Novel RGD-like molecules based on the tyrosine template: Design, synthesis, and biological evaluation on isolated integrins αVβ 3/αIIbβ3 and in cellular adhesion tests

Article abstract of DOI:10.1016/j.bmc.2004.07.055

RGD (Arg-Gly-Asp) peptidomimetics have been designed for covalent anchorage on biomaterials. The tyrosine template was thus equipped with (i) a basic side chain of various flexibility, (ii) an acidic side chain, which incorporated the XPS fluorine tag, and (iii) a spacer-arm terminated by a primary amine for surface grafting. The most active compounds showed IC₅₀ values in the nanomolar range versus isolated human integrins α_Vβ ₃ and α_IIbβ₃. Preincubation of CaCo2 cells with soluble peptidomimetics (2 and 19a) prevented cellular adhesion on culture plates coated with vitronectin. On the other hand, peptidomimetics (19a and 19b) immobilized on a poly(ethylene)terephthalate membrane (PET) promoted CaCo2 cells adhesion. A modeling study at the ab initio level in MINI-1′ basis allowed to compare the various synthetic ligands of integrins and to propose novel pharmacophore structures.

Full text of DOI:10.1016/j.bmc.2004.07.055

Bioorganic & Medicinal Chemistry 12 (2004) 5379–5393
Novel RGD-like molecules based on the tyrosine template:
design, synthesis, and biological evaluation on isolated integrins
aVb3/aIIbb3 and in cellular adhesion tests
Stephane Biltresse,a Mireille Attolini,a Georges Dive,b Alex Cordi,c Gordon C. Tuckerc and
Jacqueline Marchand-Brynaerta,
*
a
´ ´ ´
Unite de Chimie Organique et Medicinale, Universite catholique de Louvain, Batiment Lavoisier, Place Louis Pasteur, 1,
B-1348Louvain-la-Neuve, Belgium
ˆ
b
´
´
´
`
ˆ
Centre d’Ingenierie des Proteines, Universite de Liege, Batiment de Chimie B6, B-4000 Sart-Tilman, Belgium
cInstitut de Recherches Servier, Rue des Moulineaux 11, F-92150 Suresnes, France
Received 4 June 2004; accepted 23 July 2004
Available online 23 August 2004
Abstract—RGD (Arg-Gly-Asp) peptidomimetics have been designed for covalent anchorage on biomaterials. The tyrosine template
was thus equipped with (i) a basic side chain of various flexibility, (ii) an acidic side chain, which incorporated the XPS fluorine tag,
and (iii) a spacer-arm terminated by a primary amine for surface grafting. The most active compounds showed IC50 values in the
nanomolar range versus isolated human integrins aVb3 and aIIbb3. Preincubation of CaCo2 cells with soluble peptidomimetics (2
and 19a) prevented cellular adhesion on culture plates coated with vitronectin. On the other hand, peptidomimetics (19a and
19b) immobilized on a poly(ethylene)terephthalate membrane (PET) promoted CaCo2 cells adhesion. A modeling study at the
ab initio level in MINI-10 basis allowed to compare the various synthetic ligands of integrins and to propose novel pharmacophore
structures.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
RGD (Arg-Gly-Asp) active sequence of the extracellular
matrix (ECM) proteins. For our purpose, the designed
molecules have to be equipped with a spacer-arm allow-
ing further surface anchorage on the PET membranes
used as cell culture substrates. In this paper, we report
on the design, synthesis, and evaluation of novel RGD
peptidomimetics based on the (L)-tyrosine template der-
ivatized with three functionalized chains, respectively,
the mimic of the Arg basic residue, the mimic of the
Asp carboxyl function, and the spacer-arm. The pres-
ence of this last structural feature raised unexpected
activities against isolated integrins.
Modern clinical medicine relies increasingly upon man-
made materials, called biomaterials, for providing im-
plants, prostheses, and artificial organs.1 Over the last
10years, tailored substrates for tissue engineering
emerged as an important field of biomaterials.2 In this
context, various supports were purposely surface modi-
fied with a view to promote mammalian cells adhesion.3
We are interested in the biocompatibilization of
poly(ethylene terephthalate) (PET) track-etched mem-
branes by the covalent grafting of integrin ligands.4
The preparation of long-term stable biomaterials re-
quired the use of synthetic biologically active com-
pounds, for surface modification, instead of the
natural adhesive proteins and derived peptides.5
Accordingly, we considered nonpeptide mimics of the
2. Results and discussion
2.1. Design
Integrins are cell adhesion receptors intensively studied
over the last years concerning their structures and func-
tions.6 They are considered as pharmaceutical targets in
a number of therapeutic areas, especially those involved
in thrombosis, cancer, and osteoporosis treatments.7
Keywords: RGD peptidomimetic; Tyrosine template; Integrin aVb3;
Integrin aIIbb3; Cell adhesion.
Corresponding author. Tel.: +32 10 472740; fax: +32 10 474168;
e-mail: marchand@chim.ucl.ac.be
*
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.055

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S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
The integrin aVb3 (called vitronectin receptor), is ex-
pressed on endothelial, smooth muscle, bone, and epi-
thelial cells, and mediates cellular adhesion to various
ECM and serum proteins via the RGD motive.8 Con-
strained cyclic peptides involving this RGD sequence
(Fig. 1) showed higher affinity and selectivity (for in-
stance, versus the platelet integrin aIIbb3) than flexible
linear peptides;9 they were thus used as reference tem-
plates for the design of nonpeptide RGD mimics. In-
deed, the development of peptidomimetics constitutes
actually a major strategy for the discovery of new leads
(potential drugs) in medicinal chemistry.10 A lot of
works are devoted to the search of potent, selective,
and orally bioavailable antagonists of integrin aVb3.11
In the field of ꢀintelligentꢁ materials, RGD peptidomi-
metics were practically not considered for improving cell
adhesive properties of substrates,4 while both linear and
cyclic RGD peptides were abundantly used.3c This could
be due to the relatively hard synthetic investment re-
quired, which discourages the traditional materials
chemist! However, the demands for application of
RGD-like molecules in tissue engineering are less severe
than in the therapeutic field since selectivity and ADME
properties (adsorption–distribution–metabolism–excre-
tion) should be of little or none importance. But, on
the other hand, the molecules need an anchorage arm,
which does not disturb their capacity of binding to
integrin.
tural features required for the construction of our
aVb3 ligands from tyrosine are as follows: (i) a basic
moiety (Arg mimic) fixed in ortho-position; (ii) a carb-
oxylic function (Asp mimic), which is in fact the tyrosine
acid; (iii) a lipophilic substituent fixed on the a-amino
function via preferably a sulfonamide linkage as sug-
gested by recent publications11c,15––this substituent will
display a fluorine tag useful for quantitative analysis of
the biomaterials surface by X-ray photoelectron spectro-
scopy (XPS);4,14 (iv) a spacer-arm fixed on the aromatic
hydroxyl function. The choice of the basic moiety
resulted from molecular modeling and comparison with
the reference cyclic peptide c[RGDfV]. We have previ-
ously shown that the peptidomimetic 114 fulfilled our
requirements, but due to the flexibility and length of
the side chain bearing the basic function (guanidine),
this molecule could be fitted also onto the cyclic peptide
DMP728 (Figs. 1 and 2). Experimentally, 1 was demon-
strated to be an inhibitor of platelet aggregation (antag-
onist of aIIbb3).14 We have now designed the
peptidomimetic 2, as better aVb3 ligand, in which the
guanidine function (Arg mimic) is fixed on the shorter
and somewhat rigid isonipecotic motive. Also, the bulk-
iness of the lipophilic fluorine tag was increased. The
geometry of compound 2 (with R = Me, in place of
the spacer-arm) was fully optimized at the approximate
quantum chemistry level AM1; the molecule was studied
as an isolated neutral entity, and not as a zwitterionic
form, in order to avoid internal self-folded conforma-
tions. The conformer of low energy (Fig. 3) could be fit-
ted onto c[RGDfV]: the respective carboxyl and
guanidyl functions superposed well. The fitting was
definitively less good considering DMP728 as reference.
Indeed, in structure 1, the distance between the two
pharmacophoric groups was of 13 bonds, but of 11
bonds only in structure 2. This is in agreement with
the previous works of several companies: transforma-
tion of aIIbb3 antagonists into aVb3 antagonists was
achieved using compounds with a shorter distance be-
tween the carboxyl and guanidinyl groups and inserting
a hydrophobic residue next to the carboxyl function.16
Several rigid scaffolds have been used to construct aVb3
antagonists mimicking the RGD ligands.12 We have se-
lected the (L)-tyrosine template, initially exploited by the
Merckꢁs group for the synthesis of aIIbb3 antagonists,13
because the aromatic nucleus offers three points of func-
tionalization: the a-aminoacid chain, the phenolic hyd-
roxyl function, and its ortho-position after a sequence
of nitration–reduction–acylation reactions.14 The struc-
NH2
HN
HN
O
CO2H
O
H
N
NH
H2N
HN
O
Me
N
O
HN
O
O
NH
HN
RO
CO2H
CF3
N
H
HN
O
Antagonist of αΙΙbβ3 (platelet): DMP728 (IC50 = 20 nM)
1, R = Me
R = spacer-arm
H2N
NH
O
HN
N
HN
NH
O
H2N
HN
O
CO2H
N
HN
CO2H
SO2
O
A
N
O
NH
RO
N
H
O
Antagonist of αVβ3 (cells): c [RGDfV] ; A = H ( IC50 = 50 nM)
c [RGDf(N-Me)V] ; A = Me (IC50 = 3 nM)
F3C
2, R = Me
R = spacer-arm
Figure 1. Cyclic peptides as integrin antagonists.
Figure 2. RGD peptidomimetics based on the tyrosine template.

