The phenolic group of the potent μ and κ opioid morphinan agonist/antagonists cyclorphan and butorphan was replaced by phenylamino and benzylamino groups including compounds with p-substituents in the benzene ring. These compounds are highly potent μ and κ ligands, e. g. p-methoxyphenylaminocyclorphan showing a Ki of 0.026 nM at the mu and a Ki of 0.03 nM at the kappa receptor. Phenyl carbamates and phenylureas were synthesized and investigated. Selective o-formylation of butorphan and levorphanol was achieved. This reaction opened the way to a large set of 2-substituted 3-hydroxymorphinans, including 2-hydroxymethyl-, 2-aminomethyl-, and N-substituted 2-aminomethyl-3-hydroxymorphinans. Bivalent ligands bridged in the 2-position were also synthesized and connected with secondary and tertiary aminomethyl groups, amide bonds or hydroxymethylene groups, respectively. Although most of the 2-substituted morphinans showed considerably lower affinities compared to their parent compounds, the bivalent ligand approach led to significantly higher affinities compared to the univalent aminomethylmorphinans.
Addition of reversine to dividing cells ejects Mad1 and the RZZ complex from unattached kinetochores and prevents resolution of incorrect chromosome–microtubule attachments (see also related papers by Hewitt et al. and Maciejowski et al. in this issue).
The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD–ZWILCH–ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome–microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery.
The activation of the human A3 adenosine receptor (AR) by a wide range of N6-substituted adenosine derivatives was studied in intact CHO cells stably expressing this receptor. Selectivity of binding at rat and human ARs was also determined. Among N6-alkyl substitutions, small N6-alkyl groups were associated with selectivity for human A3ARs vs. rat A3ARs, and multiple points of branching were associated with decreased hA3AR efficacy. N6-Cycloalkyl-substituted adenosines were full (≤5 carbons) or partial (≥6 carbons) hA3AR agonists. N6-(endo-Norbornyl)adenosine 13 was the most selective for both rat and human A1ARs. Numerous N6-arylmethyl analogues, including substituted benzyl, tended to be more potent in binding to A1 and A3 vs. A2AARs (with variable degrees of partial to full A3AR agonisms). A chloro substituent decreased the efficacy depending on its position on the benzyl ring. The A3AR affinity and efficacy of N6-arylethyl adenosines depended highly on stereochemistry, steric bulk, and ring constraints. Stereoselectivity of binding was demonstrated for N6-(R-1-phenylethyl)adenosine vs. N6-(S-1-phenylethyl)adenosine, as well as for the N6-(1-phenyl-2-pentyl)adenosine, at the rat, but not human A3AR. Interestingly, DPMA, a potent agonist for the A2AAR (Ki = 4 nM), was demonstrated to be a moderately potent antagonist for the human A3AR (Ki = 106 nM). N6-[(1S,2R)-2-Phenyl-1-cyclopropyl]adenosine 48 was 1100-fold more potent in binding to human (Ki = 0.63 nM) than rat A3ARs. Dual acting A1/A3 agonists (N6-3-chlorobenzyl- 29, N6-(S-1-phenylethyl)- 39, and 2-chloro-N6-(R-phenylisopropyl)adenosine 53) might be useful for cardioprotection.
Purines; Nucleosides; GPCR; Cyclic AMP; Receptor binding; Structure–activity relationships
(N)-Methanocarba nucleosides containing bicyclo[3.1.0]hexane replacement of the ribose ring previously demonstrated selectivity as A3 adenosine receptor (AR) agonists (5′-uronamides) or antagonists (5′-truncated). Here, these two series were modified in parallel at the adenine C2 position. N6-3-Chlorobenzyl-5′-N-methyluronamides derivatives with functionalized 2-alkynyl chains of varying length terminating in a reactive carboxylate, ester, or amine group were full, potent human A3AR agonists. Flexibility of chain substitution allowed the conjugation with a fluorescent cyanine dye (Cy5) and biotin, resulting in binding Ki values of 17 and 36 nM, respectively. The distal end of the chain was predicted by homology modeling to bind at the A3AR extracellular regions. Corresponding l-nucleosides were nearly inactive in AR binding. In the 5′-truncated nucleoside series, 2-Cl analogues were more potent at A3AR than 2-H and 2-F, functional efficacy in adenylate cyclase inhibition varied, and introduction of a 2-alkynyl chain greatly reduced affinity. SAR parallels between the two series lost stringency at distal positions. The most potent and selective novel compounds were amine congener 15 (Ki = 2.1 nM) and truncated partial agonist 22 (Ki = 4.9 nM).
