Ribose ring-constrained nucleosides and nucleotides to act at cell-surface purine recesptors have been designed and synthesized. At the P2Y(1) nucleotide receptor and the A(3) adenosine receptor (AR) the North envelope conformation of ribose is highly preferred. We have applied mutagenesis and rhodopsin-based homology modeling to the study of purine receptors and used the structural insights gained to assist in the design of novel ligands. Two subgroups of P2Y receptors have been defined, containing different sets of cationic residues for coordinating the phosphate groups. Modeling/mutagenesis of adenosine receptors has focused on determinants of intrinsic efficacy in adenosine derivatives and on a conserved Trp residue (6.48) which is involved in the activation process. The clinical use of adenosine agonists as cytoprotective agents has been limited by the widespread occurrence of ARs, thus, leading to undesirable side effects of exogenously administered adenosine derivatives. In order to overcome the inherent nonselectivity of activating the native receptors, we have introduced the concept of neoceptors. By this strategy, intended for eventual use in gene therapy, the putative ligand binding site of a G protein-coupled receptor is reengineered for activation by synthetic agonists (neoligands) built to have a structural complementarity. Using a rational design process we have identified neoceptor-neoligand pairs which are pharmacologically orthogonal with respect to the native species.

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2′-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2′-amino-2′-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2′-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3–30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPαS and 2′-deoxy-UTPαS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A(2A) adenosine receptor (AR). Adenosine derivatives functionalized at the 5', 2, and N-6 positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5'-(2-aminoethyl)uronamide but not 5'-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N-6(2-methylbenzyl) group enhanced affinity at the Q89D- and N181 D- but not the T88D-A(2A)AR. Amino groups placed near the 2- or N-6-position only slightly affected the binding to mutant receptors. The 5'-hydrazide MRS3412 was 670-and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A(2A)AR compared to the wild-type. Thus, we identified and modeled pairs of A(2A)AR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.

A series of ring-constrained (N)-methanocarba-5'-uronamide 2,N(6)-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 A(3)AR agonists. Selected compounds were compared in binding to the rat A(3)AR to assess their viability for testing in rat disease models. The N(6)-(3-chlorobenzyl) and N(6)-(3-bromobenzyl) analogues displayed K(i) values at the human A(3)AR of 0.29 and 0.38 nM, respectively. Other subnanomolar affinities were observed for the following N(6) derivatives: 2,5-dichlorobenzyl, 5-iodo-2-methoxybenzyl, trans-2-phenyl-1-cyclopropyl, and 2,2-diphenylethyl. Selectivity for the human A(3)AR in comparison to the A(1)AR was the following (fold): the N(6)-(2,2-diphenylethyl) analogue 34 (1900), the N(6)-(2,5-dimethoxybenzyl) analogue 26 (1200), the N(6)-(2,5-dichlorobenzyl) and N(6)-(2-phenyl-1-cyclopropyl) analogues 20 and 33 (1000), and the N(6)-(3-substituted benzyl) analogues 17, 18, 28, and 29 (700-900). Typically, even greater selectivity ratios were obtained in comparison with the A(2A) and A(2B)ARs. The (N)-methanocarba-5'-uronamide analogues were full agonists at the A(3)AR, as indicated by the inhibition of forskolin-stimluated adenylate cyclase at a concentration of 10 microM. The N(6)-(2,2-diphenylethyl) derivative was an A(3)AR 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 A(3)AR affinity in the 9-riboside series, including those that reduce intrinsic efficacy, may be adapted to the (N)-methanocarba nucleoside series of full agonists.

