Cyclic RGD peptides containing azabicycloalkane reverse-turn mimics

Article abstract of DOI:10.1002/hlca.200290017

The Fmoc-protected lactams 3 and 4 were used to prepare cyclo(Arg-Gly-Asp-lactam) 1 and cyclo(Arg-Gly-Asp-Phe-lactam) 2, which contain the Arg-Gly-Asp (RGD) recognition motif. Their solid-phase synthesis, conformational analysis, and binding to purified α

Full text of DOI:10.1002/hlca.200290017

Helvetica Chimica Acta Vol. 85 (2002)
4353
Cyclic RGD Peptides Containing Azabicycloalkane Reverse-Turn
Mimics
by Laura Belvisia)b), Andrea Caporalea), Matteo Colomboa), Leonardo Manzonic), Donatella Potenzaa)b),
Carlo Scolastico*a)b), Massimo Castorinad), Matilde Catid), Giuseppe Gianninid), and Claudio Pisano*d)
a
¡
) Dipartimento di Chimica Organica e Industriale, Universita degli Studi di Milano, via G. Venezian 21,
I-20133 Milano (fax: ‡39-0250314072; e-mail: carlo.scolastico@unimi.it)
b) Centro Interdisciplinare Studi bio-molecolari e applicazioni Industriali (CISI),
¡
Universita degli Studi di Milano, via Fratelli Cervi 93, I-20090 Segrate (MI)
c) CNR-Istituto di Scienze e Tecnologie Molecolari, via C. Golgi 19, I-20133 Milano
d) Research and Development, Sigma-Tau, via Pontina Km 30,400, I-00040 Pomezia
Dedicated to Professor Dieter Seebach on the occasion of his 65th birthday
The Fmoc-protected lactams 3 and 4 were used to prepare cyclo(Arg-Gly-Asp-lactam) 1 and cyclo(Arg-
Gly-Asp-Phe-lactam) 2, which contain the Arg-Gly-Asp (RGD) recognition motif. Their solid-phase synthesis,
conformational analysis, and binding to purified aVb3 and aVb5 integrins are reported. Compound 1 was found to
act as an active and selective inhibitor of the aVb5 integrin.
Introduction. Integrins constitute a large family of a/b heterodimeric cell-surface,
transmembrane receptors, which play a major role in cell/cell and cell/matrix adhesive
interactions [1]. The term −integrin× reflects their role in integrating cell adhesion and
migration with the cytoskeleton [2]. Integrins are involved in fundamental cellular
processes and also contribute to the genesis and/or progression of many common
diseases, including neoplasia, tumor metastasis, tumor-induced angiogenesis, immune
dysfunction, viral infections, osteoporosis, inflammatory disorders, and coagulopathies
[3]. Each noncovalently-linked a/b combination has its own binding specificity and
signaling properties. Many integrins recognize polypeptide domains that contain the
Arg-Gly-Asp (RGD) amino acid sequence present in several extracellular matrix
adhesive proteins. Although RGD peptides inhibit ligand binding to integrins with an
RGD recognition specificity, these receptors can discriminate well among RGD-
containing ligands. The context of the RGD sequence (flanking residues, three-
dimensional presentation, and individual features of the integrin binding pockets)
determine the specificity and the efficacy of interaction [4]. It is, therefore, a major
challenge to identify compounds that can discriminate between RGD-binding integrins
implicated in human pathologies.
Among such RGD-dependent integrins, the vitronectin receptors aVb3 and aVb5
have recently received increasing attention as therapeutic targets because of their
critical role in tumor-induced angiogenesis and metastasis formation [5].
Endothelial cells in the angiogenic vessels within solid tumors express several
proteins that are absent or hardly detectable in established blood vessels, including aV
integrins and receptors for certain angiogenic growth factors. For instance, aVb3 is not

4354
Helvetica Chimica Acta Vol. 85 (2002)
generally expressed on normal endothelial cells (EC), but it is significantly up-
regulated on activated EC and in metastatic tumor cells. Furthermore, antagonists of
aVb3, including cyclic RGD peptides and monoclonal antibodies, significantly inhibited
vessel development and tumor growth induced by cytokines and solid tumor fragments
[6]. It is noteworthy that aVb3 antagonists have very little effect on pre-existing blood
vessels, indicating the usefulness of targeting this receptor for therapeutic benefit
without adverse side effects. Recent studies have focused on the related integrin aVb5 in
angiogenesis. Apparently, there are two major angiogenic pathways activated by two
different growth factors and mediated by aVb3 and aVb5, respectively [7]. Therefore,
selective antagonists of aVb3 and/or aVb5 may be useful in blocking tumor-induced
angiogenesis.
Cyclic RGD peptides have been developed by different groups as active and
selective integrin antagonists that compete with matrix molecules for specific integrin
receptors [8]. An efficient procedure of spatial screening, performed by Kessler and co-
workers [9] and based on libraries of stereoisomeric cyclic peptides, led to the highly
active aVb3-selective first-generation cyclic pentapeptide cyclo(Arg-Gly-Asp-d-Phe-
Val). Extensive modifications of this lead structure with different peptidomimetics and
carbohydrate scaffolds have been performed, and new potent antagonists have been
identified [10]. Distinct turn mimetics and carbohydrate amino acids were introduced
by replacing the d-Phe-Val unit, which was hypothesized to adopt abII'-turn backbone
geometry and, hence, force the RGD sequence into a kinked, aVb3-selective
conformation. Systematic derivatization of the lead peptide resulted in the N-alkylated
cyclopeptide cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (A) [11], which has entered
clinical phase II studies as an angiogenesis inhibitor (code EMD121974, Merck
KGaA) [12].
O
N
O
CH3
H
N
N
H
NH2
N
H
O
O
O
H
NH
HO
N
N
H
O
A (EMD121974)
Recently, our group has reported a library of cyclic RGD pentapeptide mimics
based on azabicycloalkane scaffolds of type B [13]. Stereoisomeric 6,5- and 7,5-fused
bicyclic lactams with different reverse-turn mimetic properties [14] were exploited as
dipeptide analogs for the synthesis of a library of the general formula cyclo(Arg-Gly-
Asp-lactam) (C). This library was found to contain specific high-affinity ligands for
aVb3 integrin, which are presently being evaluated as very promising anti-angiogenic
drugs. Among the peptides tested, ST1646 (D) showed the highest affinity to aVb3,
inhibiting echistatin binding to aVb3 with an IC50 of 3.7 Æ 0.6 nm [13].

