αvβ3 integrin-targeting Arg-Gly-Asp (RGD) peptidomimetics containing oligoethylene glycol (OEG) spacers

Article abstract of DOI:10.1021/jm901133z

RGD peptides are used in biomaterials science for surface modifications with a view to elicit selective cellular responses. Our objective is to replace peptides by small peptidomimetics acting similarly. We designed novel molecules targeting α_vβ₃ integrin and featuring spacer-arms (for surface grafting), which do not disturb the biological activity, from (L) N-(3-(trifluoromethyl)benzenesulfonyl) tyrosine used as scaffold. Various Arg-mimics were fixed on the phenol function, and the ortho position was used for the coupling of OEG spacers. All peptidomimetics were active in the nM range in a binding test toward human α_vβ ₃ integrin (IC₅₀ = 0.1 to 1.7 nM) and selective versus platelet integrin α_IIbβ₃. Selected compounds revealed excellent ability to inhibit bone cells adhesion on vitronectin. Modeling and docking studies were performed for comparing the most active RGD peptidomimetic to cilengitide, i.e., cyclo-[RGDfN(Me)V]-. Lastly, the adhesion of endothelial cells on a cultivation support grafted with RGD peptidomimetics was significantly improved. 2009 American Chemical Society.

Full text of DOI:10.1021/jm901133z

J. Med. Chem. 2009, 52, 7029–7043 7029
DOI: 10.1021/jm901133z
rvβ3 Integrin-Targeting Arg-Gly-Asp (RGD) Peptidomimetics Containing Oligoethylene Glycol
(OEG) Spacers
†
‡
§
§
,^
€
ꢀ ꢀ
Vincent Rerat, Georges Dive, Alex A. Cordi, Gordon C. Tucker, Reine Bareille, Joelle Amedee, Laurence Bordenave,
and Jacqueline Marchand-Brynaert*,†
†Uniteꢀ de Chimie Organique et Meꢀdicinale, Universiteꢀ Catholique de Louvain, Ba^timent Lavoisier, Place L. Pasteur 1, 1348 Louvain-la-Neuve,
Belgium, ‡Centre d’Ingeꢀnierie des Proteꢀines, Universiteꢀ de Lieꢁge, Ba^timent B6, Alleꢀe de la Chimie, 4000 Sart-Tilman, Belgium, §Institut de
Recherches Servier, Rue des Moulineaux 11, 92150 Suresnes, France, INSERM, U577, UniversiteꢀVictor Segalen Bordeaux 2, Rue Leꢀo Saignat
146, 33076 Bordeaux Cedex, France, and ^CIC-IT Biomateꢀriaux, INSERM, Pessac, F-33604 France; CHU Bordeaux, Ho^pital Xavier Arnozan,
Pessac, 33604, France
Received June 9, 2009
RGD peptides are used in biomaterials science for surface modifications with a view to elicit selective
cellular responses. Our objective is to replace peptides by small peptidomimetics acting similarly. We
designed novel molecules targeting Rvβ3 integrin and featuring spacer-arms (for surface grafting), which
do not disturb the biological activity, from (L) N-(3-(trifluoromethyl)benzenesulfonyl) tyrosine used as
scaffold. Various Arg-mimics were fixed on the phenol function, and the ortho position was used for the
coupling of OEG spacers. All peptidomimetics were active in the nM range in a binding test toward
human Rvβ3 integrin (IC50 = 0.1 to 1.7 nM) and selective versus platelet integrin RIIbβ3. Selected
compounds revealed excellent ability to inhibit bone cells adhesion on vitronectin. Modeling and
docking studies were performed for comparing the most active RGD peptidomimetic to cilengitide, i.e.,
cyclo-[RGDfN(Me)V]-. Lastly, the adhesion of endothelial cells on a cultivation support grafted with
RGD peptidomimetics was significantly improved.
Introduction
of proliferating endothelium, and tumor invasiveness.7-10
Therefore, antagonists of Rvβ3 receptor, namely RGD cyclic
peptides11 and RGD peptidomimetics,12,13 are currently devel-
oped as potential drugs for the treatment of osteoporosis,
restenosis, ocular disease, and cancer.14-16 This is exemplified
with cilengitide, i.e., cyclo-[RGDfN(Me)V]-, actually under
clinical trials.11 RGD-based strategies are also considered for
the selective delivery of therapeutics and imaging agents to
tumor cells.17-19 Last, biomaterials for stimulated cell adhesion
are prepared by surface modification with RGD molecules.20-22
Controlling the interface between cells and solid substrates is
of major concern in tissue engineering and regenerative med-
icine.23 In this context, we are interested in the covalent
attachment of RGD peptidomimetics on the surface of inert
polymer materials such as poly(ethylene terephthalate) (PET),
with a view to promote cellular adhesion in the absence of
serum or ECM proteins.24,25 The development of such a
strategy requires peptidomimetic molecules featuring an an-
chorage arm having a position on the molecular scaffold that
does not disturb the biological activity. In this article, we
describe the synthesis and the validation of novel RGD
peptidomimetics, containing OEG (oligoethylene glycol)
spacers, and constructed on the (L)-tyrosine scaffold, for
targeting Rvβ3 integrin. Our aim is the development of molec-
ular devices useful in biomaterials research principally, but also
in drug or imaging agent delivery and immunotherapy.26,27
RGD (Arg-Gly-Asp)-based ligands of Rvβ3 integrin are
intensively investigated in topics related to pathology, phar-
macology, and materials science. The RGD peptide sequence
has been recognized 25 years ago as the cell attachment site in
various extracellular matrix (ECMa) proteins.1,2 At the same
time, cellular receptors of ECM proteins have been identified
and most have been classified in the integrin family. These
receptors are heterodimeric transmembrane glycoproteins
formed by the association of R and β subunits.3-6 They
mediate cell adhesion and migration phenomena.
The Rvβ3 integrin (also called vitronectin receptor) is mainly
involved in adhesion of osteoclasts and osteoblasts to bone
matrix, migration of vascular smooth muscle cells, angiogenesis
*To whom correspondence should be addressed. Phone: þ32 (0)10 47
27 40. Fax: þ32 (0)10 47 41 68. E-mail: jacqueline.marchand@uclouvain.
be.
a Abbreviations: ACN, acetonitrile; Boc, tert-butyloxylcarbonyl;
DMEM, Dulbecco’s Modified Eagle Medium; DMF, dimethylforma-
mide; DMSO, dimethylsulfoxide; EC, endothelial cell; ECM, extracel-
lular matrix; EDTA, ethylenediaminetetraacetic acid; EG, ethylene
glycol; FCS, fetal calf serum; FtN, phthalimide; GRGDS, glycine-
arginine-glycine-aspartic acid-serine; HOP, human osteoprogenitor;
HSV, human saphenous vein; IMDM, Iscove Modified Dulbecco’s
Medium; IC50, half maximal inhibitory concentration; MIDAS, metal
ion dependent adhesion site; OEG, olygoethylene glycol; PB, phosphate
buffer; PBS, phosphate buffered saline; PET, poly(ethylene tereph-
thalate); PS, polystyrene; PyBop,: benzotriazol-1-yl-oxy-tris-(dime-
thylamino)phosphonium hexafluorophosphate; RGD, arginine-gly-
cine-aspartic acid; RHF, restricted Hartree-Fock; TCPS, tissue culture
polystyrene; TFA, trifluoroacetic acid; THF, tetrahydrofuran; XPS,
X-ray photoelectron spectroscopy.
Results and Discussion
Design. The promotion of cellular adhesion on biocompa-
tible polymer substrates is generally achieved either by coating
r
2009 American Chemical Society
Published on Web 10/27/2009
pubs.acs.org/jmc

7030 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Rerat et al.
Figure 2. Second generation of peptidomimetics (n = 1, 2; p = 2, 5;
X = NH2).
Figure 1. First generation of peptidomimetics based on the (L)-
tyrosine scaffold.
Scheme 1. General Synthesis of Graftable Peptidomimeticsa
the materials with ECM proteins and RGD-containing large
peptides20 or by surface derivatization with small (cyclic)
RGD peptides28,29 and peptidomimetics (i.e., nonpeptide
mimics of the RGD sequence constrained in the bioactive
conformation). This latter approach is quite underemployed
from the literature,25 most probably because it requires
expertise and consistent efforts in organic synthesis. As far
as inorganic (bio)materials are concerned, the coating of
titanium with thiol-terminated RGD peptidomimetics has
been reported by Kessler et al.30
Starting from the (L)-tyrosine scaffold,31,32 we have already
prepared RGD peptidomimetics possessing a free carboxyl
function (Asp-mimic) and a guanidine function (Arg-mimic)
˚
at a distance of about 13 A on the one hand, and an
anchorage-arm (fixed on aromatic OH) for surface grafting
on materials on the other hand.33,34 Disappointingly, such
molecules, exemplified by A and B (Figure 1), were poorly
active against Rvβ3, and nonselective versus RIIbβ3, the integ-
rin receptor expressed on blood platelets.35 Nevertheless, PET
membranes surface-derivatized with peptidomimetics A and
B showed improved adhesive properties in CaCo2 cell culture
systems.24,25 In the course of this preliminary work, evalua-
tion of the parent compound C (Figure 2) against Rvβ3 and
R
IIbβ3 integrins gave an unexpected result: this (L)-tyrosine
derivative was a modest antagonist of both receptors.
The O-aminopropyl chain, initially considered as the
spacer-arm, seemed to play the role of the basic function
mimicking the Arg residue. We decided thus to investigate
a novel family of RGD peptidomimetics derived from (L)-
tyrosine in which the relative positions of the Arg-mimic and
the spacer-arm were exchanged, as shown in the general
structure D (Figure 2). The hydrophobic moiety next to the
Asp-mimic36,37 remained the meta-trifluoromethyl-benzene-
sulfonamide group featuring a useful fluorine-tag for XPS
(X-ray photoelectron spectroscopy) analysis of the final
biomaterials.25 The basic motifs R were chosen among the
library of Arg bioisosters usually found in Rvβ3 antagonists
from the recent literature,13,38-40 i.e., cyclic amidines, amino-
pyridines, and tetrahydronaphthyridines. Last, R,ω-functio-
nalized oligoethylene glycols (OEG) were selected as spacer-
arms due to the particular properties of OEG molecules well
recognized in materials sciences: water solubility, molecular
flexibility, stability in biological medium, and antifouling
capacity.41
a Steps: (a) Mitsunobu coupling, (b) reduction by hydrogenation,
(c) acylation with OEG-spacer, (d) deprotection.
Chemical Synthesis. A convergent strategy was set up
for the synthesis of the target-molecules D (Figure 2): a key
intermediate, namely t-butyl 3-(4-hydroxy-3-nitro-phenyl)-2
(S)-(3-trifluoromethyl-benzenesulfonylamino)propionate (1),
was successively equipped with Arg-mimics via a Mitsunobu
reaction on the tryrosine-OH and with OEG-spacers via a
peptide coupling on the tyrosine-NH2 obtained by reduction
of tyrosine-NO2 precursors (Scheme 1). The basic motifs were
connected by using either a propyl (n = 1) or a butyl chain
(n = 2), in view of adjusting the distance between the acidic
42
˚
and basic moieties to about 12-14 A. The length of the
spacer was of three (p = 2) or six (p = 5) ethyleneglycol units,
according to a previous study,43 and the terminal function for
grafting on materials was a primary amine.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7031
Scheme 2. Synthesis of the Basic Arms as Arg-Mimics (See Supporting Information)
The precursor 1 was already described34 (45% yield in two
steps from commercial t-butyl tyrosinate; 1 g scale), but as we
needed large quantities, a practical synthesis was developed
starting from (L)-tyrosine (see Supporting Information).
Briefly, t-butyl tyrosinate was obtained by transesterifica-
tion with t-butyl acetate in the presence of perchloric acid;
sulfonylation was performed with meta-trifluoromethyl-
benzenesulfonyl chloride at 0 ꢀC in THF-DMF mixture
(8:1, v/v)44 in the presence of solid sodium carbonate; nitra-
tion was realized with nitric acid in acetic acid (0.25 M
solution) at 16 ꢀC (90% yield on two steps, 20 g scale).
The required ω-functionalized propanols and butanols
(with the basic groups) for Mitsunobu coupling to phenol
1 (Scheme 1, step a) were prepared by conventional routes
depicted in Scheme 2 (see Supporting Information).45-57
O-Alkylation of 1 with alcohols 10a,b (eq 1), 12b (eq 2), 13a,
b (eq 3), 14a,b (eq 4), 17b (eq 7), and 18b (eq 8), in the presence
of diisopropyl azodicarboxylate and triphenylphosphine
(THF, 20 ꢀC, 17 h), readily gave compounds 2a-i, collected
in Table 1. The Mitsunobu etherification failed when using
alcohols 12a (eq 2), 15a,b (eq 5), 16a,b (eq 6), 17a (eq 7), 18a
(eq 8), 19a (eq 9), and 19b (eq 10) due to the rapid intramo-
lecular cyclization of the activated alcohols or unfavorable
steric effects. Therefore, the desired compounds 2-3 were
obtained via indirect routes. For instance, in the case of
2-amino-3-pyridine carboxaldehyde ((L)-proline, EtOH, re-
flux, 24 h)54 gave the naphthyridine 3c (entry 15). Similarly,
reaction of 1 with 21 yielded 2l (entry 12), in which dioxolane
deprotection (FeCl3 on SiO2, acetone, 20 ꢀC, 2 h)58 affor-
€
ded 3d (entry 16). Last, Friedlander heterocyclization let to 3e
(entry 17). Etherification of phenol 1 with N-phthalimido-3-
amino-propan-1-ol (22, eq 12) produced 2j (entry 10); FtN-
deprotection (N2H4, EtOH, 80 ꢀC, 2 h) gave intermediate 3a
(entry 13), followed by N-guanidylation with di-Boc thiourea
in the presence of Mukaiyama salt ((BocNH)2CS, 2-chloro-1-
methyl-pyridinium iodide, Et3N, DMF, 20 ꢀC, 1 h),59 which
furnished compound 3b (entry 14).
The series of O-alkylated derivatives 2-3 was submitted to
hydrogenation for transforming the aromatic NO2 group into
NH2 function (Scheme 1, step b). The resulting anilines 4 are
collected in Table 2. In several cases, the basic substituents
could simultaneously suffer reduction giving the required
Arg-mimics: catalytic hydrogenation over palladium on char-
coal reduced the naphthyridine motif into tetrahydro-
naphthyridine (entries 11-12); catalytic hydrogenation in
acidic medium reduced the pyrimidine motif into six-mem-
bered cyclic amidine together with t-butyl ester cleavage
(entries 3-4); treatment with ammonium formate and Pd/C
catalyst60 reduced pyridine oxide into pyridine (entries 5-6).
Finally, simultaneous deprotection of t-butyl ester and N-Boc
groups was readily performed by reaction of compounds 4
(except 4c and 4d, already deprotected) with trifluoroacetic
acid (TFA-CH2Cl2 (1:1), 20 ꢀC, 2 h), giving the RGD
peptidomimetics 5 listed in Table 4 (entries 1-7 and 12-16)
and considered for biological evaluation (see next section).
€
naphthyridine derivatives 3, the precursors of Friedlander
heterocyclization were introduced first, namely 3-acetyl-1-
propanol (20) and 4-(2-methyl-[1,3] dioxolan-2-yl)butan-1-
ol (21, eq 11). Thus under Mitsunobu conditions, reaction
of 1 with 20 furnished 2k (entry 11); further condensation with

