Discovery of subnanomolar arginine-glycine-aspartate-based αvβ3/αvβ5 integrin binders embedding 4-aminoproline residues

Article abstract of DOI:10.1021/jm701214z

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity α_vβ₃/α_vβ ₅ integrin binders [IC_50h(α_vβ ₃) 0.03-5.12 nM; IC_50h(α_vβ ₅) 0.88-154 nM]. A total of eight cyclopeptides of type cyclo-[-Arg-Gly-Asp-Amp-], 5-12, were assembled by a standard solid-phase peptide synthesis protocol that involved the C2-carboxyl and C4-amino functionalities of the proline scaffolds, leaving the N^α- nuclear site untouched. Functionalization of this vacant proline site with either alkyl or acyl substituents proved feasible, with significant benefit to the integrin binding capabilities of the ligands. Notably, six out of eight cyclopeptide inhibitors, 5-7 and 9-11, showed moderate yet significant selectivity toward the α_vβ₃ receptor. The three-dimensional structure in water was determined by NMR techniques and molecular dynamics calculations. Docking studies to the X-ray crystal structure of the extracellular segment of integrin α_vβ₃ complexed with reference compound 1 were also performed on selected analogues to highlight the structural features required for potent ligand binding affinity.

Full text of DOI:10.1021/jm701214z

SUPPORTING INFORMATION
Discovery of Sub-Nanomolar Arginine-Glycine-Aspartate-Based
αVβ3/αVβ5 Integrin Binders Embedding 4-Aminoproline Residues
Franca Zanardi,*,‡ Paola Burreddu,† Gloria Rassu,† Luciana Auzzas,† Lucia Battistini,‡ Claudio Curti,‡
Andrea Sartori,‡ Giuseppe Nicastro,*,# Gloria Menchi,§ Nicoletta Cini,§,$ Anna Bottonocetti,$ Silvia
Raspanti,$ and Giovanni Casiraghi*,‡
†
‡Dipartimento Farmaceutico, Università di Parma, Viale G. P. Usberti 27A, I-43100 Parma, Italy, Istituto di Chimica
#
Biomolecolare del CNR, Traversa La Crucca 3, I-07040 Li Punti, Sassari, Italy, Centro Interdipartimentale Misure “G.
§
Casnati”, Università di Parma, Viale G. P. Usberti 23A, I-43100 Parma, Italy, Dipartimento di Chimica Organica “Ugo
Shiff”, Università degli Studi di Firenze, Polo Scientifico di Sesto e Agraria, Via della Lastruccia 13, I-50019 Sesto
$
Fiorentino, Italy, and Dipartimento di Fisiopatologia Clinica, Unità di Medicina Nucleare, Università degli Studi di
Firenze, Viale Pieraccini 6, I-50134 Firenze, Italy
franca.zanardi@unipr.it
giovanni.casiraghi@unipr.it
gnicast@nimr.mrc.ac.uk
N
O
NH
HN
O
HN
N
NH2
H
NH
HN
O
HO2C
O
αVβ3 IC50h: 0.08 nM
αVβ5 IC50h: 0.88 nM
Contents
• General methods
S2
• Experimental procedures and spectroscopic data for compounds 15-28
• Elemental analyses of intermediary and target compounds (Table S1)
S3
S15
• Copies of 1H NMR spectra (600 MHz, D2O) of cyclopeptides 5-12 (Figures S1-S8)
S16
S20
• Concentration-response curves of cyclopeptides 5-12 to αVβ3 and αVβ5 (Figures S9-S16)
• Schematic view of observed interactions between ligand 9 and the αVβ3 receptor (Fig. S17) S28
• Surface representations of the αVβ3 binding site with ligands 5, 9, and 10 (Figure S18) S29
• Superposition of docked 5, 9, and 10 to the X-ray αVβ3-bound conformation of 1 (Figure S19) S30
• References S31
S1

General. All solid-phase synthesis chemicals purchased from commercial sources were dried in
dessicator before use; all other chemicals were used as supplied without further purification. Apart from
NMP, all organic solvents were dried and freshly distilled before use according to literature procedures.
cTrt resin (2-chlorotrityl chloride polymer bound, 100-300 mesh, nominal loading ~1.6 mmol/g,
calculated loading 1.55 mmol/g) was from Fluka; coupling reagents were from either Bachem or
Aldrich. All moisture sensitive reactions were carried out under a positive pressure of nitrogen or
argon.
TNBS test was performed according to the following procedure. A few resin beads were sampled and
accurately washed with ethanol. The sample was then placed in a vial and 1 drop of a 10% solution of
DIEA in DMF and 1 drop of 1% TNBS in DMF were added. The sample was then observed under a
microscope and colour changes noted. The TNBS test is considered 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 remain colourless.
Thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated plates (Merck) with
visualization under short-wavelength UV light or by dipping the plates with molybdate reagent
(H2O/concentrated H2SO4/(NH4)6Mo7O24·4H2O/Ce2(SO4)3 90/10/25/1 v/v/w/w) followed by heating.
Flash chromatography was performed on 40-63 µm silica gel (Merck) using the indicated solvent
mixtures. Analytical reversed-phase HPLC was performed on a SpectraSystem P2000 apparatus
(Thermo Separation Products, UV detection 220 or 254 nm) equipped with a C18-10µm column
(Discovery BIO Wide Pore, 250 × 4.6 mm). Semipreparative reversed-phase HPLC was performed on a
Prostar 210 apparatus (Varian, UV detection at 220 or 254 nm) equipped with a C18-10µm column
(Discovery BIO Wide Pore, 250 × 10 mm). Direct infusion ESI-MS spectra were recorded on API
150EX apparatus (Applied Biosystems). High-resolution mass spectrometry measurements (HRMS)
were performed on a APEX IIIQ Fourier transform mass spectrometer (Bruker) equipped with an
S2

