ES2371425T3 - CRYSTAL FORMS OF A DIMETHYLPHENYL COMPOUND. - Google Patents

Abstract

Crystalline free base form of ester 1- [2- (4 - {[(R) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] Biphenyl-2-ylcarbamic acid piperidin-4-yl.

Description

Crystal forms of a compound of dimethylphenyl

Background of the invention Field of the Invention

The present invention relates to new forms Crystalline free base of a dimethylphenyl compound, whose compound is expected to be useful as a therapeutic agent for the treatment of lung diseases. This invention relates also to pharmaceutical compositions that contain or are prepared from this crystalline form, the processes and intermediate products useful for preparing such forms crystalline, and such crystalline forms for use in, for example, The treatment of a lung disease.

WO 2004/074246 describes the compound of the example 72, which has a structure close to that of the present compound for use in the treatment of lung diseases.

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State of the art

PCT International Shared Ownership Application No. 2007/009925, requested on the same date as this, and provisional application No. 60 / 794,702, requested on April 25, 2006, disclose new dialkylphenyl compounds that are useful as therapeutic agents for treat lung diseases, such as chronic obstructive pulmonary disease (COPD) and asthma. In particular, the ester compound 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4 Biphenyl-2-ylcarbamic acid -yl is specifically described in this application as having activity of both muscarinic antagonist and β2-adrenergic receptor agonist. The chemical structure of the 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- ester Biphenyl-2-ylcarbamic acid is represented by formula I:

one

Therapeutic agents useful for treating lung diseases are advantageously administered in a manner Direct in the respiratory tract by inhalation. In this regard, Several types of pharmaceutical devices have been developed inhalation to administer therapeutic agents by inhalation including dry powder inhalers (DPI), inhalers dispensers (MDI) and nebulizer inhalers. When preparing pharmaceutical compositions and formulations for use in such devices, it is highly desirable to have a crystalline form of therapeutic agent that is not hygroscopic or deliquescent and that have a relatively high melting point (i.e., greater than approximately 130 ° C) thus allowing the material to micronize without significant decomposition or loss of its crystallinity.

No crystalline forms of the compound of the formula I above. Therefore, there is a need for a stable and non-deliquescent crystalline form of compound of the formula I having an acceptable level of hygroscopicity and a relatively high melting point.

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Summary of the invention

The present invention relates to crystalline forms of ester-free base 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5- Biphenyl-2-ylcarbamic acid dimethylphenylcarbamoyl) ethyl] piperidine-4-yl or a solvate thereof. Surprisingly, it has been discovered that such free base crystalline forms of the compound of the formula I are not deliquescent even when exposed to atmospheric moisture. In addition, such crystalline forms have an acceptable level of hygroscopicity and a high melting point, for example, greater than about 130 ° C.

In a particular aspect, this invention relates to a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} Biphenyl-2-ylcarbamic acid -2,5-dimethylphenyl-carbamoyl) ethyl] piperidin-4-yl or a solvate thereof in micronized form.

Among other uses, a crystalline free base form of the compound of the formula I serves to prepare pharmaceutical compositions that are expected to be useful for treating lung diseases. Accordingly, in another aspect of its composition, the present invention relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a crystalline free base form of ester 1- [2- (4 - {[( R ) - Biphenyl-2-ylcarbamic acid 2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl or a solvate thereof.

If desired, the crystalline forms of the The present invention can be administered in combination with others therapeutic agents, such as an anti-inflammatory agent Steroid

Accordingly, in another aspect of its composition, this invention relates to a pharmaceutical composition comprising (a) a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- ( Biphenyl-2-ylcarbamic acid 3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidin-4-yl acid or a solvate thereof; and (b) a second therapeutic agent. In yet another aspect of its composition, this invention relates to a pharmaceutical composition comprising (a) a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3- Biphenyl-2-ylcarbamic acid formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidin-4-yl acid or a solvate thereof; and (c) a pharmaceutically acceptable carrier.

In still another aspect of its composition, this invention relates to a combination of therapeutic agents, the combination comprising (a) a crystalline free base form of ester 1- [2- (4 - {[( R ) - Biphenyl-2-ylcarbamic acid 2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl acid or solvate thereof; and (b) a second therapeutic agent. In another aspect of its composition, this invention relates to a combination of pharmaceutical compositions, the combination comprising (a) a pharmaceutical composition comprising a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-Formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl of biphenyl-2-ylcarbamic acid or a solvate thereof and a support pharmaceutically acceptable; and (b) a pharmaceutical composition comprising a second therapeutic agent and a pharmaceutically acceptable carrier.

The compound of formula I has both β2 adrenergic receptor agonist activity as muscarinic receptor antagonist activity. Therefore, free base crystalline forms of the compound of formula I serve as therapeutic agents to treat lung diseases, such as asthma and lung disease chronic obstructive

Therefore, in one of its aspects, this invention relates to a crystalline free base form of a compound of the formula I for use in a method for treating a lung disease, the method comprising administering to a patient in need of treatment a quantity therapeutically effective form of a crystalline free base of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenyl- carbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid or a solvate thereof.

This invention also relates to a crystalline free base form of a compound of the formula I for use in a method for treating chronic obstructive pulmonary disease or asthma, the method comprising administering to a patient a therapeutically effective amount of a form. Crystalline free base of ester 1- [2- (4 - {[( R ) -2- (3- formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4 - biphenyl-2-ylcarbamic acid or a solvate thereof.

Additionally, in another aspect of its methods, this invention relates to a crystalline free base form of a compound of the formula I for use in a method for producing bronchodilation in a mammal, the method comprising administering to a mammal a quantity which results in bronchodilation of a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5- Biphenyl-2-ylcarbamic acid dimethylphenyl-carbamoyl) ethyl] piperidin-4-yl or a solvate thereof.

