Nucleoside modification and concerted mutagenesis of the human A3 adenosine receptor to probe interactions between the 2-position of adenosine analogs and Gln167 in the second extracellular loop

Residues of the second extracellular loop are believed to be important for ligand recognition in adenosine receptors. Molecular modeling studies have suggested that one such residue, Gln167 of the human A3 receptor, is in proximity to the C2 moiety of some adenosine analogs when bound. Here this putative interaction was systematically explored using a neoceptor strategy, i.e., by site-directed mutagenesis and examination of the affinities of nucleosides modified to have complementary functionality. Gln167 was mutated to Ala, Glu, and Arg, while the 2-position of several adenosine analogs was substituted with amine or carboxylic acid groups. All compounds tested lost affinity to the mutant receptors in comparison to the wild type. However, comparing affinities among the mutant receptors, several compounds bearing charge at the 2-position demonstrated preferential affinity for the mutant receptor bearing a residue of complementary charge. 13, with a positively-charged C2 moiety, displayed an 8.5-fold increase in affinity at the Q167E mutant receptor versus the Q167R mutant receptor Preferential affinity for specific mutant receptors was also observed for 8 and 12. The data suggests that a direct contact is made between the C2 substituent of some charged ligands and the mutant receptor bearing the opposite charge at position 167.     

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract

Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A₁ rat brain adenosine receptor, follows the order N⁷ > N³ > N¹. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.     

Deaza-analogues of Adenosine and 2-Chloroadenosine as Agonists of Adenosine Receptors

Abstract

A variety of adenosine analogues have been recently evaluated in order Lo find more potent and selective agonists on adenosine receptors. The most potent adenosine analogues acting on A₁ receptor, a high affinity receptor inhibitory to adenylate cyclase, are N⁶-substituted compounds. So ⁶-cyclohexyladenosine (CHA) and ⁶-L-phenylisopropyladenosine (L-PIA) are extremely potent agonists on A₂ receptor, whereas they are relatively weak agonists on A receptor, a lower affinity receptor which is stirnulatory to cyclase, and they have no effect on the adenosine P site.     

Adenosine receptors in the central nervous system: relationship to the central actions of methylxanthines

Adenosine has a significant role in many functions of the central nervous system. Behaviorally, adenosine and adenosine analogs have marked depressant effects. Electrophysiologically, adenosine reduces spontaneous neuronal activity and inhibits transsynaptic potentials $\text{via}$ interaction with extracellular receptors. Biochemically, adenosine inhibits adenylate cyclase $\text{via}$ a “high” affinity receptor, and activates adenylate cyclase $\text{via}$ a “low” affinity receptor. These receptors, called “A₁” and “A₂” respectively, show differing profiles for activation by adenosine analogs. Radioactive N⁶-cyclohexyladenosine binds selectively to the “high” affinity receptor. One major class of antagonists is known at adenosine receptors: the alkylxanthines, including caffeine and theophylline. Radioactive 1,3-diethyl-8-phenylxanthine, a particularly potent antagonist, appears to bind to both low and high affinity adenosine receptors. Behavioral, electrophysiological, and biochemical effects of alkylxanthines are consistent with the hypothesis that the central stimulatory actions of caffeine and theophylline are due in large part to antagonism of central adenosine receptors.     

3′-Amidated 3′-Deoxyxylofuranose Analogues of N 6-−Cyclopentyladenosines: a New Class of Non-Xanthine Antagonists at the Adenosine A1 Receptor.

Abstract

Full adenosine A₁ receptor agonists like CPA and other N ⁶-^?substituted adenosine analogs have previously been shown to become partial agonists upon deletion of the 3′-hydroxyl moiety. The present study further explored the C-3′ site for modification. The modest affinity at A₁ and A_2a receptors found in the 3′-amido-3′-deoxyxyIofuranosyladenine series prompted us to synthesize the corresponding N ⁶-^?cyclopentyl derivatives, which proved to exhibit potent antagonistic behaviour at the A₁ receptors. This represents a new perspective in the purinergic field.     

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.     

Differentiating Adenosine Receptors and Adenosine Uptake Sites in Brain

Abstract

The characteristics of adenosine receptors and adenosine uptake sites in brain are presented. High affinity adenosine receptors of the A₁ type bind [³H]cyclohexyladenosine ([³H]CHA) and [³ H]diethyl-phenyl-xanthine ([³H]DPX) with 10^?9 potency while adenosine uptake sites are labeled 10^?10 potency with [³ H]nitrobenzyl-thioinosine ([³H]NBI). NBI does not inhibit either [³H]CHA (agonist) or [³H]DPX (antagonist) binding to adenosine receptors in brain cortical membranes and conversely CHA and other adenosine receptor ligands are very poor inhibitors of [³H]NBI binding to adenosine uptake sites. A number of other differences between the receptor and uptake site are discussed which provide rather strong evidence that these two sites are quite distinct and that the labeled ligands used represent specific probes for each site.     