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
5381
H2N
CO2tBu
O2N
CO2tBu
i
HN
NHCOCF3
40%
(3 steps)
SO2
MeO
MeO
4
3
F3C
NHBoc
N
BocN
CO2H
ii
H
N
CO2tBu
53%
BocHN
BocN
O
HN
SO2
MeO
N
5
6
F3C
O
H2N
H
N
N
CO2H
SO2
HN
HN
iii
MeO
100%
n CF CO H
3 2
Figure 3. Fitting of compound 2 onto c[RGDfV]. Atoms coloring is as
follows: C, gray; O, red; N, blue; F, green; S, yellow; H, white.
F3C
2
Scheme 1. Synthesis of the reference peptidomimetic (without spacer-
arm). Reagents and conditions: (i) see Ref. 14; (ii) PYBOP, DIEA,
DMF, 48h, 20ꢁC; (iii) TFA–CH2Cl2 (1:1), 2h, 20ꢁC.
2.2. Chemistry
The peptidomimetic devoid of spacer-arm was first syn-
thetized (2, R = Me, Fig. 2); this molecule was required
as reference for the biological evaluations. The starting
material 3 was prepared in three steps from (L)-tyrosine
t-butyl ester by known methods,14 namely N-trifluoro-
acetylation (95%), aromatic nitration (90%), and O-
methylation under modified Mitsunobu conditions
(87%).17 The key intermediate 4 was then obtained by
trifluoroacetamide hydrolysis (90%), sulfonylation with
m-trifluoromethyl-benzenesulfonyl chloride (56%), and
reduction of the nitro function by catalytic hydrogena-
tion (70%)14 (Scheme 1).
without competition with N-alkylation of the sulfon-
amide function, provided that the alkylation reagent is
bulky enough;14,17 it was not the case for the simple
methylation, which requires the temporary NH2
masking as trifluoroacetamide (see Scheme 1). Thus,
CO2tBu
NH2
CO2tBu
ii
i
NH
95%
48%
HO
HO
SO2Ar
8
7
O2N
HO
O2N
R1O
CO2tBu
CO2tBu
iii or iv
56% - 60%
After protection of isonipecotic acid as benzyl ester, the
guanidine substituent was introduced by reaction with
N,N0-bis-(t-butoxycarbonyl)-3,5-dimethylpyrazolyl-1-
carboxamidine.18 Hydrogenation over palladium on
charcoal furnished the reagent 5 in an overall yield of
40% for three steps. This acid 5 was coupled to the tyro-
sine derivative 4 under activation by PYBOP.19 The
fully protected RGD mimic 6 was isolated in 53% yield
after chromatographic purification; its structure was
established by the usual spectroscopies (see Section 4).
Lastly, the treatment with trifluoroacetic acid cleaved
simultaneously all the protections to give compound 2
(100% yield) as TFA salt (Scheme 1).
NH
NH
SO2Ar
SO2Ar
9
10
NHBoc
a, R1
b, R1
=
=
NHBoc
O
O
O
R2
H2N
CO2tBu
HN
CO2tBu
vi
42 - 69%
v
HN
100%
R1O
HN
SO2Ar
R1O
SO2Ar
11 (crude)
12 - 18 (Table 1)
O
R4
HN
R3O
CO2H
vii
100%
NH
n.CF3CO2H
We next considered the synthesis of the related pepti-
domimetics equipped with a spacer-arm. We selected a
short linker (aminopropyl motive) and a longer hydro-
philic linker (2-[2-(2-aminoethoxy)ethoxy]ethyl motive)
on the basis of a previous study of PET membrane reac-
tivity and covalent grafting of molecular probes with
various linkers.20 (L)-Tyrosine-t-butyl ester (7) was sul-
fonylated as usual (compound 8) before aromatic nitra-
SO2
19 - 25 (Table 1)
CF3
Scheme 2. Synthesis of peptidomimetics with spacer-arms. Reagents
and conditions: (i) ArSO2Cl, pyridine, CH2Cl2, 5h, 20ꢁC; (ii) HNO3
(1equiv), HOAc, 16ꢁC; (iii) R1Br (1equiv), K2CO3, 18-crown-6,
CH3CN, reflux, 18h; (iv) R1OH (1equiv), DIAD, dppe, THF, 24h,
20ꢁC; (v) PtO2 (5%), H2 (30psi), EtOAc, 20ꢁC, 18h; (vi) PYBOP,
DIEA, DMF, 48h, 20ꢁC; (vii) TFA–CH2Cl2 (1:1), 2h, 20ꢁC.
tion to furnish the intermediate 9 (Scheme 2).
Etherification of the phenol moiety could be realized

5382
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
N-(t-butoxycarbonyl)-3-bromopropylamine20 was cou-
pled to 9 under Williamson conditions to give the ether
10a in 56% yield after column chromatography. On the
other hand, 2-[2-(2-t-butoxycarbonylaminoethoxy)eth-
oxy]ethanol20 reacted well under modified Mitsunobu
conditions;17 pure compound 10b could be isolated in
60% yield (Scheme 2). The next step was the nitro group
reduction by hydrogenation: anilines 11a,b were charac-
We have also prepared related peptidomimetics with
different basicity/lipophilicity of the Arg mimic residue.
N-Boc isonipecotic acid, N-methyl isonipecotic acid,21
isonicotinic acid and 4-pyridylacetic acid were coupled
to the key intermediate 11a (Scheme 2) to furnish,
respectively, compounds 13a, 14a, 15a, and 16a in mod-
erate yields (Table 1, entries 3–6), and the corresponding
peptidomimetics 20a, 21a, 22a, and 23a after deprotec-
tion (Table 1, entries 11–14). The flexibility (conforma-
tional mobility) and length of the Arg mimic have
been varied as well, with the synthesis of compounds
24a and 25a (from the precursors 17a and 18a) obtained
by the coupling of 3-aminopropionic acid and 12-ami-
nododecanoic acid, respectively (Table 1, entries 7–8
and 15–16).
1
terized in H NMR by an important shielding of the
aromatic proton in ortho-position with respect to NH2
(from 7.56ppm in 10 to 6.39ppm in 11). The crude ani-
lines 11a,b were reacted with acid 5 (see Scheme 1) in the
presence of PYBOP as previously. Protected peptidomi-
metics 12a,b (Table 1, entries 1 and 2) were recovered in,
respectively, 48% and 42% yield, and well characterized
by NMR spectroscopy (see Section 4); amine acylation
provoked a significant deshielding of the ortho aromatic
proton, which appeared at 8ppm. As before, treatment
with TFA quantitatively yielded the final compounds
19a and 19b (Table 1, entries 9 and 10).
2.3. Biological evaluation
We have evaluated the ability of the lead compound 2
(no spacer-arm) to inhibit cellular adhesion in a compet-
Table 1. Structures of the side chains and yields of compounds
Entry
1
Compd
R1/R3
R2/R4
%
BocN
12a
48
NHBoc
N
BocHN
BocN
O
2
12b
N
42
NHBoc
2
BocHN
3
4
5
13a
14a
15a
Boc
Me
N
N
53
56
52
NHBoc
NHBoc
NHBoc
N
6
16a
56
N
NHBoc
7
8
17a
18a
45
69
BocHN
NHBoc
NHBoc
BocHN
HN
9
N
N
9
19a
19b
Quantitative
Quantitative
NH
2
H N
2
HN
O
10
NH
2
2
H N
2
H
N
11
12
13
20a
21a
22a
Quantitative
Quantitative
Quantitative
NH
2
Me
N
NH
2
N
NH
NH
2
14
23a
Quantitative
N
2
15
16
24a
25a
Quantitative
Quantitative
H N
2
NH
NH
2
H2N
2
9
17
18
26a
27a
––a
––a
NH
NH
2
2
a Compounds with no acyl side chain; R4CONH was thus replaced with NH2 or NO2.
R5
CO2H
26a R5 = NH2
27a R5 = NO2
NHSO2Ar
O
H2N

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
5383
Table 2. Activity of compounds against isolated integrins (IC50 values
are given in nM)a
itive test versus vitronectin, the natural ligand of inte-
grin aVb3. The assay was realized with CaCo2 cells (hu-
man epithelial cells from colon adenocarcinoma); they
express the target integrin and are routinely cultivated
in our laboratory.4,22 Cells were preincubated in the
presence of the synthetic integrin ligand 2 at different
concentrations, and then inoculated on culture plates
of polystyrene (PS) coated with vitronectin. After
15min at 37ꢁC, the nonadherent cells were removed
by suction and the adherent cells were assayed by a
colorimetric enzymatic test based on their N-acetyl-b-
D-glucosaminidase activity.23 Incubation with 4-nitro-
phenyl-2-acetamido-2-deoxy-b-D-glucopyranoside (en-
zyme substrate) produced p-nitrophenolate detected at
405nm. Results indicated that compound 2 is active at
the level of 10ꢀ7–10ꢀ6 M. At such concentration, the tar-
get integrin is filled up with the antagonist and, there-
fore, could not interact with the adhesive protein
coating the culture plate. In the same experiment,
RGDS peptide was active at the level of 10ꢀ6 M. At this
stage, we concluded that our designed molecule 2 was
well endowed with the required properties for substrate
biocompatibilization. Since the spacer-arm could possi-
bly disturb the activity, we similarly assayed com-
pound 19a (=2 + aminopropyl linker). Surprisingly, this
compound was more active than 2 (10ꢀ8–10ꢀ7 M,
Fig. 4). This result prompted us to examine more finely
the effect of the spacer-arm.
Entry
Compd
aVb3
aIIbb3
1
2
19b
1312
765
63
6913
5
2
3
19a
20a
11
16
4
55
5
21a
22a
154
114
64
3.8
27
6
7
23a
24a
7.8
28
8
73
9
25a
26a
127
41
16
20
10
11
12
13
27a
c[RGDf(N-Me)V]
BIBU-52
636
195
––
0.4
––
2.6
a Mean of at least two experiments with comparable results.
HN
CO2H
BIBU-52 =
O
H2N
N
H
O
active against the platelet integrin, but not 19b, which
offers a second basic residue (Table 2, entries 1 and 2).
All the other tested compounds 19a–26a are character-
ized by the presence of the aminopropyl chain fixed on
the phenol function. It appeared that this structural mo-
tive was in fact responsible for the activity, since all
compounds were active at the nanomolar level against
aVb3 and, to a slightly higher extent, against aIIbb3,
but with a poor selectivity (Table 2, entries 3–10).
Compounds listed in Table 2 were evaluated in a mole-
cular binding test with purified human integrin aVb3 in
the presence of vitronectin ligand. The selectivity was
controlled versus purified human integrin aIIbb3, in the
presence of fibrinogen ligand. Integrins were adsorbed
on PS test plates. The synthetic antagonists (in various
concentrations) were added in the presence of the natu-
ral ligands labeled with biotin. After incubation and
washings, the amount of bound ligands was assayed
with an antibody against biotin coupled to peroxidase
and colorimetric reaction. Values are given in equivalent
dose of compound producing 50% inhibition of natural
ligand (IC50). The reference compounds used in our tests
were the most active cyclopeptide (aVb3 antagonist)9d
and the anti-platelet BIBU-52 (aIIbb3 antagonist,
Fradafiban).24
We speculated that our molecules could offer two possi-
ble Arg mimics, namely the isonipecotic basic function
(A) and the aminopropyl residue (B, Fig. 5); in both
cases, the acid and basic pharmacophores are separated
with the same number of bonds. However, experimen-
tally, structures B were found to be the most active ones,
as well illustrated with the comparison between com-
pounds 2 and 26a (Table 2, entries 1 and 10), which
11
10
12
R'
N
9
O
8
7
1
3
5
HN
CO2H
2
4
6
Results showed that the lead compound 2 (no spacer-
arm) and compound 19b equipped with the longer
spacer-arm are active against aVb3 at the micromolar
level. As predicted from molecular modeling, 2 was less
NH
R-O
SO2Ar
A
R = Me, 2
R = Me, 19b
/
1.2
1
1
3
5
R-HN
CO2H
2
4
6
11
9
0.8
0.6
0.4
0.2
NH
7
H2N
O
SO2Ar
10
8
12
B
0
R = H, 26a
R = H, 19a - 25a
-Log [19a]
/
9
8
7
6
5
Figure 4. Inhibition of cellular adhesion by compound 19a in solution.
Figure 5. Two possible Arg mimics (Ar = mCF3–C6H4).