Helvetica Chimica Acta Vol. 85 (2002)
4355
O
O
N
N
COOH
n
N
Arg
Gly
Arg
Gly
n
O
O
O
HN
HN
Asp
NH2
Asp
B (n = 1,2)
C (n = 1,2)
D (ST1646)
To further address the process of spatial screening, the original library was
expanded to include new members, modified either at the scaffold moiety (1) or in the
ring size by introducing a flanking hydrophobic amino acid (2). This allowed us to
address both the effect of conformational changes in the RGD fragment and that of
adjacent amino acids or substituents. To explore the presence of secondary interaction
sites with integrins, peptides 3, featuring a quaternary stereogenic center and a benzyl
group, and 4 were employed. Remarkably, computational and spectroscopic studies
revealed that both 3 and 4 are effective reverse- and b-turn mimics [14].
7
8
9
7
8
9
O
O
6
6
N
1
2
5
4
5
N
1
2
4
Arg
Gly
Arg
Gly
3
3
O
O
Ph
NH
Phe
HN
10
Asp
Asp
1
2
O
O
N
N
OH
OH
O
O
Ph
NHFmoc
NHFmoc
3
4
Herein, we report the solid-phase synthesis, the conformational analysis by
spectroscopic and computational means, and the in vitro ability of these new RGD
cyclic pseudopeptides to compete for the binding of [125I]-echistatin to purified aVb3
and aVb5 integrins.
Results and Discussion. 1. Synthesis. The preparation of 1 and 2 was accomplished
by solid-phase synthesis of the linear peptide sequences, using the (9H-fluoren-9-yl)-
methoxycarbonyl (Fmoc) protection strategy, followed by cyclization and side-chain
deprotection in solution (Schemes 1 and 2). The Arg and Asp side-chains were
protected with the Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) and t-Bu groups,
respectively. Both these protecting groups are removed under acidic conditions and are,
thus, compatible with the Fmoc strategy. In turn, this choice suggested the use of the
SasrinTM [15] resin as the solid support. The Sasrin linker is highly labile under mildly

4356
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 1
Fmoc-Gly-OH
HO
+
i) – iii)
Coupling of:
Fmoc-Arg(Pmc)-OH
Fmoc-lactam-OH (3)
Fmoc-Asp(t-Bu)-OH
iv), v), iii), then
vi), v), iii), then
vii), v), iii)
H-Asp(t-Bu)-lactam-Arg(Pmc)-Gly-O-Sasrin (5)
viii)
H-Asp(t-Bu)-lactam-Arg(Pmc)-Gly-OH (6)
ix)
O
O
N
N
Pmc
x)
Arg
Arg
O
O
Ph
Ph
NH
NH
Gly
Gly
Asp
Asp
t-Bu
1
7
i) HOBt, DIC, DMAP, DMF. ii) Ac2O, DMAP. iii) Piperidine, DMF. iv) Fmoc-Arg(Pmc)-OH, HOAt, DIC,
CH2Cl2/DMF 2 :1. v) 1-Acetyl-1H-imidazole, CH2Cl2. vi) Fmoc-lactam-OH (3), HATU, HOAt, 2,4,6-collidine,
CH2Cl2, DMF 3 :1. vii) Fmoc-Asp(t-Bu)-OH, HATU, HOAt, 2,4,6-collidine, CH2Cl2, DMF 3 :1. viii) 1% TFA/
CH2Cl2. ix) HATU, HOAt, 2,4,6-collidine, DMF. x) TFA, scavengers; then HCl.
acidic conditions (1% TFA in CH2Cl2), and cleavage of the peptide can be achieved
without affecting the protected side-chains.
The bicyclic compounds 3 [16] and 4 [14] were prepared by following our published
procedures and were N-terminally protected as fluorenyl carbamates, using the Fmoc-
O-succinimide (Fmoc-O-NSu) reagent.
The sequence for the synthesis of 1 is shown in Scheme 1. N-Fmoc-glycine was
anchored to the Sasrin resin by the DIC/HOBt/DMAP protocol1), followed by capping
with Ac2O and DMAP. Deprotection of the amino groups was achieved under standard
conditions with a 20% piperidine solution in DMF. Condensation of the N-Fmoc-
1
)
For abbrevations, see the Exper. Part.

Helvetica Chimica Acta Vol. 85 (2002)
4357
Arg(Pmc)-OH residue was performed with the DIC/HOAt protocol, while condensa-
tion of 3 and N-Fmoc-Asp(t-Bu)-OH was accomplished according to Carpino×s
method with HATU/HOAt/2,4,6-collidine [17]. After capping with 1-acetyl-1H-
imidazole and N-deprotection, the linear H-Asp(t-Bu)-lactam-Arg(Pmc)-Gly-O-
Sasrin (5) was selectively cleaved from the resin by treatment with a 1% solution of
TFA in CH2Cl2, providing the linear peptide 6, which was purified by size-exclusion
chromatography and then cyclized in dilute DMF solution. The side-chain-protected
cyclic product 7 was obtained in 35% yield after purification by flash chromatography
(FC). The side-chain-protecting groups were then removed with TFA in the presence
of ion scavengers. The resulting TFA salt was purified by HPLC and then transformed
into the corresponding chloride 1 with gaseous HCl.
For the synthesis of 2, the linear peptide 8 was constructed, and cleaved from the
resin to furnish 9, which was cyclized to the cyclohexapeptide 10. The latter was, in turn,
deprotected and transformed to 2 as shown in Scheme 2. The resulting cyclic RGD
peptides 1 and 2 were tested and stored as chloride salts.
2. Biological Evaluation. The cyclic peptides 1 and 2 were examined in vitro for their
abilities to compete with [125I]-echistatin for binding to the purified aVb3 and aVb5
receptors (Table 1). It has been demonstrated that both purified and membrane-bound
integrins aVb3 and aVb5 bind with very high affinity to echistatin, which can be inhibited
efficiently by linear and cyclic RGD-containing peptides [18]. Affinities of compounds
c(RGDfV), EMD 121974 (A), and ST1646 (D) for the aVb3 and aVb5 integrins have
been determined in the same assays. The in vitro binding data of the RGD peptides to
purified receptors revealed that ST1646 (Table 1, Entry 4) showed the highest affinity
to aVb3 and aVb5 among the peptide analogs incorporating lactam scaffolds. Interest-
ingly, the affinity of the cyclic RGD pseudopentapeptide D for the integrin aVb3 in this
kind of assay was almost 5 and 50 times higher than the affinity of the lead structures
EMD 121974 (Table 1, Entry 3) and c(RGDfV) (Entry 2), respectively. Despite the
PhCH2 substituent or the Phe side chain, the new compounds 1 (Table 1, Entry 5) and 2
(Entry 6) were less active than the original lead structures with respect to the inhibition
of echistatin binding to both the aVb3 and aVb5 receptors. Thereby, the cyclopentapep-
tide analog 1 has a higher inhibitory activity on echistatin binding than the
cyclohexapeptide analog 2, and, remarkably, both compounds possess significant
selectivities towards the aVb5 receptor. In particular, 1 shows a 200-fold higher
inhibitory activity for echistatin binding to aVb5 than for binding to aVb3 (Table 1,
Entry 5). Structural effects of the bicyclic templates and of the cyclopeptide size on the
conformation of the RGD sequence can be examined to explore the spatial
requirements of the antagonist pharmacophore for the selective inhibition of either
aVb3 or aVb5.
3. Conformational Analysis. The structural basis for the divalent cation-dependent
binding of heterodimeric a/b integrins to their ligands, which contain the RGD
sequence, is unknown. Very recently, the crystal structure of the extracellular segment
of integrin aVb3 in complex with the cyclic pentapeptide ligand EMD121974 (A) has
been reported [19]. Examination of the three-dimensional structure of this antagonist
bound to the integrin aVb3 (Protein Data Bank, Entry 1L5G) reveals a conformation
characterized by an inverse g-turn with Asp at position (i ‡ 1) and by a distorted bII'-
turn with Gly and Asp at the (i ‡ 1) and (i ‡ 2) positions (Fig. 1,a). A distance between

