Putative role of the adenosine A(3) receptor in the antiproliferative action of N (6)-(2-isopentenyl)adenosine.

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N (6)-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A(3) receptor (hA(3)R) ligands with affinities in the high nanomolar range (K (i) values of 159 and 649 nM, respectively). These values were comparable to the observed K (i) value of adenosine on hA(3)R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A(3)R. In a functional assay in Chinese hamster ovary cells transfected with hA(3)R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A(3)R antagonist VUF5574. Both IPA and reference A(3)R agonist 2-chloro-N (6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A(3)R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A(3)R-independent mechanism, as was previously reported for other A(3)R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A(3)R.

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.

2,6,8-trisubstituted 1-deazapurines as adenosine receptor antagonists.

In this study we developed a refined pharmacophore model for antagonists of the human adenosine A1 receptor, based on features of known pyrimidine and purine derivatives. The adoption of these updated criteria assisted us in synthesizing a series of 1-deazapurines with consistently high affinity as inverse agonists for the adenosine A1 receptor. These 1-deazapurines (otherwise known as 3H-imidazo[4,5-b]pyridines) were substituted at their 2- and 6-positions, yielding a series with five of the derivatives displaying Ki values in the subnanomolar range. The most potent of these, compound 10 (LUF 5978), displayed an affinity of 0.55 nM at the human adenosine A1 receptor with >300-fold and 45-fold selectivity toward A2A and A3 receptors, respectively. Compound 14 (LUF 5981, Ki = 0.90 nM) appeared to have the best overall selectivity with respect to adenosine A2A (>200-fold) and A3 (700-fold) receptors.

2,6-disubstituted and 2,6,8-trisubstituted purines as adenosine receptor antagonists.

Purines have long been exploited as adenosine receptor antagonists. The substitution pattern about the purine ring has been well investigated, and certain criteria have become almost a prerequisite for good affinity at the adenosine A(1) receptor. The adaptation of the pharmacophore and the initial series of pyrimidines developed in an earlier publication resulted in a series of purines with an entirely new substitution pattern. One compound in particular, 8-cyclopentyl-2,6-diphenylpurine (31, LUF 5962) has been shown to be very promising with an affinity of 0.29 nM at the human adenosine A(1) receptor.

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 CPA's 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.

A series of ligands displaying a remarkable agonistic-antagonistic profile at the adenosine A1 receptor.

Adenosine receptor agonists are usually variations of the natural ligand, adenosine. The ribose moiety in the ligand has previously been shown to be of great importance for the agonistic effects of the compound. In this paper, we present a series of nonadenosine ligands selective for the adenosine A(1) receptor with an extraordinary pharmacological profile. 2-Amino-4-benzo[1,3]dioxol-5-yl-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile (70, LUF 5853) shows full agonistic behavior comparable with the reference compound CPA, while also displaying comparable receptor binding affinity (K(i) = 11 nM). In contrast, compound 58 (2-amino-4-(3-trifluoromethylphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5948) has a binding affinity of 14 nM and acts as an inverse agonist. Also present within this same series are compounds that show neutral antagonism of the adenosine A(1) receptor, for example compound 65 (2-amino-4-(4-difluoromethoxyphenyl)-6-(2-hydroxyethylsulfanyl)pyridine-3,5-dicarbonitrile, LUF 5826).

Synthesis and biological evaluation of 2-aminothiazoles and their amide derivatives on human adenosine receptors. Lack of effect of 2-aminothiazoles as allosteric enhancers.

A number of 2-aminothiazoles (2a-e) and their amide derivatives (4-10) were prepared. The 2-aminothiazoles themselves were tested as allosteric enhancers of agonist binding to human adenosine A(1) receptors. In a variety of experimental set-ups the compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiazoles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4-6) or aliphatic nature (7-10). Some of the compounds emerged as moderately active antagonists on human adenosine A(1) and/or A(2A) receptors with lower or negligible potency at adenosine A(3) receptors.

Inhibition of nucleoside transport proteins by C8-alkylamine-substituted purines.

4-nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. Here we report on the synthesis and the biological evaluation of compounds that are less polar than NBTI. Compound screening in our laboratory indicated that introduction of an alkylamine substituent at the C(8)-position of N(6)-cyclopentyladenosine (CPA, 2) led to an increment in affinity for the transport protein. It was investigated whether this would also apply for NBTI derivatives. Two series of C(8)-alkylamine-substituted compounds were prepared, one in which the nitro group was absent (46-58) and another in which the ribose moiety was replaced by a benzyl group (72-75). Comparison of the biological data of these compounds with 6-benzylthioinosine (4, K(i) = 53 nM) and 9-benzyl-6-(4-nitrobenzylsulfanyl)purine (59, K(i) = 135 nM) confirmed the hypothesis. The K(i) values improved upon elongation of the alkylamine chain from methylamine to n-hexylamine with an optimum for n-pentylamine (50, K(i) = 2.3 nM). Substitution with 2-methylbutylamine (52), cyclopropylamine (53), cyclopentylamine (54, 72), and cyclohexylamine (55, 73) revealed that the presence of a bulky group enhanced the affinity. The presence of tertiary amines obtained by substitution with pyrrolidine, piperidine, and morpholine gave only poor results. For both series substitution with cyclopentylamine was most effective. Compound 54 (LUF5942) proved the most active, showing a comparable affinity (K(i) = 0.64 nM) to NBTI but a significantly lower polar surface area.

