Insights into Structure-Activity Relationships of 3-Arylhydrazonoindolin-2-One Derivatives for Their Multitarget Activity on ß-Amyloid Aggregation and Neurotoxicity.

Despite the controversial outcomes of clinical trials executed so far, the prevention of ß-amyloid (Aß) deposition and neurotoxicity by small molecule inhibitors of Aß aggregation remains a target intensively pursued in the search of effective drugs for treating Alzheimer's disease (AD) and related neurodegeneration syndromes. As a continuation of previous studies, a series of new 3-(2-arylhydrazono)indolin-2-one derivatives was synthesized and assayed, investigating the effects of substitutions on both the indole core and arylhydrazone moiety. Compared with the reference compound 1, we disclosed equipotent derivatives bearing alkyl substituents at the indole nitrogen, and fairly tolerated bioisosteric replacements at the arylhydrazone moiety. For most of the investigated compounds, the inhibition of Aß40 aggregation (expressed as pIC50) was found to be correlated with lipophilicity, as assessed by a reversed-phase HPLC method, through a bilinear relationship. The N¹-cyclopropyl derivative 28 was tested in cell-based assays of Aß42 oligomer toxicity and oxidative stress induced by hydrogen peroxide, showing significant cytoprotective effects. This study confirmed the versatility of isatin in preparing multitarget small molecules affecting different biochemical pathways involved in AD.

Mannich base approach to 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone: A water-soluble prodrug for a multitarget inhibition of cholinesterases, beta-amyloid fibrillization and oligomer-induced cytotoxicity.

Targeting protein aggregation for the therapy of neurodegenerative diseases remains elusive for medicinal chemists, despite a number of small molecules known to interfere in amyloidogenesis, particularly of amyloid beta (Aß) protein. Starting from previous findings in the antiaggregating activity of a class of indolin-2-ones inhibiting Aß fibrillization, 5-methoxyisatin 3-(4-isopropylphenyl)hydrazone 1 was identified as a multitarget inhibitor of Aß aggregation and cholinesterases with IC50s in the low µM range. With the aim of increasing aqueous solubility, a Mannich-base functionalization led to the synthesis of N-methylpiperazine derivative 2. At acidic pH, an outstanding solubility increase of 2 over the parent compound 1 was proved through a turbidimetric method. HPLC analysis revealed an improved stability of the Mannich base 2 at pH2 along with a rapid release of 1 in human serum as well as an outstanding hydrolytic stability of the parent hydrazone. Coincubation of Aß1-42 with 2 resulted in the accumulation of low MW oligomers, as detected with PICUP assay. Cell assays on SH-SY5Y cells revealed that 2 exerts strong cytoprotective effects in both cell viability and radical quenching assays, mainly related to its active metabolite 1. These findings show that 2 drives the formation of non-toxic, off-pathway Aß oligomers unable to trigger the amyloid cascade and toxicity.

Investigation on the influence of (Z)-3-(2-(3-chlorophenyl)hydrazono)-5,6-dihydroxyindolin-2-one (PT2) on ß-amyloid(1-40) aggregation and toxicity.

In Alzheimer's disease (AD), native Aß 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 Aß aggregation cascade. We compared data from DLS, 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 Aß as non-toxic, ß-sheet arranged, ThT-insensitive protofilaments. It also strongly reduced cellular toxicity caused by Aß and showed good antioxidant properties in two radical scavenging tests. Taken together, these data confirmed that PT2 is a small molecule inhibitor of Aß oligomerization and toxicity, displaying also additional activity as antioxidant.

Design, synthesis and biological evaluation of benzo[e][1,2,4]triazin-7(1H)-one and [1,2,4]-triazino[5,6,1-jk]carbazol-6-one derivatives as dual inhibitors of beta-amyloid aggregation and acetyl/butyryl cholinesterase.

