Azide-based in situ preparation of fused heterocyclic imines and their multicomponent reactions.

Structurally diverse pyrroles, indoles and imidazoles bearing an N-ω-azidoalkyl moiety and an aldehyde or ketone function were prepared and successfully introduced into in situ imine generation via the intramolecular Staudinger/aza-Wittig tandem reaction. Reduction of the generated imines led to medicinally relevant nitrogen-containing fused heterocycles such as tetrahydropyrrolo[1,2-a]pyrazines and diazepines. Rare 8-membered hexahydropyrrolo[1,2-a][1,4]diazocine and 9-membered dihydro-4,8-(metheno)pyrrolo[1,2-a][1,4]diazacycloundecine were also synthesized. In addition, several one-pot transformations involving cyclic anhydrides or isocyanides (Castagnoli-Cushman, Ugi and azido-Ugi reactions) were added to the Staudinger/aza-Wittig sequence to afford novel fused polyheterocyclic delta-lactams, cyclic bisamides and tetrazoles in a multicomponent fashion. The synthesized compounds were profiled against human Trace Amine-Associated Receptor 1 (hTAAR1), and the best performing compound showed low nanomolar agonistic activity with an EC50 of 0.025 µM.

Discovery of a Novel Chemo-Type for TAAR1 Agonism via Molecular Modeling.

The search for novel effective TAAR1 ligands continues to draw great attention due to the wide range of pharmacological applications related to TAAR1 targeting. Herein, molecular docking studies of known TAAR1 ligands, characterized by an oxazoline core, have been performed in order to identify novel promising chemo-types for the discovery of more active TAAR1 agonists. In particular, the oxazoline-based compound S18616 has been taken as a reference compound for the computational study, leading to the development of quite flat and conformationally locked ligands. The choice of a "Y-shape" conformation was suggested for the design of TAAR1 ligands, interacting with the protein cavity delimited by ASP103 and aromatic residues such as PHE186, PHE195, PHE268, and PHE267. The obtained results allowed us to preliminary in silico screen an in-house series of pyrimidinone-benzimidazoles (1a-10a) as a novel scaffold to target TAAR1. Combined ligand-based (LBCM) and structure based (SBCM) computational methods suggested the biological evaluation of compounds 1a-10a, leading to the identification of derivatives 1a-3a (hTAAR1 EC50 = 526.3-657.4 nM) as promising novel TAAR1 agonists.

Discovery and In Vivo Efficacy of Trace Amine-Associated Receptor 1 (TAAR1) Agonist 4-(2-Aminoethyl)-N-(3,5-dimethylphenyl)piperidine-1-carboxamide Hydrochloride (AP163) for the Treatment of Psychotic Disorders.

Starting from a screening hit, a set of analogs was synthesized based on a 4-(2-aminoethyl)piperidine core not associated previously with trace amine-associated receptor 1 (TAAR1) modulation in the literature. Several structure-activity relationship generalizations have been drawn from the observed data, some of which were corroborated by molecular modeling against the crystal structure of TAAR1. The four most active compounds (EC50 for TAAR1 agonistic activity ranging from 0.033 to 0.112 µM) were nominated for evaluation in vivo. The dopamine transporter knockout (DAT-KO) rat model of dopamine-dependent hyperlocomotion was used to evaluate compounds' efficacy in vivo. Out of four compounds, only one compound (AP163) displayed a statistically significant and dose-dependent reduction in hyperlocomotion in DAT-KO rats. As such, compound AP163 represents a viable lead for further preclinical characterization as a potential novel treatment option for disorders associated with increased dopaminergic function, such as schizophrenia.

The Expression of Trace Amine-Associated Receptors (TAARs) in Breast Cancer Is Coincident with the Expression of Neuroactive Ligand-Receptor Systems and Depends on Tumor Intrinsic Subtype.

Currently, the contribution of trace amine-associated receptors (TAARs) to breast cancer (BC) is recognized, but their associations with various pathological characteristics are not yet understood. There is accumulated transcriptomic data for BC tumors, which are represented in publicly accessible databases. We estimated TAARs' (including TAAR1, TAAR2, TAAR5, TAAR6, TAAR8, and TAAR9) associations with BC stage, grade, and molecular subtypes in these data and identified that the expression of all TAARs was associated with more unfavorable cancer subtypes, including basal-like and HER2-positive tumors. Also, the significant upregulation of all TAARs was demonstrated in circulating tumor cells compared to the metastatic lesions. Considering that co-expressed genes are more likely to be involved in the same biologic processes, we analyzed genes that are co-expressed with TAARs in BC. These gene sets were enriched with the genes of the olfactory transduction pathway and neuroactive ligand-receptor interaction participants. TAARs are co-expressed with G-protein-coupled receptors of monoamine neurotransmitters including dopamine, norepinephrine, and serotonin as well as with other neuroactive ligand-specific receptors. Since TAAR1 is able to modulate the activity of monoamine receptors that are involved in the regulation of BC growth, TAAR1 and potentially other TAARs may be regarded as prospective therapeutic targets for breast cancer.

Discovery of Guanfacine as a Novel TAAR1 Agonist: A Combination Strategy through Molecular Modeling Studies and Biological Assays.

Trace amine-associated receptor 1 (TAAR1) is an attractive target for the design of innovative drugs to be applied in diverse pharmacological settings. Due to a non-negligible structural similarity with endogenous ligands, most of the agonists developed so far resulted in being affected by a low selectivity for TAAR1 with respect to other monoaminergic G protein-coupled receptors, like the adrenoreceptors. This study utilized comparative molecular docking studies and quantitative-structure activity relationship (QSAR) analyses to unveil key structural differences between TAAR1 and alpha2-adrenoreceptor (α2-ADR), with the aim to design novel TAAR1 agonists characterized by a higher selectivity profile and reduced off-target effects. While the presence of hydrophobic motives is encouraged towards both the two receptors, the introduction of polar/positively charged groups and the ligand conformation deeply affect the TAAR1 or α2-ADR putative selectivity. These computational methods allowed the identification of the α2A-ADR agonist guanfacine as an attractive TAAR1-targeting lead compound, demonstrating nanomolar activity in vitro. In vivo exploration of the efficacy of guanfacine showed that it is able to decrease the locomotor activity of dopamine transporter knockout (DAT-KO) rats. Therefore, guanfacine can be considered as an interesting template molecule worthy of structural optimization. The dual activity of guanfacine on both α2-ADR and TAAR1 signaling and the related crosstalk between the two pathways will deserve more in-depth investigation.

Increased dopamine transmission and adult neurogenesis in trace amine-associated receptor 5 (TAAR5) knockout mice.

Trace amine-associated receptors (TAARs) are a class of sensory G protein-coupled receptors that detect biogenic amines, products of decarboxylation of amino acids. The majority of TAARs (TAAR2-TAAR9) have been described mainly in the olfactory epithelium and considered to be olfactory receptors sensing innate odors. However, there is recent evidence that one of the members of this family, TAAR5, is expressed also in the limbic brain areas receiving projection from the olfactory system and involved in the regulation of emotions. In this study, we further characterized a mouse line lacking TAAR5 (TAAR5 knockout, TAAR5-KO mice) that express beta-galactosidase mapping TAAR5 expression. We found that in TAAR5-KO mice the number of dopamine neurons, the striatal levels of dopamine and its metabolites, as well as striatal levels of GDNF mRNA, are elevated indicating a potential increase in dopamine neuron proliferation. Furthermore, an analysis of TAAR5 beta-galactosidase expression revealed that TAAR5 is present in the major neurogenic areas of the brain such as the subventricular zone (SVZ), the subgranular zone (SGZ) and the less characterized potentially neurogenic zone surrounding the 3rd ventricle. Direct analysis of neurogenesis by using specific markers doublecortin (DCX) and proliferating cell nuclear antigen (PCNA) revealed at least 2-fold increase in the number of proliferating neurons in the SVZ and SGZ of TAAR5-KO mice, but no such markers were detected in mutant or control mice in the areas surrounding the 3rd ventricle. These observations indicate that TAAR5 involved not only in regulation of emotional status but also adult neurogenesis and dopamine transmission. Thus, future TAAR5 antagonists may exert not only antidepressant and/or anxiolytic action but may also provide new treatment opportunity for neurodegenerative disorders such as Parkinson's disease.

Novel 1-Amidino-4-Phenylpiperazines as Potent Agonists at Human TAAR1 Receptor: Rational Design, Synthesis, Biological Evaluation and Molecular Docking Studies.

Targeting trace amine-associated receptor 1 (TAAR1) receptor continues to offer an intriguing opportunity to develop innovative therapies in different pharmacological settings. Pursuing our endeavors in the search for effective and safe human TAAR1 (hTAAR1) ligands, we synthesized a new series of 1-amidino-4-phenylpiperazine derivatives (1-16) based on the application of a combined pharmacophore model/scaffold simplification strategy for an in-house series of biguanide-based TAAR1 agonists. Most of the novel compounds proved to be more effective than their prototypes, showing nanomolar EC50 values in functional activity at hTAAR1 and low general cytotoxicity (CC50 > 80 µM) when tested on the Vero-76 cell line. In this new series, the main determinant for TAAR1 agonism ability appears to result from the appropriate combination between the steric size and position of the substituents on the phenyl ring rather than from their different electronic nature, since both electron-withdrawing and electron donor groups are permitted. In particular, the ortho-substitution seems to impose a more appropriate spatial geometry to the molecule that entails an enhanced TAAR1 potency profile, as experienced, in the following order, by compounds 15 (2,3-diCl, EC50 = 20 nM), 2 (2-CH3, EC50 = 30 nM), 6 (2-OCH3, EC50 = 93 nM) and 3 (2-Cl, EC50 = 160 nM). Apart from the interest in them as valuable leads for the development of promising hTAAR1 agonists, these simple small molecules have further allowed us to identify the minimal structural requirements for producing an efficient hTAAR1 targeting ability.

Increased subventricular zone-derived cortical neurogenesis after ischemic lesion.

In adult rodents stroke enhances neurogenesis resulting in the addition of neurons to forebrain regions such as striatum or cortex where postnatal neurogenesis under normal conditions plays a negligible role. In the cortex, new neurons are generated either from local cortical precursors that are activated by stroke or from precursors residing in the subventricular zone (SVZ) of lateral ventricles that under normal conditions supply neuroblasts by and large only for the olfactory bulb. In this study we used 5HT3A-EGFP transgenic mice in which all neuroblasts originating in the SVZ are EGFP-labeled. We induced stroke in these mice and by combination of EGFP detection with BrdU injections we labeled all post-stroke-generated SVZ-derived neuroblasts. We showed an increase in SVZ-derived neuroblasts 14 and 35 days after stroke in the ipsilateral hemisphere. Post-stroke-generated SVZ-derived neuroblasts migrated to the cortex and survived for at least 35 days representing 2% of BrdU-positive cells in peri-infarct area where they differentiate into mature neurons. Thus, stroke enhances SVZ neurogenesis and attracts newborn neurons to the injury zone.