A new class of pyrazolo[5,1-c][1,2,4]triazines as γ-aminobutyric type A (GABAA) receptor subtype ligand: synthesis and pharmacological evaluation.

A comparison between compounds with pyrazolo[1,5-a]pyrimidine structure (series 4-6) and pyrazolo[5,1-c][1,2,4]triazine core (series 9) as ligands at GABAA-receptor subtype, was evaluated. Moreover, for pyrazolotriazine derivatives having binding recognition, the interaction on recombinant rat α(1-3,5) GABAA receptor subtypes, was performed. Among these latter, emerge compounds 9c, 9k, 9l, 9m and 9n as α1-selective and 9h as α2-selective ligands.

Synthesis and pharmacological evaluation of pyrazolo[1,5-a]pyrimidin-7(4H)-one derivatives as potential GABAA-R ligands.

The synthesis of a new series of 6-phenyl- and 6-benzylpyrazolo[1,5-a]pyrimidin-7(4H)-ones 2a-g and 3a-g, strictly related to derivatives with pyrazolobenzotriazine (PBT) and pyrazoloquinazoline (PQ) scaffold, was realized. The in vitro GABAA-receptor subtype affinity was evaluated and from preliminary pharmacological studies, compound 3g shows anxiolytic-like effect at 10-30mg/kg.

Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors.

Human neutrophil elastase (HNE) is an important target for the development of novel and selective inhibitors to treat inflammatory diseases, especially pulmonary pathologies. Here, we report the synthesis, structure-activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with an isoxazol-5(2H)-one scaffold. The most potent compound (2o) had a good balance between HNE inhibitory activity (IC50 value =20 nM) and chemical stability in aqueous buffer (t1/2=8.9 h). Analysis of reaction kinetics revealed that the most potent isoxazolone derivatives were reversible competitive inhibitors of HNE. Furthermore, since compounds 2o and 2s contain two carbonyl groups (2-N-CO and 5-CO) as possible points of attack for Ser195, the amino acid of the active site responsible for the nucleophilic attack, docking studies allowed us to clarify the different roles played by these groups.

Synthesis of Five- and Six-Membered N-Phenylacetamido Substituted Heterocycles as Formyl Peptide Receptor Agonists.

Preclinical Research Formyl peptide receptors (FPRs) are G-protein-coupled receptors that play an important role in the regulation of inflammatory process and cellular dysfunction. In humans, three different isoforms are expressed (FPR1, FPR2, and FPR3). FPR2 appears to be directly involved in the resolution of inflammation, an active process carried out by specific pro-resolving mediators that modulate specific receptors. Previously, we identified 2-arylacetamido pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of mixed-agonists for the three isoforms. Here, we report a new series of 2-arylacetamido pyridazinones substituted at position 5 and their development as FPR agonists. We also synthesized a new series of 2-oxothiazolones bearing a 4-bromophenylacetamido fragment, which was fundamental for activity in the pyridazinone series. The compounds of most interest were 4a, a potent, mixed FPR agonist recognized by all three isotypes (FPR1 EC50 = 19 nM, FPR2 EC50 = 43 nM, FPR3 EC50 = 40 nM), and 4b, which had potent activity and a preference for FPR2 (EC50 = 13 nM). These novel compounds may represent valuable tools for studying FPR activation and signaling. Drug Dev Res 78 : 49-62, 2017. © 2016 Wiley Periodicals, Inc.

2-Arylacetamido-4-phenylamino-5-substituted pyridazinones as formyl peptide receptors agonists.

N-Formyl peptide receptors (FPRs: FPR1, FPR2, and FPR3) are G protein-coupled receptors that play key roles in modulating immune cells. FPRs represent potentially important therapeutic targets for the development of drugs that could enhance endogenous anti-inflammation systems associated with various pathologies, thereby reducing the progression of inflammatory conditions. Previously, we identified 2-arylacetamide pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of FPR1/FPR2-dual agonists and several mixed-agonists for the three FPR isoforms. Here, we report a new series of 2-arylacetamido-4-aniline pyridazin-3(2H)-ones substituted in position 5 as a further development of these FPR agonists. Chemical manipulation presented in this work resulted in mixed FPR agonists 8a, 13a and 27b, which had EC50 values in nanomolar range. In particular, compound 8a showed a preference for FPR1 (EC50=45nM), while 13a and 27b showed a moderate preference for FPR2 (EC50=35 and 61nM, respectively). Thus, these compounds may represent valuable tools for studying FPR activation and signaling.

Cinnoline derivatives as human neutrophil elastase inhibitors.

