Fast Solution-Phase and Liquid-Phase Peptide Syntheses (SolPSS and LPPS) Mediated by Biomimetic Cyclic Propylphosphonic Anhydride (T3P®).

The growing applications of peptide-based therapeutics require the development of efficient protocols from the perspective of an industrial scale-up. T3P® (cyclic propylphosphonic anhydride) promotes amidation in the solution-phase through a biomimetic approach, similar to the activation of carboxylic moiety catalyzed by ATP-grasp enzymes in metabolic pathways. The T3P® induced coupling reaction was applied in this study to the solution-phase peptide synthesis (SolPPS). Peptide bond formation occurred in a few minutes with high efficiency and no epimerization, generating water-soluble by-products, both using N-Boc or N-Fmoc amino acids. The optimized protocol, which was successfully applied to the iterative synthesis of a pentapeptide, also allowed for a decrease in the solvent volume, thus improving process sustainability. The protocol was finally extended to the liquid-phase peptide synthesis (LPPS), where the isolation of the peptide was performed using precipitation, thus also showing the suitability of this coupling reagent to this emerging technique.

Application of Af4-Multidetection to Liraglutide in Its Formulation: Preserving and Representing Native Aggregation.

Aggregation is among the most critical parameters affecting the pharmacological and safety profile of peptide Active Pharmaceutical Ingredients (APIs). For this reason, it is of utmost importance to define the exact aggregation state of peptide drugs, particularly when the API is marketed as a ready-to-use solution. Consequently, appropriate non-destructive techniques able to replicate the peptide environment must be employed. In our work, we exploited Asymmetrical Flow Field-Flow Fractionation (AF4), connected to UV, dRI, fluorescence, and MALS detectors, to fully characterize the aggregation state of Liraglutide, a peptide API used for the treatment of diabetes type 2 and chronic obesity. In previous studies, Liraglutide was hypothesized to assemble into hexa-octamers in phosphate buffer, but no information on its behavior in the formulation medium was provided up to now. The method used allowed researchers to work using formulation as the mobile phase with excellent recoveries and LoQ/LoD, discerning between stable and degraded samples, and detecting, when present, aggregates up to 108 Da. The native state of Liraglutide was assessed and found to be an association into pentamers, with a non-spherical conformation. Combined to benchmark analyses, the sameness study was complete and descriptive, also giving insight on the aggregation process and covalent/non-covalent aggregate types.

Stable and Biocompatible Monodispersion of C60 in Water by Peptides.

The lack of solubility in water and the formation of aggregates hamper many opportunities for technological exploitation of C60. Here, different peptides were designed and synthesized with the aim of monomolecular dispersion of C60 in water. Phenylalanines were used as recognizing moieties, able to interact with C60 through π-π stacking, while a varying number of glycines were used as spacers, to connect the two terminal phenylalanines. The best performance in the dispersion of C60 was obtained with the FGGGF peptidic nanotweezer at a pH of 12. A full characterization of this adduct was carried out. The peptides disperse C60 in water with high efficiency, and the solutions are stable for months both in pure water and in physiological environments. NMR measurements demonstrated the ability of the peptides to interact with C60. AFM measurements showed that C60 is monodispersed. Electrospray ionization mass spectrometry determined a stoichiometry of C60@(FGGGF)4. Molecular dynamics simulations showed that the peptides assemble around the C60 cage, like a candy in its paper wrapper, creating a supramolecular host able to accept C60 in the cavity. The peptide-wrapped C60 is fully biocompatible and the C60 "dark toxicity" is eliminated. C60@(FGGGF)4 shows visible light-induced reactive oxygen species (ROS) generation at physiological saline concentrations and reduction of the HeLa cell viability in response to visible light irradiation.

Synthesis of α/ß dipeptides containing linear or cyclic α-dehydro-ß-amino acids as scaffolds for bioactive compounds.