This invention also relates to a crystalline free base form of a compound of the formula I for use in a method of antagonizing a muscarinic receptor and agonizing a β2 adrenergic receptor in a mammal, the method comprising administering to the mammal a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine -4-yl of biphenyl-2-ylcarbamic acid or a solvate thereof.

This invention also relates to processes for preparing a free base form of the compound of the formula I. Accordingly, in another of its aspects, this invention relates to a process for preparing a crystalline free base form of ester 1- [ Biphenyl-2-ylcarbamic acid 2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl I, the process comprising:

(to)

provide a solution of 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- biphenyl-2-ylcarbamic acid in a diluent;

(b)

add an antagonistic solvent to the solution of step (a) to produce a point of cloudiness

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Optionally, this process also includes the step of:

(C)

add a seed crystal of a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5- Biphenyl-2-ylcarbamic acid dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl to the product of step (b). In a particular embodiment of these processes, the diluent is methanol and the antagonistic solvent is water.

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Furthermore, this invention relates to a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2, Biphenyl-2-ylcarbamic acid 5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl or a solvate thereof for use in therapy. This invention also relates to the use of this form of crystalline free base for the manufacture of a medicament for the treatment of a lung disease. Other aspects and embodiments of this invention are described herein.

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Brief description of the drawings

Several aspects of this are illustrated invention with reference to the accompanying drawings.

Figures 1, 2 and 3 show the X-ray powder diffraction patterns (PXRD) for Forms I, II and III, respectively, of the crystalline free ester base 1- [2- (4 - {[( R ) -2- (3-Formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl of biphenyl-2-ylcarbamic acid.

Figures 4, 5 and 6 show the traces of a differential scanning calorimetry (DSC) for Forms I, II and III, respectively, of the crystalline free ester base 1- [2- (4 - {[( R ) Biphenyl-2-ylcarbamic acid -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl.

Figures 7, 8 and 9 show the traces of a thermal gravimetric analysis (TGA) for forms I, II and III, respectively, of the crystalline free ester base 1- [2- (4 - {[( R ) Biphenyl-2-ylcarbamic acid -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl.

Figures 10, 11 and 12 show the traces of a dynamic moisture absorption (DMS) for Forms I, II and III, respectively, of the crystalline free ester base 1- [2- (4 - {[( R ) Biphenyl-2-ylcarbamic acid -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidine-4-yl.

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Detailed description of the invention

This invention relates to crystalline forms of ester-free base 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl ) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid or a solvate thereof. The active therapeutic agent in these salts (ie, the compound of formula I) contains a chiral center having the configuration ( R ). However, those skilled in the art will understand that there may be small amounts of the stereoisomer (S) in the compositions of this invention unless otherwise indicated, provided that the presence of said isomer does not eliminate any utility of the composition as a whole. .

The compound of formula I has been named using AutoNom software (MDL, San Leandro, California) available in the market.

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Definitions

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

The term "melting point" as used here means the temperature at which a flow is observed of maximum endothermic heat by differential calorimetry of swept.

The term "mean mass diameter" or "MMD" when used to refer to particles, means that diameter in which half the mass of the particles is contained in particles with a larger diameter and half is contained in particles with a smaller diameter.

The term "micronized" or "in form micronized "means particles in which at least approximately 90 percent of the particles have a diameter of less than about 10 µm unless it is indicate otherwise.

The term "solvate" means a complex or aggregate formed by one or more molecules of a solute, that is, a free base form of the compound of formula I and one or more Solvent molecules. Such solvates usually have a relationship substantially fixed molar of solute and solvent. That term It also includes clathrates, including clathrates with water. The Representative solvents include, by way of example, water, methanol, ethanol, isopropanol, acetic acid and the like. When he Solvent is water, the solvate formed is a hydrate.

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

The term "treat" or "treatment" as used here means the treatment of a disease or medical condition (such as COPD) in a patient such as a mammal (particularly a human) that includes:

(to)

prevent the disease from happening or medical condition, that is, the prophylactic treatment of a patient;

(b)

improve the disease or condition medical, that is, eliminating or causing regression of the disease or medical condition in a patient;

(C)

suppress the disease or condition medical, that is, delaying or stopping the development of disease or medical condition in a patient; or

(d)

relieve disease symptoms or medical condition in a patient.

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The term "unit dosage form" means a physically differentiated unit suitable for the dosage to a patient, that is, each unit containing a predetermined amount of a salt of the invention calculated for produce the desired therapeutic effect either alone or in combination with one or more additional units. For example, such forms of Unit dosage can be dry powder capsules for inhaler, a dosage of a metered dose inhaler, capsules, tablets, pills, and the like.

All other terms used here have its normal meaning as understood by experts in the field to The one they belong to.

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Free-form crystalline forms representative of the invention

It has been found that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4 - Biphenyl-2-ylcarbamic acid -yl exists in at least three different free base crystalline forms. For the purposes of this invention, these forms are identified herein as Form I, Form II and Form III. These crystalline forms of the compound of the formula I can be distinguished by at least the following properties
dades:

Form I:

A trace of differential scanning calorimetry (DSC) that exhibits a peak in the endothermic heat flux of about 132 ° C to about 138 ° C; and a pattern of X-ray powder diffraction (PXRD) that has significant peaks diffraction, among other peaks, in values 2 \ theta of approximately 17.7 ± 0.3, 18.2 ± 0.3, 21.7 ± 0.3 and 23.2 ± 0.3.

