Study on selectin blocker. 8. Lead discovery of a non-sugar antagonist using a 3D-pharmacophore model

Article abstract of DOI:10.1021/jm990342j

We have developed a pharmacophore model of a ligand/E-selectin complex to screen drug candidates for selectin blockers. In a series of sugar mimetic studies of the E-selectin ligand, sialyl Lewis X (sLe(x)), we have already found a potent compound, a sulfated Le(x) analogue (1), and also have proposed how compound I binds to E-selectin (Tsujishita, H.; Hiramatsu, Y.; Kondo, N.; Ohmoto, H.; Kondo, H.; Kiso, M.; Hasegawa, A. J. Med. Chem. 1997, 40, 362-369). To find drug candidates that fit into the binding pocket of E- selectin, we constructed an original 3D-pharmacophore model from structural information of a compound 1/E-selectin complex model and screened lead compounds for selectin blockers using a commercially available database ACD- 3D. As a result, we discovered a lead compound (2) containing good selectin inhibitory activity, and in addition, we succeeded to preliminarily optimize it to a more active lead compound (3) with micromolar IC₅₀ values, based on the 3D-pharmacophore model investigation. This methodology using the 3D- pharmacophore model could be applicable as a pre-screen system for selectin blockers.

Full text of DOI:10.1021/jm990342j

1476
J . Med. Chem. 2000, 43, 1476-1483
Stu d y on Selectin Block er . 8. Lea d Discover y of a Non -Su ga r An ta gon ist Usin g a
3D-P h a r m a cop h or e Mod el
Yasuyuki Hiramatsu,* Takahiro Tsukida, Yoshiyuki Nakai, Yoshimasa Inoue, and Hirosato Kondo*
Department of Chemistry, Nippon Organon K.K., R&D Laboratories, 1-5-90 Tomobuchi-Cho, Miyakojima-Ku,
Osaka 534-0016, J apan
Received December 6, 1999
We have developed a pharmacophore model of a ligand/E-selectin complex to screen drug
candidates for selectin blockers. In a series of sugar mimetic studies of the E-selectin ligand,
sialyl Lewis X (sLex), we have already found a potent compound, a sulfated Lex analogue (1),
and also have proposed how compound 1 binds to E-selectin (Tsujishita, H.; Hiramatsu, Y.;
Kondo, N.; Ohmoto, H.; Kondo, H.; Kiso, M.; Hasegawa, A. J . Med. Chem. 1997, 40, 362-369).
To find drug candidates that fit into the binding pocket of E-selectin, we constructed an original
3D-pharmacophore model from structural information of a compound 1/E-selectin complex model
and screened lead compounds for selectin blockers using a commercially available database
ACD-3D. As a result, we discovered a lead compound (2) containing good selectin inhibitory
activity, and in addition, we succeeded to preliminarily optimize it to a more active lead
compound (3) with micromolar IC50 values, based on the 3D-pharmacophore model investigation.
This methodology using the 3D-pharmacophore model could be applicable as a pre-screen system
for selectin blockers.
In tr od u ction
Recently, it was clarified that oligosaccharides play
an important role in signal transduction processes
involved in cell-cell adhesion.1 In addition, syntheses2
and biological3 studies of oligosaccharides have made
remarkable progress. Proteins interacting with oligosac-
charides have been recognized as targets for new drug
discovery. However, drug development of a carbohydrate
itself is hindered by the complication that the synthesis
of complex oligosaccharides is a rather expensive and
difficult process. Thus, current studies of biologically
F igu r e 1. Chemical structure and IC50 of sLex toward
E-selectin.
active oligosaccharides focus on the mimic into variable
low molecular compounds. However, there are very few
general methodologies for sugar mimetics.
Understanding the selectin-sLex interaction is sig-
nificant for discovery of good selectin antagonists.
