C. Gennari, L. Belvisi, U. Piarulli et al.
Procedure for diketopiperazine methyl ester hydrolysis: A) Diketopiper-
azine methyl ester (1 equiv) was dissolved in THF and the mixture was
cooled to 08C. A solution of LiOH·H2O (2.5 equiv) in H2O was added
dropwise. The resulting solution was stirred for 1 h at 08C. Then, main-
taining the temperature at 08C, the mixture was acidified with HCl (1m)
to pH 1–2, and extracted with CH2Cl2 (4ꢄ). The collected organic phases
were dried over Na2SO4 and volatiles were removed under reduced pres-
sure. Either DKP-4 or DKP-6 were afforded as a white foam (100%).
(Pierce, Rockford, IL). After washing, the plates were incubated for 1 h
at room temperature with streptavidin–biotinylated peroxidase complex
(Amersham Biosciences, Uppsala, Sweden) followed by 30 min incuba-
tion with substrate reagent solution (100 mL; R&D Systems, Minneapolis,
MN) before stopping the reaction by addition of H2SO4 (2N, 50 mL). Ab-
sorbance at 415 nm was read in a Synergyꢅ HT multi-detection micro-
plate reader (BioTek Instruments, Inc.). Each data point is the result of
the average of triplicate wells and was analyzed by nonlinear regression
analysis with Prism GraphPad program. Each experiment was repeated
in triplicate.
B) Diketopiperazine methyl ester (1 equiv) was dissolved in THF. The
mixture was cooled to 08C and a solution of LiOH (2.7m) in H2O2 (35%
v/v) was added dropwise. The mixture was stirred for an additional
30 min at 08C, then warmed to room temperature and stirred for 7 h.
After addition of Na2SO3 (6 equiv) the reaction mixture was diluted with
THF/H2O (1:1). KHSO4 (1m) was then added until pH 1–2 was reached,
and the mixture was extracted with DCM (4ꢄ). The collected organic
phases were dried over Na2SO4 and volatiles removed under reduced
pressure, to afford crude DKP-8 as a yellowish solid. The crude product
was dissolved in EtOAc and extracted with a saturated NaHCO3 aqueous
solution; collected aqueous layers were acidified with KHSO4 (1m) to
reach pH 1–2, and extracted with DCM (4ꢄ). Collected organic phases
were dried over Na2SO4 and volatiles were removed under reduced pres-
sure, to afford DKP-8 as a white foam (90%).
General procedure for Mtr and OtBu ester removal: Protected macrolac-
tams were treated with TFA (0.01m) in the presence of ion scavengers:
thioanisole (5%), ethanedithiol (3%), anisole (2%). After TFA removal,
under reduced pressure, the residue was dissolved in a 1:1 mixture of di-
AHCTUNGERTGiNNUN sopropyl ether/water. Phases were separated and the aqueous layer was
washed several times with diisopropyl ether. The aqueous phase was con-
centrated under reduced pressure to give the crude product, which was
purified by HPLC to give the desired compound as white solid (60–
80%).
NMR spectroscopy studies: NMR spectroscopy experiments were per-
formed at a temperature of 298 K on Bruker Avance 400 and 600 MHz
spectrometers. All proton and carbon chemical shifts were assigned un-
ambiguously. The NMR experiments were carried out in a D2O/H2O 1:9
mixture in order to observe amide protons. Two-dimensional experiments
(TOCSY, NOESY, and HSQC) were carried out on samples of cyclic
RGD peptidomimetics 9, 10, 27–32 at a concentration range of 3–6 mm.
NOESY experiments were performed at 0.7 or 0.8 s. The water resonance
was saturated with the excitation sculpting sequence from the Bruker li-
brary. The conformations of the cyclic pentapeptides were analyzed with
respect to hydrogen bonding of amide protons (VT-NMR spectroscopy)
and NOE contacts.
General procedure for Boc deprotection reactions: A half volume of
TFA was added to a solution of the N-Boc-protected amino acid or pep-
tide in CH2Cl2 (0.13m). The reaction mixture was stirred for 2 h at room
temperature and then concentrated at reduced pressure. Excess TFA was
azeotropically removed from the residue with toluene. Diethyl ether was
added to the residue and the resulting suspension was evaporated under
reduced pressure to afford the corresponding TFA salt.
General procedure for coupling reactions: HATU (1.2 equiv), HOAt
(1.2 equiv) and DIPEA (4 equiv) were added successively to a solution
of the N-protected amino acid in DMF, under a nitrogen atmosphere and
at 08C. After 30 min, a solution of the N-deprotected TFA salt of the
peptide in DMF was added and the reaction mixture was stirred at 08C
for 1 h and at room temperature, overnight. The mixture was then diluted
with EtOAc and consecutively washed with KHSO4 (1m; 2ꢄ), a saturated
NaHCO3 aqueous solution (2ꢄ), brine (2ꢄ), and dried over Na2SO4. Vol-
atiles were evaporated under reduced pressure to afford the crude prod-
uct.