5384
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
H2N
HN
will be fully described and discussed in another
publication.25
N
O
HN
O
CO2H
SO2
2.4. Modeling
NH
OH
[spacer]
HN
PET
PET
The unexpected results collected in the binding assays
(see Table 2) with isolated integrins raised up question-
ing about our starting design. Not only the nature of the
Arg mimic has to be considered, but also its anchorage
position of the aromatic template. This has been exam-
ined again by molecular modeling taking into account
the spacer-arm.
i, ii
F3C
Scheme 3. Surface chemistry. Reagents and conditions: (i) TsCl,
Pyridine, acetone, 60ꢁC; (ii) H2N–[spacer]–ꢀRGDꢁ, PBS–CH3CN (1:1),
20ꢁC.
The geometry of compound 19a was fully optimized at
the approximate quantum chemistry level AM1 and also
at the ab initio level in MINI-10 basis. Remarkably,
three energy minima have been located named I
(folded), II (extended), and III (p–p stacked) (Fig. 6).
Their relative energies are summarized in Table 4.
As expected, the ab initio calculations gives a better
description of the intramolecular interactions as con-
formations II and III are slightly more stable than
I. The thermochemistry data are derived from the ana-
lytical frequency calculation at 1atm and 298.15K. In
the compact III structure, the entropy term is lower than
in the extended conformation thus inducing an inversion
in the relative variation of free energy between the two
conformers. In the folded conformer I, a hydrogen bond
was formed between one proton of the aminopropyl
belong unambiguously to series A and B, respectively.
The binding results are thus fully consistent with the
previous cellular tests showing that 19a was a better
adhesion inhibitor, in solution, than 2.
We next examined the adhesive effect induced by pepti-
domimetics 19a (short spacer) and 19b (long spacer)
when they are immobilized on PET membrane. The
compounds were covalently fixed on the material surface
via the hydroxyl chain ends of the polymer according to
known procedures.20 After surface activation by tosyla-
tion, the polymer membrane was incubated with solu-
tions of compounds 19a and 19b. Surface grafting
should normally occur via the most nucleophilic basic
function of the peptidomimetics, that is the primary
amine of the propyl- and [2-(2-ethoxy)ethoxy]ethyl side
chains (Scheme 3), thus displaying the structural fea-
tures of the designed compound 2 for cell interaction
(see Fig. 2). The amounts of grafted biologically active
molecules were determined by XPS analysis, from the
F/C atomic ratio, as previously described.4 CaCo2 cells
were cultivated on the native PET membrane and on the
surface-modified membranes during 2h at 37ꢁC in a
synthetic medium devoid of added proteins. As refer-
ence for the most adhesive surface, we considered the
support coated with a large amount of vitronectin
(3lg/cm2). The adherent cells were fixed and stained,
and the samples were examined with a phase contrast
microscope. The percentages of surface occupancy were
determined by image analysis (Table 3). Results clearly
showed that immobilized peptidomimetics behaved as
good to excellent promoters of cellular adhesion: 62%
of the vitronectin performance could be reached with
only 120pmol/cm2 of grafted 19b compared to 3lg/
mL/cm2 of coated protein. These results (and others)
CF3
H
O
S
N
O
HN
H
OH
O
O
R-HN
I (Folded)
CF3
O
O
S
HN
OH
O
O
R-HN
H2N
II (Extended)
F3C
Table 3. CaCo2 cell adhesion on surface modified PET membranes
O
Sample
Fixed ꢀRGDꢁ Surface
Relative
occupancy performance
(pmol/cm2)
S
O
(%)
(%)
6
NH
OH
1
2
3
4
Native PET
membrane
PET coated
with vitronectin
PET grafted
with 19a
2 ( 0.7)
O
O
33.3 ( 2.4) 100
R-HN
H2N
70
8.7 ( 1.2)
26
62
III (π−π stacked)
Figure 6. Optimized geometries of compound 19a.
PET grafted
with 19b
120
20.7 ( 2.7)

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
Table 4. Conformer relative energies of compound 19a (see Fig. 6)
5385
ety. The selectivity of RGD-like compounds depends on
the backbone conformations, the distances, and orienta-
tions of the charged side chains, and the presence of
hydrophobic moieties flanking the Asp mimic. Vitronec-
tin receptor (integrin aVb3) shares the same b subunit as
the fibrinogen receptor (integrin aIIbb3). Due to the close
relationship between these two receptors, they were of-
ten used as targets for selectivity studies. Recently, Fus-
ton et al.26 proposed a binding model for nonpeptide
antagonists of integrin aVb3, based on the crystal struc-
ture27 of the extracellular segment of this integrin and
docking experiments. They identified strong interactions
between the basic nitrogen of antagonists (such as com-
pound 27) and the D150 residue of aV subunit on the
one hand, and between the ligandsꢁ carboxylic acid
and the R214 residue of b3 subunit, on the other hand.
Moreover, a p–p stacking interaction was found be-
tween the aryl motive of sulfonamide group (if present)
and the Y178 residue of aV subunit. All the potent and
selective compounds tested in this model were character-
ized by a distance of 12 bonds between their charged
endings; when the molecules adopt an extended confor-
mation, this corresponds to the most favorable interac-
tions with aVb3 receptor. As negative control, the
authors showed that compound 28 (Fig. 7), displaying
a distance of 15 bonds between the charged endings,
cannot fit into their model. Experimentally, 28 is a po-
tent and selective antagonist of platelet integrin aIIbb3.
Conformer
AM1
DE
MINI-10
DG
DE
I (folded)
II (extended)
III (p–p stacked)
0.00
3.31
3.32
0.00
ꢀ1.98
ꢀ2.87
0.00
ꢀ3.96
ꢀ3.56
Values are given in kcalmolꢀ1
.
˚
Table 5. Distances (A) between the pharmacophoric groups, measured
from the MINI-10 conformers
Compd Conformer
a C(acid)–N(guanidine) a C(acid)–
N(spacer)
19a
2
I (folded)
II (extended)
10.03, 12.17, 12.28
9.45, 11.57, 11.70
6.54
8.63
8.68
III (p–p stacked) 9.81, 11.78, 12.18
II (extended) 10.00, 11.74, 12.37
III (p–p stacked) 10.27, 12.40, 12.53
––
––
6.68
26a
19b
I (folded)
I (folded)
II (extended)
c[RGDfV]
DMP728
––
10.17, 12.37, 12.38
10.09, 12.27, 12.29
9.52, 10.05, 11.51
10.50, 11.40, 11.60
11.04
16.32
NH2 and one oxygen of the sulfonamide SO2 group. The
distances between the carbon a to the acid and each of
the nitrogens of the basic residues were measured on
the MINI-10 optimized structures, showing that a large
˚
range of values, between 6.5 and 12.3A (Table 5), could
be covered depending on the basic residue and con-
former considered. Similar treatment of the cyclic pep-
tides c[RGDfV] and DMP728 gave distances of 9.5–
The compounds (19a–24a) we designed for materials
surface modification, in view to promote cell adhesion,
are characterized by one acid terminus and two basic
termini, separated, respectively, by 11 bonds whatever
the basic moiety being considered (see Fig. 5), except
in compounds 19b, 24a, and 25a showing distances of
11 bonds for one pair of endings, but of 16, 10, and
18 bonds, respectively, for the other one (see Table 1
for structures). The recorded activities against aVb3
(IC50 values between 50 and 150nM for 19a–25a, see
Table 2) could reasonably fit with the previous model,
considering 26a as the common pharmacophore.
However, the lack of selectivity and the systematically
higher activity against aIIbb3 was unexpected, and
˚
11.6A. This could explain the fact that 19a was almost
equally recognized by integrins aVb3 and aIIbb3 (Table
2, entry 3). For comparison, compounds 2 (the less ac-
tive against aVb3 and aIIbb3; entry 1), 26a (the most ac-
tive against aVb3, entry 10) and 19b (the most active and
selective against aIIbb3; entry 2) were similarly analyzed
(Table 5). In this last case, the long distance between the
acid and the amine of the spacer-arm was not surprising
since these functions are separated by 16 bonds com-
pared to 11 bonds in all the other situations. This amine
function most probably played the role of the basic
pharmacophore for integrin aIIbb3, but not for aVb3. Be-
side this value, the other distances for 2, 26a, and 19b
˚
were comprised between 8.2 and 12.5A, as for 19a.
NHSO2Ph
CO2H
N
H2N
H
N
From the molecular modeling study, we concluded that
activities and selectivities are hardly predictable in the
case of relatively flexible structures displaying different
conformers with almost equal probability, and that the
Arg mimic could be fixed on the tyrosine aromatic tem-
plate either in ortho of the hydroxyl group or on this
function itself. Accordingly, permutation of the relative
positions of the linker and the basic residue is now under
investigation in our group.
O
27 : IC50 = 0.08 nM (αVβ3)
IC50 = 1600 nM (αΙΙββ3)
O
NHSO2Ph
CO2H
HN
H
N
O
3. Conclusion
28 : IC50 = 10 nM (αΙΙbβ3)
IC50 > 1000 nM (αVβ3)
Usually, the design of RGD peptidomimetics as integrin
antagonists considers a rigid template, which links a
guanidine-type functionality and a carboxylic acid moi-
Figure 7. Molecules considered in the aVb3 binding model of Feuston
et al.26

5386
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
unpredictable on the basis of the actual knowledge on
integrin structures.6c Maybe, not only critical distances
between charged endings have to be taken in account,
but also the global charge of the potential ligands. The
aniline (or anilide) function of compounds 26a and
19a–25a should also play a role in both receptor binding
since the nitro derivative 27a appeared about 10-fold
less active (Table 2, entry 11). A nitro-aromatic template
was rarely considered in the previous literature.28 Our
results contradicted the generally accepted dogma of
ꢀ15-bond distanceꢁ between the basic and acidic residues
for optimum recognition by integrin aIIbb3: in our
hands, a ꢀ11-bond distanceꢁ gave excellent results too.
Amongst the novel potent aIIbb3 antagonists disclosed
in the present study (21a: IC50 = 3.8nM; 19b:
IC50 = 5nM), compound 19b could be of interest for
further development due to its selectivity versus aVb3
(Table 2, entry 2); this molecule, structurally related to
Tirofiban 29,29 displays the basic residue at the end of
a triethylene glycol chain; to our knowledge, such a
flexible, hydrophilic, structural motive has not been
exploited in the previous works about RGD mimics.
ther improved, application to materials used for bone
regeneration could now be envisaged.7f,31
4. Experimental
4.1. General
The reagents (analytical grade) were purchased from
Acros, Aldrich, or Fluka. The solvents were distilled,
after drying as follows: acetonitrile, dichloromethane,
triethylamine, and pyridine, over calcium hydride; tetra-
hydrofuran, over sodium.
The thin layer chromatographies were carried out on sil-
ica gel 60 plates F254 (Merck, 0.2mm thick); visualiza-
tion was effected with UV light, iodine vapor, a spray
of ninhydrin in ethanol or a spray of potassium perman-
ganate (3g), and potassium carbonate (20g) in aqueous
acetic acid (1%, 300mL). The column chromatographies
(under normal pressure) were carried out with Merck sil-
ica gel 60 of 70–230 mesh ASTM, and the flash chromat-
ographies, with Merck silica gel 60 of 230–400 mesh
ASTM.
Our compounds active against aVb3 (19a to 26a) are
structurally related to the tyrosine-based antagonists
discovered by the Merck company,9,16 such as 30 (Fig.
8). Thus possible improvement of our design should in-
volve (i) the one bond lengthening of the basic arm, (ii)
the use of aminoheterocycles as basic residues,11a,c,30
and (iii) the fixation of the polymer linker in ortho-posi-
tion considering the phenol ring. Nevertheless, the ac-
tual RGD peptidomimetics 19a and 19b, with the
polymer linker fixed on the phenol hydroxyl function,
were found to be good to excellent promoters of CaCo2
cells adhesion after their grafting on PET culture sub-
strates. Most probably, the surface displayed RGD
mimic was structure A, and not the more active pharma-
cophore B according to the molecular binding assays
(see Fig. 5). However, differences between molecular
and cellular tests could be observed, since cellular
adhesion is a highly complex phenomenon involving,
notably, lateral clustering of integrins for signal
transduction.6,7f
The melting points were determined with an Electro-
thermal microscope and are uncorrected. The IR spectra
were taken with a Bio-Rad FTS 135 instrument, and cal-
1
ibrated with polystyrene (1601cmꢀ1). The H and 13C
NMR spectra were recorded on Varian Gemini 200 (at
200MHz for proton and 50MHz for carbon), or Bruker
AM-500 spectrometers (at 500MHz for proton and
125MHz for carbon); the chemical shifts are recorded
in ppm (d) downfield from tetramethylsilane (internal
standard), or sodium 2,2-dimethyl-2-silapentane-5-sulfo-
nate (DSS) for the spectra recorded in D2O. The cou-
pling constant values are given in Hertz. The attribu-
tions were established by selective decoupling
experiments. The carbon of Aryl-CF3 was not visible
in the 13C NMR spectra. The mass spectra were ob-
tained on a Finnigan-MAT TSQ-70 instrument at
70eV or with a Xenon ION TECH 8KV apparatus.
The modes were EI (electronic impact), FAB (fast atom
bombardment), APCI (atmospheric pressure chemical
ionization), and ESI (electron-spray ionization). The
microanalyses were performed at the Christopher Ingold
Laboratories of the University College, London. The
HRMS were performed at the University of Mons-Hai-
naut (Belgium) on a VG-AutoSpec-Q equipment (Fisons
Instruments, Manchester).
We have thus achieved our goal of PET biocompatibili-
zation for in vitro cell culture by using small synthetic
RGD-like molecules instead of ECM proteins (like
vitronectin). Even if our system could be possibly fur-
CO2H
HN
4.2. Chemistry
NH
O
SO2 nBu
4.2.1. t-Butyl O-methyl-meta-amino-N-[3-(trifluoro-
methyl)phenylsulfonyl]-(L)-tyrosinate (4). This com-
pound, prepared according to Ref. 14, was obtained as
a colorless oil. Rf 0.3 (DCM/EA, 9:1); IR (film) m
3275, 2980, 1734, 1617, 1517, 1438, 1327, 1280, 1232,
29: Tirofiban
IC : 10 nM (α β )
50
IIb 3
CO2H
NH
N
HN
1
1161, 1132, 1105cmꢀ1; H NMR (CDCl3, 200MHz) d
O
SO2
NH
8.06 (s, 1H), 7.95 (d, J = 7.7, 1H), 7.80 (d, J = 8.1,
1H), 7.60 (dd, J = 8.1; 7.7, 1H), 6.65 (d, J = 8.1, 1H),
6.52 (d, J = 1.9, 1H), 6.47 (dd, J = 8.1; 1.9, 1H), 5.30
(d, J = 9.2, 1H), 4.13 (m, 1H), 3.85 (br s, 2H), 3.80 (s,
30: IC : ~1 nM (α β )
50
V 3
Figure 8. Integrin antagonists based on the tyrosine template.

