HK1194362A - Preparation of (r,r)-fenoterol and (r,r)- or (r,s)-fenoterol analogues and use in treating congestive heart failure - Google Patents

Description

Preparation of (R, R) -fenoterol and (R, R) -or (R, S) -fenoterol analogues and their use in the treatment of congestive heart failure

The present application is a divisional application of international application PCT/US2007/075731, filed in china on 27.3.2009, with application No. 200780036155.9, entitled preparation of (R, R) -fenoterol and (R, R) -or (R, S) -fenoterol analogues and their use in the treatment of congestive heart failure.

Technical Field

The present disclosure relates to the field of pharmaceutical compositions, and in particular to the preparation of (R, R) -fenoterol and (R, R) -or (R, S) -fenoterol analogues and their use in the treatment of congestive heart failure.

Background

Fenoterol, 5- [ 1-hydroxy-2 [ [2- (4-hydroxyphenyl) -1-methylethyl ] -amino ] ethyl ] -1, 2-benzenediol, is a β 2-adrenergic receptor agonist, which is traditionally used to treat pulmonary disorders such as asthma. The drug has two chiral (asymmetric) carbons, each of which can be independently arranged in either the R or S configuration, such that the drug exists in different known stereoisomeric forms (R, R), (R, S), (S, R), or (S, S). The commercially available form of fenoterol is a racemic mixture of the (R, R) -and (S, S) -compounds.

Fenoterol is known to bind to and activate β 2-adrenergic receptors as an agonist. This activity has led to its clinical use in the treatment of asthma, as the activity of this agonist is to dilate the constricted airways. Additional therapeutic applications of fenoterol remain to be thoroughly explored. Pharmacological studies of this class of drugs, including fenoterol, have shown that only one of the enantiomers is responsible for producing bronchorelaxation effects. For example, studies have shown that the major bronchorelaxing activity of racemic (±) -fenoterol is in the (R, R) -isomer of fenoterol. In addition, it has recently become apparent that inactive enantiomers may be associated with side effects. For example, the diastereomer (S, S) -fenoterol has been shown to cause adverse side effects or development of tolerance associated with β 2-adrenergic receptor agonist therapy

It would therefore be advantageous to provide fenoterol compositions that are effective in treating conditions such as asthma, chronic obstructive pulmonary disease, or congestive heart failure, but have reduced side effects such as hypersensitivity and drug resistance (tolerance).

Disclosure of Invention

The present disclosure relates to the discovery of fenoterol analogues that are highly potent in binding to the β 2-adrenergic receptor. Exemplary chemical structures of these analogs are provided throughout the present disclosure. For example, such compounds are represented by the following general formula:

wherein R is₁-R₃Independently hydrogen, acyl, alkoxycarbonyl, aminocarbonyl (carbamoyl), or a combination thereof;

R₄is hydrogen or lower alkyl;

R₅is a lower alkyl group, and is,

or

X, Y therein¹、Y²And Y³Independently hydrogen, -OR₆and-NR₇R₈；

R₆Independently hydrogen, lower alkyl, acyl, alkoxycarbonyl or aminocarbonyl; and

R₇and R₈Independently hydrogen, lower alkyl, alkoxycarbonyl, acyl or aminocarbonyl.

Also provided are pharmaceutical compositions comprising the (R, R) -fenoterol and fenoterol analogs and methods of using the (R, R) -fenoterol and fenoterol analogs. For example, the disclosed (R, R) -fenoterol and (R, R) -or (R, S) -fenoterol analogs (e.g., (R, R) -methoxyfenoterol, (R, R) -naphthylfenoterol and (R, S) -naphthylfenoterol) are effective in treating a disorder of the heart or a pulmonary disorder.

The above and other features and advantages will become apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

Drawings

FIG. 1 shows the chromatographic separation of (S, S) -and (R, R) -fenoterol.

Fig. 2A provides the uv spectra of (R, R) -and (S, S) -fenoterol.

Figure 2B illustrates the circular dichroism spectra of (R, R) -and (S, S) -fenoterol.

FIG. 3 provides the [2 ], [ R, R ] -fenoterol produced by adding (R, R) -fenoterol to the running buffer⁺H]Front edge (frontal) chromatographic elution profile of' - (±) -fenoterol.

Fig. 4A is a graph including dose-response curves generated by treating freshly isolated rat ventricular myocytes with (±) -fenoterol, (R, R) -fenoterol or (S, S) -fenoterol.

FIG. 4B is a graph comprising T produced in freshly isolated rat ventricular myocytes by treatment with (±) -fenoterol, (R, R) -fenoterol or (S, S) -fenoterol_50%Graph of relaxed dose-response curve.

Figure 5 illustrates the chemical structures of stereoisomers of fenoterol and fenoterol analogues (compounds 2-7).

FIG. 6 illustrates the chemical structures of compounds 47-51.

Figure 7 illustrates the effect of fenoterol and fenoterol analogues on cell contraction within single ventricular myocytes.

Fig. 8 is a graph illustrating the time-dependent mean plasma concentrations of (R, R) -fenoterol, (R, R) -methoxyfenoterol, and (R, S) -naphthylfenoterol after administration (5 mg/mL).

Detailed description of several embodiments

I. Introduction to the design reside in

The present disclosure provides fenoterol analogues that bind to the β 2-adrenergic receptor and have activity comparable to or greater than that of fenoterol. In one embodiment, the optically active fenoterol analogue is substantially purified from the racemic mixture. For example, the optically active fenoterol analogue is purified to comprise greater than 90%, typically greater than 95%, of the composition. These analogs are useful in the treatment of pulmonary disorders such as asthma and chronic obstructive pulmonary disease that have previously been treated with (±) -fenoterol. The use of the disclosed fenoterol analogues having equivalent to (±) -fenoterol to higher potency may reduce the side effects previously observed with (±) -fenoterol. For example, the use of lower concentrations of fenoterol analogues to obtain therapeutically effective responses is expected to reduce side effects such as hypersensitivity and drug resistance (tolerance) observed with the use of commercially available (±) -fenoterol. In addition, purification of the analogs removes impurities, such as inactive enantiomers that can cause these side effects.

The disclosure also shows that (R, R) -fenoterol is an active ingredient of the commercially available (±) -fenoterol. It is specifically contemplated that (R, R) -fenoterol and the disclosed (R, R) -and (R, S) -fenoterol analogs are useful in the treatment of cardiac disorders such as congestive heart failure. The use of substantially optically pure (R, R) -fenoterol or (R, R) -or (R, S) -fenoterol analogues for the treatment of congestive heart failure is expected to reduce the incidence of side effects caused by the less physiologically active forms of the drug.

Abbreviations and terms

AR adrenergic receptors

Circular dichroism of CD

CoMFA comparative molecular field analysis

HPLC, high performance liquid chromatography

IAM-PC (immobilized Artificial Membrane chromatography) carrier

ICYP:[¹²⁵I]Cyanopindolol

UV, ultraviolet radiation

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs. The definition of Terms commonly used in Chemical nomenclature can be found in The McGraw-Hill Dictionary of Chemical Terms,1985 and The Condensed Chemical Dictionary, 1981. As used herein, the singular terms "a", "an" and "the" include the plural reference unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Additionally, the term "comprising" as used herein means "comprising" and, thus, "A or B" means comprising A, comprising B, or comprising both A and B.

Unless otherwise indicated, any numerical value, whether or not stated with the terms "about" or "approximately," refers to approximate reproduction. The materials, methods, and examples described herein are illustrative only and not intended to be limiting. Any molecular weight values or molecular mass values are approximate and are provided for descriptive purposes only. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and described in various general and more specific references that are cited and discussed throughout the present specification. See, for example, Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press,2002, pp.360-361, 1084-1085; smith and March, March's advanced organic Chemistry: Reactions, mechanics, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic chemistry, accommodating Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978.

To aid in the review of the various embodiments disclosed herein, the following provides a description of the terms:

acyl group: a group of formula RC (O) -wherein R is an organic radical.

Acyloxy group: a group having the structure-OC (O) R, wherein R may be an optionally substituted alkyl or an optionally substituted aryl. "lower acyloxy" are those groups wherein R contains 1 to 10 (e.g., 1 to 6) carbon atoms.

Alkoxy groups: a group (or substituent) having the structure-O-R, wherein R is a substituted or unsubstituted alkyl group. Methoxy (-OCH)₃) Are exemplary alkoxy groups. In substituted alkoxy, R is alkyl substituted with a non-interfering substituent. "Thioalkoxy" means-S-R, wherein R is substituted or unsubstituted alkyl. "haloalkoxy" means a group-OR, wherein R is haloalkyl.

Alkoxycarbonyl group: a group of formula-C (O) OR, wherein R may be an optionally substituted alkyl OR an optionally substituted aryl. "lower alkoxycarbonyl" are those groups in which R contains 1 to 10 (e.g., 1 to 6) carbon atoms.

Alkyl groups: acyclic, saturated, branched, or straight chain hydrocarbyl groups, unless otherwise specified, containing 1 to 15 carbon atoms; for example, from 1 to 10, from 1 to 6, or from 1 to 4 carbon atoms. The term includes groups such as methyl, ethyl, n-propyl, isopropyl, isobutyl, t-butyl, pentyl, heptyl, octyl, nonyl, decyl or dodecyl. The term "lower alkyl" refers to an alkyl group containing 1 to 10 carbon atoms. Unless expressly indicated as "unsubstituted alkyl," alkyl "may be unsubstituted or substituted. The alkyl group may be substituted with one or more substituents (e.g., up to two substituents for each methylene group in the alkyl chain). Exemplary alkyl substituents include, for example, amino, amide, sulfonamide, halogen, cyano, carboxyl, hydroxyl, mercapto, trifluoromethyl, alkyl, alkoxy (e.g., methoxy), alkylthio, thioalkoxy, arylalkyl, heteroaryl, alkylamino, dialkylamino, alkylsulfanyl (sulfono), keto, or other functionality.

Aminocarbonyl (carbamoyl): a group of formula-OCN (R) R '-wherein R and R' are independently from each other hydrogen or lower alkyl.

Asthma: asthma is a respiratory disease in which the airway is usually constricted, inflamed, and lined with excess mucus in response to one or more "cues," such as exposure to environmental stimuli (or allergens), cold air, exercise, or emotional stress. This narrowing of the airway causes symptoms such as wheezing, shortness of breath, chest tightness, and coughing. The disorder is a chronic or recurrent inflammatory condition in which the airways progress to an increased response to various stimuli, characterised by bronchial hyper-responsiveness, inflammation, increased mucus production and intermittent airway obstruction.

Carbamate: a group of formula-OC (O) N (R) -wherein R is H or an aliphatic group such as lower alkyl or aralkyl.

Cardiac disorders or diseases: generally, cardiac disorders/diseases are a class of disorders/diseases involving the heart and/or blood vessels (arteries and veins). In particular embodiments, the cardiac condition/disease comprises congestive heart failure.

Chronic obstructive pulmonary disease: a group of respiratory diseases, including chronic bronchitis, emphysema and bronchiectasis, are characterized by airflow obstruction or limitation that is not fully reversible. Airflow limitation is generally progressive and is associated with an abnormal inflammatory response of the lungs to toxic particles or gases.

Congestive heart failure: heart failure in which the heart is unable to maintain adequate blood circulation within the body tissues or to pump out venous blood returning to the heart through veins.

Derivative (A): a chemical that is distinguished from other chemicals by one or more functional groups. Preferably, the derivative (e.g. fenoterol analogue) retains the biological activity of the molecule from which it is derived (such as fenoterol).

Isomers: compounds that have the same molecular formula but differ in the nature or order of bonding of their atoms or the spatial arrangement of their atoms are referred to as "isomers". Isomers that differ in their arrangement in atomic space are referred to as "stereoisomers". Stereoisomers that are non-mirror images of each other are referred to as "diastereomers", and those that are non-overlapping mirror images of each other are referred to as "enantiomers". When a compound has an asymmetric center, for example, if a carbon atom is bonded to four different groups, a pair of enantiomers may be present. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and described by the R and S-sequence rules of Cahn and Prelog, or by the manner in which molecules rotate the plane of polarized light and are designated as dextrorotatory or levorotatory (i.e., the (+) or (-) isomers, respectively). The chiral compounds may exist as individual enantiomers or as mixtures of enantiomers. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures".

The compounds described herein may have one or more asymmetric centers; such compounds are thus available in the form of (R) -or (S) -stereoisomers or mixtures thereof. Unless otherwise indicated, the specification or nomenclature of a particular compound in the specification and claims is intended to include the individual enantiomers and mixtures thereof, racemates, and the like. Methods for determining stereochemistry and methods for separating stereoisomers are well known in the art (see, e.g., March, advanced organic Chemistry,4th edition, New York: John Wiley and Sons,1992, Chapter 4).

Optionally: "optional" or "optionally" means that the subsequently described event or circumstance may not necessarily occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

A pharmaceutically acceptable carrier: the pharmaceutically acceptable carriers (vehicles) used in the present disclosure are conventional. Remington's Pharmaceutical Sciences, e.w. martin, Mack Publishing co., Easton, PA,19th Edition (1995) describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds or molecules, such as one or more nucleic acid molecules, proteins, or antibodies and other agents that bind to these proteins.

In general, the nature of the carrier will vary with the particular mode of administration employed. For example, parenteral formulations typically include injection solutions comprising pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as vehicles. For solid compositions (e.g., powder, pill, tablet or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Phenyl group: the phenyl group may be unsubstituted or substituted with one, two or three substituents independently selected from: alkyl, heteroalkyl, aliphatic, heteroaliphatic, thioalkoxy, halo, haloalkyl (such as-CF)₃) Nitro, cyano, -OR (wherein R is hydrogen OR alkyl), -N (R) R '(wherein R and R' are independently of each other hydrogen OR alkyl), -COOR (wherein R is hydrogen OR alkyl) OR-C (O) N (R ') R "(wherein R' and R" are independently selected from hydrogen OR alkyl).

Pulmonary disorders or diseases: generally, a pulmonary disorder/disease includes any disorder/disease associated with the lung. In particular embodiments, the pulmonary disorder/disease includes asthma and chronic obstructive pulmonary disease.

Purification of: the term "purified" does not require absolute purity; but rather include relative terms. Thus, for example, a purified formulation is one in which the (R, R) -enantiomer of a desired component, such as fenoterol, is more enriched than it was in a previous environment, such as a (±) -fenoterol mixture. A desired component, such as the (R, R) -enantiomer of fenoterol, e.g., a sample, is considered pure when at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% by weight is made up of the desired component. The purity of the compound can be determined, for example, by High Performance Liquid Chromatography (HPLC) or other conventional methods. In one embodiment, the particular enantiomer of the fenoterol analogue is purified to exhibit greater than 90%, often greater than 95%, relative to the other enantiomer present in the purified preparation. In other cases, the purified preparation may be substantially homogeneous, with less than 1% of the other stereoisomers.

The compounds described herein may be obtained in purified form or purified by any means known in the art including silica gel and/or alumina chromatography. See, for example, Introduction to model Liquid Chromatography,2nd Edition, Snyder and Kirkland, New York, John Wiley and Sons, 1979; and Thin layer chromatography, edited by Stahl, New York, Springer Verlag, 1969. In one embodiment, the compound comprises a purified fenoterol or fenoterol analog having a purity of at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% by weight of the sample relative to other impurities. In another embodiment, the compound comprises at least two purified stereoisomers, each having a purity of at least about 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% by weight of the sample relative to other impurities. For example, the compounds can include substantially purified (R, R) -fenoterol analogs and substantially purified (R, S) -fenoterol analogs.

Subject: the term "subject" includes human and veterinary subjects, e.g., humans, non-human primates, dogs, cats, horses, rats, mice, and cattle. Similarly, the term mammal includes both human and non-human mammals.

Treatment: with respect to a disease, the term includes (1) preventing the disease, e.g., causing the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to exposure to the disease but has not yet experienced or exhibited symptoms of the disease, (2) inhibiting the disease, e.g., arresting the development of the disease or clinical symptoms thereof, or (3) relieving the disease, e.g., causing regression of the disease or clinical symptoms thereof.

A therapeutically effective amount of: the amount of a particular agent that is capable of achieving a desired effect in a subject being treated with that agent. For example, the therapeutically effective amount can be an amount of (R, R) -fenoterol or an (R, R) -or (R, S) -fenoterol analogue that is useful in preventing, reducing, and/or inhibiting, and/or treating a cardiac disorder such as congestive heart failure. Ideally, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce, and/or inhibit, and/or treat a condition in a subject without causing a significant cytotoxic effect in the subject. The amount of the agent that can be used to prevent, reduce, and/or inhibit, and/or treat the condition in the subject will vary depending on the subject being treated, the severity of the condition, and the method of administration of the therapeutic composition.

(R, R) -fenoterol and fenoterol analogues

A. Chemical structure

Some exemplary fenoterol analogues disclosed herein have the following formula:

wherein R is₁-R₃Independently hydrogen, acyl, alkoxycarbonyl, aminocarbonyl, or a combination thereof;

R₄is hydrogen or lower alkyl;

R₅is a lower alkyl group, and is,

or

Wherein X and Y are independently selected from hydrogen, lower-OR₆and-NR₇R₈；

R₆Is lower alkyl or acyl; and

R₇and R₈Independently hydrogen, lower alkyl, alkoxycarbonyl, acyl or ammoniaAn alkylcarbonyl group.

Continuing with the general formula for the fenoterol analog above, Y may be — OH.

In one embodiment, R₅Is a 1-naphthyl or 2-naphthyl derivative optionally having 1,2 or 3 substituents. Such R₅Examples of groups are represented by the formula:

wherein Y is¹、Y²And Y³Independently of one another is hydrogen, lower-OR₆and-NR₇R₈；

R₆Independently at each occurrence, is selected from the group consisting of lower alkyl and acyl; and

R₇and R₈Independently hydrogen, lower alkyl, alkoxycarbonyl, acyl or aminocarbonyl (carbamoyl). In a specific compound, Y¹、Y²And Y³is-OCH₃。

In particular, R₅Groups include those represented by the formula:

wherein R is₆Is a lower alkyl group such as methyl, ethyl, propyl or isopropyl, or an acyl group such as acetyl.

Exemplary R₅The groups include:

in one embodiment, R₄Is lower alkyl and R₅The method comprises the following steps:

or

Wherein X and Y are independently selected from H, lower alkyl-OR₆and-NR₇R₈；

R₆Is a lower alkyl group; and

R₇and R₈Independently hydrogen or lower alkyl.

In further embodiments, R₄Selected from ethyl, n-propyl and isopropyl and R₅Has the following structure:

wherein X is H, -OR₆or-NR₇R₈. For example, R₆Can be methyl or R₇And R₈Is hydrogen.