Although only a crystal structure of E-selectin itself has
We took notice of sialyl Lewis X (sLex), a ligand of
selectin that is one of the cell adhesion molecules, as a
functional carbohydrate4 (Figure 1). It was reported that
selectin-sLex interaction led to acute and/or chronic
inflammatory diseases.5 Therefore, selectins are be-
lieved to be involved in the progression of the clinical
manifestations of inflammatory disease. We have un-
dertaken the discovery of a selectin antagonist based
on sLex carbohydrate mimicking.6
been determined,8 a ligand/E-selectin complex structure
has not been experimentally clarified yet. On the basis
of the point mutation of E-selectin9 and/or SAR study10
of sLex, an E-selectin-sLex binding mode has been
predicted by several groups.11
We have also constructed a complex model of E-
selectin and a potent selectin blocker, sulfo Lex deriva-
tive 1.12 Using this model, essential groups and desir-
able positions of compound 1 necessary for binding to
E-selectin were predicted. On the basis of this bound
conformation, we found a potential compound which
replaced the lactose moiety of compound 1 with Ser-
Glu dipeptide.6e This result might indicate that our
pharmacophore model could be a potential tool to find
other selectin antagonists.
To clarify the utility of pharmacophore models, a 3D-
database search13 is often performed. In general, 3D-
database searches make it possible to generate a new
scaffold, so it would be useful to discover a new lead
and/or mimic an interesting skeleton.
By the way, drug discovery is a very complex process.
Many different technologies can play a role in the rapid
discovery of lead compounds, such as molecular model-
ing, a combination of combinatorial chemistry/high-
throughput screening, and so on. For example, molec-
ular modeling could be one of the useful tools for lead
optimization and/or lead generation of biologically active
compounds.7 Actually, computer-docking studies are
often used to simulate good inhibitors and/or promising
antagonists.
* To whom correspondence should be addressed: Hirosato Kondo,
Ph.D. Tel: (06)6921-8920. Fax: (06)6921-9078. E-mail: Kondo.Hirosato@
organon.co.jp.
At present, there are very few examples which showed
the way to predict low molecular compounds that fit into
10.1021/jm990342j CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/25/2000

Study on Selectin Blocker
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1477
F igu r e 2. Chemical structure and IC50 of compound 1 (left). From the molecular modeling investigation, three critical interactions
of 1 toward E-selectin are clarified. On the basis of the pharmacophore, Ser-Glu dipeptide derivative (right) is found.
the sugar binding pocket using the modeling studies.
Therefore, we have investigated the pre-screening of
structurally novel compounds which can fit into the sLex
binding pocket using the 3D-database search method.
In this paper, we describe the construction of a 3D-
pharmacophore model of selectin antagonists and the
lead discovery using its 3D-pharmacophore model.
ible search option. The fitness of compounds toward the
pharmacophore model was investigated using five con-
formers in each compound. These conformers were
obtained by rotating single bonds. Figure 3 shows the
positions of the three functional groups and the dis-
tances between the groups as the query.
Scr een in g of a Hit Com p ou n d 2 a n d Its in Vitr o
Activity a ga in st E-Selectin Bin d in g. As a result of
the 3D-database search, we found compound 2 which
apparently showed inhibitory activity against E-selectin.
Although its potency (IC50 ) 1.2 mM) was weaker than
that of compound 1 (IC50 ) 0.28 mM; Figure 4),6b
compound 2 seemed to be a very potential lead com-
pound, because it was a completely non-sugar compound
and it would have some advantages, such as ease of
synthesis, oral availability, and pharmacokinetic profile.
Next, we tried to investigate the optimization of com-
pound 2.
Resu lts a n d Discu ssion
3D-P h a r m a cop h or e Mod el a n d 3D-Da t a b a se
Sea r ch . Our previous studies12 of molecular dynamics
simulations of a compound 1/E-selectin complex sug-
gested the existence of three essential binding sites of
compound 1 for E-selectin, as follows (Figure 2): (1) the
C-2 and C-3 hydroxy groups of fucose coordinate to
calcium; (2) the branched alkyl chain of compound 1
interacts with two hydrophobic regions on the surface
of E-selectin; (3) the negatively charged group of the
ligand interacts with the basic residues of E-selectin.
Namely, it is considered that shape, functional groups,
hydrogen bonding, and hydrophobic interaction affect
how well compounds bind to the selectin protein. Next,
we constructed a pharmacophore model which can
conserve the three essential groups to the desirable
positions for E-selectin binding. On the basis of this
pharmacophore, we have already found an active com-
pound which replaced the lactose moiety of compound
1 with Ser-Glu dipeptide6e (Figure 2). This result
indicates that our pharmacophore model could have
potential to find other selectin antagonists.
