Combination of multicomponent KA2 and Pauson-Khand reactions: short synthesis of spirocyclic pyrrolocyclopentenones.

The Cu-catalyzed multicomponent ketone-amine-alkyne (KA2) reaction was combined with a Pauson-Khand cycloaddition to give access of unprecedented constrained spirocyclic pyrrolocyclopentenone derivatives following a DOS couple-pair approach. The polyfunctional molecular scaffolds were tested on the cyclopentenone reactivity to further expand the skeletal diversity, demonstrating the utility of this combined approach in generating novel spiro compounds as starting material for the generation of chemical libraries. The chemoinformatics characterization of the newly-synthesized molecules gave evidence about structural and physicochemical properties with respect to a set of blockbuster drugs, and showed that such scaffolds are drug-like but more spherical and three-dimensional in character than the drugs.

Bicyclic acetals: biological relevance, scaffold analysis, and applications in diversity-oriented synthesis.

Natural products (NPs) have been shown to be an extraordinary source of bioactive compounds and three-dimensional complex frameworks that can be useful to produce high-value molecular collections that are able to address "undruggable" and difficult therapeutic targets. Bicyclic acetals are particularly relevant for these purposes, given their key role in several biological interactions and the structural and stereochemical diversity that comes from the many possible ring combinations. To investigate this topological diversity, we have systematically characterized in a systematic and detailed manner fused, spiro and bridged bicyclic acetals in a large set of NPs, highlighting the great potential of bicyclic acetals in Diversity-Oriented Synthesis (DOS). Accordingly, a summary of some recent efforts on the development of acetal-containing small molecule collections through DOS approaches is herein reported.

Identification of highly potent and selective MMP2 inhibitors addressing the S1' subsite with d-proline-based compounds.

MMP2 and MMP9, also called gelatinases, play a primary role in the angiogenic switch, as a fundamental step of tumor progression, and show high degree of structural similarity. Clinically successful gelatinase inhibitors need to be highly selective as opposite effects have been found for the two enzymes, and the S1' subsite is the major driver to attain selective and potent inhibitors. The synthesis of d-proline-derived hydroxamic acids containing diverse appendages at the amino group, varying in length and decoration allowed to give insight on the MMP2/MMP9 selectivity around the S1' subsite, resulting in the identification of sub-nanomolar compounds with high selectivity up to 730. Molecular docking studies revealed the existence of an additional hydrophobic channel at the bottom of S1' subsite for MMP2 enzyme useful to drive selectivity towards such gelatinase.

Triazole RGD antagonist reverts TGFß1-induced endothelial-to-mesenchymal transition in endothelial precursor cells.

Fibrosis is the dramatic consequence of a dysregulated reparative process in which activated fibroblasts (myofibroblasts) and Transforming Growth Factor ß1 (TGFß1) play a central role. When exposed to TGFß1, fibroblast and epithelial cells differentiate in myofibroblasts; in addition, endothelial cells may undergo endothelial-to-mesenchymal transition (EndoMT) and actively participate to the progression of fibrosis. Recently, the role of αv integrins, which recognize the Arg-Gly-Asp (RGD) tripeptide, in the release and signal transduction activation of TGFß1 became evident. In this study, we present a class of triazole-derived RGD antagonists that interact with αvß3 integrin. Above different compounds, the RGD-2 specifically interferes with integrin-dependent TGFß1 EndoMT in Endothelial Colony-Forming Cells (ECPCs) derived from circulating Endothelial Precursor Cells (ECPCs). The RGD-2 decreases the amount of membrane-associated TGFß1, and reduces both ALK5/TGFß1 type I receptor expression and Smad2 phosphorylation in ECPCs. We found that RGD-2 antagonist reverts EndoMT, reducing α-smooth muscle actin (α-SMA) and vimentin expression in differentiated ECPCs. Our results outline the critical role of integrin in fibrosis progression and account for the opportunity of using integrins as target for anti-fibrotic therapeutic treatment.

Short synthesis of polyfunctional sp3-rich threonine-derived morpholine scaffolds.

A convenient synthesis of novel complex morpholines was achieved by a two-step process involving a Petasis three-component coupling reaction of glycolaldehyde, organoboronic acid and different amines, followed by an acid- or base-mediated intramolecular cyclization. The use of threonine derivatives with glycolaldehyde in the Petasis reaction has been studied and successfully applied in the process, achieving morpholines with a higher fraction of sp3 carbon atoms compared to blockbuster drugs.

Design and synthesis of bicyclic acetals as Beta Secretase (BACE1) inhibitors.

Taking advantage of the structural similarity between aspartic proteases, small-molecule peptidomimetic inhibitors that already showed activity towards Secreted Aspartic Protease 2 as anti-Candida agents and HIV protease inhibitors were exploited as potential BACE1 inhibitors. A focused library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was synthesized and assayed towards BACE1 enzyme, resulting in the identification of a thiolactam-containing hit compound possessing IC50 in the low micromolar range, and confirming the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Insight to the binding mode of triazole RGD-peptidomimetics to integrin-rich cancer cells by NMR and molecular modeling.

