Cyclic RGD and isoDGR Integrin Ligands Containing cis-2-amino-1-cyclopentanecarboxylic (cis-β-ACPC) Scaffolds

Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) and fibronectin (α5β1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.


Materials and methods
All commercially available reagents were used as received; DMF was anhydrified according to GP0. Non solid-phase reactions were monitored by analytical thin layer chromatography (TLC) using silica gel 60 F254 pre-coated glass plates (0.20 mm thickness). Visualization was accomplished by irradiation with a UV lamp and/or staining with a potassium permanganate alkaline solution or with a ninhydrin solution. Solid phase steps was followed by LR-Mass. Flash column chromatography was performed according to the method of Still and co-workers 1 using Chromagel 60 ACC (40-63 m) silica gel. Proton NMR spectra were recorded on a spectrometer operating at 500 MHz. Carbon NMR spectra were recorded on a spectrometer operating at 125 MHz, with complete proton decoupling. The following abbreviations are used to describe spin multiplicity s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, bs = broad signal, dd = doublet of doublet, ddd = doublet of doublet of doublet, ddt = doublet of doublet of triplet. Carbon chemical shifts are reported in ppm relative to TMS with the respective solvent resonance as the internal standard. ESI-MS spectra were recorded on the ion trap mass spectrometer Finnigan LCQ Advantage.

DMF anhydrification
GP0: A flask was charged with CaH2 (around 5 g/L) and DMF. The mixture was stirred at room temperature overnight. DMF was subsequently transferred into a flask under N2 and distilled under reduced pressure (around 20 mBar). DMF was stored under N2 in a dark brown flask over molecular sieves (3Å). SPPS was manually accomplished using a shaker. Fmoc strategy and 2-chlorotritylchloride resin (100-200 mesh, 1% DVB; loading: 1.51 mmol/g resin) were used. Each coupling step consisted in: i) Activation of the Fmoc-protected amino acid, ii) Addition of the activated amino acid to the resin at the shaker to effect the coupling reaction; iii) Following steps of capping, deprotection and washing.

Resin preparation and storage
The resin was weighted in a 10 mL vial and swelled (GP1); at the end of this operation, the resin was ready for the SPPS. When necessary, beads were stored at 4 °C under nitrogen after being washed several times with DCM and dried at the high vacuum pump.

GP1:
The resin was weighted in a vial, suspended in DCM (approximately 10 mL per gram of resin) and shaken for 30 min. The solvent was dried from the resin and, at the end of this operation, the resin was ready for the SPPS. This step was repeated every time before starting all the procedures on the resin (if the resin was previously dried under vacuum and stored at 4°C).
Loading of the first amino acid and capping of the resin GP2: The resin was suspended in DCM (approximately 14 mL per gram of resin); Fmoc-AA-OH (2.0 equiv.) and DIPEA (0.3 equiv.) were sequentially added. The resin was shaken for 10 min then DIPEA (0.5 equiv.) was added and the resin was shaken for 1h 30min; after this time the solvent was removed. The resin was washed with DCM (3 × 2 mL), then methanol was added (approximately 1.5 mL per gram of resin) and the resin was shaken for 15 min. The solvent was removed and the resin was washed with DCM (3 × 2 mL), DMF (3 × 2 mL), DCM again (3 × 2 mL), and methanol (1 × 2 mL).

Amino acid activation and coupling
GP3: To a 0.14 M solution of the amino acid (1.5 equiv.) in dry DMF under N2 at 0 °C, TBTU (1.5 equiv. in DMF) and DIPEA (3.0 equiv. in DMF) were added. The mixture was stirred under these conditions for 30-40 min and then the solution was added to the resin. The resin was shaken at r.t. for 3 h then the solvent was removed. Each coupling step was performed twice times. After the second coupling, the resin was washed with DCM (3 × 2 mL), DMF (3 × 2 mL), and DCM again (3 × 2 mL).

