Raffaele Colombo

Cyclic RGD-Peptidomimetics Containing Bifunctional Diketopiperazine Scaffolds as New Potent Integrin Ligands

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)β(3) and α(v)β(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cβ(Arg)-Cβ(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)β(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.

Synthesis and Biological Evaluation (in Vitro and in Vivo) of Cyclic Arginine–Glycine–Aspartate (RGD) Peptidomimetic–Paclitaxel Conjugates Targeting Integrin αVβ3

A small library of integrin ligand-paclitaxel conjugates 10-13 was synthesized with the aim of using the tumor-homing cyclo[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel-RGD constructs 10-13 inhibited biotinylated vitronectin binding to the purified αVβ3 integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin αVβ3, making them attractive to be tested in in vivo models. cyclo[DKP-f3-RGD]-PTX 11 displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound 11 exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used.

PhthalaPhos: Chiral Supramolecular Ligands for Enantioselective Rhodium‐Catalyzed Hydrogenation Reactions

Chemists have largely taken inspiration from Nature in the development of new approaches to synthetic challenges. Combinatorial chemistry stems from the concept of evolution, whereby random mutation of a chemical structure gives rise to libraries of compounds, from which an optimal lead can be found with high probability. On the other hand, Nature makes wide use of noncovalent interactions to build its complex supramolecular architectures and to achieve efficient and selective transformations. In recent years, combinatorial and supramolecular approaches to the development of new ligands for asymmetric catalysis has gained momentum. 2d] The term “supramolecular ligand” encompasses all ligands possessing, besides the atom(s) coordinating to the catalytic metal atom, an additional functionality capable of noncovalent interactions (mainly hydrogen or coordinative bonds) which can play the following roles: 1) self-assembly of two monodentate ligands to form a so-called supramolecular bidentate ligand; 2) binding the substrate(s) in proximity to the catalytic metal center in analogy to metalloenzymes. Among the different kinds of noncovalent interactions that have been used so far for developing supramolecular ligands, hydrogen bonds are arguably the most practical and efficient for several reasons: 1) functional groups capable of hydrogen bonding (e.g., amides, ureas, guanidines) are stable and relatively easy to introduce; 2) hydrogen bonds are created dynamically and reversibly in the reaction medium (where catalysis is to take place), are capable of self-repair when broken, and often coexist with other interactions in a “noninvasive” manner. As a result of our continued interest in developing supramolecular ligands, we report herein the design and synthesis of a novel class of chiral monodentate phosphite ligands, named PhthalaPhos, which contain a phthalic acid primary diamide moiety (Scheme 1). The phthalamidic group

General and Chemoselective N-Transacylation of Secondary Amides by Means of Perfluorinated Anhydrides†