A series of ring-constrained (N)-methanocarba-5′-uronamide 2,N6-disubstituted adenine nucleosides have been synthesized via Mitsunobu condensation of the nucleobase precursor with a pseudosugar ring containing a 5′-ester functionality. Following appropriate functionalization of the adenine ring, the ester group was converted to the 5′-N-methylamide. The compounds, mainly 2-chloro substituted derivatives, were tested in both binding and functional assays at human adenosine receptors (ARs), and many were found to be highly potent and selective A3AR agonists. Selected compounds were compared in binding to the rat A3AR to assess their viability for testing in rat disease models. The N6-(3-chlorobenzyl) and N6-(3-bromobenzyl) analogues displayed Ki values at the human A3AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N6 derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A3AR in comparison to the A1AR was (fold): the N6-(2,2-diphenylethyl) analogue 34 (1900), the N6-(2,5-dimethoxybenzyl) analogue 26 (1200), the N6-(2,5-dichlorobenzyl) and N6-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N6-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700–900). Typically, even greater selectivity ratios were obtained in comparison with the A2A and A2BARs. The (N)-methanocarba-5′-uronamide analogues were full agonists at the A3AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 µM. The N6-(2,2-diphenylethyl) derivative was an A3AR agonist in the (N)-methanocarba-5′-uronamide series, although it was an antagonist in the ribose series. Thus, many of the previously known groups that enhance A3AR affinity in the 9-riboside series, including those that reducing intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.
We modified a series of (N)-methanocarba nucleoside 5 -uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A3 adenosine receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized to couple an octadiynyl A3AR agonist to azido-containing fluorescent, chemically reactive, biotinylated, and other moieties with retention of selective binding to the A3AR. A bifunctional thiol-reactive crosslinking reagent was introduced. The most potent and selective novel compound was a 1-adamantyl derivative (Ki 6.5 nM), although some of the click products had Ki values in the range of 200–400 nM. Other potent, selective derivatives (Ki at A3AR in nM) were intended as possible receptor affinity labels: 3-nitro-4-fluorophenyl (10.6), α-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocyanate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were measured, indicating that this class of click adducts varied from partial to full A3AR agonist compared to other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent and selective A3AR agonists to reporter groups of diverse structure and to carrier moieties.
G protein-coupled receptor; purines; azide; structure activity relationship; radioligand binding
We previously synthesized a series of potent and selective A3 adenosine receptor (AR) agonists (North-methanocarba nucleoside 5′-uronamides) containing dialkyne groups on extended adenine C2 substituents. We coupled the distal alkyne of a 2-octadiynyl nucleoside by Cu(I)-catalyzed “click” chemistry to azide-derivatized G4 (fourth-generation) PAMAM dendrimers to form triazoles. A3AR activation was preserved in these multivalent conjugates, which bound with apparent Ki 0.1–0.3 nM. They were substituted with nucleoside moieties, solely or in combination with water-solubilizing carboxylic acid groups derived from hexynoic acid. A comparison with various amide-linked dendrimers showed that triazole-linked conjugates displayed selectivity and enhanced A3AR affinity. We prepared a PAMAM dendrimer containing equiproportioned peripheral azido and amino groups for conjugation of multiple ligands. A bifunctional conjugate activated both A3 and P2Y14 receptors (via amide-linked uridine-5′-diphosphoglucuronic acid), with selectivity in comparison to other ARs and P2Y receptors. This is the first example of targeting two different GPCRs with the same dendrimer conjugate, which is intended for activation of heteromeric GPCR aggregates. Synergistic effects of activating multiple GPCRs with a single dendrimer conjugate might be useful in disease treatment.
G protein; coupled receptor; purines; alkyne; azide; radioligand binding; dendrimer
Derivatives of the herpes simplex thymidine kinase inhibitor HBPG (2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine) have been synthesized, and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG, and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published x-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogs with bulky tertiary amino substituents, were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 gM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.