GP4:
To a solution of the scaffold (1.5 equiv.) in dry DMF (solution 0.14 M) under N2 at 0 °C, HATU (1.5 equiv. in DMF) and DIPEA (3.0 equiv. in DMF) were added. The mixture was stirred under these conditions for 45 min and then the solution was added to the resin. The resin was shaken at r.t. for 7 h then the solvent was removed. After the coupling, the resin was washed with DCM (3 × 2 mL), DMF (3 × 2 mL), and DCM again (3 × 2 mL).
Capping procedure GP5: A 1M solution of Ac2O (50 equiv.) and pyridine (50 equiv.) in DMF (approximately 30 mL per gram of unsubstituted resin) was added to the resin. The mixture was shaken at r.t. for 40 min then solution was removed and beads were washed with DMF (3 × 2 mL).
Fmoc removal procedure GP6: Deprotection of the N-terminal Fmoc group was achieved using a solution of 2% DBU and 2% piperidine (v/v) in DMF (approximately 30 mL per gram of the unsusbstituted resin). The solution was added to the resin that was shaken for 15 min then the solution was removed. Two deprotection steps were carried out. At the end of each step, the solvent was removed. At the end of the second step, beads were sequentially washed with DMF (6 × 2 mL) and DCM (3 × 2 mL).

GP7:
The resin was washed with DCM (3 × 2 mL) and then treated with 1% trifluoroacetic acid in DCM (15.0 mL per gram of the not substituted resin) for 5 min each time. Treatment was repeated as long as the peptide was cleaved from the resin (approximately 10 times), checking by TLC (staining with a ninhydrin solution). Combined solutions were evaporated under reduced pressure and then in vacuum. The crude obtained was used without purification in the macrolactamization step.

General procedures for solution-phase synthesis
Macrolactamization GP8: The linear precursor (1 equiv.) was dissolved in dry DMF (5 mM solution) and transferred into a syringe. HATU (3.0 equiv.) was dissolved in the same volume of dry DMF and transferred into a second syringe. These two solutions were added slowly using two syringe pumps to a stirred solution of DIPEA (6.0 equiv.) and HATU (0.1 equiv.) in half of the previous volume of dry DMF at a rate of 0.7mL/h. Once the addition was complete, the mixture was stirred for 30 min. The solvent was evaporated under reduced pressure at a temperature lower than 35°C. Crude was taken up with ethyl acetate (25 mL), sequentially washed with a solution 1M of KHSO4, a saturated solution of NaHCO3 and brine. The organic layer was dried over Na2SO4 anhydrous and volatiles were removed under reduced pressure. The crude dissolved in DCM:MeOH (9:1) and filtered over silica gel.

GP9:
The crude fully protected macrolactam was treated for 8 h with a solution (around 21 mL/mg of protected peptide) of trifluoroacetic acid (95%) at room temperature in the presence of water (2.5%) and triisopropylsilane (2.5%). After volatiles removal under reduced pressure, the residue was taken up with a 1:1 mixture of diethyl ether/water. Phases were separated and the aqueous layer was washed several times with diethyl ether. The aqueous phase was concentrated under reduced pressure and then the solution was freeze-dried. The crude was purified by preparative HPLC.
For the isoDGR compound (10), HPLC purifications were performed on a Dionex Ultimate 3000 instrument equipped with a Dionex RS Variable Wavelength Detector (column: Atlantis Prep T3 OBDTM 5 TM 19 x 100 mm). The crude reaction mixture was dissolved in water/Acetonitrile (7:3) and the solution was filtered (polypropylene, 0.45 m, 13 mm ø, PK/100) and injected in the HPLC, affording the purified product.

RP-HPLC analysis
Purity analysis for each of the compounds was carried out on a Dionex Ultimate 3000 instrument equipped with a Dionex RS Variable Wavelength Detector (column: Atlantis Prep T3 OBDTM 5 TM 19 x 100 mm). 0.8 mg of purified product was dissolved in 0.5 mL of H2O and 0.2 mL of acetonitrile and was injected using gradient: 100% H2O + 0.1% CF3COOH/0% CH3CN + 0.1% CF3COOH to 50% H2O + 0.1% CF3COOH/50% CH3CN + 0.1% CF3COOH in 11 min. The analysis of the integrals and the relative percentage of purity was performed with the software Cromeleon 6.80 SR11 Build 3161.

