KR20060136436A - Aryl aniline derivatives as beta2 adrenergic receptor agonists - Google Patents

Abstract

The present invention provides novel beta 2-adrenergic receptor agonist compounds. The present invention also relates to pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with beta 2-adrenergic receptor activity, and methods of making such compounds and Provided are useful intermediates for the preparation of the same compounds.

Beta-2 adrenergic receptor, beta-2 adrenergic receptor agonist, chronic obstructive pulmonary disease, aryl aniline.

Description

Aryl aniline derivatives as beta2 adrenergic receptor agonists

The present invention relates to novel β ₂ adrenergic receptor agonists (β ₂ adrenergic receptor agonists). The present invention also provides pharmaceutical compositions comprising such compounds, methods of using such compounds to treat diseases associated with β ₂ adrenergic receptor activity, and methods of making such compounds and useful therefor. It is about intermediates.

β ₂ adrenergic receptor agonists are recognized as effective drugs for the treatment of asthma and chronic obstructive pulmonary disease (including pulmonary diseases such as chronic bronchitis and emphysema). β ₂ adrenergic receptor agonists are also useful in the treatment of pre-term labor and also potentially useful in the treatment of neurological and heart diseases. Despite the success achieved by some β ₂ adrenergic receptor agonists, currently used agents show less than desirable levels of action, efficacy, selectivity, and / or onset. Thus, there is a need for additional β ₂ adrenergic receptor agonists with improved properties, such as improved duration of action, efficacy, selectivity, and / or initiation.

The present invention provides novel compounds having β ₂ adrenergic receptor agonist activity. Among other properties, the compounds of the present invention have been found to be potent and selective β2 adrenergic receptor agonists. In addition, the compounds of the present invention have been found to have surprisingly long durations of action, which are expected to allow for once daily administration, or even lower frequency of administration.

Accordingly, the present invention is directed to compounds of formula (I):

(I)

In the above,

Each R ¹ , R ² , R ³ , and R ⁴ is independently selected from hydrogen, hydroxy, amino, halo, —CH ₂ 0H and —NHCHO, or taken together R ¹ and R ² (R ¹ and R ² taken together) is NHC (═O) CH═CH—, —CH═CHC (═O) NH—, —NHC (═O) S—; And -SC (= 0) NH-;

R ⁵ and R ⁶ is one of the _- [XC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ^13, and

R ⁵ and R ⁶ and one of which is hydrogen, hydroxy, C _{1 -} is selected from _4-alkyl, the C _{1 - - 4} alkoxy, and C _{1 4} alkyl is optionally substituted with halo,

Each X is independently selected from -0-, -NH-, -S-, -NHS0 _2- , -S0 ₂ NH-, -NHC (= 0)-, and -C (= 0) NH-;

Each ^{R l0, R 11, R 12} , and R ¹³ are independently hydrogen or C _{1 - 4} alkyl and; or

R ^l0 And R ^11, together with the nitrogen atom to which they are attached, or R ^l0 is a nitrogen atom and the adjacent C ₁ coupling it _- together with the nitrogen atom to which they are attached, or R ¹² and R ¹³ together with the ₆ alkylenyl carbon atoms, or R ¹² is a nitrogen atom and the adjacent C ₁ coupling it _- contains additional heteroatoms (heteroatom) selected, having a 5 to 7 ring atoms, from optionally oxygen, nitrogen and sulfur with the ₆ alkylenyl carbon atoms form a heterocyclic or heteroaryl ring, and the nitrogen may optionally -S (0) ₂ -C ₁ in the _{- 4} and substituted with alkyl;

n is 1, 2, or 3; And

Each R ^7, R ^8, and R ⁹ are independently selected from hydrogen or C _{l - 6} alkyl, a compound of formula (I);

Or pharmaceutically acceptable salts or solvates or stereoisomers thereof.

The invention also provides pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier. The invention also provides combinations comprising the compound of the invention and one or more other therapeutic agents and pharmaceutical compositions comprising such combinations.

The invention also provides a method of treating a mammal having a disease or condition associated with β ₂ adrenergic receptor activity (eg, lung disease such as asthma or chronic obstructive pulmonary disease, premature birth, neurological disease, heart disease or inflammation). And the method comprises administering to said mammal a therapeutically effective amount of a compound of the invention. The invention also provides a method of treatment comprising administering a therapeutically effective amount of a compound of the invention and a combination of one or more other therapeutic agents.

The invention also provides a method of treating a mammal having a disease or condition associated with β ₂ adrenergic receptor activity, the method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition of the invention.

The compounds of the present invention may also be used as research tools, ie, to study biological systems or samples, or as a tool to study the activity of other chemical compounds. Thus, in accordance with another aspect of the present invention, the present invention relates to a compound of formula (I), a pharmaceutically acceptable salt or solvate or stereoisomer thereof for Provides a method for use as a research tool to find ₂ adrenergic receptor agonists.

In separate individual embodiments, the present invention also provides the synthetic methods and intermediates described herein, useful for preparing the compounds of the present invention.

The invention also provides for treating a mammal having a disease or condition associated with β ₂ adrenergic receptor activity (eg, lung disease such as asthma or chronic obstructive pulmonary disease, premature birth, neurological disease, heart disease, or inflammation). The use of the compounds of the invention in the preparation of a formulation or drug, as well as the compounds of the invention described herein for use in medical therapy.

The present invention provides novel aryl aniline β ₂ adrenergic receptor agonists of formula (I), or pharmaceutically acceptable salts or solvates or stereoisomers thereof. The following substituents and values provide representative examples of various aspects of the invention. These representative values are intended to further define such aspects and are not intended to exclude other values or to limit the scope of the invention.

In a specific embodiment of the invention, R ¹ is halo, -CH ₂ 0H, or -NHCHO.

In other specific embodiments of the invention, R ¹ is chloro, -CH ₂ OH, or -NHCHO; Or R ¹ is —CH ₂ 0H or —NHCHO.

In a specific embodiment of the invention, R ² is hydrogen.

In a specific embodiment of the invention, R ³ is hydroxy or amino.

In specific embodiments of the invention, R ⁴ is hydrogen or halo; Or R ⁴ is hydrogen or chloro.

In a specific embodiment of the invention, R ¹ is -NHCHO, R ³ is hydroxy, and R ² and R ⁴ are each hydrogen.

In another specific embodiment of the invention, R ¹ and R ² taken together are —NHC (═O) CH═CH— or —CH═CHC (═O) NH—, R ³ is hydroxy and R ⁴ is Hydrogen.

In another specific embodiment of the invention, R ¹ is -CH ₂ 0H, R ³ is hydroxy and R ² and R ⁴ are each hydrogen.

In another specific embodiment of the invention, R ¹ and R ⁴ are chloro, R ³ is amino and R ² is hydrogen.

In another specific embodiment of the invention, R ¹ and R ² taken together are —NHC (═O) S— or —SC (═O) NH—, R ³ is hydroxy and R ⁴ is hydrogen.

In a specific aspect of the invention, R ⁵ or R ⁶ is _- [XC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ and at the, n, X, R ^l0 and R ^ll are as defined in formula (I) same.

In still another specific aspect of the invention, R ⁵ or R ⁶ is _- [OC ₁ - ₆ alkylenyl; in _n -NR ¹⁰ R ¹¹ wherein, R ¹⁰ and R ¹¹ are as defined in formula (I), n is 1 or 2;

In still another specific aspect of the invention, R ⁵ or R ⁶ is -OC _{1 - 6} alkylenyl, and -NR ¹⁰ R ^11, in formula, each of R ¹⁰ and R ¹¹ are independently hydrogen or C _{1 - 4} is alkyl. Representative values of R ⁵ or R ⁶ include —O (CH ₂ ) ₂ NH ₂ , —O (CH ₂ ) ₃ N (CH ₃ ) ₂ , —O (CH ₂ ) ₄ NH ₂ , and —OCH _2. C (CH ₃ ) ₂ NH ₂ , including but not limited to.

In still another specific aspect of the invention, R ⁵ or R ⁶ is -OC _{1 - 6} and alkylenyl -NR ¹⁰ R ^11, in formula, R ^l0 and R ¹¹ is a piperazinyl ring together with the nitrogen atom to which they are attached Form. For example, R ⁵ or R ⁶ is —O (CH ₂ ) ₂ -1-piperazinyl.

In still another specific aspect of the invention, R ⁵ or R ⁶ is C _{1 - 6} alkylenyl, and -NR ¹² R ^13, R ¹³ and R ^l2 in formula are defined as in formula (I).

In still another specific aspect of the invention, R ⁵ or R ⁶ is C _{1 - 6} alkylenyl -NR ¹² R ¹³ and, in formula, each of R ^l2 and R ¹³ are independently hydrogen or C _{1 - 4} is alkyl. Representative values of R ⁵ or R ⁶ within this embodiment include — (CH ₂ ) ₂ NH ₂ , — (CH ₂ ) ₂ N (CH ₃ ) ₂ , and —CH ₂ C (CH ₃ ) ₂ NH ₂ ; It is not limited to this.

In still another specific aspect of the invention, R ⁵ or R ⁶ is C _{1 - 4} alkyl, optionally substituted with halo C _{1 - 4} alkyl, for, for example, CF ₃ and; Or R ⁵ or R ⁶ is C _{1 - 4} alkoxy, e.g., -OCH _3, and; Or R ⁵ or R ⁶ is hydrogen; Or R ⁵ or R ⁶ is hydroxy.

In a specific embodiment of the invention, R ⁷ is hydrogen.

In a specific embodiment of the invention, R ⁸ is hydrogen.

In a specific embodiment of the invention, R ⁹ is hydrogen.

In one aspect, the present invention provides a compound of formula (II)

(II)

In the formula:

R ¹ is —CH ₂ 0H or —NHCHO and R ² is hydrogen; Or R ¹ and R ² taken together are —NHC (═O) CH═CH— or —CH═CHC (═O) NH—;

R ⁵ and R ⁶ One of the _- [OC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ^13, and

R ⁵ and R ⁶ Of the other is hydrogen, hydroxy, C _{1 - 4} alkoxy, is selected from C _{1 -4} alkyl, said C _{1 - 4} alkyl is optionally substituted with halo,

Each R ^l0 , R ¹¹ , R ^l2 And R ¹³ is independently hydrogen or C _{1 - 4} alkyl and; or

R ^l0 And R ¹¹ is or R ^l0 is a nitrogen atom and the adjacent C ₁ binding it together with the nitrogen atom to which they are _attached-is, or R ¹² and R ¹³ together with the ₆ alkylenyl carbon atoms together with the nitrogen atom to which they are attached, or R ¹² is it-bonded nitrogen atom and the adjacent C _{1 -} with a ₆ alkylenyl carbon atoms, having a 5 to 7 ring atoms, optionally heterocyclic, including the additional heteroatom selected from oxygen, nitrogen and sulfur or forms a heteroaryl ring, the nitrogen is optionally -S (0) ₂ -C ₁ in the _{- 4} and substituted with alkyl;

n is 1 or 2, a compound of formula (II);

Or pharmaceutically acceptable salts or solvates or stereoisomers thereof.

In yet another aspect, the present invention R ⁵ is _- [OC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ^13, and R ⁶ is selected from the compounds of formula (II) hydrogen Provide them.

In yet another aspect, the present invention R ⁵ is C _{1 - 4} alkoxy and R ⁶ is - in formula ₆ alkylenyl _{^{-NR 12 R 13 (- [OC}} 1 - 6 alkylenyl] _n -NR ¹⁰ R ¹¹ or C ₁ Providing compounds of II).

In another embodiment, the present invention provides a compound of formula (II),

R ⁵ is -OC _{1 - 6} alkylenyl -NR ¹⁰ R ¹¹ and C _{1 - 6} alkylenyl is selected from -NR ¹² R ^13, R ⁶ is hydrogen; or

R ⁵ is C _{1 - 6} and alkylenyl -NR ¹² R ^13, _{- 4} alkoxy and R ⁶ is -C ₁

^Wherein R ^l0 , R ¹¹ , R ^l2 And R ¹³ is independently hydrogen or C _{1 - 4} alkyl or R ^l0 and R ¹¹ are provides compounds of formula (II) to form a piperazinyl ring together with the nitrogen atom to which they are attached.

Certain groups of compounds within the scope of the present invention are groups in which R ¹ and R ² taken together are —NHC (═O) CH═CH— or —CH═CHC (═O) NH—.

Another specific group of compounds within the scope of the present invention R ⁵ is -OC _{1 - 6} alkylenyl -NR ¹⁰ R ^11, and R ⁶ is a hydrogen group.

In another particular aspect, the present invention provides variables R ^l , R ² , R ⁵ And R ⁶ provide compounds of formula (II), taking on the values listed in Table 1 below.

Table 1

Specific references may be made to the following compounds:

5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- 8-hydroxy-l H -quinolin-2-one;

8-hydroxy-5-[( R ) -1-hydroxy-2- (2- {4- [4- (2-piperazin-1-yl-ethoxy) -phenylamino] -phenyl} -ethyl Amino) -ethyl] -l H -quinolin-2-one;

5-[( R ) -2- (2- {4- [4- (2-amino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy- H -quinolin-2-one;

5-[( R ) -2- (2- {4- [4- (2-dimethylamino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy -l H -quinolin-2-one;

5-[( R ) -2- (2- {4- [3- (2-Amino-ethyl) -4-methoxy-phenylamino] -phenyl} -ethylamino) -l-hydroxy-ethyl]- 8-hydroxy-l H -quinolin-2-one; The chemical naming follows the nomenclature of AutoNom, an automatic naming program provided by MDL Information Systems, GmbH (Frankfurt, Germany).

As described above, the compounds of the present invention comprise one or more chiral centers. Accordingly, the present invention, unless otherwise indicated, racemic mixtures, pure stereoisomers (ie, individual enantiomers or diastereomers), and mixtures of such isomers predominantly stereoisomers (stereoisomer-enriched) mixtures). Where specific stereoisomers are indicated, those of ordinary skill in the art to which this invention pertains will appreciate that trace amounts of other stereoisomers in the compositions of the invention, unless the use of the entire composition is excluded by the presence of other isomers, It will be appreciated that it can exist.

