Truncated N6-substituted-(N)-methanocarba-adenosine derivatives
with 2-hexynyl substitution
were synthesized to examine parallels with corresponding 4′-thioadenosines.
Hydrophobic N6 and/or C2 substituents were tolerated in
A3AR binding, but only an unsubstituted 6-amino group with
a C2-hexynyl group promoted high hA2AAR affinity. A small
hydrophobic alkyl (4b and 4c) or N6-cycloalkyl group (4d) showed
excellent binding affinity at the hA3AR and was better
than an unsubstituted free amino group (4a). A3AR affinities of 3-halobenzylamine derivatives 4f–4i did not differ significantly, with Ki values of 7.8–16.0 nM. N6-Methyl derivative 4b (Ki = 4.9 nM) was a highly selective, low efficacy partial A3AR agonist. All compounds were screened for renoprotective effects
in human TGF-β1-stimulated mProx tubular cells, a kidney fibrosis
model. Most compounds strongly inhibited TGF-β1-induced collagen
I upregulation, and their A3AR binding affinities were
proportional to antifibrotic effects; 4b was most potent
(IC50 = 0.83 μM), indicating its potential as a good
therapeutic candidate for treating renal fibrosis.
We synthesized homologated truncated 4′-thioadenosine analogues 3 in which a methylene (CH2) group was inserted in place of the glycosidic bond of a potent and selective A3 adenosine receptor antagonist 2. The analogues were designed to induce maximum binding interaction in the binding site of the A3 adenosine receptor. However, all homologated nucleosides were devoid of binding affinity at all subtypes of adenosine receptors, indicating that free rotation through the single bond allowed the compound to adopt an indefinite number of conformations, disrupting the favorable binding interaction essential for receptor recognition.
homologation; A3 adenosine receptor; binding affinity; truncated 4′-thioadenosine
Novel D- and L-4′-thioadenosine derivatives lacking the 4′-hydroxymethyl moiety were synthesized, starting from D-mannose and D-gulonic γ-lactone, respectively, as potent and selective species-independent A3 adenosine receptor (AR) antagonists. Among the novel 4′-truncated 2-H nucleosides tested, a N6-(3-chlorobenzyl) derivative 7c was the most potent at the human A3 AR (Ki = 1.5 nM), but a N6-(3-bromobenzyl) derivative 7d showed the optimal species-independent binding affinity.
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Xanthine and adenosine derivatives, known to bind to recombinant rat A3 adenosine receptors stably expressed in Chinese hamster ovary cells, were characterized in a functional assay consisting of activation of A3 receptor-stimulated binding of [35S]GTPγS in rat RBL-2H3 cell membranes. 1,3-Dibutylxanthine-7-riboside-5′-N-methylcarboxamide (DBXRM, 7b), previously shown to inhibit adenylyl cyclase via rat A3 receptors with full efficacy, appeared to be a partial agonist at the rat A3 receptor of RBL-2H3 cells. Full agonists, such as Cl-IB-MECA or I-AB-MECA, were more potent and effective than the partial agonist DBXRM in causing desensitization of rat A3 receptors, as indicated by loss of [35S]GTPγS binding. At A1 receptors, antagonism of agonist-elicited inhibition of rat adipocyte adenylyl cyclase was observed for several xanthine-7-riboside derivatives that had been shown to be full agonists at rat A3 receptors. A new xanthine riboside (3′-deoxyDBXRM, 7c) was synthesized and found to be a partial agonist at rat A3 receptors and an antagonist at rat A1 receptors. Thus, it is possible for the same compound to stimulate one adenosine receptor subtype (A3) and block another subtype (A1) within the same species.
xanthines; adenosine derivatives; nucleosides; adenylyl cyclase; guanine nucleotides
On the basis of potent and selective binding affinity of truncated 4′-thioadenosine derivatives at the human A3 adenosine receptor (AR), their bioisosteric 4′-oxo derivatives were designed and synthesized from commercially available 2,3-O-isopropylidene-d-erythrono lactone. The derivatives tested in AR binding assays were substituted at the C2 and N6 positions. All synthesized nucleosides exhibited potent and selective binding affinity at the human A3 AR. They were less potent than the corresponding 4′-thio analogues, but showed higher selectivity to other subtypes. The 2-Cl series generally were better than the 2-H series in view of binding affinity and selectivity. Among compounds tested, compound 5d (X = Cl, R = 3-bromobenzyl) showed the highest binding affinity (Ki = 13.0±6.9 nM) at the hA3 AR with high selectivity (at least 1000-fold) in comparison to other AR subtypes. Like the corresponding truncated 4′-thio series, compound 5d antagonized the action of an agonist to inhibit forskolin-stimulated adenylate cyclase in hA3 AR-expressing CHO cells. Although the 4′-oxo series were less potent than the 4′-thio series, this class of human A3 AR antagonists is also regarded as another good template for the design of A3 AR antagonists and for further drug development.
