Click-chemistry-derived triazole ligands of arginine-glycine-aspartate (RGD) integrins with a broad capacity to inhibit adhesion of melanoma cells and both in vitro and in vivo angiogenesis

Article abstract of DOI:10.1021/jm100754z

A click chemistry approach was applied for the discovery of triazole-based arginine-glycine-aspartate (RGD) mimetics by Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction, which showed binding affinity properties toward α_vβ₃/α_vβ₅ integrins. Biological assays showed compound 18 capable of binding α_vβ₃ integrin with nanomolar affinity according to a two-sites model, and molecular modeling studies revealed a peculiar π-stacking interaction between the triazole ring and Tyr178 side chain. Accordingly, compound 18 inhibited the adhesion of integrin-expressing human melanoma cells to RGD-containing proteins of the extracellular matrix, such as vitronectin, fibronectin, and osteopontin, and also angiogenesis in in vitro and in vivo experimental models. The relevant biological effects exerted by compound 18 suggest its potential application as an antiangiogenic agent in the diagnosis and therapy of tumors where α_vβ₃ integrin expression is up-regulated.

Full text of DOI:10.1021/jm100754z

J. Med. Chem. 2010, 53, 7119–7128 7119
DOI: 10.1021/jm100754z
Click-Chemistry-Derived Triazole Ligands of Arginine-Glycine-Aspartate (RGD) Integrins with a
Broad Capacity To Inhibit Adhesion of Melanoma Cells and Both in Vitro and in Vivo Angiogenesis
Andrea Trabocchi,†,‡ Gloria Menchi,†,‡ Nicoletta Cini,†,‡,§, Francesca Bianchini,‡,^, Silvia Raspanti,‡,§, Anna Bottoncetti,‡,§,
Alberto Pupi,‡,§, Lido Calorini,*,‡,^, and Antonio Guarna*,†,‡
†Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 13, 50019 Sesto Fiorentino, Florence, Italy,
‡Interdepartmental Center for Preclinical Development of Molecular Imaging (CISPIM), University of Florence, Viale Morgagni 85,
50134 Florence, Italy, §Department of Clinical Physiopathology, Nuclear Medicine Unit, University of Florence, Italy,
Istituto Toscano Tumori, Via T. Alderotti 26N, 50139 Florence, Italy, and ^Department of Experimental Pathology and Oncology,
University of Florence, Viale Morgagni 50, 50134 Florence, Italy
Received June 21, 2010
A click chemistry approach was applied for the discovery of triazole-based arginine-glycine-aspartate
(RGD) mimetics by Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction, which showed binding
affinity properties toward Rvβ3/Rvβ5 integrins. Biological assays showed compound 18 capable of
binding Rvβ3 integrin with nanomolar affinity according to a two-sites model, and molecular modeling
studies revealed a peculiar π-stacking interaction between the triazole ring and Tyr178 side chain.
Accordingly, compound 18 inhibited the adhesion of integrin-expressing human melanoma cells to
RGD-containing proteins of the extracellular matrix, such as vitronectin, fibronectin, and osteopontin,
and also angiogenesis in in vitro and in vivo experimental models. The relevant biological effects exerted
by compound 18 suggest its potential application as an antiangiogenic agent in the diagnosis and
therapy of tumors where Rvβ3 integrin expression is up-regulated.
Introduction
expression of Rvβ3 integrin in melanocytes correlates with the
invasive phase of human melanoma, namely, the vertical
growth phase (VGP).7 Rvβ3 integrin was found to be localized
at the invasive front of melanoma, where it may regulate the
activity of matrix-degrading proteases, such as matrix metallo-
proteases and urokinase-type plasminogen activator, and cell
motility.8 Moreover, Rvβ3 integrin is not only involved in tumor
cell movement but also is essential in the regulation of endo-
thelial cell growth and differentiation, and angiogenesis is
crucial for tumor growth and dissemination. An important
finding demonstrated that, unlike quiescent endothelium, endo-
thelial cells of tumor vasculature express a high level of Rvβ3
integrin. Thus, the involvement of Rvβ3 integrin in tumor cell
invasion and angiogenesis has inspired scientists to search RGD
mimetics to be used for targeting growth and dissemination of
cancer cells as well as to inhibit tumor neoangiogenesis.9 The
RGD recognition site can be mimicked by linear and cyclic
peptides, and their specificity can be modulated by the sequence
and structure of such peptides. Rvβ3 antagonists, including
RGD-containing peptides, have been successfully applied as
inhibitors of blood vessel development and tumor growth.10
Moreover, non-peptide RGD peptidomimetics have been
developed as integrin ligands.11
Integrin receptors constitute a large family of proteins with
structural characteristics of noncovalent heterodimeric glyco-
proteins formed of R and β subunits.1 One important recogni-
tion site for many integrins is the arginine-glycine-aspartic
acid (RGDa) tripeptide sequence,2 found in a series of peptide-
based ligands such as vitronectin, fibronectin, and osteopontin.
Regarding RGD-dependent integrins, Rvβ3 and Rvβ5 receptors
have received increasing attention as therapeutic targets, as they
are expressed in various cell types and are involved in inflam-
matory and tumor-related processes.3 Rvβ3 integrin expression
is up-regulated in many solid tumors and contribute to the
mechanisms involved in tumor growth and metastatic dissemi-
nation.4,5 During the progression from a benign melanocytic
disease to a metastatic malignant melanoma, melanocytes
undergo a series of changes in the expression of cell-surface
molecules, including Rvβ3 integrin.6 In particular, an enhanced
*To whom correspondence should be addressed. For L.C.: þ39 055
4598207; fax, þ39 055 4598900; e-mail, lido.calorini@unifi.it. For A.G.:
phone, þ39 055 4573481; fax, þ39 055 4573569; e-mail, antonio.
guarna@unifi.it.
a Abbreviations: RGD, Arg-Gly-Asp; VGP, vertical growth phase;
HIV, human immunodeficiency virus; SARS, severe acute respiratory
syndrome; SEM, standard error of the mean; compd, compound;
MIDAS, metal-ion dependent adhesion site; PDB, Protein Data Bank;
RT-PCR, real time protein chain reaction; OPN, osteopontin; VN,
vitronectin; FN, fibronectin; HUVEC, human umbilical vein endo-
thelial cells; IL-1β, interleukin-1β; bFGF, basic fibroblast growth factor;
ESI-MSMS, electropray ionization tandem mass spectrometry; EGM-2,
endothelial cell growth medium-2; EDTA, ethylenediaminetetraacetic
acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; FACS,
fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate;
PBS, Phosphate buffered saline.
Fragment-based assembly is a recently developed drug dis-
covery approach that enables high-throughput identification of
small molecule inhibitors using a minimal number of com-
pounds as building blocks.12 The approach is powerful espe-
cially against protein targets that possess multiple binding
pockets in their active sites. Among different methods proposed
to assist the assembly of compounds, “click chemistry”, pioneered
by Sharpless et al.,13 is highly versatile, and it has become
r
2010 American Chemical Society
Published on Web 09/01/2010
pubs.acs.org/jmc

7120 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Trabocchi et al.
Chart 1. Selected Azides and Alkynes for the Click Chemistry
Reaction
Scheme 1. Representative Synthesis of Triazole-Based RGD
Peptidomimetics: Preparation of Compound 18
Chart 2. Panel of Triazole-Containing RGD Peptidomimetics
increasingly popular for generating new chemical entities in
medicinal chemistry, in bioconjugating strategies, and in mate-
rial chemistry. The mild and regioselective features of the Cu-
catalyzed Huisgen 1,3-dipolar cycloaddition are particularly
suited for developing combinatorial libraries containing the
triazole ring, which has been applied successfully as amide bond
isostere in peptidomimetic approaches. For example, it has been
used in several drug discovery issues, including the generation of
inhibitors against HIV protease, SARS 3CL protease, R-fuco-
sidase, sulfotransferase, and R-1,3-fucosyltransferase.14
We envisaged using this strategy for generating RGD
mimetics by virtue of combining azides and alkynes bearing
Asp and Arg isosteres, respectively. Accordingly, the triazole
ring was conceived as the Gly mimetic, thus acting as a spacer.