In further embodiments, R₅Has the following structure:

in other embodiments, R₄Selected from methyl, ethyl, n-propyl and isopropyl and R₅To represent：

For R₁-R₃Examples of suitable groups that can be cleaved in vivo to provide hydroxyl groups include, but are not limited to, acyl groups, acyloxy groups, and alkoxycarbonyl groups. Compounds having such cleavable groups are referred to as "prodrugs". The term "prodrug" as used herein refers to a compound that includes a substituent that can be converted (e.g., by hydrolysis) to a hydroxyl group in vivo. Various forms of prodrugs are known in the art, for example, as discussed below: bundgaard, (ed.), Design of produgs, Elsevier (1985); widder et al (ed.), Methods in enzymology, Vol.4, Academic Press (1985); Krogsgaard-Larsen et al (eds.), Design and Application of produgs, Textbook of Drug Design and development, Chapter5,113191 (1991); bundgaard et al, Journal of drug delivery Reviews,8:138 (1992); bundgaard, Pharmaceutical Sciences,77:285et seq (1988); and Higuchi and Stella (eds.) Prodrugs as Novel drug delivery Systems, American Chemical Society (1975).

Exemplary (R, R) -compounds have the following chemical structure:

x and R₁-R₃As defined above.

Further exemplary (R, R) -compounds have the following chemical structure:

exemplary (R, S) -compounds have the following chemical structure:

wherein X and R₁-R₃As defined above.

Further exemplary (R, S) -compounds have the following chemical structure:

exemplary (S, R) -compounds have the following chemical structure:

wherein X and R₁-R₃As defined above.

Exemplary (S, S) -compounds have the following chemical structure:

wherein X and R₁-R₃As defined above.

Examples illustrating the chemical structures of the various stereoisomers of fenoterol are provided below.

Particular process embodiments contemplate the use of solvates (e.g., hydrates), pharmaceutically acceptable salts, and/or different physical forms of (R, R) -fenoterol and any fenoterol analogs described herein.

1. Solvates, salts and physical forms

"solvate" refers to a physical combination of a compound and one or more solvent molecules. The physical conjugates include varying degrees of ionic and covalent bonding, including, for example, covalent adducts and hydrogen bonding solvates. In some cases, solvates can be isolated, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" includes both solution phase and isolatable solvates. Typical solvates include compounds that bind to ethanol, compounds that bind to methanol, and the like. A "hydrate" is a solvate in which the solvent molecule is water.

The disclosed compounds also include salts, including mixed and/or internal salts, if several salt-forming groups are present. Salts are generally non-toxic pharmaceutically acceptable salts. The salts can be of any type (inorganic and inorganic), such as fumarate, hydrobromide, hydrochloride, sulphate and phosphate salts. In one embodiment, the salt comprises a nonmetal (e.g., halogen) from group VII of the periodic Table. For example, the compound may be provided in the form of a hydrobromide salt.

Additional examples of salt-forming groups include, but are not limited to, carboxy, phosphonate, or borate groups, which can form salts with suitable bases. These salts may include, for example, nontoxic metal cations derived from metals of groups IA, IB, IIA and IIB of the periodic Table of elements. In one embodiment, alkali metal cations such as lithium, sodium or potassium ions, or alkaline earth metal cations such as magnesium or calcium ions may be used. The salt may also be a zinc or ammonium cation. Salts may also be formed with suitable organic amines, such as unsubstituted or hydroxy-substituted mono-, di-or tri-alkylamines, especially mono-, di-or tri-alkylamines, or with quaternary ammonium compounds, e.g. with N-methyl-N-ethylamine, diethylamine, triethylamine, mono-, di-or tri- (2-hydroxy-lower alkyl) amines, such as mono-, di-or tri- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine or tri (hydroxymethyl) methylamine, N, N-di-lower alkyl-N- (hydroxy-lower alkyl) amines, such as N, N-dimethyl-N- (2-hydroxyethyl) amine or tri- (2-hydroxyethyl) amine or N-methyl-D-glutamine Or quaternary ammonium compounds such as tetrabutylammonium salts.

Exemplary compounds disclosed herein have at least one basic group that can form an acid-base with a mineral acid. Examples of basic groups include, but are not limited to, amino or imino groups. Examples of inorganic acids that can form salts with these basic groups include, but are not limited to, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid. The basic groups can also be organic carboxylic, sulfonic (sulfonic acids) or phosphonic acids or N-substituted sulfamic acid salts, such as acetic, propionic, glycolic, succinic, maleic, hydroxymaleic, methylmaleic, fumaric, malic, tartaric, gluconic, glucaric, glucuronic, citric, benzoic, cinnamic, mandelic, salicylic, 4-aminosalicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, 4' -methylenebis (3-hydroxy-2-naphthoic), nicotinic or isonicotinic acid, and, in addition, salts with amino acids, such as with alpha-amino acids, also with methanesulfonic, ethanesulfonic, 2-hydroxymethanesulfonic, ethane-1, 2-disulfonic, benzenedisulfonic, 4-methylbenzenesulfonic, nicotinic, or isonicotinic acid, Naphthalene-2-sulfonic acid, 2-or 3-phosphoglyceric acid, glucose-6-phosphate or N-cyclohexylsulfamic acid (having the structure of cyclamic acid) or with other acidic organic compounds such as ascorbic acid. In a preferred embodiment of the invention, the fenoterol is provided in the form of the hydrobromide salt and the exemplary fenoterol analogue is provided in the form of the fumarate salt thereof.

Additional counterions for the formation of pharmaceutically acceptable salts can be found in Remington's pharmaceutical Sciences,19th Edition, Mack Publishing Company, Easton, Pa, 1995. In one aspect, the use of pharmaceutically acceptable salts can also be used to adjust the osmotic pressure of the composition.

In certain embodiments, the compounds used in the methods are provided in polymorphic forms. Thus, the compound may be provided in two or more physical forms such as different crystalline, liquid crystalline or amorphous (amorphous) forms.

2. Pharmaceutical use

Any of the above compounds (e.g., (R, R) -fenoterol or fenoterol analogs or hydrates or pharmaceutically acceptable salts thereof) or combinations thereof are intended for use in the preparation of a medicament for stimulating the β 2-adrenergic receptor or for treating disorders of the lung and heart (such as asthma and congestive heart failure) in a subject. Formulations suitable for such drugs, subjects who may benefit from the described and other related features, are described elsewhere herein.

B. Synthesis method

The disclosed fenoterol analogues can be synthesized by any method known in the art. Numerous general references are available which provide conventionally known chemical synthetic routes and conditions for synthesizing the disclosed compounds. (see, for example, Smith and March, March's advanced Organic Chemistry: Reactions, mechanics, and Structure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbook of Practical Organic Chemistry, Including quick analytical Analysis, Fourth Edition, NewYork: Longman, 1978).

The compounds described herein can be purified by any means known in the art, including chromatography, such as HPLC, preparative thin layer chromatography, flash column chromatography, and ion exchange chromatography. Any suitable stationary phase may be used, including normal and reverse phase and ionic resins. The most typical disclosed compounds are purified by both hollow column chromatography and preparative chromatography.

A suitable exemplary synthesis of the fenoterol analogue is provided below:

route I: an exemplary synthesis of 4 stereoisomers of 1-6 includes coupling of an epoxide formed from (R) -or (S) -3',5' -dibenzyloxyphenylbromohydrin with the appropriate (R) -or (S) -isomer of benzyl-protected 2-amino-3-benzylpropane (1-5) or with the (R) -or (S) -isomer of N-benzyl-2-aminoheptane (6).

Route II: exemplary syntheses of (R) -7 and (S) -7 using 2-phenylethylamine. The resulting compound can be deprotected by hydrogenation with Pd/C and purified as fumarate.

Scheme III describes an exemplary synthesis of the chiral building block used in scheme II. (R) -and (S) -3',5' -dibenzyloxyphenyl-bromohydrin enantiomers through the use of boron-dimethyl sulfide complex (BH)₃SCH₃) And (1R,2S) -or (1S,2R) -cis-1-amino-2-indanol, to prepare the desired (R) -and (S) -1-benzylaminopropane by enantioselective crystallization of rac-2-benzylaminopropane using (R) -or (S) -mandelic acid as counter ion.

Pharmaceutical compositions

The disclosed (R, R) -fenoterol and fenoterol analogues are useful at least in the treatment of pulmonary disorders such as asthma and Chronic Obstructive Pulmonary Disease (COPD) and cardiac disorders such as congestive heart failure. Accordingly, also described herein are pharmaceutical compositions comprising at least one of the disclosed fenoterol compounds or analogs.

The formulation of pharmaceutical compositions is well known in the art. For example, Remington's pharmaceutical Sciences, e.w. martin, Mack Publishing co., Easton, PA,19th Edition,1995 describes exemplary formulations (components thereof) suitable for drug delivery of (R, R) -fenoterol and the disclosed fenoterol analogues. Pharmaceutical compositions comprising at least one of these compounds are formulated for use in human or veterinary medicine. The particular formulation of the disclosed pharmaceutical compositions can vary depending, for example, upon the mode of administration (e.g., oral or parenteral) and/or the disease to be treated (e.g., a pulmonary disorder or a cardiac disorder such as congestive heart failure). In some embodiments, the formulation includes a pharmaceutically acceptable carrier in addition to at least one active ingredient, such as a fenoterol compound.

Pharmaceutically acceptable carriers for use in the disclosed methods and compositions are common in the art. The nature of the pharmaceutical carrier will vary depending upon the particular mode of administration employed. For example, parenteral formulations typically include injectable liquids, including pharmaceutically and physiologically acceptable liquids, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as vehicles. For solid compositions such as powder, pill, tablet or capsule forms, conventional non-toxic solid carriers may be included, such as pharmaceutical grades of mannitol, lactose, starch or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may optionally contain minor amounts of non-toxic auxiliary substances or excipients such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like; for example, sodium acetate or sorbitan monolaurate. Other non-limiting excipients include non-ionic solubilizers such as polyoxyethylene castor oil or proteins such as human serum albumin or plasma formulations.

The disclosed pharmaceutical compositions can be formulated as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are non-toxic salts of the compounds in free base form that possess the desired pharmacological activity of the free base. These salts may be derived from inorganic or organic acids. Non-limiting examples of suitable inorganic acids are hydrochloric acid, nitric acid, hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid. Non-limiting examples of suitable organic acids are acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, salicylic acid, formic acid, trichloroacetic acid, trifluoroacetic acid, gluconic acid, aspartic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, and the like. A list of other suitable pharmaceutically acceptable salts is given in Remington's Pharmaceutical Sciences,19th Edition, Mack Publishing Company, Easton, Pa, 1995. The pharmaceutically acceptable salts may also be used to adjust the osmotic pressure of the composition.

The dosage form of the disclosed pharmaceutical compositions can be determined by the mode of administration selected. For example, oral dosage forms may be employed in addition to injectable liquids. Oral dosage forms may be liquid dosage forms such as syrups, solutions or suspensions, or solid dosage forms such as powders, pills, tablets or capsules. Methods for preparing such dosage forms are known and will be apparent to those skilled in the art.

Certain embodiments of pharmaceutical compositions comprising the disclosed compounds can be formulated in unit dosage forms suitable for the individual administration of precise dosages. The amount of active ingredient, e.g., (R, R) -fenoterol, administered will vary depending on the subject being treated, the severity of the condition and the mode of administration, and is known to those skilled in the art. Within these ranges, the formulation to be administered comprises some of the extracts or compounds disclosed herein in an amount to achieve the desired effect in the subject being treated. In particular embodiments, for oral administration, the compositions are provided in the form of tablets containing from about 1.0 to about 50mg of the active ingredient, particularly containing about 2.0 mg, about 2.5 mg, 5mg, about 10mg, or about 50mg of the active form, for the symptomatic adjustment of a subject being treated. In an exemplary oral dosing regimen, a tablet containing from about 1mg to about 50mg (e.g., from about 2mg to about 10 mg) of the active ingredient is administered 2-4 times per day, such as 2,3, or 4 times.

V. method of use

The present disclosure includes methods of treating disorders including pulmonary and cardiac disorders. In some embodiments, the pulmonary disorder is asthma or chronic obstructive pulmonary disease. In other embodiments, the cardiac disorder is congestive heart failure.

The disclosed methods comprise administering (R, R) -fenoterol or disclosed fenoterol analogues (and optionally one or more other agents) to a subject in a pharmaceutically acceptable carrier and in an amount effective to treat a pulmonary and/or cardiac disorder. Routes of administration that can be used in the disclosed methods include, but are not limited to, oral and parenteral routes, such as intravenous (iv), intraperitoneal (ip), rectal, topical, ocular, nasal and transdermal routes. The formulations for these dosage forms are as described above.

The effective amount of (R, R) -fenoterol or disclosed fenoterol analogue will vary depending upon, at least, the particular method of use, the subject being treated, the severity of the condition, and the mode of administration of the therapeutic composition. A "therapeutically effective amount" of a composition is an amount of a particular compound sufficient to achieve a desired effect in the subject being treated. For example, the therapeutically effective amount can be the amount of (R, R) -fenoterol necessary to prevent, inhibit, reduce or ameliorate a pulmonary and/or cardiac disorder and/or one or more symptoms of the disorder in the subject. Ideally, a therapeutically effective amount of (R, R) -fenoterol or disclosed fenoterol analogue is an amount sufficient to prevent, inhibit, reduce or ameliorate one or more symptoms of a pulmonary and/or cardiac disorder and/or condition without causing a significant cytotoxic effect on host cells.

The therapeutically effective dose of the disclosed fenoterol or pharmaceutical composition may be determined by one skilled in the art with the aim of achieving the disclosed applicable compounds with a minimum of up to EC in the examples herein₅₀The concentration of (c). Examples of dosage ranges are from about 0.001 to about 10 mg/kg body weight, administered orally, in a single dose or in divided doses. In particular embodiments, the dosage range is from about 0.005 to about 5mg/kg body weight, orally, in a single dose or in divided doses (assuming an average body weight of about 70 kg; for humans having a body weight more or less than the average body weight, respectivelyAdjust the value). For oral administration, the compositions are provided, for example, in the form of tablets containing from about 1.0 to about 50mg of the active ingredient, particularly about 2.5 mg, 5mg, about 10mg, or about 50mg of the active ingredient, for symptomatic adjustment of the dosage to the subject being treated. In an exemplary oral dosing regimen, tablets containing from about 1mg to about 50mg of the active ingredient are administered 2-4 times per day, such as 2,3 or 4 times.

The particular dose level and frequency of dosage administration for a particular subject will vary and will depend upon a variety of factors including the activity of the particular compound, the metabolic stability and duration of action of the compound, the age, body weight, general health, sex and diet of the subject, the mode and time of administration, the rate of excretion, the drug combination, and the severity of the condition in the subject being treated.

The presently disclosed subject matter is further illustrated by the following non-limiting examples.

Detailed Description

Example 1

Materials and methods

And (3) a reagent. Phenylmethylsulfonyl fluoride (PMSF), benzamidine, leupeptin, pepstatin A, MgCl₂EDTA, Trizma-hydrochloride (Tris-HCl), (±) -propranolol and Minimal Essential Medium (MEM) were obtained from Sigma Aldrich (st. Egg lecithin Lipids (PC) were obtained from Avanti Polar Lipids (Alabaster, AL). (+ -) -fenoterol was purchased from Sigma Aldrich, and [ (+ ]. alpha. ], ] [ (+ ], ]³H]- (+) -fenoterol was obtained from Amersham Biosciences (Boston, MA). Organic solvents n-hexane, isopropanol and triethylamine were obtained from CarloErba (Milan, Italy) as ultra pure HPLC grade solvents. Fetal bovine serum and penicillin-streptomycin were purchased from Life technologies (Gaithersburg, MD), and [, [2 ]¹²⁵I]- (. + -.) -Iodocyanopinolol (ICYP) was purchased from NEN Life Science Products, Inc. (Boston, MA).

Preparation and characterization of (R, R) -fenoterol and (S, S) -fenoterol. (R, R) -fenoterol and (S, S) -fenoterol are prepared from (±) -fenoterol using an HPLC technique using an HPLC column (25cm × 0.46cm i.d.) comprising a tris- (3, 5-dimethylphenylcarbamate) chiral stationary phase (A), (B), (CAD CSP, chip Technologies, WestChester, Pa; chip LPAK is a trademark of Daicel Chemical Industries Ltd., Exton, Pa.). The chromatographic system consists of:PU-980 solvent delivery System, andan MD-910 multi-wavelength monitor set at λ =230nm and connected to a computer workstation; JASCO is a registered trademark of JASCO, inc., Tokyo, Japan. A Rheodyne type 7125 syringe with a 20 μ l sample loop tube was used for injection of 0.2-0.3 mg (±) -fenoterol onto the chromatographic system. The mobile phase was n-hexane/isopropanol (88/12v/v) containing 0.1% triethylamine at a flow rate of 1 ml/min and the temperature of the system was maintained at 25 ℃ using a column heater/cooler (model 7955, Jones Chromatography ltd., UK). The separated (R, R) -fenoterol and (S, S) -fenoterol were collected at a 10ml score as their respective peaks eluted from the column. 2 ml of the intermediate fraction were collected and discarded to improve the enantiomeric purity of the collected isomer.

The stereochemical configuration of the resolved (R, R) -fenoterol and (S, S) -fenoterol is used inCircular Dichroism (CD) measurements obtained with a J-800 spectroscopic polarimeter. The (R, R) -fenoterol and (S, S) -fenoterol were dissolved in isopropanol and the measurements were obtained using a 1 cm path length at room temperature.

Immobilized beta 2-AR front edgeChromatography. Liquid chromatography columns comprising immobilized β 2-AR were prepared using the techniques described above (Beigid et al, anal. chem.,76: 7187-. In summary, cell membranes were obtained from a HEK293 cell line that had been transfected with cDNA encoding human β 2-AR. Columns were constructed using aliquots of cell pellet suspensions corresponding to 5-7 mg total protein as determined according to the micro BCA method. Membranes were prepared in 10ml of buffer consisting of: comprising MgCl₂(2mM) Tris-HCl [50mM, pH7.4]Benzamidine (1mM), leupeptin (0.03mM), pepstatin (0.005mM) and EDTA (1 mM).

A 180 mg aliquot of the immobilized artificial membrane chromatography carrier (IAM-PC,12 micron particle size,pore size, obtained from Regis Chemical co., Morton Grove, IL) and 80 μ M PC were added to the membrane preparation and the resulting mixture was stirred at room temperature for 3 hours, transferred (5 cm length) to a nitrocellulose dialysis membrane (MW cut off 10,000Da, pierce Chemical, Rockford, IL) and placed in 1L of dialysis buffer at 40 ℃ for 24 hours, which had the following composition: Tris-HCl [50mM, pH7.4 ] containing EDTA (1mM)]，MgCl₂(2mM), NaCl (300mM) and PMSF (0.2 mM). The dialysis step was repeated twice with fresh buffer.

After dialysis, the mixture was centrifuged at 120x g for 3 minutes, the supernatant was discarded and the IAM carrier beads containing the membrane with immobilized receptor were collected. The beads were resuspended in 2 ml of chromatography flow buffer consisting of EDTA (1mM) and MgCl₂(2mM) Tris-HCl [10mM, pH7.4]The suspension was pumped out of the HR5/2 chromatographic glass column (Amersham Pharmacia Biotech, Uppsala, Sweden) using a peristaltic pump at a flow rate of 0.3 ml/min. The end adapters were assembled to give a total gel bed volume of 0.4 ml. The column was stored at 4 ℃ when not in use.