In particular, the compounds of the present invention comprise chiral centers in alkylene carbons to which a hydroxy group is bound in formulas (I) to (II). If a mixture of stereoisomers is employed, it is advantageous that the amount of stereoisomers in (R) orientation in the chiral center with hydroxy groups is greater than the amount of corresponding (S) stereoisomers. Comparing stereoisomers of the same compound, the (R) stereoisomer is preferred over the (S) stereoisomer.

Definitions

When describing the compounds, compositions and methods of the present invention, the following terms have the following meanings, unless indicated otherwise.

The term "alkyl" refers to a monovalent saturated hydrocarbon which may be straight or branched or combinations thereof. Unless defined otherwise, such alkyl groups typically contain from 1 to 10 carbon atoms. Representative alkyl groups are, for example, methyl, ethyl, n -propyl, isopropyl, n -butyl, sec -butyl, isobutyl, tert -butyl, n -pentyl, n -hexyl, n -heptyl, n -octyl, n − Nonyl, n -decyl and the like.

The term "alkoxy" means a monovalent functional group -O-alkyl, wherein alkyl is as defined above. Representative alkoxy groups include, for example, methoxy, ethoxy, propoxy, butoxy, and the like.

The term "alkenyl" refers to a monovalent unsaturated hydrocarbon group containing one or more carbon-carbon double bonds, typically one or two carbon-carbon double bonds, which may be straight or branched or combinations thereof. do. Unless defined otherwise, such alkenyl groups typically contain 2 to 10 carbon atoms. Representative alkenyl groups are, for example, vinyl, allyl, isopropenyl, but-2-enyl, n -pent-2-enyl, n -hex-2-enyl, n -hept-2-enyl, n -oct- 2-enyl, n -non-2-enyl, n -dec-4-enyl, n -deck-2,4-dienyl and the like.

The term "alkynyl" refers to a monovalent unsaturated hydrocarbon group containing one or more carbon-carbon triple bonds, typically one carbon-carbon triple bond, which may be straight or branched or combinations thereof. Unless defined otherwise, such alkynyl groups typically contain 2 to 10 carbon atoms. Representative alkynyl groups include, for example, ethynyl, propargyl, but-2-ynyl and the like.

The term "alkylenyl" refers to a divalent saturated hydrocarbon group which may be straight or branched or combinations thereof. Unless defined otherwise, such alkylenyl groups typically contain 1 to 10 carbon atoms. Representative alkylenyl groups are, for example, methylene, ethylene, n -propylene, n -butylene, propane-1,2-diyl (1-methylethylene), 2-methylpropane-1,2-diyl (1,1- Dimethylethylene) and the like.

The term "heteroaryl" refers to one or more heteroatoms (typically one to three heteroatoms) having one ring or two fused rings and selected from nitrogen, oxygen, and sulfur in the ring. It means the monovalent aromatic containing. Unless defined otherwise, such heteroaryl groups typically comprise a total of 5 to 10 ring atoms. Representative heteroaryl groups are, for example, pyrrolyl, isoxazolyl, isothiazolyl, pyrazolyl, pyridyl (or, equivalently, pyridinyl), oxazolyl, oxadizolyl, thiadiazolyl, thiazolyl, imidazolyl , Triazolyl, tetrazolyl, furanyl, triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, quinolyl, indole Reel, isoquinolyl, and the like, wherein the point of attachment is any available carbon or nitrogen ring atom.

A "heterocyclyl" or "heterocyclic ring" can be a single ring or a plurality of rings (ie fused or bridged) and selected from nitrogen, oxygen, and sulfur It means a monovalent saturated or partially unsaturated cyclic non-aromatic group containing the above heteroatoms (typically 1 to 3 heteroatoms). Unless defined otherwise, such heterocyclyl groups typically comprise a total of 5 to 10 ring atoms. Representative heterocyclyl groups are, for example, pyrrolidinyl, pyrrridinyl, piperazinyl, imidazolidinyl, morpholinyl, indolin-3-yl, 2-imidazolinyl, 1,2,3, 4-tetrahydroisoquinolin-2-yl, quinuclidinyl, and the like.

The term "halo" means fluoro, chloro, bromo, or iodo.

As used herein, the term "treatment" refers to the treatment of a disease or medical condition in a patient, such as a mammal (especially a human), which:

(a) preventing the occurrence of a disease or medical condition, ie prophylactic treatment of a patient;

(b) ameliorating the disease or medical condition, ie, removing or causing regression of the disease or medical condition in the patient;

(c) inhibiting the disease or medical condition, ie, slowing or arresting the occurrence of the disease or medical condition in a patient; or

(d) alleviating symptoms of a disease or medical condition in a patient.

The term "therapeutically effective amount" means an amount sufficient to effect treatment when administered to a patient in need thereof.

"β ₂ adrenergic receptor activity associated with the disease or condition _{(disease or condition associated with β 2} adrenergic receptor activity)" that phrase β2 adrenergic includes all medical conditions that are mitigated by treatment with the receptor agonist and, β ₂ adrenergic It includes all disease states and / or symptoms presently recognized or found in the future that are associated with receptor activity. Such disease states include, but are not limited to, neurological and heart diseases, as well as lung diseases such as asthma and chronic obstructive pulmonary disease (chronic bronchitis and emphysema). β2 adrenergic receptor activity is also known to be associated with preterm birth (see US Pat. No. 5,872,126) and some infectious diseases (see WO 99/30703 and US Pat. No. 5,290,815).

The term "pharmaceutically-acceptable salt" means a salt prepared from an acceptable base or acid for administration to a patient, such as a mammal. Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and pharmaceutically acceptable inorganic or organic acids.

Salts derived from pharmaceutically acceptable acids include acetates, benzenesulfonates, benzoates, camposulfonates, citrates, ethanesulfonates, fumarates, gluconates, glutamate, hydrobromide, hydrochlorides, lactates, Maleate, malate, mandelate, methanesulfonate, mucic, nitrate, pantothenate, phosphate, succinate, sulfate, tartarate, p-toluenesulfonate, kinapoic (1-hydroxy-2- Naphthoic acid) (xinafoic (1-hydroxy-2-naphthoic acid)), 1,5-naphthalene disulfonate, cinnamate, and the like. Of particular interest are salts derived from fumaric acid, hydrobromic acid, hydrochloric acid, acetic acid, sulfuric acid, methanesulfonic acid, 1,5-naphthalene disulfonic acid, xinafoic, oxalic acid, tartaric acid, and 4-methyl-cinnamic acid.

Salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, potassium, sodium, zinc and the like. Salts derived from pharmaceutically acceptable organic bases include substituted amines, cyclic amines, arginine, betaine, caffeine, choline, N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydravamin, isopropylamine, lysine, methylglucamine, morpholine, piperazine Primary, secondary and tertiary including piperadine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, naturally occurring amines such as tromethamine, and the like. Salts of amines; and the like.

The term "solvate" refers to a complex or aggregate formed by one or more solute molecules, that is, a compound of the present invention or a pharmaceutically acceptable salt thereof, and one or more solvent molecules. it means. Such solvates are typically crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include, for example, water, methanol, ethanol, isopropanol, acetic acid and the like. If the solvent is water, the solvate formed is a hydrate.

The term "pharmaceutically-acceptable salt or solvate of stereoisomers thereof" refers to solvates of pharmaceutically acceptable salts of the stereoisomers of the compounds of formula (I), It is intended to include all permutations of salts, solvates and stereoisomers.

The term "leaving group" refers to a functional group or atom that can be substituted by another functional group or atom in a substitution reaction such as a nucleophilic substitution reaction. By way of example, representative leaving groups include chloro, bromo and iodo groups; Sulfonic ester groups such as mesylate, tosylate, brosylate, nosylate and the like; And acyloxy groups such as acetoxy, trifluoroacetoxy and the like.

The term "amino-protecting group" means a protecting group suitable for preventing unwanted reactions in amino nitrogen. Representative amino-protecting groups include formyl; Acyl groups such as alkanoyl groups such as acetyl; alkoxycarbonyl groups such as tert -butoxycarbonyl (Boc); Arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); Arylmethyl groups such as benzyl (Bn), trityl (Tr), and 1,1-di (4'-methoxyphenyl) methyl; Silyl groups such as trimethylsilyl (TMS) and tert -butyldimethylsilyl (TBS); And the like, but are not limited thereto.

The term "hydroxy-protecting group" means a protecting group suitable for preventing unwanted reactions in the hydroxy group. Representative hydroxy-protecting groups include alkyl groups such as methyl, ethyl and tert -butyl; Acyl groups such as alkanoyl groups such as acetyl; Arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (benzhydryl, DPM); Silyl groups such as trimethylsilyl (TMS) and tert -butyldimethylsilyl (TBS); And equivalents thereof, but is not limited thereto.

General Synthesis Procedures

Compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. While particular aspects of the invention are described in the following tables, those skilled in the art to which this invention belongs may use the methods described herein or those skilled in the art to which the invention pertains. It will be appreciated that it can be prepared using other methods, reagents and starting materials. Given that conventional or desirable process conditions (ie reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.), it is noted that other process conditions may also be used unless otherwise stated. Will be understood. Optimum reaction conditions vary depending on the particular reactants or solvent employed, but such conditions can be determined by routine optimization procedures by one of ordinary skill in the art to which this invention belongs.

In addition, as will be apparent to those skilled in the art, conventional protecting groups may be needed to prevent certain functional groups from carrying out unwanted reactions. The selection of suitable protecting groups for particular functional groups and the selection of suitable conditions for protection and deprotection are known to those skilled in the art. For example, numerous protecting groups, and their introduction and removal, are described in TW Greene and GM Wuts, Protecting Groups in Organic Synthesis , Third Edition, Wiley, New York, 1999 , and references cited herein.

In one synthetic method, the compounds of formulas (I) and (II) are prepared as illustrated in Table A. (The substituents and variables shown in the following diagrams have the definitions provided above, unless otherwise indicated.)

Chart A

In the table, P ¹ represents a hydroxy-protecting group, P ² represents a hydroxy protecting group and L represents a leaving group such as bromo.

As shown in Table A, compounds of formula I are first reacted with aryl amines ( 2 ) to provide intermediates of formula 3 . Typically, this reaction is carried out in an organic solvent with heating in the presence of a base and transition metal catalyst and an arylphosphine ligand. A useful catalyst for bonding aryl groups to aryl amines is tris (dibenzylideneacetone) dipalladium (O) with lac- 2,2'-bis (diphenylphosphino) -1,1'-binaftil. The reaction is typically heated at a temperature between about 50 ° C. and about 120 ° C. for a time between about 0.25 hours and about 12 hours. The protecting group P ¹ is a silyl protecting group which is usually removed from the intermediate of formula 3 using a fluorine or acid reagent to provide the intermediate of formula 4 . The protecting group P ² is usually a benzyl protecting group, which is usually removed from the intermediate of formula 4 by hydrogenation with a palladium on carbon catalyst to give the product.

An alternative method of preparing Intermediate 3 is illustrated in Table B.

Chart B

The conditions for coupling of intermediates 5 and 6 of Table B to produce intermediate 3 are typically the same as those used for binding of intermediates 1 and 2 in Table A.

However, another alternative method of preparing intermediate 3 is illustrated in Table C.

Chart C

The reaction of Table C is usually carried out in a polar aprotic solvent in the presence of a base. Typical suitable solvents include dimethyl sulfoxide, dimethyl formamide, dimethylacetamide and the like. The reaction is typically heated at a temperature between about 60 ° C. and about 140 ° C. for a time between about 0.25 hours and about 4 hours.

Compounds of formulas 1 and 7 that are employed in the reactions described in this application are known in the art to which the present invention pertains, for example US Pat. Nos. 6,653,323 B2 and 6,670,376 B1, incorporated herein by reference. And the procedures described in the references herein. Intermediate 5 can be prepared by reacting intermediate 7 with 2- (4-aminophenyl) ethylamine with heating in an aprotic solvent.

Intermediates 2 and 6 are prepared from commercially available or readily available starting materials. For example, R ⁵ is _- [OC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ and R ⁶ can be made by hydrogen one time, the general formula 2 intermediate 2 'is in the process of Chart D,

Chart D

In the Figure ^5a is R is -OR ^5a - is defined to be a _{- [6} alkylenyl _{^{OC 1] n -NR 10 R 11}} . As an example of suitable reaction conditions for table D, the reaction is carried out in dimethylsulfoxide in the presence of sodium hydride.

For example, R ⁵ is C _{1 - 6} alkylenyl _n -NR ¹² R ¹³ and R ⁶ can be made by hydrogen one time, the process of the general formula 2 of the intermediate 2 'is Table E,

Chart E

In the Table R ^5b is C _{1 - 6} is an alkenyl group.

Intermediates of formula 8 may be prepared by reacting the intermediate of formula 2 with phenethylamine substituted with a leaving group at the 4-position of the phenyl ring, for example 4-bromophenethylamine.

Further details of the specific reaction conditions and other procedures for preparing the representative compounds of the present invention or intermediates thereof are described in the Examples below.

Thus, in a method embodiment, the present invention provides a method of preparing a compound of formula (I), or a salt or stereoisomer or protected derivative thereof, wherein the method comprises:

(a) (i) a compound of formula (III):

Compounds of the following formula (IV):

React in the presence of a transition metal catalyst; or

(ii) with a compound of formula (V):

A compound of formula (VI)

React,

Wherein P ¹ is a hydroxy-protecting group, each R ^la , R ^2a , R ^3a And R ^4a is independently R ¹ , R ² , R ³ of formula (I) And R ⁴ is defined as being the same, or P ² is —OP ² which is a hydroxy protecting group; One of A and B is a leaving group and the other of A and B is -NH ₂ ; L is a leaving group; R ⁵ , R ⁶ , R ⁷ , R ⁸ And R ⁹ is defined as in formula (I), thereby providing a compound of formula (VII);

(b) removing the protecting group P ¹ to provide a compound of formula (VIII):

; 및

; And

(c) any R ^la , R ^2a , R ^3a , Or when R ^4a is —OP ² , removing protecting group P ² to provide a compound of formula (I), or a salt or stereoisomer thereof.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions comprising the compounds of the invention. Thus, the compounds, preferably in the form of pharmaceutically acceptable salts, may be formulated in any suitable dosage form such as oral or parenteral administration or administration by inhalation.