Specifically, based on the reference integrin ligand cilengitide,
azides containing a carboxylic group and an aromatic ring were
taken into account as mimetics of both Asp and D-Phe phar-
macophoric moieties, whereas alkynes differing in length and
molecular shape were conceived as Arg mimetics (Chart 1).
for binding to isolated, purified Rvβ3 and Rvβ5 integrins
originated from human placenta15 was evaluated in solid-
phase receptor assays.16 Competition studies were carried
out using a fixed concentration of the radioligand (0.05 and
0.1 nM for Rvβ3 and Rvβ5 receptors, respectively) and a range
of concentrations (100 μM to 0.01 nM) of the tested mole-
cules. The IC50 ( SEM values (nM) were calculated as the
concentration of compound required for 50% inhibition of
radioligand binding, as estimated by the GraphPad Prism
program, and the results are reported in Table 1.
IC50 values showed a binding affinity in the micromolar
range for compounds 17, 18, and 20 toward Rvβ3 and Rvβ5
Results
Synthesis. Triazole-based RGD ligands were achieved
using click chemistry and specifically by combining azides
1-3 and alkynes 4-8 (Chart 1) in the Cu-catalyzed azide-
alkyne cycloaddition, followed by acid-mediated hydrolysis
of the protecting groups of side chain isosteres (see Scheme 1
for a representative synthesis).
Table 1. Inhibition of [125I]Echistatin Specific Binding to Purified
Human Integrin Proteins RVβ3 and RVβ5 by Triazole-Containing
RGD Peptidomimetics
Rvβ3
IC50 (μM)
Rvβ5
IC50 (μM)
compd
Hill
Hill
9
>10
>10
>10
>10
Interestingly, the Cu-catalyzed reaction with these com-
ponents proved to proceed in poor yields when the metal was
used in catalytic amounts. Thus, a full equivalent of the
copper salt was used, and longer reaction times were neces-
sary to drive the reaction to completion, compared to
standard conditions usually employed for reported applica-
tions of the click chemistry. This effect could be possibly due
to the azide component, which inactivated the Cu catalyst by
virtue of complexation reactions. The process allowed the
rapid preparation of a library of triazole-containing RGD
mimetics in 43-68% yields, as shown in Chart 2.
10
11
12
13
14
15
16
17
18
19
20
21
22
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
46.3 ( 9.7
1.78 ( 0.30
4.38 ( 0.51
1.02 ( 0.12
>10
-0.899
-0.806
-0.919
-0.798
3.47 ( 0.76
1.35 ( 0.25
>10
-0.567
-0.381
4.96 ( 0.83
>10
>10
-0.877
2.42 ( 0.3
>10
-1.037
-0.810
Receptor Binding Assays. The ability of triazole-contain-
ing RGD peptidomimetics to compete with [125I]echistatin
1.27 ( 0.18

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7121
Table 2. Inhibition of [125I]Echistatin Specific Binding to Purified
Human Integrin Proteins RVβ3 and RVβ5 by Selected Triazole-Containing
RGD Peptidomimetics According to a Two-Sites Model
compd
IC50,1 (nM)
%
1
IC50,2 (μM)
%
2
Rvβ3
17
18
1070 ( 210
16.4 ( 2.0
55
35
70.7 ( 41
3.42 ( 0.21
45
65
Rvβ5
59
18
0.405 ( 0.27
4.24 ( 4.8
41
integrins, whereas compounds 15, 16, and 22 showed a more
adaptive behavior only for Rvβ5. Compounds 9-12 did not
exhibit any binding affinity toward Rvβ3 and Rvβ5 integrins,
suggesting the N-aryl moiety to impair the potency of triazole-
containing RGD ligands. The configuration of the unique
stereocenter proved to be crucial, as compound 18 showed the
best binding affinity toward Rvβ3, whereas the corresponsing
enantiomer, i.e., compound 15, did not show any activity toward
such integrin. Such configurational specificity was reversed
when the Arg isostere was changed to a guanidinylpiperazino
derivative, as shown by binding affinities of compounds 20-22.
Also, homologated compounds 16-18 demonstrated a relation-
ship between the binding affinity and the distance between Arg
and Asp isosteres, thus allowing for the selection of 18 as the
optimal ligand. The Rvβ3 inhibition curves of active compounds
showed Hill slope values different from unity, indicating the
binding curve to be better described by a two-sites model, as a
consequence of binding of the peptidomimetic compound with
two conformational states of integrins, in agreement with other
ligands already reported in the literature.17 Thus, the analysis of
the data with a nonlinear fitting indicated that these ligands bind
to Rvβ3 receptor according to a two-sites binding model (Table 2,
Figure 1). It is possible that this effect could be due to a different
affinity for the conformational states assumed by the integrins.18
The transition from low- (bent form) to high-affinity (extended
form) conformation has been described to be induced by natural
ligands,19 and also by most ligand-mimetic antagonists.20 It was
reported that some antagonists, such as the disintegrin echista-
tin, bind to the active and inactive forms with similar affinity,
whereas others bind to the active form with higher affinity than
to the inactive form.21 Our results indicated compound 18 as a
better ligand for Rvβ3 integrin and to possess a selectivity by 2
orders of magnitude for Rvβ3 with respect to Rvβ5.
Molecular Modeling. A docking simulation using com-
pound 18 and the available crystallographic data of Rvβ3
integrin gave further insight into the possible binding modes of
the selected ligand to Rvβ3 at the molecular level. The crystal
structure of the complex formed by c[RGDf(Me)V] (cilengitide)
and the extracellular fragment of Rvβ3 (PDB code 1L5G)
provides a general mode of interaction between the integrin
and its ligands.20 The Asp carboxylate and Arg guanidinium
moieties of RGD-based ligands are the two key structural
elements for the receptor recognition. In fact, the carboxylate
group interacts with the metal-ion-dependent adhesion site
(MIDAS) consisting of Mn2þ ions and Ser121/Ser123, whereas
the Arg guanidinium group is responsible for salt bridge inter-
actions with the side chains of Asp218 and Asp150. Also,
additional ligand-receptor contacts engage Tyr122 in hydro-
phobic π-stacking interactions and engage Asn215 and Arg216
in hydrogen-bonding contacts. The docking program Auto-
dock 4.0.122 was used to evaluate the binding energies of the
global minimum conformer of 18 as a ligand for Rvβ3, and
docked conformations were analyzed by taking into account the
Figure 1. Inhibition curves of compound 18 toward Rvβ3 and Rvβ5
integrins.
binding interactions observed in the crystal structure of the
ligand-protein complex. Docking results of compound 18
showed a main cluster of conformations displaying the typical
binding mode of RGD cyclopeptide-based ligands (Figure 2,
top, structures in green and yellow). Specifically, the main
cluster showed the canonical binding mode consisting of
Asp218/Asp150/guanidine and MIDAS/Ser121/carboxylate
interactions. Interestingly, peptidomimetic 18 showed a some-
what different accommodation in the site, as the guanidinium
group of the Arg side chain isostere experienced a mono-
dentate interaction with both Asp218 and Asp150 instead of a
bidentate interaction with Asp218. Moreover, a characteristic
π-stacking interaction between the triazole ring and Tyr178
aromatic side chain was found in all the conformations of the
main cluster, thusaccounting foranadditionalstabilization of
the ligand-receptor interaction (Figure 2, top). The analysis
of the main cluster of docked conformations revealed a
possible equilibrium between two conformers characterized
by a π-stacking interaction between the aromatic ring of 18
and Tyr122 side chain (yellow structure, Figure 2, top right)
and by a hydrogen bond between the Arg216 carbonyl group
and the amide bond of 18 (green structure, Figure 2, top left),
respectively.