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
5387
3H), 2.91 (m, 2H), 1.25 (s, 9H); 13C NMR (CDCl3,
50MHz) d 169.86, 146.91, 141.53, 135.28, 131.26,
130.59, 129.78, 129.21, 127.65, 124.31, 120.03, 116.64,
110.47, 82.87, 57.38, 55.56, 38.89, 27.79; MS (FAB)
m/e 475 (C21H25O5N2F3S+1), 474, 419, 401.
4.2.4. t-Butyl N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-
tyrosinate (8). To a solution of (L)-tyrosine t-butylester
(2g, 8.42mmol) in CH2Cl2 (100mL) were added, succes-
sively, pyridine (0.7mL, 8.42mmol, 1equiv), and meta-
trifluoromethyl-benzenesulfonyl chloride (1.35mL,
8.42mmol, 1equiv). The mixture was stirred at room
temperature for 5h, then washed with brine and water.
After drying (MgSO4), concentration, and chromatogra-
phy (silica gel), the sulfonamide 8 was recovered as a
pale yellow solid. Yield: 1.79g (48%); mp 95–95.2ꢁC;
Rf 0.6 (DCM/EA, 9:1); IR (KBr) 3477, 3266, 3083,
2980, 2938, 2857, 2792, 1732, 1614, 1517, 1437, 1327,
4.2.2. t-Butyl O-methyl-meta-[N-(N,N0-di-t-butoxycarbo-
nyl)guanidino-isonipecotyl]amino-N-[3-(trifluoromethyl)-
phenylsulfonyl]-(L)-tyrosinate (6). To a suspension of
acid 5 (430mg, 1.16mmol) and PYBOP (603mg,
1equiv) in DMF (18mL), stirred under argon atmos-
phere, was added diisopropylethylamine (0.396mL,
2equiv). After complete dissolution, aniline 4 (550mg,
1equiv), in solution in DMF (2mL), was added in one
portion. The mixture was stirred for 2days at room tem-
perature. After dilution with ethyl acetate, the solution
was washed with water and then with an aqueous solu-
tion of NH4Cl. The organic layer was dried over
MgSO4, concentrated, and purified by column chroma-
tography on silica gel to furnish 6 as a white solid. Yield:
528mg (55%); mp 104.0–104.2ꢁC; Rf 0.2 (DCM/EA,
9:1); IR (KBr) m 3260, 2978, 1748, 1683, 1635, 1539,
1
1159cmꢀ1; H NMR (CDCl3, 200MHz) d 8.07 (s, 1H),
7.96 (d, J = 8.0, 1H), 7.80 (d, J = 8.0, 1H), 7.61 (dd,
J = 8.0; 8.0, 1H), 7.00 (d, J = 8.5, 2H), 6.70 (d, J = 8.5,
2H), 5.28 (d, J = 9.4, 1H, NHSO2), 5.15 (br s, 1H,
OH), 4.12 (m, 1H), 2.97 (m, 2H), 1.25 (s, 9H); 13C
NMR (CDCl3, 50MHz) d 169.9, 155.02, 141.44,
131.35, 130.83, 130.55, 129.88, 129.35, 127.04, 124.34,
115.50, 83.19, 57.40, 38.77, 27.78; MS (APCI) m/e (%)
444 (100, Mꢀ1), 388 (36), 342 (6), 209 (59); Anal. Calcd
for C20H22O5NSF3: C, 53.93; H, 4.98; N, 3.14; S, 7.20.
Found: C, 53.26; H, 4.92; N, 3.12; S, 7.12.
1
1506, 1327, 1161, 1133, 954, 802cmꢀ1; H NMR (CD-
Cl3, 500MHz) d 10.17 (s, NH–C(O)guan), 8.14 (s,
1H), 7.99 (s, 1H), 7.95 (d, J = 8.0, 1H), 7.75 (s, NH–
C(O) amide), 7.74 (d, J = 8.0, 1H), 7.56 (dd, J = 8.0;
8.0, 1H), 6.84 (d, J = 8.4, 1H), 6.73 (d, J = 8.4, 1H),
5.25 (d, J = 9.5, NHSO2), 4.21 (m, 2H), 4.05 (ddd,
J = 9.5; 7.0; 5.2, 1H), 3.85 (s, 3H), 3.10 (m, 2H), 3.0
(dd, J = 5.2; 13.6, 1H), 2.86 (dd, J = 7.0; 13.6, 1H),
2.52 (m, 1H), 1.97–1.91 (m, 4H), 1.49 (s, 18H), 1.25 (s,
9H); 13C NMR (CDCl3, 125MHz) d 171.58, 169.63,
154.91, 146.75, 141.11, 131.35, 130.51, 129.55, 128.96,
127.59, 127.17, 124.74, 124.10, 120.55, 109.6, 83.01,
57.11, 55.56, 46.40, 43.93, 38.67, 28.02, 27.57; MS
(FAB) m/e (%) 828 (24, M+1), 628 (24), 572 (80), 318
(28), 149 (40), 136 (100), 77 (80), 57 (56); Anal. Calcd
for C38H52O10N5F3S: C, 55.13; H, 6.33; N, 8.46. Found:
C, 54.92; H, 6.72; N, 8.59.
4.2.5. t-Butyl meta-nitro-N-[3-(trifluoromethyl)phenyl-
sulfonyl]-(L)-tyrosinate (9). To a solution of 8 (1.5g,
3.37mmol) in acetic acid (100mL), nitric acid 90%
(200lL, 3.40mmol) diluted in acetic acid (20mL) was
added dropwise. The mixture was maintained at 16–
20ꢁC during the addition. After reactive consumption
(TLC control), the crude mixture was poured onto ice.
The aqueous phase was extracted with CH2Cl2. The or-
ganic phase was dried (MgSO4), concentrated under
vacuum, and purified by column chromatography on sil-
ica gel to give 9 as a yellow solid. Yield: 1.57g (95%); Rf
0.8 (DCM/EA, 95:5); mp 80.4–80.6ꢁC; IR (KBr) m 3274,
2980, 2926, 2875, 1733, 1632, 1540, 1431, 1327, 1162,
1071cmꢀ1 1H NMR (CDCl3, 200MHz) d 10.43 (s,
;
1H, OH), 8.03 (s, 1H), 7.97 (d, J = 7.7, 1H), 7.83 (d,
J = 2.2, 1H), 7.79 (d, J = 7.8, 1H), 7.60 (dd, J = 7.7;
7.7, 1H), 7.43 (dd, J = 8.5; 2.2, 1H), 7.03 (d, J = 8.5,
1H), 5.43 (d, J = 9.2, 1H, NHSO2), 4.09 (m, 1H), 3.09
(dd, J = 5.5; 14.1, 1H), 2.95 (dd, J = 6.8; 14.1, 1H),
1,26 (s, 1H); 13C NMR (CDCl3, 50MHz) d 169.44,
154.18, 141.32, 139.11, 133.27, 131.35, 130.54, 129.99,
129.35, 128.01, 125.54, 124.11, 120.17, 83.74, 57.18,
38.12, 27.69; MS (FAB) m/e (%) 489 (100, Mꢀ1), 306
(88), 199 (32), 153 (60). Anal. Calcd for
C20H21O7N2SF3: C, 48.98; H, 4.32; N, 5.71; S, 6.54.
Found: C, 49.09; H, 4.17; N, 5.49; S, 6.36.
4.2.3. O-Methyl-meta-(N-guanidino-isonipecotyl)amino-
N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine
(2).
Compound 6 (50g, 0.06mmol) was dissolved in an ice-
cold 1:1 mixture (1mL) of dichloromethane (DCM)
and trifluoroacetic acid (TFA). The mixture was stirred
for 2h at 20ꢁC, then concentrated under vacuum. The
residue was triturated in dry ether to furnish 2 (bis-tri-
fluoroacetic salt) as a yellow gum. Yield: 48mg
(100%); 1H NMR (DMSO, 500MHz) d 8.98 (s, 1H,
NHCO), 8.58 (d, J = 9.1, 1H, NHSO2), 7.89 (s, 1H),
7.85 (d, J = 8.2, 1H), 7.78 (d, J = 8.2, 1H), 7.75 (d,
J = 2.1, 1H), 7.62 (dd, J = 8.2; 8.2, 1H), 7.43 (m, 3H,
NH2, NH), 6.79 (dd, J = 8.5; 2.1, 1H), 6.76 (d, J = 8.5,
1H), 3.88 (m, 2H), 3.87 (ddd, J = 10.0; 4.7; 9.1, 1H),
3.77 (s, 3H), 3.04 (m, 2H), 2.87 (m, 1H), 2.87 (dd,
J = 14.0; 4.7, 1H), 2.60 (dd, J = 14.0; 10.0, 1H),
1.84 (m, 2H), 1.57 (m, 2H); 13C NMR (DMSO,
125MHz) d 172.41, 172.09, 155.76, 148.10, 142.24,
130.25, 130.12, 129.31, 128.64, 128.25, 126.87, 124.90,
122.25, 121.73, 110.50, 57.81, 55.55, 44.80, 41.12,
37.31, 27.84; MS (ESI) m/e (%) 589 (44, M+ H2O),
572 (100, M+1); HRMS: calcd for C24H29N5O6F3S:
572.1791. Found: 572.1792.
4.2.6. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-nitro-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyro-
sinate (10a). To a solution of 9 (2.32g, 4.8mmol) in CH3
CN (180mL), under argon atmosphere, were added N-
Boc-3-bromopropylamine (1.13g, 1equiv), potassium
carbonate (0.66g), and [18-c-6] crown-ether (1.25g).
The mixture was refluxed, under stirring, for 22h.
CH3CN was removed under vacuum; the residue was
dissolved in CH2Cl2, and washed with 0.1N HCl and
water. Drying over MgSO4, concentration, and chroma-
tography on silica gel gave 10 as a yellow oil. Yield:
1.74g (56%); Rf 0.55 (DCM/EA, 8:1); IR (film) m 3422,

