Design, synthesis and biological evaluation of 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one derivatives as potent ß2-adrenoceptor agonists.

A series of ß2-adrenoceptor agonists with an 8-(2-amino-1-hydroxyethyl)-6-hydroxy-1,4-benzoxazine-3(4H)-one moiety is presented. The stimulatory effects of the compounds on human ß2-adrenoceptor and ß1-adrenoceptor were characterized by a cell-based assay. Their smooth muscle relaxant activities were tested on isolated guinea pig trachea. Most of the compounds were found to be potent and selective agonists of the ß2-adrenoceptor. One of the compounds, (R)-18c, possessed a strong ß2-adrenoceptor agonistic effect with an EC50 value of 24 pM. It produced a full and potent airway smooth muscle relaxant effect same as olodaterol. Its onset of action was 3.5 min and its duration of action was more than 12 h in an in vitro guinea pig trachea model of bronchodilation. These results suggest that (R)-18c is a potential candidate for long-acting ß2-AR agonists.

The synthesis of isotopologues of AZD7307: A selective ß2 -adrenoreceptor agonist.

A medicinal chemistry program to develop potent and selective LABA compounds required the synthesis of both carbon-14 and stable-isotope labelled materials.  Carbon-14 labelled AZD7307 was successfully synthesised in 6 steps from [14C]chloroacetyl chloride in an overall radiochemical yield of 10%. In addition, the synthetic route of a stable labelled isotopomer of AZD7307 is also described and synthesised in four linear steps from [13C6]cyclohexylamine hydrochloride in an overall yield of 12%.

Discovery of ß-arrestin-biased ß2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

ß-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). ß-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the ß-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of ß2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their ß-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or the cardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter ß-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and ß-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of ß-arrestin-biased ß2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

Antilipolytic effects of 1,8-naphthyridine derivatives ß-adrenoceptor antagonists in rat white adipocytes.

The rat fat cell ß-adrenoceptors were investigated by studying the effects of new 1,8-naphthyridine derivatives synthesized starting from 7-amino-2-chloro-3-phenyl-1,8-naphthyridine on lipolysis induced by isoprenaline, and alprenolol. Lipolysis induced by isoprenaline agonist was competitively antagonized by the 1,8-naphthyridine analogue with a 7-hydroxy-2-(4'-methoxybenzylamine)-6-nitro-3-phenyl substituent designated as 3. In contrast, 10, 50, and 100 µm of 7-methoxy and 7-ethoxy derivatives did not modify the concentration-response curve of isoprenaline. A rightward shift of the curve was, however, observed with 50 µm of a 7-methoxy-2-(4'-methoxybenzylamine)-6-amino-3-phenyl substituent designated as 10. The selective ß1 -AR antagonist, 7-hydroxy-4-morpholinomethyl-2-piperazino-1,8-naphthyridine slightly reduced isoprenaline-induced lipolysis only at high doses. Alprenolol-mediated lipolytic effect was antagonized by derivative 3, 10 and the selective ß3 -AR antagonist SR 59,230A, but resistant to the selective ß1 -AR antagonist 7-hydroxy-4-morpholinomethyl-2-piperazino-1,8-naphthyridine. The results provide preliminary pharmacological evidence for the antilipolytic effect of the newly synthesized 1,8-naphthyridine derivatives on rat fat cells. The analogues designated as 3 and 10 were the most potent antagonists of this series.

Efficient synthesis of D6 -clenproperol and D6 -cimaterol using deuterium isopropylamine as labelled precursor.

This report presents an efficient synthesis of D6 -clenproperol and D6 -cimaterol with 99.5% and 99.7% isotopic abundance in acceptable yields and excellent chemical purities with deuterium isopropylamine as labelled precursor. Their structures and the isotope-abundance were confirmed by proton nuclear magnetic resonance and liquid chromatography-mass spectrometry.

From libraries to candidate: the discovery of new ultra long-acting dibasic ß2-adrenoceptor agonists.