4358
Helvetica Chimica Acta Vol. 85 (2002)
Scheme 2
Fmoc-Gly-OH
HO
+
i) – iii)
Coupling of:
Fmoc-Arg(Pmc)-OH
Fmoc-lactam-OH (4)
Fmoc-Phe-OH
iv), v), iii), then
vi), v), iii), then
vii), v), iii), then
viii), v), iii)
Fmoc-Asp(t-Bu)-OH
H-Asp(t-Bu)-Phe-lactam-Arg(Pmc)-Gly-O-Sasrin (8)
ix)
H-Asp(t-Bu)-Phe-lactam-Arg(Pmc)-Gly-OH (9)
x)
O
O
N
N
Pmc
Arg
Arg
xi)
O
O
NH
Phe
NH
Phe
Gly
Gly
Asp
t-Bu
Asp
2
10
i) HOBt, DIC, DMAP, DMF. ii) Ac2O, DMAP. iii) Piperidine, DMF. iv) Fmoc-Arg(Pmc)-OH, HOAt, DIC,
CH2Cl2, DMF 2 :1. v) 1-Acetyl-1H-imidazole, CH2Cl2. vi) Fmoc-lactam-OH (4), HATU, HOAt, 2,4,6-collidine,
CH2Cl2, DMF 3 :1. vii) Fmoc-Phe-OH, HATU, HOAt, 2,4,6-collidine, CH2Cl2, DMF 3 :1. viii) Fmoc-Asp-
(t-Bu)-OH, HATU, HOAt, 2,4,6-collidine, CH2Cl2, DMF 3 :1. ix) 1% TFA/CH2Cl2. x) HATU, HOAt, 2,4,6-
collidine, DMF. xi) TFA, scavengers; then HCl.
the Cb atoms of Asp and Arg of 8.9 ä is observed in this pentapeptide binding
conformation. Contrary to what had been assumed previously [9][20], the inhibition of
aVb3 integrin does not require a strong kink of the RGD sequence, which is
characterized by a distance between the Cb-atoms of Asp and Arg shorter than 7 ä.
Structure determination of the cyclic RGD pentapeptide EMD121974 by NMR
spectroscopy, distance-geometry calculations, and molecular-dynamics simulations led
to similar conclusions [11] but also revealed some conformational flexibility. The crystal

Helvetica Chimica Acta Vol. 85 (2002)
4359
Table 1. Inhibition of [125I]-Echistatin Binding to aVb3 and aVb5 Receptorsa)
Entry
Compound
IC50 [nm] for aVb3
IC50 [nm] for aVb5
1
2
3
4
5
6
Echistatin
c(RGDfV)
EMD 121974 (A)
ST1646 (D)
1
2
0.28 Æ 0.08
195.9 Æ 16.8
18.9 Æ 3.1
3.7 Æ 0.6
0.29 Æ 0.02
0.11 Æ 0.03
0.13 Æ 0.01
1.39 Æ 0.2
4.12 Æ 1.1
470 Æ 17
787.1 Æ 54.6
> 105
a) IC50 values were calculated as the conc. of compds. required for 50% inhibition of echistatin binding as
estimated by the Allfit program. All values are the mean (Æstandard deviation) of triplicate determinations.
Fig. 1. a) X-Ray, aVb3-bound conformation of EMD121974 (A, cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val)) from
1L5G [19]. b) Conformation of ST1646 (D) sampled during the 10-ns MC/SD simulation after energy
minimization.
structure of the peptideÀintegrin complex provides the exact conformation of
EMD121974 (A) bound to aVb3 integrin and can serve as a basis for understanding
the general mode of interaction of integrins with other RGD-containing ligands.
4. Computational Studies. Conformational preferences of EMD121974 (A), ST1646
(D), 1, and 2 have been investigated by molecular-mechanics calculations according to
the following two-step protocol: Monte Carlo/Energy Minimization (MC/EM)
conformational searches [21] with the implicit water GB/SA solvation model [22] of
the AGA (Ala-Gly-Ala) cyclopeptide analogues containing Me groups instead of the
Arg and Asp side chains; Monte Carlo/Stochastic Dynamics (MC/SD) free simulations
[23] with the implicit water GB/SA solvation model [22] of the complete RGD cyclic
peptides, starting from the cyclopeptide backbone geometries determined by the
previous step.
The conformational preferences exhibited by the conformers calculated for the
simplified AGA compounds agree very well with the results provided by the free MC/
SD simulations on the RGD cyclopeptides, in terms of population of backbone
geometries, intramolecular H-bonds, and Cb ¥¥¥ Cb distances.
Compound EMD121974 (A) was found to exhibit a Cb(Arg) ¥¥¥ Cb(Asp) average
distance of 8.5 ä. According to the corresponding H-bonds, the inverse g-turn at Asp
and the distorted bII'-turn at Gly-Asp are populated 30% and 12%, respectively, during