Although compound 2 was picked up by the 3D-
database search of the pharmacophore model derived
from the compound 1/E-selectin complex model (Figure
5), it is not clear that the bound conformation of
compound 2 screened by the pharmacophore model
could be identical with the stable conformation of
compound 2 in the presence of E-selectin. Certainly, the
precision of search conditions was a little rough. There-
fore, we constructed the complex model of compound
2/E-selectin and investigated the binding possibility of
compound 2 for E-selectin.
P r elim in a r y Op tim iza tion of th e P ossible Bou n d
Con for m a tion of Com p ou n d 2. The compound 2/E-
selectin complex model was constructed by superimpos-
ing the three functional groups of compound 2 based
on the compound 1/E-selectin complex model. These
graphical manipulations and representations were per-
formed by Midas-plus 2.015 and Sybyl 6.5.16 The con-
structed model showed the following features (Figure
6): (1) one of the carboxylic acids of isophthalic acid
would coordinate to calcium; (2) the carboxylic acid in
the benzoic acid moiety would form the electrostatic
interaction with Arg97 and/or Lys99; (3) a C17 long
alkyl chain would interact with the shallow hydrophobic
region consisted of Lys114, Ala9, Tyr49, etc., on E-
selectin. In this model, it was also found that one of the
two carboxylic acids in the isophthalic acid moiety did
not interact with the surface of the protein and was not
Thus, we tried to find other novel selectin antagonists
using the 3D-database searching method. First, we
selected the ACD-3D database which was a com-
mercially available reagent database. Unfortunately,
there were no compounds containing all three essential
groups necessary for binding (fucose, negatively charged
group, and hydrophobic part) among the database, so
we constructed a 3D-pharmacophore model containing
other groups which could play identical roles with the
three essential groups: (1) the fucose unit, as a calcium
ligand, would be replaced with a carboxylic acid;14 (2)
the hydrophobic part, branched alkyl chain, would be
replaced with a single alkyl chain; (3) the negatively
charged group, a sulfonic acid, would be replaced with
a carboxylic acid (Figure 3).
A 3D-database search was performed using the ISIS-
3D13f software package with the conformationally flex-

1478 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hiramatsu et al.
F igu r e 3. Retrieved relative positions of the functional groups from the complex model between compound 1 and E-selectin.
Next arrow shows the pharmacophore model which is involved in the general groups (fucose to carboxylic acid, branched alkyl
chain to single alkyl chain, sulfonic acid to carboxylic acid) that replaced the functional groups in compound 1. Using this model,
the 3D-database search was performed.
F igu r e 4. Chemical structure and IC50 of one of the hit
compounds, compound 2, toward E-selectin.
F igu r e 6. Complex model of compound 2 and E-selectin.
Thick colored atoms show compound 2. Thin colored atoms
show E-selectin amino acid residues. Only 2 and the important
residues of E-selectin are shown. This model was constructed
based on the compound 1/E-selectin complex model. The three
functional groups of compound 2 superimposed with the
corresponding groups in the bound conformation of compound
1.
Tyr48 on E-selectin. Accordingly, the original complex
model constructed here showed that compound 2 could
well bind to E-selectin.
However, it was experimentally shown that the
potency of compound 2 was weaker than that of com-
F igu r e 5. Superimposition of the bound conformation of
pound 1. Thus, to clarify the reason for the potency of
compound 2 being weaker than that of compound 1, we
should estimate the binding energy between the com-
pound 2/protein complex. But, in fact, it is difficult to
estimate these interactions precisely and quickly (such
as the coordination to metal ion and hydrophobic
compounds 1 and 2. The circles indicate the corresponded
functional groups.
directed to the solvent area, but the benzene linked to
the long alkyl chain seemed to interact with the
hydrophobic surface consisted of Tyr44, Pro46, and

Study on Selectin Blocker
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1479
conformational analysis of unit 1. First, we defined two
dihedral angles of unit 1, φ and ψ, and calculated the
conformational energies every 10° of the dihedral angles
(Figure 8). Figure 8 shows that the active conformation
of unit 1 (φ ) 80° and ψ ) 200°) did not agree with the
stable ones. This result seemed to depend on an
unfavorable repulsion between the carboxylic acid of
benzoic acid and a carboxylic acid of isophthalic acid.
