Modulation of αvβ3- and α5β1-integrin-mediated adhesion by dehydro-β-amino acids containing peptidomimetics

Article abstract of DOI:10.1016/j.ejmech.2013.05.050

A novel class of low molecular weight ligands of α_vβ ₃ and α₅β₁ integrins, that possess a dehydro-β-amino acid as conformationally constrained core, linked to the pharmacophoric moieties mimicking the RGD recognition sequence, have been synthesized through a very simple protocol. Cell adhesion assays and integrin-mediated signaling activation experiments suggested a good affinity of these compounds toward both integrin receptors. Moreover, further elongation with two glycine units allowed to obtain an excellent dual inhibitor. Structural models for α_vβ₃ integrin-ligand binding con firmed that the dehydro-β-amino derivatives are able to act as an electrostatic clamp by establishing several stabilizing interactions with the receptor.

Full text of DOI:10.1016/j.ejmech.2013.05.050

Accepted Manuscript
Modulation of α β - and α β -integrin mediated adhesion by dehydro-β-amino acids
v 3
5 1
containing peptidomimetics
Alessandra Tolomelli, Monica Baiula, Laura Belvisi, Angelo Viola, Luca Gentilucci,
Stefano Troisi, Samantha Deianira Dattoli, Santi Spampinato, Monica Civera, Eusebio
Juaristi, Margarita Escudero
PII:
S0223-5234(13)00373-5
10.1016/j.ejmech.2013.05.050
EJMECH 6233
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 February 2013
Revised Date: 18 April 2013
Accepted Date: 22 May 2013
Please cite this article as: A. Tolomelli, M. Baiula, L. Belvisi, A. Viola, L. Gentilucci, S. Troisi, S.D. Dattoli,
S. Spampinato, M. Civera, E. Juaristi, M. Escudero, Modulation of α β - and α β -integrin mediated
v 3
5 1
adhesion by dehydro-β-amino acids containing peptidomimetics, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.05.050.
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Highlights Tolomelli et al..docx
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Highlights
 Dehydro--amino acid derivatives were synthesized as RGD mimetics  Cell adhesion assays
suggested good affinity toward αvβ3 and α5β1 integrins  Selected compounds showed ability to
inhibit integrin-mediated signalling activation  Docking experiments were performed to verify
ligand-receptor interactions.  The low molecular weight and the simple synthetic route may
represent an advantage on other ligands

REVISED Tolomelli et al-EJMECH-D-00225.docx
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Modulation ofv3- and 51-integrin mediated adhesion by dehydro--amino
acids containing peptidomimetics
Alessandra Tolomelli,*[a] Monica Baiula,*[b] Laura Belvisi,[c] Angelo Viola,[a] Luca Gentilucci,[a]
Stefano Troisi,[a] Samantha Deianira Dattoli,[b] Santi Spampinato,[b] Monica Civera[c], Eusebio
Juaristi[d] and Margarita Escudero[d]
[a] Department of Chemistry “G.Ciamician”, University of Bologna, Via Selmi 2, 40126, Bologna, Italy, Fax:
(+39)0512099456, e-mail: alessandra.tolomelli@unibo.it
[b] Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, Italy, e-mail:
monica.baiula@unibo.it
[c] Department of Chemistry, University of Milan , Via Golgi 19, 20133, Milan, Italy
[d]Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico
Nacional 2508, Col. San Pedro Zacatenco, 07360 México
Abstract
A novel class of low molecular weight ligands of αvβ3 and α5β1 integrins, that possess a dehydro--
amino acid as conformationally constrained core, linked to the pharmacophoric moieties mimicking
the RGD recognition sequence, have been synthesized through a very simple protocol. Cell
adhesion assays and integrin-mediated signaling activation experiments suggested a good affinity of
these compounds toward both integrin receptors. Moreover, further elongation with two glycine
units allowed to obtain an excellent dual inhibitor. Structural models for αvβ3 integrin-ligand
binding confirmed that the dehydro--amino derivatives are able to act as an electrostatic clamp by
establishing several stabilizing interactions with the receptor.
Keywords: • Dehydro--amino acids, Peptidomimetic, Cell adhesion, Signalling, Drug discovery
Introduction
In the last few decades, antiangiogenic therapy has been extensively studied since
neovascularization is essential for tumour growth and development,[1] but the benefit of this
approach is often quite limited, since inhibition of one angiogenetic pathway may turn into
activation of a parallel mechanism.[2] It has been demonstrated that, during the angiogenesis
process, spreading endothelial cells express different markers, which are completely absent in
normal capillaries.[3] Among them, αvβ3 and α5β1 integrins are over-expressed in novel vessels
surrounding tumour cells, therefore drugs having these integrins as targets could lead to a
synergistic effect in vessel generation without altering normal blood vessels.[4]
Integrin αvβ3 is not generally expressed on epithelial cells and is present only at low levels on a
subset of B cells, some cells of macrophage lineage, smooth muscle cells, and activated endothelial

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cells.[5] On the other hand, this integrin is also expressed on certain invasive tumours including
metastatic melanoma and late-stage glioblastoma.[6] In vivo, integrins αvβ3 and α5β1 are
simultaneously present on the endothelial cells of tumour blood vessels and of tissues exposed to
angiogenic growth factors.[7] Integrin α5β1 is largely selective for fibronectin, which is a
component of the provisional matrix expressed by endothelial cells during wound healing or tumour
angiogenesis.[7,8] Integrin αvβ3, in contrast, is a promiscuous integrin with the potential to mediate
migration on a host of extracellular matrix proteins with arginine-glycine-aspartic acid moieties,
such as vitronectin, fibrinogen, collagen, von Willebrand’s factor, and others.[9] To maintain
orderly outgrowth of blood vessels, this ability of αvβ3 to promote migration in most extracellular
matrix contexts must be regulated at cellular level through precise mechanisms. Thus, ligation of
integrin α5β1 by endothelial cell fibronectin is one means by which αvβ3 functions are regulated
during angiogenesis in vivo.[10] Involvement and cross-talking of the two receptors makes the
discovery of ligands capable to tune or inhibit the activity of both of them (dual inhibitors) highly
desirable.[11]
The identification of key recognition motifs within integrin ligands is the starting point for the
development of antagonists. Both αvβ3 and α5β1 integrins bind to ECM components through
recognition of the Arg-Gly-Asp (RGD) tripeptide sequence. The X-ray analysis of the complex
between αvβ3 integrin and c(RGDf-NMe-V) ligand (Cilengitide) shows that binding occurs mainly
through electrostatic interactions.[12] Concerning α5β1 integrin, the crystal structure of the receptor
in complex with a Fab fragment of the anti-1 inhibitory antibody SG/19, both in the presence and
in the absence of an RGD containing peptide, has been published, revealing a high similarity with
αvβ3 integrin complex.[13]
Recently, great interest has been paid also to integrin conformational changes and their role in
activation and signaling. Although the exact mechanism is still not completely disclosed, it is
generally accepted that ligand-receptor interaction induces switch from a low-affinity bent
conformation, to an high-affinity extended one, passing through an intermediate inactive
conformation having closed headpiece. It has been reported that small molecule ligands, that may
only partially match with the binding site, show a different effect when compared with classical
RGD ligands [14]. Carboxylate coordination with MIDAS (metal ion-dependent adhesion site)
metal ion induces swing out to the active conformation, while no changes occurs for binding with
molecules linking only to the  subunit.
In previous papers, we have identified a series of αvβ3/ α5β1 integrin dual ligands, sharing as a
common feature the presence of an unsaturated α- or β-amino acid fragment, inserted into a cyclic

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backbone (Figure 1, compounds A and B).[15] The introduction of a β-amino acid into RGD-
containing cyclic peptide has been already reported in the literature with the aim to stabilize distinct
conformations, having a β-turn with centrally positioned glycine.[16] We recently reported
isoxazoline-containing ligands having bioactivity in the nanomolar range and we hypothized that
the restricted conformation introduced by the dehydro-β-amino acid and the cyclic structure could
give a favorable alignment of both the basic and carboxylate moieties, meeting the crucial
requirements for affinity and selectivity (Figure 1, compound B). Next, our aim was to minimize
molecular complexity and synthetic steps, in order to obtain low molecular weight compounds.
Figure 1. αvβ3/ α5β1 integrin ligands containing an unsaturated α- or β-amino acid fragment (A,B) and novel dehydro-β-
amino acid structures (C).
In the study reported herein, the synthesis and biological evaluation of a collection of dehydro--
amino acid containing molecules has been developed (Figure 1, compound C). Some compounds
from the library, that have a shorter length than that generally required for RGD mimicking, have
been studied also with the aim to observe the effect of a partial ligand/receptor interaction on
adhesion and intracellular signaling. Moreover, on the basis of our recent results in the design and
synthesis of ligands containing cyclic dehydro--amino acids, we wanted to verify if the linear
dehydro--amino acid could be a useful scaffold and lead to the preparation of bioactive
derivatives, thus representing the minimal peptidomimetic structures for αvβ3 and α5β1 integrins
mediated adhesion regulation.

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Results and Discussion
Chemistry
Dehydro--amino acid containing molecules, with different variations at the nature and length of
the side chains were synthesized through SN2’ substitution of chiral carbonate-esters or amides and
analyzed as integrin antagonists. At first, the small library of N-p-aminobenzyl-dehydro--amino
acids 3a-d was prepared through an easy two step procedure starting form carbonates 1a-d (Scheme
1). The regio- and stereoselective synthesis of amino esters 2a-d was based on our previously
reported results and has been described in due papers, where each compound has been accurately
analyzed.[17]
Scheme 1. i. p-aminobenzylamine, refluxing CH3CN; ii. TFA, DCM, r.t.
Removal of the ester protection to obtain free amino acids 3a-d was performed by treatment with an
excess of trifluoroacetic acid in DCM at room temperature (77-95% yield). Although the
compounds 3a-d have a shorter length than that generally required for RGD mimetic ligands, their
low molecular weight and the respect of the commonly accepted rules for bioavailability,[18]
suggested that they could represent valuable lead compounds. On the other hand, the shorter length
may lead to a partial ligand/receptor interaction and to an uncommon effect on adhesion and
intracellular signaling.
The glycine containing derivatives 8a-b have been obtained through the SN2’ substitution on the
appropriate racemic Boc-amides 6a-b (Scheme 2).

