The cyclo[DKP-isoDGR] peptidomimetics 2-5, containing bifunctional diketopiperazine (DKP) scaffolds that differ in the configuration of the two DKP stereocenters and in the substitution at the DKP nitrogen atoms, were prepared and examined in vitro in competitive binding assays with purified αv β3 and αv β5 integrin receptors. IC50 values ranged from low nanomolar (ligand 3) to submicromolar with αv β3 integrin. The biological activities of ligands cyclo[DKP3-RGD] 1 and cyclo[DKP3-isoDGR] 3, bearing the same bifunctional DKP scaffold and showing similar αV β3 integrin binding values, were compared in terms of their cellular effects in human U373 glioblastoma cells. Compounds 1 and 3 displayed overlapping inhibitory effects on the FAK/Akt integrin activated transduction pathway and on integrin-mediated cell infiltration processes, and qualify therefore, despite the different RGD and isoDGR sequences, as integrin antagonists. Both compounds induced apoptosis in glioma cells after 72 hour treatment.

Herein we report the first example of an isoDGR–drug conjugate (2), designed to release paclitaxel selectively within cancer cells expressing integrin αVβ3. Conjugate 2 was synthesized by connecting the isoDGR peptidomimetic 5 with paclitaxel via the lysosomally cleavable Val–Ala dipeptide linker. Conjugate 2 displayed a low nanomolar affinity for the purified integrin αVβ3 receptor (IC50=11.0 nm). The tumor targeting ability of conjugate 2 was assessed in vitro in anti‐proliferative assays on two isogenic cancer cell lines characterized by different integrin αVβ3 expression: human glioblastoma U87 (αVβ3+) and U87 β3‐KO (αVβ3−). The isoDGR‐PTX conjugate 2 displayed a remarkable targeting index (TI=9.9), especially when compared to the strictly related RGD‐PTX conjugate 4 (TI=2.4).

Alzheimer's disease is a neurodegenerative disorder that is linked to oligomerization and fibrillization of different amyloid β‐peptide isoforms. Among these amyloid peptides, Aβ1–42 is considered the most aggregative and neurotoxic species. We report herein the synthesis of four β‐sheet mimics composed of a peptidomimetic arm based on a 5‐amino‐2‐methoxy benzhydrazide derivative, a 2,5‐diketopiperazine scaffold (either cis‐DKP or trans‐DKP) and a tetrapeptide sequence (either Gly‐Val‐Val‐Ile, GVVI, or Lys‐Leu‐Val‐Phe, KLVF). The derivatives containing the cis‐DKP were shown by NMR and computational studies to adopt a stable β‐hairpin conformation in solution, whereas the trans‐DKP scaffold promoted the formation of extended structures. The activity of these compounds in modulating the aggregation of Aβ1–42 peptide was investigated by conducting Thioflavin T fluorescence assays to measure the kinetics of aggregation. Capillary electrophoresis (CE) and transmission electron microscopy (TEM) were then used to monitor the formation of small soluble Aβ oligomers and higher molecular weight and insoluble Aβ aggregates, respectively. As a result, small hairpin mimics containing the cis‐DKP scaffold were found to prevent the formation of small Aβ1–42 oligomeric and neurotoxic species.

Detailed process safety investigation of an old process revealed hidden, previously unnoticed danger. The synthesis of morpholine-adduct 2 was performed under reaction conditions very close to triggering a potentially dangerous runaway reaction. Process modifications such as dilution with an inert solvent allowed gaining better control of the process. Ultimately, improvement in both quality and yield was obtained.

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.