Freeze-drying
Each product was dissolved in water and frozen with dry ice: the freeze-drying was carried out at least for 48 h at −50 °C using the instrument 5Pascal Lio5P DGT.
Exact amounts of the amino acids and coupling reagents used for SPPS are reported in in Table  S1 for 13 and in Table S2 for 14.
Exact amounts of the amino acids and coupling reagents used for SPPS are reported in Table  S3.

Cell adhesion assays with WM-115 human epithelial cancer cells
For the competition assay, 96-well ELISA-plates were immobilized overnight at 37 °C with recombinant vitronectin in PBS (1 μg/mL), afterwards blocked with 2 % (w/v) BSA in PBS.
WM-115 cells were washed with MEM (Minimum Essential Medium)-buffer, detached with trypsin and resuspended to a cell density of 1 × 10 5 cells/mL. WM-115 cells were then incubated with fluorescein diacetate for 30 min at 37 °C in the dark, washed with MEM medium and resuspended with MEM medium containing CaCl2 and MgCl2 (2 mM) and incubated on ice for 30 min in the dark.
The cell suspension was added to the peptide solutions to give concentrations from 0.5 mM to nanomolar (dilution row [mM]: 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.00195313, 0.00097565, 0.00048828, 0.00024414). Cells and peptide were incubated for 30 min at 37° C in the dark and afterwards added to the 96-well ELISA-plates previously coated with vitronectin and incubated for 1 h at 37 °C in the dark. Unbound cells were aspirated, and bound cells were washed three times with MEM medium and fluorescence measured (λex: 485 nm, λem: 514 nm) with an Infinite™ 200 Microplate Reader (Tecan, Männedorf, Switzerland).
The IC50 values were determined four times per peptide in two independent assays here reported, and the final values are the arithmetic mean ± the standard deviation (SD) of the two independent assays. The reference peptide c(RGDfV) 1b was tested together with the peptides.
Peptide 1° Assay M 2° Assay M SP190 (8) 52 98 SP179 (9) 117 132 SP225 (10) > 300 > 300 c(RGDfV) (1b) 4.0 7.1 Figure S4. 1st Assay. Figure S5. 2nd Assay. Docking Calculations Figure S6. Docking best pose of Cilengitide (green C atoms) in the crystal structure of the extracellular domain of αvβ3 integrin (α unit blue, β unit red) overlaid on the bound conformation of Cilengitide (yellow). The RMSD calculated on heavy atoms is 0.3287 Å. Only selected integrin residues involved in interactions with the ligand are shown. The metal ion at MIDAS is shown as a red CPK sphere. Figure S7. Docking best pose of compound 8 (Type II, green C atoms, Gscore = -8.48 kcal/mol) in the crystal structure of the extracellular domain of αvβ3 integrin (α unit blue, β unit red) overlaid on the bound conformation of Cilengitide (yellow). Only selected integrin residues involved in interactions with the ligand are shown. The metal ion at MIDAS is shown as a red CPK sphere. Figure S8. Docking best pose of compound 9 (Type I, green C atoms, Gscore = -9.86 kcal/mol) in the crystal structure of the extracellular domain of αvβ3 integrin (α unit blue, β unit red) overlaid on the bound conformation of Cilengitide (yellow). Only selected integrin residues involved in interactions with the ligand are shown. The metal ion at MIDAS is shown as a red CPK sphere. Figure S9. Docking best pose of compound 10 (Type I', green C atoms, Gscore = -5.74 kcal/mol) in the crystal structure of the extracellular domain of αvβ3 integrin (α unit blue, β unit red) overlaid on the bound conformation of Cilengitide (yellow). Only selected integrin residues involved in interactions with the ligand are shown. The metal ion at MIDAS is shown as a red CPK sphere.