external electrospray ion source.
Melting points were determined with an optical thermomicroscope Optiphot2-Pol (Nikon) and are
uncorrected. Optical rotations were measured using a Perkin-Elmer model 341 polarimeter at ambient
temperature using a 100-mm cell with a 1-mL capacity and are given in units of 10-1 deg cm2 g-1.
Elemental analyses were performed by the Microanalytical Laboratory of the University of Parma.
Routine NMR spectra were recorded on Avance 300 (Bruker) or Mercury Plus MP-400 (Varian)
NMR spectrometers. Chemical shifts (δ) are reported in parts per million (ppm) with TMS (CDCl3),
CD2HOD, and HOD resonance peaks set at 0, 3.31, and 4.80 ppm, respectively. For sample preparation
and NMR experiments of target cyclopeptides 5-12, see the Experimental Section in the main text.
Fmoc-Asp(But)-OH, Fmoc-Arg(Pmc)-OH, and Fmoc-Gly-OH were purchased from Fluka or
Bachem. Trans-4-hydroxy-L-proline (13) was purchased from Fluka. Cis-4-hydroxy-D-proline (14) was
prepared from 13 in a 65% yield according to a known three-step procedure (Ac2O, AcOH; then 2N
HCl; then Ag2CO3).1 Compound 14 is also commercially available from Fluka.
(2S,4R)-1-(tert-Butoxycarbonyl)-4-hydroxyproline Benzyl Ester (15). Typical Procedure. To a
solution of triethylamine (4 mL)
Boc
Boc
BnBr,
Cs2CO3
Boc2O,
Et3N
NH
N
N
and methanol (36 mL) was added
HO
HO
HO
CO2H
CO2H
CO2Bn
13
13a
15
trans-4-hydroxy-L-proline
(13)
(2.0 g, 15.3 mmol) and di-tert-butyl dicarbonate (6.66 g, 30.5 mmol). After being refluxed for 2 h, the
reaction mixture was allowed to cool to room temperature and the solvent removed in vacuo. The
residue was cooled to 0 °C, followed by addition of NaH2PO4 (150 mg), and the solution was acidified
with diluted HCl to pH 2. The mixture was stirred at 0 °C for 30 min. Then it was extracted with
EtOAc (4 ×), and the combined organic layers were collected, dried (MgSO4), and filtered. Evaporating
off the solvent under reduced pressure afforded trans-N-(tert-butoxycarbonyl)-4-hydroxy-L-proline
(13a) (3.0 g, 85%) as a colourless foam and used directly for the next step: [α]25 –77 (c 0.8, H2O);
D
S3

1H NMR (300 MHz, CD3OD) δ 4.40 (m, 1H), 4.32 (t, J = 8.0 Hz, 1H), 3.56 (dd, J = 11.5, 4.1 Hz, 1H),
13
3.45 (m, 1H), 2.28 (m, 1H), 2.07 (m, 1H), 1.48 and 1.45 (2s, 9H); C NMR (75.4 MHz, CD3OD) δ
176.0 (Cq), 147.9 (Cq), 81.7 (Cq), 70.1 (CH), 59.4 (CH), 55.6 (CH2), 40.1 (CH2), 28.5 (3C, CH3). The
spectral and chiro-optical characteristics of this compound fully matched those reported for the
commercially available material (Fluka).
Proline 13a (3.0, 13.0 mmol) was dissolved in methanol (50 mL) and the solution was cooled to 0 °C.
Aqueous caesium carbonate (2.12 g in 32 mL H2O) was added. The solution was concentrated and
sufficient DMF was added to azeotrope the water, leaving a white solid which was dissolved in DMF
(60 mL). Benzyl bromide (1.5 mL, 13.0 mmol) was added at 0 °C and the mixture was stirred
vigorously at room temperature for 20 h. The reaction mixture was concentrated in vacuo, dissolved in
EtOAc (40 mL), and washed with water (2 ×) and saturated aqueous sodium chloride (2 ×). The organic
layer was dried (MgSO4), filtered, and concentrated in vacuo to produce a residue which was purified
by silica gel flash chromatography (hexanes/EtOAc 50:50) affording benzylproline 15 as a colorless oil
1
(3.97 g, 95%): [α]25 –61.3 (c 4.0, CHCl3); [lit.2 [α]25 –63.34 (c 1, CHCl3)]; H NMR (300 MHz,
D
D
CDCl3, mixture of rotamers) δ 7.30 (m, 5H, Ph), 5.15 (m, 2H, CH2Ph), 4.45 (dd, J = 8.8, 1.7 Hz, 1H,
H2), 4.37 (m, 1H, H4), 3.74 (bs, 1H, OH), 3.55 (dd, J = 11.6, 4.3 Hz, 1H, H5A), 3.42 (m, 1H, H5B),
13
2.24 (m, 1H, H3A), 1.97 (m, 1H, H3B), 1.40 and 1.24 (2s, 9H, But); C NMR (75.4 MHz, CDCl3,
mixture of rotamers, major isomer) δ 173.03 (Cq, CO2R), 154.1 (Cq, Boc), 135.5 (Cq, Ph), 128.8 (2C,
CH, Ph), 128.4 (2C, CH, Ph), 128.1 (CH, Ph), 80.4 (Cq, Boc), 69.1 (CH, C4), 66.8 (CH2, CH2Ph), 58.1
(CH, C2), 54.7 (CH2, C5), 39.1 (CH2, C3), 28.4 (3C, CH3, Boc). Anal. (C17H23NO5): C, H, N.
(2R,4R)-1-(tert-Butoxycarbonyl)-4-hydroxyproline Benzyl Ester (16). The title compound was
prepared from hydroxyproline 14
Boc
Boc
BnBr,
Cs2CO3
Boc2O,
Et3N
NH
N
N
(1.0 g, 7.63 mmol) according to
the two-step procedure utilized to
HO
HO
HO
CO2H
CO2H
CO2Bn
14
14a
16
S4

