Central prostaglandin D2 exhibits anxiolytic-like activity via the DP1 receptor in mice

We found that prostaglandin (PG) D₂, the most abundant PG produced in the central nervous system (CNS), exhibited anxiolytic-like activity at a dose of 10–100 pmol/mouse after intracerebroventricular (i.c.v.) administration in the elevated plus-maze test in mice. A DP₁ receptor-selective agonist, BW245C, mimicked the anxiolytic-like activity of PGD₂, while a DP₂ receptor agonist 13,14-dihydro-15-keto-PGD₂ was inactive. The anxiolytic-like activity of PGD₂ was blocked by a DP₁ antagonist, BWA868C, suggesting that PGD₂-induced anxiolytic-like activity was mediated by the DP₁ receptor. Adenosine A_2A or GABA_A receptor antagonists, SCH58261 or bicuculline, respectively, also blocked its anxiolytic-like activity. Taken together, centrally administered PGD₂ may induce anxiolytic-like activity via the A_2A and GABA_A receptors, downstream of the DP₁ receptor.     

Kaempferol from the leaves of Apocynum venetum possesses anxiolytic activities in the elevated plus maze test in mice

The present work evaluated the anxiolytic activity of an aqueous extract of Apocynum venetum L. (Apocynaceae) and bioguided its fractionation using the elevated plus maze (EPM) in mice as a model of anxiety. A single treatment of AV extract markedly increased the percentage time spent on the open arms of the EPM in two distinct concentration ranges of 22.5–30 and 100–125 mg/kg p.o., respectively, indicating a putative anxiolytic-like activity. Fractions showing anxiolytic effects in concentrations equal to 30 or 125 mg/kg of whole extract were antagonized using the benzodiazepine antagonist flumazenil (3 mg/kg i.p.) or the 5-HT_1A receptor antagonist WAY-100635 (0.5 mg/kg i.p.). All active fractions in a concentration equal to 125 mg/kg were effectively blocked by the benzodiazepine antagonist flumazenil, while the anxiolytic activities of fractions in the lower dose equivalent to 30 mg/kg of whole extract were inhibited by the 5-HT_1A receptor antagonist WAY-100635. Through further separation of AV fractions it was possible to isolate and characterize the flavonol kaempferol which showed an anxiolytic-like activity in concentrations from 0.02 to 1.0 mg/kg p.o. The anxiolytic activity of kaempferol was partially antagonized by concomitant administration of flumazenil, but not by WAY-100635. In conclusion, our study clearly demonstrates that AV extract possesses anxiolytic-like activity and that at least one of its flavonoids, kaempferol, can elicit the same kind of neuropharmacological activity.     

Rubimetide (Met-Arg-Trp) derived from Rubisco exhibits anxiolytic-like activity via the DP1 receptor in male ddY mice

In this study, we found that Met-Arg-Trp (rubimetide), which had been isolated as a hypotensive peptide from a pepsin-pancreatin digest of spinach ribulose bisphosphate carboxylase/oxygenase (Rubisco), has anxiolytic-like activity in the elevated plus-maze test at a dose of 0.1mg/kg (i.p.) or 1.0mg/kg (p.o.) in mice with p<0.01 and p<0.05, respectively. The anxiolytic-like activity of rubimetide (0.1mg/kg, i.p.) was blocked by BW A868C (60microg/kg, i.p.), an antagonist for the DP1 receptor, suggesting the anxiolytic-like activity of rubimetide is mediated by prostaglandin D2 and the DP1 receptor.     

Synthesis and SAR studies of trisubstituted purinones as potent and selective adenosine A2A receptor antagonists

A series of trisubstituted purinones was synthesized and evaluated as A_2A receptor antagonists. The A_2A structure–activity relationships at the three substituted positions were studied and selectivity against the A₁ receptor was investigated. One antagonist 12o exhibits a K_i of 9 nM in an A_2A binding assay, a K_b of 18 nM in an A_2A cAMP functional assay, and is 220-fold selective over the A₁ receptor.     

