Val, Phe, Met,
Ile, Leu). This binding mode demonstrates that the molecule
part between the polar headgroup and the hydrophobic tail, the
so-called “spacer” group, fulfills its purpose. This group is
arranged in a way that it sticks out from the binding pocket of
the polar groups, and so it keeps the hydrophobic molecule part
from this region away. Thereby the alkyl chains can bind
themselves within somewhat more distant hydrophobic areas
of the E-selectin surface.
Overall, when we compare the proposed binding mode of
our inhibitors to that of the known natural ligand sLex (Figure
1), we suppose that the two carboxylic acids act as replacements
for the critical fucose and sialic acid subunits of sLex, with a
diarylamino scaffold replacing the lactosamine structure. This
proposed binding mode is similar to that discussed by Kogan
et al.36 for the mannose-containing inhibitor bimosiamose. If
the mode of binding is indeed similar, our series of compounds
suggest that the proposed calcium binding mannose residue of
bimosiamose can be replaced by a simple carboxylic acid,
Our proposed model of the binding mode calculated by the
software for the diphenylamino-based inhibitor 2a to E-selectin
is illustrated in Figure 6. It shows that compounds with a
diphenylamino structure can likewise bind to E-selectin accord-
ing to the forecasts of the pharmacophore model. A carboxylic

5996 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Ulbrich et al.
resulting in a completely novel sLex mimetic containing no more
carbohydrates.
were washed with 3 × 100 µL of PBS+, a flat-polished flow
chamber (Glycotech) was placed on the surface of the well and
MM6 or PMNs, suspended at 1 × 106 cells/mL in RPMI medium
plus 1% FCS, were perfused through the flow chamber at a constant
shear stress of 1 dyn/cm2 for 4 min. Rolling and adherent leukocytes
were visualized and counted in each well in four separate randomly
chosen fields within the first minute, applying an inverted phase
contrast microscope connected to a video camera (Kappa CF 15/
2) and a video recorder (Panasonic SVHS TL700). All experiments
were repeated two or three times. IC50 values were calculated with
the program GRAFIT, Erithacus Software Ltd., U.K.
Proteose Peptone Induced Mouse Peritonitis. Male C57BL/6
mice were subjected to intraperitoneal injection of 3 mL of 3%
protease peptone (Oxoid Ltd.) 1 h after ip treatment with 100 µL
of a suspension of test compound (50 mg/mL) in corn oil. Vehicle
only was used as control. Proteose peptone is a breakdown product
of proteins that has been widely shown to possess strong capacity
to stimulate leukocyte recruitment to extravascular tissue. After 6
h, leukocytes were harvested from the peritoneum with 3 mL of
cold HBSS containing 5 mM EDTA. Samples were centrifuged
(2000 rpm for 5 min), labeled with 5 µL of PE conjugated rat
antimouse macrophage/monocyte antibody (MOMA-2, Immu-
noKontact), antimouse PMN antibody (anti-PMN, ImmunoKontact),
and antimouse T-lymphocyte antibody (anti-CD2, ImmunoKontact)
for 20 min at 4 °C, washed twice, and resuspended in HBSS. Gating
for leukocyte subpopulations was based on forward and side scatter
parameters and FL-1.
Description of Docking Programs and Settings. Molecular
models of candidate molecules were generated by using the ab
initio, flexible docking procedure of the ICM (version 3.0)
molecular modeling program by Molsoft. Structural coordinates for
the “receptor” were derived from the cocrystal structure of sLex
bound to the E-selectin from which the ligand was removed.
Receptor grid maps were calculated with a grid spacing of 0.5 Å.
The grid was defined in such a way that it included the ligand and
key residues plus 3-5 Å in each of the x, y, z directions. For the
ligands, 2D to 3D conversion is followed by energy minimization
using the MMFF option as a force field. The docking algorithm
implemented in ICM (version 3.0) optimizes the entire ligand in
the receptor field, applying a multistart Monte Carlo minimization
procedure in internal coordinate space. The Metropolis temperature
for accepting or rejecting a new pose was set to 600 K. The number
of Monte Carlo steps and iterations of the local energy minimization
was determined automatically by an adaptive algorithm depending
on the size and number of flexible torsions in the ligand. More
details can be found in refs 38 and 39.
