The failure of the ongoing therapeutic protocols to treat neurodegenerative diseases (NDs), has been linked to the multifactorial nature of NDs connoted by a complex network of, often unrelated, cellular events. Although the etiopahtogenesis of many NDs is still obscure, growing evidence suggested that mitochondrial dysfunction, metal dyshomeostasis, oxidative stress, protein misfolding and dysregulated signaling pathways play a pivotal role in ND. The lack of disease-modifying therapies in NDs claimed for a new medicinal chemistry approach rooted on the rational design of molecular entities able to modulate multiple and aberrant biochemical mechanisms. Among the altered biochemical mechanisms, a key role of two enzymes (AChE and MAO) in the onset and progression of neural disorders has been supported by several experimental proofs. AChE is responsible for the catalytic degradation of acetylcholine and MAO is involved in the catabolism of several endogenous and exogenous amines including many neurotransmitters. The possibility of blocking simultaneously the activity of these two enzymes might restore a proper balance of neurotransmitter levels and, in addition, exert an additional beneficial effect by MAO inhibition that reduces the production of hydrogen peroxide thus avoiding the formation of reactive oxygen species.(1) Among naturally occurring heterocycles, coumarins have been largely explored as MAO inhibitors.(2) and reported also as dual binding sites AChE inhibitors.(3) As a further extension of previous investigations,(4) herein we report the design of coumarin-based dual inhibitors of AChE and MAO-B that bear small/medium sized amino groups at position 4 and a proper substituent at position 7. This design was grounded on previous 3D-QSAR studies that indicated positions 4 and 7 as preferred spatial regions, to assure a strong and selective binding at MAO-B. Moreover, structural modifications at position 4 aimed at an adequate modulation of the pharmacokinetic molecular properties, in particular, brain targeting and lipophilic balance for an in vivo activity at the CNS. The careful exploration of position 4 with basic, linear, unhindered, hydrogen bonding donor amino groups afforded promising dual inhibitors with IC50s in the low nanomolar range for MAO-B and low micromolar range for AChE.

Matrix metalloproteinases (MMPs) are a family of structurally related zinc-containing endopeptidases involved in tissue remodelling and degradation of the extracellular matrix. The failure of common synthetic inhibitors makes the design of new selective and potent MMP inhibitors an extreme challenge in health care for the treatment of various pathological states such as inflammation, arthritis, and cancer. In this view, an over-expression of MMP-2 is supposed to be responsible for the occurrence of many different human tumours and inflammatory processes involving the hydrolysis of the type IV collagen, the main component of the basement membrane. A series of studies therefore focused on the design of new potential inhibitors biased towards MMP-2: campaigns of molecular virtual screening of several large chemical libraries resulted in a number of attractive hits. Interestingly, a shortlist of alloxan-like structures was selected with inhibition constants in the nM range. In this respect, we investigated a series of complexes of MMP-2 with alloxan inhibitors by thermodynamic integration in all atoms molecular dynamics simulations. We thus obtained quantitative differences in binding free energies for a list of alloxan compounds. On this basis, we were able to elucidate the molecular rationale for the remarkable inhibition exerted by these compounds with the ultimate aim of driving the synthesis of new more potent and selective derivatives that are at present awaiting for further experimental investigations through enzymatic assays.

Alzheimer’s disease (AD) onset and progression are associated with the dysregulation of multiple and complex physiological processes and a successful therapeutic approach should therefore address more than one target. Two new chemical entities, the easily accessible heterocyclic scaffolds 1,3-diphenylbenzo [e][1,2,4]triazin-7(1H)-one (benzotriazinone I) and 2-phenyl-6H-[1,2,4]triazino[5,6,1-jk]carbazol-6-one (triazafluoranthenone II), were explored for their multitarget-directed inhibition of beta-amyloid (Ab) fibrillization and acetyl- (AChE) and/or butyryl- (BChE) cholinesterase, three valuable targets for AD therapy. Introduction of appropriate amine substituents at positions 6 and 5 on scaffold I and II, respectively, allowed the preparation of a series of compounds that were tested as Ab1e40 aggregation and cholinesterase inhibitors. Potent inhibitors of Ab self-aggregation were discovered and among them benzotriazinone 7 exhibited an outstanding IC50 equal to 0.37 mM. Compounds bearing a basic amine linked to the heterocyclic scaffold through a linear alkyl chain of varying length also afforded good ChE inhibitors. In particular, benzotriazinone 24 and triazafluoranthenone 38 were endowed with an interesting multiple activity, the former displaying IC50 values of 1.4, 1.5 and 1.9 mM on Ab aggregation and AChE and BChE inhibition, respectively, and the latter showing IC50 values of 1.4 and an outstanding 0.025 mM in the Ab aggregation and BChE inhibition, respectively. Benzotriazinone 24 and triazafluoranthenone 29, selected owing to their suitable aqueous solubility and Ab aggregation inhibition, were submitted to a time course kinetic assay followed with thioflavin T (ThT) spectrofluorimetry, circular dichroism (CD) and transmission electron microscopy (TEM). Experimental data indicated that 24 acted at a low concentration ratio (10 mM 24 vs. 50 mM Ab), stabilizing the unstructured Ab peptide and inhibiting fibrillogenesis, and that 29 also acted as fibrillization inhibitor, but likely enhancing and stabilizing the b-sheet arrangement of Ab to yield protofibrillar species as detected by TEM.

