Synthesis of Functionalized 6,5- and 7,5-Azabicycloalkane Amino Acids by Metathesis Reactions.

Azabicyclo[4.3.0]- and [5.3.0]alkanone amino acid derivatives were easily prepared by submitting the same starting dipeptide to a direct ring-closing enyne metathesis or an ethylene-mediated cross-enyne metathesis/ring-closing metathesis, respectively. The reactivity of the newly synthesized 6,5- and 7,5-fused bicyclic scaffolds was then investigated to obtain variously functionalized derivatives with potential applications in the field of peptides/peptidomimetics.

Synthesis of Variously Functionalized Azabicycloalkane Scaffolds by Domino Metathesis Reactions.

7,5-Fused azabicycloalkane scaffolds, carrying a quaternary stereocenter at C3 position of the lactam ring, can act as effective reverse-turn mimics and have proven to be useful intermediates for the preparation of Arg-Gly-Asp (RGD)-based cyclopentapeptides (cRGD) with nanomolar activity as αvß3/αvß5 integrin antagonists. Here, we report the synthesis of new azabicycloalkane scaffolds endowed at the C6 position with a para-substituted phenethyl side chain, which could be exploited to obtain cRGD-based bioconjugates that may find promising applications in anticancer therapy. By performing a domino cross enyne metathesis/ring-closing metathesis (CEYM/RCM) in the presence of styrene derivatives, followed by catalytic hydrogenation of the diene system, we easily converted a dipeptide precursor into the desired C6-functionalized azabicycloalkane scaffolds. The presence of a suitably protected p-amino group on the styrene moiety could be exploited, after deprotection, either to directly conjugate a bioactive compound or to introduce a suitable spacer between the cRGD unit and the bioactive compound.

Effect of tetracyclines on the dynamics of formation and destructuration of beta2-microglobulin amyloid fibrils.

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of ß2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits ß2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble ß2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

Stereoselective Pd-catalyzed synthesis of quaternary α-D-C-mannosyl-(S)-amino acids.

In this paper, we report the stereoselective synthesis of α-D-C-mannosyl-(S)-amino acids exploiting, as a key step, an allylic alkylation of glycal-derived π-allyl Pd(II) intermediates, prepared by oxidative addition of Pd(0) species to 2,3-unsaturated pyranosides (pseudoglycals). The reaction of 4,6-di-O-acetyl α-pseudoglucal carbonate 10a with racemic alanine-, valine-, and phenylalanine-derived azlactones gave the corresponding (4S)-4-α-D-C-mannosyl-2-phenyloxazol-5(4H)-ones as the major diastereoisomers in high yields. The final α-D-C-mannosyl-(S)-amino acids were obtained in a few steps comprising highly diastereoselective dihydroxylation of the glucal derivative double bond followed by the one-pot hydrolysis of the benzamido and acetate protecting groups. Main features of this method are the conciseness of the synthetic sequence, the high diastereoselection of the allylic alkylation step, the use of racemic α-amino acids as starting material, and the good overall yields.

An RGD small-molecule integrin antagonist induces detachment-mediated anoikis in glioma cancer stem cells.

The malignancy of glioblastoma (GB) is primarily due to the ability of glioma cancer stem cells (GSC) to disseminate into surrounding brain tissues, despite surgery and chemotherapy, and to form new tumoral masses. Members of the RGD-binding integrin family, which recognize the arginine-glycine-aspartic acid (RGD) sequence present in components of the extracellular matrix, and which serve a crucial function in the dissemination of GCS, are overexpressed in GB. Small-molecule integrin antagonists (SMIAs) designed to recognize RGD-integrins may therefore be an effective tool for decreasing GB infiltration and recurrence. In the present study, in vitro pro-apoptotic and infiltrative effects elicited by the SMIA 1a­RGD in human GSC were investigated. Reverse transcription-quantitative polymerase chain reaction analysis revealed that, compared with normal human astrocytes, GSC grown on laminin-coated dishes overexpressed stemness markers as well as αvß3 and αvß5 integrins. In addition, dissociated GSC were identified to exhibit tumorigenic capacity when injected into immunodeficient mice. Using annexin/fluorescence-activated cell sorting analysis and ELISA nucleosome assays, it was identified that treatment of GSC with 25 µM 1a­RGD for 48 h elicited detachment­dependent anoikis not accompanied by necrosis-dependent cell death. A colorimetric proliferation assay indicated that 1a­RGD did not affect cell viability, but that, instead, it markedly inhibited GSC migration as assessed using a Transwell assay. Western blot experiments revealed a decrease in focal adhesion kinase and protein kinase B phosphorylation with a concomitant increase in caspase-9 and -3/7 activity following 1a­RGD treatment, suggesting that the pro-anoikis effects of 1a­RGD may be mediated by these molecular mechanisms. Western blot analysis revealed no changes in specific markers of autophagy, suggesting further that 1a­RGD-induced cell death is primarily sustained by anoikis-associated mechanisms. In conclusion, the results of the present study indicate that SMIA have potential as a therapeutic tool for decreasing GSC dissemination.

