Structure-activity relationships at human and rat A2B adenosine receptors of xanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions

Abstract

In the search for improved selective antagonist ligands of the A2B adenosine receptor, which have the potential as antiasthmatic or antidiabetic drugs, we have synthesized and screened a variety of alkylxanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions. Competition for 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine) binding in membranes of stably transfected HEK-293 cells revealed uniformly higher affinity (<10-fold) of these xanthines for human than for rat A2B adenosine receptors. Binding to rat brain membranes expressing A1 and A2A adenosine receptors revealed greater A2B selectivity over A2A than A1 receptors. Substitution at the 1-position with 2-phenylethyl (or alkyl/olefinic groups) and at N-3 with hydrogen or methyl favored A2B selectivity. Relative to enprofylline 2b, pentoxifylline 35 was equipotent and 1-propylxanthine 3 was >13-fold more potent at human A2B receptors. Most N-7 substituents did not enhance affinity over hydrogen, except for 7-(2-chloroethyl), which enhanced the affinity of theophylline by 6.5-fold to 800 nM. The A2B receptor affinity-enhancing effects of 7-(2-chloroethyl) vs 7-methyl were comparable to the known enhancement produced by an 8-aryl substitution. Among 8-phenyl analogues, a larger alkyl group at the 1-position than at the 3-position favored affinity at the human A2B receptor, as indicated by 1-allyl-3-methyl-8-phenylxanthine, with a K(i) value of 37 nM. Substitution on the 8-phenyl ring indicated that an electron-rich ring was preferred for A2B receptor binding. In conclusion, new leads for the design of xanthines substituted in the 1-, 3-, 7-, and 8-positions as A2B receptor-selective antagonists have been identified.

Figure 1.

Competition by 7-substituted xanthines for ¹²⁵I-ABOPX binding to recombinant human A_2B receptors. The indicated antagonists (see Table 1) were incubated to equilibrium (2 h) in 100 μL with radioligand (0.7 nM ¹²⁵I-ABOPX) and human HEK-A_2B cell membranes (25 μg of membrane protein). Each point is the mean ± SEM of triplicate determinations. Where not shown, the SEM is smaller than the symbol. K_i values derived from triplicate experiments are summarized in Table 1.

Figure 2.

Correlation in the affinities of various xanthines for binding to human and rat A_2B adenosine receptors. The pK_i values of compounds that were evaluated for binding to rat and human receptors (Table 2) are plotted and fit by linear regression.

Scheme 1.

Synthesis of Xanthine Derivatives 14b–d^{a a} Reagents: (a) 10% NaOH, EtOH, R–Br; (b) NaNO₂, 6 N HCl, AcOH; (c) Na₂S₂O₄; (d) HC(OMe)₃.

Scheme 2.

Synthesis of Xanthine Derivatives 19b,c^{a a} Reagents: (a) NaH, Br(CH₂)_nCl, DMF.

Scheme 3.

Synthesis of Xanthine Derivatives 64 and 65^{a a} Reagents: (a) NaH, Cl₂(CH₂)₂, DMF.