Form II:

A trace of differential scanning calorimetry (DSC) that exhibits a peak in the endothermic heat flux of about 142 ° C to about 150 ° C; and a pattern of X-ray powder diffraction (PXRD) that has significant peaks diffraction, among other peaks, in values 2 \ theta of approximately 20.7 ± 0.3, 21.6 ± 0.3, 22.5 ± 0.3 and 23.2 ± 0.3.

Form III:

A trace of differential scanning calorimetry (DSC) that exhibits a peak in the endothermic heat flux of about 134 ° C to about 140 ° C; and a pattern of X-ray powder diffraction (PXRD) that has significant peaks diffraction, among other peaks, in values 2 \ theta of approximately 15.50 ± 0.3, 18.10 ± 0.3, 19.20 ± 0.3 and 21.80 ± 0.3.

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The present invention relates to crystalline forms of ester-free base 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5- Biphenyl-2-ylcarbamic acid dimethylphenylcarbamoyl) ethyl] piperidin-4-yl that are prepared by crystallization of 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) - Biphenyl-2-ylcarbamic acid 2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl or a solvate thereof.

In a preparation method, the forms Crystalline free base of the compound of the formula I are prepared by forming a suspension of the compound in a solvent and the resulting suspension is stirred for a period of time Enough to form the crystalline material. Normally, the suspensions are stirred for approximately 24 until approximately 72 hours at room temperature (that is, about 20 ° C to 30 ° C). Generally, the suspension will comprise between about 35 mg and about 45 mg, that is, about 40 mg of compound per milliliter of solvent In some cases, the choice of solvent will determine what form of crystalline free base is produced from the suspension. For example, when the solvent is acetonitrile, it produces Form I as the predominant form. Alternatively, when the solvent is isopropanol, Form II is produced as the predominant form. Additionally, when the solvent is ethanol, it It produces Form III as the predominant form. Besides of solvent, other factors may affect the solid form that produces.

In another method of preparation, ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) 2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine is dissolved -4-yl of biphenyl-2-ylcarbamic acid in a diluent, such as methanol, ethanol, isopropanol, ethyl acetate, acetonitrile and the like, and an antagonistic solvent (antisolvent), such as water and the like, is added, resulting in formation of the crystalline free base. Usually; The crystallization process can be performed at room temperature or by heating the mixture and then allowing it to cool to room temperature. In a particular embodiment of this method, the diluent is methanol and the antagonistic solvent is water.

The 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester Biphenyl-2-ylcarbamic used in this invention can easily be prepared from commercially available materials and reagents using the procedures described in the Examples below; or using the procedures described in the US patent application. UU of shared ownership described in the background section of this application.

A process to prepare a free base form Crystalline of this invention may optionally include the use of a seed crystal to predominantly produce a base form particular free. For example, using a seed crystal of the Form II, a crystalline free base form of a compound of the formula I having essentially the same form as the seed crystal, that is, Form II. Such seed crystals can be used when initially forming the free base form crystalline or can be used to recrystallize a form crystalline or partially crystalline free base.

Normally, seed crystals are prepared by slow crystallization without stirring and without applying refrigeration. TO by way of example, to obtain seed crystals, normally dissolves the compound of formula I in a diluent at a Sufficient temperature to provide dissolution. For example, him compound of formula I is dissolved in a 1: 2 mixture of acetonitrile and water at between approximately 75 ° C and approximately 80 ° C and then allowed to cool to room temperature for approximately 12 hours to 24 hours to provide a way to crystalline free base. In another embodiment, methanol is used containing about 40% to about 80% water per volume as diluent. The resulting crystals are isolated by filtration or other conventional means. Alternatively, the seed crystals can be obtained from a previous preparation of crystalline material

In a particular embodiment of the recrystallization process using seed crystals, a form free base of the compound of the formula I is dissolved in methanol (typically between about 120 ml and about 160 ml, including 140 ml, of methanol per gram of compound) at room temperature and added water until the cloud point is reached. Then add seed crystals (usually between about 1 and 5 mg of seed crystal per gram of compound). The resulting mixture it is stirred at room temperature for the time necessary to that the compound of the formula I crystallize. The crystals resulting are isolated using conventional procedures, as filtration It will be taken into account that temperatures specific ones used for the crystallization process or recrystallization will be selected depending on the character of the diluent and the concentration of the crystalline salt in the diluent. In addition, the recrystallization process can be performed using evaporation or an antagonistic solvent, instead of cooling, to facilitate crystallization.

In one aspect of this invention, the forms Free base crystallines of this invention are defined by their physical and / or spectral characteristics. Therefore, in a embodiment, this invention relates to a base form crystalline free of the compound of the formula I characterized by a X-ray powder diffraction pattern (PXRD) that has peaks significant diffraction in values 2 \ of approximately 17.7 ± 0.3, 18.2 ± 0.3, 21.7 ± 0.3 and 23.2 ± 0.3. In a particular aspect of this embodiment, this invention is refers to a crystalline free base form characterized by a X-ray powder diffraction pattern in which the positions of the peaks correspond substantially to those shown in the Figure 1.

In another embodiment, this invention is refers to a crystalline free base form of the compound of the formula I characterized by an x-ray diffraction pattern in powder (PXRD) that has significant peaks of diffraction in values 2 de of about 20.70 ± 0.3, 21.60 ± 0.3, 22.50 ± 0.3 and 23.2 ± 0.3. In a particular aspect of this form In this embodiment, this invention relates to a free base form crystalline characterized by an x-ray diffraction pattern in dust in which the positions of the peaks correspond substantially with those shown in Figure 2.