convert 13 into 15. After the first step, cis-N-(tert-butoxycarbonyl)-4-hydroxy-D-proline (14a) (85%)
was obtained as a white powder and used directly for the following step: [α]25D +46.1 (c 5.0, MeOH);
1
[lit.3 [α]22 +47.1 (c 2.29, EtOH)]; H NMR (300 MHz, CDCl3, mixture of rotamers) δ 7.60 (bs, 2H),
D
13
4.31 (m, 1H), 4.20 (bd, J = 8.2 Hz, 1H), 3.42 (m, 2H), 2.0-2.3 (m, 2H), 1.34 and 1.31 (2s, 9H); C
NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer) δ 175.7 (Cq), 154.2 (Cq), 80.7 (Cq), 69.3
(CH), 57.8 (CH), 54.2 (CH2), 38.0 (CH2), 27.9 (3C). Proline 14a is also commercially available from
Flamma Spa, Italy. After the second step, compound 16 (1.96 g, 80% yield from 14) was obtained as a
1
colorless oil: [α]25 +10.38 (c 2.0, CHCl3); H NMR (300 MHz, CDCl3, mixture of rotamers) δ 7.39
D
(m, 5H, Ph), 5.1-5.4 (m, 2H, CH2Ph), 4.3-4.5 (m, 2H, H2, H4), 3.5-3.8 (m, 2H, H5A, H5B), 3.29 and
3.19 (2d, J = 10.0 and 9.9 Hz, 1H, OH), 2.35 (m, 1H, H3A), 2.12 (bd, J = 12.5 Hz, 1H, H3B), 1.49 and
13
1.36 (2s, 9H, But); C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer) δ 173.9 (Cq,
CO2R), 153.5 (Cq, Boc), 135.1 (Cq, Ph), 128.4 (2C, CH, Ph), 128.2 (2C, CH, Ph), 128.1 (CH, Ph), 80.1
(Cq, Boc), 69.6 (CH, C4), 67.0 (CH2, CH2Ph), 57.8 (CH, C2), 54.9 (CH2, C5), 38.4 (CH2, C3), 28.2
(3C, CH3, Boc). Anal. (C17H23NO5): C, H, N.
(2S,4S)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (17). Typical Procedure. A solution
of DEAD (7.9 mL of a 40% solution in toluene,
Boc
Boc
DPPA, PPh3,
DEAD
18.20 mmol) and DPPA (3.9 mL, 18.20 mmol) in
THF (15 mL) was added dropwise over 30 min to a
solution of proline 15 (1.95 g, 6.07 mmol) and PPh3
N
N
N3
HO
CO2Bn
CO2Bn
17
15
(4.77 g, 18.20 mmol) in THF (50 mL) at 0 °C under argon. The mixture was stirred for 24 h at room
temperature. After addition of EtOH (20 mL), the solvent was concentrated to dryness in vacuo. The
residue was purified by silica gel flash chromatography (hexanes/EtOAc 80:20 to 70:30) to afford
azidoproline 17 (2.06 g, 98%) as a yellowish oil: [α]25 –40.1 (c 1.0, CHCl3); [lit.4 [α]D –32.2 (c 1.5,
D
CH2Cl2)]; 1H NMR (300 MHz, CDCl3, mixture of rotamers) δ 7.31 (m, 5H, Ph), 5.21 (m, 2H, CH2Ph),
4.50 (dd, J = 8.9, 3.5 Hz, 0.4H, H2), 4.38 (dd, J = 8.9, 3.9 Hz, 0.6H, H2), 4.16 (m, 1H, H4), 3.73
S5

(ddd, J = 18.0, 11.7, 6.1 Hz, 1H, H5A), 3.50 (ddd, J = 17.8, 11.7, 3.7 Hz, 1H, H5B), 2.46 (m, 1H, H3A),
2.20 (dt, J = 13.5, 3.8 Hz, 1H, H3B), 1.48 and 1.36 (2s, 9H, But); 13C NMR (75.4 MHz, CDCl3, mixture
of rotamers, major isomer, DEPT assignment) δ 171.4 (Cq), 153.7 (Cq), 135.6 (Cq), 128.6 (2C, CH),
128.5 (2C, CH), 128.2 (CH), 80.6 (Cq), 67.1 (CH2), 58.3 (CH), 57.8 (CH), 51.4 (CH2), 36.1 (CH2),
28.1 (3C, CH3). Anal. (C17H22N4O4): C, H, N.
(2S,4R)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (18). Typical Procedure. Proline 15
(2.0 g, 6.22 mmol) and CBr4 (2.13
CBr4,
Diphos
Boc
Boc
NaN3,
DMF
Boc
N
N
N
g, 6.41 mmol) were dissolved in dry
THF (18 mL). Then, Diphos5 (1.49
HO
CO2Bn
Br
N3
CO2Bn
CO2Bn
15
15a
18
g, 3.73 mmol) was added. The reaction was allowed to stir at room temperature for 20 h, at which time
the suspension was filtered through a Celite pad. The pad was rinsed with THF and the filtrate was
evaporated to give a yellow oil. The crude mixture was purified by silica gel flash chromatography
(hexanes/EtOAc 50:50) to afford a bromoproline intermediate 15a (2.0 g, 84%) as a pale yellow oil:
[α]25D –40.8 (c 1.0, CHCl3); [lit.4 [α]D –41.3 (c 1.5, CH2Cl2)]; 1H NMR (300 MHz, CDCl3, mixture of
rotamers) δ 7.20 (m, 5H), 4.9-5.1 (m, 2H), 4.10-4.30 (m, 2H), 3.85 (m, 1H), 3.55 (m, 1H), 2.5-3.7 (m,
13
1H), 2.28 (m, 1H), 1.35 and 1.21 (2s, 9H); C NMR (75.4 MHz, CDCl3, mixture of rotamers, major
isomer, DEPT assignment) δ 171.6 (Cq), 153.8 (Cq), 135.8 (Cq), 128.8 (2C, CH), 128.7 (2C, CH),
128.3 (CH), 80.8 (Cq), 67.2 (CH2), 58.5 (CH), 55.9 (CH), 42.8 (CH2), 41.1 (CH2), 28.5 (3C, CH3).
Bromoproline intermediate 15a (2.0 g, 5.22 mmol) and NaN3 (1.70 g, 26.1 mmol) were suspended in
75 mL of dry DMF. This mixture was heated at 55 °C in an oil bath for 24 h. The mixture was allowed
to cool to room temperature, whereupon it was poured into an ice cold water. The mixture was
extracted with EtOAc (3 ×), and the combined organic layers were washed with H2O and brine, dried
(MgSO4), filtered, and concentrated to give azidoproline 18 (1.75 g, 97%) as a colorless oil: [α]25
–
D
1
46.1 (c 1.0, CHCl3); [lit.4 [α]D –49.3 (c 1.0, CH2Cl2)]; H NMR (300 MHz, CDCl3, mixture of
S6

