Novel thienocycloalkylpyridazinones as useful scaffolds for acetylcholinesterase inhibition and serotonin 5-HT6 receptor interaction.

A library of eighteen thienocycloalkylpyridazinones was synthesized for human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibition and serotonin 5-HT6 receptor subtype interaction by following a multitarget-directed ligand approach (MTDL), as a suitable strategy for treatment of Alzheimer's disease (AD). The novel compounds featured a tricyclic scaffold, namely thieno[3,2-h]cinnolinone, thienocyclopentapyridazinone and thienocycloheptapyridazinone, connected through alkyl chains of variable length to proper amine moieties, most often represented by N-benzylpiperazine or 1-(phenylsulfonyl)-4-(piperazin-1-ylmethyl)-1H-indole as structural elements addressing AChE and 5-HT6 interaction, respectively. Our study highlighted the versatility of thienocycloalkylpyridazinones as useful architectures for AChE interaction, with several N-benzylpiperazine-based analogues emerging as potent and selective hAChE inhibitors with IC50 in the 0.17-1.23 µM range, exhibiting low to poor activity for hBChE (IC50 = 4.13-9.70 µM). The introduction of 5-HT6 structural moiety phenylsulfonylindole in place of N-benzylpiperazine, in tandem with a pentamethylene linker, gave potent 5-HT6 thieno[3,2-h]cinnolinone and thienocyclopentapyridazinone-based ligands both displaying hAChE inhibition in the low micromolar range and unappreciable activity towards hBChE. While docking studies provided a rational structural explanation for AChE/BChE enzyme and 5-HT6 receptor interaction, in silico prediction of ADME properties of tested compounds suggested further optimization for development of such compounds in the field of MTDL for AD.

RNAseq Analysis of Novel 1,3,4-Oxadiazole Chalcogen Analogues Reveals Anti-Tubulin Properties on Cancer Cell Lines.

1,3,4-Oxadiazole derivatives are among the most studied anticancer drugs. Previous studies have analyzed the action of different 1,3,4-oxadiazole derivatives and their effects on cancer cells. This study investigated the characterization of two new compounds named 6 and 14 on HeLa and PC-3 cancer cell lines. Based on the previously obtained IC50, cell cycle effects were monitored by flow cytometry. RNA sequencing (RNAseq) was performed to identify differentially expressed genes, followed by functional annotation using gene ontology (GO), KEGG signaling pathway enrichment, and protein-protein interaction (PPI) network analyses. The tubulin polymerization assay was used to analyze the interaction of both compounds with tubulin. The results showed that 6 and 14 strongly inhibited the proliferation of cancer cells by arresting them in the G2/M phase of the cell cycle. Transcriptome analysis showed that exposure of HeLa and PC-3 cells to the compounds caused a marked reprograming of gene expression. Functional enrichment analysis indicated that differentially expressed genes were significantly enriched throughout the cell cycle and cancer-related biological processes. Furthermore, PPI network, hub gene, and CMap analyses revealed that compounds 14 and 6 shared target genes with established microtubule inhibitors, indicating points of similarity between the two molecules and microtubule inhibitors in terms of the mechanism of action. They were also able to influence the polymerization process of tubulin, suggesting the potential of these new compounds to be used as efficient chemotherapeutic agents.

Target identification of a novel unsymmetrical 1,3,4-oxadiazole derivative with antiproliferative properties.

1,3,4-Oxadiazole derivatives are widely used in research on antineoplastic drugs. Recently, we discovered a novel unsymmetrical 1,3,4-oxadiazole compound with antiproliferative properties called 2j. To further investigate its possible targets and molecular mechanisms, RNA-seq was performed and the differentially expressed genes (DEGs) were obtained after treatment. Data were analyzed using functional (Gene Ontology term) and pathway (Kyoto Encyclopedia of Genes and Genomes) enrichment of the DEGs. The hub genes were determined by the analysis of protein-protein interaction networks. The connectivity map (CMap) information provided insight into the model action of antitumor small molecule drugs. Hub genes have been identified through function gene networks using STRING analysis. The small molecular targets obtained by CMap comparison showed that 2j is a tubulin inhibitor and it acts mainly affecting tumor cells through the cell cycle, FoxO signaling pathway, apoptotic, and p53 signaling pathways. The possible targets of 2j could be TUBA1A and TUBA4A. Molecular docking results indicated that 2j interacts at the colchicine-binding site on tubulin.

