Structure-based design of multitargeting ChEs-MAO B inhibitors based on phenyl ring bioisosteres: AChE/BChE selectivity switch and drug-like characterization.

A structure-based drug design approach was focused on incorporating phenyl ring heterocyclic bioisosteres into coumarin derivative 1, previously reported as potent dual AChE-MAO B inhibitor, with the aim of improving drug-like features. Structure-activity relationships highlighted that bioisosteric rings were tolerated by hMAO B enzymatic cleft more than hAChE. Interestingly, linker homologation at the basic nitrogen enabled selectivity to switch from hAChE to hBChE. In the present work, we identified thiophene-based isosteres 7 and 15 as dual AChE-MAO B (IC50 = 261 and 15 nM, respectively) and BChE-MAO B (IC50 = 375 and 20 nM, respectively) inhibitors, respectively. Both 7 and 15 were moderately water-soluble and membrane-permeant agents by passive diffusion (PAMPA-HDM). Moreover, they were able to counteract oxidative damage induced by both H2O2 and 6-OHDA in SH-SY5Y cells and predicted to penetrate into CNS in a cell-based model mimicking blood-brain barrier. Molecular dynamics (MD) simulations shed light on key differences in AChE and BChE recognition processes promoted by the basic chain homologation from 7 to 15.

Novel 6-hydroxybenzothiazol-2-carboxamides as potent and selective monoamine oxidase B inhibitors endowed with neuroprotective activity.

In neurodegenerative diseases, using a single molecule that can exert multiple effects to modify the disease may have superior activity over the classical "one molecule-one target" approach. Herein, we describe the discovery of 6-hydroxybenzothiazol-2-carboxamides as highly potent and selective MAO-B inhibitors. Variation of the amide substituent led to several potent compounds having diverse side chains with cyclohexylamide 40 displaying the highest potency towards MAO-B (IC50 = 11 nM). To discover new compounds with extended efficacy against neurotoxic mechanisms in neurodegenerative diseases, MAO-B inhibitors were screened against PHF6, R3 tau, cellular tau and α-synuclein (α-syn) aggregation. We identified the phenethylamide 30 as a multipotent inhibitor of MAO-B (IC50 = 41 nM) and α-syn and tau aggregation. It showed no cytotoxic effects on SH-SY5Y neuroblastoma cells, while also providing neuroprotection against toxicities induced by α-syn and tau. The evaluation of key physicochemical and in vitro-ADME properties revealed a great potential as drug-like small molecules with multitarget neuroprotective activity.

Bioisosteric replacement based on 1,2,4-oxadiazoles in the discovery of 1H-indazole-bearing neuroprotective MAO B inhibitors.

Following a hybridization strategy, a series of 5-substituted-1H-indazoles were designed and evaluated in vitro as inhibitors of human monoamine oxidase (hMAO) A and B. Among structural modifications, the bioisostere-based introduction of 1,2,4-oxadiazole ring returned the most potent and selective human MAO B inhibitor (compound 20, IC50 = 52 nM, SI > 192). The most promising inhibitors were studied in cell-based neuroprotection models of SH-SY5Y and astrocytes line against H2O2. Moreover, preliminary drug-like features (aqueous solubility at pH 7.4; hydrolytic stability at acidic and neutral pH) were assessed for selected 1,2,4-oxadiazoles and compared to amide analogues through RP-HPLC methods. Molecular docking simulations highlighted the crucial role of molecular flexibility in providing a better shape complementarity for compound 20 within MAO B enzymatic cleft than rigid analogue 18. Enzymatic kinetics analysis along with thermal stability curves (Tm shift = +2.9 °C) provided clues of a tight-binding mechanism for hMAO B inhibition by 20.

Hansch-Type QSAR Models for the Rational Design of MAO Inhibitors: Basic Principles and Methodology.

Hansch-type regression analysis enables the derivation of quantitative structure-activity relationship (QSAR) equations correlating bioactivity data with physicochemical parameters accounting for hydrophobicity, electronic properties, and steric effects of molecules or functional groups (substituents). Two datasets of MAO A and B inhibitors were enrolled in prototypical workflows employing multiparametric stepwise regression analysis, which includes linear and nonlinear (generally quadratic) terms. The optimal choice of variables (and/or combinations thereof) along with statistical validation yielded two robust equations describing MAO B potency and B/A selectivity, which included three and one parameter(s), respectively, and explained more than 80% of y-variance (r2) with low standard deviation (s) and good statistical significance (F, Fisher value).