By way of example, the compound may be mixed with conventional pharmaceutical carriers and excipients and used in the form of powders, tablets, capsules, elixirs, suspensions, syrups, wafers, and equivalents thereof. Such pharmaceutical compositions will comprise from about 0.05% to about 90% by weight of the active compound, more generally from about 0.1 to about 30% of the active compound. Pharmaceutical compositions may include common carriers and excipients such as corn starch or gelatin, lactose, magnesium sulfate, magnesium stearate, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, and alginic acid. Disintegrators commonly used in the formulations of the present invention include croscarmellose, microcrystalline cellulose, corn starch, starch glycolate and alginic acid.

Liquid compositions generally consist of a suitable liquid carrier (s), for example a suspension or solution of a non-aqueous solvent such as ethanol, glycerin, sorbitol, polyethylene glycol, oils or a compound or pharmaceutically acceptable salt in water. And optionally suspending agents, solubilizing agents (eg cyclodextrins), preservatives, surfactants, wetting agents, flavoring agents or coloring agents. Alternatively, liquid formulations may be prepared from reconstitutable powders.

For example, a powder comprising the active compound, suspending agent, sucrose and sweetener can be reconstituted with water to form a suspension; Syrups can be prepared from powders comprising the active ingredient, sucrose and sweeteners.

The composition in the form of a tablet may be prepared using any suitable pharmaceutical carrier (s) commonly used to prepare solid compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and binders such as polyvinylpyrrolidone. Colors included in a portion of the colored film coating or carrier are also provided in the tablet. In addition, the active compounds can be prepared in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix.

The composition in the form of a capsule may be prepared by incorporating the active compound and excipients into hard gelatin capsules using, for example, general encapsulation procedures. Alternatively, a semi-solid matrix of active compound and high molecular weight polyethylene glycol is prepared and filled into hard gelatin capsules; Alternatively, a solution of the active compound in polyethylene glycol or a suspension in edible oil such as liquid paraffin or fractionated coconut oil may be prepared and filled into soft gelatin capsules.

Tablet binders that may be included are acacia, methylcellulose, sodium carboxymethylcellulose, poly-vinylpyrrolidone (povidone), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose. Lubricants that can be used are magnesium stearate or other metallic stearates, stearic acid, silicone fluids, talc, waxes, oils and colloidal silica.

Flavoring agents such as peppermint, methyl salicylate (oil of wintergreen), cherry flavors and the like can also be used. In addition, it may be desirable to add colorants to make the dosage form more attractive or to help identify the product.

The compounds of the present invention and their pharmaceutically acceptable salts that are active when administered parenterally can be formulated for intramuscular, intrathecal, or intravenous administration.

Conventional compositions for intramuscular or intrathecal administration will consist of a suspension or solution of the active ingredient in oil, for example arachis oil or sesame oil. Conventional compositions for intravenous or intrathecal administration will consist of sterile isotonic aqueous solutions comprising, for example, the active ingredient with dextrose or sodium chloride, or a mixture of dextrose and sodium chloride. Other examples include lactated Ringer's injection, lactic acid-added Ringer's + dextrose injection, normosol-M and dextrose, isolyte E, acylated Ringer's injection (acylated Ringer's injection), and equivalents thereof. Optionally, co-solvents such as polyethylene glycol; Chelating agents such as ethylenediamine tetraacetic acid; Solubilizers such as cyclodextrins; And antioxidants such as sodium metasulfite may be included in the formulation. Alternatively, the solution may be lyophilized and then reconstituted with a suitable solvent just prior to administration.

The compounds of the present invention and their pharmaceutically acceptable salts that are active when administered topically can be prepared in transdermal compositions or transdermal delivery devices (“patches”). Such compositions include, for example, backing, active compound reservoirs, control membranes, liners, and contact adhesives. Such transdermal patches can be used to continuously or discontinuously inject the compounds of the invention in controlled amounts. The preparation and use of transdermal patches for the delivery of pharmaceutical agents are well known in the art. See, for example, US Pat. No. 5,023,252. Such patches can be prepared for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

One preferred mode of administering the compounds of the present invention is inhalation. Inhalation is an effective means of delivering agents directly to the respiratory tract. There are three general categories of pharmaceutical inhalation devices: nebulizer inhalers, dry powder inhalers (DPI), and metered-dose inhalers (MDI). Conventional nebulizer devices produce a stream of high velocity air that causes the therapeutic agent to be sprayed with a mist to be delivered to the patient's respiratory tract. The therapeutic agent is prepared in a liquid formulation, such as a solution or suspension of respirable sized micronized particles, wherein 'micronized' typically refers to having at least about 90% of particles having a diameter of less than about 10 μm. Is defined.

Typical formulations for use in conventional nebulizer devices are aqueous isotonic solutions of pharmaceutical salts of the active agent at concentrations between about 0.05 μg / mL and about 1 mg / mL. Suitable nebulizer devices are provided commercially, for example by PARI GmbH (Stanberg, Germany). Other nebulizer devices are disclosed, for example, in US Pat. No. 6,123,068.

DPI typically administers a therapeutic agent in a free-flowing powder formulation that can be dispersed in the patient's air-stream during inspiration. Alternative DPI devices using external energy sources to disperse the powder are also being developed. To achieve a free flowing powder state, the therapeutic agent may be formulated with a suitable excipient (eg, lactic acid or starch). Dry powder formulations are prepared, for example, by combining dried lactose particles with finely formulated particles of a suitable formulation of a compound of the invention, typically a pharmaceutically acceptable salt (ie, active agent), and dry blending. Can be. Alternatively, the active agent may be formulated without excipients. The formulation is loaded into a dry powder dispenser or capsule for use with suction cartridges or dry powder delivery device.

Examples of commercially available DPI delivery devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, eg, US Pat. No. 5,035,237); Diskus from GlaxoSmithKline (see, eg, US Pat. No. 6,378,519); Turbuhaler (AstraZeneca, Wilmington, DE) (see, eg, US Pat. No. 4,524,769); And Rotahaler (GlaxoSmithKline) (see, eg, US Pat. No. 4,353,365). Further examples of suitable DPI devices are disclosed in US Pat. Nos. 5,415,162, 5,239,993, and 5,715,810 and the references included herein.

MDI typically uses a compressed propellant gas to release a measured amount of therapeutic agent. Formulations for MDI administration include solutions or suspensions of the active ingredient in liquefied propellants. Chlorofluorocarbons such as CCl ₃ F have been commonly used as propellants, but because of the deleterious effects of such agents on the ozone layer, 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1 Formulations have been developed using hydrofluoroalkanes (HFA) such as, 1,2,3,3,3-heptafluoro-n-propane (HFA 227). Additional components of HFA formulations for MDI administration include co-solvents such as ethanol or pentane and surfactants such as sorbitan trioleate, oleic acid, lecithin, and glycerin. (See, eg, US Pat. No. 5,225,183, EP 0717987 A2, and International Patent Publication No. 92/22286.)

Thus, suitable formulations for MDI administration include from about 0.001% to about 2% by weight of the current crystalline formulation, from about 0% to about 20% by weight of ethanol, and from about 0% to 5% by weight of the surfactant; The rest may be HFA propellants. In one approach to preparing the formulation, the cooled or pressurized hydrofluoroalkane is added to a vial comprising the crystalline formulation of the invention, ethanol (if present), and surfactant (if present). To prepare a suspension, a pharmaceutical salt is provided as micronized particles. The formulation is loaded into an aerosol canister that forms part of the MDI device. Examples of MDI devices developed specifically for use with HFA propellants are provided in US Pat. Nos. 6,006,745 and 6,143,227.

In an alternative method of preparation, the suspension formulation is prepared by spray drying a coating of the surfactant onto the micronized particles of the pharmaceutical salt of the active compound. (See, eg, WO 99/53901 and WO 00/61108) For further examples of methods for preparing respirable particles and formulations and devices suitable for inhalation administration, see US Pat. Nos. 6,268,533, 5,983,956. , 5,874,063, and 6,221,398 and WO 99/55319 and WO 00/30614.

It will be appreciated that any formulation (ie free base, pharmaceutical salt, or solvate) of the compounds of the invention suitable for a particular dosage form may be used in the pharmaceutical compositions discussed above.

The active compounds are expected to be administered in a therapeutically effective amount over a wide range of dosage ranges. However, the amount of compound actually administered will be determined by the physician taking into account relevant conditions such as the condition to be treated, the route of administration chosen, the actual compound administered and its relative activity, the age, weight and response of the individual patient, the severity of the patient's symptoms, and the like. will be.

The compound may be administered at periodic doses: weekly, several times per week, daily, or several times daily. Treatment regimens may require administration for extended periods of time, for example weeks or months, or treatment regimens may require long term administration. Suitable dosages for oral administration range from about 0.05 μg / day to about 100 mg / day, preferably 0.5 to 1000 μg / day.

Suitable dosages of therapeutic agents for inhalation administration are in the general range of about 0.05 μg / day to about 1000 μg / day, preferably 0.1 μg / day to 500 μg / day. The proportion of active agent delivered to the lung characteristic of the particular delivery devices is considered to determine suitable dosages for inhalation administration.

Among other properties, the compounds of the present invention have been found to be potent and selective agonists of β ₂ adrenergic receptors. In particular, the compounds of the present invention show excellent selectivity for β ₂ adrenergic receptors over β ₁ and β ₃ adrenergic receptors. In addition, the compounds of the present invention have been found to have surprising and unexpected duration of action. As described in the following biological assays, the compounds of the present invention showed a duration of action of 24 hours or more in an animal model of bronchoprotection.

Accordingly, the present invention provides a method of treating a disease or condition in a mammal associated with β ₂ adrenergic receptor activity, said method comprising the steps of a compound of the invention or a pharmaceutical composition comprising a compound of the invention. Administering a therapeutically effective amount.

Active agents of the invention may also be used as part of a combination that additionally includes one or more other therapeutic agents. For example, the agents of the present invention may be anti-inflammatory agents (eg, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs)), anticholinergic agents (particularly muscarinic receptor antagonists), other β ₂ adrenergic receptors It may be administered with one or more therapeutic agents selected from agonists, anti-infectives (eg antibiotics or antiviral agents) or antihistamines. The present invention thus provides, in another embodiment, a combination comprising a compound of the invention and one or more therapeutic agents, such as anti-inflammatory, anticholinergic, another β ₂ adrenergic receptor agonist, anti-infective or antihistamine do.

Other therapeutic agents may be used in the form of pharmaceutically acceptable salts or solvates. If appropriate, other therapeutic agents may be used as optically pure stereoisomers.

Suitable anti-inflammatory agents include corticosteroids and NSAIDs. Suitable corticosteroids that can be used with the compounds of the present invention are oral and inhaled corticosteroids having anti-inflammatory activity and their pro-drugs. Examples are methyl prednisolone, prednisolone dexamethasone, fluticasone propionate, 6α, 9α-difluoro-17α-[(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl-3-oxo- Androsa-1,4-diene-17β-carbothioic acid S -fluoromethyl ester, 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-andros -1,4-diene-17β-carbothioic acid S- (2-oxo-tetrahydro-furan-3S-yl) ester, biclomethasone esters (e.g., 17-propionate ester or 17, 21-dipropionate esters), budesonide, flunisolid, mometasone esters (e.g., furoate esters), triamcinolone acetonide, loflefonide, ciclesonide, butyxocort propionate, RPR-106541, and ST-126. Preferred corticosteroids are fluticasone propionate, 6α, 9α-difluoro-11β-hydroxy-16α-methyl-17α-[(4-methyl-1,3-thiazole-5-carbonyl) oxy ] -3-oxo-andros-1,4-diene-17β-carbothioic acid S -fluoromethyl ester and 6α, 9α-difluoro-17α-[(2-furanylcarbonyl) oxy] -11β -Hydroxy-16α-methyl-3-oxo-androsa-1,4-diene-17β-carbothioic acid S -fluoromethyl ester, more preferably 6α, 9α-difluoro-17α -[(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl-3-oxo-andros-1,4-diene-17β-carbothioic acid S -fluoromethyl ester.

Suitable NSAIDs include sodium chromoglycate; Nedrochromyl sodium; Phosphodiesterase (PDE) inhibitors (eg, theophylline, PDE4 inhibitors or mixed PDE3 / PDE4 inhibitors); Leukotriene antagonists (eg montelukast); Inhibitors of leukotriene synthesis; iNOS inhibitors; Protease inhibitors such as tryptase and elastase inhibitors; Beta-2 integrin antagonists and adenosine receptor agonists or antagonists (eg, adenosine 2a agonists); Cytokine antagonists (eg, interleukin antibodies (αIL antibodies), in particular chemokine antagonists such as αIL-4 therapy, αIL-13 therapy or combinations thereof); Or cytokine synthesis inhibitors. Other suitable β ₂ adrenergic receptor agonists include salmeterol (eg kinapoate), salbutamol (eg sulphate or free base), formoterol (eg fumarate) , Phenoterol or terbutalin and salts thereof.

Also of note is the use of the active agents of the invention in combination with phosphodiesterase 4 (PDE4) inhibitors or mixed PDE3 / PDE4 inhibitors. Representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3 / PDE4 inhibitors include cis 4-cyano-4- (3-cyclopentyloxy-4-methoxyphenyl) cyclohexane-l-carboxylic acid, 2- Carbomethoxy-4-cyano-4- (3-cyclopropylmethoxy-4-difluoromethoxyphenyl) cyclohexane-l-one; Cis- [4-cyano-4- (3-cyclopropylmethoxy-4-difluoromethoxyphenyl) cyclohexan-1-ol]; Cis -4-cyano-4- [3- (cyclopentyloxy) -4-methoxyphenyl] cyclohexane-1-carboxylic acid and equivalents thereof, or pharmaceutically acceptable salts thereof. Other representative PDE4 or mixed PDE4 / PDE3 inhibitors include AWD-12-281 (elbion); NCS-613 (INSERM); D-4418 from Chiroscience and Schering-Plough; CI-1018 or PD-168787 (Pfizer); Benzodioxol compounds disclosed in WO 99/16766 (Kyowa Hakko); V-11294A (Napp); Roflumilast (Byk-Gulden); Phthalazinone compounds (Byk-Gulden) disclosed in WO 99/47505; K-34 from Byk-Gulden; Fumapentrin (Byk-Gulden, now Altana); Arophylline (Almirall-Prodesfarma); VM554 / UM565 from Vernalis; T-440 from Tanabe Seiyaku; And T2585 (Tanabe Seiyaku).