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Scheme 2: i. TFA, DCM, r.t.; ii. Gly-OtBu, HBTU, TEA, DCM, r.t.; iii. Boc2O, DMAP, TEA, THF, r.t.; iv. p-
aminobenzylamine, refluxing CH3CN, 16h; v. H3PO4, DCM, r.t.; vi. p-aminobenzylamine, refluxing CH3CN, 4 days;
vii. N-Boc- Gly, HBTU, DIPEA, DMF, r.t.; viii. H3PO4, DCM, r.t.
To this purpose, selective removal of the t-butyl ester moiety in 1a-b (95% yield), followed by
coupling with t-butyl glycinate afforded carbonates 5a-b in 60÷65% yield. The Boc protection on
the amide nitrogen was then introduced to obtain 6a-b (90% yield), with the aim to enhance the
reactivity of the double bond in the substitution reaction thanks to the electron withdrawing effect.
The substitution reaction on the N-Boc-derivatives gave different results depending on the side
chain. Treatment of 6b with p-aminobenzylamine in refluxing acetonitrile afforded indeed the
corresponding amino derivative 7b in 65% yield when the reaction was stopped after 16h. After
prolonged reaction times (4 days), spontaneous intramolecular cyclization to compound 9b occurred
(60% yield). On the contrary, starting from 6a, compound 7a was isolated in 70% yield, and no
traces of the cyclic derivative could be detected even after longer refluxing times. Simultaneous
removal of the ester and carbamate protections by treatment of 7a-b in a phosphoric
acid/dichloromethane biphasic mixture, allowed to obtain 8a-b in 80÷95% yield.
The different behaviors of 7a and 7b in the intramolecular cyclization can be ascribed to the
different conformational arrangement induced by the steric hinderance of methyl and isopropyl
chains. The rotation of the C2-C3 bond in 7 that may occur to minimize steric hinderance, displays
for 7b the amine nitrogen in the proper position to act as nucleophile on the carbamate carboxylic
group (Figure 2).

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Figure 2: Proposed conformational arrangements of major isomers Z-7a and Z-7b, explaining the alternative behaviors
in intramolecular cyclization.
Since this reaction affords dihydropyrimidindiones, that have been successfully introduced as rigid
scaffolds in bioactive molecules of considerable interest for the pharmaceutical industry,[19] this
process deserves further investigation. The effect of the carbamate leaving group, amine
nucleophile, C3 stereochemistry and amino acid chain will be evaluated by computational studies
and reported in due course.
To explore whether the cyclic structure could be suitable for the interaction with the receptor,
dihydropyrimidindione 9b was further decorated with a second glycine residue. Compound 10b was
isolated in 90% yield, following the usual coupling protocol and then deprotected to afford the free
amino acid 11b (80% yield). The spontaneous cyclization occurring during the substitution reaction
on N-Boc amide, suggested that inversion of the steps in the synthetic protocol could allow the
easier isolation of the linear compound. Thus, compound 12b was isolated in 84% yield by
treatment of 2b with Fmoc-Gly-Cl in the presence of triethylamine. After removal of the tert-butyl
ester, coupling with Gly-Otbu under the usual peptide synthesis conditions, allowed to obtain 13b
in 55% overall yield. Selective removal of Fmoc and t-Butyl protection afforded 14b in 60% yield.

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Scheme 3: i. Fmoc-Gly-Cl, TEA, DCM, r.t; ii. TFA, DCM, r.t.; iii. Gly-OtBu, HBTU, TEA, CH3CN, r.t.; iv. H3PO4,
DCM, r.t.; v. Piperidine, CH3OH, r.t.
Pharmacology
Inhibition of v3 and 51 integrin-mediated cell adhesion
For adhesion assays, cells were seeded onto plates coated with different substrata and allowed to
adhere to determine the number of adherent cells in the presence of increasing concentrations of test
compounds. The mechanisms by which extracellular matrix components generate intracellular
signals through integrins involves increased phosphorylation of intracellular messengers:
phosphorylation of ERK plays a central role in fibronectin-mediated signalling through
integrins.[20] Under these conditions, no significant cell adhesion was observed for bovine serum
albumin (BSA)-coated plates (negative control) or nonspecific substrate-coated plates (i.e., collagen
I for SK-MEL-24 expressing αvβ3 integrin and poly-l-lysine for K562 expressing α5β1 integrin; data
not shown). The ability of the new compounds to inhibit the adhesion of SK-MEL-24 and K562
cells to fibronectin was compared with that of the standard compounds, Gly-Arg-Gly-Asp-Thr-Pro
and H-Gly-Arg-Gly-Asp-Asn-Pro-OH.[21] The results are summarized in Table 1.
The shorter molecules reported in entries 1-4 showed unexpected activity and selectivity depending
on the substituent in position 3. Treatment with methyl derivative 3a did not affect αvβ3 integrin-
mediated cell adhesion, but excellent affinity towards α5β1 integrin was observed. On the other
hand, the introduction of the isopropylic group in 3b, gave better results since comparable affinity
could be observed towards both integrins (entry 2).

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Table 1. αvβ3 integrin and α5β1 integrin-mediated cell adhesion to fibronectin in the presence of dehydro--amino acid
containing RGD mimetics.
IC50 (M-10-6)[a]
Entry
Compound
αvβ3
>100
α5β1
0.019 ± 0.003
9.8 ± 0.6
4.7 ± 0.2
2.1 ± 0.8
9.8 ± 0.3
0.22 ± 0.05
>100
1
2
3a
1.49 ± 0.05
3.3 ± 0.3
3b
3
3c
4
0.83 ± 0.08
0.12 ± 0.03
2.0 ± 0.1
3d
5
8a
6
8b
7
>100
11b
8
0.27 ± 0.06
0.00062 ± 0.00003
0.23 ± 0.03
0.17 ± 0.03
0.93 ± 0.08
>100
14b
9
Gly-Arg-Gly-Asp-Thr-Pro [b]
H-Gly-Arg-Gly-Asp-Asn-Pro-OH [b]
10
[a] Values are means of three experiments. [b] Reference compounds. See reference 15b and 20.
Increased activity towards α5β1 integrin was observed for thiophenyl derivative 3c (entry 3), while
for 3d a preferential affinity towards αvβ3 receptor could be observed (entry 4). Anyway, the
difference in the affinity among the members of this class of compounds is not sufficient to deduce
strong structure-activity relationship effects. Moreover, these molecules are quite smaller than the
usual RGD-mimetics as cilengitide, and for this reason difference in the steric hinderance of the
side chain may not be crucial for receptor affinity. Entries 5 and 6 exemplify elongated ligands anti-
adhesion activity. Compound 8b showed an increased α5β1 integrin-mediated inhibitory activity
respect to the corresponding shorter analogue 3b. On the contrary, compound 8a displayed good
activity and a strong preference towards αvβ3 integrin, behaving as a selective not dual inhibitor.
Introduction of a second further glycine unit allowed elongation of the molecule. Moreover in this
ligand the interaction with the  chain of the receptor should no longer occur through the aniline
group but with the glycine amino moiety. These modification allowed to obtain the most potent
dual antagonist 14b (entry 8). Dramatic decrease in affinity towards both integrins occurred for
compound 11b (entry 7), thus suggesting that the conformational constraint introduced by the cyclic
scaffold doesn’t allow pharmacophor’s proper orientation.
Effect of dehydro-β-amino acid containing RGD mimetics on fibronectin-induced ERK
phosphorylation in DAOY or K562 cells
Cells are dependent upon integrin-mediated adhesion to the extracellular matrix for proliferation
and survival. Integrins regulate these processes partially through control of extracellular signal-

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regulated kinases 1 and 2 (ERK1/2). The ERK1/2 cascade impacts on cell proliferation and
survival, and this pathway is regulated by cell adhesion signaling.[22] SK-MEL-24 melanoma cells
harbour mutated B-Raf and this mutated form determines constitutively activation of ERK1/2
pathway and therefore adhesion-dependent regulation of ERK1/2 activity is by-passed.[23] As this
pathway is impaired SK-MEL-24 cells cannot be used to study any influence of the assayed
compounds on fibronectin-induced ERK phosphorylation.
Therefore, to investigate the effect of the most effective compounds 8a and 14b on intracellular
integrin-mediated signaling activation we employed DAOY medulloblastoma cells, which express
αvβ3 integrin (Figure 3).[24] Compounds 8a and 14b showed a similar potency as cell adhesion
inhibitors in DAOY cells (data not shown).
b
a
'
'
0
0
' +
' +
0
a)
2
0
pd14
om
FN9
l
tr
ompd8
FN9
FN1
FN9
C
c
c
~44 kDa
~42 kDa
pERK1/2
~44 kDa
~42 kDa
tERK1/2
b)
70
60
50
40
30
20
10
0
*
p42
p44
*
**
**
**
**
'
b
0'
8a
Ctrl
14
N 90
pd
N 12
F
pd
m
F
m
'+co
'+co
N 90
N 90
F
F
Figure 3. Compounds 8a and 14b prevent fibronectin-induced phosphorylation of ERK1/2. DAOY cells were serum-
starved in DMEM for 16 h; cells were then pre-incubated with compounds 8a and 14b (10μM) for 30 min and then
plated on fibronectin (FN, 10μg/ml) or kept in suspension without any compound (Ctrl). After 90 min, cells were lysed,
and lysates were analysed by Western blotting using an antibody directed against phosphorylated ERK1/2 (pERK1/2) or
total ERK1/2 (tERK1/2). a) Western blotting shows that cells plated on FN for 90 min had a much stronger signal for
phosphorylated ERK than control cells. Pre-incubation with compounds 8a and 14b caused a significant decrease in the
amount of phosphorylated ERK. b) Densitometric analysis of the bands (mean ± SEM; n=6): p42 (□), p44 (■); the
amount of pERK is normalized to the total amount of ERK. *p<0.001 vs Ctrl, **p<0.001 vs FN90’ (Newman-Keuls test
after ANOVA).