The column containing the immobilized beta 2-AR stationary phase was placed in a chromatography system consisting of an HPLC pump (10-AD, Shimadzu Inc., Columbia, MD),A manual FPLC syringe (Amersham Biotechnology, Uppsala, Sweden), a packed immobilized receptor column and an online radioactive flow detector (IN/US, Tampa, FL) with a 50 μ L sample loop tube, all components were connected sequentially. In the forward chromatographic study, samples of 5-7 ml were continuously withdrawn until the elution profile showed a plateau region. The flow buffer composition was as follows: comprising EDTA (1mM) and MgCl₂(2mM) and 0.05nM³H]Tris-HCl [10mM, pH7.4 ] of- (+) -fenoterol as labeling ligand]. (R, R) -fenoterol or (S, S) -fenoterol was added to the running buffer at successive concentrations of 0.1, 80.0, 240 and 700nM and applied to the column. Between each injection, the immobilized receptor column was equilibrated with about 80ml of flow buffer without the addition of (R, R) -fenoterol or (S, S) -fenoterol, respectively. All chromatographic studies were performed at a flow rate of 0.2 ml/min at room temperature.

The data were analyzed using non-linear equation (1) to determine the number of binding sites and dissociation constants,

where Vi is the elution volume of the solute, Vmin is the elution volume at the saturation point, P is the number of available binding sites, M is the concentration of the marker ligand, and Kd is the dissociation constant of the ligand.

Ligand-displacement binding. In person24 hours after adenovirus infection of beta 2-AR HEK293 cells were harvested in cell lysis buffer containing EGTA [5mM ] 5mM]Tris-HCl [5mM, pH7.4 ]]And homogenized on ice 15 times, the samples were centrifuged at 30,000Xg for 15 minutes to pellet the cells, and the membranes were resuspended in a binding buffer comprising NaCl (120mM), KCl (5.4mM), CaCl₂(1.8mM)、MgCl₂Tris-HCl [20mM, pH7.4 ] in (0.8mM) and glucose (5mM)]And stored in small portions at-80 ℃. Using a saturating amount (1-300pM) of a β -AR specific ligand [ beta ], [ beta ] -AR¹²⁵I]Cyanopindolol (ICYP) binding assays were performed on 5-10 μ g of membrane protein. For competitive binding, 5-10. mu.g of membrane protein was treated with 50. mu.M of GTP. gamma_s(non-hydrolyzable guanosine triphosphate) and then reacted with¹²⁵ICYP (50pM) and different concentrations of fenoterol or its isomer were incubated in a total volume of 250 μ L. Nonspecific binding was determined in the presence of 20. mu.M propranolol. The reaction was carried out in 250. mu.L of binding buffer at 37 ℃ for 1 hour. Termination of the binding reaction was performed as follows: adding ice-cooled Tris-HCl [10mM, pH7.4 ]]Into a membrane suspension, followed by rapid vacuum filtration through a glass fiber filter (Whatman GF/C). Each filter was supplemented with another 7mL of ice-cooled Tris-HCl [10mM, pH7.4 ]]Washed three times. The radioactivity of the wet filters was measured in a gamma particle counting tube. All assays were performed in duplicate and receptor density was normalized to milligrams of membrane protein. The Ka and the number of maximum binding sites (Bmax) in ICYP were determined by Scatchard analysis of the saturation binding equipotential lines. Data from competitive binding studies were used as GRAPHPADThe Software (GRAPHPAD PRISM is a registered trademark of GraphPad Software, inc., San Diego, CA) was analyzed using either a 1-site or a 2-site competition binding curve.

Example 2

Purification and characterization of (R, R) -fenoterol and (S, S) -fenoterol

This example illustrates the resolution of (R, R) -fenoterol and (S, S) -fenoterol from (±) -fenoterol to high enantiomeric purity.

(±) -fenoterol was separated into its component enantiomers (R, R) -fenoterol and (S, S) -fenoterol on AD-CSP using the chromatographic conditions described in example 1. As shown in fig. 1, both stereoisomers are resolved using an enantioselective factor (α)1.21 and a resolution factor (Rs) 1.06. Since a tail off of the chromatographic peak was observed, 2 ml of the intermediate fraction was collected and discarded. The collected peaks were analyzed using the same chromatographic conditions and the data showed that both enantiomers were prepared with >97% stereochemical purity.

The assignment of the absolute configuration of the separated fractions is done using its chiral optical (chiral) properties. The Ultraviolet (UV) spectra of both fractions contained the same maximum at about 280 and 230, indicating that both enantiomers have the same UV chromophore. For the less retained enantiomeric fraction, the Circular Dichroism (CD) spectrum shows negative CD bands at about 280 and 215nm, while the spectrum shows positive values at 230 and 200 nm. The sign of the CD band is opposite for the majority of the retained fenoterol fractions, confirming the enantiomeric nature of the two fractions. The lowest energy UV and circular dichroism spectra of the two enantiomeric fractions are shown in fig. 2A and 2B, respectively. The less retained chromatographic fraction showed negative CD bands at about 280nm, while the circular dichroism spectrum of the most retained chromatographic fraction contained positive CD bands at the same wavelength (fig. 2B). These results indicate that each fraction contains one of the enantiomers of fenoterol.

The sign of the lowest energy CD band was used to assign the absolute configuration of the isolated fenoterol enantiomer by applying the Brewster-Buta/Smith-Fontana sector rule for chiral benzyl derivatives (Brewster and Buta, J.Am.chem.Soc,88: 2233-. This sector rule is used to predict the interaction with benzyl compounds bearing a hydroxyl or amino moiety¹L_bThe sign of the CD band involved in electronic transitions and has been applied primarily to conformationally variable aromatic compounds containing a single stereogenic center. In the case of fenoterol, there are two stereogenic centersHowever, it is believed that the observed optical rotation is determined primarily by the arylcarbinol moiety due to the distance between the aromatic ring and the stereogenic center. Applying this rule allows the attribution to derive: the absolute configuration of the fenoterol enantiomer contained in the less retained fraction, which showed a negative CD band at 280nm, was (S, S), and the absolute configuration of the fenoterol enantiomer contained in the most retained fraction, which showed a positive CD band at 280nm, was (R, R). This assignment was confirmed by the separately synthesized (S, S) -fenoterol and (R, R) -fenoterol.

These studies indicate that (R, R) -fenoterol and (S, S) -fenoterol can be isolated from (±) -fenoterol to achieve high enantiomeric purity.

Example 3

Chromatographic assay for the binding of (R, R) -fenoterol and (5,5) -fenoterol to immobilized beta 2-AR

This example illustrates that (R, R) -fenoterol is responsible for the β 2-AR binding of the clinically used drug (±) -fenoterol.

The preparation, characterization and application of liquid chromatography stationary phases comprising immobilized membranes obtained from the β 2-AR HEK-293 cell line have been previously reported (Beigi et al, anal. chem.,76:7187-7193, 2004). For example, Beigi et al (anal. chem.,76:7187-7193,2004) describe that frontal displacement chromatography can be used to determine the dissociation constants (K) for the binding of two beta 2AR antagonists, (S) -propranolol and CGP12177A to immobilized beta 2-AR_d). Zonal displacement chromatography using CGP12177A as the marker ligand showed that the immobilized β 2-AR retained its enantioselectivity, since addition of (S) -propranolol to flow gave a larger displacement than addition of (R) -propranolol (supra). Addition of (±) -fenoterol to the mobile phase also showed a conformational change in the immobilized β 2-AR (supra). Agonist-induced β 2-AR and the conformational change of most G protein-coupled receptors from the resting state to the activated state have been described (Ghanoui et al, Proc. Natl. Acad. ScL. U.S.A.,98: 5997-.

Now, the immobilized beta 2-AR column contains [ beta ], [ beta ] -AR as a marker³H]Fenoterol in running buffer equilibration before starting the displacement study. It is hypothesized that binding data calculated using frontal displacement chromatography reflects the binding of (R, R) -fenoterol and (S, S) -fenoterol to the activated state of the receptor. In the leading edge chromatography, the initial flat part of the chromatogram represents the binding of the marker ligand specific for the immobilized target (β 2-AR in this study) and non-specific binding to other parts on the immobilized membrane fragment. Saturation of specific binding sites results in a breakthrough front after the plateau indicating the establishment of a new equilibrium. Adding a second compound to the mobile phase will shift the chromatogram to the left if the compound competes with the marker ligand for binding to β 2-AR. The relationship between the magnitude of this movement and the labeled ligand concentration can be used to calculate the binding affinity of the displacer to the target and the number of active binding sites. This method has recently been reviewed (Moaddel and Wainer, anal. chem. actata, 546:97-105,2006).

As shown in FIG. 3 (Curve 1), the [2 ] is added to the running buffer³H]Fenoterol gave the expected leading edge chromatograms. The sequential addition of increasing concentrations of (R, R) -fenoterol to the running buffer produced a corresponding shift in the chromatogram towards smaller retention volumes (fig. 3, curves 2-4). The magnitude and shift of the corresponding concentration of (R, R) -fenoterol were analyzed using equation 1, and the calculated dissociation constant K_dNumber of binding sites [ P ] 472nM and available]176 pmol/column, r²=0.9999(n=T)。

The sequential addition of increasing concentrations of (S, S) -fenoterol to the running buffer did not produce a corresponding shift in the chromatogram towards shorter retention times. Thus, (S, S) -fenoterol had no significant affinity for immobilized β 2-AR.

To confirm the chromatographic results, standard membrane binding studies were performed using membranes obtained from the same HEK-293 cell line (to construct an immobilized β 2-AR column). The data reflect the (mean + -SD) K with a single binding site for (R, R) -fenoterol_d=457±55nM (n =4), for (S, S) -fenoterol, K_d109,000 ± 10,400nM (n = 4). These data show that frontal affinity chromatography on immobilized cell membranes can be used to determine the magnitude of enantioselectivity in binding to ligands of the target receptor. In addition, the results from both frontal affinity chromatography and ligand competitive binding studies indicate that (R, R) -fenoterol is responsible for the β 2-AR binding of the clinically used drug (+) -fenoterol.

Example 4

Effect of (R, R) -fenoterol and (S, S) -fenoterol on myocardial contractility

This example illustrates that (R, R) -fenoterol and (S, S) -fenoterol differentially activate the β 2-adrenergic receptor/stimulatory heterotrimeric G-protein involved in cell contractile signaling (AR/G)_s)。

To determine whether (R, R) -fenoterol and (S, S) -fenoterol differentially activated β 2-AR/GS signaling in the regulation of cell contractility, freshly isolated adult rat cardiomyocytes were perfused with various concentrations of (R, R) -fenoterol or (S, S) -fenoterol. These studies were performed using the methods described previously (Zhou et al, MoI. Pharmacol.,200,58: 887-. In summary, individual ventricular myocytes were isolated from 2-4 month old rat hearts by standard enzymatic techniques. The isolated cells were resuspended in HEPES buffer solution [20mM, pH7.4 ]]The buffer comprises NaCl (137mM), KCl (5.4mM), MgCl₂(1.2mM)，NaH₂PO₄(1.0mM)，CaCl₂(1.0mM) and glucose (20 mM). All studies were performed in cell isolates within 8 hours.

The cells were placed on the stage of an inverted microscope (Zeiss model IM-35, Zeiss, Thornwood, NY), perfused with HEPES-buffer at a flow rate of 1.8 ml/min, and electrically stimulated at 230 ℃ at 0.5 Hz. Cell length was monitored by optical edge-tracking using a photodiode array (Model1024SAQ, reticulon, Boston, MA) with a temporal resolution of 3 milliseconds. Cell contraction was measured by measuring the percentage of shortening of the cell length after electrical stimulation.

Adding (R, R) -fenoterol (10)^-8To 10^-5M), resulting in a significantly improved inotropic effect and a significant dose-response curve shift upward relative to (±) -fenoterol (fig. 4A). Increases from 265 + -11.6% to 306 + -11.8% resting cell length (p) by maximal contractile response<0.05) and EC₅₀Reduced from-7.0 + -0.2 to-7.1 + -0.2 log [ M ]](p<0.05) is described. In contrast, (S, S) -fenoterol had only a minor inotropic effect (fig. 4B).

Cardiomyocyte contractility studies indicated that (R, R) -fenoterol was responsible for the β 2-AR agonist activity observed in cardiomyocytes.

Example 5

Synthesis of

The general process is as follows: all reactions were carried out using commercial grade reagents and solvents. Tetrahydrofuran (THF) was dried by refluxing with sodium and benzophenone. The dichloromethane was dried by refluxing with calcium hydride. The UV spectrum was recorded on a Cary50Concentration spectrophotometer. Optical rotation measurements were performed on RudolphResearch Autopol IV. NMR spectra were recorded on a Varian Mercury VMX300-MHz spectrophotometer using tetramethylsilane as an internal standard. NMR peak split numbers are reported using the following abbreviations: s, singlet; d, double line; t, triplet; q, quartet; p, quintuple peak; m, multiplet; apt, obvious; and br, broad peak. Low resolution Mass Spectrometry in Finnigan LCQ equipped with Electrospray (ESI) and Atmospheric Pressure Chemical Ionization (APCI) probes^DuoLC MS/MS atmospheric pressure chemical ionization (API) quadrupole ion trap MS system. Analytical HPLC data were obtained using a Waters2690 SearationsModule with PDA detection. Method (a): ThermoHypersil BDS 100X4.6 mm C18 column, H₂O/CH₃CN/TFA. Method (b): brown Phenyl Spheri-5100X 4.6mm, water/acetonitrile/TFA. Method (c): vydac 150X 4mm C18 column, H₂O/isopropanol/TFA. Method (d):AD-H250×10mm,95/5/0.05CH₃CN/isopropanol/diethylamine. Merck silica gel (230-.

3',5' -dibenzyloxy-alpha-bromoacetophenone (46). 2.4mL (46mmol) of Br₂CHCl at 45mL₃The solution in (4) was added dropwise over 1 hour to a stirred solution of 9.66g (29mmol) of 3',5' -dibenzyloxyacetophenone (45) in 40mL of CHCl₃Of the solution in (1), the resulting solution was warmed to room temperature over 1 hour with good stirring, then poured into 100mL of cold water and transferred to a separatory funnel where CHCl was separated₃Fractions, washed with brine solution and dried (Na)₂SO₄) Filtered and concentrated to 10.8g, the material was applied to 500g silica gel using CHCl₃Elution afforded 2.65g (22%) of compound 46 as a white solid.¹H NMR(CDCl₃)δ4.39(s,2H),5.08(s,4H),6.85(t,1H,J=2.1Hz),7.20(d,2H,J=2.4Hz),7.31-7.44(m,10H)。

Used for carrying out enantioselective reduction on the compound 46 to obtain the 3',5' -dibenzyloxyphenyl bromoalcohol [ (R) -8, (S) -8]General procedure (2). 0.06mL (0.316mmol,10mol%) of 5.0M boron-dimethyl sulfide complex (BH) in diethyl ether under argon₃SCH₃) To a solution of 25mg (0.16mmol,5mol%) of the appropriate cis-1-amino-2-indanol in 3mL of anhydrous THF is added in one portion, and this material is added to a solution of 1.3g (3.16mmol) of 3',5' -dibenzyloxy- α -bromoacetophenone in 20mL of anhydrous THF under argon over 30 minutes, while 0.45mL of 5.0M boron-dimethylsulfide complex is added in 0.05mL portions. The resulting solution was stirred under argon for 2 hours and then quenched with 3mL of methanol with controlled evolution of gas. The solvent was removed in vacuo and the residue was taken up in 30mL of CHCl₃Neutralized and washed with 25mL of 0.2M sulfuric acid, then with 20mL of brine, then dried (Na)₂SO₄) Filtered and evaporated.

(R) - (-) -3',5' -dibenzyloxyphenyl bromohydrin [ R-8 ]]. Selective catalytic reduction using (1R,2S) - (+) -cis-1-amino-2-indanol as enantiomerPreparation of the preparation gave 1.02g (78%) of (R) -8 as a white fine powder.¹H NMR(CDCl₃)δ3.44(dd,1H,J=9.0,10.5Hz),3.55(dd,1H,J=3.3,10.5Hz),4.79(dd,1H,J=3.3,8.7Hz),4.97(s,4H),6.51(t,1H,J=2.4Hz),6.57(d,2H,J=1.8Hz),7.21-7.38(m,10H);[α]_D=-12.1°(c=1.0MeOH)。

(S) - (+) -3',5' -dibenzyloxyphenyl bromohydrin [ (S) -8)]. Prepared using (1S,2R) - (-) -cis-1-amino-2-indanol as an enantioselective reduction catalyst, to give 1.07g (82%) of (S) -8 as a fine white powder.¹H NMR(CDCl₃)δ3.43(dd,1H,J=9.0,10.5Hz),3.55(dd,1H,J=3.3,10.5Hz),4.78(dd,1H,J=3.3,8.7Hz),4.96(s,4H),6.50(t,1H,J=2.4Hz),6.57(d,2H,J=1.8Hz),7.21-7.39(m,10H);[α]_D=+11.8°(c=0.90MeOH)。

4-benzyloxyacetophenone (34). To 10.0g (41.3mmol) of 4-benzyloxyphenylacetic acid (31) were added 20mL of acetic anhydride and 20mL of pyridine, which were heated under argon atmosphere under reflux for 6 hours with stirring, the solvent was evaporated, and the residue was dissolved in CHCl₃(50mL) and washed with 1N NaOH (2X 50mL), and the organic layer was dried (MgSO 4)₄) Filtration and evaporation gave 11.8g of an amber oil. Vacuum distillation of 0.1mm Hg in an oil bath set at 170 ℃ followed by CH with 8/2₂Cl₂Chromatography on silica gel eluting with hexane gave 2.68g (27%).¹H NMR(CDCl₃)δ2.14(s,3H),3.63(s,2H),5.05(s,2H),6.94(d,2H,J=8.7Hz),7.10(d,2H,J=8.7Hz),7.26-7.47(m,5H)。

Acetophenone (35). A solution of 20.4g (0.15mol) of phenylacetic acid, acetic anhydride (70mL) and pyridine (70mL) was heated to reflux under argon for 6h with stirring, the solvent was evaporated and the residue was dissolved in CHCl₃(100mL), the organic layer was washed with 1N NaOH (2X 100mL) and dried (MgSO₄) Filtration and evaporation gave 20.4 g. Vacuum distillation at 0.1mm Hg in an oil bath set at 160 deg.C, followed by hexane/CH with 1/1₂Cl₂Chromatography on silica gel eluted gave 5.5g (27%).¹H NMR(CDCl₃)δ2.15(s,3H),3.70(s,2H),7.20-7.36(m,5H)。

1-naphthalen-1-yl-propan-2-one (36). A solution of 37.2g (20mmol) of naphthoic acid (33), acetic anhydride (100mL) and pyridine (100mL) was heated under reflux with stirring under argon for 6 hours, the solvent was evaporated, and the residue was dissolved in CHCl₃(200mL), the organic layer was washed with 1N NaOH (2X 150mL) and dried (MgSO)₄) Filtration and evaporation gave 34.6 g. Vacuum distillation at 0.5mm Hg in an oil bath set at 170 deg.C followed by hexane/CH with 1/1₂Cl₂Chromatography on silica gel eluted gave 9.7g (26%).¹H NMR(CDCl₃)δ2.11(s,3H),4.12(s,2H),7.40-7.53(m,4H),7.81(d,1H,J=8.4Hz),7.87-7.90(m,2H)。

General procedure for the preparation of 2-benzylaminopropane (37-39, 42, 43). To a temperature of 0 ℃ in CH₂Cl₂(c =0.5M) to the appropriate ketone (1eq) was added glacial acetic acid (1eq), followed by benzylamine (1eq) and NaBH (AcO)₃(1.4eq), the reaction mixture was warmed to room temperature and stirred under argon for 20 hours, the reaction mixture was cooled (ice bath), 10% NaOH (5eq) was added dropwise, and CH was extracted₂Cl₂Washed with brine, and then the product was dried (Na)₂SO₄) Filtered and evaporated.

