Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective β2-adrenergic receptor agonists

doi:10.1016/j.bmc.2009.11.062

Bioorganic & Medicinal Chemistry

Volume 18, Issue 2, 15 January 2010, Pages 728-736

https://doi.org/10.1016/j.bmc.2009.11.062 Get rights and content

Abstract

Purpose: To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the β₂-adrenergic receptor. Results: Out of 21 computationally designed structures 6 compounds were synthesized and characterized for β₂-AR binding affinities, subtype selectivities and functional activities. Conclusion: the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K_iβ₂-AR = 0.28 μm, K_iβ₁-AR/K_iβ₂-AR = 573, EC_50cAMP = 3.9 nm, EC_50cardio = 16 nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure.

Graphical abstract

Introduction

In the initial studies in this series, (R,R)-fenoterol, Fig. 1, a potent and selective β₂-adrenoceptor (β₂-AR) agonist was identified as a potential drug for the treatment of congestive heart failure.1, 2 (R,R)-Fenoterol is currently entering initial clinical trials for use in this disease. Based on the potential therapeutic use of (R,R)-fenoterol, the stereoisomers of fenoterol and a series of fenoterol analogues were synthesized and tested for β₂-AR activity and selectivity.³ Three of the 26 compounds investigated in the latter study had submicromolar affinity for the β₂-AR, a >40-fold selectivity for the β₂-AR relative to the β₁-AR and were full agonists of the β₂-AR. One of these compounds, (R,R)-methoxyfenoterol was also identified as a potential candidate for drug development and preliminary pharmacokinetic and toxicity studies have been initiated.

The binding affinities, K_i values, of the 26 fenoterol analogues were used to develop a comparative molecular field analysis model, CoMFA(n = 26), to describe and predict the binding of the stereoisomers of fenoterol and fenoterol analogues to the β₂-AR.³ The CoMFA(n = 26) model explained the role of stereoconfiguration and structural modifications of studied molecules on observed β₂-AR affinities. The model was also consistent with earlier findings from other laboratories,4, 5 which indicated that the β₂-AR selectivity of the fenoterol analogues was due to interactions of the aminoalkyl portion of the molecules with different sites within the transmembrane (TM) components of the β₂-AR. These interactions included hydrogen bond formation between the p-oxygen moiety on the phenyl ring in fenoterol and methoxyfenoterol and tyrosine 308 (Y308) in TM7 and/or histidine 296 (H296) in TM6 and π–π and/or π-hydrogen bond interactions between the aromatic system of the ligand and aromatic residues located in this area of the binding site. In addition, the data from the previous study³ and an earlier study⁶ indicated that the binding process of the fenoterol analogues with the β₂-AR included the interaction of the chiral center of the aminoalkyl portion with a sterically-restricted site on the receptor.

In this study, the CoMFA(n = 26) model was used to design 21 additional fenoterol analogues as potential selective and potent β₂-AR agonists. Based on the CoMFA(n = 26) model and the indicated binding interactions, three sites on the aminoalkyl portion of the fenoterol molecule were chosen for alteration, Figure 1. The modifications were utilized to explore the effect on β₂-AR binding affinity, selectivity and agonist activity of the steric bulk at the second chiral center, of a 1-napthyl versus a 2-napthyl substituent, and of the effect of a substituent at the 4-position of a 1-naphthyl substituent. A subset of 6 compounds, composed of the (R,R)- and (R,S)- isomers of 3 of the designed compounds, was synthesized using the previously described approach.³ The β₂-AR and β₁-AR binding affinities and the EC₅₀ values associated with induced cAMP accumulation (EC_50cAMP) were determined. The (R,R)-isomers of the subset were tested for their activity in a previously described cardiomyocyte contractility model³ and their EC_50cardio values determined.

Section snippets

Results

In the previous study, the CoMFA(n = 26) model was derived using the experimentally determined β₂-AR binding affinities of a training set of 26 fenoterol derivatives and stereoisomers.³ The validity parameters of the model were high (R² = 0.920, Q² = 0.847, F = 60.380) and the standard error of prediction was low (SEP = 0.309). In the current study, the CoMFA(n = 26) model was used to estimate the pK_i values for the (R,R)-isomers of a set of 12 new molecular structures, and the (R,S)-, (S,R)- and

Discussion

The results of this study demonstrate that the CoMFA(n = 26) model can be used to design selective β₂-AR agonists based upon fenoterol or compounds that contain interactive sites equivalent to those found on the aminoalkyl portion of the fenoterol molecule. This design strategy is reflected by the synthesis and properties of the β₂-AR agonist (R,R)-54, which is highly selective (K_iβ₁/K_iβ₂ = 573) and effective (EC₅₀ = 16 nM, cardiomyocyte contractility).

The revised molecular model, CoMFA(n = 32) is

β₂-AR binding assays

Compounds synthesized in this study were tested up to three times each to determine their binding affinities at the β₁- and β₂-ARs following a previously described approach.³ In brief, β₂-AR binding was conducted on membranes derived from HEK cells containing human β₂-AR (provided by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto, CA). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.05% penicillin–streptomycin with 400 μg/mL G418. β

Acknowledgments

This work was supported in part by funds from the National Institute on Aging, Intramural Research Program, NIA Contract N01-AG-3-1009 and the Foundation for Polish Science (FOCUS 4/2006 programme)

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(R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a highly-selective β₂ adrenergic receptor (β₂-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R′)-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R′)-MNF positively correlated with the β₂-AR expression levels across tested cell lines. The anti-motility activity of (R,R′)-MNF was inhibited by the β₂-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R′)-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R′)-MNF caused significant inhibition of cell growth in β₂-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon β₂-AR activation with (R,R′)-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified β₂-AR as a novel potential target for melanoma management, and (R,R′)-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in β₂-AR-expressing lesions.
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Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective β2-adrenergic receptor agonists

Abstract

Graphical abstract

Introduction

Section snippets

Results

Discussion

β2-AR binding assays

Acknowledgments

Trends Pharmacol. Sci.

J. Biol. Chem.

J. Biol. Chem.

Chirality

J. Med. Chem.

Mol. Pharmacol.

Mol. Pharmacol.

J. Med. Chem.

Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective β₂-adrenergic receptor agonists

β₂-AR binding assays