A number of C-3 spirocyclic 2-benzazepine analogs of a-phenyl-N-tert-butyl nitrone (PBN) were synthesized and tested for their activity in protecting rat brain mitochondria and dopaminergic (DA) neurons against 6-hydroxydopamine (6-OHDA), a toxin inducing destruction of the DA nigrostriatal pathway in rodent models of Parkinson’s disease. The newly synthesized nitrone derivatives were firstly investigated for their activity in decreasing the level of hydroxyl radicals generated during 6-OHDA oxidation, and inhibit lipid peroxidation (TBARS assay) and protein carbonyl content (PCC) in rat brain mitochondria. Most of the studied 2-benzazepine nitrones showed inhibitory potencies in both TBARS and PCC assays at least two magnitude orders higher than that of PBN. The data obtained usefully complemented the known structure–activity relationships. In particular, 5 and 10, bearing C-3 spiro cyclopentyl and tetrahydropyranyl moieties, respectively, at 8 mM concentration proved to be significantly more effective than PBN in protecting cultured DA neurons exposed to 6-OHDA, which alone causes about 45% cell loss in 24 h. In addition, we found that 5 inhibited butyrylcholinesterase with an IC50 value of 16.8 mM, which would enhance its potential as neuroprotective agent in Alzheimer’s neurodegeneration. These findings extend the utility of benzazepine-based PBN analogs in the treatment of age-related free radical-mediated disorders.

New chloro-substituted biarylmethoxyphenyl piperidine-4-carboxamides were synthesized and assayed in vitro as inhibitors of the blood coagulation enzymes factor Xa (fXa) and thrombin. An investigation of effects of the amidine and isopropyl groups attached at the piperidine nitrogen and 5-(halogenoaryl)isoxazol-3-yl groups as biaryl substituents led us to identify new compounds which proved to be selective fXa inhibitors, with inhibition constants in the low nanomolar range. The most potent compound 21e, that incorporates 2-Cl-thiophen-5-yl group as the P1 motif and 1-isopropylpiperidine P4 group, inhibited fXa with K(i) value of 0.3nM and very high selectivity over thrombin and some other tested serine proteases, achieving moderate levels of anticoagulant activity in the low micromolar range, as assessed by the prothrombin time clotting assay (PT(2)=3.30μM). Based on reliable docking simulations, molecular modeling provided a rationale for interpreting structure-activity relationships. The predicted binding modes highlighted the structural requirements for addressing the subsites S1 and S4 of the fXa enzyme.

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.

We recently synthesized novel glucose-conjugated dual factor Xa (fXa) and thrombin (fIIa) inhibitors 3, bearing 5-chlorothien-2-yl and 1-isopropylpiperidine moieties as binders of the S1 and S3/S4 enzymes’ pockets, respectively, which showed potential for use in the treatment of thrombotic diseases. In particular, the β-d-glucosyl-bearing derivative proved to be a competitive inhibitor with high potency against fXa (Ki = 0.09 nM) and fIIa (Ki = 100 nM), and in vitro/ex vivo micromolar anticoagulant potency.1 Despite the narrower binding site groove of fIIa, the inhibitory potency of the glucosyl derivative, compared to the parent glucosyl-lacking compound 1, increases against fIIa (110-fold) much more than against fXa (7-fold). Experimental deconstruction of the most potent inhibitor molecule into smaller fragments, synthesized and tested, provided us with significant insights into the enzymes’ affinity contributions of the P1 and P3/P4 moieties, and a C3-alkyl-linked -d-glucose fragment (PG). To understand the inhibitors’ binding modes to fIIa, the crystal structures of human thrombin in complex with two glucose-based compounds were solved (pdb codes: 4NZE and 4N3L), and the crystallographic results will be presented and discussed.

The β-D-glucose-containing compound 3, bearing 2-chlorothiophene and 1-isopropylpiperidine moieties as binders of the S1 and S4 pockets, respectively, proved to be potent competitive inhibitor of factor Xa (fXa, Ki = 0.090 nM) and thrombin (fIIa, Ki = 100 nM). The potency of 3 increases, over the parent compound 1, against fIIa (110-fold) much more than against fXa (7-fold). Experimental deconstruction of 3 into smaller fragments revealed a binding cooperativity of the P3/P4 and C3-alkyl-linked -D-glucose fragments, stronger in fIIa (15.5 kJ•mol-1) than in fXa (2.8 kJ•mol-1). The crystal structure of human fIIa in complex with 3 revealed a binding mode including a strong H-bond network between the glucose O1’, O3’ and O5’ and two critical residues, namely R221a and K224, belonging to the Na+-binding site which may allosterically perturb the specificity sites. The potential of 3 as antithrombotic agent was supported by its ability to inhibit thrombin generation and to stimulate fibrinolysis at submicromolar concentration.