In this study, we report BF066, a novel adenine derivative, inhibits platelet activation and thrombosis via the adenosine receptor (A2A) activation and phosphodiesterase (PDE) inhibition. BF066 inhibits platelet aggregation and ATP releasing induced by multiple platelet agonists in a dose-dependent manner. The inhibition of BF066 on ADP-induced aggregation is potentiated by adenosine and can be dramatically antagonized by the A2A antagonist SCH58261. BF066 also inhibits the PDE activity and platelet spreading on fibrinogen. In FeCl3-injured mouse mesenteric arterial thrombosis model, BF066 prevents thrombus formation effectively, similar to clopidogrel. Intriguingly, at dose achieving similar antithrombotic effect compared to clopidogrel, BF066 does not increase bleeding significantly. Taken together, these results suggest that BF066 may be an effective and safe antiplatelet agent targeting both PDE and A2A. Considering the successful use of combined antiplatelet therapy, BF066 may be further developed as a novel dual target antiplatelet agent.
We have recently reported the discovery of numerous new compounds that are selective inhibitors of all of the subtypes of the adenosine receptor family via a pharmacophore database searching and screening strategy. During the course of this work we made the unexpected discovery of a potent A2B receptor antagonist, 4-methyl-7-methoxyquinazolyl-2-(2′-amino-4′-imidazolinone) (38, CMB 6446), which showed selectivity for this receptor and functioned as an antagonist, with a binding Ki value of 112 nM. We explored the effects of both substituent- and ring-structural variations on the receptor affinity in this series of derivatives, which were found to be mostly non-selective adenosine receptor ligands with Ki values in the micromolar range. Since no enhancement of A2B receptor affinity of 38 was achieved, the previously reported pharmacophore-based searching strategy yielded the most potent and selective structurally-related hit in the database originally searched.
Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective.
A3 adenosine receptor (A3AR) ligands have been modified to optimize their interaction with the A3AR. Most of these modifications have been made to the N6 and C2 positions of adenine as well as the ribose moiety, and using a combination of these substitutions leads to the most efficacious, selective, and potent ligands. A3AR agonists such as IB-MECA and Cl-IB-MECA are now advancing into Phase II clinical trials for treatments targeting diseases such as cancer, arthritis, and psoriasis. Also, a wide number of compounds exerting high potency and selectivity in antagonizing the human (h)A3AR have been discovered. These molecules are generally characterized by a notable structural diversity, taking into account that aromatic nitrogen-containing monocyclic (thiazoles and thiadiazoles), bicyclic (isoquinoline, quinozalines, (aza)adenines), tricyclic systems (pyrazoloquinolines, triazoloquinoxalines, pyrazolotriazolopyrimidines, triazolopurines, tricyclic xanthines) and nucleoside derivatives have been identified as potent and selective A3AR antagonists. Probably due to the “enigmatic” physiological role of A3AR, whose activation may produce opposite effects (for example, concerning tissue protection in inflammatory and cancer cells) and may produce effects that are species dependent, only a few molecules have reached preclinical investigation. Indeed, the most advanced A3AR antagonists remain in preclinical testing. Among the antagonists described above, compound OT-7999 is expected to enter clinical trials for the treatment of glaucoma, while several thiazole derivatives are in development as antiallergic, antiasthmatic and/or antiinflammatory drugs.
A3 adenosine receptor; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Purines; Structure activity relationship; Nucleoside; G protein-coupled receptor; Neoceptor
Excitatory glutamatergic synapses in the hippocampal CA1 region of rats are potently inhibited by purines, including adenosine, ATP, and ATP analogs. Adenosine receptors are A1 known to mediate at least part of the response to adenine nucleotides, either because adenine nucleotides activate A1 receptors directly, or activate them secondarily upon the nucleotides’ conversion to adenosine. In the present studies, the inhibitory effects of adenosine, ATP, the purportedly stable ATP analog adenosine-5′-O-(3-thio)triphosphate (ATPγS), and cyclic AMP were examined in mice with a null mutation in the adenosine A1 receptor gene. ATPγS displaced the binding of A1-selective ligands to intact brain sections and brain homogenates from adenosine A1 receptor wild-type animals. In homogenates, but not in intact brain sections, this displacement was abolished by adenosine deaminase. In hippocampal slices from wild-type mice, purines abolished synaptic responses, but slices from mice lacking functional A1 receptors showed no synaptic modulation by adenosine, ATP, cAMP, or ATPγS. In slices from heterozygous mice the dose-response curve for both adenosine and ATP was shifted to the right. In all cases, inhibition of synaptic responses by purines could be blocked by prior treatment with the competitive adenosine A1 receptor antagonist 8-cyclopentyltheophylline. Taken together, these results show that even supposedly stable adenine nucleotides are rapidly converted to adenosine at sites close to the A1 receptor, and that inhibition of synaptic transmission by purine nucleotides is mediated exclusively by A1 receptors.
