CN101239971B - Biphenyl derivatives - Google Patents

Abstract

The present invention provides biphenyl derivatives of formula I: wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷W, a, b and c are as defined in the specification, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. The biphenyl derivative of the present invention has both beta₂Adrenergic receptor agonist activity in turn has muscarinic receptor antagonist activity, and therefore, such biphenyl derivatives are useful in the treatment of pulmonary diseases, such as chronic obstructive pulmonary disease and asthma.

Description

Biphenyl derivatives

This application is a divisional application of a Chinese patent application with an application date of February 13, 2004, an application number of 200480006528.4, and an invention title of "biphenyl derivatives".

technical field

The present invention relates to novel biphenyl derivatives useful in the treatment of pulmonary diseases. The present invention also relates to pharmaceutical compositions containing such biphenyl derivatives, methods and intermediates for preparing such biphenyl derivatives, and methods of using such biphenyl derivatives for the treatment of pulmonary diseases.

Background technique

Lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) are often treated with bronchodilators. A widely used class of bronchodilators consists of _β2 adrenergic receptor (adrenoceptor) agonists such as albuterol, formoterol and salmeterol. These compounds are usually administered by inhalation. Another class of bronchodilators consists of muscarinic receptor antagonists (anticholinergic compounds), such as ipratropium and tiotropium. These compounds are also usually administered by inhalation.

Pharmaceutical compositions comprising both _β2 adrenergic receptor agonists and muscarinic receptor antagonists for the treatment of pulmonary diseases are also known in the art. For example, US Patent No. 6,433,027 discloses pharmaceutical compositions comprising a muscarinic receptor antagonist such as tiotropium bromide and a _β2 adrenergic receptor agonist such as formoterol fumarate.

Although compounds having _β2 adrenergic receptor agonist or muscarinic receptor antagonist activity are known, no previous disclosures have demonstrated activity as both _β2 adrenergic receptor agonists and muscarinic receptor antagonists. an active compound. Compounds that possess both _β2 adrenergic receptor agonist and muscarinic receptor antagonist activity are highly desirable because such bifunctional compounds are able to provide bronchodilation through two independent modes of action, while only having Single molecule pharmacokinetics.

Summary of the invention

The present invention provides novel biphenyl derivatives useful in the treatment of pulmonary diseases. Compounds of the present invention have been found to possess, among other properties, both _β2 adrenergic receptor agonist and muscarinic receptor antagonist activity.

Accordingly, in one of its compositional aspects, the present invention relates to compounds of formula I:

in

a is an integer of 0 or 1-3;

Each R ¹ is independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^1a , -C(O)OR ^1b , -SR ^1c , -S(O)R ^1d , -S(O) ₂ R ^1e and NR ^1f R ^1g ;

R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^{1f ,} and R ^1g are each independently hydrogen, (1-4C)alkyl, or phenyl-(1-4C)alkyl;

b is an integer of 0 or 1-3;

R ² are each independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^2a , -C(O)OR ^2b , -SR ^2c , -S(O)R ^2d , -S(O)2R ^2e and -NR ^2f R ^2g ;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^{2f ,} and R ^2g are each independently hydrogen, (1-4C)alkyl, or phenyl-(1-4C)alkyl;

W is connected to the 3- or 4-position relative to the nitrogen atom in the piperidine ring and represents O or NW ^a ;

W ^a is hydrogen or (1-4C) alkyl;

C is an integer of 0 or 1-4;

Each R ³ is independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^3a , -C(O)OR ^3b , -SR ^3c , -S(O)R ^3d , -S(O) ₂ R ^3e and -NR ^3f R ^3g ; or two R ³ groups combined to form (1-3C) Alkylene, (2-3C)alkenylene or oxirane-2,3-diyl;

R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^{3f ,} and R ^3g are independently hydrogen or (1-4C)alkyl;

R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -(A ¹ ) _e -(R ^4b ) _f -Q-(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -

in

d, e, f, g, h and i are each independently selected from 0 and 1;

R ^4a , R ^4b , R ^4c and R ^4d are each independently selected from (1-10C) alkylene, (2-10C) alkenylene and (2-10C) alkynylene, wherein each alkylene, The alkenylene or alkynylene group is unsubstituted or substituted with 1-5 independently selected from (1-4C)alkyl, fluoro, hydroxy, phenyl and phenyl-(1-4C)alkyl base substitution;

A ¹ and A ² are each independently selected from (3-7C) cycloalkylene, (6-10C) arylene, -O-(6-10C) arylene, (6-10C) arylene- O-, (2-9C) heteroarylene, -O-(2-9C) heteroarylene, (2-9C) heteroarylene-O- and (3-6C) heterocyclylene, wherein Each cycloalkylene group is unsubstituted or substituted with 1-4 substituents independently selected from (1-4C)alkyl, and each arylene, heteroarylene or heterocyclylene group is unsubstituted or independently selected from halogen, (1-4C)alkyl, (1-4C)alkoxy, -S-(1-4C)alkyl, -S(O)-(1-4C )alkyl, -S(O) _2- (1-4C)alkyl, -C(O)O(1-4C)alkyl, carboxy, cyano, hydroxyl, nitro, trifluoromethyl and trifluoro 1-4 substituents of methoxy are substituted;

Q is selected from a bond, -O-, -C(O)O-, -OC(O)-, -S-, -S(O)-, -S(O) ₂ -, -N(Q ^a )C (O)-, -C(O)N(Q ^b )-, -N(Q ^c )S(O) ₂ -, -S(O) ₂ N(Q ^d )-, -N(Q ^e )C (O)N(Q ^f )-, -N(Q ^g )S(O) ₂ N(Q ^h )-, -OC(O)N(Q ⁱ )-, -N(Q ^j )C(O) O-and-N(Q ^k );

Q ^a , Q ^b , Q ^c , Q ^d , Q ^e , Q ^f , Q ^g , Q ^h , Q ⁱ , Q ^j and Q ^k are each independently selected from hydrogen, (1-6C)alkyl, A ³ and (1-4C)alkylene-A ⁴ , wherein the alkyl group is unsubstituted or substituted by 1-3 substituents independently selected from fluorine, hydroxyl and (1-4C)alkoxy; or with The nitrogen atoms connected to them and the group R ^4b or R ^4c together form a 4-6 membered nitrogen heterocycloalkyl group;

A ³ and A ⁴ are each independently selected from (3-6C) cycloalkyl, (6-10C) aryl, (2-9C) heteroaryl and (3-6C) heterocyclyl, wherein each cycloalkane The group is unsubstituted or substituted by 1-4 substituents independently selected from (1-4C)alkyl, and each aryl, heteroaryl or heterocyclyl group is unsubstituted or independently 1-4 substituents selected from halogen, (1-4C) alkyl and (1-4C) alkoxy;

The condition is that the number of adjacent atoms on the shortest chain between the two nitrogen atoms connected by ^R is in the range of 4-16;

^R represents hydrogen or (1-4C) alkyl;

R ⁶ is -NR ^6a CR ^6b (O) or -CR ^6C R ^6d OR ^6e and R ⁷ is hydrogen; or R ⁶ and R ⁷ together form -NR ^7a C(O)-CR ^7b =CR ^7c -, -CR ^7d = CR ^7e -C(O)-NR ^7f -, -NR ^7g C(O)-CR ^7h R ⁷ⁱ -CR ^7j R ^7k -or -CR ^7l R ^7m -CR ⁷ⁿ R ^7o -C(O)-NR ^7p- ;

R ^6a , R ^6b , R ^6c , R ^{6d ,} and R ^6e are each independently hydrogen or (1-4C)alkyl; and

^R7a , ^R7b , ^R7c , ^R7d , ^R7e , ^R7f , ^R7g , R7h, ^R7i , ^R7j , ^R7k , ^{R7l , R7m} , ^R7n , ^R7o and ^R7p are each independently is hydrogen or (1-4C) alkyl;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another aspect of its composition, the present invention relates to compounds of formula II:

in

R ⁴ is as defined herein (including any specific or preferred embodiments);

W stands for O or NH;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

In yet another aspect of its composition, the present invention relates to compounds of formula III:

in

R ⁴ is as defined herein (including any specific or preferred embodiments);

W stands for O or NH;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

In yet another aspect of its composition, the present invention relates to compounds of formula IV:

in

R ⁴ is as defined herein (including any specific or preferred embodiments);

W stands for O or NH;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

In another aspect of its composition aspect, the present invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. Such pharmaceutical compositions may optionally contain other therapeutic agents. Accordingly, in one embodiment, the present invention is directed to a pharmaceutical composition, wherein the composition further comprises a therapeutically effective amount of a steroidal anti-inflammatory drug, such as a corticosteroid.

The compounds of the present invention possess both activities of _β2 adrenergic receptor agonists and muscarinic receptor antagonists. Compounds of formula I are therefore useful in the treatment of pulmonary diseases such as asthma and chronic obstructive pulmonary disease.

Accordingly, in one of its method aspects, the present invention is directed to a method of treating a pulmonary disease comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Furthermore, in another aspect of its method aspects, the present invention relates to a method of providing bronchodilation in a patient, the method comprising administering to a patient in need of bronchodilation a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvent thereof compounds or stereoisomers.

The present invention also relates to a method of treating chronic obstructive pulmonary disease or asthma, the method comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate or stereoisomer thereof to a patient in need thereof.

Since the compounds of the present invention possess both _β2 adrenergic receptor agonist and muscarinic receptor antagonist activity, such compounds are also suitable as research tools. Therefore, in yet another aspect of its method aspects, the present invention relates to the use of a compound of formula I, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof, as a research tool to study biological systems or samples, or to discover A method for novel chemical compounds that are both active as _beta2- adrenergic agonists and muscarinic receptor antagonists.

The present invention also relates to processes and novel intermediates suitable for the preparation of compounds of formula I or pharmaceutically acceptable salts or solvates or stereoisomers thereof. Accordingly, in another aspect of its method aspects, the present invention relates to a method of preparing a compound of formula I, the method comprising:

(a) reacting a compound of formula 1 or a salt thereof with a compound of formula 2 ;

(b) reacting a compound of formula 3 or a salt thereof with a compound of formula 4 ;

(c) coupling a compound of formula 5 with a compound of formula 6 ;

(d) For a compound of formula I wherein R represents a hydrogen atom, reacting a compound ^of formula 3 with a compound of formula 7 or a hydrate thereof in the presence of a reducing agent;

(e) reacting a compound of formula 1 with a compound of formula 8 or a hydrate thereof in the presence of a reducing agent;

(f) reacting a compound of formula 9 with a compound of formula 10 ; or

(g) reacting a compound of formula 11 or a hydrate thereof with a compound of formula 10 in the presence of a reducing agent;

Any protecting groups are then removed to form compounds of formula I; wherein compounds of formulas 1-11 are as defined herein.

In one embodiment, the above method further comprises the step of forming a pharmaceutically acceptable salt of the compound of formula I. In another embodiment, the invention relates to other methods described herein; and to products prepared by any of the methods described herein.

The present invention also relates to compounds of formula I, or pharmaceutically acceptable salts or solvates or stereoisomers thereof, for use in therapy or as a medicament.

In addition, the present invention relates to the use of the compound of formula I or a pharmaceutically acceptable salt or solvate or stereoisomer thereof for the preparation of medicaments; especially for the preparation of medicaments for the treatment of pulmonary diseases.

Detailed description of the invention

In one of its compositional aspects, the present invention relates to novel biphenyl derivatives of formula I or pharmaceutically acceptable salts or solvates or stereoisomers thereof. These compounds contain one or more chiral centers and therefore, unless otherwise indicated, the present invention relates to racemic mixtures; pure stereoisomers (ie, enantiomers or diastereomers); stereoisomer-enriched body mixtures, etc. Unless otherwise indicated, when a particular stereoisomer is shown or named herein, those skilled in the art will appreciate that minor amounts of other stereoisomers may be present in the compositions of the present invention, provided that the usefulness of the composition as a whole is not compromised The presence of such other isomers is eliminated.

In particular, compounds of formula I contain a chiral center at the carbon atom indicated by the symbol * in the following formula:

In one embodiment of the invention, the carbon atom designated by the symbol * has the (R) configuration. In this embodiment, it is preferred that the compound of formula I has the (R) configuration at the carbon atom designated by the symbol * or is enriched in the form of a stereoisomer having the (R) configuration at this carbon atom. In another embodiment of the present invention, the carbon atom designated by the symbol * has the (S) configuration. In this embodiment, it is preferred that the compound of formula I has the (S) configuration at the carbon atom designated by the symbol * or is enriched in the form of a stereoisomer having the (S) configuration at this carbon atom. In some cases, in order to optimize the _β2 adrenergic agonist activity of the compounds of the invention, it is preferred that the carbon atom designated by the symbol * has the (R) configuration.

Compounds of formula I also contain a number of basic groups (eg amino groups), and thus compounds of formula I can exist as a free base or in various salt forms. All such salt forms are included within the scope of the present invention. Furthermore, solvates of compounds of formula I or salts thereof are included within the scope of the present invention.

Furthermore, unless otherwise indicated, all cis-trans or E/Z isomers (geometric isomers), tautomeric forms and topoisomeric forms of the compounds of formula I are included within the scope of the present invention when applicable .

The nomenclature employed herein for naming compounds of the invention and intermediates thereof is generally derived using commercially available AutoNom software (MDL, San Leandro, California). Typically, compounds of formula I wherein W is O are designated as ester derivatives of biphenyl-2-ylcarbamic acid; and compounds of formula I wherein W is ^NWa are designated as urea derivatives.

Representative implementation

The following substituents and values are intended to provide representative examples of aspects and embodiments of the invention. These representative values are provided to further define and illustrate these aspects and embodiments and are not intended to exclude other embodiments or limit the scope of the invention. In this regard, the recitation of a preference for a particular value or substituent is not intended in any way to exclude other values or substituents from the invention unless specifically stated otherwise.

In a particular embodiment of the compound of formula I, a and b are independently 0, 1 or 2; In one embodiment, both a and b are 0.

When present, each ^R can be at the 2, 3, 4, 5 or 6-position of the phenyl ring to which it is attached. In one embodiment, R ¹ is each independently selected from (1-4C)alkyl, halogen, -OR ^1a and -NR ^1f R ^1g ; for example methyl, fluoro, chloro, bromo, hydroxy, methoxy, Amino, methylamino, dimethylamino, etc. A particular value for ^R1 is fluorine or chlorine.

When present, each ^R2 can be at the 3, 4, 5 or 6-position of the phenylene ring to which it is attached (the carbon atom on the phenylene ring attached to the nitrogen atom is position 1). In one embodiment, R ² are each independently selected from (1-4C)alkyl, halogen, -OR ^2a and -NR ^2f R ^2g ; for example methyl, fluoro, chloro, bromo, hydroxy, methoxy, Amino, methylamino, dimethylamino, etc. A particular value for ^R2 is fluorine or chlorine.

Each of R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f and R ^1g and R ^2a , R ^2b , R ^2c , R 2d , R ^2e , ^{R 2f and} R ^2g when in R ¹ and R respectively When used in ² , independently hydrogen, (1-4C) alkyl or phenyl-(1-4C) alkyl; for example hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl or benzyl. In one embodiment, these groups are independently hydrogen or (1-3C)alkyl. In another embodiment, these groups are independently hydrogen, methyl or ethyl.

In one embodiment of the invention W is O. In another embodiment, W is NW ^a .

In general, it has been found that compounds in which W represents O exhibit a particularly high affinity for muscarinic and _β2 adrenergic receptors. Therefore, in a particular embodiment of the invention, W preferably represents O.

When referring to W, particular mention may be made of compounds wherein W is attached to the piperidine ring at the 4-position relative to the nitrogen atom of the piperidine ring.

When W is ^NWa , ^Wa is hydrogen or (1-4C)alkyl; for example hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert butyl. In one embodiment, W ^a is hydrogen or (1-3C)alkyl. In another embodiment W ^a is hydrogen, methyl or ethyl; eg hydrogen or methyl. In another embodiment, W ^a is hydrogen and NW ^a is NH.

In particular embodiments of compounds of formula I, c is 0, 1 or 2; In one embodiment, c is 0.

In one embodiment, each ^R3 is at the 3, 4 or 5-position of the piperidine ring (wherein the nitrogen atom of the piperidine ring is the 1 position). In another embodiment, ^R3 is at the 4-position of the piperidine ring. In particular aspects of these embodiments, ^each R is independently selected from (1-4C)alkyl; for example methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl. In another aspect, each ^R3 is independently methyl or ethyl.

In another embodiment, ^R3 is at the 1-position of the piperidine ring, ie on the nitrogen atom of the piperidine ring, thus forming a quaternary ammonium salt. In a particular aspect of this embodiment, ^each R is independently selected from (1-4C)alkyl; for example methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, iso Butyl and tert-butyl. In another aspect, each ^R3 is independently methyl or ethyl.

In yet another embodiment, two ^R groups combine to form a (1-3C)alkylene or (2-3C)alkenylene group. For example, two ^R3 groups at the 2 and 6-positions on the piperidine ring can combine to form an ethylene bridge (i.e., the piperidine ring and ^R3 groups form 8-azabicyclo[3.2.1]octane ring); or the two ^R3 groups at the 1 and 4-positions on the piperidine ring can combine to form an ethylene bridge (i.e., the piperidine ring and the ^R3 group form a 1-azabicyclo[2.2.2] octane ring). In this embodiment, other ^R3 groups as defined herein may also be present.

In yet another embodiment, two ^R3 groups combine to form an oxirane-2,3-diyl group. For example, two ^R3 groups at the 2 and 6-positions on the piperidine ring can combine to form a 3-oxatricyclo[ ^3.3.1.02,4 ]nonane ring). In this embodiment, other ^R3 groups as defined herein may also be present.

Each of R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^3f and R ^3g when used in R ³ is independently hydrogen or (1-4C)alkyl; eg hydrogen, methyl, ethyl , n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl. In one embodiment, these groups are independently hydrogen or (1-3C)alkyl. In another embodiment, these groups are independently hydrogen, methyl or ethyl.

In one embodiment of the compound of formula I, ^R is hydrogen or (1-4C)alkyl; for example hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl base and tert-butyl. In another embodiment, each R ⁵ is independently hydrogen, methyl or ethyl. In particular embodiments, R ⁵ is hydrogen.

In one embodiment of the invention, R ⁶ is -NR ^6a CR ^6b (O) and R ⁷ is hydrogen, wherein R ^6a and R ^6b are each independently hydrogen or (1-4C)alkyl, such as hydrogen, Methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl. In one embodiment, these groups are independently hydrogen or (1-3C)alkyl. In another embodiment, these groups are independently hydrogen, methyl or ethyl. In this embodiment, a particular value for ^R6 is -NHCHO.

In another embodiment, R ⁶ and R ⁷ are taken together to form -NR ^7a C(O)-CR ^7b =CR ^7c -, -CR ^7d =CR ^7e -C(O)-NR ^7f -, -NR ^7g C( O)-CR ^7h R ⁷ⁱ -CR ^7j R ^7k -or -CR ^7l R ^7m -CR ⁷ⁿ R ^7o -C(O)-NR ^7p -; wherein R ^7a , R ^7b , R ^7c , R ^7d , R ^7e , R ^7f , R ^7g , R ^7h , R ⁷ⁱ , R ^7j , R ^7k , R ^7l , R ^7m , R ⁷ⁿ , R ^7o and R ^7P are each independently hydrogen or (1-4C)alkyl; for example hydrogen, Methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl and tert-butyl. In one embodiment, these groups are independently hydrogen or (1-3C)alkyl. In another embodiment, these groups are independently hydrogen, methyl or ethyl. In this embodiment, specific values for ^R6 and ^R7 are such that ^R6 and ^R7 together form -NHC(O)-CH=CH-, -CH=CH-C(O)-NH-, _-CH2 -CH ₂ -C(O)NH- or -NHC(O)-CH ₂ -CH ₂ -; including where R ⁶ and R ⁷ together form -NHC(O)-CH=CH- or -CH=CH-C (O)-NH-; and in particular, wherein R ⁶ and R ⁷ together form -NHC(O)-CH=CH- (i.e. the nitrogen atom is attached to R ⁶ and the carbon atom is attached to R ⁷ , with R ⁶ and R ⁷ linked hydroxybenzene rings together form an 8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl group).

In the compound of formula I, ^R is a divalent group of the formula:

-(R ^4a ) _d -(A ¹ ) _e -(R ^4b ) _f -Q-(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -

wherein R ^4a , A ¹ , R ^4b , Q, R ^4c , A ² , R ^4d , d, e, f, g, h and i are as defined herein. In the compounds of the invention, the value of each of the components R ^4a , A ¹ , R ^4b , Q, R ^4c , A ² and R ^4d is chosen such that on the shortest chain between the two nitrogen atoms to which R ⁴ is attached The number of adjacent atoms is in the range of 4-16, (in particular, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16); including 8, 9, 10 , 11, 12, 13 or 14; for example 8, 9, 10 or 11; or 9 or 10. When selecting values for each variable in R ⁴ , those skilled in the art will understand that the values should be selected such that a chemically stable group is formed.

When determining the number of adjoining atoms in the shortest chain between the two nitrogen atoms to which ^R4 is attached, start from the first atom of the ^R4 group adjacent to the nitrogen of the piperidine ring, to the number of atoms adjacent to the aminohydroxyethyl group The nitrogen ends with the last atom on the ^R4 group, counting consecutively each adjacent atom in the chain. When two or more chains are possible, the shortest chain is used to determine the number of adjacent atoms. As shown below, for example, when R ⁴ is -(CH ₂ ) ₂ -NHC(O)-CH ₂ -(benzene-1,4-ylidene)-CH ₂ -, from the adjacent piperidine ring as shown below Beginning with the first atom in the ^R4 group of nitrogen and ending with the last atom in the ^R4 group of nitrogen adjacent to the aminohydroxyethyl group, there are 10 adjacent atoms in the shortest chain counted consecutively:

In ^one embodiment of R, R ^is selected from (1-10C) alkylene, (2-10C) alkenylene and (2-10C) alkynylene, wherein the alkylene group is unsubstituted Or substituted by 1 or 2 substituents independently selected from (1-4C)alkyl, hydroxyl and phenyl. Representative examples of specific values for R ^4a are -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -, -(CH ₂ ) ₆ - , -(CH ₂ ) ₇ -, -(CH ₂ ) ₈ -, -(CH ₂ ) ₉ -, -(CH ₂ ) ₁₀ -, -(CH ₂ )CH(CH ₃ )-, -(CH ₂ ) C(CH ₃ ) ₂ - and -(CH ₂ ) ₂ C(phenyl) ₂ -. In another aspect, R ^4a is -(CH ₂ )C(=CH ₂ )-.

In one embodiment, d is 1.

In one embodiment, ^A is an optionally substituted (3-7C)cycloalkylene group; including cyclohexylene groups such as cyclohex-1,4-ylidene and cyclohex-1,3 - a subunit; and a cyclopentylidene group, such as a cyclopent-1,3-ylidene group.

In another embodiment, ^A is an optionally substituted (6-10C)arylene group including phenylene groups such as benzene-1,4-ylene, benzene-1,3-ylene and benzene-1,2-ylidene groups; and naphthylene groups such as naphthalene-1,4-ylidene and naphthalene-1,5-ylidene groups.

In another embodiment of the invention ^A is optionally substituted (2-9C)heteroarylene groups including pyridinylene groups such as pyridin-1,4-ylidene; furylylene Groups such as furan-2,5-ylidene and furan-2,4-ylidene; thienylene groups such as thiophene-2,5-ylidene and thiophene-2,4-ylidene; and pyrrole groups, such as pyrrole-2,5-ylidene and pyrrole-2,4-ylidene.

In another embodiment, ^A is an optionally substituted (3-6C)heterocyclylene group, including piperidinylene groups, such as piperidin-1,4-ylidene; and pyrrolidine A radical group, such as pyrrolidin-2,5-ylidene.

In particular embodiments, ^A is optionally substituted phenylene, thienylene, cyclopentylene, cyclohexylene or piperidylene.

In one embodiment, e is 0.

In a particular embodiment, R ^4b is (1-5C)alkylene. Representative examples of specific values for R ^4b are -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -; including methylene, ethylene and propylene.

In one embodiment, f is 0.

In a particular embodiment, Q is selected from a bond, -N(Q ^a )C(O)-, -C(O)N(Q ^b )-, -N(Q ^c )S(O) ₂ -, - S(O) ₂ N(Q ^d )-, -N(Q ^e )C(O)N(Q ^f )-, -OC(O)N(Q ⁱ )-, -N(Q ^j )C(O )O- or -N( ^Qk ); for example where Q is a bond, -N( ^Qa )C(O)- or -C(O)N( ^Qb )-. Representative examples of specific values for Q are bond, O, NH, -C(O)NH-, -C(O)N( _CH3 )-, -NHC(O)-, -N( _CH3 ) C(O)-, -S(O) ₂ NH-, -S(O) ₂ N(CH ₃ )-, -NHS(O) ₂ -, -N(CH ₃ )S(O) ₂ - and - NHC(O)NH-. Another example of a value for Q is -C(O)(piperidin-1,4-ylidene) with R ^4c .

In one embodiment, ^Qa , ^Qb , Qc, ^Qd , ^Qe , ^Qf , ^Qg , ^Qh , ^Qi , ^{Qj ,} and ^Qk are each independently selected from hydrogen and (1-6C) Alkyl, wherein the alkyl group is substituted or substituted with 1-3 substituents independently selected from fluoro, hydroxy and (1-4C)alkoxy. For example, Qa, ^Qb , ^Qc , ^Qd , ^Qe , ^Qf , ^Qg , ^Qh , Qi, ^{Qj ,} and ^Qk are each independently ^selected from hydrogen, and (1-3C)alkyl, Includes hydrogen, methyl, ethyl, n-propyl and isopropyl. An example of the value of each of Q ^a , Q ^b , Q ^c , Q ^d , Q ^e , Q ^f , Q ^g , Q ^h , Q ⁱ , Q ^{j ,} and Q ^k is hydrogen.

In another embodiment, Q ^a , Q ^b , Q ^c , Q ^d , Q ^e , Q ^f , Q ^g , Q ^h , Q ⁱ , Q ^j and Q ^k are connected to the nitrogen atom to which they are attached and R ^4b or R ^4c together form a 4-6 membered azacycloalkylene group. For example, Qa and ^Qb together with the nitrogen atom to ^which they are attached and ^R4b or ^R4c form a piperidin-4-ylidene group. By way of illustration, when Q represents -N(Q ^a )C(O)- and Q ^a together with the nitrogen atom to which it is attached and the group R ^4b form a piperidin-4-ylidene group, R ⁴ is of the formula Group:

Similarly, when Q represents -C(O)N(Q ^b )- and Q ^b together with the nitrogen atom to which it is attached and the group R ^4c forms piperidin-4-ylidene, R ⁴ is a group of the formula :

In a particular embodiment, R ^4c is (1-5C)alkylene. Representative examples of specific values for R ⁴ are -CH ₂ -, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -; including methylene, ethylene and propylene.

In one embodiment, ^A is an optionally substituted (3-7C)cycloalkylene group; including cyclohexylene groups such as cyclohex-1,4-ylidene and cyclohex-1,3 - a subunit; and a cyclopentylidene group, such as a cyclopentylidene 1,3-ylidene group.

In another embodiment, ^A is an optionally substituted (6-10C)arylene group including phenylene groups such as benzene-1,4-ylene, benzene-1,3-ylene and benzene-1,2-ylidene groups; and naphthylene groups such as naphthalene-1,4-ylidene and naphthalene-1,5-ylidene groups.

In another embodiment, A is ^optionally a substituted (2-9C)heteroarylene group, including a pyridinylene group, such as pyridin-1,4-ylidene; a furylylene group, For example furan-2,5-ylidene and furan-2,4-ylidene; thienylene groups such as thiophene-2,5-ylidene and thiophene-2,4-ylidene; and pyrrolylene groups such as Pyrrole-2,5-ylidene and pyrrole-2,4-ylidene.

In yet another embodiment, ^A is an optionally substituted (3-6C)heterocyclylene group, including piperidinylene groups, such as piperidin-1,4-ylidene; and pyrrole Alkyl groups such as pyrrolidin-2,5-ylidene.

In particular embodiments, ^A is optionally substituted phenylene, thienylene, cyclopentylene, cyclohexylene or piperidylene.

By way of illustration, each or both of ^A1 or ^A2 may be phenylene, such as benzene-1,4-ylidene or benzene-1,3-ylidene, wherein the phenylene group is unsubstituted or replaced by independently selected from halogen, (1-4C)alkyl, (1-4C)alkoxy, -S-(1-4C)alkyl, -S(O)-(1-4C)alkyl, -S (O) ₂ -(1-4C)alkyl, -C(O)O(1-4C)alkyl, carboxyl, cyano, hydroxyl, nitro, trifluoromethyl and trifluoromethoxy 1- 4 substituents are substituted. Representative examples include benzene-1,3-ylidene, benzene-1,4-ylidene, 4-chloro-benzene-1,3-ylidene, 6-chloro-benzene-1,3-ylidene, 4-methyl-benzene-1,3-ylidene, 2-fluoro-benzene-1,4-ylidene, 2-chloro-benzene-1,4-ylidene, 2-bromo-benzene-1,4- Subunit, 2-iodo-benzene-1,4-ylidene, 2-methylbenzene-1,4-ylidene, 2-methoxybenzene-1,4-ylidene, 2-trifluoromethoxy Benzene-1,4-ylidene, 3-nitrobenzene-1,4-ylidene, 3-chlorobenzene-1,4-ylidene, 2,5-difluorobenzene-1,4-ylidene, 2 , 6-dichlorobenzene-1,4-ylidene, 2,6-diiodo-benzene 1,4-ylidene, 2-chloro-6-methylbenzene-1,4-ylidene, 2-chloro- 5-methoxy-benzene-1,4-ylidene, 2,3,5,6-tetrafluorobenzene-1,4-ylidene.

Alternatively, A ¹ or A ² or both may be cyclopentylene or cyclohexylene; wherein the cyclopentylene or cyclohexylene group is unsubstituted or substituted with (1-4C)alkyl. Representative examples include cis-cyclopent-1,3-ylidene, trans-cyclopent-1,3-ylidene, cis-cyclohexa-1,4-ylidene and trans-cyclohexylene-1, 4-base. A ¹ or A ² or both may also be optionally substituted thienylene or piperidinylene, eg, thiophene-2,5-ylidene or piperidin-1,4-ylidene.

In one embodiment, R ^is selected from (1-10C) alkylene, (2-10C) alkenylene and (2-10C) alkynylene, wherein the alkylene is unsubstituted or independently selected from Substituted by 1 or 2 substituents from (1-4C)alkyl, hydroxy and phenyl. Representative examples of specific values for R ^4d are -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ - , -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ -, -(CH ₂ ) ₈ -, -(CH ₂ ) ₉ -, -(CH ₂ ) ₁₀ - and -(CH ₂ )CH(CH ₃ )-(CH ₂ )-C(CH ₃ ) ₂ -(CH ₂ ) ₂ -.

In a particular embodiment, R ⁴ is a divalent group of the formula: -(R ^4a ) _d - wherein R ^4a is (4-10C)alkylene. In one aspect of this embodiment, R ⁴ is a divalent group of the formula: -(CH ₂ ) _j -, where j is 8, 9 or 10. Examples of specific values for R ⁴ in this embodiment are -(CH ₂ ) ₄ -, -(CH ₂ ) ₅ -, -(CH ₂ ) ₆ -, -(CH ₂ ) ₇ -, -(CH ₂ ) ₈ -, -(CH ₂ ) ₉ - and -(CH ₂ ) ₁₀ -; including -(CH ₂ ) ₈ -, -(CH ₂ ) ₉ - and -(CH ₂ ) ₁₀ -.

In another particular embodiment, R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -(A ² ) _h -(R ^4d ) _i -

Wherein R ^4a is (1-10C) alkylene, such as -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -; A ² is (6-10C) arylene, such as Benzene-1,4-ylidene or benzene-1,3-ylidene, or (2-9C)heteroarylene, such as thiophene-2,5-ylidene or thiophene-2,4-ylidene; and R ^4d is (1-10C)alkylene, eg -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -. Examples of specific values for R ⁴ in this embodiment are -(CH ₂ )-(benzene-1,4-ylidene)-(CH ₂ )-; -(CH ₂ )-(benzene-1,4- -(CH ₂ ) ₂ -; -(CH ₂ )-(benzene-1,4-ylidene)-(CH ₂ ) ₃ -; -(CH ₂ ) ₂ -(benzene-1,4- base)-(CH ₂ )-;-(CH ₂ ) _2- (benzene-1,4-ylidene)-(CH ₂ ) ₂ -;-(CH ₂ ) _2- (benzene-1,4- )-(CH ₂ ) ₃ -;-(CH ₂ ) _3- (benzene-1,4-ylidene)-(CH ₂ )-;-(CH ₂ ) _3- (benzene-1,4-ylidene) -(CH ₂ ) ₂ -; -(CH ₂ ) ₃ -(benzene-1,4-ylidene)-(CH ₂ ) ₃ -, -(CH ₂ ) ₄ -(benzene-1,4-ylidene) -(CH ₂ )-; -(CH ₂ ) ₄ -(benzene-1,4-ylidene)-(CH ₂ ) ₂ - and -(CH ₂ ) ₄ -(benzene-1,4-ylidene)- (CH ₂ ) ₃ -.

In yet another particular embodiment, R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -Q-(A ² ) _h -(R ^4d ) _i -

Wherein Q is -O- or -N(Q ^k )-; Q ^k is hydrogen or (1-3C) alkyl, such as methyl or ethyl; R ^4a is (1-10C) alkylene, such as -( CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -; A ² is (6-10C)arylene, for example benzene-1,4-ylidene or benzene-1,3-ylidene group, or (2-9C)heteroarylene, such as thiophene-2,5-ylidene or thiophene-2,4-ylidene; and R ^4d is (1-10C)alkylene, such as -(CH ₂ )-, -(CH ₂ ) ₂ -, -(CH ₂ ) ₃ -. Examples of specific values for R ⁴ in this embodiment are -(CH ₂ ) ₂ -O-(benzene-1,4-ylidene)-(CH ₂ )-; -(CH ₂ ) ₂ -O-( Benzene-1,4-ylidene)-(CH ₂ ) ₂ -;-(CH ₂ ) ₂ -O-(Benzene-1,4-ylidene)-(CH ₂ ) ₃ -;-(CH ₂ ) ₃ -O-(Benzene-1,4-ylidene)-(CH ₂ )-;-(CH ₂ ) ₃ -O-(Benzene-1,4-ylidene)-(CH ₂ ) ₂ -;-(CH ₂ ) ₃ -O-(benzene-1,4-ylidene)-(CH ₂ ) ₃ -; -(CH ₂ ) ₂ -NH-(benzene-1,4-ylidene)-(CH ₂ )-; -(CH ₂ ) ₂ -NH-(benzene-1,4-ylidene)-(CH ₂ ) ₂ -; -(CH ₂ ) ₂ -NH-(benzene-1,4-ylidene)-(CH ₂ ) ₃ -; -(CH ₂ ) ₃ -NH-(benzene-1,4-ylidene)-(CH ₂ )-;-(CH ₂ ) ₃ -NH-(benzene-1,4-ylidene)- (CH ₂ ) ₂ - and -(CH ₂ ) ₃ -NH-(benzene-1,4-ylidene)-(CH ₂ ) ₃ -.

In yet another particular embodiment, R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -(A ¹ ) _e (R ^4b )-Q-(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -

wherein Q is -N(Q ^a )C(O)- or -C(O)N(Q ^b )-. In this embodiment, particular values for ^R are of the formula:

wherein m is an integer of 2-10; and n is an integer of 2-10; with the proviso that m+n is an integer of 4-12. In this formula for R ⁴ , d and g are 1 and e, f, h and i are 0; and R ^4a is -(CH ₂ ) _m -, R ^4c is -(CH ₂ ) _n - and Q is - C(O)NH-. Particular values of m are 2 or 3; and particular values of n are 4, 5 or 6.

Another special value for ^R4 is the following formula:

wherein o is an integer of 2-7; and p is an integer of 1-6; with the proviso that o+p is an integer of 3-8. In this formula for ^R4 , d, h and i are 1 and e, f and g are 0; and ^R4a is -( _CH2 )o-, ^A2 is benzene-1,4-ylidene, ^R4d is -(CH ₂ ) _p - and Q is -C(O)NH-. The particular value of o is 2 or 3; and the particular value of p is 1 or 2. In this embodiment, the benzene-1,4-ylidene group may optionally be substituted as defined herein for ^A2 .

Another special value for ^R4 is the following formula:

wherein q is an integer of 2-6; r is an integer of 1-5; and s is an integer of 1-5; with the proviso that q+r+s is an integer of 4-8. In this formula for R ⁴ , d, g, h and i are 1 and e and f are 0; and R ^4a is -(CH ₂ ) _q -, R ^4c is -(CH ₂ ) _r -, A ² is 1,4-Phenylene, R ^4d is -(CH ₂ ) _s - and Q is -C(O)NH-. The particular value of q is 2 or 3; the particular value of r is 1 or 2; and the particular value of s is 1 or 2. In this embodiment, the benzene-1,4-ylidene group may optionally be substituted as defined herein for ^A2 .

