N-Sulfenylation of ß-Lactams: Radical Reaction of N-Bromo-azetidinones by TEMPO Catalysis.

Azetidinones with a sulfenyl group on the ß-lactam nitrogen atom show interesting biological activities as antimicrobial agents and enzyme inhibitors. We report in the present study a versatile synthesis of N-sulfenylated azetidinones starting from the corresponding N-bromo derivatives by means of the (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) radical as the catalyst and disulfides. Preparation of N-halo-azetidinones was studied and optimized. The reactivity of N-bromo-azetidinone 2a as a model compound in the presence of TEMPO radical was investigated by NMR and electron paramagnetic resonance (EPR) spectroscopy studies. Optimization of the reaction conditions allowed the access of N-alkylthio- or N-arylthio-azetidinones from 55 to 92% yields, and the method exhibited a good substrate scope.

Chemoenzymatic enantioselective route to get (+) and (-) 4-acetoxy-azetidin-2-one by lipase-catalysed kinetic resolution and their applications.

4-Acetoxy-azetidin-2-one is an extremely useful intermediate widely applied for the synthesis of several biologically active ß-lactam compounds. However, it is available as a racemic mixture that could limit its application in the synthesis of enantiopure products. Herein we evaluated the use of lipases in a kinetic resolution (KR) process to finally obtain 4-acetoxy-zetidin-2-one as separated pure enantiomers. From a preliminary screening on a set of commercial enzymes, Pseudomonas fluorescens emerged as the most suitable lipase that allowed to obtain good conversions and excellent enantiomeric excesses. On the enantiomerically pure 4-acetoxy-azetidin-2-ones some nucleophilic substitutions and N-thio-alkylation reactions were tested in order to evaluate the stereochemical integrity at the C-4 position.

Combining Biologically Active ß-Lactams Integrin Agonists with Poly(l-lactic acid) Nanofibers: Enhancement of Human Mesenchymal Stem Cell Adhesion.

Regulating stem cell adhesion and growth onto functionalized biomaterial scaffolds is an important issue in the field of tissue engineering and regenerative medicine. In this study, new electrospun scaffolds of poly(l-lactic acid) (PLLA), as bioresorbable polymer, and ß-lactam compounds agonists of selected integrins, as functional components with cell adhesive properties, are designed. The new ß-lactam-PLLA scaffolds contribute significantly in guiding protein translation involved in human bone marrow mesenchymal stem cells (hBM-MSC) adhesion and integrin gene expression. Scanning electron microscopy, confocal laser scanning microscopy, and Western Blot analyses reveal that GM18-PLLA shows the best results, promoting cell adhesion by significantly driving changes in focal adhesion proteins distribution (ß1 integrin and vinculin) and activation (pFAK), with a notable increase of GM18-targets subunits integrin gene expression, α4 and ß1. These novel functionalized submicrometric fibrous scaffolds demonstrate, for the first time, the powerful combination of selective ß-lactams agonists of integrins with biomimetic scaffolds, suggesting a designed rule that could be suitably applied to tissue repair and regeneration.

Chiral ß-lactam-based integrin ligands through Lipase-catalysed kinetic resolution and their enantioselective receptor response.

Obtainment and testing of pure enantiomers are of great importance for bioactive compounds, because of the assessed implications of enantioselectivity in receptor-mediated responses. Herein we evaluated the use of biocatalysis to obtain enantiomerically pure ß-lactam intermediates further exploited in the synthesis of novel integrin ligands as single enantiomers. From a preliminary screening on a set of commercially available hydrolases, Burkholderia Cepacia Lipase (BCL) emerged as a suitable and highly performing enzyme for the kinetic resolution of a racemic azetidinone, key intermediate for the synthesis of novel agonists of integrins. Upon optimization of the biocatalytic protocol in terms of enzymes, acylating agents and procedures, the two ß-lactam enantiomers were obtained in excellent enantiomeric excesses (94% and 98% ee). Synthetic elaborations on the separated enantiomers allowed the synthesis of four chiral ß-lactams which were evaluated in cell adhesion assays on Jurkat cell line expressing α4ß1 integrin, and K562 cell line expressing α5ß1 integrin. Biological tests revealed that only (S)-enantiomers maintained the agonist activity of racemates with a nanomolar potency, and a specific enantio-recognition by integrin receptors was demonstrated.