4360
Helvetica Chimica Acta Vol. 85 (2002)
the 10-ns MC/SD simulation (Table 2). Remarkably, compound ST1646 (D) showed a
reduced conformational mobility and a preferred cyclopeptide conformation very
similar to the X-ray binding conformation of EMD121974 (A) (see Fig. 1). The lowest-
energy conformer of the simplified AGA cyclopeptide is characterized by an inverse g-
turn at Asp and by a distorted bII'-turn at Gly-Asp. According to the corresponding H-
bonds, these turns are populated 33% and 50%, respectively, during the 10-ns MC/SD
simulation of ST1646 (Table 2). The average of the Cb(Arg) ¥¥¥ Cb(Asp) distance is
8.5 ä over the same trajectory.
Table 2. Population of H-Bonds During the 10-ns MC/SD Simulations in H2O (GB/SA model)
Donor
Acceptor
Turn
H-Bond [%]a)
EMD121974
ST1646
1
2
NH(lactam)
NH(lactam)
NH(Arg)
NH(Gly)
NH(Asp)
NH(Arg)
NH(Phe)
NH(Phe)
NH(lactam)
NH(lactam)
CˆO(Gly)
CˆO(Arg)
CˆO(Asp)
CˆO(lactam)
CˆO(Arg)
CˆO(Phe)
CˆO(Gly)
CˆO(Arg)
CˆO(Gly)
CˆO(Asp)
inv. g (Asp)
bII' (Gly-Asp)
bII' (lactam)c)
bI (Pro-Arg)d)
g (Gly)
bII' (lactam)
inv. g (Asp)
bII' (Gly-Asp)
b (Asp-Phe)e)
inv. g (Phe)
30
33
10
0 ‡ 12b)
6 ‡ 13b)
0 ‡ 25b)
23
0 ‡ 50b)
0 ‡ 2b)
1 ‡ 12b)
11
0 ‡ 10b)
62 ‡ 18b)
35 ‡ 28b)
44
14 ‡ 32b)
14
16 ‡ 26b)
12
11 ‡ 14b)
19 ‡ 13b)
8
a) The term − H-bond× reflects the percentage of conformations sampled during the simulation in which the
NH ¥¥¥ O distance was smaller than 2.5 ä, the NÀH ¥¥¥ O angle larger than 1208, and the H ¥¥¥ OˆC angle larger
than 908. b) Percentage of conformations featuring a distorted b-turn in which the H ¥¥¥ O distance lies between
2.5 ä and 4 ä. c) For compound EMD121974, the bII'-turn is centered at d-Phe-(NMe)Val. d) For compound
EMD121974, the bI-turn is centered at (NMe)Val-Arg. e) Classification of the b-turn corresponding to this 10-
membered ring H-bond is difficult due to the distortion of dihedral angles from ideal values [29].
An energy-minimized conformation of ST1646 (D) from the 10-ns MC/SD
simulation, featuring the binding requirements, is shown in Fig. 1,b. The root-mean-
square (RMS) deviation in rigid superimposition between this conformation and the X-
ray structure of bound EMD121974 (A) is only 0.19 ä for the backbone atoms of the
RGD sequence.
A strong kink of the RGD sequence, characterized by a Cb(Arg) ¥¥¥ Cb(Asp)
distance shorter than 7 ä, is shown by the minimum-energy conformations of the
simplified AGA cyclopeptide analog of compound 1. The bicyclic dipeptide mimic 3
adopts the (i ‡ 1) and (i ‡ 2) position of a bII'-turn, Gly is in the (i ‡ 1) position of a g-
turn at the opposite side, and Arg may occupy the (i ‡ 2) position of a bI-turn, forcing
the RGD sequence into a kinked backbone conformation. The corresponding H-bonds
are populated throughout the whole MC/SD simulation of 1, and a Cb(Arg) ¥¥¥ Cb(Asp)
average distance of 6.5 ä is obtained (Table 2). An energy-minimized conformation of
1 from the 10-ns MC/SD simulation, featuring the two consecutive b-turns, the g-turn at
Gly, and the corresponding H-bonds, is shown in Fig. 2.
The calculated structures of the cyclic hexapeptide 2 showed the typical b/b
conformations. However, conformational flexibility was observed and three preferred
geometries were identified. The bicyclic lactam 4 adopts the (i ‡ 1) and (i ‡ 2) position

Helvetica Chimica Acta Vol. 85 (2002)
4361
Fig. 2. Conformation of 1 sampled during the 10-ns MC/SD simulation after energy minimization
of a bII'-turn, and the dipeptide Gly-Asp is located in the (i ‡ 1) and (i ‡ 2) position of a
bII'-turn on the opposite side (Fig. 3,a). A g-turn with Gly at the (i ‡ 1) position may
complete this b/b arrangement, forcing a kink of the RGD sequence, characterized by a
Cb(Arg) ¥¥¥ Cb(Asp) distance slightly shorter than 8 ä. A second geometry features two
consecutive b-turns (bII' with dipeptide mimic at (i ‡ 1) and (i ‡ 2) position, and bI
with Arg at (i ‡ 2) position) and one g-turn at Gly (Fig. 3,b). A strong kink of the
RGD sequence, characterized by a Cb(Arg) ¥¥¥ Cb(Asp) distance shorter than 7 ä, is
typical for this geometry. Finally, the cyclic hexapeptide 2 can adopt a different
conformation consisting of two opposed b-turns located at Pro-Arg and Asp-Phe,
respectively, which forces the RGD sequence in a fairly extended conformation
(Fig. 3,c).
Fig. 3. Snapshots of the trajectory of 2 sampled during the 10-ns MC/SD simulation after energy minimization
5. NMR Spectroscopy. The intrinsic folding propensity of peptides is determined by
several factors, including torsional strain and other nonbonded repulsion that develop
as the backbone folds back upon itself, as well as by the conformational entropy loss
that accompanies folding. The solvation forces are expected to exert relatively little
influence on the intrinsic folding propensity when the intramolecular H-bonds are
competitive with the intermolecular H-bonds also in polar solvents; in this case, the
insights gained from NMR analysis in organic solvents should apply to aqueous solution

4362
Helvetica Chimica Acta Vol. 85 (2002)
as well. Thus, the folding properties of cyclopeptide 1 were initially investigated by
studying the protected pentapeptide mimic 7 by 1H-NMR in CDCl3 solution. Study of
H-bond-driven processes in unpolar solvents must begin with control experiments to
determine the lowest concentration at which intramolecular H-bonding occurs [24].
The NMR data discussed below were obtained at 2 mm concentration and should
reflect effects of only intramolecular H-bonding (see Exper. Part and Table 3). The
chemical shift of NH(Gly) of compound 7 is 7.85 ppm, which is typical for an amide
proton in the H-bonded state. NOESY data provided further evidence of the folded
conformation. The NOE between NH(Arg) and NH(Gly) is consistent with the
conformation depicted in Fig. 2, where these amide protons are buried. NH(Arg) forms
a 10-membered-ring H-bond with CˆO(Asp), and NH(Gly) forms an additional b-
turn involving the CˆO group of the bicyclic lactam. The NOEs observed between
NH(lactam) and Hb(Asp), as well as between NH(lactam) and benzylic protons,
suggest a conformation in which NH(lactam) is not involved in an internal H-bond.
Table 3. 1H- and 13C-NMR Chemical Shifts d [ppm] of 7 in CDCl3 at 300 K (trivial numbering, see Formula 1)
Residue
NH
Ha
Hb
Hg
Hd
Ca
Cb
Cg
Cd
Arg
Gly
Asp
6.99
7.85
6.92
4.61
4.36/3.45
4.74
2.0/1.7
1.63/1.54
3.27
52.36
45.39
51.10
29.5
25.6
41.1
2.64/2.77
36.89
Residue
Lactam
NH
H(4)
H(5)
2.2/1.55
C(5)
31.7
H(6)
2.45
H(7)
1.71/1.53
C(7)
H(8)
1.92/1.59
C(8)
H(9)
4.28
H(10)
3.27/3.09
C(10)
44.66
6.91
2.17/1.44
C(4)
C(3)
46.09
C(6)
62.25
C(9)
60.16
28.9
25.7
27.6
The chemical shift of protons H(6) and H(9) of the bicyclic scaffold were found to
be very sensitive to the presence of the benzyl group. The corresponding resonances at
2.45 and 4.28 ppm were shifted upfield by 1.16 and 0.25 ppm, respectively, relative to
the corresponding protons in the analogous scaffold lacking the benzylic substituent
[25]. Therefore, the protected pentapeptide 7 most likely adopts a compact
conformation.
The formation of one or two amide/amide H-bonds in noncompetitive solvents
provided a significant but not overwhelming effect on folding. Subsequently, we
showed that analogous phenomena are manifested in aqueous solution with the
deprotected pentapeptide 1.
1
The conformational behavior of 1 was elucidated on the basis of its H-NMR
spectral characteristics in D2O (see Exper. Part and Table 4). Amide protons that
participate in a relevant H-bond within a secondary or tertiary structural element (a-
helix, b-sheet, reverse turns) exchange much more slowly with D2O than do the amide
protons exposed to bulk solvent. Amide H/D exchange rates facilitate the identification
of such amide protons involved in intramolecular H-bonding. In the case of 1, the NMR
signal of the NH(Arg) proton could still be detected in D2O over a period of 60 min,
and this behavior suggests that it is involved in a strong intramolecular H-bond.