Moreover, the compound 2/E-selectin complex model
showed that the unfavorable carboxylic acid in isoph-
thalic acid would not participate in binding with E-
selectin. Thus, if this unnecessary carboxylic acid in
isophthalic acid was removed from unit 1, the active
bound conformation could agree with the stable one
(Figure 9). To clarify our hypothesis, we designed unit
2 and performed the conformational analysis in the
same way. As a result, it was of interest to note that
the most stable conformation of unit 2 was almost
identical to the active bound conformation of compound
2 as shown in Figure 9. To confirm the adequacy of our
design in experiment, we synthesized a decarboxy
compound 3 and a tricarboxyl compound 4 with very
close structures to compound 2 and estimated the
inhibitory activities of compounds 2-4. Although we
also tried the synthesis of the decarboxy derivative of
compound 2, we could not synthesize it.
F igu r e 7. Retrieved compound 2 (left) from the complex
model with E-selectin in Figure 6. After minimization, the
right conformation was generated. The conformation of the
biphenyl ether moiety has changed (in the circle). The long
alkyl moiety was omitted during the minimization. The
minimization was performed by MMFF9417 force field attached
with Sybyl6.5 until the root mean square of gradients was
below 0.5 kcal/(mol Å). We used a nonbonded cutoff of 8 Å and
dielectric constant ꢀ ) 1.
interaction) using the current calculation technology.
Therefore, we focused on the bound conformation of
compound 2 in the presence of E-selectin and speculated
that the desirable bound conformation of compound 2
against the target protein would not be so stable;
namely the stable conformation of compound 2 would
be much different from the bound conformation toward
E-selectin. Thus, to clarify our hypothesis, we investi-
gated an energy minimization of the assumed bound
conformation in compound 2. As a result, the conforma-
tion of the biphenyl ether moiety has well changed as
shown in Figure 7. This finding indicates that the
conformation of this moiety would not be so stable.
Namely, a structural modification of this moiety, which
makes its initial bound conformation so stable, would
be important for lead optimization. Therefore, we have
undertaken the conformational analysis of this biphenyl
ether moiety in detail.
Ch em istr y. For the syntheses of compounds 3 and
4, the condensation of 5a ,b with p-nitrobenzoyl chloride
in the presence of triethylamine in chloroform afforded
compounds 6a ,b, in 54-73% yields. Next, compounds
6a ,b were subjected to hydrogenolysis in the presence
of a catalyst, 10% palladium-carbon, under hydrogen
atmosphere followed by the acyl condensation with
stearoyl chloride in the presence of triethylamine in
chloroform to afford compounds 7a ,b, in 31-58% yields.
Finally, compounds 7a ,b were transformed in good
yields, by removal of the protecting groups, into the
desired compounds 3 and 4 (Scheme 1).
Con for m a tion a l An a lysis of Com p ou n d 2 a n d
Design for a Str on g Selectin Block er . From the
above description of the structural characteristics of
compound 2, we focused on the 5-(4-carboxyphenoxy)-
isophthalic acid moiety (unit 1) and investigated the
In Vitr o Eva lu a tion of Com p ou n d s 3 a n d 4. As a
result of the in vitro assay,6a compound 3 showed
F igu r e 8. Potential energy surface of unit 1, as the axis of abscissa φ and the axis of spindle ψ. Each contour represents a
relative energy from the most stable region. The white region indicates the most stable region with increasing thickness of color
the growth in relative energy. We calculated the conformational energies every 10° of the dihedral angles using the molecular
orbital program package MOPAC93.18 This calculation was performed using the AM119 method and keywords STEP and POINT.
White open circle shows the putative active conformation of unit 1 in compound 2.

1480 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hiramatsu et al.
F igu r e 9. On the basis of the result of conformational analysis of unit 1, we designed unit 2 (decarboxylic acid derivative).
Potential energy surface of unit 2 is shown. The white region indicates the most stable region with increasing thickness of color
the growth in relative energy. Each contour represents a relative energy from the most stable region. This calculation was performed
the same way as in Figure 8. Closed circle shows the putative active conformation of unit 2.