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.

Poly(l-lactic acid) Scaffold Releasing an α4ß1 Integrin Agonist Promotes Nonfibrotic Skin Wound Healing in Diabetic Mice.

Skin wound healing is a highly complex process that continues to represent a major medical problem, due to chronic nonhealing wounds in several classes of patients and to possible fibrotic complications, which compromise the function of the dermis. Integrins are transmembrane receptors that play key roles in this process and that offer a recognized druggable target. Our group recently synthesized GM18, a specific agonist for α4ß1, an integrin that plays a role in skin immunity and in the migration of neutrophils, also regulating the differentiated state of fibroblasts. GM18 can be combined with poly(l-lactic acid) (PLLA) nanofibers to provide a controlled release of this agonist, resulting in a medication particularly suitable for skin wounds. In this study, we first optimized a GM18-PLLA nanofiber combination with a 7-day sustained release for use as skin wound medication. When tested in an experimental pressure ulcer in diabetic mice, a model for chronic nonhealing wounds, both soluble and GM18-PLLA formulations accelerated wound healing, as well as regulated extracellular matrix synthesis toward a nonfibrotic molecular signature. In vitro experiments using the adhesion test showed fibroblasts to be a principal GM18 cellular target, which we then used as an in vitro model to explore possible mechanisms of GM18 action. Our results suggest that the observed antifibrotic behavior of GM18 may exert a dual action on fibroblasts at the α4ß1 binding site and that GM18 may prevent profibrotic EDA-fibronectin-α4ß1 binding and activate outside-in signaling of the ERK1/2 pathways, a critical component of the wound healing process.

Laccase-Carrying Polylactic Acid Electrospun Fibers, Advantages and Limitations in Bio-Oxidation of Amines and Alcohols.

Laccases are oxidative enzymes that could be good candidates for the functionalization of biopolymers with several applications as biosensors for the determination of bioactive amine and alcohols, for bioremediation of industrial wastewater, and for greener catalysts in oxidation reactions in organic synthesis, especially used for non-phenolic compounds in combination with redox mediators in the so-called Laccase Mediator System (LMS). In this work, we describe the immobilization of Laccase from Trametes versicolor (LTv) in poly-L-lactic acid (PLLA) nanofibers and its application in LMS oxidation reactions. The PLLA-LTv catalysts were successfully produced by electrospinning of a water-in-oil emulsion with an optimized method. Different enzyme loadings (1.6, 3.2, and 5.1% w/w) were explored, and the obtained mats were thoroughly characterized. The actual amount of the enzyme in the fibers and the eventual enzyme leaching in different solvents were evaluated. Finally, the PLLA-LTv mats were successfully applied as such in the oxidation reaction of catechol, and in the LMS method with TEMPO as mediator in the oxidation of amines with the advantage of easier work-up procedures by the immobilized enzyme. However, the PLLA-LTv failed the oxidation of alcohols with respect to the free enzyme. A tentative explanation was provided.

Peptide Mediated Adhesion to Beta-Lactam Ring of Equine Mesenchymal Stem Cells: A Pilot Study.

Regenerative medicine applied to skin lesions is a field in constant improvement. The use of biomaterials with integrin agonists could promote cell adhesion increasing tissue repair processes. The aim of this pilot study was to analyze the effect of an α4ß1 integrin agonist on cell adhesion of equine adipose tissue (AT) and Wharton's jelly (WJ) derived MSCs and to investigate their adhesion ability to GM18 incorporated poly L-lactic acid (PLLA) scaffolds. Adhesion assays were performed after culturing AT- and WJ-MSCs with GM18 coating or soluble GM18. Cell adhesion on GM18 containing PLLA scaffolds after 20 min co-incubation was assessed by HCS. Soluble GM18 affects the adhesion of equine AT- and WJ-MSCs, even if its effect is variable between donors. Adhesion to PLLA scaffolds containing GM18 is not significantly influenced by GM18 for AT-MSCs after 20 min or 24 h of culture and for WJ-MSCs after 20 min, but increased cell adhesion by 15% GM18 after 24 h. In conclusion, the α4ß1 integrin agonist GM18 affects equine AT- and WJ-MSCs adhesion ability with a donor-related variability. These preliminary results represent a first step in the study of equine MSCs adhesion to PLLA scaffolds containing GM18, suggesting that WJ-MSCs might be more suitable than AT-MSCs. However, the results need to be confirmed by increasing the number of samples before drawing definite conclusions.