Biological screening of (hetero)aromatic compounds allowed the identification of some novel inhibitors of Ab1–40 aggregation, bearing indane and indole rings as common scaffolds. Molecular decoration of lead compounds led to inhibitors exhibiting a potency, measured by the Thioflavin T fluorimetric assay, ranging from high to low micromolar IC50. The 2-(p-isopropylphenyldiazenylmethylene)indolone derivative 6c resulted as the most potent aggregation inhibitor exhibiting an IC50 of 1.4 mM, with complete lack of fibril formation as confirmed by transmission electron microscopy. Structure–activity relationships suggested that binding to the Ab peptide may be largely guided by p-stacking and hydrogen bond interactions.

Acetylcholinesterase (AChE) inhibitors still comprise the majority of the marketed drugs for Alzheimer's disease (AD). The structural arrangement of the enzyme, which features a narrow gorge that separates the catalytic and peripheral anionic subsites (CAS and PAS, respectively), inspired the development of bivalent ligands that are able to bind and block the catalytic activity of the CAS as well as the role of the PAS in beta amyloid (Aβ) fibrillogenesis. With the aim of discovering novel AChE dual binders with improved drug-likeness, homo- and heterodimers containing 2H-chromen-2-one building blocks were developed. By exploring diverse linkages of neutral and protonatable amino moieties through aliphatic spacers of different length, a nanomolar bivalent AChE inhibitor was identified (3-[2-(4-[(dimethylamino)methyl]-2-oxo-2H-chromen-7-yloxy)ethoxy]-6,7-dimethoxy-2H-chromen-2-one (6 d), IC50 =59 nm) from originally weakly active fragments. To assess the potential against AD, the disease-related biological properties of 6 d were investigated. It performed mixed-type AChE enzyme kinetics (inhibition constant Ki =68 nm) and inhibited Aβ self-aggregation. Moreover, it displayed an outstanding ability to protect SH-SY5Y cells from Aβ1-42 damage.

Since Hansch's extra thermodynamic multi-parameter approach, originally coined as Linear Free Energy Relationship, great efforts in medicinal chemistry have been made to properly estimate the binding free energy. Despite the often small amount, its value is however very critical in determining a successful binding. As a result, its correct estimate may provide a guide for a prospective rational drug design. The calculation of the absolute binding free energies is however a very challenging task as it requires a rigorous treatment of a number of physical terms that are both very time demanding and to some extent not immediately interpretable. In view of this, the introduction of some numerical approximations has permitted to develop the so called Linear Interaction Energy method that, at present, constitutes the best compromise among accuracy, speed of computation and easy interpretation. The initially developed Linear Interaction Energy method was subsequently revisited and several important improvements have been made. Significant examples are the Extended Linear Response, the surface generalized Born LIE, the molecular mechanics generalized Born surface area, the linear interaction energy in continuum electrostatics as well as its quantum mechanics variant. Principles and selected applications of these methods will be herein reviewed.

Matrix metalloproteinases (MMP) are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. Hence, the development of potent and selective inhibitors targeting these enzymes continues to be eagerly sought. In this paper, a number of alloxan-based compounds, initially conceived to bias other therapeutically relevant enzymes, were rationally modified and successfully repurposed to inhibit MMP-2 (also named gelatinase A) in the nanomolar range. Importantly, the alloxan core makes its debut as zinc binding group since it ensures a stable tetrahedral coordination of the catalytic zinc ion in concert with the three histidines of the HExxHxxGxxH metzincin signature motif, further stabilized by a hydrogen bond with the glutamate residue belonging to the same motif. The molecular decoration of the alloxan core with a biphenyl privileged structure allowed to sample the deep S1′ specificity pocket of MMP-2 and to relate the high affinity towards this enzyme with the chance of forming a hydrogen bond network with the backbone of Leu116 and Asn147 and the side chains of Tyr144, Thr145 and Arg149 at the bottom of the pocket. The effect of even slight structural changes in determining the interaction at the S1′ subsite of MMP-2 as well as the nature and strength of the binding is elucidated via molecular dynamics simulations and free energy calculations. Among the herein presented compounds, the highest affinity (pIC50 = 7.06) is found for BAM, a compound exhibiting also selectivity (>20) towards MMP-2, as compared to MMP-9, the other member of the gelatinases.

