Design, synthesis, and evaluation of fused heterocyclic analogs of SCH 58261 as adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A_2A receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A_2A receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson’s Disease, and has aqueous solubility of 100 μM at physiological pH.     

Biaryl and heteroaryl derivatives of SCH 58261 as potent and selective adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist, which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. We report the design and synthesis of biaryl and heteroaryl analogs of SCH 58261 which improve the A_2A receptor binding selectivity as well as the pharmacokinetic properties of SCH 58261. In particular, the quinoline 25 has excellent A_2A receptor in vitro binding affinity and selectivity, sustained rat plasma levels upon oral dosing, and is active orally in a rat behavioral assay.     

Synthesis and structure-activity relationship of 1H-indole-3-carboxylic acid pyridine-3-ylamides: a novel series of 5-HT2C receptor antagonists

A novel series of 1H-indole-3-carboxylic acid pyridine-3-ylamides were synthesized and identified to show high affinity and selectivity for 5-HT_2C receptor. Among them, 1H-indole-3-carboxylic acid[6-(2-chloro-pyridin-3-yloxy)-pyridin-3-yl]-amide (15k) exhibits the highest affinity (IC₅₀ = 0.5 nM) with an excellent selectivity (>2000 times) over other serotonin (5-HT_1A, 5-HT_2A, and 5-HT₆) and dopamine (D₂–D₄) receptors.     

Interaction of the ubiquitin carboxyl terminal esterase L1 with α2-adrenergic receptors inhibits agonist-mediated p44/42 MAP kinase activation

Neuroprotective effects of α₂-adrenergic receptor (AR) agonists are mediated via the α_2AAR subtype, but the molecular mechanisms underlying these actions are still not elucidated. A two-hybrid screen was performed to identify new proteins that may control α₂AR receptor function and trafficking. This screen identified the ubiquitin carboxyl-terminal hydrolase-L1 (Uch-L1), a protein associated with Parkinson's disease, as α₂AR interacting protein. This interaction was confirmed and evaluated by GST pull down assays demonstrating that Uch-L1 binds preferentially to the α_2AAR subtype and only with less affinity to α_2BAR and α_2CAR. Co-immunoprecipitation of epitope-tagged proteins confirmed the specificity of this interaction in vivo. Moreover, co-transfection of a truncated G-protein coupled receptor kinase-DNA preventing α₂AR phosphorylation led to an increased signal-strength of coimmunoprecipitated Uch-L1. Confocal laser microscopy showed that interaction of α_2AAR and Uch-L1 occurred in the cytoplasm. α₂AR agonist mediated activation of p44/42 MAP Kinase was drastically decreased in the presence of Uch-L1 indicating a functional relevance of this interaction. These findings may present a mechanism contributing to subtype-specific α₂AR trafficking and a potential pathway for the neuroprotective effects of α₂AR agonists.     

Tryptamine-based human β3-adrenergic receptor agonists. Part 2: SAR of the methylene derivatives

A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human β₃-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the α-position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for β₃-AR with excellent subtype selectivity.     

PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity

Potent and selective adenosine A₁ receptor (A₁AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a K_i value of 0.96 nM for PQ-69 in cloned hA₁ receptor, which was 217-fold more selective compared with hA_2A receptors and >1,000-fold selectivity for hA₁ over hA₃ receptor. The results obtained from [³⁵S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A₁AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A₁AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t_1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A₁AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A₁AR function, and it could be developed as a potential therapeutic agent.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.     

Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid terminal fragment. The compounds were assessed on their affinity for 5-HT_1A and ??₁-adrenoceptors and evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to investigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one described by Barbaro et al. for ??₁-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT_1A receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT_1A receptors in nanomolar range (7-610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT_1A was observed for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity (16.6 nM) and selectivity (5.72) for ??₁-AR.     

5-Substituted 2-benzylidene-1-tetralone analogues as A1 and/or A2A antagonists for the potential treatment of neurological conditions

