Alison J. Ford

Synthesis and Structure−Activity Relationships of Long-acting β2 Adrenergic Receptor Agonists Incorporating Metabolic Inactivation: An Antedrug Approach

A series of saligenin beta(2) adrenoceptor agonist antedrugs having high clearance were prepared by reacting a protected saligenin oxazolidinone with protected hydroxyethoxyalkoxyalkyl bromides, followed by removal of the hydroxy-protecting group, alkylation, and final deprotection. The compounds were screened for beta(2), beta(1), and beta(3) agonist activity in CHO cells. The onset and duration of action in vitro of selected compounds were assessed on isolated superfused guinea pig trachea. Compound 13f had high potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f (vilanterol) were identified that had suitable properties for inhaled administration. A proposed binding mode for 13f to the beta(2)-receptor is presented.

In vitro pharmacological characterization of vilanterol, a novel long-acting β2-adrenoceptor agonist with 24-hour duration of action.

Vilanterol trifenatate (vilanterol) is a novel, long-acting β2-adrenoceptor (β2-AR) agonist with 24 h activity. In this study, we describe the preclinical pharmacological profile of vilanterol using radioligand binding and cAMP studies in recombinant assays as well as human and guinea pig tissue systems to characterize β2-AR binding and functional properties. Vilanterol displayed a subnanomolar affinity for the β2-AR that was comparable with that of salmeterol but higher than olodaterol, formoterol, and indacaterol. In cAMP functional activity studies, vilanterol demonstrated similar selectivity as salmeterol for β2- over β1-AR and β3-AR, but a significantly improved selectivity profile than formoterol and indacaterol. Vilanterol also showed a level of intrinsic efficacy that was comparable to indacaterol but significantly greater than that of salmeterol. In cellular cAMP production and tissue-based studies measuring persistence and reassertion, vilanterol had a persistence of action comparable with indacaterol and longer than formoterol. In addition, vilanterol demonstrated reassertion activity in both cell and tissue systems that was comparable with salmeterol and indacaterol but longer than formoterol. In human airways, vilanterol was shown to have a faster onset and longer duration of action than salmeterol, exhibiting a significant level of bronchodilation 22 h after treatment. From these investigations, the data for vilanterol are consistent, showing that it is a novel, potent, and selective β2-AR receptor agonist with a long duration of action. This pharmacological profile combined with clinical data is consistent with once a day dosing of vilanterol in the treatment of both asthma and chronic obstructive pulmonary disease (COPD).

Synthesis and Structure-Activity Relationships of Long-acting β2 Adrenergic Receptor Agonists Incorporating Arylsulfonamide Groups

A series of saligenin alkoxyalkylphenylsulfonamide beta(2) adrenoceptor agonists were prepared by reacting a protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction, hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the para- and the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues. The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding mode for 29b to the beta(2)-receptor is presented.

Internalization of the chemokine receptor CCR4 can be evoked by orthosteric and allosteric receptor antagonists

The chemokine receptor CCR4 has at least two natural agonist ligands, MDC (CCL22) and TARC (CCL17) which bind to the same orthosteric site with a similar affinity. Both ligands are known to evoke chemotaxis of CCR4-bearing T cells and also elicit CCR4 receptor internalization. A series of small molecule allosteric antagonists have been described which displace the agonist ligand, and inhibit chemotaxis. The aim of this study was to determine which cellular coupling pathways are involved in internalization, and if antagonists binding to the CCR4 receptor could themselves evoke receptor internalization. CCL22 binding coupled CCR4 efficiently to β-arrestin and stimulated GTPγS binding however CCL17 did not couple to β-arrestin and only partially stimulated GTPγS binding. CCL22 potently induced internalization of almost all cell surface CCR4, while CCL17 showed only weak effects. We describe four small molecule antagonists that were demonstrated to bind to two distinct allosteric sites on the CCR4 receptor, and while both classes inhibited agonist ligand binding and chemotaxis, one of the allosteric sites also evoked receptor internalization. Furthermore, we also characterize an N-terminally truncated version of CCL22 which acts as a competitive antagonist at the orthosteric site, and surprisingly also evokes receptor internalization without demonstrating any agonist activity. Collectively this study demonstrates that orthosteric and allosteric antagonists of the CCR4 receptor are capable of evoking receptor internalization, providing a novel strategy for drug discovery against this class of target.

Synthesis and Structure–Activity Relationships of Indazole Arylsulfonamides as Allosteric CC-Chemokine Receptor 4 (CCR4) Antagonists

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

Discovery of a Rapidly Metabolized, Long-Acting β2 Adrenergic Receptor Agonist with a Short Onset Time Incorporating a Sulfone Group Suitable for Once-Daily Dosing

A series of novel, potent, and selective human β2 adrenoceptor agonists incorporating a sulfone moiety on the terminal right-hand-side phenyl ring of (R)-salmeterol is presented. Sulfone 10b had salmeterol-like potency and selectivity profile, long duration of action on guinea pig trachea, and longer than salmeterol duration of action in vivo, suitable for once-daily dosing. It had lower than salmeterol oral absorption in rat, lower bioavailability in rat and dog, and a high turnover in human hepatocytes. It was metabolized in human hepatocytes by hydroxylation, oxidation, cleavage, and conjugation; most of the metabolites would be expected to have reduced or no β2 activity. The 4-biphenylsulfonic acid was identified as a crystalline, non-hygroscopic salt of 10b, suitable for inhaled delivery. Furthermore, it was free of any genetic toxicity issues and was considered as a backup to vilanterol.

Lead optimisation of the N1 substituent of a novel series of indazole arylsulfonamides as CCR4 antagonists and identification of a candidate for clinical investigation.

Synthesis and preliminary SAR of the N1 substituent of a novel series of indazole sulfonamide chemokine receptor 4 (CCR4) antagonist is reported. Compound 7r was identified for further development.

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

A series of novel, potent and selective human β(2) 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 12h in guinea pigs in vivo at its EC(90) 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.

The discovery of long-acting saligenin β2 adrenergic receptor agonists incorporating a urea group

A series of novel, potent and selective human β(2) adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.

Lead identification of benzimidazolone and azabenzimidazolone arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists

A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. Hits were required to be more potent than an existing indazole series, have better physicochemical properties (clogP <3.5, chrom logD₇.₄ <5.3 and CLND solubility >116 μg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however, these analogues were inactive in the whole blood assay (pA₂ <5). Azabenzimidazolone analogues were all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight (389) and chrom logD₇.₄ (2.4), high LE (0.43), and solubility (152 μg/mL). In addition, 38 had human serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.