Another special value for ^R4 is the following formula:

wherein t is an integer of 2-10; and u is an integer of 2-10; with the proviso that t+u is an integer of 4-12. In this formula for R ⁴ , d and g are 1 and e, f, h and i are 0; and R ^4a is -(CH ₂ ) _t -, R ^4c is -(CH ₂ ) _u - and Q is - NHC(O)-. Specific values for t are 2 or 3; and specific values for u are 4, 5 or 6.

Another special value for ^R4 is the following formula:

wherein v is an integer of 2-7; and w is an integer of 1-6; with the proviso that v+w is an integer of 3-8. In this formula for ^R4 , d, h and i are 1 and e, f and g are 0; and ^R4a is -( _CH2 )v-, ^A2 is 1,4-phenylene, ^R4d is -(CH ₂ ) _w - and Q is -NHC(O)-. The specific value of v is 2 or 3; and the specific value of w is 1 or 2. In this embodiment, the benzene-1,4-ylidene group may optionally be substituted as defined herein for ^A2 .

Another special value for ^R4 is the following formula:

wherein x is an integer of 2-6; y is an integer of 1-5; and z is an integer of 1-5; with the proviso that x+y+z is an integer of 4-8. In this formula for R ⁴ , d, g, h and i are 1 and e and f are 0; and R ^4a is -(CH ₂ ) _x -, R ^4c is -(CH ₂ ) _y -, A ² is 1,4-Phenylene, R ^4d is -(CH ₂ ) _z - and Q is -NHC(O)-. The particular value of X is 2 or 3; the particular value of y is 1 or 2; and the particular value of z is 1 or 2. In this embodiment, the benzene-1,4-ylidene group may optionally be substituted as defined herein for ^A2 .

By way of further illustration, ^R can be selected from:

-(CH ₂ ) ₇ -;

-(CH ₂ ) ₈ -;

-(CH ₂ ) ₉ -;

-(CH ₂ ) ₁₀ -;

-(CH ₂ ) ₁₁ -;

-(CH ₂ ) ₂ C(O)NH(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)NH(CH ₂ ) ₅ -;

-(CH ₂ ) ₃ NRC(O)NH(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (cyclohexyl-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(cyclopent-1,3-ylidene)-;

-(CH ₂ ) ₂ NHC(O)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

1-[-(CH ₂ ) ₂ C(O)](piperidin-4-yl)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ NHC(O)(trans-cyclohexyl-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(cis-cyclopent-1,3-ylidene)-;

-(CH ₂ )NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

1-[-(CH ₂ ) ₂ NHC(O)](piperidin-4-yl)(CH ₂ ) ₂ -;

-CH ₂ (benzene-1,4-ylidene)NH(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (pyridin-2,6-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(cis-cyclohexyl-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(trans-cyclohexyl-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(cis-cyclopent-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(trans-cyclohexyl-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)C*H(CH ₃ )-((S)-isomer);

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)C*H(CH ₃ )-((R)-isomer);

2-[(S)-(-CH ₂ -](pyrrolidin-1-yl)C(O)(CH ₂ ) ₄ -;

2-[(S)-(-CH ₂ -](pyrrolidin-1-yl)C(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(4-chlorobenzene-1,3-ylidene)CH ₂ -;

-CH ₂ (2-fluorobenzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(4-methylbenzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(6-chlorobenzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-chlorobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2,6-dichlorobenzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)NHCH ₂ (benzene-1,3-ylidene)CH ₂ -;

4-[-CH ₂ -](piperidin-1-yl)C(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)N(CH ₂ CH ₃ )(benzene-1,4-ylidene)CH ₂ -;

1-[-(CH ₂ ) ₂ NHC(O)](piperidin-4-yl)-;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ NHC(O)(thiophene-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(3-nitrobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(trans-cyclohexyl-1,4-ylidene)-;

1-[-CH ₂ -(2-fluorobenzene-1,3-ylidene)CH ₂ ](piperidin-4-yl)-;

5-[-(CH ₂ ) ₂ NHC(O)](pyridin-2-yl)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ (thiophene-2,5-ylidene)(CH ₂ ) ₃ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-CH ₂ (benzene-1,2-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

1-[-CH ₂ (2-fluorobenzene-1,3-ylidene) CH ₂ ](piperidin-4-yl)(CH ₂ ) ₂ -;

1-[-CH ₂ (2-fluorobenzene-1,3-ylidene) CH ₂ ](piperidin-4-yl) CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(3-chlorobenzene-1,4-ylidene)CH ₂ -;

-(CH2) ₂ C(O)NH(2-(CF ₃ O-)benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₃ (benzene-1,3-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ S(O) ₂ NH(CH ₂ ) ₅ -;

-CH ₂ (benzene-1,3-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-iodobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-chloro-5-methoxybenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-chloro-6-methylbenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ N(CH ₃ )S(O) ₂ (benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-bromobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₃ (benzene-1,4-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ (benzene-1,2-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

1-[-CH ₂ (2-fluorobenzene-1,3-ylidene) CH ₂ ](piperidin-4-yl)(CH ₂ ) ₃ -;

-(CH ₂ ) ₂ C(O)NH(2-methoxybenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₅ NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

4-[-(CH ₂ ) ₂ -](piperidin-1-yl)(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)CH(CH ₃ )CH ₂ -;

-(CH ₂ ) ₂ -(trans-cyclohexyl-1,4-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-fluorobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,3-ylidene)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ C(O)NH(2,5-difluorobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

1-[-CH ₂ (pyridin-2,6-ylidene) CH ₂ ](piperidin-4-yl) CH ₂ -;

-(CH ₂ ) ₃ NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ NH(naphthalene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ O(benzene-1,4-ylidene)CH ₂ -;

1-[-(CH ₂ ) ₃ ](piperidin-4-yl)CH ₂ -;

4-[-(CH ₂ ) ₂ -](piperidin-1-yl)C(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₃ (benzene-1,4-ylidene)NHC(O)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ O(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

2-[-(CH ₂ ) ₂ ](benzimidazol-5-yl)CH ₂ -;

-(CH ₂ ) ₂ -(trans-cyclohexyl-1,4-ylidene)NHC(O)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ -(trans-cyclohexyl-1,4-ylidene)NHC(O)(CH ₂ ) ₄ -;

-(CH ₂ ) ₂ -(trans-cyclohexyl-1,4-ylidene)NHC(O)(CH ₂ ) ₅ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ NHC(O)NH(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )(CH ₂ ) ₂ (cis-cyclohexyl-1,4-ylidene)-;

-(CH ₂ ) ₂ C(O)NH(2,3,5,6-tetrafluorobenzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2,6-diiodobenzene-1,4-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(CH ₂ ) ₃ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(CH ₂ ) ₄ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)NHCH ₂ (benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(2-methylbenzene-1,4-ylidene)CH ₂ -;

1-[-(CH ₂ ) ₃ O(benzene-1,4-ylidene)(CH ₂ ) ₂ ](piperidin-4-yl)CH ₂ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (benzene-1,3-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ O(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)CH ₂ O(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)CH ₂ O(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(furan-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)(thiophene-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ O(benzene-1,4-ylidene)OCH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,2-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohex-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)(furan-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)(thiophene-2,5-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)CH ₂ O(benzene-1,2-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)CH ₂ O(benzene-1,3-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)CH ₂ O(benzene-1,4-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(furan-2,5-ylidene)CH ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)(thiophene-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,2-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)CH ₂ O(benzene-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)(furan-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (benzene-1,4-ylidene)NHC(O)(thiophene-2,5-ylidene)CH ₂ -;

-(CH ₂ ) ₂ (trans-cyclohexyl-1,4-ylidene)NHC(O)(benzene-1,3-ylidene)CH ₂ -;

-(CH ₂ ) ₃ O(benzene-1,3-ylidene)CH ₂ -;

-CH ₂ CH(OH)CH ₂ NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₄ NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)CH ₂ NHC(O)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ NHC(O)CH ₂ -;

-(CH ₂ ) ₂ C(O)NHCH ₂ (trans-cyclohexyl-1,4-ylidene)CH ₂ -;

-(CH ₂ ) ₂ NHC(O)(CH ₂ ) ₅ -;

-(CH ₂ ) ₂ O(benzene-1,3-ylidene)OCH ₂ -;

-(CH ₂ ) ₂ O(benzene-1,2-ylidene)OCH ₂ -;

-CH ₂ (benzene-1,2-ylidene)O(benzene-1,2-ylidene)CH ₂ -;

-(CH ₂ ) ₂ C(O)NH(CH ₂ ) ₆ -;

-(CH ₂ ) ₃ (benzene-1,4-ylidene)(CH ₂ ) ₃ -;

-(CH ₂ ) ₃ (benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₄ (benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ (furan-2,5-ylidene)(CH ₂ ) ₃ -;

-(CH ₂ ) ₂ N(CH ₃ )C(O)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

4-[-(CH ₂ ) ₂ ](piperidin-1-yl)C(O)NH(benzene-1,4-ylidene)(CH ₂ ) ₂ -;

-(CH ₂ ) ₃ (benzene-1,3-ylidene)(CH ₂ ) ₃ -;

-(CH ₂ ) ₃ (tetrahydrofuran-2,5-ylidene)(CH ₂ ) ₃ -; and

-(CH ₂ ) ₂ O(benzene-1,4-ylidene)C(O)(CH ₂ ) ₂ -.

Representative subgeneric groups

The following general formulas and groups are intended to provide representative examples of aspects and embodiments of the invention, and unless otherwise indicated, they do not exclude other embodiments or limit the scope of the invention.

One particular group of compounds of formula I are those disclosed in US provisional application 60/447,843, filed February 14,2003. This group includes compounds of formula I; wherein:

a is an integer of 0 or 1-3;

Each R ¹ is independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^1a , -C(O)OR ^1b , SR ^1c , -S(O)R ^1d , -S(O) ₂ R ^1e and -NR ^1f R ^1g ;

R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^{1f ,} and R ^1g are each independently hydrogen or (1-4C)alkyl;

b is an integer of 0 or 1-3;

R ² are each independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^2a , -C(O)OR ^2b , SR ^2c , -S(O)R ^2d , -S(O) ₂ R ^2e and NR ^2f R ^2g ;

R ^2a , R ^2b , R ^2c , R ^2d , R ²³ , R ^2f and R ^2g are each independently hydrogen or (1-4C)alkyl;

W is connected to the 3- or 4-position relative to the nitrogen atom on the piperidine ring, and represents O or NW ^a ;

W ^a is hydrogen or (1-4C) alkyl;

c is an integer of 0 or 1-4;

Each ^of R is a substituent on carbon, independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano radical, halogen, -OR ^3a , -C(O)OR ^3b , SR ^3c , -S(O)R ^3d , -S(O) ₂ R ^3e and NR ^3f R ^3g ;

R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^{3f ,} and R ^3g are each independently hydrogen or (1-4C)alkyl;

R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -(A ¹ ) _e -(R ^4b ) _f -Q-(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -

in

d, e, f, g, h and i are each independently selected from 0 and 1;

R ^4a , R ^4b , R ^4c and R ^4d are each independently selected from (1-10C) alkylene, (2-10C) alkenylene and (2-10C) alkynylene, wherein each alkylene, Alkenylene or alkynylene groups are unsubstituted or substituted with 1-5 independently selected from (1-4C)alkyl, fluoro, hydroxy, phenyl and phenyl(1-4C)-alkyl base substitution;

A ^{and A} are each independently selected from (3-7C) cycloalkylene, (6-10C) arylene, (2-9C) heteroarylene and (3-6C) heterocyclylene; wherein Each cycloalkylene group is unsubstituted or substituted with 1-4 substituents independently selected from (1-4C)alkyl and each arylene, heteroarylene or heterocyclylene group is Unsubstituted or substituted by 1-4 substituents independently selected from halogen, (1-4C) alkyl and (1-4C) alkoxy;

Q is selected from a bond, -O-, -C(O)O, -OC(O)-, -S-, -S(O)-, -S(O) ₂ -, -N(Q ^a )C (O)-, -C(O)N(Q ^b )-, -N(Q ^c )S(O) ₂ -, -S(O) ₂ N(Q ^d )-, -N(Q ^e )C (O)N(Q ^f )-, -N(Q ^g )S(O) ₂ N(Q ^h )-, -OC(O)N(Q ⁱ )- and -N(Q ^j )C(O) O-;

Q ^a , Q ^b , Q ^c , Q ^d , Q ^e , Q ^f , Q ^g , Q ^h , Q ⁱ and Q ^j are each independently selected from hydrogen, (1-6C)alkyl, A ³ and (1- 4C) alkylene-A ⁴ ; wherein the alkyl group is unsubstituted or substituted by 1-3 substituents independently selected from fluorine, hydroxyl and (1-4C) alkoxy; or linked to them The nitrogen atom and the group R ^4b or R ^4c together form a 4-6 membered nitrogen heterocycloalkyl group;

A ³ and A ⁴ are each independently selected from (3-6C) cycloalkyl, (6-10C) aryl, (2-9C) heteroaryl and (3-6C) heterocyclyl; wherein each cycloalkane The radical is unsubstituted or substituted with 1-4 substituents independently selected from (1-4C)alkyl and each aryl, heteroaryl or heterocyclyl group is unsubstituted or independently selected from Substituted by 1-4 substituents from halogen, (1-4C) alkyl and (1-4C) alkoxy;

provided that the number of adjacent atoms in the shortest chain between the two nitrogen atoms to which ^R4 is attached is in the range of 8-14;

^R represents hydrogen or (1-4C) alkyl;

R ⁶ is -NR ^6a CR ^6b (O) and R ⁷ is hydrogen, or R ⁶ and R ⁷ together form -NR ^7a C(O)-CR ^7b =CR ^7c -, -CR ^7d =CR ^7e -C(O )-NR ^7f -, -NR ^7g C(O)-CR ^7h R ⁷ⁱ -CR ^7j R ^7k - or -CR ^7l R ^7m -CR ⁷ⁿ R ^7o -C(O)-NR ^7p -;

R ^6a and R ^6b are each independently hydrogen or (1-4C)alkyl; and

^{Each independently _ _ _ _ _ _ _ _ _ _ _ _ _ _} is hydrogen or (1-4C) alkyl;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those disclosed in US provisional application 60/467,035, filed May 1,2003. This group of compounds includes compounds of formula I; wherein:

a is an integer of 0 or 1-3;

Each R ¹ is independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^1a , -C(O)OR ^1b , SR ^1c , -S(O)R ^1d , -S(O) ₂ R ^1e and -NR ^1f R ^1g ;

R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^{1f ,} and R ^1g are each independently hydrogen or (1-4C)alkyl;

b is an integer of 0 or 1-3;

R ² are each independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano, halogen, -OR ^2a , -C(O)OR ^2b , SR ^2c , -S(O)R ^2d , -S(O) ₂ R ^2e and NR ^2f R ^2g ;

R ^2a , R ^2b , R ^2c , R ^2d , R ^2e , R ^{2f ,} and R ^2g are each independently hydrogen or (1-4C)alkyl;

W is connected to the 3- or 4-position relative to the nitrogen atom on the piperidine ring, and represents O or NW ^a ;

W ^a is hydrogen or (1-4C) alkyl;

C is an integer of 0 or 1-4;

Each ^of R is a substituent on carbon, independently selected from (1-4C) alkyl, (2-4C) alkenyl, (2-4C) alkynyl, (3-6C) cycloalkyl, cyano radical, halogen, -OR ^3a , -C(O)OR ^3b , SR ^3c , -S(O)R ^3d , -S(O) ₂ R ^3e and NR ^3f R ^3g ;

R ^3a , R ^3b , R ^3c , R ^3d , R ^3e , R ^{3f ,} and R ^3g are each independently hydrogen or (1-4C)alkyl;

R ⁴ is a divalent group of the formula:

-(R ^4a ) _d -(A ¹ ) _e -(R ^4b ) _f -Q-(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -

in

d, e, f, g, h and i are each independently selected from 0 and 1;

R ^4a , R ^4b , R ^4c and R ^4d are each independently selected from (1-10C) alkylene, (2-10C) alkenylene and (2-10C) alkynylene, wherein each alkylene, Alkenylene or alkynylene groups are unsubstituted or substituted with 1-5 independently selected from (1-4C)alkyl, fluoro, hydroxy, phenyl and phenyl(1-4C)-alkyl base substitution;

A ^{and A} are each independently selected from (3-7C) cycloalkylene, (6-10C) arylene, (2-9C) heteroarylene and (3-6C) heterocyclylene; wherein Each cycloalkylene group is unsubstituted or substituted with 1-4 substituents independently selected from (1-4C)alkyl and each arylene, heteroarylene or heterocyclylene group is Unsubstituted or substituted by 1-4 substituents independently selected from halogen, (1-4C) alkyl and (1-4C) alkoxy;

Q is selected from a bond, -O-, -C(O)O, -OC(O)-, -S-, -S(O)-, -S(O) ₂ -, -N(Q ^a )C (O)-, -C(O)N(Q ^b )-, -N(Q ^c )S(O) ₂ -, -S(O) ₂ N(Q ^d )-, -N(Q ^e )C (O)N(Q ^f )-, -N(Q ^g )S(O) ₂ N(Q ^h )-, -OC(O)N(Q ⁱ )- and -N(Q ^j )C(O) O-;

Q ^a , Q ^b , Q ^c , Q ^d , Q ^e , Q ^f , Q ^g , Q ^h , Q ⁱ and Q ^j are each independently selected from hydrogen, (1-6C)alkyl, A ³ and (1- 4C) alkylene-A ⁴ ; wherein the alkyl group is unsubstituted or substituted by 1-3 substituents independently selected from fluorine, hydroxyl and (1-4C) alkoxy; or linked to them The nitrogen atom and the group R ^4b or R ^4c together form a 4-6 membered nitrogen heterocycloalkyl group;

A ³ and A ⁴ are each independently selected from (3-6C) cycloalkyl, (6-10C) aryl, (2-9C) heteroaryl and (3-6C) heterocyclyl; wherein each cycloalkane The radical is unsubstituted or substituted with 1-4 substituents independently selected from (1-4C)alkyl and each aryl, heteroaryl or heterocyclyl group is unsubstituted or independently selected from Substituted by 1-4 substituents from halogen, (1-4C) alkyl and (1-4C) alkoxy;

Provided that the number of adjacent atoms in the shortest chain between the two nitrogen atoms connected by ^R is in the range of 4-14;

^R represents hydrogen or (1-4C) alkyl;

R ⁶ is -NR ^6a CR ^6b (O) or CR ^6c R ^6d OR ^6e and R ⁷ is hydrogen, or R ⁶ and R ⁷ together form -NR ^7a C(O)-CR ^7b = CR ^7c -, -CR ^7d =CR ^7e -C(O)-NR ^7f -, -NR ^7g C(O)-CR ^7h R ⁷ⁱ -CR ^7j R ^7k -or -CR ^7l R ^7m -CR ⁷ⁿ R ^7o -C(O)-NR ^7p -;

R ^6a , R ^6b , R ^6c , R ^6d , and R ^6e are independently hydrogen or (1-4C)alkyl; and

^{Each independently _ _ _ _ _ _ _ _ _ _ _ _ _ _} is hydrogen or (1-4C) alkyl;

or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W is O; W is attached at the 4-position of the piperidine ^ring ; R ⁵ is hydrogen; ⁶ and ^R7 are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W ^is NH; W is attached at the 4-position of the piperidine ring; R ⁵ is hydrogen; ⁶ and ^R7 are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Yet another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W is O; W is attached at the 4-position of the piperidine ring; R ⁴ is -(CH ₂ ) _j -, wherein j is 8, 9 or 10; R ⁵ is hydrogen; and R ⁶ and R ⁷ are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W is NH; W is attached at the 4-position of the piperidine ring; R ⁴ is -(CH ₂ ) _j - , wherein j is 8, 9 or 10; R ⁵ is hydrogen; and R ⁶ and R ⁷ are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Yet another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W is O; W is attached at the 4-position of the piperidine ring; R ⁴ is -(CH ₂ ) ₂ -C(O)NH-( _CH2 ) ₅ ; ^R5 is hydrogen; and ^R6 and ^R7 are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those wherein: a is 0; b is 0; c is 0; W is NH; W is attached at the 4-position of the piperidine ring; R ⁴ is -(CH ₂ ) ₂ - C(O)NH—(CH ₂ ) ₅ ; R ⁵ is hydrogen; and R ⁶ and R ⁷ are as defined herein; or a pharmaceutically acceptable salt or solvate or stereoisomer thereof.

Another specific group of compounds of formula I are those of formula II as defined herein; or pharmaceutically acceptable salts or solvates or stereoisomers thereof.

Another specific group of compounds of formula I are those of formula III as defined herein; or pharmaceutically acceptable salts or solvates or stereoisomers thereof.

Another specific group of compounds of formula I are those of formula IV as defined herein, or pharmaceutically acceptable salts or solvates or stereoisomers thereof.

Another specific group of compounds of formula I are those of formula II, III or IV as defined herein, wherein the piperidine ring is substituted at the 4-position by a methyl group; or a pharmaceutically acceptable salt or solvate thereof or Stereoisomers.

Another specific group of compounds of formula I are compounds of formula V:

Wherein W, R ⁴ , R ⁶ and R ⁷ are as defined in Table 1; or a pharmaceutically acceptable salt or solvate thereof.

Table 1

1 In Tables I-III, "(racemic)" means that the compound is racemic at the chiral carbon having a hydroxyl group in Formula V, VI or VII.

2 For this group, the nitrogen atom is attached to ^R6 and the carbon atom is attached to ^R7 .

Another special group of compounds of formula I are compounds of formula VI:

Wherein W, R ^1A , R ^1B , R ^1C , R ^2A , R ² B , R ⁴ , R ⁶ and R ⁷ are as defined in Table II; or a pharmaceutically acceptable salt or solvate thereof.

Table II

Another specific group of compounds of formula I are compounds of formula VII:

Wherein W, R ⁴ , R ⁶ and R ⁷ are as defined in Table III; or a pharmaceutically acceptable salt or solvate thereof.

Table III

definition

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

The term "alkyl" refers to a monovalent saturated hydrocarbon group which may be straight or branched. Unless otherwise defined, such alkyl groups typically contain 1-10 carbon atoms. Representative alkyl groups include, by way of 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, etc.

The term "alkylene" refers to a divalent saturated hydrocarbon group which may be straight or branched. Unless otherwise defined, such alkylene groups typically contain 1-10 carbon atoms. Representative alkylene groups include, by way of example, methylene, ethane-1,2-diyl (“ethylene”), propane-1,2-diyl, propane-1,3- Diyl, butane-1,4-diyl, pentane-1,5-diyl, etc.

The term "alkoxy" refers to a monovalent group of formula (alkyl)-O-, wherein alkyl is as defined herein. Representative alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, tert-butoxy Base etc.

The term "alkenyl" refers to a monovalent unsaturated hydrocarbon radical, which may be straight or branched, having at least 1, usually 1, 2 or 3 carbon-carbon double bonds. Unless otherwise defined, such alkenyl groups typically contain 2-10 carbon atoms. Representative alkenyl groups include, by way of example, ethenyl, n-propenyl, isopropenyl, n-but-2-enyl, n-hex-3-enyl, etc. The term "alkenylene" refers to a divalent alkenyl base group.

The term "alkynyl" refers to a monovalent unsaturated hydrocarbon radical, which may be straight or branched, having at least 1, usually 1, 2 or 3 carbon-carbon triple bonds. Unless otherwise defined, such alkynyl groups typically contain 2-10 carbon atoms. Representative alkynyl groups include, by way of example, ethynyl, n-propynyl, n-but-2-ynyl, n-hex-3-ynyl, and the like. The term "alkynylene" refers to a divalent alkynyl group.

The term "aryl" refers to a monovalent aromatic hydrocarbon having a single ring (ie, phenyl) or fused rings (ie, naphthalene). Unless otherwise defined, such aryl groups typically contain 6-10 carbon ring atoms. Representative aryl groups include, by way of example, phenyl and naphthalen-1-yl, naphthalen-2-yl, and the like. The term "arylene" refers to a divalent aryl group.

The term "azacycloalkyl" refers to a monovalent heterocyclic ring containing one nitrogen atom, ie a cycloalkyl group in which one carbon atom is replaced by a nitrogen atom. Unless otherwise defined, such azacycloalkyl groups typically contain 2-9 carbon atoms. Representative examples of azacycloalkyl groups are pyrrolidinyl and piperidinyl groups. The term "azacycloalkylene" refers to a divalent azacycloalkyl group. Representative examples of azacycloalkylene groups are pyrrolidinylene and piperidinylene groups.

The term "cycloalkyl" refers to a monovalent saturated carbocyclic hydrocarbon group. Unless otherwise defined, such cycloalkyl groups typically contain 3-10 carbon atoms. Representative cycloalkyl groups include, by way of example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "cycloalkylene" refers to a divalent cycloalkyl group.

The term "halogen" refers to fluorine, chlorine, bromine and iodine.

唑，呋喃，噻吩，三唑，吡唑，异

唑，异噻唑，吡啶，吡嗪，哒嗪，嘧啶，三嗪，吲哚，苯并呋喃，苯并噻吩，苯并咪唑，苯并噻唑，喹啉，异喹啉，喹唑啉，喹

啉等，其中连接点是在任何可用的碳或氮环原子。术语“亚杂芳基”指二价杂芳基基团。The term "heteroaryl" refers to a monovalent aryl group having a single ring or two fused rings and containing in the ring at least one (usually 1-3 heteroatoms) heteroatom selected from nitrogen, oxygen or sulfur. Unless otherwise defined, such heteroaryl groups typically contain 5-10 total ring atoms. Representative heteroaryl groups include, by way of example, the monovalent species pyrrole, imidazole, thiazole,

Azole, furan, thiophene, triazole, pyrazole, iso

Azole, isothiazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, indole, benzofuran, benzothiophene, benzimidazole, benzothiazole, quinoline, isoquinoline, quinazoline, quinoline

phenoline, etc., where the point of attachment is at any available carbon or nitrogen ring atom. The term "heteroarylene" refers to a divalent heteroaryl group.

烷、吗啉、硫代吗啉、哌嗪、3-吡咯林等，其中连接点是在任何可用的碳或氮环原子。术语“亚杂环基”指二价杂环基或杂环基团。The term "heterocyclyl" or "heterocycle" refers to a monovalent saturated ring having a single ring or multiple fused rings and containing at least one heteroatom (usually 1-3 heteroatoms) selected from nitrogen, oxygen or sulfur in the ring. or unsaturated (non-aromatic) groups. Unless otherwise defined, such heterocyclic groups typically contain 2-9 total ring carbon atoms. Representative heterocyclic groups include, by way of example, the monovalent species of pyrrolidine, imidazolidine, pyrazolidine, piperidine, 1,4-di

alkane, morpholine, thiomorpholine, piperazine, 3-pyrrolene, etc., wherein the point of attachment is at any available carbon or nitrogen ring atom. The term "heterocyclylene" refers to a divalent heterocyclic group or a heterocyclic group.

When a particular term is used herein to refer to a particular number of carbon atoms, the number of carbon atoms is shown in parentheses preceding the term. For example, the term "(1-4C)alkyl" refers to an alkyl group having 1-4 carbon atoms.

The term "pharmaceutically acceptable salt" refers to a salt that is acceptable for administration to a patient, such as a mammal (eg, a salt that has acceptable mammalian safety for a given dosage regimen). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, bile Alkali, N, N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylammonia Phenoline, N-Ethylpiperidine, Glucosamine, Glucosamine, Histidine, Hypamine, Isopropylamine, Lysine, Methylglucamine, Morpholine, Piperadine, Piperazine , Polyamine resin, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. Salts from pharmaceutically acceptable acids include acetate, ascorbate, besylate, benzoate, camphorsulfonate, citrate, ethanesulfonate, edisulfonate, fumarate Salt, Gentisate, Gluconate, Glucuronate, Glutamate, Hippurate, Hydrobromide, Hydrochloride, Isethionate, Lactate, Lactobionate, Maleate, malate, mandelate, methanesulfonate, mucate, naphthalenesulfonate, naphthalene-1,5-disulfonate, naphthalene-2,6-disulfonate, smoke Alkaline salt, nitrate, orotate, pamoate, pantothenate, phosphate, succinate, sulfate, tartrate, p-toluenesulfonate, xinafoate, etc. Particularly preferred are citrates, hydrobromides, hydrochlorides, isethionates, maleates, naphthalene-1,5-disulfonates, phosphates, sulfates and tartrates.

The term "salt thereof" refers to a compound formed when a hydrogen of an acid is replaced by a cation such as a metal cation or an organic cation or the like. Preferably, the salt is a pharmaceutically acceptable salt, but this need not be the case for salts of intermediate compounds which are not administered to the patient.

The term "solvate" refers to a complex or aggregate formed by one or more molecules of a solute, ie, a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. Such "solvates" are generally crystalline solids having a substantially fixed molar ratio of solute and solvent. Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid, and the like. When the solvent is water, the solvate formed is a hydrate.

It will be understood that the term "or a pharmaceutically acceptable salt or solvate or stereoisomer thereof" is meant to include all permutations of salts, solvates and stereoisomers, for example pharmaceutically acceptable stereoisomers of compounds of formula I Solvates of accepted salts.

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

The term "treatment (gerund)" or "treatment (noun)" as used herein refers to the treatment of a disease or medical condition (eg COPD) in a patient, such as a mammal, especially a human, which includes:

(a) prevent a disease or medical condition from occurring, i.e. treat patients prophylactically;

(b) ameliorating a disease or medical condition, that is, eliminating or causing regression of a disease or medical condition in a patient;

(c) contain a disease or medical condition, that is, delay or stop the development of a disease or medical condition in a patient; or

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

The term "leaving group" refers to a functional group or atom capable of being replaced by another functional group or atom in a substitution reaction, eg, a nucleophilic substitution reaction. By way of example, representative leaving groups include chloro, bromo, and iodo groups; sulfonate groups such as mesylate, tosylate, brosylate, nitrobenzenesulfonic acid; esters and the like; and acyloxy groups such as acetoxy, trifluoroacetoxy and the like.

The term "protected derivatives thereof" refers to derivatives of a particular compound in which one or more functional groups of the compound are protected from participation in unwanted reactions with a protecting or capping group. Functional groups that may be protected include, by way of example, carboxylic acid groups, amino groups, hydroxyl groups, thiol groups, carbonyl groups, and the like. Representative protecting groups for carboxylic acids include esters (e.g., p-methoxybenzyl ester), amides, and hydrazides; for amino groups, carbamates (e.g., tert-butoxycarbonyl) and Amides; for hydroxyl groups, ethers and esters; for thiol groups, thioethers and thioesters; for carbonyl groups, acetals and ketals; etc. Such protecting groups are well known to those skilled in the art and are described, for example, in T.W. Greene and G.M. Wuts, Protecting groups in Organic Synthesis, Third Edition, Wiley, New York, 1999 and references cited therein.

The term "amino-protecting group" refers to a protecting group suitable to prevent undesired reactions at an amino group. Representative amino-protecting groups include, but are not limited to, tert-butoxycarbonyl (BOC), trityl (Tr), benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc ), formyl, trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), etc.

The term "carboxy-protecting group" refers to a protecting group suitable for preventing undesired reactions at the carboxyl group. Representative carboxy-protecting groups include, but are not limited to, esters such as methyl, ethyl, tert-butyl, benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), diphenylmethyl (diphenylmethyl, DPM), etc.

The term "hydroxy-protecting group" refers to a protecting group suitable to prevent undesired reactions at the hydroxy group. Representative hydroxy-protecting groups include, but are not limited to, silyl groups, including tri(1-6C)alkylsilyl groups such as trimethylsilyl (TMS), triethyl Silyl (TES), tert-butyldimethylsilyl (TBS), etc.; esters (acyl groups) include (1-6C) alkanoyl groups, such as formyl, acetyl, etc.; arylformyl radical groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), diphenylmethyl (diphenylmethyl, DPM) and the like. Furthermore, two hydroxyl groups can also be protected as an alkylene group, such as ylidine, for example formed by reaction with a ketone, such as acetone.

General Synthesis Method

The biphenyl derivatives of the present invention can be prepared from readily available starting materials using the general methods and procedures described below or by application of other information readily available to one of ordinary skill in the art. Although specific embodiments of the invention will be shown or described herein, those skilled in the art will recognize that all embodiments or aspects of the invention can be prepared using the methods described herein or by application of other methods known to those skilled in the art. Methods, reagents and starting materials can also be prepared. It will also be understood that where typical or preferred process conditions (ie, reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. While optimum reaction conditions may vary with the particular reactants or solvent employed, such conditions can be readily determined by one skilled in the art by routine optimization procedures.

Furthermore, it will be apparent to those of ordinary skill in the art that conventional protecting groups may be necessary or desirable to prevent undesired reactions of particular functional groups. The selection of suitable protecting groups for a particular functional group and suitable conditions for the protection and deprotection of such functional groups are well known in the art. Protecting groups other than those illustrated in the procedures described herein may be employed if desired. For example, a number of protecting groups, and their introduction and removal, are described in T.W. Greene and G.M. Wuts, Protecting groups in Organic Synthesis, Third Edition, Wiley, New York, 1999 and references cited therein.

By way of illustration, the biphenyl derivatives of the present invention can be prepared by a process comprising:

(a) make formula 1 compound:

Or its salt; React with formula 2 compound:

Wherein X ¹ represents a leaving group, P ¹ and P ² each independently represent a hydrogen atom or a hydroxyl-protecting group;

(b) make formula 3 compound

Or its salt; React with formula 4 compound:

Wherein X ² represents a leaving group, P ³ and P ⁴ each independently represent a hydrogen atom or a hydroxyl-protecting group;

(c) make formula 5 compound

Coupling with compound of formula 6 :

wherein X ^Qa and X ^Qb each independently represent a functional group coupled to form group Q, p ^5a represents a hydrogen atom or an amino-protecting group; and p ^5b and p ⁶ each independently represent a hydrogen atom or a hydroxyl-protecting group ;

(d) For the compound of formula ^I wherein R represents a hydrogen atom, the compound of formula 3 and the compound of formula 7 are made in the presence of a reducing agent:

or its hydrate (such as glyoxal) reaction, wherein P ⁷ represents a hydrogen atom or a hydroxyl-protecting group;

(e) in the presence of a reducing agent, make the compound of formula 1 and the compound of formula 8 :

or its hydrate reaction, wherein p ⁸ and P ⁹ each independently represent a hydrogen atom or a hydroxyl-protecting group, p ¹⁰ represents a hydrogen atom or an amino-protecting group, and R ^4' represents a residue, which is connected to The carbons together provide the group R ⁴ upon completion of the reaction;

(f) make the compound of formula 9 :

Wherein ^X represents a leaving group, which reacts with the compound of formula 10 :

wherein P ¹¹ and p ¹² each independently represent a hydrogen atom or a hydroxyl-protecting group, and p ¹³ represents a hydrogen atom or an amino-protecting group; or

(g) in the presence of a reducing agent, make the compound of formula 11 :

or a hydrate thereof; wherein R ^4' represents a residue which, together with the carbon to which it is attached, provides a group R ⁴ upon completion of the reaction; reacted with a compound of formula 10 ; and then

Removal of any protecting group P ¹ , p ² , P ³ , P ⁴ , P ^5a , P ^5b , P ⁶ , P ⁷ , P ⁸ , P ⁹ , P ¹⁰ , P ¹¹ , P ¹² or P ¹³ provides formula I compound; and optionally, forming a pharmaceutically acceptable salt thereof.

In general, if a salt of one of the starting materials is used in the above process, such as an acid addition salt, this salt is usually neutralized before or during the reaction. This neutralization is generally accomplished by contacting the salt with one molar equivalent of base per molar equivalent of acid addition salt.

In method (a), ie the reaction between compounds of formulas 1 and 2 , the leaving group represented by ^X can be, for example, a halogen, such as chlorine, bromine or iodine, or a sulfonate group, such as methanesulfonic acid ester or tosylate. Groups P ¹ and P ² may for example be trimethylsilyl and benzyl, respectively. The reaction is usually carried out in an inert diluent, such as acetonitrile, in the presence of a base. For example, the reaction can be carried out in the presence of a tertiary amine, such as diisopropylethylamine. Typically, the reaction is carried out at a temperature in the range of 0°C to 100°C until the reaction is substantially complete. The reaction product is then isolated using conventional methods such as extraction, recrystallization, chromatography and the like.