Suitable anticholinergic agents are compounds which act as antagonists at the muscarinic receptor, in particular compounds which are antagonists of the M ₁ , M ₂ , or M ₃ receptors, or combinations thereof. Representative compounds include alkaloids of belladonna plants, such as atropine, scopolamine, homatropin, hydroxyamine; The compounds are generally administered as salts which are tertiary amines.

Formulations of these drugs, in particular salts, are readily available from a number of commercial sources or can be prepared from literature data through:

Atropine-CAS-51-55-8 or CAS-51-48-1 (anhydride form), atropine sulfate-CAS-5908-99-6; Atropine oxide-CAS-4438-22-6 or its hydrochloride-CAS-4574-60-1 and methylatropine nitrate-CAS-52-88-0.

Homatropin-CAS-87-00-3, hydrobromide-CAS-51-56-9, methyl bromide salt-CAS-80-49-9.

Hydroxyamine ( d, l ) -CAS-101-31-5, hydrobromide-CAS-306-03-6 and sulphate salt-CAS-6835-16-1.

Scopolamine-CAS-51-34-3, hydrobromide-CAS-6533-68-2, methyl bromide salt-CAS-155-41-9.

Preferred anticholinergic agents are ipratropium (eg as its bromide), oxytropium (eg as its bromide) and tiotropium (eg its bromide) sold under the trade name Atrovent. (CAS-139404-48-1). Also of interest are the following compounds: methanetellin (CAS-53-46-3), propantelin bromide (CAS-50-34-9), anisotropin methyl bromide or Valpin 50 (CAS-80-50-2), clidinium bromide (Quarzan, CAS-3485-62-9), copyrrolate (Robinul), isopropamide iodide (CAS-71-81-8), Phenzolate bromide (US Pat. No. 2,918,408), tridihexetyl chloride (Pathilone, CAS-4310-35-4), and hexocyclium methylsulfate (Tral, CAS-115-63-9). In addition, cyclopentolate hydrochloride (CAS-5870-29-1), trophamide (CAS-1508-75-4), trihexyfenidyl hydrochloride (CAS-144-11-6), pyrene Zepin (CAS-29868-97-1), tellenzepine (CAS-80880-90-9), AF-DX 116, or methoxtamine, and WO 01/04118, the disclosure of which is incorporated herein by reference compound.

Suitable antihistamines (also referred to as H ₁ -receptor antagonists) include one or more of a number of antagonists known to inhibit H ₁ receptors and to be safe for human use. All are reversible and competitive inhibitors of the interaction of histamine with H ₁ -receptors. The majority of these inhibitors, which are mostly first generation antagonists, are characterized by ethanolamines, ethylenediamines, and alkylamines based on their core structures. Other first generation antihistamines also include compounds that can be characterized as based on piperizine and phenothiazines. Non-sedating second generation antagonists have a similar structure-activity relationship in that they have a core ethylene group (alkylamines) or resemble tertiary amine groups with piperizine or piperidine. Representative antagonists include:

Ethanolamines: carvinoxamine maleate, clemastine fumarate, diphenylhydramine hydrochloride, and dimenhydrinate.

Ethylenediamines: pyrylamine amritate, tripelennamin HCl, and tripelennamin citrate.

Alkylamines: salts thereof such as chlorpheniramine and maleate salts, and acrivastin.

Piperazines: hydroxyzin HCl, hydroxyzin pamoate, cyclizin HCl, cyclizin lactate, meclizin HCl, and cetirizine HCl.

Piperidines: astemizole, levocarvastin HCl, rollatadine or descarboethoxy analogs thereof, and terfenadine and fexofenadine hydrochloride or another pharmaceutically acceptable salt.

Azelastine hydrochloride is another H ₁ receptor antagonist that can be used in combination with a compound of the present invention.

Examples of preferred anti-histamines include metapyrylene and rollatadine.

The present invention therefore provides, in a further aspect, a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer and corticosteroid thereof. In particular, the present invention provides that the corticosteroid is fluticasone propionate or the corticosteroid is 6α, 9α-difluoro-17α-[(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl 3-oxo-andros-1,4-diene-17β-carbothioic acid S -fluoromethyl ester or 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propy A combination is provided that is onyloxy-andros-1,4-diene-17β-carbothioic acid S- (2-oxo-tetrahydro-furan-3S-yl) ester.

The present invention therefore provides, in a further aspect, a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer thereof and a PDE4 inhibitor.

The present invention therefore provides, in a further aspect, a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer and anticholinergic agent thereof.

The present invention therefore provides in a further aspect a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer and antihistamine.

The present invention therefore provides in a further aspect a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer thereof, in combination with a PDE4 inhibitor and a corticosteroid.

The present invention therefore provides, in a further aspect, a combination comprising a compound of formula (I), or a pharmaceutically acceptable salt or solvate or stereoisomer thereof, together with an anticholinergic and corticosteroid.

As used in the above combinations, the term “a compound of formula (I)” refers to a compound of formula (II) and preferred groups thereof, and any individually disclosed compound or compound Include them.

Thus, the pharmaceutical compositions of the present invention may optionally comprise a compound of formula (I) or a pharmaceutically acceptable salt or solvate or stereoisomer thereof in combination with one or more therapeutic agents, as described above.

Individual compounds of the combinations of the present invention may be formulated separately or together in one pharmaceutical composition. Individual compounds may be administered sequentially or simultaneously in separate or combined pharmaceutical agents. Suitable dosages of known therapeutic agents will be readily understood by those skilled in the art to which this invention pertains. The methods of treatment of the present invention thus comprise administering such compounds in separate or combined pharmaceutical formulations sequentially or simultaneously.

Thus, according to a further aspect, the present invention provides a method of treating a disease or condition associated with β ₂ adrenergic receptor activity in a mammal, said method comprising the steps of: Administering a therapeutically effective amount of an acceptable salt or solvate or stereoisomer and one or more other therapeutic agents.

Since the compounds of the invention are β reading ₂ adrenergic receptor agonists, compounds such as those also a research tool for studying or discover new β ₂ adrenergic receptor agonist biological systems or samples having β ₂ adrenergic receptors Useful as In addition, since the compounds of the present invention show selectivity for β ₂ adrenergic receptors relative to binding and functional activity in receptors of other β adrenergic subtypes, such compounds are also β ₂ in biological systems or samples. It is useful to study the effects of selective agonism of adrenergic receptors. Any suitable biological system or sample having β ₂ adrenergic receptors can be employed in such studies that can be performed in vitro or in vivo . Representative biological systems or samples useful for such studies include cells, cell extracts, plasma membranes, tissue samples, mammals (mouses, mice, guinea pigs, rabbits, dogs, pigs, etc.). Including, but not limited to.

biological system comprising a β ₂ adrenergic receptor, or the sample is in contact with the positive (agonizing amount) for enhancing the β ₂ adrenergic receptors, the compounds of the invention. The effect of advancing β ₂ adrenergic receptors can be attributed to radioligand binding measurements and functional tests, for example, the measurement of ligand-mediated changes or similar properties in intracellular cyclic adenosine monophosphate (cAMP) described below. Determination using conventional procedures and apparatus such as The amount of β ₂ adrenergic receptor-impression of a compound of the invention will typically range from 1 nanomolar to about 100 nanomolar. When the compounds of the present invention are used as a research tool for discovering novel β ₂ adrenergic receptor agonists, the present invention also, as separate embodiments, generates and targets comparative data (using suitable measurements). Analysis of test data to identify compounds.

The following non-limiting examples illustrate representative pharmaceutical compositions of the present invention. Additional suitable carriers for formulations of the active compounds of the invention may be found in Remington: The Science and Practice of Pharmacy , 20 th Edition , Lippincott Williams & Wilkins, Philadelphia, PA, 2000.

1 is an x-ray powder diffraction pattern of the product of Example 17b.

2 is a differential scanning calorimetry trace of the product of Example 17b.

3 is an x-ray powder diffraction pattern of the product of Example 17c.

4 is a differential scanning calorimeter result of the product of Example 17c.

5 is an x-ray powder diffraction pattern of the product of Example 17d.

6 is a differential scanning calorimeter result of the product of Example 17d.

Formulation Example  A

This example illustrates the preparation of an exemplary pharmaceutical composition for oral administration of a compound of the present invention:

ingredient Amount per tablet (mg) Active compound One Lactose, spray dried 148 Magnesium stearate 2

The ingredients were mixed and placed in hard-shell gelatin capsules.

Formulation Example  B

This example illustrates the preparation of another representative pharmaceutical composition for oral administration of a compound of the present invention:

ingredient Amount per tablet (mg) Active compound One Corn starch 50 Lactose 145 Magnesium stearate 5

The components were mixed finely and pressed into single scored tablets.

Formulation Example  C

This example illustrates the preparation of an exemplary pharmaceutical composition for oral administration of a compound of the present invention.

An oral suspension having the following composition was prepared.

ingredient Active compound 3 mg Fumaric acid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.1 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum K (Vanderbilt Co.) 1.0 g Flavor 0.035 mL coloring agent 0.5 mg Distilled water Adjust total volume to 100 mL (q.s. to 100 mL)

Formulation Example  D

This example illustrates the preparation of an exemplary pharmaceutical composition comprising a compound of the present invention.

Injectable formulations were prepared having the following composition and buffered to pH 4:

ingredient Active compound 0.1 mg Sodium Acetate Buffer Solution (0.4M) 2.0 mL HCl (1N) Fit to pH4 Water (distilled, sterilized) Adjust total volume to 20 mL

Formulation Example  E

This example illustrates the preparation of representative pharmaceutical compositions for injection of the compounds of the invention.

Reconstituted solution was prepared by adding 20 mL of sterile water to 1 mg of the compound of the present invention. Prior to use, the solution was diluted with 200 mL of intravenous fluid compatible with the active compound. Such sap is selected from 5% dextrose solution, 0.9% sodium chloride solution, or a mixture of 5% dextrose and 0.9% sodium chloride. Other examples are Ringer's injection with lactic acid, Ringer's with lactic acid plus 5% dextrose injection, normosol-M and 5% dextrose, isolite E, and acylated Ringer's injection.

Formulation Example  F

This example illustrates the preparation of a representative pharmaceutical composition for topical application of a compound of the invention.

ingredient Volume (g) Active compound 0.2-10 Span 60 2 Twin 60 2 Mineral oil 5 Petrolatum 10 Methyl paraban 0.15 Profile paraban 0.05 BHA (Butylated Hydroxy Anisole) 0.01 water Fit to 100

All components, except water, were combined under stirring and heated to 60 ° C. Sufficient water was added at 60 ° C. under vigorous stirring to make the components oily and then water to make 100 g.

Formulation Example  G

This example illustrates the preparation of an exemplary pharmaceutical composition comprising a compound of the present invention.

Aqueous aerosol formulations for use in nebulizers were prepared by dissolving 0.1 mg of the pharmaceutical salt of the active compound in 0.9% sodium chloride solution acidified with citric acid. The mixture was stirred and sonicated until the active salt dissolved. The pH of the solution was adjusted from pH 3 to pH 8 by the slow addition of NaOH.

Formulation Example  H

This example illustrates the preparation of a dry powder formulation comprising a compound of the invention for use in a suction cartridge.

Gelatin inhalation cartridges were filled with a pharmaceutical composition having the following ingredients:

ingredient Mg per cartridge Pharmaceutical salts of active compounds 0.2 Lactose 25

Pharmaceutical salts of the active compounds were micronized prior to blending with lactose. The contents of the cartridges were administered using a powder inhaler.

Formulation Example  I

This example illustrates the preparation of a dry powder formulation comprising a compound of the invention for use in a dry powder inhalation device.

A pharmaceutical composition was prepared having a bulk formulation ratio of 1: 200 micronized pharmaceutical salts to lactose. The composition was filled in a dry powder inhalation device capable of delivering an active drug component in an amount between about 10 μg and about 100 μg per dose.

Formulation Example  J

This example illustrates the preparation of a formulation comprising a compound of the invention for use in a metered dose inhaler.

5 g of the active compound in a colloidal solution formed from 0.5 g of trihalose and 0.5 g of lecithin dissolved in 100 mL of demineralized water as micronized particles having an average size of less than 10 μm to obtain a pharmaceutical salt of the active compound 5 A suspension comprising%, 0.5% lecithin, and 0.5% trihalose was prepared. The suspension was spray dried and the result was micronized into particles having an average diameter of less than 1.5 μm. The particles were loaded into canisters with pressurized 1,1,1,2-tetrafluoroethane.

Formulation Example  K

This example illustrates the preparation of a formulation comprising a compound of the invention for use in a metered dose inhaler.

In a solution formed from 0.2 g of lecithin dissolved in 200 mL of demineralized water, 10 g of the active compound was dispersed as micronized particles having an average size of less than 10 μm to give a suspension comprising 5% of the pharmaceutical salt of the active compound and 0.1% of lecithin. Prepared. The suspension was spray dried and the result was micronized into particles having an average diameter of less than 1.5 μm. The particles were loaded into canisters with pressurized 1,1,1,2,3,3,3-heptafluoro-n-propane.

Biological measurements

The compounds of the present invention and their pharmaceutically acceptable salts exhibit biological activity and are useful for medical treatment. The ability of a compound to bind β ₂ adrenergic receptors and their selectivity, agonist potency, and intrinsic activity were demonstrated using the following tests AB or other tests known in the art to which this invention pertains. Can be demonstrated.

