Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β₂-adrenergic receptor

Abstract

The β₂-adrenergic receptor (β₂-AR) agonist [³H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (K_i values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [³H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the K_i values obtained using [³H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [³H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [³H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [³H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.

Introduction

(R,R′)-Fenoterol ((R,R′)-1, Table 1) is a potent and selective agonist of the β₂-adrenergic receptor (β₂-AR), which has entered clinical trials for the treatment of congestive heart failure. The potential therapeutic utility of (R,R′)-1 is based upon its 43-fold selectivity for the β₂-AR relative to the β₁-AR, determined as binding affinities (K_i values) that were calculated using the β₂-AR antagonist [³H]-CGP-12177 as the marker ligand.¹ (R,R′)-1 also selectively couples to G_s proteins² and produces a 300% increase in contractile response in isolated rat ventricular myocytes.1, 2

Using (R,R′)-1 as the scaffold, a project was initiated to study the effect of stereochemistry and the structure of the aminoalkyl moiety of the molecule on β₂-AR binding affinity and selectivity as well as activity using induced stimulation of cAMP accumulation (EC_50cAMP) and cadiomyocyte contractility (EC_50cardio) as the functional markers.1, 3 In the first stage of the project 26 compounds were synthesized and the K_iβ2-AR data used to develop a Comparative Molecular Field Analysis (CoMFA) model¹, which was then used for virtual in silico prediction of binding affinities for a set of 21 new molecular structures.³ Six of the designed molecules were synthesized and their experimentally determined K_iβ2-AR values were in good agreement with the theoretical prediction and the data obtained using the additional compounds were used to refine the CoMFA model. While the expected K_iβ2-AR values were observed for the designed compounds, two of the derivatives, (R,R′)-52 and (R,R′)-54 had unexpectedly high β₂-AR subtype selectivity with K_iβ1-AR/K_iβ2-AR of 334 and 573, respectively.³ The functional activities of a subset of the compounds were also determined and relationships between K_iβ2-AR and EC_50cAMP and K_iβ2-AR and EC_50cardio were observed for (R,R′)-1, (R,R′)-2 and (R,R′)-54 suggesting that the CoMFA model could be used to design compounds for use in the treatment of congestive heart failure. However, when (R,R′)-52 was studied, the compound had a >100-fold lower activity in the cardiomyocyte contractility model while the relationship between K_iβ2-AR and EC_50cAMP was consistent with the other tested compounds. The results suggested that the substitution of an ethyl group on the aminoalkyl moiety of (R,R′)-1 had a profound effect on the interaction of (R,R′)-52 with a conformation(s) of β₂-AR and that this effect was not reflected by the K_iβ2-AR values obtained using [³H]-CGP-12177 as the marker ligand or the CoMFA model constructed using these K_iβ2-AR values.

Based on the data obtained with (R,R′)-52, we have explored the effect of steric bulk at the α′-carbon of the aminoalkyl moiety on binding affinity and functional activity using 4′-methoxyfenoterol (2) as the scaffold. The alkyl length of the substituent on the α′-carbon of the aminoalkyl moiety was varied from 0 (compound 67) to 3 carbon atoms (compounds 65, 66), Table 1, and the K_iβ2-AR values were determined using [³H]-CGP-12177 and [³H]-(R,R′)-2 as the marker ligands. [³H]-(R,R′)-2 was used for this study as it has been previously demonstrated that this radioligand binds with high affinity to an agonist conformation of the β₂-AR⁴, and that K_iβ2-AR values for the fenoterol analogs, including (R,R′)-52, determined using [³H]-(R,R′)-2 modeled their EC_50cAMP values better than the data obtained using [³H]-CGP-12177.⁴ The EC_50cAMP values of the newly synthesized compounds were also determined. The data were used to refine the CoMFA model developed using the K_iβ2-AR values obtained with [³H]-CGP-12177 as the marker ligand and to develop a new CoMFA model with the K_iβ2-AR values obtained when [³H]-(R,R′)-2 was the marker ligand. The new CoMFA model reflects binding to an agonist-stabilized conformation of the β₂-AR. The results demonstrate that there are subtle but significant differences between the two models and suggest that the use of multiple models can be beneficial in the design of new chemical entities with specific pharmacological properties.