In yet another embodiment, this invention refers to a crystalline free base form of the compound of the formula I characterized by an x-ray diffraction pattern in powder (PXRD) that has significant peaks of diffraction in values 2 de of about 15.50 ± 0.3, 18.10 ± 0.3, 19.20 ± 0.3 and 21.80 ± 0.3. In a particular aspect of this embodiment, this invention relates to a base form crystalline free characterized by a pattern of lightning diffraction x powder in which the positions of the peaks correspond substantially with those shown in Figure 3.

In yet another embodiment, this invention refers to a crystalline free base form of the compound of the formula I that has a corresponding DSC trace substantially with the trace shown in Figure 4. In another form In this embodiment, this invention relates to a free base form crystalline compound of the formula I having a DSC trace that corresponds substantially to the trace shown in Figure 5. In yet another embodiment, this invention relates to a crystalline free base form of the compound of the formula I which has a DSC trace that substantially corresponds to the trace shown in Figure 6.

It has been shown that a free base form Form II crystalline has a reversible profile of absorption / desorption with a weight gain of less than approximately 0.5% in the humidity range of 40% humidity relative to 75% relative humidity.

These properties of the crystalline forms of Free base of this invention are illustrated in more detail in the Examples below.

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Compositions, combinations and pharmaceutical formulations

The free base crystalline forms of this invention are usually administered to a patient in the form of a pharmaceutical composition or formulation. Such compositions Pharmaceuticals can be administered to the patient by any means of acceptable administration including, but not limited to, modes administration by inhalation, oral, nasal, topical (including transdermal) and parenteral.

Therefore, in one of the aspects of its compositions, this invention relates to a composition Pharmaceutical comprising a pharmaceutical carrier or excipient acceptable and a crystalline free base form of the compound of the formula I. Optionally, such pharmaceutical compositions may contain other therapeutic and / or formulation agents if want.

The pharmaceutical compositions of this invention typically contain a therapeutically amount effective of a crystalline free base form of the compound of the formula I. Those skilled in the art will recognize, however, that a pharmaceutical composition may contain more than one amount therapeutically effective, that is, bulk compositions, or less than a therapeutically effective amount, that is, dose Individual units designed for multiple administration to achieve a therapeutically effective amount. Normally the pharmaceutical composition will contain from about 0.01 to approximately 95 percent by weight of the therapeutic agent, including, from about 0.01 to about 30 per weight percent, such as from about 0.01 to approximately 10 weight percent of the therapeutic agent.

Any support or excipient can be used conventional in the pharmaceutical compositions of this invention. The choice of a particular carrier or excipient or combinations of supports and excipients, will depend on the mode of administration that be using to treat a patient or type of medical condition or state of particular disease. In this regard, the preparation of a pharmaceutical composition suitable for a particular mode of administration falls within the competence of technical experts Pharmaceuticals

The carriers or excipients used in the pharmaceutical compositions of this invention are commercially available. For example, such materials can be purchased from Sigma (St. Louis, MO). As another example, conventional formulation techniques are well known to those skilled in the art as illustrated by Remington's theories: The Science and Practice of Pharmacy, 20th edition, Lippincott Williams & White, Baltimore, Maryland (2000) and HC Ansel et al ., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th edition, Lippincott Williams & White, Baltimore, Maryland (1999).

Representative examples of materials that they can serve as pharmaceutically acceptable carriers include, although not exclusively, the following: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as starch corn and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose 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, seed oil cotton, 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 oleate ethyl and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) acid alginic; (16) pyrogen-free water; (17) saline solution isotonic; (18) Ringer's solution; (19) ethyl alcohol; (twenty) phosphate buffer solutions; (21) compressed propellant gases, such as chlorofluorocarbons and hydrofluorocarbons; and (22) others compatible non-toxic substances used in compositions Pharmaceuticals

The pharmaceutical compositions of this invention are usually prepared by mixing or combining carefully and thoroughly a crystalline free base form of the invention with a pharmaceutically acceptable support and one or more ingredients optional If needed or desired, mix evenly The resulting combined can be formed or loaded into tablets, capsules, pills, bottles, cartridges, dispensers and the like using conventional procedures and equipment.

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In one embodiment, the compositions Pharmaceuticals of this invention are suitable for administration. inhaled Pharmaceutical compositions suitable for Inhaled administration will normally be in the form of an aerosol or powder. Such compositions are generally administered using well-known administration devices, such as a nebulizer inhaler, a metered dose inhaler (MDI), an inhaler of dry powder (DPI) or similar delivery device.

In a specific embodiment of this invention, the pharmaceutical composition comprising the agent Therapeutic is administered by inhalation using an inhaler nebulizer Such nebulizer devices usually produce a high velocity air stream that makes the composition pharmaceutical comprising the therapeutic agent is sprayed as a mist that is carried to the patient's respiratory tract. In Consequently, when formulated for use in an inhaler nebulizer, the therapeutic agent is usually dissolved in a support suitable to form a solution. Alternatively, the agent therapeutic can be micronized and combined with a support suitable for forming a suspension of micronized particles. The nebulizer devices suitable for administering agents Therapeutic by inhalation are well known by experts in the Matter or such devices are commercially available. By example, nebulizer devices or representative products include the Respimat Softmist (Boehringer Ingelheim) inhaler; he AERx pulmonary administration system (Aradigm Corp.); he PARI LC Plus reusable nebulizer (Pari GmbH); and the like

A representative pharmaceutical composition for use in a nebulizer inhaler comprises an aqueous solution isotonic comprising from about 0.05 µg / ml to approximately 10 mg / ml of a compound of the formula I. Such compositions are typically prepared by dissolving a form of crystalline free base of the compound of formula I in solution isotonic saline In one embodiment, this solution has a pH of about 4 to about 6.