7032 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Table 1. O-Alkylation of Key Intermediate 1 (Step a)
Rerat et al.
Table 2. Reduction of Nitro Intermediates 2-3 (Step b)
a Simultaneous t-butyl ester deprotection is observed (conditions in
noted). b After filtrationon celite. c Isolated product by chromatography.
d A = Pd/C (10%), H2 (1 bar), MeOH, 18 h, 20 ꢀC; B = HClaq (35%)-
-
AcOH (1:9, v/v), Pd/C (10%), H2 (3 bar), 2 h, 20 ꢀC; C = NH4þHCO2
(10 equiv), Pd/C (10%), EtOH, 12 h, reflux.
a Chromatography. b Obtained from 2j by hydrazinolysis. c Obtained
from 3a by guanidylation. d Obtained from 2k by Friedlander reaction.
€
23a and 23b reacted with 4g and 4k (0.9 equiv, DMF, 40 ꢀC,
18 h) to furnish the amides 6a,b (p = 2) and 7a,b (p = 5),
respectively (Table 3, entries 1-4). (2-(2-(2-Methoxy-ethoxy)-
ethoxy)-ethoxy)-aceticacid(24)66 (spacer-arm devoid of term-
inalaminefunction) was similarly coupled to4gand 4k, giving
compounds 6c,d (entries 5-6). For biological evaluation, the
precursors 6-7 were fully deprotected by treatment with TFA
(50% in CH2Cl2, 20 ꢀC, 2 h) to afford the RGD peptidomi-
metics 8-9 (Table 4, entries 9-11 and 18-20). Last, the
aromatic NH2 function of intermediates 4g and 4k has been
acylated (CH3COCl, Et3N, CH2Cl2, 20 ꢀC, 1 h) and the result-
ing acetamides (4m,n; 62-66%) were deprotected as usual,
giving 5m and 5n for evaluation (Table 4, entries 8 and 17).
e
f
€
Obtained from 2l by deprotection. Obtained from 3d by Friedlander
reaction.
Two OEG spacer-arms were prepared following conventional
chemistry (see Supporting Information). Briefly, N-Boc pro-
tected 2-(2-(2-aminoethoxy)ethoxy)ethanol61,62 and t-butyl 17-
hydroxy-3,6,9,12,15-pentaoxaheptadecylcarbamate63 were O-al-
kylated with methyl bromoacetate or ethyl diazoacetate and the
resulting esters were saponified to give the corresponding acids
23a (p = 2)64 and 23b (p = 5)65 used for coupling with aniline
derivatives 4 (see Scheme 1, step c). In the presence of benzotri-
azol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluoropho-
sphate (PyBOP) as activating agent (Et3N, DMF, 20 ꢀC, 2 h),

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7033
Table 3. Coupling of Spacer-Arms (Step c)
First of all, their aniline function has been masked to ensure
that this function does not make part of the pharmacophore
(compounds 5m and 5n, entries 8 and 17). Indeed, the acylated
peptidomimetics 5m,n were almost similarly active as their
parent compounds but with a drop of selectivity. Fortunately,
in both cases, the grafting of spacer-arms with three or six EG
units on molecules 5g and 5k maintained their excellent activ-
ities: IC50 values of the graftable peptidomimetics 8a, 9a (entries
9-10) and 8b, 9b (entries 18-19) ranged within 0.8-0.9 nM
and 0.3-0.7 nM, respectively. Here again, a control experiment
consisting in the replacement of the NH2 spacer end-group with
a methoxy group (compounds 8c and 8d, entries 11 and 20)
showed that this amino function is not involved in the Rvβ3
recognition process. Thus, all the RGD peptidomimetics con-
taining OEG spacers (8a, 9a, 8c, 8b, 9b, 8d) remain active in the
nM range against Rvβ3, but their selectivity versus RIIbβ3
diminishes, as compared to previously published active mole-
cules designed to be orally bioavailable.67
Selected peptidomimetics, namely 5g, 5n, and 8b, have been
evaluated in solution for their ability to inhibit cellular adhesion
in a competitive test versus vitronectin. The assay was realized
with HOP (human osteoprogenitor) cells.68,69 Cells were pre-
incubated with tested molecules in serum-free medium to satu-
rate the integrin receptors and then seeded on polystyrene (PS)
plates coated with vitronectin. Cell attachment was measured
by a colorimetric method.70 For positive control (100%
attachment), cells nonincubated with peptidomimetics were
inoculated on the culture plates. GRGDS (Gly-Arg-Gly-Asp-
Ser) pentapeptide was used as reference ligand.20,71 Results of
Figure 3 show that all tested compounds are active to inhibit cell
adhesion on PS plates coated with vitronectin. Peptidomimetic
5g (devoid of spacer-arm) is active against integrins in the μM
and nM ranges. In the same experiment, GRGDS was active in
the same ranges, but the latter absorbance ratios were signifi-
cantly superior to those of peptidomimetic 5g (p < 0.01).
Peptidomimetic 5m (devoid of spacer-arm) appears also active
in this assay based on living cells as a whole “sensor”, but its
corresponding graftable peptidomimetic 8b (equipped with the
spacer-arm) has lost the activity at the nanomolar concentra-
tions. The effect of the spacer-arm has been examined by
computational studies.
Biological Evaluation. Our design and synthesis of tyro-
sine-based RGD peptidomimetics afforded a library of 20
compounds which were evaluated for binding affinity to-
ward human Rvβ3 integrin by using a competitive assay
already described.34 Briefly, the purified integrin was incu-
bated with biotinylated vitronectin (natural ligand) in excess
in the presence of various concentrations of the tested
compound. The amount of bound natural ligand was mea-
sured, indirectly, by incubation with a biotin-directed anti-
body coupled to alkaline phosphatase and reading at 405 nm
of the amount of p-nitrophenol liberated by enzymatic
hydrolysis of p-nitrophenylphosphate. The structurally close
platelet integrin RIIbβ3 was also considered in this study,
using a similar colorimetric binding test with fibrinogen as
the competitive natural ligand. Results were expressed in
IC50 values for comparison (Table 4).
Compounds 4c-d (entries 3-4), 5e-g (entries 5-7), and
5h-l (entries 12-16) are all active in the nM range, thus
confirming the right position of the Arg-mimics on the
tyrosine aromatic core. The selected choice of Arg-mimics
was also judicious because the simple NH2 substituent con-
ferred only modest activity (compounds 5a-b, entries 1-2).
The most active molecules featured either 2-amino-4-methyl-
1-pyridin-2-yl or tetrahydro-1-naphthyridin-2-yl substituents
(= R group) with respectively a butyl (n = 2) or a propyl chain
(n = 1). These RGD peptidomimetics, namely 5g (entry 7) and
5k (entry 15), were also highly selective for Rvβ3 versus RIIbβ3.
They were thus chosen for the anchorage of the spacer-arms.
Modeling Study. A conformational study of selected com-
pounds, namely 4c, 4d, 5e, 5f, 5g, 5h, and 5k (see structures in
Table 4), has been performed and the critical distances
between Arg- and Asp-mimics have been compared to those
of cilengitide (cyclo-[RGDfN(Me)V]-). All the geometry
degrees of freedom were fully optimized at the RHF level
using the double-ζ polarized basis set 6-31G(d). For all
compounds, at least two conformations could be located: a
folded one with a potential π stacking between the two
aromatic rings and an extended one which reduces the
interactions between the bulky sulfonamide group and the
R motif of the Arg-mimic. Energy differences (ΔE) between
the folded (reference) and the extended conformers are given
in Table 5. The extended conformers are generally more
stable, except in the case of 4c and 5k. The distances between
the carbon bearing the carboxyl group and each of the
nitrogen atoms of the basic motifs have been determined
(Table 5). For the extended conformations, the values ranged
˚
within 11-14 A, as required for an optimal interaction
with Rvβ3 receptor. Our molecules compare with the cyclic
peptide cilengitide for which the bioactive conformation is
known from X-ray data.72 Their extended conformations are
in good agreement with the three-point pharmacophore

7034 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Table 4. RGD Peptidomimetics for Biological Evaluation
Rerat et al.
a Mean of at least two experiments. b Selectivity (S) = IC50
and 120 nM (RIIbβ3).71
R
IIbβ3/IC50 Rvβ3. c For comparison, GRGDS pentapeptide IC50 values are 750 nM (Rvβ3)
proposed by Moitessier et al. on the basis of geometric
considerations.73
the Arg- and Asp-mimics and the orientation of the space-arm
alongside the V residue of cilengitide (Figure 5), thus well
positioned for pointing out the receptor recognition pocket.
To further confirm the design of our peptidomimetic, 5k
(extended conformer) was docked in the active site of Rvβ3,
obtained from the X-ray crystal structure of the extracellular
segment of the Rvβ3 integrin complexed with cilengitide.74,75 The
goodness of fit appeared very interesting as the naphthyridinyl
part can be in direct electrostatic interaction with the Asp218
residue (from the Rv subunit) and the carboxylic head is charge
paired with the Arg214 residue (from the β3 subunit) and Mn2þ
ion (from the metal ion dependent adhesion site, MIDAS)
(Figure 6). As the molecule 5k is rather flexible, a complemen-
tary optimization has been performed starting from several
conformations which can be fitted in the active site. Remarkably,
Because of the size of the systems, the graftable peptido-
mimetics 8a and 8b (see structures in Table 4) were studied
in MINI1 basis. Two accessible extended conformations
were found, referred to as Exten 1 (reference) and Exten 2
(Figure 4). Exten 1 features the spacer-arm perpendicularly
to the tyrosine scaffold, while Exten 2 is stabilized by a
hydrogen bonding between arylamide NH and arylether O
due to a rotation of the spacer-arm. As shown in Table 6, the
presence of a spacer-arm did not disturb the characteristic
distances between Arg- and Asp-mimics.
The conformer Exten 1 of 8b was superimposed on the
crystallographic structure of cyclo-[RGDfN(Me)V]- extracted
from its complex with the receptor. This shows a good fitting of

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7035
Figure 3. Inhibition of HOP cell attachment on vitronectin by
GRGDS peptide and the synthesized peptidomimetics.
Table 5. Conformational Study of Peptidomimetics without Spacer-Arm
distances
C-CO2H/N (A)
a
˚
compd
(Table 4)
ΔEc
folded extended
conformation conformation
entry
1
(kcal mol-1
)
3
4c
þ0.532
7.09; 8.17;
9.12
10.92; 12.05;
13.18
2
4d
-2.239
10.12; 10.89;
12.05
12.03; 13.77;
14.23
3
4
5
6
7
8
5e
-0.054
-1.413
-1.195
-1.678
þ1.563
6.97; 7.71
9.00; 10.56
8.89; 10.42
10.07; 11.81
6.90; 7.14
10.93; 13.11
12.03; 14.13
12.03; 14.13
12.03; 14.13
11.96; 14.16
5f
5g
5h
5k
cilengitideb
10.99; 11.62; 12.57
a Distances between C (bearing the carboxyl group) and each N atom
of the Arg-mimic. b Structure from X-ray data of the complex with the
receptor. c ΔE = Eextended - Efolded (RHF/6-31G(d)).
Table 6. Conformational Study of Graftable Peptidomimetics
a
˚
Distances C-CO2H / N (A)
entry compd (Table 4)
Exten 1
Exten 2
ΔEb (kcal mol-1
)
3
1
2
8a
8b
12.25; 14.45 12.22; 14.41
11.64; 13.50 11.50; 13.23
7.41
7.31
Figure 4. Two conformers of 8b obtained by calculation in MINI-
10 basis.
a Distances between C (bearing the carboxyl group) and each N atom
of the Arg-mimic. b ΔE = ΔEexten1 - ΔEexten2 (MINI-10).
the reoptimized minima remained in the local conformation
without significant modifications of the dihedral angles defining
the principal skeleton in an energetic range less than 3 kcal
3
mol-1. The main difference is only the rotation of the phenyl-
sulfonamide fragment (Figure 7). In the same way, with a view
to validate our docking simulation, cyclo-[RGDfN(Me)V]-
was optimized at the RHF/6-31G(d) level. Again, several con-
formations could be located and the observed X-ray conforma-
tion remained a local minimum when reoptimized (see
Supporting Information).
Application to a Cell Culture System. A poly(ethylene
terephthalate) (PET) track-etched membrane currently used
as cell culture support24,25 was derivatized with peptidomi-
metic molecules 8b, 9a, 9b, and GRGDS peptide by surface
covalent grafting. The native membrane was activated by a
wet chemistry treatment with trifluorotriazine76 and then
incubated with the adhesive molecules to furnish the supports
named PET-g-8b, PET-g-9a, PET-g-9b, and PET-g-RGD,
respectively. The rates of surface functionalization were de-
termined by X-ray photoelectron spectroscopy (XPS)43 and
the recorded percentages of grafting converted into pmol/cm2
of apparent surface for comparing the samples.
Figure 5. Superimposition of the conformer exten1 of 8b and
cilengitide.