The synthesis of α/ß dipeptides containing linear or cyclic α-dehydro-ß-amino acids has been performed starting from alkylidene acetacetamides, which were obtained from α-amino esters via Ir-catalyzed allylic amination. Differently hindered carbonates were synthesized via a protocol involving chemoselective Luche's reduction, acylation, and allylic amination. Depending on the nature of the selected α-amino acid, we observed strong influence on the product regiochemistry due to the carbonate size and the amino-acid side chain. In particular, complete regioselectivity was observed in the aminic allylation of carbonates deriving from amino acids possessing a methylene unit in ß-position. On the contrary, methyl carbonates deriving from ß-branched amino acid afforded different results depending on the hindrance of the carbonate. Moreover, spontaneous cyclization was observed for carbamate-containing intermediates, allowing to obtain peptidomimetic polyfunctionalized dihydropyrimidine-2,4-dione. Finally, by inverting the order of reduction/acylation steps on the starting alkylidene acetoacetamides, the formation of polyfunctionalized 1,3-oxazinane-2,4-dione was obtained demonstrating the wide applications of these substrates for the preparation of bioactive peptidomimetics.

Integrin Ligands with α/ß-Hybrid Peptide Structure: Design, Bioactivity, and Conformational Aspects.

Integrins are cell surface receptors for proteins of the extracellular matrix and plasma-borne adhesive proteins. Their involvement in diverse pathologies prompted medicinal chemists to develop small-molecule antagonists, and very often such molecules are peptidomimetics designed on the basis of the short native ligand-integrin recognition motifs. This review deals with peptidomimetic integrin ligands composed of α- and ß-amino acids. The roles exerted by the ß-amino acid components are discussed in terms of biological activity, bioavailability, and selectivity. Special attention is paid to the synthetic accessibility and efficiency of conformationally constrained heterocyclic scaffolds incorporating α/ß-amino acid span.

Highly stable atropisomers by electrophilic amination of a chiral γ-lactam within the synthesis of an elusive conformationally restricted analogue of α-methylhomoserine.

Starting from chiral-protected 4-hydroxymethyl pyrrolidin-2-ones, the otherwise elusive 3,4-trans-3,3,4-trisubstituted isosteres of α-methyl homoserine, tethered on a γ-lactam ring, were prepared exploiting stereoselective electrophilic aminations. These reactions led to the isolation and characterization of a novel type of atropisomers, exceedingly stable at room temperature, that were directly converted to the desired products by a novel non-reductive N-N bond cleavage reaction.

Conjecturing about Small-Molecule Agonists and Antagonists of α4ß1 Integrin: From Mechanistic Insight to Potential Therapeutic Applications.

Integrins are heterodimeric cell-surface receptors that regulate cell-cell adhesion and cellular functions through bidirectional signaling. On the other hand, anomalous trafficking of integrins is also implicated in severe pathologies as cancer, thrombosis, inflammation, allergies, and multiple sclerosis. For this reason, they are attractive candidates as drug targets. However, despite promising preclinical data, several anti-integrin drugs failed in late-stage clinical trials for chronic indications, with paradoxical side effects. One possible reason is that, at low concentration, ligands proposed as antagonists may also act as partial agonists. Hence, the comprehension of the specific structural features for ligands' agonism or antagonism is currently of the utmost interest. For α4ß1 integrin, the situation is particularly obscure because neither the crystallographic nor the cryo-EM structures are known. In addition, very few potent and selective agonists are available for investigating the mechanism at the basis of the receptor activation. In this account, we discuss the physiological role of α4ß1 integrin and the related pathologies, and review the few agonists. Finally, we speculate on plausible models to explain agonism vs. antagonism by comparison with RGD-binding integrins and by analysis of computational simulations performed with homology or hybrid receptor structures.

In-peptide synthesis of di-oxazolidinone and dehydroamino acid-oxazolidinone motifs as ß-turn inducers.

Small and easy-to-do mimetics of ß-turns are of great interest to interfere with protein-protein recognition events mediated by ß-turn recognition motifs. We propose a straightforward procedure for constraining the conformation of tetrapeptides lacking a pre-formed scaffold. According to the stereochemistry array, N-Ts tetrapeptides including Thr or PhSer (phenylserine) at the positions 2 or 3 gave rise in a single step to the sequences Oxd(2)-Oxd(3) or ΔAbu(2)-Oxd(3) (Oxd, oxazolidin-2-one; ΔAbu, 2,3-dehydro-2-aminobutyric). These pseudo-Pro residues displayed highly constrained ϕ, ψ, and χ dihedral angles, and induced clear ß-turns or inverse turns of type I or II, as determined by extensive spectroscopic and computational analyses.

