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1.  (N)-Methanocarba 2,N6-Disubstituted Adenine Nucleosides as Highly Potent and Selective A3 Adenosine Receptor Agonists 
Journal of medicinal chemistry  2005;48(6):1745-1758.
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.
PMCID: PMC3463111  PMID: 15771421
2.  Nucleotide analogues containing 2-oxa-bicyclo[2.2.1]heptane and L-α-threofuranosyl ring systems: interactions with P2Y receptors 
Bioorganic & medicinal chemistry  2004;12(21):5619-5630.
The ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor has been successfully substituted with a rigid methanocarba ring system, leading to the conclusion that the North (N) ring conformation is preferred in receptor binding. Similarly, at P2Y2 and P2Y4 receptors, nucleotides constrained in the (N) conformation interact equipotently with the corresponding ribosides. We now have synthesized and examined as P2Y receptor ligands nucleotide analogues substituted with two novel ring systems: (1) a (N) locked-carbocyclic (cLNA) derivative containing the oxabicyclo[2.2.1]heptane ring system and (2) L-α-threofuranosyl derivatives. We have also compared potencies and preferred conformations of these nucleotides with the known anhydrohexitol-containing P2Y1 receptor antagonist MRS2283. A cLNA bisphosphate derivative MRS2584 21 displayed a Ki value of 22.5nM in binding to the human P2Y1 receptor, and antagonized the stimulation of PLC by the potent P2Y1 receptor agonist 2-methylthio-ADP (30nM) with an IC50 of 650nM. The parent cLNA nucleoside bound only weakly to an adenosine receptor (A3). Thus, this ring system afforded some P2Y receptor selectivity. A L-α-threofuranosyl bisphosphate derivative 9 displayed an IC50 of 15.3μM for inhibition of 2-methylthio-ADP-stimulated PLC activity. L-α-Threofuranosyl-UTP 13 was a P2Y receptor agonist with a preference for P2Y2 (EC50 = 9.9μM) versus P2Y4 receptors. The P2Y1 receptor binding modes, including rotational angles, were estimated using molecular modeling and receptor docking.
PMCID: PMC3402881  PMID: 15465340
Nucleoside; Purine; Pyrimidine; G protein-coupled receptor; Carbocyclic; Phospholipase C; Radioligand binding; Molecular model
3.  Search for New Purine- and Ribose-Modified Adenosine Analogues as Selective Agonists and Antagonists at Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(7):1174-1188.
The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2′-, 3′-, and 5′-deoxy; 2′- and 3′-O-methyl; 2′-deoxy 2′-fluoro; 6′-thio; 5′-uronamide; carbocyclic; 4′- or 3′-methyl; and inversion of configuration). (−)- and (+)-5′-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6′-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(β-L-2′-deoxy-6′-thiolyxofuranoside) displayed a Ki value of 8 μM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropylxanthine). 2-Chloro-2′-deoxyadenosine and 2-chloroadenin-9-yl(β-D-6′-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 μM, respectively. The A2a selective agonist 2-(1-hexynyl)-5′-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4′-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4′-methyladenosine-5′-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5′-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.
PMCID: PMC3457658  PMID: 7707320
4.  Selective A3 Adenosine Receptor Antagonists Derived from Nucleosides Containing a Bicyclo[3.1.0]hexane Ring System 
Bioorganic & medicinal chemistry  2008;16(18):8546-8556.
We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH, however these derivatives did not display the desired activity as selective A3 AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A3 AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b – 39b (N6-3-halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of 0.7 − 1.4 nM. In a functional assay of [35S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a KB of 8.9 nM. Thus, a highly potent and selective series of A3AR antagonists has been described.
PMCID: PMC2593936  PMID: 18752961
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase
5.  2-Substitution of Adenine Nucleotide Analogues Containing a Bicyclo[3.1.0]hexane Ring System Locked in a Northern Conformation: Enhanced Potency as P2Y1 Receptor Antagonists 
Journal of medicinal chemistry  2003;46(23):4974-4987.
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.
PMCID: PMC3408611  PMID: 14584948
6.  Northern Ring Conformation of Methanocarba-Adenosine 5′-Triphosphate Required for Activation of P2X Receptors 
Drug development research  2004;61(4):227-232.
Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
Replacement of the ribose moiety of adenosine 5′-triphosphate (ATP) with a carbocyclic ring constrained in either the Northern (N) or Southern (S) conformation produces agonists with widely differing activities at P2Y receptors (Kim et al. [2002] J Med Chem 45:208–218). We have used whole cell patch clamp recording to investigate the agonist activity of these two methanocarba analogs of ATP at four different P2X receptors (P2X1, P2X2, P2X3, and P2X2/3). On dorsal root ganglion neurons, (N) methanocarba-ATP ((1′S,2′R,3′S,4′R,5′S)-4-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicyclo[ 3.1.0]hexane-2,3-diol; MRS 2340) activated rapidly-desensitizing (P2X3) and slowly-desensitizing (P2X2/3) receptors with a similar potency to ATP. In contrast, (S) methanocarba-ATP ((±)-5-(6-amino-9H-purin-9-yl)-1-[triphosphoryloxymethyl] bicycle [3.1.0]hexane-2,3-diol MRS 2312) was devoid of agonist activity. On nodose ganglion neurones, that express mainly P2X2/3 receptors, ATP evoked a slowly desensitizing inward current with an EC50 value of 26 μM. MRS 2340 was an effective agonist, but less potent than ATP, while MRS 2312 at concentrations up to 100 μM produced a barely detectable response. On mammalian cell lines expressing recombinant hP2X1 and hP2X2 receptors, MRS 2340 evoked inward currents similar in amplitude to those produced by the same concentration of ATP or α,β-mATP. In contrast, MRS 2312 failed to give a detectable response. Although the conformation of the ribose affects agonist activity at P2Y receptors, there is a strong requirement for the (N) conformation for the activation of these P2X receptors. Furthermore, the region of the agonist binding site that accommodates the ribose moiety appears to be highly conserved among different P2X receptors. Drug Dev Res 61:227–232, 2004.
PMCID: PMC3402081  PMID: 22833693
ATP; P2X receptors; structure activity relationship
7.  Structure–Activity Relationships of 9-Alkyladenine and Ribose-Modified Adenosine Derivatives at Rat A3 Adenosine Receptors† 
Journal of medicinal chemistry  1995;38(10):1720-1735.
9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5′-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5′-(N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3–6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(β-d-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 µM. 2-Chloro-9-[2-amino-2,3-dideoxy-β-d-5-(methylcarbamoyl)-arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3′-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3′-OH and 4′-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2′,3′-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric “P” site.
PMCID: PMC3445626  PMID: 7752196
8.  Truncated Nucleosides as A3 Adenosine Receptor Ligands: Combined 2-Arylethynyl and Bicyclohexane Substitutions 
ACS medicinal chemistry letters  2012;3(7):596-601.
C2-Arylethynyladenosine-5′-N-methyluronamides containing a bicyclo[3.1.0]hexane ((N)-methanocarba) ring are selective A3 adenosine receptor (AR) agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing alkyl or alkylaryl groups at the N6 position were low-efficacy agonists or antagonists of the human A3AR with high selectivity. Higher hA3AR affinity was associated with N6-methyl and ethyl (Ki 3–6 nM), than with N6-arylalkyl groups. However, combined C2-phenylethynyl and N6-2-phenylethyl substitutions in selective antagonist 15 provided a Ki of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-p-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated by bulky N6 groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with general preservation of A3AR selectivity, especially with small N6 groups, but reduced efficacy in A3AR-induced inhibition of adenylate cyclase.
PMCID: PMC3491360  PMID: 23145215
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenosine receptor
9.  Truncated Nucleosides as A3 Adenosine Receptor Ligands: Combined 2-Arylethynyl and Bicyclohexane Substitutions 
ACS Medicinal Chemistry Letters  2012;3(7):596-601.
C2-Arylethynyladenosine-5′-N-methyluronamides containing a bicyclo[3.1.0]hexane [(N)-methanocarba] ring are selective A3 adenosine receptor (AR) agonists. Similar 4′-truncated C2-arylethynyl-(N)-methanocarba nucleosides containing alkyl or alkylaryl groups at the N6 position were low-efficacy agonists or antagonists of the human A3AR with high selectivity. Higher hA3AR affinity was associated with N6-methyl and ethyl (Ki = 3–6 nM) than with N6-arylalkyl groups. However, combined C2-phenylethynyl and N6-2-phenylethyl substitutions in selective antagonist 15 provided a Ki of 20 nM. Differences between 4′-truncated and nontruncated analogues of extended C2-p-biphenylethynyl substitution suggested a ligand reorientation in AR binding, dominated by bulky N6 groups in analogues lacking a stabilizing 5′-uronamide moiety. Thus, 4′-truncation of C2-arylethynyl-(N)-methanocarba adenosine derivatives is compatible with general preservation of A3AR selectivity, especially with small N6 groups, but reduced efficacy in A3AR-induced inhibition of adenylate cyclase.