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 (Aß) 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 Aß(1-40) aggregation and cholinesterase inhibitors. Potent inhibitors of Aß self-aggregation were discovered and among them benzotriazinone 7 exhibited an outstanding IC(50) equal to 0.37 µM. 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 IC(50) values of 1.4, 1.5 and 1.9 µM on Aß aggregation and AChE and BChE inhibition, respectively, and the latter showing IC(50) values of 1.4 and an outstanding 0.025 µM in the Aß aggregation and BChE inhibition, respectively. Benzotriazinone 24 and triazafluoranthenone 29, selected owing to their suitable aqueous solubility and Aß 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 µM 24 vs. 50 µM Aß), stabilizing the unstructured Aß peptide and inhibiting fibrillogenesis, and that 29 also acted as fibrillization inhibitor, but likely enhancing and stabilizing the ß-sheet arrangement of Aß to yield protofibrillar species as detected by TEM.

ß-D-glucosyl conjugates of highly potent inhibitors of blood coagulation factor Xa bearing 2-chorothiophene as a P1 motif.

We synthesized a novel O-glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5-chlorothien-2-yl moiety and 1-isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A ß-D-glucosyl unit was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of 1. The synthesized ß-D-glucose-based compound 16 achieved picomolar inhibitory potency against human fXa (K(i)=60 pM) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1-isopropylpiperidine (ΔΔG=29.7-30.5 kJ mol(-1)), which should bind to the aromatic S4 pocket through efficient cation-π and C-H···π interactions. Instead, the C3-alkyl-linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9-3.8 kJ mol(-1) . Compound 16 showed sub-micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT(2) and aPTT(2) equal to 0.135 and 0.389 µM, respectively). Although compound 16 was 1.4-fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6-fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg(-1)).

Synthesis and biophysical evaluation of arylhydrazono-1H-2-indolinones as ß-amyloid aggregation inhibitors.

A series of isatin-3-arylhydrazones were synthesized and evaluated in vitro as inhibitors of Aß(1-40) aggregation using a thioflavin T fluorescence method. An exploration of the effects on Aß(1-40) 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 IC(50) 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 4'- and 3'-position, respectively, of the phenylhydrazone moiety. Two derivatives are noteworthy, namely 18 (IC(50) = 0.4 µM) and 42 (IC(50) = 1.1 µM). The in vitro effects of the highly active, water soluble, compound 42 on the temporal evolution of Aß(1-40) 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.

Design, synthesis and biological evaluation of indane-2-arylhydrazinylmethylene-1,3-diones and indol-2-aryldiazenylmethylene-3-ones as beta-amyloid aggregation inhibitors.

Biological screening of (hetero)aromatic compounds allowed the identification of some novel inhibitors of Abeta(1-40) 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 IC(50). The 2-(p-isopropylphenyldiazenylmethylene)indolone derivative 6c resulted as the most potent aggregation inhibitor exhibiting an IC(50) of 1.4 muM, with complete lack of fibril formation as confirmed by transmission electron microscopy. Structure-activity relationships suggested that binding to the Abeta peptide may be largely guided by pi-stacking and hydrogen bond interactions.

1,3-Dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines as potent A2B adenosine receptor antagonists: design, synthesis, structure-affinity and structure-selectivity relationships.

A number of 1,3-dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines were prepared and evaluated as ligands of recombinant human adenosine receptors (hARs). Several 1,3-dipropyl derivatives endowed with nanomolar binding affinity at hA(2B) receptors, but poor selectivity over hA(2A), hA(1) and hA(3) AR subtypes were identified. A comparison with the corresponding 7-OH- and 7,9-unsubstituted-deazaxanthines revealed that 9-OH-9-deazaxanthines are more potent hA(2B) ligands with lower partition coefficients and higher water solubility compared to the other two congeneric classes of deazaxanthines. An optimization of the para-substituent of the 8-phenyl ring of 9-OH-9-deazaxanthines led to the discovery of compound 38, which exhibited outstanding hA(2B) affinity (Ki=1.0 nM), good selectivity over hA(2A), hA(1) and hA(3) (selectivity indices=100, 79 and 1290, respectively) and excellent antagonist potency in a functional assay on rat A(2B) (pA(2B)=9.33).

Design, synthesis, and biological evaluation of glycine-based molecular tongs as inhibitors of Abeta1-40 aggregation in vitro.