Summary
Novel and potent small-molecule pan-selectin inhibitors have
been developed. A combination of synthesis and structure-based
design allowed rapid optimization, resulting in compounds with
in vivo activity. We exploited the three minimal criteria that
are essential for activity: (1) a calcium ion binding moiety, (2)
a negatively charged group, and (3) a hydrophobic tail structure
(Figure 3). Of those that were tested, the presence of two
carboxylic acids was found to be optimal. One carboxylic acid
of a benzoic acid derivative coordinates to calcium and the other
undergoes electrostatic interactions with Arg97 (E-selectin) and/
or Lys99 (P-selectin). As several different spacers were tolerated,
we believe that a number of templates can be used to combine
the carboxylic acid groups.
Taken together, the ligand binding glyco-ELISA assays, the
cell-selectin flow chamber assay, and the static cell-cell assay
systems proved to be essential for lead optimization and seemed
to give more predictive results in terms of identifying com-
pounds with in vivo activity. Several members of this series
were found to be active E-, P-, and L-selectin inhibitors. One
of the more potent derivatives, compound 2a, identified as
having good activity and negligible toxicity in vitro, was shown
to reduce inflammation in vivo. This novel class of nonglyco-
sidic and nonpeptidic pan-selectin blockers opens up new
possibilities for the development of therapeutic agents in
inflammatory disease. In the course of our ongoing research
project, further investigations are currently undertaken.
Experimental Section
Static Assay. The adhesion assay was performed as described
previously.37 Briefly, BAEC monolayers 7 days after seeding,
passage 1-3, were grown in 24-well plates. Prior to assay, the
BAEC samples were examined microscopically to confirm the
integrity and uniformity of the monolayers. After the culture
medium was removed and the endothelial cell monolayer was
washed with DPBS (2 × 1 mL/well at 37 °C), the cells were treated
with cell culture medium containing 20 ng/mL recombinant tumor
necrosis factor R (TNFR) for 4 or 16 h and the respective test
substance was dissolved in DMSO or vehicle for 20 min (in other
settings for 4 or 16 h) at 37 °C/5% CO2. The BAEC samples were
washed with DPBS (2 × 1 mL/well at 37 °C) before 900 µL of
culture medium and 100 µL of PMN suspension (1 × 106 cells/
mL) per well were added and incubated for 30 min at 37 °C/5%
CO2. Nonadherent PMNs were removed by washing with DPBS
(3 × 1 mL/well at 37 °C). Culture medium (1 mL/well at 37 °C)
was added prior to microscopic examination. (In inhibition experi-
ments, various concentrations of drugs were used (1 × 10-4 to 1
× 10-7 mol/L).) After incubation with cytokines or drugs, the cell
viability of BAEC was confirmed by trypan blue exclusion and
additional microscopic examination. IC50 values were calculated
using the program GRAFIT, Erithacus Software Ltd., U.K.
E-Selectin-Cell Adhesion Assays. The ability of compounds to
inhibit the adhesion of MM6 cells to pure selectin proteins was
measured using a “cell-selectin” assay. Recombinant soluble
E-selectin protein purchased from R&D Systems (Minneapolis,
MN) was diluted to 5 µg/mL in Dulbecco’s PBS containing calcium
and magnesium ions (PBS+). Plates coated with recombinant
E-selectin were produced by adding 50 µL of the protein (5 µg/
mL) to the center of a well in a six-well plate and stored overnight
at 4 °C. The selectin protein was omitted from negative control
(“background”) wells. The plates were washed with 3 × 100 µL
of PBS+ and incubated for 60 min with 100 µL of a solution of
the test compounds (or vehicle controls) in RPMI (containing 1%
FCS and DMSO) in the final test concentration. After the plates
6-Bromohexanoic Acid N,N-Bis(4-carboxyphenyl)amide (12a).