1- (4-benzyloxy) -2-benzylaminopropane (37). Prepared from 4-benzyloxy-acetophenone (34;2.0g,8.3mmol) to give 2.61g (95%) as a tan solid.¹H NMR(CDCl₃)δ1.10(d,3H,J=6.3Hz),2.50-2.58(m,1H),2.68-2.77(m,1H),2.82-2.89(m,1H),3.75(dd,2H,J=12Hz,J=30Hz),5.05(s,2H),6.90(d,2H,J=8.7Hz),7.04(d,2H,J=8.7Hz),7.17-7.42(m,10H);MS(APCI+)m/z(rel):332(100)。

1-phenyl-2-benzylaminopropane (38). Prepared from acetophenone (35; 5.5g,41mmol) to give 8.4g (91%) as a tan solid.¹H NMR(CDCl₃)δ1.09(d,3H,J=6.3Hz),2.61-2.81(m,2H),2.92(m,1H),3.80(dd,2H),7.14-7.30(m,10H);MS(APCI+)m/z(rel):226(100)。

1- (1' -naphthyl) -2-benzylaminopropane (39). From 1-naphthalen-1-yl-propan-2-one (36; 5.0)g,27.1mmol) to yield 7.0g (94%) as a tan solid.¹H NMR(CDCl₃)δ1.14(d,3H,J=6.0Hz),3.02-3.18(m,2H),3.27(m,1H),3.80(dd,2H,J=13.2,43.8Hz),7.13-7.23(m,5H),7.31-7.48(m,4H),7.73(d,1H,J=7.8Hz),7.83-7.86(m,1H),7.96-7.99(m,1H);MS(APCI+)m/z(rel):276(100)。

1- (4' -methoxyphenyl) -2-benzylaminopropane (42). Prepared from 4-methoxyacetophenone (40;2.75g,13.1mmol) to give 2.31g (97%).¹H NMR(CDCl₃)δ1.10(d,3H,J=6.3Hz),2.56-2.75(m,2H),2.90(m,1H),3.79(s,1H),3.79(m,2H,J=13.2Hz),6.82(d,2H,J=8.7Hz),7.07(d,2H,J=8.7Hz),7.18-7.32(m,5H);MS(APCI+)m/z(rel):256(100)。

1- (4' -nitrophenyl) -2-benzylaminopropane (43). Prepared from 4-nitroacetophenone (41;4.95g,28mmol) to give 7.32g (98%) as an amber oil.¹H NMR(CDCl₃)δ1.60(d,3H,J=6.3Hz),2.73-2.85(m,1H),3.00-3.12(m,2H),3.86(dd,2H,J=26Hz,J=60Hz),7.23-7.40(m,5H),7.30(d,2H,J=9.0Hz),8.14(d,2H,J=8.7Hz).MS(APCI+)m/z(rel):271(100)。

2-benzylaminopropane [ (R) -10-14, (S) -10-14]General procedure for the enantiomeric separation of (a). The appropriate racemic 2-benzylaminopropane (1eq) was combined with the appropriate optically active mandelic acid (1eq) in methanol (c =0.5M) and refluxed until the solution became homogeneous, then cooled to room temperature, the crystals were filtered, collected, crystallized, and recrystallized twice from methanol (c =0.3M) to give the optically active 2-benzylaminopropane mandelate salt by heating at 10% K₂CO₃And CHCl₃Partitioning mandelate salt to convert salt to free amine for NMR and optical data collection, drying organic extract (Na)₂SO₄) And evaporated.

(R) - (-) -1- (4' -benzyloxy) -2-benzylaminopropane [ (R) -10]. 2.13g (6.42mmol) of 1- (4-benzyloxy) -2-benzylaminopropane (37) are reacted with 972mg (6.42mmol) of (R) - (-) -mandelic acid to yield 295mg (28% based on enantiomeric abundance) of the free amine after work-up.¹H NMR(CDCl₃)δ1.12(d,3H,J=6.3Hz),2.58-2.78(m,2H),2.82-2.91(m,1H),3.75(dd,2H,J=12Hz,J=30Hz)),5.07(s,2H),6.93(d,2H,J=8.7Hz),7.10(d,2H,J=8.7Hz),7.21-7.42(m,10H);MS(APCI+)m/z(rel):332(100);[α]_D=-19.1°(c=1.4,MeOH)。

(S) - (+) -1- (4' -benzyloxy) -2-benzylaminopropane [ (S) -10]. The wash solution obtained at the separation from (R) -10 was concentrated and purified in 50mL of chloroform and 50mL of 10% K₂CO₃The organic was washed with brine and dried (Na)₂SO₄) Filtered and evaporated to 1.70g (5.1mmol), the organics are refluxed with 782mg (5.1mmol) of (S) - (+) -mandelic acid (as before) and crystallized 3 times to give 670mg of (S) -amine- (S) -mandelate salt. (S) -amine (S) -mandelate salt triturated in ether then 30mL chloroform and 20mL10% K₂CO₃Was partitioned between water and the organic isolate was washed with brine and then dried (Na)₂SO₄) Filtered and evaporated to yield 366mg of free amine (33% based on enantiomeric abundance).¹H NMR(CDCl₃)δ1.10(d,3H,J=6.3Hz),2.58-2.78(m,2H),2.82-2.91(m,1H),3.76(dd,2H,J=12,30Hz),5.06(s,2H),6.93(d,2H,J=8.7Hz),7.09(d,2H,J=8.7Hz),7.21-7.42(m,10H);MS(APCI+)m/z(rel):332(100);[α]_D=+19.2°(c=1.5MeOH)。

(R) - (-) -1- (4' -methoxyphenyl) -2-benzylaminopropane [ (R) -11]. A sample of 3.02g (11.8mmol) of 1- (4' -methoxyphenyl) -2-benzylaminopropane (42) is reacted with 1.8g (11.8mmol) of (R) - (+) -mandelic acid to yield after work-up 530mg (35%, based on enantiomeric abundance) of the free amine.¹H NMR(CDCl₃)δ1.10(d,3H,J=6.3Hz),2.57-2.76(m,2H),2.88-2.94(m,1H),3.79(s,3H),3.72-3.88(m,2H),6.82(d,2H,J=8.7Hz),7.07(d,2H,J=8.4Hz),7.15-7.31(m,5H);MS(APCI+)m/z(rel):256(100);[α]_D=-30.4°(c=1.25MeOH)。

(S) - (+) -1- (4' -methoxyphenyl) -2-benzylaminopropane [ (S) -11]. A sample of 3.36g (13.2mmol) of the racemate 1- (4' -methoxyphenyl) -2-benzylaminopropane (42) is reacted with 2.0g (13.2mmol) of (R) - (-) -mandelic acid to yield, after work-up, 740mg (44%, based on enantiomeric abundance) of the free amine.¹H NMR,(CDCl₃)δ1.10(d,3H,J=6.2Hz),2.55-2.76(m,2H),2.88-2.95(m,1H),3.73-3.88(m,2H),3.79(s,3H),6.80(d,2H,J=8.7Hz),7.08(d,2H,J=8.4Hz),7.15-7.30(m,5H);MS(APCI+)m/z(rel):256(100);[α]_D=+30.5°(c=1.1MeOH)。

(R) - (-) -1- (4' -nitrophenyl) -2-benzylaminopropane [ (R) -12)]. A sample of 2.0g (7.3mmol) of 1- (4' -nitrophenyl) -2-benzylaminopropane (43) was reacted with 1.13g (7.3mmol) of (S) - (+) -mandelic acid to yield 486mg (49% based on enantiomeric abundance) of the free amine after work-up.¹H NMR(CDCl₃)δ1.60(d,3H,J=6.3Hz),2.73-2.85(m,1H),3.00-3.12(m,2H),3.86(dd,2H,J=26Hz,J=60Hz),7.23-7.40(m,5H),7.30(d,2H,J=9.0Hz),8.14(d,2H,J=8.7Hz);MS(APCI+)m/z(rel):271(100);[α]_D=-9.3°(c=1.0MeOH)。

(S) - (+) -1- (4' -nitrophenyl) -2-benzylaminopropane [ (S) -12]. A sample of 2.0g (7.3mmol) of 1- (4' -nitrophenyl) -2-benzylaminopropane (43) was reacted with 1.13g (7.3mmol) of (R) - (-) -mandelic acid to yield after work-up 640mg (65% based on enantiomeric abundance) of the free amine.¹H NMR(CDCl₃)δ1.60(d,3H,J=6.3Hz),2.73-2.85(m,1H),3.00-3.12(m,2H),3.86(dd,2H,J=26,60Hz),7.23-7.40(m,5H),7.30(d,2H,J=9.0Hz),8.14(d,2H,J=8.7Hz);MS(APCI+)m/z(rel):271(100);[α]_D=+8.2°(c=1.0MeOH)。

(R) - (-) -1-phenyl-2-benzylaminopropane [ (R) -13)]. A sample of 2.62g (11.6mmol) of 1-phenyl-2-benzylaminopropane (38) was reacted with 1.77g (11.6mmol) of (S) - (+) -mandelic acid to give after work-up 747mg (57% based on enantiomeric abundance) of the free amine.¹HNMR(CDCl₃)δ1.13(d,3H,J=6.0Hz),2.62-2.84(m,2H),2.92-2.99(m,1H),3.81(dd,2H,J=13.2,34.5Hz)7.14-7.29(m,10H);MS(APCI+)m/z(rel):226(100);[α]_D=-24.5°(c=1.10MeOH)。

(S) - (+) -1-phenyl-2-benzylaminopropane [ (S) -13)]. A sample of 5.0g (22.2mmol) of racemic 1-phenyl-2-benzylaminopropane (38) is reacted with 3.4g (22.2mmol) of (R) - (-) -mandelic acid to yield after workup 2.15g (86%, based on p-mandelic acid)Enantiomeric abundance).¹H NMR(CDCl₃)δ1.11(d,3H,J=6.0Hz),2.62-2.84(m,2H),2.92-2.99(m,1H),3.81(dd,2H,J=13.2,34.5Hz),7.14-7.29(m,5H);MS(APCI+)m/z(rel):226(100);[α]_D=+18.2°(c=0.85MeOH)。

(R) - (-) -1- (1' -naphthyl) -2-benzylaminopropane [ (R) -14)]. The wash recovered from the separation of (S) -14 was concentrated and washed with 40mL of chloroform and 40mL of 10% K₂CO₃The organic isolate was washed with 20mL brine and then dried (Na)₂SO₄) This gave 1.16g (4.2mmol) of the free amine, which was reacted with 640mg (4.2mmol) of (S) - (+) -mandelic acid to give 588mg (46%, based on enantiomeric abundance) of the free amine.¹H NMR(CDCl₃)δ1.07(d,3H,J=6.0Hz),3.02-3.18(m,2H),3.27(m,1H),3.74(dd,2H,J=13.2,30.9Hz),7.13-7.23(m,5H),7.31-7.48(m,4H),7.73(d,1H,J=7.8Hz),7.83-7.86(m,1H),7.96-7.99(m,1H);MS(APCI+)m/z(rel):276(100);[α]_D=-5.8°(c=1.0MeOH)。

(S) - (+) -1- (1' -naphthyl) -2-benzylaminopropane [ (S) -14)]. A sample of 2.6g (9.4mmol) of 1- (1' -naphthyl) -2-benzylaminopropane (39) is reacted with 1.44g (9.4mmol) of (R) - (-) -mandelic acid to yield after work-up 420mg (21% based on enantiomeric abundance) of the free amine.¹H NMR(CDCl₃)δ1.07(d,3H,J=6.0Hz),3.02-3.18(m,2H),3.27(m,1H),3.74(dd,2H,J=13.2,30.9Hz),7.13-7.23(m,5H),7.31-7.48(m,4H),7.73(d,1H,J=7.8Hz),7.83-7.86(m,1H),7.96-7.99(m,1H);MS(APCI+)m/z(rel):276(100);[α]_D=+6.3°(c=1.0MeOH)。

(R) - (-) -2-benzylaminoheptane [ (R) -15)]. A sample of 0.65mL (4.4mmol) of (R) - (-) -2-aminoheptane (R-44), 0.44mL (4.4mmol) of benzaldehyde and 0.1mL of HOAc in 40mL of CH₂Cl₂Are combined and then cooled to 0 ℃ and 2.75mg (13mmol) of sodium triacetoxyborohydride are added in one portion to the reaction mixture, which is stirred at room temperature under argon for 28 hours, and the reaction mixture is stirred with 30mL of CH₂Cl₂Diluted, cooled in an ice bath and 80mL of 5% NaOH in water was added, the fractions were separated and the organics were dried (Na)₂SO₄) And evaporated to give 638mg (71%) of (R) -15.¹H NMR(CDCl₃)δ0.88(m,3H),1.08(d,3HJ=6.6Hz),1.20-1.39(m,6H),1.41-1.67(m,2H),3.62-3.77(m,1H),3.75(p,2H,J=12Hz),7.17-7.41(m,5H);MS(APCI+)m/z(rel):206(100);[α]_D=+6.9°(c=1.0,MeOH)。

(S) - (+) -2-benzylaminoheptane [ (S) -15]. A sample of 0.15mL (1mmol) of (S) - (+) -2-aminoheptane ((S) -44), 0.1mL (1mmol) of benzaldehyde and 0.1mL of HOAc in 10mL of CH₂Cl₂Combined and cooled to 0 ℃ then 650mg (3mmol) of sodium triacetoxyborohydride are added in one portion, the reaction mixture is stirred at room temperature under argon for 28 hours, the mixture is diluted with 10mL of dichloromethane, cooled in an ice bath and 20mL of 5% NaOH in water are added, the fractions are separated and the organics are dried (Na₂SO₄) And evaporated to give 154mg (70%).¹H NMR(CDCl₃)δ0.88(m,3H),1.08(d,3H J=6.6Hz),1.19-1.37(m,6H),1.41-1.67(m,2H),3.62-3.77(m,1H),3.75(p,2H,J=12Hz),7.17-7.41(m,5H);MS(APCI+)m/z(rel):206(100);[α]_D=+7.8°(c=1.0,MeOH)。

Preparation of fenoterol analogues, process a. To form the epoxide, the appropriate 3',5' -dibenzyloxyphenylbromohydrin ((R) -8) or (S) -8(1eq) is reacted with K₂CO₃(1.4eq) combined in 1:1 THF/MeOH (c =0.3M) and stirred at room temperature for 2 hours under argon atmosphere, the solvent was removed, the residue partitioned between toluene and water, the toluene fraction was separated, dried (Na)₂SO₄) Filtered and evaporated. The residue is dissolved with the appropriate free benzylamine (R) -or (S) -10-15, 28(0.95eq) in a sufficient amount of toluene and evaporated again in vacuo to remove traces of water, the resulting colourless residue is heated to 120 ℃ for 20 hours under an argon atmosphere, cooled and passed through¹H NMR and mass spectroscopy to confirm ligation. The residue was dissolved in EtOH (C =0.07M) with heating and transferred to a Parr flask, hydrogenated in a flask under 50psi of hydrogen using 10% (wt) Pd/C (10mg catalyst/65 mg bromohydrin) for 24 hours, the complete debenzylation being confirmed by mass spectroscopy. The mixture was filtered through siliconKieselguhr, the filter cake was rinsed with isopropanol, the filtrate was concentrated, the residue was dissolved in 1:1 isopropanol/EtOH (c =0.2M) and refluxed with 0.5eq of fumaric acid for 30 minutes, the reaction was cooled and the solvent was removed, and the crude material was purified by open column chromatography or preparative chromatography.

Column separation of (R, R) -1 and (S, S) -1, Process B. A75 mg sample of fenoterol HBr was dissolved in 1.5mL of 95/5/0.05CH₃CN/isopropyl alcohol/HNEt₂Neutralizing and injecting 100. mu.L intoAD-H10X 250mm5 μm semi-preparative columns using Waters2690Separations Module, PDA set at 280 nm. Elution solvent is 95/5/0.05CH₃CN/isopropyl alcohol/HNEt₂5 mL/min. The retention times of the (S, S) and (R, R) isomers were 4.8 minutes and 7.8 minutes, respectively.

(R, R) - (-) -fenoterol [ (R, R) -1)]. Obtained according to procedure B, collected after evaporation to yield 40 mg.¹H NMR(CD₃OD)δ1.05(d,3H,J=6.3Hz),2.49(q,1H,J=6.9Hz),2.62-2.74(m,2H),2.80-2.91(m,2H),4.55(dd,1H,J=5.1,J=3.3Hz),6.16(t,1H,J=2.4Hz),6.27(d,2H,J=2.1Hz),6.68(d,2H,J=8.4Hz),6.94(d,2H,J=8.4Hz);¹³C NMR(CD₃CN)δ20.3,43.2,55.1,55.2,72.4,102.2,105.4,116.0,131.3,131.8,147.4,156.2,159.0;UV(MeOH)λmax279nm(ε2,760),225(12,900),204(32,600);MS(APCI+)m/z(rel):304(100,M+H);[α]_D= 29.0 ° (concentration =0.2% MeOH); HPLC (a)0.1% diethylamine in H₂In O, 0.50mL/min,254nm, t_R2.90min,99% pure, (d) t_R7.8min,>99% pure.

(S, S) - (+) -fenoterol [ (S, S) -1)]. Obtained according to procedure B to yield 35mg after evaporation.¹H NMR(CD₃OD)δ1.05(d,3H,J=6.6Hz),2.49(q,1H,J=7.2Hz),2.62-2.76(m,2H),2.80-2.94(m,2H),4.55(dd,1H,J=4.8,J=3.3Hz),6.16(t,1H,J=2.1Hz),6.27(d,2H,J=2.4Hz),6.68(d,2H,J=8.4Hz),6.94(d,2H,J=8.4Hz);¹³C(CD₃CN)δ20.3,43.2,55.0,55.2,72.4,102.2,105.4,116.0,131.3,131.8,147.4,156.2,159.0;UV(MeOH)λmax279nm(ε2,680),224(12,700),204(32,800);MS(APCI+)m/z(rel):304(100,M+H);[α]_D= 28.5 ° (concentration =0.20% MeOH); HPLC (a)0.1% diethylamine in H₂In O, 0.50mL/min,254nm, t_R2.72min,>99% pure, (d) t_R4.8min,>99% pure.