A comparison of minimum energy conformations of 18
with the binding mode of the cyclopentapeptide reference
ligand, c[RGDf(Me)V] (Figure 2, bottom, cyan structure),
suggested a similar arrangement in the receptor’s cavity,
though displaying different secondary interactions. Specifi-
cally, the global minimum conformer of compound 18 (green
structure) showed a hydrogen-bonding interaction with the
Arg216 carbonyl group and a good superimposition of car-
boxylate and guanidino groups to Asp and Arg side chains of
c[RGDf(Me)V] (Figure 2, bottom left), though not mimicking
the hydrophobic π-stacking between D-Phe and Tyr122 aro-
matic side chains (Figure 2, bottom). On the contrary, the
second low-energy conformation of compound 18 (Figure 2,
bottom right, yellow structure), though displaying a worse

7122 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Trabocchi et al.
Figure 2. Top: Compound 18 (green to the left, yellow to the right) docked into the binding region of Rvβ3, highlighting the protein residues
(magenta) that form the key interactions. Bottom: Overlap between the reference ligand c[RGDf(Me)V] (cyan) and compound 18 (green to the
left, yellow to the right) in the binding region of Rvβ3 integrin. Nonpolar hydrogen atoms are omitted for clarity.
Figure 3. Integrin expression in human melanoma cells: RT-PCR
analysis of integrin subunits in A375P and A375M melanoma cells (A);
flow cytometry determination of Rvβ3-positive cells in A375M line (B).
Figure 4. Inhibition values (%) of integrin-mediated A375M cell
adhesion to osteopontin (OPN), vitronectin (VN), and fibronectin
arrangement of pharmacophoric Asp and Arg isosteres,
showed the π-stacking interaction with Tyr 122 side chain in
(FN) induced by compound 18, tested in the absence (A) and in the
analogy with the reference cyclopeptide (cyan).
presence (B) of Mn2þ. ni = no inhibition detected.
Cell Biology Assays. Inhibition of Tumor Cell Adhesion to
Proteins of the Extracellular Matrix. A series of experiments
carried out using semiquantitative RT-PCR analysis allowed us
to demonstrate that A375M human melanoma cells, a high lung
metastatic variant of A375P cells isolated in hard agarose,23
express a wide variety of integrin subunits (RV, β3, β5, R5, β1)
(Figure 3A) and a high level of Rvβ3 heterodimer, (Figure 3B).
A375M melanoma line may well represent a suitable model of
malignant cells to investigate the effects of RGD mimetics.
A375M cells have been used to test the inhibitory capacity of
compound 18, used at 10, 1.0, and 0.1 μM, for tumor cell
binding to important RGD-containing substrates such as
vitronectin, fibronectin, and osteopontin.
Considering that the capacity of the cells to adhere to different
substrates might be dependent on the ligand-binding affinity
related to either the bent or the extended form of integrins, we
tested the effects of compound 18 on the adhesion of tumor cells
to RGD-containing substrates in the presence (Figure 4A) or in
the absence of Mn2þ (Figure 4B). Mn2þ activates integrins

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7123
Figure 5. Integrin expression in HUVEC: RT-PCR analysis of integrin subunits in HUVEC and IL-1β-stimulated HUVEC (A); flow
cytometry determination of Rvβ3-positive cells in HUVEC and IL-1β-stimulated HUVEC (B).
directly by interacting with cation coordination sites in the βA
domain, thus switching integrins from the bent form that binds
ligands with low affinity to the extended form that binds ligands
and carrying out outside-in signaling.24 The results clearly
showed that compound 18, in the presence of Mn2þ, significantly
inhibited the binding of melanoma cells to both vitronectin (with
a dose-dependent effect) and osteopontin (even at nanomolar
concentration), whereas it displayed an incomplete inhibition
toward the adhesion of the cells to fibronectin. These results
suggested that compound 18 displays a high affinity for Rvβ3 and
Rvβ5 integrins and recognizes the RGD sequence exposed by
vitronectin and osteopontin. Moreover, the findings that com-
pound 18, in the absence of Mn2þ, inhibited the binding of
melanoma cells to osteopontin, although at a concentration of
Figure 6. Inhibition values (%) of integrin-mediated IL-1β-
stimulated HUVEC adhesion to osteopontin (OPN), vitronectin
(VN), and fibronectin (FN) induced by compound 18.
the product of 10 and 1 μM, and displayed no effect on the adhe-
sion of tumor cells on vitronectin indicated that compound 18
displays a particular affinity for the activated form of Rvβ3. The
fact that the capacity of compound 18 to inhibit the adhesion of
tumor cells on fibronectin was not modified by Mn2þ (inhibition
in the region of 30-40%) revealed that the affinity of compound
18 is independent of the conformation of the receptor.
Inhibition of HUVEC Adhesion. RT-PCR (Figure 5A) and
FACS analyses (Figure 5B) of the HUVEC (human umbili-
cal vein endothelial cells) grown in the presence or in the
absence of IL-1β indicate that these cells express integrin
subunits (Rv, β3, β5, R5, β1) and Rvβ3 heterodimer, mainly
after IL-1β stimulation. IL-1β stimulated HUVEC mimic the
activated endothelial cells found in tumor angiogenesis.
Before the evaluation of the potential antiangiogenic
capacity of compound 18, we determined the capacity of
this compound to inhibit the adhesion of HUVEC to vitro-
nectin, fibronectin, or osteopontin (Figure 6). Compound 18
suppressed the adhesion of IL-1β-stimulated HUVEC to
vitronectin and osteopontin only partially (inhibition in the
region of 50%), whereas a stronger suppression was found
on fibronectin (inhibition in the region of 80%). This effect
was at variance with the partial inhibition promoted by
compound 18 on melanoma cell adhesion to fibronectin.
Figure 7. Inhibition of in vitro tube formation by HUVEC (A) and
Effect of RGD Antagonists on in Vitro and in Vivo Angio-
genesis. We examined the ability of compound 18 to repress
in vivo vessel invasion into Matrigel plugs (B), induced by com-
pound 18.
angiogenesis by studying the tube formation by endothelial
cells seeded on a Matrigel layer and the in vivo vessel
invasion into Matrigel plugs. We found that compound 18
(final concentration of 0.1 μM) inhibited the tube formation
by HUVEC seeded in Matrigel (Figure 7A). We confirmed
the in vitro antiangiogenic effect of compound 18, demon-
strating that this compound may inhibit vessel invasion into
Matrigel plugs enriched in angiogenic growth factors, such
as bFGF (Figure 7B).

7124 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Trabocchi et al.
Discussion
added a 0.9 M sodium ascorbate solution (1.1 mL/mmol, 1
equiv) and a 0.3 M solution of Cu(OAc)2 (3 mL/mmol, 1 equiv).
The mixture was stirred at room temperature for 2 days. The
organic phase was extracted three times with CH2Cl2, washed
with 5% NaHCO3 solution and brine, then dried over Na2SO4,
and evaporated. The crude mixture was purified through flash
chromatography, thus obtaining the protected product in pure
form. The title compounds were obtained as hydrochloride salts
from the corresponding protected derivatives by acid catalyzed
hydrolysis at room temperature for 16 h with 3 M HCl solution
(5 mL/mmol).
Cu-catalyzed Huisgen cycloaddition reaction between
azidesandalkynesbearingcarboxylate andguanidinogroups,
respectively, allowed for the generation of a pool of triazole-
based RGD mimetics, some of which showed binding affinity
properties toward Rvβ3/Rvβ5 integrins. In particular, com-
pound 18 was selected as a potent ligand for Rvβ3 integrin.