The binding features of a novel class of 'click chemistry'-derived RGD mimics with integrin ligand capability were studied toward αvß3 integrin using STD-NMR techniques on intact integrin-rich ECV340 bladder cancer cell line. STD is useful to identify which moieties of the ligand are closest to the receptor in the bound state. The NMR data were integrated with competitive binding assays to the purified αvß3 receptor and were interpreted with the aid of docking calculations. The involvement of the triazole hydrogen atom in the interaction with the receptor was evinced for all compounds but 2, in agreement with docking studies showing a certain proximity between triazole and Tyr178. Moreover, the interaction of the hydroxylated ligands with the receptor was not as extended as in the compounds belonging to the corresponding series, with the exception of compound 4 having 2-aminobenzimidazole as the arginine bioisostere, in agreement with biological assay results showing reduced binding capability for the hydroxylated peptidomimetics.

Identification of Novel Human Breast Carcinoma (MDA-MB-231) Cell Growth Modulators from a Carbohydrate-Based Diversity Oriented Synthesis Library.

The application of a cell-based growth inhibition on a library of skeletally different glycomimetics allowed for the selection of a hexahydro-2H-furo[3,2-b][1,4]oxazine compound as candidate inhibitors of MDA-MB-231 cell growth. Subsequent synthesis of analogue compounds and preliminary biological studies validated the selection of a valuable hit compound with a novel polyhydroxylated structure for the modulation of the breast carcinoma cell cycle mechanism.

Skeletal diversity from carbohydrates: use of mannose for the diversity-oriented synthesis of polyhydroxylated compounds.

The application of d-mannose as a multipurpose building block from the chiral pool enabled the diversity-oriented synthesis of an array of cyclic and bicyclic scaffolds with polyhydroxylated appendages with the aim to expand the skeletal diversity in the panorama of glycopeptidomimetic compounds.

Two-step one-pot synthesis of dihydropyrazinones as Xaa-Ser dipeptide isosteres through morpholine acetal rearrangement.

The synthesis of the uncommon dihydropyrazinone ring was accomplished by a two-step one pot process taking advantage of the ring rearrangement of N-acylated morpholine acetal derived from serine under acidic treatment in the presence of 2,6-lutidine. The mechanism involves an N-acyl iminium intermediate resulting from morpholine acetal ring opening, which occurs after a nucleophilic attack of the amino acid nitrogen atom to the acetal carbonyl atom. X-Ray diffraction analysis of the dihydropyrazinone, which may be exploited as a constrained Xaa-Ser dipeptide isostere, showed a planar assembly and the internal side-chain in axial orientation with respect to the cyclic molecular scaffold.

Radiosynthesis and micro-SPECT analysis of triazole-based RGD integrin ligands as non-peptide molecular imaging probes for angiogenesis.

Taking advantage of click chemistry, we synthesized triazole-containing RGD peptidomimetics capable of binding to αvß3 integrin with diverse potency, and selected (125)I-labeled compounds proved to interact in vitro and in vivo with αvß3 integrin expressed by melanoma cells. Two (125)I-compounds containing either 2-aminobenzimidazole or 2-aminopyridine groups as the arginine bioisostere with the capacity to selectively bind cells of highly expressing αvß3 melanoma xenografts were found using micro-SPECT imaging studies.

Peptidomimetics as protein arginine deiminase 4 (PAD4) inhibitors.

The protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme, which catalyses the irreversible conversion of peptidyl-arginines into peptidyl-citrullines and plays an important role in several diseases such as in the rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Creutzfeldt-Jacob's disease and cancer. In this study, we report the inhibition profiles and computational docking toward the PAD4 enzyme of a series of 1,2,3-triazole peptidomimetic-based derivatives incorporating the ß-phenylalanine and guanidine scaffolds. Several effective, low micromolar PAD4 inhibitors are reported in this study.

One-pot sequential Ti-/Cu-catalysis for tandem amidation/Ullmann-type cyclization: synthesis of model benzodiazepine(di)ones promoted by microwave irradiation.

The application of sequential Ti-/Cu-catalysis in the model one-pot synthesis of benzodiazepine(di)ones promoted by microwave irradiation demonstrates the expediency of dual catalysis in coupling-cyclization methods useful for diversity-oriented synthesis.

Cyclic glycopeptidomimetics through a versatile sugar-based scaffold.

Cyclic peptidomimetics are attracting structures to obtain a distinct, bioactive conformation. Even more attractive are sugar-containing cyclic peptidomimetics which present turn structures induced by the pyranose ring when incorporated in cyclic peptides. The use of a new and versatile saccharidic scaffold to achieve sugar-based peptidomimetics is here reported together with the successful synthesis of diastereomerically pure cyclic SAA peptidomimetics 15 and 16.