(R, S) - (-) -fenoterol fumarate [ (R, S) -1-]. Prepared according to procedure A from (R) -8 and (S) -10 to yield 168mg (64%).¹H NMR(CD₃OD)δ1.22(d,3H,J=6.6Hz),2.64(dd,1H,J=9.9Hz,J=13.2Hz),3.01-3.51(m,4H),4.79(dd,1H,J=3.0Hz,J=9.9Hz),6.23(t,1H,J=2.4Hz),6.36(d,2H,J=2.1Hz),6.75(s,1H),6.76(d,2H,J=8.4Hz),7.05(d,2H,J=8.1Hz);¹³C NMR(CD₃OD)δ16.2,39.1,52.5,57.4,70.4,103.4,105.3,116.7,127.8,131.4,135.2,144.6,157.7,160.0,168.2;UV(MeOH)λmax278nm(ε2,520),205(27,900);MS(ESI+)m/z(rel):304(100,M+H);[α]_D=7.5 ° (concentration =0.75% MeOH), HPLC (a)70/30/0.05.1.00mL/min,282nm, t_R1.35min,>99% pure, (b)50/50/0.05.1.0mL,0.50mL/min,254nm, t_R2.72min,>99% pure, (d) t_R4.8min,1.00mL/min,280nm,t_R2.10min,97.5% pure.

(S, R) - (+) -fenoterol fumarate [ (S, R) -1)]. Prepared according to procedure A from (S) -8 and (R) -10 to yield 104mg (39%).¹H NMR(CD₃OD)δ1.22(d,3H,J=6.6Hz),2.64(dd,1H,J=9.9Hz,J=13.5Hz),3.47-3.04(m,4H),4.80(dd,1H,J=2.7,J=9.6Hz),6.23(t,1H,J=2.4Hz),6.36(d,2H,J=2.1Hz),6.75(s,1H),6.76(d,2H,J=8.4Hz),7.05(d,2H,J=8.4Hz);¹³C NMR(CD₃OD)δ16.2,39.1,52.5,57.4,70.4,103.4,105.3,116.7,127.8,131.4,135.2,144.6,157.7,159.9,168.2;UV(MeOH)λmax278nm(ε2,640),202(36,600);MS(ESI+)m/z(rel):304(100,M+H),413(10);[α]_D= 6.4 ° (concentration =0.50% MeOH), HPLC (a)70/30/0.05,1.00mL/min,282nm, t_R1.35min,95.9% pure, (b)50/50/0.05,1.0mL/min,280nm, t_R2.06min,99% pure.

(R, R) - (-) -1-p-methoxyphenyl-2- (beta-3 ',5' -dihydroxy-phenyl-beta-oxy) ethylamino-propane fumarate [ (R, R) -2]. Prepared according to procedure A from (R) -8 and (R) -11 to yield 172mg (38%).¹H NMR(CD₃OD)δ1.08(d,3H,J=6.3Hz),3.05-2.56(m,5H),4.57(dd.IH,J=8.4,5.4Hz),6.16(m,1H),6.26(d,2H,J=2.7Hz),6.81(d,2H,J=8.7Hz),7.03(d,2H J=8.7Hz);¹³C NMR(CD₃OD)δ18.8,42.3,54.5,55.6,56.0,72.6,103.0,105.4,115.0,131.1,131.2,131.3,146.2,159.8,159.9;UV(MeOH)λmax277nm(ε3,590),224(17,700),207(29,500);MS(ESI+)m/z(rel):318(100,M+H);[α]_D=-24.9°(c=0.8MeOH);HPLC:(a)70/30/0.05,1.0mL/min,282nm,t_R1.54min,96.5% pure, (b)50/50/0.05,2.0mL/min,276nm, t_R1.51min,95.9% pure.

(S, S) - (+) -1-p-methoxyphenyl-2- (. beta. -3',5' -dihydroxy-phenyl-. beta. -oxy) ethylamino-propane fumarate [ (S, S) -2]. Prepared according to procedure A from (S) -8 and (S) -11 to provide 318mg (53%).¹H NMR(CD₃OD) δ 1.15(d,3H, J =6.0Hz),2.58-3.22(m,5H),3.77(s,3H),4.68(dd,1H, J =4.8,8.4Hz),6.18(t,1H, J =2.1Hz),6.31(d,2H, J =2.1Hz),2.23(s,0.5H, fumarate), 6.84(d,2H, J =8.7Hz),7.10(d,2H, J =9.0Hz);¹³C NMR(CD₃OD)δ16.1,39.9,52.4,54.5,55.3,70.4,101.9,104.2,114.0,129.2,130.1,144.4,158.7,158.9;UV(MeOH)λmax277nm(ε2,100),224(11,00),205(22,700);MS(ESI+)m/z(rel):318(100,M+H);[α]_D= 28.6 ° (c =0.95MeOH), HPLC (a)70/30/0.05,1.0mL/min,282nm, preparation 1.67min,96.0% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.51min,97.1% pure.

(R, S) - (-) -1-p-methoxyphenyl-2- (beta-3 ',5' -dihydroxy-phenyl-beta-oxy) ethylaminopropane fumarate [ (R, S) -2]. Prepared according to procedure A from (R) -8 and (S) -11 to yield 160mg (38%).¹H NMR(CD₃OD) δ 1.20(d,3H, J =6.6Hz),2.62-2.71(m,1H),2.98-3.20(m,3H),3.30-3.42(m,2H),4.73-4.81(m,1H),6.21(m,2H),3.35(m,2H),6.71(s,0.5H, fumarate), 6.56-6.89(m,2H),7.11-7.19(m,2H);¹³C NMR(CD₃OD)δ15.6,38.5,51.8,54.5,55.9,69.7,102.1,104.1,114.1,128.5,130.2,136.0,143.8,158.8,159.1;UV(MeOH)λmax277nm(ε4,100),224(21,400),203(50,600);MS(ESI+)m/z(rel):318(100,M+H);[α]_D=-7.2°(c=1.5MeOH);HPLC:(a)70/30/0.05,1.00mL/min,282nm,t_R1.40min,99% pure;(b)50/50/0.05,2.0mL/min,276nm,t_R1.51min,96.1% pure.

(S, R) - (+) -1-p-methoxyphenyl-2- (. beta. -3',5' -dihydroxyphenyl-. beta. -oxy) ethylaminopropane fumarate [ (S, R) -2]. Prepared according to procedure A from (S) -8 and (R) -11 to yield 200mg (51%).¹H NMR(CD₃OD)δ1.12(d,3H,J=6.0Hz),2.58-3.13(m,5H),3.77(s,3H),4.62(dd,1H,J=3.6,9.0Hz),6.15(m,1H),6.30(d,2H,J=1.8Hz),6.85(d,2H,J=8.7Hz),7.11(d,2H,J=8.7Hz);¹³C NMR(CD₃OD)δ18.2,41.4,54.1,55.7,56.5,64.7,103.0,105.3,115.1,130.7,131.3,145.9,159.8,160.0;UV(MeOH)λmax277nm(ε3,150),224(3,310),205(30,600);MS(ESI+)m/z(rel):318(100,M+H);[α]_D= 14.1 ° (c =0.95MeOH), HPLC (a)70/30/0.05,1.00mL/min,282nm, preparation 1.42min,97.7% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.52min,97.8% pure.

(R, R) - (-) -5- {2- [2- (4-aminophenyl) -1-methylethylamino group]-1-hydroxyethyl } -1, 3-benzenediol fumarate [ (R, R) -3]. Prepared according to procedure A from (R) -8 and (R) -12 to yield 88mg (42%).¹H NMR(CD₃OD) δ 1.23(m,3H),2.70-3.24(m,4H),3.54(m,1H),4.84(dd,1H, J =3.3,9.6Hz),6.23(t,1H, J =2.4Hz),6.38(d,2H, J =2.1Hz),6.75(s,2H, fumarate), 7.35(dd,4H, J =8.1,21.0Hz), 13C (CD)₃OD)δ15.5,39.6,52.7,56.6,70.3,103.4,105.3,123.8,132.0,132.1,135.2,137.5,144.7,160.0,168.1;UV(MeOH)λmax284nm(ε1,520),206(21,700);MS(ESI+)m/z(rel):303(100,M+H);[α]_D= 6.8 ° (concentration =1.0% MeOH); HPLC (a)80/20/0.05,0.70mL/min,276nm, t_R2.07min,95.5% pure, (b)50/50/0.05,1.0mL/min,282nm, t_R2.60,97.16% pure.

(S, S) - (+) -5- {2- [2- (4-aminophenyl) -1-methylethylamino group]1-hydroxyethyl } -1, 3-benzenediol fumarate [ (5,5) -3]. Prepared according to procedure A from (S) -8 and (S) -12 to yield 56mg (25%).¹H NMR(CD₃OD) δ 1.23(m,3H),2.62-3.27(m,4H),3.55(m,1H),4.74-4.88(m,1H),6.22(t,1H, J =1.8Hz),6.37(d,2H, J =2.4Hz),6.75(s,2H, fumarate), 7.32(dd,4H, J =8.7,25.8Hz);¹³C NMR(CD₃OD)δ15.5,39.6,52.5,56.7,70.7,103.4,105.3,123.3,131.8,132.0,135.2,136.9,144.7,160.0,168.1;UV(MeOH)λmax284nm(ε1,720),207(28,400);MS(ESI+)m/z(rel):303(100,M+H),329(20);[α]_D= 11.1 ° (concentration =0.50% MeOH), HPLC (a)80/20/0.05,0.7mL/min,276nm, preparation 2.01min,<99% pure, (b)50/50/0.05,1.0mL/min,282nm, preparation 2.50min,99.4% pure.

(R, S) - (-) -5- {2- [2- (4-aminophenyl) -1-methylethylamino group]-1-hydroxyethyl } -1, 3-benzenediol fumarate [ (R, S) -3]. Prepared according to procedure A from (R) -8 and (S) -12 to yield 72mg (35%).¹H NMR(CD₃OD) δ 1.23(m,3H),2.73-3.24(m,4H),3.51(m,1H),4.80(dd,1H, J =2.7,9.6Hz),6.22(t,1H, J =2.1Hz),6.36(d,2H, J =2.4Hz),6.75(s,2H, fumarate), 7.32(dd,4H, J =8.4,25.2Hz);¹³CNMR(CD₃OD)δ16.1,39.12,5.16,56.9,70.4,103.4,105.3,123.4,132.0,132.0,135.2,136.8,144.6,160.,168.10;UV(MeOH)λmax284nm(ε1,620),205(27,200);MS(ESI+)m/z(rel):303(100,M+H),134(14);[α]_D=7.5 ° (concentration =0.50% MeOH), HPLC (a)80/20/0.05,0.7mL/min,276nm, t_R2.08min,95.0% pure, (b)50/50/0.05,1.0mL/min,282nm, t_R2.51min,97.4% pure.

(S, R) - (+) -5- {2- [2- (4-aminophenyl) -1-methylethylamino group]-1-hydroxyethyl } -1, 3-benzenediol fumarate [ (S, R) -3]. Prepared according to procedure A from (S) -8 and (R) -12 to yield 93mg (42%).¹H NMR(CD₃OD) δ 1.23(d,3H, J =6.3Hz),2.70-3.78(m,4H),3.42-3.62(m,1H),4.80(dd,1H, J =3.0,9.9Hz),6.22(t,1H, J =2.1Hz),6.37(d,2H, J =2.1Hz),6.75(s,2H, fumarate), 7.33(dd,4H, J =8.4,26.7Hz);¹³C NMR(CD₃OD)δ16.2,39.1,52.6,56.9,70.5,103.4,105.3,123.5,132.1,133.7,135.2,137.1,144.7,160.0,168.1UV(MeOH)λmax284nm(ε8,230),207(100,000);MS(ESI+)m/z(rel):303(100,M+H),134(18);[CC]d = +11.4 ° (concentration =0.50% MeOH), HPLC (a)70/30/0.05,1.00mL/min,280nm, t_R1.45min,99% pure, (b)50/50/0.05,1.0mL/min,282nm, t_R2.63min,95.33% pure.

(R, R) - (-) -5- [ 1-hydroxy-2- (1-methyl-2-phenylethylamino) ethyl]1, 3-Benzenediol fumarate [ (R, R) -4-]. According to Process A, from (R) -8 and(R) -13 preparation, yielding 92mg (26%).¹H NMR(CD₃OD) δ 1.22(m,3H),2.68-3.28(m,2H),3.10-3.28(m,2H),3.53(br-m,1H),4.75-4.80(m,1H),6.24(t,1H, J =2.4Hz),6.38(d,2H, J =2.1Hz),6.75(s,1H, fumarate), 7.22-7.33(m,5H);¹³C NMR(CD₃OD)δ15.5,40.3,56.9,70.2,103.4,105.3,128.3,129.9,130.3,135.2,137.3,144.6,144.6,159.9,168.1;UV(MeOH)λmax277nm(ε926),204(18,700);MS(APCI+)m/z288(100,M+H);[α]_D= 21.2 ° (concentration =0.85% MeOH), HPLC (a)50/50/0.05,1.00mL/min,282nm, preparation 1.73min, 99% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.46min,97.5% pure.

(S, S) - (+) -5- [ 1-hydroxy-2- (1-methyl-2-phenylethylamino) ethyl]1, 3-Benzenediol fumarate [ (S, S) -4-]. Prepared according to procedure A from (S) -8 and (S) -13 to yield 184mg (51%).¹H NMR(CD₃OD) δ l.21(m,3H),2.70-3.13(m,2H),3.15-3.23(m,2H),3.54(br-m,1H),4.79-4.86(m,1H),6.24(t,1H, J =2.1Hz),6.39(t,2H, J =2.7Hz),6.76(s,1H, fumarate), 7.22-7.32(m,5H);¹³C NMR(CD₃OD)δ15.5,40.3,56.9,70.2,103.4,105.3,128.3,129.9,130.3,135.1,137.3,144.6,144.6,159.9,168.1UV(MeOH)λmax278nm(ε1,510),207(26,600);MS(APCI+)m/z288(100,M+H);[α]_D= 19.3 ° (concentration =0.90% MeOH), HPLC (a)50/50/0.05,1.00mL/min,282nm, preparation 1.49min, 98.4% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.35min,99% pure.

(R, S) - (-) -5- [ 1-hydroxy-2- (1-methyl-2-phenylethylamino) ethyl]1, 3-Benzenediol fumarate [ (R, S) -4-]. Prepared according to procedure A from (R) -8 and (S) -13 to yield 170mg (45%).¹H NMR(CD₃OD) δ 1.22(m,3H),2.68-3.28(m,2H),3.13-3.28(m,2H),3.53(br-m,1H),4.76-4.80(m,1H),6.23(t,1H, J =2.1Hz),6.37(t,2H, J =3.0Hz),6.75(s,1H, fumarate), 7.24-7.37(m,5H);¹³C NMR(CD₃OD)δ16.3,24.2,39.8,57.2,70.5,103.4,105.3,128.4,130.0,130.4,135.2,137.4,144.6,160.1UV(MeOH)λmax278nm(ε1,110),205(31,000);MS(APCI+)m/z(rel):288(100,M+H);[α]_D=6.9 ° (concentration =0.85% MeOH), HPLC (a)50/50/0.05,1.00mL/min,282nm, preparation 1.53min,99% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.46min,98.5% pure.

(S, R) - (+) -5- [ 1-hydroxy-2- (1-methyl-2-phenylethylamino) ethyl]1, 3-Benzenediol fumarate [ (S, R) -4-]. Prepared according to procedure A from (S) -8 and (R) -13 to yield 212mg (59%).¹H NMR(CD₃OD) δ 1.22(m,3H),2.72(dd,1H J =10.2,13.2Hz),3.11(dd,1H, J =10.2,12.6Hz),3.18-3.27(m,2H),3.48-3.61(m,1H),4.83(dd,1H, J =3.3,9.9Hz),6.22(t,1H, J =2.4Hz),6.36(d,2H, J =2.4Hz),6.75(s,1H, fumarate), 7.24-7.37(m,5H);¹³C NMR(CD₃OD)δ16.3,24.2,39.8,57.2,70.5,103.4,105.3,128.4,130.0,130.4,135.2,137.4,144.6,160.1;UV(MeOH)λmax278nm(ε1,680),206(35,500);MS(APCI+)m/z(rel):288(100,M+H),270(19,M-OH);[α]_D= 9.1 ° (concentration =1.1%, MeOH), HPLC (a)50/50/0.05,1.00mL/min,282nm, preparation 1.51min,99% pure, (b)50/50/0.05,2.0mL/min,276nm, preparation 1.43min,99% pure.

(R, R) - (-) -5- { 1-hydroxy-2- [ 1-methyl-2- (1-naphthyl) ethylamino]Ethyl } -1, 3-benzenediol fumarate [ (R, R) -5]. Prepared according to procedure A from (R) -8 and (R) -14 to yield 135mg (46%).¹H NMR(CD₃OD)δ1.18-1.23(m,3H),3.16-3.34(m,lH,2H),3.69-3.74(m,2H),4.78-4.80(m,1H),6.23(t,1H,J=2.4Hz),6.38(m,2H),7.41-7.61(m,4H),7.83(d,1H,J=7.5Hz),7.90(d,1H,J=7.8Hz),8.10(m,1H);¹³C NMR(CD₃OD) delta 16.2,37.2,54.5,56.1,70.3,103.4,105.3,124.3,126.5,127.0,127.7,129.3,130.1,133.2,135.2,135.6,144.7,160.1,168.2, UV (MeOH) lambda max282nm (. epsilon.5, 860),224(50,900),208(35,500), MS (APCI +) M/z (rel), 338(100, M + H),169(15, fragment) (. alpha.) [ alpha. + H ], (E) ]]_D= 20.4 (concentration =0.50% MeOH), HPLC (a)60/40/0.05,1.00mL/min,282nm, preparation 2.08min,95.7% pure, (b)50/50/0.05,1.5mL/min,282nm, preparation 2.20min,99% pure.

(S, S) - (+) -5- { 1-hydroxy-2- [ 1-methyl-2- (1-naphthyl) ethylamino]Ethyl } -1, 3-benzenediol fumarate [ (S, S) -5%]. Prepared according to procedure A from (S) -8 and (S) -14 to give 118mg (40%).¹H NMR(CD₃OD)δ1.13-1.17(m,3H),3.14-3.26(m,1H,2H),3.61-3.76(m,2H),4.44-4.75(m,1H),6.18(t,1H,J=2.4Hz),6.33(m,2H),7.36-7.52(m,4H),7.77(dd,1H,J=1.8,7.5Hz),7.84(d,1H,J=8.1Hz),8.04(t,1H,J=8.4Hz);¹³C NMR(CD₃OD) delta 16.1,37.1,54.4,56.0,70.3,103.3,105.3,124.3,126.5,127.0,127.7,129.2,130.0,133.2,135.2,135.7,144.5,160.0,168.1, UV (MeOH) lambda max282nm (epsilon 6,210),223(56,400),208(42,700), MS (APCI +) M/z (rel) 338(100, M + H),169(8, fragment) (. alpha.), [ alpha., (epsilon. alpha. max) 282 (epsilon. 6,210), and [ alpha]_D= 20.0 ° (concentration =1.1% MeOH), HPLC (a)60/40/0.05,1.00mL/min,282nm, preparation 2.35min,98.9% pure, (b)50/50/0.05,1.5mL/min,282nm, preparation 2.26min,97.2% pure.

