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.

Dopamine D4 receptor subtype activation reduces the rat cardiac parasympathetic discharge.

The dopaminergic system influences the heart rhythm by inhibiting the rat cardiac sympathetic and parasympathetic neurotransmissions through activation of D2-like receptors (encompassing the D2, D3, and D4 subtypes). Whereas D2 receptor subtype activation results in cardiac sympatho-inhibition, the dopamine receptor subtypes involved in rat cardiac vago-inhibition remain unknown. Hence, this study investigated the specific functional role of the D2-like receptor subtypes (D2, D3, and/or D4) inhibiting the rat heart cholinergic drive. For this purpose, male Wistar rats were pithed and prepared for cardiac vagal stimulation. Bradycardic responses were obtained by electrical stimulation of vagal fibres (3, 6, 9 Hz; n = 100) or i.v. acetylcholine (ACh; 1, 5, 10 µg/kg; n = 15). Expression of D2, D3, and D4 receptors was studied in left and right atrium samples by PCR (n = 4). Intravenous injections of quinpirole (D2-like agonist; 1-30 µg/kg), but not of SFK-38393 (D1-like agonist; 1-30 µg/kg), dose-dependently inhibited the vagally induced bradycardia. The vago-inhibition induced by quinpirole (which failed to affect the bradycardia to i.v. ACh) was unchanged after i.v. injections of the antagonists L-741,626 (D2; 100 µg/kg) or SB-277011-A (D3; 100 µg/kg), but it was abolished by L-745,870 (D4; 100 µg/kg). mRNA levels of D2, D3, and D4 receptor subtype were detected in the left and right rat atria. Our results suggest that the quinpirole-induced vagolytic effect involves prejunctional D4 receptor subtypes, located in the left and right atria. This provides new evidence on the relevance of D4 receptor modulating the heart parasympathetic control.

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.

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews.

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 µL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development.

Norepinephrine activates dopamine D4 receptors in the rat lateral habenula.

The lateral habenula (LHb) is involved in reward and aversion and is reciprocally connected with dopamine (DA)-containing brain regions, including the ventral tegmental area (VTA). We used a multidisciplinary approach to examine the properties of DA afferents to the LHb in the rat. We find that >90% of VTA tyrosine hydroxylase (TH) neurons projecting to the LHb lack vesicular monoamine transporter 2 (VMAT2) mRNA, and there is little coexpression of TH and VMAT2 protein in this mesohabenular pathway. Consistent with this, electrical stimulation of LHb did not evoke DA-like signals, assessed with fast-scan cyclic voltammetry. However, electrophysiological currents that were inhibited by L741,742, a DA-D4-receptor antagonist, were observed in LHb neurons when DA uptake or degradation was blocked. To prevent DA activation of D4 receptors, we repeated this experiment in LHb slices from DA-depleted rats. However, this did not disrupt D4 receptor activation initiated by the dopamine transporter inhibitor, GBR12935. As the LHb is also targeted by noradrenergic afferents, we examined whether GBR12935 activation of DA-D4 receptors occurred in slices depleted of norepinephrine (NE). Unlike DA, NE depletion prevented the activation of DA-D4 receptors. Moreover, direct application of NE elicited currents in LHb neurons that were blocked by L741,742, and GBR12935 was found to be a more effective blocker of NE uptake than the NE-selective transport inhibitor nisoxetine. These findings demonstrate that NE is released in the rat LHb under basal conditions and that it activates DA-D4 receptors. Therefore, NE may be an important regulator of LHb function.

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).

Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a novel series of (R)-4,4-difluoropiperidine core scaffold as dopamine receptor 4 (D4) antagonists. A series of compounds from this scaffold are highly potent against the D4 receptor and selective against the other dopamine receptors. In addition, we were able to confirm the active isomer as the (R)-enantiomer via an X-ray crystal structure.

Structure and dynamics of DRD4 bound to an agonist and an antagonist using in silico approaches.