On the basis of a bioisosteric rationale, 4′-thionucleoside analogues of IB-MECA, which is a potent and selective A3 adenosine receptor agonist (AR), were synthesized from d-gulonic acid γ-lactone. The 4′-thio analogue (5h) of IB-MECA showed extremely high binding affinity (Ki = 0.25 nM) at the human A3AR and was more potent than IB-MECA (Ki = 1.4 nM). Bulky substituents at the 5′-uronamide position, such as cyclohexyl and 2- methylbenzyl, in this series of 2-H nucleoside derivatives were tolerated in A3AR binding, although small alkyl analogues were more potent.
A3 adenosine receptor; 4’-thionucleosides; agonist; binding affinity
The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-l,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H]CGS21680 (2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]adenosine-5′-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversibly binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.
We recently reported that 2-substitution of N6-benzyladenosine-5'-uronamides greatly enhances selectivity of agonists for rat A3 adenosine receptors J. Med. Chem.
1994, 37, 3614–3621). Specifically, 2-Chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (2-CI-IB-MECA), which displayed a K1 value of 0.33 nM, is the most selective for A3 receptors yet reported with selectivity versus A1 and A2a receptors of 2500- and 1400-fold, respectively. In order to obtain pharmacological tools for the study of A3 adenosine receptors, two routes for radiolabeling of 2-CI-IB-MECA through incorporation of tritium at the 5'-methylamido group were compared. One route formed a 2',3'-protected nucleoside 5'-carboxylic acid (9), which was condensed with methylamine and deprotected. The more efficient synthesis started from D-ribose and provided 2-CI-IB-MECA (12) in six steps with an overall yield of 5.6 %. Tritium was introduced in the penultimate step by heating N6-(3-iodobenzyl)-2-chloro-2',3'-di-O-acetyl-5'-(methoxycarbonyl)adenosine (17) with [3H]methylamine in methanol at 60 °C for 2 h. The specific activity of [3H]2-CI-IB-MECA was 29 Ci/mmol with a radiochemical purity of 99%.
Adenosine Derivatives; Radioligands; Adenosine Receptors; Tritium; Nucleosides
The affinity and efficacy at four subtypes (A1, A2A, A2B and A3) of human adenosine receptors (ARs) of a wide range of 2-substituted adenosine derivatives were evaluated using radioligand binding assays and a cyclic AMP functional assay in intact CHO cells stably expressing these receptors. Similar to previous studies of the N6-position, several 2-substituents were found to be critical structural determinants for the A3AR activation. The following adenosine 2-ethers were moderately potent partial agonists (Ki, nM): benzyl (117), 3-chlorobenzyl (72), 2-(3-chlorophenyl)ethyl (41), and 2-(2-naphthyl)ethyl (130). The following adenosine 2-ethers were A3AR antagonists: 2,2-diphenylethyl, 2-(2-norbornan)ethyl, R- and S-2-phenylbutyl, and 2-(2-chlorophenyl)ethyl. 2-(S-2-Phenylbutyloxy)a-denosine as an A3AR antagonist right-shifted the concentration–response curve for the inhibition by NECA of cyclic AMP accumulation with a KB value of 212 nM, which is similar to its binding affinity (Ki = 175 nM). These 2-substituted adenosine derivatives were generally less potent at the A1AR in comparison to the A3AR, but fully efficacious, with binding Ki values over 100 nM. The 2-phenylethyl moiety resulted in higher A3AR affinity (Ki in nM) when linked to the 2-position of adenosine through an ether group (54), than when linked through an amine (310) or thioether (1960). 2-[2-(l-Naphthyl)ethyloxy]adenosine (Ki = 3.8 nM) was found to be the most potent and selective (>50-fold) A2A agonist in this series. Mixed A2A/A3AR agonists have been identified. Interestingly, although most of these compounds were extremely weak at the A2BAR, 2-[2-(2-naphthyl)ethyloxy]adenosine (EC50 = 1.4 µM) and 2-[2-(2-thienyl)-ethyloxy]adenosine (EC50 = 1.8 (M) were found to be relatively potent A2B agonists, although less potent than NECA (EC50 = 140 nM).