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DAOY cells were serum-starved in DMEM for 16 h; thereafter, they were detached and pre-
incubated with 8a or 14b integrin antagonists for 30 min and plated for 90 min on fibronectin. As
shown in Figure 3, in cells exposed to fibronectin, a significant increase in phosphorylated ERK1/2
is observed after 90 min, while after 120 min ERK1/2 activation is completely abolished (ERK1/2
phosphorylation was not detected in DAOY cells exposed for 30 or 60 minutes to fibronectin; data
not shown). Pre-incubation with both compounds 8a or 14b caused a significant decrease in the
amount of fibronectin-induced ERK phosphorylation in DAOY cells.
To investigate the effect of the most effective compounds 3a and 14b on intracellular α5β1 integrin-
mediated signalling activation we employed K562 cells. As previously described, K562 cells were
serum-starved in RPMI-1640 containing 1% fetal bovine serum (FBS) for 16 h; thereafter, they
were pre-incubated with compound 3a or 14b (10μM) for 60 min in suspension and plated for 60
min on fibronectin. In agreement with other studies,[25] the ERK signaling is activated in K562
cells exposed to phorbol-12-myristate-13-acetate positive control (PMA, 65 nm; Figure 4).
b
4
a
1
3
d
d
' +
'
' +
p
p
a)
0
0'
0
6
A
A
6
6
60
m
m
rl
N
N
N
o
N
t
o
c
F
c
F
F
F
PM
PM
C
~44 kDa
~42 kDa
pERK1/2
~44 kDa
~42 kDa
tERK1/2
b)
70
p42
p44
60
50
40
30
20
10
0
*
*
*
*
*
*
*
*
*
*
**
**
'
'
l
b
a
r
Ct
0
0
N6
4
3
MA
MA
1
d
N6
P
P
d
p
F
F
p
m
m
o
c
o
c
+
'
+
'
0
0
N6
N6
F
F
Figure 4. Compound 14b and 3a prevent fibronectin-induced phosphorylation of ERK1/2. K562 cells were serum-
starved in RPMI+1% FBS for 16 h; cells were then pre-incubated with compound 14b or 3a (10μM) for 60 min and
then plated on fibronectin (FN, 10μg/ml) or kept in suspension (Ctrl). PMA (65nM) treated cells were used as positive
control. After 60 min, cells were lysed, and lysates were analysed by Western blotting using an antibody directed
against phosphorylated ERK1/2 (pERK1/2) or total ERK1/2 (tERK1/2). a) Western blotting shows that cells plated on
FN for 60 min had a much stronger signal for phosphorylated ERK than control cells. Pre-incubation with compound
14b or 3a caused a significant decrease in the amount of phosphorylated ERK in K562 cells. b) Densitometric analysis

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of the bands (mean ± SEM; n=6): p42 (□), p44 (■); the amount of pERK is normalized to the total amount of ERK.
*p<0.001 vs Ctrl, **p<0.001 vs FN60’ (Newman-Keuls test after ANOVA).
Cells exposed to PMA or fibronectin showed a significant increase in phosphorylated ERK after 60
min whereas compound 14b was able to revert fibronectin-induced ERK1/2 phosphorylation
increment (Figure 4). On the contrary, compound 3a was not able to block ERK pathway activation.
The behaviour of compound 3a, that is ineffective towards αvβ3 integrin-mediated cell adhesion and
shows high affinity for α5β1 integrin but is not able to block signalling, suggest that probably, it
could act through a different mechanism of action that deserves more investigation in future studies.
Integrins and their endogenous ligands are large and complex molecules and so there is the potential
for competitive antagonists to interact with more than one site on the integrin to block ligand
binding and to prevent the downstream sequela of integrin activation.[26] It is conceivable that
small molecules, like 3a described in this study, can prevent fibronectin-mediated cell adhesion in
cells expressing α5β1 integrin, whereas they are not capable to block fibronectin-induced cell
signaling. Integrin-mediated adhesion often occurs under tensile forces such as fluid flow or
myosin-mediated contractions that cells exert to sample the rigidity of their surroundings. Thus, to
enable mechanosensing, integrins could not be constitutively active.[27] These events are mediated
by fibronectin and seem to be antagonized by the compound 14b. On the contrary, compound 3a
may not prevent fibronectin-initiated cell signaling which requires more complex integrin
conformational modifications.
In the literature examples of small molecule ligands that may only partially interact with the binding
site and for this reason show an uncommon effect when compared with classical RGD ligands, have
been reported [14a]. Moreover, several studies have also described compounds that could be
classified as allosteric antagonist or allosteric effector: these molecules could not directly compete
with the binding of integrin specific ligands.[28]
Molecular Docking
In order to rationalize, on a molecular basis, the inhibition activity observed for the dehydro--
amino acid containing RGD mimetics in the αvβ3-mediated cell adhesion assays, computational
models for the binding of 8b and 14b to the αvβ3 integrin were built using a docking approach
previously set up and validated.[29]
Starting from the X-ray structure of the extracellular segment of integrin αvβ3 in complex with the
cyclic pentapeptide ligand Cilengitide (Protein Data Bank entry 1L5G),[12a] automated docking
calculations were carried out using the Glide program,[30] according to the procedure described in

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the experimental section. The docking protocol was successful in reproducing the crystallographic
binding mode of Cilengitide at the interface of the  and  subunits, forming all the specific
electrostatic and H-bond interactions. The docking procedure was then applied to small libraries of
cyclic and linear RGD peptidomimetics to evaluate their ability to properly fit the receptor site. A
nice agreement between docking results and experimental biological data was observed.[29,31] In
all the calculations, the experimentally observed binding mode of Cilengitide with αvβ3 integrin was
taken as a reference model for the analysis of the docking results in terms of ligand-protein
interactions.
In this work, the Glide docking protocol was applied to compounds 8b and 14b to generate
structural models for the interaction with the ligand binding site of the αvβ3 integrin receptor.
Automated docking calculations of the (3R,Z) isomers of both compounds produced top-ranked
poses conserving the key polar interactions of the X-ray complex.
In the best pose of compound (3R,Z)-8b (Figure 5), the ligand carboxylate group is coordinated to
the metal cation in the metal-ion-dependent adhesion site (MIDAS) region of the β3 subunit, and the
ligand aromatic amine moiety interacts with the negatively charged side chain of Asp218 in the αv
subunit, although the fit with the Cilengitide Arg guanidinium group is lacking.
Figure 5. Docking best pose of compound (3R,Z)-8b (atom colour ball and stick representation) into the crystal
structure of the extracellular domain of αvβ3 integrin ( unit blue and  unit red ribbon representation) overlaid on the
bound conformation of Cilengitide (green tube representation). Only selected integrin residues involved in the
interactions with the ligand are shown. The metal ions at MIDAS and ADMIDAS are shown as magenta CPK spheres.
Non polar hydrogens hidden for better visual representation, intermolecular hydrogen bonds visible (green dashed
lines).

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Further stabilizing interactions occur in the docking poses of 8b, involving the formation of
hydrogen bonds between the ligand carboxylate group and the backbone amide hydrogens of
Asn215 and Tyr122 in the β unit. A ring stacking effect between the ligand 4-amino aromatic group
and the αv -Tyr178 side chain is also observed in the binding modes calculated for compound 8b.
In the best pose of compound (3R,Z)-14b (Figure 6), the acid and basic pharmacophoric groups act
like an electrostatic clamp, interacting with charged regions of the receptor binding site. The
elongation of the molecule by the introduction of an additional glycine unit on the aromatic amine
of 8b allows to achieve the pharmacophoric distance required for RGD mimicking and a good fit
with the receptor-bound structure of Cilengitide. Moreover, in ligand 14b the interaction with the
negatively charged carboxylate of Asp218 in the αv subunit not only occurs through the aromatic
amide moiety but also through the positively charged glycine amino terminal group. In addition,
this amino group forms a hydrogen bond with the side chain amide carbonyl of Gln180. Similarly to
what is observed for compound 8b, one carboxylate oxygen of ligand 14b is coordinated to the
metal cation in the MIDAS region of the  unit, while the second carboxylate oxygen forms
hydrogen bonds with the backbone amides of Asn215 and Tyr122 in the  unit. Further stabilizing
interactions observed in the calculated binding modes of 14b involve a H-bond between the ligand
Gly amide and the backbone carbonyl of Asn215, and ring stacking between the ligand aromatic
group and the αv -Tyr178 side chain.
Figure 6. Docking best pose of compound (3R,Z)-14b (atom colour ball and stick representation) into the crystal
structure of the extracellular domain of αvβ3 integrin ( unit blue and  unit red ribbon representation) overlaid on the
bound conformation of Cilengitide (green tube representation). Only selected integrin residues involved in the
interactions with the ligand are shown. The metal ions at MIDAS and ADMIDAS are shown as magenta CPK spheres.
Non polar hydrogens hidden for better visual representation, intermolecular hydrogen bonds visible (green dashed
lines).