A series of N-terminus benzamides of glycine-based symmetric peptides, linked to m-xylylenediamine and 3,4'-oxydianiline spacers, were prepared and tested as inhibitors of beta-amyloid peptide Abeta(1-40) aggregation in vitro. Compounds with good anti-aggregating activity were detected. Polyphenolic amides showed the highest anti-aggregating activity, with IC(50) values in the micromolar range. Structure-activity relationships suggested that pi-pi stacking and hydrogen-bonding interactions play a key role in the inhibition of Abeta(1-40) self-assembly leading to amyloid fibrils.

Synthesis and monoamine oxidase inhibitory activity of new pyridazine-, pyrimidine- and 1,2,4-triazine-containing tricyclic derivatives.

A number of condensed azines, mostly belonging to the families of indeno-fused pyridazines (1), pyrimidines (2, 3), and 1,2,4-triazines (4, 5), were synthesized and evaluated in vitro as monoamine oxidase (MAO) A and B inhibitors. Most of them showed higher inhibition potency toward MAO-B, the most effective one being 3-(3-nitrophenyl)-9H-indeno[1,2-e] [1,2,4]triazin-9-one (4c), which displayed an IC50 value of 80 nM and proved to be 10-fold more potent than its [2,1-e] fusion isomer 5. Replacing the 3-phenyl group of the known indeno[1,2-c]pyridazin-5-one MAO-B inhibitors with a flexible phenoxymethyl group enhanced the inhibitory potency. The inhibition data highlighted the importance of the aza-heterocyclic scaffold in affecting the MAO isoform selectivity. The 3-phenyl derivatives with type 1, 4, and 5 scaffolds were inhibitors of MAO-B with little or no MAO-A effect, whereas 2- or 3-phenyl derivatives of type 2 and 3 pyrimidine-containing fusion isomers inhibited both isoenzymes with a structure-dependent preference toward MAO-A.

Synthesis, central and peripheral benzodiazepine receptor affinity of pyrazole and pyrazole-containing polycyclic derivatives.

A series of new pyrazole-condensed 6,5,5 tricyclic compounds were synthesized and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Some 1-aryl-5-phenylpyrazole derivatives were also prepared and tested for comparison with their corresponding rigid tricyclic analogs. Among the newly synthesized 1-aryl-1,4-dihydro-indeno[1,2-c]pyrazoles bearing both an ethoxycarbonyl group at position 3 and a carbonyl function at the position 4, compound 4b emerged as a new potent (IC(50) = 26.4 nM) and selective CBR ligand. The 4-oxo-1-aryl-1,4-dihydro-indeno[1,2-c]pyrazole diethylamide derivative 14a was instead identified as a relatively potent (IC(50) = 124 nM) but highly selective PBR ligand.

Synthesis and biological evaluation of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines as new ligands of central and peripheral benzodiazepine receptors.

A large number of pyridazino[4,3-b]indoles and indeno[1,2-c]pyridazines were synthesised and tested to evaluate their binding affinities at both central (CBR) and peripheral (PBR) benzodiazepine receptors. Relatively good PBR binding affinities were found for ligands belonging to the 3-arylmethyloxy-pyridazinoindole series, whereas only 2-aryl-indenopyridazines 7a, 8a and 10a display a weak binding affinity for CBR. To find out the main structural determinants affecting PBR affinity, a molecular modelling study based on the comparative analysis of the three-dimensional properties of four properly selected derivatives 24a, 3b, 18a and 10d, with those of highly active and selective PBR ligands, taken as reference, was performed.

Synthesis and antibacterial activity of pyridazino[4,3-b]indole-4-carboxylic acids carrying different substituents at N-2.

The synthesis and the in vitro evaluation of antibacterial activity of new pyridazino[4,3-b]indole-4-carboxylic acids 2-4, 6 against some selected representative of Gram-positive and Gram-negative bacteria are reported. The role of the lipophilicity in the modulation of the antibacterial activity of the tested compounds is discussed. All the synthesized compounds appear quite weak against Gram-positive bacteria, whereas have no significant activity against Gram-negative bacteria. Only derivative 2g possesses an interesting activity against Gram-positive bacteria.