Asymmetric total synthesis and identification of tetrahydroprotoberberine derivatives as new antipsychotic agents possessing a dopamine D1, D2 and serotonin 5-HT1A multi-action profile

An effective and rapid method for the microwave-assisted preparation of the key intermediate for the total synthesis of tetrahydroprotoberberines (THPBs) including l-stepholidine (l-SPD) was developed. Thirty-one THPB derivatives with diverse substituents on A and D ring were synthesized, and their binding affinity to dopamine D₁, D₂ and serotonin 5-HT_1A and 5-HT_2A receptors were determined. Compounds 18k and 18m were identified as partial agonists at the D₁ receptor with K_i values of 50 and 6.3 nM, while both compounds act as D₂ receptor antagonists (K_i = 305 and 145 nM, respectively) and 5-HT_1A receptor full agonists (K_i = 149 and 908 nM, respectively). These two THPBs compounds exerted antipsychotic actions in animal models. Further electrophysiological studies employing single-unit recording in intact animals demonstrated that 18k-excited dopaminergic (DA) neurons are associated with its 5-HT_1A receptor agonistic activity. These results suggest that these two compounds targeted to multiple neurotransmitter receptors may present novel lead drugs with new pharmacological profiles for the treatment of schizophrenia.     

Synthesis and in vivo evaluation of [18F]2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)-dione ([18F]FECUMI-101) as an imaging probe for 5-HT1A receptor agonist in nonhuman primates

The 5-HT_1AR partial agonist PET radiotracer, [¹¹C]CUMI-101, has advantages over an antagonist radiotracer as it binds preferentially to the high affinity state of the receptor and thereby provides more functionally meaningful information. The major drawback of C-11 tracers is the lack of cyclotron facility in many health care centers thereby limiting widespread clinical or research use. We identified the fluoroethyl derivative, 2-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione (FECUMI-101) (K_i = 0.1 nM; E_max = 77%; EC₅₀ = 0.65 nM) as a partial agonist 5-HT_1AR ligand of the parent ligand CUMI-101. FECUMI-101 is radiolabeled with F-18 by O-fluoroethylation of the corresponding desmethyl analogue (1) with [¹⁸F]fluoroethyltosylate in DMSO in the presence of 1.6 equiv of K₂CO₃ in 45 ± 5% yield (EOS). PET shows [¹⁸F]FECUMI-101 binds specifically to 5-HT_1AR enriched brain regions of baboon. The specificity of [¹⁸F]FECUMI-101 binding to 5-HT_1AR was confirmed by challenge studies with the known 5-HT_1AR ligand WAY100635. These findings indicate that [¹⁸F]FECUMI-101 can be a viable agonist ligand for the in vivo quantification of high affinity 5-HT_1AR with PET.     

Synthesis of novel 7-imino-2-thioxo-3,7-dihydro-2H-thiazolo [4,5-d] pyrimidine derivatives as adenosine A2A receptor antagonists

Novel bicyclic thiazolopyrimidine compounds (15–26) were synthesized to develop adenosine A_2A receptor (A_2AR) antagonist for the treatment of Parkinson’s disease (PD). The binding affinity of the compounds (15–26) with A_2AR was evaluated using radioligand binding assay on isolated membranes from stably transfected HEK293 cells. Selectivity of the compounds towards A_2AR was assessed by comparing their binding affinities with A₁ receptors (A₁R). cAMP concentrations were measured from HEK293 cells treated with compounds (15–26) as compared to NECA (A_2AR agonist). The compound (16) possessed strongest A_2AR binding affinity (K_i value = 0.0038 nM) and selectivity (737-fold) versus A₁R. Decrease in A_2AR-coupled release of endogenous cAMP from HEK293 cells treated with compounds (15–26) is evocative of their potential as A_2AR antagonist.     

Synthesis and antinociceptive properties of N-phenyl-N-(1-(2-(thiophen-2-yl)ethyl)azepane-4-yl)propionamide in the mouse tail-flick and hot-plate tests

The goals of this study, were to synthesize N-phenyl-N-(1-(2-(thiophen-2-yl)ethyl)azepane-4-yl)propionamide (1c) and determine its antinociceptive properties. The effect of clonidine on 1c antinociception and the involvement of opioid, α₂-adrenergic, and I₂ imidazoline receptors in 1c antinociception were studied. Also examined was the effect of an endothelin ET_A receptor antagonist on 1c antinociception. Synthesis of 1c was accomplished in two steps using modifications of previously reported methods. Antinociceptive (tail-flick and hot-plate) latencies were measured in male Swiss Webster mice treated with 1c; antagonists + 1c; clonidine + 1c; or antagonists + clonidine + 1c. Mice were pretreated with naloxone (opioid antagonist), yohimbine (α₂-adrenoceptor antagonist), idazoxan (α₂-adrenoceptor/I₂-imidazoline antagonist), BU224 (I₂-imidazoline antagonist) or BQ123 (endothelin ET_A receptor antagonist) to study the involvement of these receptors. Compound 1c produced a dose-dependent increase in antinociceptive latencies; ED₅₀ values were 0.15 mg/kg and 0.16 mg/kg, respectively, in the tail flick and hot plate tests. Naloxone, but not yohimbine, idazoxan or BU224, blocked 1c antinociception. Neither clonidine nor BQ123 potentiated 1c antinociception. Results demonstrate that 1c is 15-times more potent than morphine. The antinociceptive effect of 1c is mediated through opioid receptors. The α₂-adrenergic, I₂-imidazoline and endothelin ET_A receptors are not involved in 1c antinociception.     