Molecular docking studies gave insight into a RGD-like
binding mode of compound 18 toward Rvβ3 integrin, also
showing a peculiar π-stacking interaction between the triazole
ring and Tyr178 side chain. Also, cluster analysis of docking
calculations revealed two possible equilibrating conforma-
tions characterized by a π-stacking interaction between the
aromatic ring of 18 and Tyr122 side chain and by a hydrogen
bond between the Arg216 carbonyl group and the amide bond
of 18, respectively.
For cell biology experiments, we used A375M melanoma
cells, a highly metastatic cell line expressing a variety of integrin
receptors, which can adhere to fibronectin, osteopontin, and
vitronectin with the same ability (data not shown). Metastatic
tumor cells have redundant pathways to interact with cells and
proteins of the extracellular matrix, including through the
heparin binding domain also present on fibronectin, osteo-
pontin, and vitronectin molecule. Thus, the suppression of
tumor cell adhesion to a specific substrate by RGD antagonists
might be sometimes incomplete. We showed that compound 18
inhibited the adhesion of human melanoma cells to extracellular
matrix proteins, such as vitronectin, fibronection, and osteo-
pontin. In particular, compound 18 proved to be more specific
as an Rvβ3/Rvβ5 integrin antagonist in tumor cells, in view of the
strong inhibition of the adhesion of A375M melanoma cells to
vitronectin and osteopontin. The high affinity of compound 18
for Rvβ3/Rvβ5 was also revealed by the observation that com-
pound 18 inhibited the adhesion of tumor cells to vitronectin
only in the presence of Mn2þ, when integrins are in the activated
form. A special interest was deserved on the capacity of
compound 18 to inhibit tumor cell adhesion to osteopontin
even at nanomolar concentration. Osteopontin represents a
molecular biomarker of the invasion and dissemination in
tumors of breast, stomach, lung, prostate, colon; moreover,
the Rvβ3/osteopontin interaction promotes the activation of
osteoclasts, thus representing a possible mechanism of meta-
static lytic lesions in bone,24b Compound 18 might have a role in
the future in the diagnosis and treatment of bone metastases.
Compound 18 also repressed the adhesion of IL-1β-stimulated
HUVEC to vitronectin and osteopontin, although the most
relevant inhibitory effect was found using fibronectin as a
substrate. Furthermore, compound 18 blocked angiogenesis
in in vitro and in vivo experimental models.
{[2-(4-Guanidinomethyl[1,2,3]triazol-1-yl)acetyl]phenylamino}-
acetic Acid Hydrochloride (9). Following the general procedure,
alkyne 6 (165 mg, 0.55 mmol) and azide 1 (160 mg, 0.55 mmol) in
H2O/t-BuOH 1:1 (2 mL) gave, after workup and purification by
flash chromatography (EtOAc/petroleum ether 2:1, Rf = 0.56),
Pg-9 (177 mg, 55%) as a yellow oil. 1H NMR (CDCl3, 200 MHz)
δ 11.41 (br, 1 H), 8.71 (br, 1 H), 7.68 (s, 1 H), 7.40 (m, 5 H), 4.92
(s, 2 H), 4.66 (d, J = 6.0 Hz, 2 H), 4.23 (s, 2 H), 1.45 (s, 9 H), 1.42
(s, 9 H), 1.41 (s, 9 H). 13C NMR (CDCl3, 50 MHz) δ 167.1 (s),
164.9 (s), 163.0 (s), 155.6 (s), 152.5 (s), 143.4 (s), 140.5 (s), 130.0
(d, 2 C), 129.0 (d), 127.7 (d, 2 C), 123.7 (d), 82.9 (s), 82.1 (s), 79.0
(s), 52.2 (t), 50.9 (t), 36.2 (t), 28.1 (q, 3 C), 27.8 (q, 6 C). The
hydrolysis of Pg-9 allowed us to obtain the final product 9 as a
1
pale orange solid. H NMR (D2O, 400 MHz) δ 7.76 (s, 1 H),
7.43-7.35 (m, 6 H), 5.11 (s, 2 H), 4.39 (s, 2 H), 4.36 (s, 2 H). 13C
NMR (D2O, 50 MHz) δ 174.6 (s), 170.3 (s), 159.4 (s), 145.6 (s),
142.3 (s), 132.9 (d, 2C), 132.2 (d), 130.0 (d, 2 C), 128.0 (d), 54.5
(t), 54.3 (t), 38.8 (t). ESI-MSMS m/z 332 (M - Hþ, 8.6), 315 (70),
245 (80), 199 (100). Anal. Calcd for C14H18ClN7O3: C, 45.72; H,
4.93; N, 26.66. Found: C, 45.81; H, 4.99; N, 26.54.
({2-[4-(2-Guanidinoethyl)[1,2,3]triazol-1-yl]acetyl}phenylamino)-
acetic Acid Hydrochloride (10). Following the general proce-
dure, alkyne 7 (200 mg, 0.64 mmol) and azide 1 (186 mg, 0.64
mmol) in H2O/t-BuOH 1:1 (2.6 mL) gave, after workup and
purification by flash chromatography (EtOAc/petroleum ether
4:1, Rf = 0.69), Pg-10 (228 mg, 59%) as a white solid. 1H NMR
(CDCl3, 200 MHz) δ 7.62 (s, 1 H), 7.44 (m, 5 H), 4.91 (s, 2 H),
4.27 (s, 2 H), 4.18 (t, J = 7.2 Hz, 2 H), 3.02 (t, J = 7.2 Hz, 2 H),
1.48 (s, 9 H), 1.46 (s, 9 H), 1.44 (s, 9 H). 13C NMR (CDCl3, 50
MHz) δ 167.1 (s), 165.1 (s), 163.1 (s), 160.0 (s), 154.6 (s), 143.2
(s), 140.7 (s), 130.1 (d, 2 C), 129.0 (d), 127.9 (d, 2 C), 123.3 (d),
83.9 (s), 82.2 (s), 78.7 (s), 52.4 (t), 50.9 (t), 44.3 (t), 28.4 (q, 3 C),
28.1 (q, 3 C), 28.0 (q, 3 C), 25.4 (t). The hydrolysis of Pg-10
allowed us to obtain the final product 10 as a yellowish solid. 1H
NMR (D2O, 400 MHz) δ 7.68 (s, 1 H), 7.44-7.37 (m, 5 H), 5.10
(s, 2 H), 4.38 (s, 2 H), 3.37 (t, J = 6.2 Hz, 2 H), 2.87 (t, J = 6.2
Hz, 2 H). 13C NMR (D2O, 50 MHz) δ 171.8 (s), 167.4 (s), 156.5
(s), 142.8 (s), 139.6 (s), 130.2 (d, 2 C), 129.5 (d), 127.3 (d, 3 C),
52.0 (t, 2 C), 40.4 (t), 24.2 (t). ESI-MSMS m/z 346 (M - Hþ, 22),
329 (52), 301 (100). Anal. Calcd for C15H20ClN7O3: C, 47.18; H,
5.28; N, 25.68. Found: C, 47.24; H, 5.33; N, 25.53.