A solution of 11a (150 mg, 0.324 mmol) in THF (8 mL) was stirred
with an aqueous lithium hydroxide solution (0.5 M, 1.95 mL, 0.97
mmol) solution at 5 °C for 8 h and at 15 °C for another 16 h.
Subsequently, water (20 mL) was added and the mixture was
extracted with ether twice. The organic extracts were discarded,
and the aqueous layer was acidified to pH 1 by dropwise addition
of aqueous hydrochloric acid (1.0 M) and extracted with ether (3
× 8 mL). The combined organic layers were washed with water
and brine, dried over MgSO4, and concentrated. The crude product
was recrystallized from methanol to yield 114 mg (81%) of 12a as
1
a colorless solid. Mp 150-151 °C; H NMR (DMSO-d6) δ 13.09
(sbr, 2H), 7.97 (d, 8.1 Hz, 4H), 7.45 (d, 8.1 Hz, 4H), 3.49 (t, 6.6
Hz, 2H), 2.24 (t, 7.4 Hz, 2H), 1.78-1.65 (m, 2H), 1.65-1.50 (m,
2H), 1.39-1.27 (m, 2H); MS (FD) m/z 435 (M+ for Br81) and 433
(M+ for Br79). Anal. (C20H20BrNO5) C, H, N.
6-Bromohexanoic Acid N,N-3,4′-Bis(carboxyphenyl)amide
(12b). Dimethyl ester 11b (250 mg, 0.541 mmol) in THF (13 mL)
was stirred with an aqueous lithium hydroxide solution (0.5 M,
3.06 mL, 1.53 mmol), employing the procedure for the preparation
of dicarboxylic acid 12a to give 201 mg (86%) of 12b as a colorless
1
solid. Mp 190-191 °C; H NMR (DMSO-d6) δ 11.71 (sbr, 2H),
7.96 (d, 8.4 Hz, 2H), 7.87 (d, 7.2 Hz, 1H), 7.84 (s, 1H), 7.70-7.41
(m, 4H), 3.48 (t, 6.7 Hz, 2H), 2.22 (t, 7.4 Hz, 2H), 1.78-1.65 (m,

Pan-Selectin Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 5997
2H), 1.65-1.50 (m, 2H), 1.39-1.27 (m, 2H); MS (FD) m/z 435
(M+ for Br81) and 433 (M+ for Br79). Anal. (C20H20BrNO5) C, H,
N.
(471 mg, 0.73 mmol) in THF (18 mL) was treated with an aqueous
solution of lithium hydroxide (0.5 M, 4.38 mL, 2.19 mmol),
applying the procedure reported for the preparation of dicarboxylic
acid 1a to achieve 339 mg (75%) of 1f as a colorless solid. Mp
6-(3,5-Didecyloxyphenoxy)hexanoic Acid N,N-Bis(4-carbox-
yphenyl)amide (1a). 13a (500 mg, 0.635 mmol) was dissolved in
THF (16 mL) and stirred with an aqueous lithium hydroxide
solution (0.5 M, 3.81 mL, 1.90 mmol) at 5 °C for 8 h and at 15 °C
for another 16 h. Subsequently, water (50 mL) was added and the
mixture was extracted with ether once. A suitable separation of
the layers was achieved by storing the emulsion in a refrigerator at
5 °C overnight. The ethereal extract was separated and discarded.
The remaining aqueous layer was acidified to pH 1 by the dropwise
addition of aqueous hydrochloric acid (1.0 M solution) and extracted
with ether. The combined organic layers were washed with water
and brine, dried over MgSO4, and concentrated. The crude product
was recrystallized from methanol to yield 428 mg (89%) of 1a as
a colorless solid. Mp 158-160 °C; IR (KBr) 3030, 2890, 2820,
1
167-168 °C; H NMR (CDCl3) δ 8.08 (d, 8.6 Hz, 2H), 8.01 (d,
8.4 Hz, 2H), 7.29 (d, 8.4 Hz, 2H), 7.13 (t, 7.9 Hz, 1H), 7.10 (d,
8.4 Hz, 2H), 6.50-6.40 (m, 3H), 5.01 (s, 2H), 3.94-3.85 (m, 4H),
2.17 (t, 7.4 Hz, 2H), 1.81-1.64 (m, 6H), 1.47-1.20 (m, 16H), 0.87
(t, 6.7 Hz, 2H); MS (FD) m/z 618 (M+). Anal. (C37H47NO7) C, H,
N.