A small-molecule RGD-integrin antagonist inhibits cell adhesion, cell migration and induces anoikis in glioblastoma cells.

In cancer cells integrins modulate important cellular events that regulate the metastasic cascade which involves detachment from the tumor mass, dissemination and attachment to the oncogenic niche. The α5ß1, αvß3 and αvß5 integrins are widely expressed in different cancer types and recognize the tripeptide Arg-Gly-Asp (RGD) motif present in several extracellular matrix proteins. In human glioblastoma, αvß3 integrin expression correlates with tumor grade, suggesting that this integrin may play a crucial role in the highly infiltrative behavior of high grade gliomas. However, few selective RGD-like antagonists have been developed and few studies have investigated their effects in in vitro models of human glioblastoma. In this study, we investigated several cellular effects and the underlying molecular mechanisms exerted by a new small-molecule RGD antagonist, 1a-RGD, in the U251 and U373 human glioblastoma cell lines. Treatment with 1a-RGD (20 µM) demonstrated a weak effect on cell viability and cell proliferation but strongly inhibited cell attachment and cell migration together with actin cytoskeleton disassembly. Prolonged 1a-RGD treatment (72 h) induced anoikis, assessed by Annexin staining and nucleosome assay, particularly in the detached cells. When integrin-linked transduction pathways were investigated, 1aRGD was found to exert a marked reduction in focal adhesion kinase (FAK) phosphorylation without affecting the AKT- and ERK-dependent pathways. Our data indicate that 1a-RGD, probably via modulation of the FAK-dependent pathway, inhibits cell migration and attachment and induces anoikis in glioblastoma cells. This novel finding suggests that the development of an RGD-like molecule may represent a promising tool for the pharmacological approach aimed at reducing the malignancy of glioblastoma cells.

Small molecule integrin antagonists in cancer therapy.

Integrins are a large family of dimeric receptors composed by alpha and beta subunits that, once bound to extra-cellular matrix (ECM) proteins, regulate a variety of cellular processes such as cell motility, migration, and proliferation. The integrins transduce signals from inside-out and outside-in the cell, thus representing the cellular link to the external environment. For these properties, integrin activation has been involved in pathological processes like tumor growth and metastasis formation. Recent advances in the elucidation of the crystallographic structures of the alphavbeta3 and alphaIIbeta3 integrins are promoting studies focused to the search of small molecule antagonists that can block the integrin binding to ECM and inhibit the biological effects exerted by these receptors. In this review we will focus on small molecule antagonists of alphavbeta3 and alphavbeta5 integrin as tools for cancer therapy while other integrins will only be briefly mentioned. Cilengitide (cyclic peptidic alphavbeta3 and alphavbeta5 antagonist) is currently in clinical trials for anti cancer therapy. Combination of integrin alphavbeta3 antagonists and other traditional therapeutic approaches may represent a future strategy to inhibit tumor growth and metastasis spreading.

A potent integrin antagonist from a small library of cyclic RGD pentapeptide mimics including benzyl-substituted azabicycloalkane amino acids.

A small library of cyclic RGD pentapeptide mimics, including benzyl-substituted azabicycloalkane amino acids, was synthesized with the aim of developing active and selective integrin antagonists. In vitro binding assays established one particular compound with affinity for both the alpha(v)beta(3) and the alpha(v)beta(5) integrins. The synthesis in solution and the in vitro screening of these RGD derivatives, as well as the determination of the conformational properties of the integrin ligands by spectroscopic and computational methods are described.

Bradykinin antagonists modified with dipeptide mimetic beta-turn inducers.

Bradykinin (BK) is involved in a wide variety of pathophysiological processes. Potent BK peptide antagonists can be developed introducing constrained unnatural amino acids, necessary to force the secondary structure of the molecule. In this paper, we report a structure-activity relationship study of two peptide analogues of the potent B2 antagonist HOE 140 by replacing the D-Tic-Oic dipeptide with conformationally constrained dipeptide mimetic beta-turn inducers.