Molecular Delivery of Cytotoxic Agents via Integrin Activation.

Integrins are cell adhesion receptors overexpressed in tumor cells. A direct inhibition of integrins was investigated, but the best inhibitors performed poorly in clinical trials. A gained attention towards these receptors arouse because they could be target for a selective transport of cytotoxic agents. Several active-targeting systems have been developed to use integrins as a selective cell entrance for some antitumor agents. The aim of this review paper is to report on the most recent results on covalent conjugates between integrin ligands and antitumor drugs. Cytotoxic drugs thus conjugated through specific linker to integrin ligands, mainly RGD peptides, demonstrated that the covalent conjugates were more selective against tumor cells and hopefully with fewer side effects than the free drugs.

Selective Integrin Ligands Promote Cell Internalization of the Antineoplastic Agent Fluorouracil.

Drug conjugates consisting of an antineoplastic drug and a targeting receptor ligand could be effective to overcome the heavy side effects of unselective anticancer agents. To address this need, we report here the results of a project aimed to study agonist and antagonist integrin ligands as targeting head of molecular cargoes for the selective delivery of 5-fluorouracil (5-FU) to cancer or noncancer cells. Initially, two fluorescent ß-lactam-based integrin ligands were synthesized and tested for an effective and selective internalization mediated by α4ß1 or α5ß1 integrins in Jurkat and K562 cells, respectively. No cellular uptake was observed for both fluorescent compounds in HEK293 noncancerous control cells. Afterward, three conjugates composed of the ß-lactam-based integrin ligand, suitable linkers, and 5-FU were realized. The best compound E, acting as α5ß1 integrin agonist, is able to selectively deliver 5-FU into tumor cells, successfully leading to cancer cell death.

Strontium substituted hydroxyapatite with ß-lactam integrin agonists to enhance mesenchymal cells adhesion and to promote bone regeneration.

Multi-functionalization of calcium phosphates to get delivery systems of therapeutic agents is gaining increasing relevance for the development of functional biomaterials aimed to solve problems related to disorders of the muscolo-skeletal system. In this regard, we functionalized Strontium substituted hydroxyapatite (SrHA) with some ß-lactam integrin agonists to develop materials with enhanced properties in promoting cell adhesion and activation of intracellular signaling as well as in counteracting abnormal bone resorption. For this purpose, we selected two monocyclic ß-lactams on the basis of their activities towards specific integrins on promoting cell adhesion and signalling. The amount of ß-lactams loaded on SrHA could be modulated on changing the polarity of the loading solution, from 3.5-24 wt% for compound 1 and from 3.2-8.4 wt% for compound 2. Studies on the release of the ß-lactams from the functionalized SrHA in aqueous medium showed an initial burst followed by a steady-release that ensures a small but constant amount of the compounds over time. The new composites were fully characterized. Co-culture of human primary mesenchymal stem cells (hMSC) and human primary osteoclast (OC) demonstrated that the presence of ß-lactams on SrHA favors hMSC adhesion and viability, as well as differentiation towards osteoblastic lineage. Moreover, the ß-lactams were found to enhance the inhibitory role of Strontium on osteoclast viability and differentiation.

Nerve Growth Factor Biodelivery: A Limiting Step in Moving Toward Extensive Clinical Application?