A twenty-year journey exploring coumarin-based derivatives as bioactive molecules.

The coumarin core (i.e., 1-benzopyran-2 (2H)-one) is a structural motif highly recurrent in both natural products and bioactive molecules. Indeed, depending on the substituents and branching positions around the byciclic core, coumarin-containing compounds have shown diverse pharmacological activities, ranging from anticoagulant activities to anti-inflammatory, antimicrobial, anti-HIV and antitumor effects. In this survey, we have reported the main scientific results of the 20-years investigation on the coumarin core, exploited by the research group headed by Prof. Angelo Carotti (Bari, Italy) either as a scaffold or a pharmacophore moiety in designing novel biologically active small molecules.

Evaluation of Novel Guanidino-Containing Isonipecotamide Inhibitors of Blood Coagulation Factors against SARS-CoV-2 Virus Infection.

Coagulation factor Xa (fXa) and thrombin (thr) are widely expressed in pulmonary tissues, where they may catalyze, together with the transmembrane serine protease 2 (TMPRSS2), the coronaviruses spike protein (SP) cleavage and activation, thus enhancing the SP binding to ACE2 and cell infection. In this study, we evaluate in vitro the ability of approved (i.e., dabigatran and rivaroxaban) and newly synthesized isonipecotamide-based reversible inhibitors of fXa/thr (cmpds 1-3) to hinder the SARS-CoV-2 infectivity of VERO cells. Nafamostat, which is a guanidine/amidine antithrombin and antiplasmin agent, disclosed as a covalent inhibitor of TMPRSS2, was also evaluated. While dabigatran and rivaroxaban at 100 µM concentration did not show any effect on SARS-CoV-2 infection, the virus preincubation with new guanidino-containing fXa-selective inhibitors 1 and 3 did decrease viral infectivity of VERO cells at subtoxic doses. When the cells were pre-incubated with 3, a reversible nanomolar inhibitor of fXa (Ki = 15 nM) showing the best in silico docking score toward TMPRSS2 (pdb 7MEQ), the SARS-CoV-2 infectivity was completely inhibited at 100 µM (p < 0.0001), where the cytopathic effect was just about 10%. The inhibitory effects of 3 on SARS-CoV-2 infection was evident (ca. 30%) at lower concentrations (3-50 µM). The covalent TMPRSS2 and the selective inhibitor nafamostat mesylate, although showing some effect (15-20% inhibition), did not achieve statistically significant activity against SARS-CoV-2 infection in the whole range of test concentrations (3-100 µM). These findings suggest that direct inhibitors of the main serine proteases of the blood coagulation cascade may have potential in SARS-CoV-2 drug discovery. Furthermore, they prove that basic amidino-containing fXa inhibitors with a higher docking score towards TMPRSS2 may be considered hits for optimizing novel small molecules protecting guest cells from SARS-CoV-2 infection.

Design, synthesis and biological evaluation of light-driven on-off multitarget AChE and MAO-B inhibitors.

Neurodegenerative diseases are multifactorial disorders characterized by protein misfolding, oxidative stress, and neuroinflammation, finally resulting in neuronal loss and cognitive dysfunctions. Nowadays, an attractive strategy to improve the classical treatments is the development of multitarget-directed molecules able to synergistically interact with different enzymes and/or receptors. In addition, an interesting tool to refine personalized therapies may arise from the use of bioactive species able to modify their activity as a result of light irradiation. To this aim, we designed and synthesized a small library of cinnamic acid-inspired isomeric compounds with light modulated activity able to inhibit acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B), with remarkable selectivity over butyrylcholinesterase (BChE) and MAO-A, which have been investigated as the enzyme targets related to Alzheimer's disease (AD). The inhibitory activities were evaluated for the pure E-diastereomers and the E/Z-diastereomer mixtures, obtained upon UV irradiation. Molecular docking studies were carried out to rationalize the differences in the inhibition potency of the E and Z diastereomers of the best performing analogue 1c. Our preliminary findings may open-up the way for developing innovative multitarget photo-switch drugs against neurodegenerative diseases.

Probing Fluorinated Motifs onto Dual AChE-MAO B Inhibitors: Rational Design, Synthesis, Biological Evaluation, and Early-ADME Studies.