Sch em e 1a
a
Conditions: (a) p-nitrobenzoyl chloride, Et3N/CHCl3, 54-73%; (b) 10% Pd-C/1,4-dioxane-MeOH; (c) stearoyl chloride, Et3N/CHCl3,
31-58% from 6a ,b; (d) 1 N NaOH/1,4-dioxane-MeOH, 22-97%.
stronger inhibitory activity (IC50 ) 86 µM) toward
E-selectin than compound 1 (Figure 10), while the
inhibitory activity of compound 4 was very weak (IC50
3 could bind to E-selectin, as predicted by the complex
model of compound 2/E-selectin (Figure 6). In addition,
a fucose necessary20 for calcium binding would be
replaced with a carboxylic acid that was a simple
functional group. However, the precise contribution of
the three functional groups of compound 3 for the
binding to the protein was not clarified. To clarify this
point, we need to perform a detailed SAR investigation
of compound 3 derivatives and/or an estimation of
binding energy between compound 3/E-selectin based
> 500 µM), the same as that for compound 2 (IC50
)
1200 µM). Namely, as speculated for the inhibitory
activities of compounds 3 and 4 from the conformational
analysis, it was found that the strong activity of
compound 3 would be due to the stability of the bound
conformation toward E-selectin. This finding would also
suggest that the three functional groups of compound

Study on Selectin Blocker
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8 1481
bance at 405 nm was measured. Percent binding was calcu-
lated by the following equation:
% binding ) (X - C/ A - C) × 100
wherein X is the absorbance of wells containing the test
compounds at each concentration, C is the absorbance of wells
not containing the selectin-Ig and test compounds, and A is
the absorbance of control wells not containing the test com-
pounds. The results of inhibitory activities are presented in
figures as IC50 values. The number of replicates is 2.
Dim eth yl 3-Nitr o-3′,4-oxyd iben zoa te. To a solution of
methyl 4-fluoro-3-nitrobenzoate (1.99 g, 10 mM) and methyl
3-hydroxybenzoate (1.52 g, 10 mM) in DMF (20 mL) was added
K2CO3 (1.38 g, 10 mM), and the mixture was stirred for 6 h at
room temperature. AcOEt was added to the solution, and the
mixture was washed with water and brine successively, dried
over Na2SO4, and concentrated. To the resulting residue was
added n-hexane and Et2O, and the precipitate was filtered to
afford the title compound (3.04 g, 91.8%) as a crystal: 1H NMR
(CDCl3) δ 3.91 (s, 3H), 3.96 (s, 3H), 6.99 (d, 1H, J ) 8.7 Hz),
7.31 (dd, 1H, J ) 8.1, 2.5 Hz), 7.51 (t, 1H, J ) 7.9 Hz), 7.74 (s,
1H), 7.93 (d, 1H, J ) 8.2 Hz), 8.14 (ddd, 1H, J ) 0.5, 2.2, 8.8
Hz), 8.62 (d, 1H, J ) 2.2 Hz).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 6a ,b : Di-
m eth yl 3-(4-Nitr oben zoyla m in o)-3′,4-oxyd iben zoa te (6a ).
To a solution of dimethyl 3-nitro-3′,4-oxydibenzoate (4.37 g,
13.19 mM) in MeOH (100 mL) and 1,4-Dioxane (50 mL) was
added 10% Pd-C (100 mg), and the mixture was stirred for 1
h under 3 atmospheric pressure of hydrogen at room temper-
ature. The precipitate was filtered off, and the filtrate was
concentrated in vacuo to afford dimethyl 3-amino-3′,4-oxy-
dibenzoate (5a ) as a syrup.
On the other hand, to p-nitrobenzoic acid (0.90 g, 5.39 mM)
was added thionyl chloride (20 g), and the mixture was stirred
for 2 h at 50 °C and then for 1 h at 75 °C. The mixture was
coevaporated with toluene to give p-nitrobenzoyl chloride as
a syrup. The resulting residue and 5a (864 mg, 2.87 mM) were
dissolved in CHCl3 (20 mL), triethylamine (653 mg, 6.45 mM)
was added to the solution, and the mixture was stirred for 20
h at room temperature. CHCl3 (50 mL) was added to the
solution, and the mixture was washed with 1 N HCl, saturated
sodium carbonate and brine successively, dried over MgSO4,
and concentrated. The resulting precipitate was filtered to
afford 6a (700 mg, 54.3%) as a colorless crystal: 1H NMR
(DMSO-d6) δ 3.83 (s, 3H), 3.87 (s, 3H), 7.07 (d, 1H, J ) 8.6
Hz), 7.35-7.45 (m, 1H), 7.50-7.60 (m, 2H), 7.77 (d, 1H, J )
7.6 Hz), 7.85 (dd, 1H, J ) 2.2, 8.7 Hz), 8.07 (d, 2H, J ) 8.6
Hz), 8.32 (d, 2H, J ) 8.6 Hz), 8.35-8.43 (m, 1H), 10.37 (s, 1H).