({2-[4-(3-Guanidinopropyl)[1,2,3]triazol-1-yl]acetyl}phenyl-
amino)acetic Acid Hydrochloride (11). Following the general
procedure, alkyne 8 (197 mg, 0.61 mmol) and azide 1 (176 mg,
0.61 mmol) in H2O/t-BuOH 1:1 (2.4 mL) gave, after workup and
purification by flash chromatography (EtOAc/petroleum ether
4:1, Rf = 0.75), Pg-11 (232 mg, 62%) as a yellow oil. 1H NMR
(CDCl3, 200 MHz) δ 9.30 (br, 2 H), 7.63 (s, 1 H), 7.48 (m, 5 H),
4.92 (s, 2 H), 4.27 (s, 2 H), 3.95 (t, J = 7.3 Hz, 2 H), 2.74 (t, J =
7.5 Hz, 2 H), 1.95 (m, 2 H), 1.48 (s, 9 H), 1.47 (s, 9 H), 1.43 (s,
9 H). 13C NMR (CDCl3, 50 MHz) δ 167.0 (s), 165.1 (s), 163.4 (s),
160.4 (s), 154.7 (s), 146.3 (s), 140.6 (s), 130.7 (d, 2 C), 129.0 (d),
127.8 (d, 2 C), 122.7 (d), 83.7 (s), 82.1 (s), 78.5 (s), 52.3 (t), 51.0
(t), 44.1 (t), 28.3 (q, 3 C), 28.1 (q, 3 C), 28.0 (q, 3 C), 23.0 (t), 21.8
(t). The hydrolysis of Pg-11 allowed us to obtain the final
product 10 as a yellow solid. 1H NMR (D2O, 400 MHz) δ
7.64 (s, 1 H), 7.44-7.36 (m, 5 H), 5.10 (s, 2 H), 4.38 (s, 2 H), 3.09
In conclusion, our results demonstrated that compound 18,
as a hit RGD mimetic, is capable of exerting relevant bio-
logical effects on integrin receptors associated with the plasma
membrane of tumor cells (i.e., Rvβ3, Rvβ5) as well as endo-
thelial cells, which is a requirement for its future applica-
tions as an antiangiogenic agent in anticancer diagnosis and
therapeutic strategies.
Experimental Section
Combustion analysis was used to determine purity. All tested
compounds were >95% pure.
General Click Chemistry and Hydrolysis Procedure for Com-
pounds 9-22. To a stirred solution of alkyne (1 equiv) and azide
(1 equiv) in H2O/t-BuOH 1:1 (4 mL/mmol) were successively
(t, J = 6.6 Hz, 2 H), 2.66 (t, J = 7.4 Hz, 2 H), 1.83 (m, 2 H). 13
NMR (D2O, 50 MHz) δ 174.7 (s), 170.3 (s), 159.4 (s), 142.3 (s),
C

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7125
139.6 (s), 132.9 (d, 2 C), 132.2 (d), 130.0 (d, 2 C), 128.1 (d), 54.6
(t, 2 C), 42.9 (t), 29.9 (t), 24.0 (t). ESI-MSMS m/z 360.2 (M -
Hþ, 0.6), 343 (54), 315 (16), 301 (26), 227 (100). Anal. Calcd for
C16H22ClN7O3: C, 48.55; H, 5.60; N, 24.77. Found: C, 48.72; H,
5.64; N, 24.65.
(R)-3-{2-[4-(3-Guanidinopropyl)[1,2,3]triazol-1-yl]acetylamino}-
3-phenylpropionic Acid Hydrochloride (15). Following the gen-
eral procedure, alkyne 8 (153 mg, 0.47 mmol) and azide 3a (124
mg, 0.47 mmol) in H2O/t-BuOH 1:1 (2 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 4:1, Rf = 0.38), Pg-15 (153 mg, 55%) as a yellow oil. 1H
NMR (CDCl3, 200 MHz) δ 9.34 (br, 1 H), 7.28-7.20 (m, 6 H),
5.38 (m, 1 H), 5.05 (s, 2 H), 3.90 (m, 2 H), 3.55 (s, 3 H), 2.79 (m, 4
H), 1.98 (m, 2 H), 1.49 (s, 9 H), 1.46 (s, 9 H). 13C NMR (CDCl3,
50 MHz) δ 170.6 (s), 164.5 (s), 163.3 (s), 159.8 (s), 154.6 (s), 147.7
(s), 139.6 (s), 128.5 (d, 2 C), 127.5 (d), 126.0 (d, 2 C), 122.8 (d),
83.8 (s), 78.8 (s), 52.7 (t), 51.8 (q), 50.0 (d), 43.9 (t), 39.8 (t), 28.3
(q, 3 C), 28.0 (q, 3 C), 27.7 (t), 22.9 (t). The hydrolysis of Pg-15
allowed us to obtain the final product 15 as a yellow solid.
[R]24D þ56.9 (c 0.39, H2O). 1H NMR (D2O, 400 MHz) δ 7.68 (s,
1 H), 7.31-7.26 (m, 5 H), 5.19 (pt, J = 7.2 Hz, 1 H), 5.12 (d, J =
3.6 Hz, 2 H), 3.07 (t, J = 6.8, 2 H), 2.86 (d, J = 7.2 Hz, 2 H), 2.68
(t, J = 7.6 Hz, 2 H), 1.83 (m, 2 H). 13C NMR (D2O, 50 MHz) δ
177.0 (s), 168.8 (s), 159.2 (s), 142.4 (s, 2 C), 131.5 (d, 2 C), 130.7
(d), 129.6 (d), 128.8 (d, 2 C), 55.3 (t), 53.4 (d), 42.9 (t), 42.5 (t),
29.8 (t), 23.9 (t). ESI-MSMS m/z 374.2 (M - Hþ, 1.8), 357 (100),
315 (82). Anal. Calcd for C17H24ClN7O3: C, 49.82; H, 5.90; N,
23.92. Found: C, 49.91; H, 5.97; N, 23.75.
[(2-{4-[2-(4-Carbamimidoylpiperazin-1-yl)ethyl][1,2,3]triazol-
1-yl}acetyl)phenylamino]acetic Acid Hydrochloride (12). Fol-
lowing the general procedure, alkyne 5 (372 mg, 0.98 mmol)
and azide 1 (284 mg, 0.98 mmol) in H2O/t-BuOH 1:1 (4 mL)
gave, after workup and purification by flash chromatography
(CH2Cl2/MeOH 12:1, Rf = 0.46), Pg-12 (450 mg, 68%) as an
orange solid. 1H NMR (CDCl3, 200 MHz) δ 7.59 (s, 1 H), 7.45
(m, 5 H), 4.94 (s, 2 H), 4.28 (s, 2 H), 3.62 (m, 4 H), 2.90 (m, 2 H),
2.70 (m, 2 H), 2.57 (m, 4 H), 1.49 (s, 9 H), 1.47 (s, 9 H), 1.44 (s, 9
H). The hydrolysis of Pg-12 gave the final product 12 as an
orange solid. 1H NMR (D2O, 400 MHz) δ 7.88 (s, 1 H),
7.39-7.32 (m, 5 H), 5.06 (s, 2 H), 4.30 (m, 2 H), 3.94 (m, 2 H),
3.62 (m, 2 H), 3.51 (m, 2 H), 3.44 (m, 4 H), 3.12 (m, 4 H). 13C
NMR (D2O, 50 MHz) δ 171.9 (s), 167.8 (s), 163.0 (s), 156.7 (s),
139.8 (s), 130.4 (d, 2 C), 129.7 (d), 127.5 (d, 3 C), 55.7 (t), 51.9 (t),
50.8 (t, 2 C), 48.7 (t), 42.7 (t, 2 C), 40.8 (t). ESI-MSMS m/z 415.2
(M - Hþ, 16), 398 (69), 373 (36), 282 (100), 265 (15). Anal. Calcd
for C19H27ClN8O3: C, 50.61; H, 6.04; N, 24.85. Found: C, 50.78;
H, 6.08; N, 24.68.