rotamers) δ 7.27 (m, 5H), 5.18 (m, 2H), 4.45 (dd, J = 8.5, 6.6 Hz, 0.4H), 4.28 (t, J = 7.5 Hz, 0.6H), 4.05
13
(m, 1H), 3.3-3.6 (m, 2H), 2.20 (m, 1H), 2.05 (m, 1H), 1.40 and 1.30 (2s, 9H); C NMR (75.4 MHz,
CDCl3, mixture of rotamers, major isomer, DEPT assignment) δ 171.9 (Cq), 153.9 (Cq), 135.6 (Cq),
128.6 (2C, CH), 128.5 (2C, CH), 128.2 (CH), 80.6 (Cq), 67.0 (CH2), 58.9 (CH), 57.8 (CH), 51.4 (CH2),
36.5 (CH2), 28.3 (3C, CH3). Anal. (C17H22N4O4): C, H, N.
(2R,4S)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (19). The title compound was
prepared from hydroxyproline 16 (1.0 g, 3.11
Boc
Boc
DPPA, PPh3,
DEAD
mmol) according to the procedure utilized to
convert 15 into 17. After purification by silica gel
flash chromatography (hexanes/EtOAc 80:20 to
N
N
N3
HO
CO2Bn
CO2Bn
19
16
70:30), the title compound 19 (1.04 g, 96%) was obtained as a colorless oil: [α]25 +48.2 (c 1.1,
D
1
13
CHCl3). The H and C NMR data fully matched those reported for the enantiomeric counterpart 18.
Anal. (C17H22N4O4): C, H, N.
(2R,4R)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (20). The title compound was
prepared from hydroxyproline 16
CBr4,
Diphos
Boc
Boc
NaN3,
DMF
Boc
N
N
N
(1.0 g, 3.11 mmol) according to the
two-step procedure utilized to
HO
CO2Bn
Br
N3
CO2Bn
CO2Bn
16
16a
20
convert 15 into 18. After the first step, bromoproline intermediate 16a4 (1.02 g, 85%) was obtained as a
1
white powder: [α]25 +51.55 (c 2.0, CHCl3); H NMR (300 MHz, CDCl3, mixture of rotamers) δ 7.35
D
(m, 5H), 5.1-5.4 (m, 2H), 4.4-4.7 (m, 2H), 3.7-4.0 (m, 2H), 2.60 (m, 1H), 2.40 (m, 1H), 1.48 and 1.37
(2s, 9H); 13C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer, DEPT assignment) δ 172.1
(Cq), 153.4 (Cq), 135.3 (Cq), 128.6 (2C, CH), 128.5 (2C, CH), 128.1 (CH), 80.7 (Cq), 67.0 (CH2), 58.2
(CH), 55.9 (CH), 44.3 (CH2), 41.4 (CH2), 28.2 (3C, CH3). After the second step, the title compound 204
1
13
(883 mg, 82% from 16) was obtained as a colorless oil: [α]25 +45.8 (c 2.0, CHCl3). The H and C
D
S7

NMR data fully matched those reported for the enantiomeric counterpart 17. Anal. (C17H22N4O4): C, H,
N.
(2S,4S)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (21). Typical
Procedure. Azidoproline 17 (0.5 g,
Boc
FmocOSu,
aq Na2CO3
Boc
Boc
N
N
N
H2, Pd/C
H2N
1.44 mmol) was dissolved in
MeOH (30 mL) and palladium on
carbon (10%, 50 mg) was added.
HN
Fmoc
N3
CO2Bn
CO2H
CO2H
21
17
17a
The reaction vessel was evacuated by aspirator and thoroughly purged with hydrogen (three times), and
the resulting heterogeneous mixture was stirred under a hydrogen balloon for 24 h at room temperature.
The mixture was filtered through a pad of Celite and the pad was washed with MeOH. The filtrate was
concentrated in vacuo to give crude N-Boc-4-aminoproline intermediate 17a (332 mg, 100%) which
was used as such in the following step: a white powder; [α]25 +14.3 (c 1.0, H2O); [lit.6 [α]D +21 (c
D
0.24, H2O)]; 1H NMR (300 MHz, D2O, mixture of rotamers) δ 4.07 (dd, J = 9.4, 2.8 Hz, 1H), 3.90 (m,
1H), 3.62 (m, 2H), 2.57 (ddd, J = 14.9, 9.3, 5.8 Hz, 1H), 2.02 (dt, J = 14.5, 2.8 Hz, 1H), 1.36 and 1.32
(2s, 9H); 13C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer, DEPT assignment) δ 177.1
(Cq), 154.9 (Cq), 83.1 (Cq), 58.2 (CH), 49.3 (CH2), 48.7 (CH), 33.5 (CH2), 27.5 (3C, CH3).
N-Boc-4-aminoproline intermediate 17a (322 mg, 1.44 mmol) was dissolved in THF (3 mL), and
10% aqueous Na2CO3 solution (4 mL) was added. FmocOSu (486 mg, 1.44 mmol) dissolved in THF
(10 mL) was then added to the solution pre-cooled to 0 °C. The reaction mixture was stirred for 2 h at
room temperature and concentrated in vacuo to leave a residue which was dissolved in EtOAc (10 mL)
and treated with saturated aq NH4Cl solution. The mixture was extracted with EtOAc (3 ×) and the
organic layers were collected, dried over MgSO4, filtered, and concentrated to afford a crude residue
which was purified by silica gel flash chromatography (EtOAc/MeOH 70:30) to afford aminoproline 21
(586 mg, 90%) as a white solid: mp 168 °C; [α]25D –17.2 (c 0.87, MeOH); 1H NMR (300 MHz, CDCl3,
S8