7036 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Rerat et al.
Figure 6. Docking of 5k in the active site of integrin Rvβ3. The R and β subunits of integrin are colored yellow and pink, respectively. 5k is colored
purple. Oxygen atoms are red, nitrogen atoms are blue, hydrogen atoms are white, and fluorine atoms are cyan. The Mnþ2 ion is shown as a green sphere.
Figure 8. HSVEC attachment on surface-modified PET substrates.
(HSV) collected after surgical coronary bypass. Cells were
cultured under specific conditions and used in the fourth
passage.77 Cells were seeded on native PET membrane and
surface modified supports, in the absence of serum, at the
density of 50000 HSVECs per cm2 for 1, 3, and 6 h. There-
after, nonadherent cells were removed by washings and
adherent cells assayed by a quantitative colorimetric test.70
Results presented in Figure 8 are expressed in percentages of
absorbance related to untreated PET, set at 100%. This blank
sample (native PET) is systematically included in each test run
for comparing the treated PET samples. The PET-g-RGD
support is considered as the adhesive control (C) for evaluating
the effects of the nonpeptide mimics. The naphthyridin pepti-
domimetic 8b equipped with a three EG-units spacer showed a
significant effect of surface concentration increase (from 69 to
89 pmol/cm2) on cell attachment at 6 h (p < 0.001, see columns
2 and 3). For similar surface densities, the naphthyridin
peptidomimetic 9b featuring a longer spacer-arm (i.e., six
EG-units) was significantly more active at 1, 3, and 6 h for
promoting HSVECs adhesion (p < 0.004, see columns 2 and
4). But increasing the surface density of 9b did not improve
further the performances at 6 h (from 51 to 103 pmol/cm2,
see columns 4 and 5). Last, for similar surface densities
(103-109 pmol/cm2) and the same length of the spacer-arm
(six EG-units), the 4-methyl-2-amino-pyridine peptidomi-
metic 9a was less efficient than the naphthyridin one 9b at
Figure 7. Conformer 5k obtained from docking (A) and reopti-
mized (B).
3 and 6 h (see columns 5 and 6). Regarding the control surface
(column 1), the PET-g-9b support with a functionalization rate
of about 50 pmol/cm2 (column 4) appears of particular interest
for further studies. This novel substrate allows an increase
The modified surfaces were investigated with human en-
dothelial cells (EC) isolated from human saphenous veins

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7037
of cellular attachment of 200-250% in a serum-free culture
medium, in comparison with the commercial (native) PET
membrane, while the performance of PET-g-RGD (about
85 pmol/cm2) is lower (150-180%).
Shimatzu FTIR 8400S equipment; products were deposited as
thin films on NaCl crystal. Low resolution mass spectra were
obtained with a TSQ 7000 spectrometer from Finnigan in CI
(chemical ionization) mode and with a LCQ or quantum
spectrometer from Finnigan in ESI (electronspray ionization)
mode at 70 eV and APCI (atmospheric pressure chemical
ionization) mode at 100 eV. High resolution mass spectra
(HRMS) were recorded at the University of Mons-Hainaut,
laboratory of professor R. Flammang. Melting points were
measured on a Buchi B-540 apparatus calibrated with benzoic
acid and are uncorrected. Rotations were measured on a Perkin-
Elmer 241 MC apparatus using sodium D ray, at 25 ꢀC; con-
centrations are given in g/100 mL. Elemental analyses were
made in the Christopher Ingold laboratories (Department of
Chemistry, Imperial College, London). Analytical thin layer
chromatography (TLC) was carried out on Merck TLC plates
coated with silica gel 60 F254 (0.25 mm layer thickness); visua-
lization was carried out using either a UV lamp (254 nm), or
KMnO4, phosphomolybdic acid (10% in ethanol), and ninhy-
drin (10% in ethanol) as indicators. Flash chromatography was
performed with Merck 60 silica gel (230-400 mesh ASTM).
Solvent mixtures used for TLC and flash chromatography are
reported in v/v total. Lyophilization of aqueous solutions was
realized on a Alpha 2-4LD-plus equipment from Christ
(Osterode, Germany). The purity of tested compounds (at least
95%) was established by analytical HPLC using a Waters
(Belgium) equipment (Waters 600 pump, Waters 600 multi-
solvent controller, 996 photodiode array detector, injection
system of Mistral, Spark Holland and EMPOWER software),
and a chiralcel OD-H column (5 μm) from Daicel Chemical
Industries (Illkirch, France) of 250 mm ꢀ 4.6 mm (internal
diameter). Elution was made with a mixture of hexane (HPLC
grade Chromasolv, Sigma-Aldrich) and i-propyl alcohol
(HPLC grade HiPerSolv Chromanorm, VWR), in linear gradi-
ent mode (from Hex/i-PrOH 75:25 to Hex/i-PrOH 40:60 over
30 min) at a flow rate of 1 mL/min and temperature of 22 ꢀC
(detection at 254 nm).
Conclusion
The idea that RGD-peptidomimetic molecules could be
equipped with a spacer-arm without disturbing their capacity
to bind the Rvβ3 integrin has been successfully exploited in the
case of ligands based on the tyrosine scaffold. This scaffold
was initially considered by the Merck groupfor the research of
platelet receptor RIIbβ3 antagonists,78 giving tirofibane as
cardiovascular drug, and more recently used by Kessler
et al. for the design of selective R5β1 ligands,42,52 potentially
useful in the field of antiangiogenic cancer therapy. Both
receptors, RIIbβ3 and R5β1 feature high sequence similarity
with Rvβ3 integrin.
Practically, we have introduced the spacer-arm on the
tyrosine aromatic ring thanks to a simple sequence of reac-
tions involving nitration/reduction/acylation. It is worth not-
ing that this position of functionalization is unusual. Other
studies devoted to bifunctional ligands targeting tumor cells,
in the fields of drug delivery and imaging contrast agents,
made use of the lipophilic moiety (i.e., the sulfonamide group
of piperazine-based RGD peptidomimetics) to couple the
spacer.79-81
All the synthesized compounds showed natural ligand
displacement competence in the nM range for Rvβ3, with a
variable selectivity versus RIIbβ3 (S = 1.4 103 for 5k and 0.83
102 for 8b; S = 1.8 102 for 5g and 0.3 102 for 8a, see Table 4).
The drop of selectivity related to the presence of OEG spacers
could not be presently explained. Although Rvβ3/RIIbβ3 selec-
tivity is an important parameter for therapeutic applica-
tions, this aspect is not a limiting factor when peptidomimetics
are devoted to ex vivo surface modification of biomaterials.
Similarlytothe reference peptideGRGDS, peptidomimetics5
and 8 were able to inhibit HOP cell adhesion on a vitronectin-
coated support.
Computational studies have confirmed the good fitting of
our graftable RGD-peptidomimetics into the target receptor
and the possible positioning of the spacer-arm outside the
binding site. This is exactly the requirement for the develop-
ment of molecules 8a,b and 9a,b in the field cell adhesive
materials with a view of endothelialisation of vascular
grafts.77 Preliminary results of HSV (human saphenous vein)
cell adhesion on poly(ethylene terephthalate) (PET) mem-
branes grafted with peptidomimetics demonstrated the good
performance of one substrate, namely PET-g-9b. Further
works are in progress for studying EC proliferation and
response under physiological shear stress conditions in flow
chambers.
Selected compounds are fully described below. For the others,
namely 2b, 2c, 2d, 2e, 2f, 2h, 2i, 2j, 2l, 3a, 3b, 3d, 3e, 4b, 4c, 4d, 4e,
4f, 4h, 4i, 4j, 4l, 6c, 6d, 5b, 5e, 5f, 5h, 5i, 5j, and 5k, see Supporting
Information.
General Procedure for Coupling Alcohols to Key Intermediate
1 (Table 1). Tyrosine scaffold 1 (0.5 g, 1.02 mmol, 1 equiv) and
alcohol (from Scheme 2, 1.12 mmol, 1.1 equiv) were dissolved in
dry THF (4 mL) under argon atmosphere, and cooled at 0 ꢀC.
Ph3P (0.4 g, 1.53 mmol, 1.5 equiv) and then DIAD (0.3 mL,
1.43 mmol, 1.4 equiv) were added dropwise. The stirred mixture
was allowed to reach slowly room temperature and further left
for 1-12 h at 20 ꢀC. Concentration under vacuum and flash
chromatography on silica gel gave the coupled product 2 of
Table 1.
(S)-t-Butyl 3-(4-(4-(t-Butoxycarbonyl(4-methylpyridin-2-yl)-
amino)butoxy)-3-nitrophenyl)-2-(3-(trifluoromethyl)phenylsulfon-
amido)propanoate (2g). The title compound was obtained from
17b (0.250 g, 0.89 mmol) as a pale-yellow oil (0.424 g, 63%). Rf
(nHex/EtOAc 6:4) = 0.6. IR 2978, 1701, 1533, 1327, 1161 cm-1
.
1H NMR (500 MHz, CDCl3) δ 1.26 (s, 9 H), 1.5 (s, 9 H), 1.83 (m,
4 H), 2.33(s, 3 H), 2.99(m, 1 H), 3.07(m, 1 H), 3.98 (t, J = 6.4Hz,
2 H), 4.06 (m, 3 H), 5.26 (d, J = 6.5 Hz, SO2NH, 1 H), 6.84 (d,
J = 5.1 Hz, 1 H), 6.93 (d, J = 8.6 Hz, 1 H), 7.35 (dd, J = 8.6, 2.2
Hz, 1 H), 7.4 (s, 1 H), 7.55 (s, 1 H), 7.61 (t, J = 7.8 Hz, 1 H), 7.79
(d, J = 7.8 Hz, 1 H), 7.96 (d, J = 7.9 Hz, 1 H), 8.04 (s, 1 H), 8.21
(d, J = 5.1 Hz, 1 H). 13C NMR (125 MHz, CDCl3) δ 21, 25.25,
26.25, 27.55, 28.18, 37.98, 46.15, 56.68, 69.23, 80.8, 83.63, 114.38,
120.57, 120.88, 124.08, 124.7 (CF3), 126.41, 127.18, 129.24,
129.76, 130.34, 131.55, 135.44, 139.08, 140.95, 147.18, 148.14,
151.54, 154.18, 154.46, 169.23. MS (APCI) m/z 697, 641. HRMS
C35H43F3N4O9S calcdfor [M þ Na]þ, 775.2601;found, 775.2628.
(S)-t-Butyl 3-(3-Nitro-4-(4-oxo-pentyloxy)phenyl)-2-(3-(trifluoro-
methyl)phenylsulfonamido)propanoate (2k). The title compound was
Experimental Section
Chemistry. Reagents were purchased from Aldrich or Acros
Organics and used as received. Anhydrous solvents were pur-
chased from Fluka. Reactions needing anhydrous conditions
were performed in flame-dried glassware, placed under an argon
atmosphere. 1H and 13C NMR spectra were recorded on Bruker
300 Ultra Shield and Bruker AM 500 spectrometers, using
deuterated solvents from Rocc SA (Belgium) and TMS
(tetramethylsilane) as internal standard. Patterns are designed
as s (singlet), br s (broad singlet), d (doublet), dd (doublet of
doublet), t (triplet), q (quartet), m (multiplet). Coupling con-
stants (J) are given in hertz (Hz). IR spectra were recorded on a