In another specific embodiment of this invention, the pharmaceutical composition comprising the agent Therapeutic is administered by inhalation using an inhaler of dry powder These dry powder inhalers usually administer the therapeutic agent as a loose powder that disperses in a patient air flow during inspiration. To achieve a loose powder, the therapeutic agent is usually formulated with a Suitable excipient such as lactose, starch, mannitol, dextrose, acid polylactic (PLA), polylactide-co-glycolides (PLGA) or combinations thereof. Normally, the therapeutic agent is micronized and combined with a suitable support to form a suitable mixture for inhalation.

A representative pharmaceutical composition for use in a dry powder inhaler comprises ground particles dried and micronized lactose in a free base form crystalline compound of the formula I.

This dry powder formulation can be made, for example, combining lactose with the therapeutic agent and then dry mixing the components. Alternatively, if If desired, the therapeutic agent can be formulated without excipient. The pharmaceutical composition is then normally loaded in a dry powder dispenser or in cartridges or inhalation capsules for use with a dry powder delivery device.

Dry powder inhaler administering devices suitable for administering therapeutic agents by inhalation are well known to those skilled in the art or such devices are commercially available. For example, administrator devices for dry powder inhalers or representative products include
Aeolizer (Novartis); Airmax (IVAX); ClickHaler (Innovata Biomed); Diskhaler (GlaxoSmithKIine); Diskus / Accuhaler
(GlaxoSmithKIine); Easyhaler (Orion Pharma); Eclipse (Aventis); FlowCaps (Hovione); Handihaler (Boehringer
Ingelheim); Pulvinal (Chiesi); Rotahaler (GlaxoSmithKIine); SkyeHaler / Certihaler (SkyePharma); Twisthaler
(Schering-Plow); Turbuhaler (AstraZeneca); Ultrahaler (Aventis) and the like.

In another specific embodiment more than this invention, the pharmaceutical composition comprising the agent Therapeutic is administered by inhalation using an inhaler dispenser These dosing inhalers typically discharge a measured amount of the therapeutic agent using a gas compressed propellant Consequently, the compositions pharmaceuticals administered using a metered dose inhaler usually comprise a solution or suspension of the agent therapeutic in a liquid propellant. You can use any liquid propellant including chlorofluorocarbons, such as CCl 3 F, and hydrofluoroalkanes (HFA), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and 1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Whereas chlorofluorocarbons affect the ozone layer, formulations are generally preferred containing HFA. Other optional components of formulations of HFA include cosolvents, such as ethanol or pentane and surfactants, such as sorbitan trioleate, oleic acid, Lecithin and glycerin.

A representative pharmaceutical composition for use in a dosing inhaler comprises from approximately 0.01% to about 5% by weight of a free base form crystalline of the compound of the formula I, from about 0% up to about 20% by weight ethanol and from about 0% up to about 5% by weight of surfactant with the rest being an HFA propellant.

Such compositions are usually prepared by adding hydrofluoroalkane cooled or pressurized to a suitable container containing the therapeutic agent, ethanol (if present) and the surfactant (if present). To prepare a suspension, the therapeutic agent is micronized and then combined with the propellant Then the formulation is loaded in an aerosol can which is part of a metering device inhaler.

Dosing inhaler devices Suitable for administering therapeutic agents by inhalation are well known to those skilled in the art or such devices They are commercially available. For example, the devices Dosing inhalers or products include the inhaler system AeroBid (Forest Pharmaceuticals); Atrovent inhalation spray (Boehringer Ingelheim); Flovent (GlaxoSmithKIine); the inhaler Maxair (3M); the Proventil (Schering) inhaler; the spray of Serevent inhalation (GlaxoSmithKIine); and the like

In another embodiment, the compositions Pharmaceuticals of this invention are suitable for administration. oral. Pharmaceutical compositions suitable for oral administration may be in the form of capsules, tablets, pills, lozenges, stamps, dragees, powders, granules; or as a solution or suspension in an aqueous or non-aqueous liquid, or as a liquid emulsion of oil in water or water in oil, or as an elixir or syrup; and the like; each containing an amount predetermined form of a crystalline free base form of the present invention as active ingredient.

When intended for oral administration in a solid dosage form (i.e., in the form of capsules, tablets, pills and the like), the pharmaceutical compositions of this invention will normally comprise a crystalline free base form of the present invention as an active ingredient. and one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. Optionally or alternatively, such solid dosage forms may also comprise: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol and / or silicic acid; (2) binders, such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and / or sodium carbonate; (5) dissolution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and / or glycerol monostearate; (8) absorbents, such as kaolin and / or bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and / or mixtures thereof (10); coloring agents and (11) buffering agents. In the pharmaceutical compositions of this invention, release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulphite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and the like include those used for enteric coatings, such as cellulose acetate phthalate (CAP), polyvinyl acetate phthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylic acid-acid ester copolymers methacrylic, cellulose acetate trimellitate (CAT), carboxymethylcellulose cellulose (CMEC), hydroxypropylmethyl cellulose acetate succinate (HPMCAS), and
Similar.

If desired, the pharmaceutical compositions of The present invention can also be formulated to provide a slow or controlled release of the active substance using, by way of example, hydroxypropyl methylcellulose in variable proportions, or matrices of other polymers, liposomes and / or microspheres

In addition, the pharmaceutical compositions of the invention, optionally, may contain opacifying agents and they can be formulated so that they release the active substance only, or preferably, in a certain portion of the tract gastrointestinal, optionally, in a delayed manner. Examples of inclusion compositions that can be used include polymeric substances and waxes. The active substance can also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, elixirs and syrups. These liquid dosage forms usually comprise the active ingredient and an inert diluent, such as, for example, water or other solvents, solubilizing and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (e.g., cottonseed oils, peanut, corn, wheat germ, olive, castor and sesame), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. The suspensions, in addition to the active ingredient, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. same. Obviously, when administered as a solution in the form of a liquid dose, the crystalline free base form is no longer in crystalline form but is dissolved in the support
liquid.