Compounds that can effectively inhibit the proteolytic activity of human neutrophil elastase (HNE) represent promising therapeutics for treatment of inflammatory diseases. We present here the synthesis, structure-activity relationship analysis, and biological evaluation of a new series of HNE inhibitors with a cinnoline scaffold. These compounds exhibited HNE inhibitory activity but had lower potency compared to N-benzoylindazoles previously reported by us. On the other hand, they exhibited increased stability in aqueous solution. The most potent compound, 18a, had a good balance between HNE inhibitory activity (IC50 value = 56 nM) and chemical stability (t1/2 = 114 min). Analysis of reaction kinetics revealed that these cinnoline derivatives were reversible competitive inhibitors of HNE. Furthermore, molecular docking studies of the active products into the HNE binding site revealed two types of HNE inhibitors: molecules with cinnolin-4(1H)-one scaffold, which were attacked by the HNE Ser195 hydroxyl group at the amido moiety, and cinnoline derivatives containing an ester function at C-4, which is the point of attack of Ser195.

Pyrazolo[1,5-a]quinazoline scaffold as 5-deaza analogue of pyrazolo[5,1-c][1,2,4]benzotriazine system: synthesis of new derivatives, biological activity on GABAA receptor subtype and molecular dynamic study.

To investigate the binding affinity of GABAA receptor subtype, new pyrazolo [1,5-a]quinazolines were designed, synthesized, and in vitro evaluated. These compounds, 5-deaza analogues of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives which were already studied in our research group, permit us to evaluate the relevance of the nitrogen or the oxygen atom at 5-position of the tricyclic scaffold. Molecular dynamic study was done on a set of the new and known ligands to rationalize and to explain the lack of affinity on the 4- or 5-substituted new derivative. In fact, from biological results, it can be found that the only 5-unsubstituted new derivative, compound 15, has receptor recognition (Ki = 834.7 nM).

Synthesis and Pharmacological Evaluation of Indole Derivatives as Deaza Analogues of Potent Human Neutrophil Elastase Inhibitors.

Preclinical Research A number of N-benzoylindoles were designed and synthesized as deaza analogs of previously reported potent and selective HNE inhibitors with an indazole scaffold. The new compounds containing substituents and functions that were most active in the previous series were active in the micromolar range (the most potent had IC50 = 3.8 µM) or inactive. These results demonstrated the importance of N-2 in the indazole nucleus. Docking studies performed on several compounds containing the same substituents but with an indole or an indazole scaffold, respectively, highlight interesting aspects concerning the molecule orientation and H-bonding interactions, which could help to explain the lower activity of this new series. Drug Dev Res, 2016. © 2016 Wiley Periodicals, Inc.

Synthesis of five and six-membered heterocycles bearing an arylpiperazinylalkyl side chain as orally active antinociceptive agents.

A number of heterocycles bearing an arylpiperazinylalkyl side chain and structurally related to the previously described lead ET1 (4-amino-6-methyl-2-[3-(4-p-tolylpiperazin-1-yl)propyl]-5-vinylpyridazin-3(2H)-one) was synthesized and tested for their antinociceptive activity in Writhing Test. Many compounds, tested at doses of 20-40 mg/kg po were able to reduce the number of abdominal constrictions by more than 47% and, in same cases, the potency is comparable to lead ET1 as for 5e, 24a, 27b and 27c. The analgesia induced by the active compounds was completely prevented by pretreatment with α2-antagonist yohimbine, confirming the involvement of the adrenergic system in the mechanism of action for these new compounds.

Synthesis of novel cognition enhancers with pyrazolo[5,1-c][1,2,4]benzotriazine core acting at γ-aminobutyric acid type A (GABA(A)) receptor.

Memory dysfunction associated with aging, neurodegenerative and psychiatric disorders represents an increasing medical need. Advances in research exploring the biological mechanisms underlying learning and memory have opened new potential approaches for development of memory-enhancing therapies addressed to selective neuronal targets. In this work, we synthesized some derivatives with a pyrazolo[5,1-c][1,2,4]benzotriazine core to identify ligands on GABAA receptors subtype (benzodiazepine site on GABAA-receptor) endowed with the potential of enhancing cognition activity without the side effects usually associated with non-selective GABAA modulators. In fact, there is much evidence that GABAA-R (γ-aminobutyric acid, type A receptor) subtype ligands have relevance in learning and memory. In vitro and in vivo tests have been performed. Pharmacological data indicate that compounds 7, 13, 14 and 22 act as dual functional modulators of GABAA-Rs (promnemonic and anxiolytic agents) while only compounds 3 and 10 stand out as selectively displaying good antiamnesic and procognitive activity (1 and 3 mg/kg, respectively).

Development of ligands at γ-aminobutyrric acid type A (GABAA) receptor subtype as new agents for pain relief.