Current anticoagulant therapy of venous thromboembolism (VTE) is based on parenterally administered heparins and orally administered vitamin K antagonists (e.g., warfarin), but narrow therapeutic window and side effects, such as bleeding, diet and genetic makeup influence, are associated with their use [1]. Recently, key serine proteases of the blood coagulation cascade, such as thrombin (thr) and factor Xa (fXa), have emerged as promising targets for anticoagulants, and indeed several direct inhibitors of thr (e.g., argatroban, dabigatran) and fXa (e.g., rivaroxaban, apixaban) have been introduced in therapy or in advanced clinical trials [2,3]. Some years ago we investigated the isonipecotanilide scaffold for new thr/fXa inhibitors [4]. Further optimization studies led us to develop new benzyloxy derivatives of N-(phenyl)-1-(pyridin-4-yl)piperidine-4-carboxamide, one of them (i.e., the 3-F analog, see below) showing low nanomolar Ki (thr) value, high selectivity against other serine proteases and good anticoagulant activity as measured by the activated partial thromboplastin time (aPTT) test. Physicochemical profiles of the newly synthesized compounds were assessed and their potential oral bioavailability estimated, by measuring effective permeability coefficients using PAMPA (Parallel Artificial Membrane Permeability Assay).

Starting from the cholinergic hypothesis, which originally implied a major role of acetylcholinesterase (AChE) in the cognitive impairment of the Alzheimer’s disease (AD), the role of butyrylcholinesterase (BChE) has progressively become more crucial in the AD development and progression. Indeed, it has been shown that the levels of AChE in the AD brain decrease by as much as 90%, whilst the levels of BChE, mainly in the G1 form (i.e., globular form of monomer structure), increase, suggesting that inhibition of BChE may represent a privileged target to develop new drugs for treating neurodegenerative diseases. As a matter of fact, in the last years several efforts have been made to identify selective BChE inhibitors, such as tricyclic cymserine analogs, which proved beneficial in vivo in animal models, most likely by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Previously, we reported the ChE inhibition activity of novel medium-sized nitrogen-containing heterocycles (e.g., tetrahydroazocines) fused on indole, which showed ChE inhibition activity. Herein, we explore the ChE inhibition activity of a series of 3,4,5,6-tetrahydroazepino[4,3-b]indole derivatives, some of which proved to be highly potent and selective BChE inhibitors, with low toxicity as assessed in vitro on neuroblastoma cell cultures. Among the newly synthesized compounds, the lactam derivative 2 showed the highest BChE inhibition potency (IC50 = 1.5 nM), whereas further investigation showed that the above tricyclic system could provide a promising scaffold for new multimodal derivatives with potential in the treatment of neurodegenerative disorders.

Several 6-substituted 3,4,5,6-tetrahydroazepino[4,3-b]indol-1(2H)-one (THAI) derivatives were synthesized and evaluated for their activity as cholinesterase (ChE) inhibitors. The most potent inhibitors were identified among 6-(2-phenylethyl)-THAI derivatives, and in particular compounds 12b and 12d proved to be very active against human BChE (IC50 = 13 and 1.8 nM, respectively), with 1000-fold selectivity over AChE. Structure-activity relationships highlighted critical features (e.g., ring fusion [4,3-b], integrity of the lactam CONH function) and favorable physicochemical properties of the 6-(2-phenylethyl) group (i.e., optimal position, size and lipophilicity of phenyl substituents). The effects of a number of compounds against NMDA-induced SH-SY5Y neuronal cell injury were also evaluated. Treatment with 12b increased cell viability in SH-SY5Y cells pretreated with 250 μM NMDA, with significant effects (P < 0.05) at concentrations between 0.5 and 5 μM. These findings suggest that THAI can be used as a scaffold for developing new drug leads for the treatment of Alzheimer-type neurodegeneration syndrome.