Adenosine A₁ and A_2A receptors are attracting great interest as drug targets for their role in cognitive and motor deficits, respectively. Antagonism of both these adenosine receptors may offer therapeutic benefits in complex neurological diseases, such as Alzheimer’s and Parkinson’s disease. The aim of this study was to explore the affinity and selectivity of 2-benzylidene-1-tetralone derivatives as adenosine A₁ and A_2A receptor antagonists. Several 5-hydroxy substituted 2-benzylidene-1-tetralone analogues with substituents on ring B were synthesized and assessed as antagonists of the adenosine A₁ and A_2A receptors via radioligand binding assays. The results indicated that hydroxy substitution in the meta and para position of phenyl ring B, displayed the highest selectivity and affinity for the adenosine A₁ receptor with K_i values in the low micromolar range. Replacement of ring B with a 2-amino-pyrimidine moiety led to compound 12 with an increase of affinity and selectivity for the adenosine A_2A receptor. These substitution patterns led to enhanced adenosine A₁ and A_2A receptor binding affinity. The para-substituted 5-hydroxy analogue 3 behaved as an adenosine A₁ receptor antagonists in a GTP shift assay performed with rat whole brain membranes expressing adenosine A₁ receptors. In conclusion, compounds 3 and 12, showed the best adenosine A₁ and A_2A receptor affinity respectively, and therefore represent novel adenosine receptor antagonists that may have potential with further structural modifications as drug candidates for neurological disorders.     

EFFECT OF ULCERATIVE COLITIS ACTIVITY ON PLASMA CONCENTRATION OF TRANSFORMING GROWTH FACTOR β1

Enhanced expression of transforming growth factor-β₁(TGF-β₁) demonstrated in human colonic mucosa of patients with ulcerative colitis (UC), indicates its possible significance in the pathogenesis of this disease. The aim of this study was to evaluate plasma TGF-β₁concentration in patients with different degrees of colonic mucosal injury, as a possible indicator of ulcerative colitis activity. TGF-β₁concentration was measured with an enzyme immunoassay (EIA) in plasma of 45 patients with endoscopically confirmed UC. Values observed in UC patients (40.5±15.9 ng/ml) were significantly higher than in healthy people (18.3±11.6 ng/ml) and higher than in patients with irritable colon syndrome (ICS), (20.5±13.6 ng/ml). The highest plasma TGF-β₁(58.6±112.1 ng/ml) was in patients with the severe UC course. TGF-β₁level analysed in all UC patients revealed significant positive correlation with scored degree of mucosal injury (r=0.396;P<0.01). Among other possible laboratory markers of the disease activity, only C-reactive protein concentration demonstrated significant correlation. Enhanced production of TGF-β₁can be related to inflammation activity. Measurement of plasma TGF-β₁may be considered as a biomarker of the disease activity.     

Adenosine A2A Receptor Signaling and Golf Assembly Show a Specific Requirement for the γ7 Subtype in the Striatum

The adenosine A_2A receptor (A_2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein α_olf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ₇ subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ₇ protein controls the hierarchial assembly of a specific G-protein α_olfβ₂γ₇ heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A_2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ₇ subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A_2AR G-protein α_olfβ₂γ₇ interface as a possible therapeutic target for Parkinson disease.     

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABA_A receptor (GABA_AR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABA_AR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABA_AR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABA_AR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABA_AR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABA_AR α3 interacts with gephyrin, GABA_AR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABA_AR α2 and collybistin or GABA_AR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABA_AR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABA_AR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABA_AR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABA_AR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABA_ARs, but not GlyRs or other GABA_AR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABA_ARs containing the α2 subunit.     

4-Aminopiperidine ureas as potent selective agonists of the human β3-Adrenergic receptor

The preparation and structure–activity relationships (SARs) of potent agonists of the human β₃-adrenergic receptor (AR) derived from a 4-aminopiperidine scaffold are described. Examples combine human β₃-AR potency with selectivity over human β₁-AR and/or human β₂-AR agonism. Compound 29s was identified as a potent (EC₅₀=1 nM) and selective (greater than 400-fold over β₁- with no β₂-AR agonism) full β₃-AR agonist with in vivo activity in a transgenic mouse model of thermogenesis.     

Potent and selective α1A adrenoceptor partial agonists—Novel imidazole frameworks

Novel imidazole frameworks have been identified as potent partial agonists of the α_1A adrenergic receptor, with good selectivity over the α_1B, α_1D and α_2A receptor sub-types. Nitrile 28 possessed attractive CNS drug-like properties with good membrane permeability and no P-pg mediated efflux. 28 also possessed excellent solubility, metabolic stability and wide ligand selectivity.     