The identification of compounds with selective anxiolytic-like effects, exerted through the benzodiazepine site on γ-aminobutyric acid type A (GABA(A)) receptors, and that show pronounced antihyperalgesia in several pain models, has oriented research towards the development of new agents for the relief of pain. Starting from our previously reported ligands at the benzodiazepine site on GABA(A) receptors showing selective anxiolytic-like effects, we have designed new compounds with the aim of identifying those devoid of the typical side effects of the classical benzodiazepines. Our preliminary results indicate that compounds 4, 10(±) and 11 have a very promising antihyperalgesic profile in different animal pain models (peripheral mono-neuropathy, STZ-induced hyperalgesia). In particular 11 exhibits high potency since it exerted its protective effect starting from the dose of 3mg/kg po, after single injection.

New 3-, 8-disubstituted pyrazolo[5,1-c][1,2,4]benzotriazines useful for studying the interaction with the HBp-3 area (hydrogen bond point area) in the benzodiazepine site on the γ-aminobutyric acid type A (GABAA) receptor.

The pharmacophoric model using ADLR procedure, based on pyrazolo[5,1-c][1,2,4]benzotriazine system, studied in our laboratory, allowed us to identify the essential interaction points (HBp-1, HBp-2, and Lp-1) and the important areas for affinity modulation (HBp-3 and Lp-2) for binding recognition at benzodiazepine (Bzs) site of GABA(A) receptors (GABA(A)-Rs). In this work ADLR method is used to rationalize the affinity data of 23 new compounds and to improve the knowledge on HBp-3 area, hydrogen bond area. Among these new compounds emerge the pyrrolo derivatives (18, 25, 28, 34, and 37) for their good affinity value (14.9>K(i)(nM)>63.0). In the orientations proposed by ADLR, the NH moiety of the pyrrole ring, independently of the position in the pyrazolobenzotriazine core, fits in HBp-3 area and points out the acceptor feature of this hydrogen bond area, already known as donor area. Unexpectedly, the oxygen atom of the furane ring does not form efficient hydrogen bond with the same area, probably for an imperfect distance. The size of substituent at position 8 is important but not necessary for the receptor recognition, in fact the interdependence between the features of the 3- and 8-substituent's is again verified, (i.e., compound 20 vs 32).

Synthesis of new pyrazolo[5,1-c][1,2,4] benzotriazines, pyrazolo[5,1-c]pyrido[4,3-e][1,2,4] triazines and their open analogues as cytotoxic agents in normoxic and hypoxic conditions.

The synthesis and antitumor activity in normoxic and hypoxic conditions of a series of pyrazolo[5,1-c][1,2,4]benzotriazine and its related analogues are reported. All compounds were tested on human colorectal adenocarcinoma cell line HCT-8 and for compounds 15 and 20, which show to have selective cytotoxicity in hypoxic and in normoxic conditions respectively, ROS production, cell cycle, and DNA fragmentation were measured. This preliminary study encouraged us to consider 15 and 20 as interesting leads for further optimization.

Novel 3-aroylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted, ligands at GABAA/benzodiazepine receptor complex: synthesis, pharmacological and molecular modeling studies.

The synthesis and binding studies of a series of 3-acylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides 8-substituted are reported. High-affinity ligands at benzodiazepine site on GABA(A) receptor complex (GABA(A)/BzR complex) were obtained when the 3-aroyl substituent is represented by a five-member heteroaroyl ring (furoyl-, thenoyl-, and pyrroyl-). Moreover the type of heteroaroyl ring at position 3 influences the feature of the substituent at position 8 to obtain high-affinity ligands: a 'hydrogen-bond acceptor ring' at position 3 is synergic with an electron donor substituent at position 8, while a 'hydrogen-bond donor ring' is synergic with a withdrawing substituent. Compounds 8a, 9b, and 11 were deeply studied in vivo for their pharmacological effects considering six potential benzodiazepine actions: motor coordination, anticonvulsant action, spontaneous motor activity and explorative activity, anxiolytic-like effects, mouse learning and memory modulation, and ethanol-potentiating action. To rationalize and qualitatively interpret the GABA(A)/Bz binding affinities of compounds 8a and 11, a dynamic molecular modeling study has been performed, with the aim of assessing the preferred geometry of protein-ligand complex.

Identification of a New Pyrazolo[1,5-a]quinazoline Ligand Highly Affine to γ-Aminobutyric Type A (GABAA) Receptor Subtype with Anxiolytic-Like and Antihyperalgesic Activity.

Compounds that can act on GABAA receptor subtype in a selective manner, without the side effects of classical benzodiazepine ligands, represent promising therapeutic tools in neurological disorder as well as for relief of pain or in comorbidity of anxiety states and depression. Continuing our research on GABAA receptor subtype ligands, here is reported the synthesis of a series of pyrazolo[1,5-a]quinazoline 3- and/or 8-substituted as 5-deaza analogues of previous reported pyrazolo[5,1-c][1,2,4]benzotriazine, already identified as selective GABAA receptor subtype ligands endowed with anxiolytic-like and antihyperalgesic action or enhancer cognition. Between the new compounds stands out 12b for its high affinity value (Ki = 0.27 nM) and for its anxiolytic-like and ability to relieve neuropathic painful conditions evaluated in CCI and STZ murine model.