Human dopamine receptor D4 (DRD4), a member of G-protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. To assess the conformational dynamics induced upon ligand binding, all-atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region--ICL3, is significantly dynamic in nature, mainly due to the side-chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine-bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands.

Inhibition of dengue virus replication by a class of small-molecule compounds that antagonize dopamine receptor d4 and downstream mitogen-activated protein kinase signaling.

UNLABELLED: Dengue viruses (DENV) are endemic pathogens of tropical and subtropical regions that cause significant morbidity and mortality worldwide. To date, no vaccines or antiviral therapeutics have been approved for combating DENV-associated disease. In this paper, we describe a class of tricyclic small-molecule compounds-dihydrodibenzothiepines (DHBTs), identified through high-throughput screening-with potent inhibitory activity against DENV serotype 2. SKI-417616, a highly active representative of this class, displayed activity against all four serotypes of DENV, as well as against a related flavivirus, West Nile virus (WNV), and an alphavirus, Sindbis virus (SINV). This compound was characterized to determine its mechanism of antiviral activity. Investigation of the stage of the viral life cycle affected revealed that an early event in the life cycle is inhibited. Due to the structural similarity of the DHBTs to known antagonists of the dopamine and serotonin receptors, we explored the roles of two of these receptors, serotonin receptor 2A (5HTR2A) and the D4 dopamine receptor (DRD4), in DENV infection. Antagonism of DRD4 and subsequent downstream phosphorylation of epidermal growth factor receptor (EGFR)-related kinase (ERK) were found to impact DENV infection negatively, and blockade of signaling through this network was confirmed as the mechanism of anti-DENV activity for this class of compounds. IMPORTANCE: The dengue viruses are mosquito-borne, reemerging human pathogens that are the etiological agents of a spectrum of febrile diseases. Currently, there are no approved therapeutic treatments for dengue-associated disease, nor is there a vaccine. This study identifies a small molecule, SKI-417616, with potent anti-dengue virus activity. Further analysis revealed that SKI-417616 acts through antagonism of the host cell dopamine D4 receptor and subsequent repression of the ERK phosphorylation pathway. These results suggest that SKI-417616, or other compounds targeting the same cellular pathways, may have therapeutic potential for the treatment of dengue virus infections.

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors.

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations--a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1-induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.

Dopamine D4 receptor excitation of lateral habenula neurons via multiple cellular mechanisms.

Glutamatergic lateral habenula (LHb) output communicates negative motivational valence to ventral tegmental area (VTA) dopamine (DA) neurons via activation of the rostromedial tegmental nucleus (RMTg). However, the LHb also receives a poorly understood DA input from the VTA, which we hypothesized constitutes an important feedback loop regulating DA responses to stimuli. Using whole-cell electrophysiology in rat brain slices, we find that DA initiates a depolarizing inward current (I(DAi)) and increases spontaneous firing in 32% of LHb neurons. I(DAi) was also observed upon application of amphetamine or the DA uptake blockers cocaine or GBR12935, indicating involvement of endogenous DA. I(DAi) was blocked by D4 receptor (D4R) antagonists (L745,870 or L741,742), and mimicked by a selective D4R agonist (A412997). I(DAi) was associated with increased whole-cell conductance and was blocked by Cs+ or a selective blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channel, ZD7288. I(DAi) was also associated with a depolarizing shift in half-activation voltage for the hyperpolarization-activated cation current (Ih) mediated by HCN channels. Recordings from LHb neurons containing fluorescent retrograde tracers revealed that I(DAi) was observed only in cells projecting to the RMTg and not the VTA. In parallel with direct depolarization, DA also strongly increased synaptic glutamate release and reduced synaptic GABA release onto LHb cells. These results demonstrate that DA can excite glutamatergic LHb output to RMTg via multiple cellular mechanisms. Since the RMTg strongly inhibits midbrain DA neurons, activation of LHb output to RMTg by DA represents a negative feedback loop that may dampen DA neuron output following activation.