Adenosine receptors; Purines; Nucleosides; GPCR; Efficacy; Structure–activity relationships
A variety of non-xanthine heterocycles were found to be antagonists of binding of [3H]phenylisopropyladenosine to rat brain A1-adenosine receptors and of activation of adenylate cyclase via interaction of N-ethylcarboxarnidoadenosine with A2-adenosine receptors in human platelet and rat pheochromocytoma cell membranes. The pyrazolopyridines tracazolate, cartazolate and etazolate were several fold more potent than theophylline at both A1- and A2-adenosine receptors. The pyrazolopyridines, however, were still many fold less potent than 8-phenyltheophylline and other 8-phenyl-1,3-dialkylxanthines. A structurally related N6-substituted 9-methyladenine was also a potent adenosine antagonist with selectivity for A1 receptors. None of several aryl-substituted heterocycles, including a thiazolopyrimidine, imidazopyridines, benzimidazoles, a pyrazoloquinoline, a mesoionic xanthine analog and a triazolopyridazine exhibited the high potency typical of 8-phenyl-1,3-dialkylxanthines. A furyl-substituted triazoloquinazoline was very potent at both A1 and A2 receptors. A pteridin-2,4-dione, 1,3-dipropyllumazine, was somewhat less potent than theophylline at A1- and A2-adenosine receptors, whereas 1,3-dimethyllumazine was much less potent. A benzopteridin-2,4-dione, alloxazine, was somewhat more potent than theophylline. Other heterocycles with antagonist activity were the dibenzazepine carbamazepine and β-carboline-3-ethyl carboxylate. The phenylimidazoline clonidine had no activity, whereas a related dihydroxyphenylimidazoline was a weak non-competitive adenosine antagonist.
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.
Adenosine receptors; Antagonist binding; Ligand-based homology modeling; Molecular modeling
The structure activity relationship (SAR) of 1,2,4-triazolo[1,5-a]-1,3,5-triazine derivatives related to ZM241385 as antagonists of the A2A adenosine receptor (AR) was explored through the synthesis of analogues substituted at the 5 position. The A2A AR X-ray structure was used to propose a structural basis for the activity and selectivity of the analogues and to direct the synthetic design strategy to provide access to solvent-exposed regions. Thus, we have identified a point of substitution for the attachment of solubilizing groups to enhance both aqueous solubility and physicochemical properties, maintaining potent interactions with the A2A AR and, in some cases, receptor subtype selectivity. Among the most potent and selective novel compounds were a long-chain ether-containing amine congener 20 (Ki 11.5 nM) and its urethane-protected derivative 14 (Ki 17.8 nM). Compounds 20 and 31 (Ki 11.5 and 16.9 nM, respectively) were readily water soluble up to 10 mM. The analogues were docked in the crystallographic structure of the hA2A AR and in a homology model of the hA3 AR, and the per residue electrostatic and hydrophobic contributions to the binding were assessed and stabilizing factors were proposed.
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylyl cyclase
In the last 5 years, many efforts have been conducted searching potent and selective human A3 adenosine antagonists. In this field several different classes of compounds, possessing very good affinity (nM range) and with a broad range of selectivity, have been proposed. Recently, our group synthesized a new series of pyrazolo-triazolo-pyrimidines bearing different substitutions at the N5 and N8 positions, which have been described as highly potent and selective human A3 adenosine receptor antagonists. The present review summarizes available data and provides an overview of the structure–activity relationships found for this class of human A3 adenosine receptor antagonists.