(R, S) - (-) -5- { 1-hydroxy-2- [ 1-methyl-2- (1-naphthyl) ethylamino]Ethyl } -1, 3-benzenediol fumarate [ (R, S) -5]. Prepared according to procedure A from (R) -8 and (S) -14, to yield 114mg (39%).¹H NMR(CD₃OD) δ 1.08-1.11(m,3H),3.02-3.24(m,1H,2H),3.54-3.68(m,2H),4.45-4.75(m,1H),6.11(t,1H, J =1.8Hz),6.26(m,2H),6.63(s,2H fumarate), 7.28-7.48(m,4H),7.70(d,1H, J =7.5Hz),7.77(d,1H, J =7.8Hz),7.97(t,1H, J =7.8Hz);¹³C NMR(CD₃OD) delta 16.0,37.1,52.5,56.0,70.4,103.4,105.3,124.4,126.5,127.0,127.6,129.2,130.1,133.1,135.2,135.5,144.7,159.9,168.2, UV (MeOH) lambda max281nm (. epsilon.12, 600),224(61,900),204(47,200), MS (APCI +) M/z (rel), 338(100, M + H),190(15, fragment) (. alpha.) [ alpha.. alpha.]_D= 11.3 ° (concentration =0.85% MeOH), HPLC (a)60/40/0.05,1.00mL/min,282nm, t_R2.30min,98.6% pure, (b)50/50/0.05,1.5mL/min,282nm, t_R2.36min,99% pure.

(S, R) - (+) -5- { 1-hydroxy-2- [ 1-methyl-2- (1-naphthyl) ethylamino]Ethyl } -1, 3-benzenediol fumarate [ (S, R) -5]. Prepared according to procedure A from (S) -8 and (R) -14 to yield 123mg (42%).¹H NMR(CD₃OD) δ 1.18-1.22(m,3H),3.10-3.28(m,1H,2H),3.69-3.78(m,2H),4.45-4.75(m,1H),6.23(t,1H, J =2.1Hz),6.39(m,2H),6.73(s,2H fumarate), 7.39-7.59(m,4H),7.80(d,1H, J =7.5Hz),7.88(d,1H, J =7.8Hz),8.01(t,1H, J =9.0Hz);¹³C NMR(CD₃OD)δ16.4,37.4,52.5,56.2,70.6,103.4,105.3,124.4,126.5,127.0,129.3,130.1,133.1,133.4,135.6,136.3,144.8,160.0,171.4;UV(MeOH)λmax282nm(ε7,740),224(70,900),206(55,800);MS(ESI+)m/z(rel):338(100,M+H);[α]_D= 15.5 (concentration =1.0% MeOH) HPLC (a)60/40/0.05,1.0mL/min,282nm,t_R1.95,95.7% pure, (b)50/50/0.05,1.5mL/min,282nm, t_R2.29min,95.7% pure.

(R, R) - (-) -5- [ 1-hydroxy-2- (1-methylhexylamino) ethyl]1, 3-Benzenediol fumarate [ (R, R) -6]. Prepared according to procedure A from (R) -8 and (R) -15 to yield 45mg (29%).¹HNMR(CD₃OD) δ 0.920(t,3H, J =6.9Hz),1.30(d,3H, J =6.9Hz),1.29-1.64(m,8H),3.01-3.18(m,2H),3.14-3.30(m,1H),4.80(dd,1H, J =3.3,9.6Hz),6.22(t,1H, J =2.1Hz),6.36(d,2H, J =2.4Hz),6.75(s,1H, fumarate);¹³C NMR(CD₃OD)δ14.3,16.0,23.5,26.2,32.6,34.2,52.1,55.7,70.2,103.3,105.3,135.2,144.7,160.0,168.0;UV(MeOH)λmax278nm(ε931),203nm(20,100);MS(ESI+)m/z(rel):268(100,M+H);[α]_D= 8.8 ° (concentration =1.1% MeOH); HPLC (c)70/30/0.1,1.0mL/min,276nm, t_R2.18min,96.6% pure, (b)50/50/0.05,1.0mL/min,279nm, t_R2.06min,98.9% pure.

(S, S) - (+) -5- [ 1-hydroxy-2- (1-methylhexylamino) ethyl]1, 3-Benzenediol fumarate [ (S, S) -6]. Prepared according to procedure A from (S) -8 and (S) -15 to yield 96mg (43%).¹HNMR(CD₃OD) δ 0.923(t,3H, J =6.6Hz), 1.31(d,3H, J =6.6Hz),1.26-1.84(m,8H),2.01-3.18(m,2H),3.14-3.30(m,1H),4.81(dd,1H, J =3.3,9.6Hz),6.23(t,1H, J =2.4Hz),6.39(d,2H, J =2.1Hz),6.76(s,1H, fumarate):¹³C NMR(CD₃OD)δ14.2,16.0,23.4,26.3,32.6,34.1,52.1,55.8,70.2,103.4,105.3,135.2,144.7,159.9,168.2;UV(MeOH)λmax278nm(ε1,340),203(28,800);MS(APCI+)m/z(rel):268(100,M+H);[α]_D= 10.8 ° (concentration =0.50% MeOH); HPLC (c)70/30/0.1,1.0mL/min,276nm, t_R97.0% pure at 2.16min, (b)50/50/0.05,1.0mL/min,279nm, t_R2.11min,99% pure.

(R, S) - (-) -5- [ 1-hydroxy-2- (1-methylhexylamino) ethyl]1, 3-Benzenediol fumarate [ (R, S) -6]. Prepared according to procedure A from (R) -8 and (S) -15 to yield 83mg (38%).¹HNMR(CD₃OD) δ 0.924(m,3H), 1.32(d,3H, J =6.6Hz),1.26-1.84(m,8H),2.98-3.20(m,2H),3.32-3.22(m,1H),4.78(dd,1H, J =3.0,9.9Hz),6.23(t,1H, J =2.1Hz),6.37(d,2H, J =1.8Hz),6.76(s,1H, Rich)Salts of maleic acid);¹³CNMR(CD₃OD)δ14.2,16.4,23.4,26.2,32.6,33.5,52.2,56.0,70.4,103.4,105.3,135.2,144.7,160.0,168.1;UV(MeOH)λmax276nm(ε2,770),203(35,900);MS(APCI+)m/z(rel):268(100,M+H);[α]_D= 15.9 ° (concentration =0.70% MeOH), HPLC (c)70/30/0.1,1.0mL/min,276nm, t_R97.0% pure at 2.16min, (b)50/50/0.05,1.0mL/min,279nm, t_R2.07min,96.2% pure.

(S, R) - (+) -5- [ 1-hydroxy-2- (1-methylhexylamino) ethyl]1, 3-Benzenediol fumarate [ (S, R) -6]. Prepared according to procedure A from (S) -8 and (R) -15 to yield 81mg (38%).¹HNMR(CD₃OD) δ 0.920(t,3H, J =6.3Hz),1.32(d,3H, J =6.9Hz),1.30-1.77(m,8H),2.99-3.17(m,2H),3.23-3.26(m,1H),4.76(dd,1H, J =3.0,9.6Hz),6.22(t,1H, J =2.4Hz),6.36(d,2H, J =2.1Hz),6.75(s,1H, fumarate);¹³C NMR(CD₃OD)δ14.2,16.5,23.5,26.2,32.6,39.5,52.2,56.0,70.4,103.4,105.3,135.2,144.7,160.0,168.0;UV(MeOH)λmax278nm(ε1,440),204(29,900);MS(APCI+)m/z(rel):268(100,M+H);[α]_D= 12.7 ° (concentration =1.0% MeOH); HPLC (c)70/30/0.1,1.0mL/min,276nm, t_R2.16min,99% pure, (b)50/50/0.05,1.0mL/min,279nm, t_R2.02min,95.7% pure.

(R) - (-) -5- (1-hydroxy-2-phenylethylaminoethyl) -1, 3-benzenediol fumarate [ (R) -7]. Prepared from (R) -8 and 28 to yield 37mg (15%).¹H NMR(CD₃OD)δ2.94-3.23(m,6H),4.73(dd,1H,J=3.3,9.9Hz),6.15(t,1H,J=2.4Hz),6.29(d,2H,J=1.8Hz),7.19-7.28(m,5H),6.69(s,1H);UV(MeOH)λmax278nm(ε1,360),205(32,600);MS(APCI+)m/z(rel):274(100,M+H);[α]_D= 13.0 ° (concentration =1.0% MeOH), HPLC (a)80/20/0.05,1.00mL/min,282nm, t_R1.47min,96.7% pure, (b)50/50/0.05,1.0mL/min,272nm, t_R2.78min,95.1% pure.

(S) - (+) -5- (1-hydroxy-2-phenylethylaminoethyl) -1, 3-benzenediol fumarate [ (S) -7%]. Prepared from (S) -8 and 28 to yield 51mg (17%).¹HNMR(CD₃OD)δ2.87-3.21(m,6H),4.68(dd,1H,J=3.6,9.9Hz),6.10(t,1H,J=2.4Hz),6.24(d,2H,J=2.1Hz),6.63(s,1H),7.12-7.21(m,5H);UV(MeOH)λmax278nm(ε1,280),204(33,700);MS(APCI+)m/z(rel):274(100,M+H);[α]_D= 14.64 ° (concentration =1.1% MeOH); HPLC (a)80/20/0.05,1.00mL/min,282nm, t_R1.47min,98.6% pure, (b)50/50/0.05,1.0mL/min,272nm, t_R2.74min,98.8% pure.

(R, R) - (-) -ethyl fenoterol.

¹H NMR:(300MHz,CD₃OD):δ0.950(t,3H,J=7.5Hz),1.67(m,2H),2.83-3.18(m,4H),3.33-3.40(m,1H),3.37(s,4H),4.82(m,1H),6.24(d,1H,J=2.1Hz),6.37(d,2H,J=1.8Hz),6.73(s,2H,fum),6.76(d,2H,J=8.4Hz),7.05(d,2H,J=8.7Hz)ppm.CMR:¹³C(75MHz,CD₃OD). delta.9.43, 23.28,36.56,52.29,62.16,70.02,103.4,105.3,116.7,127.8,131.3,136.5,144.6,157.6,159.9,172.3ppm UV (methanol), lambda.max (. epsilon.). 206nm (22,500),223(12,300),278(2,460). MS (1CQ DUO ESI positive ion mass spectrometry) M/z (rel):318(100, M + H). HPLC1: column: Varian Sunfire C181004.6; 70/30/0.1 water/acetonitrile/TFA; 1.0mL/min; detection: 278nm;2.76min (fumarate, 6.99%),3.57min (90.11%); purity: 97.1%. HPLC2 column Chiralpak IA250x10, 90/10/0.05 acetonitrile/methanol/TFA, 2.0mL/min, detection 278nm, 5.26(RR isomer, 92.37%),7.11min (fumarate, 5.02%), purity 97.5%. specific rotation: [ alpha ] to]_D= 15.6 (free amine, 0.5% MeOH).

(R, S) - (-) -ethyl fenoterol.

¹H NMR:(300MHz,CD₃OD):δ0.972(t,3H,J=7.5Hz),1.70(p,2H,J=6.9Hz)),2.86-3.22(m,4H),3.32-3.37(m,1H),3.34(s,4H),4.82(m,1H),6.25(t,1H,J=2.1Hz),6.36(d,2H,J=1.8Hz),6.74(s,2H,fum),6.77(d,2H,J=8.4Hz),7.08(d,2H,J=8.7Hz)ppm.CMR:¹³C(75MHz,CD₃OD) delta 9.820,24.16,36.48,52.30,62.32,69.92,103.3,105.3,116.8,127.7,131.3,136.1,144.4,157.6,159.8,171.3ppm UV (methanol), lambda max (epsilon) 204nm (26,900),224(11,500),278(2,320). MS (LCQ DUO ESI positive ion mass spectrometry) M/z (rel) 318(100, M + H). HPLC l: column: Varian Sunfire C181004.6; 70/30/0.1 water/acetonitrile/TFA; 1.0mL/min; detection: 278nm;2.79min (fumarate, 3.34%),3.56min (96.11%); purity: 99.5% HPLC2 column Chiralpak IA250X10, 90/10/0.05 acetonitrile/methanol/TFA, 2.0mL/min, detection 278nm, 5.88(RS isomer, 97.08%),7.12min (fumarate, 2.92%); Purity>99% specific rotation [ alpha ]]_D= 7.2 (free amine, 0.5% MeOH).

C₂₂H₂₅NO₄?0.5C₄H₄O₄

¹H NMR:(300MHz,CD₃OD):δ1.22(t,3H,J=6.6Hz),3.09-3.21(m,3H),3.59-3.69(m,2H),3.99(s,3H),4.74-4.83(m,1H),6.23(t,1H,J=2.4Hz),6.37(dd,2H,J=2.4,5.7Hz),6.74(s,1H),6.86(d,1H,J=7.8Hz),7.32(d,1H,J=7.8Hz),7.48(t,1H,J=6.9Hz),7.56(t,1H,J=6.9Hz),8.02(dd,1H,J=8.4,12.0Hz),8.27(d,1H,J=8.7Hz)ppm.CMR:¹³C(75MHz,CD₃OD is delta 15.78,36.66,52.39,55.96,70.20,103.4,104.5,105.3,123.8,124.3,124.9,126.2,127.4,128.1,129.5,133.8,135.2,144.6,156.6,160.0,168.3ppm UV (methanol), lambda max (epsilon): 298nm (4,970),286(9,920),234(22,600),210(42,500). MS (1CQ DUO ESI positive ion mass spectrum) M/z (rel):368(100, M + H). specific rotation degree [ alpha ], (80, M + H)]_D= -28.8 (free amine; 0.5% MeOH).

C₂₂H₂₅NO₄·0.5C₄H₄O₄

¹H NMR:(300MHz,CD₃OD):δ1.20(t,3H,J=6.6Hz),3.07-3.21(m,3H),3.52-3.75(m,2H),3.97(s,3H),4.69-4.83(m,1H),6.24(t,1H,J=2.1Hz),6.39(dd,2H,J=2.4,5.4Hz),6.74(s,1H),6.84(d,1H,J=7.8Hz),7.31(d,1H,J=8.1Hz),7.48(t,1H,J=6.9Hz),7.56(t,1H,J=6.9Hz),8.01(dd,1H,J=8.4,13.5Hz),8.27(d,1H,J=7.8Hz)ppm.CMR:¹³C(75MHz,CD₃OD). delta.15.77, 36.64,52.37,55.94,70.46,103.4,104.5,105.3,123.8,124.3,124.9,126.2,127.4,128.1,129.4,133.8,135.5,144.7,156.6,160.0,169.0ppm.UV (methanol), lambda.max (. epsilon.). gtoren.298 nm (5,430),286(5,710),233(25,100),210(43,200). MS (1CQ DUO ESI positive ion mass spectrum) M/z (rel):368(100, M + H). Specific rotation degree [ alpha ]]_D= -15.8 (free amine; 0.5% MeOH).

The step in the synthesis of the 4 stereoisomers of 1-6 is the coupling of the epoxide formed from (R) -or (S) -3',5' -dibenzyloxyphenylbromohydrin with the appropriate (R) -or (S) -isomer of benzyl-protected 2-amino-3-benzylpropane (1-5) or with the (R) -or (S) -isomer of N-benzyl-2-aminoheptane (6), scheme I.

Route I

The synthesis of (R) -7 and (S) -7 is accomplished using 2-phenylethylamine, scheme II. This route is similar to the route developed by Trocast et al (Chirality3:443-450,1991) for the synthesis of the stereoisomer of formoterol (compound 47), FIG. 6. The resulting compound was then deprotected using hydrogenation over Pd/C and purified as fumarate.

Route II

The chiral building blocks used in the synthesis were obtained using scheme III. The (R) -and (S) -3',5' -dibenzyloxyphenyl-bromohydrin isomers were obtained as follows: using boron-dimethyl sulfide complex (BH)₃SCH₃) And (1R,2S) -or (1S,2R) -cis-1-amino-2-indanol to perform enantioselective reduction of 3, 5-dibenzyloxy-alpha-bromoacetophenone. The desired (R) -and (S) -2-benzylaminopropane were prepared as follows: enantioselective crystallization of racemic 2-benzylaminopropane was carried out using (R) -or (S) -mandelic acid as a counter ion.

Route III

Example 6

Exemplary fenoterol analogs have binding affinities for β 1 and β 2 adrenergic receptors.

This example illustrates that fenoterol analogues have equal, if not greater, binding affinity for the β 2-adrenergic receptor than fenoterol.

Each compound was tested up to three times to determine its presence at β₁-and β₂-binding affinity of adrenergic receptors. Competition curves with standard and unknown compounds included at least six concentrations (in triplicate). For each compound, a graph containing separate competition curves was prepared for the test compounds. Using GraphPadSoftware computing IC₅₀Values and Hill coefficients. Ki values were calculated using the Cheng-Prusoff transform (Biochem Pharmacol22: 3099-containing cell line 3108, 1973). In each experiment, standard compound experiments were performed simultaneously on 96-well plates. If the IC of the standard compound₅₀Values do not approach the established mean for the compound, the entire experiment is discarded and repeated again.

Beta on rat cortical membranes according to the procedure described previously₁Adrenergic receptor binding (Beer et al, biochem. Pharmacol.37:1145-1151, 1988). Briefly, male Sprague-Dawley rats weighing 250-350 g were sacrificed and their brains were rapidly removed. Cerebral cortex was dissected on ice, weighed and quickly transferred to 50ml tubes containing approximately 30ml of 50mm tris-HCl (ph 7.8, at room temperature). The tissue was homogenized using polytron and centrifuged at 20,000x g for 12 minutes at 4 ℃. The beads were again washed in the same manner and with assay buffer (20mM Tris-HCl, 10mM MgCl) per ml₂1mM EDTA, 0.1mM ascorbic acid, pH7.8) at a concentration of 20mg (initial wet weight) was resuspended. To block the beta present in cortical membrane preparations₂Site, 30nM ICI118-551 was also added to the assay buffer. To a solution containing 100. mu.l of the test drug and 100. mu.l of the solution³H]0.8 ml of homogenate was added to the wells of CGP-12177(1.4nM final concentration). After 2 hours at 25 ℃, the culture was terminated by rapid filtration. Nonspecific binding was determined by 10 μ M propranolol.

By encoding human beta₂HEK293 cells stably transfected with cDNA for AR (supplied by Dr. Brian Kobilka, Stanford Medical Center, Palo Alto, Calif.) were grown in the previously described Dulbecco's Modified Eagle Medium (DMEM) containing 10% bovine serum (FBS), 0.05% penicillin-streptomycin and 400. mu.g/ml G418(Pauwels et al, biochem. Pharmacol.42:1683-1689, 1991). Cells were scraped from 150X 25mm plates and centrifuged at 500x g for 5 minutes. The beads were homogenized in 50mM Tris-HCl, pH7.7, using Polytron, centrifuged at 27,000x g, and resuspended in the same buffer. The latter procedure was repeated and the pellets were resuspended in 25mM of a suspension containing 120mM NaCl, 5.4mM KCl, 1.8mM CaCl₂，0.8mM MgCl₂And 5mM glucose in Tris-HCl (pH 7.4). The binding assay comprises 0.3nM in a volume of 1.0mL³H]CGP-12177. Nonspecific binding was determined by 1 μ M propranolol.

