Identification of potent P2Y-purinoceptor agonists that are derivatives of adenosine 5′-monophosphate

1. A series of chain-extended 2-thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells, and the cloned human P2U-receptor stably expressed in 1321N1 human astrocytoma cells. 2. Although adenosine monophosphate itself was not an agonist in the two P2Y-purinoceptor test systems, eleven different 2-thioether-substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2-hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2-hexylthio ATP in the same system. 2-hexylthio adenosine was inactive. 3. Monophosphate analogues that were the most potent activators of the C6 cell P2Y-purinoceptor were also the most potent activators of the turkey erythrocyte P2Y-purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4. Although 2-thioether derivatives of adenosine monophosphate were potent P2Y-purinoceptor agonists no effect of these analogues on the human P2U-purinoceptor were observed. 5. These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y-purinoceptors, and provide insight for strategies for development of novel P2Y-purinoceptor agonists of high potency and selectivity

Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonists, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coil, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5–770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6–8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coli P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP

Structure activity relationships for derivatives of adenosine-5?-triphosphate as agonists at P2 purinoceptors: Heterogeneity within P2x and P2y subtypes

The structure-activity relationships for a variety of adenine nucleotide analogues at P2x- and P2Y-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N6-substituents, and a uridine base instead of adenine), the ribose moiety (2′ and 3′-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P2Y-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P2X-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the P2X- or the P2Y-purinoceptors. Certain analogues displayed selectivity or specificity within the P2X- or P2Y-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2′,3′-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P2Y-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P2X-purinoceptors. The potent agonist N6-methyl ATP and the somewhat less potent agonist 2′-deoxy-ATP were selective for P2Y-purinoceptors in the guinea pig taenia coli, but were inactive at P2X-purinoceptors and the vascular P2Y-purinoceptors. 3′-Benzylamino-3′-deoxyATP was very potent at the P2X-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P2Y receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P2X- and P2Y-purinoceptor classes

Rules of Chemospecificity of Nucleophilc Ring-opening of Dithia-/Oxathia-Phospholane Towards the Selective Synthesis of Nucleoside 5’-O-Pα-Thio/Dithio/Trithio-Phosphate Ester Conjugates

DBU-assisted nucleophilic ring-opening of both uridine-5’-(2-thio-1,3,2-dithia-phospholane), 3, and uridine-(2-thio-1,3,2-oxathia-phospholane), 8, lasted 2 min at RT and resulted in quantitative yields of uridine-5’-phosphoro-di/trithioate esters. Furthermore, it was selective for alcohol and thiol vs. amine nucleophiles. Yet, reaction of mercaptoethanol with 3, was chemo-specific for the oxygen vs. sulfur nucleophile, while for the reaction of mercaptoethanol with 8, the opposite chemo-specificity was observed, probably related to the steric hindrance in the former case. The observed chemospecificity opens facile avenue for the synthesis of nucleoside-5’-O-Pα-thio/dithio/trithio-phosphate ester derivative

Phenyl-imidazolo-cytidine Analogues: Structure–Photophysical Activity Relationship and Ability To Detect Single DNA Mismatch

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized <i>para</i>-substituted phenyl-imidazolo-cytidine (^PhImC) analogues <b>5a</b>–<b>g</b> and established a relationship between their structure and fluorescence properties. These analogues were more emissive than cytidine (λ_em 398–420 nm, Φ 0.009–0.687), and excellent correlation was found between Φ of <b>5a</b>–<b>g</b> and σ_p^– of the substituent on the phenyl-imidazolo moiety (<i>R</i>² = 0.94). Calculations suggested that the dominant tautomer of ^PhImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected ^PhImC analogues suggested a relationship between the HOMO–LUMO gap and Φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF₃-^PhImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A <i>17-fold</i> reduction of fluorescence was observed for the <i>G-matched</i> duplex vs <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b>, while for <i>A-mismatched</i> duplex, only a <i>2-fold</i> decrease was observed. Furthermore, since the quantum yield of <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b>:<b>ODN­(G)</b> was reduced 17-fold vs that of a single strand, whereas that of <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b>:<b>ORN­(G)</b> was reduced only 3.8-fold, <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b> appears to be a DNA-selective probe. We conclude that the <b>ODN­(CF</b>_<b>3</b><b>-</b>^<b>Ph</b><b>ImdC)</b> probe, exhibiting emission sensitivity upon single nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing

Methylenediphosphonotetrathioate: Synthesis, Characterization, and Chemical Properties

Metal chelators are potential therapeutic agents for treating diseases such as Wilson’s and Alzheimer’s where the pathology involves an excess of metal-ions (Cu­(II) and Zn­(II)/Cu­(II)/Fe­(II/III), respectively). In addition to the high affinity of the metal-ion to the chelators, metal selectivity of the chelators is essential to achieve the therapeutic goal, that is, the successful removal of excess of harmful metal-ions in a physiological extracellular medium rich in alkali and alkali earth metal-ions. For this purpose, we synthesized a novel chelator, methylenediphosphonotetrathioate (MDPT) which is the tetrathio analogue of methylenediphosphonic acid (MDP). MDPT was synthesized from bis-methylene­(phosphonicdichloride) in a 3-step synthesis and a 31% overall yield. MDPT formed a stable complex with Zn­(II) (log <i>K</i> = 10.84), which is 10⁷ times more stable than the corresponding Ca­(II) complex. Moreover, the MDPT-Zn­(II) complex was 50-fold more stable than the MDP-Zn­(II) complex. In addition, MDPT was found to inhibit the Cu­(I)-catalyzed Fenton reaction (IC₅₀ 26 μM) 2.5 times more potently than a Fe­(II)-catalyzed Fenton reaction, and 2.5 times more potently than EDTA (IC₅₀ 64 μM) in the Cu­(I)/H₂O₂ system, as monitored by electron spin resonance (ESR). Furthermore, MDPT was found to be relatively stable in both acidic (pD 1.9, <i>t</i>_¹/₂ = 71.5 h) and basic media (pD 12.4, <i>t</i>_¹/₂ = 81 h) as monitored by ³¹P/¹H NMR. However, MDPT was not stable in air because of intramolecular oxidation and disulfide formation (33% oxidation after 27 h). In conclusion, MDPT was found to be a water-soluble chelator showing a clear preference to soft/borderline metal-ions and a remarkable selectivity to those metal-ions vs Ca­(II) ions. The relative sensitivity of MDPT to oxidation may limit its use; however, the application of MDPT in acidic or basic media will increase its lifetime