2,4,6-trisubstituted pyrimidines as a new class of selective adenosine A1 receptor antagonists.

Adenosine receptor antagonists usually possess a bi- or tricyclic heteroaromatic structure at their core with varying substitution patterns to achieve selectivity and/or greater affinity. Taking into account molecular modeling results from a series of potent adenosine A1 receptor antagonists, a pharmacophore was derived from which we show that a monocyclic core can be equally effective. To achieve a compound that may act at the CNS we propose imposing a restriction related to its polar surface area (PSA). In consequence, we have synthesized two novel series of pyrimidines, possessing good potency at the adenosine A1 receptor and desirable PSA values. In particular, compound 30 (LUF 5735) displays excellent A1 affinity (Ki = 4 nM) and selectivity (< or =50% displacement of 1 muM concentrations of the radioligand at the other three adenosine receptors) and has a PSA value of 53 A2.

Inhibition of nucleoside transport by new analogues of 4-nitrobenzylthioinosine: replacement of the ribose moiety by substituted benzyl groups.

4-Nitrobenzylthioinosine (NBTI, 1) is a well-known inhibitor for the nucleoside transport protein ENT1. However, its highly polar nature is unfavorable for oral absorption and/or penetration into the CNS. In the search for compounds with lower polarity than NBTI we replaced its ribose moiety by substituted benzyl groups. Halogen, hydroxyl, (trifluoro)methyl(-oxy), nitro, and amine functionalities were among the substituents at the benzyl group. In general, substitution of the benzyl group resulted in a lower affinity for ENT1. Only 2-hydroxyl substitution showed a higher affinity. Most likely this is the result of hydrogen bonding. Substitution at the 2-position of the benzyl group with aryl groups was also addressed. Compared to parent compound carrying a 2-phenylbenzyl group, all synthesized analogues gave higher affinities. Introduction of fluoro, trifluoromethyl, methoxy, and hydroxyl groups at the phenyl group clearly showed that addition to the 4-position was preferable. Despite the highly different character of a ribose and a benzyl group, Ki values in the low nanomolar range were obtained for the benzyl-substituted derivatives. Compound 35, LUF5919, and compound 60, LUF5929, displayed the highest affinity (Ki = 39 nM for both compounds), having a polar surface area of 101 A2 and 85 A2, respectively.

New, non-adenosine, high-potency agonists for the human adenosine A2B receptor with an improved selectivity profile compared to the reference agonist N-ethylcarboxamidoadenosine.

The adenosine A(2B) receptor is the least well characterized of the four known adenosine receptor subtypes because of the absence of potent, selective agonists. Here, we present five non-adenosine agonists. Among them, 2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile, 17, LUF5834, is a high-efficacy partial agonist with EC(50) = 12 nM and 45-fold selectivity over the adenosine A(3) receptor but lacking selectivity versus the A(1) and A(2A) subtypes. Compound 18, LUF5835, the 3-hydroxyphenyl analogue, is a full agonist with EC(50) = 10 nM.

The mouse brain adenosine A1 receptor: functional expression and pharmacology.

The adenosinergic system is involved in many important physiological functions. Adenosine exerts its extracellular effects through four types of G-protein-coupled receptors: A(1), A(2A), A(2B) and A(3). Adenosine acts as an important regulator of metabolic processes. In the brain adenosine mediates prominent neuroprotective functions via the adenosine A(1) receptor. Whereas the pharmacological characteristics of the rat and human adenosine A(1) receptor have been intensively studied, the mouse adenosine A(1) receptor has not yet been characterised. Accordingly, we have cloned the mouse brain adenosine A(1) receptor and present here a pharmacological characterisation of the mouse adenosine A(1) receptor using functional studies and radioligand binding assays. The results show that the binding affinities of several ligands for the mouse adenosine A(1) receptor are similar to the affinities for the rat and human adenosine A(1) receptor with some exceptions.

Synthesis and biological evaluation of disubstituted N6-cyclopentyladenine analogues: the search for a neutral antagonist with high affinity for the adenosine A1 receptor.