5388
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
3275, 2981, 1714, 1625, 1535, 1458, 1438, 1369, 1353,
1H), 7.75 (d, J = 8.0, 1H), 7.55 (dd, J = 8.0; 8.0, 1H),
6.58 (d, J = 7.9, 1H), 6.44 (s, 1H), 6.39 (d, J = 7.9,
1H), 5.75 (br d, 1H, NHSO2), 4.91 (br s, NHCO), 4.05
(m, 1H), 3.96 (t, J = 6.2, 2H), 3.60 (br s, 2H, NH2),
3.31 (m, 2H), 2.88 (dd, J = 6.0; 14.0, 1H), 2.80 (dd,
J = 6.0; 14.0, 1H), 1.96 (t, J = 6.2, 2H), 1.42 (s, 9H),
1.22 (s, 9H), 13C NMR (CDCl3, 50MHz) d 170.97,
157.23, 146.82, 142.68, 137.59, 131.69, 130.89, 130.34,
129.05, 125.44, 120.48, 117.33, 112.74, 83.96,
80.20, 67.05, 58.46, 40.07, 38.10, 31.01, 29.62, 28.92;
MS (APCI) m/e (%) 619 (25), 618 (40
M+1, C28H38F3N3O7S), 519 (25), 518 (100), 462 (45),
338(18).
1
1328, 1263, 1165cmꢀ1; H NMR (CDCl3, 200MHz) d
8.04 (s, 1H), 7.98 (d, J = 8.0, 1H), 7.81 (d, J = 8.0,
1H), 7.63 (dd, J = 8.0, 1H), 7.62 (d, J = 2.1, 1H), 7.40
(dd, J = 2.1; 8.6, 1H), 6.99 (d, J = 8.6, 1H), 5.30 (d,
J = 9.2, 1H, NHSO2), 5.07 (s, 1H, NHCO), 4.14 (t,
J = 7.3, 2H), 4.07 (m, 1H), 3.37 (m, 2H), 3.09 (dd,
J = 5.8; 14.0, 1H), 2.99 (dd, J = 6.1; 14.0, 1H), 2.04
(m, 2H), 1.44 (s, 9H), 1.21 (s, 9H); 13C NMR (CDCl3,
50MHz) d 169.01, 156.08, 151.45, 140.79, 139.09,
135.61, 131.68, 130.37, 129.85, 129.37, 127.54, 126.58,
124.09, 114.39, 83.82, 79.06, 67.85, 56.73, 38.03, 38.01,
29.03, 28.29, 27.59; MS (FAB) m/e (%) 646 (20, Mꢀ1),
546 (24), 490 (12), 386 (8), 209 (10); Anal. Calcd for
C28H36F3N3O9S: C, 51.93; H, 5.60; N, 6.49; S, 4.95.
Found: C, 51.80; H, 5.80; N, 6.25; S, 4.78.
4.3.2. t-Butyl O-[2-(2-(2-(N-t-butoxycarbonyl)-2-amino-
ethoxy)ethoxy)ethyl]-meta-amino-N-[3-(trifluoromethyl)-
phenylsulfonyl]-(L)-tyrosinate (11b). White gum; Rf 0.4
1
4.2.7. t-Butyl O-[2-(2-(2-(N-t-butoxycarbonyl)-2-amino-
ethoxy)ethoxy)ethyl]-meta-nitro-N-[3-(trifluoromethyl)-
phenylsulfonyl]-(L)-tyrosinate (10b). To a solution of 9
(1g, 2.4mmol) in THF (6mL), under argon atmosphere,
were added 2-(2-(2-t-butoxycarbonyl)aminoethoxy) eth-
oxyethanol20 (536mg, 1.1equiv) and bis(diphen-
ylphosphino) ethane (1.22g, 1.5equiv). At 0ꢁC,
diisopropyl azodicarboxylate (600lL, 618mg, 1.3equiv)
was added dropwise. The mixture was stirred for 20h at
room temperature. THF was removed under vacuum
and the crude mixture was suspended in diethylether
and left at ꢀ20ꢁC overnight. After filtration, the organic
phase was concentrated, and purified by column chro-
matography on silica gel to give 10b as a yellow oil.
Yield: 1.04g (60%); Rf 0.5 (DCM/EA, 4:1); IR (film) m
3299, 2980, 2933, 2884, 1711, 1620, 1533, 1327, 1164,
(DCM/EA, 7:3); H NMR (CDCl3, 500MHz) d 8.02
(s, 1H), 7.89 (d, J = 8.1, 1H), 7.74 (d, J = 8.1, 1H),
7.54 (dd, J = 8.1; 8.1, 1H), 6.60 (d, J = 7.8, 1H), 6.44
(d, J = 2.2, 1H), 6.38 (dd, J = 7.8; 2.2, 1H), 5.57 (d,
J = 7.9, 1H, NHSO2), 5.10 (br s, NHCO), 4.07 (m,
2H), 4.04 (ddd, J = 8.1; 6.2; 5.8, 1H), 3.80 (m, 2H),
3.66 (m, 2H), 3.62 (m, 2H), 3.58 (br s, 2H, NH2), 3.51
(m, 2H), 3.28 (m, 2H), 2.86 (dd, J = 6.2; 14.1, 1H),
2.82 (dd, J = 5.9; 14.1, 1H), 1.41 (s, 9H), 1.21 (s, 9H);
13C NMR (CDCl3, 125MHz) d 169.68, 155.90, 145.34,
141.30, 136.81, 131.26, 130.34, 129.61, 128.98, 128.22,
124.00, 119.03, 116.14, 112.56, 82.54, 79.04, 70.49,
70.12, 69.60, 68.18, 57.15, 40.25, 38.71, 28.26, 27.53;
MS (APCI) m/e (%) 693 (32), 692 (100, M+1,
C31H44F3N3O9S), 636 (30), 592 (85), 536 (16).
1
1103cmꢀ1; H NMR (CDCl3, 200MHz) d 8.03 (s, 1H),
4.4. General procedure for 11 coupling with acids
7.96 (d, J = 8.1, 1H), 7.79 (d, J = 8.1, 1H), 7.50 (dd,
J = 8.1; 8.1, 1H), 7.59 (d, J = 2.1, 1H), 7.37 (dd,
J = 8.8; 2.1, 1H), 6.99 (d, J = 8.8, 1H), 5.75 (d, J = 8,
1H, NHSO2), 5.07 (br s, NHCO), 4.23 (m, 2H), 4.06
(m, 1H), 3.89 (m, 2H), 3.73 (m, 2H), 3.63 (m, 2H),
3.53 (m, 2H), 3.28 (m, 2H), 3.06 (dd, J = 5.9; 13.8,
1H), 2.95 (dd, J = 5.9; 13.8, 1H), 1.42 (s, 9H), 1.22 (s,
9H); 13C NMR (CDCl3, 50MHz) d 169.28, 156.12,
151.62, 141.13, 135.57, 132.11, 131.45, 130.54, 130.03,
129.50, 128.07, 126.54, 124.23, 115.18, 83.82, 77.42,
71.13, 70.40, 70.29, 69.79, 69.36, 56.97, 40.43, 38.10,
28.45, 27.71; MS (FAB) m/e (%) 720 (28, Mꢀ1), 620
(48), 564 (20), 224 (20), 209 (100); Anal. Calcd for
C31H42F3N3O11SÆH2O: C, 50.32; H, 5.95; N, 5.68; S,
4.33. Found: C, 50.10; H, 6.01; N, 5.95; S, 4.17.
Acid of interest (1equiv) and PYBOP (1equiv) were sus-
pended in DMF (10mL/0.5mmol) under argon atmos-
phere. Diisopropylethylamine (2equiv) was added and
the mixture was stirred until dissolution. Then aniline
11 (1equiv) in DMF (5mL/0.5mmol) was added in
one portion. The mixture was stirred for 2days at
20ꢁC. After addition of ethyl acetate and washing with
aqueous NH4Cl and brine, the organic layer was dried
over MgSO4, and concentrated under vacuum. The
crude anilide was purified by column chromatography
on silica gel.
4.4.1. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-[N-(N,N0-di-t-butoxycarbonyl-guanidino)isonipeco-
tyl amino]-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyro-
sinate (12a). Yield from 300mg (0.486mmol) of 11a:
226mg (48%); hygroscopic beige solid; mp 74.2–
74.4ꢁC; Rf 0.3 (DCM/MeOH, 30:1); IR (KBr) m 3317,
2978, 1748, 1680, 1599, 1536, 1485, 1393, 1326, 1164,
4.3. General procedure for hydrogenation
In a Parr vessel, the nitro precursor dissolved in ethyl
acetate (1mmol/25mL) was shaked under hydrogen
atmosphere (p = 40psi) for 18h at room temperature in
the presence of PtO2 (20mg/mmol nitro compd). After
filtration and concentration, the residue (100% crude
yield) was used in the next step without purification.
1105, 954cmꢀ1 1H NMR (CDCl3, 500MHz) d 10.3
,
(NHguaBoc), 8.15 (ArNHCO), 8.11 (d, J = 1.8, 1H),
8.02 (s, 1H), 7.97 (d, J = 8.4, 1H), 7.74 (d, J = 8.4),
7.57 (dd, J = 8.4; 8.4, 1H), 6.82 (d, J = 8.4, 1H), 6.74
(d, J = 8.4, 1H), 5.18 (d, J = 9.6; 1H, NHSO2), 4.72
(NHCO2tBu), 4.17 (m, 2H), 4.08 (m, 2H), 4.07
(ddd, J = 5.1; 5.7; 9.5, 1H), 3.35 (m, 2H), 3.26 (m,
2H), 3.00 (dd, J = 5.1; 13.6, 1H), 2.9 (dd, J = 7.0; 13.6,
1H), 2.72 (m, 1H), 2.05 (m, 4H), 1.98 (m, 2H), 1.51 (s,
4.3.1. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyro-
sinate (11a). White solid; mp 61–61.2ꢁC; 1H NMR
(CDCl3, 200MHz) d 8.02 (s, 1H), 7.89 (d, J = 8.0,