When they are planned for administration orally, the pharmaceutical compositions of the invention can be packaged in a unit dosage form. The term "form unit dosage "means a unit physically differentiated suitable for dosing a patient, that is, each unit containing a predetermined amount of active agent calculated to produce the desired therapeutic effect either alone or in combination with one or more additional units. For example, such unit dosage forms may be capsules, tablets, pills and the like.

The free base crystalline forms of this invention can also be administered transdermally using transdermal administration systems and excipients. For example, a crystalline free base form can be mixed with permeation enhancers, such as propylene glycol, monolaurate polyethylene glycol, azacycloalcan-2-ones and the like, and incorporate them into a patch or similar administration system. Be they can use other excipients, including gelling agents, emulsifiers and buffers in such transdermal compositions if want.

Additionally, the crystalline base forms free of this invention can be administered parenterally, it is that is, intravenously, subcutaneously or intramuscularly. For the parenteral administration, a base form is normally dissolved crystalline free of the compound of the formula I on a support acceptable for parenteral administration, such as sterile water, saline solution, vegetable oil and the like. As an example, a intravenous composition typically comprises an aqueous solution sterile prepared from a crystalline free base form of a compound of the formula I, where the solution has a pH in the range from about 4 to about 7.

If desired, the crystalline base forms free of this invention can be administered in combination with one or more other therapeutic agents. In this form of embodiment, a crystalline free base form of this invention is either physically mixed with the therapeutic agent to form a composition containing the two agents, or each agent is present in separate and distinct compositions that are administered to the patient simultaneously or sequentially.

For example, a crystalline free base form of the compound of the formula I can be combined with the second therapeutic agent using procedures and equipment conventional to form a composition comprising a shape Crystalline free base of the compound of formula I and a second therapeutic agent In addition, therapeutic agents can be combined with a pharmaceutically acceptable carrier to form a pharmaceutical composition comprising a free base form crystalline compound of the formula I, a second agent therapeutic and a pharmaceutically acceptable support. In this way of embodiment, the components of the composition are mixed or normally combined to create a physical mixture. Mix physics is then administered in a therapeutically amount effective using any of the routes described in this memory.

Alternatively, therapeutic agents they can remain separated and differentiated before the patient administration In this embodiment, the therapeutic agents are not physically mixed before the administration but are administered simultaneously or sequentially as separate compositions. For example, a way Crystalline free base of the compound of the formula I can administered by inhalation simultaneously or sequentially with another therapeutic agent using a delivery device by inhalation using separate compartments (for example blisters) for each therapeutic agent. Alternatively, the combination can be managed using separate devices administration, that is, an administration device for Each therapeutic agent. Additionally, therapeutic agents they can be administered by different routes of administration, it is that is, one by inhalation and the other by oral administration.

Any therapeutic agent compatible with the free base crystalline forms of the present invention can be used in combination with such forms. In a particular embodiment, the second therapeutic agent is one that is effectively administered by inhalation. By way of example, representative types of therapeutic agents that can be used with the compounds of this invention include, but are not limited to, anti-inflammatory agents, such as steroidal anti-inflammatory agents (including corticosteroids and glucocorticoids), non-steroidal anti-inflammatory agents (NSAIDs) and inhibitors. PDE4; bronchodilators, such as PDE 3 inhibitors, adenosine 2b modulators and β 2 adrenergic receptor agonists; anti-infective agents, such as antibiotics against Gram-positive bacteria, antibiotics against Gram-negative bacteria, and antiviral agents; antihistamines; protease inhibitors; afferent nerve pathway blockers, such as D2 agonists and neuroquinine modulators; and muscarinic receptor antagonists (anticholinergic agents). Numerous examples of such therapeutic agents are well known in the art. The appropriate doses for the rest of therapeutic agents administered in combination with a
Compound of the invention are usually in the range of about 0.05 µg / day to 500 mg / day.

In a particular embodiment of this invention, a crystalline free base form of the compound of the formula I is administered in combination with an agent steroidal anti-inflammatory. Representative examples of agents steroidal anti-inflammatories that can be used in combination with the free base crystalline forms of this invention include, but not exclusively, dipropionate of beclomethasone; budesonide; butixocort propionate; 20R-16α, 17α- [butylidenobis (oxy)] -6α, 9α-difluoro-11β-hydroxy-17β- (methylthio) androsta-4-dien-3-one (RPR-106541); ciclesonide; dexamethasone; ester S-fluoromethyl acid 6α, 9α-difluoro-17α - [(2-furanylcarbonyl) oxy] -11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbotioic ; S-fluoromethyl acid ester 6α, 9α-difluoro-11β-hydroxy-16α-methyl-17α- [- [(4-methyl-1,3-thiazol-5-carbonyl) oxy] -3-oxoandrosta -1,4-diene-17β-carbotioic; ester (S) - (2-Oxotetrahydrofuran-3S-Ilic) of the acid 6α, 9α-difluoro-11β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbotioic; flunisolide; fluticasone propionate; methylprednisolone; furoate of mometasone; prednisolone; prednisone; rofleponide; ST-126; triamcinolone acetonide; and the like, or pharmaceutically acceptable salts thereof. These agents Steroidal anti-inflammatories are commercially available or can be prepared using procedures and reagents conventional. For example, the preparation and use of agents Steroidal anti-inflammatories is described in US Patent 6,750,210 B2, granted on June 15, 2004; US 6,759,398 B2, granted on July 6, 2004; US Patent 6,537,983, granted on March 25, 2003; US patent application publication 2002/0019378 A1, published on February 14, 2002; and the references cited therein.

