Original articleSynthesis, pharmacological and in silico evaluation of 1-(4-di-hydroxy-3,5-dioxa-4-borabicyclo[4.4.0]deca-7,9,11-trien-9-yl)-2-(tert-butylamino)ethanol, a compound designed to act as a β2 adrenoceptor agonist
Graphical abstract
The compound 1-(4-di-hydroxy-3,5-dioxa-4-borabicyclo[4.4.0]deca-7,9,11-trien-9-yl)-2-(tert-butylamino)ethanol shows greater affinity and intrinsic efficacy than salbutamol and acts as an agonist on the β2 adrenoceptor.
Introduction
Development of β2 adrenoceptor (β2AR) agonists is an attractive area of research, since their biological effects have many applications in the medical field. These drugs are used alone or in combination with corticosteroids in the treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD) [1]. In addition, β2AR agonists have been studied in relation to Alzheimer disease [2], diabetes mellitus [3] and some vascular diseases [4], [5].
Currently, only a few natural and pharmaceutical boron containing products are known, since nature lacks biosynthetic pathways to form a boron–carbon bond and probably lacks metabolic enzymes able to break such a bond down. This biological condition could represent an advantage if these molecules were employed as therapeutic agents [6], [7], [8], [9]. Among the different organic boron containing compounds, boronic acids RB(OH)2 are particularly suited for drug design owing to their low toxicity, adequate chemical stability under physiological conditions, and good lipophilicity [10]. Their strong Lewis acid character allows boronic acids to readily convert from the trigonal, planar sp2 form to anionic, tetrahedral sp3 complexes, by coordination with various nucleophilic centers. Some synthetic boron containing compounds have been shown to form very tight complexes with several serine proteases, such as chymotrypsin, β-lactamases, trypsin and thrombin [6]. In addition, X-ray crystal structures show that boronic acids inhibit these enzymes by the formation of tetrahedral adducts, analogous to the deacylation tetrahedral intermediate formed by the normal catalytic activity of these enzymes [6].
On the other hand, recent experimental and theoretical studies have suggested that some serine, threonine and tyrosine, which are amino acids that expose hydroxyl groups in their lateral chains, are implicated in β2AR activation [11], [12].
Accordingly, by taking the structural characteristics of the binding site on the β2AR into account, we designed of a set of boron containing compounds that can act as ligands for this receptor.
The β2AR is a prototype for the G-protein-coupled receptor (GPCR) family, which makes it an excellent model system for studying the mechanism of activation and signalling of such receptors [12], [13]. The catecholamine binding site of β2AR is well known and has affinity for a wealth of structurally related ligands with a diverse functionality. Recent reports have demonstrated the existence of different binding sites on the β2AR for various ligands, and have provided evidence that activation of a GPCR is a multistep process [14].
Other studies have demonstrated that the β2AR can activate not only G-proteins of the Gs type, but also the Gi- and Gq-family, and that it interacts with other proteins such as β-arrestin [12], [13], [14], [15]. This evidence is not divergent from what Kobilka and co-workers reported recently when using models developed by Rasmussen et al. [16] and Cherezov et al. [17] with X-ray crystallography, and when observing the differences in signalling from β1 and β2 adrenoceptors [18]. All these information help in the development of new ligands for the β2AR.
The aim of this study was to employ docking studies to design a compound with a better fit on the β2AR than the well-known ligands. One of the boron containing compounds with the best fit was 1-(4-di-hydroxy-3,5-dioxa-4-borabicyclo[4.4.0]deca-7,9,11-trien-9-yl)-2-(tert-butylamino)ethanol, a derivative of salbutamol whose existence had only been suggested [19] which in this paper is denominated as BR-AEA (acronym of borate salt of R-Arylethylamine), was synthesized and chemically characterized in this study. Once characterized, an attempt was made to establish its pharmacodynamic effects on β2AR, first by utilizing docking studies to examine the binding site on the receptor for all the ligands proposed. The Kd values of BR-AEA and those of other well-known ligands and boron containing compounds were compared based on these theoretical studies. After that, in vitro assays were carried out and the Kd value obtained for BR-AEA was compared with reported in vitro data for salbutamol and other well-known ligands.
