Structure Activity Relationships for Derivatives of Adenosine-5'-Triphosphate as Agonists at P(2) Purinoceptors: Heterogeneity Within P(2X) and P(2Y) Subtypes

Abstract

The structure-activity relationships for a variety of adenine nucleotide analogues at P(2x)- and P(2Y)-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N(6)-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 P(2Y)-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 P(2X)-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 P(2X)- or the P(2Y)-purinoceptors. Certain analogues displayed selectivity or specificity within the P(2X)- or P(2Y)-purinoceptor superfamilies, giving hints about possible subclasses. For example, 8-(6-aminohexylamino)ATP and 2',3'-isopropylidene-AMP were selective for endothelial Pzypurinoceptors over P(2Y)-purinoceptors in the guinea pig taenia coli, rabbit aorta, and turkey erythrocytes. These compounds were both inactive at P(2X)-purinoceptors. The potent agonist N(6)-methyl ATP and the somewhat less potent agonist 2'-deoxy-ATP were selective for P(2Y)-purinoceptors in the guinea pig taenia coli, but were inactive at P(2X)-purinoceptors and the vascular P(2Y)-purinoceptors. 3'-Benzylamino-3'-deoxyATP was very potent at the P(2X)-purinoceptors in the guinea pig vas deferens and bladder, but not in the rabbit saphenous artery and was inactive at P(2Y) receptors. These data suggest that specific compounds can be developed that can be utilized to activate putative subtypes of the P(2X)- and P(2Y)-purinoceptor classes.

Figure 1

Synthesis of a 3′,5′-cyclic analogue of α,β-rnethylene ATP.

Figure 2

Synthesis of ribose 3′-position-modified ATP analogues.

Figure 3

Guanine nucleotide-dependent activation of turkey erythrocyte phospholipase C by ATP analogues. Substitution was on the phosphate chain (A), on the purine base (B), or on the ribose moiety (C). Turkey erythrocyte ghosts were incubated for 5 min at 30°C in the presence of 1 μM GTPγS and the indicated concentrations of ATP analogues, as described in Methods. The formation of total [³H]inositol phosphates stimulated by ATP analogues is shown. The ATP analogues tested in A were: ATP, 1 (●); ADP, 2 (▲); AMP, 3 (■); α,β-Me-ATP, 4 (▼); β,γ-Me-ATP, 5 (●); AppNHp, 7 (◯); ATP-α-S (S-isomer), 8 (△); and ADP-β-S, 10 (□). For B: N⁶-Me-ATP, 12 (▲); 8-Br-ATP, 13 (■); 8-(6-aminohexyl-amino)-ATP, 14 (▼); 2-(6-cyanohexylthio)-ATP, 11b (●); adeno-sine-N1-oxide-5′-triphosphate, 15 (◆); N1,N⁶-etheno-ATP, 16 (◯); UTP, 17 (△); and 5-F-UTP, 18(□). For C: 2′-deoxy-ATP, 19 (●); 3′-deoxy-ATP, 20 (▲); 2′,3′-dideoxy-ATP, 21 (■); 3′-amino-3′-deoxy-ATP, 22 (▼); 3′-acetylamino-3′-deoxy-ATP, 23 (◆); 3′-[3-(4-hydroxyphenyl)propionylaminol-3′-deoxy-ATP, 24 (◯); 3′-ben zylamino-3′-deoxy-ATP, 25 (△); and 2′,3′-isopropylidene-AMP, 27(□).

Figure 4

Concentration-response relationships for ATP analogues causing relaxation of the carbachol-contracted guinea pig taenia coli (P_2Y-purinoceptor). Each curve is the mean of 2–5 determinations. Ordinate axis shows % relaxation of the carbachol (50 nM)-induced contraction; abscissa axis shows -log concentration of applied agonists, which are (from left to right at the 50% level): 11b, 2-(6-cyanohexyl)thio-ATP; 12, N⁶methyl-ATP; ₁₉, 2′-deoxy-ATP; 22, 3′-amino-3′-deoxy-ATP; 15, adenosine-N-oxide-5′-triphosphate; 21, 2′,3′-dideoxy-ATP.

Figure 5

Concentration-response relationships for ATP analogues causing relaxation of the rabbit aorta (endothelial P_2Y-purinoceptor). Ordinate axis shows % relaxaiion of the noradrenaline (1 μM)-induced contraction; abscissa axis shows -log concentration of applied agonist; 11b, 2-(6-cyanohexyl)thio-ATP (▲); 15, adenosine-N1-oxide-5′-triphosphate (△); 14, 8-(6-amino-hexylamino)-ATP (▽); 18, 5-fluoro-UTP (◆); 22, 3′-deoxy-3′-amino-ATP (▼). Each point is the mean of 2–4 determinations.

Figure 6

Concentration-response relationships for ATP analogues causing contraction of (A) guinea pig isolated urinary bladder detrusor muscle and (B) guinea pig isolated vas deferens (both possess P_2X-purinoceptors). Agonists: 25, 3′-benzylamino-3′-deoxy-ATP (■); 11b, 2-(6-cyanohexyl)thio-ATP (▲); 18, 5-fluoro-UTP (◆); 22, 3′-deoxy-3′-arnino-ATP (▼); 1, ATP (●); 21, 2′, 3′-dideoxy-ATP (◇). Each curve is the mean of two determinations. Ordinate axis shows % contraction relative to a standard dose of KCI (60 mM); abscissa axis shows -log concentration of applied agonist.