When used, the anti-inflammatory agent steroid is typically administered in an amount that has a therapeutically beneficial effect when administered together with a crystalline free base form of the invention. Generally the steroidal anti-inflammatory agent will be administered in an amount enough to provide from about 0.05 µg to approximately 500 µg per dose.

The following examples illustrate the Pharmaceutical compositions representative of the present invention:

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Example A Dry powder composition

A crystalline free base form is mixed micronized of the compound of the formula I (100 mg) with lactose ground (25 g) (for example, lactose in which no more than approximately 85% of the particles have an MMD of about 60 µm to about 90 µm and not less 15% of the particles have an MMD of less than 15 µm). This mixture is then loaded in individual blisters of a container with tear-off blisters in an amount sufficient to provide about 10 \ mug to about 500 \ mug of compound of formula I per dose. The contents of the blisters It is administered using a dry powder inhaler.

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Example B Dry powder composition

A crystalline free base form is mixed micronized compound of the formula I (1 g) with ground lactose (200 g) to form a bulk composition that has a relationship 1: 200 crystalline to ground lactose free base weight. The mixed composition is packaged in an inhalation device of dry powder capable of administering between about 10 µg and approximately 500 µg of the compound of formula I per dose.

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Example C Dry powder composition

A crystalline free base form is mixed micronized compound of the formula I (100 mg) and an agent Micronized steroidal anti-inflammatory (500 mg) with ground lactose (30 g) This mixture is then loaded into individual blisters of a container with blisters bleachable in an amount sufficient to provide about 10 \ mug up to about 500 mug of the compound of formula I per dose. The content of Blisters are administered using a dry powder inhaler.

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Example D Composition for dosing inhaler

A crystalline free base form is dispersed micronized of the compound of the formula I (10 g) in a solution prepared by dissolving lecithin (0.2 g) in demineralized water (200 ml) The resulting suspension is spray dried and then micronized to form a micronized composition comprising particles that have an average diameter less than about 1.5 µm. The micronized composition is then loaded into cartridges for dosing inhaler containing 1,1,1,2-tetrafluoroethane pressurized in an amount enough to provide approximately 10 \ mug until approximately 500 µg of the compound of formula I per dose when administered with the metered dose inhaler.

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Example E Nebulizer Composition

A crystalline free base form of the compound of the formula I (25 mg) is dissolved in saline solution (125 ml) buffered with citrate (pH 5). The mixture is stirred and sonicated until the compound dissolves. The pH of the solution is verified and adjusted, if necessary, to pH 5 by slowly adding aqueous IN sodium hydroxide. The solution is administered using a nebulizer device that provides between approximately
10 µg and about 500 µg of the compound of formula I per dose.

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Example F Hard gelatin capsules

Mix a base form carefully crystalline free of the compound of the formula I (50 g), lactose spray dried (440 g) and magnesium stearate (10 g). Be load the resulting composition into a hard gelatin capsule (500 mg composition per capsule) administered orally.

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Example G Oral suspension

The following ingredients are mixed comprehensively to form a suspension for administration oral:

2

The resulting suspension contains 100 mg of active substance per 10 ml of suspension. The suspension is Administer orally.

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Example H Injectable Composition

A crystalline free base form of the compound of the formula I (0.2 g) with 0.4 M of buffer solution of sodium acetate (2.0 ml). The pH of the resulting solution is adjusted at pH 4 using 0.5 N of aqueous hydrochloric acid or 0.5 N of aqueous sodium hydroxide, as necessary, after which add enough water for injection to provide a volume total of 20 ml. The mixture is then filtered with a sterile filter. (0.22 microns) to provide a sterile solution suitable for administration by injection.

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Utility

The compound of the formula I possesses both β2-adrenergic receptor agonist and muscarinic receptor antagonist activity and therefore, a crystalline free base form of the compound of the formula I is expected to be useful as a therapeutic agent to treat medical conditions mediated by β2-adrenergic receptors or muscarinic receptors, that is, medical conditions that improve with treatment with a β2-adrenergic receptor agonist or a muscarinic receptor antagonist. These medical conditions are well known to those skilled in the art as illustrated by the theories of Eglen et al ., Muscarinic Receptor Subtypes: Pharmacology and Therapuetic Potential, DN&P 10 (8), 462-469 (1997); Emilien et al ., Current Therapeutic Uses and Potential of beta-Adrenoceptor Agonists and Antagonists, European J. Clinical Pharm., 53 (6), 389-404 (1998); and the references cited therein. 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 panting bronchitis and emphysema), asthma, pulmonary fibrosis and the like. Other conditions include preterm birth, depression, congestive heart failure, skin diseases (for example, inflammatory, allergic, psoriatic and proliferative skin diseases), conditions in which it is desirable to lower peptic acidity (for example, peptic ulceration and gastric) and muscular atrophy.

Therefore, in one embodiment, This invention relates to a crystalline free base form of a compound of formula I for use in a method of treating a lung disease, the method comprising administering to a patient requiring treatment a therapeutically amount effective of a crystalline free base form of the compound of the formula I. When used to treat a disease, the forms Free base crystallines of this invention will be administered typically by aspiration in several doses per day, in one dose single a day or a single dose a week. Generally, the dose to treat a lung disease will vary between about 10 \ mug / day and about 500 \ mug / day.