7038 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Rerat et al.
obtained from 20 (0.082 mL, 0.861 mmol) as a pale-yellow oil (0.277
g, 59%). Rf (ether/nHex 85:15) = 0.46. IR 2978, 1716, 1531, 1327,
1163 cm-1. 1H NMR (300 MHz, CDCl3) δ 1.27 (s, 9 H), 2.09 (m,
2 H), 2.19 (s, 3 H), 2.73 (t, J = 6.8 Hz, 2 H), 2.99 (m, 1 H), 3.07 (m,
1 H), 4.06 (m, 3 H), 5.45 (d, J = 8.9 Hz, SO2NH, 1 H), 6.96 (d, J =
8.6Hz, 1H), 7.37(dd,J= 8.6, 2.2 Hz, 1 H), 7.6 (d, J= 2.2 Hz, 1 H),
7.63 (t, J = 7.8 Hz, 1 H), 7.8 (d, J = 7.8 Hz, 1 H), 7.98 (d, J =
7.8 Hz, 1 H), 8.04 (s, 1 H). 13C NMR (75 MHz, CDCl3) δ 23.06,
27.86, 30.29, 38.24, 39.45, 57.08, 68.47, 84, 114.73, 124.35, 126.69,
127.68, 129.66, 130.17, 130.68, 131.55, 135.81, 139.11, 141.07,
151.73, 169.38, 208.47, (CF3 not visible). MS (APCI) m/z 573
[M - H]-, 517. HRMS C25H29F3N2O8S calcd for [M þ Na]þ,
597.1494; found, 597.1481.
21, 25.44, 26.56, 27.57, 28.22, 38.76, 46.42, 56.96, 67.86, 80.64,
82.6, 111.07, 115.78, 119.16, 119.51, 120.99, 124.1, 124.7 (CF3),
127.07, 129.04, 129.6, 130.37, 131.35, 136.12, 141.13, 145.72,
147.18, 148.14, 154.18, 154.46, 169.53. MS (APCI) m/z 721 [M
- H]-, 621, 209. HRMS C35H45F3N4O7S calcd for [M þ H]þ,
723.3039; found, 723.3057. Anal. calcd (%) C, 58.17; H, 6.23; N,
7.76; S, 4.45. Found C, 58.13; H, 6.37; N, 7.75; S, 4.98.
(S)-t-Butyl 3-(3-Amino-4-(3-(5,6,7,8-tetrahydro-1,8-naphthy-
ridin-2-yl)propoxy)phenyl)-2-(3-(trifluoromethyl)phenylsulfon-
amido)propanoate (4k). The title compound was obtained
from 3c (0.150 g, 0.227 mmol), according to method A, as a
pale-brown foam (0.143 g, 98%). Rf (EtOAc/acetone 8:2) =
0.2. [R]2D0 -11 (c = 1, CHCl3). IR 2955, 1731, 1651, 1518, 1327,
1159 cm-1. 1H NMR (500 MHz, CDCl3) δ 1.21 (s, 9 H), 1.9 (m,
2 H), 2.25 (m, 2 H), 2.68 (br s, NH2, 2 H), 2.71 (t, J = 6.1 Hz, 2
H), 2.85 (m, 2 H), 2.91 (t, J = 7.4 Hz, 2 H), 3.47 (m, 2 H), 3.93
(t, J = 5.8 Hz, 2 H), 4.02 (m, 1 H), 5.5 (m, SO2NH, 1 H), 6.35
(m, 2 H), 6.43 (s, 1 H), 6.57 (d, J = 8.4 Hz, 1 H), 7.26 (d, J = 7.3
Hz, 1 H), 7.56 (t, J = 7.8 Hz, 1 H), 7.73 (d, J = 7.8 Hz, 1 H), 7.9
(S)-t-Butyl 3-(4-(3-[1,8]Naphthyridin-2-yl-propoxy)-3-nitro-
phenyl)-2-(3-(trifluoromethyl)phenylsulfonamido)propanoate (3c). A
solution of 2-amino-3-pyridin-carboxaldehyde (0.064 g, 0.517
mmol), precursor 2k (0.270 g, 0.470 mmol) and (L)-proline (0.027
g, 0.235 mmol) in EtOH (5 mL) was refluxed for 48 h under Ar
atmosphere. After concentration under vacuum, the residue was
purified by chromatography to afford the title compound (0.167 g,
49%) as a white solid. Rf (DCM/i-PrOH 98:2) = 0.4. mp =
(d, J = 7.8 Hz, 1 H), 8.01 (br s, NH, 1 H), 8.1 (br s, 1 H). 13
C
NMR (125 MHz, CDCl3) δ 19.44, 25.42, 27.56, 28.51, 30.17,
38.59, 40.91, 57.16, 66.39, 82.55, 109.98, 111.03, 115.92,
117.92, 118.92, 124, 124.7 (CF3), 127.5, 129, 129.64, 130.38,
131.35, 136.26, 139.96, 141.25, 145.33, 148.69, 152.46, 169.71.
MS (ESI) m/z 635 [M þ H]þ, 579. HRMS C31H37F3N4O5S
calcd for [M þ H]þ, 635.2515; found, 635.2525. Anal. Calcd
1
143-144 ꢀC. IR 1731, 1610, 1533, 1327, 1161 cm-1. H NMR
(500 MHz, CDCl3) δ 1.26 (s, 9 H), 2.51 (quint, J = 6.3 Hz, 2 H),
2.97 (m, 1 H), 3.05 (m, 1 H), 3.3 (t, J = 6.3 Hz, 2 H), 4.06 (m, 1 H),
4.2 (t, J = 6.3 Hz, 2 H), 5.38 (d, J = 9 Hz, SO2NH, 1 H), 6.97 (d,
J= 8.6 Hz, 1 H), 7.33 (dd, J= 8.6, 2.2 Hz, 1 H), 7.46 (d, J=8.4Hz,
1 H), 7.46 (dd, J= 7.9, 4.3 Hz, 1 H), 7.57 (d, J= 2.2 Hz, 1 H), 7.6 (t,
J= 7.8 Hz, 1 H), 7.77 (d, J= 7.8 Hz, 1 H), 7.96 (d, J= 7.8 Hz, 1 H),
8.03 (s, 1 H), 8.11 (d, J = 8.4 Hz, 1 H), 8.17 (dd, J = 7.9, 2 Hz, 1 H),
9.08 (dd, J = 4.3, 2 Hz, 1 H). 13C NMR (125 MHz, CDCl3) δ 27.45,
27.55, 34.58, 37.95, 56.73, 68.52, 83.69, 114.49, 121.06, 121.44,
123.02, 124.07, 126.33, 127.12, 129.3, 129.81, 130.34, 131.55,
135.38, 136.74, 137.03, 139.07, 140.78, 151.55, 153.2, 155.83,
for M 0.5H2O (%) C, 57.85; H, 5.90. Found C, 58.02; H, 5.82.
3
General Procedure for Acylation of 4. Precursor 4 (0.1 mmol)
dissolved in DCM (2 mL) was treated successively with Et3N
(0.11 mmol) and acetyl chloride (0.11 mmol) at 0 ꢀC under Ar
atmosphere. The mixture was stirred for 15 min at 0 ꢀC and 1 h at
20 ꢀC. Dilution with EtOAc, washing with water, drying
(MgSO4) and chromatography gave the acetanilide.
165.18, 169.03, (CF3 not visible). MS (ESI) m/z 1318 [M - H]-
ꢀ
(S)-t-Butyl 3-(3-Acetamido-4-(4-(t-butoxycarbonyl(4-methyl-
pyridin-2-yl)amino)butoxy)phenyl)-2-(3-(trifluoromethyl)phenyl-
sulfonamido)propanoate (4m). The title compound was obtained
from 4g (0.05 g) as a white foam (0.036 g, 62%). Rf (ether/n-Hex
9:1) = 0.44. 1H NMR (300 MHz, CDCl3) δ 1.27 (s, 9 H), 1.5 (s,
9 H), 1.83 (m, 4 H), 2.13 (s, 3 H), 2.34 (s, 3 H), 2.87 (m, 1 H), 3.02
(m, 1 H), 4.02 (m, 5 H), 5.2 (d, J = 9.4 Hz, SO2NH, 1 H), 6.68 (d,
J = 8.4 Hz, 1 H), 6.79 (dd, J = 8.4, 2 Hz, 1 H), 6.85 (d, J = 5 Hz,
1 H), 7.39 (s, 1 H), 7.56 (t, J = 7.9 Hz, 1 H), 7.74 (d, J = 7.9 Hz,
1 H), 7.8 (br s, NHCO, 1 H), 7.94 (d, J = 7.9 Hz, 1 H), 8.02 (s,
1 H), 8.09 (d, J = 2 Hz, 1 H), 8.2 (d, J = 5 Hz, 1 H). 13C NMR
(75 MHz, CDCl3) δ 21.34, 25.03, 25.86, 26.57, 27.9, 28.54,
38.96, 46.67, 57.42, 68.53, 81.2, 83.22, 110.82, 120.87, 121.04
121.23, 124.4, 124.88, 127.66, 127.82, 129.1, 129.87, 130.81,
131.81, 141.45, 146.45, 147.47, 148.5, 154.5, 154.7, 167.19,
169.86, (CF3 not visible). MS (ESI) m/z 787 [M þ Na]þ, 765
[M þ H]þ. HRMS C37H47F3N4O8S calcd for [M þ Na]þ,
787.2964; found, 787.2935.
(S)-t-Butyl 3-(3-Acetamido-4-(3-(5,6,7,8-tetrahydro-1,8-naphthy-
ridin-2-yl)propoxy)phenyl)-2-(3-(trifluoromethyl)phenylsulfon-
amido)propanoate (4n). The title compound was obtained from
4k (0.1 g) as a white solid (0.07 g, 66%). Rf (acetone/n-Hex
8:2) = 0.56. IR 2955, 1731, 1651, 1327, 1159 cm-1. 1H NMR
(300 MHz, CDCl3) δ 1.26 (s, 9 H), 1.91 (m, 2 H), 2.17 (m þ s,
5 H), 2.72 (m, 4 H), 2.86 (m, 1 H), 3.02 (m, 1 H), 3.39 (m, 2 H),
4.01 (t, J = 6.4 Hz, 2 H), 4.07 (m, 1 H), 4.85 (br s, SO2NH, 1 H),
5.3 (br s, NH, 1 H), 6.36 (d, J = 7.3 Hz, 1 H), 6.7 (d, J = 8.3 Hz,
1 H), 6.79 (dd, J = 8.3, 2 Hz, 1 H), 7.08 (d, J = 7.3 Hz, 1 H),
7.54 (t, J = 7.9 Hz, 1 H), 7.73 (d, J = 7.9 Hz, 1 H), 7.85 (br s,
CONH, 1 H), 7.93 (d, J = 7.9 Hz, 1 H), 8.02 (s, 1 H), 8.09 (d,
J = 2 Hz, 1 H). 13C NMR (75 MHz, CDCl3) δ 19.44, 25.14,
26.55, 27.92, 29.22, 34.1, 39.01, 41.81, 57.41, 68.29, 83.24,
111.35, 111.52, 114.04, 120.99, 124.43, 124.85, 127.71, 128.05,
129.27, 129.89, 130.82, 131.9, 137.14, 141.46, 146.49, 155.94,
156.73, 168.37, 170.05 (CF3 not visible). MS (ESI) m/z 675
[M - H]-, 209. HRMS C33H39F3N4O6S calcd for [M þ H]þ,
677.2621; found, 677.2606.
2, 659 [M - H]-, 209. HRMS C31H31F3N4O7S calcd for [M þ H]þ,
661.1944; found, 661.1949.
General Procedure for Reduction of 2-3 (Table 2). Method A.
A solution of 2 or 3 in MeOH or EtOH (0.1 mmol/3 mL)
containing Pd/C (10%) as catalyst (0.01 g/0.1 mmol product 2 or
3) was placed under H2 atmosphere (1 atm) and stirred for 18 h
at 20 ꢀC. The mixture was filtered over a short celite pad, using
MeOH (EtOH); filtrate concentration under vacuum gave
quantitatively crude 4.
Method B. A solution of 2 (0.2 mmol) in HOAc (5 mL) and
37% HClaq (0.5 mL), containing Pd/C (10%) as catalyst (0.1 g)
was introduced in a Parr flask. The mixture was hydrogenated
(Parr apparatus) for 2 h at 20 ꢀC, under a pressure of 45 psi. The
mixture was filtered on a celite pad, using MeOH-H2O. Con-
centration under vacuum and chromatography gave crude 4,
recovered by lyophilization.
Method C. A solution of 2 (0.5 mmol) and ammonium
formate (5 mmol) in EtOH (5 mL), containing Pd/C (10%) as
catalyst (0.05 g), was refluxed for 12 h under Ar atmosphere and
vigorous stirring. The mixture was filtered on a celite pad, using
EtOH. After concentration, the residue was dissolved in EtOAc
(10 mL), washed with brine (2 ꢀ 5 mL), and dried (MgSO4).
Solvent evaporation gave crude compound 4.
(S)-t-Butyl 3-(3-Amino-4-(4-(t-butoxycarbonyl(4-methylpyri-
din-2-yl)amino)butoxy)phenyl)-2-(3-(trifluoromethyl)phenylsulfon-
amido)propanoate (4g). The title compound was obtained from 2g
(0.420 g, 0.560 mmol), according to method A, as a pale-yellow oil
(0.361 g, 89%). Rf (ether/EtOAc 9:1) = 0.9. [R]2D0 -11 (c = 1.3,
1
CHCl3). IR 2975, 1703, 1325, 1159 cm-1. H NMR (500 MHz,
CDCl3) δ 1.26 (s, 9 H), 1.49 (s, 9 H), 1.79 (m, 4 H), 2.33 (s, 3 H),
2.87 (m, 2 H), 3.71 (br s, NH2, 2 H), 3.93 (t, J = 6 Hz, 2 H), 3.99 (t,
J = 7 Hz, 2 H), 4.06 (m, 1 H), 5.16 (d, J = 8.9 Hz, SO2NH, 1 H),
6.39 (dd, J = 8, 2.1 Hz, 1 H), 6.43 (d, J = 2.1 Hz, 1 H), 6.57 (d, J =
8.2 Hz, 1 H), 6.84 (d, J = 5.3 Hz, 1 H), 7.39 (s, 1 H), 7.57 (t, J = 7.8
Hz, 1 H), 7.75 (d, J = 7.8 Hz, 1 H), 7.91 (d, J = 7.8 Hz, 1 H), 8.05
(s, 1 H), 8.21 (d, J = 5.3 Hz, 1 H). 13C NMR (125 MHz, CDCl3) δ