Helvetica Chimica Acta Vol. 85 (2002)
4363
Table 4. 1H- and 13C-NMR Chemical Shifts d [ppm] of 1 in D2O at 300 K (trivial numbering, see Formula 1)
Residue
NH
7.45
Ha
Hb
Hg
Hd
Ca
Cb
Cg
Cd
Arg
Gly
Asp
4.4
3.41/4.21
4.74
1.65/1.85
1.55/1.50
3.15
52.9
44.57
50.4
28.0
25.17
40.9
2.7/2.8
35.73
Residue
Lactam
NH
H(4)
1.75
H(5)
2.15
H(6)
2.35
H(7)
1.35
H(8)
1.9
H(9)
4.25
H(10)
2.95/3.2
C(10)
43.86
C(3)
C(4)
27.0
C(5)
32.3
C(6)
59.7
C(7)
31.47
C(8)
28.27
C(9)
61.5
NOESY studies were carried out with 1 to gain additional insight into the folded
conformations adopted in D2O. The most relevant NOE enhancements were observed
between the aromatic protons (meta- and para-positions) and H(6) and H(9) of the
bicyclic scaffold. An NOE between H(6) and H(9) was also evident. These observed
NOEs and chemical shifts also suggest a compact folding conformation in which the
aromatic group is close to the bicyclic moiety (Fig. 2). So, in aqueous solution,
lipophilic interactions appear to favor the adoption of folded conformations.
The lactam moiety of the Pmc-protected hexapeptide 10 is a good b-turn inducer
1
expected to promote b-hairpin-like folding. The H-NMR spectrum of 10 at 2 mm
concentration in CDCl3 exhibited amide protons in the range of 6.9 7.98 ppm (see
Exper. Part and Table 5). These chemical shifts are characteristic for peptide backbone
NH groups strongly involved in H-bonding. Since these H-bonds cannot occur
simultaneously, we propose that 10 exists in at least three differently folded
conformations (Fig. 4). One surprising feature of the NMR data was the 1.8 ppm
downfield shift of the lactam NH of 10 relative to the reference compound 4. This
behaviour suggests that the lactam NH could participate in intramolecular H-bonding
with the Gly CˆO (10-membered ring H-bond). The long-range NOEs observed in
2 mm solution are indicated in Fig. 4. None of the conformations I III, alone, can
account for all of these NOEs; therefore, the NOESY data provide further evidence of
multiple folded conformations. The NOE between NH(Arg) and NH(Gly) could arise
from conformation I or II. The NOE between NH(Phe) and NH(Asp) is consistent
Table 5. 1H- and 13C-NMR Chemical Shifts d [ppm] of 10 in CDCl3 at 300 K (trivial numbering, see Formula 2)
Residue
NH
Ha
Hb
Hg
Hd
Ca
Cb
Cg
Cd
Arg
Gly
Asp
Phe
7.53
7.99
7.65
6.92
4.56
4.27/3.41
4.54
2.24/1.89
1.82/1.71
3.29
52.7
45.0
52.6
55.6
28.82
26.4
41.26
2.88/2.68
3.13/2.90
36.17
38.75
4.48
Residue
Lactam
NH
H(3)
3.56
H(4)
H(5)
2.16/1.80
C(6)
H(6)
3.79
H(7)
1.8/1.50
C(8)
28.6
H(8)
2.5/1.83
C(9)
H(9)
4.49
7.41
C(3)
52.0
2.21/1.91
C(5)
C(4)
28.1
C(7)
33.2
C(10)
33.1
61.2
61.59

4364
Helvetica Chimica Acta Vol. 85 (2002)
Table 6. 1H- and 13C-NMR Chemical Shifts d [ppm] of 2 in D2O at 300 K (trivial numbering, see Formula 2)
Residue
NH
NH
Ha
Hb
Hg
Hd
Ca
Cb
Cg
Cd
Arg
Gly
Asp
Phe
4.49
3.55/4.09
4.36
1.85/1.95
4.09
2.55/2.45
2.9
1.59/1.63
3.21
52.8
43.9
52.8
54.8
29.1
24.5
41.16
35.5
38.6
4.59
Residue
Lactam
H(3)
3.67
H(4)
H(5)
H(6)
3.72
H(7)
1.45/1.55
C(8)
H(8)
1.85/2.45
C(9)
H(9)
4.45
2.15/1.65
C(5)
2.15/1.85
C(6)
C(3)
51.9
C(4)
29.4
C(7)
31.8
C(10)
27.4
55.1
29.8
61.3
O
H
R
O
O
H
H
H
R
N
R
H
H
H
H
O
N
N
N
H
H
O
N
O
H
N
H
H
H N
H
O
O
N
H N
O
O
O
N H
O
N
O
O
H
N
N
H
N
H
H
H
N
N
N H
O
H
N
H
H
H
t
t
t
CO2 Bu
CO2 Bu
CO2 Bu
H
H
O
H
O
O
I
II
III
Fig. 4. Alternative folding patterns for 10 according to 1H-NMR measurements in CDCl3. Significant,
nonadjacent NOEs are indicated with arrows.
with the b-hairpin-like folding pattern III and with conformation I. Moreover, the NOE
between NH(Gly) and NH(Asp) is consistent with distorted type I, II, and III folding
patterns.
Conclusions.
A library of cyclic RGD pentapeptide mimics incorporating
stereoisomeric bicyclic lactams was expanded to include the new members 1 and 2,
modified either at the lactam moiety or with respect to the ring size by introducing a
flanking hydrophobic amino acid. The effect of these modifications on biological
activity was investigated and revealed lower activities in inhibiting the binding of [125I]-
echistatin to purified aVb3 and aVb5 integrins with respect to the potent antagonist
ST1646 (D) of the original library. However, compound 1 showed significant affinity
and selectivity towards the aVb5 integrin, inhibiting echistatin binding to this receptor
with an IC50 of 4.12 Æ 1.1 nm.
Structural effects of the bicyclic templates and of the cyclopeptide size on the
conformation of the RGD sequence were examined. The conformational analysis of
cyclic RGD peptides 1 and 2, containing azabicycloalkane reverse-turn mimics,
suggests that the pharmacophoric RGD groups do not adopt the properly kinked
orientation required for binding to the aVb3 integrin, as deduced from the recently-
solved crystal structure of the extracellular segment of integrin aVb3 in complex with