Tricyclic Pyrazole-Based Compounds as Useful Scaffolds for Cannabinoid CB1/CB2 Receptor Interaction.

Cannabinoids comprise different classes of compounds, which aroused interest in recent years because of their several pharmacological properties. Such properties include analgesic activity, bodyweight reduction, the antiemetic effect, the reduction of intraocular pressure and many others, which appear correlated to the affinity of cannabinoids towards CB1 and/or CB2 receptors. Within the search aiming to identify novel chemical scaffolds for cannabinoid receptor interaction, the CB1 antagonist/inverse agonist pyrazole-based derivative rimonabant has been modified, giving rise to several tricyclic pyrazole-based compounds, most of which endowed of high affinity and selectivity for CB1 or CB2 receptors. The aim of this review is to present the synthesis and summarize the SAR study of such tricyclic pyrazole-based compounds, evidencing, for some derivatives, their potential in the treatment of neuropathic pain, obesity or in the management of glaucoma.

Biological Effects on µ-Receptors Affinity and Selectivity of Arylpropenyl Chain Structural Modification on Diazatricyclodecane Derivatives.

Opioid analgesics are clinically used to relieve severe pain in acute postoperative and cancer pain, and also in the long term in chronic pain. The analgesic action is mediated by µ-, δ-, and κ-receptors, but currently, with few exceptions for k-agonists, µ-agonists are the only ones used in therapy. Previously synthesized compounds with diazotricyclodecane cores (DTDs) have shown their effectiveness in binding opioid receptors. Fourteen novel diazatricyclodecanes belonging to the 9-propionyl-10-substituted-9,10-diazatricyclo[4.2.1.12,5]decane (compounds 20-23, 53, 57 and 59) and 2-propionyl-7-substituted-2,7-diazatricyclo[4.4.0.03,8]decane (compounds 24-27, 54, 58 and 60) series, respectively, have been synthesized and their ability to bind to the opioid µ-, δ- and κ-receptors was evaluated. Five of these derivatives, compounds 20, 21, 24, 26 and 53, showed µ-affinity in the nanomolar range with a negligible affinity towards δ- and κ-receptors and high µ-receptor selectivity. The synthesized compounds showed µ-receptor selectivity higher than those of previously reported methylarylcinnamyl analogs.

Synthesis, biological evaluation and docking studies of a novel class of sulfur-bridged diazabicyclo[3.3.1]nonanes.

A small library of 3-thia-7,9-diazabicyclo[3.3.1]nonanes was synthesized and their opioid receptors affinity and selectivity evaluated. Among these novel sulfur-bridged compounds, the (E) 9-[3'-(3-chlorophenyl)-but-2'-en-1'-yl]-7-propionyl-3-thia-7,9-diazabicyclo[3.3.1]nonane 2i emerged as the derivative with the highest µ receptor affinity (Ki = 85 nM) and selectivity (Ki µ/δ = 58.8, Ki µ/κ > 117.6). The antinociceptive activity of 2i was also evaluated in acute thermal pain. Docking studies disclosed the specific pattern of interactions of these derivatives.

Development of Oxygen-Bridged Pyrazole-Based Structures as Cannabinoid Receptor 1 Ligands.

In this work, the synthesis of the cannabinoid receptor 1 neutral antagonists 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta[1,2-c]pyrazole-3-carboxamide 1a and its deaza N-cyclohexyl analogue 1b has led to a deepening of the structure-activity studies of this class of compounds. A series of novel 4,5-dihydrobenzo-oxa-cycloheptapyrazoles analogues of 1a,b, derivatives 1c-j, was synthesized, and their affinity towards cannabinoid receptors was determined. Representative terms were evaluated using in vitro tests and isolated organ assays. Among the derivatives, 1d and 1e resulted in the most potent CB1 receptor ligands (KiCB1 = 35 nM and 21.70 nM, respectively). Interestingly, both in vitro tests and isolated organ assays evidenced CB1 antagonist activity for the majority of the new compounds, excluding compound 1e, which showed a CB1 partial agonist behaviour. CB1 antagonist activity of 1b was further confirmed by a mouse gastrointestinal transit assay. Significant activity of the new CB1 antagonists towards food intake was showed by preliminary acute assays, evidencing the potentiality of these new derivatives in the treatment of obesity.

Novel sulfenamides and sulfonamides based on pyridazinone and pyridazine scaffolds as CB1 receptor ligand antagonists.