ACCEPTED MANUSCRIPT
Conclusion
We have reported herein a novel class of low molecular weight ligands, possessing a dehydro--
amino acid as conformationally constrained core linked to the pharmacophoric moieties mimicking
the RGD recognition sequence. The shortest ligands 3a-d were obtained through a a very simple
synthetic procedure. Although 3a showed an unusual behaviour, the other members of this class
were able to inhibit cell adhesion and integrin-mediated signalling activation with satisfactory
values. Althoug the affinity is not excellent, the low molecular weight and the minimization of
synthetic steps, may represent an advantage on other more complex ligands.
On the other hand, enhanced affinity and dual inhibition could be observed by adding two further
glycine units to the central core, as confirmed by docking experiments showing that the dehydro--
amino derivatives are able to act as electrostatic clamps by establishing several stabilizing
interactions with the receptor. These observation suggest a potential exploitation of these molecules
as lead compounds for antiangiogenetic tools development. The important contribution of integrins
to the biology of both tumour cells and tumour-associated cell types has made them an appealing
target for cancer therapy and for this reason the use of peptides containing the sequence RGD or
structural analogues able to mimic it, represents a promising strategy for anticancer therapy.
Experimental Section
General: All chemicals were purchased from commercial suppliers and used without further
purification. Anhydrous solvents were purchased in sure seal bottles over molecular sieves and used
without further drying. Flash chromatography was performed on silica gel (230-400 mesh).
DOWEX® 50WX2-200(H) ion exchange resin was used for purification of free amino acids. NMR
Spectra were recorded with Varian Gemini 200, Mercury Plus 400 or Unity Inova 600 MHz
spectrometers. Chemical shifts were reported as  values (ppm) relative to the solvent peak of
CDCl3 set at  = 7.27 (1H NMR) or  = 77.0 (13C NMR), CD3OD set at  = 3.31 (1H NMR) or  =
49.0 (13C NMR), D2O set at  = 4.79 (1H NMR). Coupling constants are given in Hz. Purity of
compounds was established by means of HPLC-MS analyses was performed on a HP1100 liquid
chromatograph equipped with Phenomenex Gemini 3-C18 column (3 particle size, 110Å pore
size, 100 X 3 mm), coupled with an electrospray ionization-mass spectrometer (LC-ESI-MS), using
H2O/CH3CN as solvent at 25 °C (positive scan 100-500 m/z, fragmentor 70V). Compounds 1a-d
and 2a-d were prepared according to a previously reported procedure. Complete characterization
can be found in ref. 17.

ACCEPTED MANUSCRIPT
General procedure for the synthesis of dehydro-β-amino acids 3a-d: Trifluoroacetic acid (7 mmol,
7 equiv.) was added to a solution of 2a-d (1 mmol) in CH2Cl2 (5mL) and the reaction mixture was
stirred at room temperature until complete consumption of the starting material. The solvent and the
excess of reagent were removed under reduced pressure. The crude residue was treated with Dowex
50WX2-200 ion exchange resin, eluting with NH4OH 0.5 M. Compounds 3a-d were isolated in 77-
95% yield after removal of the aqueous solvent.
1
3a: Yield 77%, (75:25, Z/E mixture), pale yellow solid, m.p. 96-98 °C (dec.) H NMR (CD3OD,
200 MHz) Z isomer δ 1.53 (d, J=6.8 Hz, 3H, CH3CHN),1.88 (d, J=7.2 Hz, 3H, CH3CH=C), 3.91 (d,
J=12.8 Hz, 1H, NHCH2Ph), 4.01 (d, J=12.8Hz, 1H, NHCH2Ph), 4.31 (q, J=6.8 Hz, 1H, CH3CHN),
6.80 (d, J=8.4 Hz, 2H, Ph), 7.02 (q, J=7.2 Hz, 1H, C=CHCH3), 7.23 (d, J=8.4 Hz, 2H, Ph); E
isomer δ 1.53 (d, J=6.8 Hz, 3H, CH3CHN), 2.12 (d, J=7.0 Hz, 3H, CH3CH=C), 3.81 (q, J=6.6 Hz,
1H, CH3CHN), 3.91 (d, J=12.8 Hz, 1H, NHCH2Ph), 4.01 (d, J=12.8 Hz, 1H, NHCH2Ph), 6.05 (q,
13
J=7.0 Hz, 1H, C=CHCH3), 6.80 (d, J=8.4 Hz, 2H, Ph), 7.23 (d, J=8.4 Hz, 2H, Ph). C NMR
(CD3OD, 50 MHz) δ Z isomer 14.3, 19.5, 50.9, 53.6, 117.1, 122.1, 130.6, 132.6, 139.6, 151.3,
174.3; IR (neat, cm-1) 1081, 1392, 1590, 1651, 1688, 2848, 2921, 2950, 3368, 3399. LC-MS-ESI rt
1.18 min (Z isomer), 1.53 min (E isomer), 235 (M+1), 257 (M+23), 491 (2M+23). Anal. cald. for
C13H18N2O2 (234.29): C 66.64, H 7.74, N 11.96; found C 66.40, H 7.76, N 11.99.
1
3b: Yield 95%, (70:30, Z/E mixture), white solid, m.p. 108-110 °C (dec.) H NMR (CD3OD, 200
MHz) Z isomer δ 0.80 (d, J=7.0 Hz, 3H, CH3CHCH3), 0.91 (d, J=7.0 Hz, 3H, CH3CHCH3), 1.66 (d,
J=7.2 Hz, 3H, CH3CH=C), 2.01 -2.16 (m,1H, CH3CHCH3), 3.69-3.95 (m, 3H, CHCHN e
NHCH2Ph), 6.61 (d, J=8.4 Hz, 2H, Ph), 6.94 (q, J=7.2 Hz, 1H, C=CHCH3), 7.04 (d, J=8.4 Hz, 2H,
Ph); E isomer δ 0.81 (d, J=7.0 Hz, 3H, CH3CHCH3), 0.89 (d, J=7.0 Hz, 3H, CH3CHCH3), 1.94 (d,
J=7.0 Hz, 3H, CH3CH=C), 2.01-2.16 (m,1H, CH3CHCH3), 3.69-3.95 (m, 3H, CHCHN e
NHCH2Ph), 5.77 (q, J=7.0 Hz, 1H, C=CHCH3), 6.61 (d, J=8.4 Hz, 2H, Ph), 7.06 (d, J=8.4 Hz, 2H,
13
Ph). C NMR (CD3OD, 50 MHz) δ Z isomer 14.3, 19.0, 32.6, 50.2, 62.0, 116.3, 121.0, 131.9,
132.0, 142.0, 150.3, 176.1; E isomer 14.9, 20.0, 31.8, 50.3, 62.0, 116.3, 121.0, 131.8, 132.0, 142.1,
151.0, 177.0. IR (neat, cm-1) 846, 1372, 1610, 1684, 2852, 2957, 3366, 3421. LC-MS-ESI rt 1.13
min (Z isomer), 1.59 min (E isomer), 263 (M+1), 547 (2M+23). Anal. cald. for C15H22N2O2
(262.35): C 68.67, H 8.45, N 10.68; found C 68.88, H 8.45, N 10.71.
1
3c: Yield 88%; (70:30, Z/E mixture), yellow solid, m.p. 195-197°C (dec.) H NMR (DMSO, 200
MHz) Z isomer δ 1.74 (d, J=7.4 Hz, 3H, CH3CH=C), 3.60 (d, J=13.2 Hz, 1H, NHCH2Ph), 3.72 (d,
J=13.2 Hz, 1H, NHCH2Ph), 5.02 (s, 1H,CHNH), 6.53 (d, J=8.6 Hz, 2H, Ph), 6.78 (q, J=7.4 Hz, 1H,
C=CHCH3), 7.01 (d, J=8.4Hz, 2H, Ph), 7.10 (d, 1H, thiophenyl), 7.47 (bs, 1H, thiophenyl), 7.50-