Bioisosteric H/F or CH2OH/CF2H replacement was introduced in coumarin derivatives previously characterized as dual AChE-MAO B inhibitors to probe the effects on both inhibitory potency and drug-likeness. Along with in vitro screening, we investigated early-ADME parameters related to solubility and lipophilicity (Sol7.4, CHI7.4, log D7.4), oral bioavailability and central nervous system (CNS) penetration (PAMPA-HDM and PAMPA-blood-brain barrier (BBB) assays, Caco-2 bidirectional transport study), and metabolic liability (half-lives and clearance in microsomes, inhibition of CYP3A4). Both specific and nonspecific tissue toxicities were determined in SH-SY5Y and HepG2 lines, respectively. Compound 15 bearing a -CF2H motif emerged as a water-soluble, orally bioavailable CNS-permeant potent inhibitor of both human AChE (IC50 = 550 nM) and MAO B (IC50 = 8.2 nM, B/A selectivity > 1200). Moreover, 15 behaved as a safe and metabolically stable neuroprotective agent, devoid of cytochrome liability.

First-in-Class Isonipecotamide-Based Thrombin and Cholinesterase Dual Inhibitors with Potential for Alzheimer Disease.

Recently, the direct thrombin (thr) inhibitor dabigatran has proven to be beneficial in animal models of Alzheimer's disease (AD). Aiming at discovering novel multimodal agents addressing thr and AD-related targets, a selection of previously and newly synthesized potent thr and factor Xa (fXa) inhibitors were virtually screened by the Multi-fingerprint Similarity Searching aLgorithm (MuSSeL) web server. The N-phenyl-1-(pyridin-4-yl)piperidine-4-carboxamide derivative 1, which has already been experimentally shown to inhibit thr with a Ki value of 6 nM, has been flagged by a new, upcoming release of MuSSeL as a binder of cholinesterase (ChE) isoforms (acetyl- and butyrylcholinesterase, AChE and BChE), as well as thr, fXa, and other enzymes and receptors. Interestingly, the inhibition potency of 1 was predicted by the MuSSeL platform to fall within the low-to-submicromolar range and this was confirmed by experimental Ki values, which were found equal to 0.058 and 6.95 µM for eeAChE and eqBChE, respectively. Thirty analogs of 1 were then assayed as inhibitors of thr, fXa, AChE, and BChE to increase our knowledge of their structure-activity relationships, while the molecular determinants responsible for the multiple activities towards the target enzymes were rationally investigated by molecular cross-docking screening.

Evaluation of Water-Soluble Mannich Base Prodrugs of 2,3,4,5-Tetrahydroazepino[4,3-b]indol-1(6H)-one as Multitarget-Directed Agents for Alzheimer's Disease.

Different Mannich base derivatives have been studied with the aim of addressing the poor aqueous solubility of the recently disclosed 6-phenethyl-2,3,4,5-tetrahydroazepino[4,3-b]indol-1(6H)-one (1), a human butyrylcholinesterase inhibitor (hBChE, IC50 13 nM) and protective agent in NMDA-induced neurotoxicity, in in vivo assays. The N-(4-methylpiperazin-1-yl)methyl derivative 2 c showed a 50-fold increase in solubility in pH 7.4-buffered solution, high stability in serum and (half-life >24 h) and rapid (<3 min) conversion to 1 at acidic pH. Although less active than 1, 2 c retained moderate hBChE inhibition (IC50 =3.35 µM) and a significant protective effect against NMDA-induced neurotoxicity at 0.1 µM. Moreover, 2 c resulted a weaker serum albumin binder than 1, could pass the blood-brain barrier, and exerted negligible cytotoxicity on HepG2 cells. These findings suggest that 2 c could be a water-soluble prodrug candidate of 1 for oral administration or a slow-release injectable derivative in in vivoAlzheimer's disease models.

Chasing ChEs-MAO B Multi-Targeting 4-Aminomethyl-7-Benzyloxy-2H-Chromen-2-ones.

A series of 4-aminomethyl-7-benzyloxy-2H-chromen-2-ones was investigated with the aim of identifying multiple inhibitors of cholinesterases (acetyl- and butyryl-, AChE and BChE) and monoamine oxidase B (MAO B) as potential anti-Alzheimer molecules. Starting from a previously reported potent MAO B inhibitor (3), we studied single-point modifications at the benzyloxy or at the basic moiety. The in vitro screening highlighted triple-acting compounds (6, 8, 9, 16, 20) showing nanomolar and selective MAO B inhibition along with IC50 against ChEs at the low micromolar level. Enzyme kinetics analysis toward AChE and docking simulations on the target enzymes were run in order to get insight into the mechanism of action and plausible binding modes.