mixture of rotamers) δ 7.78 (d, J = 7.4 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.34
(t, J = 7.3 Hz, 2H), 5.77 (bd, J = 5.9 Hz, 1H), 4.51 (d, J = 8.3 Hz, 1H), 4.25-4.45 (m, 3H), 4.22 (t, J =
7.1 Hz, 1H), 3.58 (m, 2H), 2.49 (bd, J = 13.6 Hz, 1H), 2.37 (m, 1H), 1.52 and 1.46 (2s, 9H); 13C NMR
(75.4 MHz, CDCl3, HSQC assignment) δ 180.9 (Cq), 155.7 (2C, Cq), 143.5 (2C, Cq), 141.0 (2C, Cq),
127.5 (2C, CH), 126.9 (2C, CH), 124.9 (2C, CH), 119.8 (2C, CH), 82.9 (Cq), 66.9 (CH2), 58.7 (CH),
53.9 (CH2), 50.5 (CH), 46.9 (CH), 32.4 (CH2), 28.1 (3C, CH3). Anal. (C25H28N2O6): C, H, N. The title
amino acid is also commercially available from Sigma-Aldrich or NeoMPS.
(2S,4S)-1-Benzoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (22). Typical Procedure. A
O
O
O
FmocOSu,
aq Na2CO3
PhCO2H,
DIC, HOBt
H2,
Pd/C
Boc
N
NH
N
Ph
N
Ph
CO2H
N
Ph
CO2H
TFA
HN
N3
CO2Bn N3
CO2Bn
CO2Bn
N3
H2N
Fmoc
17b
17c
17d
17
22
solution of azidoproline 17 (0.5 g, 1.44 mmol) in dry DCM (10 mL) was cooled to 0 °C and treated
with anhydrous TFA (2 mL). After the mixture was stirred at room temperature for 2 h, saturated aq
NaHCO3 solution was added until neutral pH. The mixture was extracted with EtOAc (4 ×) and the
organic layers were collected, dried over MgSO4, filtered, and concentrated to afford a crude residue
which was purified by silica gel flash chromatography (EtOAc/MeOH 96:4) to afford the Nα-
deprotected azidoproline intermediate 17b (294 mg, 83%) as a colorless oil: [α]25 –42.5 (c 1.0,
D
1
CHCl3); H NMR (300 MHz, CDCl3) δ 7.34 (m, 5H, Ph), 5.21 (1/2 ABq, J = 12.2 Hz, 1H, CH2Ph),
5.16 (1/2 ABq, J = 12.2 Hz, 1H, CH2Ph), 4.05 (ddt, J = 6.0, 4.8, 2.5 Hz, 1H, H4), 3.83 (dd, J = 9.5, 4.3
Hz, 1H, H2), 3.13 (ddd, J = 12.0, 2.3, 1.4 Hz, 1H, H5A), 2.96 (dd, J = 12.0, 4.8 Hz, 1H, H5B), 2.64 (bs,
1H, NH), 2.33 (ddd, J = 14.0, 9.5, 6.0 Hz, 1H, H3A), 2.11 (dddd, J = 14.0, 4.2, 2.7, 1.4 Hz, 1H, H3B);
13C NMR (75.4 MHz, CDCl3, HSQC assignment) δ 173.9 (Cq), 135.5 (Cq), 128.6 (2C, CH), 128.5
(CH), 128.3 (2C, CH), 67.1 (CH2), 61.3 (CH), 58.9 (CH), 52.6 (CH2), 36.0 (CH2).
Azidoproline intermediate 17b (294 mg, 1.20 mmol) was dissolved in DCM (10 mL) and the
S9

resulting solution was cooled to 0 °C and treated with benzoic acid (176 mg, 1.44 mmol), HOBt (195
mg, 1.44 mmol), and DIC (0.26 mL, 1.68 mmol). After stirring at room temperature for 2 h, the
reaction mixture was filtered to take off the solid precipitate. The organic layer was washed with aq
NaHCO3 solution (3 ×), dried over MgSO4, filtered, and concentrated to afford a crude residue which
was purified by silica gel flash chromatography (hexanes/EtOAc 40:60). Nα-Benzoyl azidoproline
intermediate 17c (370 mg, 88%) was obtained as an oil. Compound 17c (370 mg, 1.06 mmol) was
transformed into the title amino acid 22 via the two-step sequence previously described to convert 17
into 21. Compound 22 (425 mg, 88%, over two steps, corresponding to a 64% yield from 17): a white
1
solid; mp 165 °C; [α]25 –47.0 (c 1.0, MeOH); H NMR (300 MHz, CDCl3, mixture of rotamers) δ
D
7.76 (d, J = 7.4 Hz, 2H), 7.4-7.6 (m, 8H), 7.30 (m, 3H), 5.85 (bd, J = 6.7 Hz, 1H), 4.93 (t, J = 5.5 Hz,
1H), 4.3-4.6 (m, 3H), 4.19 (t, J = 6.8 Hz, 1H), 3.90 (dd, J = 11.8, 6.0 Hz, 1H), 3.60 (bd, J = 10.1 Hz,
13
1H), 2.51 (m, 2H); C NMR (75.4 MHz, CDCl3, HSQC assignment) δ 185.8 (Cq), 172.8 (Cq), 155.7
(Cq), 143.7 (2C, Cq), 141.3 (2C, Cq), 131.3 (Cq), 128.7 (2C, CH), 127.8 (2C, CH), 127.3 (CH), 127.1
(4C, CH), 125.1 (2C, CH), 120.0 (2C, CH), 67.1 (CH2), 59.8 (CH), 56.7 (CH2), 50.8 (CH), 47.1 (CH),
32.4 (CH2). Anal. (C27H24N2O5): C, H, N. The title amino acid is commercially available from
NeoMPS.
(2S,4S)-1-Propanoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (23). The title compound
O
O
O
FmocOSu,
aq Na2CO3
EtCO2H,
DIC, HOBt
H2,
Pd/C
Boc
N
NH
N
N
N
TFA
HN
N3
CO2Bn N3
CO2Bn
CO2Bn
CO2H
CO2H
N3
H2N
Fmoc
17b
17e
17f
17
23
was prepared from azidoproline 17 (0.5 g, 1.44 mmol) according to the procedure followed for the
transformation of 17 into 22 and using propanoic acid instead of benzoic acid. Compound 23 (365 mg,
62% yield from 17): a white foam; [α]25D –9.1 (c 4.1, MeOH); 1H NMR (400 MHz, CD3OD, mixture of
rotamers) δ 7.81 (d, J = 7.2 Hz, 2H), 7.65 (bd, J = 6.0 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.2
Hz, 2H), 4.3-4.5 (m, 3H), 4.24 (m, 2H), 3.91 (m, 1H), 3.40 (m, 1H), 2.57 (m, 1H), 2.38 (m, 2H), 1.95
S10

