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. 1993 Nov 26;36(24):3937-46.
doi: 10.1021/jm00076a023.

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

B Fischer  1 , J L Boyer, C H Hoyle, A U Ziganshin, A L Brizzolara, G E Knight, J Zimmet, G Burnstock, T K Harden, K A Jacobson
Affiliations

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

B Fischer et al. J Med Chem. .

Abstract

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 agonist, 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 coli, 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 coil 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.

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Figures

Figure 1
Figure 1
Synthesis of 2-thioether and N6-methyl-ATP analogues. Compound a in this scheme (25–30) refers to R = H, and compound b refers to R = CH3. Compound 27a was synthesized by procedures previously described., Conditions were: (i) m-chloroperbenzoic acid, 3 days, room temperature; (ii) (1) 5 N NaOH, (2) CS2, MeOH, H2O,120 °C; (iii) RBr, Et3N, DMF; (iv) (1) POCl3, (2) (Bu3NH+)2P2O7H2; (v) (1) POCl3, (2) (Bu3NH+)2-PO4H2. Adenine 5′-triphosphate derivatives synthesized by this method but not shown in figure: 2-(hexylthio)-, 8; 2[(2-phenylethyl)thio]-, 12; and 2-(cyclohexylthio)-, 16. Adenine 5′-monophosphate derivatives also isolated as byproducts of the phosphorylation reactions: 2-(hexenylthio)-, 11; 2-[[2-nitrophenypethyl]thio]-, 14; 2-(6-cyanohexylthio)-, 18; and 2-(6-cyanohexylthio)-N6-methyl-, 24.
Figure 2
Figure 2
Synthesis of terminally functionalized 2-(alkylthio)-ATP analogues, compounds 19–21.
Figure 3
Figure 3
Concentration-dependent stimulation of inositol phosphate formation by 2-thioether derivatives of ATP. Membranes from [3H]inositol-labeled erythrocytes were incubated for 5 min at 30 °C in the presence of the indicated concentrations of ATP, 1 (■); 2-(methylthio)-ATP, 6 (○); 2-(hexylthio)-ATP, 8 (△); 2-(hexenylthio)-ATP, 9 (□); 2-[(phenylethyl)thio]-ATP, 12 (◊); 2-(cyclohexylthio)-ATP, 16 (▽); 2-[(cyanohexyl)thio]-ATP, 17 (●); or 2-[[(p-nitrophenyl)ethyl]thio]-ATP, 13 (▲). Incubation was in the presence of 1µM GTPγS as described in Materials and Methods. The data shown are the average of three to eight experiments carried out in duplicate using different membrane preparations. The average cpm of [3H]inositol phosphates produced in the presence of 1 µM GTPγS alone was 400 cpm (0%). The maximal (100%) level of [3H]inositol phosphates in the presence of GTPγS and adenine nucleotide analogues was at least 5000 cpm with all membrane preparations tested.
Figure 4
Figure 4
Concentration-dependent stimulation of inositol phosphate formation by N6- and 2-thioether derivatives of ATP. Membranes from [3H]inositol-labeled erythrocytes were incubated in the presence of ATP, 1 (○); N6-methyl-ATP, 22 (●); 2-(hexenylthio)-ATP, 9 (△); N6-methyl-2-(hexenylthio)-ATP, 23 (▲); and N6-methyl-2-(hexenylthio)-AMP, 24 (□). Incubation was for 5 min at 30 °C in the presence of 1 µM GTPγS. Data shown are from a representative experiment repeated at least three times with similar results. [3H] Inositol phosphate accumulation in the presence of 1µM GTPγS alone was 250 cpm (0%). Maximal levels (100%) of [3H]inositol phosphate accumulation in the presence of GTPγS and a maximal concentration of N6-methyl-2-(hexenylthio)-ATP was 9150 cpm.
Figure 5
Figure 5
Phospholipase C activity of 2-thioether analogues of ATP and ADP. Membranes from [3H]inositol-labeled erythrocytes were incubated in the presence of the indicated concentrations of ADP, 2 (■); ATP, 1 (□); 2-(methylthio)-ADP, 7 (●); 2-(methylthio)-ATP, 6 (○); 2-(hexenylthio)-ADP, 10 (▲); or 2-(hexenylthio)-ATP, 9 (△). Incubation was for 5 min at 30 °C in the presence of 1 µM GTPγS as described under Materials and Methods. The data shown are from a representative experiment repeated at least three times using different membrane preparations. [3H]Inositol phosphate accumulation in the presence of GTPγS alone was 200–400 cpm (0%). Maximal levels of [3H) inositol phosphates produced by ADP and ATP analogues in the presence of GTPγS (100%) were identical within the same membrane preparation with values ranging from 5000 to 9000 cpm.
Figure 6
Figure 6
Comparative effects of 2-thioether analogues of AMP and ATP on phospholipase C activity in turkey erythrocyte membranes. Membranes from [3H]inositol-labeled erythrocytes were incubated for 5 min at 30 °C in the presence of the indicated concentrations of AMP, 3 (□); ATP, 1 (■); 2-[(cyanohexyl)thio]-AMP, 18 (○); 2-[(cyanohexyl)thio]-ATP, 17 (●); 2-[[(p-nitrophenyl)ethyl]thiol]-AMP, 14 (△); 2-[[(p-nitrophenyl)ethyl]thio]-ATP, 14 (▲). Incubation was in the presence of 1µM GTPγS as described under Materials and Methods. Results shown are from a representative experiment repeated at least three times using different membrane preparations. [3H]inositol phosphate accumulation in the presence of 1µM GTPγS alone was 200 cpm (0%). Maximal levels (100%) of [3H] inositol phosphate accumulation in the presence of GTPγS and a maximal concentration of agonist, e.g. 1µM 2-[(cyanohexyl)thio]-ATP, was 6000 cpm.
Figure 7
Figure 7
Concentration—response relationships for ATP and its derivatives causing relaxation of the carbachol-contracted guinea pig taenia coli (P2Y-purinoceptor). All curves are the mean of two determinations except for 1 (n = 38), 8 (n = 4), 11 (n = 3), and 14 (n = 5). ATP was tested on all the preparations that the derivatives were tested on. Ordinate axis shows the percentage relaxation of the carbachol-induced contraction; abscissa axis shows −log[agonist]. The pD2 value for 2MeSATP is 8.0 ± 0.15 (ref 33).
Figure 8
Figure 8
Correlation between turkey erythrocyte P2Y-purinoceptor agonism and guinea pig taenia coli P2Y-purinoceptor agonism, represented by pK0.5 and pD2 values, respectively. The line shows the linear regression of pD2 on pK0.5, which had a correlation coefficient, r, of 0.960 (P < 0.001) for the equation y = 0.5x + 3.59. None of these compounds had significant activity at P2X-purinoceptors (see Table I). The pD2 value for 2-(methylthio)-ATP (2MeSATP) was taken from the literature.
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