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
5389
18H), 1.43 (s, 9H), 1.29 (s, 9H); 13C NMR (CDCl3,
125MHz) d 171.96, 169.61, 156.05, 146.1, 141.1,
131.35, 130.52, 129.58, 129.01, 127.65, 127.60, 124.73,
121.36, 111.00, 82.98, 79.34, 64.65, 57.08, 38.68, 36.70,
28.29, 27.97, 27.35; MS (FAB) m/e (%) 971 (88, M+1),
771 (88), 715 (44), 615 (20), 462 (32), 275 (96); Anal.
Calcd for C45H65F3N6O12SÆ0.7H2O: C, 54.90; H, 6.75;
N, 8.54; S, 3.25. Found: C, 54.41; H, 6.71; N, 8.02; S,
3.43.
4.4.4. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-[N-(methyl)isonipecotyl]amino-N-[3-(trifluorometh-
yl)phenylsulfonyl]-(L)-tyrosinate (14a). Yield from
125mg (0.202mmol) of 11a: 84mg (56%); pale yellow
1
solid; mp 84.2–84.6ꢁC; Rf 0.2 (DCM/MeOH, 95:5); H
NMR (CDCl3, 500MHz) d 8.22 (br s, ArNHCO), 7.99
(s, 1H), 7.96 (s, 1H), 7.93 (d, J = 8.0, 1H), 7.74 (d,
J = 8.0, 1H), 7.56 (dd, J = 8.0; 8.0, 1H), 6.80 (d,
J = 8.4, 1H), 6.72 (d, J = 8.4, 1H), 4.88 (br s, 1H,
NHSO2), 4.5 (br s, 1H, NHBoc), 4.05 (m, 1H), 4.03
(m, 1H), 3.44 (m, 2H), 3.31 (m, 2H), 2.98 (m, 2H),
2.97 (dd, J = 5.6; 13.6, 1H), 2.84 (dd, J = 7.0; 13.6,
1H), 2.78 (m, 1H), 2.77 (s, 3H), 2.10 (m, 4H), 1.98 (m,
2H), 1.4 (s, 9H), 1.25 (s, 9H); 13C NMR (CDCl3,
125MHz) d 172.24, 169.74, 156.39, 147.0, 141.02,
131.23, 130.43, 129.76, 129.06, 127.59, 126.90, 125.28,
123.84, 122.21, 111.26, 82.92, 79.23, 65.33, 57.41,
54.02, 44.43, 39.76, 38.43, 36.97, 29.26, 28.23, 27.49,
26.62; MS (FAB) m/e (%) 741 (70, Mꢀ1), 667 (95),
641 (25), 584 (20), 481 (18), 209 (100); Anal. Calcd for
C35H49O8N4SF3: C, 56.59; H, 6.65; N, 7.54. Found: C,
56.37; H, 6.96; N, 7.18.
4.4.2. t-Butyl O-[2-(2-(2-N-t-butoxycarbonyl)-2-amino-
ethoxy)ethoxyethyl]-meta-[N-(N,N0-di-t-butoxycarbonyl-
guanidino)isonipecotyl]amino-N-[3-(trifluoromethyl)phen-
ylsulfonyl]-(L)-tyrosinate (12b). Yield from 384mg
(0.555mmol) of 11b: 243mg (42%); hygroscopic white
solid; mp 65.2–65.4ꢁC; Rf 0.3 (DCM/EA, 1:1); IR
(KBr) m 2987, 2927, 2851, 2373, 2325, 1651, 1317,
1260, 1227, 1146, 1118, 959cmꢀ1 1H NMR (CDCl3,
;
500MHz) d 8.11 (d, J = 1.8, 1H), 8.00 (s, 1H), 7.94 (d,
1H), 7.74 (d, 1H), 7.56 (dd, 1H), 6.82 (dd, J = 8.4; 1.8,
1H), 6.76 (d, J = 8.4, 1H), 5.02 (br s, 1H, NHBoc),
4.24 (m, 2H), 4.15 (m, 2H), 4.04 (dd, J = 5.2; 7.1, 1H),
3.83 (m, 2H), 3.68 (m, 2H), 3.63 (m, 2H), 3.53 (m,
2H), 3.29 (m, 2H), 3.07 (m, 2H), 3.00 (dd, J = 14.0;
5.2, 1H), 2.86 (dd, J = 14.0; 7.1, 1H), 2.54 (m, 1H),
1.96 (m, 2H), 1.89 (m, 2H), 1.48 (s, 18H), 1.40 (s, 9H),
1.24 (s, 9H); 13C NMR (CDCl3, 125MHz) d 171.86,
169.62, 155.85, 154.90, 146.12, 141.16, 131.29, 130.44,
129.52, 128.91, 128.46, 128.00, 124.76, 124.04, 121.10,
112.23, 82.90, 81.84, 79.40, 70.42, 70.14, 70.02, 69.35,
68.49, 57.06, 46.25, 43.13, 40.14, 38.70, 28.28, 28.25,
28.08, 27.95, 27.50; MS (APCI) m/e (%) 1045 (12,
M+1), 945 (4), 871 (100), 845 (6), 728 (50); Anal. Calcd
for C48H71F3N6O14SÆ3H2O: C, 52.41; H, 7.01; N, 7.64.
Found: C, 52.22; H, 7.03; N, 7.53.
4.4.5. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-(4-pyridylcarbonyl)amino-N-[3-(trifluoromethyl)phen-
ylsulfonyl]-(L)-tyrosinate (15a). Yield from 126mg
(0.202mmol) of 11a: 76mg (52%); orange solid; mp
51.4–51.6ꢁC; Rf 0.3 (DCM/MeOH, 30:1); IR (KBr) m
3419, 3068, 2985, 2938, 2875, 1734, 1704, 1677, 1594,
1530, 1472, 1432, 1310, 1252, 1160cmꢀ1 1H NMR
,
(CDCl3, 500MHz) d 8.90 (s, 1H, ArNHCO), 8.83 (br
s, 2H), 8.22 (d, J = 1.8, 1H), 8.02 (s, 1H), 7.98 (d,
J = 7.9, 1H), 7.91 (br s, 2H), 7.74 (d, J = 7.9, 1H), 7.57
(dd, J = 7.9; 7.9, 1H), 6.92 (dd, J = 8.4; 1.8, 1H), 6.79
(d, J = 8.4, 1H), 5.43 (br d, 1H, NHSO2), 4.70 (br s,
1H, NHCO2), 4.11 (m, 1H), 4.10 (m, 2H), 3.39 (m,
2H), 3.05 (dd, J = 5.2; 14.1, 1H), 2.92 (dd, J = 7.0;
14.1; 1H), 2.01 (m, 2H), 1.39 (s, 9H), 1.28 (s, 9H); 13C
NMR (CDCl3, 125MHz) d 169.57, 162.59, 155.98,
149.84, 146.72, 142.53, 141.16, 131.39, 130.49, 129.59,
128.97, 127.84, 126.93, 125.78, 124.1, 121.61, 121.28,
110.84, 83.02, 79.47, 65.18, 57.09, 38.67, 37.00, 29.68,
28.22, 27.58; MS (APCI) m/e (%) 724 (55, M+1), 723
(100), 667 (70), 623 (90), 567 (65); Anal. Calcd for
C34H41F3N4O8S: C, 56.50; H, 5.72; N, 7.75. Found: C,
56.27; H, 5.83; N, 8.01.
4.4.3. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-[N-(t-butoxycarbonyl)-isonipecotyl]amino-N-[3-(tri-
fluoromethyl)phenylsulfonyl]-(L)-tyrosinate (13a). Yield
from 157mg (0.25mmol) of 11a: 110mg (53%); white so-
lid; mp 60.4–60.6ꢁC; Rf 0.4 (DCM/MeOH, 30:1); IR
(KBr) m 3357, 2979, 2924, 2868, 1742, 1682, 1591,
1537, 1420, 1362, 1325, 1248, 1154, 1127cmꢀ1 1H
;
NMR (CDCl3, 500MHz) d 8.17 (br s, 1H, NarHCO),
8.13 (d, J = 1.8, 1H), 8.01 (s, 1H), 7.96 (d, J = 7.9,
1H), 7.74 (d, J = 7.9, 1H), 7.57 (dd, J = 7.9; 7.9, 1H),
6.82 (d, J = 8.4, 1H), 6.74 (d, J = 8.4, 1H), 5.24 (d,
J = 9.4, 1H, NHSO2), 4.66 (br s, 1H, NHCO2), 4.15
(m, 2H), 4.06 (ddd, J = 7.0; 5.2; 9.4, 1H), 4.06 (m,
2H), 3.34 (m, 2H), 3.00 (dd, J = 5.2; 14.1, 1H), 2.88
(dd, J = 7.0; 14.1, 1H), 2.82 (m, 2H), 2.57 (m, 1H),
1.96 (m, 2H), 1.90 (m, 2H), 1.72 (m, 2H), 1.47 (s, 9H),
1.43 (s, 9H), 1.26 (s, 9H); 13C NMR (CDCl3,
125MHz) d 172.72, 169.61, 156.00, 154.64, 146.04,
141.14, 131.39, 130.50, 129.56, 128.95, 127.7, 124.6,
124.11, 121.31, 110.94, 82.98, 79.48, 79.41, 64.88,
57.10, 43.85, 43.12, 38.71, 36.79, 29.57, 28.57, 28.44,
28.35, 28.28, 27.56; MS (APCI) m/e (%) 830 (41,
M+1), 730 (50), 729 (100), 673 (95), 658 (20), 629 (14),
518 (20); Anal. Calcd for C39H55F3N4O10S: C, 56.51;
H, 6.69; N, 6.75; S, 3.87. Found: C, 56.00; H, 6.55; N,
6.33; S, 3.82.
4.4.6. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-(4-pyridylacetyl)amino-N-[3-(trifluoromethyl)phenyl-
sulfonyl]-(L)-tyrosinate (16a). Yield from 200mg
(0.324mmol) of 11a: 137mg (56%); hygroscopic yellow
solid; mp 71.8–72.0ꢁC; Rf 0.25 (DCM/MeOH, 30:1);
IR (KBr) m 3376, 3315, 3052, 2979, 2927, 2875, 1734,
1686, 1588, 1536, 1432, 1362, 1322, 1252, 1150cmꢀ1
;
1H NMR (CDCl3, 500MHz) d 8.59 (br s, 2H), 8.34
(br s, 1H, ArNHCO), 8.07 (d, J = 1.8, 1H), 7.99 (s,
1H), 7.94 (d, J = 7.9, 1H), 7.72 (d, J = 7.9, 1H), 7.54
(dd, J = 7.9; 7.9, 1H), 7.39 (br s, 2H), 6.82 (dd,
J = 1.8; 8.4, 1H), 6.70 (d, J = 8.4, 1H), 5.34 (d, J = 9.4,
1H, NHSO2), 4.80 (br s, 1H, NHCO2), 4.04 (ddd,
J = 7.0; 5.2; 9.4, 1H), 3.97 (m, 2H), 3.83 (s, 2H), 3.23
(m, 2H), 2.99 (dd, J = 5.2; 14.0, 1H), 2.85 (dd, J = 7.0;
14.0, 1H), 1.84 (m, 2H), 1.43 (s, 9H), 1.23 (s, 9H); 13C