A series of sulfenamide and sulfonamide derivatives was synthesized and evaluated for the affinity at CB1 and CB2 receptors. The N-bornyl-S-(5,6-di-p-tolylpyridazin-3-yl)-sulfenamide, compound 11, displayed good affinity and high selectivity for CB1 receptors (Ki values of 44.6 nM for CB1 receptors and >40 µM for CB2 receptors, respectively). The N-isopinocampheyl-sulfenamide 12 and its sulfonamide analogue 22 showed similar selectivity for CB1 receptors with Ki values of 75.5 and 73.2 nM, respectively. These novel compounds behave as antagonists/inverse agonists at CB1 receptor in the [35S]-GTPγS binding assays, and none showed adequate predictive blood-brain barrier permeation, exhibiting low estimated LD50. However, testing compound 12 in a supraspinal analgesic test (hot-plate) revealed that it was as effective as the classic CB1 receptor antagonist rimonabant, in reversing the analgesic effect of a cannabinoid agonist.

Synthesis, molecular modeling and SAR study of novel pyrazolo[5,1-f][1,6]naphthyridines as CB2 receptor antagonists/inverse agonists.

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB1 and CB2 receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a-i) or a 2,4-dichlorophenyl (8j) motif at the C5-position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C2-position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB2 receptor affinity (Ki=33nM) and a high degree of selectivity (KiCB1/KiCB2=173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, Ki=53nM) and the myrtanyl derivative 8j (Ki=67nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB2 inverse agonists.

Novel diazabicycloalkane delta opioid agonists.

Here we report the investigation of diazabicycloalkane cores as potential new scaffolds for the development of novel analogues of the previously reported diazatricyclodecane selective delta (δ) opioid agonists, as conformationally constrained homologues of the reference δ agonist (+)-4-[(αR)-α((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80). In particular, we have simplified the diazatricyclodecane motif of δ opioid agonist prototype 1a with bridged bicyclic cores. 3,6-diazabicyclo[3.1.1]heptane, 3,8-diazabicyclo[3.2.1]octane, 3,9-diazabicyclo[3.3.1]nonane, 3,9-diazabicyclo[4.2.1]nonane, and 3,10-diazabicyclo[4.3.1]decane were adopted as core motifs of the novel derivatives. The compounds were synthesized and biologically assayed as racemic (3-5) or diastereoisomeric (6,7) mixtures. All the novel compounds 3-7 showed δ agonism behaviour and remarkable affinity to δ receptors. Amongst the novel derivatives, 3,8-diazabicyclo[3.2.1]octane based compound 4 evidenced improved δ affinity and selectivity relative to SNC80.

NESS06SM reduces body weight with an improved profile relative to SR141716A.

We have recently synthesized a new series of 4,5-dihydrobenzo-oxa-cycloheptapyrazole derivatives with the aim to discover novel CB1 antagonist agents characterized by anti-obesity activity comparable to that of SR141716A but with reduced adverse effects such as anxiety and depression. Within the novel class, the CB1 antagonist 8-chloro-1-(2,4-dichlorophenyl)-N-piperidin-1-yl-4,5-dihydrobenzo-1H-6-oxa-cyclohepta(1,2-c)pyrazole-3-carboxamide (NESS06SM) has been selected as lead compound. We found that NESS06SM is a CB1 neutral antagonist, characterized by poor blood-brain barrier permeability. Moreover, NESS06SM chronic treatment determined both anti-obesity effect and cardiovascular risk factor improvement in C57BL/6N Diet Induced Obesity (DIO) mice fed with fat diet (FD mice). In fact, the mRNA gene expression in Central Nervous System (CNS) and peripheral tissues by real time PCR, showed a significant increase of orexigenic peptides and a decrease of anorexigenic peptides elicited by NESS06SM treatment, compared to control mice fed with the same diet. Moreover, in contrast to SR141716A treatment, the chronic administration of NESS06SM did not change mRNA expression of both monoaminergic transporters and neurotrophins highly related with anxiety and mood disorders. Our results suggest that NESS06SM reduces body weight and it can restore the disrupted expression profile of genes linked to the hunger-satiety circuit without altering monoaminergic transmission probably avoiding SR141716A side effects. Therefore the novel CB1 neutral antagonist could represent a useful candidate agent for the treatment of obesity and its metabolic complications.

Novel 1,3,4-oxadiazole chalcogen analogues: Synthesis and cytotoxic activity.