Dual blockade of the A1 and A2A adenosine receptor prevents amyloid beta toxicity in neuroblastoma cells exposed to aluminum chloride

In a previous work we have shown that exposure to aluminum (Al) chloride (AlCl₃) enhanced the neurotoxicity of the amyloid beta_25-35 fragment (Abeta_25-35) in neuroblastoma cells and affected the expression of Alzheimer's disease (AD)-related genes. Caffein, a compound endowed with beneficial effects against AD, exerts neuroprotection primarily through its antagonist activity on A_2A adenosine receptors (A_2AR), although it also inhibits A₁Rs with similar potency. Still, studies on the specific involvement of these receptors in neuroprotection in a model of combined neurotoxicity (Abeta_25-35 + AlCl₃) are missing. To address this issue, cultured SH-SY5Y cells exposed to Abeta_25-35 + AlCl₃ were assessed for cell viability, morphology, intracellular ROS activity and expression of apoptosis-, stress- and AD-related proteins. To define the role of A₁R and A_2ARs, pretreatment with caffein, specific receptor antagonists (DPCPX or SCH58261) or siRNA-mediated gene knockdown were delivered. Results indicate that AlCl₃ treatment exacerbated Abeta_25-35 toxicity, increased ROS production, lipid peroxidation, β-secretase-1 (BACE1) and amyloid precursor protein (APP). Interestingly, SCH58261 successfully prevented toxicity associated to Abeta_25-35 only, whereas pretreatment with both DPCPX and SCH58261 was required to fully avert Abeta_25-35 + AlCl₃-induced damage, suggesting that A₁Rs might also be critically involved in protection during combined toxicity. The effects of caffein were mimicked by both N-acetyl cysteine, an antioxidant, and desferrioxamine, likely acting through distinct mechanisms. Altogether, our data establish a novel protective function associated with A₁R inhibition in the setting of combined Abeta_25-35 + AlCl₃ neurotoxicity, and expand our current knowledge on the potential beneficial role of caffein to prevent AD progression in subjects environmentally exposed to aluminum.     

Exploring the 2- and 5-positions of the pyrazolo[4,3-d]pyrimidin-7-amino scaffold to target human A1 and A2A adenosine receptors

A new series of 7-aminopyrazolo[4,3-d]pyrimidine derivatives (1–31) were synthesized to evaluate some structural modifications at the 2- and 5-positions aimed at shifting affinity towards the human (h) A_2A adenosine receptor (AR) or both hA_2A and hA₁ ARs. The most active compounds were those featured by a 2-furyl or 5-methylfuran-2-yl moiety at position 5, combined with a benzyl or a substituted-benzyl group at position 2. Several of these derivatives (22–31) displayed nanomolar affinity for the hA_2A AR (K_i = 3.62–57 nM) and slightly lower for the hA₁ ARs, thus showing different degrees (3–22 fold) of hA_2A versus hA₁ selectivity. In particular, the 2-(2-methoxybenzyl)-5-(5-methylfuran-2-yl) derivative 25 possessed the highest hA_2A and hA₁ AR affinities (K_i = 3.62 nM and 18 nM, respectively) and behaved as potent antagonist at both these receptors (cAMP assays). Its 2-(2-hydroxybenzyl) analog 26 also showed a high affinity for the hA_2A AR (K_i = 5.26 nM) and was 22-fold selective versus the hA₁ subtype. Molecular docking investigations performed at the hA_2A AR crystal structure and at a homology model of the hA₁ AR allowed us to represent the hypothetical binding mode of our derivatives and to rationalize the observed SARs.     

