2',3'-O-Substituted ATP derivatives as potent antagonists of purinergic P2X3 receptors and potential analgesic agents

Abstract

Blocking membrane currents evoked by the activation of purinergic P2X3 receptors localized on nociceptive neurons represents a promising strategy for the development of agents useful for the treatment of chronic pain conditions. Among compounds endowed with such antagonistic action, 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) is an ATP analogue, whose inhibitory activity on P2X receptors has been previously reported. Based on the results of molecular modelling studies performed with homology models of the P2X3 receptor, novel adenosine nucleotide analogues bearing cycloalkyl or arylalkyl substituents replacing the trinitrophenyl moiety of TNP-ATP were designed and synthesized. These new compounds were functionally evaluated on native P2X3 receptors from mouse trigeminal ganglion (TG) sensory neurons using patch clamp recordings under voltage clamp configuration. Our data show that some of these molecules are potent (nanomolar range) and reversible inhibitors of P2X3 receptors, without any apparent effect on trigeminal GABA_A and 5-HT₃ receptors, whose membrane currents were unaffected by the tested compounds.

Keywords: ATP derivatives; Molecular modelling; P2X3 receptor antagonists; Patch clamp; Purine derivatives; Purinergic receptors.

Fig. 1

Ligands of the P2X3 receptor

Fig. 2

a, b Docking conformation (schematic description in panel b) of TNP-ATP at the human P2X3 binding site. Main residues for the ligand-receptor interaction are indicated. c, d Design of new ligands; docking conformation of the compound 4 (schematic description in panel d)

Scheme 1

Reagents and conditions: a, cyclohexanone, p-toluenesulfonic acid, 4 Å molecular sieves, 70 °C, 48 h; b, i. phosphorus oxychloride, dry trimethyl phosphate, 0 °C, 4 h; ii. 1 M TEAB, 0 °C to r. t., 15 min; c, i. phosphorus oxychloride, dry trimethyl phosphate, 0 °C, 4 h; ii. bis-(tri-n-butylammonium) pyrophosphate solution in dry DMF, 0 °C, 10 min; iii. 1 M TEAB, 0 °C to r. t., 15 min

Scheme 2

Reagents and conditions: a, benzaldehyde, p-toluenesulfonic acid, 4 Å molecular sieves, 70 °C, 4 h. b, i. phosphorus oxychloride, dry trimethyl phosphate, 0 °C, 4 h; ii. 1 M TEAB, 0 °C to r. t., 15 min; c, i. phosphorus oxychloride, dry trimethyl phosphate, 0 °C, 4 h; ii. bis-(tri-n-butylammonium) pyrophosphate solution in dry DMF, 0 °C, 10 min; iii. 1 M TEAB, 0 °C to r. t., 15 min

Fig. 3

NOESY-1D NMR characterization for the configuration assignment of epimers 6 and 5

Fig. 4

Effect of the application of compound 10 on currents evoked by α,β-meATP at native mouse P2X3 receptors expressed by TG sensory neurons. The current evoked by 10 μM α,β-meATP was taken as control peak current and corresponds at a near-maximal response [37, 42]. After 5-min application of 10 (1 and 0.1 μM concentrations via fast superperfusion), subsequent application of 10 μM α,β-meATP induced smaller peak currents or no response at all. After 5-min washout, the current amplitude was almost completely restored. Figure 4 shows representative traces of the fast desensitizing inward currents evoked by 10 μM α,β-meATP and the effect of the synthesized compound 10 applied at 1 and 0.1 μM concentrations

Fig. 5

Dose-inhibition curves of P2X3-mediated currents for compounds 4, 9 and 10 built by applying different concentrations of antagonists using the same concentration of agonist (10 μM α,β-meATP). Data points from n = 5–10 cells, fitted with Hill equation (Eq. 2), asterisk indicates p < 0.05, two-sample t test

Fig. 6

Concentration-response curves for α,β-meATP-evoked P2X3 currents in control and with pre-application of compounds 4, 10 and AF-353 (0.05 μM for 4 and 10; 0.01 μM for AF-353). The agonist was applied at 1, 3, 10, 30 and 100 μM concentrations. Data for each point are obtained from 5 to 11 cells, asterisk indicates p < 0.05, two-sample t test

Fig. 7

Desensitization of P2X3 currents after application of compound 10. Superimposed current traces from the same cell in control and after compound 10 (0.03 μM, 3 min) demonstrate no significant change in current decay time. Control current is induced by 10 μM α,β-meATP, current response after 10 treatment is evoked by 30 μM α,β-meATP. Histograms on the right show values of desensitization time constant τ₁ (mean ± S.E.); n cells = 7, p = 0.41681, paired t test

Fig. 8

Effect of compounds 4, 9 and 10 on GABA_A and 5-HT receptors. a Diagram on the left summarizes mean normalized peak amplitude values for currents evoked by 10 μM GABA (2 s pulses) in control and after 5-min application of compounds 4, 10 or 9 (10 μM); n = 4, 6 and 5 for compounds 4, 10 and 9, respectively. Representative traces on the right show GABA-mediated currents in control and after treatment with compound 10, recorded from the same cell. b Diagram on the left shows mean normalized peak amplitudes for currents evoked by 10 μM 5-HT (2 s pulses) in control and after 5-min application of compounds 4, 10 or 9 (10 μM); n = 4. Examples of 5-HT-mediated current traces (recorded from a single cell) in control and after compound 10 application are presented on the right. p > 0.05 for each compound/receptor type, paired t test