Human C1orf27 protein interacts with α2A-adrenergic receptor and regulates its anterograde transport

The molecular mechanisms underlying the anterograde surface transport of G protein–coupled receptors (GPCRs) after their synthesis in the endoplasmic reticulum (ER) are not well defined. In C. elegans, odorant response abnormal 4 has been implicated in the delivery of olfactory GPCRs to the cilia of chemosensory neurons. However, the function and regulation of its human homolog, C1orf27, in GPCR transport or in general membrane trafficking remain unknown. Here, we demonstrate that siRNA-mediated knockdown of C1orf27 markedly impedes the ER-to-Golgi export kinetics of newly synthesized α_2A-adrenergic receptor (α_2A-AR), a prototypic GPCR, with the half-time being prolonged by more than 65%, in mammalian cells in retention using the selective hooks assays. Using modified bioluminescence resonance energy transfer assays and ELISAs, we also show that C1orf27 knockdown significantly inhibits the surface transport of α_2A-AR. Similarly, C1orf27 knockout by CRISPR-Cas9 markedly suppresses the ER–Golgi-surface transport of α_2A-AR. In addition, we demonstrate that C1orf27 depletion attenuates the export of β₂-AR and dopamine D2 receptor but not of epidermal growth factor receptor. We further show that C1orf27 physically associates with α_2A-AR, specifically via its third intracellular loop and C terminus. Taken together, these data demonstrate an important role of C1orf27 in the trafficking of nascent GPCRs from the ER to the cell surface through the Golgi and provide novel insights into the regulation of the biosynthesis and anterograde transport of the GPCR family members.     

An analog of 1α,25-dihydroxy-19-norvitamin D3 with the 1α-hydroxy group fixed in the axial position lacks biological activity in vitro

The relationship between the A-ring chair conformation of vitamin D compounds and their ability to bind the vitamin D receptor (VDR) has long attracted the attention of many researchers. It was established that in the crystalline complexes of hVDRmt with the natural hormone, 1α,25-dihydroxyvitamin D₃ (1), and its side-chain analogs the vitamins exist in β-chair form with an equatorial orientation of 1α-OH. However, with all these ligands the interconversion between both A-ring forms would be possible in solution. In an attempt to verify the conformation of vitamin D compounds required for binding the VDR we prepared analog 4, characterized by the presence of an axial 1α-hydroxy group. Since the additional ring connecting 3β-oxygen and C-2 prevents A-ring conformational flexibility, the synthesized vitamin 4 can exist exclusively in the α-chair form. The geometrical isomer 5 with a free 3β-OH group was also obtained. The analog 5 binds very poorly to VDR, whereas the vitamin 4 is practically devoid of binding ability. Both compounds also show very low HL-60-differentiating activity. When tested in vivo in mice the analogs 4 and 5 exhibit significant calcemic responses with analog 4 showing more activity than analog 5.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.     

Discovery of benzothiazole-based adenosine A2B receptor antagonists with improved A2A selectivity

The highly potent but modestly selective N-(2-amino-4-methoxy-benzothiazol-7-yl)-N-ethyl-acetamide derivative 2 was selected as the starting point for the design of novel selective A_2B antagonists, due to its excellent potency, and good drug-like properties. A series of compounds containing nonaromatic amides or ureas of five- or six-membered rings, and also bearing an m-trifluoromethyl-phenyl group (shown to impart superior potency) was prepared and evaluated for their selectivity against the A_2A and A₁ receptors. This work resulted in the identification of compound 30, with excellent potency and high selectivity against both A_2A and A₁ receptors.     

Lipid rafts constrain basal α1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the α_1A-adrenergic receptor (α_1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α_1AAR signaling. Unexpectedly, MCD increased α_1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α_1AAR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal α_1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α_1AAR signaling following stimulation.     

Functional expression and sites of gene transcription of a novel α subunit of the GABAA receptor in rat brain

Two α subunits of the gabaa receptor in rat brain have been identified by molecular cloning. The deduced polypeptide sequences share major characteristics with other chemically gated ion channel proteins. One polypeptide represents the rat homologue of the α3 subunit previously cloned from bovine brain [14], while the other polypeptide is a yet unknown subunit, termed α5. When coexpressed with the β1 subunit in Xenopus oocytes the receptors containing the α5 subunit revealed a higher sensitivity to GABA than receptors expressed from α1 + β1 subunits or α3 + β1 subunits (K_a = 1 μM, 13 μM and 14 μM, respectively). The α5 subunit was expressed only in a few brain areas such as cerebral cortex, hippocampal formation and olfactory bulb granular layer as shown by in situ hybridization histochemistry. Since the mRNA of the α5 subunit was colocalized with the αl and α3 subunits only in cerebral cortex and in the hippocampal formation the α5 subunit may be part of distinct GABA_A receptors in neuronal populations within the olfactory bulb.     

A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid,Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes

Propofol, an intravenous anesthetic, is a positive modulator of the GABA_A receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABA_A receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABA_A receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABA_A receptor subunit protection. This investigation demonstrates striking interfacial GABA_A receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity.