2- and 8-alkynyl-9-ethyladenines: Synthesis and biological activity at human and rat adenosine receptors

The synthesis of a series of 9-ethyladenine derivatives bearing alkynyl chains in 2- or 8-position was undertaken, based on the observation that replacement of the sugar moiety in adenosine derivatives with alkyl groups led to adenosine receptor antagonists. All the synthesized compounds were tested for their affinity at human and rat A1, A2A, and A3 adenosine receptors in binding assays; the activity at the human A2B receptor was determined in adenylyl cyclase experiments. Biological data showed that the 2-alkynyl derivatives possess good affinity and are slightly selective for the human A2A receptor. The same compounds tested on the rat A1 and A2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A1, A2A, and A2B receptors proved to be lower than that of the corresponding 2-alkynyl derivatives. On the contrary, the affinity of the same compounds for the human A3 receptor was improved, resulting in A3 selectivity. As in the case of the 2-alkynyl-substituted compounds, the 8-alkynyl derivatives showed decreased affinity for rat receptors. However, it is worthwhile to note that the 8-phenylethynyl-9-ethyladenine was the most active compound of the two series (Ki in the nanomolar range) at both the human and rat A3 subtype. Docking experiments of the 2- and 8-phenylethynyl-9-ethyladenines, at a rhodopsin-based homology model, gave a rational explanation of the preference of the human A3 receptor for the 8-substituted compound

Erratum to: Molecular modelling study of 2-phenylethynyladenosine (PEAdo) derivatives as highly selective A3 adenosine receptor ligands

A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N6- and 4′position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assay (A2B). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A3 receptors, resulting in compounds endowed with high affinity and selectivity for the A3 subtype. Additional substitution of the N6- and 4′position increases both A3 affinity and selectivity. The results showed that the new compounds have a good affinity for the A3 receptor and in particular, the N6-methoxy-2-phenylethynyl-5′N-methylcarboxamidoadenosine, with a Ki at A3 of 1.9 nM and a selectivity A1/A3 and A2A/A3 of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A3 adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A3 receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A3 receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A1, A2A and A3 adenosine receptors and in adenylyl cyclase experiments for their potency at the human A2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N6 position of 9-propyladenine significantly increased binding affinity at the human A1 and A3 adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N6 position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles

[3H]Adenine is a suitable radioligand for the labeling of G protein-coupled adenine receptors but shows high affinity to bacterial contaminations in buffer solutions

[3H]Adenine has previously been used to label the newly discovered G protein-coupled murine adenine receptors. Recent reports have questioned the suitability of [3H]adenine for adenine receptor binding studies because of curious results, e.g. high specific binding even in the absence of mammalian protein. In this study, we showed that specific [3H]adenine binding to various mammalian membrane preparations increased linearly with protein concentration. Furthermore, we found that Tris-buffer solutions typically used for radioligand binding studies (50 mM, pH 7.4) that have not been freshly prepared but stored at 4°C for some time may contain bacterial contaminations that exhibit high affinity binding for [3H]adenine. Specific binding is abolished by heating the contaminated buffer or filtering it through 0.2-μm filters. Three different, aerobic, gram-negative bacteria were isolated from a contaminated buffer solution and identified as Achromobacter xylosoxidans, A. denitrificans, and Acinetobacter lwoffii. A. xylosoxidans, a common bacterium that can cause nosocomial infections, showed a particularly high affinity for [3H]adenine in the low nanomolar range. Structure–activity relationships revealed that hypoxanthine also bound with high affinity to A. xylosoxidans, whereas other nucleobases (uracil, xanthine) and nucleosides (adenosine, uridine) did not. The nature of the labeled site in bacteria is not known, but preliminary results indicate that it may be a high-affinity purine transporter. We conclude that [3H]adenine is a well-suitable radioligand for adenine receptor binding studies but that bacterial contamination of the employed buffer solutions must be avoided

Pyrazolo-triazolo-pyrimidines as adenosine receptor antagonists: Effect of the N-5 bond type on the affinity and selectivity at the four adenosine receptor subtypes