6-(3-Decyloxyphenoxy)hexanoic Acid N-(4-Carboxybenzyl)-
N-(3-carboxyphenyl)amide (1g). Dimethyl ester 13g (336 mg,
0.520 mmol) was dissolved in THF (13 mL) and stirred with an
aqueous lithium hydroxide solution (0.5 M, 3.12 mL, 1.56 mmol)
following the procedure for the preparation of dicarboxylic acid
1a so that an amount of 258 mg (80%) of 1g was obtained as a
colorless solid. Mp 134-135 °C; 1H NMR (CDCl3) δ 8.06 (d, 8.1
Hz, 1H), 8.01 (d, 8.1 Hz, 2H), 7.67 (s, 1H), 7.47 (t, 7.9 Hz, 1H),
7.32-7.26 overlapping signals (7.29, d, 8.4 Hz, 2H and m, 1H),
7.12 (t, 8.1 Hz, 1H), 6.49-6.39 (m, 3H), 4.99 (s, 2H), 3.94-3.84
(m, 4H), 2.13 (t, 7.4 Hz, 2H), 1.81-1.63 (m, 6H), 1.49-1.21 (m,
16H), 0.87 (t, 6.7 Hz, 3H); MS (FD) m/z 618 (M+). Anal. (C37H47-
NO7) C, H, N.
1
2640, 2520, 1660, 1580 cm-1; H NMR (CDCl3) δ 8.13 (d, 8.6
Hz, 4H), 7.33 (d, 8.6 Hz, 4H), 6.06 (t, 2.1 Hz, 1H), 6.03 (d, 2.1
Hz, 2H), 3.88 (d, 6.4 Hz, 6H), 2.33 (t, 7.2 Hz, 2H), 1.81-1.67 (m,
8H), 1.51-1.21 (m, 30H), 0.87 (t, 6.7 Hz, 6H); MS (FD) m/z 760
(M+). Anal. (C46H65NO8) C, H, N.
6-(3,5-Didecyloxyphenoxy)hexanoic Acid N,N-3,4′-Bis(car-
boxyphenyl)amide (1b). Dimethyl ester 13b (463 mg, 0.588 mmol)
in THF (15 mL) was stirred with an aqueous lithium hydroxide
solution (0.5 M, 3.53 mL, 1.76 mmol) using the procedure described
for the preparation of dicarboxylic acid 1a to give 300 mg (67%)
of 1b as a colorless solid. 1H NMR (DMSO-d6, 500 MHz, 323 K)
δ 7.95 (d, 8.6 Hz, 2H), 7.87 (d, 7.8 Hz, 1H), 7.83 (t, 2.0 Hz, 1H),
7.60 (d, 7.8 Hz, 1H), 7.53 (t, 7.8 Hz, 1H), 7.43 (d, 8.6 Hz, 2H),
6.01 (s, 3H), 3.90-3.83 (m, 6H), 2.24 (t, 7.4 Hz, 2H), 1.69-1.55
(m, 8H), 1.41-1.19 (m, 30H), 0.84 (t, 6.9 Hz, 6H); MS (FD) m/z
760 (M+). Anal. (C46H65NO8) C, H, N.
4-[N-(6-(3,5-Bis(decyloxy)phenoxy)hexyl)-N-(4-(carboxy-
phenyl)amino]benzoic Acid (2a). A solution of dimethyl ester 21a
(123 mg, 0.160 mmol) in THF (5 mL) was stirred with an aqueous
potassium hydroxide solution (0.5 M, 0.96 mL, 0.480 mmol) at
room temperature according to the procedure for the preparation
1
of 1a to give 86 mg (73%) of 2a as a colorless solid. H NMR
(CDCl3) δ 8.03 (d, 8.8 Hz, 4H), 7.10 (d, 8.8 Hz, 4H), 6.09-6.03
(m, 3H), 3.94-3.78 overlapping signals (3.89, t, 6.4 Hz, 6H and
m, 2H), 1.81-1.67 (m, 8H), 1.54-1.19 (m, 32H), 0.88 (t, 6.7 Hz,
6H); MS (FD) m/z 746 (M+). Anal. (C46H67NO7) C, H, N.
