Discovery and characterization of benzyloxy piperidine based dopamine 4 receptor antagonists.

The dopamine receptor 4 (D4R) is highly expressed in both motor, associative and limbic subdivisions of the cortico-basal ganglia network. Due to the distribution in the brain, there is mounting evidence pointing to a role for the D4R in the modulation of this network and its subsequent involvement in l-DOPA induced dyskinesias in Parkinson's disease. As part of our continued effort in the discovery of novel D4R antagonists, we report the discovery and characterization of a new 3- or 4-benzyloxypiperidine scaffold as D4R antagonists. We report several D4R selective compounds (>30-fold vs. other dopamine receptor subtypes) with improved in vitro and in vivo stability over previously reported D4R antagonists.

Design, synthesis and preliminary bioactivity evaluation of bitopic benzopyranomorpholine analogues as selective dopamine D3 receptor ligands as anti-drug addiction therapeutic agents.

Three series of bitopic benzopyranomorpholine analogues were designed, synthesized, and evaluated as a novel class of selective ligands for the dopamine D3 receptor. Binding affinities of target compounds were determined using the method of radioligand binding assay. Most compounds demonstrated considerable binding affinities and selectivity for D3 receptor. Besides, the compounds were screened for their ability to alleviate withdrawal symptoms of opioid addiction in animal behavioral models. The results showed that compound 20h displayed nanomolar affinity for the D3R, and exhibited anti-drug addiction efficacy in the animal model of of naloxone-induced withdrawal symptoms in morphine-dependent mice.

Synthesis, in silico, and in vitro studies of novel dopamine D2 and D3 receptor ligands.

Dopamine is an important neurotransmitter in the human brain and its altered concentrations can lead to various neurological diseases. We studied the binding of novel compounds at the dopamine D2 (D2 R) and D3 (D3 R) receptor subtypes, which belong to the D2 -like receptor family. The synthesis, in silico, and in vitro characterization of 10 dopamine receptor ligands were performed. Novel ligands were docked into the D2 R and D3 R crystal structures to examine the precise binding mode. A quantum mechanics/molecular mechanics study was performed to gain insights into the nature of the intermolecular interactions between the newly introduced pentafluorosulfanyl (SF5 ) moiety and D2 R and D3 R. A radioligand displacement assay determined that all of the ligands showed moderate-to-low nanomolar affinities at D2 R and D3 R, with a slight preference for D3 R, which was confirmed in the in silico studies. N-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl}-4-(pentafluoro-λ6-sulfanyl)benzamide (7i) showed the highest D3 R affinity and selectivity (pKi values of 7.14 [D2 R] and 8.42 [D3 R]).

Recent findings leading to the discovery of selective dopamine D4 receptor ligands for the treatment of widespread diseases.

Since its discovery, the dopamine D4 receptor (D4R) has been suggested to be an attractive target for the treatment of neuropsychiatric diseases. Novel findings have renewed the interest in such a receptor as an emerging target for the management of different diseases, including cancer, Parkinson's disease, alcohol or substance use disorders, eating disorders, erectile dysfunction and cognitive deficits. The recently resolved crystal structures of D4R in complexes with the potent ligands nemonapride and L-745870 strongly improved the knowledge on the molecular mechanisms involving the D4R functions and may help medicinal chemists in drug design. This review is focused on the recent development of the subtype selective D4R ligands belonging to classical or new chemotypes. Moreover, ligands showing functional selectivity toward G protein activation or ß-arrestin recruitment and the effects of selective D4R ligands on the above-mentioned diseases are discussed.

Quantitative analysis of fluorescent ligand binding to dopamine D3 receptors using live-cell microscopy.

Dopamine receptors are G protein-coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright-field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it into a data management software aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS-Cy3B was synthesized by conjugating a dopaminergic antagonist N-(p-aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS-Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the membrane tools software, enables the detection and quantification of ligand binding with a high-throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the screening of novel unlabeled dopaminergic ligands. We propose that this simple yet more native-like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.

Synthesis and dopamine receptor pharmacological evaluations on ring C ortho halogenated 1-phenylbenzazepines.