Cystic fibrosis (CF) is a common, lethal genetic disease, which is due to mutations in the CFTR gene. The CF lung expresses a profoundly proinflammatory phenotype, due to constitutive hypersecretion of IL-8 from epithelial cells lining the airways. In a systematic search for candidate drugs that might be used therapeutically to suppress IL-8 secretion from these cells, we have identified a potent and efficacious series of amphiphilic pyridinium salts. The most potent of these salts is MRS2481, an (R)-1-phenylpropionic acid ester, with an IC50 of ca. 1μM. We have synthesized 21 analogues of MRS2481, which have proven sufficient to develop a preliminary structure–activity relationship (SAR). For optimal activity, we have found that the ester must be connected to the pyridinium derivative by an eight-carbon chain. An optical isomer of the lead compound, containing an (S)-1-phenylpropionic acid ester, has been found to be a much less active. The mechanism of action of MRS2481 appears to involve inhibition of signaling of the NFκB and AP-1 transcription factors to the IL-8 promoter. MRS2481 is a potent inhibitor of TNFα-induced phosphorylation and proteosomal destruction of IκBα. Inasmuch as IκBα is the principal inhibitor of the NFκB signaling pathway, preservation of intact IκBα would serve to keep the IL-8 promoter silent. We also find that MRS2481 blocks TNFα-activated phosphorylation of JNK, the c-JUN kinase. The IL-8 promoter is also activated by an AP-1 site, which requires a phospho-c-JUN/c-FOS dimer for activity. We therefore interpret these data to suggest that the mechanism of MRS2481 action is to inhibit both NFκB and AP-1 signaling on the IL-8 promoter. Given the medicinally promising properties of water-solubility, potency in the low μM concentration range, and high efficacy, we anticipate that MRS2481, or a further optimized derivative, may find an important place in the armamentarium of pharmaceutical strategies yet to be arrayed against the inflammatory phenotype of the CF lung.

Ring-constrained adenosine analogues have been designed to act as dual agonists at tissue-protective A(1) and A(3) adenosine receptors (ARs). 9-Ribosides transformed into the ring-constrained (N)-methanocarba-2-chloro-5'-uronamides consistently lost affinity at A(1)/A(2A)ARs and gained at A(3)AR. Among 9-riboside derivatives, only N(6)-cyclopentyl and 7-norbornyl moieties were extrapolated for mixed A(1)/A(3) selectivity and rat/human A(3)AR equipotency. Consequently, 2 was balanced in affinity and potency at A(1)/A(3)ARs as envisioned and dramatically protected in an intact heart model of global ischemia and reperfusion.

(-)-Cannabidiol (CBD) is a major, non psychotropic constituent of cannabis. It has been shown to cause numerous physiological effects of therapeutic importance. We have reported that CBD derivatives in both enantiomeric series are of pharmaceutical interest. Here we describe the syntheses of the major CBD metabolites, (-)-7-hydroxy- CBD and (-)-CBD-7-oic acid and their dimethylheptyl ( DMH) homologs, as well as of the corresponding compounds in the enantiomeric (+)-CBD series. The starting materials were the respective CBD enantiomers and their DMH homologs. The binding of these compounds to the CB1 and CB2 cannabinoid receptors are compared. Surprisingly, contrary to the compounds in the (-) series, which do not bind to the receptors, most of the derivatives in the (+) series bind to the CB1 receptor in the low nanomole range. Some of these compounds also bind weakly to the CB2 receptor.

In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X(1) (derivatives of pyridoxal phosphates and suramin), P2X(3)(A-317491), P2X(7) (derivatives of the isoquinoline KN-62), P2Y(1)(nucleotide analogues MRS 2179 and MRS 2279), P2Y(2)(thiouracil derivatives such as AR-C126313), and P2Y(12)(nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y(1)receptors. The dinucleotide INS 37217 potently activates the P2Y(2)receptor. UTP-gamma-S and UDP-beta-S are selective agonists for P2Y(2)/P2Y(4)and P2Y(6)receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y(1)receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.

OBJECTIVE:To explore the antiarthritic potential of a novel synthetic cannabinoid acid, Hebrew University-320 (HU-320), in the DBA/1 mouse model of arthritis, and to investigate in vitro antiinflammatory and immunosuppressive effects of HU-320 on macrophages and lymphocytes.METHODS:DBA/1 mice were immunized with bovine type II collagen (CII) to induce arthritis and then injected intraperitoneally daily with HU-320. The effects of treatment on arthritic changes in hind feet were assessed clinically and histologically, and draining lymph node responses to CII were assayed. Murine splenic and human blood lymphocytes were cultured to study the effect of HU-320 on polyclonal mitogenic stimulation. Macrophage cultures were set up to evaluate in vitro effects of HU-320 on production of tumor necrosis factor alpha (TNF alpha) and reactive oxygen intermediates (ROIs). The effect of HU-320 administration on lipopolysaccharide-induced serum TNF levels was assayed using C57BL/6 mice. Bioactive TNF production was measured using BALB/c clone 7 target cells. Evaluation of HU-320 psychoactivity was performed using established laboratory tests on Sabra mice.RESULTS:Systemic daily administration of 1 and 2 mg/kg HU-320 ameliorated established CII-induced arthritis. Hind foot joints of treated mice were protected from pathologic damage. CII-specific and polyclonal responses of murine and human lymphocytes were down-modulated. HU-320 inhibited production of TNF from mouse macrophages and of ROIs from RAW 264.7 cells and suppressed the rise in serum TNF level following endotoxin challenge. HU-320 administration yielded no adverse psychotropic effects in mice.CONCLUSION:Our studies show that the novel synthetic cannabinoid acid HU-320 has strong antiinflammatory and immunosuppressive properties while demonstrating no psychoactive effects. The profound suppressive effects on cellular immune responses and on the production of proinflammatory mediators all indicate its usefulness as a novel nonpsychoactive, synthetic antiinflammatory product.