4-[N-(4-Carboxylbenzyl)-N-(6-bis(3,5-decyloxy)phenoxy)hex-
ylamino]benzoic Acid (2b). Dimethyl ester 21b (301 mg, 0.38
mmol) was dissolved in THF (5 mL) and treated with an aqueous
potassium hydroxide solution (0.5 M, 2.28 mL, 1.14 mmol) at room
temperature following the procedure for the preparation of 1a to
give 196 mg (68%) of 2b as a colorless resin. 1H NMR (CDCl3) δ
7.99 (d, 8.3 Hz, 2H), 7.85 (d, 9.0 Hz, 2H), 7.21 (d, 8.3 Hz, 2H),
6.55 (d, 9.1 Hz, 2H), 5.99-5.90 (m, 3H), 4.61 (s, 2H), 3.86-3.79
(m, 6H), 3.44 (t, 6.9 Hz, 2H), 1.73-1.62 (m, 6H), 1.40-1.30 (m,
2H), 1.19-0.97 (m, 32H), 0.80 (t, 6.9 Hz, 6H); MS (EI) m/z 761
(M+ + 1). Anal. (C47H69NO7) C, H, N.
O-[-6-(3,5-Didecyloxyphenyloxy)hexyl]-4,4′-bis(carboxy-
phenyl)methanone Oxime (3a). 25a (500 mg, 0.623 mmol) was
dissolved in a mixture of ethanol (5.10 mL) and THF (12.3 mL)
and stirred with an aqueous sodium hydroxide solution (0.5 M,
3.75 mL) at room temperature for 24 h. Subsequently, water (30
mL) was added and the mixture was extracted with Et2O once.
The ether extract was discarded. The aqueous layer was acidified
to pH 6-5 by the dropwise addition of 1 M aqueous hydrochloric
acid and extracted with ether. The combined ethereal extracts were
washed with water and brine and dried over Na2SO4 to provide
458 mg (95%) of 3a as a colorless resin. 1H NMR (CDCl3) δ 8.21
(d, 8.4 Hz, 2H), 8.09 (d, 8.4 Hz, 2H), 7.58 (d, 8.4 Hz, 2H), 7.45
(d, 8.4 Hz, 2H), 6.05 (s, 3H), 4.24 (t, 6.4 Hz, 2H), 3.89 (t, 6.4 Hz,
6H), 1.81-1.68 (m, 8H), 1.51-1.22 (m, 32H), 0.87 (t, 6.7 Hz, 6H);
MS (FD) m/z 774 (M+). Anal. (C47H67NO8) C, H, N.
6-(3,5-Didecyloxyphenoxy)hexanoic Acid N-(4-Carboxy-
phenyl)-N-(3,4-dicarboxyphenyl)amide (1c). A solution of the
trimethyl ester 13c (239 mg, 0.28 mmol) in THF (11 mL) was
treated with an aqueous solution of lithium hydroxide (0.5 M, 2.26
mL, 1.13 mmol) according to the procedure described for the
preparation of compound 1a so that an amount of 161 mg (71%)
1
of 1c was isolated as a colorless resin. H NMR (CDCl3) δ 8.07
(d, 8.1 Hz, 2H), 7.81 (d, 8.1 Hz, 1H), 7.57-7.47 (m, 2H), 7.30 (d,
8.1 Hz, 2H), 6.05 (t, 1.9 Hz, 1H), 6.02 (d, 1.9 Hz, 2H), 3.94-3.81
(m, 6H), 2.38-2.24 (m, 2H), 1.82-1.64 (m, 8H), 1.47-1.18 (m,
30H), 0.86 (t, 6.7 Hz, 6H); MS (FD) m/z 805 (M+ + 1). Anal.