PMCID: PMC3491360  PMID: 23145215
G protein-coupled receptor; purines; molecular modeling; structure−activity relationship; radioligand binding; adenosine receptor
10.  Polyamidoamine (PAMAM) dendrimer conjugate specifically activates the A3 adenosine receptor to improve post-ischemic/reperfusion function in isolated mouse hearts 
BMC Pharmacology  2011;11:11.
When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR.
A PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific.
Covalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.
PMCID: PMC3247180  PMID: 22039965
11.  Agonists and antagonists for P2 receptors 
Novartis Foundation symposium  2006;276:58-281.
Recent work has identified nucleotide agonists selective for P2Y1, P2Y2 and P2Y6 receptors and nucleotide antagonists selective for P2Y1, P2Y12 and P2X1 receptors. Selective non-nucleotide antagonists have been reported for P2Y1, P2Y2, P2Y6, P2Y12, P2Y13, P2X2/3/P2X3 and P2X7 receptors. For example, the dinucleotide INS 37217 (Up4dC) potently activates the P2Y2 receptor, and the non-nucleotide antagonist A-317491 is selective for P2X2/3/P2X3 receptors. Nucleotide analogues in which the ribose moiety is substituted by a variety of novel ring systems, including conformation-ally locked moieties, have been synthesized as ligands for P2Y receptors. The focus on conformational factors of the ribose-like moiety allows the inclusion of general modifications that lead to enhanced potency and selectivity. At P2Y1,2,4,11 receptors, there is a preference for the North conformation as indicated with (N)-methanocarba analogues. The P2Y1 antagonist MRS2500 inhibited ADP-induced human platelet aggregation with an IC50 of 0.95 nM. MRS2365, an (N)-methanocarba analogue of 2-MeSADP, displayed potency (EC50) of 0.4 nM at the P2Y1 receptor, with >10 000-fold selectivity in comparison to P2Y12 and P2Y13 receptors. At P2Y6 receptors there is a dramatic preference for the South conformation. Three-dimensional structures of P2Y receptors have been deduced from structure activity relationships (SAR), mutagenesis and modelling studies. Detailed three-dimensional structures of P2X receptors have not yet been proposed.
PMCID: PMC4321821  PMID: 16805423
12.  2-Chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate is a selective high affinity P2Y1 receptor antagonist 
British Journal of Pharmacology  2002;135(8):2004-2010.
We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y1 receptor and that modification of the ribose in these analogues is tolerated in the P2Y1 receptor binding pharmacophore.Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety.MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pKB=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y1 receptor (pKB=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y1 receptor since MRS2279 had no effect on activation of the human P2Y2, P2Y4, P2Y6, or P2Y11 receptors by their cognate agonists.MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation.In contrast, the P2Y1 receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affnity (pKB=8.05) similar to that observed at the human P2Y1 receptor heterologously expressed in 1321N1 cells.Taken together these results illustrate selective high affinity antagonism of the P2Y1 receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.
PMCID: PMC1573330  PMID: 11959804
P2Y receptors; P2Y1 receptor antagonist; 2-chloro N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate; phospholipase C; adenylyl cyclase; platelet aggregation
13.  Structural determinants of efficacy at A3 adenosine receptors: modification of the ribose moiety 
Biochemical pharmacology  2004;67(5):893-901.
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.
PMCID: PMC3150582  PMID: 15104242
Nucleosides; A3 adenosine receptor agonist; A3 adenosine receptor antagonist; Adenylyl cyclase; Phospholipase C; Partial agonist
14.  Functionalized Congeners of A3 Adenosine Receptor-Selective Nucleosides Containing a Bicyclo[3.1.0]hexane Ring System† 
Journal of medicinal chemistry  2009;52(23):7580-7592.
(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).