A series of 1-phenylbenzazepines containing bromine or chlorine substituents at the ortho position of the appended phenyl ring (2'-monosubstituted or 2',6'- disubstituted patterns) were synthesized and evaluated for affinity towards dopamine D1R, D2R and D5R. As is typical of the 1-phenylbenzazepine scaffold, the compounds displayed selectivity towards D1R and D5R; analogs generally lacked affinity for D2R. Interestingly, 2',6'-dichloro substituted analogs showed modest D5R versus D1R selectivity whereas this selectivity was reversed in compounds with a 2'-halo substitution pattern. Compound 10a was identified as a D1R antagonist (Ki = 14 nM; IC50 = 9.4 nM).

Design and Synthesis of Bitopic 2-Phenylcyclopropylmethylamine (PCPMA) Derivatives as Selective Dopamine D3 Receptor Ligands.

2-Phenylcyclopropylmethylamine (PCPMA) analogues have been reported as selective serotonin 2C agonists. On the basis of the same scaffold, we designed and synthesized a series of bitopic derivatives as dopamine D3R ligands. A number of these new compounds show a high binding affinity for D3R with excellent selectivity. Compound (1R,2R)-22e and its enantiomer (1S,2S)-22e show a comparable binding affinity for the D3R, but the former is a potent D3R agonist, while the latter acts as an antagonist. Molecular docking studies revealed different binding poses of the PCPMA moiety within the orthosteric binding pocket of the D3R, which might explain the different functional profiles of the enantiomers. Compound (1R,2R)-30q shows a high binding affinity for the D3R (Ki = 2.2 nM) along with good selectivity, as well as good bioavailability and brain penetration properties in mice. These results reveal that the PCPMA scaffold may serve as a privileged scaffold for the design of aminergic GPCR ligands.

Structure-based development of caged dopamine D2/D3 receptor antagonists.

Dopamine is a neurotransmitter of great physiological relevance. Disorders in dopaminergic signal transduction are associated with psychiatric and neurological pathologies such as Parkinson's disease, schizophrenia and substance abuse. Therefore, a detailed understanding of dopaminergic neurotransmission may provide access to novel therapeutic strategies for the treatment of these diseases. Caged compounds with photoremovable groups represent molecular tools to investigate a biological target with high spatiotemporal resolution. Based on the crystal structure of the D3 receptor in complex with eticlopride, we have developed caged D2/D3 receptor ligands by rational design. We initially found that eticlopride, a widely used D2/D3 receptor antagonist, was photolabile and therefore is not suitable for caging. Subtle structural modification of the pharmacophore led us to the photostable antagonist dechloroeticlopride, which was chemically transformed into caged ligands. Among those, the 2-nitrobenzyl derivative 4 (MG307) showed excellent photochemical stability, pharmacological behavior and decaging properties when interacting with dopamine receptor-expressing cells.

Huntington's Disease: A Review of the Known PET Imaging Biomarkers and Targeting Radiotracers.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a CAG expansion mutation in the huntingtin gene. As a result, intranuclear inclusions of mutant huntingtin protein are formed, which damage striatal medium spiny neurons (MSNs). A review of Positron Emission Tomography (PET) studies relating to HD was performed, including clinical and preclinical data. PET is a powerful tool for visualisation of the HD pathology by non-invasive imaging of specific radiopharmaceuticals, which provide a detailed molecular snapshot of complex mechanistic pathways within the brain. Nowadays, radiochemists are equipped with an impressive arsenal of radioligands to accurately recognise particular receptors of interest. These include key biomarkers of HD: adenosine, cannabinoid, dopaminergic and glutamateric receptors, microglial activation, phosphodiesterase 10 A and synaptic vesicle proteins. This review aims to provide a radiochemical picture of the recent developments in the field of HD PET, with significant attention devoted to radiosynthetic routes towards the tracers relevant to this disease.

Inclusion of enclosed hydration effects in the binding free energy estimation of dopamine D3 receptor complexes.