5390
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
NMR (CDCl3, 125MHz) d 169.57, 167.13, 156.18,
149.46, 146.16, 144.71, 141.11, 131.31, 130.47, 129.58,
128.97, 127.67, 127.44, 125.08, 124.89, 124.08, 121.19,
111.02, 82.96, 79.41, 64.84, 57.08, 43.74, 38.67, 36.74,
29.52, 28.26, 27.53; MS (APCI) m/e (%) 738 (80,
M+1), 737 (100), 681 (95), 625 (15), 518 (10), 258 (50);
Anal. Calcd for C35H43F3N4O8SÆH2O: C, 55.64; H,
5.96; N, 7.42; S, 4.23. Found: C, 55.22; H, 5.99; N,
7.40; S, 3.96.
under vacuum. The deprotected product was quantita-
tively recovered as nTFA salt (gum or foam).
4.5.1. O-(3-Aminopropyl)-meta-[N-(guanidino)isonipecot-
yl]amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyro-
1
sine (19a). H NMR (D2O, 500 MHz) d 7.87 (s, 1H),
7.85 (d, J = 8.2, 1H), 7.76 (d, J = 8.2, 1H), 7.55 (dd,
J = 8.2; 8.2, 1H), 7.26 (d, J = 2.1, 1H), 6.90 (dd, J =
8.5; 2.1, 1H), 6.76 (d, J = 8.5, 1H), 4.12 (dd, J = 4.1;
11.1, 1H), 4.09 (t, J = 7.5, 2H), 3.95 (m, 2H), 3.21
(m, 2H), 3.19 (m, 2H), 3.12 (dd, J = 4.1; 13.7, 1H),
2.83 (m, 1H), 2.69 (dd, J = 11.1; 13.7, 1H), 2.17 (m,
2H), 2.02 (m, 2H), 1.77 (m, 2H); 13C NMR (D2O,
125MHz) d 178.26, 177.67, 158.64, 151.77, 142.62,
133.16, 132.75, 132.65, 132.06, 131.36, 130.17, 127.59,
127.43, 125.90, 126.02, 115.15, 68.13, 60.40, 47.73,
44.55, 39.70, 39.29, 30.11, 29.17; MS (APCI) m/e (%)
616 (8, M+1), 615 (30), 283 (16), 282 (56); HRMS
(ESI): calcd for C26H34N6O6F3S: 615.2213. Found:
615.2238.
4.4.7. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-[3-(N-t-butoxycarbonyl)aminopropanoyl]amino-N-
[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosinate (17a).
Yield from 218mg (0.353mmol) of 11a: 125mg (45%);
1
white foam; Rf 0.4 (DCM/EA, 5:1); H NMR (CDCl3,
500MHz) d 8.22 (br s, 1H, ArNHCO), 8.09 (d,
J = 1.8, 1H), 8.02 (s, 1H), 7.97 (d, J = 7.9, 1H), 7.75
(d, J = 7.9, 1H), 7.58 (dd, J = 7.9; 7.9, 1H), 6.82 (dd,
J = 8.3; 1.8, 1H), 6.74 (d, J = 8.3, 1H), 5.33 (br s, 1H,
NHBoc), 5.22 (d, J = 9.3, 1H, NHSO2), 4.74 (br s,
NHBoc), 4.07 (m, 1H), 4.07 (t, J = 7.3, 2H), 3.47 (m,
J = 6.3; 6.0, 2H), 3.34 (m, J = 7.3; 6.0, 2H), 3.00 (dd,
J = 5.2; 14.0, 1H), 2.88 (dd, J = 6.8; 14.0, 1H), 2.68 (t,
J = 6.8, 2H), 1.97 (m, J = 7.3, 2H), 1.43 (s, 9H), 1.41
(s, 9H), 1.27 (s, 9H); 13C NMR (CDCl3, 125MHz) d
169.91, 169.55, 156.07, 155.89, 146.01, 141.13, 131.39,
130.47, 129.58, 128.95, 127.71, 127.62, 124.70, 124.10,
121.19, 111.10, 82.96, 79.40, 79.06, 65.13, 57.04, 38.71,
37.07, 36.86, 36.61, 29.55, 28.29, 28.27, 27.56; MS
(APCI) m/e (%) 789 (14, M+1, C36H51F3N4O10S), 689
(94), 633 (66), 589 (30), 533 (18).
4.5.2.
O-[2-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)]-meta-
[N-(guanidino)isonipecotyl]amino-N-[3-(trifluoromethyl)-
phenylsulfonyl]-(L)-tyrosine (19b). 1H NMR (D2O,
500MHz) d 7.86 (s, 1H), 7.83 (d, J = 8.4, 1H), 7.76 (d,
J = 8.4, 1H), 7.54 (d, J = 1.8, 1H), 7.54 (dd, J = 8.4;
8.4, 1H), 6.85 (dd, J = 8.5; 1.8, 1H), 6.73 (d, J = 8.5,
1H), 4.17 (dd, J = 10.8; 4.2, 1H), 3.98 (m, 2H), 3.96
(m, 2H), 3.85 (m, 4H), 3.83 (m, 2H), 3.28 (m, 2H),
3.26 (m, 2H), 3.14 (dd, J = 4.2; 14.0, 1H), 2.92 (m,
1H), 2.72 (dd, J = 10.8; 14.0, 1H), 2.07 (m, 2H), 1.83
(m, 2H); 13C NMR (D2O, 125MHz) d: 175.26, 175.00,
156.08, 148.09, 140.30, 130.53, 130.18, 130.01, 129.36,
128.62, 126.54, 125.63, 123.40, 123.31, 112.20, 69.88,
69.67, 69.24, 67.38, 66.56, 57.92, 45.19, 42.12, 39.25,
36.85, 27.60; MS (ESI) m/e (%) 690 (80, M+1), 689
(100); HRMS (ESI): calcd for C29H40F3N6O8S:
689.2593. Found: 689.2580.
4.4.8. t-Butyl O-[N-(t-butoxycarbonyl)-3-aminopropyl]-
meta-[12-(N-t-butoxycarbonyl)aminododecanoyl amino]-
N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosinate (18a).
Yield from 131mg (0.212mmol) of 11a: 134mg (69%);
white foam; Rf 0.4 (DCM/MeOH, 20:1); 1H NMR
(CDCl3, 500MHz) d 8.14 (d, J = 1.8, 1H), 8.04 (br s,
1H, ArNHCO), 8.00 (s, 1H), 7.94 (d, J = 7.9, 1H),
7.74 (d, J = 7.9, 1H), 7.58 (dd, J = 7.9; 7.9, 1H), 6.80
(dd, J = 8.5; 1.8, 1H), 6.72 (d, J = 8.5, 1H), 5.26 (d,
J = 9.1, NHSO2), 4.70 (br s, 1H, NHBoc), 4.50 (br s,
1H, NHBoc), 4.07 (t, J = 7.3, 2H), 4.07 (m, 1H), 3.34
(m, 2H), 3.09 (m, 2H), 2.99 (dd, J = 14.1; 5.3, 1H),
2.87 (dd, J = 14.1; 6.8, 1H), 2.45 (t, J = 7.3, 2H), 1.97
(m, 2H), 1.68 (m, 2H), 1.45 (m, 2H), 1.43 (s, 18H),
1.36 (m, 2H), 1.30 (m, 2H), 1.27 (m, 10H), 1.25 (s,
9H); 13C NMR (CDCl3, 125MHz) d 171.45, 169.65,
155.92, 155.82, 145.88, 141.12, 131.29, 130.50, 129.56,
128.91, 127.97, 127.64, 124.32, 124.10, 121.04, 110.88,
82.89, 79.31, 78.87, 65.22, 57.09, 40.51, 38.70, 37.67,
36.94, 29.93, 29.58, 29.40, 29.38, 29.34, 29.28, 29.15,
29.11, 28.30, 28.24, 27.56, 26.68, 25.57; MS (APCI) m/
e (%) 916 (5, M+1, C45H69F3N4O10S), 815 (100), 759
(35), 715 (4), 659 (13).
4.5.3. O-(3-Aminopropyl)-meta-(isonipecotyl)amino-N-[3-
(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine (20a). 1H
NMR (D2O, 500MHz) d 7.92 (s, 1H), 7.90 (d, J = 7.9,
1H), 7.81 (d, J = 7.9, 1H), 7.62 (dd, J = 7.9; 7.9, 1H),
7.38 (d, J = 1.8, 1H), 6.95 (dd, J = 8.4; 1.8, 1H), 6.82
(d, J = 8.4, 1H), 4.20 (dd, J = 10.6; 4.6, 1H), 4.17 (m,
2H), 3.66 (m, 2H), 3.29 (m, 2H), 3.21 (m, 2H), 3.20
(dd, J = 14.3; 4.6, 1H), 2.96 (m, 1H), 2.76 (dd,
J = 14.3; 10.6, 1H), 2.29 (m, 2H), 2.27 (m, 2H), 2.04
(m, 2H); 13C NMR (D2O, 125MHz) d 175.02, 174.66,
149.04, 140.08, 130.60, 130.30, 130.06, 129.48, 128.67,
127.49, 124.82, 124.74, 123.40, 112.46, 65.52, 57.79,
43.20, 40.25, 37.15, 36.71, 26.64, 25.26; MS (APCI)
m/e (%) 575 (15), 574 (25, M+1), 573 (100), 283 (18),
282 (85); HRMS (ESI): calcd for C25H32N4O6F3S:
573.1995. Found: 573.2018.
4.5. General procedure for deprotection
4.5.4.
O-(3-Aminopropyl)-meta-[N-(methyl)isonipeco-
tyl]amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyro-
sine (21a). 1H NMR (D2O, 500MHz) d 7.83 (s, 1H), 7.79
(d, J = 7.9, 1H), 7.70 (d, J = 7.9, 1H), 7.50 (dd, J = 7.9;
7.9, 1H), 7.21 (d, J = 2.0, 1H), 6.85 (dd, J = 8.4; 2.0,
1H), 6.71 (d, J = 8.4, 1H), 4.07 (dd, J = 4.1; 11.1, 1H),
4.04 (t, J = 7.1, 1H), 3.58 (m, 2H), 3.12 (t, J = 7.1,
In a 1:1 (v/v) mixture of CH2Cl2/trifluoroacetic acid
(TFA) cooled at 0ꢁC, was added the protected com-
pound (0.05mmol/mL). The mixture was stirred at room
temperature for 4h. After concentration, the crude resi-
due was extracted with cold diethylether, then dried