Morpholine-based RGD-cyclopentapeptides as alphavbeta3/alphavbeta5 integrin ligands: role of configuration towards receptor binding affinity.

Two c[RGDfX] cyclopeptides, having either L- or D-morpholine-3-COOH (Mor) as the X amino acid were developed as ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins. Biological assays showed only d-Mor-containing cyclopentapeptide capable to bind alpha(v)beta(3) integrin with a low nanomolar affinity according to a two-site model, thus revealing a connection between the configuration of Mor and the preferred binding to alpha(v)beta(3) integrin. Conformational analysis showed different structural preferences for the two peptides induced by the two enantiomeric cyclic amino acids, suggesting a role of the stereochemistry of Mor on the overall peptide conformation and on the presentation of the pharmacophoric Arg and Asp side chains.

Discovery of subnanomolar arginine-glycine-aspartate-based alphaVbeta3/alphaVbeta5 integrin binders embedding 4-aminoproline residues.

The embodiment of 4-aminoproline residues (Amp) into the arginine-glycine-aspartate (RGD) sequence led to the discovery of a novel class of high-affinity alpha Vbeta 3/alpha Vbeta 5 integrin binders [IC 50 h (alpha Vbeta 3) 0.03-5.12 nM; IC 50 h (alpha Vbeta 5) 0.88-154 nM]. A total of eight cyclopeptides of type cyclo-[-Arg-Gly-Asp-Amp-], 5- 12, were assembled by a standard solid-phase peptide synthesis protocol that involved the C2-carboxyl and C4-amino functionalities of the proline scaffolds, leaving the N (alpha)-nuclear site untouched. Functionalization of this vacant proline site with either alkyl or acyl substituents proved feasible, with significant benefit to the integrin binding capabilities of the ligands. Notably, six out of eight cyclopeptide inhibitors, 5- 7 and 9- 11, showed moderate yet significant selectivity toward the alpha Vbeta 3 receptor. The three-dimensional structure in water was determined by NMR techniques and molecular dynamics calculations. Docking studies to the X-ray crystal structure of the extracellular segment of integrin alpha Vbeta 3 complexed with reference compound 1 were also performed on selected analogues to highlight the structural features required for potent ligand binding affinity.

Diversity-Oriented Synthesis and Chemoinformatic Analysis of the Molecular Diversity of sp3-Rich Morpholine Peptidomimetics.

Diversity-Oriented Synthesis (DOS) consists of generating structurally diverse compounds from a complexity-generating reaction followed by cyclization steps and appendage diversity. DOS has gathered interest to systematically explore the chemical space by generating high-quality small-molecule collections as probes to investigate biological pathways. The generation of heterocycles using amino acid and sugar derivatives as building blocks is a powerful approach to access chemical and geometrical diversity thanks to the high number of stereocenters and the polyfunctionality of such compounds. Our efforts in this field are focused on the generation of diversity-oriented molecules of peptidomimetic nature as a tool addressing protein-protein interactions, taking advantage of amino acid- and sugar-derived polyfunctional building blocks to be applied in couple-pair synthetic approaches. In this paper, the combination of diversity-oriented synthesis and chemoinformatics analysis of chemical space and molecular diversity of heterocyclic peptidomimetics are reported, with particular interest toward carbohydrate- and amino acid-derived morpholine scaffolds with a higher fraction of sp3 carbon atoms. Also, the chemoinformatic analysis of chemical space and molecular diversity of 186 morpholine peptidomimetics is outlined.

Synthesis, biological activity, and three-dimensional quantitative structure-activity relationship model for a series of benzo[c]quinolizin-3-ones, nonsteroidal inhibitors of human steroid 5alpha-reductase 1.

New 5alpha-reductase 1 (5alphaR-1) inhibitors were designed to complete a consistent set of analogues suitable for a 3D QSAR study. These compounds were synthesized by a modification of the aza-Robinson annulation, further functionalized by Pd-catalyzed cross-coupling processes, and were tested with human 5alphaR-1 expressed in Chinese hamster ovary 1827 cells. It turned out that the potency of the resulting inhibitors was strongly dependent on the type of substitution at the 8 position, with the IC(50) values ranging from 8.1 to 1050 nM. The construction of this homogeneous set of molecules allowed a 3D QSAR study. In particular, comparative molecular field analysis (CoMFA) was used to correlate the potency of the inhibitors with their physicochemical features. Highly accurate evaluations of the atomic point charges were carried out by means of quantum chemical calculations at the DFT/B3LYP level of theory followed by the RESP fitting procedure. It turned out that increasing the reliability of electrostatic parameters greatly affected the statistical results of the QSAR analysis. The 3D QSAR model proposed could be very useful in the further development of 5alphaR-1 inhibitors, which are suitable candidates to be evaluated as drugs in the treatment of 5alphaR-1 related diseases such as acne and alopecia in men and hirsutism in women.