The discovery of long-acting saligenin β2 adrenergic receptor agonists incorporating hydantoin or uracil rings

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Abstract

A series of novel, potent and selective human β2 adrenoceptor agonists incorporating a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long duration of action in vitro on guinea pig trachea, and 12 h in guinea pigs in vivo at its EC90 25 μM. It had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in both rats and dogs (2% and 5%, respectively). An improved method for measuring the absorbed fraction of analogues dosed to rats, which considers the glucuronidated fraction is presented. Compound 12a was metabolised in human liver microsomes and hepatocytes to the active hydantoic acid 12m.

Introduction

Asthma is a chronic disease affecting 300 million people worldwide, characterised by an increase in inflammatory cell populations in the epithelium and submucosa of the airways.1 There are two main components of asthma pathophysiology, airway inflammation and smooth muscle dysfunction, which are treated by two major categories of medicines: anti-inflammatory drugs and bronchodilators. Inhaled corticosteroids are used to treat the inflammatory component of asthma. Inhaled β2-agonists are the most effective bronchodilators, offering proven benefits in reducing the burden of this disease.2, 3 Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the US, affecting 5% of its population. Bronchodilators such as inhaled β2-agonists and muscarinic antagonists are currently the mainstay of treatment for COPD and combinations with inhaled corticosteroids are known to reduce the incidence of exacerbations. There are two classes of β2-agonists: the short-acting (first generation) agonists, such as salbutamol (1) (Chart 1), which are used in rescue therapy, and the long-acting (second generation) agonists which are used in maintenance therapy. The short-acting agonists have a rapid onset of action and relieve symptoms for 3–6 h. The two currently prescribed inhaled long-acting β2-agonists are salmeterol (2) and formoterol (3). Salmeterol has lower intrinsic activity than salbutamol, a delayed onset of action, and 12 h duration, which is independent of dose.4, 5 In contrast, formoterol has high intrinsic efficacy, onset time similar to salbutamol, and a dose-dependent duration of action.5, 6 In the last 10 years there has been great interest within the pharmaceutical industry in the discovery of a third generation, once daily β2 adrenoceptor agonist to be used in new combination therapies for the treatment of asthma and COPD. Novartis have recently published data on their clinical candidate, indacaterol (4),7 which has now been approved for the treatment of COPD in Europe. A review by Glossop and Price summarises the progress up to 2006 made by pharmaceutical companies in identifying inhaled β2-agonists with extended duration of action.8 The Pfizer group have published three papers9, 10, 11 describing their earlier candidates, and more recently on their clinical candidate PF-610355 (5).12 Our group has published two papers one on sulfonamides, including our first candidate 6,13 and the second on antedrugs, including the clinical candidate vilanterol (7).14 Finally the Boehringer-Ingelheim group have published three papers including their clinical candidate olodaterol (8).15, 16, 17

A major fraction of the dose (80–90%) of an inhaled drug is swallowed and liable to be absorbed from the gastro-intestinal tract.18 Thus, one approach to improve the therapeutic index could be to alter the physicochemical properties of the drug and make it less prone to absorption.13 A second approach (the antedrug approach) could be to introduce metabolic instability to the molecule to facilitate its conversion to inactive metabolites following systemic absorption from either the GI tract or the lung.14 Better still, a combination of the two approaches may potentially deal with both the inhaled and swallowed fractions of each dose. In this report we present our studies in identifying alternative, non-sulfonamide, β2 adrenoceptor agonists with reduced oral absorption. Some of the physicochemical parameters that influence oral absorption are molecular weight, lipophilicity, membrane permeability, the number of hydrogen-bond donors and acceptors, conformational flexibility and solubility.19 Substitution on the right-hand side phenyl ring of salmeterol with the polar sulfonamide group was found to enhance β2 agonist activity.13 It was hypothesised that an alternative polar substituent, such as a heterocycle, with increased number of hydrogen-bond donors and acceptors which contravened the Lipinski rules,20 might be expected to show reduced oral bioavailability. Hydantoin and uracil rings, which possess two hydrogen-bond acceptor groups and two hydrogen-bond donors, were considered as good starting points for investigation. We have demonstrated that introduction of the polar sulfonamide group brings about longer duration of action.13 It was therefore hypothesised that introduction of the hydantoin or uracil ring might also bind in a similar way to the sulfonamide group of 6 and hence have similarly long duration of action.

Section snippets

Chemistry

The aminoalcohol 9 with the R configuration was prepared by reduction of the ketone 1021 with borane-dimethylsulfide and (R)-2-methyl-CBS-oxazaborolidine as catalyst, followed by catalytic hydrogenation of the resulting azido alcohol 11 (Scheme 1). The enantiomeric ratio of 11 was determined by 1H NMR spectroscopy using Pirkle’s alcohol22 at 750 MHz and found to be 9:1.

The β2-adrenoceptor agonists 12 were prepared using the synthetic route outlined in Scheme 2, which involved Sonogashira

Results and discussion

Compounds in Table 1 were tested for their ability to cause cyclic AMP accumulation in Chinese hamster ovary (CHO) cells transfected with human β1, β2 or β3 adrenoceptors. Agonist activity was assessed by measuring changes in intracellular cyclic AMP, and the potency is reported as pEC50 values (negative log10 molar concentration for half maximal response ± SEM). The efficacy of the test compounds was expressed as intrinsic activity (IA), which is defined as the maximal response of the test

Conclusion

Incorporation of a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol has provided a series of novel, very potent and selective human β2 adrenoceptor agonists. Hydantoin 12a had long duration of action in vitro on guinea pig trachea, and 12 h in guinea pigs in vivo at its EC90 25 μM. It had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in both rats and dogs (2% and 5%, respectively). However 12a was metabolised in

Experimental

Organic solutions were dried over anhydrous MgSO4. TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates. Products were visualised under UV light and/or by staining with aqueous KMnO4 solution. LCMS analysis was conducted on a Supelcosil LCABZ+PLUS column (3.3 cm × 4.6 mm) eluting with 0.1% formic acid and 0.01 M ammonium acetate in water (solvent A), and 0.05% formic acid and 5% water in acetonitrile (solvent B), using the following elution gradient 0 0.7 min 0% B, 0.7 4.2 min 100% B, 4.2–5.3 min

Acknowledgments

We thank Mr. Bill J. Leavens for collecting the HRMS data, Mr. Inder Bhamra for scaling up the preparation of 10, Miss Isobel Hackney and Miss Sara C. Hughes for the in vivo work on 12a.

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