Abbreviations

% Eff% Efficacy

ATCC American Type Culture Collection

BSA Bovine Serum Albumin

cAMP adenosine 3 ': 5'-cyclic monophosphate

DMEM Dulbecco's Modified Eagle's Medium

DMSO dimethyl sulfoxide

EDTA ethylenediaminetetraacetic acid

Emax Maximum Efficacy

FBS Fetal bovine serum

Gly glycine

HEK-293 Human embryonic kidney-293

PBS Phosphate Buffered Saline

rpm rpm

Tris Tris (hydroxymethyl) aminomethane

Human β _One  Or β ₂ Adrenaline  Expressing receptors

Membrane Preparation from Cells

Cloned human β ₁ or HEK-293-derived cell lines stably expressing β ₂ adrenergic receptors were cultured to near confluency in DMEM containing 10% of dialyzed FBS in the presence of 500 μg / mL of Geneticin. . Monolayers of cells were harvested with Versene 1: 5,000 (0.2 g / L EDTA in PBS) using a cell scraper. Cells were pelleted by centrifugation at 1,000 rpm and cell pellets were stored frozen at -80 ° C or immediately prepared membranes. To prepare, the cell pellets were prepared in lysis buffer (10 mM Tris / HCL pH 7.4 @ 4 ° C., 1 tablet per 50 mL buffer (Roche cat. # 1697498, Roche Molecular Biochemicals, Indianapolis, IN). Resuspend in Protease Inhibitor Cocktail Tablets with 2 mM EDTA "and homogenize using a tight-fitting Dounce glass homogenizer (20 strokes) on ice. Homogenate the homogenate at 20,000 xg The pellet was washed once with lysis buffer and resuspended and centrifuged as described above The finally obtained pellet was membrane buffer (75 mM Tris / HCl pH 7.4, 12.5 mM MgCl ₂ , 1 mM EDTA @ 25 ° C.) The protein concentration of the membrane suspension was determined by the Bradford method (Bradford MM., Analytical Biochemistry , 1976 , 72, 248-54) .The membranes were aliquoted in -80 ° C. Stored frozen.

Test A

Human β _One  And β ₂ Adrenaline  For receptors

Radioligand  Combined measurement

For membranes containing human β ₂ adrenergic receptors, 5 μg of membrane protein or for membranes containing human β ₁ adrenergic receptors, 2.5 μg of membrane protein was measured in assay buffer (75 mM Tris / HCl pH). Binding assays were performed on 96-well microtiter plates at 100 μl total measurement volume in 7.4 @ 25 ° C., 12.5 mM MgCl ₂ , 1 mM EDTA, 0.2% BSA). Saturation binding studies to determine K _d values of radioligand [ ³ H] dihydroalpronolol (NET-720,100 Ci / mmol, PerkinElmer Life Sciences) at 10 different concentrations in the range of 0.01 nM-200 nM Inc., Boston, Mass.). Displacement measurements for determination of pK _i values of compounds were performed at 10 different concentrations in the range of 1 nM and 40 pM-10 μM with [ ³ H] dihydroalprenolol. Compounds are dissolved in dissolving buffer (25 mM Gly-HCl pH 3.0 with 50% DMSO) at a concentration of 10 mM, then diluted to 1 mM with 50 mM Gly-HCl pH 3.0 and therefrom. Dilutions were made sequentially with test buffer. Non-specific binding was determined in the presence of 10 μM unlabeled alprenolol. The measurements were left for 90 minutes at room temperature and terminated by rapid filtration through GF / B glass filter plates (Packard BioScience Co., Meriden, CT) that were presoaked in 0.3% polyethyleneimine. Filter plates were washed three times with filtration buffer (75 mM Tris / HCl pH 7.4 @ 4 ° C., 12.5 mM MgCl ₂ , 1 mM EDTA) to remove unbound radioactive activity. Plates were dried and 50 μl Microscint-20 liquid scintillation fluid (Packard BioScience Co., Meriden, CT) was added and plates were measured with a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden, CT). Binding data was analyzed by nonlinear regression analysis using the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) Using a 3-parameter model for one-site competition. The curve minimum was fixed to the value for nonspecific binding, as determined in the presence of 10 μM alprenolol. K _i values for the compounds are determined from the IC ₅₀ values and the K _d values of the radioligand observed using the Cheng-Prusoff equation (Cheng Y, and Prusoff WH., Biochemical Pharmacology , 1973 , 22,23, 3099-108). Calculated. Receptor subtype selectivity was calculated as the ratio of K _i (β ₁ ) / K _i (β ₂ ). Compounds of the invention are β ₁ adrenergic K _i (β ₁ )> K _i (β ₂ ), with stronger binding at β ₂ adrenergic receptor than at receptor, with selectivity higher than about 30.

Test B

Each human β _One Adrenaline  Receptor, β ₂ Adrenaline  Receptor, and β ₃ Adrenaline  Receptors Heterogeneously ( heterologously )

By expressing cell lines Whole cell (whole cell)

cAMP Flash Plate ( Flashplate Prosecutors

HEK-293 cell lines stably expressing cloned human β ₁ adrenergic receptor (clone H34.1) were about 70-90% in media consisting of 500 μg / mL of geneticin and DMEM supplemented with 10% FBS Incubated to the confluence point. HEK-293 cell lines stably expressing cloned human β ₂ adrenergic receptor (clone H24.14) were incubated in the same medium to complete confluence. CHO-K1 cell lines stably expressing cloned human β ₃ adrenergic receptor (clone H34.1) were 800 μg / mL in every fifth passage in Ham's F-12 medium supplemented with 10% FBS. Incubation was made to about 70-90% confluence with the addition of geneticin. On the day before the measurement was performed, the cultures were transferred to the same growth-medium without antibiotics.

cAMP measurements were performed in a radioimmuneassay format using a Flashplate Adenylyl Cyclase Activation Assay System with ¹²⁵ I-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.).

On the day of the measurement, cells were washed once with PBS, harvested with Versene 1: 5,000 (0.2 g / L EDTA in PBS) and counted. Cells were pelleted by centrifugation at 1,000 rpm and resuspended in stimulation buffer preheated to 37 ° C. For cells expressing β ₁ adrenergic receptor, 10 nM ICI 118,551 was added to a stimula buffer and the cells were left at 37 ° C. for 10 minutes. For cells expressing β ₁ adrenergic receptor, β ₂ adrenergic receptor, β ₃ adrenergic receptor, the cells were used at final concentrations of 30,000, 40,000 and 70,000 cells / well, respectively. Compounds were dissolved in DMSO at a concentration of 10 mM and diluted to 50 mM Gly-HCl pH 3.0 to 1 mM, from which the measurement buffer (75 mM Tris / HCl pH 7.4 @ 25 ° C, 12.5 mM MgCl ₂ , 1 mM EDTA, 0.2% Diluted sequentially with BSA). Compounds were tested at 11 different concentrations ranging from 10 μM to 9.5 pM. The reactions were left at 37 ° C. for 10 minutes and stopped by the addition of 100 μl of ice-cold detection buffer. Plates were sealed and left overnight at 4 ° C. and measured the following day with a topcount scintillation counter (Packard BioScience Co., Meriden, CT). The amount of cAMP produced per mL of reaction was calculated based on the counts observed in samples and cAMP standards, as described in the manufacturer's user manual. Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) Using a 3-parameter model for sigmoidal dose-response (Hill). Slope = 1). Agonist potency was expressed as pEC ₅₀ .

Compounds of the invention showed strong activity at the β ₂ adrenergic receptor in this measurement, evidenced by pEC ₅₀ in excess of about 9. In addition, the tested compounds showed selectivity in functional activity at the β ₂ receptor as compared to functional activity at the β ₁ and β ₃ receptors. In particular, the compounds of the invention showed a ratio of _{EC 50 (β 3) / EC} 50 (β 2) greater than the ratio and 50 of _{EC 50 (β 1) / EC} 50 (β 2) greater than about 10 .

Test C

Human β ₂ Adrenaline  Endogenously expressing receptors

lungs Epithelial cell line  by Whole cell cAMP Flash Plate  Measure field

To determine the strength and potency (unique activity) of agonists in cell lines expressing endogenous levels of β ₂ adrenergic receptors, human lung epithelial cell line (BEAS-2B) was used (ATCC CRL-9609, American Type Culture Collection, Manassas, VA (January B, et al., British Journal of Pharmacology , 1998, 123 , 4,701-11). Cells were cultured to 75-90% confluence in complete serum-free medium (LHC-9 medium with epinephrine and retinoic acid, cat # 181-500, Biosource International, Camarillo, Calif.). The day before the measurement, the medium was replaced with LHC-8 (without epinephrine or retinoic acid, cat # 141-500, Biosource International, Camarillo, CA).

cAMP measurements were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with ¹²⁵ I-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.) according to the manufacturer's instructions.

On the day of the measurement, cells were washed once with PBS, scraped and scraped with 5 mM EDTA in PBS and counted. Cells were pelleted by centrifugation at 1,000 rpm and resuspended at a final concentration of 600,000 cells / mL in stimulation buffer preheated to 37 ° C. Cells were used at the final concentration of 30,000 cells / well in the measurement. Compounds are dissolved in a dissolving buffer (25 mM Gly-HCl pH 3.0 with 50% DMSO) at a concentration of 10 mM and diluted to 50 mM Gly-HCl pH 3.0 to 1 mM, from which the measurement buffer ( diluted sequentially with assay buffer) (75 mMTris / HCl pH 7.4 @ 25 ° C., 12.5 mM MgCl ₂ , 1 mM EDTA, 0.2% BSA).

In the measurement, compounds were tested at 10 different concentrations ranging from 10 μM to 40 pM. The maximum reaction amount was determined in the presence of 10 μM isoproterenol. The reactions were left at 37 ° C. for 10 minutes and stopped by addition of 100 μl of cooled detection buffer solution. The plates were sealed and left overnight at 4 ° C. and measured with a topcount scintillation counter (Packard BioScience Co., Meriden, CT) the next morning. The amount of cAMP produced per mL of reaction was calculated based on the counts observed in samples and cAMP standards, as described in the manufacturer's user manual. Data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, Calif.) Using a four-parameter model for sigmoidal dose-responses with varying slopes. Compounds of the invention tested in this measurement showed pEC ₅ > 8.

Compound efficacy (% Eff) was calculated from the observed Emax (top of the fitted curve) and the maximum response obtained for 10 μM isoproterenol, expressed as% Eff relative to isoproterenol. . The compounds tested showed% Eff greater than about 50.

Test d

Guinea Pig  Model for Acetylcholine-Induced Bronchial Spasm

Bronchial protection Bronchoprotection ) Measurement

Groups consisting of six male guinea pigs (Duncan-Hartley (HsdPoc: DH) Harlan, Madison, Wi.) Weighing 250-350 g were individually identified by cage number. During the study, the animals were randomly ( ad libitum ) provides access to feed and water.

Test compounds were administered via inhalation over 10 minutes in a whole-body exposure dosing chamber (R & S Molds, San Carlos, Calif.). The dosing chambers were arranged such that the aerosol could be delivered to six separate chambers simultaneously from the central manifold. After 60 minutes of acclimation and 10 minutes of exposure to nebulized water (WFI) for administration, the guinea pigs were exposed to the aerosol of the test compound or vehicle (WFI). These aerosols are propelled by a gas mixture (C0 ₂ = 5%, 0 ₂ = 21% and N ₂ = 74%) at a pressure of 22 psi (Model 22F51, PARI Respiratory Equipment, Inc. Midlothian, VA) ) From aqueous solutions. At this operating pressure the gas flow rate through the nebulizer was about 3 L / min. The resulting aerosols are injected into the chambers by positive pressure. Dilution air was not used during the delivery of the aerosolized solutions. During 10 minutes of nebulization, about 1.8 mL of solution was nebulized. This was determined by gravimetric comparison of pre- and post-atomization weights of the filled nebulizer.

The bronchial protective effect of the compounds administered through inhalation was assessed using whole body plethysmography 1.5, 24, 48, and 72 hours after administration. 45 minutes before the start of pulmonary evaluation, each guinea pig was injected by intramuscular injection of ketamine (43.75 mg / kg), xylazine (3.50 mg / kg) and acepromazine (1.05 mg / kg). Anesthetized. After surgical shaving and washing with 70% alcohol, a midline incision was made to the ventral aspect of the neck. The jugular vein was then isolated and saline-filled polyethylene catheter (PE-) for intravenous injection of 0.1 mg / mL solution of acetylcholine (Ach) (Sigma-Aldrich, St. Louis, MO) in saline solution. 50, Becton Dickinson, Sparks, MD). The trachea was then excised and the catheter inserted into a 14G Teflon tube (# NE-014, Small Parts, Miami Lakes, FL). If necessary, anesthesia was maintained by additional intramuscular injections of the anesthetic cocktail described above. The depth of anesthesia was monitored and the depth of anesthesia was adjusted if the animal responded to a pinch of the foot or if the respiration rate was greater than 100 breaths / minute.

After completing the cannulation, the animal is placed on a volumetric recorder (# PLY3114, Buxco Electronics, Inc., Sharon, CT) and the esophageal pressure cannula (P) is used to measure pulmonary driving pressure ( pressure ). esophageal pressure cannula was inserted. A Teflon tracheal tube was attached to the opening of the volumetric recorder to allow the guinea pig to breathe indoor air outside the chamber. The chamber was sealed. Using a heating lamp, the lungs of the guinea pig are placed in a 10 mL calibration syringe (# 5520 Series, Hans Rudolph, Kansas City) to maintain body temperature and to prevent the lower airways from collapsing and the animal undergoing hyperventilation. , MO) was inflated three times (3 times) with 4 mL of air.

If the baseline values are determined to be within 0.3-0.9 mL / cm H ₂ 0 for compliance and within 0.1-0.199 cm H ₂ 0 / mL for resistance, Lung testing was initiated. The Buxco lung measurement computer program enabled the collection and derivation of lung measurements. Running this program initiated experimental protocols and data collection. The volume change over time in the volumetric recorder with each breath was measured with a Buxco pressure transducer. By integration of the signal for the passage of time it was measured for every respiratory per flow rate (flow). This signal, along with changes in closed drive pressure, collected using a Sensym pressure transducer (# TRD4100), was connected to the data acquisition interfaces (# SFT3400 and SFT3813) via a Buxco (MAX 2270) preamplifier. All other lung parameters were derived from these two inputs.