Article
General Procedure for the Coupling of Spacer-Arms (Table 3).
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7039
26.4, 27.88, 28.6, 29.12, 33.3, 38.83, 40.46, 41.76, 57.49, 67.83,
70.1-71, 83.23, 111.39, 111.56, 114.85, 121.71, 124.36, 125.53,
126.85, 127.8, 129.33, 129.98, 130.82, 131.72, 137.86, 141.46,
147.23, 155.27, 155.96, 156.61, 168.26, 169.65, (CF3 not visible).
MS (ESI) m/z 922 [M - H]-. HRMS C44H60F3N5O11S calcd for
[M þ Na]þ, 964.3860; found, 964.3831.
Acid 23 or 24 (see Scheme 1, 0.2 mmol) dissolved in DMF (1 mL)
was treated successively with PyBOP (0.011 g, 0.2 mmol) and
Et3N (0.027 mL, 0.2 mmol) at 20 ꢀC under Ar atmosphere. After
2 h, precursor 4 (0.18 mmol) dissolved in DMF (0.3 mL) was
added dropwise with a syringe, and the mixture was stirred for
18 h at 20 ꢀC. After dilution with brine (5 mL), the solution was
extracted with ether (3 ꢀ 5 mL) and EtOAc (1 ꢀ 5 mL). The
combined organic phases were washed with brine (5 mL), dried
over MgSO4, and concentrated under vacuum. The residue
(brown oil) was purified by chromatography on silica gel.
(S)-t-Butyl 3-(3-(2-(2-(2-(2-(t-Butoxycarbonyl)aminoethoxy)-
ethoxy)ethoxy)acetamido)-4-(4-(4-methylpyridin-2-yl-t-butoxy-
carbonylamino)butoxy)phenyl-2-(3-(trifluoromethyl)phenylsulfon-
anmido)propanoate (6a). The title compound was obtained from
23a (0.046 g) and 4g (0.1 g) as a white foam (0.052 g, 37%). Rf
(EtOAc/acetone 8:2) = 0.53. IR 2936, 1701, 1327, 1163 cm-1. 1H
NMR (500 MHz, CDCl3) δ 1.27 (s, 9 H), 1.44 (s, 9 H), 1.49 (s,
9 H), 1.84 (m, 4 H), 2.34 (s, 3 H), 2.88 (m, 1 H), 3.01(m, 1 H), 3.28
(m, 2 H), 3.5 (t, J = 5.8 Hz, 2 H), 3.61-3.75 (m, 8 H), 4.02 (m,
4 H), 4.07 (m, 1 H), 4.12 (s, 2 H), 5.07 (br s, BocNH, 1 H), 5.32 (d,
J = 10 Hz, SO2NH, 1 H), 6.72 (d, J = 8.3 Hz, 1 H), 6.85 (dd, J =
8.3, 2 Hz, 1 H), 6.87 (d, J = 5.4 Hz, 1 H), 7.4 (s, 1 H), 7.57 (t, J =
7.8 Hz, 1 H), 7.75 (d, J = 7.8 Hz, 1 H), 7.95 (d, J = 7.8 Hz, 1 H),
8.02 (s, 1 H), 8.13 (d, J = 2 Hz, 1 H), 8.2 (d, J = 5.4 Hz, 1 H), 8.9
(br s, CONH, 1 H). 13C NMR (125 MHz, CDCl3) δ 21.42, 25.53,
26.79, 27.91, 28.51, 28.64, 38.95, 40.52, 46.54, 57.39, 68.36,
70.37-71.2, 71.35, 81.43, 83.25, 111.12, 120.6, 120.98, 121.18,
124.41, 125.32, 127.13, 127.76, 129.31, 129.91, 130.81, 131.78,
141.47, 146.93, 147.12, 148.38, 154.38, 154.42, 156.22, 167.78,
169.94, (CF3 not visible). MS (ESI) m/z 1010 [M - H]-, 910.
HRMSC48H68F3N5O13S calcd for [M þ Na]þ, 1034.4384; found,
1034.4384.
(S)-t-Butyl 3-(3-(2-(2-(2-(2-(2-(2-(2-(t-Butoxycarbonyl) amino-
ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)acetamido)-4-(4-(4-
methylpyridin-2-yl-t-butoxycarbonylamino)butoxy)phenyl-2-(3-(tri-
fluoromethyl)phenylsulfonanmido)propanoate (7a). The title com-
pound was obtained from 23b (0.171 g) and 4g (0.219 g) as a pale-
yellow foam (0.165 g, 50%). Rf (EtOAc/acetone 9:1) = 0.57. IR
2932, 1700, 1541, 1327, 1163 cm-1. 1H NMR (500 MHz, CDCl3) δ
1.25 (s, 9 H), 1.43 (s, 9 H), 1.49 (s, 9 H), 1.79 (m, 2 H), 1.85 (m, 2 H),
2.34 (s, 3 H), 2.88 (m, 1 H), 3.01 (m, 1 H), 3.3 (m, 2 H), 3.54 (t, J =
5.1 Hz, 2 H), 3.61-3.75 (m, 20 H), 4 (m, 4 H), 4.07 (m, 1 H), 4.17 (s,
2H), 5.14(brs, BocNH, 1H), 5.44(brs, SO2NH, 1H), 6.71(d, J=
8.3 Hz, 1 H), 6.81 (dd, J= 8.3, 2 Hz, 1 H), 6.86 (d, J=5.4Hz, 1H),
7.37 (s, 1 H), 7.58 (t, J = 7.8 Hz, 1 H), 7.75 (d, J = 7.8 Hz, 1 H),
7.92 (d, J = 7.8 Hz, 1 H), 8 (s, 1 H), 8.13 (s, 1 H), 8.2 (d, J = 5.4 Hz,
1 H), 8.7 (s, CONH, 1 H). 13C NMR (125 MHz, CDCl3) δ 21.27,
25.56, 26.47, 27.82, 28.47, 28.53, 38.98, 40.29, 46.57, 57.48, 68.52,
69.7-70.78, 71.3, 79.53, 81.14, 83.21, 111.1, 120.94, 121.28, 122,
124.24, 125.39, 126.89, 127.55, 129.33, 130.02, 130.73, 131.76,
141.32, 147.03, 147.47, 148.51, 154.4, 154.7, 155, 167.19, 169.86,
(CF3 not visible). MS (ESI) m/z 1142 [M - H]-. HRMS
C54H80F3N5O16S calcd for [M þ Na]þ, 1166.5171; found,
1166.5159.
(S)-t-Butyl 3-(3-(2-(2-(2-(2-(2-(2-(2-(t-Butoxycarbonyl) amino-
ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)acetamido)-4-(3-
(5,6,7,8-tetrahydro-1,8,naphthyridin-2-yl)propoxy)phenyl)-2-(3-
(trifluoromethyl)phenylsulfonamido)propanoate (7b). The title
compound was obtained from 23b (0.171 g) and 4k (0.344 g)
as a very viscous colorless oil (0.187 g, 33%). Rf (EtOAc/
acetone 1:1) = 0.6. IR 2926, 1704, 1327, 1161 cm-1 1H
.
NMR (500 MHz, CDCl3) δ 1.26 (s, 9 H), 1.45 (s, 9 H), 1.89
(m, 2 H), 2.19 (m, 2 H), 2.69 (t, J = 6.2 Hz, 2 H), 2.74 (t, J =
7.4 Hz, 2 H), 2.86-3.02 (m, 2 H), 3.31 (m, 2 H), 3.39 (m, 2 H),
3.51-3.8 (m, 22 H), 3.99 (t, J = 6.4 Hz, 2 H), 4.07 (m, 1 H), 4.13
(s, 2 H), 4.85 (br s, BocNH, 1 H), 5.35 (br s, SO2NH, 1 H), 6.36
(d, J = 7.3 Hz, 1 H), 6.7 (d, J = 8.3 Hz, 1 H), 6.79 (dd, J = 8.3,
2 Hz, 1 H), 7.08 (d, J = 7.3 Hz, 1 H), 7.54 (t, J = 7.9 Hz, 1 H),
7.73 (d, J = 7.9 Hz, 1 H), 7.85 (br s, CONH, 1 H), 7.93 (d, J =
7.9 Hz, 1 H), 8.02 (s, 1 H), 8.13 (d, J = 2 Hz, 1 H), 8.99 (br s,
NH-CdN, 1 H). 13C NMR (75 MHz, CDCl3) δ 21.61, 26.53,
27.86, 28.6, 29.22, 33.98, 38.88, 40.51, 41.74, 57.45, 67.91,
70.3-71.3, 83.12, 111.09, 111.55, 113.74, 120.74, 124.33,
125.18, 127.08, 127.67, 129.18, 129.83, 130.73, 131.72, 136.88,
141.49, 146.88, 156.06, 156.91, 156.93, 167.71, 170.03, (CF3 not
visible). MS (ESI) m/z 1054 [M - H]-. HRMS C50H72F3-
N5O14S calcd for [M þ H]þ, 1056.4827; found, 1056.4845.
General Procedure for Boc Deprotection (Table 4). Boc-pro-
tected compound (0.1 g) dissolved in DCM (1 mL) was treated
with TFA (1 mL) during 2 h at 20 ꢀC. Concentration under
vacuum quantitatively gave the peptidomimetic (for testing) as
TFA salt. Compounds are stored in the fridge (-18 ꢀC) as TFA
salt. Neutralization could be performed by dissolution in DCM
(0.1 g/5 mL DCM), washing with phosphate buffer (pH 8, 2 ꢀ
1 mL), drying (MgSO4), and concentration (yellow oils).
(S)-3-(3-Amino-4-(4-(4-methylpyridin-2-ylamino)butoxy)phenyl)-
2-(3-(trifluoromethyl)phenylsulfonamido)propanoic Acid (5g). 1H
NMR (500 MHz, CD3OD) δ 1.96 (m, 4 H), 2.4 (s, 3 H), 2.86 (m,
1 H), 3.12 (m, 1 H), 3.44 (t, J = 6.6 Hz, 2 H), 4.11 (m, 1 H), 4.16 (t,
J = 7.8 Hz, 2 H), 6.73 (dd, J = 6.7, 1.2 Hz, 1 H), 6.88 (s, 1 H), 7.04
(d, J= 8.4 Hz, 1 H), 7.22 (dd, J=8.4, 2Hz, 1H), 7.25(d,J=2Hz,
1 H), 7.69 (m, 2 H), 7.86 (d, J = 7.9 Hz, 1 H), 7.96 (d, J = 7.9 Hz,
1 H), 8.02 (s, 1 H). 13C NMR (125 MHz, CD3OD) δ 22.81, 26.74,
28.24, 39.69, 43.71, 59.66, 70.35, 113.65, 114.66, 116.33, 121.28,
125.68, 126.78, 130.9, 131.93, 132.02, 132.4, 132.86, 133.35, 136.39,
144.49, 144.5, 153.12, 155.07, 174.34. HRMS C26H29F3N4O5Scalcd
for [M þ H]þ, 567.1889; found, 567.1882.
(S)-3-(3-Amino-4-(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-
butoxy)phenyl)-2-(3-(trifluoromethyl)phenylsulfonamido)propanoic
Acid (5l). 1H NMR (500 MHz, CD3OD) δ 1.93 (m, 6 H), 2.83 (m,
5 H), 3.1 (m, 1 H), 3.49 (t, J = 5.6 Hz, 2 H), 4.1 (m, 1 H), 4.15 (m,
2 H), 6.63 (d, J = 7.3 Hz, 1 H), 7.05 (d, J = 9 Hz, 1 H), 7.25 (m,
2 H), 7.57 (d, J = 7.3 Hz, 1 H), 7.69 (t, J = 7.9 Hz, 1 H), 7.86 (d,
J = 7.9Hz, 1 H), 7.98 (d, J = 7.9Hz, 1 H), 8.03 (s, 1 H). 13CNMR
(125 MHz, CD3OD) δ 21.38, 27.19, 27.34, 30.26, 34.1, 39.84,
43.06, 59.66, 70.36, 112.51, 114.62, 121.52, 124.64, 125.76, 126.59,
130.92, 132.04, 132.44, 132.96, 133.27, 143.63, 144.58, 150.58,
153.04, 153.76, 174.43, (C-CF3 not visible). HRMS C28H31F3-
N4O5S calcd for [M þ H]þ, 593.2046; found, 593.2022.
(S)-t-Butyl 3-(3-(2-(2-(2-(2-(t-Butoxycarbonyl)aminoethoxy)-
ethoxy)ethoxy)acetamido)-4-(3-(5,6,7,8-tetrahydro-1,8-naphthyr-
idin-2-yl)propoxy)phenyl-2-(3(trifluoromethyl)phenylsulfonamido)-
propanoate (6b). The title compound was obtained from 23a
(0.058 g) and 4k (0.110 g) as a white foam (0.059 g, 37%). Rf
(EtOAc/acetone 6:4) = 0.57. IR 2931, 1684, 1327, 1161 cm-1. 1H
NMR (500 MHz, CDCl3) δ 1.26 (s, 9 H), 1.42 (s, 9 H), 1.91 (m,
2 H), 2.19 (m, 2 H), 2.71 (t, J = 6.2 Hz, 2 H), 2.75 (t, J = 7.4 Hz,
2 H), 2.85-3.04 (m, 2 H), 3.28 (m, 2 H), 3.41 (m, 2 H), 3.46-3.8
(m, 10 H), 3.98 (t, J = 6.4 Hz, 2 H), 4.01 (m, 1 H), 4.13 (s, 2 H),
5.05-5.4 (m, SO2NH þ BocNH, 2 H), 6.35 (d, J = 7.2 Hz, 1 H),
6.71 (d, J = 8.3 Hz, 1 H), 6.82 (d, J = 8.3 Hz, 1 H), 7.12 (d, J =
7.2 Hz, 1 H), 7.56 (t, J = 7.9 Hz, 1 H), 7.74 (d, J = 7.9 Hz, 1 H),
7.94 (d, J = 7.9 Hz, 1 H), 8.03 (s, 1 H), 8.14 (s, 1 H), 8.9 (br s,
CONH þ HN-CdN, 2 H). 13C NMR (75 MHz, CDCl3) δ 21.23,
(S)-3-(3-Acetamido-4-(4-(4-methylpyridin-2-ylamino)butoxy)-
phenyl)-2-(3-(trifluoromethyl)phenylsulfonamido)propanoic Acid
1
(5m). H NMR (500 MHz, CD3OD) δ 1.93 (m, 4 H), 2.14 (s,
3 H), 2.38 (s, 3 H), 2.75 (m, 1 H), 3.02 (m, 1 H), 3.4 (t, J = 7 Hz,
2 H), 4.07 (m, 3 H), 6.73 (d, J = 6.6 Hz, 1 H), 6.8 (d, J = 8.5 Hz,
1 H), 6.82 (s, 1 H), 6.88 (dd, J = 8.5, 2.1 Hz, 1 H), 7.58 (m, 2 H),
7.67 (d, J = 6.6 Hz, 1 H), 7.8 (d, J = 7.9 Hz, 1 H), 7.85 (d, J =
7.9 Hz, 1 H), 7.95 (s, 1 H). 13C NMR (125 MHz, CD3OD) δ
22.82, 24.55, 26.87, 28.31, 40, 43.72, 60, 69.83, 113.66, 113.67,

