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. 2002 Jan 3;45(1):208-18.
doi: 10.1021/jm010369e.

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors

Hak Sung Kim  1 , R Gnana Ravi, Victor E Marquez, Savitri Maddileti, Anna-Karin Wihlborg, David Erlinge, Malin Malmsjö, José L Boyer, T Kendall Harden, Kenneth A Jacobson
Affiliations

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors

Hak Sung Kim et al. J Med Chem. .

Abstract

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.

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Figures

Figure 1
Figure 1
Effects of (N)-methanocarba or (S)-methanocarba modification of ATP on PLC activity in 1321N1 astrocytoma cells expressing either hP2Y1 (A) or hP2Y2 (B) receptors. Concentration-dependent stimulation of inositol phosphate formation by adenine nucleotides (triphosphates), compounds 3a (▲), 3b (○), and 8a (▼). Membranes from [3H]inositol-labeled membranes were incubated for 5 min at 30 °C in the presence of the indicated concentrations of agonist. The data shown are typical curves for at least three experiments carried out in duplicate using different membrane preparations.
Figure 2
Figure 2
Effects of the (N)-methanocarba modification in uracil nucleotides (triphosphates) on activation of PLC in 1321N1 astrocytoma cells expressing either hP2Y2 (A) or hP2Y4 (B) receptors, showing concentration-dependent stimulation of inositol phosphate formation by compounds 5a (○) and 5b (●). Membranes from [3H]inositol-labeled membranes were incubated for 5 min at 30 °C in the presence of the indicated concentrations of agonist. The data shown are typical curves for at least three experiments carried out in duplicate using different membrane preparations.
Figure 3
Figure 3
Effects of the (N)-methanocarba modification in uracil nucleotides (diphosphates) on activation of PLC in 1321N1 astrocytoma cells expressing hP2Y6 receptors, showing concentration-dependent stimulation of inositol phosphate formation by compound 6b (●) but not by compound 6a (○). Membranes from [3H]inositol-labeled membranes were incubated for 5 min at 30 °C in the presence of the indicated concentrations of nucleotide. The data shown are typical curves for at least three experiments carried out in duplicate using different membrane preparations.
Figure 4
Figure 4
Vasodilatory responses to UTP (▲) and 5a (■) in the rat mesenteric artery. The uracil nucleotides were added after P2X receptor desensitization with 10 µM α,β-meATP. Relaxation is expressed as a percentage of an initial contraction induced by 1 µM norepinephrine. Data are shown as means ± sem of 5–7 vessel segments.
Figure 5
Figure 5
Vasocontractile responses to uracil nucleotides UTP (▲) and 5a (■) in the rat mesenteric artery. The uracil nucleotides were added after P2X receptor desensitization with 10 µM α,β-meATP. L-NAME (0.1 mM) was present to inhibit the release of NO. Data are shown as means ± sem of 2–4 vessel segments, except for the highest concentration of 5a, where n= 1.
Figure 6
Figure 6
Vasocontractile responses to UDP (△), 6a (□), and UDP in the presence of 10 µM 6a (◇) in the rat mesenteric artery. The uracil nucleotides were added after P2X receptor desensitization with 10 µM α,β-meATP. L-NAME (0.1 mM) was present to inhibit the release of NO. Data are shown as means ± sem of 2–4 vessel segments.
Scheme 1
Scheme 1
General Method for Stepwise Phosphorylation of (N)-Methanocarba Nucleosides: Formation of Di- and Triphosphates via the Phosphorimidazolidate Intermediate, IIa a Reagents: (i) 1,1′-Carbonyldiimidazole, DMF, room temperature, 6 h and then 5% aqueous triethylamine, room temperature, 2 h. (ii) Tributylammonium phosphate, DMF, room temperature, 2 days, 43%. (iii) Tributylammonium pyrophosphate, DMF, 3 days, 6–40%.
Scheme 2
Scheme 2
Synthesis of (N)-Methanocarba Adenosine 5′-Phosphate Derivativesa a Reagents: (i) DEAD, Ph3P, THF (ref 40). (ii) NH3/i-PrOH, 95%. (iii) Palladium black, HCO2H, methanol, 76%. (iv) Di-t-butyl N,N-diethylphosphoramidite, tetrazole, THF, room temperature, 6 h and then m-CPBA, −78 °C, room temperature, 78%. (v) DOWEX 50 × 8–200, methanol, 60–70 °C, 69%. (vi) (1) 1,1′Carbonyldiimidazole, (2) triethylamine/MeOH, (3) tributylammonium pyrophosphate, 20%.
Scheme 3
Scheme 3
Synthesis of (N)-Methanocarba-uridine 5′-Monophosphate, 7a, and 5′-Di- and Triphosphates, 6a and 5aa a Reagents: (i) (a) EtOCH=CHC(=O)NCO, benzene, room temperature, 10 min, 89%; (b) 2 N H2SO4, MeOH, reflux, 30 min, 67%. (ii) Pd black, HCO2H, methanol, 74%. (iii) p-Toluene sulfonic acid, acetone, room temperature, 4 h, 86%. (iv) Di-t-butyl N,N-diethylphosphoramidite, tetrazole, THF, room temperature, 20 min and then m-CPBA, −78 °C, room temperature, 79%. (v) DOWEX 50 × 8–200, methanol, 60–70 °C, 86%. (vi) (1) 1,1′-Carbonyldiimidazole, (2) triethylamine/MeOH, (3) tributylammonium phosphate or pyrophosphate, DMF, 2–3 days, 41% (6a), 38% (5a).
Scheme 4
Scheme 4
Synthesis of (S)-Methanocarba Adenosine 5′-Phosphate Derivativesa a Reagents: (i) POCl3, trimethyl phosphate, 60%. (ii) 1,1′-Carbonyldiimidazole, DMF, room temperature, 6 h and then 5% aqueous triethylamine, room temperature, 2 h. (iii) Tributylammonium pyrophosphate, DMF, 3 days, 30%.
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