In Alzheimer's disease (AD), native Aβ protein monomers aggregate through the formation of a variety of water-soluble, toxic oligomers, ultimately leading to insoluble fibrillar deposits. The inhibition of oligomers formation and/or their dissociation into non-toxic monomers, are considered an attractive strategy for the prevention and treatment of AD. A number of studies have demonstrated that small molecules, containing single or multiple (hetero)aromatic rings, can inhibit protein aggregation, being potentially effective in AD treatment. Starting from previously reported data on the antiamyloidogenic activity of a series of 3-hydrazonoindolinones, compound PT2 was selected to deeply investigate the inhibitory mechanism in the Aβ aggregation cascade. We compared data from DLS, 1H, 13C, 15N HSQC NMR, CD, TEM and ThT fluorescence measures to ascertain the interactions with amyloidogenic species formed in vitro during the aggregation process, and confirmed this feature with cell viability tests on HeLa cultured cells. PT2 was effective in disrupting toxic oligomers and mature amyloid fibrils, stabilizing Aβ as non-toxic, β-sheet arranged, ThT-insensitive protofilaments. It also strongly reduced cellular toxicity caused by Aβ and showed good antioxidant properties in two radical scavenging tests. Taken together, these data confirmed that PT2 is a small molecule inhibitor of Aβ oligomerization and toxicity, displaying also additional activity as antioxidant.

The enantiomer separation of a number of racemic 7-[(1-alkylpiperidin-3-yl)methoxy]coumarin derivatives, some of which show outstanding in vitro multitarget neuroprotective activities, was successfully achieved on a polysaccharide-based chiral stationary phase, bearing amylose tris(3,5-dimethylphenylcarbamate) as chiral selector, in normal polar mode (methanol and acetonitrile as the mobile phases). The majority of the screened selectands, especially those bearing 1-(3-X-benzyl)piperidin-3-yl moieties, showed baseline enantiomer separations, and compound 8 (X = NO2) was the best resolved (α = 2.01; RS = 4.27). Linear free-energy relationships, usefully complemented by molecular docking calculations, proved the key role in enantioselective retention of aromatic interactions between π-donor moieties in the chiral selector and π-acceptor moieties in selectand, strengthened by hydrogen bond interaction between a hydrogen bond donor in the chiral selector and the hydrogen bond acceptor group(s) in the selectand. Statistically reliable equations highlighted the importance of the substituent's size and substitution pattern (meta better than para) to affect the enantiorecognition of the title compounds. The chromatographic data support the scalability of the optimized experimental conditions for preparative purposes. This article is protected by copyright. All rights reserved.

A series of isatin-3-arylhydrazones were synthesized and evaluated in vitro as inhibitors of Ab1e40 aggregation using a thioflavin T fluorescence method. An exploration of the effects on Ab1e40 aggregation of a number of diverse substituents at phenylhydrazone group and 5,6- positions of the indolinone nucleus led us to single out some new anti-aggregating compounds with IC50 values in the low micromolar range. The most active compounds carry methoxy- or hydroxy- substituents in the indolinone 5,6-positions and lipophilic groups such as iPr and Cl at 40- and 30-position, respectively, of the phenylhydrazone moiety. Two derivatives are noteworthy, namely 18 (IC50 ¼ 0.4 mM) and 42 (IC50 ¼ 1.1 mM). The in vitro effects of the highly active, water soluble, compound 42 on the temporal evolution of Ab1e40 fibrils formation were further investigated by circular dichroism spectroscopy, transmission electron microscopy and dynamic light scattering studies, which clearly showed that this compound delayed and lowered the amyloid fibril formation.

The socioeconomic burden of multi-factorial pathologies, such as neurodegenerative diseases (NDs), is enormous worldwide. Unfortunately, no proven disease-modifying therapy is available yet and in most cases (e.g., Alzheimer's and Parkinson's disease) the approved drugs exert only palliative and symptomatic effects. Nowadays, an emerging strategy for the discovery of disease-modifying drugs is based on the multi-target directed ligand (MTDL) design, an innovative shift from the traditional approach one-drug-one-target to the more ambitious one-drug-more-targets goal. Herein, we review the discovery strategy, the mechanism of action and the biopharmacological evaluation of multipotent ligands exhibiting monoamine oxidase (MAO) inhibition as the core activity with a potential for the treatment of NDs. In particular, MAO inhibitors exhibiting additional acetylcholinesterase (AChE) or nitric oxide synthase (NOS) inhibition, or ion chelation/antioxidant-radical scavenging/anti-inflammatory/A2A receptor antagonist/APP processing modulating activities have been thoroughly examined.

The present invention relates to the use of compounds having a galloyl benzamide structure in the treatment and/or prevention of medical conditions mediated through c-Jun N-terminal kinases (JNKs) and to pharmaceutical compositions comprising said compounds.