Compounds of formula I are generally known in the art or may be prepared from commercially available starting materials using known procedures. For example, compounds of formula 1 can be prepared by deprotecting compounds of formula 12 :

wherein P ¹⁴ represents an amino-protecting group, such as a benzyl group. By way of illustration, benzyl groups can be readily removed by reduction using, for example, hydrogen or ammonium formate and a Group VIII metal catalyst, such as palladium on carbon. When W represents ^NWa , the hydrogenation is conveniently carried out using Pearlman's catalyst (ie Pd(OH) ₂ ).

The compound of formula 12 can be obtained by making the isocyanate compound of formula 13 :

Prepared by reaction with a compound of formula 14 :

Compounds of formula 2 can be prepared by various methods described herein or by methods known to those skilled in the art. For example, the hydroxyl group of the compound of formula 23 below can be easily converted into a leaving group using well-known reagents and methods. By way of illustration, hydroxyl groups can be converted to halo groups using inorganic acid halides such as thionyl chloride, phosphorus trichloride, phosphorus tribromide, phosphorus oxychloride, etc., or hydrohalic acids such as hydrobromic acid.

In method (b), that is, the reaction of a compound of formula 3 with a compound of formula 4 , the leaving group represented by ^X can be, for example, a halogen, such as chlorine, bromine or iodine, or a sulfonate group, such as mesylate or tosylate. Groups ^P3 and ^P4 can be, for example, tert-butyldimethylsilyl and benzyl, respectively. The reaction is usually carried out in the presence of a base, such as sodium bicarbonate and an alkali metal iodide, such as sodium iodide. Typically, the reaction is carried out in an inert diluent, such as tetrahydrofuran, at a temperature of 25°C to 100°C until the reaction is substantially complete. The reaction product is then isolated using conventional methods such as extraction, recrystallization, chromatography and the like.

Compounds of formula 3 are prepared by deprotecting compounds of formula 15 :

wherein one or both of P ¹⁵ and P ¹⁶ independently represent a protecting group, such as tert-butoxycarbonyl, and any remaining represent a hydrogen atom. For example the tert-butoxycarbonyl group can be removed by treating the protected compound with trifluoroacetic acid.

Compounds of formula 15 can be prepared by reacting compounds of formula 1 with compounds of formula 16 :

X ³ -R ⁴ -NP ¹⁵ P ¹⁶

16

wherein ^X represents a leaving group such as a halogen such as chlorine, bromine or iodine, or a sulfonate group such as mesylate or tosylate. The reaction is generally carried out by reacting a compound of formula 1 with a compound of formula 16 in an inert diluent, such as acetonitrile, DMF or mixtures thereof, at a temperature ranging from about 0°C to about 100°C until the reaction is substantially complete.

Alternatively, compounds of formula 3 can be obtained by reductive amination of compounds of formula 11 . The reductive amination can be carried out by reacting a compound of formula 11 with, for example, benzylamine and hydrogen in the presence of palladium on carbon.

Compounds of formula 11 can be prepared by oxidation of the corresponding alcohol of formula 17 using a suitable oxidizing agent such as sulfur trioxide pyridine complex and dimethylsulfoxide.

The oxidation is generally carried out in an inert solvent, such as dichloromethane, in the presence of a tertiary amine, such as diisopropylethylamine, at a temperature of from about -20°C to about 25°C.

Compounds of formula 17 can be prepared by reacting compounds of formula 1 with compounds of formula 18 :

X ⁴ -R ⁴ -OH

18

wherein ^X4 represents a leaving group such as a halogen such as chlorine, bromine or iodine, or a sulfonate group such as mesylate or tosylate.

The compound of formula 4 can be obtained by compounding the compound of formula 19 :

Prepared by reaction with a reducing agent such as borane. This reduction can be carried out, if desired, in the presence of a chiral catalyst to provide the compound of formula 4 in chiral form. For example, the compound of formula 19 can be formed from (R)-(+)-α, α-biphenyl-2-pyrrolidinemethanol and trimethylboroxane; or from (S)-(-)-α , α-biphenyl-2-pyrrolidinemethanol and trimethylboroxane were reduced in the presence of a chiral catalyst. The resulting hydroxyl group can then be protected with a hydroxyl protecting group ^-P3 , for example by reaction with tert-butyldimethylsilyl trifluoromethanesulfonate.

The compound of formula 19 wherein ^X represents a bromine atom can be obtained by ordering the compound of formula 20 in the presence of a Lewis acid such as boron trifluoride diethyl etherate:

Prepared by reaction with bromine. Compounds of formula 20 are well known in the art and can be prepared by known methods using commercially available starting materials and reagents.

Referring to method (c), ie the reaction of a compound of formula 5 with a compound of formula 6 , it will be understood that the groups X ^Qa and X ^Qb should be chosen so as to provide the desired group Q at the completion of the reaction. For example, when the desired group Q is an amide group, ie -N(Q ^a )C(O)- or -C(O)N(Q ^b ), one of X ^Qa and X ^Qb may be an amino group (ie, -NHQ ^a or -NHQ ^b ) and the other may be a carboxyl group (ie, -COOH) or their reactive derivatives (eg, acid halides, such as acid chlorides or acid bromides). The groups P ^5a , P ^5b and P ⁶ can be, for example, benzyl, trimethylsilyl and benzyl, respectively. When Q is an amide group, the reaction can be carried out under conventional amide coupling conditions. When the similar group Q is a sulfonamide, namely -N(Q ^c )S(O) ₂ -or -S(O) ₂ N(Q ^d )-, one of X ^Qa and X ^Qb may be an amino group, -NHQ ^c or NHQ ^d and the other may be a sulfonyl halide group (eg sulfonyl chloride or sulfonyl bromide).

Compounds of formula 5 can be prepared by reacting compounds of formula 1 with compounds of formula 21 :

X ⁵ -(R ^4a ) _d -(A ¹ ) _e -(R ^4b ) _f -X ^Qa′

twenty one

wherein X represents a leaving group including a ^halogen such as chlorine, bromine or iodine and a sulfonate such as mesylate or tosylate; and X ^Qa' represents X ^Qa such as a carboxyl group or an amino group NHQ ^a , or their protected derivatives, such as (1-6C)alkoxycarbonylamino groups or tert-butoxycarbonylamino groups. The reaction is generally carried out by methods analogous to those used to prepare compounds of formula 3 , followed by removal of any protecting groups in X ^Qa' .

Compounds of formula 6 can be prepared by reacting compounds of formula 4 with compounds of formula 22 :

X ^Qb′ -(R ^4c ) _g -(A ² ) _h -(R ^4d ) _i -X ⁶

twenty two

wherein ^X represents a leaving group including a halogen such as chlorine, bromine or iodine and a sulfonate such as mesylate or tosylate; and X ^Qb' represents X ^Qb such as a carboxyl group or an amino group NHQ ^b , or their protected derivatives, such as (1-6C)alkoxycarbonylamino groups or tert-butoxycarbonylamino groups. The reaction is generally carried out by methods analogous to those used to prepare compounds of formula 3 , followed by removal of any protecting groups in X ^Qb' .

Referring to method (d), ie the reaction of a compound of formula 3 with a compound of formula 7 , any suitable reducing agent may be employed in this reaction. For example, the reducing agent can be hydrogen in the presence of a Group VIII metal catalyst, such as palladium on carbon; or a metal hydride reagent, such as sodium triacetoxyborohydride. The group P ⁷ can be, for example, benzyl. The reaction is generally carried out in a mixture of an inert diluent and a protic solvent such as dichloroethane and methanol at a temperature in the range of 0°C to 100°C until the reaction is substantially complete.

Compounds of formula 7 in the form of hydrates can be prepared by conventional methods, for example by dibromination of compounds of formula 19 (where ^X in this case can also be hydrogen), followed by hydrolysis of the resulting dibromide to form its glyoxal or hydrate. For example, a compound of formula 19 can be reacted with hydrogen bromide followed by hydrolysis with water to form the corresponding glyoxal hydrate.

Referring to method (e), ie the reaction of a compound of formula 1 with a compound of formula 8 , any suitable reducing agent may be employed in this reaction. For example, the reducing agent can be hydrogen in the presence of a Group VIII metal catalyst, such as palladium on carbon; or a metal hydride reagent, such as sodium triacetoxyborohydride. The groups P ⁸ , P ⁹ and P ¹⁰ can be, for example, trimethylsilyl, benzyl and benzyl, respectively. The reaction is generally carried out in an inert diluent and a protic solvent such as dichloroethane and methanol at a temperature in the range of 0°C to 100°C until the reaction is substantially complete.

Compounds of formula 8 can be prepared by oxidation of compounds of formula 23 with any suitable oxidizing agent such as sulfur trioxide pyridine complex and dimethylsulfoxide.

The reaction is generally carried out in the presence of a tertiary amine such as diisopropylethylamine at a temperature in the range of about -20°C to about 25°C until the oxidation is substantially complete.

Compounds of formula 23 can be prepared by reacting compounds of formula 10 with compounds of formula 24 :

HO-R ⁴ -X ⁷

twenty four

wherein ^X7 represents a leaving group including halogen such as chlorine, bromine or iodine and sulfonate such as mesylate or tosylate.

Referring to method (f), i.e. the reaction of a compound of formula 9 with a compound of formula 10 , the leaving group represented by ^X can be, for example, a halogen such as chlorine, bromine or iodine, or a sulfonate group such as mesylate or Tosylate. The groups P ¹¹ , P ¹² and P ¹³ can be, for example, trimethylsilyl, benzyl and benzyl, respectively. The reaction is usually carried out in an inert diluent such as acetonitrile in the presence of a suitable base. For example the reaction is usually carried out in the presence of a tertiary amine such as diisopropylethylamine. Typically, the reaction is carried out at a temperature in the range of 0°C to 100°C until the reaction is substantially complete.

Compounds of formula 9 can be prepared starting from compounds of formula 1 by procedures analogous to methods (a)-(e) herein. Alternatively, compounds _of formula 10 can be prepared from compounds of formula 4 by reaction with amines of formula ^P13NH2 .

Referring to method (g), the reaction of a compound of formula 11 with a compound of formula 10 , any suitable reducing agent may be employed in this reaction. For example, the reducing agent can be hydrogen in the presence of a Group VIII metal catalyst, such as palladium on carbon; or a metal hydride reagent, such as sodium triacetoxyborohydride. The groups P ¹¹ , P ¹² and P ¹³ can be, for example, tert-butyldimethylsilyl, benzyl and benzyl, respectively. The reaction is generally carried out in an inert diluent and a protic solvent such as dichloroethane and methanol at a temperature in the range of 0°C to 100°C until the reaction is substantially complete.

Compounds of formula 11 are readily prepared by oxidation of the corresponding alcohol or hydrolysis of the corresponding acetal. Any suitable oxidizing agent can be used in this reaction to provide the aldehyde, such as sulfur trioxide pyridine complex and dimethylsulfoxide. The acetal can be hydrolyzed using aqueous acid under conventional conditions to provide the aldehyde.

In certain embodiments, certain specific compounds of formula I are prepared by a process comprising:

(h) deprotecting the compound of formula 25 :

Wherein P ¹⁷ represents a hydrogen atom or an amino-protecting group; P ¹⁸ , P ¹⁹ and P ²⁰ each independently represent a hydrogen atom or a hydroxyl-protecting group; the condition is that at least one of P ¹⁷ , P ¹⁸ , P ¹⁹ or P ²⁰ One is a protecting group;

(i) deprotecting the compound of formula 26 :

wherein P ²¹ represents a hydrogen atom or an amino-protecting group; and P ²² and P ²³ each independently represent a hydrogen atom or a hydroxyl-protecting group; with the proviso that at least one of P ²¹ , P ²² or p ²³ is a protecting group ;or

(j) deprotecting the compound of formula 27 :

wherein P ²⁴ represents a hydrogen atom or an amino-protecting group; and P ²⁵ and P ²⁶ each independently represent a hydrogen atom or a hydroxyl-protecting group; with the proviso that at least one of P ²⁴ , P ²⁵ or p ²⁶ is a protecting group .

A compound of formula I is provided, and optionally a pharmaceutically acceptable salt of a compound of formula I is formed.

Referring to method (h), examples of specific values for P ¹⁷ , P ¹⁸ , P ¹⁹ and P ²⁰ are: P ¹⁷ is hydrogen or benzyl; P ¹⁸ is hydrogen or tert-butyldimethylsilyl; P ¹⁹ and P ²⁰ are hydrogen or benzyl, or together propylene (propylene). In this method, the benzyl protecting group is conveniently removed by catalytic hydrogenation in the presence of a Group VIII metal catalyst such as palladium on carbon; Trihydrofluoride treatment is conveniently removed; propylene groups are conveniently removed by treatment with an acid such as trifluoroacetic acid.

Compounds of formula 25 can be prepared by the methods described herein, for example by methods (a)-(g). Alternatively, the compound of formula 25 can be obtained by making the compound of formula 28 :

wherein R ⁸ represents -CH ₂ OP ¹⁹ , -CHO, -COOH or -C(O)O(1-6C)alkoxy, for example carbomethoxy, R ⁹ represents -OP ¹⁸ and R ¹⁰ represents a hydrogen atom, or R ⁹ and R ¹⁰ together represent =O, prepared by reaction with a reducing agent. The reaction may employ any suitable reducing agent, including, by way of example, metal hydride reducing agents such as sodium borohydride, lithium aluminum hydride, and the like.

Compounds of formula 28 wherein R ⁹ and R ¹⁰ together represent a =O group can be readily obtained by making compounds of formula 29

or its salt is prepared by reacting with a compound of formula 30 :

Wherein ^X represents a leaving group, such as bromine.

Referring to method (i), examples of specific values for P ²¹ , P ²² and P ²³ are: P ²¹ is hydrogen or benzyl; P ²² is hydrogen or tert-butyldimethylsilyl; P ²³ is hydrogen or benzyl. In this method, the benzyl protecting group is conveniently removed by catalytic hydrogenation in the presence of a Group VIII metal catalyst such as palladium on carbon; Trihydrofluoride treatment is conveniently removed. Compounds of formula 26 can be prepared by the methods described herein, for example by methods (a)-(g).

Referring to method (j), examples of specific values for P ²⁴ , P ²⁵ and P ²⁶ are: P ²⁴ is hydrogen or benzyl; P ²⁵ is hydrogen or tert-butyldimethylsilyl; P ²⁶ is hydrogen or benzyl. In this method, the benzyl protecting group is conveniently removed by catalytic hydrogenation in the presence of a Group VIII metal catalyst such as palladium on carbon; Trihydrofluoride treatment is conveniently removed. Compounds of formula 27 can be prepared by the methods described herein, for example by methods (a)-(g).

Also, wherein R ⁶ and R ⁷ together form the formula -NR ^7g C(O)-CR ^7h R ⁷ⁱ -CR ^7j R ^7k - or -CR ^7l R ^7m -CR ⁷ⁿ R ^7o -C(O)-NR ^7p - Compound I can be prepared by reduction of the corresponding compound of formula I wherein R ⁶ and R ⁷ together form -NR ^7a C(O)-CR ^7b =CR ^7c - or -CR ^7d =CR ^7e -C(O)-NR ^7f - Prepared, for example, by catalytic hydrogenation as described in Example 6 below.

Further details regarding specific reaction conditions and other methods used to prepare representative compounds of the invention or intermediates thereof are described in the Examples below.

Pharmaceutical Compositions and Formulations

The biphenyl derivatives of the present invention are usually administered to patients in the form of pharmaceutical compositions or preparations. Such pharmaceutical compositions may be administered to a patient by any acceptable route of administration, including, but not limited to, inhalational, oral, nasal, topical (including transdermal), and parenteral. It should be understood that any form of the compound of the invention (ie, free base, pharmaceutically acceptable salt, solvate, etc.) suitable for a particular mode of administration may be used in the pharmaceutical compositions discussed herein.

Accordingly, in one of its compositional aspects, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. Optionally, such pharmaceutical compositions may contain other therapeutic and/or formulation substances, if desired.

The pharmaceutical compositions of the invention generally comprise a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. Typically, such pharmaceutical compositions will comprise from about 0.01 to about 95% by weight active agent; including from about 0.01 to about 30% by weight; for example from about 0.01 to about 10% by weight active agent.

Any conventional carrier or excipient may be used in the compositions of the invention. The choice of a particular carrier or excipient or combination of carriers or excipients will depend on the mode of administration or the type of medical condition or disease state being used to treat a particular patient. In this regard, the preparation of suitable pharmaceutical compositions for a particular mode of administration is well within the purview of those skilled in the pharmaceutical arts. In addition, the ingredients used in this composition are commercially available, e.g., from Sigma, P.O. Box 14508, St. Louis, MO 63178. By way of further illustration, general formulation techniques are described in Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott Williams & White, Baltimore, Maryland (2000); and H.C. Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed. , described in Lippincott Williams & White, Baltimore, Maryland (1999).

Representative examples of pharmaceutically acceptable carriers include, but are not limited to, the following: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) Cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; ( 8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers such as magnesium hydroxide (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; (21) Compressed propellant gases, such as chlorofluorocarbons and hydrofluorocarbons; and (22) other non-toxic compatible substances used in pharmaceutical compositions.

The compositions of the invention are generally prepared by thoroughly and intimately mixing or blending a compound of the invention and a pharmaceutically acceptable carrier and one or more optional ingredients. The resulting homogeneously mixed mixture can be shaped or loaded, if necessary or desired, into tablets, capsules, pills, jars, cartridges, etc., using conventional methods and equipment.

In one embodiment, the pharmaceutical composition of the invention is suitable for administration by inhalation. Suitable pharmaceutical compositions for administration by inhalation are usually in aerosol or powder form. Such compositions are usually administered using well-known delivery devices, such as nebulizer inhalers, metered dose inhalers (MDI), dry powder inhalers (DPI) or similar delivery devices.

In a particular embodiment of the invention, the pharmaceutical composition comprising the active agent is administered by inhalation using a nebuliser inhaler. Such nebulizer devices typically generate a high velocity gas flow which causes the pharmaceutical composition comprising the active agent to be sprayed into the patient's respiratory tract as a mist. Thus, when prepared for use in a nebulizer inhaler, the active agent will generally be dissolved in a suitable carrier to form a solution. Alternatively, the active agent may be micronized and combined with a suitable carrier to form a suspension of micronized particles of respirable size, micronization generally being defined as about 90% or more of the particles having a diameter of less than about 10 μm. Suitable nebulizer devices are commercially available, eg from PARI GmbH (Starnberg, Germany). Other nebulizers include the Respimat (Boehringer Ingelheim) and those disclosed, for example, in U.S. Patent No. 6,123,068 and WO 97/12687.

Representative pharmaceutical compositions for use in nebulizer inhalers include isotonic formulations comprising from about 0.05 μg/mL to about 10 mg/mL of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof or a stereoisomer thereof. aqueous solution.

In another particular embodiment of the invention, the pharmaceutical composition comprising the active agent is administered by inhalation using a dry powder inhaler. Such dry powder inhalers typically administer the active agent in the form of a free-flowing powder that is dispersed in the patient's airstream during inhalation. The active agent is usually formulated with suitable excipients such as lactose or starch in order to obtain a free-flowing powder.

Examples of representative pharmaceutical compositions for dry powder inhalers include dry lactose having a particle size between about 1 μm and about 100 μm, and a compound of formula I or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. Micronized particles.

Such dry powder formulations can be prepared, for example, by admixing lactose and the active agent and then dry blending the ingredients. Alternatively, the active agents can be formulated without excipients, if desired. The pharmaceutical composition is then typically filled into a dry powder dispenser, or into an inhalation cartridge or capsule for use with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler (GlaxoSmithKline, Research Triangle Park, NC) (see, for example, U.S. Patent No. 5,035,237); Diskus (GlaxoSmithKline) (see, for example, U.S. Patent No. 6,378,519; (see, for example, U.S. Patent No. 4,524,769); Rotahaler (GlaxoSmithKline) (see, for example, U.S. Patent No. 4,353,365) and Handihaler (Boehringer Ingelheim). Additional examples of suitable DPI devices are in U.S. Patent Nos. 5,415,162, 5,239,993 and 5,715,810, among others Described in the cited references.

In yet another particular embodiment of the present invention, the pharmaceutical composition comprising the active agent is administered by inhalation using a metered dose inhaler. Such metered dose inhalers typically utilize compressed propellant gas to deliver a measured amount of the active agent, or a pharmaceutically acceptable salt thereof. Accordingly, pharmaceutical compositions administered by metered dose inhalers generally comprise solutions or suspensions in liquefied propellants. Any suitable liquefied propellant may be used, including chlorofluorocarbons such as _CCl3F and hydrofluoroalkanes (HFAs) such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1, 1,2,3,3,3-Heptafluoro-n-propane, (HFA 227). Formulations comprising HFAs are generally preferred due to the involvement of chlorofluorocarbons which affect the ozone layer. Additional optional ingredients for HFA formulations include co-solvents, such as ethanol or pentane, and surfactants, such as sorbitan trioleate, oleic acid, lecithin, and glycerin. See, eg, US Patent No. 5,225,183, EP 0717987 A2 and WO92/22286.

A representative pharmaceutical composition for a metered dose inhaler comprises from about 0.01% to about 5% by weight of a compound of formula I or a pharmaceutically acceptable salt or solvate or stereoisomer thereof; from about 0% to about 2% ethanol by weight; and from about 0% to about 5% by weight surfactant; the remainder being HFA propellant.

Such compositions are generally prepared by adding ice-cold or pressurized hydrofluoroalkane to a suitable vessel containing the active agent, ethanol, if present, and surfactant, if present. To prepare suspensions, the active agent is micronized and mixed with a propellant. The formulation is then filled into an aerosol can, which forms part of a metered dose inhaler device. Examples of metered dose inhaler devices developed specifically for use with HFA propellants are provided in US Patent Nos. 6,006,745 and 6,143,277. Alternatively the suspension formulation can be prepared by spray drying a coating of surfactant on micronized particles of active agent. See for example WO 99/53901 and WO 00/61108.

For other examples of methods and formulations for preparing inhalable particles and devices suitable for administration by inhalation see U.S. Pat.

In another embodiment, the pharmaceutical composition of the present invention is suitable for oral administration. Suitable pharmaceutical compositions for oral administration may be in the form of capsules, tablets, pills, lozenges, cachets, dragees, powders, granules; or as solutions or suspensions in aqueous or anhydrous liquids or as an oil-in-water or water-in-oil emulsion; or as an elixir or syrup, etc.; each containing a predetermined amount of the compound of the invention as the active ingredient.

When intended for oral administration in solid dosage form (i.e., as a capsule, tablet, pill, etc.), the pharmaceutical compositions of the invention will generally comprise, as the active ingredient, a compound of the invention and one or more pharmaceutically acceptable Carriers such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also contain: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as Carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants such as glycerin; (4) disintegrants such as agar-agar, calcium carbonate, potato or tapioca Starch, alginic acid, specific silicates and/or sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and/or or glyceryl monostearate; (8) absorbents such as kaolin and/or bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl sulfate sodium and/or mixtures thereof; (10) coloring agents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the present invention. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbic acid palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as Citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Coatings for tablets, capsules, pills, etc. include those used for enteric coatings such as cellulose acetate peptide (CAP), polyvinyl acetate phthalate (PVAP), hypromellose Phthalates, methacrylic acid-methacrylate copolymer, cellulose acetate-1,2,4-trimesate (CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropylmethyl Cellulose acetate succinate (HPMCAS), etc.

The pharmaceutical compositions of the present invention may also be formulated, if desired, to provide slow or controlled release of the active ingredient, by way of example, using hydroxypropylmethylcellulose in varying proportions; or other polymer matrices, liposomes and/or microparticles. ball.

Furthermore, the pharmaceutical compositions of the present invention may optionally contain opacifying agents and may be formulated so that they release the active ingredients only or preferentially, in a certain part of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that may be employed include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Suitable liquid dosage forms for oral administration include, by way of example, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Such liquid dosage forms usually contain the active ingredient and an inert diluent, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-Butanediol, fatty acid esters of oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofuran alcohol, polyethylene glycol, and sorbitan and their mixtures. Suspensions may contain, in addition to the active ingredient, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar- Agar and tragacanth and mixtures thereof.

When intended for oral administration, the pharmaceutical compositions of the invention are preferably packaged in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable for administration to a patient, ie each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic effect alone or in combination with one or more additional units. For example, such unit dosage forms can be capsules, tablets, pills, and the like.

The compounds of the present invention can also be administered transdermally using known transdermal delivery systems and excipients. For example, a compound of the invention can be mixed with a penetration enhancer, such as propylene glycol, polyethylene glycol monolaurate, azacycloalkan-2-one, and the like, and incorporated into a patch or similar delivery system. Additional excipients including gelling agents, emulsifying agents and buffering agents may, if desired, be used in such transdermal compositions.

The pharmaceutical composition of the present invention may also contain other therapeutic agents co-administered with the compound of formula I, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. For example, the pharmaceutical composition of the present invention may further comprise one or more selected from other bronchodilators (such as PDE ₃ inhibitors, adenosine 2b modulators and _β2 adrenoceptor agonists); anti-inflammatory agents (such as steroidal anti-inflammatory agents such as corticosteroids; non-steroidal anti-inflammatory agents (NSAIDs) and PDE ₄ inhibitors); other muscarinic receptor antagonists (i.e. anticholinergics); anti-infective agents (e.g. gram-positive and gram-negative antibiotics or antivirals); antihistamines; protease inhibitors; and afferent blockers such as _D2 agonists and neurokinin modulators. Other therapeutic agents may be used in the form of pharmaceutically acceptable salts or solvates. Furthermore, other therapeutic agents may be used as optically pure stereoisomers, if appropriate.

Representative beta2-adrenoceptor agonists (in addition to the compounds of the present invention) that can be used in combination with the compounds of the present invention include, but are not limited to, salmeterol, salbutamol, formoterol, salmethanol, fenote Luo, terbutaline, salbutamol, isotaline, orcinarin, bitoterol, pirbuterol, levalbuterol, etc., or their pharmaceutically acceptable salts. Other _β2 adrenoceptor agonists that may be used in combination with the compounds of the present invention include, but are not limited to, 3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-( Hydroxymethyl)-phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamide and 3-(3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy -3-(Hydroxymethyl)phenyl]ethyl}-amino)heptyl]oxy}-propyl)benzenesulfonamide and related compounds disclosed in WO02/066422 published on August 29, 2002; 3- [3-(4-{[6-([(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl]amino)hexyl]oxy}butyl)- Phenyl]imidazolidine-2,4-dione and related compounds disclosed in WO 02/070490 published on September 12, 2002; 3-(4-{[6-({(2R)-2-[ 3-(Formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide; 3-(4-{[6-({(2S)- 2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide, 3-(4-{[6-({( 2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide, N-(tert-butyl) -3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxygenbutyl) Benzenesulfonamide, N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl }amino)-hexyl]oxy}butyl)-benzenesulfonamide, N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formyl Amino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide and related compounds disclosed in WO02/076933 published on October 3, 2002; 4- {(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl) Phenol and related compounds disclosed in WO03/024439 published March 27, 2003; and their pharmaceutically acceptable salts. When used, the _β2 -adrenoceptor agonist will be present in the pharmaceutical composition in a therapeutically effective amount. Typically, the _β2 -adrenoceptor agonist is present in an amount sufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used in combination with the compounds of this invention include, but are not limited to, methylprednisolone, prednisolone, dexamethasone, fluticasone propionate, 6,9-difluoro- 17-[(2-furylcarbonyl)oxy]-11-hydroxy-16-methyl-3-oxandrost-1,4-diene-17-thiohydroxy acid S-fluoromethyl ester, 6, 9-Difluoro-11-hydroxy-16-methyl-3-oxo-17-propionyloxy-androst-1,4-diene-17-thiocarboxylic acid S-(2-oxo-tetrahydrofuran- 3S-yl) esters, beclomethasone esters (eg, 17-propionate or 17,21-dipropionate), budesonide, flunisolide, mometasone esters (eg, furoate), triamcinolone , rofluconide, ciclesonide, buticot propionate, RPR-106541, ST-126, etc., or their pharmaceutically acceptable salts. When used, the steroidal anti-inflammatory agent will be present in the pharmaceutical composition in a therapeutically effective amount. Typically, the steroidal anti-inflammatory agent will be present in an amount sufficient to provide from about 0.05 μg to about 500 μg per dose.

Other suitable combinations include, for example, other anti-inflammatory agents such as NSAIDs (e.g. cromolyn sodium; nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors) ); leukotriene antagonists (such as 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 (e.g. adenosine 2a agonists); cytokine antagonists (e.g. chemokine antagonists such as interleukin antibodies (IL antibodies), in particular, IL-4 therapy, IL-13 therapy or a combination thereof); or an inhibitor of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors or mixed PDE3/PDE4 inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, cis-4-cyano-4-(3- Cyclopentyloxy-4-methoxyphenyl)cyclohexane-1-carboxylic acid, 2-methoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethane oxyphenyl)cyclohexane-1-one; cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexane-1-ol ]; cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid, etc., or their pharmaceutically acceptable salts. Other representative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281 (elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough); CI-1018 or PD-168787 ( Pfizer); Benzodioxole compounds disclosed in WO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp); Roflumilast (Byk-Gulden); Pthalazinone compound disclosed in WO99/47505 (Byk-Gulden); Promafentrine (Byk-Gulden, now Altana); Aloephylline (Almirall-Prodesfarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku) and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents) that can be used in combination with the compounds of the present invention (in addition to the compounds of the present invention) include, but are not limited to, atropine, atropine sulfate, oxytropine, atropine methylnitrate , Homatropine hydrobromide, hyoscyamine hydrobromide (d, l), scopolamine hydrobromide, ipratropium bromide, oxitropium bromide, tiotropium bromide, methylamine bromide, propantheline bromide, cintropine methyl bromide, clidinium bromide, copyrrolate (glycopyrrolate), isopropyl iodide, mepentyl bromide, triethylhexylanilinium chloride (Pathilone), thiocyclonium, hydrochloric acid Cyclopentone, tropicamide, trihexyphenidyl hydrochloride, pirenzepine, tirenzepine, AF-DX 116, and mesotramine, etc., or their pharmaceutically acceptable salts; or, those compounds listed as salts, their alternative pharmaceutically acceptable salts.

Representative antihistamines (i.e., H1-receptor antagonists) that may be used in combination with the compounds of this invention include, but are not limited to, ethanolamines such as carbinoxamine maleate, clemastine fumarate, benzene Dimenhydrinate and dimenhydrinate; ethylenediamines, such as mepyramine maleate (amleate), benzylpyramine hydrochloride, and benzhydrin citrate; alkylamines, such as chlorpheniramine and avalan statins; piperazines such as hydroxyzine hydrochloride, hydroxyzine pamoate, phenmeclizine hydrochloride, cyclizine lactate, meclizine hydrochloride, and cetirizine hydrochloride; piperidines such as astemizole, Levocabastine hydrochloride, loratadine or its descarboethoxy analogs, terfenadine and fexonadine hydrochloride; azelastine hydrochloride, etc., or their pharmaceutically acceptable salts; Alternatively, those compounds are listed as salts, their alternative pharmaceutically acceptable salts.

Suitable dosages of other therapeutic agents to be administered in combination with the compounds of this invention range from about 0.05 g per day to about 100 mg per day.

The following formulations illustrate representative pharmaceutical compositions of the invention:

Formulation Example A

Dry powders for administration by inhalation are prepared as follows:

Ingredients

Compound of the present invention 0.2mg

Lactose 25mg

Representative method: the compound of the present invention is micronized and mixed with lactose. This blended mixture is filled into gelatin inhalation cartridges. Administer the contents of the cartridge with a powder inhaler.

Formulation Example B

Dry powder formulations for use in dry powder inhalation devices are prepared as follows:

A representative method: prepare a pharmaceutical composition in which the ratio of the micronized compound of the present invention and the mixed preparation of lactose is 1:200. The composition is filled into a dry powder inhaler device capable of delivering about 10 μg to 100 μg of a compound of the invention per dose.

Formulation Example C

A dry powder for administration by inhalation in a metered dose inhaler is prepared as follows:

A representative method: a compound containing 5 wt% of the compound of the invention and 0.1 Suspension of wt% lecithin. The suspension was spray dried and the resulting material was micronized to particles with an average diameter of less than 1.5 μm. The granules are loaded into a cartridge with pressurized 1,1,1,2-tetrafluoroethane.

Formulation Example D

Pharmaceutical compositions for metered dose inhalers are prepared as follows:

Representative method: prepared by dispersing 5 g of the active ingredient in the form of micronized particles with an average particle size of less than 10 μm in a colloidal solution formed by dissolving 0.5 g of trehalose and 0.5 g of lecithin in 100 mL of demineralized water containing 5% Suspension of the compound of the present invention, 0.5% lecithin and 0.5% trehalose. The suspension was spray dried and the resulting material was micronized to particles with an average diameter of less than 1.5 μm. The pellets were loaded into a tank with pressurized 1,1,1,2-tetrafluoroethane.

Formulation Example E

A pharmaceutical composition for use in a nebulizer inhaler is prepared as follows:

Representative method : Aqueous aerosol formulations for nebulizers are prepared by dissolving 0.1 mg of a compound of the invention in 1 mL of 0.9% sodium chloride solution acidified with citric acid. Stir and sonicate the mixture until the active ingredient is dissolved. The pH of the solution was adjusted to a value in the range of 3-8 by slow dropwise addition of NaOH.

Formulation Example F

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients

Compound of the present invention 250mg

Lactose (spray dried) 200mg

Magnesium Stearate 10mg

Representative method : The ingredients are mixed thoroughly and filled into hard gelatin capsules (460 mg of composition per capsule).

Formulation Example G

Suspensions for oral administration are prepared as follows:

Ingredients

Compound of the present invention 1.0g

Fumaric acid 0.5g

Sodium chloride 2.0g

Methylparaben 0.15g

Propylparaben 0.05g

Granulated sugar 25.5g

Sorbitol (70% solution) 12.85g

Veegum k (Vanderbilt Co.) 1.0g

Flavoring agent 0.035mL

Coloring agent 0.5mg

Distilled water Appropriate amount to 100mL

Representative method : The ingredients are mixed to form a suspension containing 100 mg of active ingredient per 10 mL of suspension.

Formulation Example H

Injectable formulations are prepared as follows:

Ingredients

Compound of the present invention 0.2g

Sodium acetate buffer solution (0.4M) 2.0mL

HCl (0.5N) or NaOH (0.5N) appropriate to pH4

Water (distilled, sterile) Appropriate to 20mL

Representative method : Mix the above ingredients and adjust the pH to 4±0.5 with 0.5N HCl or 0.5N NaOH.

Practicality

Biphenyl derivatives of the present invention have both activities of _β2 adrenergic receptor agonists and muscarinic receptor antagonists, therefore, this compound is suitable for the treatment of receptors induced by _β2 adrenergic receptors or muscarinic receptors. Body-mediated medical conditions, ie, medical conditions that can be alleviated by the use of _β2 adrenergic receptor agonists or muscarinic receptor antagonists. Such medical conditions include, by way of example, pulmonary disorders or diseases associated with reversible airway obstruction, such as chronic obstructive pulmonary disease (eg, chronic and wheezing bronchitis and emphysema), asthma, pulmonary fibrosis, and the like. Other conditions that can be treated include premature birth, depression, congestive heart failure, skin disorders (e.g., inflammatory, allergic, psoriasis, and proliferative skin diseases), conditions in which lowering of digestive acidity is desirable (e.g., peptic and gastric ulcers ) and muscular dystrophy.

Accordingly, in one embodiment, the present invention is directed to a method of treating a pulmonary disease comprising administering to a patient in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate or stereoisomer thereof. When used in the treatment of pulmonary disease, the compounds of the invention are generally administered by inhalation in multiple daily doses, single daily doses or single weekly doses. Typically, dosages for the treatment of pulmonary disease range from about 10 μg/day to about 200 μg/day.

When administered by inhalation, the compounds of the invention generally have a bronchodilation effect. Accordingly, in another aspect of its method aspects, the present invention is directed to a method of providing bronchodilation in a patient, the method comprising administering to a patient in need of bronchodilation a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvent thereof compounds or stereoisomers. Typically, doses to provide bronchodilation will range from about 10 μg/day to about 200 μg/day.

In one embodiment, the present invention relates to a method of treating chronic obstructive pulmonary disease or asthma, the method comprising administering a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt or solvate or stereoisomer thereof to a patient in need thereof . When used to treat COPD or asthma, the compounds of the invention will generally be administered by inhalation in multiple daily doses or in a single daily dose. Typically, dosages for the treatment of COPD or asthma will be in the range of 10 μg/day to 200 μg/day.

As used herein, COPD includes chronic obstructive bronchitis and emphysema (see eg Barnes, Chronic Obstructive Pulmonary disease, N Engl J Med 2000:343:269-78).