7040 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Rerat et al.
116.4, 125.54, 126.31, 128.38, 128.56, 130.58, 130.83, 131.9,
132.36, 133, 133.5, 136.26, 144.69, 151.07, 154.96, 172.63,
174.94, (CF3 not visible). HRMS C28H31F3N4O6S calcd for
[M þ H]þ, 609.1995; found, 609.1998.
7.77 (d, J = 7.5 Hz, 1 H), 7.93 (m, 3 H). 13C NMR (75 MHz,
CD3OD) δ 22.32, 27.81, 30.79, 33.35, 41.06, 41.51, 42.99, 61.99,
68.78, 69.48, 72.76-72.81, 112.57, 113.28, 118.78, 123.47,
125.63, 128.02, 128.06, 130.6, 131.64, 131.99, 132.24, 132.92,
141.11, 144.54, 149.23, 154.25, 156.16, 171.14, 177.99, (CF3 not
visible). HRMS C41H56F3N5O12S calcd for [M þ H]þ,
900.3677; found, 900.3699.
(S)-3-(3-Acetamido-4-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-
2-yl)propoxy)phenyl)-2-(3-(trifluoromethyl)phenylsulfonamido)-
1
propanoic Acid (5n). H NMR (500 MHz, CD3OD) δ 1.92 (m,
2 H), 2.17 (s, 3 H), 2.21(m, 2 H), 2.78 (m, 3 H), 2.91 (t, J = 7.4 Hz,
2 H), 3.02 (m, 1 H), 3.45 (t, J = 5.4 Hz, 2 H), 4.03 (t, J = 5.8 Hz,
2 H), 4.09 (m, 1 H), 6.63 (d, J = 7.4 Hz, 1 H), 6.77 (d, J = 8.4 Hz,
1 H), 6.9 (dd, J = 8.4, 1.8 Hz, 1 H), 7.56 (m, 2 H), 7.61 (t, J =
7.8 Hz, 1 H), 7.8 (d, J = 7.8 Hz, 1 H), 7.87 (d, J = 7.8 Hz, 1 H),
7.96 (s, 1 H). 13C NMR (125 MHz, CD3OD) δ 21.3, 24.6, 27.25,
30.03, 31.2, 40.02, 43.14, 60, 68.57, 112.85, 113.58, 121.7, 125.63,
126.67, 128.43, 128.67, 130.64, 131.1, 131.9, 132.41, 133.05,
143.69, 144.71, 149.79, 151.08, 153.57, 172.8, 174.84, (CF3 not
visible). HRMS C29H31F3N4O6S calcd for [M þ H]þ, 621.1995;
found, 621.1985.
Evaluations. Binding to Isolated Integrins. The human Rvβ3
integrins (purified from placenta) were purchased from Chemi-
con International Inc. (Temecula, CA), and RIIbβ3 integrins
were purified from platelets by affinity chromatography, as
described.82 The integrins were diluted to 500 ng/mL (Rvβ3) or
1 μg/mL (RIIbβ3) in a Tris binding buffer (2 mM CaCl2, 1 mM
MgCl2 and MnCl2, pH 7.5), and absorbed onto 96-well Costar
(Bethesda, MD) binding plates (100 μL per well). After a one-
hour incubation at room temperature, the plates were incubated
for another hour with a blocking solution of 1% albumin to
prevent nonspecific binding. The plates were then washed three
times in binding buffer containing 0.1% albumin. The physio-
logical ligands vitronectin (BD Biosciences, Le Pont de Claix,
France) for Rvβ3 and fibrinogen (Sigma) for RIIbβ3 were labeled
with N-hydroxysuccinimido-biotin. Stock solutions of com-
pounds were solubilized at 10 mM in dimethylsulfoxide
(DMSO). All compounds were aliquoted and stored at
-20 ꢀC. Adsorbed integrins were then incubated for 30 min
with their corresponding soluble biotinylated ligand in excess, in
the binding buffer containing 0.1% albumin and in the presence
of various concentrations of tested compounds. After several
washings, the amount of bound ligands was indirectly estimated
after the sequential incubation with an antibody against biotin
coupled to alkaline phosphatase and para-nitrophenyl phos-
phate for readout at 405 nm in a colorimetric assay. The
concentration of compound corresponding to 50% inhibition
(IC50) was inferred from the dose-response curves of at least
two pooled experiments and used to compare the products.
Human OsteoProgenitor (HOP) Cell Cultures. HOP cells
were isolated from human bone marrow stromal cells
(HBMSC) according to Vilamitjana et al.,83 with some mod-
ifications.84 Briefly, human bone marrow was obtained by
aspiration from the iliac crest of healthy donors (30-70 y/o)
undergoing hip prosthesis surgery after traumatic shock. Cells
were separated into single suspension by sequentially passing
the suspension through syringes fitted with 16, 18, and 21 gauge
needles. After centrifugation for 15 min at 800g, the pellet was
resuspended in Iscove Modified Dulbecco’s Medium (IMDM,
Gibco) supplemented with 10% (v/v) fetal calf serum (FCS,
Gibco) and 10-8 M dexamethasone (Sigma). Cells were then
plated into 75 cm2 cell culture flasks (Nunc) and incubated in a
humidified atmosphere of 95% air and 5% CO2 at 37 ꢀC. For
two weeks, the complete medium was supplemented with 10-8
dexamethasone and then every 3 days with IMDM containing
10% FCS (v/v). Subculturing was performed using 0.2% (w/v)
trypsin, 5 mM EDTA. Cells arising from the second subculture
were used in these experiments to get a homogeneous cell
population in terms of cell phenotype. Alkaline phosphatase
and Cbfa1/runx2 gene expression were assessed by real time
quantitative PCR to control the osteoblastic phenotype.
(S)-3-(3-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)acetamido)-
4-(4-(4-methylpyridin-2-ylamino)butoxy)phenyl)-2-(3-(trifluoro-
methyl)phenylsulfonamido)propanoic Acid (8a). H NMR (500
1
MHz, CD3OD) δ 1.93 (m, 2 H), 1.98 (m, 2 H), 2.38 (s, 3 H), 2.74
(m, 1 H), 3.05 (m, 3 H), 3.43 (t, J = 6.8 Hz, 2 H), 3.60-3.85 (m,
10 H), 4.08 (m, 3 H), 4.18 (s, 2 H), 6.73 (dd, J = 6.6, 1.3 Hz, 1 H),
6.84 (m, 2 H), 6.91 (dd, J = 8.3, 2 Hz, 1 H), 7.61 (t, J = 7.8 Hz,
1 H), 7.7 (d, J = 6.6 Hz, 1 H), 7.81 (d, J = 7.8 Hz, 1 H), 7.89 (d,
J = 7.8 Hz, 1 H), 7.9 (bs, 2 H). 13C NMR (125 MHz, CD3OD) δ
22.85, 26.84, 28.45, 40.13, 41.44, 43.66, 60.08, 68.79, 69.84,
71.8-72.9, 113.4, 113.41, 116.42, 123.65, 125.5, 127.97, 128.26,
130.61, 131.24, 131.89, 132.38, 133, 136.43, 144.6, 149.57, 155,
155.1, 171.3, 174.96, (CF3 not visible). HRMS C34H45F3N5O9S
calcd for [M þ H]þ, 756.2890; found, 756.2886.
(S)-3-(3-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)acetamido)-
4-(3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propoxy)phenyl)-
2-(3-(trifluoromethyl)phenylsulfonamido)propanoic Acid (8b).
1H NMR (500 MHz, CD3OD) δ 1.92 (m, 2 H), 2.14 (m, 2 H),
2.75 (t, J = 6.2 Hz, 2 H), 2.81 (m, 3 H), 2.96 (m, 1 H), 3.03 (t, J =
5.2 Hz, 2 H), 3.4 (t, J = 5.1 Hz, 2 H), 3.6-3.79 (m, 10 H), 3.88
(m, 1 H), 4.03 (t, J = 5.8 Hz, 2 H), 4.15 (s, 2 H), 6.59 (d, J =
7.3 Hz, 1 H), 6.78 (d, J = 8.3 Hz, 1 H), 6.9 (dd, J = 8.3, 2 Hz,
1 H), 7.44 (d, J = 7.4 Hz, 1 H), 7.62 (t, J = 7.8 Hz, 1 H), 7.8 (d,
J = 7.8 Hz, 1 H), 7.89 (d, J = 2 Hz, 1 H), 7.96 (d, J = 7.8 Hz,
1 H), 8 (s, 1 H). 13C NMR (125 MHz, CD3OD) δ 21.46, 27.36,
30.36, 31.49, 40.79, 41.38, 42.75, 61.6, 68.8, 69.19, 72.09-72.84,
112.23, 113.28, 120.93, 124.05, 125.62, 128.1, 128.17, 130.63,
131.7, 131.99, 132.59, 142.79, 144.47, 149.23, 150.61, 155.06,
171.14, 177.99, (C-CF3 not visible). HRMS C35H44F3N5O19S
calcd for [M þ H]þ, 768.2890; found, 768.2883.
(S)-3-(3-(20-Amino-3,6,9,12,15,18-hexaoxaicosanamido)-4-(4-
(4-methylpyridin-2-ylamino)butoxy)phenyl)-2-(3-(trifluoromethyl)-
phenylsulfonamido)propanoic Acid (9a). 1H NMR (500 MHz,
CD3OD) δ 1.93-1.99 (m, 4 H), 2.38 (s, 3 H), 2.74 (m, 1 H), 3.05
(m, 3 H), 3.13 (t, J = 5 Hz, 2 H), 3.43 (m, 2 H), 3.59-3.81 (m,
20 H), 4.08 (m, 3 H), 4.18 (s, 2 H), 6.73 (dd, J = 6.5, 1.3 Hz, 1 H),
6.83 (m, 2 H), 6.91 (m, 1 H), 7.61 (m, 1 H), 7.7 (m, 1 H), 7.8 (d, J =
7.8 Hz, 1 H), 7.86 (d, J = 7.8 Hz, 1 H), 7.9 (m, 2 H). 13C NMR
(125 MHz, CD3OD) δ 22.85, 26.87, 28.45, 40.13, 41.52, 43.68,
60.11, 68.76, 69.84, 71.8-72.88, 113.4, 113.42, 116.42, 123.57,
125.5, 127.93, 128.26, 130.61, 131.18, 131.9, 132.37, 132.95, 136.43,
144.7, 149.54, 154.9, 154.94, 171.28, 174.94, (CF3 not visible).
HRMS C40H56F3N5O12S calcd for [M þ H]þ, 888.3677; found,
888.3674.
(S)-3-(3-(20-Amino-3,6,9,12,15,18-hexaoxaicosanamido)-4-(3-
(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propoxy)phenyl)-2-(3-
(trifluoromethyl)phenylsulfonamido)propanoic Acid (9b). 1H
NMR (300 MHz, CD3OD) δ 1.87 (m, 2 H), 2.15 (m, 2 H),
2.73 (m, 5 H), 3.01 (m, 1 H), 3.11 (t, J = 5.1 Hz, 2 H), 3.38 (t, J =
5.3 Hz, 2 H), 3.54-3.87 (m, 22 H), 4.03 (m, 3 H), 4.18 (s, 2 H),
6.45 (d, J = 7.2 Hz, 1 H), 6.76 (d, J = 8.4 Hz, 1 H), 7.26 (d, J =
8.4 Hz, 1 H), 7.27 (d, J = 7.2 Hz, 1 H), 7.6 (t, J = 7.5 Hz, 1 H),
Competition and Cell Adhesion Assay. The 96-well polystyr-
ene plates (Nunc) were coated with vitronectin from human
plasma (Sigma) dissolved at 5 μg/mL in IMDM 1 mM CaCl2.
The coated plates (30 μL per well) were conditioned 2 h at 37 ꢀC
and then rinsed in PBS before use. Peptidomimetics were
prepared at the following final concentrations: 10-4, 10-6
,
10-8, 10-10, 10-12 M. HOP (32000 cells/mL) were incubated
with peptidomimetics 5g, 5n, and 8b in serum-free IMDM to
saturate the integrin receptors on the cell surface for 1 h at 37 ꢀC
under gentle shaking before seeding. For each competition
assay, 100 μL of the cell suspension were inoculated onto
the vitronectin-coated well, and the competitive adhesion was
allowed to take place for 30 min at 37 ꢀC.

