Development of novel pyridazinone-based adenosine receptor ligands

With the aim of finding new adenosine receptor (AR) ligands, a preliminary investigation focusing on the thieno[2,3-d]pyridazin-5(4H)-one scaffold was undertaken. The synthesized compounds 1–11 were evaluated for their binding at hA₁, hA_2A and hA₃ ARs and efficacy at hA_2B subtype in order to determine the affinity at the human adenosine receptor subtypes. Small structural changes on this scaffold highly influenced affinity; compound 5 (5-ethyl-7-(thiazol-2-yl)thieno[2,3-d]pyridazin-4(5H)-one) emerged as the best of this series. The simplicity of the synthetic process, the capability of the scaffold to be easily decorated, together with the predicted ADME properties confirm the role of these compounds as promising hits. A molecular docking investigation at the hA₁AR crystal structure was performed to rationalize the SARs of the herein reported thienopyridazinones.     

Development of novel adenosine receptor ligands based on the 3-amidocoumarin scaffold

With the aim of finding new adenosine receptor (AR) ligands presenting the 3-amidocoumarin scaffold, a study focusing on the discovery of new chemical entities was carried out. The synthesized compounds 1–8 were evaluated in radioligand binding (A₁, A_2A and A₃) and adenylyl cyclase activity (A_2B) assays in order to determine their affinity for human AR subtypes. The 3-benzamide derivative 4 showed the highest affinity of the whole series and was more than 30-fold selective for the A₃ AR (K_i = 3.24 μM). The current study supported that small structural changes in this scaffold allowed modulating the affinity resulting in novel promising classes of A₁, A_2A, and/or A₃ AR ligands. We also performed docking calculations in hA_2A and hA₃ to identify the hypothetical binding mode for the most active compounds. In addition, some ADME properties were calculated in order to better understand the potential of these compounds as drug candidates.     

Synthesis and structural characterization of solvated calcium amides containing bulky silylamide ligands

A series of solvated calcium bis(amides) with the general formula Ca[N(R)(SiMe₃)]₂(solv)_x (R = -SiMe₂t-Bu, -SiPh₂t-Bu, and -SiPh₃) was prepared from the reaction of CaI₂ with 2 equiv of the corresponding potassium amide. Solvent ligands used in this study include THF, pyridine (py), hexamethylphosphoranide (HMPA), and 4-dimethylaminopyridine (DMAP). The coordinated THF in Ca[N(SiMe₂t-Bu)(SiMe₃)]₂(THF)₂ could be replaced by stronger donating ligands. When -N[(SiPh₃)(SiMe₃)] was used as the amide ligand again a bis(THF) adduct was formed. Quite interestingly, when the more sterically-demanding ligand -N[(SiPh₂t-Bu)(SiMe₃)] ligand was used only one THF molecule could coordinate to Ca, leading to a rare example of a tri-coordinate Ca bis(amide). One of the starting amides, [KN(SiMe₂t-Bu)(SiMe₃)]₂, was isolated and found to adopt a dimeric structure in the solid state. All complexes were characterized with ¹H NMR, ¹³C NMR, elemental analyses, and X-ray crystallography when appropriate.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

Estrogen receptor beta selective ligands: discovery and SAR of novel heterocyclic ligands

A series of ligands with varying heterocyclic cores and substituents that display a range of selectivity’s (up to >100x) for ER-β over ER- are reported.     

Design and evaluation of xanthine based adenosine receptor antagonists: Potential hypoxia targeted immunotherapies

Molecular modeling techniques were applied to the design, synthesis and optimization of a new series of xanthine based adenosine A_2A receptor antagonists. The optimized lead compound was converted to a PEG derivative and a functional in vitro bioassay used to confirm efficacy. Additionally, the PEGylated version showed enhanced aqueous solubility and was inert to photoisomerization, a known limitation of existing antagonists of this class.     

8-Substituted 2-alkynyl-N9-propargyladenines as A2A adenosine receptor antagonists

Structure–activity relationships of 2-alkynyladenine derivatives were explored by varying substituents at the 9-, 8- and 2-positions of the purine moiety in order to optimize A_2A adenosine receptor antagonist activity in vitro. A propargyl group at the 9-position was found to be important for A_2A antagonist activity, and the introduction of a halogen, aryl, or heteroaryl at the 8-position further enhanced activity. A series of 8-substituted 2-alkynyl-N⁹-propargyladenine derivatives exhibited potent antagonist activity, with IC₅₀ values in the low nM range. Compound 4a from this series was found to be orally active at a dose of 3 mg/kg in a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model of Parkinson’s disease.     