S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
5391
2H), 3.08 (dd, J = 4.1; 13.7, 1H), 3.03 (m, 2H), 2.84 (s,
3H), 2.77 (tt, J = 12.2; 3.8, 1H), 2.63 (dd, J = 11.1;
13.7, 1H), 2.14 (m, 2H), 2.13 (m, 2H), 1.92 (m, 2H);
13C NMR (D2O, 125MHz) d 177.61, 177.08, 151.81,
142.61, 133.30, 132.79, 132.67, 132.12, 131.37, 130.30,
127.6, 127.31, 126.04, 115.19, 68.14, 60.40, 56.22,
45.91, 42.36, 39.70, 39.29, 29.19, 28.88; MS (APCI)
m/e (%) 589 (8), 588 (25, M+1), 587 (100), 283 (8), 282
(40); HRMS (ESI): calcd for C26H34N4O6F3S:
587.2151. Found: 587.2139.
8.0, 1H), 7.44 (br s, 1H), 6.80 (br d, J = 8.4, 1H), 6.70 (d,
J = 8.4, 1H), 4.00 (m, 2H+1H), 3.18 (m, 2H), 3.07 (t,
J = 6.9, 2H), 2.89 (dd, J = 14.0; 5.0, 1H), 2.70 (dd,
J = 14.0; 10.0, 1H), 2.33 (t, J = 6.9, 2H), 2.07 (m, 2H),
1.55 (m, 4H), 1.25 (m, 14H); 13C NMR (CD3OD,
50MHz) d 174.17, 174.13, 161.50, 143.93, 134.0,
131.64, 131.19, 130.58, 129.90, 127.86, 126.01, 125.96,
120.18, 114.45, 66.96, 59.19, 39.28, 38.60, 37.85, 30.56,
30–25 (several lines); MS (ESI) m/e (%) 659 (100,
M+1), 299 (14), 285 (15); HRMS (ESI): calcd for
C31H46N4O6F3S: 659.3090. Found: 659.3119.
4.5.5.
O-(3-Aminopropyl)-meta-(4-pyridylcarbonyl)-
amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine
(22a). 1H NMR (D2O, 500MHz) d 8.72 (d, J = 6.4, 2H),
8.00 (d, J = 6.4, 2H), 7.77 (s, 1H), 7.76 (d, J = 8.0, 1H),
7.60 (d, J = 8.0, 1H), 7.45 (dd, J = 8.0; 8.0, 1H), 7.25 (d,
J = 1.8, 1H), 6.83 (dd, J = 8.4; 1.8, 1H), 6.70 (d, J = 8.4,
1H), 4.05 (dd, J = 3.8; 11.0, 1H), 4.05 (t, J = 7.3, 2H),
3.14 (t, J = 7.3, 2H), 3.05 (dd, J = 3.8; 13.7, 1H), 2.60
(dd, J = 11.0; 13.7, 1H), 2.13 (tt, J = 7.3; 7.3, 2H); 13C
NMR (D2O, 125MHz) d 177.59, 171.34, 159.13,
151.34, 143.49, 142.64, 133.08, 132.7, 132.6, 131.9,
131.32, 130.17, 130.90, 127.35, 126.95, 125.96, 115.05,
68.23, 60.34, 39.72, 39.23, 29.17; MS (APCI) m/e (%)
569 (8), 568 (25, M+1), 567 (100); HRMS (ESI): calcd
for C25H26N4O6F3S: 567.1525. Found: 567.1508.
4.5.9.
O-(3-Aminopropyl)-meta-amino-N-[3-(trifluoro-
methyl)phenylsulfonyl]-(L)-tyrosine (26a). 1H NMR
(CD3OD, 200MHz) d 7.99 (s, 1H), 7.94 (d, J = 8.0,
1H), 7.82 (d, J = 8.0, 1H), 7.66 (dd, J = 8.0; 8.0, 1H),
7.01 (s, 1H), 6.93 (m, 2H), 4.15 (t, J = 7.0, 2H), 4.05
(m, 1H), 3.18 (t, J = 7, 2H), 3.03 (dd, J = 5.1; 13.9,
1H), 2.81 (dd, J = 9; 13.9, 1H), 2.16 (m, 2H); 13C
NMR (CD3OD, 50MHz) d 174.60, 150.46, 143.99,
131.77, 131.29, 130.63, 130.09, 128.15, 128.0, 126.17,
125.0, 124.90, 113.15, 66.81, 59.24, 39.32, 38.54, 26.94;
MS (ESI) m/e (%) 462 (100, M+1), 209 (10); HRMS
(ESI): calcd for C19H23N3O5F3S: 462.1311. Found:
462.1298.
4.5.10.
O-(3-Aminopropyl)-meta-nitro-N-[3-(trifluoro-
4.5.6. O-(3-Aminopropyl)-meta-(4-pyridylacetyl) amino-
N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine (23a).
1H NMR (D2O, 500MHz) d 8.87 (m, 2H), 8.16 (m,
2H), 7.88 (m, 1H), 7.87 (s, 1H), 7.76 (m, 1H), 7.57 (m,
1H), 7.41 (d, J = 1.8, 1H), 6.94 (dd, J = 8.4; 1.8, 1H),
6.81 (d, J = 8.4, 1H), 4.18 (dd, J = 10.8; 4.3, 1H), 4.16
(m, 2H), 3.31 (m, 2H), 3.16 (dd, J = 14.1; 4.3, 1H),
2.72 (dd, J = 14.1; 10.8, 1H), 2.30 (m, 2H); 13C NMR
(D2O, 125MHz) d 174.97, 168.56, 156.53, 148.56,
140.87, 140.06, 130.39, 129.96, 129.32, 128.59, 128.30,
127.36, 124.79, 124.11, 123.28, 112.27, 65.57, 57.76,
41.96, 37.13, 36.64, 26.59; MS (APCI) m/e (%) 583
(10), 582 (30, M+1), 581 (100), 463 (8), 462 (45), 283
(22), 282 (100); HRMS (ESI): calcd for C26H28N4O6F3S:
581.1682. Found: 581.1707.
methyl)phenylsulfonyl]-(L)-tyrosine (27a). 1H NMR
(CD3OD, 200MHz) d 8.01 (s, 1H), 7.95 (d, J = 7.9,
1H), 7.85 (d, J = 7.9, 1H), 7.72 (d, J = 2.1, 1H), 7.65
(dd, J = 7.9; 7.9, 1H), 7.45 (dd, J = 2.2; 6.8, 1H), 7.14
(d, J = 6.8, 1H), 4.33 (m, 2H), 4.15 (m, 1H), 3.29 (m,
2H), 3.10 (m, 1H), 2.83 (m, 1H), 2.20 (m, 2H); 13C
(CD3OD, 50MHz) d 173.61, 152.20, 144.04, 140.51,
137.09, 131.63, 131.50, 131.39, 131.28, 130.12, 127.80,
124.88, 115.97, 68.69, 58.79, 39.18, 38.55, 28.10; MS
(ESI) m/e (%) 492 (100, M+1), 271 (24); HRMS (ESI):
calcd for C19H21N3O7F3S: 492.1052. Found: 492.1049.
4.6. Biological evaluation
4.6.1. Binding assay. The avb3 integrin purified from hu-
man placenta was purchased from Chemicon Interna-
tional Inc., (Temecula, CA, USA). The aIIbb3 integrin
was purified from human platelets by affinity chroma-
tography, as described in Ref. 32. Solutions containing
the integrins, diluted to 500ng/mL (avb3) or 1lg/mL
(aIIbb3) in a Tris binding buffer (2mM CaCl2, 1mM
MgCl2, and MnCl2, pH7.5), were transferred to 96-well
Costar binding plates (100lL per well). After a 1h incu-
bation at room temperature, the plates were incubated
for another hour with a blocking solution of 1% albu-
min to prevent nonspecific binding. The physiological
ligands vitronectin (for avb3) and fibrinogen (for aIIbb3)
were labeled with biotin. The plates were washed three
times in binding buffer containing 0.1% albumin and ad-
sorbed integrins were then incubated for 30min with
their corresponding soluble ligands in excess, in the
binding buffer containing 0.1% albumin and in the
presence of various concentrations of compounds. After
several washings, the amount of bound ligands was indi-
rectly estimated after the sequential incubation with an
antibody against biotin coupled to alkaline phosphatase
4.5.7.
O-(3-Aminopropyl)-meta-(3-aminopropanoyl)-
amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine
(24a). 1H NMR (D2O, 500MHz) d 7.92 (s, 1H), 7.92 (d,
J = 8.0, 1H), 7.81 (d, J = 8.0, 1H), 7.62 (dd, J = 8.0; 8.0,
1H), 7.38 (d, J = 1.8, 1H), 6.95 (dd, J = 8.4; 1.8, 1H),
6.82 (d, J = 8.4, 1H), 4.20 (dd, J = 10.6; 4.6, 1H), 4.17
(m, 2H), 3.86 (m, 2H), 3.29 (m, 2H), 3.21 (dd,
J = 14.3; 4.6, 1H), 3.01 (m, 2H), 2.76 (dd, J = 14.3,
10.6, 1H), 2.27 (m, 2H); 13C NMR (D2O, 125MHz) d
175.02, 174.66, 149.04, 140.08, 130.60, 130.20, 130.06,
129.48, 128.67, 127.49, 124.82, 124.74, 123.40, 123.30,
112.46, 65.52, 57.79, 37.62, 37.22, 37.15, 36.71, 26.64;
MS (ESI) m/e (%) 533 (100, M+1), 159 (4); HRMS
(ESI): calcd for C22H28N4O6F3S: 533.1682. Found:
533.1680.
4.5.8. O-(3-Aminopropyl)-meta-(12-aminododecanoyl)-
amino-N-[3-(trifluoromethyl)phenylsulfonyl]-(L)-tyrosine
(25a). 1H NMR (CD3 OD, 200MHz) d 7.84 (s, 1H), 7.77
(d, J = 8.0, 1H), 7.70 (d, J = 8.0, 1H), 7.49 (dd, J = 8.0;

5392
S. Biltresse et al. / Bioorg. Med. Chem. 12 (2004) 5379–5393
and para-nitrophenyl phosphate for a readout at 405nm
in a colorimetric assay. The concentration of com-
pound corresponding to 50% inhibition (IC50) was
inferred from the dose–response curves of at least
two pooled experiments and used to compare the
products.
Blue (0.25% w/v) + 45% (v/v) methanol + 9% (v/v) acetic
acid. The staining solution was removed by suction and
the inserts were washed successively with AcOH in
methanol–water (7mL AcOH + 10mL MeOH + 83 mL
Milli-Q water), and water (twice). The samples were
mounted on glass slides and observed with a phase con-
trast microscope (Labovert, Leitz; original magnifica-
tion of ·250 to ·400) equipped with a color video
camera (JVC TK 1280E) coupled to a Macintosh AV
840 computer. The surface occupied by the cells was
analyzed using the NIH 1.54 Image software. For each
sample, three zones were analyzed, selected by systemic
sampling on the culture area. The results given are the
average of 2 · 3 measurements performed with two sub-
strates similarly treated. The standard deviation is indi-
cated in parentheses.
4.6.2. Cellular assay. The CaCo2 cells from the Ameri-
can-Type Culture Collection (ATCC, Rockville, MD)
were cultured at 37ꢁC in a water-saturated incubator
with an atmosphere of 5% CO2 in air, in 175cm3 flasks
(Becton Dickinson, Lincoln Park, NJ) precoated for 2h
at 37ꢁC with type I collagen (30lg/mL in PBS; Vitrogen
100, Collagen Corp., Palto Alto, CA). The cells were
progressively adapted to S-BDM (synthetic basal de-
fined medium) hormone-defined nutritive medium as de-
scribed in Ref. 22.
4.7. Theoretical33
The aVb3 antagonist activity of peptidomimetics in solu-
tion has been evaluated in competition with vitronectin.
96-Well polystyrene plate (Falcon) was coated with hu-
man vitronectin (Sigma) dissolved at 10lg/mL in PBS
buffer containing Ca2+ and Mg2+ ions (PBS++: 16.86g
Na2HPO4Æ2H2O and 0.826g NaH2PO4ÆH2O in 1L
Milli-Q water; addition of 0.132g CaCl2Æ2H2O, and
0.100g MgCl2Æ6H2O). The coated plate was conditioned
overnight at 4ꢁC and then rinsed twice with PBS++ be-
fore use. CaCo2 cells (250,000 cells) in S-BDM medium
were preincubated in the presence of a peptidomimetic
(or RGDS peptide), at various concentrations, during
30min at 37ꢁC (atmosphere of 5% CO2 in air). The con-
centrations were from 10ꢀ3 to 10ꢀ9 M. The pretreated
cells were inoculated into the coated wells at
50,000cells/well in S-BDM medium. After 15min at
37ꢁC, the medium was removed by suction and each
well was gently rinsed with PBS++ to eliminate nonad-
herent cells. A solution of 4-nitrophenyl-2-acetamido-2-
deoxy-b-D-glucopyranoside (Acros, 7.5mM) and Triton
X-100 (0.5%) was added in each well (60lL/well). After
2h of incubation at 37ꢁC, the enzymatic reaction (of cel-
lular N-acetyl-b-D-glucosaminidase) was stopped by the
addition of a solution of glycine (150mM) and EDTA
(5mM) at 90lL/well. Absorbance (A) due to the enzy-
matic formation of 4-nitrophenolate was measured at
405nm with a Bio-Rad FTS 135 instrument. Results
of Figure 4 are the average of three independent
measurements (standard deviation indicated on the
graph).
All the calculations have been performed using the
Gaussian 98 A7 suite of programs.34 The starting geom-
etries were sketched drawn by standard fragments and
then completely optimized following all the 3Nꢀ6 de-
grees of freedom.
Acknowledgements
This work was supported by the FNRS (Belgium), the
FRIA (Belgium), and the communaute franc¸aise de Bel-
gique (ARC contract no. 99/04-240). J.M-B. and G.D.
are senior research associates of FNRS. The authors
thank Professors Y.-J. Schneider (UCL, Belgium), P.
Rouxhet (UCL, Belgium) and M. Raes (FUNDP, Bel-
gium) for useful discussions, and Dr. R. Touillaux
(UCL, Belgium) for 500MHz NMR analysis. The tech-
nical help of A. Genton (IdRS, France) is gratefully
acknowledged.
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