When used to treat pulmonary disease, the compounds of the invention are optionally administered in combination with other therapeutic agents. Especially in combination with a compound of the invention and a steroidal anti-inflammatory agent such as a corticosteroid, the pharmaceutical composition of the invention can provide a triple therapy with only two active ingredients, namely a _β2 adrenergic receptor agonist, a muscarinic Receptor antagonist and anti-inflammatory activity. Since pharmaceutical compositions comprising two active ingredients are generally easier to prepare than compositions comprising three active ingredients, such two-component compositions are significantly preferred over compositions comprising three active ingredients. Accordingly, in certain embodiments, the pharmaceutical compositions and methods of the invention further comprise a therapeutically effective amount of a steroidal anti-inflammatory agent.

The compounds of the present invention exhibit both muscarinic receptor antagonist and _β2 adrenergic receptor agonist activity. Therefore, among other properties, compounds of particular interest are those that demonstrate an inhibition constant _K value at _M3 muscarinic receptor binding and an _EC50 value of _β2 adrenergic receptor agonist activity of less than about 100 nM; especially Those less than 10 nM. In addition to these compounds, compounds of particular interest include muscarinic activity expressed with an inhibitory constant _K at _M3 muscarinic receptor binding approximately equal to that expressed with half the in vitro assay described herein or in a similar assay. The _EC50 of the maximal effective concentration expresses that of the _β2 adrenergic agonist activity of the compound. For example, compounds of particular interest are those in which the ratio of the inhibition constant Ki for binding to the _M3 muscarinic receptor to the _EC50 for the _β2 adrenergic receptor is from about 30:1 to about 1:30; including about 20:1 to about 1:20; for example those of about 10:1 to about 1:10.

In one aspect of its method aspects, the present invention also provides a method of treating a pulmonary disease comprising administering to a patient in need of treatment a therapeutically effective amount of a combination of a muscarinic receptor antagonist and a _β2 adrenergic receptor agonist. active compound. In particular embodiments of this method, the administered compound has an _M3 muscarinic receptor inhibition constant K1 _of less than about 100 nM and an _EC50 for _β2 adrenergic receptor agonism of less than about 100 nM. In another embodiment, the method of treating a pulmonary disease comprises administering a therapeutically effective amount of a compound having a ratio of the inhibitory constant _K for _M3 muscarinic receptors to the _EC50 for _beta2 adrenergic receptor agonism of about 30:1 to about 1:30.

Since the compounds of the present invention have both _β2 adrenergic agonist activity and muscarinic receptor antagonist activity, such compounds are also suitable for investigation or research of compounds with _β2 adrenergic receptors or muscarinic receptors. As a research tool for biological systems or samples, or as a research tool for discovering new compounds having both _β2 adrenergic receptor agonist activity and muscarinic receptor antagonist activity. Such a biological system or sample may comprise _β2 adrenergic receptors and/or muscarinic receptors. Any suitable biological system or sample having _β2 adrenergic and/or muscarinic receptors can be used in such studies, which can be performed in vitro or in vivo. Representative biological systems or samples suitable for such studies include, but are not limited to, cells, cell extracts, plasma membranes, tissue samples, mammals (e.g., mice, rats, guinea pigs, rabbits, dogs, pigs, etc.) wait.

测量)。本发明化合物将激动或引起β₂肾上腺素能受体的活化并在上述列出的任何功能试验或类似性质的试验中拮抗或减少毒蕈碱受体的活化。在这些研究中采用的化合物的量将通常在约0.1纳摩尔至约100纳摩尔的范围内。In this embodiment, a biological system or sample comprising a _β2 adrenergic receptor or a muscarinic receptor is treated with a _β2 adrenergic receptor agonistic or muscarinic receptor antagonistic amount of a compound of the invention touch. Effects are then determined using conventional methods and equipment, such as radioligand binding assays and functional assays. Such functional assays include ligand-mediated changes in intracellular cyclic adenosine monophosphate (cAMP), ligand-mediated changes in the activity of the enzyme adenylyl cyclase (which synthesizes cAMP), ligand-mediated Body-catalyzed exchange of [ ³⁵ S]GTPS for GDP introduces guanosine 5′-O-(-thio)triphosphate ([ ³⁵ S]GTPS) into isolated membrane changes, ligand-mediated free cell Intrinsic calcium ion changes (e.g. with Fluorescence Integrated Imaging Plate Reader or FLIPR from Molecular Devices, Inc.

Measurement). Compounds of the invention will agonize or cause activation of _β2 adrenergic receptors and antagonize or reduce activation of muscarinic receptors in any of the functional assays listed above or assays of a similar nature. The amount of compound employed in these studies will generally range from about 0.1 nanomolar to about 100 nanomolar.

In addition, the compounds of the present invention can be used as research tools for the discovery of new compounds having both _β2 adrenergic receptor agonist and muscarinic receptor antagonist activity. In this embodiment, _β2 adrenergic receptor and muscarinic receptor binding data (e.g., determined by in vitro radioligand displacement assays) for a test compound or group of test compounds are compared to _β2 receptor binding data for compounds of the invention. Adrenergic receptor and muscarinic receptor binding data are compared to identify those test compounds that have about the same or better binding to _β2 adrenergic receptors and/or muscarinic receptors, if any if. This aspect of the invention includes, as separate embodiments, the generation of comparative data (using appropriate assays) and analysis of assay data to identify test compounds of interest.

In certain instances, the compounds of the invention may possess weak muscarinic receptor antagonist activity or weak _β2 adrenergic receptor agonist activity. In these cases, those of ordinary skill in the art will recognize that such compounds still have utility primarily as _β2 adrenergic receptor agonists or muscarinic receptor antagonists, respectively.

The properties and utility of the compounds of the present invention can be demonstrated using various in vitro and in vivo assays well known to those skilled in the art. For example, representative assays are described in further detail in the Examples below.

Example

The following Preparations and Examples are provided to illustrate specific embodiments of the invention. However, unless otherwise indicated, these particular embodiments do not limit the scope of the invention in any way.

Unless otherwise indicated, the following abbreviations have the following meanings, and any other abbreviations used herein that are not defined have their standard meanings:

AC adenylyl cyclase

Acetylcholine

ATCC American Type Culture Collection

BSA bovine serum albumin

cAMP3′-5′cyclic adenosine monophosphate

CHO Chinese Hamster Ovary

chimpanzee M ₅ receptor of cM ₅ clone

DCM dichloromethane (i.e. methylene chloride)

DIPEAN, N-Diisopropylethylamine

dPBS Dulbecco's Phosphate Buffered Saline

DMEM Dulbecco's Modified Eagle's Medium

DMSO dimethyl sulfoxide

EDTA ethylenediaminetetraacetic acid

Emax maximum performance

EtOAc ethyl acetate

EtOH ethanol

FBS Fetal Bovine Serum

FLIPR Fluorometric Imaging Plate Reader

Gly glycine

HATU O-(7-Azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HBSS Hank's buffered saline solution

HEK human embryonic kidney cells

HEPES4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

Human _M1 receptor of _hM1 clone

Human _M2 receptor of _hM2 clone

Human _M3 receptor of _hM3 clone

Human _M4 receptor of _hM4 clone

Human _M5 receptor of _hM5 clone

HPLC High Performance Liquid Chromatography

IBMX 3-isobutyl-1-methylxanthine

%Eff Percentage efficacy

PBS Phosphate Buffered Saline

PyBOP Benzotriazol-1-yloxytripyrrolidinylphosphonium hexafluorophosphate

rpm revolutions per minute

TFA Trifluoroacetic acid

THF Tetrahydrofuran

Tris Tris(hydroxymethyl)aminomethane

Unless otherwise noted, reagents, starting materials and solvents were purchased from commercial suppliers (eg Aldrich, Fluka, Sigma, etc.) and used without further purification.

In the following examples, HPLC analysis was performed using an Agilent (Palo Alto, CA) Series 1100 instrument with a Zorbax Bonus RP 2.1 x 50 mm column (C14 column) supplied by Agilent with a particle size of 3.5 microns. Detection is by UV absorption at 214nm. HPLC 10-70 data was acquired with a flow rate of 0.5 mL/min 10%-70% B over 6 minutes. Mobile phase A is 2%-98%-0.1% ACN-water-TFA; mobile phase B is 90%-10%-0.1% CAN-water-TFA. Using mobile phases A and B above, HPLC 5-35 data and HPLC 10-90 data were acquired with a 5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were acquired with an Applied Biosystems (Foster City, CA) model API-150EX instrument. LCMS 10-90 data were acquired with a 10%-90% mobile phase B over a 5 min gradient.

Small scale purifications were performed using the API 150EX Prep Workstation system from Applied Biosystems. The mobile phases were A: water + 0.05% v/v TFA; and B: acetonitrile + 0.05% v/v TFA. For alignment (typically about 3-50 mg recovered sample size) the following conditions were used: 20 mL/min flow rate; 15 min gradient and 20 mm x 50 mm Prism RP column (Thermo Hypersil-Keystone, Bellefonte, PA) with 5 micron particles. For larger scale purifications (typically greater than 100 mg crude sample), the following conditions were used: 60 ml/min flow rate; 30 min gradient and 41.4 mm x 250 mm Microsorb BDS column (Varian, Palo Alto, CA) with 10 micron particles.

The specific rotation (called [α] ²⁰ _D ) of chiral compounds was measured at 20° C. using a Jasco polarimeter (model P-1010) with a tungsten-halogen light source and a 589 nm filter.

preparation 1

N-1,1'-biphenyl-2-yl-N'-4-(1-benzyl)piperidinyl urea

Biphenyl-2-isocyanate (50 g, 256 mmol) was dissolved in acetonitrile (400 mL) at room temperature. After cooling to 0°C, a solution of 4-amino-N-benzylpiperidine (48.8 g, 256 mmol) in acetonitrile (400 mL) was added over 5 minutes. Precipitation was observed immediately. After 15 minutes, acetonitrile (600 mL) was added and the resulting viscous mixture was stirred at 35 °C for 12 h. The solid was then filtered off and washed with cold acetonitrile, then dried in vacuo to afford the title compound (100 g, 98% yield). MS m/ _z for _C25H27N3O : [M+H ⁺ ] calcd. 386.22; found 386.3 _.

preparation 2

N-1,1'-biphenyl-2-yl-N'-4-piperidinyl urea

The product from Preparation 1 (20 g, 52 mmol) was dissolved in a mixture of anhydrous methanol and anhydrous DMF (3:1, 800 mL). Aqueous hydrochloric acid (0.75 mL of a 37% concentrated solution, 7.6 mmol) was added to the solution and nitrogen was bubbled vigorously for 20 minutes. Pearlman's catalyst (Pd(OH) ₂ , 5 g) was added under nitrogen flow before placing the reaction mixture under a hydrogen atmosphere (balloon). The reaction mixture was stirred for 4 days, then passed twice through a pad of celite to remove the catalyst. The solvent was then removed under reduced pressure to afford the title compound (13 g, 85% yield). MS m/ _z for _C18H21N3O : [M+H ⁺ ] calcd. 296.17; found 296.0 _.

Alternatively, N-1,1'-N-1,1'- Biphenyl-2-yl-N'-4-piperidinylurea (reaction monitored by LCMS). The reaction mixture was cooled to 50°C, and ethanol (500 mL) was added, followed by the slow addition of 6M hydrochloric acid (95 mL).

The reaction mixture was cooled to room temperature. Ammonium formate (48.4 g, 768 mmol) was added to the reaction mixture prior to the addition of palladium (10 wt. % (dry basis) on activated carbon) (10 g) and nitrogen was bubbled vigorously through the solution for 20 minutes. The reaction mixture was heated at 40 °C for 12 h, then filtered through a pad of celite and the solvent was removed under reduced pressure. To the crude residue was added 1M hydrochloric acid (20 mL) and 10N sodium hydroxide to adjust the pH to 12. The aqueous layer was extracted with ethyl acetate (2 x 80 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure to afford the title compound as a solid (71.7 g, 95% yield). MS m/ _z for _C18H21N3O : [M+H ⁺ ] calcd. 296.17; found 296.0 _.

preparation 3

N-1,1'-biphenyl-2-yl-N'-4-[1-(9-hydroxynonyl)]piperidinyl urea

9-Bromo-1-nonanol (4.84 g, 21.7 mmol) was added to the product of Preparation 2 (5.8 g, 19.7 mmol) and diisopropylethylamine (10.29 mL, 59.1 mmol) in acetonitrile (99 mL ) in a stirred solution. The reaction mixture was heated at 50 °C for 8 h. The reaction mixture was then cooled and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (100 mL), washed with saturated aqueous sodium bicarbonate (2 x 50 mL) and dried (magnesium sulfate). The solvent was removed under reduced pressure. The crude product was purified by flash chromatography (dichloromethane:methanol:ammonia system) to afford the title compound (7.1 g, 16.2 mmol, 82% yield).

preparation 4

N-1,1'-biphenyl-2-yl-N'-4-[1-(9-oxononyl)]piperidinyl urea

Dimethylsulfoxide (490 μL, 6.9 mmol) followed by diisopropylethylamine (324 μL, 3.45 mmol) was added to the product of Preparation 3 (500 mg, 1.15 mmol) in dichloro solution in methane (11.5 mL). The reaction mixture was stirred at -15°C for 15 minutes, then sulfur trioxide pyridine complex (549 mg, 3.45 mmol) was added portionwise. The reaction mixture was stirred at -15°C for 1 h, then water (10 mL) was added. The organic phase was then separated, washed with water (10 mL), and dried (sodium sulfate). The solvent was removed under reduced pressure to obtain the title compound (475 mg, 1.09 mmol, 95% yield). HPLC (10-70) Rt = 3.39.

Preparation 5

N-1,1'-biphenyl-2-yl-N'-4-[1-(9-aminononyl)]piperidinyl urea

Palladium (10 wt.% (dry basis) on activated carbon) (1.5 g) was added to the product of Preparation 4 (1.58 g, 3.63 mmol) and benzylamine (516 μL, 4.72 mmol) in methanol (36.3 mL) in the stirred solution. The reaction mixture was placed under a hydrogen atmosphere. After stirring for 12 h, the reaction mixture was filtered through a pad of celite and washed with methanol (10 mL). The solvent was removed under reduced pressure to obtain the title compound (1.50 g, 3.45 mmol, 95% yield). HPLC (10-70 _{) Rt} = 2.35; MS _m / _z for _C27H40N4O1 : [M+H ⁺ ] calcd. 437.06; found 437.5.

Preparation 6

8-Benzyloxy-5-(2,2-dihydroxyacetyl)-1H-quinolin-2-one

(a) 8-Acetoxy-1H-quinolin-2-one

8-Hydroxyquinoline-N-oxide (160.0 g, 1.0 mol) and acetic anhydride (800 mL, 8.4 mol), commercially available from Aldrich, Milwaukee, WI, were heated at 100° C. for 3 h, then cooled in ice. The product was collected on a Buchner funnel, washed with acetic anhydride (2 x 100 mL) and dried under reduced pressure to give 8-acetoxy-1 H-quinolin-2-one (144 g) as a solid.

(b) 5-acetyl-8-hydroxy-1H-quinolin-2-one

A slurry of aluminum chloride (85.7 g, 640 mmol) in 1,2-dichloroethane (280 mL) was cooled in ice and the product of step (a) (56.8 g, 280 mmol) was added. The mixture was warmed to room temperature and then heated at 85°C. After 30 minutes, acetyl chloride (1.5 mL, 21 mmol) was added and the mixture was heated for an additional 60 minutes. The reaction mixture was then cooled and added to 1N hydrochloric acid (3 L) at 0°C with good stirring. After stirring for 2 h, the solid was collected on a Buchner funnel, washed with water (3 x 250 mL) and dried under reduced pressure. The crude product (135 g) isolated from multiple batches was combined and triturated with dichloromethane (4 L) for 6 h. The product was collected on a Buchner funnel and dried under reduced pressure to give 5-acetyl-8-hydroxy-2(1H)-quinolinone (121 g).

(c) 5-acetyl-8-benzyloxy-1H-quinolin-2-one

To the product of step (b) (37.7 g, 186 mmol) was added N,N-dimethylformamide (200 mL) and potassium carbonate (34.5 g, 250 mmol), followed by benzyl bromide (31.8 g, 186 mmol). The mixture was stirred at room temperature for 2.25 hours, then poured into saturated sodium chloride (3.5 L) at 0°C and stirred for 1 hour. The product was collected and dried on a Buchner funnel for 1 hour, the resulting solid was dissolved in dichloromethane (2 L), and the mixture was dried over sodium sulfate. The solution was filtered through a pad of celite and washed with dichloromethane (5 x 200 mL). The combined filtrates were then concentrated to dryness and the resulting solid was triturated with diethyl ether (500 mL) for 2 h. The product was collected on a Buchner funnel, washed with diethyl ether (2 x 250 mL) and dried under reduced pressure to give 5-acetyl-8-benzyloxy-1 H-quinolin-2-one (44 g) as a powder.

(d) 8-benzyloxy-5-(2,2-dihydroxyacetyl)-1H-quinolin-2-one

To a slurry of the product from step (c) (10.0 g, 34.1 mmol) in DMSO (60 mL) was added 48% w/w hydrobromic acid solution (11.8 mL, 102.3 mmol). The mixture was warmed to 60 °C for 16 h, then allowed to cool to room temperature. Water (100 mL) was added and the resulting slurry was stirred at room temperature for 0.5 h before cooling to 0 °C. The product was collected on a Buchner funnel and dried under reduced pressure to give 8-benzyloxy-5-(2,2-dihydroxyacetyl)-1H-quinolin-2-one (12.2 g) as a solid.

Preparation 7

1-(1-{9-[2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-hydroxyethylamino]nonyl}piperidine-4 -yl)-3-biphenyl-2-ylurea

The product of Preparation 5 (183 mg, 0.42 mmol) and the product of Preparation 6 (149 mg, 0.46 mmol) were stirred in dichloroethane (4.2 mL) for 2 h at room temperature. Sodium triacetoxyborohydride (267 mg, 1.26 mmol) was then added and the reaction mixture was stirred for a further 12 h.

The reaction mixture was diluted with dichloromethane (10 mL), washed with saturated aqueous sodium bicarbonate (10 mL), dried (magnesium sulfate), and the solvent was removed under reduced pressure. The crude reaction mixture was purified by flash chromatography (5-10% methanol in dichloromethane, 0.5% ammonium hydroxide) to afford the title compound (144 mg, 0.20 mmol, 48% yield). HPLC (10-70 _{) Rt = 3.48; MS m/z for C45H55N5O4 : [} M+H ⁺ ] calcd. 730.4; found 730.7.

Example 1

1-biphenyl-2-yl-3-(1-{9-[2-hydroxyl-2-(8-hydroxyl-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino ]nonyl}piperidin-4-yl)urea

Palladium (10 wt.% (dry basis) on activated carbon) (63 mg) was added to a stirred solution of the product from Preparation 7 (144 mg, 0.20 mmol) in methanol (2 mL) and the reaction mixture was placed under an atmosphere of hydrogen . After stirring for 12 h, the reaction mixture was filtered through a pad of celite, washed with methanol (2 mL), and the solvent was removed under reduced pressure. The resulting residue was purified by preparative HPLC to give the title compound (10 mg). HPLC _{(10-70) Rt = 2.8; MS m/z for C38H49N5O4 : [ M +} H ⁺ ] calcd. 640.3; found 640.5.

Preparation 8

Biphenyl-2-ylpiperidin-4-ylcarbamate

Biphenyl-2-isocyanate (97.5 g, 521 mmol), both purchased from Aldrich, Milwaukee, WI, and 4-hydroxy-1-benzylpiperidine (105 g, 549 mmol) were heated together at 70° C. for 12 h , the formation of biphenyl-2-ylcarbamate-1-benzylpiperidin-4-yl ester during this period was monitored by LCMS. The reaction mixture was then cooled to 50°C and ethanol (1 L) was added followed by the slow addition of 6M hydrochloric acid (191 mL). The reaction mixture was then cooled to room temperature, ammonium formate (98.5 g, 1.56 mol) was added, and nitrogen was bubbled vigorously through the solution for 20 minutes. Palladium (10 wt.% (dry basis) on activated carbon) (20 g) was then added. The reaction mixture was heated at 40 °C for 12 h, then filtered through a pad of celite. The solvent was then removed under reduced pressure and 1M hydrochloric acid (40 mL) was added to the crude residue. Sodium hydroxide (10N) was then added to adjust the pH to 12. The aqueous layer was extracted with ethyl acetate (2 x 150 mL) and dried (magnesium sulfate), then the solvent was removed under reduced pressure to give the title compound (155 g, 100%). HPLC (10-70) _{Rt =} 2.52; MS _m /z for _Ci8H20N2O2 : [M+H ⁺ ] calcd. 297.15; found _297.3 .

Preparation 9

N,N-(di-tert-butoxycarbonyl)-9-bromononylamine

A solution of di-tert-butoxycarbonylamine (3.15 g, 14.5 mmol) in N,N-dimethylformamide (0.28 mL) was cooled to 0° C. for about 10 min. 60% sodium hydride in mineral oil (0.58 g, 14.5 mmol) was added and the reaction mixture was stirred at 0 °C for 10 min. The reaction mixture was removed from the ice bath and allowed to warm to room temperature for about 30 min. The reaction mixture was then cooled back to 0 °C and a solution of 1,9-dibromononane (2.46 mL, 12.1 mmol) in dimethylformamide (100 mL) was added. The reaction mixture was stirred overnight at room temperature. After 24 h, MS analysis showed the reaction was complete. The reaction mixture was concentrated to dryness and diluted with ethyl acetate (100 mL). The organic layer was washed with saturated sodium bicarbonate (2 x 100 mL), brine (100 mL), dried (magnesium sulfate) and concentrated under reduced pressure to give the crude product, which was purified by silica gel chromatography using 5% ethyl acetate in hexanes Crude product, the title compound was obtained. MS m _{/ z for C19H36N1O4Br} : [M+H ⁺ ] calcd. 423.18; found 423.

Prepare 10

1-(9-di-tert-butoxycarbonylamino)nonyl]piperidin-4-ylcarbamate

A mixture of acetonitrile and N,N dimethylformamide (50 mL) was added to the product of Preparation 8 (3.0 g, 10.1 mmol) and the product of Preparation 9 (5.1 g, 12.2 mmol) and triethylamine (1.42 mL, 10.1 mmol) )middle. The reaction mixture was stirred at room temperature for 24 h and monitored by LCMS analysis. The reaction mixture was then concentrated and diluted with ethyl acetate (50 mL). The organic layer was washed with saturated sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic phase was then dried over magnesium sulfate and concentrated to give 6.5 g of a crude oil. The oil was purified by chromatography on silica gel with 1:1 hexane/ethyl acetate to give the title compound (3g). MS m/ _{z for C37H55N3O6} : [M+H ⁺ ] calcd. 638.41; found ₆₃₉ .

Preparation 11

1-(9-aminononyl)piperidin-4-yl biphenyl-2-ylcarbamate

Trifluoroacetic acid (11 mL) was added to a solution of the product from Preparation 10 (7.2 g, 11.3 mmol) in dichloromethane (56 mL). After 12 h, LCMS analysis showed the reaction was complete. The reaction mixture was then concentrated to dryness and diluted with ethyl acetate (75 mL). Sodium hydroxide (1 N) was then added until the pH of the mixture reached 14. The organic phase was then collected and washed with saturated sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic phase was then dried over magnesium sulfate and concentrated to give the title compound (5.5 g). MS m/ _{z for C27H39N3O2} : [M+H ⁺ ] calcd. _438.30 ; found 439.

Preparation 12

Biphenyl-2-ylcarbamate 1-{9-[2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-hydroxyethylamino]nonane yl}piperidin-4-yl ester

The product from Preparation 11 (196 mg, 0.43 mmol) was dissolved in dichloroethane (4 mL) and sodium triacetoxyborohydride (101 mg, 0.48 mmol) was added. The reaction mixture was stirred at room temperature for about 10 min then 8-benzyloxy-5-(2,2-dihydroxyacetyl)-1H-quinolin-2-one (Preparation 6) (141 mg, 0.43 mmol) was added. LCMS analysis showed the reaction was complete after 2h. Methanol (1 mL) was added to the reaction mixture, followed by the slow addition of sodium borohydride (18 mg, 0.48 mmol). After 1 hour, LCMS analysis showed the reaction was complete. The reaction mixture was then quenched with aqueous ammonium chloride, and the mixture was extracted with dichloromethane. The organic phase was washed with saturated sodium bicarbonate (2 x 50 mL) and brine (10 mL). The organic phase was then dried over magnesium sulfate and concentrated to give 315 mg of a yellow solid. The solid was purified by silica gel chromatography with 10% methanol in dichloromethane to give the title compound (64 mg). MS m/ _{z for C43H55N4O5} : [M+H ⁺ ] calcd. 730.40; found ₇₃₁ .

Example 2

Biphenyl-2-ylcarbamate 1-{9-[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]nonyl} Piperidin-4-yl ester ditrifluoroacetate

A solution of the product of Preparation 12 (64 mg, 0.09 mmol) in methanol (450 mL) was bubbled with nitrogen. Palladium on carbon (10%, 10 mg) was then added and the reaction mixture was placed under a balloon containing hydrogen and stirred. LCMS analysis showed the reaction was complete after 9 h. The reaction mixture was then filtered and the filtrate was concentrated to give a yellow brittle solid. The solid was purified by preparative HPLC (5-35 over 60 min) to afford the title compound (19 mg). MS m/ _{z for C38H48N4O5} : [M+H ⁺ ] calcd. 641.36; found ₆₄₁ .

Preparation 13

8-Benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilyloxy)ethyl]-1H-quinolin-2-one

(a) 8-Benzyloxy-5-(2-bromoacetyl)-1H-quinolin-2-one

5-Acetyl-8-benzyloxy-1H-quinolin-2-one (Preparation 6) (20.0 g, 68.2 mmol) was dissolved in dichloromethane (200 mL) and cooled to 0 °C. Boron trifluoride diethyl etherate (10.4 mL, 82.0 mmol) was added via syringe and the mixture was warmed to room temperature to give a thick suspension. The suspension was heated at 45°C (oil bath) and a solution of bromine (11.5 g, 72.0 mmol) in dichloromethane (100 mL) was added over 40 minutes. The mixture was kept at 45 °C for another 15 min and then cooled to room temperature. The mixture was concentrated under reduced pressure, then triturated with 10% aqueous sodium carbonate (200 mL) for 1 hour. The solid was collected on a Buchner funnel, washed with water (4 x 100 mL) and dried under reduced pressure. The two products were combined for purification. The crude product (52 g) was triturated with 50% methanol in chloroform (500 mL) for 1 hour. The product was collected on a Buchner funnel, washed with 50% methanol in chloroform (2 x 50 mL) and methanol (2 x 50 mL). The solid was dried under reduced pressure to give the title compound (34.1 g) as a powder.

(b) 8-benzyloxy-5-((R)-2-bromo-1-hydroxyethyl)-1H-quinolin-2-one

(R)-(+)-α,α-Diphenyl prolinol (30.0 g, 117 mmol) and trimethylboroxane (11.1 mL, 78 mmol) were combined in toluene (300 mL) and stirred at room temperature for 30 min. The mixture was placed in a 150°C oil bath and the liquid was distilled off. Toluene was added in 20 mL aliquots and distillation continued for 4 h. A total of 300 mL of toluene was added. The mixture was then cooled to room temperature. A 500 [mu]L aliquot was evaporated to dryness and weighed (246 mg) to determine that the catalyst concentration was 1.8M.

8-Benzyloxy 5-(2-bromoacetyl)-1H-quinolin-2-one (90.0 g, 243 mmol) was placed under nitrogen, tetrahydrofuran (900 mL) was added followed by the above catalyst (1.8 M in toluene medium, 15mL, 27mmol). The suspension was cooled to -10±5°C in an ice/isopropanol bath. Borane (1.0 M in THF, 294 mL, 294 mmol) was added over 4 h. The reaction was then stirred at -10°C for an additional 45 minutes before methanol (250 mL) was added slowly. The mixture was concentrated in vacuo and the residue was dissolved in boiling acetonitrile (1.3 L), filtered while hot, then cooled to room temperature. The crystals were filtered, washed with acetonitrile and dried in vacuo to afford the title compound (72.5 g, 196 mmol, 81% yield, 95% enantiomeric excess, 95% purity by HPLC).

(c) 8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilyloxy)ethyl]-1H-quinolin-2-one

To the product of step (b) (70.2 g, 189 mmol) was added N,N dimethylformamide (260 mL) and the mixture was cooled in an ice bath under nitrogen. 2,6-Lutidine (40.3 g, 376 mmol) was added over 5 minutes, followed by tert-butyldimethylsilyl trifluoromethanesulfonate (99.8 g, 378 mmol) slowly while maintaining the temperature at 20 °C the following. The mixture was allowed to warm to room temperature for 45 minutes. Methanol (45 mL) was added dropwise to the mixture over 10 minutes, and the mixture was partitioned between ethyl acetate/cyclohexane (1:1, 500 mL) and water/brine (1:1, 500 mL). The organics were washed twice more with water/brine (1:1, 500 mL each). The combined organics were evaporated under reduced pressure to give a light yellow oil. Two separate portions of cyclohexane (400 mL) were added to the oil and distillation continued until a thick white slurry formed. Cyclohexane (300 mL) was added to the slurry, and the obtained white crystals were filtered, washed with cyclohexane (300 mL), and dried under reduced pressure to obtain the title compound (75.4 g, 151 mmol, 80% yield, 98.6% enantiomeric excess).

Preparation 14

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert Butyldimethylsilyloxy)ethylamino]nonyl}piperidin-4-yl ester

The product from Preparation 13 (3.9 g, 8.17 mmol) was added to a solution of the product from Preparation 11 (5.0 g, 11.4 mmol) in THF (20 mL), followed by sodium bicarbonate (2.0 g, 24.5 mmol) and sodium iodide (1.8 g, 12.2 mmol). The reaction mixture was heated to 80 °C for 72 h. The reaction mixture was then cooled, diluted with dichloromethane (20 mL) and the organic phase was washed with saturated sodium bicarbonate (2 x 50 mL) and brine (50 mL). The organic phase was then dried (magnesium sulfate) and concentrated to give 6.5 g of crude product. Chromatography on silica gel eluting with 3% methanol in dichloromethane afforded the title compound (1.4 g, 21% yield).

Preparation 15

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-hydroxyethyl Nylamino]nonyl}piperidin-4-yl ester

Triethylamine hydrofluoride (376 pL, 2.3 mmol) was added to a solution of the product of Preparation 14 (1.3 g, 1.5 mmol) in THF (8 mL), and the reaction mixture was stirred at room temperature. After 5 h, the completion of the reaction was determined by LCMS analysis. The reaction mixture was quenched with 1N NaOH until the pH was 14, then diluted with ethyl acetate (20 mL), washed with 1N NaOH (20 mL) and brine (20 mL). The organic phase was then separated, dried over magnesium sulfate and concentrated to give the title compound (1.1 g).

Example 3

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)ethyl Amino]nonyl}piperidin-4-yl ester ditrifluoroacetate

A solution of the product of Preparation 15 (1.1 g, 1.5 mmol) was purged with nitrogen and palladium on carbon (10%, 110 mg) was added. The reaction mixture was stirred under balloon pressure hydrogen. LCMS analysis showed the reaction was complete after 9 h. The reaction mixture was then filtered and concentrated to give a yellow solid. The solid was purified by preparative HPLC (5-30 over 60 min) to give the title compound (510 mg). MS m/ _{z for C38H48N4O5} : [M+H ⁺ ] calcd. 641.36; found ₆₄₁ . HPLC Methods 10-70: 3.207. [α] ²⁰ _D = -23.6 (c = 1.0 mg/mL, water).

Example 3A

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)ethyl Amino]nonyl}piperidin-4-yl ester ditrifluoroacetate

Alternatively, the title compound was prepared as follows:

(a) 9-bromononanal

To a 100-mL round bottom flask equipped with a magnetic stirrer, addition funnel, and temperature controller was added 9-bromononanol (8.92 g, 40 mmol) and dichloromethane (30 mL) under nitrogen. The resulting mixture was cooled to 5 °C and a solution of sodium bicarbonate (0.47 g, 5.6 mmol) and potassium bromide (0.48 g, 4 mmol) in water (10 mL) was added. Add 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO) (63mg, 0.4mmol), then add 10-13% bleach solution dropwise at a certain rate through the addition funnel ( 27 mL), so that the temperature was maintained at about 8°C (+/- 2°C) with an ice-cold bath (over about 40 min). After the bleach addition was complete, the mixture was stirred for 30 minutes. While maintaining the temperature at about 0°C. A solution of sodium bisulfite (1.54 g) in water (10 mL) was added and the resulting mixture was stirred at room temperature for 30 minutes. The mixture layers were then separated and the milky aqueous layer was extracted with dichloromethane (1 x 20 mL). The combined dichloromethane layers were then washed with water (1 x 30 mL), dried ( _MgSO4 ), filtered and concentrated under reduced pressure to afford the title intermediate (8.3 g, 94% yield), which was used in the next step without further purification .

(b) 9-bromo-1,1-dimethoxynonane

烷(0.2mL，0.8mmol)中的溶液，回流得到的混合物3h。然后将反应混合物冷却至室温并加入固体碳酸氢钠(100mg，1.2mmol)。减压浓缩得到的混合物至其原始体积的四分之一，然后加入乙酸乙酯(50mL)。用水(2×40mL)洗涤有机层，干燥(MgSO₄)，过滤，减压浓缩得到呈液体的标题中间体(8.44g，(产率97％))，其被用于下一步骤而无需进一步纯化。To a 100 mL round bottom flask was added 9-bromononanal (7.2 g, 32.5 mmol), methanol (30 mL) and trimethyl-n-formate (4 mL, 36.5 mmol). Add 4N hydrochloric acid in two

solution in alkanes (0.2 mL, 0.8 mmol), and the resulting mixture was refluxed for 3 h. The reaction mixture was then cooled to room temperature and solid sodium bicarbonate (100 mg, 1.2 mmol) was added. The resulting mixture was concentrated under reduced pressure to a quarter of its original volume, then ethyl acetate (50 mL) was added. The organic layer was washed with water (2 x 40 mL), dried (MgSO ₄ ), filtered, and concentrated under reduced pressure to afford the title intermediate as a liquid (8.44 g, (97% yield)), which was used in the next step without further purification.

(c) Biphenyl-2-ylcarbamate 1-(9,9-dimethoxynonyl)piperidin-4-yl ester

To a 50 mL three necked round bottom flask was added piperidin-4-yl biphenyl-2-ylcarbamate (1 g, 3.38 mmol) and acetonitrile (10 mL) to form a slurry. To this slurry was added 9-bromo-1,1-dimethoxynonane (1.1 g, 1.3 mmol) and triethylamine (0.57 g, 4.1 mmol) and the resulting mixture was heated at 65 °C for 6 h (by HPLC The reaction was monitored until <5% starting material). The reaction mixture was then cooled to room temperature, at which point the mixture formed a thick slurry. Water (5 mL) was added, the mixture was filtered and the solid collected on a coarse fritted glass filter. The solid was washed with a premixed solution of acetonitrile (10 mL) and water (5 mL), then with another premixed solution of acetonitrile (10 mL) and water (2 mL). The resulting solid was air dried to provide the title intermediate (1.37 g, 84%, >96% purity by LC, 1HNMR) as a white solid.

(d) 1-(9-oxononyl)piperidin-4-yl biphenyl-2-ylcarbamate

To a 500 mL round bottom flask with a magnetic stirrer was added 1-(9,9-dimethoxynonyl)piperidin-4-yl biphenyl-2ylcarbamate (7.7 g, 15.9 mmol), Acetonitrile (70 mL) and aqueous 1M hydrochloric acid (70 mL) were then added. The resulting mixture was stirred at room temperature for 1 h, then dichloromethane (200 mL) was added. The mixture was stirred for 15 min, then the layers were separated. The organic layer was dried ( _MgSO4 ), filtered and concentrated under reduced pressure to give the title intermediate (6.8g) which was used in the next step without further purification.

(e) Biphenyl-2-ylcarbamate 1-(9-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline-5- base)-2-(tert-butyldimethylsilyloxy)ethyl]amino}nonyl)-piperidin-4-yl ester

Into a 300 mL round bottom flask was added 5-[(R)-2-benzylamino-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy-1H-quinoline-2- Ketone (5 g, 9.73 mmol), dichloromethane (100 mL) and glacial acetic acid (0.6 mL, 10 mmol). The mixture was cooled to 0° C. using an ice bath, and 1-(9-oxononyl)piperidin-4-yl biphenyl-2-ylcarbamate (4.6 g, 9.73 mmol) was added with stirring. The mixture was stirred at 0°C for 30 minutes, then sodium triacetoxyborohydride (6.15 g, 29 mmol) was added in portions over 15 minutes. The reaction mixture was stirred at 0°C-10°C for 2 hours, then saturated aqueous sodium bicarbonate (50 mL) was added and the mixture was stirred for 15 minutes. The layers were then separated, the organic layer was washed with 5% aqueous sodium chloride (50 mL), dried (MgSO ₄ ), filtered and concentrated under reduced pressure to afford the title intermediate (8.5 g, 80% purity by HPLC), which was used without Further purification.