Investigation of the GnRH antagonist degarelix isomerization in biological matrices.

One of the main objectives of peptide drug design is the improvement of peptide pharmacokinetics with maintaining biological activity, which can be achieved by the complex modifications of the primary structure of the peptides. However, these changes often lead to the formation of peculiar impurities in the peptide drugs and their metabolites, which require the development of advanced analytical methods to properly assess their content. Here, we investigated the degradation of the potent long-acting GnRH antagonist degarelix in various biologic media by the tailor-made HPLC method, which allows precise determination of 5-Aph(Hyd)-degarelix isomer, an impurity found in the degarelix active pharmaceutical ingredient (API) during its manufacturing. Unexpectedly, we discovered a rapid and irreversible conversion of degarelix API into the corresponding hydantoin isomer in serum, suggesting that this impurity can be also a potential drug metabolite in vivo. This finding underlines the importance of the development of more accurate and performing analytical techniques to correctly characterize the chemical composition of the manufactured drugs and their behavior under physiological conditions.

Expedient synthesis of pseudo-Pro-containing peptides: towards constrained peptidomimetics and foldamers.

The reaction of sulfonyl peptides containing L- or D-configured Ser or Thr with bis(succinimidyl) carbonate in the presence of a catalytic amount of a base affords, in solution or in the solid phase, the corresponding peptides with one or two, consecutive or alternate oxazolidin-2-ones (Oxd). The Oxd ring can be regarded to as a pseudo-Pro with an exclusively trans conformation of the preceding peptide bond; homochiral Oxd-containing peptides adopt extended conformations, while the presence of a D-configured Oxd favours folded conformations.

Fast MacMillan's Imidazolidinone-Catalyzed Enantioselective Synthesis of Polyfunctionalized 4-Isoxazoline Scaffolds.

The enantioselective 1,3-dipolar cycloaddition of nitrones and arylpropionaldehydes to generate highly functionalized scaffolds for application in drug discovery was herein investigated. The use of a second-generation MacMillan catalyst as hydrochloride salt consistently accelerated the reaction speed, allowing a decrease in the reaction time up to >100 times, still affording 4-isoxazolines with good to excellent enantiomeric ratios at room temperature. As a proof of concept, further functionalization of the isoxazoline core through Pd-catalyzed cross-coupling was performed, generating differently functionalized chemical architectures in high yield.

A straightforward route to enantiopure 2-substituted-3,4-dehydro-ß-proline via ring closing metathesis.

The synthesis of unusual cyclic amino acids, that may be envisaged as proline analogs, is an area of great interest for their potential applications as scaffolds for the design of bioactive peptidomimetics or units for the creation of novel foldamers. We have carried out the preparation of cyclic dehydro-ß-amino acids starting from allylic carbonates via a two-step allylic amination/ring closing metathesis (RCM) protocol. The introduction of the allylamino moiety has been carried out either without a catalyst, through an S(N)2' reaction, or in the presence of iridium complexes. The backbone of the allylamino intermediates contains two unsaturations, thus suggesting that RCM could be a valuable tool for the preparation of dihydropyrrole scaffolds. A similar reaction has been already reported in the literature for racemic aromatic-substituted substrates, but no examples of enantiopure derivatives bearing aliphatic chains have been reported. The reaction was optimized by testing different Grubbs' catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-ß-amino acids as central core of peptidomimetics, the malonate chain was also used to protect nitrogen prior to RCM.

Therapeutic Peptides Targeting PPI in Clinical Development: Overview, Mechanism of Action and Perspectives.

Targeting protein-protein interactions (PPIs) has been recently recognized as an emerging therapeutic approach for several diseases. Up today, more than half a million PPI dysregulations have been found to be involved in pathological events. The dynamic nature of these processes and the involvement of large protein surfaces discouraged anyway the scientific community in considering them promising therapeutic targets. More recently peptide drugs received renewed attention since drug discovery has offered a broad range of structural diverse sequences, moving from traditionally endogenous peptides to sequences possessing improved pharmaceutical profiles. About 70 peptides are currently on the marked but several others are in clinical development. In this review we want to report the update on these novel APIs, focusing our attention on the molecules in clinical development, representing the direct consequence of the drug discovery process of the last 10 years. The comprehensive collection will be classified in function of the structural characteristics (native, analogous, heterologous) and on the basis of the therapeutic targets. The mechanism of interference on PPI will also be reported to offer useful information for novel peptide design.