Nerve growth factor (NGF) was the first-discovered member of the neurotrophin family, a class of bioactive molecules which exerts powerful biological effects on the CNS and other peripheral tissues, not only during development, but also during adulthood. While these molecules have long been regarded as potential drugs to combat acute and chronic neurodegenerative processes, as evidenced by the extensive data on their neuroprotective properties, their clinical application has been hindered by their unexpected side effects, as well as by difficulties in defining appropriate dosing and administration strategies. This paper reviews aspects related to the endogenous production of NGF in healthy and pathological conditions, along with conventional and biomaterial-assisted delivery strategies, in an attempt to clarify the impediments to the clinical application of this powerful molecule.

N-Thio-ß-lactams targeting L,D-transpeptidase-2, with activity against drug-resistant strains of Mycobacterium tuberculosis.

Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic ß-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-ß-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-ß-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated in vitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds.

Chemoenzymatic synthesis of (2S)-2-arylpropanols through a dynamic kinetic resolution of 2-arylpropanals with alcohol dehydrogenases.

We applied Horse Liver Alcohol Dehydrogenase (HLADH) to the enantioselective synthesis of six (2S)-2-arylpropanols, useful intermediates in the synthesis of Profens. The influence of substrate structure and reaction conditions on yields and enantioselectivity were investigated. The high yields and high enantioselectivity towards the (S)-enantiomer obtained in the bioreduction of 2-arylpropionic aldehydes, clearly indicate the achievement of a DKR process through a combination of an enzyme-catalyzed kinetic reduction with a chemical base-catalyzed racemization of the unreacted aldehydes. The racemization step is represented by the keto-enol equilibrium of the aldehyde and can be controlled by modulating pH and reaction conditions.

Integrin-Targeting Peptides for the Design of Functional Cell-Responsive Biomaterials.

Integrins are a family of cell surface receptors crucial to fundamental cellular functions such as adhesion, signaling, and viability, deeply involved in a variety of diseases, including the initiation and progression of cancer, of coronary, inflammatory, or autoimmune diseases. The natural ligands of integrins are glycoproteins expressed on the cell surface or proteins of the extracellular matrix. For this reason, short peptides or peptidomimetic sequences that reproduce the integrin-binding motives have attracted much attention as potential drugs. When challenged in clinical trials, these peptides/peptidomimetics let to contrasting and disappointing results. In the search for alternative utilizations, the integrin peptide ligands have been conjugated onto nanoparticles, materials, or drugs and drug carrier systems, for specific recognition or delivery of drugs to cells overexpressing the targeted integrins. Recent research in peptidic integrin ligands is exploring new opportunities, in particular for the design of nanostructured, micro-fabricated, cell-responsive, stimuli-responsive, smart materials.

Leukocyte Integrin Antagonists as a Novel Option to Treat Dry Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a complex multifactorial degenerative disease that leads to irreversible blindness. AMD affects the macula, the central part of the retina responsible for sharp central vision. Retinal pigment epithelium (RPE) is the main cellular type affected in dry AMD. RPE cells form a monolayer between the choroid and the neuroretina and are in close functional relationship with photoreceptors; moreover, RPE cells are part of the blood retina barrier that is disrupted in ocular diseases such as AMD. During ocular inflammation lymphocytes and macrophages are recruited, contact RPE and produce pro-inflammatory cytokines, which play an important role in AMD pathogenesis. The interaction between RPE and immune cells is mediated by leukocyte integrins, heterodimeric transmembrane receptors, and adhesion molecules, including VCAM-1 and ICAM-1. Within this frame, this study aimed to characterize RPE-leukocytes interaction and to investigate any potentially beneficial effects induced by integrin antagonists (DS-70, MN27 and SR714), developed in previous studies. ARPE-19 cells were co-cultured for different incubation times with Jurkat cells and apoptosis and necrosis levels were analyzed by flow cytometry. Moreover, we measured the mRNA levels of the pro-inflammatory cytokine IL-1ß and the expression of adhesion molecules VCAM-1 and ICAM-1. We found that RPE-lymphocyte interaction increased apoptosis and necrosis levels in RPE cells and the expression of IL-1ß. This interaction was mediated by the binding of α4ß1 and αLß2 integrins to VCAM-1 and ICAM-1, respectively. The blockade of RPE-lymphocyte interaction with blocking antibodies highlighted the pivotal role played by integrins. Therefore, α4ß1 and αLß2 integrin antagonists were employed to disrupt RPE-lymphocyte crosstalk. Small molecule integrin antagonists proved to be effective in reducing RPE cell death and expression of IL-1ß, demonstrating that integrin antagonists could protect RPE cells from detrimental effects induced by the interaction with immune cells recruited to the retina. Overall, the leukocyte integrin antagonists employed in the present study may represent a novel opportunity to develop new drugs to fight dry AMD.