ACCEPTED MANUSCRIPT
7.54 (m, 1H, thiophenyl); E isomer δ 1.96 (d, J=6.8 Hz, 3H, CH3CH=C), 3.60 (d, J=13.2 Hz, 1H,
NHCH2Ph), 3.72 (d, J=13.2 Hz, 1H, NHCH2Ph), 4.60 (s, 1H,CHNH), 5.89 (q, J=6.8 Hz, 1H,
C=CHCH3), 6.53 (d, J=8.6 Hz, 2H, Ph), 7.01 (d, J=8.4Hz, 2H, Ph), 7.10 (d, 1H, thiophenyl), 7.47
(bs, 1H, thiophenyl), 7.50-7.54 (m, 1H, thiophenyl); 13C NMR (CD3OD, 50 MHz) δ Z isomer 14.4,
51.2, 56.4, 117.1, 121.7, 126.2, 126.7, 128.7, 130.0, 132.8, 132.9, 134.3, 138.8, 140.5, 151.3, 174.6;
E isomer 16.7, 50.5, 56.4, 117.1, 122.0, 126.3, 126.7, 128.7, 130.0, 132.6, 132.9, 134.3, 138.6,
140.5, 153.3, 170.7; IR (neat, cm-1) 760, 823, 1027, 1160, 1368, 1442, 1678, 1741, 2824, 2924,
2978, 3364, 3437. LC-MS-ESI rt 1.22 min (E isomer), 1.63 min (Z isomer), 303 (M+1). Anal. cald.
for C16H18N2O2S (302.39): C 63.55, H 6.00, N 9.26, S 10.60; found C 63.32, H 5.99, N 9.24, S
10.57.
3d: Yield 78%; (75:25, Z/E mixture), pale yellow solid, m.p. 134-136 °C (dec.) 1H NMR (CD3OD,
200 MHz) Z isomer δ 0.80-1.36 (m, 6H, cyclohexyl), 1.64-1.88 (m, 4H, cyclohexyl), 1.77 (d, J=7.2
Hz, 3H, CH3CH=C), 1.99-2.01 (m, 1H, cyclohexyl), 3.89-4.11 (m, 3H, CHCHN, NHCH2Ph), 6.83
(d, J=7.0 Hz, 2H, Ph), 7.22 (d, J=7.0 Hz, 2H, Ph), 7.34 (q, J=7.4 Hz,1H, C=CHCH3); E isomer δ
0.80-1.36 (m, 6H, cyclohexyl), 1.64-1.88 (m, 4H, cyclohexyl), 1.99-2.01 (m, 1H, cyclohexyl), 2.13
(d, J=6.8 Hz, 3H, CH3CH=C), 3.89-4.11 (m, 3H, CHCHN,NHCH2Ph), 6.33 (q, J=6.8 Hz,1H,
C=CHCH3), 6.83 (d, J=7.0 Hz, 2H, Ph), 7.22 (d, J=7.0 Hz, 2H, Ph). 13C NMR (CD3OD, 50 MHz) Z
isomer δ 15.1, 27.8 (2C), 27.9 (2C), 32.0, 43.1, 51.1, 62.3, 117.1, 121.9, 131.3, 132.7, 141.7, 151.2,
175.7; E isomer δ 15.6, 27.8 (2C), 28.0 (2C), 30.7, 42.1, 51.0, 62.3, 117.1, 122.1, 131.3, 132.7,
140.8, 151.2, 175.7; IR (neat, cm-1) 1095, 1178, 1370, 1455, 1614, 1651, 2841, 2922, 2939, 3307,
3409. LC-MS-ESI rt 1.68 min (Z isomer), 1.80 min (E isomer), 303 (M+1), 325 (M+23), 341
(M+39), 627 (2M+23). Anal. cald. for C18H26N2O2 (302.41): C 71.49, H 8.67, N 9.26; found C
71.25, H 8.69, N 9.24.
Procedure for the preparation of the acid 4a-b. To a stirred solution of the carbonate 1a-b (0.2
mmol) in CH2Cl2 (2 mL) at 0°C, trifluoroacetic acid (15 equiv, 3 mmol, 223 μL) was added. After
2h, the mixture was washed twice with water (5 mL), the organic layer was dried over Na2SO4, and
solvent was removed under reduced pressure.
1
4a: Yield 95%, pale yellow solid, (75:25, Z/E mixture), H NMR (CDCl3, 400 MHz) Z isomer δ
1.57 (d, J=6.8 Hz, 3H, CH3CHO), 2.05 (d, J=7.2 Hz, 3H, CH3CH=C), 3.79 (s, 3H, OCH3), 5.76 (q,
J=6.8 Hz, 1H, CH3CHO), 7.20 (q, J=7.2 Hz, 1H, C=CHCH3); E isomer δ 1.49 (d, J=6.8 Hz, 3H,
CH3CHO), 1.92 (d, J=7.2Hz, 3H, CH3CH=C), 5.53 (q, J=6.8 Hz, 1H, CH3CHO), 6.67 (q, J=7.2 Hz,
13
1H, C=CHCH3). IR (neat, cm-1) 1105, 1172, 1288, 1463, 1671, 1750, 2855, 2915, 2961, 3317. C
NMR (CDCl3, 200 MHz) δ Z isomer 14.3, 19.5, 50.9, 53.6, 117.1, 130.6, 139.6, 151.3, 174.3. LC-

ACCEPTED MANUSCRIPT
MS-ESI rt 1.22 min , 189 (M+1). Anal. cald. for C8H12O5 (188.18): C 51.06, H 6.43; found C 50.95,
H 6.45.
1
4b: Yield 95%; (>99:1, Z/E mixture), pale yellow solid, H NMR (CDCl3, 200 MHz)  0.97 (d,
J=5.4 Hz, 6H, CH3CHCH3), 1.40 (d, J=6.6 Hz, 3H, CH3CHO), 3.25-3.42 (m, 1H, CH3CHCH3),
3.70 (s, 3H, CH3OCO), 5.46 (q, J=6.6 Hz, 1H, CH3CHO), 6.11 (d, J=10.0 Hz, 1H, =CHCH), 10.41
13
(bs, 1H, CO2H); C NMR (CDCl3, 50 MHz) δ 20.1, 21.9, 31.8, 54.4, 73.3, 128.9, 142.5, 154.7,
171.4; IR (neat, cm-1) 1010, 1168, 1287, 1691, 1742, 2845, 2921, 2961, 3323. LC-ESI-MS rt 6.65
min, m/z 216 (M), 239 (M+Na). Anal. cald. for C10H16O5 (216.23): C 55.55, H 7.46; found C 55.57,
H 7.45.
Procedure for the preparation of the amide 5a-b. To a stirred solution of the acid 4a-b (0.2 mmol)
in dry CH2Cl2 (2 mL), under nitrogen atmosphere, EDCI (1.2 equiv, 0,24 mmol, 46 mg) and
triethylamine (2.4 equiv, 0.48 mmol, 67 μL) were added. After 30 min HOBT (1.2 equiv, 0,24
mmol, 33 mg) and glycine t-butyl ester hydrochloride (1.2 equiv, 0,24 mmol, 41 mg) were added.
The solution was stirred overnight and then the mixture was diluited with CH2Cl2 and washed twice
with acidic water (5 mL) and twice with basic water (5 mL). The two phases were separated, the
organic layer was dried over Na2SO4, and solvent was removed under reduced pressure. Compound
5a-b was isolated by flash chromatography on silica gel (cyclohexane/ethyl acetate 80/20 as eluent).
1
5a: Yield 60%; (75:25, Z/E mixture), yellow oil, H NMR (CDCl3, 200 MHz) δ 1.41 (s, 9H, tBu),
1.43 (d, J=6.6 Hz, 3H, CH3CHO), 1.83 (d, J=7.2 Hz, 3H, CH3CH=C), 3.74 (s, 3H, OCH3), 3.94-
4.01 (m, 2H, NHCH2CO), 5.25 (q, J=6.8 Hz, 1H, CHO), 5.96 (q, J=7.2 Hz, 1H, C=CHCH3), 6.61
13
(bt, 1H, NH). C NMR (CDCl3, 50 MHz) δ 15.0, 20.0, 28.0 (3C), 42.0, 54.8, 76.1, 82.2, 131.8,
137.3, 155.1, 167.3, 168.8. IR (neat, cm-1) 938, 1040, 1158, 1267, 1371, 1524, 1612, 1674, 1744,
1750, 28661, 2926, 2971, 3336. LC-MS-ESI rt 6.8 min, m/z 301 (M+1). Anal. cald. for C14H23NO6
(301.34): C 55.80, H 7.69, N 4.65; found C 55.60, H 7.68, N 4.66.
5b: Yield 65%; (>99:1, Z/E mixture), yellow oil, 1H NMR (CDCl3, 200 MHz) 1.01 (d, J=6.6 Hz,
6H, CH3CHCH3), 1.45 (d, J=7.0 Hz, 3H, CH3CHO), 1.49 (s, 9H, OC(CH3)3), 2.70-2.88 (m, 1H,
CH3CHCH3), 3.79 (s, 3H, CH3OCO), 4.00 (d, J=4.8 Hz , 2H, NHCH2), 5.27 (q, J=6.6 Hz, 1H,
13
CH3CHO), 5.67 (d, J=10.2 Hz, 1H, CHCHC), 6.57 (bt, 1H, NH); C NMR (CDCl3, 50 MHz) δ
19.9, 22.7 (2C), 26.8, 28.0 (3C), 41.9, 54.8, 76.0, 82.2, 131.8, 137.3, 155.1, 167.3, 168.8; IR (neat,
cm-1) 942, 1046, 1157, 1267, 1367, 1442, 1519, 1605, 1667, 1736, 1750, 2861, 2924, 2960, 3348.
LC-ESI-MS rt 10.82 min, m/z 329 (M), 352 (M+Na); Anal. cald. for C16H27NO6 (329.39): C 58.34,
H 8.26, N 4.25; found C 58.20, H 8.28, N 4.26.