A3 Adenosine Receptor; Antagonists; Truncated Adenosine; Structure-Activity Relationships
Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases.
lithiation-mediated stannyl transfer; structure-activity relationship; adenosine receptors; truncated adenosine; palladium-catalyzed cross coupling; dual-acting ligands
An alternative approach to overcome the inherent lack of specificity of conventional agonist therapy can be the reengineering of the GPCRs and their agonists. A reengineered receptor (neoceptor) could be selectively activated by a modified agonist, but not by the endogenous agonist. Assisted by rhodopsin-based molecular modeling, we pinpointed mutations of the A3 adenosine receptor (AR) for selective affinity enhancement following complementary modifications of adenosine. Ribose modifications examined included, at 3′: amino, aminomethyl, azido, guanidino, ureido; and at 5′: uronamido, azidodeoxy. N6-variations included: 3-iodobenzyl, 5-chloro-2-methyloxybenzyl, and methyl. An N6-3-iodobenzyl-3′-ureido adenosine derivative 10 activated phospholipase C in COS-7 cells (EC50=0.18 μM) or phospholipase D in chick primary cardiomyocytes mediated by a mutant (H272E), but not the wild-type, A3AR. The affinity enhancements for 10 and the corresponding 3′-acetamidomethyl analogue 6 were >100-fold and >20-fold, respectively. 10 concentration-dependently protected cardiomyocytes transfected with the neoceptor against hypoxia. Unlike 10, adenosine activated the wild-type A3AR (EC50 of 1.0 μM), but had no effect on the H272E mutant A3AR (100 μM). Compound 10 was inactive at human A1, A2A, and A2BARs. The orthogonal pair comprising an engineered receptor and a modified agonist should be useful for elucidating signaling pathways and could be therapeutically applied to diseases following organ-targeted delivery of the neoceptor gene.
1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.
Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of P2Y1 receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem.
2000, 43, 829–842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y1 receptor and by using the radiolabeled antagonist [3H]2-chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine 3′,5′-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y1 receptor was an (N)-methanocarba N6-methyl-2-iodo analogue 12, which displayed a Ki value in competition for binding of [3H]5 of 0.79 nM and a KB value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y1 receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y1 receptors. An enzymatic method of synthesis of the 3′,5′-bisphosphate from the corresponding 3′-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.
The truncated C2- and C8-substituted 4′-thioadenosine derivatives 4a−d were synthesized from d-mannose, using palladium-catalyzed cross-coupling reactions as key steps. In this study, an A3 adenosine receptor (AR) antagonist, truncated 4′-thioadenosine derivative 3, was successfully converted into a potent A2A AR agonist 4a (Ki = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N6-substituted. However, C8-substitution greatly reduced binding affinity at the human A2A AR. All synthesized compounds 4a−d maintained their affinity at the human A3 AR, but 4a was found to be a competitive A3 AR antagonist/A2A AR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted 4′-thioadenosine derivatives 4a and 4b can serve as novel templates for the development of new A2A AR ligands.
A2A adenosine receptor agonists; truncated 2-hexynyl-4′-thioadenosine; palladium-catalyzed cross-coupling reactions; binding mode
The truncated C2- and C8-substituted-4′-thioadenosine derivatives 4a-d were synthesized from D-mannose, using palladium-catalyzed cross coupling reactions as key steps. In this study, an A3 adenosine receptor (AR) antagonist, truncated 4′-thioadenosine derivative 3 was successfully converted into a potent A2AAR agonist 4a (Ki = 7.19 ± 0.6 nM) by appending a 2-hexynyl group at the C2-position of a derivative of 3 that was N6-substituted. However, C8-substitution greatly reduced binding affinity at the human A2AAR. All synthesized compounds 4a-d maintained their affinity at the human A3AR, but 4a was found to be a competitive A3AR antagonist/A2AAR agonist in cyclic AMP assays. This study indicates that the truncated C2-substituted-4′-thioadenosine derivatives 4a and 4b can serve as a novel template for the development of new A2AAR ligands.