(m, 1H), 1.13 (m, 3H); 13C NMR (100 MHz, CD3OD) δ 176.0 (Cq), 160.7 (Cq), 159.0 (Cq), 146.1 (2C,
Cq), 143.4 (2C, Cq), 129.6 (2C, CH), 129.0 (2C, CH), 127.0 (2C, CH), 121.8 (2C, CH), 68.7 (CH2),
60.1 (CH), 53.9 (CH2), 53.5 (CH), 52.4 (CH), 36.5 (CH2), 29.3 (CH2), 9.9 (CH3). Anal. (C23H24N2O5):
C, H, N.
(2S,4S)-1-Heptyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (24). A solution of azidoproline
FmocOSu,
aq Na2CO3
heptanal,
NaBH(OAc)3
H2,
Pd/C
Boc
N
N
NH
N
N
6
6
6
TFA
HN
N3
N3
CO2H
CO2Bn
CO2Bn
CO2Bn
CO2H
N3
H2N
Fmoc
17b
17
24
17g
17h
17 (0.5 g, 1.44 mmol) in dry DCM (10 mL) was cooled to 0 °C and treated with anhydrous TFA (2
mL). After the mixture was stirred at room temperature for 2 h, saturated aq NaHCO3 solution was
added until neutral pH. The mixture was extracted with EtOAc (4 ×) and the organic layers were
collected, dried over MgSO4, filtered, and concentrated to afford a crude residue which was purified by
silica gel flash chromatography (EtOAc/MeOH 96:4) to afford Nα-deprotected azidoproline
intermediate 17b (294 mg, 83%), whose optical and spectral characteristics are reported above (see
transformation of compound 17 into 22, first step).
To an ice-cooled solution7 of azidoproline intermediate 17b (294 mg, 1.19 mmol) in DCE (10 mL)
were added freshly distilled heptanal (0.20 mL, 1.43 mmol) and sodium triacetoxyborohydride
[NaBH(OAc)3] (353 mg, 1.67 mmol). The resulting mixture was stirred at room temperature for 1 h,
and quenched with saturated aq NaHCO3 solution until neutral pH. Extraction with EtOAc (4 ×), drying
of the organic layers over MgSO4, filtration, and evaporation of the solvent in vacuo gave a crude
residue, which was purified by silica gel flash chromatography (hexanes/EtOAc 80:20) to yield N-
heptyl azidoproline 17g (348 mg, 85%) as an oil; 1H NMR (300 MHz, CDCl3) δ 7.30 (m, 5H), 5.21 (m,
2H), 4.01 (m, 1H), 3.28 (dd, J = 8.9, 6.3 Hz, 1H), 3.23 (dd, J = 10.3, 1.8 Hz, 1H), 2.73 (dt, J = 12.3, 8.0
Hz, 1H), 2.62 (dd, J = 9.9, 5.7 Hz, 1H), 2.51 (dt, J = 13.3, 8.7 Hz, 1H), 2.39 (dt, J = 11.6, 7.0 Hz, 1H),
13
2.12 (ddd, J = 13.9, 5.4, 2.5 Hz, 1H), 1.45 (m, 2H), 1.2-1.4 (m, 8H), 0.75 (m, 3H); C NMR (75.4
S11