In the last few years, many efforts have been made to search for potent and selective human A3 adenosine antagonists. In particular, one of the most promising human A3 adenosine receptor antagonists is represented by the pyrazolo-triazolo-pyrimidine family. This class of compounds has been strongly investigated from the point of view of structure-activity relationships. In particular, it has been observed that fundamental requisites for having both potency and selectivity at the human A3 adenosine receptors are the presence of a small substituent at the N8 position and an unsubstitued phenyl carbamoyl moiety at the N5 position. In this study, we report the role of the N5-bond type on the affinity and selectivity at the four adenosine receptor subtypes. The observed structure-activity relationships of this class of antagonists are also exhaustively rationalized using the recently published ligand-based homology modeling approach

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A2B receptors of native human and rodent cell lines were investigated using [3H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [3H]PSB-298 showed saturable and reversible binding. It exhibited a KD value of 60 ± 1 nM and limited capacity (Bmax = 3.511 fmol per milligram protein) at recombinant human adenosine A2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A2B receptors. Adenosine A2B receptors were shown to be the major adenosine A2 receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [3H]PSB-298 is a selective radioligand for adenosine A2B binding sites in this cell line

Recent improvements in the development of A2B adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A1, A2A, A2B and A3 (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N6-, C2-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA1Ki = 1050 nM, hA2AKi = 1550 nM, hA2B EC50 = 82 nM, hA3Ki > 5 μM) and its 2-chloro analogue 23 (hA1Ki = 3500 nM, hA2AKi = 4950 nM, hA2B EC50 = 210 nM, hA3Ki > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA1, hA2A, hA3 EC50 > 10 μM; hA2B EC50 = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis

Adenosine induces growth-cone turning of sensory neurons

The formation of appropriate connections between neurons and their specific targets is an essential step during development and repair of the nervous system. Growth cones are located at the leading edges of the growing neurites and respond to environmental cues in order to be guided to their final targets. Directional information can be coded by concentration gradients of substrate-bound or diffusible-guidance molecules. Here we show that concentration gradients of adenosine stimulate growth cones of sensory neurons (dorsal root ganglia) from chicken embryos to turn towards the adenosine source. This response is mediated by adenosine receptors. The subsequent signal transduction process involves cAMP. It may be speculated that the in vivo function of this response is concerned with the formation or the repair and regeneration of the peripheral nervous system

Management of Patients with Advanced Prostate Cancer: Report from the Advanced Prostate Cancer Consensus Conference 2021.

BACKGROUND: Innovations in treatments, imaging, and molecular characterisation in advanced prostate cancer have improved outcomes, but various areas of management still lack high-level evidence to inform clinical practice. The 2021 Advanced Prostate Cancer Consensus Conference (APCCC) addressed some of these questions to supplement guidelines that are based on level 1 evidence. OBJECTIVE: To present the voting results from APCCC 2021. DESIGN, SETTING, AND PARTICIPANTS: The experts identified three major areas of controversy related to management of advanced prostate cancer: newly diagnosed metastatic hormone-sensitive prostate cancer (mHSPC), the use of prostate-specific membrane antigen ligands in diagnostics and therapy, and molecular characterisation of tissue and blood. A panel of 86 international prostate cancer experts developed the programme and the consensus questions. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The panel voted publicly but anonymously on 107 pre-defined questions, which were developed by both voting and non-voting panel members prior to the conference following a modified Delphi process. RESULTS AND LIMITATIONS: The voting reflected the opinions of panellists and did not incorporate a standard literature review or formal meta-analysis. The answer options for the consensus questions received varying degrees of support from panellists, as reflected in this article and the detailed voting results reported in the Supplementary material. CONCLUSIONS: These voting results from a panel of experts in advanced prostate cancer can help clinicians and patients to navigate controversial areas of management for which high-level evidence is scant. However, diagnostic and treatment decisions should always be individualised according to patient characteristics, such as the extent and location of disease, prior treatment(s), comorbidities, patient preferences, and treatment recommendations, and should also incorporate current and emerging clinical evidence and logistic and economic constraints. Enrolment in clinical trials should be strongly encouraged. Importantly, APCCC 2021 once again identified salient questions that merit evaluation in specifically designed trials. PATIENT SUMMARY: The Advanced Prostate Cancer Consensus Conference is a forum for discussing current diagnosis and treatment options for patients with advanced prostate cancer. An expert panel votes on predefined questions focused on the most clinically relevant areas for treatment of advanced prostate cancer for which there are gaps in knowledge. The voting results provide a practical guide to help clinicians in discussing treatment options with patients as part of shared decision-making