PMCID: PMC3109436  PMID: 19499950
15.  Partial Agonists for A3 Adenosine Receptors 
Selective agonists for A3 adenosine receptors (ARs) could potentially be therapeutic agents for a variety of disorders, including brain and heart ischemic conditions, while partial agonists may have advantages over full agonists as a result of an increased selectivity of action. A number of structural determinants for A3AR activation have recently been identified, including the N6-benzyl group, methanocarba substitution of ribose, 2-chloro and 2-fluoro substituents, various 2’- and 3’-substitutions and 4’-thio substitution of oxygen. The 2-chloro substitution of CPA and R-PIA led to A3 antagonism (CCPA) and partial agonism (Cl-R-PIA). 2-Chloroadenosine was a full agonist, while 2-fluoroadenosine was a partial agonist. Both 2’- and 3’- substitutions have a pronounced effect on its efficacy, although the effect of 2’-substitution was more dramatic. The 4-thio substitution of oxygen may also diminish efficacy, depending on other substitutions. Both N6-methyl and N6-benzyl groups may contribute to the A3 affinity and selectivity; however, an N6-benzyl group but not an N6-methyl group diminishes A3AR efficacy. N6-benzyl substituted adenosine derivatives have similar potency for human and rat A3ARS while N6-methyl substitution was preferable for the human A3AR. The combination of 2-chloro and N6-benzyl substitutions appeared to reduce efficacy further than either modification alone. The A2AAR agonist DPMA was shown to be an antagonist for the human A3AR. Thus, the efficacy of adenosine derivatives at the A3AR appears to be more sensitive to small structural changes than at other subtypes. Potent and selective partial agonists for the A3AR could be identified by screening known adenosine derivatives and by modifying adenosine and the adenosine derivatives.
PMCID: PMC3425644  PMID: 15078216
16.  2-Substitution of N6-Benzyladenosine-5′-uronamides Enhances Selectivity for A3 Adenosine Receptors 
Journal of medicinal chemistry  1994;37(21):3614-3621.
Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5′-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636–646), were synthesized starting from methyl β-d-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5′-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na+-independent adenosine transporter, as indicated in displacement of [3H]N6-(4-nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5′-position and a 3-iodobenzyl group at the N6-position.
PMCID: PMC3468333  PMID: 7932588
17.  Click Modification in the N6 Region of A3 Adenosine Receptor-Selective Carbocyclic Nucleosides for Dendrimeric Tethering that Preserves Pharmacophore Recognition 
Bioconjugate Chemistry  2012;23(2):232-247.
Adenosine derivatives were modified with alkynyl groups on N6 substituents for linkage to carriers using Cu(I)-catalyzed click chemistry. Two parallel series, both containing a rigid North-methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose, behaved as A3 adenosine receptor (AR) agonists: (5′-methyluronamides) or partial agonists (4′-truncated). Terminal alkynyl groups on a chain at the 3 position of a N6-benzyl group or simply through a N6–propargyl group were coupled to azido derivatives, which included both small molecules and G4 (fourth-generation) multivalent poly(amidoamine) (PAMAM) dendrimers, to form 1,2,3-triazolyl linkers. The small molecular triazoles probed the tolerance in A3AR binding of distal, sterically bulky groups such as 1-adamantyl. Terminal 4-fluoro-3-nitrophenyl groups anticipated nucleophilic substitution for chain extension and 18F radiolabeling. N6-(4-Fluoro-3-nitrophenyl)-triazolylmethyl derivative 32 displayed a Ki of 9.1 nM at A3AR with ~1000-fold subtype selectivity. Multivalent conjugates additionally containing click-linked water-solubilizing polyethylene glycol groups potently activated A3AR in the 5′-methyluronamide, but not 4′ truncated series. N6-Benzyl nucleoside conjugate 43 (apparent Ki 24 nM) maintained binding affinity of the monomer better than a N6-triazolylmethyl derivative. Thus, the N6 region of 5′-methyluronamide derivatives, as modeled in receptor docking, is suitable for functionalization and tethering by click chemistry to achieve high A3AR agonist affinity and enhanced selectivity.
PMCID: PMC3291892  PMID: 22175234
G protein-coupled receptor; PAMAM dendrimer; purines; structure activity relationship; molecular modeling; adenylate cyclase
18.  Functionalized Congeners of P2Y1 Receptor Antagonists: 2-Alkynyl (N)-Methanocarba 2′-Deoxyadenosine 3′,5′-Bisphosphate Analogues and Conjugation to a Polyamidoamine (PAMAM) Dendrimer Carrier 
Bioconjugate chemistry  2010;21(7):1190-1205.
The P2Y1 receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphates containing extended 2-alkynyl chains was designed and binding affinity at the human (h) P2Y1 receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (Ki 23 nM) and extended amine congener 15 (Ki 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended ε-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y1 receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y1 receptor modeling and ligand docking. Attempted P2Y1 antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e. antithrombotic action.
PMCID: PMC2912410  PMID: 20565071
19.  Activity of Novel Adenine Nucleotide Derivatives as Agonists and Antagonists at Recombinant Rat P2X Receptors 
Drug development research  2000;49(4):253-259.