Helvetica Chimica Acta Vol. 85 (2002)
4365
the cyclic pentapeptide ligand EMD121974 (A) [19]. In addition, improper arrange-
ment of the phenyl ring might result in a weaker hydrophobic interaction between
integrin and cyclopeptide. A greater tolerance in the conformation of the RGD
fragment and the role of adjacent hydrophobic substituents are likely to be
hypothesized for the interaction with the aVb5 receptor.
Experimental Part
General. Abbreviations: DIC: N,N'-diisopropylcarbodiimide, DIPEA: N-ethyl-N,N-diisopropylamine,
DMAP: 4-(dimethylamino)pyridine, HOAt: 3-hydroxy-3H-[1,2,3]-triazolo[4,5-b]pyridine, HOBt: 1-hydroxy-
1H-benzotriazole, HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate,
TNBS: 2,4,6-trinitrobenzenesulfonic acid. Reagents and solvents: solvents were dried by standard procedures,
and reactions requiring anh. conditions were performed under Ar or N2. Sasrin resin (200 400 mesh,
1.02 mmol/g) was purchased from Bachem. All solvents used for solid-phase syntheses were of HPLC quality or
Analytical Reagent grade and were dried over molecular sieves before use. All solid-phase reactions were
carried out on a wrist shaker. The TNBS test was performed as follows: a few resin beads were sampled and
washed several times with EtOH. The sample was then placed in a vial, and 1 drop of a 10% soln. of DIPEA in
DMF plus 1 drop of 1% TNBS in DMF were added. The sample was then examined, and color changes were
noted. The TNBS test was considered to be positive (presence of free amino groups) when the resin beads turn
orange or red within 1 min, and negative (no free amino groups) when the beads remained colorless. Optical
rotations [a]D were measured in cells of 1 dm pathlength and 1 ml capacity with a Perkin-Elmer 241 polarimeter.
Thin-layer chromatography (TLC) was carried out with precoated Merck silica-gel F-254 plates. Flash
1
chromatography (FC) was carried out with Macherey-Nagel silica gel 60 (230 400 mesh). H- and 13C-NMR
Spectra were recorded at 300 K on a Bruker AVANCE-400 spectrometer. Chemical shifts d are expressed in ppm
relative to internal Me4Si as standard. Elemental analyses were performed by the staff of the microanalytical
laboratory of our department.
10-[3-(Guanidino)propyl]-3,6,9,12,19-pentaoxo-1-(phenylmethyl)-2,5,8,11,20-pentazatricyclo[11.5.2.016,20]-
icosane-4-acetic Acid (1; cyclo(Arg-Gly-Asp-lactam). In a solid-phase reaction vessel, Sasrin resin (0.59 mmol)
was suspended in a soln. of N-Fmoc-Gly-OH (1.77 mmol), HOBt (1.77 mmol), DIC (1.77 mmol), and DMAP
(0.18 mmol) in DMF (10 ml) for 18 h. The soln. was drained, and the resin was washed with DMF (3 Â 10 ml)
and CH2Cl2 (3 Â 10 ml). Potentially unreacted OH groups were capped by treatment with a soln. of Ac2O
(1.18 mmol) and DMAP (0.59 mmol) in DMF (12 ml) for 2 h. The soln. was drained, and the resin was washed
with DMF (3 Â 10 ml) and CH2Cl2 (3 Â 10 ml). The resulting N-Fmoc-Gly-O-Sasrin resin (0.59 mmol) was
treated with a 20% piperidine/DMF soln. (10 ml, 1 Â 3 min, 2 Â 17 min). The soln. was drained, and the resin
was washed with DMF (3 Â 10 ml), MeOH (2 Â 10 ml), and CH2Cl2 (3 Â 10 ml). The deprotection was assessed
by performing a TNBS test. N-Fmoc-Arg(Pmc)-OH (1.77 mmol) and HOAt (1.77 mmol) were dissolved in
CH2Cl2/DMF 2 :1 (24 ml). At 08, DIC (1.77 mmol) was added dropwise to this soln. The resulting mixture was
stirred for 10 min at this temp. and for 10 more min at r.t. and added to the resin. This mixture was shaken at r.t.
for 2.5 h. The soln. was drained, and the resin washed with DMF (3 Â 10 ml) and CH2Cl2 (3 Â 10 ml). The
success of the coupling was assessed by performing a TNBS test. Unreacted amino groups were capped by
treatment with a soln. of 1-acetyl-1H-imidazole (5.1 mmol) in CH2Cl2 (12 ml) for 2 h. The soln. was drained,
and the resin was washed with CH2Cl2 (3 Â 10 ml). The N-Fmoc-Arg(Pmc)-Gly-O-Sasrin resin (0.59 mmol)
was treated with a 20% piperidine/DMF soln. (10 ml, 1 Â 3 min, 2 Â 17 min). The soln. was drained, and the
resin was washed with DMF (3 Â 10 ml), MeOH (2 Â 10 ml), and CH2Cl2 (3 Â 10 ml). The deprotection was
assessed by performing a TNBS test. The resin was suspended in a soln. of N-Fmoc-lactam-OH (3) (0.59 mmol)
and treated with HATU (1.18 mmol), HOAt (1.18 mmol), and 2,4,6-collidine (1.12 mmol) in DMF/CH2Cl2 3 :1
(13 ml) for 18 h. The soln. was drained, and the resin was washed with DMF (3 Â 10 ml), MeOH (2 Â 10 ml),
and CH2Cl2 (3 Â 10 ml). The success of the coupling was assessed by performing a TNBS test. Unreacted amino
groups were capped by treatment with a soln. of 1-acetyl-1H-imidazole (5.1 mmol) in CH2Cl2 (12 ml) for 2 h.
The soln. was drained, and the resin was washed with CH2Cl2 (3 Â 10 ml). The N-Fmoc-lactam-Arg(Pmc)-Gly-
O-Sasrin resin (0.59 mmol) was treated with a 20% piperidine/DMF soln. (10 ml, 1 Â 3 min, 2 Â 17 min). The
soln. was drained, and the resin was washed with DMF (3 Â 10 ml), MeOH (2 Â 10 ml), and CH2Cl2 (3 Â
10 ml). The deprotection was assessed by performing a TNBS test. The resin was suspended in a soln. of N-
Fmoc-Asp(t-Bu)-OH (2.36 mmol) and treated with HATU (2.36 mmol), HOAt (2.36 mmol), and 2,4,6-

