Synthesis and pharmacological characterization of a new series of 5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazine derivatives as adenosine receptor antagonists: A preliminary inspection of ligand-receptor recognition process

Abstract

A new series of triazolotriazines variously substituted at the C5 and N7 (5-25) positions was synthesized and fully characterized at the four adenosine receptor (AR) subtypes. In particular, arylacetyl or arylcarbamoyl moieties were introduced at the N7 position, which enhanced affinity at the hA(2B) and hA(3) ARs, respectively, when utilized on the pyrazolo-triazolopyrimidine nucleus as we reported in the past. In general, compounds with a free amino group at the 7 position (5, 6), showed good affinity at the rat (r) A(2A) AR (range 18.3-96.5nM), while the introduction of a phenylcarbamoyl moiety at the N7 position (12, 19, 24) slightly increased the affinity at the hA(3) AR (range 311-633nM) with respect to the unsubstituted derivatives. The binding profiles of the synthesized analogues seemed to correlate with the substitutions at the C5 and N7 positions. At the hA(2B) AR, derivative 5, which contained a free amino group at the 7 position, was the most potent (EC(50) 3.42microM) and could represent a starting point for searching new non-xanthine hA(2B) AR antagonists. Molecular models of the rA(2A) and hA(3) ARs were constructed by homology to the recently reported crystallographic structure of the hA(2A) AR. A preliminary receptor-driven structure-activity relationship (SAR) based on the analysis of antagonist docking has been provided.

Figure 1

Multiple sequence alignment of hA_2A receptor, rat A_2A receptor and hA₃ receptor. Conserved residues in all the sequences are identified by stars. Differences in sequences of rA_2A and hA₃ compared to hA_2A are shown in boldface type.

Figure 2

Structure superimposition of crystallographic pose (in magenta) and docking pose (in green) of ZM241385 inside the rA_2A AR binding site. Side chains of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.

Figure 3

Structure superimposition of docked conformations of compound 5 (in red, K_i rA_2AAR = 18.3 ± 3.4) and compound 18 (in magenta, K_i rA_2AAR = 38.9 ± 3.5) inside the rA_2A AR binding pocket. Side chains of some amino acids important for ligand recognition and H-bonding interactions are highlighted. Hydrogen atoms are not displayed.

Figure 4

Electrostatic interaction energy (in kcal/mol) between the ligand and each single amino acid involved in ligand recognition calculated from: (A) crystallographic binding mode of ZM241385 inside the hA_2A receptor (PDB code: 3EML); (B) hypothetical binding mode of ZM241385 inside the rA_2A receptor obtained after docking simulations; (C) hypothetical binding mode of compound 5 inside the rA_2A receptor obtained after docking simulations; and (D) hypothetical binding mode of compound 18 inside the rA_2A receptor obtained after docking simulations.

Figure 5

Hydrophobic interaction energy (in arbitrary hydrophobic unit) between the ligand and each single amino acid involved in ligand recognition calculated from: (A) crystallographic binding mode of ZM241385 inside the hA_2A receptor (PDB code: 3EML); (B) hypothetical binding mode of ZM241385 inside the rA_2A receptor obtained after docking simulations; (C) hypothetical binding mode of compound 5 inside the rA_2A receptor obtained after docking simulations; and (D) hypothetical binding mode of compound 18 inside the rA_2A receptor obtained after docking simulations.

Scheme 1

Reagents: (i) EtOH, dimethylamine, 120 °C sealed tube, 4 h; (ii) R–N=C=O, dioxane, Et₃N, reflux, 12 h; (iii) R–COCl, dioxane, reflux, 12 h.

Chart 1

Pyrazolo-triazolopyrimidine derivatives as A_2A and A₃ AR antagonists.

Chart 2

Triazolotriazine derivatives as AR antagonists.

Chart 3

Molecular simplification approach applied for the design of the novel triazolotriazine analogs.

Chart 4

Summary of the most relevant SAR features of the novel triazolotriazine analogs.