2-Phenylhydroxypropynyladenosine Derivatives as High Potent Agonists at A2B Adenosine Receptor Subtype

Abstract

Adenosine derivatives bearing in 2-position the (R,S)- phenylhydroxypropynyl chain were evaluated for their potency at human A_2B adenosine receptor, stably transfected on CHO cells, on the basis that (R,S)-2-phenylhydroxy-propynyl-5′-N-ethylcarboxyamidoadenosine [(R,S)-PHPNECA] was found to be a good agonist at the A_2B receptor subtype. Biological studies demonstrated that the presence of small alkyl groups in N ⁶-position of these molecules are well tolerated, whereas large groups abolished A_2B potency. On the other hand, the presence of an ethyl group in the 4′-carboxamido function seems to be optimal, the (S)-PHPNECA resulting the most potent agonist at A_2B receptor reported so far.     

Strategically Functionalized Adenosines: Agonists for Adenosine Receptors

Abstract

The syntheses of three classes of adenosine analogues involving cyclosubstitution at the 6-position and functionalization at the 2-position are reported. The target molecules synthesized are stable with respect to hydrolytic deamination by mammalian adenosine deaminase, and, because of major structural changes at the 2- and 6-positions, these compounds are expected to be poor phosphorylation substrates for the kinases. Adenosine receptor binding data reveal that several of the compounds synthesized show excellent A₁ receptor affinity and A₂/A₁ selectivity.     

Design and Synthesis of A3 Adenosine Receptor Ligands, 2′-Fluoro Analogues of Cl-IB-MECA

Abstract

Synthesis of 2′-deoxy-2′-fluoro-N ⁶-substituted adenosines as bioisosteres of Cl-IB-MECA and their binding affinities to A₃ adenosine receptor are described.     

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).     

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1 Receptor in the Rat Cortex

Abstract

The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [³H]XCC and [³H]XAC², and [³H]CPX³) which bind with high and selective affinity to the adenosine A₁ receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 8216⁵, represents the first reported non-xanthine structure that potently blocks adenosine receptors⁶. CGS 15943 has nanomolar affinity for both A₁ and A₂ receptor subtypes⁶. However, in contrast to many of the xanthine adenosine receptor antagonists, CGS 15943 is not a phosphodiesterase inhibitor and does not interact with adenosine transporter sites⁶. This compound is a potent and selective adenosine receptor antagonist in vivo ⁷ with a solubility/affinity ratio of greater than 1000⁷. In the present studies, the binding of [³H]CGS 15943 to the adenosine A₁ receptor was characterized.     

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, A_2A, and A₃ adenosine receptors in binding assays; the activity at the human A_2B 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 A_2A receptor. The same compounds tested on the rat A₁ and A_2A subtypes showed in general lower affinity for both receptors. On the other hand, the affinity of the 8-alkynyl derivatives at the human A₁, A_2A, and A_2B 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 (K_i 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.     

Synthesis of Novel Peptidyl Adenosine Antibiotic Analogs

A small library of peptidyl adenosine antibiotic analogs was synthesized, under the Pilot Scale Library Program of the NIH Roadmap initiative, from 2′,3′-O-isoproylideneadenosine-5′-carboxylic acid 2 in excellent yield. The coupling of the amino terminus of L-2-aminophenylbutyric methyl ester to a free 5′-carboxylic acid moiety of 2 followed by sodium hydroxide treatment led to carboxylic acid analog 4. Hydrolysis of this latter gave unprotected nucleoside analog 5. Intermediate 4 served as the precursor for the preparation of novel peptidyl adenosine analogs 6–18 in good yields and high purity through peptide coupling reactions to diverse amine derivatives. No marked anticancer and antimalaria activity was noted on preliminary cellular testing; however these analogs should be useful candidates for other types of biological activity.     

NMR studies of new arginine vasopressin analogs modified with α-2-indanylglycine enantiomers at position 2 bound to sodium dodecyl sulfate micelles

In this paper, we use NMR spectroscopy and molecular modeling to examine four new vasopressin analogs modified with α-2-indanylglycine (Igl) at position 2, [L-Igl²]AVP (I), [D-Igl²]AVP (II), [Mpa¹,L-Igl²]AVP (III) and [Mpa¹,D-Igl²]AVP (IV), embedded in a sodium dodecyl sulfate (SDS) micelle. All the analogs display antiuterotonic activity. In addition, the analogs with D-Igl reveal antipressor properties.Each analog exhibits the tendency to adopt β-turns at positions 2, 3 and/or 3, 4, which is characteristic of oxytocin-like peptides. Mutual arrangement of aromatic residues at positions 2 and 3 has been found to be crucial for binding antagonists with the OT and V_1a receptors. The orientation of the Gln⁴ side chain seems to be important for the V_1a receptor affinity. In each of the peptides studied, the Gln⁴ side chain is folded back over the ring moiety. However, it lies on the opposite face of the tocin moiety in analogs with L and D enantiomers of Igl.     