Confined hydration and conformational flexibility are some of the challenges encountered for the rational design of selective antagonists of G-protein coupled receptors. We present a set of C3-substituted (-)-stepholidine derivatives as potent binders of the dopamine D3 receptor. The compounds are characterized biochemically, as well as by computer modeling using a novel molecular dynamics-based alchemical binding free energy approach which incorporates the effect of the displacement of enclosed water molecules from the binding site. The free energy of displacement of specific hydration sites is obtained using the Hydration Site Analysis method with explicit solvation. This work underscores the critical role of confined hydration and conformational reorganization in the molecular recognition mechanism of dopamine receptors and illustrates the potential of binding free energy models to represent these key phenomena.

Synthesis, Characterisation and In Vitro Permeation, Dissolution and Cytotoxic Evaluation of Ruthenium(II)-Liganded Sulpiride and Amino Alcohol.

Sulpiride (SPR) is a selective antagonist of central dopamine receptors but has limited clinical use due to its poor pharmacokinetics. The aim of this study was to investigate how metal ligation to SPR may improve its solubility, intestinal permeability and prolong its half-life. The synthesis and characterisation of ternary metal complexes [Ru(p -cymene)(L)(SPR)]PF6 (L1 = (R)-(+)-2-amino-3-phenyl-1-propanol, L2 = ethanolamine, L3 = (S)-(+)-2-amino-1-propanol, L4 = 3-amino-1-propanol, L5 = (S)-(+)-2-pyrrolidinemethanol) are described in this work. The stability constant of the [Ru(p -cymene)(SPR)] complex was determined using Job's method. The obtained value revealed higher stability of the metal complex in the physiological pH than in an acidic environment such as the stomach. The ternary metal complexes were characterised by elemental analysis, Fourier transform infrared spectroscopy (FT-IR), 1H and 13C nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermal analyses, Ultraviolet-Visible (UV-Vis). Solubility studies showed higher aqueous solubility for complexed SPR than the free drug. Dissolution profiles of SPR from the metal complexes exhibited slower dissolution rate of the drug. Permeation studies through the pig's intestine revealed enhanced membrane permeation of the complexed drug. In vitro methyl thiazolyl tetrazolium (MTT) assay showed no noticeable toxic effects of the ternary metal complexes on Caco-2 cell line.

Design and synthesis of bridged piperidine and piperazine isosteres.

We have developed versatile methods toward the synthesis of a variety of piperidine/piperazine bridged isosteres of pridopidine. The compounds were assessed against the D2 receptor in agonist and antagonist modes and against the D4 receptor in agonist mode. hERG Binding and the ADME profiles were studied.

1-[3-(4-Butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1) as a Model for the Rational Design of a Novel Class of Brain Penetrant Ligands with High Affinity and Selectivity for Dopamine D4 Receptor.

In the present article, the M1 mAChR bitopic agonist 1-[3-(4-butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1, 1) has been demonstrated to show unexpected D4R selectivity over D2R and D3R and to behave as a D4R antagonist. To better understand the structural features required for the selective interaction with the D4R and to obtain compounds unable to activate mAChRs, the aliphatic butyl chain and the piperidine nucleus of 1 were modified, affording compounds 2-14. The 4-benzylpiperidine 9 and the 4-phenylpiperazine 12 showed high D4R affinity and selectivity not only over the other D2-like subtypes, but also over M1-M5 mAChRs. Derivative 12 was also highly selective over some selected off-targets. This compound showed biased behavior, potently and partially activating Gi protein and inhibiting ß-arrestin2 recruitment in functional studies. Pharmacokinetic studies demonstrated that it was characterized by a relevant brain penetration. Therefore, 12 might be a useful tool to better clarify the role played by D4R in disorders in which this subtype is involved.

Highly Selective Dopamine D3 Receptor (D3R) Antagonists and Partial Agonists Based on Eticlopride and the D3R Crystal Structure: New Leads for Opioid Dependence Treatment.