Novel 3,8- and 8,9-disubstituted N(6)-cyclopentyladenine derivatives were synthesised in moderate overall yield from 6-chloropurine. The derivatives were made in an attempt to find a new neutral antagonist with high affinity for adenosine A(1) receptors. N(6)-Cyclopentyl-9-methyladenine (N-0840) was used as a lead compound. Binding affinities of the new analogues were determined for human adenosine A(1) and A(3) receptors. Their intrinsic activity was assessed in [35S]GTPgammaS binding experiments. Elongation of the 9-methyl of N-0840 to a 9-propyl substituent was very well tolerated. A 9-benzyl group, on the other hand, caused a decrease in adenosine A(1) receptor affinity. Next, the 8-position was examined in detail, and affinity was increased with appropriate substitution. Most derivatives were A(1)-selective and 20 of the new compounds (6-9, 15-21, 23-26, 28, 31, 33, 35, and 36) had higher adenosine A(1) receptor affinity than the reference substance, N-0840. Compound 31 (N(6)-cyclopentyl-8-(N-methylisopropylamino)-9-methyladenine, LUF 5608) had the highest adenosine A(1) receptor affinity, 7.7 nM. In the [35S]GTPgammaS binding experiments, derivatives 5, 14, 22, 23, 25, 26, 33 and 34 did not significantly change basal [35S]GTPgammaS binding, thus behaving as neutral antagonists. Moreover, four of these compounds (23, 25, 26, and 33) displayed a 4- to 10-fold increased adenosine A(1) receptor affinity (75-206 nM) compared to N-0840 (852 nM). In summary, we synthesised a range of N-0840 analogues with higher affinity for adenosine A(1) receptors. In addition, four new derivatives, LUF 5666 (23), LUF 5668 (25), LUF 5669 (26) and LUF 5674 (33), behaved as neutral antagonists when tested in [35S]GTPgammaS binding studies. Thus, these compounds have improved characteristics as neutral adenosine A(1) receptor antagonists.

N6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA), a very selective agonist with high affinity for the human adenosine A1 receptor.

Four subtypes of adenosine receptors are currently known, that is, A(1), A(2A), A(2B), and A(3) receptors. Interestingly, quite substantial species differences exist especially between human and rat A(3) receptors. As a result, ligands such as CCPA, which are very selective for the rat A(1) receptor versus the human A(3) receptor, are substantially less selective when the human A(1) and A(3) receptors are compared. New 2-substituted and 2,N(6)-disubstituted adenosines were synthesized, and their affinities for the human adenosine A(1), A(2A), A(2B), and A(3) receptors were determined. Although large substituents on the C2-position are generally thought to yield adenosine A(2A) receptor selective ligands, the reported series of 2-triazeno-substituted adenosines had a very high affinity for the A(1) receptor. For example, 2-(3-phenylaminocarbonyltriazene-1-yl)adenosine had an affinity of 6.1 +/- 1.3 nM for the human adenosine A(1) receptor. Introduction of a diphenethyl substituent at the N(6)-position of this compound resulted in a high-affinity agonist, 3.1 +/- 0.9 nM, for the human adenosine A(1) receptor with 316- and 45-fold selectivity versus the human A(2A) and human A(3) receptors, respectively. The most selective, high-affinity human adenosine A(1) receptor agonist was the disubstituted compound N(6)-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA). TCPA had an affinity of 2.8 +/- 0.8 nM for the human adenosine A(1) receptor and was 75-fold and 214-fold selective versus the human A(2A) and human A(3) receptors, respectively. In addition, TCPA was a full agonist and inhibited the forskolin-induced cAMP production of CHO cells stably transfected with the human adenosine A(1) receptor with an IC(50) of 1.5 +/- 0.5 nM.

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.

2,5'-Disubstituted adenosine derivatives: evaluation of selectivity and efficacy for the adenosine A(1), A(2A), and A(3) receptor.

Novel 2,5'-disubstituted adenosine derivatives were synthesized in good overall yields starting from commercially available guanosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. E(max) values were determined for the stimulation or inhibition of cAMP production in CHO cells expressing human adenosine A(2A) (EC(50) values as well) or A(3) receptors, respectively. The compounds displayed affinities in the nanomolar range for both the adenosine A(2A) and A(3) receptor, without substantial preference for either receptor. The derivatives with a 2-(1-hexynyl) group had the highest affinities for both receptors; compound 4 (2-(1-hexynyl)adenosine) had the highest affinity for the adenosine A(2A) receptor with a K(i) value of 6 nM (A(3)/A(2A) selectivity ratio of approximately 3), whereas compound 37 (2-(1-hexynyl)-5'-S-methyl-5'-thioadenosine) had the highest affinity for the adenosine A(3) receptor with a K(i) value of 15 nM (A(2A)/A(3) selectivity ratio of 4). In general, compounds with a relatively small 5'-S-alkyl-5'-thio substituent (methyl-5'-thio) displayed the highest affinities for both the adenosine A(2A) and A(3) receptor; the larger ones (n- or i-propyl-5'-thio) increased the selectivity for the adenosine A(3) receptor. The novel compounds were also evaluated in cAMP assays for their (partial) agonistic behavior. Overall, the disubstituted derivatives behaved as partial agonists for both the adenosine A(2A) and A(3) receptor. The compounds showed somewhat higher intrinsic activities on the adenosine A(2A) receptor than on the A(3) receptor. Compounds 37, 40 and 45, 48, with either a 5'-S-methyl-5'-thio or a 5'-S-i-propyl-5'-thio substituent had the lowest intrinsic activities on the adenosine A(2A) receptor. For the A(3) receptor, compounds 34, 35, 38, 39, and 46, 47, with a 5'-S-ethyl-5'-thio or a 5'-S-n-propyl-5'-thio substituent had the lowest intrinsic activities.