ACCEPTED MANUSCRIPT
Procedure for the preparation of the N-BOC-protected amide 6a-b. To a stirred solution of the
amide 5a-b (0.2 mmol) in dry THF (1 mL), DMAP (0.2 equiv, 0.04 mmol, 5mg), triethylamine (1.2
equiv, 0.24 mmol, 34 μL) and (BOC)2O (1.3 equiv, 0.26 mmol, 60 μL) were added. The solution
was stirred overnight and then the solvent was removed under reduced pressure. The residue was
diluted with ethyl acetate (10 mL) and washed twice with water (5 mL). The two phases were
separated, the organic layer was dried over Na2SO4, and solvent was removed under reduced
pressure. Compound 6a-b was isolated by flash chromatography on silica gel (cyclohexane/ethyl
acetate 95/5 as eluent).
6a: Yield 88%; (70:30, Z/E mixture), yellow oil, 1H NMR (CDCl3, 200 MHz) δ 1.42 (s, 18H, tBu),
1.46 (d, J=6.6 Hz, 3H, CH3CHO), 1.72 (d, J=7.2 Hz, 3H, CH3CH=C), 3.69 (s, 3H, OCH3), 4.19-
13
4.41 (m, 2H, NHCH2CO), 5.41 (q, J=6.6 Hz, 1H, CHO), 5.80 (q, J=7.2 Hz, 1H, C=CHCH3). C
NMR (CDCl3, 50 MHz) δ14.6, 19.2, 27.7 (3C), 28.0 (3C), 45.9, 54.6, 74.8, 81.8, 83.8, 126.7, 137.6,
151.4, 155.1, 167.6, 169.8; IR (neat, cm-1) 790, 849, 1034, 1160, 1448, 1592, 1657, 1679, 1746,
1762, 2866, 2928, 2979. LC-MS-ESI rt 11.0 min, m/z 424 (M+Na), 440 (M+K). Anal. cald. for
C19H31NO8 (401.45): C 56.84, H 7.78, N 3.49; found C 57.00, H 7.76, N 3.48.
1
6b: Yield 90%; (70:30, Z/E mixture), yellow oil, H NMR (CDCl3, 200 MHz)  0.99 (d, J=6.6 Hz,
6H, CH3CHCH3), 1.42 (d, J=6.6 Hz, 3H, CH3CHO), 1.48 (s, 18H, OC(CH3)3), 2.41-2.49 (m, 1H,
CH3CHCH3), 3.75 (s, 3H, CH3OCO), 4.28 (d, J=16.8 Hz , 1H, NCH2), 4.45 (d, J=16.8 Hz , 1H,
NCH2), 5.42-5.53 (m, 2H, CH3CHO, CHCHC); 13C NMR (CDCl3, 50 MHz) δ 19.2, 22.6 (2C), 27.2,
27.8 (3C), 28.0 (3C), 45.9, 54.5, 74.7, 81.8, 83.8, 126.7, 137.6, 151.4, 155.1, 167.6, 169.8; IR (neat,
cm-1) 745, 848, 1030, 1159, 1442, 1588, 1652, 1677, 1743, 1756, 2859, 2928, 2978. LC-ESI-MS rt
12.26 min, m/z 429 (M), 452 (M+Na); Anal. cald. for C21H35NO8 (429.50): C 58.72, H 8.21, N
3.26; found C 58.78, H 8.19, N 3.25.
Procedure for the preparation of the amino derivative 7a-b. To a stirred solution of 6a-b (0.2
mmol) in dry CH3CN (2 mL), under nitrogen atmosphere, 4-aminomethylaniline (1.5 equiv, 0.3
mmol, 34 μL) was added. The solution was refluxed for 16h and then the solvent was removed
under reduced pressure. Compound 7a-b was isolated by flash chromatography on silica gel
(cyclohexane/ethyl acetate 95/5 as eluent).
7a: Yield 70%; (81:19, Z/E mixture), yellow sticky oil, 1H NMR (CDCl3, 200 MHz) δ 1.39 (s, 18H,
tBu), 1.48 (d, J=7.2 Hz, 3H, CH3CHN),1.73 (d, J=7.2 Hz, 3H, CH3CH=C), 3.90 (t, J=6.2 Hz, 2H,
NHCH2CO), 4.44 (s, 2H, CH2Ph), 5.03 (q, J=7.2 Hz, 1H, CHN), 6.15 (q, J=7.2 Hz, 1H,
13
C=CHCH3), 6.50 (bt, NH), 6.59 (d, J=8.4 Hz, 2H, Ph), 7.01 (d, J=8.4 Hz, 2H, Ph). C NMR

ACCEPTED MANUSCRIPT
(CDCl3, 50 MHz) δ 13.6, 18.0, 28.1(3C), 28.4 (3C), 42.2, 47.3, 50.1, 79.8, 82.1, 115.0, 127.9,
130.7, 131.3, 139.6, 144.9, 155.9, 169.1, 169.9. IR (neat, cm-1) 741, 848, 1163, 1371, 1458, 1519,
1673, 1718, 1741, 2864, 2887, 2907, 2962, 3061, 3379, 3464. LC-MS-ESI rt 9.1 min, m/z 448
(M+1); Anal. cald. for C24H37N3O5 (447.57): C 64.41, H 8.33, N 9.39; found C 64.35, H 8.33, N
9.36.
1
7b: Yield 65%; (78:22, Z/E mixture), yellow sticky oil, H NMR (CDCl3, 200 MHz)  0.83 (d,
J=6.6 Hz, 6H, CH3CHCH3), 1.42 (d, J=7.4 Hz, 3H, CH3CHC), 1.47 (s, 18H, OC(CH3)3), 2.60-2.81
(m, 1H, CH3CHCH3), 3.59 (bs, 2H, NH2), 3.83-3.98 (m, 3H, NCH2, CHNHCO), 4.34-4.61 (m, 2H,
CH2Ph), 6.61 (d, J=8.0 Hz, 2H, Ph), 6.83 (q, J=7.4 Hz, 1H, CCHCH3), 7.01 (d, J=8.0 Hz, 2H, Ph);
13C NMR (CDCl3, 50 MHz) δ 13.5, 20.2 (2C), 28.1 (3C), 28.5 (3C), 31.8, 42.2, 47.3, 50.1, 79.8,
82.1, 115.0, 127.9, 130.7, 131.3, 139.6, 144.9, 156.0, 169.1, 169.9; IR (neat, cm-1) 737, 847, 1157,
1367, 1463, 1519, 1674, 1715, 1738, 2863, 2902, 2962, 3047, 3373, 3467. LC-ESI-MS rt 9.15 min,
m/z 476 (M+1), 498 (M+Na); Anal. cald. for C26H41N3O5 (475.62): C 65.66, H 8.69, N 8.83; found
C 65.58, H 8.70, N 8.86.
Procedure for the preparation of the amino acid 8a-b. To a stirred solution of compound 7a-b (0.2
mmol), in CH2Cl2 (0.5mL), H3PO4 (85% solution in water, 5 equiv, 1 mmol, 69 μL) was added.
After 3h the organic solvent was removed under reduced pressure and the residue was treated with
Dowex 50WX2-200 ion exchange resin, eluting with NH4OH 0.5 M. Compound 8a-b was isolated
after removal of the aqueous solvent.
1
8a: Yield 80%; (77:23, Z/E mixture), pale yellow solid, m.p. 92-94 °C (dec.) H NMR (CD3OD,
200 MHz) δ 1.38 (d, J=7.0 Hz, 3H, CH3CHN),1.79 (d, J=6.8 Hz, 3H, CH3CH=C), 3.70-4.25 (m,
5H, NHCH2CO + CH2Ph + CHN), 6.70-6.74 (m, 3H, Ph + C=CHCH3), 7.01-7.25 (m, 3H, NH+ Ph).
13C NMR (CD3OD, 50 MHz) δ 14.1, 18.7 , 44.7, 49.8, 53.7, 115.1 (2C), 128.0, 130.4, 132.0 (2C),
137.8, 144.6, 164.2, 179.7; IR (neat, cm-1) 1109, 1215, 1299, 1467, 1578, 1662, 1746, 2824, 2956,
3026, 3325, 3366. LC-MS-ESI rt 0.98 min, m/z 292 (M+1). Anal. cald. for C15H21N3O3 (291.35): C
61.84, H 7.27, N 14.42; found C 62.02, H 7.25, N 14.43.
1
8b: Yield 95%; (80:20, Z/E mixture), pale yellow solid, m.p. 96-98 °C (dec.) H NMR (CD3OD,
200 MHz)  0.93 (d, J=6.6 Hz, 6H, CH3CHCH3), 1.82 (d, J=7.2 Hz, 3H, CH3CHC), 2.18-2.28 (m,
1H, CH3CHCH3), 3.83-4.38 (m, 5H, CHNH, NHCH2, CH2Ph), 6.74 (d, J=8.4 Hz, 2H, Ph), 6.90 (q,
13
J=7.2 Hz, 1H, CCHCH3), 7.21 (d, J=8.4 Hz, 2H, Ph); C NMR (CD3OD, 50 MHz) δ 14.3, 19.9
(2C), 31.9, 46.9, 50.9, 62.4, 116.3 (2C), 121.1, 128.8, 132.0 (2C), 140.7, 148.5, 161.6, 178.2; IR
(neat, cm-1) 1107, 1206, 1289, 1455, 1574, 1662, 1750, 2821, 2842, 2926, 2981, 3015, 3317, 3368.

