Structures of Ezomycins A1 and A2

The structures of ezomycins A₁. and A₂, antifungal antibiotics produced by a strain of Streptomyces, were determined as 1 and 2, respectively, by degradative and spectrometric studies.     

Design,synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor

The development of adenosine A_2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A_2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A_2A receptor. Compound F1, with an affinity of 1?μm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184?μm) without being cytotoxic at 100?μm. This compound, along with low-molecular weight compound D1 (K_i?=?10?μm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.     

Effects of Adenosine (ADO) and A1 and A2 Ado Agonists on Calcium Uptake and cAMP Levels in Cultured Rat Mesangial Cells (MC)

Abstract

MC exhibits A₁ and A₂ receptors with opposite actions on cAMP formation and ⁴⁵Ca²+ uptake. ADO 10^?4 M activated both second messengers, but neither A₁ nor A₂ receptors seem to be involved in these ADO-induced effects.     

The Synthesis and Biological activity of a Highly Selective Adenosine A2a. Receptor Agonist

Abstract

Three novel nucleosides 1, 2, and 3 were prepared that contained side chains at the 2-position of adenosine. Compound 1 was shown to be the most selective A_2a receptor agonist reported to date having an A₁/A₂ ratio of 2400. In addition, compound 1 was shown to reduce blood pressure in rats and dogs with only minimal effects on heart rate.     

New cardiac expression of two adenosine-2A receptor isoforms in dysfunctioning minipigs

Purpose: Eight A_2AR variants are reported in humans while no A_2AR isoforms in pigs. The aim of this study was to evaluate potential isoforms presence in cardiac pig tissue to better define possible involvement of A_2AR in the cardiovascular pathophysiology.

Materials and methods: In adult male minipigs (n?=?4) left ventricular dysfunction (LVD) was induced by pacing at 200 bpm in the right ventricular (RV) apex. In these animals and in sham operated pigs (C-SHAM, n?=?4) cardiac tissue was collected from LV-septal wall (LV-SW)-close to pacing site-and from lateral (opposite) site (LV-OSW). A_2AR specific primers, derived from Sus scrofa AY772412 sequence, were used for Real-Time PCR. The DNA was sequenced using the Sanger method. Histological analysis was also performed.

Results: In LV-SW of LVD minipigs the A_2AR melting curves were characterized by a sharp peak between 87 and 91?°C (short isoform, 1–94?bp) on the right of the principal peak corresponding to a long A_2AR isoform (GenBank: JQ229674.1) 1–213?bp. As for C-SHAM only one peak was observed in LV-OSW region of LVD animals. The short isoform had an alternative promoter region and a specific translated protein. Histology showed in LVD-LV-SW prominent Purkinje cells compared to LV-OSW and C-SHAM. No difference in A_2AR expression was observed between LVD animals and C-SHAM although a slight decrease was observed in LVD-LV-OSW.

Conclusions: The presence of two different isoforms in the myocardium close to the insertion of pacing is suggestive of a differential state-specific expression of A_2AR in cardiac tissue.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A₁ and/or A_2A receptor subtypes. On the whole, the novel derivatives 1–24 shared scarce or no affinities for the off-target hA_2B and hA₃ ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (K_i =?150?nM) and the best selectivity for the hA_2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA_2A (K_i =?123?nM) and A₁ AR affinity (K_i =?25?nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (K_i =?11?nM) and good selectivity for the hA₁ AR. The 5-(N⁴-substituted-piperazin-1-yl) derivatives 15–24 bind the hA₁ AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

Adenosine A2A receptor deficiency prevents p38MAPK activation and apoptosis of mouse hippocampal cells in the chronic hypoxic-hypercapnia model

ABSTRACT

This study aims to study the effects of adenosine A_2A receptor (A_2AR) on hippocampal cell apoptosis and the putative mechanisms in a mouse model of chronic hypoxic-hypercapnia. Wild-type (WT) or A_2AR knockout (A_2AR KO) mice were randomly divided into normal control (NC) groups and chronic hypoxic-hypercapnia (4HH) groups. Compared with their corresponding NC groups (WT-NC and KO-NC), the apoptosis index (AI), caspase-3 activity, Bax mRNA and P-p38 protein expression in the hippocampus of 4HH groups (WT-4HH and KO-4HH) were significantly increased, while Bcl2 mRNA expression was significantly decreased (P < 0.05). Moreover, A_2AR deficiency significantly rescued the effect of chronic hypoxic-hypercapnia on apoptosis when compared with the WT-4HH group (P < 0.05). A_2AR deficiency inhibits hippocampal cell apoptosis in mice exposed to chronic hypoxic-hypercapnia, which might be associated with dampened p38 MAPK activation and Bax mRNA expression, and augmented Bcl-2 mRNA expression.     