(f) Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxyl-2-oxo-1,2 -Dihydroquinolin-5-yl)ethylamino]nonyl}piperidin-4-yl ester

To a 200 mL round bottom flask was added the intermediate from Step E (8.5 g, 9 mmol), ethanol (100 mL) and glacial acetic acid (0.54 mL, 18 mmol), and the mixture was stirred until the solids dissolved. The reaction mixture was purged with hydrogen for 5 min, then 10% palladium on carbon (1.7 g) was added. The mixture was stirred at room temperature while slowly bubbling hydrogen into the reaction mixture until >95% conversion was observed by HPLC (ca. 8-9 h). The mixture was then filtered through a pad of celite and the solvent was removed under reduced pressure. Chromatography on silica gel (15 g silica gel/1 g crude product) using 5% MeOH/0.5% NH ₄ OH in DCM (10×150 mL), 8% MeOH/0.5% NH ₄ OH in DCM (10×150 mL) The residue was purified with 10% MeOH/0.5% _NH4OH in DCM (10 x 150 mL). The appropriate fractions were combined and the solvent was removed under reduced pressure while maintaining the temperature <35°C to afford the title intermediate (4.05 g, 97% purity).

(g) Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxyl-2-(8-hydroxyl-2-oxo-1,2-dihydroquinolin-5-yl) Ethylamino]nonyl}piperidin-4-yl ester

To a 200 mL round bottom flask was added the intermediate from Step F (4.05 g, 5.36 mmol) and dichloromethane (80 mL) and the resulting mixture was stirred until the solids dissolved. Triethylamine trihydrofluoride (2.6 mL, 16 mmol) was added and stirring was continued for 18-20 h under nitrogen. Methanol (20 mL) was added, then saturated aqueous sodium bicarbonate (50 mL) was added slowly and the mixture was stirred for 15 minutes. The layers were then separated, washed with saturated aqueous sodium chloride (20 mL), dried ( _MgSO4 ), filtered and concentrated under reduced pressure to afford the title compound (3.5 g, 98% pure by HPLC) as a yellow solid.

Example 3B

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino ]nonyl}piperidin-4-yl ester naphthalene-1,5-disulfonate

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxyl-2-(8-hydroxyl-2-oxo-1,2-dihydroquinolin-5-yl)ethyl Amino]nonyl}piperidin-4-yl ester (1.0 g, 1.56 mmol, free base) was dissolved in methanol (10 mL; low water content). A solution of naphthalene-1,5-disulfonic acid (0.45 g, 1.56 mmol) in methanol (5 mL; low water content) was added and the reaction mixture was stirred at 30° C. for two hours, then at room temperature overnight (18 h). The resulting thick slurry was filtered, and the filter cake was washed with methanol (5 mL), then dried to afford the title compound (1.16 g, 80% yield) as an off-white crystalline solid.

Preparation 16

N-{2-Benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilyloxy)ethyl]phenyl}-formamide

(R)-2-Bromo-1-(3-carboxamido-4-benzyloxyphenyl)ethanol (9.9 g, 28 mmol) was dissolved in dimethylformamide (36 mL). Add imidazole (2.3 g, 34 mmol) and tert-butyldimethylsilyl chloride (4.7 g, 31 mmol). The solution was stirred under nitrogen atmosphere for 72h. Additional imidazole (0.39 g, 5.7 mmol) and tert-butyldimethylsilyl chloride (0.64 g, 4.3 mmol) were added and the reaction was stirred for a further 20 h. The reaction mixture was then diluted with a mixture of isopropyl acetate (53 mL) and hexanes (27 mL) and transferred into a separatory funnel. The organic layer was washed twice with a mixture of water (27 mL) and saturated aqueous sodium chloride (27 mL), followed by a final wash with saturated aqueous sodium chloride (27 mL). The organic layer was dried over sodium sulfate. Silica gel (23.6 g) and hexane (27 mL) were added and the suspension was stirred for 10 minutes. The solids were removed by filtration and the filtrate was concentrated in vacuo. Crystallization of the residue from hexane (45 mL) afforded 8.85 g of the title compound (19 mmol, 68%) as a solid. MS m/z _{for C22H30NO3SiBr} : [M+H ⁺ ] calcd. 464.1; found _464.2 .

Can be as described in U.S. Patent No. 6,268,533 B1; Or R. Hett et al., Organic Process Research and Development, 1998, 2: 96-99; Or use and describe in Hong et al., Tetrahedron Lett., 1994, 35: 6631 or using a method similar to that described in US Patent No. 5,495,054 to prepare the starting material - (R)-2-bromo-1-(3-carboxamido-4-benzyloxyphenyl)ethanol.

Preparation 17

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(4-benzyloxy-3-formylaminophenyl)-2-(tert-butyldimethylsilyloxy )ethylamino]nonyl}piperidin-4-yl ester

The product of Preparation 16 (500 mg, 1.008 mmol) and sodium iodide (243 mg, 1.62 mmol) were stirred in tetrahydrofuran (0.5 mL) for 15 min at room temperature. The product of Preparation 11 (564mg, 1.29mmol) and sodium bicarbonate (272mg, 3.24mmol) were then added and the reaction mixture was heated at 80°C for 24h. The reaction mixture was then allowed to cool. Water (2 mL) was then added, and the mixture was extracted with dichloromethane (2 x 2 mL). The combined organic extracts were washed with 1M hydrochloric acid (2 x 1 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (5-10% methanol/dichloromethane) to afford the title compound (360 mg, 0.44 mmol, 41% yield). HPLC ( _{10-70) Rt = 4.96; MS m/z for C49H68N4O5 : [ M +} H ⁺ ] calcd. 821.51; found 821.9.

Preparation 18

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(4-benzyloxy-3-formylaminophenyl)-2-hydroxyethylamino]nonyl}piperidine- 4-yl ester

To a stirred solution of the product from Preparation 17 (360 mg, 0.44 mmol) in tetrahydrofuran (2.2 mL) was added triethylamine trihydrofluoride (108 μL, 0.66 mmol) at room temperature. The reaction mixture was stirred for 24 h, then diluted with dichloromethane (5 mL) and washed with 1M hydrochloric acid (2 mL) and saturated aqueous sodium bicarbonate (2 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude title compound was used directly in the next step without further purification. HPLC _{(10-70) Rt = 4.6; MS m/z for C43H54N4O5 : [ M +} H ⁺ ] calcd. 707.43; found 707.8.

Example 4

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-(3-formylamino-4-hydroxyphenyl)-2-hydroxyethylamino]nonyl}piperidine-4- ditrifluoroacetate

Palladium (10 wt.% (dry basis) on charcoal) (124 mg) was added to a stirred solution of the product of Preparation 18 (311 mg, 0.44 mmol) in ethanol (4 mL) and the reaction mixture was placed under an atmosphere of hydrogen. After stirring for 12 h, the reaction mixture was filtered through a pad of celite, washed with methanol (2 mL) and the solvent was removed under reduced pressure. The resulting residue was purified by preparative HPLC to give the title compound (41 mg). HPLC _{(10-70) Rt = 3.0; MS m/z for C36H48N4O5 : [ M +} H ⁺ ] calcd. 617.39; found 617.5.

Example 5

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2,3,4-tetrahydroquinolin-5-yl )ethylamino]nonyl}piperidin-4-yl ester ditrifluoroacetate

Palladium (10 wt.% (dry basis) on activated carbon) (80 mg) was added to a stirred solution of the product of Example 3 (80 mg, 0.11 mmol) in ethanol (1.1 mL) and the reaction mixture was placed under an atmosphere of hydrogen . The reaction mixture was stirred for 12 h, then filtered through a pad of celite, washed with methanol (2 mL) and the solvent was removed under reduced pressure. The crude material was resubjected to the above conditions to ensure completion of the reaction. The resulting crude residue was purified by preparative HPLC to give the title compound (6 mg). HPLC (10-70) _Rt = 3.23; MS _m / _z for _{C38H50N4O5 :} [M+H ⁺ ] calcd. 643.39; found 643.7.

Preparation 19

3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid methyl ester

Methyl 3-bromopropionate (553 μL, 5.07 mmol) was added to a stirred solution of the product of Preparation 8 (1.00 g, 3.38 mmol) and DIPEA (1.76 mL, 10.1 mmol) in acetonitrile (34 mL) at 50 °C , and the reaction mixture was heated at 50 °C overnight. The solvent was then removed under reduced pressure, and the residue was dissolved in dichloromethane (30 mL). The resulting solution was washed with saturated aqueous sodium bicarbonate (10 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (5-10% MeOH/DCM) to give the title compound (905 mg, 70%).

Preparation 20

3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propanoic acid

A stirred solution of the product of Preparation 19 (902 mg, 2.37 mmol) and lithium hydroxide (171 mg, 7.11 mmol) in 50% THF/water (24 mL) was heated at 30°C overnight, then acidified with concentrated hydrochloric acid and lyophilized to afford the title compound (~100% yield, also contains LiCl salt).

Preparation 21

{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-(tert-butyldimethyl-silyloxy)ethyl tert-Butylamino]pentyl}carbamate

The product of Preparation 13 (600 mg, 1.23 mmol) and N-tert-butoxycarbonyl-1,5-diaminopentane (622 mg, 3.07 mmol) were dissolved in dimethylsulfoxide (1.23 mL) and heated to 105 ℃6h. The reaction mixture was then cooled and diluted with ethyl acetate (10 mL) and washed with saturated aqueous sodium bicarbonate (4 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (5-10% methanol/dichloromethane) to afford the title compound (-100% yield).

Preparation 22

5-[(R)-2-(5-aminopentylamino)-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

A solution of the product from Preparation 21 (800 mg, 1.31 mmol) in trifluoroacetic acid/dichloromethane (25%, 12 mL) was stirred at room temperature for 1 hour. The solvent was then removed under reduced pressure and the crude residue was dissolved in dichloromethane (15 mL) and washed with 1 N sodium hydroxide (8 mL). The organic phase was separated, dried (magnesium sulfate) and the solvent removed under reduced pressure to afford the title compound (509 mg, 81% yield over 2 steps).

Preparation 23

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2 -(tert-Butyldimethylsilyloxy)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-yl ester

To the product of Preparation 20 (417 mg, 1.13 mmol) and HATU (430 mg, 1.13 mmol) was added the product of Preparation 22 (458 mg, 0.90 mmol) in DMF (1.8 mL) followed by DIPEA (204 μL, 1.17 mmol). The reaction mixture was stirred at 50 °C for 12 h, then the solvent was removed under reduced pressure. The crude residue was dissolved in dichloromethane (10 mL). The resulting solution was washed with saturated aqueous sodium bicarbonate (4 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (5-10% methanol/dichloromethane and 0.5% _NH4OH ) to afford the title compound (240 mg, 31% yield).

Preparation 24

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2 -Hydroxyethylamino]pentylcarbamoyl}ethyl)piperidin-4-yl ester

To a stirred solution of the product of Preparation 23 (240 mg, 0.28 mmol) in dichloromethane (2.8 mL) was added triethylamine trihydrofluoride (91 μL, 0.56 mmol). The reaction mixture was stirred for 10 h, then diluted with dichloromethane (10 mL). The resulting solution was then washed with saturated aqueous sodium bicarbonate (5 mL), then the organic phase was dried (magnesium sulfate), and the solvent was removed under reduced pressure to obtain the title compound (209 mg, yield 100%).

Example 6

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl) Ethylamino]pentylcarbamoyl}ethyl)piperidin-4-yl ester, ditrifluoroacetate

To a stirred solution of the product of Preparation 24 (209 mg, 0.28 mmol) in ethanol (2.8 mL) was added palladium (10 wt.% (dry basis) on activated carbon) (81 mg) and the reaction mixture was placed under an atmosphere of hydrogen and stir overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (58 mg). HPLC (10-70) R _t = 2.30; MS m/z for C ₃₇ H ₄₅ N ₅ O ₆ : [M+H+] calcd. 656.34; found 656.6; [α] ²⁰ _D = -6.5(c = 1.0 mg/mL, water).

Example 6A

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl) Ethylamino]pentylcarbamoyl}ethyl)piperidin-4-yl ester

Alternatively, the title compound can be prepared as follows:

(a) 5-chloropentanal

To a 2 L three necked round bottom flask equipped with a magnetic stirrer, addition funnel and temperature controller was added 5-chloropentanol (53 g, 0.433 mol) and dichloromethane (300 mL) under nitrogen.

The mixture was cooled to 5°C and a solution of sodium bicarbonate (5 g, 0.059 mol) and potassium bromide (5.1 g, 0.043 mol) in water (225 mL) was added. 2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO) (63 mg, 0.4 mmol) was added, followed by 10-13% bleach solution (275 mL) dropwise through the addition funnel at a certain rate, In order to maintain the temperature at about 8°C (+/- 2°C) with an ice-cold bath (over about 45 min.). After the bleach addition was complete, the mixture was stirred for 30 min. By maintaining the temperature at approximately 5°C. A solution of sodium bisulfite (4 g) in water (30 mL) was added and the resulting mixture was stirred at room temperature for 30 min. The layers of the mixture were then separated, and the aqueous layer was extracted with dichloromethane (1 x 50 mL). The combined dichloromethane layers were then washed with water (1 x 50 mL), dried ( _MgSO4 ), filtered and concentrated under reduced pressure to afford the title compound (53 g). Distillation of the product at 65°C/8 torr afforded the title compound (31.16 g) as an orange oil (GC purity 70-80%).

The product was further purified by adding the crude material (4 g) to a mixture of ethanol (920 mL), ethyl acetate (12 mL) and water (4 mL). Sodium bisulfite (4 g) was added and the mixture was heated to reflux for 4 h, then cooled to room temperature and stirred at room temperature for 14 h to form a very thick slurry. The solid was filtered on a coarse glass filter, washed with solvent mixture (5 mL), and the solid was dried on the filter to give 8.4 g of the bisulfite adduct. This material was then added to MTBE (20 mL) and 1 N aqueous sodium hydroxide solution (45 mL) was added with vigorous stirring. The resulting biphasic mixture was stirred vigorously until all solids had dissolved (ca. 15 min), then the layers were separated. The aqueous layer was extracted with MTBE (20 mL) and the combined MTBE layers were dried ( _MgSO4 ), filtered and concentrated to afford 3.46 g of the title compound as a primary colored liquid (GC purity >90%).

(b) 5-[(R)-2-[benzyl-(5-chloropentyl)amino]-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy- 1H-quinolin-2-one

To a 1 L three necked round bottom flask was added the product of Preparation 28 (48.4 g, 94 mmol), dichloromethane (400 mL) and glacial acetic acid (11.3 mL). The mixture was stirred at 0°C (ice bath), the product from step (a) (12.5 g, 103.6 mmol) was added and stirring continued for 15 minutes. Sodium triacetoxyborohydride (59.8 g, 282 mmol) was then added in portions over 15 minutes, and the resulting mixture was stirred at 0°C-10°C for 2h. Then saturated sodium bicarbonate solution (200 mL) was added slowly (gas evolution) and stirring was continued for 15 min. The pH was then adjusted to about 9 with solid sodium carbonate and the layers were separated. The organic layer was washed with 5% aqueous sodium chloride (200 mL), dried ( _MgSO4 ), filtered and concentrated under reduced pressure to afford the title compound (53 g).

(c) 5-[(R)-2-[(5-N, N-diformylaminopentyl)benzylamino]-1-(tert-butyldimethyl-silyloxy)ethyl]- 8-Benzyloxy-1H-quinolin-2-one

To a stirred solution of the product of step (b) (26.5 g, 42.8 mmol) in 1-methyl-2-pyrrolidone (175 mL) was added sodium diformylamide (6.1 g, 64.2 mmol) and sodium iodide (2.13 g, 14.3 mmol). The reaction flask was flushed with nitrogen and the mixture was heated at 65 °C for 8 h. The mixture was then cooled to room temperature and water (300 mL) and ethyl acetate (100 mL) were added. The mixture was stirred for 10 minutes. The aqueous layer was extracted with ethyl acetate (150 mL), the combined organic layers were washed with water (300 mL), 50% brine solution (300 mL), water (300 mL), dried (MgSO ₄ ), filtered and concentrated to give the title compound (23.3 g).

(d) 5-[(R)-2-[(5-aminopentyl)benzylamino]-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy-1H -Quinolin-2-one

To a stirred solution of the product from step (c) (10.5 g, 16 mmol) in methanol (75 mL) was added p-toluenesulfonic acid (7.42 g. 39 mmol). The resulting mixture was heated at 40 °C for 15 h, then concentrated under reduced pressure to about half its volume. Methanol (70 mL) was added and the mixture was heated at 50 °C for 2 h, then concentrated under reduced pressure. Water (100 mL), methanol (50 mL) and MTBE (100 mL) were added, the mixture was stirred for 15 minutes, then the layers were separated. To the aqueous layer were added 1N aqueous sodium hydroxide solution (45 mL) and MTBE (100 mL), and the mixture was stirred for 15 min. The layers were then separated and the aqueous layer was extracted with MTBE (100 mL). The combined MTBE layers were dried ( _MgSO4 ), filtered and concentrated to give the title compound (7.3g) as a yellow oil. This material contained about 13% (HPLC) of the corresponding des-tert-butyldimethylsilyl compound.

(e) 3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionic acid

To a solution of the product from Preparation 8 (50 g, 67.6 mmol) in dichloromethane (500 mL) was added acrylic acid (15.05 mL, 100 mmol). The resulting mixture was heated at reflux at 50 °C for 18 h, then the solvent was removed. Methanol (600 mL) was added, and the mixture was heated at 75 °C for 2 h, then cooled to room temperature to form a thick slurry. The solid was collected by filtration, washed with methanol (50 mL), and air dried to give the title compound (61 g, >96% purity) as a white powder.

(f) Biphenyl-2-ylcarbamate 1-[2-(5-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline -5-yl)-2-(tert-butyldimethylsilyloxy)ethyl]amino}-pentylcarbamoyl)ethyl]piperidin-4-yl ester

A mixture of the product of step (e) (3.68 g, 10 mmol) and N,N dimethylformamide (50 mL) was heated at 60 °C until the solids were completely dissolved, then cooled to room temperature. The product of step (d) (6 g, 10 mmol) and diisopropylethylamine (3.5 mL) were added and the reaction mixture was cooled to 0°C. A portion of PyBOP (6.25 g, 12 mmol) was added and the reaction mixture was stirred at 0°C to room temperature for 2 hours. The reaction mixture was then poured into cold water (500 mL) with stirring and the pH of the resulting mixture was adjusted to approximately 2 using aqueous 1M hydrochloric acid. The mixture was stirred for 15 minutes, then the solid was collected by filtration, washed with water (100 mL) and dried to give the title compound (8.7 g, HPLC purity >95%) as an off-white solid.

(g) Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-hydroxyl-2-(8-hydroxyl-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]pentylcarbamoyl}ethyl)piperidin-4-yl ester

The product of step (f) can be deprotected using essentially the same procedures as those described in Preparation 24 and Example 6 to obtain the title compound.

Preparation 25

2-(N-Benzyloxycarbonyl-N-methylamino)acetaldehyde

(a) 2-(N-Benzyloxycarbonyl-N-methylamino)ethanol

Benzyl chloroformate (19 g, 111.1 mmol) in THF (20 mL) was added dropwise to 2-(methylamino)ethanol (10 g, 133.3 mmol) in THF (100 mL) and sodium carbonate at 0 °C over 15 min solution in aqueous solution (100 mL). The reaction mixture was stirred at 0 °C for 12 h, then extracted with EtOAc (2 x 200 mL). The organic layer was washed with aqueous sodium carbonate (200 mL), dried (potassium carbonate), and the solvent was removed under reduced pressure to afford the title compound (22.5 g, 97% yield).

(b) 2-(N-Benzyloxycarbonyl-N-methylamino)acetaldehyde

DMSO (71 mL, 1 mol) and DIPEA (87.1 mL, 0.5 mol) were added to a stirred solution of the product from step (a) (20.9 g, 0.1 mol) in dichloromethane (200 mL) at -10°C. The reaction mixture was stirred at -10°C for 15 min, then sulfur trioxide pyridine complex (79.6 g, 0.5 mol) was added, and the resulting mixture was stirred for 1 h. The reaction mixture was quenched by dropwise addition of 1M hydrochloric acid (200 mL). The organic layer was separated and washed with saturated aqueous sodium bicarbonate (100 mL), brine (100 mL), dried (potassium carbonate) and solvent removed under reduced pressure to afford the title compound (20.7 g, ~100% yield).

Preparation 26

1-[2-(methylamino)ethyl]piperidin-4-yl biphenyl-2-ylcarbamate

To a stirred solution of the product of Preparation 25 (20.7 g, 100 mmol) and the product of Preparation 8 (25 g, 84.7 mmol) in MeOH (200 mL) was added sodium triacetoxyborohydride (21.2 g, 100 mmol). The reaction mixture was stirred at room temperature for 12 h, then quenched with 2M hydrochloric acid and the solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate (200 mL), washed with saturated aqueous sodium bicarbonate (100 mL) and brine (50 mL), then dried (magnesium sulfate) and the solvent was removed under reduced pressure. Purification of the crude residue by column chromatography (50-90% EtOAc/hexanes) gave biphenyl-2-ylcarbamate 1-[2-(benzyloxycarbonyl-methylamino)ethyl] as an oil Piperidin-4-yl ester.

The oil was dissolved in methanol (100 mL) and palladium (10 wt.% (dry basis) on charcoal) (5 g) was added. The reaction mixture was stirred under hydrogen (30 psi) for 12 h, then filtered through celite, washed with methanol, and the solvent was evaporated to give the title compound (13.2 g, 44% yield).

Preparation 27

1-{2-[(6-bromohexanoyl)methylamino]ethyl}piperidin-4-ylcarbamate

6-Bromohexanoyl chloride (3.23 mL, 21.1 mmol) was added to a stirred solution of the product of Preparation 26 (6.2 g, 17.6 mmol) and DIPEA (6.13 mL, 35.2 mmol) in dichloroethane (170 mL) middle. The reaction mixture was stirred for 1 h, then diluted with EtOAc (250 mL), and washed with saturated aqueous sodium bicarbonate (2 x 200 mL) and brine (200 mL), then dried (MgSO4). The solvent was removed under reduced pressure to obtain the title compound (6.6 g, 73% yield).

Preparation 28

8-Benzyloxy-5-[(R)-2-(N-benzylamino)-1-(tert-butyldimethylsilyloxy)ethyl]-1H-quinolin-2-one

A stirred solution of the product of Preparation 13 (1.00 g, 2.05 mmol) and benzylamine (493 μL, 4.51 mmol) in DMSO (1.7 mL) was heated at 105 °C for 4 h. The reaction mixture was allowed to cool, then diluted with EtOAc (10 mL), washed with saturated aqueous ammonium chloride (5 mL) and 1 N sodium hydroxide (5 mL), dried (MgSO ₄ ) and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (50% BtOAc/hexanes) to afford the title compound (700 mg, 67%). MS m _{/ z for C31H38N2O3Si} : [M+H+] calcd. 515.27; found 515.5.

Preparation 29

Biphenyl-2-ylcarbamate 1-{2-[(6-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline-5 -yl)-2-(tert-butyldimethylsilyloxy)ethyl]amino}hexanoyl)-methylamino]ethyl}piperidin-4-yl ester

To a stirred solution of the product from Preparation 28 (807 mg, 1.57 mmol) and DIPEA (819 μL, 4.7 mmol) in acetonitrile (3.14 mL) was added the product from Preparation 27 (995 mg, 1.88 mmol). The reaction mixture was heated to 80 °C for 24 h. The solvent was removed under reduced pressure, the residue was dissolved in EtOAc (10 mL), then washed with saturated aqueous sodium bicarbonate (5 mL), dried (magnesium sulfate), and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (4-6% MeOH/DCM) to afford the title compound (452 mg, 30% yield).

Prepare 30

Biphenyl-2-ylcarbamate 1-{2-[(6-{benzyl-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline-5 -yl)-2-hydroxyethyl]amino}hexanoyl)methylamino]ethyl}piperidin-4-yl ester

To a stirred solution of the product from Preparation 29 (452 mg, 0.47 mmol) in dichloromethane (4.7 mL) was added triethylamine trihydrofluoride (116 μL, 0.71 mmol). The reaction mixture was stirred for 10 h. It was then diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was then dried ( _MgSO4 ) and the solvent removed under reduced pressure to afford the title compound (100% yield).

Example 7

Biphenyl-2-ylcarbamate 1-[2-({6-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl )ethylamino]hexanoyl}methylamino)ethyl]piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product of Preparation 30 (400 mg, 0.47 mmol) in ethanol (4.7 mL) was added palladium (10 wt.% (dry basis) on activated carbon) (160 mg) and the reaction mixture was placed under an atmosphere of hydrogen and stir overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (73 mg). HPLC _{(10-70) Rt = 2.33; MS m/z for C38H47N5O6 : [ M +} H+] calcd. 670.36; [α] ²⁰ _D = -9.4 (c = 1.0 mg/mL, water).

Preparation 31

1-[2-(4-(Aminomethyl)phenylcarbamoyl)-ethyl]piperidin-4-ylcarbamate

To 4-(N-tert-butoxycarbonylaminomethyl)aniline (756mg, 3.4mmol), the product of Preparation 20 (1.5g, 4.08mmol) and HATU (1.55g, 4.08mmol) in DMF (6.8mL) To the stirred solution of DIPEA (770 μL, 4.42 mmol) was added. The reaction mixture was stirred overnight at 50 °C, then the solvent was removed under reduced pressure. The obtained residue was dissolved in dichloromethane (20 mL), and washed with saturated aqueous sodium bicarbonate (10 mL). The organic phase was then dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (5-10% MeOH/DCM) to give a solid, which was dissolved in TFA/DCM (25%, 30 mL) and stirred at room temperature for 2 h. The solvent was then removed under reduced pressure, and the crude residue was dissolved in dichloromethane (30 mL), and washed with 1N sodium hydroxide (15 mL). The organic phase was separated, dried (magnesium sulfate) and the solvent removed under reduced pressure to give the title compound (1.5 g, 94% over 2 steps).

Preparation 32

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)- 2-(tert-Butyldimethylsilyloxy)ethylamino]methyl}phenylcarbamoyl)ethyl]-piperidin-4-yl ester

The product of Preparation 31 (489 mg, 1.04 mmol), the product of Preparation 13 (610 mg, 1.25 mmol), sodium bicarbonate (262 mg, 3.12 mmol) and sodium iodide (203 mg, 1.35 mmol) were dissolved in THF (0.52 mL) The solution was heated at 80 °C for 12 h. The reaction mixture was diluted with dichloromethane (10 mL), washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was dried ( _MgSO4 ) and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (10% MeOH/DCM) to give the title compound (687 mg, 77% yield) as a solid.

Preparation 33

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)- 2-Hydroxyethylamino]methyl}phenylcarbamoyl)ethyl]piperidin-4-yl ester

To a stirred solution of the product from Preparation 32 (687 mg, 0.8 mmol) in dichloromethane (8 mL) was added triethylamine trihydrofluoride (261 μL, 1.6 mmol). The reaction mixture was stirred for 10 h, then diluted with dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL). The organic phase was then dried (magnesium sulfate) and the solvent was removed under reduced pressure to afford the title compound (500 mg, 81% yield).

Example 8

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl )ethylamino]methyl}phenylcarbamoyl)ethyl]piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product of Preparation 33 (500 mg, 0.65 mmol) in ethanol (6.5 mL) was added palladium (10 wt.% (dry basis) on activated carbon) (200 mg) and the reaction mixture was placed under hydrogen under atmosphere and stir overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (81 mg, 2 TFA salt). HPLC (10-70 _{) Rt} = 2.41; MS _m / _z for _C39H41N5O6 : [M+H+] calcd. 676.32; found 676.5.

Preparation 34

1-(2-tert-butoxycarbonylaminoethyl)piperidin-4-yl biphenyl-2-ylcarbamate

To a stirred solution of the product from Preparation 8 (2.00 g, 6.76 mmol) and DIPEA (3.54 mL, 20.3 mmol) in acetonitrile (67.6 mL) was added 2-tert-butoxycarbonylaminoethyl bromide (1.82 g, 8.11 mmol), and the reaction mixture was heated at 50 °C overnight.

The solvent was then removed under reduced pressure, and the residue was dissolved in dichloromethane (60 mL) and washed with saturated aqueous sodium bicarbonate (30 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (5% MeOH/DCM) to afford the title compound (2.32 g, 78% yield) as a solid.

Preparation 35

1-(2-aminoethyl)piperidin-4-yl biphenyl-2-ylcarbamate

The product from Preparation 34 was dissolved in TFA/DCM (25%, 52 mL) and stirred at room temperature for 2 h. The solvent was then removed under reduced pressure and the crude residue was dissolved in dichloromethane (30 mL) and washed with 1N sodium hydroxide (15 mL). The organic phase was separated, dried (magnesium sulfate) and the solvent was removed under reduced pressure to afford the title compound (1.61 g, 90% yield).

Preparation 36

1-[2-(4-Aminomethylbenzoylamino)ethyl]piperidin-4-ylcarbamate

To a stirred solution of the product of Preparation 35 (339 mg, 1 mmol), 4-(tert-butoxycarbonylaminomethyl)benzoic acid (301 mg, 1.2 mmol) and HATU (456 mg, 1.2 mmol) in DMF (2 mL) DIPEA (226 μL, 1.3 mmol) was added. The reaction mixture was stirred overnight at room temperature, then the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was dissolved in TFA/DCM (25%, 10 mL), and the mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the crude residue was dissolved in dichloromethane (15 mL) and washed with 1N sodium hydroxide (5 mL). The organic phase was separated, dried (magnesium sulfate) and the solvent removed under reduced pressure to afford the title compound (472 mg, -100% over 2 steps).

Preparation 37

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)- 2-(tert-Butyldimethylsilyloxy)ethylamino]methyl}benzoylamino)ethyl]-piperidin-4-yl ester

The product of Preparation 36 (520 mg, 1.1 mmol), the product of Preparation 13 (634 mg, 1.3 mmol), sodium bicarbonate (277 mg, 3.3 mmol) and sodium iodide (215 mg, 1.43 mmol) were dissolved in THF (0.55 mL) The solution was heated at 80 °C for 12 h. The reaction mixture was then diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was then dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (5-10% MeOH/DCM) to afford the title compound (316 mg, 33% yield) as a solid.

Preparation 38

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)- 2-Hydroxyethylamino]methyl}benzoylamino)ethyl]piperidin-4-yl ester

To a stirred solution of the product from Preparation 37 (316 mg, 0.36 mmol) in dichloromethane (3.6 mL) was added triethylamine trihydrofluoride (117 μL, 0.72 mmol). The reaction mixture was stirred for 10 h, then diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate (5 mL). Drying ( _MgSO4 ) of the organic phase and removal of the solvent under reduced pressure afforded the title compound which was used directly in the next step (100% yield).

Example 9

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl )ethylamino]methyl}benzoylamino)ethyl]piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product of Preparation 38 (275 mg, 0.36 mmol) in ethanol (3.6 mL) was added palladium (10 wt.% (dry basis) on activated carbon) (275 mg) and the reaction mixture was placed under an atmosphere of hydrogen and stir overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (6 mg, 2 TFA salt). HPLC (10-70 _{) Rt} = 2.26; MS _m / _z for _C39H41N5O6 : [M+H ⁺ ] calcd. 676.32; found 676.5.

Preparation 39

1-(2-aminoethyl)piperidin-4-yl biphenyl-2-ylcarbamate

2-tert-Butoxycarbonylaminoethyl bromide (1.22 g, 5.44 mmol) was added to the product of Preparation 8 (1.46 g, 4.95 mmol) and diisopropylethylamine (1.03 mL, 5.94 mmol) in acetonitrile ( 24mL) in the solution. The reaction mixture was stirred at 65°C for 12 hours at which time MS analysis indicated the reaction was complete. The reaction mixture was concentrated to dryness, then dichloromethane (10 mL) was added. Trifluoroacetic acid was added to the mixture and the mixture was stirred at room temperature for 4 hours, at which time MS analysis indicated the reaction was complete. The mixture was then concentrated to half its volume and 1 N sodium hydroxide was added to the solution until the pH was adjusted to 14. The organic layer was washed with brine, then dried over magnesium sulfate and filtered. The filtrate was concentrated to give 1.6 g of the title compound as a solid. MS m/ _{z for C20H25N3O2} : [M+H ⁺ ] calcd. _340.2 ;

Preparation 40

5-[(R)-2-(5-aminopentylamino)-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy-1H-quinolin-2-one

N-tert-butoxycarbonyl-1,5-diaminopentane (1.04 g, 5.12 mmol) was added to a solution of the product from Preparation 13 (1.00 g, 2.05 mmol) in dimethylsulfoxide (2 mL). The solution was stirred at 75°C for 12 hours at which time LCMS analysis indicated the reaction was complete. The reaction mixture was then concentrated to dryness in vacuo. Dichloromethane (2 mL) was added to the residue, followed by trifluoroacetic acid (1 mL). The solution was stirred at room temperature for about 3 hours at which time MS analysis indicated the reaction was complete. The mixture was then concentrated to half its volume and 1 N sodium hydroxide was added to the solution until the pH was adjusted to 14. The organic layer was collected, washed with brine, then dried over magnesium sulfate, then concentrated to give 782 mg of the title compound as an oil. MS m _/ z for _C29H43N3O3Si : [ _M +H ⁺ ] calcd. _510.8 ;

Preparation 41

Biphenyl-2-ylcarbamate 1-[2-(3-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl )-2-(tert-Butyldimethylsilyloxy)ethylamino]pentyl}-ureido)ethyl]piperidin-4-yl ester

Carbonyldiimidazole (127 mg, 0.78 mmol) was added to a solution of the product of Preparation 39 (266 mg, 0.78 mmol) in dimethylformamide (4 mL), and the resulting mixture was stirred at room temperature for 3 hours. After 3 hours, the product of Preparation 40 (399 mg, 0.78 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 12 hours, at which time LCMS analysis determined the reaction was complete. The reaction mixture was concentrated in vacuo and the residue was diluted with ethyl acetate (5 mL). Wash twice with saturated sodium bicarbonate (5 mL), then brine (5 mL). The organic layer was dried over magnesium sulfate, filtered and concentrated to give 597 mg of the title compound as a solid which was used without further purification. MS m/z for _C50H66N6O6Si : [M+H ⁺ ] calcd _{. 875.5} ; found 875 _.

Preparation 42

Biphenyl-2-ylcarbamate 1-[2-(3-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl )-2-Hydroxyethylamino]pentyl}ureido)ethyl]piperidin-4-yl ester

Triethylamine trihydrofluoride (0.16 mL, 1.02 mmol) was added to a solution of the product of Preparation 41 (597 mg, 0.68 mmol) in tetrahydrofuran (3.4 mL) and the mixture was stirred at room temperature for about 12 hours, at which point the chromatin was detected by MS The analysis determined that the reaction was complete. The reaction mixture was diluted with ethyl acetate (5 mL) and the mixture was washed with 1 N sodium hydroxide (5 mL), brine, dried over magnesium sulfate, concentrated to give 417 mg of the title compound as a solid. MS m _{/ z for C44H51N6O6} : [M+H ⁺ ] calcd. 760.4;

Example 10

Biphenyl-2-ylcarbamate 1-[2-(3-{5-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]pentyl}ureido)ethyl]piperidin-4-yl ester ditrifluoroacetate

A solution of the product of Preparation 42 (417 mg, 0.55 mmol) in ethanol (3 mL) was purged with nitrogen for about 10 min. Palladium (10 wt.% (dry basis) on charcoal) (200 mg) was added and the solution was flushed with nitrogen for about 10 minutes. The flask was vacuum purged, then filled three times with nitrogen, and then a hydrogen-filled balloon was placed over the flask. The reaction mixture was stirred under hydrogen for 12 hours at which time the reaction was determined to be complete by MS analysis. The reaction mixture was then filtered and the organic filtrate was concentrated and purified by HPLC (10-35% over 60 min) to afford 146 mg of the title compound as a powder. MS m/ _{z for C37H46N6O6 :} [M+H ⁺ ] calcd. _671.4 ; HPLC (10-70) _Rt = 2.6 min.

Example 11

Biphenyl-2-ylcarbamate 1-[3-(3-{5-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]pentyl}ureido)propyl]piperidin-4-yl ester ditrifluoroacetate

Using the above procedure in Preparations 39-42 and Example 10, and substituting 3-tert-butoxycarbonylaminopropan-1-yl bromide for 2-tert-butoxycarbonylaminoethyl bromide in Preparation 39, Preparation of the title compound. MS m/ _{z for C38H48N6O6} : [M+H ⁺ ] calcd. 685.4; found ₆₈₄ . HPLC (1070) _Rt = 2.6 min.

