A3 adenosine receptor agonists containing dopamine moieties for enhanced interspecies affinity.

Following our study of 4'-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5'-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (∼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and ß-arrestin 2 (ßarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.

Dual Actions of A2A and A3 Adenosine Receptor Ligand Prevents Obstruction-Induced Kidney Fibrosis in Mice.

Kidney fibrosis is the final outcome of chronic kidney disease (CKD). Adenosine plays a significant role in protection against cellular damage by activating four subtypes of adenosine receptors (ARs), A1AR, A2AAR, A2BAR, and A3AR. A2AAR agonists protect against inflammation, and A3AR antagonists effectively inhibit the formation of fibrosis. Here, we showed for the first time that LJ-4459, a newly synthesized dual-acting ligand that is an A2AAR agonist and an A3AR antagonist, prevents the progression of tubulointerstitial fibrosis. Unilateral ureteral obstruction (UUO) surgery was performed on 6-week-old male C57BL/6 mice. LJ-4459 (1 and 10 mg/kg) was orally administered for 7 days, started at 1 day before UUO surgery. Pretreatment with LJ-4459 improved kidney morphology and prevented the progression of tubular injury as shown by decreases in urinary kidney injury molecular-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) excretion. Obstruction-induced tubulointerstitial fibrosis was attenuated by LJ-4459, as shown by a decrease in fibrotic protein expression in the kidney. LJ-4459 also inhibited inflammation and oxidative stress in the obstructed kidney, with reduced macrophage infiltration, reduced levels of pro-inflammatory cytokines, as well as reduced levels of reactive oxygen species (ROS). These data demonstrate that LJ-4459 has potential as a therapeutic agent against the progression of tubulointerstitial fibrosis.

Synthesis and evaluation of adenosine derivatives as A1, A2A, A2B and A3 adenosine receptor ligands containing boron clusters as phenyl isosteres and selective A3 agonists.

A series of adenosine and 2'-deoxyadenosine pairs modified with a 1,12-dicarba-closo-dodecaborane cluster or alternatively with a phenyl group at the same position was synthesized, and their affinity was determined at A1, A2A, A2B and A3 adenosine receptors (ARs). While AR affinity differences were noted, a general tendency to preferentially bind A3 AR over other ARs was observed for most tested ligands. In particular, 5'-ethylcarbamoyl-N6-(3-phenylpropyl)adenosine (18), N6-(3-phenylpropyl)-2-chloroadenosine (24) and N6-(3-phenylpropyl)adenosine (40) showed nanomolar A3 affinity (Ki 4.5, 6.4 and 7.5 nM, respectively). Among the boron cluster-containing compounds, the highest A3 affinity (Ki 206 nM) was for adenosine derivative 41 modified at C2. In the matched molecular pairs, analogs bearing boron clusters were found to show lower binding affinity for adenosine receptors than the corresponding phenyl analogs. Nevertheless, interestingly, several boron cluster modified adenosine ligands showed significantly higher A3 receptor selectivity than the corresponding phenyl analogs: 7vs. 8, 15vs. 16, 17vs. 18.

Assessment of biased agonism at the A3 adenosine receptor using ß-arrestin and miniGαi recruitment assays.

The A3 adenosine receptor (A3AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable ß-arrestin 2 (ßarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGαi protein to activated A3AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N6-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the ßarr2 or miniGαi pathway.

7-Amino-2-aryl/hetero-aryl-5-oxo-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles: Synthesis and adenosine receptor binding studies.

A series of novel 7-amino-5-oxo-2-substituted-aryl/hetero-aryl-5,8-dihydro[1,2,4]triazolo[1,5-a]pyridine-6-carbonitriles (4a-4t) was synthesized, characterized and evaluated for their binding affinity and selectivity towards hA1 , hA2A , hA2B and hA3 adenosine receptors (ARs). Compound 4a with a phenyl ring at 2-position of the triazolo moiety of the scaffold showed high affinity and selectivity for hA1 AR (Ki hA1  = 0.076 µM, hA2A  = 25.6 µM and hA3  > 100 µM). Introduction of various electron donating and withdrawing groups at different positions of the phenyl ring resulted in drastic reduction in affinity and selectivity towards all the ARs, except compound 4b with a 4-hydroxyphenyl group at 2-position. Interestingly, the replacement of the phenyl ring with a smaller heterocyclic thiophene ring (π excessive system) resulted in further improvement of affinity for hA1 AR of compound 4t (Ki hA1  = 0.051 µM, hA2A  = 9.01 µM and hA3  > 13.9 µM) while retaining the significant selectivity against all other AR subtypes similar to compound 4a. The encouraging results for compounds 4a and 4t indicate that substitution at 2-position of the scaffold with π-excessive systems other than thiophene may lead to even more potent and selective hA1 AR antagonists.

Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding.

Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

The synthesis of a series of adenosine A3 receptor agonists.

A series of 1'-(6-aminopurin-9-yl)-1'-deoxy-N-methyl-ß-d-ribofuranuronamides that were characterised by 2-dialkylamino-7-methyloxazolo[4,5-b]pyridin-5-ylmethyl substituents on N6 of interest for screening as selective adenosine A3 receptor agonists, have been synthesised. This work involved the synthesis of 2-dialkylamino-5-aminomethyl-7-methyloxazolo[4,5-b]pyridines and analogues that were coupled with the known 1'-(6-chloropurin-9-yl)-1'-deoxy-N-methyl-ß-d-ribofuranuronamide. The oxazolo[4,5-b]pyridines were synthesized by regioselective functionalisation of 2,4-dimethylpyridine N-oxides. The regioselectivities of these reactions were found to depend upon the nature of the heterocycle with 2-dimethylamino-5,7-dimethyloxazolo[4,5-b]pyridine-N-oxide undergoing regioselective functionalisation at the 7-methyl group on reaction with trifluoroacetic anhydride in contrast to the reaction of 4,6-dimethyl-3-hydroxypyridine-N-oxide with acetic anhydride that resulted in functionalisation of the 6-methyl group. To optimise selectivity for the A3 receptor, 5-aminomethyl-7-bromo-2-dimethylamino-4-[(3-methylisoxazol-5-yl)methoxy]benzo[d]oxazole was synthesised and coupled with the 1'-(6-chloropurin-9-yl)-1'-deoxy-N-methyl-ß-d-ribofuranuronamide. The products were active as selective adenosine A3 agonists.

In vivo phenotypic screening for treating chronic neuropathic pain: modification of C2-arylethynyl group of conformationally constrained A3 adenosine receptor agonists.

(N)-Methanocarba adenosine 5'-methyluronamides containing 2-arylethynyl groups were synthesized as A3 adenosine receptor (AR) agonists and screened in vivo (po) for reduction of neuropathic pain. A small N(6)-methyl group maintained binding affinity, with human > mouse A3AR and MW < 500 and other favorable physicochemical properties. Emax (maximal efficacy in a mouse chronic constriction injury pain model) of previously characterized A3AR agonist, 2-(3,4-difluorophenylethynyl)-N(6)-(3-chlorobenzyl) derivative 6a, MRS5698, was surpassed. More efficacious analogues (in vivo) contained the following C2-arylethynyl groups: pyrazin-2-yl 23 (binding Ki, hA3AR, nM 1.8), fur-2-yl 27 (0.6), thien-2-yl 32 (0.6) and its 5-chloro 33, MRS5980 (0.7) and 5-bromo 34 (0.4) equivalents, and physiologically unstable ferrocene 36, MRS5979 (2.7). 33 and 36 displayed particularly long in vivo duration (>3 h). Selected analogues were docked to an A3AR homology model to explore the environment of receptor-bound C2 and N(6) groups. Various analogues bound with µM affinity at off-target biogenic amine (M2, 5HT2A, ß3, 5HT2B, 5HT2C, and α2C) or other receptors. Thus, we have expanded the structural range of orally active A3AR agonists for chronic pain treatment.

Synthesis and evaluation of N6-substituted apioadenosines as potential adenosine A3 receptor modulators.

Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A3AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of ß-D-apio-D-furano- and α-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N(6) of ß-D-apio-D-furanoadenosine afforded an A3AR antagonist (10c, Ki=0.98 µM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki=3.07 µM). The structural basis for this difference was examined by docking to an A3AR model; the antagonist lacked a crucial interaction with Thr94.

Synthesis and anti-renal fibrosis activity of conformationally locked truncated 2-hexynyl-N(6)-substituted-(N)-methanocarba-nucleosides as A3 adenosine receptor antagonists and partial agonists.

Truncated N(6)-substituted-(N)-methanocarba-adenosine derivatives with 2-hexynyl substitution were synthesized to examine parallels with corresponding 4'-thioadenosines. Hydrophobic N(6) 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 N(6)-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. N(6)-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-ß1-stimulated mProx tubular cells, a kidney fibrosis model. Most compounds strongly inhibited TGF-ß1-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.

Rational design of sulfonated A3 adenosine receptor-selective nucleosides as pharmacological tools to study chronic neuropathic pain.