(R)-3-[2-(4-Guanidinomethyl[1,2,3]triazol-1-yl)acetylamino]-
3-phenylpropionic Acid Hydrochloride (13). Following the gen-
eral procedure, alkyne 6 (301 mg, 1.01 mmol) and azide 3b (308
mg, 1.01 mmol) in H2O/t-BuOH 1:1 (4 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 1:1, Rf = 0.25), Pg-13 (273 mg, 45%) as a colorless oil. 1H
NMR (CDCl3, 200 MHz) δ 11.42 (br, 1 H), 8.77 (t, J = 5.2 Hz, 1
H), 7.73 (s, 1 H), 7.48 (d, J = 8.0 Hz, 1 H), 7.30-7.16 (m, 5 H),
5.32 (m, 1 H), 5.02 (s, 2 H), 4.68 (d, J = 5.2 Hz, 2 H), 2.79 (m, 2
H), 1.45 (s, 9 H), 1.41 (s, 9 H), 1.29 (s, 9 H). 13C NMR (CDCl3, 50
MHz) δ 169.8 (s), 164.1 (s), 163.1 (s), 155.8 (s), 152.8 (s), 144.3
(s), 139.7 (s), 128.5 (d, 2 C), 127.6 (d), 126.1 (d, 2 C), 123.8 (d),
83.1 (s), 81.4 (s), 79.3 (s), 52.8 (t), 50.2 (d), 41.0 (t), 36.3 (t), 28.2
(q, 3 C), 27.9 (q, 3 C), 27.8 (q, 3 C). The hydrolysis of Pg-13 gave
the final product 13 as white solid. [R]24D þ73.2 (c 0.23, H2O).
1H NMR (D2O, 400 MHz) δ 7.86 (s, 1 H), 7.31-7.22 (m, 5 H),
5.15 (s, 2 H), 5.12 (t, J = 6.8 Hz, 2 H), 4.42 (s, 2 H), 2.64 (d, J =
6.8 Hz, 2 H). 13C NMR (D2O, 50 MHz) δ 180.4 (s), 169.4 (s),
159.5 (s), 145.9 (s), 143.5 (s), 131.4 (d, 2 C), 130.3 (d), 128.8 (d, 2
C), 127.7 (d), 54.8 (t), 54.6 (d), 45.6 (t), 38.9 (t). ESI-MSMS m/z
346 (M - Hþ, 5.6), 329 (100), 304 (36), 259 (48). Anal. Calcd for
C15H20ClN7O3: C, 47.18; H, 5.28; N, 25.68. Found: C, 47.26; H,
5.35; N, 25.57.
(R)-3-{2-[4-(2-Guanidinoethyl)[1,2,3]triazol-1-yl]acetylamino}-
3-phenylpropionic Acid Hydrochloride (14). Following the gen-
eral procedure, alkyne 7 (200 mg, 0.64 mmol) and azide 3a (168
mg, 0.64 mmol) in H2O/t-BuOH 1:1 (2.6 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 4:1, Rf = 0.50), Pg-14 (177 mg, 48%) as a yellow oil. 1H
NMR (CDCl3, 200 MHz) δ 9.24 (br, 1 H), 7.68 (s, 1 H),
7.30-7.17 (m, 5 H), 5.38 (m, 1 H), 5.02 (s, 2 H), 4.17 (t, J =
7.3 Hz, 2 H), 3.58 (s, 3 H), 3.06 (t, J = 7.3 Hz, 2 H), 2.81 (m, 2 H),
1.49 (s, 9 H), 1.48 (s, 9 H). 13C NMR (CDCl3, 50 MHz) δ 170.7
(s), 164.3 (s), 163.5 (s), 160.0 (s), 154.5 (s), 145.6 (s), 139.5 (s),
128.6 (d, 2 C), 127.7 (d), 125.9 (d, 2 C), 123.3 (d), 84.0 (s), 78.7 (s),
52.9 (t), 51.9 (q), 50.1 (d), 44.2 (t), 39.7 (t), 28.5 (q, 3 C), 28.0 (q, 3
C), 25.4 (t). The hydrolysis of Pg-14 allowed us to obtain the
final product 14 as a yellow solid. [R]24D þ60.9 (c 0.32, H2O). 1H
NMR (D2O, 400 MHz) δ 7.72 (s, 1 H), 7.32-7.28 (m, 5 H), 5.20
(m, 1 H), 5.14 (d, J = 5.2 Hz, 2 H), 3.38 (t, J = 6.4 Hz, 2 H),
3.89-2.85 (m, 4 H). 13C NMR (D2O, 50 MHz) δ 173.5 (s), 165.8
(s), 155.7 (s), 138.9 (s, 2 C), 128.0 (d, 2 C), 127.1 (d), 126.0 (d),
125.3 (d, 2 C), 51.3 (t), 49.8 (d), 39.4 (t), 38.9 (t), 23.3 (t). ESI-
MSMS m/z 360.2 (M - Hþ, 1.8), 343 (65), 315 (100). Anal.
Calcd for C16H22ClN7O3: C, 48.55; H, 5.60; N, 24.77. Found: C,
48.70; H, 5.68; N, 24.65.
(S)-3-[2-(4-Guanidinomethyl[1,2,3]triazol-1-yl)acetylamino]-
3-phenylpropionic Acid Hydrochloride (16). Following the gen-
eral procedure, alkyne 6 (195 mg, 0.66 mmol) and azide 2b (200
mg, 0.66 mmol) in H2O/t-BuOH 1:1 (2.6 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 1:1, Rf = 0.25), Pg-16 (388 mg, 64%). The hydrolysis of
protected Pg-16 compound gave the final product 16 as a
yellowish solid with similar NMR data as reported for com-
pound 13. [R]24 -66.3 (c 0.2, H2O). Anal. Calcd for C15H20-
D
ClN7O3: C, 47.18; H, 5.28; N, 25.68. Found: C, 47.29; H, 5.38;
N, 25.59.
(S)-3-{2-[4-(2-Guanidinoethyl)[1,2,3]triazol-1-yl]acetylamino}-
3-phenylpropionic Acid Hydrochloride (17). Following the gen-
eral procedure, alkyne 7 (140 mg, 0.45 mmol) and azide 2a (118
mg, 0.45 mmol) in H2O/t-BuOH 1:1 (2 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 4:1, Rf = 0.50), Pg-17 (203 mg, 55%). The hydrolysis of
protected Pg-17 compound gave the final product 17 as a
yellowish solid with similar NMR data as reported for com-
pound 14. [R]24D -63.2 (c 0.27, H2O). Anal. Calcd for C16H22-
ClN7O3: C, 48.55; H, 5.60; N, 24.77. Found: C, 48.72; H, 5.69;
N, 24.64.
(S)-3-{2-[4-(3-Guanidinopropyl)[1,2,3]triazol-1-yl]acetylamino}-
3-phenylpropionic Acid Hydrochloride (18). Following the gen-
eral procedure, alkyne 8 (200 mg, 0.61 mmol) and azide 2a (160
mg, 0.61 mmol) in H2O/t-BuOH 1:1 (2.5 mL) gave, after workup
and purification by flash chromatography (EtOAc/petroleum
ether 4:1, Rf = 0.38), Pg-18 (215 mg, 60%). The hydrolysis of
protected Pg-18 compound gave the final product 18 as a
yellowish solid with similar NMR data as reported for com-
pound 15. [R]24D -63.5 (c 0.57, H2O). Anal. Calcd for C17H24-
ClN7O3: C, 49.82; H, 5.90; N, 23.92. Found: C, 49.90; H, 5.94;
N, 23.71.