Central pharmacological activity of a new piperazine derivative: 4-(1-phenyl-1h-pyrazol-4-ylmethyl)-piperazine-1-carboxylic acid ethyl ester

AimsOur study focuses on the design and synthesis of a new piperazinic derivate, 4-(1-phenyl-1h-Pyrazol-4-Ylmethyl)-Piperazine-1-Carboxylic Acid Ethyl ester (LQFM008), and evaluation of its anxiolytic-like profile in Swiss mice.Main methodsLQFM008 was evaluated in a screening test of the central nervous system including the rota-rod, sodium pentobarbital-induced sleep, open field, elevated plus maze and light–dark box tests.Key findingsLQFM008 induced convulsions at the dose of 1.1 mmol/kg (i.p., s.c. or p.o.). LQFM008 up to 400 μmol/kg had no effect in the rota rod test. In the open field test, LQFM008 increased the number of crossings and the time spent at the central area as well as the sleeping time in sodium pentobarbital-induced sleep. In the elevated plus maze and light–dark box tests, this compound showed an anxiolytic-like activity. This anxiolytic-like activity was antagonized by NAN-190 (5-HT_1A antagonist) but not by flumazenil (benzodiazepine antagonist).SignificanceThe compound LQFM008 showed anxiolytic-like activity which may involve serotonergic pathway.     

Discovery of a new series of 5-HT1A receptor agonists

Starting from compounds previously identified as α₁-adrenoceptor antagonists that were also found to bind to the 5-HT_1A receptor, in an attempt to separate the two activities, a new series of 5-HT_1A receptor agonists was identified and shown to have high potency and/or high selectivity. Of these, compound 13, which combines high selectivity (5-HT_1A/α₁ = 151) and good agonist potency (pD₂ = 7.82; E_max = 76), was found to be the most interesting.     

The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety

In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3′,5′-(CF₃)₂Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[d-Cys-Gly-Phe-Met-Pro-d-Cys]-Trp-NH-[3′,5′-(CF₃)₂Bzl]) was found as a highly selective δ opioid agonist over μ receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24 h half life in rat plasma.     

Endothelin-1 and angiotensin II modulate rate and contraction amplitude in a subpopulation of mouse embryonic stem cell-derived cardiomyocyte-containing bodies

Embryonic stem cell-derived cardiomyocytes (ESC-CMs) have applications in understanding cardiac disease pathophysiology, pharmacology, and toxicology. Comprehensive characterization of their basic physiological and pharmacological properties is critical in determining the suitability of ESC-CMs as models of cardiac activity. In this study we use video microscopy and quantitative PCR to investigate the responses of mouse ESC-CMs to adrenoceptor, muscarinic, angiotensin II (Ang II), and endothelin-1 (ET-1) receptor activation. Isoprenaline (10 nM–10 μM) increased beating rate and contraction amplitude in all beating bodies (BBs), whereas carbachol (up to 1 μM) and the I_f channel blocker ZD-7288 (10 μM) decreased contraction frequency. ET-1 (0.01–100 nM) reduced contraction amplitude in all BBs and increased contraction frequency in 50% of BBs; these effects were blocked by the ET_A receptor antagonist BQ123 (250 nM). Ang II (0.01 nM–1 μM) increased both contraction amplitude (all BBs) and frequency (in 50% of BBs), effects blocked, respectively, by losartan (100 nM) and PD123,319 (200 nM). These results indicate the presence of functional ET_A and both AT₁ and AT₂ receptors in murine ESC-CMs, but their expression and or activity appears to be evident only in a limited set of BBs.     

2-Arylacetamido-4-phenylamino-5-substituted pyridazinones as formyl peptide receptors agonists

N-Formyl peptide receptors (FPRs: FPR1, FPR2, and FPR3) are G protein-coupled receptors that play key roles in modulating immune cells. FPRs represent potentially important therapeutic targets for the development of drugs that could enhance endogenous anti-inflammation systems associated with various pathologies, thereby reducing the progression of inflammatory conditions. Previously, we identified 2-arylacetamide pyridazin-3(2H)-ones as FPR1- or FPR2-selective agonists, as well as a large number of FPR1/FPR2-dual agonists and several mixed-agonists for the three FPR isoforms. Here, we report a new series of 2-arylacetamido-4-aniline pyridazin-3(2H)-ones substituted in position 5 as a further development of these FPR agonists. Chemical manipulation presented in this work resulted in mixed FPR agonists 8a, 13a and 27b, which had EC₅₀ values in nanomolar range. In particular, compound 8a showed a preference for FPR1 (EC₅₀ = 45 nM), while 13a and 27b showed a moderate preference for FPR2 (EC₅₀ = 35 and 61 nM, respectively). Thus, these compounds may represent valuable tools for studying FPR activation and signaling.     