(C47H65NO10) C, H, N.
6-(3,5-Didecyloxyphenoxy)hexanoic Acid N-(4-Carboxyben-
zyl)-N-(4-carboxyphenyl)amide (1d). Dimethyl ester 13d (443 mg,
0.552 mmol) dissolved in THF (14 mL) was stirred with an aqueous
solution of lithium hydroxide (0.5 M, 3.31 mL, 1.66 mmol) using
the procedure for the preparation of 1a. Thus, an amount of 343
mg (80%) of the desired dicarboxylic acid 1d was obtained as a
colorless resin. 1H NMR (CDCl3) δ 8.08 (d, 8.6 Hz, 2H), 8.01 (d,
8.4 Hz, 2H), 7.29 (d, 8.4 Hz, 2H), 7.10 (d, 8.6 Hz, 2H), 6.05 (t,
1.9 Hz, 1H), 6.02 (d, 1.9 Hz, 2H), 5.01 (s, 2H), 3.92-3.83 (m,
6H), 2.16 (t, 7.4 Hz, 2H), 1.80-1.62 (m, 8H), 1.49-1.21 (m, 30H),
0.87 (t, 6.7 Hz, 6H); MS (FD) m/z (%) 774 (M+). Anal. (C47H67-
NO8) C, H, N.
6-(3,5-Didecyloxyphenoxy)hexanoic Acid N-(4-Carboxyben-
zyl)-N-(3-carboxyphenyl)amide (1e). A solution of the dimethyl
ester 13e (425 mg, 0.530 mmol) in THF (13 mL) was treated with
an aqueous lithium hydroxide solution (0.5 M, 3.18 mL, 1.59
mmol), employing the procedure given for the preparation of
compound 1a to provide 312 mg (76%) of 1e as a colorless resin.
1H NMR (CDCl3) δ 8.05 (d, 7.9 Hz, 1H), 8.01 (d, 8.1 Hz, 2H),
7.62 (s, 1H), 7.48 (t, 7.9 Hz, 1H), 7.33-7.25 (m, 3H), 6.04 (t, 1.9
Hz, 1H), 6.01 (d, 1.9 Hz, 2H), 4.99 (s, 2H), 3.91-3.81 (m, 6H),
2.12 (t, 7.4 Hz, 2H), 1.79-1.62 (m, 8H), 1.46-1.17 (m, 30H), 0.87
(t, 6.7 Hz, 6H); MS (FD) m/z 774 (M+). Anal. (C47H67NO8) C, H,
N.
O-[-6-(3,5-Didecyloxyphenyloxy)hexyl]-3,4′-bis(carboxy-
phenyl)methanone Oxime (3b). Following the procedure for the
preparation of compound 3a, a solution of dimethyl ester 25b (341
mg, 0.425 mmol) in THF (8.5 mL) and ethanol (3.5 mL) was stirred
with an aqueous sodium hydroxide solution (0.5 M, 2.55 mL, 1.28
1
mmol) to give 287 mg (87%) of 3b as a colorless resin. H NMR
(CDCl3) δ 8.22-8.03 (m, 4H), 7.64-7.42 (m, 4H), 6.05 (s, 3H),
4.24 (t, 6.4 Hz, 2H), 3.89 (t, 6.4 Hz, 6H), 1.81-1.67 (m, 8H), 1.54-
1.21 (m, 32H), 0.87 (t, 6.7 Hz, 6H); MS (FD) m/z 774 (M+). Anal.
(C47H67NO8) C, H, N.
6-(3-Decyloxyphenoxy)hexanoic Acid N-(4-Carboxybenzyl)-
N-(4-carboxyphenyl)amide (1f). A solution of dimethyl ester 13f
O-[-6-(3-Decyloxyphenyloxy)hexyl]-4,4′-bis(carboxyphenyl)-
methanone Oxime (3c). A solution of the dimethyl ester 25c (357

5998 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20
Ulbrich et al.