The recent and precipitous increase in opioid analgesic abuse and overdose has inspired investigation of the dopamine D3 receptor (D3R) as a target for therapeutic intervention. Metabolic instability or predicted toxicity has precluded successful translation of previously reported D3R-selective antagonists to clinical use for cocaine abuse. Herein, we report a series of novel and D3R crystal structure-guided 4-phenylpiperazines with exceptionally high D3R affinities and/or selectivities with varying efficacies. Lead compound 19 was selected based on its in vitro profile: D3R Ki = 6.84 nM, 1700-fold D3R versus D2R binding selectivity, and its metabolic stability in mouse microsomes. Compound 19 inhibited oxycodone-induced hyperlocomotion in mice and reduced oxycodone-induced locomotor sensitization. In addition, pretreatment with 19 also dose-dependently inhibited the acquisition of oxycodone-induced conditioned place preference (CPP) in rats. These findings support the D3R as a target for opioid dependence treatment and compound 19 as a new lead molecule for development.

Synthesis and characterization of a series of chiral alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a series of chiral alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel potent and selective dopamine D4 receptor antagonist with selectivity against the other dopamine receptors tested (<10% inhibition at 1µM against D1, D2L, D2S, D3, and D5).

[Novel central dopaminergic agents derived from atypical di-substituted 2-aminoindane-4, 7. Synthesis and central pharmacological profile]. / Novedosos agentes dopaminérgicos centrales derivados del 2-aminoindano-4,7 disustituido atipico. Síntesis y perfil farmacológico central.

In recent decades, many compounds with central dopaminergic activity have been designed, synthesized and evaluated pharmacologically. However, it has not been possible to obtain a drug able to improve or cure diseases involving dopaminergic regulation in the central nervous system, such as Parkinson's disease and schizophrenia, among others. Taking into consideration the term "atypical pharmacophore" and from the compound 5, the aralkyl fragment was incorporated, and the compounds 10, 11, 13a-h and 14a-h were synthesized. Both the compounds 10 and 13a-h under its methoxylated form and the compounds 11 and 14a-h under the phenolic form, were evaluated to determine their pharmacologically agonistic and antagonistic effects on central dopaminergic activity. For this, the effect of intracerebroventricular injection of said compounds on the hydromineral balance and stereotyped behavior in rats, was determined. The results of the preliminary pharmacological evaluation show a centrally acting action through dopamine mechanisms, in which the compounds 10, 11, 13d-h and 14a showed responses as agonists, whereas compounds 14b-h, had responses as antagonists.

Novedosos agentes dopaminérgicos centrales derivados del 2-aminoindano-4,7 disustituido atípico. Síntesis y perfil farmacológico central / Novel central dopaminergic agents derived from atypical di-substituted 2-aminoindane-4, 7. Synthesis and central pharmacological profile

En las últimas décadas son muchos los compuestos con actividad dopaminérgica central que se han diseñado, sintetizado y evaluado farmacológicamente. A pesar de ello, no se ha logrado obtener un fármaco capaz de mejorar o curar las patologías que involucran la regulación dopaminérgica en el sistema nervioso central tales como la Enfermedad de Parkinson y la esquizofrenia, entre otras. Tomando en consideración el término de “farmacóforo atípico” y a partir del compuesto 5, se incorporó el fragmento aralquil y se sintetizaron los compuestos 10, 11, 13a-h y 14a-h. Tanto los compuestos 10 y 13a-h bajo su forma metoxilada como los compuestos 11 y 14a-h bajo su forma fenólica, fueron evaluados farmacológicamente para determinar su actividad agonística y antagonística sobre el sistema dopaminérgico central. Para ello se determinó el efecto de la inyección intracerebroventricular de dichos compuestos sobre el balance hidromineral y la conducta estereotipada en ratas. Los resultados de la evaluación farmacológica preliminar muestran una acción central a través de mecanismos dopaminérgicos, siendo que los compuestos 10, 11, 13d-h y 14a mostraron respuestas como agonistas, mientras que los compuestos 14b-h, tuvieron respuestas como antagonistas.