Preparation 43

烷6-(2-Bromo-(R)-1-tert-butyldimethylsilyloxy)ethyl-2,2-dimethyl-1,3-benzobis

alkyl

(a) 6-bromo-2,2-dimethyl-4H-benzo[1,3]di alkyl

To 5-bromo-2-hydroxybenzyl alcohol (93 g, 0.46 mol, purchased from Sigma-Aldrich) in 2.0 L of 2,2-dimethoxypropane was added 700 mL of acetone followed by zinc chloride (170 g) . After stirring for 18 hours, 1.0 M aqueous sodium hydroxide solution was added until the aqueous phase was basic. Diethyl ether was added to the slurry and the organic phase was decanted into a separatory funnel. The organic phase was washed with brine, dried over _Na2SO4 , filtered and concentrated _under reduced pressure to afford the title compound as an oil.

烷(b) 6-acetyl-2,2-dimethyl-4H-benzo[1,3]di

alkyl

The product of step (a) (110 g, 0.46 mol) in 1.0 L THF was added via dropping funnel to 236 mL (0.51 mol) of 2.14M n-butyllithium in hexane at -78°C. After 30 minutes, N-methyl-N-methoxyacetamide (71 g, 0.69 mol, from TCI) was added. After two hours, the reaction mixture was quenched with water, diluted with 2.0 L of 1.0 M aqueous phosphate buffer (pH=7.0) and extracted once with diethyl ether. The diethyl ether was washed once with brine, dried over _Na2SO4 , filtered and concentrated _under reduced pressure to give a light orange oil. This oil was dissolved in a minimal volume of ethyl acetate and diluted with hexanes to give the title compound as a crystalline solid.

烷(c) 6-bromoacetyl-2,2-dimethyl-4H-benzo[1,3]di

alkyl

To the product of step (b) (23.4 g, 0.113 mol) in 600 mL THF was added 135 mL of 1.0 M sodium hexamethyldisilazane in THF (Sigma-Aldrich) at -78°C. After 1 hour, trimethylsilyl chloride (15.8 mL, 0.124 mol) was added. After an additional 30 minutes, bromine (5.82 mL, 0.113 mol) was added. After 10 min, the reaction was quenched by diluting the reaction mixture with diethyl ether and pouring it into 500 mL of 5% aqueous _Na2SO3 premixed with 500 mL of 5% _{aqueous NaHCO3} . The phases were separated and the organic phase was washed with brine, dried over _Na2SO4 , filtered and concentrated under reduced _pressure to give the title compound as an oil which solidified upon storage in the refrigerator.

烷-6-基)乙醇(d) (R)-2-bromo-1-(2,2-dimethyl-4H-benzo[1,3]di

Alk-6-yl)ethanol

To the product of step (c) (10 g, 35.1 mmol) in 100 mL THF was added the solid catalyst from Preparation 13, step (c)(1) (0.97 g, 3.5 mmol). The solution was cooled to -20°C to -10°C, and BH ₃ -THF (35 mL, 35 mmol) diluted with 50 mL THF was added dropwise through the addition funnel. After complete addition, the reaction mixture was allowed to warm to room temperature. After 30 minutes, the reaction mixture was quenched by the slow addition of 50 mL of methanol, then concentrated to a thick oil. The oil was purified by silica gel chromatography eluting with 1:2 ethyl acetate/hexanes. Fractions were combined and concentrated to give the title compound as an off-white solid.

烷-6-基)乙氧基]-叔-丁基二甲基甲硅烷(e) [(R)-2-bromo-1-(2,2-dimethyl-4H-benzo[1,3]di

Alk-6-yl)ethoxy]-tert-butyldimethylsilane

To the product of step (d) (10 g, 34.8 mmol) and imidazole (4.7 g, 69.7 mmol) dissolved in 100 mL of DMF was added tert-butyldimethylsilyl chloride (5.78 g, 38.3 mmol). The reaction mixture was stirred for 18 hours. The reaction mixture was then partitioned between 200 mL saturated sodium chloride and 200 mL diethyl ether. The aqueous layer was extracted with 200 mL of diethyl ether. The organic layers were then combined, washed with saturated sodium chloride (3 x 100 mL), dried over MgSO ₄ and concentrated. The product was purified by silica gel chromatography, eluting with hexanes, then 5% ethyl acetate in hexanes. Desired fractions were combined and concentrated to give the title compound as an oil.

Preparation 44

Biphenyl-2-ylcarbamate 1-{9-[2-(tert-butyldimethylsilyloxy)-2-(2,2-dimethyl-4H-benzo[1,3]di Alk-6-yl)ethylamino]nonyl}piperidin-4-yl ester

The product of Preparation 43 (802 mg, 2.00 mmol) and sodium iodide (300 mg, 2.00 mmol) were stirred in tetrahydrofuran (0.77 mL) for 15 min at ambient temperature. The product of Preparation 11 (675mg, 1.54mmol) and sodium bicarbonate (388mg, 4.62mmol) were added and the reaction mixture was heated at 80°C for 24h. The reaction mixture was then cooled and water (2 mL) was added. The mixture was then extracted with dichloromethane (2 x 2 mL). The combined organic extracts were dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (5-10% methanol/dichloromethane) to give the title compound (798 mg, 1.05 mmol, 60% yield) as a solid. MS m for C ₄₅ H ₆₇ N ₃ O ₅ Si /z: [M+H ⁺ ] calculated value is 758.5; found value is 758.6.

Preparation 45

烷-6-基)-2-羟基乙基氨基]壬基}哌啶-4-基酯Biphenyl-2-ylcarbamate 1-{9-[2-(2,2-dimethyl-4H-benzo[1,3]di

Alk-6-yl)-2-hydroxyethylamino]nonyl}piperidin-4-yl ester

Triethylamine trihydrofluoride (342 μL, 2.10 mmol) was added to a stirred solution of the product of Preparation 44 (798 mg, 1.05 mmol) in dichloromethane (10.5 mL) at room temperature. The reaction mixture was stirred for 24 h, then diluted with dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (15 mL). The organic layer was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude title compound (659 mg, 1.02 mmol) was isolated as an oil and used in the next step without further purification. MS m/ _{z for C39H53N3O5} : [M+H ⁺ ] calcd. _644.4 ; found 644.8.

Example 12

Biphenyl-2-ylcarbamate 1-{9-[(R)-2-hydroxy-2-(4-hydroxy-3-hydroxymethylphenyl)ethylamino]nonyl}piperidine-4- ditrifluoroacetate

Trifluoroacetic acid (2.80 mL) was added to a stirred solution of the product of Preparation 45 (600 mg, 0.93 mmol) in THF/water (14 mL, 1 :1 ), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and dissolved in 20% MeCN/water, then purified by preparative HPLC to give the title compound (200 mg, 2TFA salt). HPLC _{(10-70) Rt = 2.76; MS m/z for C36H49N3O5 : [ M +} H ⁺ ] calcd. 604.4; found 604.8.

Preparation 46

1-[1-(9-Benzylaminononyl)piperidin-4-yl]-3-biphenyl-2-ylurea

N-Benzylamine (0.903 ml, 8.30 mmol) was added to a solution of the product of Preparation 4 (2.40 g, 5.52 mmol) in methanol (25 mL), and the resulting mixture was stirred at room temperature. After 10 minutes, sodium triacetoxyborohydride (1.75 g, 8.30 mmol) was added to the reaction mixture. The progress of the reaction was followed by HPLC analysis. After 2 h at ambient temperature, the reaction mixture was quenched with water (5 mL), then concentrated in vacuo to half its volume. The reaction mixture was diluted with dichloromethane (15 mL) and washed with 1 N sodium hydroxide (2 x 10 mL) followed by brine (5 mL). Dry the organic layer over magnesium sulfate and concentrate to give the title compound.

Preparation 47

Methyl 2-benzyloxy-5-(2-bromoacetyl)benzoate

(a) Methyl 2-benzyloxy-5-acetylbenzoate

Methyl 5-acetylsalicylate (100 g, 0.515 mol) was dissolved in acetonitrile (1 L) in a 2 L flask under reflux conditions under a nitrogen atmosphere. Potassium carbonate (213.5 g, 1.545 mol) was added in portions over 15 minutes. Benzyl bromide (67.4 mL, 0.566 mol) was added using the addition funnel over 15 minutes. The reaction was heated to 85 °C for 9 h, then filtered and rinsed with acetonitrile (100 mL). The solution was concentrated under reduced pressure to a volume of about 300 mL and partitioned between water (1 L) and ethyl acetate (1 L). The organic layer was washed with saturated sodium chloride (250 mL), dried over magnesium sulfate (75 g), then filtered and rinsed with ethyl acetate (100 mL). The organic layer was concentrated to give methyl 2-benzyloxy-5-acetylbenzoate as a solid (yield 100%).

(b) Methyl 2-benzyloxy-5-(2-bromoacetyl)benzoate

The product from step (a) (10.0 g, 35.2 mmol) was dissolved in chloroform (250 mL) in a 500 mL flask under nitrogen atmosphere. Bromine (1.63 mL, 31.7 mmol) dissolved in chloroform (50 mL) was added over 30 minutes using a dropping funnel. The reaction mixture was stirred for 2.5 h, then concentrated to a solid. Dissolve the solid in toluene (150 mL) with gentle heat, then add diethyl ether (150 mL) to give the title compound as a crystalline solid (55% yield).

Preparation 48

5-[2-(Benzyl-{9-[4-(3-biphenyl-2-ylureido)piperidin-1-yl]nonyl}amino)acetyl]-2-benzyloxybenzene Methyl formate

The product from Preparation 47 (371 mg, 1.00 mmol) was added to a solution of the product from Preparation 46 (448 mg, 0.85 mmol) in dimethylsulfoxide (4.5 mL) followed by potassium carbonate (234 mg, 1.7 mmol). The reaction mixture was stirred at 40 °C for 6 h at which time the product of Preparation 46 was no longer observed by HPLC analysis. The reaction mixture was cooled to ambient temperature and filtered, then diluted with ethanol (4 mL). Sodium borohydride (63 mg, 1.7 mmol) was added to the reaction mixture and the reaction was stirred at room temperature for 24 h. The reaction mixture was quenched with 0.5M ammonium chloride (5 mL), extracted with ethyl acetate (2 x 10 mL). The combined organic layers were washed with saturated sodium bicarbonate (10 mL), then brine (5 mL). The organic layer was dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude residue was purified by chromatography on silica gel (3% methanol in chloroform) to afford the title compound.

Preparation 49

1-[1-(9-{benzyl-[2-(4-benzyloxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}nonyl)piperidin-4-yl]- 3-biphenyl-2-ylurea

A solution of the product of Preparation 48 (163 mg, 0.20 mmol) in tetrahydrofuran (1.00 mL) was cooled to 0°C. To this mixture was added lithium aluminum hydride (1.0 M in THF; 0.50 mL, 0.50 mmol) dropwise. After 1 h, the reaction mixture was quenched with water (1 mL) and diluted with ethyl acetate (2 mL). The organic layer was washed with brine, dried over magnesium sulfate, and the organic extracts were combined and concentrated to give the title compound.

Example 13

1-biphenyl-2-yl-3-(1-{9-[2-hydroxy-2-(4-hydroxy-3-hydroxymethylphenyl)ethylamino]nonyl}piperidine-4- base) urea dihydrochloride

A solution of the product of 49 (130 mg, 0.16 mmol) in isopropanol (0.80 ml) was prepared with a nitrogen sparge for 10 minutes, then palladium (10 wt.% (dry basis)) on charcoal (60 mg) was added. The reaction flask was purged with nitrogen , and then a hydrogen-filled balloon was attached to the flask, and the reaction mixture was stirred under a hydrogen atmosphere. After 72 h, the reaction mixture was filtered and concentrated, and the residue was purified with preparative HPLC. The ditrifluoroacetate salt of the title compound obtained Dissolve in 1N hydrochloric acid (5 mL) and lyophilize to give the title compound as its dihydrochloride salt.

Prepare 50

5-[(R)-2-[(3-aminomethylcyclohexylmethyl)amino]-1-(tert-butyldimethylsilyloxy)ethyl]-8-benzyloxy-1H-quinone Lin-2-one

A stirred solution of the product of Preparation 13 (1.46 g, 3 mmol) and 1,3-cyclohexanebis(methylamine) (426 mg, 3 mmol) in DMSO (3 mL) was heated at 100 °C for 6 h. The reaction mixture was allowed to cool, then diluted with dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL). The organic layer was dried ( _MgSO4 ) and the solvent was removed under reduced pressure. The crude residue was purified by flash chromatography (10% MeOH/DCM and 0.5% _NH4OH ) to afford the title compound (775 mg, 50% yield) as a solid. MS m _{/ z for C32H47N3O3Si} : [M+H ⁺ ] calcd. 550.3; found 550.6.

Preparation 51

Biphenyl-2-ylcarbamate 1-{2-[(3-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydro-quinolin-5-yl )-2-(tert-Butyldimethylsilyloxy)ethylamino]methyl}-cyclohexylmethyl)carbamoyl]ethyl}piperidin-4-yl ester

To a stirred solution of the product of Preparation 50 (552 mg, 1.01 mmol), the product of Preparation 20 (309 mg, 0.84 mmol) and HATU (384 mg, 1.01 mmol) in DMF (1.68 mL) was added DIPEA (190 μL, 1.09 mmol) . The reaction mixture was stirred overnight at 50 °C, then the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane (20 mL), and washed with saturated aqueous sodium bicarbonate (10 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (5-10% MeOH/DCM) to afford the title compound (267 mg, 36% yield) as a solid. LCMS (10-70) _Rt = 5.04. MS m _/ z _{for C53H69N5O6Si} : [M+H ⁺ ] calcd. _900.5 ; found 900.6.

Preparation 52

Biphenyl-2-ylcarbamate 1-{2-[(3-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl) -2-Hydroxyethylamino]methyl}cyclohexylmethyl)carbamoyl]ethyl}piperidin-4-yl ester

To a stirred solution of the product from Preparation 51 (267 mg, 0.30 mmol) in dichloromethane (3 mL) was added triethylamine trihydrofluoride (98 μL, 0.6 mmol). The reaction mixture was stirred for 10 h, then it was diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure to give the title compound (236 mg, 100% yield) as a solid. MS m/ _z for _C47H55N5O6 : [M+H ⁺ ] calcd _. 786.4; found _786.5 .

Example 14

Biphenyl-2-ylcarbamate 1-{2-[(3-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5- Base) ethylamino] methyl}-cyclohexylmethyl)carbamoyl]ethyl}-piperidin-4-yl ester

Palladium (10 wt.% (dry basis) on activated carbon) (120 mg) was added to a stirred solution of the product of Preparation 52 (236 mg, 0.30 mmol) in ethanol (3 mL). The reaction mixture was placed under hydrogen atmosphere and stirred overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (27 mg, 2 TFA salt). HPLC (10-70) _Rt = 2.76. MS m/ _{z for C40H49N5O6 :} [M+H ⁺ ] calcd. 696.4; found _696.6 .

Preparation 53

1-{2-[((1R,3S)-3-aminocyclopentanecarbonyl)amino]-ethyl}piperidin-4-ylcarbamate

To the product of Preparation 39 (318 mg, 0.94 mmol), (1R,3S)-3-tert-butoxycarbonylaminocyclopentanecarboxylic acid (258 mg, 1.1 mmol) and HATU (428 mg, 1.1 mmol) in DMF (5 mL) To the stirred solution in , DIPEA (245 μL, 1.09 mmol) was added. The reaction mixture was stirred overnight at room temperature, then the solvent was removed under reduced pressure. The resulting residue was dissolved in dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL). The organic layer was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (5-10% MeOH/DCM) and then dissolved in a trifluoroacetic acid/DCM mixture (1 mL/5 mL) and stirred at room temperature for 1 h. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and washed with 1M sodium hydroxide (10 mL), dried (magnesium sulfate) and reduced solvent to afford the title compound (167 mg, 39% yield).

Preparation 54

Biphenyl-2-ylcarbamate 1-[2-({(1R,3S)-3-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline -5-yl)-2-(tert-butyldimethylsilyloxy)ethylamino]-cyclopentanecarbonyl}amino)ethyl]piperidin-4-yl ester

A stirred solution of the product of Preparation 53 (167 mg, 0.38 mmol) and the product of Preparation 13 (92 mg, 0.19 mmol) in DMSO (0.38 mL) was heated at 90 °C for 5 h. The solution was cooled and diluted with ethyl acetate (10 mL), then washed with saturated aqueous sodium bicarbonate (5 mL). The organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (5-10% MeOH/DCM) to afford the title compound (343 mg, 100% yield). LCMS (10-70) _Rt = 4.97. MS m/z for _C50H63N5O6Si : [M+H ⁺ ] calcd. _858.5 ; found _{858.8 .}

Preparation 55

Biphenyl-2-ylcarbamate 1-[2-({(IR,3S)-3-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinoline -5-yl)-2-hydroxyethylamino]cyclopentanecarbonyl}amino)ethyl]-piperidin-4-yl ester

To a stirred solution of the product of Preparation 54 (343 mg, 0.4 mmol) in THF (2 mL) was added triethylamine trihydrofluoride (130 μL, 0.8 mmol). The reaction mixture was stirred for 10 h, then diluted with EtOAc (10 mL). The reaction mixture was washed with saturated aqueous sodium bicarbonate (5 mL), then the organic phase was dried (magnesium sulfate) and the solvent was removed under reduced pressure to afford the title compound (298 mg, 100% yield) as a solid. HPLC (10-70) _Rt = 2.8. MS m/ _{z for C44H49N5O6} : [M+H ⁺ ] calcd. _744.4 ; found 744.4.

Example 15

Biphenyl-2-ylcarbamate 1-[2-({(1R,3S)-3-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2,3, 4-tetrahydroquinolin-5-yl)ethylamino]-cyclopentanecarbonyl}amino)ethyl]-piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product from Preparation 55 (236 mg, 0.40 mmol) in ethanol (3 mL) was added palladium (10 wt.% (dry basis)) on charcoal (120 mg). The reaction mixture was placed under an atmosphere of hydrogen and stirred overnight The reaction mixture was filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (3 mg, 2 TFA salt). HPLC (5-75) R _t = 2.18.C ₃₇ H ₄₅ N ₅ O ₆ MS m/z: Calcd. for [M+H ⁺ ] 656.3; found 656.2.

Preparation 56

4-(tert-butoxycarbonylaminomethyl)-2-chlorophenylamine

A stirred solution of 4-aminomethyl-2-chlorophenylamine (940 mg, 6 mmol) and di-tert-butyldicarbonate (1.44 g, 6.6 mmol) in dichloromethane (30 mL) was stirred at room temperature 4 h, at which point the reaction was complete as determined by LCMS. The reaction mixture was then washed with saturated aqueous sodium bicarbonate (15 mL) and the organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure. Recrystallization of the obtained orange solid from ethyl acetate afforded the title intermediate as a white solid (~100% yield).

Preparation 57

N-[4-(tert-butoxycarbonylaminomethyl)-2-chlorophenyl]acrylamide

To a stirred solution of the product from Preparation 56 (1.54 g, 6.0 mmol) in a mixture of diethyl ether (35 mL) and 1M sodium hydroxide (35 mL) was added acryloyl chloride (687 μL, 8.45 mmol) dropwise. After 1 h, the organic layer was separated, dried ( _Na2SO4 ) and the solvent was removed _under reduced pressure to afford the title intermediate (1.8 g, 96% yield) as a white solid.

Preparation 58

1-[2-(4-(tert-butoxycarbonylaminomethyl)-2-chlorophenylcarbamoyl)ethyl]piperidin-4-ylcarbamate

A solution of the product of Preparation 8 (1.04 g, 3.5 mmol) and the product of Preparation 57 (1.19 g, 3.85 mmol) in a mixture of dichloromethane and methanol (12 mL, 1:1) was heated at 60° C. for 12 h. The reaction mixture was allowed to cool and the solvent was removed under reduced pressure. The crude material was purified by column chromatography (5-10% MeOH/DCM) to afford the title intermediate (2.00 g, 94% yield) as a white solid.

Preparation 59

1-[2-(4-Aminomethyl-2-chlorophenylcarbamoyl)ethyl]-piperidin-4-ylcarbamate

A solution of the product of Preparation 58 (2.00 g, 3.3 mmol) in dichloromethane (24 mL) and TFA (8 mL) was stirred for 1 h, then the solvent was removed under reduced pressure. The crude reaction mixture was dissolved in dichloromethane (30 mL) and washed with 1M sodium hydroxide (2 x 30 mL). Drying ( _Na2SO4 ) of the organic layer and removal of solvent under reduced _pressure afforded the title intermediate (1.46 g, 88% yield) as an oily white solid.

Prepare 60

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)- 2-tert-butyldimethylsilyloxy)ethylamino]methyl}-2-chlorophenylcarbamoyl)-ethyl]piperidin-4-yl ester

A stirred solution of the product of Preparation 59 (1.41 g, 2.79 mmol) and the product of Preparation 13 (680 mg, 1.39 mmol) in DMSO (1.39 mL) was heated at 90 °C for 8 h and then cooled to room temperature. The reaction mixture was diluted with ethyl acetate/chloroform (20 mL, 1/1), and the organic layer was washed with saturated aqueous sodium bicarbonate (10 mL), dried (Na ₂ SO ₄ ) and the solvent was removed under reduced pressure. The resulting crude residue was purified by column chromatography (5-10% MeOH/DCM) to afford the title intermediate (1.12 g, 88% yield) as a white solid. MS m/z M+H ⁺ = 914.9.

Preparation 61

Biphenyl-2-yl-carbamic acid 1-[2-(4-{[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl) -2-Hydroxyethylamino]methyl}-2-chloro-phenylcarbamoyl)ethyl]piperidin-4-yl ester

To a stirred solution of the product of Preparation 60 (1.12 g, 1.23 mmol) in dichloromethane (12 mL) was added _Et3N.3HF (401 μL, 0.6 mmol). The reaction mixture was stirred for 10 h, then diluted with dichloromethane (10 mL). The mixture was washed with saturated aqueous sodium bicarbonate (5 mL) and the organic layer was dried (Na ₂ SO ₄ ) and the solvent was removed under reduced pressure to afford the title intermediate (959 mg, 100% yield) as a white solid. MS m/z M +H ⁺ = 800.5.

Example 16

Biphenyl-2-ylcarbamate 1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline -5-yl)ethylamino]methyl}phenylcarbamoyl)ethyl]piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product from Preparation 61 (959 mg, 1.2 mmol) in ethanol (12 mL) was added Pd/C (290 mg) and the reaction mixture was placed under an atmosphere of hydrogen and stirred overnight. The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (67 mg, 2 TFA salt). HPLC (10-70) _Rt = 2.76; MS m/z M+H ⁺ = 710.6.

Preparation 62

2-Chloroethanesulfonic acid (5-tert-butoxycarbonylaminopentyl)amide

To a stirred solution of 5-(tert-butoxycarbonylamino)pentylamine (1.00 g, 4.94 mmol) and triethylamine (689 μLg, 4.94 mmol) in dichloromethane (22 mL) was added at 0 °C 2-Chloro-1-ethanesulfonyl chloride (470 μL, 4.50 mmol). The reaction mixture was stirred at room temperature for 2 h, then washed with saturated aqueous sodium bicarbonate (15 mL). The organic layer was dried ( _Na2SO4 ) and _the solvent was removed under reduced pressure to afford the title compound (100% yield) which was used in the next step without further purification.

Preparation 63

1-[2-(5-tert-butoxycarbonylaminopentylsulfamoyl)-ethyl]piperidin-4-ylcarbamate

A solution of the product of Preparation 8 (1.33 g, 3.5 mmol) and the product of Preparation 62 (1.62 g, 4.94 mmol) in dichloromethane and methanol (22 mL, 1:1) was heated at 60° C. for 5 h. The reaction mixture was allowed to cool to room temperature and the solvent was removed under reduced pressure. The crude residue was dissolved in dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate (10 mL). The organic layer was then dried ( _Na2SO4 ) and _the solvent was removed under reduced pressure. The crude residue was purified by column chromatography (5-10% MeOH/DCM) to give the title intermediate (1.6 g, 55%) as a white solid. MS m/z M+H ⁺ = 589.6.

Preparation 64

1-[2-(5-aminopentylsulfamoyl)ethyl]piperidin-4-ylcarbamate

A solution of the product of Preparation 63 (1.6 g, 2.72 mmol) in dichloromethane (21 mL) and TFA (7 mL) was stirred for 1 h, then the solvent was removed under reduced pressure. The crude reaction mixture was dissolved in dichloromethane (30 mL) and washed with 1M sodium hydroxide (2 x 30 mL). The organic layer was dried ( _Na2SO4 ) and the solvent was removed under reduced pressure to afford the title intermediate (1.19 g, 90% yield) as _an oily white solid.

Preparation 65

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2 -(tert-Butyldimethylsilyloxy)ethylamino]pentylsulfamoyl}ethyl)piperidin-4-yl ester

A stirred solution of the product of Preparation 64 (917 mg, 1.88 mmol) and the product of Preparation 13 (460 mg, 0.94 mmol) in DMSO (0.92 mL) was heated at 90 °C for 8 h and then cooled to room temperature. The reaction mixture was diluted with ethyl acetate/chloroform ₍ 20 mL, 1/1), dried ( _Na2SO4 ) and the solvent was removed under reduced pressure. The resulting crude residue was purified by column chromatography (3-6% MeOH/DCM) to give the title intermediate (500 mg, 60% yield) as a white solid. MS m/z M+H ⁺ = 896.9.

Preparation 66

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2 -Hydroxyethylamino]pentylsulfamoyl}ethyl)piperidin-4-yl ester

To a stirred solution of the product of Preparation 65 (500 mg, 0.56 mmol) in dichloromethane (5.6 mL) was added triethylamine trihydrofluoride (183 μL, 1.12 mmol). The reaction mixture was stirred for 10 h and dichloromethane (10 mL) was added. The resulting mixture was washed with saturated aqueous sodium bicarbonate (5 mL). The organic layer was dried ( _Na2SO4 ) and the solvent was removed under reduced pressure to afford the title _intermediate (437 mg, 100% yield. MS m/z M+H ⁺ = 782.8) as a yellow solid.

Example 17

Biphenyl-2-ylcarbamate 1-(2-{5-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl) Ethylamino]pentylsulfamoyl}ethyl)piperidin-4-yl ester ditrifluoroacetate

To a stirred solution of the product of Preparation 66 (437 mg, 0.56 mmol) in ethanol/methanol (5.6 mL, 1/1 ) was added Pd/C (131 mg) and the reaction mixture was placed under hydrogen atmosphere and stirred overnight . The reaction mixture was then filtered and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (71 mg) as the bistrifluoroacetate salt. HPLC (10-70) _Rt = 2.59; MS m/z M+H ⁺ = 692.6.

Preparation 67

1-{2-[(4-formylbenzenesulfonyl)methylamino]-ethyl}piperidin-4-ylcarbamate

To a stirred solution of the product of Preparation 26 (350 mg, 1 mmol) and triethylamine (167 μL, 1.2 mmol) in dichloromethane (5 mL) was added 4-formylbenzenesulfonyl chloride (225 mg, 1.1 mmol ). After 1 h at room temperature, the reaction was monitored for completion by MS, and the reaction mixture was washed with saturated aqueous sodium bicarbonate (5 mL). The organic layer was then dried ( _Na2SO4 ) and the solvent was removed under reduced pressure _to afford the title intermediate (323 mg, 62% yield). MS m/z M+H ⁺ = 522.4.

Preparation 68

Biphenyl-2-ylcarbamate 1-{2-[(4-{[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2-oxo-1 , 2-Dihydroquinolin-5-yl)ethylamino]methyl}benzenesulfonyl)-methylamino]ethyl}piperidin-4-yl ester

5-[(R)-2-Amino-1-(tert-butyldimethylsilyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one (293 mg, 0.74 mmol) and Preparation 67 A solution of the product in dichloromethane and methanol (6.2 mL, 1/1) was stirred at room temperature for 1 h, then sodium triacetoxyborohydride (394 mg, 1.86 mmol) was added. The reaction mixture was stirred for 4 h and was determined to be complete by MS. The reaction mixture was then acidified with concentrated hydrochloric acid and the solvent removed under reduced pressure to afford the title compound which was used in the next step without further purification. MS m/z M+H ⁺ = 840.8.

Example 18

Biphenyl-2-ylcarbamate 1-{2-[(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5- Base)ethylamino]methyl}benzenesulfonyl)methylamino]ethyl}-piperidin-4-yl ester ditrifluoroacetate

A stirred solution of the product of Preparation 68 (520 mg, 0.62 mmol) in 1M hydrochloric acid (5 mL) and acetonitrile (5 mL) was heated at 60 °C for 8 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The crude residue was purified by preparative HPLC to give the title compound (220mg) as the bistrifluoroacetate salt. HPLC (10-70) _Rt = 2.77; MS m/z M+H ⁺ = 726.7.

Preparation 69

(3-Aminomethylphenyl)methanol hydrochloride

(a) (3-tert-butoxycarbonylmethylphenyl)methanol

Borane dimethyl sulfide (2.05 mL, 21.6 mmol) was added to a solution of 3-(tert-butoxycarbonylaminomethyl)benzoic acid (1.81 g, 7.20 mmol) in tetrahydrofuran (24 mL) and heated at room temperature The resulting mixture was stirred for 3 hours. The resulting mixture was then diluted with ethyl acetate (20 mL) and the layers were separated. The organic layer was washed with saturated sodium bicarbonate, saturated sodium chloride, dried over magnesium sulfate and concentrated to give the title compound (1.71 g) as a yellow oil.

(b) (3-Aminomethylphenyl)methanol hydrochloride

烷中的溶液(9mL，36mmol)，并在室温搅拌得到的混合物1h。然后浓缩反应混合物，并用二乙基醚(50mL)稀释该残留物和过滤得到呈白色固体的标题化合物(1.09g)。To the product of step (a) (1.71 g, 7.2 mmol) was added 4M hydrochloric acid in di

solution in alkanes (9 mL, 36 mmol), and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was then concentrated and the residue was diluted with diethyl ether (50 mL) and filtered to give the title compound (1.09 g) as a white solid.

Preparation 70

1-{2-[3-(3-Hydroxymethylbenzyl)ureido]ethyl}piperidin-4-ylcarbamate

A 0.2M solution of the product of Preparation 35 (760 mg, 2.24 mmol) in N,N-dimethylformamide was added dropwise to 1,1'-carbonyldiimidazole (364 mg, 2.24 mmol) and diisopropylethylamine (0.31 mL, 2.24 mmol) in N,N-dimethylformamide (11 mL) and the resulting mixture was stirred at room temperature for 2 h. Diisopropylethylamine (0.31 mL, 2.24 mmol) and the product of Preparation 69 (578 mg, 3.4 mmol) were added and the mixture was stirred at 50°C for 12 hours. The reaction mixture was then concentrated to dryness, and the residue was diluted with dichloromethane (20 mL) and the solution was washed with saturated sodium bicarbonate (2x), saturated sodium chloride, dried over magnesium sulfate, and concentrated to afford the title compound ( 1.12g). LCMS (2-90) _Rt = 4.01 min.; MSm/z M+H = 503.5.

Preparation 71

1-{2-[3-(3-formylbenzyl)ureido]ethyl}piperidin-4-yl biphenyl-2-ylcarbamate

A solution of the product of Preparation 70 (1.12 g, 2.23 mmol) in dichloromethane (11.1 mL) was cooled to 0 °C and diisopropylethylamine (1.17 mL, 6.70 mmol) and dimethylsulfoxide ( 0.949 mL, 13.4 mmol). After about 10 minutes, pyridine sulfur trioxide complex (1.06 g, 6.70 mmol) was added and the resulting mixture was stirred at 0 °C for 2 h. The reaction was then quenched with water (15 mL) and the organic layer was washed with cold water (3x), dried over magnesium sulfate and concentrated to give the title compound (609 mg) as a yellow crumbly mass. LCMS (2-90) _Rt = 4.13 min; MS m/z M+H = 501.3.

Preparation 72

Biphenyl-2-ylcarbamate 1-{2-[3-(3-{[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2- Oxy-1,2-dihydroquinolin-5-yl)ethylamino]methyl}benzyl)ureido]ethyl}-piperidin-4-yl ester

5-[(R)-2-Amino-1-(tert-butyldimethylsilyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one (575 mg, 1.40 mmol) was added to Preparation 71 A solution of the product (609 mg, 1.2 mmol) and diisopropylamine (0.25 mL, 1.40 mmol) in dichloromethane (6 mL) was stirred at room temperature for 45 min. Sodium triacetoxyborohydride (385 mg, 1.80 mmol) was then added and the mixture was stirred at room temperature for 12 h. The reaction was then quenched with 10% aqueous hydrochloric acid (5 mL) and the layers were separated. The organic layer was washed with saturated sodium bicarbonate, saturated sodium chloride, dried over magnesium sulfate and concentrated to give the title compound (1.1 g). HPLC (10-70) _Rt = 3.55 min; MS m/z M+H = 819.7.

Example 19

Biphenyl-2-ylcarbamate 1-{2-[3-(3-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline- 5-yl)ethylamino]methyl}benzyl)ureido]ethyl}piperidin-4-yl ester ditrifluoroacetate

Triethylamine trihydrofluoride (2.4 mL, 13.6 mmol) was added to a solution of the product of Preparation 72 (1.1 g, 1.36 mmol) in dichloromethane (2 mL), and the resulting mixture was stirred at room temperature for 15 h. The reaction mixture was then concentrated in vacuo to dryness, and the residue was dissolved in a 1:1 mixture of water and acetonitrile with 0.1% TFA. The mixture was purified by HPLC (5-35 over 60 min) to give (296 mg, purity 99%) of the title compound. MS m/z M+H = 705.6.

Preparation 73

1-[(E)-3-(4-nitrophenyl)allyl]piperidin-4-yl biphenyl-2-ylcarbamate

The product of Preparation 8 (2.96 g, 0.01 mol) and p-nitrocinnamaldehyde (1.77 g, 0.01 mol) were stirred in 50 mL of dichloromethane for 2 h. Sodium triacetoxyborohydride (6.33 g, 0.03 mol) was added and the resulting mixture was stirred for 2 h. The reaction was then quenched with 10 mL of water and the mixture was diluted with dichloromethane (100 mL). The organic layer was washed with saturated sodium bicarbonate (2x), brine, dried over _Na2SO4 , filtered and concentrated to give the title compound (3.8 g, 80% yield ₎ as a yellow foam.

Preparation 74

1-[3-(4-aminophenyl)propyl]piperidin-4-yl biphenyl-2-ylcarbamate

The product of Preparation 73 (2.5 g, 5.4 mmol) was dissolved in 100 mL of ethanol and the resulting solution was purged with nitrogen for 30 minutes. Then, palladium on carbon (2.5 g; 50% w/w water; 10% Pd; 1.1 mmol Pd) was added while degassing with nitrogen. The mixture was then placed under hydrogen (50 psi) until no more hydrogen was consumed (-30 minutes). The mixture was then purged with nitrogen, filtered through celite and concentrated. The residue was dissolved in ethyl acetate and the mixture was washed with saturated sodium bicarbonate (2x), brine, dried ( _Na2SO4 ), filtered and concentrated to afford the title compound (2.08 _g , 90% yield). MS m/z M+H = 430.5.

Preparation 75

Biphenyl-2-ylcarbamate 1-{3-[4-(4-{2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydro-quinoline -5-yl)-2-(tert-butyldimethylsilyloxy)ethylamino]ethyl}phenylamino)-phenyl]propyl}piperidin-4-yl ester

To a 25 mL round bottom flask was added the product of Preparation 74 (400 mg, 0.8 mmol); 8-Benzyloxy-5-[(R)-2-[2-(4-bromophenyl)ethylamino]-1- (tert-butyldimethylsilyloxy)ethyl]-1H-quinolin-2-one (769mg, 1.2mmol); Tris(dibenzylideneacetone)dipalladium(0) (73mg, 0.08mmol, 20 % Pd); and 2-(dicyclohexylphosphine)biphenyl (84 mg, 0.24 mmol). The mixture was purged with nitrogen, then dried, degassed toluene (8 mL, 0.1 M) was added and the resulting mixture was heated at 70 °C for 30 min.

Sodium tert-butoxide (382mg, 4.0mmol) was then added and the temperature was raised to 95°C for 4h, at which point LCMS showed complete consumption of the product from Preparation 74 and a large product peak (M+H=956.7). The reaction mixture was then cooled to room temperature and diluted with ethyl acetate. The mixture was washed with saturated sodium bicarbonate (2x), brine _, dried ( _Na2SO4 ), filtered and concentrated to give the title compound (1.5 g) which was used without further purification.

Preparation 76

Biphenyl-2-ylcarbamate 1-{3-[4-(4-{2-[(R)-2-(8-benzyloxy-2-oxo-1,2-dihydro-quinoline -5-yl)-2-hydroxyethylamino]ethyl}phenylamino)phenyl]propyl}piperidin-4-yl ester

The product of Preparation 75 was dissolved in dichloromethane (10 mL), and triethylamine trihydrofluoride (10 eq.) was added. The reaction mixture was stirred overnight, then diluted with dichloromethane, and _the organic layer was washed with saturated sodium bicarbonate (2x), brine, dried ( _Na2SO4 ), filtered and concentrated to afford 1.3 g of crude product. This material was purified by silica gel chromatography (DCM, increasing to 50% methanol) to afford the title compound (300 mg, ~75% purity) which was used without further purification.