Section snippets
Chemistry
By placing R-salbutamol in an alkaline medium with NaOH, the electrophilic attack of the boron atom of boric acid on the electron rich oxygen atoms was increased, allowing them to bind to the hydroxyl groups located in the aromatic ring, yielding BR-AEA. This derivative was synthesized as depicted in Scheme 1.
The spectroscopic data obtained (see below) show signals which correspond with the proposed structure, and these signals are in agreement with those reported for other analogous structures
Discussion
The aim of this study was the design, synthesis and characterization (chemical and pharmacodynamic) of a compound with capacity for interacting with proposed key amino acids involved in β2AR activation. Each of these residues has a hydroxyl group exposed in its lateral chains (Scheme 2). Accordingly, we considered boron containing compounds that have greater affinity for hydroxyl groups on the β2AR, and compared them to salbutamol, regarded as a selective agonist agent on this receptor [21].
Conclusion
Both the experimental data in relation to tracheal rings from guinea pigs and docking studies evidence that BR-AEA has greater affinity than classical agonists such as salbutamol for the β2AR. The key factor that probably provides BR-AEA with its experimentally observed higher affinity for the receptor than salbutamol is the larger number of interactions in the active site of the β2AR. Some of these interactions, such as that with Thr118 in TM3 and those with some residues in TM5, in which
Experimental protocols
The protocol was revised and approved by the bioethical committee of our Institution and is in accordance with the Mexican Health Law in relation with the use of experimental animals.
Acknowledgments
The present work and the scholarships for the authors were supported partially by COFAA, SIP-IPN(20080026), and CONACYT(62488). We thank Bruce Allan Larsen for his review of the use of English in the manuscript.
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2013, Advances in PharmacologyCitation Excerpt :Built upon insights gained from these structural biology studies, in silico ligand design for GPCRs has become increasingly used and has in fact yielded many new compounds, including a series of ligands active at the adenosine A2A receptor (Pastorin et al., 2010). Moreover, based on the resolution of the structure of β2-adrenoceptor, a series of potent agonists were designed using in silico approaches (Soriano-Ursua, Valencia-Hernandez, Arellano-Mendoza, Correa-Basurto, & Trujillo-Ferrara, 2009). Homology models using resolved crystal structures have also been a powerful tool for ligand identification/design for GPCRs with as yet unsolved crystal structures.
Cell-based and in-silico studies on the high intrinsic activity of two boron-containing salbutamol derivatives at the human β <inf>2</inf>- adrenoceptor
2012, Bioorganic and Medicinal ChemistryCitation Excerpt :However, in the highest affinity complexes, boric and phenylboronic acids were only able to reach a region that is shallower than the orthosteric site. The estimated affinity correlated with our previous experimental estimation and with similar docking procedures.4,5 The coefficient of determinations in linear regression for theoretical–experimental values was in the range 0.675–0.893 for all ligands tested on the four structures employed in this study, but no greater correlation was observed for agonists or antagonists (or inverse agonists) on any particular hβ2AR structure, nor did agonists have higher affinity on putative active models or antagonists on inactive models.
β <inf>2</inf>-Adrenergic activity of 6-methoxykaempferol-3-O-glucoside on rat uterus: In vitro and in silico studies
2011, European Journal of PharmacologyThe Use of GPCR Structures in Drug Design
2011, Advances in PharmacologyCitation Excerpt :In particular, the presence of a less bulky amino acid (Val169) in the ECL2 of adenosine A3 seemed to have a key influence over molecules containing larger substituents on the amino group of the heterocyclic core, modulating potency, and selectivity for the A2A receptor versus A3. Soriano-Ursua and colleagues have rationally designed boron-containing analogues of β2AR agonists using molecular docking against the β2 receptor binding site (Soriano-Ursua et al., 2009). The new ligands, inspired by the crystal structures, were shown to be more potent than the corresponding diol agonists from which they were derived in a functional assay (relaxation of isolated GP tracheal rings) and were competitively antagonized by β2AR antagonists.