Libraries of dibasic compounds designed around the molecular scaffold of the DA(2)/ß(2) dual agonist sibenadet (Viozan™) have yielded a number of promising starting points that have been further optimised into novel potent and selective target molecules with required pharmacokinetic properties. From a shortlist, 31 was discovered as a novel, high potency, and highly efficacious ß(2)-agonist with high selectivity and a duration of action commensurable with once daily dosing.

Synthesis and characterization of high-affinity 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-labeled fluorescent ligands for human ß-adrenoceptors.

The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human ß-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity ß-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log K(D) of -9.53 and -8.46 as an antagonist of functional ß2- and ß1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human ß2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent ß2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) ( J. Cardiovasc. Pharmacol.1983, 5, 430-437. ).

Inhalation by design: novel ultra-long-acting ß(2)-adrenoreceptor agonists for inhaled once-daily treatment of asthma and chronic obstructive pulmonary disease that utilize a sulfonamide agonist headgroup.

A novel series of potent and selective sulfonamide derived ß(2)-adrenoreceptor agonists are described that exhibit potential as inhaled ultra-long-acting bronchodilators for the treatment of asthma and chronic obstructive pulmonary disease. Analogues from this series mediate very long-lasting smooth muscle relaxation in guinea pig tracheal strips. The sulfonamide agonist headgroup confers high levels of intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in the solid state. Optimization of pharmacokinetic properties was achieved through targeted introduction of a phenolic moiety to support rapid phase II clearance, thereby minimizing systemic exposure following inhalation and reducing systemically mediated adverse events. Compound 38 (PF-610355) is identified as a clinical candidate from this series, with in vivo duration of action studies confirming its potential for once-daily use in humans. Compound 38 is currently in advanced phase II clinical studies.

Synthesis and structure-activity relationships of long-acting beta2 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.

Effects of beta3-adrenergic receptor activation on rat urinary bladder hyperactivity induced by ovariectomy.

Voiding dysfunctions, including increased voiding frequency, urgency, or incontinence, are prevalent in the postmenopausal population. Beta(3)-adrenergic receptor (beta(3)AR) agonists, which relax bladder smooth muscle, are being developed to treat these conditions. We utilized the rat ovariectomy (OVX) model to investigate the effect of ovarian hormone depletion on bladder function and the potential for beta(3)AR agonists to treat bladder hyperactivity in this setting. OVX increased voiding frequency and decreased bladder capacity by approximately 25% in awake rats and induced irregular cystometrograms in urethane-anesthetized rats. Reverse transcription-polymerase chain reaction revealed three betaARs subtypes (beta(1,2,3)) in bladder tissue, and immunostaining indicated beta(3)AR localization in urothelium and detrusor. Receptor expression was not different in OVX and SHAM rats. The beta(3)AR agonist selectivity of BRL37344 [(+/-)-(R(*),R(*))-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium hydrate], TAK-677 [(3-((2R)-(((2R)-(3-chlorophenyl)-2-hydroxyethyl)amino)propyl)-1H-indol-7-yloxy)acetic acid], and FK175 [acetic acid, 2-[[(8S)-8-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]-6,7,8,9-tetrahydro-5H-benzocyclohepten-2-yl]oxy], ethyl ester, hydrochloride] was confirmed by examining the relative potency for elevation of cAMP in CHOK1 cells overexpressing the various rat betaARs. Intravenous injection of each of the beta(3)AR agonists (0.1-500 microg/kg) in anesthetized rats decreased voiding frequency, bladder pressure, and amplitude of bladder contractions. In bladder strips, beta(3)AR agonists (10(-12)-10(-4) M) decreased baseline tone and reduced spontaneous contractions. BRL37344 (5 mg/kg) and TAK-677 (5 mg/kg) injected intraperitoneally in awake rats decreased voiding frequency by 40 to 70%. These effects were not altered by OVX. The results indicate that OVX-induced bladder dysfunction, including decreased bladder capacity and increased voiding frequency, is not associated with changes in beta(3)AR expression or the bladder inhibitory effects of beta(3)AR agonists. This suggests that beta(3)AR agonists should prove effective for the treatment of overactive bladder symptoms in the postmenopausal population.

Synthesis and pharmacological characterization of beta2-adrenergic agonist enantiomers: zilpaterol.

