Preoperative ultrasonography can correctly identify nonrecurrent ILN, allowing earlier nerve identification and prevention of injuries.

Canataxpropellane belongs to the medicinally important taxane diterpene family. The most prominent congener, Taxol, is one of the most commonly used anticancer agent in clinics today. Canataxpropellane exhibits a taxane skeleton with three additional transannular C–C bonds, resulting in a total of six contiguous quaternary carbons, of which four are located on a cyclobutane ring. Unfortunately, isolation of canataxpropellane from natural sources is inefficient. Here, we report a total synthesis of (–)-canataxpropellane in 26 steps and 0.5% overall yield from a known intermediate corresponding to 29 steps from commercial material. The core structure of the (–)-canataxpropellane (2) was assembled in two steps using a Diels–Alder/ortho-alkene-arene photocycloaddition sequence. Enantioselectivity was introduced by designing chiral siloxanes to serve as auxiliaries in the Diels–Alder reaction.

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.

The interaction of a small library of cyclic RGD (Arg-Gly-Asp) peptidomimetics with αVβ6 integrin has been investigated by means of competitive solid phase binding assays to the isolated receptor and docking calculations in the crystal structure of the αVβ6 binding site. To this aim, a rigid receptor-flexible ligand docking protocol has been set up and then applied to predict the binding mode of the cyclic RGD peptidomimetics to αVβ6 integrin. Although the RGD interaction with αVβ6 recapitulates the RGD binding mode observed in αVβ3, differences between the integrin binding pockets can strongly affect the ligand binding ability. In general, the peptidomimetics exhibited IC50 values for integrin αVβ6 (i.e., the concentration of compound required for 50% inhibition of biotinylated fibronectin binding to isolated αVβ6 integrin) in the nanomolar range (77–345 nM), about 10–100 times higher than those for the related αVβ3 receptor, with a single notable ligand displaying a low nanomolar IC50 value (2.3 nM). Insights from the properties of the binding pocket combined with the analysis of the docking poses provided a rationale for ligand recognition and selectivity.

A dual-action ligand targeting both integrin αVβ3 and vascular endothelial growth factor receptors (VEGFRs), was synthesized via conjugation of a cyclic peptidomimetic αVβ3 Arg-Gly-Asp (RGD) ligand with a decapentapeptide. The latter was obtained from a known VEGFR antagonist by acetylation at the Lys13 side chain. Functionalization of the precursor ligands was carried out in solution and in the solid phase, affording two fragments: an alkyne VEGFR ligand and the azide integrin αVβ3 ligand, which were conjugated by click chemistry. Circular dichroism studies confirmed that both the RGD and VEGFR ligand portions of the dual-action compound substantially adopt the biologically active conformation. In vitro binding assays on isolated integrin αVβ3 and VEGFR-1 showed that the dual-action conjugate retains a good level of affinity for both its target receptors, although with one order of magnitude (10/20 times) decrease in potency. The dual-action ligand strongly inhibited the VEGF-induced morphogenesis in Human Umbilical Vein Endothelial Cells (HUVECs). Remarkably, its efficiency in preventing the formation of new blood vessels was similar to that of the original individual ligands, despite the worse affinity towards integrin αVβ3 and VEGFR-1.

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).

Theranostic RGD-camptothecin conjugates, possessing a disulfide linker and a fluorescent naphthalimide moiety, were synthesized and biologically evaluated. The conjugates showed nanomolar affinity for the purified a V b 3 -integrin receptor. For antiproliferative assays, the U87 human glioblastoma were chosen as a V b 3 -expressing cells, whereas a non a V b 3 -expressing clone (U87 b 3 -KO) was generated as negative control. Although the U87 b 3 -KO cells treated with the conjugates showed a statistically significant reduced fluorescence intensity (in the range 7-12 %) compared to the parental U87, internalization of the conjugates was clearly observed in both cell lines. Stability studies showed premature cleavage of the disulfide linker in the cell media, with consequent release of free camptothecin. Consistent with the results of the internalization and stability studies, the conjugates did not show significant selectivity against the U87 cells compared to the U87 b 3 -KO clone.

Hybrid cyclic α/β-peptides, in which one or more β-amino acids are incorporated into the backbone, are gaining increasing interest as potential therapeutics, thanks to their ability to achieve enhanced binding affinities for a biological target through pre-organization in solution. The in silico prediction of their three dimensional structure through strategies such as MD simulations would substantially advance the rational design process. However, whether the molecular mechanics force fields are accurate in sampling highly constrained cyclopeptides containing β-amino acids remains to be verified. Here, we present a systematic assessment of the ability of 8 widely used force fields to reproduce 79 NMR observables (including chemical shifts and 3J scalar couplings) on five cyclic α/β-peptides that contain the integrin recognition motif isoDGR. Most of the investigated force fields, which include force fields from AMBER, OPLS, CHARMM and GROMOS families, display very good agreement with experimental 3J(HN,Hα), suggesting that MD simulations could be an appropriate tool in the rational design of therapeutic cyclic α-peptides. However, for NMR observables directly related to β-amino acids, we observed a poor agreement with experiments and a remarkable dependence of our evaluation on the choice of Karplus parameters. The force field weaknesses herein unveiled might constitute a source of inspiration for further force field optimization.