Article
Cell attachment was measured by a modified colorimetric
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7041
cells/cm2 in serum-free medium DMEM (Dulbecco’s Modified
Eagle Medium without phenol red), for 1, 3, and 6 h (n = 3). For
each incubation time, cell attachment was measured by a
colorimetric method according to Landegren et al.70 with some
modifications. The nonadherent cells were removed by washing
with PBS 0.1 M pH 7.4. Chromogenic substrate solution of
500 μL (see above, HOP assay) was added for 2 h at 37 ꢀC in
humidified atmosphere. Reaction was stopped (see above, HOP
assay), and the resulting chromophore was measured spectro-
photometrically at 405 nm. The positive and negative controls
for cell adhesion were the native tissue culture PET and the
tissue culture polystyrene (TCPS) coated with agarose (2%)
respectively. Results of Figure 8 are expressed as % of absor-
bance considering native PET as 100%. The U Mann-Whitney
nonparametric test was used to test significance.
method according to Landegren et al.70 After the nonadherent
cells were removed by washings with PBS 0.1 M pH 7.4, 500 μL
of chromogenic substrate solution (7.5 mM substrate (p-nitro-
phenyl N-acetyl β-D-glucosaminide); 0.1 M Na citrate; pH = 5;
5% (v/v) Triton-X 100) were added for 2 h at 37 ꢀC in humidified
atmosphere. The reaction was stopped with 5 mM EDTA/ 50
mM glycine pH 10.4. The resulting chromophore was measured
spectrophotometrically at 405 nm. The controls of cell adhesion
were the followings: cells nonincubated in the presence of
peptidomimetics then inoculated on culture plates coated with
vitronectin (positive controls) and cells incubated in the pre-
sence of GRGDS (Sigma) at the same final concentrations then
seeded on coated plates. Experiments were performed at least
twice (n = 6 wells per concentration). Results are expressed as
absorbance ratios of positive controls.
The U Mann-Whitney nonparametric test was used to test
significancy (p < 0.05).
Acknowledgment. This work was supported by the FRS-
FNRS (Belgium) and FRIA-FNRS (Belgium), and by UCL
(Louvain-la-Neuve) and ULg (Sart-Tilman) for computa-
tional facilities. V.R. is FRIA fellow, and G.D. and J.M.-B.
are senior research associates of FRS-FNRS. Sabrina De-
vouge (UCL) and Annie Genton (Servier) are acknowledged
for technical assistance.
Calculations. The geometry of the molecules has been fully
optimized at the RHF level using the polarized double ζ basis set
6-31G(d)85 and the minimal basis MINI-10 for the compound 8a
and 8b.86 All the calculations have been performed with the
Gaussian 03 program.87
Cell Culture System. Substrate Functionalization. Track-
etched microporous membrane of PET (cyclopore) used as cell
culture support was purchased from Whatman SA (Louvain-
la-Neuve, Belgium). Samples for surface modification were cut
in disks of 13 mm diameter and placed in a 24-well PS plate.
Samples were washed with acetonitrile (ACN) (1 mL/sample, 10
min shaking) then activated by immersion in a freshly prepared
1 M solution of trifluorotriazine (1 mL/sample) for 2 or 3 h at
30 or 40 ꢀC. After washing with ACN, the samples were
incubated in a 10-3 M solution of adhesive molecules
(graftable peptidomimetics and pentapeptide GRGDS) in phos-
phate buffer pH 8 (PB)-ACN (1:1, v/v; 1 mL/sample) for 17 h at
20 ꢀC. The samples were washed successively (1 mL/sample)
with PB-ACN, H2O-ACN, 1 N HCl-ACN, and H2O-ACN. For
XPS analysis, samples were dried under high vacuum. Blank
samples were prepared as above, but with omitting either the
activation step, or the incubation step. The analyses were
performed on a Kratos Axis Ultra spectrometer (Kratos Ana-
lytical, Manchester, UK) as previously described.88 The deriva-
tization rates were calculated from the experimental F/C atomic
ratios (for peptidomimetics) and the N/C atomic ratio (for
pentapeptide).43 Results of covalent grafting were validated by
comparison with the blank samples showing less than 0.01% of
contamination by adsorbed peptidomimetic/peptide.
Endothelial Cell Culture. Mature endothelial cells were iso-
lated from human saphenous veins (HSVEC) collected after
surgical coronary bypass according to the French legislation.
The samples were kept in Hanks’ balanced salt solution supple-
mented with heparin (50 UI/mL; Sanofi Synthelabo, Paris,
France) and transferred to the laboratory for isolation of
endothelial cells. Cells were harvested as described by Golledge
et al.89 The isolated cells were seeded on culture flasks coated
with 0.2% gelatin (w/v) and grown in a complete culture
medium composed of M199 medium with glutamax (Invitro-
gen Corp, Gergy Pontoise, France) supplemented with 20%
FCS (Eurobio, les Ulis, France), heparin 50 IU/mL (Sanofi
Synthelabo, Paris, France), gentamycin 50 μg/mL (Biomedica,
Boussens, France), 10 ng/mL bFGF (Sigma Aldrich, St Quentin
Fallavier, France). Media were changed three times a week.
Cells were used for attachment experiments at passage 4.
Endothelial Cell Attachment. A pool of HSV cells from three
donors was used. Disks of native and functionalized PET
membranes were sterilized with ethanol (70%, 10 min), rinsed
with phosphate buffered saline (PBS) three times (0.1 M, pH
7.4), and placed in 24-well tissue culture plates (Corning);
samples were immobilized by the mean of glass rings. HSVECs
were seeded on the surface of PET disks at the density of 5 ꢀ 104
Supporting Information Available: Large scale synthesis of
precursor 1 (t-butyl 3-(4-hydroxy-3-nitro-phenyl)-2 (S)-(3-tri-
fluoromethyl-benzene sulfonylamino)propionate) and HPLC
control of its enantiomeric purity, synthesis of ω-functionalized
propanols and butanols bearing the basic groups (or precursors
of Arg-mimics) and their spectroscopic data (10a, 10b, 12a, 12b,
13a, 13b, 14a, 14b, 15a, 15b, 16a, 16b, 17a, 17b, 18a, 18b, 19a,
19b, 21, 22; see Scheme 2), the synthesis and characterization of
the OEG spacer-arms (23a, 23b, 24) and the complete descrip-
tions of compounds cited in Tables 1-4 (except those included
in the Experimental Section-Chemistry) are provided. Details
of computational studies on cilengitide are also given). This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Pierschbacher, M. D.; Ruoslahti, E. Cell attachment activity of
fibronectin can be duplicated by small synthetic fragments of the
molecule. Nature 1984, 309, 30–33.
(2) Ruoslahti, E. The RGD story: a personnal account. Matrix Biol.
2003, 22, 459–465.
(3) Tamkun, J. W.; DeSimone, D. W.; Fonda, D.; Patel, R. S.; Buck,
C.; Horwitz, A. F.; Hynes, R. O. Structure of integrin, a glycopro-
tein involved in the transmembrane linkage between fibronectin
and actin. Cell 1986, 46, 271–282.
(4) Humphries, M. J. Integrin structure. Biochem. Soc. Trans. 2000, 28,
311–340.
(5) Takagi, J.; Petre, B. M.; Walz, T.; Springer, T. A. Global con-
formational rearrangements in integrin extracellular domains in
outside-in and outside-out signaling. Cell 2002, 110, 599–611.
(6) Hynes, R. O. Integrins: bidirectional, allosteric signaling machines.
Cell 2002, 110, 673–687.
(7) Brooks, P. C.; Clark, R. A. F.; Cheresh, D. A. Requirement of
vascular integrin Rvβ3 for angiogenesis. Science 1994, 264, 569–571.
(8) Duong, L. T.; Rodan, G. A. Integrin-mediated signaling in the
regulation of osteoclast adhesion and activation. Front. Biosci.
1998, 3, d757–d768.
(9) Petitclerc, E.; Stromblad, S.; Von Schakscha, T. L.; Mitjans, F.;
Piutlats, J.; Montgommery, A. M.; Cheresh, D. A.; Brooks, P. C.
Integrin Rvβ3 promotes M21 melanoma growth in human skin by
regulating tumor cell survival. Cancer Res. 1999, 59, 2724–2730.
(10) Kumar, C. C. Integrin Rvβ3 as a therapeutic target for blocking
tumor-induced angiogenesis. Curr. Drug Targets 2003, 4, 123–131.
(11) Dechantstreiter, M. A.; Planker, E.; Mathae, B.; Lohof, E.; Hoel-
zemann, G.; Jonczyk, A.; Goodman, S. L.; Kessler, H. N-Methy-
lated cyclic RGD peptides as highly active and selective Rvβ3
integrin antagonists. J. Med. Chem. 1999, 42, 3033–3040.
(12) Keenan, R. M.; Miller, W. H.; Kwon, C.; Ali, F. E.; Callahan, J. F.;
Calvo, R. R.; Hwang, S.-M.; Kopple, K. D.; Peishoff, C. E.;