Dim eth yl 5-[4-Meth oxyca r bon yl-2-(4-n itr oben zoyla m i-
n o)p h en oxy]isop h th a la te (6b). Compound 6b was prepared
from dimethyl 5-[3-amino-4-methoxycarbonylphenoxy]iso-
phthalate (3.0 g, 8.35 mM) and p-nitrobenzoic acid (2.0 g, 11.97
mM) as described above: yield 72.9%; mp 226-229 °C; 1H NMR
(DMSO-d6) δ 3.87 (s, 6H), 3.88 (s, 3H), 7.18 (d, 1H, J ) 8.6
Hz), 7.82 (d, 2H, J ) 1.5 Hz), 7.87 (dd, 1H, J ) 2.2, 8.6 Hz),
8.06 (d, 2H, J ) 8.9 Hz), 8.25 (t, 1H, J ) 1.5 Hz), 8.31 (d, 2H,
J ) 8.8 Hz), 8.39 (d, 1H, J ) 2.1 Hz), 10.4 (s, 1H).
Gen er a l P r oced u r e for t h e P r ep a r a t ion of 7a ,b : Di-
m eth yl 3-(4-Octa d eca n oyla m in oben zoyla m in o)-3′,4-oxy-
d iben zoa te (7a ). To a solution of 6a (650 mg, 1.44 mM) in
MeOH-1,4-dioxane (10-10 mL) was added 10% Pd-C (100
mg), and the mixture was stirred for 2 h under hydrogen
atmosphere at room temperature. The precipitate was filtered
off, and the filtrate was concentrated in vacuo to afford
dimethyl 3-aminobenzoylamino-3′,4-oxydibenzoate as a syrup.
On the other hand, to stearic acid sodium salt (607 mg, 1.98
mM) was added thionyl chloride (20 mL), and the mixture was
stirred for 2 h at room temperature. The mixture was coevapo-
rated with toluene to give stearoyl chloride as a syrup. To a
solution of stearoyl chloride in CHCl3 (10 mL) was added a
solution of dimethyl 3-aminobenzoylamino-3′,4-oxydibenzoate
in CHCl3 (10 mL) and triethylamine (240 mg, 2.37 mM), and
then the mixture was stirred for 21 h at room temperature.
CHCl3 (50 mL) was added to the solution, and the mixture
F igu r e 10. Chemical structure and IC50 of compounds 3 and
4 toward E-selectin.
on the docking study. These works are now under
investigation.
In conclusion, we discovered the structurally novel
selectin blocker 2 based on the 3D-pharmacophore
model investigation. Namely, this pharmacophore would
have potential to find a new selectin blocker and the
3D-database search using this pharmacophore model
could be responsible for carbohydrate mimicking. In
addition, we succeeded in optimizing the lead compound
2 preliminarily and obtained the potent selectin an-
tagonist, compound 3. Further optimization of com-
pound 3 is in progress. Thus, we could present a
methodology for the discovery of a new class of selectin
blocker.
Exp er im en ta l Section
In h ibition Assa y of Selectin -sLex Bin d in gs. The con-
struction of the selectin-immunoglobulin was carried out
according to a previous paper.6a
A solution of sLex pentasaccharide ceramide analogue in a
1:1 mixture of methanol and distilled water was pipetted into
microtiter plate wells (96 wells; Falcon PRO-BIND) at 100
pmol/50 µL/well and was adsorbed by evaporating the solvent.
The wells were washed twice with distilled water, blocked with
5% BSA (bovine serum albumin)-1 mM CaCl2/50 mM imida-
zole buffer (pH 7.2) for 1 h at room temperature, and washed
three times with 50 mM imidazole buffer (pH 7.2).
Separately, a 1:1 volumetric mixture of a 1:500 dilution in
1% BSA-1 mM CaCl2/50 mM imidazole buffer (pH 7.2) of
biotinylated goat F(ab′)2 anti-human IgG(g)/streptavidin-
alkaline phosphatase (Zymed Lab Inc.) and a selectin-immu-
noglobuline fusion protein (selectin-Ig) was incubated at room
temperature for 30 min to form a complex. The test compounds
were dissolved in DMSO at 10 mM and finally diluted by 1
mM CaCl2/50 mM imidazole buffer (pH 7.2) to final concentra-
tions at 1000, 500, 250, 125, 62.5, 31.3, 15.6, and 7.8 µM,
respectively. Reactant solutions were prepared by incubating
30 µL of this solution at each concentration with 30 µL of the
above complex solution for 30 min at room temperature. This
reactant solution was then added to the above microtiter wells
at 50 µL/well and incubated at 37 °C for 45 min. The wells
were washed three times with 50 mM imidazole buffer (pH
7.2) and diluted water, respectively, followed by addition of
p-nitrophenyl phosphate (1 mg/mL) and 0.01% MgCl2 in 1 M
diethanolamine (pH 9.8) at 50 µL/well. The reactant mixture
was developed for 120 min at room temperature, and absor-

1482 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 8
Hiramatsu et al.