1,2,3,4-Tetrahydroisoquinoline/2H-chromen-2-one conjugates as nanomolar P-glycoprotein inhibitors: Molecular determinants for affinity and selectivity over multidrug resistance associated protein 1.

A series of coniugates bearing a 1,2,3,4-tetrahydroisoquinoline motif linked to substituted 7-hydroxy-2H-chromen-2-ones was synthesized and assayed through calcein-AM test in Madin-Darby Canine Kidney (MDCK) cells overexpressing P-glycoprotein (P-gp) and closely related multidrug resistance associated protein 1 (MRP1) to probe the interference with efflux mechanisms mediated by P-gp and MRP1, respectively. A number of substituents at C3 and C4 of coumarin nucleus along with differently sized and shaped spacers was enrolled to investigate the effects of focused structural modifications over affinity and selectivity. Linker length and flexibility played a key role in enhancing P-gp affinity as proved by the most potent P-gp modulator (3h, IC50 = 70 nM). A phenyl ring within the spacer (3k, 3l, 3o) and bulkier groups (Br in 3r, Ph in 3u) at coumarin C3 led to derivatives showing nanomolar activity (160 nM < IC50 < 280 nM) along with outstanding selectivity over MRP1 (SI > 350). Molecular docking calculations carried out on a human MDR1 homology model structure contributed to gain insights into the ligands' binding modes. Some compounds (3d, 3h, 3l, 3r, 3t, 3u) reversed MDR thereby restoring doxorubicin cytotoxicity when co-administered with the drug into MDCK-MDR1 cells.

Investigating alkyl nitrates as nitric oxide releasing precursors of multitarget acetylcholinesterase-monoamine oxidase B inhibitors.

Herein we envisaged the possibility of exploiting alkyl nitrates as precursors of alcohol-bearing dual inhibitors targeting acetylcholinesterase (AChE) and monoamine oxidase B (MAO B), key enzymes in neurodegenerative syndromes such as Alzheimer's disease (AD), through biotransformation unmasking an alcoholic function upon nitric oxide (NO) release. The cooperation to neuroprotection of low fluxes of NO and target enzymes' inhibition by the alcohol metabolites might return a multitargeting effect. The in vitro screening towards ChEs and MAOs of a collection of 21 primary alcohols disclosed a subset of dual inhibitors, among which three diverse chemotypes were selected to study the corresponding nitrates. Nitrate 14 proved to be a brain permeant, potent AChE-MAO B inhibitor by itself. Moreover, it protected human SH-SY5Y lines against rotenone and hydrogen peroxide with a poor inherent cytotoxicity and showed a slow conversion profile to its alcohol metabolite 9d that still behaved as bimodal and neuroprotective molecule.

Natural Scaffolds with Multi-Target Activity for the Potential Treatment of Alzheimer's Disease.

A few symptomatic drugs are currently available for Alzheimer's Disease (AD) therapy, but these molecules are only able to temporary improve the cognitive capacity of the patients if administered in the first stages of the pathology. Recently, important advances have been achieved about the knowledge of this complex condition, which is now considered a multi-factorial disease. Researchers are, thus, more oriented toward the preparation of molecules being able to contemporaneously act on different pathological features. To date, the inhibition of acetylcholinesterase (AChE) and of ß-amyloid (Aß) aggregation as well as the antioxidant activity and the removal and/or redistribution of metal ions at the level of the nervous system are the most common investigated targets for the treatment of AD. Since many natural compounds show multiple biological properties, a series of secondary metabolites of plants or fungi with suitable structural characteristics have been selected and assayed in order to evaluate their potential role in the preparation of multi-target agents. Out of six compounds evaluated, 1 showed the best activity as an antioxidant (EC50 = 2.6 ± 0.2 µmol/µmol of DPPH) while compound 2 proved to be effective in the inhibition of AChE (IC50 = 6.86 ± 0.67 µM) and Aß1⁻40 aggregation (IC50 = 74 ± 1 µM). Furthermore, compound 6 inhibited BChE (IC50 = 1.75 ± 0.59 µM) with a good selectivity toward AChE (IC50 = 86.0 ± 15.0 µM). Moreover, preliminary tests on metal chelation suggested a possible interaction between compounds 1, 3 and 4 and copper (II). Molecules with the best multi-target profiles will be used as starting hit compounds to appropriately address future studies of Structure-Activity Relationships (SARs).

Structure-property relationship study of the HPLC enantioselective retention of neuroprotective 7-[(1-alkylpiperidin-3-yl)methoxy]coumarin derivatives on an amylose-based chiral stationary phase.

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 a 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, have 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.