A small library of novel 1,3,4-oxadiazole bioisosteres was synthesized and their cytotoxic activity evaluated in vitro. Five of the new derivatives (3, 6, 11, 14 and 15) showed high potency against different human cancer cell lines, with 14 being the most interesting compound endowed with IC50 ranging from 0.005 to 0.091 µM. Preliminary SAR studies have suggested that the-chlorine atom in ortho position of the phenyl ring on the 1,3,4-selenadiazole is important for antitumor potency in vitro. Notably, these new compounds showed stronger anti-tumor activity than the previously synthesized and published oxadiazole lead compound 2. Furthermore, the cytotoxic effect was only relevant in tumor cells compared to human primary cells. These results suggest that the nature of the selenadiazole and thiadiazole rings may be even more important for antitumor potency in vitro than the nature of the previously described oxadiazole. All five compounds resulted in a G2/M arrest of the cell cycle and activated an apoptotic response. The colony formation assay showed the long-term effect of the compounds on tumor lines in vitro. Immunofluorescence analysis of ß-tubulin indicated that all compounds interacted with micro-tubulin organization and mitotic spindle formation causing aberrant cell formation. For these reasons, the new molecules 3, 6, 11, 14 and 15 could be good candidates in preventive and chemotherapeutic strategies.

Synthesis and SAR study of new phenylimidazole-pyrazolo[1,5-c]quinazolines as potent phosphodiesterase 10A inhibitors.

A series of phenylimidazole-pyrazolo[1,5-c]quinazolines 1a-q was designed, synthesized and characterised as a novel class of potent phophodiesterase 10A (PDE10A) inhibitors. In this series, 2,9-dimethyl-5-(2-(1-methyl-4-phenyl-1H-imidazol-2-yl)ethyl)pyrazolo[1,5-c]quinazoline (1q) showed the highest affinity for PDE10A enzyme (IC(50)=16nM).

Preliminary Anti-Coxsackie Activity of Novel 1-[4-(5,6-dimethyl(H)- 1H(2H)-benzotriazol-1(2)-yl)phenyl]-3-alkyl(aryl)ureas.

BACKGROUND: Coxsackievirus infections are associated with cases of aseptic meningitis, encephalitis, myocarditis, and some chronic disease. METHODS: A series of benzo[d][1,2,3]triazol-1(2)-yl derivatives (here named benzotriazol-1(2)-yl) (4a-i, 5a-h, 6a-e, g, i, j and 7a-f, h-j) were designed, synthesized and in vitro evaluated for cytotoxicity and antiviral activity against two important human enteroviruses (HEVs) members of the Picornaviridae family [Coxsackievirus B 5 (CVB-5) and Poliovirus 1 (Sb-1)]. RESULTS: Compounds 4c (CC50 >100 µM; EC50 = 9 µM), 5g (CC50 >100 µM; EC50 = 8 µM), and 6a (CC50 >100 µM; EC50 = 10 µM) were found active against CVB-5. With the aim of evaluating the selectivity of action of this class of compounds, a wide spectrum of RNA (positive- and negativesense), double-stranded (dsRNA) or DNA viruses were also assayed. For none of them, significant antiviral activity was determined. CONCLUSION: These results point towards a selective activity against CVB-5, an important human pathogen that causes both acute and chronic diseases in infants, young children, and immunocompromised patients.

5,6-Dichloro-2-phenyl-benzotriazoles: New Potent Inhibitors of Orthohantavirus.

Orthohantaviruses, previously known as hantaviruses (family Hantaviridae, order Bunyavirales), are emerging zoonoses hosted by different rodent and insectivore species. Orthohantaviruses are transmitted by aerosolized excreta (urine, saliva and feces) of their reservoir hosts. When transmitted to humans, they cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe and hantavirus (cardio) pulmonary syndrome (HPS) in the Americas. Clinical studies have shown that early treatments of HFRS patients with ribavirin (RBV) improve prognosis. Nevertheless, there is the need for urgent development of specific antiviral drugs. In the search for new RNA virus inhibitors, we recently identified a series of variously substituted 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazole derivatives active against the human respiratory syncytial virus (HRSV). Interestingly, several 2-phenyl-benzotriazoles resulted in fairly potent inhibitors of the Hantaan virus in a chemiluminescence focus reduction assay (C-FRA) showing an EC50 = 4-5 µM, ten-fold more active than ribavirin. Currently, there are no FDA approved drugs for the treatment of orthohantavirus infections. Antiviral activities and cytotoxicity profiles suggest that 5,6-dichloro-1(2)-phenyl-1(2)H-benzo[d][1,2,3]triazoles could be promising candidates for further investigation as a potential treatment of hantaviral diseases.