A2A adenosine receptor agonists; truncated 2-hexynyl-4′-thioadenosine; palladium-catalyzed cross coupling reactions; binding mode
We have found previously that structural features of adenosine derivatives, particularly at the N6- and 2-positions of adenine, determine the intrinsic efficacy as A3 adenosine receptor (AR) agonists. Here, we have probed this phenomenon with respect to the ribose moiety using a series of ribose-modified adenosine derivatives, examining binding affinity and activation of the human A3 AR expressed in CHO cells. Both 2′- and 3′-hydroxyl groups in the ribose moiety contribute to A3 AR binding and activation, with 2′-OH being more essential. Thus, the 2′-fluoro substitution eliminated both binding and activation, while a 3′-fluoro substitution led to only a partial reduction of potency and efficacy at the A3 AR. A 5′-uronamide group, known to restore full efficacy in other derivatives, failed to fully overcome the diminished efficacy of 3′-fluoro derivatives. The 4′-thio substitution, which generally enhanced A3 AR potency and selectivity, resulted in 5′-CH2OH analogues (10 and 12) which were partial agonists of the A3 AR. Interestingly, the shifting of the N6-(3-iodobenzyl)adenine moiety from the 1′- to 4′-position had a minor influence on A3 AR selectivity, but transformed 15 into a potent antagonist (16) (Ki = 4.3 nM). Compound 16 antagonized human A3 AR agonist-induced inhibition of cyclic AMP with a KB value of 3.0 nM. A novel apio analogue (20) of neplanocin A, was a full A3 AR agonist. The affinities of selected, novel analogues at rat ARs were examined, revealing species differences. In summary, critical structural determinants for human A3 AR activation have been identified, which should prove useful for further understanding the mechanism of receptor activation and development of more potent and selective full agonists, partial agonists and antagonists for A3 ARs.
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
His272 (7.43) in the seventh transmembrane domain (TM7) of the human A3 adenosine receptor (AR) interacts with the 3′ position of nucleosides, based on selective affinity enhancement at a H272E mutant A3 AR (neoceptor) of 3′-ureido, but not 3′-OH, adenosine analogues. Here, mutation of the analogous H278 of the human A1 AR to Ala, Asp, Glu, or Leu enhanced the affinity of novel 2′- and 3′-ureido adenosine analogues, such as 10 (N6-cyclopentyl-3′-ureido-3′-deoxyadenosine), by >100-fold, while decreasing the affinity or potency of adenosine and other 3′-OH adenosine analogues. His278 mutant receptors produced a similar enhancement regardless of the charge character of the substituted residue, implicating steric rather than electrostatic factors in the gain of function, a hypothesis supported by rhodopsin-based molecular modeling. It was also demonstrated that this interaction was orientationally specific; i.e., mutations at the neighboring Thr277 did not enhance the affinity for a series of 2′- and 3′-ureido nucleosides. Additionally, H-bonding groups placed on substituents at the N6 or 5′ position demonstrated no enhancement in the mutant receptors. These reengineered human A1 ARs revealed orthogonality similar to that of the A3 but not the A2A AR, in which mutation of the corresponding residue, His278, to Asp did not enhance nucleoside affinity. Functionally, the H278D A1 AR was detectable only in a measure of membrane potential and not in calcium mobilization. This neoceptor approach should be useful for the validation of molecular modeling and the dissection of promiscuous GPCR signaling.
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.
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 highly selective A3 receptor agonist, 4′-thio-Cl-IB-MECA was successfully converted into selective A3 receptor antagonists by appending a second N-alkyl group on the 5′-uronamide position. This result indicates that the hydrogen bonding ability of the 5′-uronamide is essential for the conformational change required for the receptor activation. Among compounds tested, a N6-(3-bromobenzyl) derivative with 5′-dimethyluronamide exhibited the highest binding affinity (Ki = 9.32 nM) at the human A3 AR with very-low binding affinities to other AR subtypes.
On the basis of high binding affinity of 3'-aminoadenosine derivatives 2b at the human A3 adenosine receptor (AR), 3'-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A3AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A3AR.
3'-acetamidoadenosines; human A3 adenosine receptor; hydrogen bonding donor; hydroboration-oxidation; steric effects
The highly selective agonists of the A3 adenosine receptor (AR), Cl-IB-MECA (2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine) and its 4′-thio analogue, were successfully converted into selective antagonists simply by appending a second N-methyl group on the 5′-uronamide position. The 2-chloro-5′-(N,N-dimethyl)uronamido analogues bound to, but did not activate the human A3AR, with Ki values of 29 nM (4′-O) and 15 (4′-S) nM, showing >100-fold selectivity over A1, A2A, and A2BARs. Competitive antagonism was demonstrated by Schild analysis. The 2-(dimethylamino)-5′-(N,N-dimethyl)uronamido substitution also retained A3AR selectivity but lowered affinity.
nucleoside; G protein-coupled receptor; adenylyl cyclase; molecular modeling; radioligand binding; AR, adenosine receptor; CGS21680, 2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine; CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine; CPA, N6-cyclopentyladenosine; DMEM, Dulbecco’s modified Eagle’s medium; I-AB-MECA, N6-(4-amino-3-iodobenzyl)-5′-N-methylcarboxamidoadenosine; NECA, 5′-N-ethylcarboxamidoadenosine; PIA, N6-(phenylisopropyl)adenosine; PTLC, preparative thin layer chromatography