Strategy, Management and Health PolicyVenture Capital Enabling TechnologyPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV
The effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2-thioether groups) or antagonist (additional phosphate moieties at the 3′- or 2′-position) properties at P2Y1 receptors were examined at recombinant rat P2X1, P2X2, P2X3, and P2X4 receptors expressed in Xenopus oocytes. The potency of P2Y1 agonists HT-AMP (2-(hexylthio)adenosine-5′-monophosphate) and PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5′-triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X sub-units. HT-AMP was 5-fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM). MRS 2179 (N6-methyl-2′-deoxyadenosine 3′, 5-bisphosphate, a potent P2Y1 receptor antagonist) inhibited ATP-evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X3 receptors, with an IC50 value of 12.9 ± 0.1 μM, and was inactive at rat P2X2 and P2X4 receptors. Thus, MRS 2179 was 11-fold and 130-fold selective for P2Y1 receptors vs. P2X1 and P2X3 receptors, respectively. MRS 2209, the corresponding 3′-deoxy-2′-phosphate isomer, was inactive at rat P2X1 receptors, thus demonstrating its greater selectivity as a P2Y1 receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y1 antagonists with 2- and 6-substitutions), the phosphate (a 3′,5′-cyclic diphosphate, inactive at P2Y1 receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X1 and P2X3 receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y1 receptors, since they were inactive as agonist or antagonist at P2X1 and P2X3 receptors.
PMCID: PMC3393598  PMID: 22791931
ion channel; oocytes; purines; ATP derivatives; bisphosphates; deoxyadenosine derivatives
20.  Semi-Rational Design of (N)-Methanocarba Nucleosides as Dual Acting A1 and A3Adenosine Receptor Agonists: Novel Prototypes for Cardioprotection 
Journal of medicinal chemistry  2005;48(26):8103-8107.
Ring-constrained adenosine analogues have been designed to act as dualagonists at tissue-protective A1 and A3 adenosine receptors (ARs). 9-Ribosides transformed into the ring-constrained (N)-methanocarba-2-chloro-5′-uronamides consistently lost affinity at A1/A2AARs and gained at A3AR. Among 9-riboside derivatives, only N6-cyclopentyl and 7-norbornyl moieties were extrapolated for mixed A1/A3 selectivity and rat/human A3AR equipotency. Consequently, 2 was balanced in affinity and potency at A1/A3ARs as envisioned and dramatically protected in an intact heart model of global ischemia and reperfusion.
PMCID: PMC2597460  PMID: 16366590
21.  Structure-Guided Design of A3 Adenosine Receptor-Selective Nucleosides: Combination of 2-Arylethynyl and Bicyclo[3.1.0]hexane Substitutions 
Journal of Medicinal Chemistry  2012;55(10):4847-4860.
(N)-Methanocarba adenosine 5′-methyluronamides containing known A3 AR (adenosine receptor)-enhancing modifications, i.e. 2-(arylethynyl)adenine and N6-methyl or N6-(3-substituted-benzyl), were nanomolar full agonists of human (h) A3AR and highly selective (Ki ~0.6 nM, N6-methyl 2-(halophenylethynyl) analogues 13, 14). Combined 2-arylethynyl-N6-3-chlorobenzyl substitutions preserved A3AR affinity/selectivity in the (N)-methanocarba series (e.g. 3,4-difluoro full agonist MRS5698 31, Ki 3 nM, human and mouse A3) better than for ribosides. Polyaromatic 2-ethynyl N6-3-chlorobenzyl analogues, such as potent linearly extended 2-p-biphenylethynyl MRS5679 34 (Ki hA3 3.1 nM; A1, A2A: inactive) and fluorescent 1-pyrene adduct MRS5704 35 (Ki hA3 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 A2AAR, with a predicted helical rearrangement requiring an agonist-specific outward displacement of TM2 resembling opsin. Thus, X-ray structure of related A2AAR is useful in guiding design of new A3AR agonists.