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N6-substitutions known to enhance human A3AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A1, A2A, A2B, and A3ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA3AR affinity and efficacy in the cases of N6-(3-iodobenzyl) and N6-(trans-2-phenyl-1-cyclopropyl), for which a full A3AR agonist was converted into a selective antagonist; the 2-cyano-N6-methyl analogue was a full A3AR agonist. The combination of N6-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A1AR. The environment surrounding the 2-position within the putative A3AR binding site was explored using rhodopsin-based homology modeling and ligand docking.

We synthesized phenyl ring-substituted analogues of N(6)-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A(3)AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA(3)AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A(3)AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A(3)AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A(3)AR binding. Other related N(6)-substituted adenosine derivatives were included for comparison. Although the N(6)-(2-phenyl-1-cyclopropyl) derivatives were full A(3)AR agonists, several other derivatives had greatly reduced efficacy. N(6)-Cyclopropyladenosine was an A(3)AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N(6)-(2,2-Diphenylethyl)adenosine was an A(3)AR antagonist, and either adding a bond between the two phenyl rings (N(6)-9-fluorenylmethyl) or shortening the ethyl moiety (N(6)-diphenylmethyl) restored efficacy. A QSAR study of the N(6) region provided a model that was complementary to the putative A(3)AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A(3)AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.

Activation of the P2Y1 nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y1 receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC50 value of 0.95nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC50 values of 62.8nM and 1.5μM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y1 receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca2+. No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y1 receptor antagonists with the proaggregatory P2Y12 receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y1 receptor, and MRS2500 is the most potent such antagonist yet reported.

The effect of 2,2′-pyridylisatogen tosylate (PIT) on the human P2Y1 receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y1 receptors in a non-competitive and concentration-dependent manner. The IC50 for reduction of the maximal agonist effect was 0.14μM. In contrast, MRS2179, a competitive P2Y1 receptor antagonist, parallel-shifted the agonist concentration–response curve to the right. PIT also concentration-dependently blocked the P2Y1 receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y1 receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y1 receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y1 receptor, [3H] MRS2279. It was found that PIT (0.01–10μM) did not inhibit [3H] MRS2279 binding to the human P2Y1 receptor. PIT (10μM) had no effect on the competition for [3H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y1 receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y2, P2Y4, P2Y6, P2Y11 and P2Y12 receptors. Thus, PIT selectively and non-competitively blocked P2Y1 receptor signaling without affecting nucleotide binding.

Publisher Summary This chapter presents an overview of A 3 adenosine receptors. Extracellular adenosine is involved in many cytoprotective functions of the body, including conditioning the heart against ischemia, counteracting the damaging effects of excitotoxicity and seizure activity in the brain, and suppressing an excessive immune and inflammatory response. There are four subtypes of adenosine receptors of which the A 3 receptor was identified as a result of its cloning from various species. The A 3 receptor is activated endogenously by higher concentrations of adenosine that are required for the activation of A 1 /A 2A receptors. The effector mechanisms are the inhibition of adenylate cyclase and stimulation of phospholipase C. The A 3 receptor is distributed at low, diffuse levels in the brain and in the human periphery, where it is present in lungs, liver, heart, and immune cells such as eosinophils. Therapeutic interests related to A 3 receptors are anti-inflammatory, cardioprotective, cerebroprotective anticancer, and antiglaucoma. A mouse line lacking the A 3 receptor demonstrated that this is a nonlethal mutation that has inflammatory, cardiovascular, and behavioral consequences. The chapter discusses the concepts related to A 3 receptor structure and explains A 3 receptor agonists and A 3 receptor antagonists.