Computer-Aided Design and Biological Evaluation of Diazaspirocyclic D4R Antagonists.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra, resulting in motor dysfunction. Current treatments are primarily centered around enhancing dopamine signaling or providing dopamine replacement therapy and face limitations such as reduced efficacy over time and adverse side effects. To address these challenges, we identified selective dopamine receptor subtype 4 (D4R) antagonists not previously reported as potential adjuvants for PD management. In this study, a library screening and artificial neural network quantitative structure-activity relationship (QSAR) modeling with experimentally driven library design resulted in a class of spirocyclic compounds to identify candidate D4R antagonists. However, developing selective D4R antagonists suitable for clinical translation remains a challenge.

Discovery of highly potent and selective D4 ligands by interactive SAR study.

A series of thienylmethylphenylpiperazins was synthesized and tested for affinity towards the five subtypes of dopaminergic receptors. Compound 5f showed more than 1000 folds selectivity to D4 receptors; analogue 5e showed the highest affinity to D4 receptors with Ki 3.9 nM. An interactive SAR approach was adopted and lead to compound 14a with Ki (D4) as low as 0.03 nM. Molecular docking studies showed a potential, first to report arene cation interaction between the D4 unique residue Arg-186 and the ligands' arene moiety, explaining the importance of having a strong negative electrostatic potential at this area of the compound structure.

Discovery of dopamine D4 receptor antagonists with planar chirality.

Employing the D4 selective phenylpiperazine 2 as a lead compound, planar chiral analogs with paracyclophane substructure were synthesized and evaluated for their ability to bind and activate dopamine receptors. The study revealed that the introduction of a [2.2]paracyclophane moiety is tolerated by dopamine receptors of the D2 family. Subtype selectivity for D4 and ligand efficacy depend on the absolute configuration of the test compounds. Whereas the achiral single-layered lead 2 and the double-layered paracyclophane (R)-3 showed partial agonist properties, the enantiomer (S)-3 behaved as a neutral antagonist.

Evaluation of N-phenyl homopiperazine analogs as potential dopamine D3 receptor selective ligands.

A series of N-(2-methoxyphenyl)homopiperazine analogs was prepared and their affinities for dopamine D2, D3, and D4 receptors were measured using competitive radioligand binding assays. Several ligands exhibited high binding affinity and selectivity for the D3 dopamine receptor compared to the D2 receptor subtype. Compounds 11a, 11b, 11c, 11f, 11j and 11k had K(i) values ranging from 0.7 to 3.9 nM for the D3 receptor with 30- to 170-fold selectivity for the D3 versus D2 receptor. Calculated logP values (logP=2.6-3.6) are within the desired range for passive transport across the blood-brain barrier. When the binding and the intrinsic efficacy of these phenylhomopiperazines was compared to those of previously published phenylpiperazine analogues, it was found that (a) affinity at D2 and D3 dopamine receptors generally decreased, (b) the D3 receptor binding selectivity (D2:D3 K(i) value ratio) decreased and, (c) the intrinsic efficacy, measured using a forskolin-dependent adenylyl cyclase inhibition assay, generally increased.

Aberrant dopamine D2-like receptor function in a rodent model of schizophrenia.

Based on the observation that antipsychotic medications display antagonist properties at dopamine D2-like receptors, aberrant dopamine signaling has been proposed to underlie psychosis in patients with schizophrenia. Thus, it is not surprising that considerable research has been devoted to understanding the mechanisms involved in the antipsychotic action of these compounds. It is important to note that the majority of these studies have been performed in "normal" experimental animals. Given that these animals do not possess the aberrant neuronal information processing typically associated with schizophrenia, the aim of the current study was to examine the dopamine D2 receptor system in a rodent model of schizophrenia. Here, we demonstrate that methylazoxymethanol acetate (MAM)-treated rats display an enhanced effect of quinpirole on dopamine neuron activity and an aberrant locomotor response to D2-like receptor activation, suggesting changes in postsynaptic D2-like receptor function. To better understand the mechanisms underlying the enhanced response to D2-like ligands in MAM-treated rats, we examined the expression of D2, D3, and dopamine transporter mRNA in the nucleus accumbens and ventral tegmental area by quantitative reverse transcription-polymerase chain reaction. MAM-treated rats displayed a significant increase in dopamine D3 receptor mRNA expression in the nucleus accumbens with no significant changes in the expression of the D2 receptor. Taken together, these data demonstrate robust alterations in dopamine D2-like receptor function in a rodent model of schizophrenia and provide evidence that preclinical studies examining the mechanisms of antipsychotic drug action should be performed in animal models that mirror aspects of the abnormal neuronal transmission thought to underlie symptoms of schizophrenia.