According to the above method, the binding affinity expressed in Ki values is determined as follows: by using a slaveEncoding human beta₂Membranes obtained from HEK293 cell lines stably transfected with cDNA for the-AR (Pauwels et al, biochem. Pharmacol.42:1683-³H]CGP-12177 acts as a labeled ligand. Using GraphPadThe software calculates the resultant IC for each test compound₅₀Values and Hill coefficients, and calculating Ki using the Cheng-Prusoff transform (biochem Pharmacol22: 3099-containing 3108, 1973):

Ki=IC₅₀/(1+L/Kd)+Eqn.1.

wherein: l is [2 ]³H]The concentration of CGP-12177, and Kd are [, [2 ]³H]Binding affinity of CGP-12177. Each test compound was tested in triplicate.

Stereoisomer pairs beta of Compounds 1-4 and 6₂The relative binding affinity of the AR is R, R>R,S>S, R ≈ S, S (FIG. 5; Table 1 below). This stereoselectivity is consistent with previously reported efficacy of formoterol stereoisomers (Trocast et al, Chiralty3:443-450,1991) and the results of binding studies using isoproterenol derivative PTFAM compound 48 (FIG. 6) (Eimerl et al, biochem. Pharmacol.36:3523-3527, 1987). With respect to compound 5, no significant difference was found between the Ki values of the R, R and R, S isomers, and thus the order was R, R = R, S>S,R>And S, S. Ki values for (R) -7 are greater than those for (S) -7, which correlates with the established pair of β -OH moieties in the stereogenic center comprising the R-configuration of the β -OH moiety₂Enantioselective binding preference for AR is consistent, see (Eimerl et al, biochem. Pharmacol.36:3523-3527,1987; Wieland et al, Proc. Natl. Acad. ScL USA93:9276-9281,1996; Kikkawa et al, MoI. Pharmacol.53:128-134,1998; and Zuurmond et al, MoI. Pharmacol.56:909-916,1999).

TABLE 1 Compound pairs synthesized in this study vs. beta₂Binding affinity of AR, converted to Ki ± sem (nm), n ═ 3. Beta of fenoterol isomer₁-and β₂Comparison of adrenergic binding affinities.

Compound (I)	Kiβ1	Kiβ2	Kiβ1/Kiβ2
				(R,R)-1	14750+2510	345+34	43
(R,S)-1	18910+2367	3695+246	5
				(S,R)-1	>100,000	10330+1406	NC
(S,S)-1	>100,000	27749+6816	NC
				(R,R)-2	21992+3096	474+35	46
(R,S)-2	30747+6499	1930+135	16
				(S,R)-2	33378+9170	5269+509	6
(S,S)-2	>100,000	15881+2723	NC
				(R,R)-3	24956+2100	2934+168	9
(R,S)-3	31324+3485	7937+397	4
				(S,R)-3	77491+3583	23125+2093	3
(S,S)-3	31440+1681	28624+906	1
				(R,R)-4	17218+1270	1864+175	9
(R,S)-4	33047+2779	6035+434	4
				(S,R)-4	>100,000	30773+3259	NC
(S,S)-4	>100,000	28749+1811	NC
				(R,R)-5	3349+125	241+38	14
(R,S)-5	15791+6269	341+23	46
				(S,R)-5	34715+9092	1784+148	19
(S,S)-5	>100,000	2535+209	NC
				(R,R)-6	10185+499	9275+902	1
(R,S)-6	>100,000	31440+1681	NC
				(S,R)-6	61295+5821	>100,000	NC
(S,S)-6	52609+1434	56420+5186	1
				(R)-7	42466+3466	10466+1461	4
(S)-7	52178+3006	20562+3721	3

When only the R, R isomers were compared, (R, R) -5 had the highest relative affinity in the test compounds, although the difference between (R, R) -5 and (R, R) -1 did not reach statistical significance, see Table 1. The only other (R, R) stereoisomer with sub-micromolar affinity is (R, R) -2, which is significantly lower than (R, R) -5 (p = 0.0051) and (R, R) -1: (R, R) -2p = 0.0291), although the average Ki value for (R, R) -2 was only 23% greater than for (R, R) -1. The minimal effect on the conversion of the-OH moiety to the methyl ether is consistent with previous data from Schirrmacher et al (bioorg. Med. chem. Lett.13:2687-92, 2003). In a previous study, racemate-1 was converted into [2 ], [2 ]¹⁸F]No significant loss of in vitro activity occurred with fluoroethoxy ether, and it was concluded that: derivatization did not alter the racemate-1 vs. beta within the precise range of experimental measurements₂-binding affinity of AR.

P.beta.expressed as Ki value₁The binding affinity of the-AR is carried out using a rat cortical membrane using [2 ]³H]CGP-12177 as a marker ligand (Beer et al, biochem. Pharmacol.37:1145-1151, 1988.). The calculated Ki value for (R, R) -5 was 3,349nM and the binding affinity of all remaining test compounds>10,000nM, see Table 1. And is derived from beta₂The data from the AR binding studies are different and there is no clear trend with respect to the stereochemistry of the compounds.

Compound p [ beta ]₂-AR and beta₁Relative selectivity of-AR employs Ki β₁/Kiβ₂The ratio of (A) to (B) was measured, and is shown in Table 1. Of particular interest is the compound having the formula p₂AR is the ratio of (R, R) -1, (R, R) -2, (R, R) -and (R, S) -5 for the four compounds with sub-micromolar affinity, 46, 43, 14 and 46 respectively. The results for (R, R) -1 and (R, R) -2 are in accordance with the previous reports on beta₂Ki β of-AR-Selective agonist (R, R) -TA-2005 (Compound 49)₁/Kiβ₂The ratio is 53 is uniform, see fig. 6.

Observed beta of (R, R) -5₂The loss of selectivity to AR is unexpected because the selectivity shown by (R, S) -5 is increased by a factor of 3 relative to (R, R) -5. Previous studies using the stereoisomer of 47 showed that both the (R, R) -and (R, S) -isomers are p β -isomer pairs₂AR relative to p₁the-ARs are all highly selective, the selectivity for the (R, R) -isomer being greater than the selectivity for the (R, S) -isomer (Trocast et al, Chirality3:443-450, 1991). This is the case for compounds 1 and 2, but the opposite is true for 5. Go back toOf interest is to note that (S, R) -5 has a similar selectivity (19 times) and that it is beta₂The affinity of-AR is only one seventh of (R, R) -5, 1783nM and 241nM, respectively.

These studies show that the (R, R) -or (R, S) -naphthylfenoterol analogues are more beta-specific than any of the fenoterol isoforms₂Adrenergic receptors all have a higher binding affinity. (R, R) -Methoxyfenoterol analogue p-beta₂The Ki of the-adrenergic receptor is similar to that of (R, R) -fenoterol. Thus, these analogs are β₂-a viable candidate for adrenergic receptor agonists and may be useful in the treatment of disorders currently treated with the commercially available (±) -fenoterol.

Example 7

Myocardial contractility studies using fenoterol analogues

This example illustrates the pair beta₂-compounds with sub-micromolar affinity for AR and the pharmacological activity of (R, S) -1 and (S, S) -1.

In these studies, the magnitude of contraction indexed by the reduction in cell length induced by electrical pacing was measured in single ventricular myocytes before and after exposure to a single dose of the test compound. The contractile response to agonists is expressed as a percentage of basal contractility, and agonists are directed against β₂Specificity of adrenergic receptors by ICI118,551 (10)^-7mol/L, Tocriscokson Ltd., Bristol, U.K.), a selective beta₂-inhibition by an AR antagonist.

All test compounds, except (S, S) -1, produced a significant contractile response, which was blocked by ICI118,551, whereas no pharmacological effect was observed for (S, S) -1, see fig. 7. These results are consistent with those of previous studies (Beigi et al, Chirality,18:822-₂Agonist activity at the AR the R-configuration is preferred at the stereogenic center comprising a beta-OH moiety, see (Eimerl et al, biochem. Pharmacol.36:3523-3527,1987; Wieland et al, Proc. Natl. Acad. ScL USA93: 9276-; and Zuurmond et al, MoI. Pharmacol.56:909-916,1999). It is interesting to note that the maximum effect is triggered by 0.1. mu.M of (R, R) -5 and (R, S) -5, whereas other active compounds require a concentration of 0.5. mu.M. In addition, although the combined data show equivalent activity for (R, R) -5 and (R, S) -5, the observed activity for (R, S) -1 was unexpected because previous studies of stereoisomers of 47 (Trocast et al, Chiralty3:443-450,1991) and 48(Eimerl et al biochem. Pharmacol.36:3523-3527,1987) indicated that the agonist activity of the (R, R) -isomer was significantly greater than the activity of the corresponding (R, S) -isomer.

Example 8

Comparative molecular field analysis

This example illustrates the analysis of the disclosed compounds using comparative molecular field analysis (CoMFA).

The disclosed compounds are analyzed using comparative molecular field analysis, which is a 3D QSAR technique that is suitable for analyzing the relative activity of stereoisomers and/or enantiomers at selected targets.

The CoMFA was performed as performed in SYBYL7.2. (tritos inc., st.louis, MO). Molecular models of all derivatives were prepared in HyperChem v.6.03(HyperCube inc., Gainesville, FL) using the model build procedure to ensure the same conformation of the backbone. The model was extracted into SYBYL and the local atomic charge calculated (Gasteiger-Huckel type). Calibration of ligand model (-C x-CH) with common Structure of two asymmetric carbon atoms within the core of the fenoterol molecule₂-NH-C*-CH₂-). Two types of molecular fields (spatial and electrostatic) in a grid around each structure: (Spaced) lattice is sampled. The distance-dependent dielectric constant was used in the electrostatic calculation, and an energy cutoff of 30 kcal/mol was set for both the spatial energy and the electrostatic energy.

The local least squares correlation equation for the resulting database extracted from the four statistically significant components was obtained for the optimal solution along with the following validation parameters: r²=0.920, F (4,21) =60.380, standard estimation error =0.223, cross validation (leave-one-out) R²= 0.847. Typically, the electrostatic field accounts for 48.1% of the illustrated variation, and the spatial field accounts for 51.9%. The resulting 3D QSAR model showed good mathematical statistical correlation with the trial number data, R²=0.920 and F =60.380, and R by cross validation²Value (Q)²) Good prediction ability as shown by =0.847 and standard prediction error (SEP) =0.309, see table 2.

TABLE 2 pK predicted by CoMFA model_d。

Derivatives of the same	pKdMeasured value	pKdPrediction value
			(R,R)-1	6.46	5.84
(R,S)-1	5.43	5.48
			(S,R)-1	4.99	5.02
(S,S)-1	4.56	4.66
			(R,R)-2	6.32	6.17
(R,S)-2	5.71	5.80
			(S,R)-2	5.28	5.34
(S,S)-2	4.80	4.99
			(R,R)-3	5.53	5.57
(R,S)-3	5.10	5.21
			(S,R)-3	4.64	4.75
(S,S)-3	4.54	4.39
			(R,R)-4	5.73	5.58
(R,S)-4	5.22	5.25
			(S,R)-4	4.51	4.75
(S,S)-4	4.54	4.43
			(R,R)-5	6.62	6.72
(R,S)-5	6.47	6.36
			(S,R)-5	5.75	5.90
(S,S)-5	5.60	5.54
			(R,R)-6	5.03	5.01
(R,S)-6	4.50	4.66
			(S,R)-6	4.00	4.19
(S,S)-6	4.25	3.84
			(R)-7	4.98	5.33
(S)-7	4.69	4.51

In the first stage, the model is used to identify the regions responsible for discriminating between stereoisomers. The CoMFA program produces several distinct asymmetric regions located closely adjacent to each chiral center. The first chiral center (carrying the beta hydroxyl group) is surrounded by an electropositive region behind the molecule. The electropositive region may be associated with hydrogen bond formation and may indicate either favorable donor properties or unfavorable acceptor properties of the pseudo acceptor. In this case, the position of the electropositive field indicates that the orientation of the β -OH moiety behind the plane of the model (S configuration at the chiral center) prevents H-bond formation with the acceptor. The electropositive region is closely related to the spatially adverse region behind the first chiral center. This additionally indicates that the model shows preferential selectivity for the beta-hydroxy group in the R configuration. Preference for the R configuration at this center is consistent with previous models and experimental data, suggesting that the R configuration favors functional activity at the β -AR receptor (see Eimerl et al, biochem. Pharmacol.36:3523-3527,1987; Wieland et al, Proc. Natl. Acad. ScL USA93:9276-9281,1996; Kikkawa et al, MoI. Pharmacol.53:128-134,1998; and Zimrmond et al, MoI. Pharmacol.56:909-916,1999).

The CoMFA model also shows the effect of a second chiral center. Preferred configurations can be derived from binding data in which the (R, R) -isomer has a higher affinity relative to its respective (R, S) -isomer for compounds 1-4 and 6, while the Ki values for (R, R) -5 and (R, S) -5 are equal, see Table 1. Thus, in this model, the more active isomers are those with a methyl moiety at the stereogenic center on the aminoalkyl moiety of the molecule pointing out of the plane of the CoMFA model diagram. This is depicted by a sterically unfavourable region behind the second chiral centre of the molecule and indicates that the R configuration is preferred at this position.

In this study, the fenoterol molecule had only the aminoalkyl moiety altered and therefore the critical CoMFA region was relevant to this aspect of the molecule. In the resulting analysis, all four regions of interaction are identified in the vicinity of the aromatic moiety and all of these can be used to generate a hypothesis as to the manner in which the studied derivatives bind.

In the model, larger ones contain close to-OH or OCH₃The electropositive region of the substituent represents the H-bonding donor nature of the pseudo-acceptor to these moieties. These interactions are responsible for the relatively higher binding affinity of O-derivative compounds 1 and 2 relative to compounds 3 and 4, where the para amino substituent should be positively charged under the experimental conditions.

The larger electronegative region and the other electropositive region, both located parallel on either side of the aromatic system, are likely to be represented at β₂-pi or pi-hydrogen bonding interaction between the AR and an electron rich aromatic moiety such as a naphthyl ring. This is consistent with compounds 1,2 and 5 having increased affinity relative to the other compounds tested in this study. The function of this interaction is suggested by the following observations: ki values for (R, R) -5 and (R, S) -5 are equal to (R, R) -1 and (R, R) -2, see Table 1.

The two spatial regions are located close to the electrostatic region and within the respective region one is favorable to volume capacity and the other is unfavorable to volume capacity. This indicates that the incorporation of the aminoalkyl moiety of the molecule is also sterically limited.

Agonist and antagonist pairs of beta₂The binding of AR has been studied using site-directed mutagenesis and molecular modeling techniques (Eimerl et al, biochem. Pharmacol.36:3523-3527,1987; Wieland et al, Proc. Natl. Acad. ScL USA93:9276-9281,1996; Kikkawa et al, MoI. Pharmacol.53:128-134,1998; Zuuurmond et al, mol. Pharmacol.56:909-916,1999; Kontoyiani et al, J.Med. chem.39:4406-4420,1996; Furse et al, J.Med. chem.46:4450-4462, 2003; and Swamith et al, J.biol. chem.686.279: 2004). It is believed that binding of the "catechol" moiety of the agonist occurs within the binding region produced by the Transmembrane (TM) helices known as TM3, TM5 and TM 6. This binding process is a sequential event that produces a conformational change leading to activation of the G protein (Furse et al, J.Med.chem.46:4450-4462, 2003). A key aspect in this process is the interaction of the hydroxyl moiety on the chiral carbon of the agonist with the Asn-293 residue in TM6, and for this interaction the R-configuration is preferred at the chiral carbon (Eimerl et al, biochem. Pharmacol.36:3523-3527,1987; Kikkawa et al, MoI. Pharmacol.53:128-134,1998; and Swamiath et al, J.biol. chem.279:686-691, 2004). Since the "catechol" moiety of the fenoterol molecule was not altered in this study, it allowed the R-configuration at the first stereogenic center to be preferred in most stable complexes in the CoMFA model.

β₂Most of the binding and functional studies of AR agonists have been performed with small N-alkyl substituents such as methyl, isopropyl and tert-butyl groups (Kontoyiannei et al, J.Med.chem.39: 4406-. However, despite the presence of these compounds in beta₂AR are active, but they are not subtype selective. This is via Ki β of compounds 49, 50 and 51₁/Kiβ₂The ratios of (A) indicate that the three ratios are 53, 1.7 and 1.3, respectively (Kikkawa, et al, mol. Pharmacol.53:128-134, 1998)). Removal of the p-methoxyphenyl moiety not only reduces selectivity, but also reduces affinity, because of the respective beta₂Ki values were 12nM, 170nM and 6300 nM. (Kikkawa, et al, mol. Pharmacol.53:128-134,1998).

Aminoalkyl substituents at beta₂The role played in AR selectivity has been studied using site-directed mutagenesis and molecular modeling techniques (Kikkawa, et al, mol. Pharmacol.53:128-134,1998); Furse et al, J. Med. chem.46:4450-4462, 2003; and Swaminath et al, J.biol.chem.279: 686-. Using (R, R) -49 as a model ligand, Kikkawa, et al, it was determined that the hydrogen bond formation between the p-methoxy oxy group of compound 49 and the hydroxyl group of tyrosine 308(Y308) located within the extracellular terminal of TM7 is β₂A source of AR selectivity (mol. Pharmacol.53:128-134,1998).

Furse and Lybrand developed beta₂A newly formed (de novo) model of AR and the use of this subtype to study the molecular complex (agonists and antagonists) of some ligands (J.Med.chem.46:4450-4462, 2003). In the structures studied, (R, R) -49 has the same aminoalkyl substituent as compound 2. (R, R) -49/beta₂The study of the-AR complex revealed that the p-methoxy group of (R, R) -49 forms a hydrogen bond with the hydroxyl group of Y308, which supports the model proposed by Kikkawa et al (mol. Pharmacol.53:128-134,1998). The distance between two oxygen atoms bonded in this model isHowever, the methoxy moiety of the ligand is also located in association with three other polar residues: histidine 296(H296) in TM6, tryptophan 109(W109) in TM3, and asparagine 312(N312) in TM7 are in close proximity, and thus each can interact with the aryl group on the aminoalkyl moiety of (R, R) -49.

In the Furse and Lybrand models, the distance between the oxygen atom of the ligand and the hydrogen atom of H296 isAnd H296 is recommended to be an alternative hydrogen bond donor for interaction with the methoxy group of (R, R) -49. Since Y308 and H296 were found to exist only in beta₂-AR at β₁The interactions of the corresponding residues found in AR, F359 and K347, H296 and Y308, respectively, have been proposed as β₁/β₂Alternative sources (Furse et al, J.Med.chem.46:4450-4462, 2003).