7042 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Rerat et al.
Samanen, J. M.; Wong, A. S.; Yuan, C.-K.; Huffman, W. F.
Discovery of potent nonpeptide vitronectin receptor (Rvβ3) an-
tagonists. J. Med. Chem. 1997, 40, 2289–2292.
(33) Boxus, T.; Touillaux, R.; Dive, G.; Marchand-Bryanert, J. Synth-
esis and evaluation of RGD peptidomimetics aimed at surface
bioderivatization of polymer substrates. Bioorg. Med. Chem. 1998,
6, 1577–1595.
(34) Biltresse, S.; Attolini, M.; Dive, G.; Cordi, A.; Tucker, G. C.;
Marchand-Brynaert, J. Novel RDG-like molecules based on the
tyrosine template: design, synthesis, and biological evaluation on
isolated integrins Rvβ3/RIIbβ3 and in cellular adhesion tests. Bioorg.
Med. Chem. 2004, 12, 5379–5393.
(35) Yamanaka, T.;Ohkudo, M.;Kuroda, S.;Nakamura, H.;Takahashi,
F.; Aoki, T.; Mihara, K.; Seki, J.; Kato, M. Design, synthesis, and
structure-activity relationships of potent GPIIb/IIIa antagonists:
discovery of FK419. Bioorg. Med. Chem. 2005, 13, 4343–4352.
(36) Duggan, M. E.; Duong, L. T.; Fisher, J. E.; Hamill, T. G.; Hoffman;
Huff, W. F. J. R.; Ihle, N. C.; Leu, C.-T.; Nagy, R. M.; Perkins, J. J.;
Rodan, S. B.; Wesolowski, G.; Whitman, D. B.; Zartman, A. E.;
Rodan, G. A.; Hartman, G. D. Nonpeptide Rvβ3 Antagonists. 1.
Transformation of a Potent, Integrin-Selective RIIbβ3 Antagonist
into a Potent Rvβ3 Antagonist. J. Med. Chem. 2000, 43, 3736–3745.
(37) Rockwell, A. L.; Rafalski, M.; Pitts, W. J.; Batt, D. G.; Petraitis,
J. J.; DeGrado, W. F.; Mousa, S.; Jadhav, P. K. Rapid synthesis of
RGD mimetics with isoxazoline scaffolds on solid phase: identifi-
cation of Rvβ3 antagonists lead compounds. Bioorg. Med. Chem.
Lett. 1999, 9, 937–942.
(13) Miller, W. H.; Keenan, R. M.; Willette, R. N.; Lark, M. W.
Identification and in vivo efficacy of small-molecule antagonists
of integrin Rvβ3 (the vitronectin receptor). Drug Discovery Today
2000, 5, 397–408.
(14) Kerr, J. S.; Slee, A. M.; Mousa, S. A. The Rv integrin antagonists as
novel anticancer agents: an update. Expert Opin. Investig. Drugs
2002, 11, 1765–1774.
(15) Coleman, P. J.; Brashear, K. M.; Askew, B. C.; Hutchinson, J. H.;
McVean, C. A.; Duong, L. T.; Feuston, B. P.; Fernandez-
Metzler, C.; Gentile, M. A.; Hartman, G. D.; Kimmel, D. B.;
Leu, C.-T.; Lipfert, L.; Merkle, K.; Pennypacker, B.; Prueksaritanont,
T.; Rodan, G. A.; Wesolowski, G. A.; Rodan, S. B.; Duggan, M.
E. Nonpeptide Rvβ3 antagonists. Part 11: Discovery and precli-
nical evaluation of potent Rvβ3 antagonists for the prevention
and treatment of osteoporosis. J. Med. Chem. 2004, 47, 4829–
4837.
(16) Dayam, R.; Aiello, F.; Deng, J.; Wu, Y.; Garofalo, A.; Chen, X.;
Neamati, N. Discovery of small molecule integrin Rvβ3 antagonists
as novel anticancer agents. J. Med. Chem. 2006, 49, 4526–4534.
(17) Thumshirn, G.; Hersel, U.; Goodman, S. L.; Kessler, H. Multi-
meric cyclic RGD peptides as potential tools for tumor targeting:
solid-phase peptide synthesis and chemoselective oxime ligation.
Chem.;Eur. J. 2003, 9, 2717–2725.
(38) Duggan, M. E.; Hutchinson, J. H. Ligands to the integrin receptor
Rvβ3. Expert Opin. Ther. Pat. 2000, 10, 1367–1383.
(39) Coleman, P. J.; Duong, L. T. Ligands to the integrin receptor Rvβ3.
Expert Opin. Ther. Pat. 2002, 12, 1009–1021.
(18) Nasongkla, N.; Shuai, X.; Ai, H.; Weinberg, B. D.; Pink, J.;
Boothman, D. A.; Gao, J. cRGD-functionalized polymer micelles
for targeted doxorubicin delivery. Angew. Chem., Int. Ed. 2004, 43,
6323–6327.
(40) Cacciari, B.; Spalluto, G. Non peptidic Rvβ3 antagonists: Recent
developments. Curr. Med. Chem. 2005, 12, 51–70.
(19) Temming, K.; Schiffelers, R. M.; Molema, G.; Kok, R. J. RGD-
based strategies for selective delivery of therapeutics and imaging
agents to the tumour vasculature. Drug Resist. Updates 2005, 8,
381–402.
(20) Hersel, U.; Dahmen, C.; Kessler, H. RGD modified polymers:
biomaterials for stimulated cell adhesion and beyond. Biomaterials
2003, 24, 4385–4415.
(21) Siebers, M. C.; ter Brugge, P. J.; Walboomers, X. F.; Jansen, J. A.
Integrins as liker proteins between osteoblasts and bone replacing
materials. A critical review. Biomaterials 2005, 26, 137–146.
(22) Lieb, E.; Hacker, M.; Tessmar, J.; Kunz-Schughart, L. A.; Fiedler,
(41) Pilkington-Miksa, M. A.; Sarkar, S.; Writer, M. J.; Barker, S. E.;
Shamlou, P. A.; Hart, S. L.; Hailes, H. C.; Tabor, A. B. Synthesis of
bifonctional integrin-binding peptides containing PEG spacers of
defined length for nonviral gene delivery. Eur. J. Org. Chem. 2008,
2900–2914.
(42) Heckmann, D.; Meyer, A.; Laufer, B.; Zahn, G.; Stragies, R.;
Kessler, H. Rational design of highly active and selective ligands
for the R5β1 integrin receptor. ChemBioChem 2008, 9, 1397–1407.
(43) Biltresse, S.;Descamps,D.;Henneuse-Boxus,C.;Marchand-Brynaert,
J. Effect of the chemical nature and the length of spacer-arms on the
covalent grafting of molecular probes at the surface of PET mem-
branes. J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 770–781.
(44) Penso, M.; Albanese, D.; Landini, D.; Lupi, V.; Tricarico, G.
Specific solvation as a tool for the N-chemoselective arylsulfonyla-
tion of tyrosine and (4-hydroxyphenyl)glycine methyl esters. Eur.
J. Org. Chem. 2003, 4513–4517.
€
J.; Dahmen, C.; Hersel, U.; Kessler, H.; Schulz, M. B.; Gopferich,
A. Mediating specific cell adhesion to low-adhesive diblock copo-
lymers by instant modification with cyclic RGD peptides. Bioma-
terials 2005, 26, 2333–2341.
(23) Ma, P. X. Biomimetic materials for tissue engineering. Adv. Drug
Delivery Rev. 2008, 60, 184–198.
(45) Brennauer, A.; Keller, M.; Freund, M.; Bernhardt, G.; Buschauer,
A. Decomposition of 1-(ω-aminoalkanoyl)guanidines under alka-
line conditions. Tetrahedron Lett. 2007, 48, 6996–6999.
(24) Marchand-Brynaert, J.; Detrait, E.; Noiset, O.; Boxus, T.; Schneider,
Y.-J.; Remacle, C. Biological evaluation of RGD peptidomimetics,
designed for the covalent derivatization of cell culture substrata, as
potential promotors of cellular adhesion. Biomaterials 1999, 20,
1773–1782.
(25) Biltresse, S.; Attolini, M.; Marchand-Bryanert, J. Cell adhesive
PET membranes by surface grafting of RGD peptidomimetics.
Biomaterials 2005, 26, 4576–4587.
(26) Li, L.-S.; Rader, C.; Matsushita, M.; Das, S.; Barbas, C. F.; Lerner,
R. A.; Sinha, S. C. Chemical adaptor immunotherapy: design,
synthesis, and evaluation of novel integrin-targeting devices.
J. Med. Chem. 2004, 47, 5630–5640.
(27) Meyer, A.; Auernheimer, J.; Modlinger, A.; Kessler, H. Targeting
RGD recognizing integrins: drug development, biomaterial re-
search, tumor imaging and targeting. Curr. Pharm. Des. 2006, 12,
2723–2747.
(46) Nguyen, C.; Ruda, G. F.; Schipani, A.; Kasinathan, G.; Leal, I.;
Musso-Buendia, A.; Kaiser, H.; Brun, R.; Ruiz-Perez, L. M.;
Sahlberg, B.-L.; Johanson, N. G.; Gonzalez-Pacanowska, D.;
Gilbert, I. Acyclic Nucleoside Analogues as Inhibitors of Plasmo-
dium falciparum dUTPase. J. Med. Chem. 2006, 49, 4183–4195.
(47) Kim, K. S.; Qian, L. Fire-retardant and intumescent compositions
for cellulosic material. Tetrahedron Lett. 1993, 34, 7677–7680.
(48) Frederick, R.; Charlier, C.; Robert, S.; Wouters, J.; Masereel, B.;
Pochet, L. Investigation of mechanism-based thrombin inhibitors:
Implications of a highly conserved water molecule for the binding
of coumarins within the S pocket. Bioorg. Med. Chem. Lett. 2006,
16, 2017–2021.
(49) Botta, M.; Corelli, F.; Maga, G.; Manetti, F.; Renzulli, M.;
Spadari, S. Research on anti-HIV-1 agents. Part 2: Solid-phase
synthesis, biological evaluation and molecular modeling studies of
2,5,6- trisubstituted-4(3H)-pyrimidinones targeting HIV-1 reverse
transcriptase. Tetrahedron 2001, 57, 8357–8367.
(28) Patel, S.; Thakar, R. G.; Wong, J.; McLeod, S. D.; Li, S. Control of
cell adhesion on poly(methyl methacrylate). Biomaterials 2006, 27,
2890–2897.
(29) Auernheimer, J.; Zukowski, D.; Dahmen, C.; Kantlehner, M.;
Enderle, A.; Goodman, S. L.; Kessler, H. Titanium implant
materials with improved biocompatibity through coating with
phosphonate-anchored cyclic RGD peptides. ChemBioChem
2005, 6, 2034–2040.
(30) Dahmen, C.; Auernheimer, J.; Meyer, A.; Enderle, A.; Goodman,
S. L.; Kessler, H. Improving implant materials by coating with
nonpeptidic, highly specific integrin ligands. Angew. Chem., Int.
Ed. 2004, 43, 6649–6652.
(50) Marugan, J. J.; Manthey, C.; Anaclerio, B.; Lafrance, L.; Lu, T.;
Markotan, T.; Leonard, K. A.; Crysler, C.; Eisennagel, S.;
Dasgupta, M.; Tomczuk, B. Design, Synthesis, and Biological
Evaluation of Novel Potent and Selective Rvβ3/RIIbβ3 Integrin
Dual Inhibitors with Improved Bioavailability. Selection of the
Molecular Core. J. Med. Chem. 2005, 48, 926–934.
(51) Geneste, H.; Kling, A.; Lauterbach, A.; Graef, C. I.; Subkowski,
T.; Hornberger, W. Preparation of dibenzo[b,e]azepines as Rvβ3
integrin ligands. Int. Patent WO 2002014320, 2002.
(31) Curley, G. P.; Blum, H.; Humphries, M. J. Integrin antagonists.
(52) Heckmann, D.; Meyer, A.; Marinelli, L.; Zahn, G.; Stragies, R.;
Kessler, H. Probing integrin selectivity: rational design of highly
active and selective ligands for the R5β1 and Rvβ3 integrin receptor.
Ang. Chem., Int. Ed. 2007, 46, 3571–3574.
(53) Lefrancois, J. M.; Heckmann, B. Preparation of pyrimidine amino
acid derivatives as antagonists of the vitronectin receptor. French
Patent FR 2870541, 2005.
Cell. Mol. Life, Sci. 1999, 56, 427–441.
(32) Hartman, G. D.; Egbertson, M. S.; Halczenko, W.; Laswell, W. L.;
Duggan, M. E.; Smith, R. L.; Naylor, A. M.; Manno, P. D.; Lynch,
R. J.; Zhang, G.; Chang, C. T.-C.; Gould, R. J. Non-peptide
fibrinogen receptor antagonists. 1. Discovery and design of exosite
inhibitors. J. Med. Chem. 1992, 35, 4640–4642.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7043
(54) Dormer, P. G.; Eng, K. K.; Farr, R. N.; Humphrey, G. R.;
McWilliams, J. C.; Reider, P. J.; Sager, J. W.; Volante, R. P.
Highly Regioselective Friedlaender Annulations with Unmodified
Ketones Employing Novel Amine Catalysts: Syntheses of 2-Sub-
stituted Quinolines, 1,8-Naphthyridines, and Related Hetero-
cycles. J. Org. Chem. 2003, 68, 467–477.
(55) Ameer, F.; Giles, R. G. F.; Green, I. R.; Nagabhushana, K. S. The
DDQ mediated cyclization products of some 2-hydroxy-3-(1’-
alkenyl)-1,4-naphthoquinones. Synth. Commun. 2002, 32, 369–380.
(56) Backes, J. R.; Koert, U. Stereoselective synthesis of the monomeric
unit of SCH 351448. Eur. J. Org. Chem. 2006, 2777–2785.
(57) Prabhakaran, P. C.; Gould, S. J.; Orr, G. R.; Coward, J. K.
Synthesis of chirally deuteriated phthalimido-propanols and eva-
luation of their absolute stereochemistry. J. Am. Chem. Soc. 1988,
110, 5779–5784.
(74) Manzoni, L.; Bassanini, M.; Belvisi, L.; Motto, I.; Scolastico, C.;
Castorina, M.; Pisano, C. Nonpeptide integrin antagonists: RGD
mimetics incorporating substituted azabicycloalkanes as amino
acid replacements. Eur. J. Org. Chem. 2007, 1309–1317.
(75) Zanardi, F.; Burreddu, P.;Rassu, G.;Auzzas, L.;Battistini, L.;Curti,
C.; Sartori, A.; Nicastro, G.; Menchi, G.; Cini, N.; Bottonocetti, A.;
Raspanti, S.; Casiraghi, G. Discovery of subnanomolar arginine-
glycine-aspartate-based Rvβ3/Rvβ5 integrin binders embedding
4-aminoproline residues. J. Med. Chem. 2008, 51, 1771–1782.
(76) Momtaz, M.; Dewez, J.-L.; Marchand-Brynaert, J. Chemical
reactivity assay and surface characterization of a poly(vinylidene
fluoride microfiltration membrane (Durapore DVPP). J. Membr.
Sci. 2005, 250, 29–37.
(77) Chollet, C.; Chanseau, C.; Remy, M.; Guignandon, A.; Bareille,
ꢁ
R.; Labrugere, C.; Bordenave, L.; Durrieu, M.-C. The effect of
(58) Kim, K. S.; Song, Y. H.; Lee, B. H.; Hahn, C. S. Efficient and
selective cleavage of acetals and ketals using ferric chloride ad-
sorbed on silica gel. J. Org. Chem. 1986, 51, 404–406.
(59) Yong, Y. F.; Kowalski, J. A.; Lipton, M. A. Facile and Efficient
Guanylation of Amines Using Thioureas and Mukaiyama’s Re-
agent. J. Org. Chem. 1997, 62, 1540–1542.
(60) Balicki, R.; Maciejewski, G. A mild and selective deoxygenation of
N-oxides with ammonium formate as a catalytic hydrogen transfer
agent. Synth. Commun. 2002, 32, 1681–1683.
(61) Salvagnini, C.; Gharbi, S.; Boxus, T.; Marchand-Brynaert, J.
Synthesis and evaluation of a small library of graftable thrombin
inhibitors derived from (L)-arginine. Eur. J. Med. Chem. 2007, 42,
37–53.
(62) Momtaz, M.; Rerat, V.; Gharbi, S.; Gerard, E.; Pourcelle, V.;
Marchand-Brynaert, J. A graftable LDV peptidomimetic: design,
synthesis and application to a blood filtration membrane. Bioorg.
Med. Chem. Lett. 2008, 18, 1084–1090.
(63) Takahashi, T.; Shiyama, T.; Hosoya, K.; Tanaka, A. Development
of chemically stable solid phases for the target isolation with
reduced nonspecific binding proteins. Bioorg. Med. Chem. Lett.
2006, 16, 447–450.
RGD density on osteoblast and endothelial cell behavior on RGD-
grafted polyethylene terephthalate surfaces. Biomaterials 2009, 30,
711–720.
(78) Hartman, G. D.; Egbertson, M. S.; Halczenko, W.; Laswell, W. L.;
Duggan, M. E.; Smith, R. L.; Naylor, A. M.; Manno, P. D.; Lynch,
R. J.; Zhang, G.; Chang, C. T.-C.; Gould, R. J. Non-peptide
fibrinogen receptor antagonists. 1. Discovery and design of exosite
inhibitors. J. Med. Chem. 1992, 35, 4640–4642.
(79) Owen, R. M.; Carlson, C. B.; Xu, J.; Mowery, P.; Fasella, E.;
Kiessling, L. L. Bifunctional ligands that target cells displaying the
Rvβ3 integrin. ChemBioChem 2007, 8, 68–82.
(80) Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.;
Kiessling, L. L. Selective tumor cell targeting using low-affinity,
multivalent interactions. ACS Chem. Biol. 2007, 2, 119–127.
(81) Dijkgraaf, I.; Kruijtzer, J. A. W.; Frielink, C.; Soede, A. C.;
Hilbers, H. W.; Oyen, W. J. G.; Corstens, F. H. M.; Liskamp, R.
M. J.; Boerman, O. C. Synthesis and biological evaluation of
potent Rvβ3 integrin receptor antagonists. Nucl. Med. Biol. 2006,
33, 953–961.
€
(82) Muller, B.; Zerwes, H. G.; Tangemann, K.; Peter, J.; Engel, J.
2-Step binding mechanism of fribrinogen to RIIbβ3 integrin recon-
stituted into planar lipid bilayers. J. Biol. Chem. 1993, 268, 6800–
6808.
(64) Adamczyk, M.; Fishpaugh, J. R.; Thiruvazhi, M. Concise synthesis
of N-protected carboxyalkyl ether amines. Org. Prep. Proc. Int.
2002, 34, 326–331.
ꢀ ꢀ
(83) Vilamitjana-Amedee, J.; Bareille, R.; Rouais, F.; Caplan, A. E.;
(65) Chapman, R. G.; Ostuni, E.; Liang, M. N.; Yan, L.; Whitesides,
G. M. Self-assembled monolayer surfaces that resist the adsorption
of biological species. Int. Patent WO 2002006407, 2002.
(66) Martinez, V.; Mecking, S.; Tassaing, T.; Besnard, M.; Moisan, S.;
Moisan, S.; Cansell, F.; Aymonier, C. Dendritic core-shell macro-
molecules soluble in supercritical carbon dioxide. Macromolecules
2006, 39, 3978–3979.
Harmand, M. F. Human bone marrow stromal cells expressing
osteoblastic phenotype in vitro. Cell. Dev. Biol. 1993, 29, 699–707.
ꢀ ꢀ
(84) Villars, F.; Guillotin, B.; Amedee, T.; Dutoya, S.; Bordenave, L.;
ꢀ ꢀ
Bareille, R.; Amedee, J. Effect of HUVEC on human osteopro-
genitor cell differentiation needs heterotypic gap junction commu-
nication. Am. J. Physiol. Cell. Physiol. 2002, 282, C775–C785.
(85) Hariharan, P. C.; Pople, J. A. The influence of polarization
functions on molecular hydrogenation energies. Theor. Chim. Acta
1973, 28, 213–217.
(86) Dive, G.; Dehareng, D.; Ghuysen, J. M. Energy analysis of small to
medium sized H-bonded complexes. Theor. Chim. Acta 1993, 85,
409–421.
€
(67) Sulyok, G. A. G.; Gibson, C.; Goodman, S. L.; Holzemann, G.;
Wiesner, M.; Kessler, H. Solid-phase synthesis of a nonpeptide
RGD mimetic library: new selective Rvβ3 integrin antagonists.
J. Med. Chem. 2001, 44, 1938–1950.
ꢀ
ꢁ
(68) Porte-Durrieu, M. C.; Guillemot, F.; Pallu, S.; Labrugere, C.;
ꢀ ꢀ
Brouillaud, B.; Bareille, R.; Amedee, J.; Barthe, N.; Dard, M.;
(87) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Montgomery, J. A.; Jr., Vreven, T.;
Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi,
J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.;
Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.;
Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.;
Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian,
H. P.; Cross, J. B.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli,
C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.;
Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck,
A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.;
Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.;
Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D.
J.; Keith, T.;Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.;
Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong,
M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02;
Gaussian, Inc.: Wallingford, CT, 2004.
Baquey, Ch. Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V:
physical characterization and interest towards human osteopro-
genitor cells adhesion. Biomaterials 2004, 25, 4837–4846.
(69) Pallu, S.; Bourget, C.; Bareille, R.; Labrugere, C.; Dard, M.;
Sewing, A.; Jonczyk, A.; Vernizeau, M.; Durrieu, M. C.;
ꢀ ꢀ
Amedee-Vilamitjana, J. The effect of cyclo-DfKRG peptide im-
mobilization on titanium on the adhesion and differentiation of
human osteoprogenitor cells. Biomaterials 2005, 26, 6932–6940.
(70) Landegren, U. Measurement of cell numbers by means of the
endogenous enzyme hexosaminidase. Applications to detection
of lymphokines and cell surface antigens. J. Immunol. Methods
1984, 67, 379–388.
€
€
(71) Pfaff, M.; Tangemann, K.; Muller, B.; Gurrath, M.; Muller, G.;
Kessler, H.; Timpl, R.; Engel, J. Selective recognition of cyclic
RGD peptides of NMR defined conformation by RIIbβ3, Rvβ3, and
R5β1 integrins. J. Biol. Chem. 1994, 269, 20233–20238.
(72) Xiong, J.-P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.;
Goodman, S. L.; Arnaout, M. Crystal structure of the extracellular
segment of integrin Rvβ3 in complex with an Arg-Gly-Asp ligand.
Science 2002, 296, 151–155.
ꢀ
(88) Pourcelle, V.; Devouge, S.; Garinot, M.; Preat, V.; Marchand-
Brynaert, J. PCL-PEG-based nanoparticles grafted with GRGDS
peptide: preparation and surface analysis by XPS. Biomacromole-
cules 2007, 8, 3977–3983.
(73) Moitessier, N.; Henry, C.; Maigret, B.; Chapleur, Y. Combining
pharmacophore search, automated docking, and molecular dy-
namics simulations as a novel strategy for flexible docking. Proof
of concept: docking of arginine-glycine-aspartic acid-like com-
pounds into the Rvβ3 binding site. J. Med. Chem. 2004, 47, 4178–
4187.
(89) Golledge, J.; Tumer, R. J.; Harley, S. L.; Springall, D. R.; Powell,
J. T. Development of an in vitro model to study the response of
saphenous vein endothelium to pulsatile arterial flow and circumfer-
ential deformation. Eur. J. Vasc. Endovasc. Surg. 1997, 13, 605–612.