Example 20

Biphenyl-2-ylcarbamate 1-{3-[4-(4-{2-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinone Phenyl-5-yl)ethylamino]ethyl}phenylamino)phenyl]propyl}piperidin-4-yl ester ditrifluoroacetate

The product of Preparation 76 (300 mg) was dissolved in 10 mL of ethanol and the mixture was purged with nitrogen for 15 minutes. Palladium on carbon (10% Pd, 50% w/w water, 0.2 equiv Pd) was added while degassing. The resulting mixture was then placed under 1 atm of hydrogen for 2 h at which time the reaction was complete as determined by LCMS. The solution was then purged with nitrogen for 15 min, then filtered through celite and concentrated. Purification by preparative HPLC afforded the title compound (59 mg, >95% pure) as the ditrifluoroacetate salt. MS m/z M+H = 752.8.

Preparation 77

1-[2-fluoro-3-(4-hydroxymethylpiperidin-1-ylmethyl)-benzyl]piperidin-4-ylcarbamate

The product of Preparation 8 (500 mg, 1.69 mmol), 2,6-bis(bromomethyl)-1-fluorobenzene (476 mg, 1.69 mmol), piperidin-4-ylmethanol (195 mg, 1.69 mmol) and potassium carbonate ( 466 mg, 3.37 mmol) was suspended in acetonitrile (5 mL) and stirred at room temperature for 18 h. The reaction mixture was then concentrated and the residue was dissolved in dichloromethane/water. The layers were separated and the organics were washed with water (2x), brine layer, dried ( _MgSO4 ) and concentrated. The crude material was purified by silica gel column chromatography eluting with 3% methanol/chloroform to give the title compound (282 mg) as a white foam. MS m/z M+H = 532.3.

Preparation 78

1-[2-Fluoro-3-(4-formylpiperidin-1-ylmethyl)benzyl]-piperidin-4-ylcarbamate

The product of Preparation 77 (282 mg, 0.53 mmol) was dissolved in dichloromethane, and to the mixture were added diisopropylethylamine (280 μL, 1.6 mmol) and dimethylsulfoxide (115 μL, 1.6 mmol). The reaction mixture was cooled at -15°C under nitrogen, and pyridine sulfur trioxide complex (255 mg, 1.6 mmol) was added, and the resulting mixture was stirred for 40 min. The reaction was then quenched with water and the layers were separated. The organic layer was washed with aqueous _NaH2PO4 (1M x 3), brine, dried ( _MgSO4 ) and concentrated to give the title _compound as a foam (253mg). MS m/z M+H = 530.4.

Example 21

Biphenyl-2-ylcarbamate 1-[2-fluoro-3-(4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline -5-yl)ethylamino]methyl}piperidin-1-ylmethyl)benzyl]piperidin-4-yl ester ditrifluoroacetate

The product of Preparation 78 (253 mg, 0.48 mmol) was dissolved in a 1:1 mixture of dichloromethane and methanol (6 mL), and to this mixture was added 5-[(R)-2-amino-1-(tert-butyl Dimethylsilyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one acetate (228mg, 0.58mmol) and sodium triacetoxyborohydride (317mg, 1.5mmol). The reaction mixture was stirred at room temperature under nitrogen for 18 h, then concentrated. The residue was dissolved in a 2:3 mixture of acetonitrile and aqueous 6N hydrochloric acid, and the mixture was heated at 55°C for 4 hours. The reaction mixture was then concentrated and the residue was dissolved in water/acetonitrile/trifluoroacetic acid (1:1:0.005) and purified by reverse phase column chromatography to give the title compound (175mg) as a white solid. MS m/z M+H = 734.5.

Preparation 79

2-[4-(3-Bromopropoxy)phenyl]ethanol

To a solution of 4-hydroxyphenethyl alcohol (4.37 g, 31.0 mmol) and potassium carbonate (6.55 g, 47.0 mmol) in acetonitrile (62.0 mL) was added 1,3 dibromopropane (31.0 mL, 316 mmol). The reaction mixture was heated to 70°C for 12 hours, then cooled to room temperature, filtered and concentrated in vacuo. The resulting oil was purified by silica gel chromatography using a 4:1 mixture of hexanes and ethyl acetate to afford the title compound (6.21 g) as a white solid.

Preparation 80

1-{3-[4-(2-Hydroxyethyl)phenoxy]propyl}piperidin-4-ylcarbamate

To a solution of the product of Preparation 79 (1.11 g, 4.30 mmol) and diisopropylethylamine (0.90 mL, 5.10 mmol) in acetonitrile (21.5 mL) was added the product of Preparation 8 (1.27 g, 4.30 mmol) and The resulting mixture was stirred at 60 °C for 12 h. The reaction mixture was then diluted with dichloromethane (20 mL) and washed with saturated sodium bicarbonate (25 mL), saturated sodium chloride (25 mL), dried over magnesium sulfate and concentrated to afford the title compound (1.98 g, 85% purity ). MS m/z M+H = 475.5.

Preparation 81

1-{3-[4-(2-Oxoethyl)phenoxy]propyl}piperidin-4-yl biphenyl-2-ylcarbamate

A solution of the product of Preparation 80 (723 mg, 1.53 mmol) and dichloromethane (75 mL) was cooled to about 5 °C and diisopropylethylamine (798 mL, 4.58 mmol) and dimethylsulfoxide (649 mL, 9.15 mmol). Pyridinesulfur trioxide (728mg, 4.58mmol) was then added and the resulting mixture was stirred at 5°C for 45min. The reaction mixture was then diluted with dichloromethane (20 mL), washed with saturated sodium bicarbonate (25 mL), saturated sodium chloride (25 mL), dried over magnesium sulfate and concentrated to give the title compound (604 mg). MS m/z M+H = 473.4.

Preparation 82

Biphenyl-2-ylcarbamate 1-[3-(4-{2-[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2-oxo- 1,2-Dihydroquinolin-5-yl)ethylamino]ethyl}-phenoxy)propyl]piperidin-4-yl ester

The product of Preparation 81 (604 mg, 1.28 mmol) was dissolved in methanol (6.4 mL) and 5-[(R)-2-amino-1-(tert-butyldimethylsilyloxy)ethyl]- 8-Hydroxy-1H-quinolin-2-one (605 mg, 1.53 mmol) and diisopropylethylamine (0.27 mL, 1.53 mmol). Sodium triacetoxyborohydride (405 mg, 1.91 mmol) was then added and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was then concentrated to dryness, and the residue was diluted with ethyl acetate (20 mL) and the solution was washed with saturated sodium bicarbonate (25 mL), saturated sodium chloride (25 mL), dried over magnesium sulfate and concentrated to afford the title compound (704mg). MS m/z M+H = 791.8.

Example 22

Biphenyl-2-ylcarbamate 1-[3-(4-{2-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]ethyl}phenoxy)propyl]piperidin-4-yl ester ditrifluoroacetate

Triethylamine trihydrofluoride (1.5 mL, 8.87 mmol) was added to a solution of the product of Preparation 82 (702 mg, 0.89 mmol) in dichloromethane (4.5 mL) and the resulting mixture was stirred at room temperature for 24 h. The mixture was then concentrated in vacuo and purified by HPLC (2-35 over 90 min) to give the title compound (92 mg) as a white powder. MS m/z M+H = 677.4.

Preparation 83

Methyl 4-iodophenyl acetate

烷(10mL)中的4N盐酸。在室温搅拌该反应混合物24h，然后减压除去溶剂得到标题化合物(5.17g，产率98％)，其被使用而无需进一步纯化。To a stirred solution of 4-iodophenylacetic acid (5.0 g, 19.1 mmol) in MeOH (200 mL) was added di

4N hydrochloric acid in alkanes (10 mL). The reaction mixture was stirred at room temperature for 24 h, then the solvent was removed under reduced pressure to afford the title compound (5.17 g, 98% yield), which was used without further purification.

Preparation 84

Methyl[4-(4-hydroxybut-1-ynyl)phenyl]acetate

To a stirred solution of the product of Preparation 83 (4.5 g, 16.3 mmol) in diethylamine (100 mL) was added but-3-yn-1-ol (1.9 mL, 32.6 mmol), Pd( _PPh3 ) ₂ Cl ₂ (500 mg, 1.63 mmol) and Cul (154 mg, 0.815 mmol) and the resulting mixture was stirred at room temperature for 17 h. The solvent was then removed under reduced pressure and the residue was dissolved in diethyl ether (200 mL) and the solution was filtered to remove salts. The solvent was then removed under reduced pressure and the crude product was purified by silica gel chromatography (60% EtOAc/hexanes) to afford the title intermediate (3.03 g, 91% yield).

Preparation 85

Methyl[4-(4-hydroxybutyl)phenyl]acetate

A stirred solution of the product of 84 (2.8 g, 12.8 mmol) in methanol (50 mL) was prepared with a nitrogen sparge, followed by the addition of 10% palladium on carbon (400 mg, 20% wt/wt). The reaction flask was then alternately placed under vacuum and flushed with nitrogen for several cycles, then stirred under hydrogen for 14 h. The reaction mixture was flushed with nitrogen, then filtered and the solvent removed under reduced pressure to afford the title compound (2.75 g, 97% yield) which was used without further purification.

Preparation 86

Methyl(4-{4-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]butyl}phenyl)acetate

(a) Methyl {4-[4-(toluene-4-sulfonyloxy)butyl]phenyl} acetate

To a stirred solution of the product of Preparation 85 (2.6 g, 12.5 mmol) in THF (100 mL) was added DABCO (2.6 g, 25.0 mmol) followed by p-toluenesulfonyl chloride (2.44 g, 13.75 mmol). The reaction mixture was stirred at room temperature for 23 h, then the solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (200 mL). The organic layer was then washed with water (2 x 100 mL), 1N hydrochloric acid (100 mL) and saturated aqueous sodium chloride (100 mL), dried (MgSO ₄ ), filtered and the solvent removed under reduced pressure to afford the title compound which was used without further purification.

(b) Methyl (4-{4-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]butyl}phenyl)acetate

To the crude product from step (a) were added DMF (50 mL), diisopropylethylamine (3.0 mL, 17.3 mmol) and the product of Preparation 8 (2.4 g, 8.1 mmol). The reaction mixture was stirred at room temperature for 18 h, then the solvent was removed under reduced pressure to afford the title compound (3.5 g, 86.3% yield). MS m/z 501.6 (MH ⁺ ), _Rf 4.89 min (10-70% ACN:water, reverse phase HPLC).

Preparation 87

1-{4-[4-(2-hydroxyethyl)phenyl]butyl}piperidin-4-yl biphenyl-2-ylcarbamate

To a stirred solution of the product of Preparation 86 (2.0 g, 4.0 mmol) in THF (100 mL) was added DIBAL (24 mL, 24 mmol, 1.0 M in THF) dropwise. After the addition was complete, the reaction mixture was stirred for 3 h, then quenched by the slow addition of methanol (until gas evolution ceased). The mixture was then stirred for 30 min before ethyl acetate (200 mL) and 1 N aqueous sodium hydroxide solution (200 mL) were added. The organic layer was separated and washed with saturated aqueous sodium chloride (100 mL), dried ( _MgSO4 ), filtered and the solvent removed under reduced pressure to afford the title compound (1.3 g, 69% yield) which was used without further purification. MS m/z 473.4 (MH ⁺ ), _Rf 4. 53 min (10-70% ACN: _H2O , reverse phase HPLC).

Example 23

Biphenyl-2-ylcarbamate 1-[2-(4-{2-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]ethyl}phenyl)ethyl]piperidin-4-yl ester

To a stirred solution of the product of Preparation 87 (500 mg, 1.06 mmol) in dichloromethane (25 mL) was added dimethylsulfoxide (0.60 mL, 10.6 mmol) and diisopropylethylamine (0.921 mL, 5.3 mmol). The reaction mixture was then cooled to -10°C and pyridinic sulfur trioxide (842 mg, 5.3 mmol) was added. The reaction mixture was stirred for 1 h, then quenched by adding water (100 mL). The mixture was stirred for 10 minutes, then the organic layer was removed and washed with saturated aqueous sodium chloride (100 mL), dried ( _MgSO4 ), and filtered.

To the filtrate was added methanol (25 mL), 5-[(R)-2-amino-1-(tert-butyldimethylsilyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one Acetate (419 mg, 1.06 mmol) and sodium triacetoxyborohydride (468 mg, 2.12 mmol). The mixture was stirred for 16 h, then concentrated, and to the resulting mixture was added a 1:1 mixture of acetonitrile and aqueous 4N hydrochloric acid (20 mL). The mixture was heated at 50 °C for 17 h, then the solvent was removed under reduced pressure. To the residue was added a 1:1 mixture of acetic acid and water (8.0 mL), and the mixture was chromatographed on reverse phase silica gel (gradient elution, 10-50% ACN/water) to give the title compound (67 mg, Yield 7% over 3 steps). MS m/z (MH+) 675.5; _Rf 3.07 (10-70% ACN:water, reverse phase HPLC).

Preparation 88

Ethyl 3-[5-(2-ethoxycarbonylvinyl)phenylthio-2-yl]acrylate

To a stirred solution of sodium hydride (2.1 g, 53 mmol, 60% in mineral oil) in THF (200 mL) was added triethylphosphonoacetate (10 mL, 50 mmol), hydrogen gas evolution was observed, and the reaction was stirred Until the deflation stops (about 30min). To this reaction mixture was added 2,5-thiophenedicarbaldehyde (3 g, 21 mmol) and the reaction mixture was stirred for 1 h. The solvent was removed under reduced pressure and the residue was dissolved in dichloromethane (200 mL). The organic layer was washed with water (100 mL), aqueous 1N hydrochloric acid (100 mL), saturated aqueous sodium chloride (100 mL), dried (MgSO ₄ ), filtered and the solvent removed under reduced pressure to afford the title compound (5.8 g, 98% yield), It was used without further purification.

Preparation 89

3-[5-(2-Ethoxycarbonylethyl)phenylthio-2-yl]propanoic acid ethyl ester

A stirred solution of the product of 88 (5.8 g, 21 mmol) in methanol (200 mL) was prepared with a nitrogen flush and 10% palladium on carbon (576 mg, 10% wt/wt) was added. The reaction flask was alternately placed under vacuum and flushed with hydrogen for 3 cycles, then the reaction mixture was stirred under hydrogen for 1 h. The mixture was then flushed with nitrogen, filtered and the solvent removed under reduced pressure to afford the title compound (5.8 g, 99% yield) which was used without further purification.

Prepare 90

3-[5-(3-Hydroxypropyl)phenylthio-2-yl]propan-1-ol

To a stirred solution of DIBAL (88 mL, 88 mmol, 1.0 M in cyclohexane) in THF (300 mL) was added the product of Preparation 89 (5.0 g, 17.6 mmol) dropwise at -78 °C. After the addition was complete, the reaction mixture was warmed to room temperature over 30 min and quenched by the slow addition of aqueous 1N hydrochloric acid (200 mL). Dichloromethane (400 mL) was added and the layers were separated. The aqueous layer was washed with dichloromethane (4 x 100 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (100 mL), dried (MgSO ₄ ), filtered and the solvent removed under reduced pressure to afford the title compound (3.0 g, yield 85%), which was used without further purification.

Preparation 91

Biphenyl-2-ylcarbamate 1-{3-[5-(3-hydroxypropyl)phenylthio-2-yl]propyl}piperidin-4-yl ester

(a) 3-[5-(3-hydroxypropyl)phenylthio-2-yl]propyl toluene-4-sulfonic acid

To a stirred solution of the product of Preparation 90 (423 mg, 2.1 mmol) in THF (20 mL) was added DABCO (420 mg, 4.2 mmol) followed by p-toluenesulfonyl chloride (442 mg, 2.3 mmol). The reaction mixture was stirred at room temperature for 2 h, then the solvent was removed under reduced pressure, and the residue was dissolved in dichloromethane (200 mL). The organic layer was washed with water (2 x 100 mL), saturated aqueous sodium chloride (100 mL), dried ( _MgSO4 ), filtered and the solvent removed under reduced pressure to give the title compound which was used without further purification.

(b) 1-{3-[5-(3-hydroxypropyl)phenylthio-2-yl]propyl}piperidin-4-ylcarbamate

To the product from step (a) was added acetonitrile (20 mL), diisopropylethylamine (0.5 mL, 2.8 mmol) and the product of Preparation 8 (626 mg, 2.11 mmol). The reaction mixture was heated to 50 °C for 20 h, then cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (5% MeOH/DCM with 0.6% _NH3 (aq)) to afford the title compound (450 mg, 44% yield). MS m/z (MH ⁺ ) 479.6; _Rf 4.15 min (10-70% ACN:water, reverse phase HPLC).

Preparation 92

Biphenyl-2-ylcarbamate 1-[3-(5-{3-[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2-oxo- 1,2-Dihydroquinolin-5-yl)ethylamino]propyl}phenylthio-2-yl)propyl-piperidin-4-yl ester

To a stirred solution of the product from Preparation 91 (450 mg, 0.94 mmol) in dichloromethane (20 mL) was added dimethylsulfoxide (0.21 mL, 3.7 mmol) and diisopropylethylamine (0.65 mL, 3.7 mmol). The mixture was cooled to -10°C and pyridinesulfur trioxide (444mg, 2.8mmol) was added. The reaction mixture was stirred for 3 h, then quenched by the addition of water (100 mL). The mixture was stirred for 10 minutes, then the organic layer was removed and washed with saturated aqueous sodium chloride (100 mL), dried ( _MgSO4 ) and filtered.

To the filtrate was added methanol (20 mL), 5-[(R)-2-amino-1-(tert-butyldimethylsilyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one acetic acid ester (368 mg, 0.93 mmol), followed by sodium triacetoxyborohydride (412 mg, 1.86 mmol). The mixture was stirred for 19 h, then the mixture was concentrated to give the title compound which was used without further purification. MS m/z (MH ⁺ ) 795.8; _Rf 4.93 min (10-70% ACN:water, reverse phase HPLC).

Example 24

Biphenyl-2-ylcarbamate 1-[3-(5-{3-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline-5 -yl)ethylamino]propyl}phenylthio-2-yl)propyl]piperidin-4-yl ester

To the crude product from Preparation 92 was added a 1:1 mixture of acetonitrile and aqueous 4N hydrochloric acid (25 mL). The mixture was heated at 50 °C for 17 h, then the solvent was removed under reduced pressure. To the residue was added a 1:1 mixture of acetic acid and water (8.0 mL) and the mixture was chromatographed on reverse phase silica gel (elution gradient, 10-50% ACN/water) to afford the title compound (135 mg, 3 Step yield 16%). MS m/z (MH ⁺ ) 681.5; _Rf 3.03 (10-70% ACN:water, reverse phase HPLC).

Preparation 93

Methyl 4-amino-5-chloro-2-methoxybenzoate

To a solution of 4-amino-5-chloro-2-methoxybenzoic acid (1.008 g, 5.0 mmol) in a mixture of toluene (9 mL) and methanol (1 mL) was added dropwise (trimethylsilyl ) diazomethane (2.0M in hexane, 3.0 mL, 6.0 mmol). The reaction mixture was then warmed to room temperature and stirred for 16 h. Excess (trimethylsilyl)diazomethane was quenched by adding acetic acid until the bright yellow color of the reaction mixture disappeared. The mixture was then concentrated in vacuo to afford the title compound as an off-white solid which was used without further purification.

Preparation 94

Methyl 4-acrylamido-5-chloro-2-methoxybenzoate

To the crude product of Preparation 93 was added dichloromethane (10 mL, 0.5 M) and triethylamine (2.1 mL, 15 mmol). The mixture was cooled to 0° C., and acryloyl chloride (812 μL, 10 mmol) was added dropwise with stirring. After 2 h, the reaction was quenched at 0 °C by the addition of methanol (ca. 2 mL), and the resulting mixture was stirred at room temperature for 15 min, then concentrated in vacuo. Dichloromethane (30 mL) and water (30 mL) were added to the residue and the mixture was mixed thoroughly. The layers were separated, and the aqueous layer was extracted with dichloromethane (20 mL). The organic layers were combined, dried ( _Na2SO4 ), filtered and the solvent removed _in vacuo to give the title compound as a brown foamy solid which was used without further purification.

Preparation 95

Methyl 4-{3-[4-(biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-5-chloro-2-methoxybenzoate

To the crude product from Preparation 94 was added the product from Preparation 8 (1.33 g, 4.5 mmol) and a mixture of THF (22.5 mL) and methanol (2.5 mL). The mixture was heated with stirring at 50°C for 16 hours, then the solvent was removed in vacuo. Chromatography (silica gel; EtOAc) of the residue afforded the title compound as an off-white foamy solid (0.82 g; _Rf = 0.4, 29% yield over 3 steps). MS m/z _566.4 ( _M +H, expected _565.20 for _C30H32ClN3O6 ).

Preparation 96

1-[2-(2-chloro-4-hydroxymethyl-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylcarbamate

To a solution of the product of Preparation 95 (0.82 mg, 1.45 mmol) in a mixture of THF (4.5 mL) and methanol (0.5 mL) was added lithium borohydride (32 mg, 1.45 mmol) at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for 41 h. The reaction was then quenched at 0° C. by the addition of 1 N aqueous hydrochloric acid until no more bubbles were observed, and the mixture was stirred for 10 min. The solvent was removed in vacuo, and the residue was dissolved in acetonitrile (ca. 2 mL). The solution was purified by prep-RP-HPLC (gradient: 2-50% acetonitrile in water with 0.05% TFA). Appropriate fractions were collected, combined and lyophilized to afford the title compound as the trifluoroacetate salt. The salt was treated with isopropyl acetate (10 mL) and 1N aqueous sodium hydroxide (10 mL) and the organic layer was collected, dried (Na ₂ SO ₄ ), filtered and the solvent removed in vacuo to give the title compound as a white foamy solid (161 mg , 21% yield). MS m/z 538.4 (M+H, _{expected 537.20} for _{C29H32ClN3O5 )} .

Preparation 97

1-[2-(2-Chloro-4-formyl-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylcarbamate

To a solution of the product from Preparation 96 (161 mg, 0.3 mmol) in dichloromethane (3 mL) was added dimethylsulfoxide (213 μL, 3.0 mmol) and diisopropylethylamine (261 μL, 1.5 mmol). The mixture was cooled to -20°C and sulfur trioxide pyridine complex (238 mg, 1.5 mmol) was added slowly. After 30 minutes, the reaction mixture was quenched by adding water (approximately 3 mL). The layers were separated and the organic layer was dried ( _Na2SO4 ), filtered and the solvent removed _in vacuo to afford the title compound as a light yellow solid. MS m _/ z 536.3 (M+H, expected for _{C29H30ClN3O5 is} 535.19) _.

Preparation 98

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2-oxo-1, 2-Dihydroquinolin-5-yl)ethylamino]methyl}-2-chloro-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-yl ester

To the product of Preparation 97 in a mixture of dichloromethane (0.5 mL) and methanol (0.5 mL) was added 5-[(R)-2-amino-1-(tert-butyldimethylsilyloxy)ethyl ]-8-Hydroxy-1H-quinolin-2-one acetate (124.1 mg, 3.1 mmol), and the resulting mixture was stirred at room temperature for 1.5 h. Sodium triacetoxyborohydride (190.7 mg, 0.9 mmol) was added and the reaction mixture was stirred at room temperature for 15 h. The reaction was quenched by adding water (-0.2 mL) and the mixture was concentrated in vacuo to give the title compound which was used without further purification. MS m/z 854.5 (M+H, _{expected 853.36} for _{C46H56ClN5O7Si )} .

Example 25

Biphenyl-2-ylcarbamate 1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline -5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-yl ester ditrifluoroacetate

To a suspension of the product of Preparation 98 in dichloromethane (1.0 mL, 0.3 M) was added triethylamine trihydrofluoride (245 μL, 1.5 mmol). The mixture was stirred at room temperature for 45 h, then the mixture was concentrated in vacuo. The residue was dissolved in a mixture of DMF (0.5 mL), acetonitrile/water (1:1 with 0.1% TFA, 0.6 mL), TFA (0.3 mL) and acetonitrile (ca. 1 mL) and purified by prep-RP-HPLC. Mixture (gradient: 2-50% acetonitrile with 0.05% TFA in water). Appropriate fractions were collected, combined and lyophilized to afford the title compound (100 mg, 34% yield, 98.7% purity by HPLC) as an off-white solid. MS m _/ z 740.5 _{(M+H, expected 739.28 for C40H42ClN5O7 ) .}

Using the methods described above and appropriate starting materials, the following compounds were prepared.

Preparation 99

Biphenyl-2-ylcarbamate 1-[2-(4-[1,3]dioxolan-2-ylphenylcarbamoyl)-ethyl]-4-methylpiperidine-4 -yl ester

Biphenyl-2-ylcarbamate 4-methylpiperidin-4-yl ester (2.73 g, 8.79 mmol) and N(4-[1,3]dioxolane A mixture of -2-yl-phenyl)acrylamide (2.05 g, 8.80 mmol) was heated in 100 mL of 1:1 methanol/dichloromethane for 1 h. The solution was then diluted with ethyl acetate and the organic layer was washed with water, brine, dried ( _MgSO4 ) and concentrated under reduced pressure to afford the title compound. MS m/z (M+ _H ) ⁺ calcd. for _{C31H35N3O5 ,} 530.6; found _530.4 .

Prepare 100

1-[2-(4-Formylphenylcarbamoyl)ethyl]-4-methylpiperidin-4-yl biphenyl-2-ylcarbamate

The product from Preparation 99 was redissolved in 40 mL of methanol and 25 mL of aqueous 1N hydrochloric acid was added. The resulting mixture was stirred overnight at room temperature and the organic solvent was removed under reduced pressure. The residue was dissolved in ethyl acetate, the organic layer was washed with water, brine, dried ( _MgSO4 ) and the solvent was removed under reduced pressure. The product was triturated with dichloromethane to give the title compound (2.47g) as a white powder. LCMS (2-90) _{Rt = 4.27} min; MS m/z _{calcd. for C29H31N3O4 (} M+H) ⁺ 486.6, found 486.5.

Preparation 101

Biphenyl-2-ylcarbamate 1-[2-(4-{[(R)-2-(tert-butyldimethylsilyloxy)-2-(8-hydroxy-2-oxo-1, 2-Dihydroquinolin-5-yl)ethylamino]methyl}phenylcarbamoyl)ethyl]-4-methyl-piperidin-4-yl ester

The product of Preparation 100 (1.70 g, 3.51 mmol) and 5-[(R)-2-amino-1-(tert-butyldimethylsilyloxy)ethyl]-8-hydroxyl-1H-quinone were mixed at room temperature A mixture of lin-2-one acetate (1.65 g, 4.19 mmol) was stirred overnight in 40 mL of 1:1 methanol and dichloromethane. A portion of sodium triacetoxyborohydride (2.23 g, 10.5 mmol) was then added and the reaction mixture was stirred at room temperature for 6 hours. The reaction was then quenched with water and diluted with ethyl acetate. The layers were separated and the organic layer was washed with saturated sodium bicarbonate, brine, dried ( _MgSO4 ) and the solvent removed under reduced pressure to afford the title compound (2.9g). MS m/z calcd. _{for C46H57N5O6Si} (M+H) ⁺ _805.0 , found _804.6 .

Example 170

Biphenyl-2-ylcarbamate[1-2-(4-{[(R)-2-hydroxyl-2-(8-hydroxyl-2-oxo-1,2-dihydroquinolin-5-yl )ethylamino]methyl}-phenylcarbamoyl)ethyl]-4-methylpiperidin-4-yl ester

The product from Preparation 101 (2.9 g, 3.6 mmol) was dissolved in 75 mL of dichloromethane and triethylamine trihydrofluoride (0.85 mL, 5.2 mmol) was added. The resulting mixture was stirred at room temperature overnight, then the solvent was removed under reduced pressure to give the crude product as an oil. The product was then dissolved in acetic acid/water (1:1) and purified by preparative HPLC to afford the title compound as an off-white solid. LCMS (2-90) _{Rt = 3.67 min.; MS m/z calcd. for C40H43N5O6 ( M} +H) ⁺ 690.8 _, found 690.3.

Using the methods described above and appropriate starting materials, the following compounds can be prepared.

Preparation 102

Biphenyl-2-ylcarbamate (R)-(1-azabicyclo[3.2.1]oct-4-yl)ester

2-biphenylisocyanate (1.00g, 5.12mmol) and (R)-(-)-3-hydroxyquinucidine (quinuclidinol) hydrochloride (921mg, 5.63mmol) together in N, N two Heat in methylformamide (2.06mL) at 110°C for 12h. The reaction mixture was cooled and diluted with ethyl acetate (15 mL), then washed with saturated aqueous sodium bicarbonate (2 x 10 mL). The organic layer was extracted with 1M hydrochloric acid (3 x 20 mL), and the combined aqueous extracts were made basic to pH 8-9 with potassium carbonate. The aqueous layer was extracted with ethyl acetate (3 x 20 mL) and the combined organic layers were dried (magnesium sulfate), and the solvent was removed under reduced pressure to afford the title compound (1.64 g, 99% yield) as a yellow oil.

Preparation 103

(R)-4-(biphenyl-2-ylcarbamoyloxy)-1-(9-bromononyl)-1-nitrogen Heterobicyclo[3.2.1]octane bromide

To a stirred solution of the product from Preparation 102 (1.21 g, 3.76 mmol) and triethylamine (1.05 mL, 7.52 mmol) in acetonitrile (18.8 mL) was added 1,9 dibromononane (994 μL, 4.89 mmol ) and the reaction mixture was heated at 50 °C for 4 h. The reaction mixture was then cooled and the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and the organic layer was washed with saturated aqueous sodium bicarbonate (10 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (10% methanol/dichloromethane, 0.5% ammonium hydroxide) to afford the title compound (1.04 g, 1.97 mmol, 52% yield).

Preparation 104

杂双环[3.2.1]辛烷溴化物(R)-1-(9-N, N-bis(tert-butoxycarbonyl)aminononyl)-4-(biphenyl-2-ylcarbamoyloxy)-1-nitrogen

Heterobicyclo[3.2.1]octane bromide

To a stirred solution of sodium hydride (60% dispersion in mineral oil) (126 mg, 3.15 mmol) in N,N-dimethylformamide (10 mL) was added at 0 °C under nitrogen atmosphere in Di-tert-butyliminodicarboxylate (513 mg, 2.36 mmol) in N,N-dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 15 minutes, then it was cooled to 0 °C and the product of Preparation 103 (1.04 g, 1.97 mmol) in N,N dimethylformamide (5 mL) was added. The reaction mixture was warmed to room temperature over 12 hours, then the solvent was removed under reduced pressure to afford the title compound which was used without further purification.

Preparation 105

杂双环[3.2.1]辛烷溴化物(R)-1-(9-aminononyl)-4-(biphenyl-2-ylcarbamoyloxy)-1-nitrogen

Heterobicyclo[3.2.1]octane bromide

The product from Preparation 104 (1.31 g, 1.97 mmol) was dissolved in dichloromethane (15 mL) and trifluoroacetic acid (5 mL) was added slowly. The reaction mixture was stirred at room temperature for 1 h, then the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (20 mL) and washed with 1M aqueous sodium hydroxide (20 mL). The organic layer was extracted with 1M hydrochloric acid (3 x 20 mL), the combined aqueous extracts were made basic with potassium carbonate and extracted with dichloromethane (3 x 20 mL). The combined organic layers were dried (magnesium sulfate) and the solvent was removed under reduced pressure to afford the title compound (210 mg, 23% yield over 2 steps).

Preparation 106

杂双环[3.2.1]辛烷溴化物(R)-1-{9-[(R)-2-(8-Benzyloxy-2-oxo-1,2-dihydroquinolin-5-yl)-2-hydroxyethylamino]-nonyl Base}-4-(biphenyl-2-ylcarbamoyloxy)-1-nitrogen

Heterobicyclo[3.2.1]octane bromide

The product of Preparation 105 (210 mg, 0.45 mmol) and sodium triacetoxyborohydride (286 mg, 1.35 mmol) were stirred in dichloroethane (4.5 mL) at room temperature for 2 h, then the product of Preparation 6 (163 mg, 0.50 mmol). The reaction mixture was stirred for 12 h, then diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate (10 mL), dried (magnesium sulfate) and the solvent was removed under reduced pressure. The crude reaction product was purified by flash chromatography (10-50% methanol/dichloromethane, 0.5% ammonium hydroxide) to afford the title compound (131 mg, 38% yield).

Example 183

杂双环[2.2.2]辛烷溴化物二三氟醋酸酯4-(biphenyl-2-ylcarbamoyloxy)-1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinoline -5-yl)-ethylamino]nonyl}-1-nitrogen

Heterobicyclo[2.2.2]octane bromide ditrifluoroacetate

To a stirred solution of the product from Preparation 105 (131 mg, 0.17 mmol) in methanol (1.8 mL) was added palladium (10 wt.% dry basis on charcoal; 39 mg) and the reaction mixture was placed under an atmosphere of hydrogen. After stirring for 12 h, the reaction mixture was filtered through a pad of celite, washed with methanol (2 mL) and the solvent was removed under reduced pressure. The resulting residue was purified by preparative HPLC to give the title compound (8 mg) as its bistrifluoroacetate salt. MS m/z 667.5.

Using the methods described herein and appropriate starting materials, the following compounds can be prepared.

Preparation A

Cell culture and membrane preparation from cells expressing human β ₁ , β ₂ or β ₃ adrenergic receptors

Chinese hamster ovary (CHO) cell lines stably expressing cloned human β ₁ , β ₂ or β ₃ adrenergic receptors, respectively, were cultured in Hams F-12 with 10% FBS in the presence of 500 μg/mL geneticin. Grow in medium until near confluency. Cell monolayers were floated with 2 mM EDTA in PBS. Cells were pelleted by centrifugation at 1000 rpm and cell pellets were either stored frozen at -80°C or membranes were prepared for immediate use. For the preparation of _β1 and _β2 receptor expressing membranes, the cell pellet was resuspended in lysis buffer (10 mM HEPES/HCl, 10 mM EDTA, pH 7.4 at 4°C) and homogenized using a tight-fitting Dounce glass. The slurry was homogenized (30 strokes) on ice. For more protease-sensitive _β3- receptor-expressing membranes, the cell pellet was supplemented with one tablet of "Complete Protease Inhibitor Cocktail with 2 mM DETA" lysis buffer per 50 mL of buffer (Roche Catalog No. 1697498, Roche Molecular Biochemicals, Indianapolis, IN). The homogenate was centrifuged at 20,000 xg, and the resulting pellet was washed once with lysis buffer by resuspension and centrifugation as described above. The final pellet was then resuspended in ice-cold binding assay buffer (75 mM Tris/HCl pH 7.4, _{12.5 mM} MgCl2, 1 mM EDTA). The protein concentration of the membrane suspension was determined by the method described in Lowry et al., 1951, Journal of Biological Chemistry, 193, 265; and Bradford, Analytical Biochemistry, 1976, 72, 248-54. All membranes were stored frozen at -80°C in aliquots or used immediately.

Preparation B

Cell Culture and Membrane Preparation from Cells Expressing Human _M1 , _M2 , _M3 and _M4 Muscarinic Receptors

CHO cell lines stably expressing cloned human hM ₁ , hM ₂ , hM ₃ and hM ₄ muscarinic receptor subtypes, respectively, were cultured in HAM's F-12 supplemented with 10% FBS and 250 μg/mL geneticin. Grow in medium until near confluency. Cells were grown in a 5% _CO2 , 37°C incubator and floated with 2mM EDTA in dPBS. Cells were harvested by centrifugation at 650 xg for 5 minutes and cell pellets were stored frozen at -80°C or membranes were prepared for immediate use. For membrane preparation, cell pellets were resuspended in lysis buffer and homogenized with a Polytron PT-2100 tissue disruptor (Kinematica AG; 20 sec x 2 disruptions). The crude membrane was centrifuged at 40,000 xg for 15 minutes at 4°C. The membrane pellet was then resuspended with resuspension buffer and homogenized again with a Polytron tissue disruptor. The protein concentration of the membrane suspension was determined by the method described in Lowry et al., 1951, Journal of Biochemistry, 193, 265. All membranes were stored frozen at -80°C in aliquots or used immediately. Aliquots of prepared _hM5 receptor membranes were purchased directly from Perkin Elmer and stored at -80°C until use.