adenosine receptors; antagonists; binding; molecular modeling; pyrazolo-triazolo-pyrimidines
In this paper, we present the biochemical and biological evaluation of N-arylmethyl-substituted iminoribitol derivatives as potential chemotherapeutic agents against trypanosomiasis. Previously, a library of 52 compounds was designed and synthesized as potent and selective inhibitors of Trypanosoma vivax inosine-adenosine-guanosine nucleoside hydrolase (IAG-NH). However, when the compounds were tested against bloodstream-form Trypanosoma brucei brucei, only one inhibitor, N-(9-deaza-adenin-9-yl)methyl-1,4-dideoxy-1,4-imino-d-ribitol (UAMC-00363), displayed significant activity (mean 50% inhibitory concentration [IC50] ± standard error, 0.49 ± 0.31 μM). Validation in an in vivo model of African trypanosomiasis showed promising results for this compound. Several experiments were performed to investigate why only UAMC-00363 showed antiparasitic activity. First, the compound library was screened against T. b. brucei IAG-NH and inosine-guanosine nucleoside hydrolase (IG-NH) to confirm the previously demonstrated inhibitory effects of the compounds on T. vivax IAG-NH. Second, to verify the uptake of these compounds by T. b. brucei, their affinities for the nucleoside P1 and nucleoside/nucleobase P2 transporters of T. b. brucei were tested. Only UAMC-00363 displayed significant affinity for the P2 transporter. It was also shown that UAMC-00363 is concentrated in the cell via at least one additional transporter, since P2 knockout mutants of T. b. brucei displayed no resistance to the compound. Consequently, no cross-resistance to the diamidine or the melaminophenyl arsenical classes of trypanocides is expected. Third, three enzymes of the purine salvage pathway of procyclic T. b. brucei (IAG-NH, IG-NH, and methylthioadenosine phosphorylase [MTAP]) were investigated using RNA interference. The findings from all these studies showed that it is probably not sufficient to target only the nucleoside hydrolase activity to block the purine salvage pathway of T. b. brucei and that, therefore, it is possible that UAMC-00363 acts on an additional target.
We have established structure-activity relationships of novel truncated D-4′-thioadenosine derivatives from d-mannose as potent and selective A3 adenosine receptor (AR) antagonists. At the human A3 AR, most of N6-substituted analogues showed high potency and selectivity and acted as pure antagonists in a cyclic AMP functional assay. Among compounds tested, 2-chloro-N6-3-chlorobenzyl and N6-3-chlorobenzyl analogues displayed very high binding affinities (Ki = 1.66 nM and 1.5 nM, respectively) at the human A3 AR. Truncated 4′-thioadenosine derivatives studied here are regarded as an excellent template for the design of novel A3 AR antagonists to act at both human and murine species.
Amide derivatives of a carboxylic acid congener of 1,3-dialkylxanthine, having a 4-[(carboxymethyl)oxy]phenyl substituent at the 8-position, have been synthesized in order to identify potent antagonists at A2-adenosine receptors stimulatory to adenylate cyclase in platelets. Distal structural features of amide-linked chains and the size of the 1,3-dialkyl groups have been varied. 1,3-Diethyl groups, more than 1,3-dimethyl or 1,3-dipropyl groups, favor A2 potency, even in the presence of extended chains attached at the 8-(p-substituted-phenyl) position. Polar groups, such as amines, on the chain simultaneously enhance water solubility and A2 potency. Among the most potent A2 ligands are an amine congener, 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-diethylxanthine, and its D-lysyl conjugate, which have KB values of 21 and 23 nM, respectively, for the antagonism of N-ethyl-adenosine-5′-uronamide-stimulated adenylate cyclase activity in human platelet membranes. Strategies for the selection and tritiation of new radioligands for use in competitive binding assays at A2-adenosine receptors have been considered.
Functionalized congeners derived from 1,3-dipropyl-8-phenylxanthine and from N6-phenyladenosine were derivatized to contain electrophilic groups (isothiocyanate, N-hydroxysuccinimide ester, maleimide, sulfonyl chloride, or α-haloacyl group) capable of reaction with nucleophiles on biopolymers. The goal was to inhibit chemically the A1 adenosine receptor by using reactive agonist and antagonist ligands. Some of the electrophilic derivatives were synthesized through acylation of amine-functionalized congeners using hetero- or homobifunctional reagents available for protein cross-linking. The affinity for A1 adenosine receptors was evaluated in competitive binding assays by using rat and bovine brain membranes. Several xanthine and adenosine thiourea derivatives prepared from 1,3- and l,4-phenylene diisothiocyanate (DITC) were potent irreversible inhibitors of adenosine receptors. Derivatives of m-DITC, at concentrations between 10 and 500 nM, irreversibly eliminated binding at 90% of the A1-receptor sites. Receptor affinity of both xanthine and adenosine derivatives containing distal phenylthiourea substituents was diminished by electron-donating groups on the ring.