4366
Helvetica Chimica Acta Vol. 85 (2002)
collidine (2.36 mmol) in DMF/CH2Cl2 3 :1 (13 ml) for 18 h. The soln. was drained, and the resin was washed
with DMF (3 Â 10 ml), MeOH (2 Â 10 ml), and CH2Cl2 (3 Â 10 ml). The success of the coupling was assessed by
performing a TNBS test. Unreacted amino groups were capped by treatment with a soln. of 1-acetyl-1H-
imidazole (5.1 mmol) in CH2Cl2 (12 ml) for 2 h. The soln. was drained, and the resin was washed with CH2Cl2
(3 Â 10 ml). The N-terminal Fmoc-protecting group was removed with 20% piperidine/DMF, and the resin was
washed with MeOH and CH2Cl2 to afford 5. Cleavage of the peptide from the resin was repeated five times each
with 1% TFA/CH2Cl2 for 3 min. The combined filtrate was neutralized with a soln. of pyridine in MeOH
(2 equiv. of pyridine for each equiv. of TFA) and evaporated to afford crude 6, which was separated from
pyridinium salts by size-exclusion chromatography (Amberlite XAD-2 resin, H2O, then MeOH), and used
without further purification. For cyclization, 6 (0.59 mmol) was dissolved in anh. DMF (100 ml) and stirred with
HATU (1.77 mmol), HOAt (1.77 mmol), and 2,4,6-collidine (1.77 mmol) for 24 h at r.t. The solvent was
evaporated under reduced pressure, and the remainder was dissolved in CH2Cl2 (12 ml) and washed with 5%
NaHCO3 soln. The org. phase was dried (Na2SO4), and the solvent was evaporated under reduced pressure.
Purification by FC (CH2Cl2/EtOH 95 :5) afforded the side-chain-protected cyclopeptide 7 (35%). [a]2D3 ˆ À10.3
1
‡
(c ˆ 0.952, CHCl3). H- and 13C-NMR: see Table 3. FAB-MS (pos. mode): 921 ([M ‡ 1] , C46H64N8O10S; calc.
920.45). Anal. calc. for C46H64N8O10S (920.45): C 52.98, H 7.00, N 12.17; found: C 52.98, H 7.00, N 12.16.
The cyclic pseudopentapeptide 7 was treated with 70 ml of a mixture of TFA/thioanisol/ethane-1,2-dithiol/
anisol 90 :5 :3 :2 for 2 h. Evaporation, titration with (i-Pr)2O, and purification by HPLC (C18 column; 0 70%
MeCN/H2O ‡ 0.1% TFA over 40 min) afforded crude 1 (79%). Excess TFA was removed under vacuum, and
the remainder was treated with gaseous HCl to afford pure 1 as a white solid ready for biological assay. [a]D23
ˆ
À70.2 (c ˆ 0.76, H2O). 1H- and 13C-NMR: see Table 4. FAB-MS (pos. mode): 599 ([M À Cl] ; calc. M
(C28H39ClN8O7): 634.26). Anal. calc. for C28H39ClN8O7 (634.26): C 52.95, H 6.19, N 17.64; found: C 52.93,
H 6.18, N 17.63.
‡
‡
1,4-Bis[(phenyl)methyl]-13-[3-(guanidino)propyl]-3,6,9,12,15,22-hexaoxo-2,5,8,11,14,23-hexazatricy-
clo[14.5.2.019,23]tricosaneacetic Acid (2; cyclo(Arg-Gly-Asp-Phe-lactam)). N-Fmoc-Gly-OH (3 equiv.) was
coupled to Sasrin resin (0.59 mmol) with DIC, HOBt, and DMAP for activation in DMF. Chain elongation was
performed with N-Fmoc-Arg(Pmc)-OH (1.77 mmol), N-Fmoc-lactam-OH (4) (0.59 mmol), N-Fmoc-Asp(t-
Bu)-OH (1.77 mmol), and N-Fmoc-Phe-OH (1.77 mmol) with HOAt/DIC for arginine and HATU/HOAt and
2,4,6-collidine for the activation of the other amino acids. The remaining operations were (unless stated
otherwise) performed as for the synthesis of 1. The N-terminal Fmoc group was removed to afford 8, which was
cleaved from the resin to give the side-chain-protected linear peptide 9. The latter was cyclized in anh. DMF
(100 ml) with a three-fold excess of HATU, HOAt, and 2,4,6-collidine for 24 h at r.t. Evaporation and
purification by FC (CH2Cl2/EtOH 95 :5) afforded the side-chain protected cyclopseudohexapeptide 10 (43%).
‡
[a]2D3 ˆ À4.96 (c ˆ 0.867, CHCl3). FAB-MS (pos. mode): 978 (M , C48H67N9O11S; calc. 978.17). Anal. calc. for
C48H67N9O11S (978.17): C 58.94, H 6.90, N 12.89; found: C 58.93, H 6.89, N 12.88.
Peptide 10 was treated with 70 ml of a mixture of TFA/thioanisol/ethane-1,2-dithiol/anisol 90 :5 :3 :2 for
2 h. Evaporation, titration with (i-Pr)2O, and purification by HPLC (C18 column; 0 70% MeCN/H2O ‡ 0.1%
TFA over 40 min) afforded 2 as the TFA salt (75%). Excess TFA was removed under vacuum, and the
remainder was treated with gaseous HCl to afford pure 2 as a white solid ready for biological assay. [a]D23
ˆ
‡
‡
À58.1 (c ˆ 0.97, H2O). 1H- and 13C-NMR: see Table 6. FAB-MS (pos. mode): 656 ([M À Cl] ; calc. M
(C30H42ClN9O8): 692.16). Anal. calc. for C30H42ClN9O8 (692.16): C 52.06, H 6.12, N 18.21; found: C 52.05,
H 6.13, N 18.22.
Solid-Phase Receptor-Binding Assay. The receptor-binding assays were performed as described in [18].
Receptors avb3 and avb5 (Chemicon International, Inc.) were diluted, resp., to 500 ng/ml and 1000 ng/well in
coating buffer (20 mm Tris ¥ HCl (pH 7.4), 150 mm NaCl, 2 mm CaCl2, 1 mm MgCl2, and 1 mm MnCl2). An
aliquot of the diluted receptors (100 ml/well) was added to 96-well microtiter plates and incubated overnight at
48. The coating soln. was removed by aspiration, and 200 ml of blocking soln. (coating buffer containing 1%
bovine serum albumin (BSA)) was added to the wells, which were incubated for an additional 2 h at r.t. After
incubation, the plates were rinsed with 200 ml of blocking soln. (3Â) and incubated with [125I]echistatin2)
(0.05 nm for avb3 and 0.1 nm for avb5) for 3 h at r.t. in the presence of diluted competing ligands. Nonspecific
binding was determined with a molar excess (1 mm) of cold echistatin. After incubation, radioactivity was
Labeled by lactoperoxidase to a specific activity of 2000 Ci mmolÀ1, purchased from Amersham
Pharmacia.
2
)

