Drug design, synthesis, and evaluation of a non-sugar-based selectin antagonist

Article abstract of DOI:10.1016/S0960-894X(01)00430-9

We have designed a series of simple rigid compounds (2) having a phenyl ring attached to three essential groups necessary for selectin binding, i.e., a fucose unit, a carboxylic acid, and the hydrophobic part. In this series of compound 2, 2a exhibited strong inhibitory activity in in vitro P-selectin mediated cell adhesion assay. The novel type of compound 2a would be a potential lead compound for selectin antagonist.

Full text of DOI:10.1016/S0960-894X(01)00430-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2365–2367
Drug Design, Synthesis, and Evaluation of a
Non-sugar-based Selectin Antagonist
Kyoko Fukunaga, Takahiro Tsukida,
Hideki Moriyama and Hirosato Kondo*
Department of Chemistry, R&D Labs, Nippon Organon K.K., Tomobuchi-cho, Miyakojima-ku, Osaka 534-0016, Japan
Received 14 May 2001; accepted 21 June 2001
Abstract—We have designed a series of simple rigid compounds (2) having a phenyl ring attached to three essential groups neces-
sary for selectin binding, i.e., a fucose unit, a carboxylic acid, and the hydrophobic part. In this series of compound 2, 2a exhibited
strong inhibitory activity in in vitro P-selectin mediated cell adhesion assay. The novel type of compound 2a would be a potential
lead compound for selectin antagonist. # 2001 Elsevier Science Ltd. All rights reserved.
Sialyl Lewis X (sLeX) is a natural carbohydrate ligand
of cell adhesion molecules such as E-, P-, and L-selec-
tins. It has been reported that the sLeX/selectin interac-
tion plays an important role in the migration of
inflammatory cells from the blood stream to inflamma-
tory sites, and the interaction participates in various
inflammatory diseases.1ꢀ8 Therefore, it is expected that
selectin inhibitors based on sLeX or sLeX mimetics
could be a new type of anti-inflammatory agent. How-
ever, drug development of a carbohydrate itself is hin-
dered by the complication that the synthesis of complex
oligosaccharides is rather expensive and difficult. Thus,
one of the current studies of biologically active oligo-
saccharides is directed to mimic sugar with low mole-
cular-weight compounds having simple and drug-like
structures (Fig. 1).
1 is characterized by the type II b-turn structure, which
is supported by NMR studies.11
We focused on the type II b-turn motif of compound 1.
If the type II b-turn structure played an important role
in orienting the desired positions of three essential
groups for binding to selectins, the b-turn motif may be
replaced by other templates to keep the desired posi-
tions for the three functional groups. To this end, we
designed a simple rigid compound (2) having a phenyl
ring attached to the three essential groups necessary for
selectin binding, that is a fucose unit, a carboxylic acid,
and the hydrophobic part.
Synthesis of compounds 2 was achieved as shown in
Scheme 1. Diethyl 5-(hydroxymethyl)-isophthalate (3)
was treated with triphenylphosphine and CBr4 in methy-
lene chloride to afford bromide 4 in 83% yield.
There has been a substantial amount of work on this
subject. Recently, we have succeeded in discovering a
Ser-Glu dipeptide inhibitor (1) for selectin/sLeX bind-
ing.9,10 From the SAR study, it has been found that
compound 1 has three essential groups for the selectin
binding: (1) the fucose unit, as a calcium ligand; (2) the
hydrophobic part with a branched alkyl chain; and (3)
the negatively charged group, a carboxylic acid for inter-
action with the target molecule.10 In addition, compound
Glycosylation of 4 with 1-thio-l-fucose derivative 512 in
the presence of 20% NaOEt gave compound 6 in 92%
yield. Hydrolysis of compound 6 with KOH (1.5 equiv)
in ethanol afforded compound 7 in 90% yield. Next,
condensation of 7 with a branched alkylamine in the
presence of 1-[3-(dimethylamino)propyl]-3-ethyl-carbo-
diimide (WSC) and 1-hydroxybenzotriazole (HOBt)
gave compound 8 in 47% yield. Deprotection of com-
pound 8 in the presence of 1 N NaOH afforded target
compound 2a. At the same time, compound 2a is a key
intermediate for compounds 2b and 2c. So, condensation
*Corresponding author. Tel.: +81-6-6921-8920; fax: +81-6-6921-
9078; e-mail: kondo.hirosata@organon.co.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00430-9