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca²⁺ activated Cl^? channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16A_inh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16A_inh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16A_inh-01 was tolerated, with most compounds showing no activity at 10?μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16A_inh-01 with 2-thiophene, had IC₅₀ ～1?μM for inhibition of TMEM16A chloride conductance.     

A1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels

AimsWe investigated the effects induced by exogenous adenosine on the spontaneous contractile activity of the longitudinal muscle of a mouse ileum, the receptor subtypes activated, the involvement of enteric nerves and whether opening of K⁺ channels was a downstream event leading to the observed effects.Main methodsMechanical responses of the mouse ileal longitudinal muscle to adenosine were examined in vitro as changes in isometric tension.Key findingsAdenosine caused a concentration-dependent reduction of the spontaneous contraction amplitude of the ileal longitudinal muscle up to its complete disappearance. This effect induced was markedly reduced by an A₁ receptor antagonist, but not by A₂ and A₃ receptor antagonists and mimicked only by the A₁ receptor agonist. Adenosine uptake inhibitors did not change adenosine potency. A₁ receptor expression was detected at the smooth muscle level. Adenosine responses were insensitive to tetrodotoxin, atropine or nitric oxide synthase inhibitor. Tetraethylammonium and iberiotoxin, BK_Ca channel blockers, significantly reduced adenosine effects, whilst 4-aminopyridine, a K_v blocker, apamin, a small conductance Ca²⁺-activated K⁺ (SK_Ca) channel blocker, charybdotoxin, an intermediate conductance Ca²⁺-activated K⁺ (IK_Ca) and BK_Ca channel blocker, or glibenclamide, an ATP-sensitive K⁺ channel blocker, had no effects. The combination of apamin plus iberiotoxin caused a reduction of the purinergic effects greater than iberiotoxin alone.SignificanceAdenosine acts as an inhibitory modulator of the contractility of mouse ileal longitudinal muscle through postjunctional A₁ receptors, which in turn would induce opening of BK_Ca and SK_Ca potassium channels. This study would provide new insight in the pharmacology of purinergic receptors involved in the modulation of the gastrointestinal contractility.     

Influence of Phospholipase A2-Prostaglawdin System Linked to A1 Adexosine Becepmb on Protein Kinase C Activity of Cultubed Glial Cells

Abstract

In cultured astrocytes phospholipase A₂ seems to be functionally linked to G₁ protein of A₁ adenosine receptors. Phospholipase A₂ inhibition and A₁-linked G₁ protein inactivation resulted in purine release increase due to protein kinase C activation.     

Two New Gibberellins A50 and A52 in Seeds of Lagenaria leucantha

Two new gibberellins A₅₀ and A₅₂ were isolated from seeds of Lagenaria leucantha Rusby var. clavata Makino. Their structures were shown to be ent-2α,3α,10,llα-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (1) and ent-2α,3α,11β20-tetrahydroxy-gibberell-16-ene-7,19-dioic acid 19,20-lactone (10), respectively.     

Pharmacological characterization of DPTN and other selective A3 adenosine receptor antagonists

The A₃ adenosine receptor (AR) is emerging as an attractive drug target. Antagonists are proposed for the potential treatment of glaucoma and asthma. However, currently available A₃AR antagonists are potent in human and some large animals, but weak or inactive in mouse and rat. In this study, we re-synthesized a previously reported A₃AR antagonist, DPTN, and evaluated its affinity and selectivity at human, mouse, and rat ARs. We showed that DPTN, indeed, is a potent A₃AR antagonist for all three species tested, albeit a little less selective for mouse and rat A₃AR in comparison to the human A₃AR. DPTN’s K_i values at respective A₁, A_2A, A_2B, and A₃ receptors were (nM) 162, 121, 230, and 1.65 (human); 411, 830, 189, and 9.61 (mouse); and 333, 1147, 163, and 8.53 (rat). Its antagonist activity at both human and mouse A₃ARs was confirmed in a cyclic AMP functional assay. Considering controversial use of currently commercially available A₃AR antagonists in rats and mice, we also re-examined other commonly used and selective A₃AR antagonists under the same experimental conditions. The K_i values of MRS1523 were shown to be 43.9, 349, and 216 nM at human, mouse, and rat A₃ARs, respectively. MRS1191 and MRS1334 showed incomplete inhibition of [¹²⁵I]I-AB-MECA binding to mouse and rat A₃ARs, while potent human A₃AR antagonists, MRS1220, MRE3008F20, PSB10, PSB-11, and VUF5574 were largely inactive. Thus, we demonstrated that DPTN and MRS1523 are among the only validated A₃AR antagonists that can be possibly used (at an appropriate concentration) in mouse or rat to confirm an A₃AR-related mechanism or function.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09823-5.     