MHz, CDCl3) δ 172.9 (Cq), 136.0 (Cq), 128.3 (3C, CH), 128.2 (2C, CH), 66.8 (CH2), 65.0 (CH), 58.9
(CH), 58.6 (CH2), 54.3 (CH2), 35.8 (CH2), 28.3 (CH2), 25.1 (CH2), 24.6 (CH2), 23.8 (CH2), 13.5 (CH3).
Anal. (C19H28N4O2): C, H, N.
Azidoproline intermediate 17g (348 mg, 1.01 mmol) was converted into the title amino acid 24 via
the two-step sequence previously described to transform 17 into 21. Compound 24 (396 mg, 87%, over
1
two steps, corresponding to a 61% yield from 17): a white foam; [α]25 –12.8 (c 4.75, MeOH); H
D
NMR (400 MHz, CD3OD, mixture of rotamers) δ 7.79 (d, J = 7.6 Hz, 2H), 7.64 (dd, J = 7.6, 2.4 Hz,
2H), 7.40 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 4.38 (m, 2H), 4.30 (m, 1H), 4.19 (t, J = 6.4 Hz,
1H), 3.80 (bt, J = 8.0 Hz, 1H), 3.60 (bd, J = 10.4 Hz, 1H), 3.29 (m, 1H), 3.22 (m, 1H), 3.01 (m, 1H),
2.75 (dt, J = 13.6, 8.0 Hz, 1H), 2.08 (m, 1H), 1.69 (m, 2H), 1.34 (m, 8H), 0.92 (m, 3H); 13C NMR (75.4
MHz, CDCl3, HSQC assignment) δ 181.3 (Cq), 162.4 (Cq), 156.0 (Cq), 143.7 (2C, Cq), 141.3 (2C,
Cq), 131.8 (Cq), 128.7 (2C, CH), 127.8 (2C, CH), 127.3 (CH), 127.1 (4C, CH), 125.1 (2C, CH), 120.0
(2C, CH), 67.1 (CH2), 59.8 (CH), 56.7 (CH2), 50.8 (CH), 47.1 (CH), 32.4 (CH2). Anal. (C27H34N2O4):
C, H, N.
(2S,4R)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (25). The title
compound was obtained from
Boc
FmocOSu,
aq Na2CO3
Boc
Boc
N
N
N
H2, Pd/C
H2N
azidoproline 18 (0.5 g, 1.44 mmol)
via the two-step sequence utilized to
convert 17 into 21. Compound 25
HN
Fmoc
N3
CO2Bn
CO2H
CO2H
25
18
18a
(573 mg, 88% from 18), a white solid: mp 190 °C; [α]25D –11.58 (c 0.95, MeOH); 1H NMR (300 MHz,
CDCl3, mixture of rotamers) δ 7.79 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.4 Hz,
2H), 7.34 (t, J = 7.4 Hz, 2H), 4.95 and 4.88 (2bs, 1H), 4.4-4.6 (m, 2H), 4.2-4.4 (m, 3H), 3.78 (m, 1H),
13
3.3-3.5 (m, 1H), 2.53 (m, 1H), 2.12 (m, 1H), 1.50 and 1.45 (2s, 9H); C NMR (75.4 MHz, CDCl3,
HSQC assignment) δ 179.2 (Cq), 156.3 (2C, Cq), 143.7 (2C, Cq), 141.4 (2C, Cq), 127.8 (2C, CH),
S12

127.1 (2C, CH), 124.9 (2C, CH), 120.0 (2C, CH), 81.1 (Cq), 66.6 (CH2), 57.9 (CH), 51.9 (CH2), 49.95
(CH), 47.2 (CH), 34.0 (CH2), 28.3 (3C, CH3). Anal. (C25H28N2O6): C, H, N. The title amino acid is also
commercially available from NeoMPS.
(2S,4R)-1-Benzoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (26). The title compound was
O
O
O
FmocOSu,
aq Na2CO3
PhCO2H,
DIC, HOBt
H2,
Pd/C
Boc
N
NH
N
Ph
N
Ph
CO2H
N
Ph
CO2H
TFA
HN
N3
CO2Bn N3
CO2Bn
CO2Bn
N3
H2N
Fmoc
18a
18b
18c
18
26
obtained from azidoproline 18 (1.0 g, 2.88 mmol) via the four-step sequence utilized to convert 17 into
22. Compound 26 (881 mg, 67% yield from 17): a glassy solid: [α]25D –14.0 (c 1.5, MeOH); 1H NMR
(400 MHz, CD3OD, mixture of rotamers) δ 7.88 (m, 2H), 7.2-7.7 (m, 11H), 4.68 (t, J = 7.2 Hz, 1H),
4.25-4.45 (m, 3H), 4.10-4.25 (m, 1H), 3.98 and 3.87 (2m, 1H), 3.63 and 3.44 (2m, 1H), 2.0-2.4 (m,
13
2H); C NMR (100 MHz, CD3OD, HSQC assignment) δ 185.8 (Cq), 173.0 (Cq), 163.9 (Cq), 157.2
(Cq), 146.0 (2C, Cq), 143.4 (2C, Cq), 132.4 (Cq), 130.3 (2C, CH), 129.6 (2C, CH), 129.2 (2C, CH),
129.0 (2C, CH), 127.0 (3C, CH), 121.8 (2C, CH), 68.4 (CH2), 61.5 (CH), 57.3 (CH2), 52.8 (CH), 48.1
(CH), 37.0 (CH2). Anal. (C27H24N2O5): C, H, N.
(2R,4S)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (27). The title
compound was obtained from azidoproline 19 (1.0 g, 2.88 mmol) via the two-step sequence utilized to
convert 17 into 21. Compound 27
Boc
FmocOSu,
aq Na2CO3
Boc
Boc
(1.1 g, 85% from 19), a white foam:
N
N
N
H2, Pd/C
H2N
1
[α]25 +9.2 (c 4.2, MeOH); H
HN
Fmoc
N3
CO2Bn
CO2H
CO2H
D
27
19
19a
NMR (400 MHz, CD3OD, mixture
of rotamers) δ 7.78 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.31 (t, J =
7.2 Hz, 2H), 4.35 (m, 2H), 4.26 (m, 2H), 4.18 (t, J = 6.4 Hz, 1H), 3.73 (dd, J = 10.4, 6.0 Hz, 1H), 3.38
13
(m, 1H), 2.53 (m, 1H), 2.0 and 2.2 (2m, 1H), 1.47 and 1.46 (2s, 9H); C NMR (100 MHz, CD3OD,
S13