L-745,870 reduces L-DOPA-induced dyskinesia in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned macaque model of Parkinson's disease.

L-DOPA-induced dyskinesia remains an unmet challenge in the treatment of Parkinson's disease (PD). Here, we investigate the potential antidyskinetic efficacy of 3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine (L-745,870), a potent and selective dopamine D(4) receptor antagonist with a good toxicology profile and an excellent safety and tolerability record in phase I/II clinical studies, for non-PD indications. Six macaques were rendered parkinsonian by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration. After induction of stable and marked dyskinesia, animals were administered acute challenges of L-745,870 in combination with L-DOPA. To guarantee D(4) selectivity at the doses used in the study, we determined the plasma, cerebrospinal fluid, and brain levels of L-745,870. Coadministration of L-745,870 (1 mg/kg) and L-DOPA significantly reduced the severity of dyskinesia, by up to 59%, in comparison with L-DOPA alone (P < 0.01). L-745,870 had no effect on the duration of antiparkinsonian benefit (ON-time) (P > 0.05). However, L-745,870 (1 mg/kg) significantly increased the duration of ON-time without disabling dyskinesia (+204%; P < 0.001) and decreased duration of ON-time with disabling dyskinesia compared with L-DOPA alone (-56%; P < 0.01). Brain levels of L-745,870 (∼600 ng/g) were within the range at which L-745,870 provides selective D(4) receptor antagonism. Plasma levels were comparable with those demonstrated to be well tolerated in human studies. These data suggest that selective D(4) receptor antagonists represent a potential therapeutic approach for L-DOPA-induced dyskinesia. It is noteworthy that L-745,870 has already undergone significant clinical development, has an excellent profile for a therapeutic candidate, and could be advanced rapidly to phase IIa clinical studies for dyskinesia in PD.

Moderate chemical modifications of WAY-100635 improve the selectivity for 5-HT1A versus D4 receptors.

The selectivity for 5-HT(1A) versus D(4) receptors is significantly increased when the basic side chain of WAY-100635 is replaced by a 4-phenylpiperazine (3e) or a 4-phenyl-1,2,3,6-tetrahydropyridine moiety (3i). The 4-phenyl-1,2,3,6-tetrahydropyridine compounds (3i-l) have a higher affinity for 5-HT(1A) receptors than do the corresponding unsubstituted phenylpiperazine analogues (3e-h). Compounds 3e and 3i appear to be selective for 5-HT(1A) receptors over other relevant receptors and still behave as neutral antagonists.

Bivalent molecular probes for dopamine D2-like receptors.

Merging two arylamidoalkyl substituted phenylpiperazines as prototypical recognition elements for dopamine D(2)-like receptors by oligoethylene glycol linkers led to a series of bivalent ligands. These dimers were investigated in comparison to their monomeric analogues for their dopamine D(2long), D(2short), D(3) and D(4) receptor binding. Radioligand binding experiments revealed strong bivalent effects for some para-substituted benzamide derivatives. For the D(3) subtype, the target compounds 32, 34 and 36 showed an up to 70-fold increase of affinity and a substantial enhancement of subtype selectivity when compared to the monovalent analogue 24. Analysis of the binding curves displayed Hill slopes very close to one indicating that the bivalent ligands displace 1equiv of radioligand. Obviously, the two pharmacophores occupy an orthosteric and an allosteric binding site rather than adopting a receptor-bridging binding mode.