In recent decades, many compounds with central dopaminergic activity have been designed, synthesized and evaluated pharmacologically. However, it has not been possible to obtain a drug able to improve or cure diseases involving dopaminergic regulation in the central nervous system, such as Parkinson’s disease and schizophrenia, among others. Taking into consideration the term “atypical pharmacophore” and from the compound 5, the aralkyl fragment was incorporated, and the compounds 10, 11, 13a-h and 14a-h were synthesized. Both the compounds 10 and 13a-h under its methoxylated form and the compounds 11 and 14a-h under the phenolic form, were evaluated to determine their pharmacologically agonistic and antagonistic effects on central dopaminergic activity. For this, the effect of intracerebroventricular injection of said compounds on the hydromineral balance and stereotyped behavior in rats, was determined. The results of the preliminary pharmacological evaluation show a centrally acting action through dopamine mechanisms, in which the compounds 10, 11, 13d-h and 14a showed responses as agonists, whereas compounds 14b-h, had responses as antagonists.

Design, synthesis and evaluation of benzo[a]thieno[3,2-g]quinolizines as novel l-SPD derivatives possessing dopamine D1, D2 and serotonin 5-HT1A multiple action profiles.

A novel scaffold derived from l-SPD with a substituted thiophene group in the D ring were designed, synthesized, and evaluated for their binding affinities at dopamine (D1, D2 and D3) and serotonin (5-HT1A and 5-HT2A) receptors. Most of the tetracyclic compounds exhibited higher affinities for D2 and 5-HT1A receptors than l-SPD, while compound 23 e showed the highest Ki value of 7.54 nM at D2 receptor which was 14 times more potent than l-SPD. Additionally, compounds 23 d and 23 e were more potent than l-SPD at D3 receptor. According to the functional assays, 23 d and 23 e were demonstrated as full antagonists at D1 and D2 receptors and full agonists at 5-HT1A receptor. Since the combination of D2 antagonism and 5-HT1A agonism is considered effective in treating both the positive and negative symptoms of schizophrenia, these novel compounds are implicated as potential therapeutic agents.

Identification of a new selective dopamine D4 receptor ligand.

The dopamine D4 receptor has been shown to play key roles in certain CNS pathologies including addiction to cigarette smoking. Thus, selective D4 ligands may be useful in treating some of these conditions. Previous studies in our laboratory have indicated that the piperazine analog of haloperidol exhibits selective and increased affinity to the DAD4 receptor subtype, in comparison to its piperidine analog. This led to further exploration of the piperazine moiety to identify new agents that are selective at the D4 receptor. Compound 27 (KiD4=0.84 nM) was the most potent of the compounds tested. However, it only had moderate selectivity for the D4 receptor. Compound 28 (KiD4=3.9 nM) while not as potent, was more discriminatory for the D4 receptor subtype. In fact, compound 28 has little or no binding affinity to any of the other four DA receptor subtypes. In addition, of the 23 CNS receptors evaluated, only two, 5HT1AR and 5HT2BR, have binding affinity constants better than 100 nM (Ki <100 nM). Compound 28 is a potentially useful D4-selective ligand for probing disease treatments involving the D4 receptor, such as assisting smoking cessation, reversing cognitive deficits in schizophrenia and treating erectile dysfunction. Thus, further optimization, functional characterization and evaluation in animal models may be warranted.

Shuttle-cargo fusion molecules of transport peptides and the hD2/3 receptor antagonist fallypride: a feasible approach to preserve ligand-receptor binding?

To determine if the conjugation of a small receptor ligand to a peptidic carrier to potentially facilitate transport across the blood-brain barrier (BBB) by "molecular Trojan horse" transcytosis is feasible, we synthesized several transport peptide-fallypride fusion molecules as model systems and determined their binding affinities to the hD2 receptor. Although they were affected by conjugation, the binding affinities were found to be still in the nanomolar range (between 1.5 and 64.2 nM). In addition, homology modeling of the receptor and docking studies for the most potent compounds were performed, elucidating the binding modes of the fusion molecules and the structure elements contributing to the observed high receptor binding. Furthermore, no interaction between the hybrid compounds and P-gp, the main excretory transporter of the BBB, was found. From these results, it can be inferred that the approach to deliver small neuroreceptor ligands across the BBB by transport peptide carriers is feasible.