Palladium Catalyst Recycling for Heck-Cassar-Sonogashira Cross-Coupling Reactions in Green Solvent/Base Blend.

The identification of a green, versatile, user-friendly, and efficient methodology is necessary to facilitate the use of Heck-Cassar-Sonogashira (HCS) cross-coupling reaction in drug discovery and industrial production in the pharmaceutical segment. The Heck-Cassar and Sonogashira protocols, using N-hydroxyethylpyrrolidone (HEP)/water/N,N,N',N'-tetramethyl guanidine (TMG) as green solvent/base mixture and sulfonated phosphine ligands, allowed to recycle the catalyst, always guaranteeing high yields and fast conversion under mild conditions, with aryl iodides, bromides, and triflates. No catalyst leakage or metal contamination of the final product were observed during the HCS recycling. To our knowledge, a turnover number (TON) up to 2375, a turnover frequency (TOF) of 158 h-1 , and a process mass intensity (PMI) around 7 that decreased around 3 after solvent, base, and palladium recovery, represent one of the best results to date using a sustainable protocol. The Heck-Cassar protocol using sSPhos was successfully applied to the telescoped synthesis of Erlotinib (TON: 1380; TOF: 46 h-1 ).

Ampicillin sodium: Isolation, identification and synthesis of the last unknown impurity after 60 years of clinical use.

Ampicillin, discovered in 1958, was the first broad spectrum semisynthetic penicillin introduced into the market. Despite its wide use not all the impurities have been identified to date. Herein, the last unknown impurity present in commercially available medicines was isolated and identified. This impurity that accounts up to 0.8 in area % by HPLC (EP 10.0) in the Reference Listed Drugs (RLD) was characterized and identified to be the 16-keto penicillin G. The structure was confirmed by comparison with a chemically synthesized sample. The determination of the Relative Response Factor (RRF) of the impurity respect to the parent drug allowed to recalculate the real amount that is consistently below the reporting threshold.

Integrin-mediated adhesive properties of neutrophils are reduced by hyperbaric oxygen therapy in patients with chronic non-healing wound.

In recent years, several studies suggested that the ability of hyperbaric oxygen therapy (HBOT) to promote healing in patients with diabetic ulcers and chronic wounds is due to the reduction of inflammatory cytokines and to a significant decrease in neutrophils recruitment to the damaged area. α4 and ß2 integrins are receptors mediating the neutrophil adhesion to the endothelium and the comprehension of the effects of hyperbaric oxygenation on their expression and functions in neutrophils could be of great importance for the design of novel therapeutic protocols focused on anti-inflammatory agents. In this study, the α4 and ß2 integrins' expression and functions have been evaluated in human primary neutrophils obtained from patients with chronic non-healing wounds and undergoing a prolonged HBOT (150 kPa per 90 minutes). The effect of a peptidomimetic α4ß1 integrin antagonist has been also analyzed under these conditions. A statistically significant decrease (68%) in ß2 integrin expression on neutrophils was observed during the treatment with HBO and maintained one month after the last treatment, while α4 integrin levels remained unchanged. However, cell adhesion function of both neutrophilic integrins α4ß1 and ß2 was significantly reduced 70 and 67%, respectively), but α4ß1 integrin was still sensitive to antagonist inhibition in the presence of fibronectin, suggesting that a combined therapy between HBOT and integrin antagonists could have greater antinflammatory efficacy.

Side chain effect in the modulation of αvß3/α5ß1 integrin activity via clickable isoxazoline-RGD-mimetics: development of molecular delivery systems.

Construction of small molecule ligand (SML) based delivery systems has been performed starting from a polyfunctionalized isoxazoline scaffold, whose αvß3 and α5ß1 integrins' potency has been already established. The synthesis of this novel class of ligands was obtained by conjugation of linkers to the heterocyclic core via Huisgen-click reaction, with the aim to use them as "shuttles" for selective delivery of diagnostic agents to cancer cells, exploring the effects of the side chains in the interaction with the target. Compounds 17b and 24 showed excellent potency towards α5ß1 integrin acting as selective antagonist and agonist respectively. Further investigations confirmed their effects on target receptor through the analysis of fibronectin-induced ERK1/2 phosphorylation. In addition, confocal microscopy analysis allowed us to follow the fate of EGFP conjugated α5ß1 integrin and 17b FITC-conjugated (compound 31) inside the cells. Moreover, the stability in water solution at different values of pH and in bovine serum confirmed the possible exploitation of these peptidomimetic molecules for pharmaceutical application.