Could Dissecting the Molecular Framework of ß-Lactam Integrin Ligands Enhance Selectivity?

By dissecting the structure of ß-lactam-based ligands, a new series of compounds was designed, synthesized, and evaluated toward integrins αvß3, α5ß1, and α4ß1. New selective ligands with antagonist or agonist activities of cell adhesion in the nanomolar range were obtained. The best agonist molecules induced significant adhesion of SK-MEL-24 cells and Saos-2 cells as a valuable model for osteoblast adhesion. These data could lead to the development of new agents to improve cellular osseointegration and bone regeneration. Molecular modeling studies on prototypic compounds and αvß3 or α5ß1 integrin supported the notion that ligand carboxylate fixing to the metal ion-dependent adhesion site in the ß-subunit can be sufficient for binding the receptors, while the aryl side chains play a role in determining the selectivity as well as agonism versus antagonism.

New polyphenolic beta-lactams with antioxidant activity.

Novel 4-alkylidene-beta-lactams with a polyphenolic side chain were synthesized and evaluated as radical scavengers. We have undertaken a detailed study of the antioxidant activity in vitro with chemical and biological testing of the new beta-lactams and of the corresponding methyl polyhydroxy benzoates. Antioxidant activity of beta-lactams and methyl benzoates was measured with the Briggs-Rauscher oscillating reaction, the TEAC (Trolox( Equivalent Antioxidant Capacity) assay, and as ability to inhibit ROS (=Reactive Oxygen Species) production on myoblast H9c2 cells. The results were discussed with regard to mechanism and correlated with structural parameters.

Antibacterial and antioxidant activities for natural and synthetic dual-active compounds.

Antimicrobial resistance is widely recognized as a grave threat to global health in the 21st century, since the past decades have seen a dramatic increase in human-pathogenic bacteria that are resistant to one or multiple antibiotics. New antimicrobial agents are urgently required, particularly in the treatment of chronic infections such as cystic fibrosis, often associated with persistent colonization by drug-resistant pathogens and epithelial damage by pulmonary oxidative stress. In such events, it would be favourable to find agents that could have antioxidant and antibacterial activities combined in one molecule. The discovery of compounds that can show a dual-target activity considerably increased in the last years, reflecting the growing confidence that this new approach could lead to better therapeutic solutions for complex multigenic diseases. The aim of this review is to report those natural and synthetic compounds displaying significant antioxidant and antibacterial activities. In recent years there has been a growing attention on plant-derived antimicrobials as an alternative to antibiotics, for their efficacy and low tendency in developing bacterial resistance. Moreover, it was found that some natural products could enhance the activity of common antibiotics displaying a synergistic effect. We then report some selected synthetic compounds with an in-built capacity to act on two targets or with the combination in a single structure of two pharmacophores with antioxidant and antibacterial activities. Recent literature instances were screened and the most promising examples of dual-active antibacterial-antioxidant molecules were highlighted.

Highly efficient asymmetric reduction of arylpropionic aldehydes by horse liver alcohol dehydrogenase through dynamic kinetic resolution.

The enantioselective synthesis of (2S)-2-phenylpropanol and (2S)-2-(4-iso-butylphenyl)propanol ((S)-Ibuprofenol) has been achieved by means of Horse Liver Alcohol Dehydrogenase (HLADH) in buffered aqueous solution or buffered organic solvent mixtures; under the reaction conditions, a dynamic kinetic resolution (DKR) process was realized with good reaction yields and enantiomeric ratios.