(R)-3-{2-[4-(4-Carbamimidoylpiperazin-1-ylmethyl)[1,2,3]tri-
azol-1-yl]acetylamino}-3-phenylpropionic Acid Hydrochloride
(19). Following the general procedure, alkyne 4 (117 mg, 0.32
mmol) and azide 3b (98 mg, 0.32 mmol) in H2O/t-BuOH 1:1 (1.5
mL) gave, after workup and purification by flash chromato-
graphy (CH2Cl2/MeOH 10:1, Rf = 0.47), Pg-19 (93 mg, 43%) as
a yellowish oil. 1H NMR (CDCl3, 200 MHz) δ 7.63 (s, 1H), 7.37
(br, 1 H), 7.20 (m, 5 H), 5.33 (m, 1 H), 5.01 (s, 2 H), 3.65 (s, 2 H),
3.51 (m, 4 H), 2.68 (m, 2 H), 2.51 (m, 4 H), 1.46 (s, 18 H), 1.28 (s,
9 H). 13C NMR (CDCl3, 50 MHz) δ 169.7 (s), 164.1 (s), 162.8 (s),
154.4 (s), 144.0 (s), 139.7 (s), 137.2 (s), 128.3 (d, 2 C), 127.5 (d),
126.1 (d, 2 C), 124.3 (d), 88.7 (s), 81.2 (s), 80.0 (s), 52.6 (t), 52.4
(t), 52.1 (t), 50.2 (d), 46.4 (t), 41.0 (t), 28.2 (q), 27.9 (q, 3 C), 27.6
(q, 3 C). The hydrolysis of Pg-19 allowed us to obtain the final

7126 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Trabocchi et al.
product 19 as a white solid. [R]24D þ49.3 (c 0.21, H2O). 1H NMR
(D2O, 400 MHz) δ 7.96 (s, 1 H), 7.33-7.27 (m, 5 H), 5.23 (s, 2 H),
5.17 (t, J = 6.8 Hz, 2 H), 3.87 (s, 2 H), 3.45 (m, 4 H), 3.21 (m, 2
H), 2.67 (m, 4 H). 13C NMR (D2O, 50 MHz) δ 180.9 (s), 169.2
(s), 158.8 (s), 143.7 (s), 143.4 (s), 131.4 (d, 2 C), 130.3 (d), 129.7
(d), 128.8 (d, 2 C), 54.8 (t), 52.9 (t), 50.8 (d), 47.0 (t), 46.2 (t), 45.6
(t). ESI-MSMS m/z 415 (M - Hþ, 32), 398 (27), 373 (100). Anal.
Calcd for C19H27ClN8O3: C, 50.61; H, 6.04; N, 24.85. Found: C,
50.71; H, 6.11; N, 24.43.
(R)-3-(2-{4-[2-(4-Carbamimidoylpiperazin-1-yl)ethyl][1,2,3]tri-
azol-1-yl}acetylamino)-3-phenylpropionic Acid Hydrochloride
(20). Following the general procedure, alkyne 5 (319 mg, 0.84
mmol) and azide 3a (221 mg, 0.84 mmol) in H2O/t-BuOH 1:1
(3.5 mL) gave, after workup and purification by flash chroma-
tography (CH2Cl2/MeOH 12:1, Rf = 0.46), Pg-20 (297 mg,
55%). 1H NMR (CDCl3, 200 MHz) δ 7.54 (s, 1 H), 7.30-7.20
(m, 5 H), 5.38 (m, 1 H), 5.03 (s, 2 H), 3.58 (m, 4 H), 3.58 (s, 3 H),
2.94 (m, 2 H), 2.81 (m, 2 H), 2.71 (m, 2 H), 2.56 (m, 4 H), 1.48 (s,
18 H). 13C NMR (CDCl3, 50 MHz) δ 170.8 (s), 164.4 (s), 162.8
(s), 154.6 (s), 146.4 (s), 139.5 (s), 137.2 (s), 128.6 (d, 2 C), 127.7
(d), 126.0 (d, 2 C), 122.8 (d), 88.7 (s), 81.2 (s), 57.3 (t), 52.9 (t),
52.5 (t), 52.0 (q), 50.0 (d), 46.8 (t), 39.7 (t), 28.2 (q, 6C), 23.3 (t).
The hydrolysis of Pg-20 gave the final product 20 as a white
solid. [R]24D þ57.4 (c 0.31, H2O). 1H NMR (D2O, 400 MHz) δ
7.81 (s, 1 H), 7.33-7.28 (m, 5 H), 5.18-5.16 (m, 3 H), 3.68 (m, 4
H), 3.54-3.41 (m, 6 H), 3.16 (t, J = 7.4 Hz, 2 H), 2.84 (d, J = 7.2
Hz, 2 H). 13C NMR (D2O, 50 MHz) δ 173.5 (s), 165.9 (s), 155.6
(s), 141.3 (s), 138.9 (s), 128.0 (d, 2 C), 127.1 (d), 125.3 (d, 2 C),
124.7 (d), 54.7 (t), 51.2 (d), 49.8(t), 41.6 (t), 39.7 (t), 38.9 (t), 18.9
(t). ESI-MSMS m/z 429.2 (M - Hþ, 33), 413 (100), 388 (30).
Anal. Calcd for C20H29ClN8O3: C, 51.66; H, 6.29; N, 24.10.
Found: C, 51.78; H, 6.34; N, 24.01.
and then competition binding studies were performed with a
fixed concentration of [125I]echistatin (0.05 and 0.1 nM for Rvβ3
and Rvβ5, respectively) and concentrations ranging from 0.01
nM to 100 μM tested compounds. All assays were performed in
triplicate in a final volume of 0.2 mL, each containing the
following species: 0.05 mL of [125I]echistatin, 0.04 mL of the
tested compound, and 0.11 mL of the blocking/binding buffer.
Nonspecific binding was defined as [125I]echistatin bound in the
presence of an excess (1 μM) of unlabeled echistatin. After 3 h
incubating at room temperature, the plate was washed three
times with the blocking/binding buffer. Then it was counted in a
Top-Count NXT microplate scintillation counter (PerkinElmer
Life Sciences, Boston, MA) using 200 μL/well of MicroScint-40
liquid scintillation (PerkinElmer Life Sciences, Boston, MA).
The IC50 values were determined by fitting the binding inhi-
bition data by nonlinear regression using GraphPad Prism
4.0 software package (GraphPad Prism, San Diego, CA). More-
over, when the Hill slope of the curves was significantly less than
unity (K<-0.80), the data were reprocessed with a two-sites
model. The displacement curves that fitted (p < 0.05) better
according to a two-sites model rather than to a one-site model
were considered significant.
Cell Lines and Culture Conditions. A375P and A375M human
melanoma cell lines used in this study were obtained from
American Type Culture Collection (ATCC, Rockville, MD).
The A375M cells were a clone isolated in hard agarose by Li
et al.23 from A375P cells and characterized by a high lung
metastatic potential. Melanoma cells were grown in Dulbecco’s
modified Eagle medium containing 4500 mg/L glucose (DMEM
4500, GIBCO) supplemented with 10% fetal calf serum (FCS) at
37 ꢀC in a humidified incubator containing 10% CO2. Then
5.0 ꢀ 105 cells were seeded in 100 mm Sarstedt dishes and
propagated every 3 days by incubation with a trypsin-EDTA
solution. The capacity of A375M cells to grow in dense agarose
was evaluated before the experiments. Cultures were periodi-
cally monitored for mycoplasma contamination .
(S)-3-{2-[4-(4-Carbamimidoylpiperazin-1-ylmethyl)[1,2,3]tri-
azol-1-yl]acetylamino}-3-phenylpropionic Acid Hydrochloride
(21). Following the general procedure, alkyne 4 (222 mg, 0.61
mmol) and azide 2b (186 mg, 0.61 mmol) in H2O/t-BuOH 1:1
(2 mL) gave, after workup and purification by flash chroma-
tography (CH2Cl2/MeOH 10:1, Rf = 0.47), Pg-21 (201 mg,
49%). The hydrolysis of protected Pg-21 compound gave the
final product 21 as a yellowish solid with similar NMR data as
Primary cultures of human umbilical vein endothelial cells
(HUVEC) were obtained from Lonza. Cells grown in an endo-
thelial cell growth medium-2 (EGM-2) containing endothelial
cell growth supplements were subcultivated using a trypsin-
EDTA solution, 1:3 split ratio. For the use in the experiments,
cells (passages 2-4) were grown to confluence in plates coated
with 1% bovine gelatin.
reported for compound 19. [R]24 -70.8 (c 0.58, H2O). Anal.