Novel O-glucosides of the recently reported potent factor Xa (fXa) inhibitors,(1) which bear 5- chlorothien-2-yl moiety and 1-isopropylpiperidine as the fragments binding the S1 and S4 enzyme pockets, respectively, were synthesized. In particular, β-D-glucose was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of the most potent inhibitor, providing a β-D-glucosyl derivative which showed picomolar inhibition potency against human fXa (Ki = 60 pM), nanomolar potency against thrombin (fIIa, Ki = 60 nM) and high selectivity over a panel of other serine proteases, including trypsin and leukocyte elastase, as well as in vitro sub-micromolar anticoagulant activity in the prothrombin time (PT) clotting assay and a statistically significant 1.6-fold prolongation of the basal PT in an ex vivo assay in mice. The crystal structures of human thrombin in complex with two highly potent glucose-based compounds were solved, which provided us with useful information on the binding modes of these inhibitors. While as expected from previous studies(2) the chlorothiophene group binds in the S1 pocket and the N1-isopropylpiperidine group in the S4 region, the sugar moiety binds in a protein region hitherto unexploited by small-molecule direct fIIa inhibitors, which is located near the S4 subsite, where the glucose O2 form strong H-bonds with two basic residues, i.e., Arg221A and Lys224.

Crystallography is a major tool for structure-driven drug design, as it allows knowledge of the 3D structure of protein targets and protein–ligand complexes. However, the route for crystal structure determination involves many steps, some of which may hamper its high-throughput use. Recent efforts have produced significant advances in experimental and computational tools and protocols. They include automatic crystallization tools, faster data collection devices, more efficient phasing methods and improved ligand-fitting procedures. The timescales of drug-discovery processes have been also reduced by using a fragment-based screening approach. Herein, the achievements in protein crystallography over the last 5 years are reviewed, and advantages and disadvantages of the fragment-based approaches to drug discovery that make use of x-ray crystallography as a primary screening method are examined. In particular, in some detail, five recent case studies pertaining to the development of new hits or leads in relevant therapeutic areas, such as cancer, immune response, inflammation, metabolic syndrome and neurology are described.

A study was carried out on the domino transformations of tetrahydrobenzofuro[2,3-c]pyridines and tetrahydrobenzofuro[3,2-c]pyridines by the action of activated alkynes in methanol and acetonitrile. This reaction is a new method for the synthesis of spiro[benzofuropyridines] showing acetylcholinesterase inhibition activity.

The design and synthesis of a new class of nonpeptide direct thrombin inhibitors, built on the structure of 1-(pyridin-4-yl)piperidine-4-carboxamide, are described. Starting from a strongly basic 1-amidinopiperidine derivative (6) showing poor thrombin (fIIa) and factor Xa (fXa) inhibition activities, anti-fIIa activity and artificial membrane permeability were considerably improved by optimizing the basic P1 and the X-substituted phenyl P4 binding moieties. Structure-activity relationship studies, usefully complemented with molecular modeling results, led us to identify compound 13b, that showed excellent fIIa inhibition (Ki = 6 nM), weak anti-Xa activity (Ki = 5.64 µM) and remarkable selectivity over other serine proteases (e.g., trypsin). Compound 13b showed in vitro anticoagulant activity in the low micromolar range and significant membrane permeability. In mice (ex vivo), 13b demonstrated anticoagulant effects at 2h after oral dosing (100 mg•kg-1), with a significant 43%-prolongation of the activated partial thromboplastin time (aPTT), over controls (P < 0.05)

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 role of butyrylcholinesterase (BChE) in the progression of Alzheimer’s disease (AD) has recently become more crucial (1). It is known that in healthy human brain acetylcholinesterase (AChE) predominates over BChE activity, but, as AD progresses, The levels of AChE in the brain decrease by as much as 90%, whilst the levels of BChE, Mainly in the G1 form (i.e., globular form of monomer structure), increase (2,3). This suggests that the inhibition of BChE may be useful in ameliorating the cholinergic transmission, which likely worsen in AD due to the BChE increased activity (4). As a matter of fact, in the AD brain, selective BChE inhibitors, such as tricyclic cymserine Analogs (1,5), have been demonstrated to have beneficial effects in vivo, probably by recovering cholinergic activity and/or by restoring AChE:BChE activity ratios to the levels observed in the healthy brain. Some years ago, we reported the ChE inhibition activity of novel annulated (e.g., pyrrole- and indole-fused) tetrahydroazocines, which showed AChE selectivity (6). Herein, we explore the ChE inhibition activity of a large series of 1,2,3,4,5,6-hexahydroazepino[4,3-b]ndole derivatives, some of which proved to be potent and selective BChE inhibitors. In particular, the lactam derivative 2 was highly active against BChE, with a subnanomolar IC50 Value. Synthesis, enzyme inhibition data and SARs are presented and discussed.