The beta-adrenergic agonist 1 (zilpaterol) is used as production enhancer in cattle. Binding experiments of separated enantiomers on recombinant human beta(2)-adrenergic and mu-opioid receptors and functional studies showed that the (-)-1 enantiomer accounts for essentially all the beta(2)-adrenergic agonist activity and that it exhibits less affinity toward the mu-opioid receptor than (+)-1, which is a mu-opioid receptor antagonist. X-ray crystallography revealed the absolute configuration of (-)-1 to be 6R,7R.

Highly enantioselective synthesis, crystal structure, and circular dichroism spectroscopy of (R)-bambuterol hydrochloride.

The present article describes the asymmetric synthesis of (R)-bambuterol hydrochloride based on 1-(3,5-dihydroxyphenyl)ethanone as starting material, which was esterified by dimethylcarbamic chloride, and brominated by copper (II) bromide. Then the carbonyl group was reduced efficiently using (-)-B-chlorodiisopinocamphenylborane [(-)-DIP-chloridetrade mark] as an asymmetrical reducing agent. Followed by epoxide ring closure with NaOH and ring expansion with tert-butylamine led to the desired product (R)-bambuterol with e.e. up to 99%. The optical properties and absolute configuration of (R)-bambuterol hydrochloride were further investigated using circular dichroism spectroscopy and X-ray single crystal analysis.

Discovery of a novel series of biphenyl benzoic acid derivatives as potent and selective human beta3-adrenergic receptor agonists with good oral bioavailability. Part I.

A novel class of biphenyl analogues containing a benzoic acid moiety based on lead compound 8i have been identified as potent and selective human beta 3 adrenergic receptor (beta 3-AR) agonists with good oral bioavailability and long plasma half-life. After further substituent effects were investigated at the terminal phenyl ring of lead compound 8i, we have discovered that more lipophilic substitution at the R position improved potency and selectivity. As a result of these studies, 10a and 10e were identified as the leading candidates with the best balance of potency, selectivity, and pharmacokinetic profiles. In addition, compounds 10a and 10e were evaluated to be efficacious for a carbachol-induced increase of intravesical pressure, such as an overactive bladder model in anesthetized dogs. This represents the first demonstrated result dealing with beta 3-AR agonists.

Potent oxindole based human beta3 adrenergic receptor agonists.

The synthesis and biological evaluation of a series of oxindole beta(3) adrenergic receptor agonists is described. A modulation of rat atrial tachycardia was observed with substitution at the 3-position of the oxindole moiety.

Asymmetric hydrogenation of alpha-primary and secondary amino ketones: efficient asymmetric syntheses of (-)-arbutamine and (-)-denopamine.

Two beta-receptor agonists (-)-denopamine and (-)-arbutamine were prepared in good yields and enantioselectivities by asymmetric hydrogenation of unprotected amino ketones for the first time by using Rh catalysts bearing electron-donating phosphine ligands. A series of alpha-primary and secondary amino ketones were synthesized and hydrogenated to produce various 1,2-amino alcohols in good yields and with good enantioselectivies. This Rh electron-donating phosphine-catalyzed asymmetric hyderogenation represents one of the most promising and convenient approaches towards the asymmetric synthesis of chiral amino alcohols.

Synthesis of the marine sponge derived beta2-adrenoceptor agonist S1319.

[reaction: see text] The marine sponge derived beta2-adrenoceptor agonist S1319 has been synthesized following a six-step linear sequence. Central to the approach employed is the formation of a 7-lithiated-2,4-dialkoxybenzothiazole intermediate obtained via a directed-lithiation/benzyne-mediated cyclization reaction. The incorporation of a tert-butyl ether residue into the cyclization precursor for the pivotal ring-closing step has been shown to significantly increase the efficiency of the reaction by the suppression of a competing directed ortho-lithiation reaction.

Synthesis of 1,2-benzisothiazolyloxypropanolamine derivatives and investigation of their activity at beta-adrenoceptors.