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension

The A_2B adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A_2BAR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A_2BAR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15–20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A_2BAR. Our data indicate varying roles for A_2BAR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.     

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A₁ adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A₁, A_2A, A_2B and A₃ adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A₁, A₃ or dual A₁/A₃ adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.     

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.     

Synthesis of a New Series of 1H‐Imidazol‐1‐yl Substituted 8‐Phenylxanthines as Adenosine Receptor Ligands

A new series of 1H‐imidazol‐1‐yl substituted 8‐phenylxanthine analogs has been synthesized to study the effects of the imidazole group on the binding affinity of compounds for adenosine receptors. Competition binding studies of these compounds were carried out in vitro with human cloned receptors using [³H]DPCPX and [³H]ZM 241385 as radioligands at A₁ and A_2A adenosine receptors, respectively. The effect of the substitution pattern of the (imidazolyl)alkoxy group on various positions of the phenyl ring at C(8) was also studied. The xanthine derivatives displayed varying degrees of affinity and selectivity towards A₁ and A_2A receptor subtypes despite a common but variedly substituted Ar C(8).     

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.     

Exploring the structural determinants of novel xanthine derivatives as A2B adenosine receptor antagonists: a computational study

Adenosine is a ubiquitous endogenous nucleoside that controls numerous physiological functions via interacting with its specific G-coupled receptors. Activation of adenosine receptors (AdoRs), particularly A_2B AdoRs promotes the release of inflammatory cytokines; reduces vascular permeabilization and induces angiogenesis, thereby making A_2B AdoR becomes a potentially pharmacological target for drug development. Presently, for investigating the structural determinants of 164 xanthine derivatives as A_2B AdoR antagonists, we performed an in silico study integrating with 3D-QSAR, docking and molecular dynamics (MD) simulation. The obtained optimal model shows strong predictability (Q²?=?0.647, R²_ncv?=?0.955, and R²_pred?=?0.848). Additionally, to explore the binding mode of the ligand with A_2B AdoR and to understand their binding mechanism, docking analysis, MD simulations (20?ns), and the calculation of binding free energy were also carried out. Finally, the structural determinants of these xanthine derivatives were identified and a total of 20 novel A_2B AdoR antagonists with improved potency were computationally designed, and their synthetic feasibility and selectivity were also evaluated. The information derived from the present study offers a better appreciation for exploring the interaction mechanism of the ligand with A_2B AdoR, which could be helpful for designing novel potent A_2B AdoR antagonists.

Communicated by Ramaswamy H. Sarma     

Synthesis and pharmacological characterization of new neuronal nicotinic acetylcholine receptor ligands derived from Sazetidine-A

The enantiomers of two analogs of Sazetidine-A as well as several other novel biosteric analogues were synthesized. Their binding affinities at three major nAChRs subtypes and selectivity profiles were determined. Though many (S)-enantiomers of Sazetidine-A analogs have high binding affinities and good subtype selectivities, it is not a general rule that (S)-enantiomers are better than their (R) counterparts. Compound 11, of which the ethynyl group was replaced by its’ bioisostere—the triazole via click chemistry, showed a high binding affinity to α4β2 subtype (K_i = 1.3 nM) and better selectivity to the α4β2 subtype over α3β4 subtype with that of Sazetidine-A. The azide compound 15, a potential photoaffinity label, showed improved high selectivity and similar binding property profile with that of Sazetidine-A. The biaryl analog 17 exhibited a much lower affinity as compared to Sazetidine-A indicating the importance of a ‘long tail’ side chain for α4β2 nAChR binding.     

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).     

Genetic and pharmacological inactivation of the adenosine A2A receptor attenuates 3-nitropropionic acid-induced striatal damage