Test method A

Radioligand binding assay of human β ₁ , β ₂ and β ₃ adrenergic receptors

Binding assays were performed in 96-well microtiter plates with a total assay volume of 100 μl, 10-15 μg _containing human Membrane protein of β1, β2, or β3 adrenergic receptors. Saturation binding studies to determine radioligand K values were performed using [ ^3H ]-dihydroalprenolol (NET-720, 100 Ci/mmol, PerkinElmerLife Sciences Inc., Boston, MA) for the _β1 and _β2 receptors. and [ ¹²⁵ I]-(-)-iodocyanopindolol (NEX-189, 220 Ci/mmol, PerkinElmer Life Sciences Inc., Boston, MA), at 10 or 11 different concentrations ranging from 0.01 nM to 20 nM ongoing. The displacement test for determining the Ki value of the test compound is to use 1nM [ ³ H]-dihydroalprenolol and 0.5nM [ ¹²⁵ I]-(-)-iodocyanopindolol for the range of 10pM-10μM 10 or 11 different concentrations of the test compound were performed. Non-specific binding was determined in the presence of 10 [mu]M propranolol. Incubate the test substance at 37°C for 1 hour, then pass through GF/B for _β1 and _β2 receptors or GF/C glass fiber filter plate for _β3 receptors pre-soaked in 0.3% polyethyleneimine (Paekard BioScience Co., Meriden, CT) rapid filtration to terminate the binding reaction. Unbound radioactivity was removed by washing filter plates three times with filtration buffer (75 mM tris/HCl pH 7.4 at 4°C, 12.5 mM _MgCl2 , 1 mM EDTA). The plate was then dried, 50 μl of Microscint-20 liquid scintillation fluid (Packard BioScience Co., Meriden, CT) added and the plate counted in a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden, CT). Binding data were analyzed by nonlinear regression analysis using a 3-parameter model of one-site competition with the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA). Curve minima were fixed at the value of non-specific binding when assayed in the presence of 10 [mu]M propranolol. Ki values of test compounds were calculated from observed _IC50 values and _{Kd values} of radioligands using the Cheng-Prusoff equation (Cheng Y, and Prusoff WH., Biochemical Pharmacology, 1973, 22, 23, 3099-108).

In this assay, a lower Ki value indicates a higher binding affinity of the test compound for the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have _Ki values for _β2 adrenergic receptors of less than about 300 nM. For example, the compounds of Examples 3 and 6 were found to have _Ki values of less than 10 nM.

Receptor subtype selectivity of a test compound can be calculated as the ratio of K _i (β ₁ )/K _i (β ₂ ) or the ratio of K _i (β ₃ )/K _i (β ₂ ), if desired. Typically, the compounds of the invention demonstrate greater binding at the _β2 adrenergic receptors than the _β1 or _β3 adrenergic receptors, i.e. K _i (β ₁ ) or K _i (β ₃ ) usually greater than K _i (β ₂ ). In general, compounds that are selective for _β2 adrenergic receptors over _β1 or _β3 adrenergic receptors are preferred; especially compounds having a selectivity of greater than about 5; and especially greater than about 8. By way of example, the compounds of Examples 3 and 6 have a K _i (β ₁ )/K _i (β ₂ ) ratio greater than 8.

Test method B

Radioligand Binding Assay for Muscarinic Receptors

Radioligand binding assays for cloned human muscarinic receptors were performed in 96-well microtiter plates in a total assay volume of 100 μl. CHO cell membranes stably expressing hM ₁ , hM ₂ , hM ₃ , hM ₄ or hm ₅ muscarinic isoforms were diluted in assay buffer to the following specific target protein concentrations (μg/well): for hM ₁ 10 μg, Similar signals (cpm) were obtained for hM ₂ 10 μg-15 μg, for hM ₃ 10 μg-20 μg, for hM ₄ 10 μg-20 μg, and for hM ₅ 10 μg-12 μg. The membrane was homogenized briefly (10 sec) using a Polytron tissue disruptor prior to the addition of the assay plate. Saturation binding studies for the determination of radioligand K _D values were performed using L-[N-methyl- ^3H ]chloromethospolamine ([ ^3H ]-NMS) (TRK666, 84.0Ci/mmol, Amersham Pharmacia Biotech, Buckinghamshire, England). Displacement assays for determining K _i values of test compounds were performed with 1 nM [ ³ H]-NMS and 11 different test compound concentrations. Test compounds were first dissolved in dilution buffer to a concentration of 400 μM and then serially diluted 5× with dilution buffer to a final concentration of 10 pM to 100 μM. The order and volumes added to the assay plate were as follows: 25 μL radioligand, 25 μL diluted test compound and 50 μL membrane. Assay plates were incubated at 37°C for 60 minutes. Binding reactions were terminated by rapid filtration through GF/B glass fiber filter plates (PerkinElmer Inc., Wellesley, MA) preconditioned in 1% BSA. The filter plates were washed three times with wash buffer (10 mM HEPES) to remove unbound radioactivity. The plate was then air dried and 50 μL of Microscint-20 liquid scintillation fluid (Perkin Elmer Inc., Wellesley, MA) was added to each well. The plate was then counted in a PerkinElmer Topcount liquid scintillation counter (PerkinElmer Inc., Wellesley, MA). Binding data were analyzed by nonlinear regression analysis using a one-site competition model with the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA). Ki values for test compounds were calculated from the observed _IC50 values and _{the KD} values of the radioligands using the Cheng-Prusoff equation (Cheng Y; Prusoff WH. (1973) Biochemical Pharmacology, 22(23):3099-108). K _i values were converted to pK _i values to determine geometric means and 95% confidence intervals. These summarized statistics were then converted back to K _i values for data reporting.

In this assay, lower _Ki values indicate higher binding affinity of the test compound for the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have _Ki values for the _M3 muscarinic receptor of less than about 300 nM. For example, the compounds of Examples 3 and 6 were found to have _Ki values of less than 10 nM.

Test method C

Whole-cell cAMP flash plate assay in CHO cell lines heterologously expressing human β ₁ , β ₂ or β ₃ adrenergic receptors

cAMP assays were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with [ ¹²⁵ I]-cAMP following the manufacturer's instructions (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, MA). For the determination of β-receptor agonist potency (EC ₅₀ ), CHO-K1 cell lines stably expressing cloned human β ₁ , β ₂ or β ₃ adrenergic receptors were incubated with 10% FBS and geneticin ( 250 μg/ml) in HAM's F-12 medium until nearly confluent. Cells were washed with PBS and detached in dPBS (Dulbecco's phosphate buffered saline, without _CaCl2 and _MgCl2 ) containing 2 mM EDTA or trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA). After counting the cells in a Coulter cell counter, pellet the cells by centrifugation at 1000 rpm and resuspend in stimulation buffer containing IBMX (PerkinElmer kit) pre-warmed to room temperature to a concentration of 1.6× ¹⁰⁶ to 2.8× 10 ⁶ cells. Approximately 60,000-80,000 cells per well were used in this assay. Test compounds (10 mM in DMSO) were diluted in Beckman Biomek-2000 in PBS containing 0.1% BSA and tested at 11 different concentrations ranging from 100 μM to 1 pM. Reactions were incubated at 37°C for 10 minutes and stopped by adding 100 μL of ice-cold detection buffer (NEN SMP004, PerkinElmer Life Sciences, Boston, MA) containing [ ¹²⁵ I]-cAMP. The amount of cAMP produced (pmol/well) was calculated based on the observed counts of the samples and the cAMP standards described in the manufacturer's user manual. Data were analyzed by nonlinear regression analysis using the sigmoidal equation with the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA). EC50 values were calculated using the Cheng-Prusoff equation (Cheng Y, and Prusoff WH., Biochemical Pharmacology, 1973, 22, 23, 3099-108).

In this assay, a lower _EC50 value indicates a higher functional activity of the test compound at the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have _EC50 values for _β2 adrenergic receptors of less than about 300 nM. For example, the compounds of Examples 3 and 6 were found to have _EC50 values of less than 10 nM.

Receptor subtype selectivity of test compounds can be calculated as the ratio of _EC50 ( _β1 )/ _EC50 ( _β2 ) or the ratio of _EC50 ( _β3 )/ _EC50 ( _β2 ), if desired. Typically, the compounds of the invention demonstrate greater functional activity at the _β2 adrenergic receptors than the _β1 or _β3 adrenergic receptors, i.e. the _EC50 ( _β1 ) or _EC50 ( _β3 ) typically Greater than EC ₅₀ (β ₂ ). In general, compounds that are selective for _β2 adrenergic receptors over _β1 or _β3 adrenergic receptors are preferred; especially compounds having a selectivity of greater than about 5; and especially greater than about 8. By way of example, the compounds of Examples 3 and 6 have an EC ₅₀ (β ₁ )/EC ₅₀ (β ₂ ) ratio greater than 10.

Test method D

Functional Assay for Antagonism of Muscarinic Receptor Subtypes

A. Blockade of Agonist-Mediated Inhibition of cAMP Accumulation

In this assay, the functional potency of test compounds is determined by measuring the ability of test compounds to block oxytremorin inhibition of forskolin-mediated cAMP accumulation in CHO-K1 cells expressing the _hM2 receptor. cAMP assays were performed in radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System ^with125I -cAMP (NEN SMP004B, PerkinElmer Life Sciences Inc., Boston, MA) following the manufacturer's instructions. Cells were rinsed once with dPBS and resuspended with trypsin-EDTA solution (0.05% trypsin/0.53 mM EDTA) as described in the cell culture and membrane preparation section above. The detached cells were washed twice by centrifugation at 650 xg in 50 mL dPBS for five minutes. The cell pellet was then resuspended in 10 mL of dPBS, and cells were counted using a Coulter Z1 binary particle counter (Beckman Coulter, Fullerton, CA). The cells ^were centrifuged again at 650xg for five minutes and resuspended in stimulation buffer to an assay concentration of ^{1.6x106-2.8x106} cells per ml.

Test compounds were first dissolved in dilution buffer (dPBS supplemented with 1 mg/mL BSA (0.1%)) to a concentration of 400 μM, and then serially diluted with dilution buffer to a final molar concentration of 100 μM to 0.1 nM. Dilute oxytremorine in a similar manner.

To determine oxytremorin inhibition of adenylate cyclase (AC) activity, 25 μL of forskolin (diluted in dPBS to a final concentration of 25 μM), 25 μL of diluted oxytremorin, and 50 μL of cells were added to agonist wells. To determine the ability of a test compound to block AC activity inhibited by oxytremorine, 25 μL of forskolin and oxytremorine (25 μM and 5 μM final concentrations, respectively, diluted in dPBS), 25 μL of diluted test compound, and 50 μL of cells Add remaining test wells.

Reactions were incubated at 37°C for 10 minutes and stopped by adding 100 μL of ice-cold detection buffer. Plates were sealed, incubated overnight at room temperature and counted the next morning on a PerkinElmer TopCount liquid scintillation counter (PerkinElmer Inc., Wellesley, MA). The amount of cAMP produced (pmol/well) was calculated based on the observed counts of the samples and the cAMP standards 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, CA) using nonlinear regression, a one-site competition equation. _Ki was calculated using the Cheng-Prusoff equation, using the _EC50 of the oxytremorine concentration-response curve and the test concentration of oxytremorine as _KD and [L], respectively.

In this assay, a lower _Ki value indicates a higher binding affinity of the test compound for the receptor being tested. Exemplary compounds of the invention tested in this assay were generally found to have a potency of less than about 300 nM for blocking the oxidotremorin inhibition of forskolin-mediated cAMP accumulation in CHO-K1 cells expressing the _hM2 receptor. _Ki value. For example, the compound of Example 3 was found to have a _Ki value of less than 10 nM.

B. Agonist-mediated blockade of [ ³⁵ S]GTPγS binding

In a second functional assay, the functional potency of test compounds was determined by determining their ability to block the binding of oxidotremorine-stimulated [ ^35S ]GTPyS in CHO-K1 cells expressing the _hM2 receptor.

At the time of use, frozen membranes were thawed and then diluted in assay buffer with a final target tissue concentration of 5-10 μg protein per well. Membranes were briefly homogenized using a Polytron PT-2100 Tissue Disruptor prior to addition to assay plates.

In each assay, the _EC90 value (effective concentration for 90% of the maximal response) for stimulation of [ ^35S ]GTPyS binding by the agonist oxytremorine was determined.

To determine the ability of test compounds to inhibit oxidotremorine-stimulated [ ³⁵ S]GTPγS binding, the following were added to each well of a 96-well plate: 25 μL of assay buffer with [ ³⁵ S]GTPγS (0.4 nM), 25 μL of oxotremorin (EC ₉₀ ) and GDP (3 uM), 25 μL of diluted test compound and 25 μL of CHO cell membrane expressing hM ₂ receptor. The assay plates were then incubated at 37°C for 60 minutes. Assay plates were filtered using a PerkinElmer 96-well harvester with GF/B filters preconditioned with 1% BSA. Rinse the plate 3 x 3 s with ice-cold wash buffer, then air or vacuum dry. Microscint-20 scintillation fluid (50 μL) was added to each well, each plate was sealed, and radioactivity was counted on a Topcounter (PerkinElmer). Data were analyzed by nonlinear regression analysis with the GraphPad Prism software package (GraphPad Software, Inc., San Diego, CA) using nonlinear regression, a one-site competition equation. _Ki was calculated using the Cheng-Prusoff equation using the _IC50 value of the concentration-response curve of the test compound and the concentration of oxytremorine in the assay as _KD and [L], the ligand concentration, respectively.

In this assay, lower _Ki values indicate higher functional activity of the test compound at the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have a K of less than about 300 nM for the blockade of oxidotremorine-stimulated [ ^35S ]GTPγS binding in CHO-K1 cells expressing the _hM2 receptor. _i value. For example, the compound of Example 3 was found to have a _Ki value of less than 10 nM.

C. Agonist-mediated blockade of calcium release via the FLIPR assay

The muscarinic receptor subtypes ( _M1 , _M3 and _M5 receptors) coupled to the _Gq protein activate the phospholipase C (PLC) pathway upon binding of an agonist to the receptor. As a result, activated PLC hydrolyzes phosphatidylinositol bisphosphate (PIP ₂ ) to diacylglycerol (DAG) and phosphatidyl-1,4,5-triphosphate (IP ₃ ), which in turn are removed from intracellular stores, the endoplasmic Reticulum and sarcoplasmic reticulum produce calcium release. The FLIPR (Molecular Devices, Sunnyvale, CA) assay exploits this increase in intracellular calcium by utilizing a calcium-sensitive dye that fluoresces when free calcium is bound (Fluo-4AM, Molecular Probes, Eugene, OR). This fluorescence event was measured in real time with FLIPR, which detected changes in fluorescence in monolayers cloned with human _M1 and _M3 and chimpanzee _M5 receptors. Antagonist potency is measured by the ability of the antagonist to inhibit the agonist-mediated increase in intracellular calcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing hM ₁ , hM ₃ and cM ₅ receptors were seeded into 96-well FLIPR plates the night before assays were performed. The inoculum was washed by Cellwash (MTXLabsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4, 2 mM calcium chloride, 2.5 mM profenacine in Hank's buffered saline solution (HBSS) free of calcium and magnesium). Cells were washed twice to remove growth medium and residual 50 μL/well of FLIPR buffer. Cells were then incubated with 50 μL/well of 4 μM FLUO-4AM (made into a 2X solution) at 37° C. under 5% carbon dioxide for 40 minutes. After the dye incubation period, cells were washed twice with FLIPR buffer, leaving a total volume of 50 μL/well.

To determine antagonist potency, the dose-dependent stimulation of intracellular Ca2 ⁺ release by oxytremorine was first determined so that antagonist potency against oxidotremorine stimulation at _EC90 concentrations could then be measured. Cells were first incubated with compound dilution buffer for 20 minutes prior to addition of agonist, which was performed by FLIPR. The EC ₉₀ values for oxytremorine were generated by combining the formula EC _F =((F/100-F)^1/H)*EC ₅₀ as detailed in the FLIPR measurements and in the Data Summary section below. Prepare an oxytremorine concentration of 3 x EC _F in the stimulation plate, and add an EC ₉₀ concentration of oxytremorine to each well of the antagonist inhibition assay plate.

The parameters used for FLIPR were: exposure time 0.4 sec, laser intensity 0.5 Watt, excitation wavelength 488 nm and emission wavelength 550 nm. Baseline was determined by measuring the change in fluorescence for 10 s prior to agonist addition. Following agonist stimulation, FLIPR continuously measures fluorescence changes every 0.5-1 s for 1.5 min to capture maximal fluorescence changes.

Fluorescence change is expressed as maximum fluorescence minus baseline fluorescence per well. Raw data were analyzed by non-linear regression on the logarithm of drug concentration with GraphPad Prism (GraphPad Software, Inc., San Diego, CA) using an embedded model for inverse dose response. _Antagonist Ki values were determined by Prism using the oxytremorine EC ₅₀ value as the _KD and the oxytremorine EC ₉₀ for the ligand concentration according to the Cheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, lower Ki values indicate higher functional activity of the test compound at the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have a K of less than about 300 nM for blocking agonist-mediated calcium release in CHO cells stably expressing _hM1 , _hM3 and _cM5 receptors. _i value. For example, the compound of Example 3 was found to have _Ki values of less than 10 nM for the hM ₁ , hM ₃ and cM ₅ receptors.

Test method E

Whole-cell cAMP fluorescent plate assay using a lung epithelial cell line endogenously expressing the human β2 _- adrenoceptor

To determine agonist potency and efficacy (intrinsic activity) in cell lines expressing endogenous levels of _β2 adrenergic receptors, a human lung epithelial cell line (BEAS-2B) (ATCC CRL-9609, American Type Culture Collection Center, Manassas, VA) (January B, et al. British Journal of Pharmacology, 1998, 123, 4, 701-11). Cells were grown to 75%-90% confluency in complete, serum-free medium (LHC-9 medium containing epinephrine and tretinoin, cat# 181-500, Biosource International, Camarillo, CA). The day before the experiment, the medium was switched to LHC-8 (no epinephrine or tretinoin cat # 141-500, Biosource International, Camarillo, CA). cAMP assays were performed in a radioimmunoassay format using the Flashplate Adenylyl Cyclase Activation Assay System with [ ¹²⁵ I]-cAMP following the manufacturer's instructions (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, MA). On the day of the assay, cells were rinsed with PBS, floated by scraping with 5 mM EDTA in PBS and counted. Cells were pelleted by centrifugation at 1000 rpm and resuspended in stimulation buffer pre-warmed to 37°C to a final concentration of 600,000 cells/mL. Cells were used in this assay at a final concentration of 100000-120000 cells/well. Test compounds were serially diluted into assay buffer (75 mM Tris/HCl pH 7.4 at 25° C., 12.5 mM MgCl ₂ , 1 mM EDTA, 0.2% BSA) in a Beckman Biomek-2000. Test compounds were tested in the assay at 11 different concentrations ranging from 10 μM to 10 pM. Reactions were incubated at 37°C for 10 minutes and stopped by adding 100 μL of ice-cold detection buffer. Plates were sealed, incubated overnight at 4°C and counted the next morning in a Topcount scintillation counter (Packard BioScience Co., Meriden, CT). The amount of cAMP produced per milliliter of reaction was calculated based on the counts observed for the samples and the cAMP standards 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, CA) using a 4-parameter model of recurve dose response.

In this assay, lower _EC50 values indicate higher functional activity of the test compound at the tested receptor. Exemplary compounds of the invention tested in this assay were generally found to have _EC50 values for _β2 adrenergic receptors of less than about 300 nM. For example, the compounds of Examples 3 and 6 were found to have _EC50 values of less than 10 nM.

If desired, test compound efficacy (% efficacy) was calculated from the observed Emax (highest point of the fitted curve) and the maximum response obtained for the isoproterenol dose-response curve and expressed as % relative to isoproterenol effect. Exemplary compounds of the invention tested in this assay generally demonstrated greater than about 40% efficacy.

Test method F

Duration of bronchoprotection in guinea pig models of acetylcholine-induced or histamine-induced bronchoconstriction

These in vivo assays were used to evaluate the bronchoprotective effect of test compounds exhibiting both muscarinic receptor antagonist and _β2 adrenergic receptor agonist activity. To isolate muscarinic antagonist activity in a model of acetylcholine-induced bronchoconstriction, animals were given propranolol, a compound that blocks beta-receptor activity, prior to administration of acetylcholine. The duration of bronchoprotection in a model of histamine-induced bronchoconstriction reflects the activity of β2 _- adrenoceptor agonists.

Groups of 6 male guinea pigs weighing 250-350 g (Duncan-Hartley (HsdPoc:DH) Harlan, Madison, WI) were individually identified by cage cards. Animals were allowed ad libitum access to food and water throughout the study.

Test compounds were administered by inhalation over 10 minutes in a systemic exposure dosing chamber (R & S Molds, San Carlos, CA). Dosing chambers are arranged so that 6 individual chamber aerosols can be administered simultaneously from a central manifold. Guinea pigs were exposed to aerosols of test compounds or vehicle (WFI). The aerosol is generated from an aqueous solution, for example, from a gas mixture (CO ₂ =5%, O ₂ =21% and N ₂ = 74%) advance. The gas flow through the nebulizer at this operating pressure was approximately 3 L/min. The generated aerosol is driven into the chamber by positive pressure. No dilution air was used during administration of the nebulized solution. During the 10 minutes of nebulization, approximately 1.8 mL of solution was nebulized. This value was calculated gravimetrically by comparing the weight of the filled nebulizer before and after nebulization.

At 1.5, 24, 48 and 72 hours post-dose, the bronchoprotective effect of the test compound administered via inhalation was assessed by whole body plethysmography.

Forty-five minutes before the start of lung evaluation, each guinea pig was anesthetized with an intramuscular injection of ketamine (43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg). After skin preparation and cleaning with 70% ethanol at the surgical site, a 2-3 cm median incision was made on the ventral side of the neck. The jugular vein was then isolated and a saline-filled polyethylene catheter (PE-50, Becton Dickinson, Sparks, MD) was inserted to allow infusion of acetylcholine (Ach) or histamine in saline. The open trachea was then dissected and a 14G Teflon tube (#NE-014, SmallParts, Miami Lakes, FL) inserted. Anesthesia was maintained by further intramuscular injections of the previously mentioned anesthetic mixture if necessary. If the animal responds to pinching its paw or if the breathing rate is greater than 100 breaths/min, monitor and adjust the depth of anesthesia.

Once intubation is complete, the animal is placed in a volume scanner (#PLY 3114, BuxcoElectronics, Inc., Sharon, CT) and an esophageal pressure catheter (PE-160, Becton Dickinson, Sparks, MD) inserted to measure lung driving pressure (pressure). A Teflon endotracheal tube was connected to the opening of the plethysmometer to allow guinea pigs to breathe air from the outside. Then seal the chamber. Body temperature was maintained using a heat lamp, and the guinea pig lungs were inflated 3 times with 4 mL of air using a 10 mL calibrated syringe (#5520 Series, Hans Rudolph, Kansas City, MO) to ensure that the lower airways did not collapse and the animal did not hyperventilate.

Lung assessments were initiated once baseline compliance was determined to be within the range of 0.3-0.9 mL/cm of water and baseline resistance was measured to be within the range of 0.1-0.199 cm of water/mL/sec. The Buxco lung measurement computer program enables the collection and derivation of lung values.

Start this procedure to initiate the trial protocol and data collection. The volume change over time that occurs with each breath within the plethysmometer is measured by a Buxco pressure transducer. By integrating this signal over time, flow measurements are calculated for each breath. This signal and lung actuation pressure changes collected with a Sensym pressure transducer (#TRD4100) were connected to a data collection interface (#’s SFT3400 and SFT3813) via a Buxco (MAX 2270) preamplifier. All other lung parameters are derived from these two inputs.

Baseline values were collected for 5 minutes before challenge of guinea pigs with Ach or histamine. When evaluating muscarinic antagonist effects, propranolol (5 mg/Kg, iv) (Sigma-Aldrich, St. Louis, MO) was administered 15 minutes before challenge with Ach. Ach (Sigma-Aldrich, St. Louis, MO) (0.1 mg/mL) was infused intravenously from a syringe pump (sp210iw, World Precision Instruments, Inc., Sarasota, FL) at the following doses and times from the start of the study 1 minute: 1.9 μg/min at 5 min, 3.8 μg/min at 10 min, 7.5 μg/min at 15 min, 15.0 μg/min at 20 min, 30 μg/min at 25 min and 30 min min 60 μg/min. Alternatively, the bronchoprotective effect of test compounds is evaluated in the acetylcholine challenge model without pretreatment with beta-blocking compounds.

When evaluating the β2 _- adrenoceptor agonist effect of the test compound, histamine (25 μg/mL) was infused intravenously from a syringe pump (Sigma-Aldrich, St. Louis, MO) 1 minute: 0.5 μg/min at 5 minutes, 0.9 μg/min at 10 minutes, 1.9 μg/min at 15 minutes, 3.8 μg/min at 20 minutes, 7.5 μg/min at 25 minutes minutes and 15 μg/min at the 30th minute. If resistance or compliance does not return to baseline values 3 min after each Ach or histamine dose, inflate the guinea pig's lungs 3 times with 4 mL of air from a 10 mL calibration syringe. Recorded lung parameters included respiratory rate (breath/min), compliance (mL/cm water), and lung resistance (cm water/mL/sec). Once pulmonary function measurements were completed at minute 35 of the protocol, the guinea pigs were removed from the plethysmometer and sacrificed by carbon dioxide asphyxiation.

Evaluate data in one of two ways:

(a) Lung resistance ( _RL , cm water/mL/sec) was calculated from the ratio of "change in pressure" to "change in flow". The _RL response to Ach (60 μg/min, 1H) was calculated for vehicle and test compound groups. The mean ACh response in vehicle treated animals at each pre-treatment time was calculated and used to calculate the percent inhibition of ACh response at each test compound dose at the corresponding pre-treatment time. The _{bronchoprotective} ID ₅₀ (inhibition of ACh (60 μg/min) bronchial The dose required for a 50% contractile response). The equation used is as follows:

Y＝Min+(Max-Min)/(1+10 ^{((log ID50-X)*Hill slope)} )

where X is the logarithm of the dose and Y is the response (percent inhibition of ACh-induced increase _{in RL} ). Y starts at Min and progressively approaches Max with a recurve shape.

(b) The amount PD ₂ , which is defined as the amount required to cause a doubling of baseline lung resistance, using the following equation (derived from the equation used in clinical practice to calculate PC ₂₀ values (see Am. ThoracicSoc, 2000) using a series of Ach or Calculated from flow and pressure derived lung resistance values during histamine challenge:

${\mathrm{<span\; class="notranslate">\; PD</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; anti</span>}\mathrm{<span\; class="notranslate">\; log</span>}<span\; class="notranslate">\; [</span>\mathrm{<span\; class="notranslate">\; log</span>}{\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; +</span>\frac{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; log</span>}\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; log</span>}\mathrm{<span\; class="notranslate">\; C</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; 2</span>}{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 0</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}<span\; class="notranslate">\; )</span>}{{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 2</span>}}<span\; class="notranslate">\; -</span>{\mathrm{<span\; class="notranslate">\; R</span>}}_{\mathrm{<span\; class="notranslate">\; 1</span>}}}<span\; class="notranslate">\; ]</span>$

in:

C ₁ = concentration of Ach or histamine before C ₂

_C2 = concentration of Ach or histamine that causes at least a 2-fold increase in lung resistance ( _RL ).

R ₀ = baseline _RL value

R ₁ = _RL value after C ₁

R ₂ = _RL value after C ₂

Statistical analysis of the data was performed using a two-tailed-Student's t-test. A P value <0.05 was considered significant.

Exemplary compounds of the invention tested in this assay generally produced dose-dependent bronchoprotection against MCh-induced bronchoconstriction and His-induced bronchoconstriction. Typically, in this test, subjects with less than about 300 μg/mL potency (ID ₅₀ at 1.5 h post-dose) on ACh-induced bronchoconstriction and less than about 300 μg/mL on His-induced bronchoconstriction Compounds are generally preferred. For example, the compounds of Examples 3 and 6 were found to have an ID50 of less than about 100 μg/mL for ACh-induced bronchoconstriction and _{an ID50} of less than about 100 μg/mL for His-induced bronchoconstriction at 1.5 hours after administration .

Furthermore, test compounds having a duration of bronchoprotective activity (PD T _1/2 ) of at least about 24 hours are generally preferred in this assay. By way of example, the compounds of Examples 3 and 6 were found to have a PD T _1/2 of at least about 24 hours after administration.

Test method G

The Einthoven Model for Measuring Ventilation Changes in Guinea Pigs

The bronchodilator activity of test compounds was evaluated in an anesthetized guinea pig model (Einthoven model) using ventilation pressure as a surrogate measure of airway resistance. See, eg, Einthoven (1892) Pfugers Arch. 51:367-445; and Mohammed et al. (2000) Pulm Pharmacol Ther. 13(6):287-92. In this model, muscarinic antagonist and _β2 agonist activity was assessed by measuring protection against methacholine (MCh) and histamine (His)-induced bronchoconstriction.

The experiment was performed using Duncan-Hartley guinea pigs (Harlan, Indianapolis, IN) weighing 300-400 g.

Test compounds or vehicle (ie sterile water) were administered by inhalation (IH) over a 10 minute period using 5 mL of dosing solution in a whole body exposure dosing chamber (R+S Molds, San Carlos, CA). Animals were exposed to an aerosol generated from a LC Star Nebulizer Set (Model 22F51, PARI Respiratory Apparatus, Inc. Midlothian, VA) at a pressure of 22 psi by Bioblend - a gas mixture (5% CO ₂ ; 21% _O2 ; and 74% _N2 ) advance. Pulmonary function was evaluated at different time points after inhalation administration.

Guinea pigs were anesthetized with an intramuscular (IM) injection of a mixture of ketamine (13.7 mg/kg)/xylazine (3.5 mg/kg)/acepromazine (1.05 mg/kg) forty-five minutes before the start of pulmonary function evaluation . Supplementary doses of this mixture (50% of the initial dose) were administered as needed. The jugular vein and carotid artery were isolated and saline-filled polyethylene catheters (micro-renathane and PE-50, respectively, Becton Dickinson, Sparks, MD) were inserted. The carotid artery was connected to a pressure transducer to allow measurement of blood pressure, and the jugular vein cannulated for IV injection of MCh or His. The free trachea was then dissected and a 14G needle (#NE-014, SmallParts, Miami Lakes, FL) inserted. Once intubated, the guinea pigs were ventilated at 100 strokes per minute using a ventilator (Model 683, Harvard Apparatus, Inc., MA) set at a dynamic volume of 1 mL per 100 g of body weight but not to exceed a volume of 2.5 mL. Ventilatory pressure (VP) was measured in the tracheal tube using a Biopac sensor connected to a Biopac (TSD 137C) preamplifier. Body temperature was maintained at 37°C using a heating pad. Before starting data collection, pentobarbital (25 mg/kg) was administered intraperitoneally (IP) to suppress spontaneous respiration and obtain a stable baseline. VP changes are recorded on the Biopac Windows data collection interface. Baseline values were collected for at least 5 minutes before guinea pigs were non-cumulatively challenged IV with 2-fold increasing amounts of bronchoconstrictor (MCh or His). When MCh was used as a bronchoconstrictor, animals were pretreated with propranolol (5 mg/kg, IV) to isolate the antimuscarinic effects of the test compound. VP changes were recorded using Acknowledge Data Collection Software (Santa Barbara, CA). After the study was completed, the animals were sacrificed.

The VP change is measured in cm of water. Change in VP (cm water) = peak pressure (post bronchoconstrictor challenge) - peak baseline pressure. MCh or His dose-response curves were fitted to linear four-parameter logistic equations using GraphPad Prism, Windows version 3.00 (GraphPad Software, San Diego, California). The equation used is as follows:

Y＝Min+(Max-Min)/(1+10 ^{(log ID50-x)*Hill slope)} )

where X is the logarithm of the dose and Y is the response. Y starts at Min and gradually approaches Max with a recurve shape.

The percent inhibition of the bronchoconstrictor response to the submaximal dose of MCh or His was calculated at each dose of the test compound using the following equation: Percent inhibition of response = 100-((peak pressure (after bronchoconstrictor challenge, treatment )-peak baseline pressure (administration group)*100%/(peak pressure (after bronchodilator attack, water)-peak baseline pressure (water)). Utilize the four-parameter logistic equation fitting inhibition curve of GraphPad software. When _ID50 (dose required to produce 50% inhibition of bronchoconstrictor response) and Emax (maximum inhibition) were also estimated when required.

The half-life (PD T _1/2 ) of the drug effect was estimated by using the bronchoprotective measures at different time points after inhalation of the test compound. Using the single-phase exponential decay equation (GraphPad Prism, version 4.00): Y=span*exponent (-K*X)+plateau Use nonlinear regression fitting to determine PD T _1/2 ; at span+plateau Starts and decays to a plateau with a rate constant K. PD T _1/2 = 0.69/K. Force the ping value back to 0.

Exemplary compounds of the invention tested in this assay generally produced dose-dependent bronchoprotection against MCh-induced and His-induced bronchoconstriction. In general, test compounds with an _ID50 of less than about 300 μg/mL for _MCh -induced bronchoconstriction and less than about 300 μg/mL for His-induced bronchoconstriction at 1.5 hours post-dose are preferred in this assay . Furthermore, test compounds having a duration of bronchoprotective activity (PD T _1/2 ) of at least about 24 hours are generally preferred in this assay.

Test method H

Inhalation guinea pig salivation test

Guinea pigs weighing 200-350 g (Charles River, Wilmington, MA) were acclimated to house guinea pig housing for at least 3 days upon arrival. Test compounds or vehicle were administered by inhalation (IH) over a 10 minute period in a pie-shaped dosing chamber (R+S Molds, San Carlos, CA). The test solution was dissolved in sterile water and administered using a nebulizer filled with 5.0 mL of the dosing solution. Confine the guinea pigs to the inhalation chamber for 30 min. During this time, the guinea pigs were confined to an area of approximately 110 cm2. This space is sufficient for the animal to turn freely, reset itself and allow grooming. After 20 minutes of acclimatization, animals were exposed to an aerosol generated from the LS Star Nebulizer Set (Model 22F51, PARI Respiratory Apparatus, Inc. Midlothian, VA) propelled by house air at 22 psi pressure. Guinea pigs were evaluated at 1.5, 6, 12, 24, 48 or 72 hours post-treatment when nebulization was complete.

Guinea pigs were anesthetized with an intramuscular (IM) injection of a mixture of ketamine 43.75 mg/kg, xylazine 3.5 mg/kg and acepromazine 1.05 mg/kg at a volume of 0.88 mL/kg one hour before the test. Animals were placed belly-up on a heated (37°C) blanket with their heads tilted at 20 degrees on a downward slope. A 4-layer 2 x 2 inch gauze pad (Nu-Gauze Universal Cotton Ball, Johnson and Johnson, Arlington, TX) was inserted into the mouth of the guinea pig. Five minutes later, the muscarinic agonist pilocarpine (3.0 mg/kg, s.c.) was administered and the gauze pads were immediately discarded and replaced with new pre-weighed gauze pads. Saliva was collected for 10 minutes, at which point the gauze pad was weighed and the recorded weight difference was used to determine the amount (mg) of saliva accumulated. The average amount of saliva collected for animals receiving vehicle and each dose of test compound was calculated. The vehicle group mean was considered 100% salivating. Results are calculated using the mean of the results (n=3 or more).

Confidence intervals (95%) were calculated for each dose at each time point using two-way ANOVA. This model is an improved version of the model described in Rechter, "Estimation of anticholinergic drug effects inmice by antagonistism against pilocarpine-induced salivation" Ata Pharmacol Toxicol, 1996, 24:243-254.

The mean weight of saliva in vehicle-treated animals was calculated at each pre-treatment time and used to calculate the percent inhibition of salivation at each dose at the corresponding pre-treatment time. Antisialotropic _ID50 (dose required to inhibit 50% of pilocarpine-induced salivation) was estimated by fitting inhibitory dose response data to a four-parameter logistic equation using GraphPad Prism, Windows version 3.00 (GraphPad Software, San Diego, California) . The equation used is as follows:

Y＝Min+(Max-Min)/(1+10 ^{(log ID50-x*Hill slope)} )

where X is the logarithm of the dose and Y is the response (percent inhibition of salivation). Y starts at Min and gradually approaches Max with a recurve shape.

The ratio of the anti-sialagogue _ID50 to the bronchoprotective _ID50 was used to calculate the apparent lung selectivity index of the test compound. In general, compounds having an apparent lung selectivity index greater than about 5 are preferred. The compound of Example 3 had an apparent lung selectivity index greater than 5 in this assay.

While the invention has been described in terms of specific aspects or embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, to the extent the patent status and rules of the application permit, all patents and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each article were individually incorporated by reference herein.

Claims (5)

1. A compound selected from the group consisting of:

or their salts. 2. The compound of claim 1, wherein the compound is:

or its salt. 3. The compound of claim 1, wherein the compound is:

or its salt. 4. The compound of claim 1, wherein the compound is:

or its salt. 5. The compound of claim 1, wherein the compound is:

or its salt.