Management of Patients with Advanced Prostate Cancer. Part I: Intermediate-/High-risk and Locally Advanced Disease, Biochemical Relapse, and Side Effects of Hormonal Treatment: Report of the Advanced Prostate Cancer Consensus Conference 2022.

BACKGROUND: Innovations in imaging and molecular characterisation and the evolution of new therapies have improved outcomes in advanced prostate cancer. Nonetheless, we continue to lack high-level evidence on a variety of clinical topics that greatly impact daily practice. To supplement evidence-based guidelines, the 2022 Advanced Prostate Cancer Consensus Conference (APCCC 2022) surveyed experts about key dilemmas in clinical management. OBJECTIVE: To present consensus voting results for select questions from APCCC 2022. DESIGN, SETTING, AND PARTICIPANTS: Before the conference, a panel of 117 international prostate cancer experts used a modified Delphi process to develop 198 multiple-choice consensus questions on (1) intermediate- and high-risk and locally advanced prostate cancer, (2) biochemical recurrence after local treatment, (3) side effects from hormonal therapies, (4) metastatic hormone-sensitive prostate cancer, (5) nonmetastatic castration-resistant prostate cancer, (6) metastatic castration-resistant prostate cancer, and (7) oligometastatic and oligoprogressive prostate cancer. Before the conference, these questions were administered via a web-based survey to the 105 physician panel members ("panellists") who directly engage in prostate cancer treatment decision-making. Herein, we present results for the 82 questions on topics 1-3. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Consensus was defined as ≥75% agreement, with strong consensus defined as ≥90% agreement. RESULTS AND LIMITATIONS: The voting results reveal varying degrees of consensus, as is discussed in this article and shown in the detailed results in the Supplementary material. The findings reflect the opinions of an international panel of experts and did not incorporate a formal literature review and meta-analysis. CONCLUSIONS: These voting results by a panel of international experts in advanced prostate cancer can help physicians and patients navigate controversial areas of clinical management for which high-level evidence is scant or conflicting. The findings can also help funders and policymakers prioritise areas for future research. Diagnostic and treatment decisions should always be individualised based on patient and cancer characteristics (disease extent and location, treatment history, comorbidities, and patient preferences) and should incorporate current and emerging clinical evidence, therapeutic guidelines, and logistic and economic factors. Enrolment in clinical trials is always strongly encouraged. Importantly, APCCC 2022 once again identified important gaps (areas of nonconsensus) that merit evaluation in specifically designed trials. PATIENT SUMMARY: The Advanced Prostate Cancer Consensus Conference (APCCC) provides a forum to discuss and debate current diagnostic and treatment options for patients with advanced prostate cancer. The conference aims to share the knowledge of international experts in prostate cancer with health care providers and patients worldwide. At each APCCC, a panel of physician experts vote in response to multiple-choice questions about their clinical opinions and approaches to managing advanced prostate cancer. This report presents voting results for the subset of questions pertaining to intermediate- and high-risk and locally advanced prostate cancer, biochemical relapse after definitive treatment, advanced (next-generation) imaging, and management of side effects caused by hormonal therapies. The results provide a practical guide to help clinicians and patients discuss treatment options as part of shared multidisciplinary decision-making. The findings may be especially useful when there is little or no high-level evidence to guide treatment decisions