The synthesis of 3-methoxy-1,2-benzisothiazole derivatives, substituted in position 5- (compounds 1-7) or 7- (compounds 8-14), with oxypropanolaminic side chains and the pharmacological investigation on their activity at beta-adrenoceptors are described. Compounds were prepared in an attempt to explore the ability of the benzisothiazole ring to interact with the beta-adrenoceptor site and to establish whether oxypropanolaminic derivatives recognise the beta3-adrenoceptor subtype. All the products were tested on rat atria, bladder and small intestine, which preferentially (but not exclusively) express beta1-, beta2- and beta3-adrenoceptors, respectively. When compared with the reference, non-specific, beta-adrenoceptor agonist isoprenaline, the products tested did not show any consistent beta-adrenoceptor agonistic activity in the different models. Most compounds relaxed smooth muscle preparations, but such effect was resistant to the blockade by propranolol (1 micromol/l), ICI 118,551 (1 micromol/l) or bupranolol (1-10 micromol/l), thus excluding that the spasmolytic effect involves any beta-adrenoceptors. When tested as antagonists, some of these products showed a concentration-dependent attenuation of the isoprenaline-induced effects in rat atria, without affecting beta-adrenoceptor-mediated relaxation in smooth muscle. These data confirm the ability of the benzisothiazole ring to interact with beta-adrenoceptors, but the substitution in 5- or 7-positions with oxypropanolaminic groups does not generate compounds endowed with specific activity at beta3-adrenoceptors. Conversely, most of these compounds behave as (specific) antagonists at beta1- (cardiac) adrenoceptors. At the maximum concentrations tested (1-100 micromol/l), these compounds also exert direct spasmolytic and negative chronotropic effects, which could be related to a blockade of Ca2+-dependent mechanisms at an intracellular level and/or an anaesthetic-like activity at plasma membranes.

QSAR analysis of thiazole benzenesulfonamide substituted 3-pyridylethanolamines as beta3-adrenergic receptor agonist.

A quantitative structure-activity relationship study on a series of substituted benzene sulfonamide-3-pyridylethanolamines with beta3-adrenergic receptor agonist activity was made using a combination of various physiochemical descriptors. Several significant equations with good co-efficients of correlation (0.930) were obtained; the two models were selected using predictive ability of equations for test set. Both models highlight some common important structural features, that is, high electrostatic potential energy and the lipophilic nature of the molecule, favorable for beta3-adrenergic receptor agonist activity.

Discovery of a novel and potent human and rat beta3-adrenergic receptor agonist, [3-[(2R)-[[(2R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1H-indol-7-yloxy]acetic acid.

In search for potent and selective beta3-adrenergic receptor (beta3-AR) agonists as potential drugs for the treatment of type II diabetes and obesity, a novel series of 1-(3-chlorophenyl)-2-aminoethanol derivatives were prepared and evaluated for their biological activity at human beta1-, beta2-, and beta3-ARs and rat beta3-AR expressed in Chinese hamster ovary (CHO) cells. Replacement of the right-hand side (RHS, benzene ring) in the 'first generation' beta3-AR agonists BRL 37344 and CL 316243 with a 1H-indole ring gave compound 31 with unique pharmacological properties among beta3-AR agonists. Initial in vitro assays showed that 31 possesses modest rat and human beta3-ARs agonistic activity. Introduction of various substituent into the indole nucleus of 31 afforded a number of compounds with good beta3-ARs agonistic activity. In particular, 90 having a carboxylic acid functionality at the 7-position of the indole nucleus showed the most potent human beta3-AR agonistic activity. Finally, optical resolution of 90 led to the identification of the most promising compound, [3-[(2R)-[[(2R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-1H-indol-7-yloxy]acetic acid (96, AJ-9677). This compound exhibited potent human beta3-AR agonistic activity (EC50=0.062 nM, IA=116%) with 210- and 103-fold selectivity over human beta2-AR and beta1-AR, respectively. Compound 96 also exhibited potent rat beta3-AR agonistic activity (EC50=0.016 nM, IA=110%). Moreover, repeated oral administration of 96 inhibited body weight gain and significantly decreased glucose, insulin, free fatty acid, and triglyceride concentrations in plasma in KK-Ay/Ta mice. On the basis of this pharmacological profile, 96 entered clinical development as a drug for the treatment of type II diabetes and obesity.