Examples of direct N-transacylations of amides are very rare and lacking in general applicability. For instance, earlier attempts at N-transacylation were performed under harsh conditions. Other procedures required prolonged treatment with an equimolar mixture of trifluoroacetic acid (TFA) and trifluoroacetic anhydride (TFAA; 100 8C, 48 h), 3] or even running the reaction in TFAA followed by the addition of a strong base, reported as necessary to abstract the hydrogen atom in the alpha position to the eliminated acyl group. Finally, some acetanilides were treated with chloroacetyl chloride under acid catalysis of zeolites or AlCl3 at 83 8C for 16 h, or in refluxing pyridine containing dimethylaminopyridine for 5 h. Thus, N-transacylations are usually accomplished by hydrolysis of the acylamides and successive re-acylation of the formed amines, a procedure that does not allow the simultaneous presence, in the amide molecule, of functional groups labile to the basic or acidic conditions of the hydrolysis. 8] Herein, we report the first direct, general, and chemoselective procedure for the N-transacylation of secondary acylamides to their perfluorinated analogues, in high yields, with perfluorinated anhydrides. Remarkably, the perfluorinated amide formed could then be directly converted to a different amide by simple treatment with the desired acyl chloride, followed by a very mild aqueous process of the reaction mixture. Our work originated while studying sialic acid 1,7-lactone 1a. Surprisingly, on reacting the lactone 1 a with heptafluorobutyric anhydride (HFBAA) to volatilize it (135 8C for 5 min, in CH3CN), [10] we did not obtain the expected derivative 1b but the lactone 2b, which could be quantitatively transformed into lactone 2a by treatment of the reaction mixture with methanol at room temperature. Prompted by these initial results, we explored the scope of this new reaction (Table 1). Because of the particular utility of the reaction in carbohydrate chemistry, we started with some sialic acid and amido sugar derivatives of interest in organic synthesis and in biological studies. In particular, we were interested in testing molecules containing groups labile to the commonly used conditions of amide hydrolysis and reacylation. In effect, our reaction conditions allowed the successful N-transacylation of several compounds containing a great variety of functional groups (often within the same molecule), such as hydroxy groups, lactones, benzyloxycarbonyls (OCbz), methyl esters, acetates, tert-butyldimethylsilyl (TBDMS) groups, and acyclic and cyclic acetals as their 2-methoxyethoxymethyl (MEM), methyl, and benzylidene derivatives (Table 1, entries 1–18). Moreover, to test possible anomerizations resulting from the perfluorinated acid liberated in the reaction, we tested aand b-glycosidic compounds as well as a b-disaccharide. Finally, we selected carbohydrates with an equatorial or an axial acetamido group, to test the possible influence of the amide geometry. The study was first performed with HFBAA, then the reaction was repeated on some representative samples with TFAA, which gave comparable results (Table 1, entries 3, 13, 14, and 17). In all cases, except for entry 7, the reaction occurred in good yields, chemoselectively, and involving exclusively the amido group independently of its equatorial or axial geometry. Other functional groups present in the treated compounds were conserved, with the exception of free hydroxy, alcoholic, or acetalic groups, which, as expected, were perfluoroacylated (mass spectrometry (MS) and NMR evidence) under the reaction conditions employed. However, they could be easily regenerated by simple, short treatment of the crude reaction mixture with a solution of aqueous TFA in THF. Only acyclic acetals appeared to be labile under the reaction conditions, as observed for the MEM group (Table 1, entry 7). Remarkably, analysis of the H NMR spectra of compounds 4, 6, 8, 10, 12, 14, and 16 clearly showed in all cases that the reaction does not modify the configuration of the anomeric centers (see the Supporting Information). The anomeric geometry for the sialic acid derivative 8a, which lacks the anomeric proton, was established on the basis of the values of the heteronuclear vicinal coupling constant (JC1,H3ax and JC2,H3ax). [12, 13]

Chemoselective synthesis of sialic acid 1,7-lactones.

The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.

Elucidation of the opening of the epoxidic ring of the 3β-acetoxy-14α,15α-epoxy-5α-cholest-8-en-7-one by methanol, using NMR techniques assisted by a conformational study through theoretical calculations

This paper demonstrates that the crystallization of 3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-cholest-8-en-7-one from methanol affords the 3beta-acetoxy-9alpha-methoxy-15alpha-hydroxycholest-8(14)-en-7-one. The structure of this steroid, which shows an apparently anomalous UV absorption maximum, is determined by high field NMR experiments, supporting the coupling constant values assignments and the NOE contacts by a conformational study through theoretical calculations at the B3LYP/6-31G* level. The computational study also justifies the observed UV absorption of the steroid, thus demonstrating the usefulness of computer chemistry in providing support for the identification of unknown compounds.

Correction to “Chemoselective Synthesis of Sialic Acid 1,7-Lactones”

A new general protocol has been prepared to address issues relating to the required excess of benzyloxycarbonyl chloride (CbzCl) due to its low concentration in the commercial reagent used for the synthesis of 2-methyl-Nacetyl-β-neuraminic acid 1,7-lactone 8, 2-benzyloxycarbonyl-Nacetyl-β-neuraminic acid 1,7-lactone 10, 2-benzyloxycarbonylN-benzyloxyacetylneuraminic acid 1,7 lactone 14, 2-benzyloxycarbonyl-N-glycolyl-β-neuraminic acid 1,7-lactone 15, and 2benzyloxycarbonyl-3-deoxy-D-glycero-D-galacto-2-nononic acid 1,7-lactone 16. The general unified procedure uses a CbzCl ratio (2.5 molar equiv) reduced in respect to that reported (9.6 mol equiv).