Base values were collected for 5 minutes, and then the guinea pigs were challenged with Ach. Ach was injected intravenously for 1 minute from a syringe pump (sp210iw, World Precision Instruments, Inc., Sarasota, FL) at the following doses and time points from the start of the experiment: 1.9 μg / min 5 minutes after the start of the experiment, 10 minutes 3.8 μg / minute, 15 minutes 7.5 μg / minute, 20 minutes 15.0 μg / minute, 25 minutes 30 μg / minute and 30 minutes 60 μg / minute). If resistance or compliance did not return to baseline values 3 minutes after each Ach administration, lungs of the guinea pig were inflated three times with 4 mL of air from a 10 mL graduated syringe. The recorded lung parameters included respiratory frequency (breath rate / minute), compliance (mL / cmH ₂ 0) and lung resistance (cmH ₂ 0 / mL per _second ) (Giles et al . , 1971). Once pulmonary function measurements were completed at the 35 minute time point in this protocol, the guinea pig was removed from the volumetric recorder and euthanized with CO ₂ choking.

Primary pulmonary resistance derived from the flow rate (flos) and pressure (pressure) for a range of Ach attack, PD ₂ which is defined as the amount of Ach the (baseline pulmonary resistance) required to doubled by using the following equation: Calculated lung resistance values. This was derived from the equation used to calculate PC ₂₀ in the hospital (Am. Thoracic Soc, 2000).

In the formula:

C _l = Second to last Ach concentration (concentration preceding C ₂ )

C ₂ = final concentration of Ach (concentration resulting in a twofold increase in lung resistance (R _L ))

R _o = default R _L value

R ₁ = C _l After R _L value

R ₂ = C ₂ After R _L value

Statistical analysis of the data was performed through one-way analysis of variance and post-hoc analysis using the Bonferroni / Dunn test. P -values <0.05 were considered significant.

Dose-response curves were fitted with a four parameter logistic equation using GraphPad Prism, version 3.00 for Windows (GraphPad Software, San Diego, California).

Y = Min + (Max-Min) / (l + 10 ^ ((1og ED ₅₀ -X) * Hill slope)),

Where X is the logarithm of the dose, Y is the response (PD ₂ ), and Y is an asymptotic approach from Min to S to Max.

Representative compounds of the invention were found to have significant bronchioprotective activity at time points 24 hours after administration.

The following synthetic examples are provided to illustrate the invention and should not be understood as limiting the scope of the invention.

Examples

General: Unless stated otherwise, reagents, starting materials and solvents are commercial sources such as Sigma-Aldrich (St. Louis, MO), JTBaker (Phillipsburg, NJ), and Honeywell Burdick and Jackson (Muskegon, MI) and used without further purification; The reactions were carried out under nitrogen atmosphere; The reaction mixtures were monitored by thin layer chromatography (silica TLC), analytical high performance liquid chromatography (analytical HPLC), or mass spectrometry; The reaction mixtures were usually purified by flash column chromatography on silica gel or preparative HPLC using the general protocol described below; NMR samples were dissolved in deuterated solvent (CD ₃ 0D, CDCl ₃ , or DMSO- d6 ) and spectra were obtained using a Varian Gemini 2000 device (300 MHz) under standard parameters; Mass spectrometric identification was performed by electrospray ionization method (ESMS) with Perkin Elmer instrument (PESCIEX API 150 EX).

Example  1: 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -8-hydroxy- l H Synthesis of -quinolin-2-one

a. Preparation of 4- (2-amino-2-methyl-propoxy) -phenylamine hydrochloride

A strongly stirred slurry of sodium hydride (60% dispersion in mineral oil, 11.32 g. 0.28 mol) in dimethylsulfoxide (400 mL) was heated at 45 ° C. for 1 hour. Thereafter, pure 2-amino-2-methyl-1-propanol (25.3 g, 1 equiv) was added to the slurry. The reaction mixture was warmed to 75 ° C. for 1 hour and cooled to 20 ° C. in an ice bath. While maintaining the temperature below 30 ° C., l-fluoro-4-nitrobenzene (40 g, 1 equiv) was added slowly, and the resulting dark red solution was stirred further at room temperature for 1 hour. The reaction was quenched with water (1000 mL), extracted with dichloromethane (500 mL) and the organic layer was washed (saturated sodium chloride aqueous solution: 1: 1 mixture of water, 1000 mL). 3M hydrochloric acid (400 mL) was added to the organic layer to precipitate the product. The resulting orange solid was then filtered off and washed with dichloromethane until the filtrate was colorless.

The solid material was immediately transferred to a hydrogenation flask. Palladium (10% w / w carbon, 50% w / w water) was added followed by methanol (500 mL). The slurry was vigorously shaken for 16 hours under 3 atmospheres of hydrogen. The catalyst is then filtered off, the solvent is removed under reduced pressure, and toluene (3 x 150 mL) to obtain the resulting title intermediate as a white solid (4.Og, 0.18 mole, 65%). It was dried by azeotroping.

b. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- ( tert -butyl-dimethyl Preparation of -silanyloxy) -ethyl] -8-benzyloxy-1 H -quinolin-2-one

Product of step a (23.2 g, 1.1 equiv) in toluene (600 mL), 8-benzyloxy-5-{( R ) -2- [2- (4-bromo-phenyl) -ethylamino-1- ( A mixture of tert -butyl-dimethyl-silanyloxy) -ethyl} -l H -quinolin-2-one hydrochloride (66.0 g, 0.1 mol), and sodium tert -butoxide (54.0 g, 5.5 equiv) was homogenized Stir at 90 ° C. until one solution is obtained. Palladium tris (dibenzylideneacetone) (1.4 g, 0.015 equiv) is added followed by lac- 2,2'-bis (diphenylphosphino) -1, l'-binaftil (2.87 g, 0.045 equiv) Was added. The reaction mixture was stirred at 90 ° C. for 3 hours and allowed to cool. The solution was washed with water (100 mL), saturated aqueous sodium chloride: 1: 1 solution of water (100 mL) and dried over sodium sulfate. Removal of solvent under reduced pressure gave the title intermediate (40 g, crude) as a dark brown solid, which was used without further purification.

c. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-benzyloxy-1 H -quinolin-2-one

The product of the previous step was treated with triethylamine trihydrofluoride (36 g) in 2-propanol (500 mL) / ethanol (200 mL) for 16 hours at room temperature. The mixture was concentrated under reduced pressure to one third of the original volume. 1 M aqueous sodium hydroxide solution (500 mL) was added followed by acetonitrile (500 mL) and isopropyl acetate (500 mL). The aqueous layer was removed and the organic layer was washed with a 1: 1 mixed solution of saturated aqueous chloride: water (400 mL) and saturated aqueous sodium chloride solution (400 mL). The organic layer was dried over sodium sulphate and the solvent was removed in vacuo to yield the title intermediate (50 g, crude) as a brown solid, which was used without further purification.

d. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-hydroxyl H -quinolin-2-one

Palladium hydroxide (10 g, 20% w / w on carbon, 50% w / w on water) was added to the product from the previous reaction followed by ethanol (500 mL). The slurry was vigorously stirred for 8 hours under hydrogen atmosphere. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to afford the title compound (40 g), which was purified by reverse phase HPLC and separated as trifluoroacetate salt by lyophilization.

Examples  2-8: Synthesis of Compounds 2-8

Trifluoroacetate salts of compounds 2-8 are prepared using procedures similar to those described in Example 1, except that in step a, 2-amino-2-methyl-1-propanol is replaced with a suitable alcohol. It was.

Compound 2: 5-[( R ) -2- (2- {4- [4- (2-amino-ethoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8 -Hydroxy-l H -quinolin-2-one: m / z [M + H ⁺ ], 475.2, calculated for C ₂₇ H _3l N ₄ 0 ₄ . Observed 475.3.

Compound 3: 5-[( R ) -2- (2- {4- [4- (3-amino-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8 -Hydroxy-l H -quinolin-2-one: m / z [M + H ⁺ ], 489.2 calculated for C ₂₈ H ₃₃ N ₄ 0 ₄ ; Observed 489.5.

Compound 4: 5-[( R ) -2- (2- {4- [4- (4-amino-butoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- 8-hydroxy-l H -quinolin-2-one: m / z [M + H ⁺ ], 503.3, calculated for C ₂₅ H ₃₅ N ₄ 0 ₄ ; Observed 503.5.

Compound 5: 5-{( R ) -2- [2- (4- {4- [2- (2-amino-ethoxy) -ethoxy] -phenylamino} -phenyl) -ethylamino] -1- Hydroxy-ethyl} -8-hydroxy-l H -quinolin-2-one: m / z [M + H ⁺ ], 519.3, calculated for C ₂₉ H ₃₅ N ₄ 0 ₅ ; Observed 519.5.

Compound 6: 8-hydroxy-5-[( R ) -1-hydroxy-2- (2- {4- [4- (2-morpholin-4-yl-ethoxy) -phenylamino] -phenyl } -Ethylamino) -ethyl] -l H -quinolin-2-one: m / z [M + H ⁺ ], 545.3, calculated for C ₃₁ H ₃₇ N ₄ 0 ₅ ; Observed 545.6.

Compound 7: 8-hydroxy-5-[( R ) -1-hydroxy-2- (2- {4- [4- (2-piperazin-1-yl-ethoxy) -phenylamino] -phenyl } -Ethylamino) -ethyl] -l H -quinolin-2-one: m / z [M + H ⁺ ] calculated for C ₃₁ H ₃₈ N ₅ 0 ₄ , 544.7; Observed 544.7.

Compound 8: 8-hydroxy-5-[( R ) -1-hydroxy-2- (2- {4- [4-pyridin-2-yl-methoxy) -phenylamino] -phenyl} -ethylamino ) -Ethyl] -l H -quinolin-2-one: m / z [M + H ⁺ ], 523.1 calculated for C ₃₁ H ₃₁ N ₄ 0 ₄ ; Observed 523. 2.

Example  9: 5-[( R ) -2- (2- {4- [4- (2-amino-2- methyl - Propoxy ) -3- Trifluoromethyl - Phenylamino ]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -8-hydroxy- l H Synthesis of -quinolin-2-one

Using procedures similar to those described in Example 1, except for replacing 1-fluoro-4-nitrobenzene in step a with 1-fluoro-4-nitro-2-trifluoromethylbenzene The compound was prepared. m / z : [M + H ⁺ ] 571.3; calculated for C ₃₀ H ₃₄ F ₃ N ₄ 0 ₄ ; observed 571.3.

Example  10: 8-hydroxy-5-{( R ) -1-hydroxy-2- [2- (4- {4- [2- (4- Methanesulfonyl Piperazin-1-yl) Ethoxy ]- Phenylamino }- Phenyl )- Ethylamino ] -Ethyl} -l H Synthesis of -quinolin-2-one

a. 8-benzyloxy-5-[( R ) -l- ( tert -butyl-dimethyl-silanyloxy) -2- (2- {4- [4- (2-piperazin-1-yl-ethoxy) Preparation of -phenylamino] -phenyl} -ethylamino) -ethyl] -1 H -quinolin-2-one

Using procedures similar to those described in Step a and Step b of Example 1, except that in step a, 2-amino-2-methyl-1-propanol was substituted with N- (2-hydroxyethyl) piperazine To prepare the title intermediate.

b. 8-benzyloxy-5-[( R ) -l- ( tert -butyl-dimethyl-silanyloxy) -2- [2- (4- {4- [2- (4-methanesulfonyl-piperazine- Preparation of l-yl) -ethoxy] -phenylamino} -phenyl) -ethylamino] -ethyl} -l H -quinolin-2-one

The product of step a (100 mg) was treated with methanesulfonyl chloride (15.3 mg, 1 equiv) in a mixture of diisopropylethylamine (0.14 mL) and tetrahydrofuran (2 mL) for 1 hour at room temperature. . Then the mixture was evaporated to yield the title intermediate.

c. 8-hydroxy-5-{( R ) -1-hydroxy-2- [2- (4- {4- [2- (4-methanesulfonyl-piperazin-1-yl) -ethoxy]- Synthesis of Phenylamino} -phenyl) -ethylamino] -ethyl} -1 H -quinolin-2-one

The intermediate of step b was converted to the title compound using procedures similar to those described in steps c and d of Example 1. m / z : [M + H ⁺ ], 622.3 calculated for C ₃₂ H ₄₀ N ₅ 0 ₆ S; Observed 622.5.

Example  11: 5-[( R ) -2- (2- {4- [4- (2-amino-ethyl)- Phenylamino ]- Phenyl } -Ethylamino) -l-hydroxy-ethyl] -8-hydroxy-l H Synthesis of -quinolin-2-one

a. 5-[( R ) -2- (2- {4- [4- (2-amino-ethyl) -phenylamino] -phenyl} -ethylamino) -1- ( tert -butyl-dimethyl-silanyloxy) Preparation of -ethyl] -8-benzyloxy-1 H -quinolin-2-one

Tris (dibenzylideneacetone) dipalladium (0) (0.028g, 0.031mmol) followed by lac -2,2'-bis (diphenylphosphino) -l, l'-binaftil (0.057g, 0.092mmol) ) 2- (4-aminophenyl) ethylamine (0.100 g, 0.740 mmol) in toluene (20 mL) at room temperature, 8-benzyloxy-5-{( R ) -2- [2- (4-bro) Mother-phenyl) -ethylamino-1- ( tert -butyl-dimethyl-silanyloxy) -ethyl} -l H -quinolin-2-one hydrochloride (0.393 g, 0.612 mmol), and sodium tert -butoxide (0.265 g, 2.75 m ° C. mol) was added to the solution. The resulting mixture was heated at 90 ° C. for 2 hours and allowed to cool. The solution was washed with water (100 mL), saturated aqueous sodium chloride solution: 1: 1 solution of water (100 mL) and dried over magnesium sulfate. Solvent was removed under reduced pressure to afford the title intermediate as a dark brown solid (0.473 g), which was used without further purification.

b. 5-[( R ) -2- (2- {4- [4- (2-amino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-benzyloxy- Preparation of 1 H -quinolin-2-one

The product of the previous step (0.473 g, 0.714 mmol) was treated with triethylamine trihydrofluoride (0.173 g, 1.07 mmol) in tetrahydrofuran (20 mL) for 16 hours at room temperature. The mixture was diluted with dichloromethane (100 mL) and water (100 mL). The resulting mixture was stirred vigorously and made basic by addition of 1N aqueous sodium hydroxide solution (pH> 10). The organic phase was washed with water (200 mL) and with saturated aqueous sodium chloride solution (200 mL). The organic phase was dried over magnesium sulfate and the solvent removed under reduced pressure to give the titled intermediate (0.500 g) as a brown solid which was used without further purification.

c. 5-[( R ) -2- (2- {4- [4- (2-amino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy- Preparation of 1 H -quinolin-2-one

Palladium (0.100 g, 10% wt. On activated carbon) was added to the solution of the product of the previous step in a 1: 1 solution (20 mL) of methanol: dichloromethane. The slurry was vigorously stirred for 16 h under hydrogen atmosphere. The reaction mixture was filtered through celite and concentrated under reduced pressure to afford the title compound, which was purified by reverse phase HPLC and separated by trifluoroacetate salt by lyophilization.