Novel Ligands Targeting α4ß1 Integrin: Therapeutic Applications and Perspectives.

Among the other members of the adhesion molecules' family, α4ß1 integrin, a heterodimeric receptor, plays a crucial role in inflammatory diseases, cancer development, metastasis and stem cell mobilization or retention. In many cases, its function in pathogenesis is not yet completely understood and investigations on ligand binding and related stabilization of active/inactive conformations still represent an important goal. For this reason, starting from the highlight of α4ß1 functions in human pathologies, we report an overview of synthetic α4ß1 integrin ligands under development as potential therapeutic agents. The small molecule library that we have selected represents a collection of lead compounds. These molecules are the object of future refinement in academic and industrial research, in order to achieve a fine tuning of α4ß1 integrin regulation for the development of novel agents against pathologies still eluding an effective solution.

Novel insights into the chemistry of an old medicine: A general degradative pathway for penicillins from a piperacillin/tazobactam stability study.

Piperacillin is a broad spectrum beta-lactam antibiotic used in combination with tazobactam for hospital-related bacterial infections. The reconstituted solutions must respect the sub-visible and visible particles specifications. It was claimed that the reformulation containing EDTA/sodium citrate was able to control the formation of an insoluble impurity responsible for the formation of particulate matter observed using Ringer Lactate as diluent. The nature of the impurities formed during the degradative process of piperacillin/tazobactam combination has been herein investigated, by exploring the effect of added excipients and pH variations. The exact structure of the isolated dimeric impurity, the penicilloic acid-piperacillin dimer, was determined through complete characterization, allowing to propose a novel degradative general pathway for beta-lactam antibiotics. The presence of EDTA resulted unnecessary to contain the formation of the insoluble impurity, since the use of sodium citrate alone allowed to avoid this drawback. Finally, the proposed mechanism was successfully applied to the design of a novel, easy and high purity procedure for the synthesis of the acetylated penicilloic acid, known related substance of piperacillin.

Investigation of the interaction between the atypical agonist c[YpwFG] and MOR.

Endogenous and exogenous opiates are currently considered the drugs of choice for treating different kinds of pain. However, their prolonged use produces several adverse symptoms, and in addition, many forms of pain are resistant to any kind of therapy. Therefore, the discovery of compounds active towards mu-opioid receptors (MORs) by alternative pharmacological mechanisms could be of value for developing novel classes of analgesics. There is evidence that some unusual molecules can bind opioid receptors, albeit lacking some of the typical opioid pharmacophoric features. In particular, the recent discovery of a few compounds that showed agonist behavior even in the absence of the primary pharmacophore, namely a protonable amine, led to a rediscussion of the importance of ionic interactions in stabilizing the ligand-receptor complex and in activating signal transduction. Very recently, we synthesized a library of cyclic analogs of the endogenous, MOR-selective agonist endomorphin-1 (YPWF-NH(2)), containing a Gly5 bridge between Tyr1 and Phe4. The cyclopeptide c[YpwFG] showed good affinity and agonist behavior. This atypical MOR agonist does not have the protonable Tyr amine. In order to gain more information about plausible mechanisms of interaction between c[YpwFG] and the opioid receptor, we synthesized a selected set of derivatives containing different bridges between Tyr1 and Phe4, and tested their affinities towards mu-opioid receptors. We performed conformational analysis of the cyclopeptides by NMR spectroscopy and molecular dynamics, and investigated plausible, unprecedented modes of interaction with the MOR by molecular docking. The successive quantum mechanics/molecular mechanics investigation of the complexes obtained by the molecular docking procedure furnished a more detailed description of the binding mode and the electronic properties of the ligands. The comparison with the binding mode of the potent agonist JOM-6 seems to indicate that the cyclic endomorphin-1 analogs interact with the receptor by way of an alternative mechanism, still maintaining the ability to activate the receptor.