Identification of peptides that antagonize formyl peptide receptor-like 1-mediated signaling

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1's agonists (Trp-Lys-Tyr-Met-Val-D-Met-CONH(2) (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH(2) (WRWWWW (WRW(4))) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW(4) inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW(4) to the FPR family, we observed that WRW(4) specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid beta42 (Abeta42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW(4) on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Abeta42 peptide in human neutrophils. Abeta42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW(4), which also completely inhibited the internalization of Abeta42 peptide in human macrophages. WRW(4) is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.     

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: Involvement of glutamic acid,dopamine, and nitric oxide

Oxytocin (5–100 ng), but not Arg⁸-vasopressin (100 ng), injected unilaterally into the bed nucleus of the stria terminalis (BNST) induces penile erection and yawning in a dose-dependent manner in male rats. The minimal effective dose was 20 ng for penile erection and 5 ng for yawning. Oxytocin responses were abolished not only by the oxytocin receptor antagonist d(CH₂)₅Tyr(Me)²-Orn⁸-vasotocin (1 μg), but also by (+) MK-801 (1 μg), an excitatory amino acid receptor antagonist of the N-methyl-d-aspartic acid (NMDA) subtype, SCH 23390 (1 μg), a D1 receptor antagonist, but not haloperidol (1 μg), a D2 receptor antagonist, and SMTC (40 μg), an inhibitor of neuronal nitric oxide synthase, injected into the BNST 15 min before oxytocin. Oxytocin-induced penile erection, but not yawning, was also abolished by CNQX (1 μg), an excitatory amino acid receptor antagonist of the AMPA subtype. In contrast, oxytocin responses were not reduced by bicuculline (20 ng), a GABA_A receptor antagonist, phaclofen (5 μg), a GABA_B receptor antagonist, CP 376395, a CRF receptor-1 antagonist (5 μg), or astressin 2B, a CRF receptor-2 antagonist (150 ng). Considering the ability of NMDA (100 ng) to induce penile erection and yawning when injected into the BNST and the available evidence showing possible interaction among oxytocin, glutamic acid, and dopamine in the BNST, oxytocin possibly activates glutamatergic neurotransmission in the BNST. This in turn leads to the activation of neural pathways projecting back to the paraventricular nucleus, medial preoptic area, ventral tegmental area, and/or ventral subiculum/amygdala, thereby inducing penile erection and yawning.     

Synthesis and evaluation of 1,2,4-triazolo[1,5-c]pyrimidine derivatives as A2A receptor-selective antagonists

Movement disorders such as Parkinson’s disease and Huntington’s disease are serious life-limiting and debilitating movement disorders. Their onset typically occurs from mid-life to late in life, and effective diagnostic techniques for detecting and following the disease course are lacking. Our goal is to develop receptor imaging agents for positron emission tomography (PET) that selectively target the most relevant subtype of adenosine receptors (AR) that are highly expressed in the striatum, that is, the A_2A AR. To further this goal, we have synthesized and characterized pharmacologically a family of high affinity A_2A AR ligands, based on the known antagonist, SCH 442416 (R = –Me), which have structural variability on the terminus (R = –Et, –i-Pr, –allyl, and others). A O-fluoroethyl analogue suitable for use as a PET tracer had a K_i value of 12.4 nM and was highly selective for the A_2A AR in comparison to the A₁ and A₃ ARs.     

9-Aminomethyl-9,10-dihydroanthracene (AMDA) analogs as structural probes for steric tolerance in 5-HT2A and H1 receptor binding sites

Synthesis, radioligand binding and molecular modeling studies of several 9-aminomethyl-9,10-dihydroanthracene (AMDA) analogs were carried out to determine the extent of the steric tolerance associated with expansion of the tricyclic ring system and amine substitution at 5-HT_2A and H₁ receptors. A mixture of (7,12-dihydrotetraphene-12-yl)methanamine and (6,11-dihydrotetracene-11-yl)methanamine in a 75–25% ratio was found to have an apparent K_i of 10 nM at the 5-HT_2A receptor. A substantial binding affinity for (7,12-dihydrotetraphene-3-methoxy-12-yl)methanamine at the 5-HT_2A receptor (K_i = 21 nM) was also observed. Interestingly, this compound was found to have 100-fold selectivity for 5-HT_2A over the H₁ receptor (K_i = 2500 nM). N-Phenylalkyl-AMDA derivatives, in which the length of the alkyl chain varied from methylene to n-butylene, were found to have only weak affinity for both 5-HT_2A and H₁ receptors (K_i = 223 to 964 nM). Our results show that large rigid annulated AMDA analogs can be sterically accommodated within the proposed 5-HT_2A binding site.