Gibberellin A59: A New Gibberellin from Canavalia gladiata

Gibberellin A₅₉ has been isolated from immature seeds of Canavalia gladiata (sword bean) and its structure was determined to be Δ²-gibberellin A₂₁ (1).     

Platelet Aggregation Inhibitory Activity of Selective A2 Adenosine Receptor Agonists

Abstract

A series of new 2-alkynyl, 2-cycloalkynyl, and 2-aralkynyl derivatives of adenosine-5′-ethyluronamide (NECA) were synthesized and evaluated in binding studies and functional assays to assess their potency and selectivity at A₂ vs A₁ receptors. The new derivatives were also tested as inhibitors of rabbit platelet aggregation induced by ADP. While the presence of an aromatic or heteroaromatic ring conjugated to the triple bond decreased antiplatelet activity, the introduction of a hydroxyl group or a heterocyclic ring on the alkynyl side chain increased the antiaggregatory activity in comparison with NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. However, the presence of an α-quaternary carbon markedly reduced the antiaggregatory potency without affecting the A₂ binding affinity, suggesting that the platelet receptor is not a typical A_2a site.     

APEC,An A2-Selective Adenosine Agonist,is a More Potent Locomotor Depressant Than N6-Cyclohexyladenosine

Abstract

The locomotor depressant effects of APEC are due to activation of central A2 adenosine receptors, while the depressant effects of NECA are dependent primarily on A₁-receptor activation. A variety of potent A₁-antagonists, including conjugates of the XAC, were screened as antagonists; 2-thio-CPX and CPT reversed effects of CHA, but not of APEC.     

Effect of Endogenous Phospholipids on the [3H]R-Pia Binding to A1 Adenosine Receptors from PIG Cerebral Cortex

Abstract

The effect of phospholipases on the membrane-bound and solubilized A₁ adenosine receptor has been studied. The results indicate that phospholipids are essential for the functionality of the A₁ adenosine receptor and that there is a co-solubilization of receptor and phospholipids.     

Brainstem 5-Hydroxytrytamine1A Binding Sites are not Down-Regulated by Agonists which Induce Tolerance in the Rat: Myoclonus and other Serotonergic Behaviors

Abstract

To study the regulation of 5-HT_1A receptors in the brainstem, the region most relevant to the serotonin syndrome and to serotonin-responsive human myoclonic disorders, we chronically treated rats with various 5-HT_1A agonists and labeled 5-HT_1A sites with [³H]8-OH-DPAT. Daily injection for 30 consecutive days of 10 mg/kg ip 8-OH-DPAT (pre- and post-synaptic 5-HT_1A agonist) significantly decreased 8-OH-DPAT-evoked flat body posture, forelimb myoclonus, and hypothermia compared to chronic vehicle injection. There was no cross tolerance to 8-OH-DPAT in rats chronically injected with ipsapirone or buspirone (presynaptic 5-HT_1A agonists). However, none of the 5HT_1A agonists significantly altered B_max of brainstem 5-HT_1A binding sites. Chronic injection with other drugs such as 1-propranolol, (±) pindolol and spiperone (5-HT_1A and 5-HT₂ antagonists), methysergide (5-HT₁ and 5-HT₂ antagonist), and agonists and antagonists at various other 5-HT receptors also had no effect on binding parameters. These data demonstrate lack of cross-tolerance between pre- and post-synaptically acting 5-HT_1A agonists and absence of down-regulation of presynaptic 5-HT_1A sites at doses which induced tolerance of 5-HT_1A-mediated behaviors of the serotonin syndrome. They suggest changes in the post-synaptic cell rather than the receptor recognition site as the mechanism of tolerance.