Since previously with respect to beta₁/β₂The selectivity study employed (R, R) -49, and the subtype selectivity of the (R, R) -stereoisomer of the compound synthesized in this study was compared to that of (R, R) -49. The data from this study suggest hydrogen bond formation between Y308 and/or H296 and that the oxygen atom on the para substituent of the agonist is involved in β₂-AR selectivity. Interactions may occur between (R, R) -1 and (R, R) -2, and Ki β of these compounds₁/Kiβ₂In a ratio of 43 and 46, respectively, which is comparable to the measured Ki β of (R, R) -49₁/Kiβ₂The ratio of (d) is 53 (g). Ki beta of Compounds 3, 4, 6 and 7₁/Kiβ₂Ratio of (A to (B)<10 and reflects the fact that: they are not capable of forming hydrogen bonds with Y308 or H296. Hydrogen bonding interactions are also through recognition around-OH or OCH₃The CoMFA model of a larger electropositive region of the nearby region of the substituent is suggested to represent the hydrogen bond donor properties of the pseudo-acceptor.

The data from this study also suggest that the aromatic moiety on the aminoalkyl moiety of the compound promotes Ki and subtype selectivity, even though the aromatic moiety is unable to form hydrogen bonds with the receptor. This is done by comparing the (R, R) -isomers of the compounds 1-5<Ki beta of 3,000nM₂Ki beta of 9,000nM with (R, R) -6₂And Ki beta of Compounds 1 to 5₁/Kiβ₂Ratio of (A to (B)>9, while compound 6 did not show subtype selectivity, as demonstrated in table 1. One possible mechanism for interpreting this data is pi-bond formation. Aromatic rings yielding pi-electron clouds can act as hydrogen bonding acceptors, although it has been estimated that the interaction will be about half as strong as normal hydrogen bonding (1evitt and Perutz J.MoI.biol.201:751-754, 1998). The higher affinity and subtype selectivity of (R, R) -5 relative to (R, R) -3 and (R, R) -4 or (R) -7 is consistent with a larger pi-electron distribution in the naphthyl ring relative to the other aromatic rings.

The CoMFA model also identifies a larger electronegative region and an additional electropositive region, both of which arePositioned parallel to the aromatic system, this being likely to be parallel to beta₂the-AR is associated with pi-pi or pi-hydrogen bonding interactions between aromatic-rich moieties such as the naphthyl ring. Using the model developed by Furse and Lybrand, with (R, R) -49 as the interacting ligand, Y308, H296, W109 and N312 were referred to as the source of possible pi-pi and/or pi-hydrogen bonding interactions. At beta₂In the AR model, the estimated distances between the p-methoxy moiety on (R, R) -49 and W109 and N312 were 4.80 angstroms and 3.45 angstroms, respectively. Since W109 and N312 are all retained as total β -AR subtypes, the interactions suggested by the CoMFA model may represent the reason for the increased affinity of (R, R) -1, (R, R) -2 and (R, R) -5 relative to the other (R, R) -isomers, but β -is not observed₁/β₂And (4) selectivity.

The data from this study and the resulting CoMFA model indicate the test compound versus β₂The binding process of the-AR involves interaction of a chiral center on the aminoalkyl moiety of the agonist with a sterically restricted site on the receptor. The existence of sterically constrained sites has been previously exploited by the development of agonists and antagonists and beta₂Data in 3D models of complexes of the AR suggest (Kobilka, MoI. Pha. n.65:1060-1062, 2004). For example, (R, R) -49 and similar compounds having a substituent on the stereogenic center on the aminoalkyl moiety that is larger than the methyl group are suggested to produce significant steric interactions that adversely affect the ligand-receptor complex.

Agonists and beta₂AR binding has been described as a multi-step, interrelated approach in which sequential interactions between agonist and receptor produce corresponding conformational changes (Kobilka, MoI. Pharm.65:1060-1062, 2004). The CoMFA model reflects the final agonist/. beta.₂AR complex and in order to discriminate the effect of a spatially restricted site it is necessary to consider the effect of the interaction with that site on the outcome of the binding process. A detailed description of the CoMFA model of the present invention is disclosed by Jozwiak et al, (J.Med.chem.,50(12):2903-2915,2007), the entire contents of which are incorporated herein by reference.

If one assumes the interaction of the "catechol" of the agonist with the binding area (first binding area) generated by TM3, TM5 and TM6, then these interactions fix the location of the aminoalkyl moiety of the agonist relative to the sterically constrained site and perhaps even generate that site. In the CoMFA model, steric constraints at this site force the methyl moiety at the chiral center of the aminoalkyl moiety to point out the plane of the model.

Due to free rotation around the N-atom, the configuration out of the chiral center with the methyl moiety is unlikely to affect the ability of the molecule to minimize interaction with spatially restricted sites. However, in the lowest energy conformation, e.g., methyl with the indicated CoMFA model plane, the orientation of the remaining fragment of the aminoalkyl moiety relative to the second binding region is affected by stereochemistry. In effect, the R and S configurations will produce a mirror image relationship with the second binding region. This site is illustrated in FIG. 5, where the catechol (first chiral center) and methyl moieties of (R, R) -5 and (R, S) -5 have been overlaid on each other in FIG. 5.

Elucidation of beta₂The research of the source of AR selectivity mainly employed (R, R) -49 and one previous study report on the effect of chirality of subtype selectivity reported that (R, R) -47 has a higher beta than (R, S) -47₂AR selectivity (Trocast et al, Chiralty3: 443-. Thus, the observed (R, R) -5 and (R, S) -5 are in beta₂Equal affinity and functional activity at AR and β of (R, S) -5₂A3-fold increase in AR selectivity is an unexpected result. One possible explanation for these results is that the naphthyl moiety of (R, S) -5 does not interact with and orient and bind to the sites defined by Y308 and H296₂-additional sites on the AR. This interaction also mediates or participates in subtype selectivity as well as increased binding affinity and agonist activity. Because of the previous beta₂the-AR selectivity model uses only the (R, R) -isomer, possibly ignoring the site.

Another explanation of the data is inspired by the "rocking tetrahedron" chiral recognition mechanism recommended by Sokolov and Zefirrov (Doklademii NaukSSSR319:1382-1383, 1991). In this molecular chiral recognition approach, the enantiomeric ligands are immobilized on the chiral selector by two binding interactions. The interaction must be non-equivalent and directional so that only one orientation is possible. The tethered enantiomers still have conformational mobility and the remainder on the chiral center will scavenge overlapping, but not identical, spatial volumes. The position and to what extent the interaction of the chiral selector with these spatial volumes occurs determines the enantioselectivity of the process. If the chirality of the chiral selector is such that the interaction is perpendicular to the plane of the ligand, no enantioselectivity is observed. As the difference from the perpendicular increases, the enantioselectivity with respect to the R or S configuration also increases.

With respect to (R, R) -5 and (R, S) -5, the interaction with the first binding region of the spatially restricted site of the CoMFA model is not two non-equivalent and directional interactions, which results in the remaining building blocks on the second chiral center being in the same, albeit mirror-image, orientation relative to the second binding region. As discussed above, the interaction of the 1-naphthyl moiety of Compound 5 with Y308 and H296 is believed to be the observed β₂-a source of AR selectivity. Ki β observed if the 1-naphthyl ring clears overlapping, but not identical, spatial volumes₂Values and subtype selectivities indicate the following meanings: 1) ki beta₂AR values represent pi-hydrogen bonding and pi-pi interactions between the 1-naphthyl moiety and Y308 and H296, and additional non-beta interactions with other residues such as W109 and N312₂-sum of AR specific interactions; 2) the steric bulk scavenged by (R, S) -5 relative to (R, R) -5 increases the probability of interaction of Y308 and H296 with the π electron cloud of the naphthyl moiety; and 3) the volume of space cleared by (R, R) -5 is increased relative to (R, S) -5 by NOT beta₂-probability of interaction of AR-specific sites.

The effect of configuration at the second chiral center and conformational base chiral selectivity is also illustrated by the affinity and subtype selectivity of (R, R) -3, (R, S) -3 and (R) -7, see Table 1. Reversal of chirality from R to S at the second chiral carbonLow in (R, S) -3/beta₂-AR Complex relative to (R, R) -3/beta₂Ki beta of the-AR Complex₂The values decreased by about 3-fold despite their Ki β₁There was no significant difference between the values. The observed increased subtype selectivity of (R, R) -3 over (R, S) -3,9 over 4, respectively, essentially reflects Ki β₂A difference in value, which may reflect a decreased likelihood of the increased conformational energy required to bring the aromatic portion of the aminoalkyl chain into contact with the positively and negatively charged regions comprising the second binding region, or when such interaction occurs.

Removal of the methyl moiety at the second chiral center and thus the chirality ((R) -7) at this site has a similar effect, since the chirality is converted from R to S at this position. Ki beta of (R) -7₂A value 32% higher than that of (R, S) -3, and therefore in beta₂There was no difference in AR selectivity, see table 1. These results suggest that the primary effect of compound 3, the R configuration at the second chiral site, is to direct the aminoalkyl chain to the second binding region, which increases the probability of interaction with that site and decreases the conformational energy required to accomplish this interaction.

The difference between compounds 3 and 5 is the steric area that is cleared by the aromatic substituent. With compound 3, the phenyl ring produces a smaller, more linear region, while with compound 5, the 1-naphthyl ring system produces a relatively larger and wider region. These differences can be used to guide the synthesis of other derivatives.

In one embodiment, (R, R) -2 and (R, S) -5 were selected as potential candidates for developing novel selectivity beta₂-an AR agonist. These compounds have increased and prolonged systemic exposure relative to the commercial racemate-1, due to changes in their molecular hydrophobicity, metabolic patterns, and transporter interactions.

Embodiments of the present invention provide pharmacophore models that can be used as structural guides to design genes with beta₂-AR selective novel compounds which can be tested for the treatment of desired conditions including congestive heart failure.

Example 9

Pharmacokinetic study of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol

This example illustrates the plasma concentrations of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol when administered Intravenously (IV) concentrated to male Sprague-Dawley rats.

(R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol were administered intravenously in a single dose of 5mg/ml into Jugular Vein Cannulated (JVC) rats (see Table 3). The calculation of the dose (mg/kg) is based on the individual body weight measured on the day of treatment. The duration of the study for the pharmacokinetic study was 6 hours. Plasma samples were collected over 6 hours at the following nine time points: prior to administration of the desired dose; 5.00-5.30 minutes after dosing; 15.00-16.30 minutes after dosing; 30.00-33.00 minutes after dosing; 60-65 minutes after dosing; 120-125 minutes after dosing; 240-245 minutes after dosing; 300-305 minutes after dosing; and 360-365 minutes after dosing. Urine was collected from 3 rats in each treatment group for 0-6 hours and 6-24 hours.

TABLE 3 plasma concentration study conditions for the measurement of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol.

Pharmacokinetic parameters of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol after intravenous administration to rats (5mg/kg) were analyzed according to the two-compartment open model (see Table 4). Drugs that follow the pharmacokinetics of the two-compartment model do not rapidly equilibrate throughout the body, as is assumed for the single-compartment model. In the case of the two-compartment model,

the drug is distributed in two compartments, a central compartment and a tissue or peripheral compartment. The central compartment represents blood, extracellular fluid and highly perfused tissue. The medicine is rapidly and uniformly distributed in the medicine

In the central compartment. The second compartment, referred to as the tissue compartment or peripheral compartment, contains tissue in which the drug equilibrates more slowly. The transfer of the drug between the two compartments is believed to occur by a primary process.

The following abbreviations are used in table 4 below: an α -macroscopic rate constant associated with the distribution phase; beta-macroscopic rate constants associated with the elimination phase; a, B-zero time intercept associated with the alpha and beta phases, respectively; AUC area under the curve; t1/2 (K10) -half-life related to rate constant K10; k10-elimination rate-rate of drug leaving the system from the central compartment; k12-rate of drug entry into tissue compartment from central compartment; k21-rate of drug entry from tissue compartment into central compartment; v1 — distribution volume of central compartment; v2 — volume of distribution of tissue compartments; vss-distribution volume at steady state; and C1-clearance.

Table 4 pharmacokinetic parameters of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol after intravenous administration to rats (5 mg/kg).

Tables 5-7 and FIG. 8 illustrate the individual plasma concentrations of (R, R) -fenoterol, (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol after IV administration to rats (5 mg/kg). Five minutes after administration to rats (5mg/kg), the mean concentration of (R, R) -fenoterol in plasma (1.34 μ g/ml) was significantly lower than that of (R, R) -methoxyfenoterol (2.12 μ g/ml) or (R, S) -naphthylfenoterol (2.11 μ g/ml).

TABLE 5 Individual plasma concentrations of (R, R) -fenoterol after intravenous administration (5 mg/kg).

TABLE 6 Individual plasma concentrations of (R, R) -methoxyfenoterol after intravenous administration (5 mg/kg).

TABLE 7 individual plasma concentrations of (R, S) -naphthylfenoterol after intravenous administration (5 mg/kg).

The above data indicate that the two derivatives, i.e., (R, R) -methoxyfenoterol and (R, S) -naphthylfenoterol, have significantly higher systemic exposure (AUC) and longer clearance than (R, R) -fenoterol, which can result in a longer acting drug. It is suggested that longer clearance times may be a result of ethyl glucuronic acid binding.

Example 10

Treatment of cardiac disorders with (R, R) -fenoterol or fenoterol analogues

Based on the teachings disclosed herein, cardiac conditions such as congestive heart failure are treated by administering a therapeutically effective dose of (R, R) -fenoterol or one or more of the fenoterol analogs disclosed above (see sections III and IV). In one embodiment, a subject who has been diagnosed with congestive heart failure is identified. After the subject selection, a therapeutically effective dose of (R, R) -fenoterol or the respective fenoterol analogue is administered to the subject. For example, a therapeutically effective dose of an (R, R) fenoterol analogue including a methoxy group or a naphthyl derivative is administered to the subject. In further embodiments, a therapeutically effective dose of the (R, S) -fenoterol analogue, including the naphthyl derivative, is administered to at least the subject. The fenoterol analogues were prepared and purified as described in section iii.b and example 5. The amount of (R, R) -fenoterol or fenoterol analogue or pharmaceutical composition thereof administered for reducing, inhibiting and/or treating congestive heart failure will vary depending on the subject being treated, the severity of the condition and the mode of administration of the therapeutic composition (see section V). Theoretically, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce and/or inhibit and/or treat a cardiac disorder (e.g., congestive heart failure) in a subject without causing substantial cytotoxic effects in the subject.

In one embodiment, (R, R) -fenoterol, disclosed fenoterol analogs (such as (R, R) -fenoterol analogs including a methoxy group or a naphthyl derivative or (R, S) -fenoterol analogs including a naphthyl derivative) or pharmaceutical compositions are provided in a single or divided dose oral administration of about 0.001 to about 10 mg/kg body weight. In a specific embodiment, the dosage is in the range of about 0.005 to about 5mg/kg body weight, administered orally in single or divided doses (assuming an average body weight of about 70 kg; for persons having a body weight more or less than the average body weight, the value is adjusted accordingly).

In certain embodiments, the disclosed fenoterol compounds or pharmaceutical compositions are provided for oral administration in the form of tablets containing about 1.0 to about 50mg of the active ingredient, specifically about 2.0 mg to about 10mg, more specifically about 2.5 mg, about 5mg, or about 10mg of the active ingredient, for symptomatic adjustment of the dosage to the subject being treated. In an exemplary oral dosage regimen, tablets containing from about 1mg to about 50mg of the active ingredient are administered 2-4 times a day. For example, tablets containing from about 1mg to about 10mg of active ingredient are administered twice daily.

Example 11

Treatment of pulmonary disorders using fenoterol analogues

According to the teachings disclosed herein, pulmonary disorders such as asthma or chronic obstructive pulmonary disease are treated by administering a therapeutically effective dose of the fenoterol analogs disclosed above (see sections III-V). In one embodiment, a subject that has been diagnosed with or displays one of the symptoms associated with asthma or chronic obstructive pulmonary disease is identified. After the subject has selected, a therapeutically effective dose of the desired fenoterol analogue is administered to the subject. For example, a therapeutically effective dose of an (R, R) -fenoterol analogue, including a methoxy or naphthyl derivative, is administered to a subject. In further embodiments, a therapeutically effective dose of the (R, S) -fenoterol analogue, including the naphthyl derivative, is administered to at least the subject. The fenoterol analogues were prepared and purified as described in section iii.b and example 5. The amount of the fenoterol analogue administered for preventing, reducing, and/or inhibiting, and/or treating the pulmonary disorder varies depending on the subject to be treated, the severity of the disorder, and the method of administration of the therapeutic composition. Theoretically, a therapeutically effective amount of an agent is an amount sufficient to prevent, reduce, and/or inhibit and/or treat a pulmonary disorder in a subject without causing substantial cytotoxic effects in the subject.

In one embodiment, (R, R) -fenoterol, disclosed fenoterol analogs (such as (R, R) -fenoterol analogs including methoxy or naphthyl derivatives or (R, S) -fenoterol analogs including naphthyl derivatives) or pharmaceutical compositions are provided in a single or divided dose oral administration of about 0.001 to about 10 mg/kg body weight. In a specific embodiment, the dosage is in the range of about 0.005 to about 5mg/kg body weight, administered orally in single or divided doses (assuming an average body weight of about 70 kg; for persons having a body weight more or less than the average body weight, the value is adjusted accordingly).

In certain embodiments, the disclosed fenoterol compounds or pharmaceutical compositions are provided for oral administration in the form of tablets containing about 1.0 to about 50mg of the active ingredient, specifically about 2.0 mg to about 10mg, more specifically about 2.5 mg, about 5mg, or about 10mg of the active ingredient, for symptomatic adjustment of the dosage to the subject being treated. In an exemplary oral dosing regimen, tablets containing from about 1mg to about 50mg of the active ingredient are administered 2-4 times per day. For example, tablets containing from about 1mg to about 10mg of active ingredient are administered twice daily.

In view of the many possible embodiments to which the principles disclosed herein may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. The scope of the present invention is defined by the appended claims, and therefore, is limited only by the scope and spirit of the claims.

Claims (10)

1. A compound represented by the formula:

wherein R is₁-R₃Independently hydrogen, acyl, C (O) OR wherein R contains 1-15 carbon atoms, OR combinations thereof;

wherein R is₄Is hydrogen or lower alkyl; and

R₅is that

Or

Wherein Y is¹、Y²And Y³Independently of one another is hydrogen, lower-OR₆and-NR₇R₈；

Wherein X is H, -OR₆or-NR₇R₈；

R₆Is hydrogen or lower alkyl; and R₇And R₈Independently hydrogen, lower alkyl, C (O) OR wherein R contains 1-15 carbon atoms, acyl OR aminocarbonyl, and wherein said compounds are optically active.

2. The compound of claim 1, wherein R₄Selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.

3. The compound of claim 1 or 2, wherein R₆Is methyl.

4. A compound according to any one of claims 1 to 3, wherein R₁-R₃Is hydrogen.

5. The compound of any one of claims 1-4, wherein the compound is an (R, R) -compound.

6. The compound of any one of claims 1-4, wherein the compound is a (R, S) -compound.

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and at least one pharmaceutically acceptable carrier.

8. Use of a therapeutically effective amount of a compound according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 in the manufacture of a medicament for treating a cardiovascular disorder such as congestive heart failure or a pulmonary disorder such as asthma or chronic obstructive pulmonary disease in a subject.

9. Use of a therapeutically effective amount of substantially optically pure (R, R) -fenoterol in the manufacture of a medicament for a subject suffering from a cardiovascular disorder.

10. The use of claim 9, wherein the cardiovascular disorder is congestive heart failure.