Synthesis of 2′-Deuterio Tubercidin and Adenosine and the Corresponding Arabino Analogs

Abstract

The 2′-deuterio arabino analogs of tubercidin and adenosine have been prepared by Swern oxidation of the 3′,5′-TPDS derivatives of tubercidin and adenosine and reduction with NaBD_4. Subsequent inversion of stereochemistry at C-2′ yielded [2′-²H]tubercidin and [2′-²H]adenosine with 98% deuterium incorporation.     

Recognition of Adenosine Receptors by Amiloride and Its Analogues

Abstract

Amiloride and its analogues displace the adenosine A, receptor ligands [³H]CPDPX and [³H]PIA from their binding sites in calf brain membranes in a GTP-insensitive manner. High [NaCl] or low pH reduces the affinity of amiloride for A, receptors, whereas the affinity of [³H]CPDPX is not affected. Notwithstanding this difference in modulation, the interaction between amiloride and A, receptors appears competitive in nature. The structure-affinity relationships differ from those for classic amiloride-sensitive Na' transport systems, indicating that a coupling between the A, receptor and one of these systems is very unlikely. Amiloride and its analogues may reprcsent a novel class of A, receptor antagonists.     

Xanthine-7-Ribosides as Adenosine Al Receptor Antagonists: Further Evidence for Adenosine's Anti Mode of Binding

Abstract

The synthesis and A₁ adenosine receptor affinity of some xanthine-7-ribosides is described. It appears that these compounds are A, receptor antagonists. The orientation of the ribose moiety, as determined by ′H-NMR spectroscopy and theoretical chemical calculations, is compared with the orientation of the ribose in the agonist adenosine. Implications for the syn vs anti modes of binding to the receptor are discussed.     

Survey of Nonxanthine Derivatives as Adenosine Receptor Ligands

Abstract

The binding affinities at rat A₁, A_2a, and A₃ adenosine receptors of a wide range of heterocyclic derivatives have been determined. Mono-, bi-, tricyclic and macrocyclic compounds were screened in binding assays, using either [³H]PIA or [³H]CGS 21680 in rat brain membranes or [¹²⁵I]AB-MECA in CHO cells stably transfected with rat A₃ receptors. Several new classes of adenosine antagonists (e. g. 5- oxoimidazopyrimidines and a pyrazoloquinazoline) were identified. Various sulfonylpiperazines, 11- hydroxytetrahydrocarbazolenine, 4H-pyrido[1,2-a]pyrimidin-one, folic acid, and cytochalasin H and J bound to A₃ receptors selectively. Moreover, cytochalasin A, which bound to A₁ adenosine receptors with Ki value of 1.9 μM, inhibited adenylyl cyclase in rat adipocytes, but not via reversible A₁ receptor binding.

     

Structural Modifications of the N-terminal Tetrapeptide Segment of [D-Ala]Deltorphin I: Effects on Opioid Receptor Affinities and Activities In Vitro and on Antinociceptive Potency

Schmidt, R., D. Menard, C. Mrestani-Klaus, N. N. Chung, C. Lemieux and P. W. Schiller. Structural modifications of the N-terminal tetrapeptide segment of [d-Ala²]deltorphin I: effects on opioid receptor affinities and activities in vitro and on antinociceptive potency. Peptides 18(10) 1615–1621, 1997.—A series of deltorphin I analogs containing d- or l-N-methylalanine (MeAla), d- or l-proline (Pro), α-aminoisobutyric acid (Aib), sarcosine (Sar) or D-tert-leucine (Tle) in place of d-Ala², or phenylalanine in place of Tyr¹, was synthesized. The opioid activity profiles of these peptides were determined in μ and δ opioid receptor-representative binding assays and bioassays in vitro as well as in the rat tail flick test in vivo. In comparison with the deltorphin I parent, both the l- and the d-MeAla²-analog were slightly more potent δ agonists in the mouse vas deferens (MDV) assay, and the d-MeAla²-analog showed two-fold higher antinociceptive potency in the analgesic test. In view of the fact that deltorphin analogs with an unsubstituted l-amino acid residue in the 2-position generally lack opioid activity, the observed high δ opioid potency of [l-MeAla²]deltorphin I is postulated to be due to the demonstrated presence of a conformer with a cis Tyr¹-MeAla² peptide bond, since the cis conformer allows for a spatial arrangement of the pharmacophoric moieties in the N-terminal tripeptide segment similar to that in active deltorphin analogs containing a d-amino acid residue in the 2-position. Substitution of Aib in the 2-position led to a compound, H-Tyr-Aib-Phe-Asp-Val-Val-Gly-NH₂, which displayed lower δ receptor affinity than the parent peptide but higher δ selectivity and, surprisingly, three times higher antinociceptive potency. The d- and l- Pro²-, Sar²- and d-Tle²-analogs showed much reduced δ receptor affinities and were inactive in the tail flick test. Replacement of Tyr¹ in deltorphin I with Phe produced a 32-fold decrease in δ receptor affinity but only a 7-fold drop in antinociceptive potency.