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was washed with 1 N HCl, saturated sodium carbonate and
brine successively, dried over MgSO4, and concentrated. The
residue was purified by thin-layer chromatography developing
with 1:1 AcOEt/n-hexane to afford 7a (310 mg, 31.3%) as a
1
colorless crystal: H NMR (CDCl3) δ 0.70-0.90 (m, 3H), 1.25
(s, 28H), 1.40-1.80 (m, 2H), 2.20-2.40 (m, 2H), 3.80-3.95 (m,
6H), 6.70-6.90 (m, 1H), 7.15-7.30 (m, 3H), 7.30-7.95 (m, 7H),
8.44 (brs, 1H), 9.24 (brs, 1H).
Dim eth yl 5-[4-Meth oxycar bon yl-2-(4-octadecan oylam i-
n oben zoyla m in o)p h en oxy]isop h th a la te (7b): yield 58.2%;
1H NMR (CDCl3) δ 0.8-0.95 (m, 3H), 1.1-1.8 (m, 30H), 2.3-
2.4 (m, 2H), 3.92 (s, 3H), 3.94 (s, 6H), 6.84 (d, 1H, J ) 8.6 Hz),
7.37 (brs, 1H), 7.64 (d, 2H, J ) 8.7 Hz), 7.77 (dd, 1H, J ) 2.1,
8.7 Hz), 7.83 (d, 1H, J ) 8.7 Hz), 7.94 (d, 2H, J ) 1.4 Hz),
8.41 (brs 1H), 8.54 (t, 1H, J ) 1.4 Hz), 9.24 (d, 1H, J ) 2.0
Hz).
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Gen er a l P r oced u r e for th e P r ep a r a tion of 3 a n d 4:
3-(4-Octa d eca n oyla m in oben zoyla m in o)-3′,4-oxyd iben zo-
ic Acid (3). 7a (300 mg, 0.44 mM) was dissolved in 1,4-
dioxane-MeOH (9-3 mL), and 1 N NaOH (4.1 mL) was added
to the solution; then the mixture was stirred for 5.5 h at room
temperature. Cold water (30 mL) was added to the mixture;
the resulting mixture was adjusted to pH 1 using concentrated
HCl and then extracted with AcOEt. The organic layer was
washed with water and brine successively, dried over MgSO4,
and concentrated. The resulting precipitate was washed with
n-hexane (30 mL) and then purified by HPLC eluting with 9:1
CH3CN/0.1% aqueous TFA to afford 3 (63 mg, 21.9%): mp 240
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1
°C dec; H NMR (DMSO-d6) δ 0.80-0.85 (m, 3H), 1.10-1.34
(m, 28H), 1.46-1.65 (m, 2H), 2.30 (t, 2H), 7.00 (d, 1H, J ) 8.6
Hz), 7.30-7.40 (m, 1H), 7.45-7.60 (m, 2H), 7.60-7.85 (m, 6H),
8.37 (d, 1H, J ) 2.2 Hz), 9.79 (s, 1H), 10.07 (s, 1H).
5-[4-Ca r boxy-2-(4-octa d eca n oyla m in oben zoyla m in o)-
p h en oxy]isop h th a lic Acid (4): yield 97.0%; mp >250 °C; 1H
NMR (DMSO-d6) δ 0.75-0.95 (m, 3H), 1.23 (s, 28H), 1.4-1.7
(m, 2H), 2.25-2.4 (m, 2H), 7.12 (d, 1H, J ) 8.6 Hz), 7.66 (d,
1H, J ) 8.8 Hz), 7.7-7.85 (m, 5H), 8.2-8.3 (m, 1H), 8.35-
8.4(m,1H), 9.84 (s, 1H), 10.07 (s, 1H).
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