(12) Kaila, N.; Thomas, B. E. Selectin inhibitors. Expert Opin. Ther. Pat.
2003, 13, 305-317.
mg, 0.553 mmol) in a mixture of THF (11 mL) and ethanol (4.5
mL) was treated with an aqueous sodium hydroxide solution (0.5
M, 3.65 mL, 1.82 mmol) using the procedure reported for the
preparation of the dicarboxylic acid 3a to provide 321 mg (94%)
(13) Ohta, S.; Inujima1, Y.; Abe, M.; Uosaki, Y.; Sato, S.; Miki, I.
Inhibition of P-selectin specific cell adhesion by a low molecular
weight, non-carbohydrate compound, KF38789. Inflammation Res.
2001, 50, 544-551.
1
of 3c as a colorless resin. H NMR (CDCl3) δ 8.20 (d, 8.4 Hz,
2H), 8.08 (d, 8.6 Hz, 2H), 7.58 (d, 8.6 Hz, 2H), 7.45 (d, 8.4 Hz,
2H), 7.15 (t, 8.1 Hz, 1H), 6.51-6.43 (m, 3H), 4.25 (t, 6.7 Hz, 2H),
3.92 (t, 6.4 Hz, 4H), 1.83-1.69 (m, 6H), 1.55-1.20 (m, 18H), 0.87
(t, 6.7 Hz, 3H); MS (FD) m/z 618 (M+). Anal. (C37H47NO7) C, H,
N.
(14) Thoma, G.; Kinzy, W.; Bruns, C.; Patton, J. T.; Magnani, J. L.;
Banteli, R. Synthesis and biological evaluation of a potent E-selectin
antagonist. J. Med. Chem. 1999, 42, 4909-4913.
(15) Tsai, C. Y.; Park, W. K.; Weitz-Schmidt, G.; Ernst, B.; Wong, C.
H. Synthesis of sialyl Lewis X mimetics using the Ugi four-
component reaction. Bioorg. Med. Chem. Lett. 1998, 8, 2333-2338.
(16) Beeh, K. M.; Beier, J.; Meyer, M.; Buhl, R.; Zahlten, R.; Wolff, G.
Bimosiamose, an inhaled small-molecule pan-selectin antagonist,
attenuates late asthmatic reactions following allergen challenge in
mild asthmatics: A randomized, double-blind, placebo-controlled
clinical cross-over-trial. Pulm. Pharmacol. Ther. 2006, 19 (4), 233-
241.
O-[-6-(3,5-Didecyloxyphenyloxy)hexyl]bis(4-chlorophenyl)-
methanone Oxime (28). A solution of 27 (234 mg, 0.878 mmol)
in dry DMF (3 mL) was treated with sodium hydride (60% in
mineral oil, 40.4 mg, 1.01 mmol) and subsequently reacted with
16 (500 mg, 0.878 mmol) in the presence of a catalytic amount of
n-Bu4NI according to the procedure described for the preparation
of compound 25a. After column chromatography (silica gel, ethyl
acetate/petroleum ether 1:15), an amount of 581 mg (88%) of 28
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1
was obtained as a colorless oil. H NMR (CDCl3) δ 7.40 (d, 8.6
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2H), 3.89 (t, 6.7 Hz, 6H), 1.81-1.66 (m, 8H), 1.52-1.21 (m, 32H),
0.88 (t, 6.7 Hz, 6H); MS (FD) m/z (%) 758 (M+ + 3, 27), 757
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Acknowledgment. This study was supported by grants from
the Deutsche Pharmazeutische Gesellschaft and Fonds der
Chemischen Industrie, the Swedish Research Council, the
Swedish Heart-Lung Foundation, and the AFA Health Fund.
H.K.U. and O.S. thank the Deutsche Forschungsgemeinschaft
(Grant UL-199/1-2 and SO 876/1-1) for financial support. We
thank the Revotar AG for their collaboration. Their work was
supported by grants of the Ministry of Economics of the State
of Brandenburg and the European Union. The authors thank
Prof. Dr. Pindur for the opportunity to perform the ICM (version
3.0) investigations.
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