Adenosine A2A receptor (A2AR) antagonism attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced dopaminergic neurodegeneration and quinolinic acid-induced excitotoxicity in the neostriatum. As A2ARs are enriched in striatum, we investigated the effect of genetic and pharmacological A2A inactivation on striatal damage produced by the mitochondrial complex II inhibitor 3-nitropriopionic acid (3-NP). 3-NP was administered to A2AR knockout (KO) and wild-type (WT) littermate mice over 5 days. Bilateral striatal lesions were analyzed from serial brain tissue sections. Whereas all of the 3-NP-treated WT mice (C57BL/6 genetic background) had bilateral striatal lesions, only one of eight of the 3-NP-treated A2AR KO mice had detectable striatal lesions. Similar attenuation of 3-NP-induced striatal damage was observed in A2AR KO mice in a 129-Steel background. In addition, the effect of pharmacological antagonism on 3-NP-induced striatal neurotoxicity was tested by pre-treatment of C57Bl/6 mice with the A2AR antagonist 8-(3-chlorostyryl) caffeine (CSC). Although bilateral striatal lesions were observed in all mice treated either with 3-NP alone or 3-NP plus vehicle, there were no demonstrable striatal lesions in mice treated with CSC (5 mg/kg) plus 3-NP and in five of six mice treated with CSC (20 mg/kg) plus 3-NP. We conclude that both genetic and pharmacological inactivation of the A2AR attenuates striatal neurotoxicity produced by 3-NP. Since the clinical and neuropathological features of 3-NP-induced striatal damage resemble those observed in Huntington's disease, the results suggest that A2AR antagonism may be a potential therapeutic strategy in Huntington's disease patients.     

Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration

1. The potential neuroprotective actions of the A₃ adenosine receptor (A₃AR) were investigated using mice with functional deletions of the A₃AR (A₃AR^–/–) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival.2. Untreated A₃AR^–/– mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques.3. A₃AR^–/– mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A₃AR^–/– mice also showed evidence of decreased nociception in the hot-plate, but not tail-flick tests. Further, A₃AR^–/– mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A₃AR^+/+) and A₃AR^–/– mice. However, the extent of neuronal death in the CA2–3 subfields was less pronounced in A₃AR^+/+ than A₃AR^–/– mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A₃AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.).4. These results indicate that pharmacologic or genetic suppression of A₃AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A₃AR agonists, A₃AR^–/– mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.     

Species dependence of A3 adenosine receptor pharmacology and function

Efforts to fully understand pharmacological differences between G protein-coupled receptor (GPCR) species homologues are generally not pursued in detail during the drug development process. To date, many GPCRs that have been successfully targeted are relatively well-conserved across species in amino acid sequence and display minimal variability of biological effects. However, the A₃ adenosine receptor (AR), an exciting drug target for a multitude of diseases associated with tissue injury, ischemia, and inflammation, displays as little as 70% sequence identity among mammalian species (e.g., rodent vs. primate) commonly used in drug development. Consequently, the pharmacological properties of synthetic A₃AR ligands vary widely, not only in binding affinity, selectivity, and signaling efficacy, but to the extent that some function as agonists in some species and antagonists in others. Numerous heterocyclic antagonists that have nM affinity at the human A₃AR are inactive or weakly active at the rat and mouse A₃ARs. Positive allosteric modulators, including the imidazo [4,5-c]quinolin-4-amine derivative LUF6000, are only active at human and some larger animal species that have been evaluated (rabbit and dog), but not rodents. A₃AR agonists evoke systemic degranulation of rodent, but not human mast cells. The rat A₃AR undergoes desensitization faster than the human A₃AR, but the human homologue can be completely re-sensitized and recycled back to the cell surface. Thus, comprehensive pharmacological evaluation and awareness of potential A₃AR species differences are critical in studies to further understand the basic biological functions of this unique AR subtype. Recombinant A₃ARs from eight different species have been pharmacologically characterized thus far. In this review, we describe in detail current knowledge of species differences in genetic identity, G protein-coupling, receptor regulation, and both orthosteric and allosteric A₃AR pharmacology.     

Synthesis and Biological Activity of Trisubstituted Adenines as A 2A Adenosine Receptor Antagonists

The discovery of new drugs for the treatment of neurodegenerative disorders, such as Parkinson's disease, has become an attractive field of research. Due to the regulation of D ₂ receptor activity by A _{2 A} adenosine receptor, potent and selective ligands of A _{2 A} subtype could be useful tools to study neurodegenerative disorders. A series of 2,8-disubstituted-9-ethyladenine derivatives was synthesized and tested in binding affinity assay at human adenosine receptors. New compounds showed good affinity and selectivity at A _{2 A} receptor versus the other subtypes. The introduction of a bromine atom in 8-position increased the affinity of these compounds, leading to ligands with K _i in the nanomolar range.     

Synthesis and biological activity of a novel class nicotinic acetylcholine receptors (nAChRs) ligands structurally related to anatoxin-a

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.