Helvetica Chimica Acta Vol. 85 (2002)
4367
determined in a g-counter. Each data point is the result of the average of triplicate wells and was analyzed by
nonlinear-regression analysis with the Allfit program.
Computational Methods. Molecular-mechanics calculations were performed within the framework of
MacroModel [26] (version 5.5) by means of the MacroModel implementation of the AMBER all-atom force
field [27] (denoted AMBER*) and the implicit water GB/SA solvation model of Still et al. [22]. The torsional
space of each AGA cyclopeptide was randomly varied with the Monte Carlo conformational search of Chang
et al. [21]. Ring-closure bonds were defined in the six-membered ring of the 6,5-fused bicyclic lactams and in the
cyclopeptide ring. Amide bonds were included among the rotatable bonds. For each search, at least 1000 starting
structures for each variable torsion angle were generated and minimized until the gradient was less than 0.05 kJ
äÀ1 molÀ1 by the truncated Newton Raphson method [28] implemented in MacroModel. Duplicate
conformations and those with an energy greater than 6 kcal molÀ1 above the global minimum were discarded.
The nature of the stationary points individuated was tested by computing the eigenvalues of the second-
derivative matrix.
Simulations of the RGD cyclic peptides were performed at 300 K with the Monte Carlo/Stochastic
Dynamics (MC/SD) hybrid simulation algorithm [23], applying the AMBER* all-atom force field and the water
GB/SA continuum solvation model. A time step of 1 fs was used for the stochastic-dynamics (SD) part of the
algorithm. The total simulation time was 10 ns for each RGD cyclopeptide, and samples were taken at 1 ps
intervals, yielding 10000 conformations for analysis.
The authors thank CNR and MURST (COFIN research programs) for financial support and CILEA for
computing facilities.
REFERENCES
[1] R. O. Hynes, Cell 1987, 48, 549; E. Ruoslahti, M. D. Pierschbacher, Science 1987, 238, 491; M. J. Humphries,
Biochem. Soc. Trans. 2000, 28, 311; B. P. Eliceiri, D. A. Cheresh, Curr. Opin. Cell Biol. 2001, 13, 563.
[2] J. A. McDonald, J. Biol. Chem. 2000, 275, 21783; A. E. Aplin, A. Howe, S. K. Alahari, R. L. Juliano,
Pharmacol. Rev. 1998, 50, 197; F. G. Giancotti, E. Ruoslahti, Science 1999, 285, 1028; F. G. Giancotti, Nat.
Cell. Biol. 2000, 2, E13.
[3] S. A. Mousa, D. A. Cheresh, Drug Discov. Today 1997, 2, 187; M. A. Arnaout, Immunol. Rev. 1990, 114,
145; R. O. Hynes, Cell 1992, 69, 11.
[4] E. F. Plow, T. A. Haas, L. Zhang, J. Loftus, J. W. Smith, J. Biol. Chem. 2000, 275, 21785; K. Suehiro, J. W.
Smith, E. F. Plow, J. Biol. Chem. 1996, 271, 10365.
[5] D. G. Stupack, D. A. Cheresh, Science×s Signal Transduction Knowledge Environment 2002, 119, pe7; J.
Folkman, Nature Med. 1995, 1, 27; J. Folkman, Nature Biotechnol. 1997, 15, 510; H. P. Hammes, M.
Brownlee, A. Jonczyk, A. Sutter, K. T. Preissner, Nature Med. 1996, 2, 529; B. P. Eliceiri, D. A. Cheresh, J.
Clin. Invest. 1999, 103, 1227.
[6] P. C. Brooks, R. A. Clark, D. A. Cheresh, Science 1994, 264, 569; M. E. Duggan, J. H. Hutchinson, Exp.
Opin. Ther. Pat. 2000, 10, 1367.
[7] M. Friedlander, P. C. Brooks, R. W. Shaffer, C. M. Kincaid, J. A. Varner, D. A. Cheresh, Science 1995, 270,
1500.
[8] R. Haubner, D. Finsinger, H. Kessler, Angew. Chem., Int. Ed. 1997, 36, 1374; A. C. Bach II, J. R. Espina,
S. A. Jackson, P. F. Stouten, J. L. Duke, S. A. Mousa, W. F. DeGrado, J. Am. Chem. Soc. 1996, 118, 293; G.
M¸ller, M. Gurrath, H. Kessler, J. Comput.-Aided Mol. Des. 1994, 8, 709; R. M. Scarborough, M. A.
Naughton, W. Teng, J. W. Rose, D. R. Phillips, L. Nannizzi, A. Arfsten, A. M. Campbell, I. F. Charo, J. Biol.
Chem. 1993, 268, 1066.
[9] R. Haubner, R. Gratias, B. Diefenbach, S. L. Goodman, A. Jonczyk, H. Kessler, J. Am. Chem. Soc. 1996,
118, 7461.
[10] E. Lohof, E. Planker, C. Mang, F. Burkhart, M. A. Dechantsreiter, R. Haubner, H.-J. Wester, M. Schwaiger,
G. Hˆlzemann, S. L. Goodman, H. Kessler, Angew. Chem., Int. Ed. 2000, 39, 2761; F. Schumann, A. M¸ller,
M. Koksch, G. M¸ller, N. Sewald, J. Am. Chem. Soc. 2000, 122, 12009; R. Haubner, W. Schmitt, G.
Hˆlzemann, S. L. Goodman, A. Jonczyk, H. Kessler, J. Am. Chem. Soc. 1996, 118, 7881.
[11] M. A. Dechantsreiter, E. Planker, B. Math‰, E. Lohof, G. Hˆlzemann, A. Jonczyk, S. L. Goodman, H.
Kessler, J. Med. Chem. 1999, 42, 3033.
[12] H. N. Lode, T. Moehler, R. Xiang, A. Jonczyk, S. D. Gillies, D. A. Cheresh, R. A. Reisfeld, Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 1591.

4368
Helvetica Chimica Acta Vol. 85 (2002)
[13] L. Belvisi, A. Bernardi, A. Checchia, L. Manzoni, D. Potenza, C. Scolastico, M. Castorina, A. Cupelli, G.
Giannini, P. Carminati, C. Pisano, Org. Lett. 2001, 3, 1001.
[14] L. Belvisi, A. Bernardi, L. Manzoni, D. Potenza, C. Scolastico, Eur. J. Org. Chem. 2000, 2563; M. Angiolini,
S. Araneo, L. Belvisi, E. Cesarotti, A. Checchia, L. Crippa, L. Manzoni, C. Scolastico, Eur. J. Org. Chem.
2000, 2571.
[15] M. Mergler, R. Tanner, J. Gosteli, P. Grogg, Tetrahedron Lett. 1988, 29, 4005.
[16] L. Colombo, M. Di Giacomo, G. Brusotti, N. Sardone, M. Angiolini, L. Belvisi, S. Maffioli, L. Manzoni, C.
Scolastico, Tetrahedron 1998, 54, 5325.
[17] L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397.
[18] C. C. Kumar, H. Nie, C. P. Rogers, M. Malkowski, E. Maxwell, J. J. Catino, L. Armstrong, J. Pharmacol.
Exp. Ther. 1997, 283, 843.
[19] J.-P. Xiong, T. Stehle, R. Zhang, A. Joachimiak, M. Frech, S. L. Goodman, M. A. Arnout, Science 2002, 296,
151.
[20] M. Pfaff, K. Tangemann, B. M¸ller, M. Gurrath, G. M¸ller, H. Kessler, R. T. J. Engel, J. Biol. Chem. 1994,
269, 20233.
[21] G. Chang, W. C. Guida, W. C. Still, J. Am. Chem. Soc. 1989, 111, 4379.
[22] W. C. Still, A. Tempczyk, R. C. Hawley, T. Hendrickson, J. Am. Chem. Soc. 1990, 112, 6127.
[23] F. Guarnieri, W. C. Still, J. Comput. Chem. 1994, 15, 1302.
[24] R. R. Gardner, G.-B. Liang, S. H. Gellman, J. Am. Chem. Soc. 1999, 121, 1806.
[25] L. Belvisi, A. Maltagliati, L. Manzoni, D. Potenza, C. Scolastico, G. Giannini, M. Marcellini, T. Riccioni, C.
Pisano, in preparation.
[26] F. Mohamadi, N. G. J. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T.
Hendrickson, W. C. Still, J. Comput. Chem. 1990, 11, 440.
[27] S. J. Weiner, P. A. Kollman, D. T. Nguyen, D. A. Case, J. Comput. Chem. 1986, 7, 230.
[28] J. W. Ponder, F. M. Richards, J. Comput. Chem. 1987, 8, 1016.
[29] G. D. Rose, L. M. Gierasch, J. A. Smith, Adv. Prot. Chem. 1985, 37, 1.
Received July 19, 2002