Example  12: 5-[( R ) -2- (2- {4- [4- (2-dimethylamino-ethyl)- Phenylamino ] -Phenyl} -ethylamino) -l-hydroxy-ethyl] -8-hydroxy-l H Synthesis of -quinolin-2-one

a. Preparation of Dimethyl- [2- (4-nitro-phenyl) -ethyl] -amine

4-nitrofenethyl bromide (1.0 g, 4.35 mmol) and N, N-diisopropylethylamine (1.69 g,) in dimethylamine hydrochloride (0.390 g, 4.78 mmol) in anhydrous dimethylformamide (20 mL) 13.05 mmol) was added to the solution. The reaction mixture was heated to 60 ° C. for 5 hours and allowed to cool. The solution was diluted with a 1: 1 solution of water: dichloromethane (200 mL) and added to a separatory funnel. The organic phase was collected and the product extracted with 1N aqueous hydrogen chloride solution. The organic phase was removed and the aqueous layer was made basic with 1N aqueous sodium hydroxide solution. The product was extracted with dichloromethane (100 mL) and washed with saturated aqueous sodium chloride solution (200 mL). The organic phase was dried over magnesium sulfate and the solvent was removed under reduced pressure to afford the title intermediate as a clear oil (0.426 g, unpurified) which was used without further purification.

b. Preparation of 4- (2-dimethylamino-ethyl) -phenylamine

Palladium (0.043 g, 10% wt. On activated carbon) was added to a solution of the previous step product (0.430 g, 2.2 mmol) in methanol (20 mL). The slurry was vigorously stirred for 6 hours under a hydrogen atmosphere. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the title intermediate (0.307 g unpurified), which was used without further purification.

c. 5-[( R ) -2- (2- {4- [4- (2-dimethylamino-ethyl) -phenylamino] -phenyl} -ethylamino) -l-hydroxy-ethyl] -8-hydroxy Synthesis of -l H -quinolin-2-one

Trifluoroacetate salt of the title compound, using procedures similar to those described in Example 11, except that 2- (4-aminophenyl) ethylamine in Step a of Example 11 was replaced with the product of the previous step Was prepared.

Example  13: 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propyl) -phenylamino]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -8-hydroxy- l H Synthesis of -quinolin-2-one

a. Preparation of 1,1-dimethyl-2- (4-nitro-phenyl) -ethylamine

α, α-dimethylphenethylamine hydrochloride (20 g, 108 mmol) was dissolved in concentrated sulfuric acid (40 mL). The solution was cooled to -10 ° C. Nitric acid (31 mL, 90%) was added dropwise over 30 minutes while maintaining the reaction temperature below -5 ° C. The solution was stirred for an additional 45 minutes, transferred to ice and warmed to room temperature overnight. The product was collected by filtration (13.7 g). An aqueous sodium hydroxide solution (lN) was added to the filtrate and the title compound was separated as a clear oil.

b. Preparation of 4- (2-amino-2-methyl-propyl) -phenylamine

Palladium (0.136 g, 10% wt. On activated carbon) was added to a solution of the product of step a (1.36 g, 7.0 mmol) in methanol (20 mL). The slurry was vigorously stirred for 6 hours under 1 atmosphere of hydrogen. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the title intermediate (1.04 g, crude), which was used without further purification.

c. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8 - the synthesis of quinolin-2-one-hydroxyphenyl -l H

Trifluoroacetate salt of the title compound, using procedures similar to those described in Example 11, except that 2- (4-aminophenyl) ethylamine in Step a of Example 11 was replaced with the product of the previous step Was prepared.

Example  14: N -{5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phen Nylamino ]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -2-hydroxy- Phenyl }- Formamide  synthesis

a. Preparation of [4- (2-amino-2-methyl-propoxy-phenyl]-[4- (2-aminoethyl) -phenyl] -amine

4- (2-amino-2-methyl-propoxy) -phenylamine (1.0 g, 5.5 mmol) in toluene (30 mL) (free base of Example 1, intermediate of step a), 4-bromophenethylamine (1.1 g, 1 equiv), and a mixture of sodium tert -butoxide (1.9 g, 3.5 equiv) were stirred at 90 ° C. until a uniform solution was obtained.

Tris (dibenzylideneacetone) dipalladium (0) (252 mg, 0.05 equiv) was added followed by lac- 2,2'-bis (diphenylphosphino) -1, l'-binaftil (516 mg, 0.015 equiv) ) Was added. The reaction mixture was stirred at 90 ° C. for 16 h and allowed to cool. The solution was diluted with ethyl acetate (100 mL) and washed with a 1: 1 solution of saturated aqueous sodium chloride: water (100 mL) and extracted with 6M hydrochloric acid (100 mL). The aqueous layer was washed with ethyl acetate (2 x 100 mL) and diluted with isopropyl acetate (100 mL). The mixture was cooled to 0 ° C. and neutralized with sodium hydroxide (13 g dissolved in 20 mL of water). The aqueous layer was removed and the organic layer was washed (1: 1 solution of saturated sodium chloride solution: water, 100 mL), dried over sodium sulfate and evaporated to afford the titled intermediate (1.3 g, crude).

b. N - {5 - [(R ) -2- (2- {4- [4- (2- amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- (tert- Butyl-dimethyl-silanyloxy) -ethyl] -2-benzyloxy-phenyl} -formamide

[5 - - ((R) -2-bromo-1- (tert-butyl-dimethyl-silanyloxy) -ethyl) -2-benzyloxy-phenyl] - dimethyl sulfoxide N contained in the seed (8mL) (2.0 g, 4.35 mmol), the product of the previous step (1.3 g, 1 equiv), potassium carbonate (2.4 g, 4 equiv) and sodium iodide (718 mg, 1.1 equiv) were heated at 140 ° C. for 20 min. The mixture was allowed to cool, diluted with water (20 mL) and extracted with ethyl acetate (2 x 20 mL). The organic layer was washed with saturated aqueous sodium chloride solution (20 mL), dried over sodium sulfate and evaporated. The residue was purified by reverse phase HPLC to give the title intermediate (500 mg, 0.73 mmol, 17% yield).

c. N- {5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy- Preparation of Ethyl] -2-hydroxy-phenyl} -formamide

Using intermediate procedures similar to those described in steps c and d of Example 1, the intermediate of step b was converted to the title compound. m / z : [M + H &lt; + &gt;], 479.3 calculated for C ₂₇ H ₃₅ N ₄ 0 ₄ ; Observed 479.3.

Example  15: 5-[( R ) -2- (2- {4- [3- (2-amino-ethyl) -4-methoxy-phenylamino]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -8-hydroxy- l H Synthesis of -quinolin-2-one

a. 5-{( R ) -2- [2- (4-Amino-phenyl) -ethylamino] -1- ( tert -butyl-dimethyl-silanyloxy) -ethyl] -8-benzyloxy-1 H -quinoline Preparation of 2-one

5- (2-Bromo-1- ( tert -butyl-dimethyl-silanyloxy) -ethyl) -8-benzyloxy-1 H -quinolin-2-one (5.0 g, contained in dimethyl sulfoxide (10 mL) 1.0 equiv) and 2- (4-aminophenyl) ethylamine (2.8 g, 2.0 equiv) were heated at 100 ° C for 12 h. The reaction mixture was cooled to 20 ° C. and the resulting red oil was diluted with ethyl acetate (200 mL) and extracted with two portions of water (200 mL). The organic layer was washed with two portions (200 mL) of a 10% aqueous solution of acetic acid: a saturated aqueous sodium chloride solution. The organic layer was basified by careful extraction with two portions of saturated aqueous sodium bicarbonate solution (200 mL) followed by saturated aqueous sodium chloride solution (200 mL). The resulting organic solution was treated with anhydrous sodium sulfate and the solvent was removed under reduced pressure to give the title intermediate (5.1 g), which was used without further purification.

b. 5-[( R ) -2- (2- {4- [3- (2-amino-ethyl) -4-methoxy-phenylamino] -phenyl} -ethylamino) -1- ( tert -butyl-dimethyl Preparation of -silanyloxy) -ethyl] -8-benzyloxy-1 H -quinolin-2-one

Previous step product in toluene (10 mL) (0.500 g, 1.0 equiv), 5-bromo-2-methoxyphenethylamine hydrobromide (0.343 g, 1.2 equiv) and sodium tert -butoxide (0.397 g, 4.5 equivalents) was stirred at 90 ° C. until a uniform solution was obtained. Palladium tris (dibenzylideneacetone) (0.0042 g, 0.05 equiv) is added followed by lac-2,2'-bis (diphenylphosphino) -1,1'-binaftil (0.0086 g, 0.15 equiv) Was added. The reaction mixture was stirred at 90 ° C. for 5 hours and allowed to cool to 20 ° C. The solution was diluted with dichloromethane (100 mL) and washed with water (2 x 100 mL). The organic solution was dried over sodium sulfate. The reaction mixture was filtered and the solvent removed under reduced pressure to yield the title intermediate (1.0 g) as a dark brown solid, which was used without further stagnation.

c. 5-[( R ) -2- (2- {4- [3- (2-amino-ethyl) -4-methoxy-phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-benzyloxy-1 H -quinolin-2-one

The crude solid (1.0 g) obtained in the previous step was treated with triethylamine trihydrofluoride (0.70 mL) in tetrahydrofuran (10 mL) for 5 hours at room temperature. The mixture was diluted with dichloromethane (100 mL) and extracted with 0.1 M NaOH (2 × 100 mL) followed by saturated aqueous sodium chloride solution (100 mL). The organic layer was treated with anhydrous sodium sulfate and the solvent was dried under reduced pressure. The resulting solid was purified by reverse phase HPLC and separated as its trifluoroacetate salt by lyophilization to obtain the title intermediate (200 mg).

d. 5-[( R ) -2- (2- {4- [3- (2-amino-ethyl) -4-methoxy-phenylamino] -phenyl} -ethylamino) -1-hydroxy) -ethyl] Preparation of -8-hydroxy-1 H -quinolin-2-one

Palladium (10 mg, 10% w / w on carbon) followed by methanol (5 mL) was added to the intermediate of the previous step (50 mg) and the slurry was vigorously stirred under hydrogen atmosphere for 5 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give the title compound (42.0 g) which was purified by reverse phase HPLC and separated as its trifluoroacetate salt by lyophilization. m / z : [M + H ⁺ ] calc'd for C ₂₈ H ₃₂ N ₄ 0 ₄ , 489.3; Observed 489.5.

Example  16: N -{5-[( R ) -2- (2- {4- [3- (2-amino-ethyl) -4-methoxy-phenylamino]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -2-hydroxy- Phenyl }- Formamide  synthesis

5- (2-Bromo-1- ( tert -butyl-dimethyl-silanyloxy) -ethyl) -8-benzyloxy-1 H -quinolin-2-one N- [5-(( R ) -2 Using procedures similar to those described in Example 15, except that it was substituted with -bromo-1- ( tert -butyl-dimethyl-silanyloxy) -ethyl) -2-benzyloxy-phenyl] -formamide The title compound was prepared. m / z [M + H ⁺ ], 465.3 calculated for C ₂₆ H ₃₂ N ₄ 0 ₄ ; Observed 465.3

Example  17: 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino]- Phenyl }- Ethylamino ) -l-hydroxy-ethyl] -8-hydroxy- l H -Salts of quinoline-2-one

a. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-hydroxy-1 H -quinolin-2-one free base

Ammonium bicarbonate (10%) aqueous solution (50 mL) in product of Example 1 in ethanol (6 mL) 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl A solution of -propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy-1 H -quinolin-2-one trifluoroacetate (1.80 g, 2.1 mmol) Was added. The solution was stirred at rt for 1 h. The resulting solid was filtered and dried under reduced pressure to give the title compound (0.84 g, 1.67 mmol, 80%) as a yellow solid.

b. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-hydroxy-1 H -quinolin-2-one sulfate

The free base (5.85 g, 11.6 mmol) prepared as in the previous step was dissolved in acetonitrile: water (200 mL: 60 mL) at 90 ° C. and filtered to remove residual solid particles. The filtrate was reheated to 90 ° C. and a concentrated sulfuric acid (2 mL) solution in acetonitrile: water (18 mL: 2 mL) was added. The solution was cooled to room temperature over 2 hours and cooled to 10 ° C. in an ice / water bath. The solid was filtered and dried under reduced pressure to give the crude title salt (5.70 g, 82%). The material (5.70 g) was redissolved in acetonitrile: water (120 mL: 410 mL) at 90 ° C. and again cooled to room temperature over 2 hours. The slurry was cooled to 10 ° C. in an ice / water bath, the solids were filtered and dried under reduced pressure to give the title salt hydrate (4.40 g, overall yield 63%) as an off-white powder. The x-ray powder diffraction (XRPD) pattern of the product is shown in FIG. 1. Differential scanning calorimeter results of the product are shown in FIG. 2.

c. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-hydroxy-1 H -quinolin-2-one nafadisylate

1,5-naphthalene-disulfonic acid tetrahydrate (33 mg, 2 equiv) in a solution of free base (23 mg, 0.046 mmol) prepared as in step a dissolved in isopropanol: water (1.8 mL: 0.2 mL) at 60 ° C ) Was added to obtain a white precipitate. The temperature was raised to 70 ° C. and 1 mL of water was further added to give a clear solution. After cooling to room temperature, the slurry was filtered and dried to give the title salt (25 mg, 63%) as a white powder. The XRPD pattern of the product is shown in FIG. 3. Differential scanning calorimeter results of the product are shown in FIG. 4.

d. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Preparation of 8-hydroxy-1 H -quinolin-2-one oxalate

Oxalic acid (50 mg, 2 equiv) was added to a solution of the free base (100 mg, 0.2 mmol) prepared as in step a dissolved in isopropanol: water (3.6 mL: 0.4 mL) at 60 ° C. to obtain a white precipitate. The temperature was raised to 70 ° C. and 4 mL of water was further added to give a clear solution. After cooling to room temperature, the slurry was filtered and dried to give the title salt (82 mg, 65%) as an ivory powder. The XRPD pattern of the product is shown in FIG. 5. Differential scanning calorimeter results of the product are shown in FIG. 6.

e. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Synthesis of 8-hydroxy-1 H -quinolin-2-one 4-methyl-cinnamate

5-[( R ) -2- (2- {4- [4- (2-Amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -l-hydroxy-ethyl]- 8-hydroxy-1 H -quinolin-2-one (0.21 g) was dissolved in a mixture of tetrahydroleuran (1.2 mL) and water (1.2 mL). At room temperature 4-methyl-cinnamic acid (0.07 g, mainly trans ) was added to the stirred solution and dissolved. After about 10 minutes, crystallization occurred. The slurry was stirred overnight and filtered. The cake was washed with aqueous tetrahydrofuran solution (THF: 1: 1 solution of water, 2 × 0.4 mL, 1 × 0.2 mL) to afford the title compound, which was dried overnight at 45 ° C. under vacuum (output: 0.207 g).