D
Calcd for C19H27ClN8O3: C, 50.61; H, 6.04; N, 24.85. Found: C,
50.69; H, 6.13; N, 24.38.
(S)-3-(2-{4-[2-(4-Carbamimidoylpiperazin-1-yl)ethyl][1,2,3]tri-
azol-1-yl}acetylamino)-3-phenylpropionic Acid Hydrochloride
(22). Following the general procedure, alkyne 5 (361 mg, 0.95
mmol) and azide 2a (250 mg, 0.95 mmol) in H2O/t-BuOH 1:1
(4 mL) gave, after workup and purification by flash chroma-
tography (CH2Cl2/MeOH 12:1, Rf = 0.46), Pg-22 (400 mg,
65%). The hydrolysis of protected Pg-22 compound gave the
final product 22 as a yellow solid with similar NMR data as
RNA Isolation and Polymerase Chain Reaction (PCR). Total
RNA extracted using RNAgents (Total RNA Isolation System,
Promega, Madison, WI) was determined spectrophotometri-
cally. Complementary DNA (cDNA) was synthesized from 2 μg
of total RNA using 1 μL of ImProm-II reverse transcriptase
(Promega). Aliquots of 2 μL of the cDNA were used for PCR
amplification. The specific primers used for the identification of
human Rv, R5, β1, β3, β5, and GAPDH are reported in Table S1
of Supporting Informations. All PCR experiments were
conducted using 0.05 U/μL Go-Taq polymerase (Promega).
Amplification was carried out on a Perkin-Elmer thermal cycler.
An amount of 8 μL of each PCR product was visualized by a 2%
agarose gel containing ethidium bromide (0.5 μg/mL) electro-
phoresis. cDNA products were evaluated on the basis of a
standard PCR marker.
Flow Cytometry Assay. Cells were detached by gentle treat-
ment with Accutase, a 0.5 mM EDTA solution, washed, and
incubated for 1 h at 4 ꢀC in the presence of anti-Rvβ3 mono-
clonal antibody (1 μg/50 μL, anti-integrin Rvβ3, clone LM609,
Millipore). Cells were then washed and incubated for 1 h at
4 ꢀC with a secondary antibody, 5 μg/mL goat antimouse IgG
conjugated with FITC (Santa Cruz Biotecnology, Inc., Santa
Cruz, CA). Rvβ3-Positive cells were analyzed at 488 nm on the
flow cytometer FACScan system (BD-FACS Canto).
reported for compound 20. [R]24 -55.2 (c 0.32, H2O). Anal.
D
Calcd for C20H29ClN8O3: C, 51.66; H, 6.29; N, 24.10. Found: C,
51.73; H, 6.36; N, 24.00.
Integrin Binding Assay. [125I]Echistatin, labeled by the lacto-
peroxidase method25 to a specific activity of 2200 Ci/mmol, was
purchased from PerkinElmer Life Sciences, Boston, MA.
Integrin proteins Rvβ3 and Rvβ5 purified from human placenta
were purchased from Chemicon International Inc., Temecula,
CA. The receptor binding assay was performed as described.16
Purified receptors Rvβ3 and Rvβ5 were diluted respectively at 500
and 1000 ng/mL in a coating buffer [20 mM Tris (pH 7.4), 150
mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2]. An
aliquot of the diluted receptors (100 μL/well) was added to a
96-well microtiter plate (Optiplate-96 HB, PerkinElmer Life
Sciences, Boston, MA) and incubated overnight at 4 ꢀC. The
plate was washed once with a blocking/binding buffer [20 mM
Tris (pH 7.4), 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM
MnCl2, 1% BSA] and incubated for an additional 2 h at room
temperature. The plate was rinsed twice with the same buffer,
Cell Adhesion Assay. Plates (96 wells) were coated with
fibronectin (1 μg/mL), osteopontin (0.5 μg/mL), or vitronectin
(10 μg/mL) by overnight incubation at 4 ꢀC. Plates were washed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7127
with PBS and then incubated at 37 ꢀC for 1 h with PBS-1%
BSA. After being washed, tumor cells or IL-1β (400 U/mL)
stimulated HUVEC were counted, resuspended in serum free
medium, and exposed to compound 18 (final concentration of
compound was 0.1, 1, or 10 μM) at 37 ꢀC for 30 min to allow the
ligand-receptor equilibrium to be reached. In the case of
melanoma cell adhesion, assays were performed in the presence
or in the absence of 2 mM MnCl2 solution.26 Cells were plated
((4-5)ꢀ104 cells/well) and incubated at 37 ꢀC for 2 h. All the
wells were washed with PBS to remove the nonadherent cells,
and 0.5% crystal violet solution in 20% methanol was added.
After 2 h of incubation at 4 ꢀC, plates were examined at 540 nm
in a counter ELX800 (Bio TEK Instruments). Experiments were
conducted in triplicate and were repeated at least three times.
In Vitro Tubular Formation by HUVEC. Matrigel (10 μg/μL,
BD Bioscience, San Jose, CA) precoated 96-well plates were
used, and HUVEC (20 000 cells/well) were seeded in the pres-
ence of compound 18 (10 μM final concentration). Cells were
incubated at 37 ꢀC for 16-18 h, and capillary-like tube struc-
tures were examined and photographed by the use of a Nikon
inverted microscope. The experiments were repeated three
times.
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sion. Cancer Res. 1990, 50, 6757–6764.
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P.; Herlyn, M.; Elder, D. E. Progression-related expression of beta3
integrin in melanomas and nevi. Hum. Pathol. 1999, 30, 562–567.
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melanoma progression: multifunctional activities associated with
alpha(v)beta3 integrin expression. Am. J. Pathol. 1998, 153, 1347–
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In Vivo Angiogenesis Assay. The 5- to 6-week-old C57Bl/6
mice were used. Matrigel solution was mixed with 50 U/mL
heparin resuspended in EGM-2 medium and compound 18.
Compound 18 was used at the final concentration of 0.1 μM.
500 μL Matrigel mixture was injected subcutaneously, and
5 days after, mice were sacrificed. Matrigel plugs were removed
and photographed with the aid of a dissecting microscope.
Acknowledgment. Financial support from Istituto Toscano
Tumori, MIUR (PRIN08, Grant 2008J4YNJY_003), and
Fondazione Roma is acknowledged.
(10) (a) Raboisson, P.; DesJarlais, R. L.; Reed, R.; Lattanze, J.;
ꢀ
Chaikin, M.; Manthey, C. L.; Tomczuk, B. E.; Marugan, J. J.
Supporting Information Available: Experimental procedures
for compounds 1-8; 1H and 13C NMR spectra of compounds
9-22; inhibition curves of [125I]echistatin specific binding to
purified human integrin proteins RVβ3 and RVβ5 by compounds
15, 16, 17, 20, and 22; and Table S1 detailing specific primers for
the identification of human Rv, R5, β1, β3, β5, and GAPDH.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Identification of novel short chain 4-substituted indoles as potent
Rvβ3 antagonist using structure-based drug design. Eur. J. Med.
Chem. 2007, 42, 334–343. (b) Ishikawa, M.; Kubota, D.; Yamamoto,
M.; Kuroda, C.; Iguchi, M.; Koyanagi, A.; Murakami, S.; Ajito, K.
Tricyclic pharmacophore-based molecules as novel integrin Rvβ3
antagonists. Part 2: Synthesis of potent Rvβ3/RIIbβ3 dual antagonists.
Bioorg. Med. Chem. 2006, 14, 2109–2130. (c) Cacciari, B.; Spalluto,
G. Non peptidic Rvβ3 antagonists: recent developments. Curr. Med.
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