A novel selective GABA(A) alpha1 receptor agonist displaying sedative and anxiolytic-like properties in rodents.

In our pursuit to identify selective ligands for Bz/GABA(A) receptor subtypes, a novel pyrazolo[1,5-a]pyrimidine derivative (4), the azaisostere of zolpidem, was synthesized and evaluated in vitro on bovine brain homogenate and on recombinant benzodiazepine receptors (alphaxbeta2/3gamma2, x = 1-3, 5) expressed in HEK293 cells. Compound 4 displayed affinity only for alpha1beta2gamma2 subtype (K(i) = 31 nM), and in an in-depth, in vivo study it revealed sedative and anxiolytic-like properties without any amnesic and myorelaxant effects in rodents.

Further studies on pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones as potent and selective human A1 adenosine receptor antagonists.

A new series of pyrazolo[1',5':1,6]pyrimido[4,5-d]pyridazin-4(3H)-ones was synthesized and tested in radioligand binding assays on human A1, A2A and A3 adenosine receptors. Most of the compounds showed high selectivity of action towards A1 receptor and high affinity with Ki values in the low nanomolar range. The pharmacological profile of the most active molecules towards A1 adenosine receptors was evaluated in cAMP functional assay. Compounds demonstrated their ability to completely counteract the effect of the agonist NECA, thus demonstrating their antagonist profile. Moreover, the most interesting compound, tested in the mouse passive avoidance, exhibited an antiamnesic effect at the doses of 10 and 30 mg/kg.

Benzodiazepine receptor ligands. 7. Synthesis and pharmacological evaluation of new 3-esters of the 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide. 3-(2-Thienylmethoxycarbonyl) derivative: an anxioselective agent in rodents.

The synthesis and binding study of new 8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 3-ester compounds are reported. A pharmacological evaluation of the high-affinity ligands 1-4 belonging to the 3-heteroarylester series is made. The 3-(2-thienylmethoxycarbonyl) derivative 4 stands out from the other heteroarylesters and is found, using nine different behavioral methods, to be a functionally selective ligand in vivo: it shows anxiolytic-like activity in the conflict models (light-dark box and plus maze test) similarly to diazepam, without any sedative and amnesic properties or interference from alcohol.

Synthesis and benzodiazepine receptor affinity of pyrazolo[1,5-a]pyrimidine derivatives. 3. New 6-(3-thienyl) series as alpha 1 selective ligands.

New 3-aryl-6-(3-thienyl)pyrazolo[1,5-a]pyrimidin-7-ones (2a-j) are synthesized and evaluated in vitro on Bz/GABA(A) receptors and on recombinant benzodiazepine receptors (alpha x beta 2/3 gamma 2; x = 1-3, 5) expressed in HEK293 cells. SAR studies on the new compounds are conducted and molecular modeling is accomplished to better investigate requirements leading to subtype selectivity. Some of the synthesized compounds are tested in vivo to explore their pharmacological effect as a consequence of their high alpha 1 beta 2 gamma 2 subtype selectivity observed in vitro.

Benzodiazepine receptor ligands: a patent review (2006-2012).

INTRODUCTION: Ligands at the benzodiazepine site of the GABA(A) receptor (GABA(A)-R) act by modulating the effect of GABA(A) (γ-aminobutyric acid). The selective modulator effects of such ligands are related to the α-subunits type (i.e., α1, α2, α3, and α5), being shown that the α1 subunit is associated with sedative, anticonvulsant and amnesic effects; whereas the α2 and α3 subunits mediate anxiolytic and myorelaxant effects. Recently it was shown the involvement of α5 subunit in pain relief, which is involved in cognitive processes of learning and memory. AREAS COVERED: This review covers patents, published from January 2006 to October 2012, on ligands for the benzodiazepine binding site of the GABA(A)-Rs. Patents filed from different companies and research groups report many series of compounds that may be used in the treatment or prevention of a large variety of neurodegenerative diseases. EXPERT OPINION: Most patents highlighted that various memory deficits, related to Alzheimer's disease, Down syndrome, mood disorders, schizophrenia, and age-related cognitive impairment may be treated using α5-selective ligands. Other aspects related to the use of allosteric modulators of the α7-nAchR and/or α5-GABA(A)-R (dual approach) for alleviating the impaired cognition and the use of α2-selective ligands for pain relief are highlighted, being particularly intriguing as new therapeutic approaches.