Synthesis and β-adrenoceptor agonist properties of (±)-1-(3',4'-dihydroxyphenoxy)-3-(3",4"-dimethoxyphenyl) ethylamino-2-propanol hydrochloride, (±)-RO363.HCl, and the (2S)-(-)-isomer

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Abstract

The synthesis of (±)-1-(3',4'-dihydroxyphenoxy)-3-(3",4"-dimethoxyphenyl)ethylamino-2-propanol hydrochloride, (±)-RO363.HCl, and the (2S)-(-)-isomer is described for the first time. The binding affinities for (±)-RO363.HCl, (2S)-(-)-RO363.HCl and a number of well known β-adrenoceptor agonists for transfected human β1-, β2- and β3-adrenoceptors expressed in Chinese hamster ovary cells have been determined and compared with the functional potencies in rat atria (β1) and trachea (β2). The results indicate that both (±)-RO363 and (2S)-(-)-RO363 are selective for the human and rat β1-adrenoceptors. The (2S)-(-)-isomer of RO363, as expected, has a higher binding affinity for the human and functional potency for rat β-adrenoceptor subtypes than the racemate. However, in contrast to the catecholamines and formoterol, the functional potency of the racemic mixture and its (-)-enantiomer are not significantly different from their binding affinity, suggesting that they are examples of partial agonists with sufficient intrinsic activity to produce full agonist responses.

Introduction

The first pharmacological reports on (±)-1-(3',4'-dihydroxyphenoxy)-3-(3",4"-dimethoxyphenyl)ethylamino-2-propanol ((±)-RO363,  figure 1 ), a synthetic catecholamine, appeared in 1978 [1] and it has been described as a potent, selective β1-adrenoceptor agonist in the guinea-pig, rat, rabbit and cat [1], [2], [3]. More recently, work carried out by Molenaar et al. [4], [5] suggests that (2S)-(-)-RO363 is a potent partial agonist for both human β1- and β3-adrenoceptors. In addition, they report that (2S)-(-)-RO363 is able to activate a novel human atrial β-adrenoceptor, which they have designated as a putative β4-adrenoceptor [4], [5].

The original synthesis of (±)-RO363, and the subsequent resolution of the (2S)-(-)- and (2R)-(+)-enantiomers, was carried out by D. Iakovidis as part of his doctoral studies [6] and has never been formally reported. In light of the renewed interest in RO363 as a research tool, we describe here the synthesis of (±)-RO363 (7,  figure 2) using a shorter (5 steps) and simpler procedure than the original 9 step process [6]. We also report the chiral synthesis of (2S)-(-)-RO363 (11,  figure 3). Both (±)-RO363 7 and (2S)-(-)-RO363 11 have been prepared as the hydrochloride salts.

The binding affinity of (±)-RO363 7 and (2S)-(-)-RO363 11 for human β1-, β2- and β3-adrenoceptors transfected and expressed in Chinese hamster ovary (CHO) cells and their functional potency in rat atria (β1) and trachea (β2) have been determined and compared to other well known β-adrenoceptor agonists. In addition, we have examined the functional potency of (-)-isoprenaline and (2S)-(-)-RO363 in guinea-pig atria and trachea.

Section snippets

Chemistry

The synthetic routes used to prepare (±)-RO363.HCl 7 and (2S)-(-)-RO363.HCl 11 are outlined in  figures 2 and 3. Commercially available 3,4-dibenzyloxybenzaldehyde 1 was oxidized with m-chloroperbenzoic acid (MCBA) [7] to produce the corresponding formate 2, which after mild hydrolysis produced the desired phenol 3.

The phenol 3 reacted with epichlorohydrin under alkaline conditions to produce the epoxide 4 (figure 2). The ring opening addition reaction of the epoxide 4 with the amine 5,

Pharmacology

The binding affinities for transfected human β1-, β2- and β3-adrenoceptors expressed in CHO cells were determined for the endogenous catecholamine (-)-noradrenaline, the non-selective agonist (-)-isoprenaline, the β1-selective agonist (±)-RO363 7 and the isomer (2S)-(-)-RO363 11, the β2-selective agonists (±)-salbutamol and (±)-formoterol and the β3-selective agonist (±)-BRL37344 (the structures of all agonists are given in  figure 1). The functional potencies of all seven agonists were also

Binding at human β-adrenoceptors

The dissociation constants (Kd) and maximal density of binding sites (Bmax) of (-)-[125I]-iodocyanopindolol (ICYP) for the transfected human β1-, β2- and β3-adrenoceptor subtypes were determined by saturation binding experiments. For human β1-adrenoceptors the Kd was 4.99 ± 0.48 pM and the Bmax was 7 127 ± fmol mg protein–1; for the β2-adrenoceptor the Kd was 8.00 ± 1.10 pM and the Bmax was 3 914 ± 583 fmol mg protein–1; and for the β3-adrenoceptor the Kd was 313 ± 93.8 pM and the Bmax was

Conclusion

(2S)-(-)-RO363 11 is unusual in that, in spite of its phenoxypropanolamine structure, it possesses high intrinsic efficacy with approximately 66-fold greater affinity for transfected human β1-adrenoceptors than (-)-isoprenaline and approximately 2-fold greater potency at stimulating chronotropic responses in rat atria (β1-mediated) than (-)-isoprenaline. Interestingly the phenoxypropanolamine agonists ((±)-RO363 7 and its (2S)-(-)-enantiomer 11), in contrast to (-)-isoprenaline,

General

Melting points were determined using a manual Gallenkamp electrothermal apparatus (range 0–400 °C) in glass capillary tubes and are uncorrected. IR spectra were recorded on a Perkin-Elmer FT/IR 1600 spectrometer. 1H-NMR spectra were recorded on a Varian EM 360 spectrometer. Chemical ionisation (C.I., methane gas) mass spectra were recorded on either a Finnigan GCQ™ or a Finnigan 4000 series GC/MS mass spectrometer. All spectra were consistent with the assigned structures. Optical rotation

Acknowledgements

The authors would like to thank Professor A. Donny Strosberg (Institut Cochin de Genetique Moleculaire, Paris, France) for providing us with the three CHO cell lines transfected with the human β1-, β2- and β3-adrenoceptors and Ms Leanne Styan for her technical assistance. This work was supported by a grant from the National Health and Medical Research Council of Australia.

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