Jacobien K. von Frijtag Drabbe Künzel

2-Amino-6-furan-2-yl-4-substituted nicotinonitriles as A2A adenosine receptor antagonists.

A 2A adenosine receptor antagonists usually have bi- or tricyclic N aromatic systems with varying substitution patterns to achieve desired receptor affinity and selectivity. Using a pharmacophore model designed by overlap of nonxanthine type of previously known A 2A antagonists, we synthesized a new class of compounds having a 2-amino nicotinonitrile core moiety. From our data, we conclude that the presence of at least one furan group rather than phenyl is beneficial for high affinity on the A 2A adenosine receptor. Compounds 39 (LUF6050) and 44 (LUF6080) of the series had K i values of 1.4 and 1.0 nM, respectively, with reasonable selectivity toward the other adenosine receptor subtypes, A 1, A 2B, and A 3. The high affinity of 44 was corroborated in a cAMP second messenger assay, yielding subnanomolar potency for this compound.

A new generation of adenosine receptor antagonists: From di- to trisubstituted aminopyrimidines

New adenosine receptor ligands were designed as hybrid structures between previously synthesized substituted dicyanopyridines and aminopyrimidines, yielding two series of cyano-substituted diphenylaminopyrimidines. We were interested in assessing the effect of this substitution pattern on both affinity and intrinsic activity, as the dicyanopyridines comprised both agonists and inverse agonists, whereas the original aminopyrimidines were exclusively inverse agonists. It was found that the new compounds were generally selective for adenosine A(1) receptors, although affinity for the adenosine A(2A) receptor was also noticed for some of the compounds. In a cAMP second messenger assay the compounds behaved as inverse agonists rather than agonists. Among the more A(1) receptor-selective compounds were 5 (LUF6048), 27 (LUF6040) and 53 (LUF6056) with K(i) values of 8.1, 1.2 and 5.7nM, respectively.

8-substituted adenosine and theophylline-7-riboside analogues as potential partial agonists for the adenosine A1 receptor

A series of 8-substituted adenosine and theophylline-7-riboside analogues (28 and 9 compounds, respectively) was tested on adenosine A1 and A2A receptors as an extensive exploration of the adenosine C8-region. Alkylamino substituents at the 8-position cause an affinity decrease for adenosine analogues, but an affinity increase for theophylline-7-riboside derivatives. The affinity decrease is probably due to a direct steric hindrance between the C8-substituent and the binding site as well as to electronic effects, not to a steric influence on the ribose moiety to adopt the anti conformation. The 8-substituents increase the affinity of theophylline-7-riboside analogues probably by binding to a lipophilic binding site. The intrinsic activity was tested in vitro for some 8-substituted adenosine analogues, by determining the GTP shift in receptor binding studies and the inhibition of adenylate cyclase in a culture of rat thyroid FRTL-5 cells, and in vivo in the rat cardiovascular system for 8-butylaminoadenosine. Thus, it was shown that 8-ethyl-, 8-butyl-, and 8-pentylamino substituted analogues of adenosine may be partial agonists in vitro, and that 8-butylaminoadenosine is a partial agonist for the rat cardiovascular A1 receptor in vivo.

2,8-Disubstituted adenosine derivatives as partial agonists for the adenosine A2A receptor.

Novel 2,8-disubstituted adenosine derivatives were synthesized in good overall yields starting from 2-iodoadenosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. Some compounds displayed good adenosine A(2A) receptor affinities, with most of the 2-(1-hexynyl)- and 2-[(E)-1-hexenyl]-substituted derivatives having K(i) values in the nanomolar range. Although the introduction of an 8-alkylamino substituents decreased the affinity for the adenosine A(2A) receptor somewhat, the selectivity for this receptor compared to A(3) was improved significantly. The 8-methylamino (12) and 8-propylamino (14) derivatives of 2-(1-hexynyl)adenosine (3), showed reasonable A(2A) receptor affinities with K(i) values of 115 and 82nM, respectively, and were 49- and 26-fold selective for the adenosine A(2A) receptor compared to the A(3) receptor. The compounds were also evaluated for their ability to stimulate the cAMP production in CHO cells expressing the human adenosine A(2A) receptor. 2-(1-Hexynyl)adenosine (3) and 2-[(E)-1-hexenyl]adenosine (4) both showed submaximal levels of produced cAMP, compared to the reference full agonist CGS 21680, and thus behaved as partial agonists. Most 8-alkylamino-substituted derivatives of 3, displayed similar cAMP production as 3, and behaved as partial agonists as well. Introduction of alkylamino groups at the 8-position of 4, showed a slight reduction of the efficacy compared to 4, and these compounds were partial agonists also.

2-Nitro analogues of adenosine and 1-deazaadenosine: synthesis and binding studies at the adenosine A1, A2A and A3 receptor subtypes.

The influence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a nitro group at the 2-position with several N6 substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with good adenosine receptor affinity, showing directable selectivity for the A1, A2A and A3 adenosine receptor subtypes.

Allosteric modulation, thermodynamics and binding to wild‐type and mutant (T277A) adenosine A1 receptors of LUF5831, a novel nonadenosine‐like agonist

The interaction of a new nonribose ligand (LUF5831) with the human adenosine A1 receptor was investigated in the present study. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild‐type (wt) and mutant (T277A) adenosine A1 receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N6‐cyclopentyladenosine (CPA), a full agonist, and 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A1 receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A1 receptor (Ki=122±22 nM), whereas CPAs affinity was negligible. The results of temperature‐dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high‐ and low‐affinity states of the receptor, respectively. The inhibition of the forskolin‐induced production of cAMP through activation of the wt adenosine A1 receptor showed that LUF5831 had a submaximal effect (37±1%) in comparison to CPA (66±5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A1 receptor.

Assessment of the enantiomeric purity of R- and S-N6-phenylisopropyladenosine (PIA): Implications for adenosine receptor subclassification

A chiral column high-performance liquid chromatographic method was developed for the assessment of the enantiomeric purity of the stereoisomers of N6-phenylisopropyladenosine (PIA). The observed chiral purity of R-PIA was greater than 99.90%, whereas S-PIA was found to contain 4.4% of the R-enantiomer. In radioligand binding studies, the observed affinity of S-PIA for the adenosine A1 receptor (IC50 240 nM) could entirely be attributed to its content of R-PIA (IC50 7.8 nM). Calculation of a theoretical IC50 of pure S-PIA for the A2 receptor yielded a value of 6700 nM, which was 35-fold higher than for R-PIA (190 nM). Concludingly, the utilization of enantiomeric impure S-PIA in the definition of adenosine receptor subclasses is questionable.