PMCID: PMC3371665  PMID: 22559880
G protein-coupled receptor; purines; molecular modeling; structure activity relationship; radioligand binding; adenylate cyclase
22.  Design of (N)-Methanocarba Adenosine 5′-Uronamides as Species-Independent A3 Receptor-Selective Agonists 
2-Chloro-5′ -N-methylcarboxamidoadenosine analogues containing the (N)-methanocarba (bicyclo[3.1.0]hexane) ring system as a ribose substitute display increased selectivity as agonists of the human A3 adenosine receptor (AR). However, the selectivity in mouse was greatly reduced due to an increased tolerance of this ring system at the mouse A1AR. Therefore, we varied substituents at the N6 and C2 positions in search of compounds that have improved A3AR selectivity and are species independent. An N6-methyl analogue was balanced in affinity at mouse A1/A3ARs, with high selectivity in comparison to the A2AAR. Substitution of the 2-chloro atom with larger and more hydrophobic substituents, such as iodo and alkynyl groups, tended to increase the A3AR selectivity (up to 430-fold) in mouse and preserve it in human. Extended and chemically functionalized alkynyl chains attached at the C2 position of the purine moiety preserved A3AR selectivity more effectively than similar chains attached at the 3 position of the N6–benzyl group.
PMCID: PMC2430186  PMID: 18424135
nucleoside; G protein-coupled receptor; mouse; adenosine receptor; radioligand binding
23.  Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  1998;41(17):3186-3201.
The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure–activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.
PMCID: PMC3474377  PMID: 9703464
24.  Structure-Based Design, Synthesis by Click Chemistry and in Vivo Activity of Highly Selective A3 Adenosine Receptor Agonists 
MedChemComm  2015;6:555-563.
2-Arylethynyl derivatives of (N)-methanocarba adenosine 5′-uronamides are selective A3AR (adenosine receptor) agonists. Here we substitute a 1,2,3-triazol-1-yl linker in place of the rigid, linear ethynyl group to eliminate its potential metabolic liability. Docking of nucleosides containing possible short linker moieties at the adenine C2 position using a hybrid molecular model of the A3AR (based on the A2AAR agonist-bound structure) correctly predicted that a triazole would maintain the A3AR selectivity, due to its ability to fit a narrow cleft in the receptor. The analogues with various N6 and C2-aryltriazolyl substitution were synthesized and characterized in binding (Ki at hA3AR 0.3 – 12 nM) and in vivo to demonstrate efficacy in controlling chronic neuropathic pain (chronic constriction injury). Among N6-methyl derivatives, a terminal pyrimidin-2-yl group in 9 (MRS7116) increased duration of action (36% pain protection at 3 h) in vivo. N6-Ethyl 5-chlorothien-2-yl analogue 15 (MRS7126) preserved in vivo efficacy (85% protection at 1 h) with short duration. Larger N6 groups, e.g. 17 (MRS7138, >90% protection at 1 and 3 h), greatly enhanced in vivo activity. Thus, we have combined structure-based methods and phenotypic screening to identify nucleoside derivatives having translational potential.
PMCID: PMC4517612  PMID: 26236460
G protein-coupled receptor; nucleoside; click chemistry; neuropathic pain; molecular modeling
25.  Development of Selective High Affinity Antagonists, Agonists, and Radioligands for the P2Y1 Receptor 
The P2Y1 receptor is a member of the P2Y family of nucleotide-activated G protein-coupled receptors, and it is an important therapeutic target based on its broad tissue distribution and essential role in platelet aggregation. We have designed a set of highly selective and diverse pharmacological tools for studying the P2Y1 receptor using a rational approach to ligand design. Based on the discovery that bisphosphate analogues of the P2Y1 receptor agonist, ADP, are partial agonists/competitive antagonists of this receptor, an iterative approach was used to develop competitive antagonists with enhanced affinity and selectivity. Halogen substitutions of the 2-position of the adenine ring provided increased affinity while an N6 methyl substitution eliminated partial agonist activity. Furthermore, various replacements of the ribose ring with symmetrically branched, phosphorylated acyclic structures revealed that the ribose is not necessary for recognition at the P2Y1 receptor. Finally, replacement of the ribose ring with a five member methanocarba ring constrained in the Northern conformation conferred dramatic increases in affinity to both P2Y1 receptor antagonists as well as agonists. These combined structural modifications have resulted in a series of selective high affinity antagonists of the P2Y1 receptor, two broadly applicable radioligands, and a high affinity agonist capable of selectively activating the P2Y1 receptor in human platelets. Complementary receptor modeling and computational ligand docking have provided a putative structural framework for the drug-receptor interactions. A similar rational approach is being applied to develop selective ligands for other subtypes of P2Y receptors.
PMCID: PMC3116925  PMID: 18673269
Adenosine-3′,5′-bisphosphate; MRS2179; MRS2279; MRS2500; P2Y receptors; P2Y1 receptor; platelet aggregation; radioligand

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