Synthesis and cytotoxicity of novel hexahydrothienocycloheptapyridazinone derivatives.

Designed as a new group of tricyclic molecules containing the thienocycloheptapyridazinone ring system, a number of 2N-substituted-hexahydrothienocycloheptapyridazinone derivatives were synthesized and their biological activity evaluated. Among the synthesized compounds, derivatives 7d and 7h were found to possess cytotoxic activity against non-small cell lung cancer and central nervous system cancer cell lines, respectively.

Novel N-aryl nicotinamide derivatives: Taking stock on 3,6-diazabicyclo[3.1.1]heptanes as ligands for neuronal acetylcholine receptors.

We designed the synthesis of a small library of 3-substituted-3,6-diazabicyclo[3.1.1]heptanes whose affinity on neuronal nicotinic receptors (nAChRs) was evaluated. Among the synthesized compounds, the 5-(3,6-diazabicyclo[3.1.1]heptane-3-yl)-N-(2-fluorophenyl)nicotinamide 43 proved to be the most interesting compound with α4ß2Ki value of 10 pM and a very high α7/α4ß2 selectivity. Furthermore, compounds 35, 39 and 43 elicited a selective partial agonist activity for α4ß2 nAChR subtype. Finally, in this paper we also report the conclusions on the 3,6-diazabicyclo[3.1.1]heptanes as ligands for nAChRs, resulting from our consolidated structure activity relationship (SAR) studies on this template.

Synthesis of 3,6-diazabicyclo[3.1.1]heptanes as novel ligands for neuronal nicotinic acetylcholine receptors.

Alpha series of novel 3,6-diazabicyclo[3.1.1]heptane derivatives 4a-f was synthesized and their affinity and selectivity towards alpha4beta2 and alpha7 nAChR subtypes were evaluated. The results of the current study revealed a number of compounds (4a, 4b and 4c) having a very high affinity for alpha4beta2 (K(i) at alpha4beta2 ranging from 0.023 to 0.056 nM) versus alpha7 nAChR subtypes; among these compounds, the 3-(6-bromopyridin-3-yl)-3,6-diazabicyclo[3.1.1]heptane 4c was found to be the most alpha7alpha4beta2 selective term in receptor binding assays (alpha7alpha4beta2=1295). Moreover, compound 4d also had high affinity for the alpha4beta2 nAChR subtype (K(i)=1.2 nM) with considerably high selectivity (alpha7/alpha4beta2=23300).

Novel pyrrolocycloalkylpyrazole analogues as CB1 ligands.

Novel 1,4-dihydropyrazolo[3,4-a]pyrrolizine-, 4,5-dihydro-1H-pyrazolo[4,3-g]indolizine- and 1,4,5,6-tetrahydropyrazolo[3,4-c]pyrrolo[1,2-a]azepine-3-carboxamide-based compounds were designed and synthesized for cannabinoid CB1 and CB2 receptor interactions. Any of the new synthesized compounds showed high affinity for CB2 receptor with Ki values superior to 314 nm, whereas some of them showed moderate affinity for CB1 receptor with Ki values inferior to 400 nm. 7-Chloro-1-(2,4-dichlorophenyl)-N-(homopiperidin-1-yl)-4,5-dihydro-1H-pyrazolo[4,3-g]indolizine-3-carboxamide (2j) exhibited good affinity for CB1 receptor (Ki CB1  = 81 nm) and the highest CB2 /CB1 selectively ratio (>12). Docking studies carried out on such compounds were performed using the hCB1 X-ray in complex with the close pyrazole analogue AM6538 and disclosed specific pattern of interactions related to the tricyclic pyrrolopyrazole scaffolds as CB1 ligands.

Synthesis, Pharmacological Evaluation, and Docking Studies of Novel Pyridazinone-Based Cannabinoid Receptor Type†2 Ligands.

In recent years, cannabinoid type 2 receptors (CB2 R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB2 R are devoid of the psychoactive effects typically observed for CB1 R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure-activity relationships for this promising scaffold with the aim to develop potent CB2 R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB2 R affinity, with Ki values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB2 R inverse agonists. Compound 22 displayed the highest CB2 R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48.