(N)-Methanocarba(bicyclo[3.1.0]hexane)adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g., blood-brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N(6)-p-sulfophenylethyl substituent would determine higher hA3AR vs mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N(6)-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki(hA3AR) = 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered ip reduced mouse chronic neuropathic pain that was ascribed to either A3AR or A1/A3AR using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine's CNS vs peripheral actions.

Structure-guided design of A(3) adenosine receptor-selective nucleosides: combination of 2-arylethynyl and bicyclo[3.1.0]hexane substitutions.

(N)-Methanocarba adenosine 5'-methyluronamides containing known A(3) AR (adenosine receptor)-enhancing modifications, i.e., 2-(arylethynyl)adenine and N(6)-methyl or N(6)-(3-substituted-benzyl), were nanomolar full agonists of human (h) A(3)AR and highly selective (K(i) ∼0.6 nM, N(6)-methyl 2-(halophenylethynyl) analogues 13 and 14). Combined 2-arylethynyl-N(6)-3-chlorobenzyl substitutions preserved A(3)AR affinity/selectivity in the (N)-methanocarba series (e.g., 3,4-difluoro full agonist MRS5698 31, K(i) 3 nM, human and mouse A(3)) better than that for ribosides. Polyaromatic 2-ethynyl N(6)-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (K(i) hA(3) 3.1 nM; A(1), A(2A), inactive) and fluorescent 1-pyrene adduct MRS5704 35 (K(i) hA(3) 68.3 nM), were conformationally rigid; receptor docking identified a large, mainly hydrophobic binding region. The vicinity of receptor-bound C2 groups was probed by homology modeling based on recent X-ray structure of an agonist-bound A(2A)AR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, the X-ray structure of related A(2A)AR is useful in guiding the design of new A(3)AR agonists.

Suppression of inflammation response by a novel A3 adenosine receptor agonist thio-Cl-IB-MECA through inhibition of Akt and NF-κB signaling.

Adenosine, a purine nucleoside, is released from metabolically active cells into extracellular space and plays an important role in various pathophysiological processes. Adenosine regulates many biological responses including inflammation by the interaction with their receptors such as A1, A(2A), A(2B), and A3. Especially, A3 adenosine receptor (A3AR) is considered to be expressed in macrophage cells. To the end, A3AR agonists have been reported to have an anti-inflammatory activity. In our continuous efforts to develop new anti-inflammatory agents, we found a novel adenosine analog, 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA), was a potent human A3AR agonist. The study was designed to investigate whether thio-Cl-IB-MECA has an anti-inflammatory potential in mouse macrophage RAW 264.7 cells and mouse sepsis model in vivo. Thio-Cl-IB-MECA exhibited an effective anti-inflammatory activity. The expression of pro-inflammatory biomarkers including inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and tumor necrosis factor (TNF-α) was suppressed by the treatment of thio-Cl-IB-MECA in the protein and mRNA levels in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Further examination revealed that thio-Cl-IB-MECA inhibited LPS-induced phosphatidylinositol 3-kinase (PI3 kinase)/Akt activation, NF-kB binding activity, and ß-catenin expression. In addition, in in vivo LPS-induced mouse endotoxemia model, thio-Cl-IB-MECA exerted the increase of survival rate compared to vehicle-treated mouse. The analysis of the protein levels of iNOS, IL-1ß, and TNF-α was also suppressed by the compound-treated groups in lung tissues. These results suggest that thio-Cl-IB-MECA might have an anti-inflammatory activity through the inhibition of pro-inflammatory cytokine expression by modulating PI3K/Akt and NF-κB signaling pathways.

Molecular modeling study on potent and selective adenosine A(3) receptor agonists.

Adenosine A(3) receptor (A(3)AR) is involved in a variety of key physio-pathological processes and its agonists are potential therapeutic agents for the treatment of rheumatoid arthritis, dry eye disorders, asthma, as anti-inflammatory agents, and in cancer therapy. Recently reported MECA (5'-N-methylcarboxamidoadenosine) derivatives bearing a methyl group in N(6)-position and an arylethynyl substituent in 2-position demonstrated to possess sub-nanomolar affinity and remarkable selectivity for the human A(3)AR, behaving as full agonists of this receptor. In this study, we made an attempt to get a rationalization of the high affinities and selectivities of these molecules for the human A(3)AR, by using adenosine receptor (AR) structural models based on the A(2A)AR crystal structure and molecular docking analysis. Post-docking analysis allowed to evaluate the ability of modeling tools in predicting AA(3)R affinity and in providing interpretation of compound substituents effect on the A(3)AR affinity and selectivity.