ACCEPTED MANUSCRIPT
LC-ESI-MS rt 1.06 min, m/z 320 (M+1), 342 (M+Na). Anal. cald. for C17H25N3O3 (319.40): C
63.93, H 7.89, N 13.16; found C 64.12, H 7.90, N 13.20.
Procedure for the preparation of the dihydropyrimidindione 9b. To a stirred solution of 6b (0.2
mmol) in dry CH3CN (2 mL), under nitrogen atmosphere, 4-aminomethylaniline (1.5 equiv, 0.3
mmol, 34 μL) was added. The solution was refluxed for 4 days and then the solvent was removed
under reduced pressure. Compound 9b was isolated by flash chromatography on silica gel
(DCM/ethyl acetate 90/10 as eluent).
1
9b: Yield 60%; (89:11, Z/E mixture), yellow sticky oil, H NMR (CDCl3, 400 MHz)  0.79 (d,
J=6.8 Hz, 3H, CH3CHCH3), 0.96 (d, J=6.8 Hz, 3H, CH3CHCH3),1.42 (s, 9H, tBu), 1.48 (d, J=7.2
Hz, 3H, CH3CH=C), 2.13 (m, 1H, CH3CHCH3), 3.77 (d, J= 7.6 Hz, 1H, CHN), 3.90 (d, J= 14.8 Hz,
1H, CH2Ph), 4.38 (d, J= 16.8 Hz, 1H, NCH2CO), 4.45 (d, J= 16.8 Hz, 1H, NCH2CO), 5.28 (d, J=
14.8 Hz, 1H, CH2Ph), 6.57 (d, J=6.8 Hz, 2H, Ph), 6.77 (q, J=7.2 Hz, 1H, CCHCH3), 6.94 (d, J=6.8
Hz, 2H, Ph); 13C NMR (CDCl3, 100 MHz) δ 14.0, 18.5, 19.7, 28.0 (3C), 33.0, 42.9, 51.0, 57.0, 81.6,
115.2 (2C), 126.1, 129.0 (2C), 130.7, 137.2, 146.0, 153.1, 164.9, 167.6. IR (neat, cm-1) 948, 1196,
1345, 1463, 1581, 1658, 1705, 2853, 2922, 2950, 3366. LC-ESI-MS rt 9.5 min, m/z 424 (M+Na),
825 (2M+Na); Anal. cald. for C22H31N3O4 (401.50): C 65.81, H 7.78, N 10.47; found C 65.84, H
7.81, N 10.51.
Procedure for the preparation of the 10b. To a stirred solution of the N-Boc-Gly (0.2 mmol, 35 mg)
in dry DMF (2 mL), under nitrogen atmosphere, N,N-diisopropylethylamine (2 equiv, 0.4 mmol, 69
L) and HBTU (1 equiv, 0.2 mmol, 76 mg) were added. After 30 min, compound 9b (1.2 equiv,
0,24 mmol, 96 mg) was added. The solution was stirred for 3h and then the mixture was diluited
with ethyl acetate and washed twice with acidic water (5 mL) and twice with basic water (5 mL).
The two phases were separated, the organic layer was dried over Na2SO4, and solvent was removed
under reduced pressure. Compound 10b was isolated by flash chromatography on silica gel
(DCM/ethyl acetate 80/20 as eluent).
1
10b: Yield 90%; (91:9, Z/E mixture), yellow oil, H NMR (CDCl3, 400 MHz)  0.77 (d, J=7.2 Hz,
3H, CH3CHCH3), 0.94 (d, J=7.2 Hz, 3H, CH3CHCH3),1.39 (s, 9H, tBu), 1.41 (s, 9H, tBu), 1.49 (d,
J=7.2 Hz, 3H, CH3CH=C), 2.10 (m, 1H, CH3CHCH3), 3.73 (d, J= 7.6 Hz, 1H, CHN), 3.84 (d, J=
6.0 Hz, 2H, CH2NHBoc), 3.98 (d, J= 15.6 Hz, 1H, NCH2Ph), 4.36 (d, J= 16.6 Hz, 1H, NCH2CO),
4.42 (d, J= 16.6 Hz, 1H, NCH2CO), 5.20 (bs, 1H, NHBoc), 5.32 (d, J= 15.6Hz, 1H, CH2Ph), 6.79
(q, J=7.2 Hz, 1H, CCHCH3), 7.09 (d, J=8.4 Hz, 2H, Ph), 7.40 (d, J=8.4 Hz, 2H, Ph), 8.10 (bs,
13
NHGlyBoc); C NMR (CDCl3, 100 MHz) δC 14.2, 18.4, 19.7, 28.0 (3C), 28.2 (3C), 33.1, 42.9,

ACCEPTED MANUSCRIPT
45.4, 51.0, 57.8, 81.7, 120.1 (2C), 127.8, 128.1 (2C), 132.5, 137.0, 137.6, 152.7, 156.5, 164.8,
167.5, 167.9. IR (neat, cm-1) 961, 1200, 1377, 1463, 1552, 1632, 1651, 1660, 1700, 2884, 2924,
2956, 3500. LC-ESI-MS rt 9.97 min, m/z 581 (M+Na). Anal. cald. for C29H42N4O7 (558.67): C
62.35, H 7.58, N 10.03; found C 62.53, H 7.58, N 10.01.
Procedure for the preparation of the amino acid 11b. To a stirred solution of compound 10b (0.2
mmol, 112 mg), in CH2Cl2 (0.5mL), H3PO4 (85% solution in water, 7.5 equiv, 1.5 mmol, 207 μL)
was added. After 12h the organic solvent was removed under reduced pressure and the residue was
treated with Dowex 50WX2-200 ion exchange resin, eluting with NH4OH 0.5 M. Compound 11b
was isolated after removal of the aqueous solvent.
1
11b: Yield 80%; (88:12, Z/E mixture), yellow solid, m.p. 130-132 °C (dec.) H NMR (D2O, 400
MHz)  1.68 (d, J=6.8 Hz, 3H, CH3CHCH3), 1.85 (d, J=6.8 Hz, 3H, CH3CHCH3), 2.55 (d, J=7.6
Hz, 3H, CH3CH=C), 3.03 (m, 1H, CH3CHCH3), 4.79 (s, 2H, CH2NH2), 5.01 (d, J= 6.8 Hz, 1H,
CHN), 5.09 (d, J= 16.4 Hz, 1H, CH2COOH), 5.11 (d, J= 16.4 Hz, 1H, CH2COOH), 5.23 (d, J=
15.8Hz, 1H, CH2Ph), 6.02 (d, J= 15.8Hz, 1H, CH2Ph), 7.79 (q, J=7.6 Hz, 1H, CCHCH3), 8.18 (d,
J=8.4 Hz, 2H, Ph), 8.33 (d, J=8.4 Hz, 2H, Ph); 13C NMR (D2O, 100 MHz) δ 13.9, 17.1, 18.3, 32.7,
41.3, 44.6, 51.2, 58.4, 121.4 (2C), 126.6, 128.0 (2C), 133.7, 135.7, 140.6, 154.0, 166.3, 167.2,
175.3. IR (neat, cm-1) 846, 1157, 1367, 1470, 1538, 1635, 1667, 1698, 2841, 2875, 2924, 2948,
3363, 3421. LC-ESI-MS rt 1.23 min, m/z 403 (M+1), 425 (M+Na). Anal. cald. for C20H26N4O5
(402.44): C 59.69, H 6.51, N 13.92; found C 59.75, H 6.48, N 13.96.
Procedure for the preparation of 12b. To a stirred solution of the 2b (0.2 mmol) in dry DCM (5
mL), triethylamine (1.2 equiv, 0.24 mmol, 34 μL) and Fmoc-Gly-Cl (1.1 equiv, 0.22 mmol, 69 μL)
were added. The solution was stirred for 12h and then washed twice with water (5 mL). The two
phases were separated, the organic layer was dried over Na2SO4, and solvent was removed under
reduced pressure. Compound 12b was isolated by flash chromatography on silica gel
(cyclohexane/ethyl acetate 90/10 as eluent).
1
12b: Yield 84%; (75:25, Z/E mixture), pale yellow solid, m.p. 95-97 °C, H NMR (CDCl3, 400
MHz) 0.74 (d, J=6.6 Hz, 3H, CH3CHCH3), 1.14 (d, J=6.6 Hz, 3H, CH3CHCH3),1.49 (s, 9H, tBu),
1.66 (d, J=7.2 Hz, 3H, CH3CH=C), 1.93 (m, 1H, CH3CHCH3), 3.08 (d, J=9.2 Hz, 1H CHN), 3.52
(d, J=12.8 Hz, 1H, CH2NHFmoc), 3.81 (d, J=12.8 Hz, 1H, CH2NHFmoc) 4.00 (bs, 2H, NCH2Ph),
4.25 (t, J= 9.6 Hz, 1H, Fmoc), 4.42 (d, J= 9.6 Hz, 2H, Fmoc), 7.14 (q, J=7.2 Hz, 1H, CCHCH3),
7.30-7.43 (m, 8H, Fmoc), 7.70 (d, J=7.2 Hz, 2H, Ph), 7.74 (d, J=7.2 Hz, 2H, Ph); 13C NMR (CDCl3,
100 MHz) δ 14.1, 20.1, 21.2, 28.2 (3C), 32.0, 46.1, 50.4, 53.4, 65.0, 80.3, 114.9, 120.6, 122.2,

ACCEPTED MANUSCRIPT
123.3, 124.5, 128.8, 129.2, 130.2, 130.6, 138.0, 144.0, 149.0, 156.2, 167.0, 168.8. IR (neat, cm-1)
1159, 1276, 1367, 1455, 1651, 1682, 1698, 1738, 2823, 2861, 2923, 2982, 3018, 3450. LC-ESI-MS
rt 5.46 min, m/z 620 (M+Na). Anal. cald. for C35H41N3O5 (597.32): C 72.34; H 7.25; N 7.03; found
C 72.62, H 7.28, N 7.05.
Procedure for the preparation of the acid 13b. To a stirred solution of 12b (0.2 mmol) in CH2Cl2 (2
mL) at 0°C, trifluoroacetic acid (15 equiv, 3 mmol, 223 μL) was added. After 2h, the mixture was
washed twice with water (5 mL), the organic layer was dried over Na2SO4, and solvent was
removed under reduced pressure. The obtained acid was diluted in dry CH2Cl2 (2 mL), under
nitrogen atmosphere, and EDCI (1.2 equiv, 0,24 mmol, 46 mg) and triethylamine (2.4 equiv, 0.48
mmol, 67 μL) were added. After 30 min HOBT (1.2 equiv, 0,24 mmol, 33 mg) and glycine t-butyl
ester hydrochloride (1.2 equiv, 0,24 mmol, 41 mg) were added. The solution was stirred overnight
and then the mixture was diluted with CH2Cl2 and washed twice with acidic water (5 mL) and twice
with basic water (5 mL). The two phases were separated, the organic layer was dried over Na2SO4,
and solvent was removed under reduced pressure. Compound 13b was isolated by flash
chromatography on silica gel (cyclohexane/ethyl acetate 90/10 as eluent).
13b: Yield 55%; (95:5, Z/E mixture), yellow solid, m.p. 119-121 °C, 1H NMR (CDCl3, 400 MHz) 
0.82 (d, J=7.2 Hz, 3H, CH3CHCH3), 1.07 (d, J=7.2 Hz, 3H, CH3CHCH3), 1.49 (s, 9H, tBu), 1.71 (d,
J=7.6 Hz, 3H, CH3CH=C), 2.10 (m, 1H, CH3CHCH3), 3.34-3.95 (m, 7H, CHN, CH2NHFmoc,
NCH2Ph, NCH2CO), 4.28 (t, J= 6.4 Hz, 1H, Fmoc), 4.42 (d, J= 6.4 Hz, 2H, Fmoc), 6.85 (q, J=7.6
Hz, 1H, CCHCH3), 7.31-7.44 (m, 6H, Fmoc), 7.55 (d, J=8.0 Hz, 2H, Ph), 7.71 (d, J=7.6 Hz, 2H,
13
Fmoc), 7.83 (d, J=8.0 Hz, 2H, Ph); C NMR (CDCl3, 100 MHz) δ 14.0, 20.1, 20.4, 28.3 (3C),
32.2, 38.8, 42.8, 48.3, 51.2, 62.4, 68.2, 82.9, 120.6, 121.0, 121.3, 122.0, 126.2, 128.7, 129.8, 130.2,
130.3, 138.9, 142.5, 145.2, 158.3, 170.5, 171.4 (2C); IR (neat, cm-1) 1161, 1278, 1368, 1463, 1651,
1673, 1684, 1698, 1732, 2822, 2861, 2918, 2980, 3020, 3391. LC-ESI-MS rt 6.54 min, m/z 655
(M+1). Anal. cald. for C38H46N4O6 (654.80): C 69.70; H 7.08; N 8.56; found C 69.89, H 7.05, N
8.58.
Procedure for the preparation of the amino acid 14b. To a stirred solution of compound 13b (0.2
mmol, 112 mg), in CH2Cl2 (0.5mL), H3PO4 (85% solution in water, 3 equiv, 0.6 mmol, 83 μL) was
added. After 16h the organic solvent was removed under reduced pressure. The residue was diluted
in methanol (2 mL), and piperidine (0.1 equiv., 0.02 mmol, 2 L) was added. After 8h the organic
solvent was removed under reduced pressure, the residue was diluted in 1N HCl (0,5 mL) and with
treated with Dowex 50WX2-200 ion exchange resin, eluting with NH4OH 0.5 M. Compound 14b
was isolated after removal of the aqueous solvent.