2366
K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2365–2367
Figure 1. Design of novel selectin antagonist.
of 2a with C-terminal protected amino acids, followed
by deprotection according to the general condition
yielded target compounds 2b and 2c in 82 and 88%
yields from 2a, respectively.
Table 1. In vitro P-selectin mediated cell adhesion assay and cell
toxicology
P-selectin cell adhesion assay and cell toxicology
Compd
% inhibition
@ 30 mM
% death
@ 30 mM
IC50
(mM)
In order to examine the inhibitory activity of com-
pounds 2a–c against selectin/sLeX binding, the biologi-
cal activities of compounds 2a–c were evaluated in an in
vitro P-selectin mediated cell adhesion assay13 and the
cell toxicities of compounds 1 and 2a–c was also inves-
tigated. As shown in Table 1, all compounds did not
show significant cell toxicity at 30 mM concentration.
2a
2b
2c
1
75.7
ꢀ0.9
31.3
42.8
1.9
5.3
2.8
1.7
10.6
NDa
ND
ND
aND, not determined.
Scheme 1.

K. Fukunaga et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2365–2367
2367
Compound 1 exhibited 42.8% inhibition at 30 mM
against P-selectin mediated cell adhesion. On the other
hand, compound 2a showed the strongest inhibition
against the cell-based assay, 75.7% inhibition at 30 mM
and IC50 value 10.6 mM. However, other compounds,
2b, 2c, were weaker than compound 2a; in particular
compound 2b did not show inhibition at all. From the
data, it was found that the length between the car-
boxylic acid and the phenyl ring of compound 2 would
be very important to interact with the target molecule
and compound 2a was found to become a good lead
compound for selectin antagonists. Optimization of
compound 2 is in progress.
gannon, P.; Wisdom, W.; Meyer, J.; Gallatin, W. M.; Oka-
sinski, G. F. J. Med. Chem. 2001, 44, 988.
7. Keramidaris, E.; Merson, T. D.; Steeber, D. A.; Tedder,
T. F.; Tang, M. L. J. Allergy Clin. Immunol. 2001, 107, 734.
8. Hafezi-Moghadam, A.; Thomas, K. L.; Prorock, A. J.;
Huo, Y.; Ley, K. J. Exp. Med. 2001, 193, 863.
9. Tsukida, T.; Hiramatsu, Y.; Tsujishita, H.; Kiyoi, T.;
Yoshida, M.; Kurokawa, K.; Moriyama, H.; Ohmoto, H.;
Wada, Y.; Saito, T.; Kondo, H. J. Med. Chem. 1997, 40, 3534.
10. Tsukida, T.; Moriyama, H.; Kurokawa, K.; Achiha, T.;
Inoue, Y.; Kondo, H. J. Med. Chem. 1998, 41, 4279.
11. Nakajima, F.; Kondo, H.; Nishimura, S. submitted for
publication.
12. Hashimoto, H.; Shimada, K.; Horita, S. Tetrahedron:
Asymmetry 1994, 5, 2351.
13. Cell adhesion assay: Anti-human IgG(Fc) mAb was dilu-
ted to 10 mg/mL in PBS and 50 mL aliquots were incubated
overnight in each well of 96-well microtiter plate (Micro-
test96TM3075; Becton Dickinson Labware, Flanklin Lakes,
NJ, USA) at 4 ꢁC. The well were washed with PBS containing
0.05% Tween 20 and blocked with 2% BSA in PBS for 1 h at
room temperature. The wells were then washed with T-PBS
and incubated with 5 mg/mL P-selectin-IgG chimera. The wells
were incubated with 1 mg/mL goat F(ab0)2 anti-human
IgG(Fc) to block Fc receptors. Compounds dissolved in
DMSO or distilled water at 10 mM were diluted by RPMI
1640 containing 10% FCS to final concentration at 0.1 mM.
BCECF-AM (5 mM; Dojindo, Osaka, Japan)-labeled HL-60
cells (1ꢂ105 cells/well) were then added to P-selectin-IgG chi-
mera coated wells and allowed to bind for 30 min at 37 ꢁC. The
wells were then filled with RPMI1640 containing 10% FCS,
and the plate was inverted and placed for 30 min at 37 ꢁC.
After removal of the unbound cells by gentle aspiration, 1%
NP-40 in PBS was added to each well and the fluorescence
intensity was measured at 490 nm of excitation and 530 nm of
emission by MTP-32F fluorescence microplate reader (Corona
Electric, Hitachinaka, Japan).
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