Computational studies: All calculations were run by using the Schrçding-
graphical interface.
Conformational analysis: Conformational preferences of the RGD pepti-
domimetics were investigated by Monte Carlo/Stochastic Dynamics (MC/
SD) hybrid simulations[32] by using the NMR spectroscopy restraints de-
rived from the experimental NOE contacts (for distance restraints used
for each calculation, see the Supporting Information). All the NOE re-
General procedure for Cbz and OBn hydrogenolytic cleavage: Protected
compound (1 equiv) was dissolved in a mixture of THF/H2O (1:1) and
Pd/C 10% (0.1 equiv) was added. The reaction mixtures were subjected
to three vacuum/hydrogen cycles and then stirred, overnight, at room
temperature under 1 bar of hydrogen. The mixture was filtered through
Celite, and the cake thus obtained was washed thoroughly with THF/
H2O (1:1). The filtrate was concentrated and dried to give the crude
product as white solid (100%).
straints were set to a distance value of 2
(Æ0.5) ꢀ with a force constant of
100 kJmolÀ1 ꢀÀ2. MC/SD simulations were performed at 300 K within the
framework of MacroModel version 9.5[36] by employing the OPLS_2001
force field[34] and the implicit water GB/SA solvation model.[33] RGD
side-chain dihedral angles were defined as internal coordinate degrees of
freedom in the Monte Carlo part of the algorithm. A time step of 1 fs
was used for the stochastic dynamics (SD) part of the algorithm for 10 ns
of simulation time. Samples were taken at 2 ps intervals during each sim-
ulation, yielding 5000 conformations for analysis. The percentages of
H bonds discussed here were calculated as percentages of conformations
sampled during the simulation in which donor H–acceptor O distance
<2.5 ꢀ (g-turn) or <4 ꢀ (b-turn).
General procedure for macrolactamization: HATU (4 equiv), HOAt
(4 equiv) and DIPEA (6 equiv) were added successively to a solution of
deprotected linear compound (1.4 mm; 1 equiv) in DMF, under a nitrogen
atmosphere at 08C. After stirring the reaction mixture at 08C for 1 h, it
was allowed to reach room temperature and stirred, overnight. DMF was
then removed under reduced pressure and the residue was purified by
flash chromatography (CH2Cl2/MeOH; for ratios used, see the Support-
ing Information) on silica gel to afford the product as white foam (31–
74%).
Molecular docking: The recently solved crystal structure of the extracel-
lular domain of the integrin avb3 receptor in complex with Cilengitide
and in the presence of the proadhesive ion Mn2+ (PDB ID: 1L5G)[5] was
used for docking studies. Docking was performed only on the globular
head of the integrin because the headgroup of integrin has been identi-
fied in the X-ray structure as the ligand-binding region. The protein
structure was setup for docking as follows. The protein was truncated to
residue sequences 41–342 for chain a and 114–347 for chain b. Due to
a lack of parameters, the Mn2+ ions in the experimental protein structure
were modeled by replacing them with Ca2+ ions. The resulting structure
was prepared by using the Protein Preparation Wizard of the graphical
user interface Maestro and the OPLSAA force field. The automated
docking calculations were performed by using Glide[37] (Grid-based
Ligand Docking with Energetics). The grid generation step started from
the extracellular fragment of X-ray structure of avb3 complex with Cilen-
gitide, as described in the protein setup section. The center of the grid
enclosing box was defined by the center of the bound ligand, as described
Solid-phase receptor-binding assay: Purified avb3 and avb5 receptors
(Chemicon International, Inc., Temecula, CA) were diluted to
0.5 mgmLÀ1 in coating buffer containing Tris-HCl (20 mmolLÀ1; pH 7.4),
NaCl (150 mmolLÀ1), MnCl2 (1 mmolLÀ1), CaCl2 (2 mmolLÀ1
) and
MgCl2 (1 mmolLÀ1). An aliquot of diluted receptors (100 mL per well)
was added to 96-well microtiter plates (Nunc MW 96F Medisorp
Straight) and incubated, overnight, at 48C. The plates were then incubat-
ed with blocking solution (coating buffer plus 1% bovine serum albumin)
for an additional 2 h at room temperature to block nonspecific binding
followed by 3 h incubation at room temperature with various concentra-
tions (10À12–10À5 m) of test compounds in the presence of vitronectine
(1 mgmLÀ1) biotinylated by using EZ-Link Sulfo-NHS-Biotinylation kit
6206
ꢃ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2012, 18, 6195 – 6207
Marchini, Mattia