ACCEPTED MANUSCRIPT
1
14b: Yield 60%; (90:10, Z/E mixture), yellow solid, m.p. 141-143°C (dec.) H NMR (CD3OD, 400
MHz)  0.78 (d, J=7.2 Hz, 3H, CH3CHCH3), 0.91 (d, J=7.2 Hz, 3H, CH3CHCH3), 1.64 (d, J=7.2
Hz, 3H, CH3CH=C), 2.21 (m, 1H, CH3CHCH3), 3.33-3.39 (m, 2H, NCH2CO), 3.94-4.02 (m, 2H,
CH2NH2) 4.04 (s, 2H, NCH2Ph,), 4.41 (d, J=7.8Hz, 1H, CHN), 6.78 (q, J=7.2 Hz, 1H, CCHCH3),
7.33 (d, J=8.4 Hz, 2H, Ph), 7.40 (d, J=8.4 Hz, 2H, Ph); 13C NMR (CD3OD, 100 MHz)  14.7, 19.7,
19.9, 31.9, 42.9, 43.4, 52.6, 67.1, 121.3 (2C), 128.5 (2C), 132.1, 132.4, 138.1, 154.0, 165.3, 168.6,
171.9. IR (neat, cm-1) 1160, 1276, 1367, 1444, 1671, 1682, 1698, 1701, 2824, 2855, 2921, 3368,
3410. LC-ESI-MS rt 1.04 min, m/z 377(M+1), 399 (M+Na). Anal. cald. for C19H28N4O4 (376.45):
C 60.62, H 7.50, N 14.88; found C 60.43, H 7.51, N 14.91.
Cell culture: SK-MEL-24 cells (American Type Culture Collection, ATCC, Rockville, MD, USA)
were routinely grown in minimum essential medium (MEM, Cambrex, Walkersville, MD, USA)
supplemented with 10% fetal bovine serum (FBS; Life technologies, Carlsbad, CA, USA),
nonessential amino acids, and sodium pyruvate. K562 cells (ATCC) were maintained as a stationary
suspension culture in RPMI-1640 and glutamine with 10% FBS. Cells were kept at 37°C under a
5% CO2 humidified atmosphere; 40 h prior to experiments, K562 cells were treated with 25 ng/mL
PMA (Phorbol 12-myristate 13-acetate, Sigma–Aldrich SRL, Milan, Italy) to induce differentiation
and to increase expression of cell surface antigens.[32] DAOY human medulloblastoma cell line (a
kind gift from Dr G. Cenacchi, University of Bologna) was propagated in Dulbecco's Modified
Eagle's Medium (DMEM; Life technologies, Carlsbad, CA, USA) supplemented with 20% (v/v)
FBS, 1% penicillin-streptomycin and 1mM glutamine (Cambrex, Walkersville, MD, USA). For all
experiments, cells were used between passage 3 and 15.
Cell adhesion assays: 96-well plates (Corning Costar, Celbio, Milan, Italy) were coated by passive
adsorption with fibronectin (10 μg/mL) or nonspecific substrate poly-l-lysine (0.002 %; Sigma–
Aldrich SRL) overnight at 4°C. Cells were counted with a hemocytometer and pre-incubated with
various concentrations of each compound for 30 min at room temperature to reach a ligand–receptor
equilibrium. At the end of the incubation time, the cells were plated (50000 cells per well) and
incubated at room temperature for 1 h. All the wells were then washed with PBS to remove
nonadherent cells, and 50 μL hexosaminidase [4-nitrophenyl-N-acetyl-β-d-glucosaminide dissolved
at a concentration of 7.5 mM in 0.09 M citrate buffer (pH 5) and mixed with an equal volume of
0.5% Triton X-100 in H2O] was added. This product is a chromogenic substrate for β-N-
acetylglucosaminidase, whereby it is transformed into 4-nitrophenol; absorbance was measured at
405 nm. The reaction was blocked by the addition of 100 μL of a stopping solution [50 mM glycine
and 5 mM EDTA (pH 10.4)], and the plates were read in a Victor2 Multilabel Counter

ACCEPTED MANUSCRIPT
(PerkinElmer, Waltham, MA, USA). Experiments were carried out in quadruplicate and repeated at
least three times. Data analysis and IC50 values were calculated using GraphPad Prism 3.0
(GraphPad Software, San Diego, CA, USA).
Western blot analysis: K562 or DAOY cells were incubated in RPMI-1640 with 1% FBS or
DMEM respectively for 16 h. Plates were coated with fibronectin (10 μg/mL) and blocked with 1%
BSA (Sigma–Aldrich SRL). Subsequently, 4x106 cells were pre-incubated with the various
compounds for 30 min and then cells were allowed to adhere for 60-90 min on fibronectin. The
cells were then lysed in mammalian protein extraction reagent (M-PER; Pierce, Rockford, IL, USA)
supplemented with Phosphatase Inhibitor Cocktail (Sigma–Aldrich SRL) for 10 min at 4°C by
gentle shaking. Cell debris was removed by centrifugation (14000 g for 15 min at 4°C), and protein
concentrations were estimated by BCA assay (Pierce, Rockford, IL, USA). Protein extracts were
denatured at 95°C for 3 min before loading and separation by 12% SDS-PAGE. The membranes
were blocked in 1% BSA and incubated for 2 h with anti-phospho-ERK 1/2 (extracellular signal-
regulated kinase 1/2) (1:2500) (Cell Signaling Technology, Danvers, MA, USA) or anti-total
ERK1/2 antibodies (1:5000) (Promega, Madison, WI, USA), and, thereafter, with anti-rabbit HRP-
conjugated secondary antibody. Digital images were acquired and analyzed according to a
previously reported method.
Molecular Docking
All calculations were run using the Schrödinger suite of programs (http://www.schrodinger.com)
through the Maestro graphical interface.
Protein Setup: The recently determined crystal structure of the extracellular domain of the integrin
v3 receptor in complex with cilengitide and in the presence of the proadhesive ion Mn2+ (PDB
entry 1L5G)[12a] was used for docking studies. Docking was performed only on the globular head
of the integrin because the headgroup of integrin has been identified in the X-ray structure as the
ligand-binding region. The protein structure was set up for docking as follows; the protein was
truncated to residues 41-342 for chain  and residues 114-347 for chain β. Due to a lack of
parameters, the Mn2+ ions in the experimental protein structure were modeled via replacement with
Ca2+ ions. The resulting structure was prepared using the Protein Preparation Wizard of the
graphical user interface Maestro and the OPLSAA force field.
Docking: The automated docking calculations were performed using Glide (Grid-based Ligand
Docking with Energetics)[30] within the framework of Impact version 4.5 in a standard precision
mode (SP). The grid generation step started from the extracellular fragment of the X-ray structure of

ACCEPTED MANUSCRIPT
the v3 complex with cilengitide, prepared as described in Protein Setup. The center of the grid-
enclosing box was defined by the center of the bound ligand. The enclosing box dimensions, which
are automatically deduced from the ligand size, fit the entire active site. For the docking step, the
size of the bounding box for placing the ligand center was set to 12 Å. No further modifications
were applied to the default settings. The Glide-Score function was used to select 20 poses for each
ligand. Glide was initially tested for its ability to reproduce the crystallized binding geometry of
cilengitide. The program was successful in reproducing the experimentally found binding mode of
this compound, as it corresponds to the best-scored pose.
Acknowledgements
This study has been carried out with the fundamental contribution of “Fondazione del Monte di
Bologna e Ravenna”, MIUR (PRIN project 2010 NRREPL: Synthesis and biomedical applications
of tumor-targeting peptidomimetics) and University of Bologna. We also thank MAE (Italian
Minister for Foreign Affair, General Direction for the Cultural Promotion and Cooperation) for
financial support to a bilateral projects between Italy and Mexico. Mr. Andrea Garelli is gratefully
acknowledged for the LC-ESI-MS analysis.
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