The crystalline product was characterized by XRPD and DSC.

f. 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- Synthesis of 8-hydroxy-1 H -quinolin-2-one 4-methyl-cinnamate

5-[(R) -2- (2- {4- [4- (2-Amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- 8-hydroxy-1 H -quinolin-2-one (1.77 g) was dissolved in a mixture of tetrahydrofuran (89 mL) and water (89 mL). 4-Methyl-cinnamic acid (6.02 g, mainly trans ) was quantified and about 1/4 was added to the free base solution followed by the seed crystals obtained in the procedure of step e. The mixture was stirred and the remaining acid was added in portions over 1.5 hours. The slurry was stirred for another 4 hours, and the slurry was filtered. The cake was washed with aqueous tetrahydrofuran solution (THF: water 1: 1, 36 mL) and then with tetrahydrofuran (2 x 18 mL) to give the title compound which was dried overnight in vacuo at 40-50 ° C. ( Output: 16.916 g).

The crystalline product was characterized by XRPD and DSC.

g. Analytical Methods

X-ray powder diffraction patterns of FIGS. 1, 3, and 5 were obtained with a Rigaku diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation. The analysis was performed with a goniometer operating in a continuous-scan mode of 3 ° per minute with a step size of 0.03 ° in the range of 2 to 45 °. Samples were prepared on quartz specimen holders in a thin layer of powdered material. The instrument was calibrated to silicon metal standards.

The differential scanning calorimetry results of FIGS. 2, 4, and 6 were obtained with a TA device model DSCQ10. Samples were placed in sealed aluminum pans for analysis and empty pans were standard. Samples were equilibrated at 30 ° C. and heated to a temperature of 300 ° C. at a rate of 5 ° C. per minute. The instrument was calibrated with indium as standard.

While the invention has been described with reference to specific embodiments thereof, those skilled in the art will appreciate that various modifications may be made and equivalents may be substituted without departing from the true principles of the invention and its scope. will be. In addition, modifications may be made to adapt a particular situation, material, composition, method, method step, or steps to the objects, principles, and scope of the invention. All such modifications are intended to be within the scope of the claims appended hereto. In addition, all documents, patents, and patent documents cited herein are hereby incorporated by reference in their entirety, as if individually incorporated by reference.

Claims (39)

As a compound of formula (I):

From above: Each R ¹ , R ² , R ³ , and R ⁴ is independently selected from hydrogen, hydroxy, amino, halo, —CH ₂ 0H and —NHCHO, or taken together, R ¹ and R ² are —NHC (═O ) CH = CH-, -CH = CHC (= 0) NH-, -NHC (= 0) S-; And -SC (= 0) NH-; R ⁵ and R ⁶ is one of the _- [XC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ^13, and The other of the R ⁵ and R ⁶ is hydrogen, hydroxy, C _{1 -} is selected from _4-alkyl, the C _{1 - - 4} alkoxy, and C _{1 4} alkyl is optionally substituted with halo, Each X is independently selected from -0-, -NH-, -S-, -NHS0 _2- , -S0 ₂ NH-, -NHC (= 0)-, and -C (= 0) NH-; Each ^{R l0, R 11, R 12} , and R ¹³ are independently hydrogen or C _{1 - 4} alkyl and; or R ^l0 And R ¹¹ is or R ^l0 is a nitrogen atom and the adjacent C ₁ binding it together with the nitrogen atom to which they are _attached-is, or R ¹² and R ¹³ together with the ₆ alkylenyl carbon atoms together with the nitrogen atom to which they are attached, or R ¹² is a nitrogen atom and the adjacent C ₁ coupling it _- contains additional heteroatoms (heteroatom) selected, having a five to seven ring atoms, from optionally oxygen, nitrogen and sulfur with the ₆ alkylenyl carbon atoms form a heterocyclic or heteroaryl ring, and the nitrogen may optionally -S (0) ₂ -C ₁ in the _{- 4} and substituted with alkyl; n is 1, 2, or 3; And Each R ^7, R ^8, and R ⁹ are independently selected from hydrogen or C _{l - 6} alkyl, a compound of formula (I); Or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. The compound of claim 1, wherein R ⁷ , R ⁸ , and R ⁹ are each hydrogen. The compound of claim 1, wherein X is —O—. The compound of claim 1, wherein the compound of formula (II) is:

(II) In the above, R ¹ is —CH ₂ 0H or —NHCHO and R ² is hydrogen; Or R ¹ and R ² taken together are —NHC (═O) CH═CH— or —CH═CHC (═O) NH—; R ⁵ and R ⁶ One of the _- [OC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ^13, and R ⁵ and R ⁶ Of the other is hydrogen, hydroxy, C _{1 - 4} is selected from alkoxy, and C _{1 -4} alkyl, said C _{1 - 4} alkyl is optionally substituted with halo, Each R ^l0 , R ¹¹ , R ^l2 And R ¹³ is independently hydrogen or C _{1 - 4} alkyl and; or R ^l0 And R ¹¹ is or R ^l0 is a nitrogen atom and the adjacent C ₁ binding it together with the nitrogen atom to which they are _attached-is, or R ¹² and R ¹³ together with the ₆ alkylenyl carbon atoms together with the nitrogen atom to which they are attached, or R ¹² is it-bonded nitrogen atom and the adjacent C _{1 -} with a ₆ alkylenyl carbon atoms, having a 5 to 7 ring atoms, optionally heterocyclic, including the additional heteroatom selected from oxygen, nitrogen and sulfur or forms a heteroaryl ring, the nitrogen is optionally -S (0) ₂ -C ₁ in the _{- 4} and substituted with alkyl; n is 1 or 2, a compound of formula (II); Or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. 5. The method of claim 4, wherein R ⁵ is _- [OC ₁ - ₆ alkylenyl] _n -NR ¹⁰ R ¹¹ or C _{1 - 6} alkylenyl -NR ¹² R ¹³ is The compound wherein R ⁶ is one of hydrogen. 5. The method of claim 4, wherein R ⁵ is C _{l - 6} is that the alkylenyl -NR ¹² R ¹³ _{- 4} alkoxy wherein R ⁶ is - [OC _1-6 alkylenyl] _n -NR ¹⁰ R ¹¹ or C ₁ compound. The method of claim 4, wherein, R ⁵ is -OC _{1 - 6} alkylenyl -NR ¹⁰ R ¹¹ And -C _{1 - 6} alkylenyl selected from -NR ¹² R ^13, and R ⁶ is hydrogen; or R ⁵ is C _{1 - 6} and alkylenyl -NR ¹² R ^13, _{- 4} alkoxy and R ⁶ is -C ₁ Each above R ¹⁰ , R ¹¹ , R ¹² And R ¹³ is independently hydrogen or C _{1 - 4} alkyl or R ^10, or taken together And R ¹¹ is a compound together with the nitrogen atom to which they are attached to form a piperazinyl ring. 8. The compound of claim 7, wherein R ¹ and R ² taken together are —NHC (═O) CH═CH— or —CH═CHC (═O) NH—. The method of claim 8, wherein R ⁵ is -OC _{1 - 6} alkylenyl -NR ¹⁰ R ^11, and R ⁶ is one of a hydrogen compound. 5. The compound of claim 4, wherein the stereochemistry at the alkylene carbon having a hydroxyl group is ( R ). The method of claim 4 wherein: 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl]- 8-hydroxy-l H -quinolin-2-one; 8-hydroxy-5-[( R ) -1-hydroxy-2- (2- {4- [4- (2-piperazin-1-yl-ethoxy) -phenylamino] -phenyl} -ethyl Amino) -ethyl] -l H -quinolin-2-one; 5-[( R ) -2- (2- {4- [4- (2-amino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy- H -quinolin-2-one; 5-[( R ) -2- (2- {4- [4- (2-dimethylamino-ethyl) -phenylamino] -phenyl} -ethylamino) -1-hydroxy-ethyl] -8-hydroxy -l H -quinolin-2-one; 5-[( R ) -2- (2- {4- [3- (2-Amino-ethyl) -4-methoxy-phenylamino] -phenyl} -ethylamino) -l-hydroxy-ethyl]- 8-hydroxy-l H -quinolin-2-one; And Pharmaceutically acceptable salts and solvates and stereoisomers thereof. The compound of claim 11, wherein 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- Hydroxy-ethyl] -8-hydroxy-l H -quinolin-2-one, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. 13. The compound of claim 12, wherein 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- Hydroxy-ethyl] -8-hydroxy-l H -quinolin-2-one sulfate. 13. The compound of claim 12, wherein 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- Hydroxy-ethyl] -8-hydroxy-l H -quinolin-2-one napadisylate. 13. The compound of claim 12, wherein 5-[( R ) -2- (2- {4- [4- (2-amino-2-methyl-propoxy) -phenylamino] -phenyl} -ethylamino) -1- Hydroxy-ethyl] -8-hydroxy-l H -quinolin-2-one oxalate. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1 to 15 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 16, wherein the composition further comprises a therapeutically effective amount of one or more other therapeutic agents. The pharmaceutical composition of claim 17, wherein the other therapeutic agent is a corticosteroid, anticholinergic, or PDE4 inhibitor. The method of claim 17, wherein the other therapeutic agent is fluticasone propionate, 6α, 9α-difluoro-17α-[(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl-3- Oxo-andros-1,4-diene-17β-carbothioic acid S -fluoromethyl ester, and 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy A pharmaceutical composition, wherein the composition is selected from -androsa-1,4-diene-17β-carbothioic acid S- (2-oxo-tetrahydrofuran-3S-yl) ester. The pharmaceutical composition of claim 16, wherein the composition is suitable for administration by inhalation. A combination comprising a compound of any one of claims 1 to 15 and one or more other therapeutic agents. The combination of claim 21, wherein the other therapeutic agent is a corticosteroid, anticholinergic, or PDE4 inhibitor. The compound of any one of claims 1 to 15 and fluticasone propionate, 6α, 9α-difluoro-17α-[(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl 3-oxo-andros-1,4-diene-17β-carbothioic acid S -fluoromethyl ester and 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propy A combination comprising a compound selected from onyloxy-andros-1,4-diene-17β-carbothioic acid S- (2-oxo-tetrahydrofuran-3S-yl) ester. The compound of any one of claims 1 to 15 for use in therapy. Use of the compound of any one of claims 1 to 15 in the manufacture of a medicament. The use of claim 25, wherein the medicament is for the treatment of a mammal having a disease or condition associated with β ₂ adrenergic receptor activity. The use of claim 26, wherein the disease or condition is a lung disease or condition. 28. The use of claim 27, wherein the lung disease or condition is asthma or chronic obstructive pulmonary disease. 27. The use of claim 26, wherein the medicament is suitable for administration by inhalation. Use of a compound of any one of claims 1 to 15 in the manufacture of a medicament administered in combination with one or more therapeutic agents for the treatment of a mammal having a disease or condition associated with β ₂ adrenergic receptor activity. 16. A process for the preparation of a compound of formula (I) according to any one of claims 1 to 15, wherein (a) (i) a compound of formula (III):

Compounds of the following formula (IV):

React in the presence of a transition metal catalyst; or (ii) with a compound of formula (V):

A compound of formula (VI)

React, In the above, P ¹ is a hydroxy-protecting group, each R ^la , R ^2a , R ^3a And R ^4a is independently R ¹ , R ² , R ³ of formula (I) And R ⁴ is defined as being the same, or P ² is —OP ² which is a hydroxy protecting group; One of A and B is a leaving group and the other of A and B is -NH ₂ ; L is a leaving group; R ⁵ , R ⁶ , R ⁷ , R ⁸ And R ⁹ is defined as in formula (I), thereby providing a compound of formula (VII);

(b) removing the protecting group P ¹ to provide a compound of formula (VIII):

; And (c) R ^la , R ^2a , R ^3a Or when one of R ^4a is —OP ² , removing P ² to provide a compound of formula (I), or a salt or stereoisomer thereof. A product made by the method of claim 31. A method of treating a mammal having a disease or condition associated with β ₂ adrenergic receptor activity, the method comprising administering to said mammal a therapeutically effective amount of a compound of any one of claims 1-15. How to do. 34. The method of claim 33, further comprising administering a therapeutically effective amount of one or more other therapeutic agents. The method of claim 34, wherein the other therapeutic agent is a corticosteroid, anticholinergic, or PDE4 inhibitor. The method of claim 33, wherein the method comprises administering the compound by inhalation. A method of treating a lung disease or condition in a mammal, the method comprising administering to said mammal a therapeutically effective amount of a compound of any one of claims 1-15. A method of treating asthma or chronic obstructive pulmonary disease in a mammal, the method comprising administering to said mammal a therapeutically effective amount of a compound of any one of claims 1-15. A method of studying a biological system or sample comprising a β ₂ adrenergic receptor, the method comprising: (a) contacting said biological system or sample with a compound of any one of claims 1-15; And (b) determining the effects brought about by the compound on the biological system or sample.

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