mixture of rotamers, major isomer, HSQC assignment) δ 179.0 (Cq), 159.1 (Cq), 156.9 (Cq), 146.1
(2C, Cq), 143.4 (2C, Cq), 129.6 (2C, CH), 129.0 (2C, CH), 127.0 (2C, CH), 121.8 (2C, CH), 82.3 (Cq),
68.5 (CH2), 61.3 (CH), 53.3 (CH2), 51.4 (CH), 50.7 (CH), 38.3 (CH2), 29.5 (3C, CH3). Anal.
(C25H28N2O6): C, H, N.
(2R,4R)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (28). The title
compound was obtained from
azidoproline 20 (0.5 g, 1.44 mmol)
via the two-step sequence utilized to
Boc
FmocOSu,
aq Na2CO3
Boc
Boc
N
N
N
H2, Pd/C
H2N
HN
Fmoc
N3
CO2Bn
CO2H
CO2H
28
20
20a
convert 17 into 21. Compound 28 (573 mg, 88% from 20), a glassy solid: [α]25D +17.5 (c 0.8, MeOH);
1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 7.71 (d, J = 7.4 Hz, 2H), 7.57 (m, 2H), 7.35 (t, J =
7.3 Hz, 2H), 7.27 (t, J = 7.3 Hz, 2H), 6.0 and 6.4 (2bs, 1H), 4.0-4.5 (m, 5H), 3.3-3.7 (m, 2H), 1.9-2.4
(m, 2H), 1.43 and 1.37 (2s, 9H); 13C NMR (100 MHz, CDCl3, HSQC assignment) δ 176.4 (Cq), 156.0
(2C, Cq), 143.9 (2C, Cq), 141.2 (2C, Cq), 127.6 (2C, CH), 127.0 (2C, CH), 125.3 (2C, CH), 119.9 (2C,
CH), 81.8 (Cq), 66.9 (CH2), 59.7 (CH), 54.1 (CH2), 50.5 (CH), 47.1 (CH), 33.8 (CH2), 28.3 (3C, CH3).
Anal. (C25H28N2O6): C, H, N.
S14

Table S1. Elemental Analyses of Intermediary and Target Compounds
Elemental Analyses
Calcd (%)
Found (%)
H
Compound
Formula
C17H28N8O6 HCl
C17H28N8O6 HCl
C17H28N8O6 HCl
C17H28N8O6 HCl
C24H42N8O6 HCl
C20H32N8O7 HCl
C24H32N8O7 HCl
C24H32N8O7 HCl
C17H23NO5
C
H
N
C
N
42.81
42.81
42.81
42.81
50.12
45.07
49.61
49.61
63.54
63.54
58.95
58.95
58.95
58.95
66.36
71.04
67.63
71.97
66.36
71.04
66.36
66.36
6.13
6.13
6.13
6.13
7.54
6.24
5.72
5.72
7.21
7.21
6.40
6.40
6.40
6.40
6.24
5.30
5.92
7.61
6.24
5.30
6.24
6.24
23.50
23.50
23.50
23.50
19.48
21.02
19.29
19.29
4.36
42.59
42.70
42.74
42.98
49.97
45.33
49.76
49.33
63.28
63.37
58.68
59.12
59.09
58.69
66.08
70.87
67.73
71.78
66.18
71.22
66.13
66.48
6.40
6.31
6.44
6.29
7.81
6.20
5.98
5.94
7.05
7.52
6.78
6.59
6.74
6.61
6.45
5.63
6.04
7.50
6.47
5.54
6.29
6.50
23.25
23.36
23.19
23.28
19.37
20.90
19.14
19.35
4.50
5
6
7
8
9
10
11
12
15
16
17
18
19
20
21
22
23
24
25
26
27
28
C17H23NO5
4.36
4.20
C17H22N4O4
16.17
16.17
16.17
16.17
6.19
16.24
16.01
16.07
16.35
6.00
C17H22N4O4
C17H22N4O4
C17H22N4O4
C25H28N2O6
C27H24N2O5
6.14
5.97
C23H24N2O5
6.86
6.63
C27H34N2O4
6.22
6.35
C25H28N2O6
6.19
5.96
C27H24N2O5
6.14
6.02
C25H28N2O6
6.19
6.07
C25H28N2O6
6.19
5.97
S15

Figure S1. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 5.
Figure S2. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 6.
S16

Figure S3. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 7.
Figure S4. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 8.
S17

Figure S5. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 9.
Figure S6. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 10.
S18

Figure S7. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 11.
Figure S8. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 12.
S19

αVβ3
αVβ5
Figure S9. Compound 5: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S20

αVβ3
αVβ5
Figure S10. Compound 6: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S21

αVβ3
αVβ5
Figure S11. Compound 7: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S22

αVβ3
αVβ5
Figure S12. Compound 8: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S23

αVβ3
αVβ5
Figure S13. Compound 9: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S24

αVβ3
αVβ5
Figure S14. Compound 10: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S25

αVβ3
αVβ5
Figure S15. Compound 11: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S26

αVβ3
αVβ5
Figure S16. Compound 12: Inhibition of [125I]-echistatin specific binding to purified human integrin
protein αVβ3 (up) and αVβ5 (down). Each point represents the mean value SEM of triplicate
determinations.
S27

Tyr166
Asp150
Tyr178
O
Arg214
HN
O
N
O
NH2
H
NH
O
Asn215
NH
HN
NH2 HN
O
HN
O
NH
H O
Mn2+
O
O
O
O
Ser123
Asp218
Figure S17. Schematic 2D representation of ligand 9 bound to the αVβ3 receptor highlighting the
protein residues that form the main interactions with the different structural units of the inhibitor. The
dashed lines represent H-bonding and salt bridges.
S28

Figure S18. Best-scored docking solution of ligands 5 (up), 9 (middle), and 10 (down) into the αVβ3
binding site (as Connolly surface) highlighting the complementarity between the ligands and the
receptor. An evident hydrophobic hollow hosting the Nα-n-heptyl and Nα-propanoyl chains of ligands 9
and 10 is visible. The protein residues involved in the ligand-receptor binding are colored in red; the
manganese ion sites are colored in magenta. Nonpolar hydrogen atoms are omitted for clarity.
S29

Figure S19. Superposition of docked structure 5 (red), 9 (blue), and 10 (green) to the X-ray αVβ3-
bound conformation of 1 (yellow). The highly active, aminoproline-based ligands 5, 9, and 10 share a
common cyclopeptide backbone arrangement, as well as similar Cβ Arg-Asp distances. Hydrogen
atoms are omitted for clarity.
S30