In one of its aspects, this invention is refers to a crystalline free base form of a compound of the formula I for use in the method of treating lung disease of chronic obstruction or asthma, the method comprising administering to a patient a therapeutically effective amount of a form of crystalline free base of the compound of the formula I. Generally, The dose to treat COPD or asthma will vary between approximately 10 \ mug / day and about 500 \ mug / day. The experts in the matter will understand that the term "COPD" includes a variety of respiratory conditions, including bronchitis and emphysema Chronic obstructive, as illustrated by the theories of Barnes, Chronic Obstructive Pulmonary Disease, N. Engl. J. Med, 2000: 343.269-78 and the references cited in the same.

When administered by inhalation, the compounds of the formula I will normally have the effect of producing bronchodilation Therefore, in another of its aspects, this invention relates to a crystalline free base form of a compound of formula I for use in a method to produce bronchodilation in a mammal, the method comprising administering to a mammal an amount that produces bronchodilation of a crystalline free base form of the compound of the formula I. Generally, the dose to produce bronchodilation will vary between about 10 \ mug / day and about 500 \ mug / day.

When used as a therapeutic agent, the free base crystalline forms of this invention are administered optionally combined with another therapeutic agent or agents. In in particular, when administering a crystalline free base form of This invention with a steroidal anti-inflammatory agent, can be get triple therapy, that is, agonist activity of the β2 adrenergic receptor, antagonist activity of the muscarinic receptor and anti-inflammatory activity, using only Two therapeutic agents. As pharmaceutical compositions (and their combinations) containing two therapeutic agents are usually easier to formulate and / or administer compared to compositions containing three therapeutic agents, such two component compositions provide an advantage significant on compositions containing three agents therapeutic Therefore, in one embodiment in particular, the compositions and pharmaceutical combinations of this invention further comprise an anti-inflammatory agent Steroid

The properties, as well as the utility of the free base crystalline forms of the compounds of the formula I, can be demonstrated using several in vitro and in vivo assays well known to those skilled in the art. For example, representative tests are described in greater detail in the following Examples.

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Examples

The following preparations and examples are provide to illustrate the embodiments and aspects specific to this invention. However, the illustration of embodiments and specific aspects not intended limit the scope of this invention in any way unless Specify specifically.

All reagents, starting materials and Solvents used in the following examples were purchased from commercial suppliers (such as Aldrich, Fluka, Sigma, and the like) and were used without further purification unless indicated another thing.

In the following examples, HPLC analysis It was normally performed using an Agilent equipment (Palo Alto, CA) 1100 series with Zorbax Bonus RP 2.1 x 50 mm columns, supplied by Agilent, (a C14 column), having a particle size of 3.5 microns The detection was by UV absorbance at 214 nm. The phase mobile "A" was 2% acetonitrile, 97.9% water and acid 0.1% trifluoroacetic acid (v / v / v); and the mobile phase "B" was 89.9% acetonitrile, 10% water and 0.1% trifluoroacetic acid (v / v / v). HPLC data (10-70) were obtained with a flow rate of 0.5 ml / minute with a gradient of 10% to 70% of the mobile phase B for a period of 6 minutes; HPLC data (5-35) were obtained with a flow rate of 0.5 ml / minute with a gradient of 5% to 35% of the mobile phase B over a period 5 minutes; and the HPLC data (10-90) is obtained with a flow rate of 0.5 ml / minute with a gradient of I0% at 90% of the mobile phase B for a period of 5 minutes.

Liquid chromatography data with Mass spectrometry (LCMS) were normally obtained with a API-150EX Applied Biosystems model equipment (Foster City, CA). The data from the LCMS 10-90 is obtained with a gradient of mobile phase B from 10% to 90% over a period of 5 minutes.

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Small-scale purifications were performed using an Applied 150EX Prep Workstation API system Biosystems The mobile phase "A" was water containing acid 0.05% (v / v) trifluoroacetic acid; and the mobile phase "B" was acetonitrile containing 0.05% (v / v) trifluoroacetic acid. For small samples (size of approximately 3 to 50 mg of the sample recovered) the following were normally used conditions: flow rate 20 ml / min; 15 minute gradients and one 20 mm x 50 mm Prism RP column with 5 micron particles (Thermo Hypersil-Keystone, Bellefonte, PA). For samples larger (i.e., greater than about 100 mg of the raw sample) the following conditions were normally used: flow rate 60 ml / min; 30 minute gradients and a 41.4 mm column x 250 mm Microsorb BDS with 10 micron particles (Varian, Stick Alto, CA).

For 1 H NMR data, they are used The following abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = width, nd = no determined.

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Example 1 Piperidin-4-Ilic acid ester biphenyl-2-ylcarbamic

They got hot biphenyl-2-isocyanate (97.5 g, 521 mmol) and 4-hydroxy-1-benzylpiperidine (105 g, 549 mmol) (both marketed by Aldrich, Milwaukee, W1) together at 70 ° C for 12 hr, during which time the ester formation 1-Benzylpiperidin-4-Ilic acid biphenyl-2-ylcarbamic by LCMS. The reaction mixture was then cooled to 50 ° C and ethanol was added. (1 L) after which 6M hydrochloric acid (191 ml) was added slowly. The reaction mixture was cooled to room temperature and ammonium format (98.5 g, 1.56 mol) was added and gas was bubbled nitrogen in the solution vigorously for 20 min. Then Palladium (10% by weight (dry) in activated carbon) (20 g) was added. The reaction mixture was heated at 40 ° C for 12 hr and then filtered through a celite support. Then the solvent was removed under reduced pressure and 1M hydrochloric acid (40 ml) was added to the raw waste Then sodium hydroxide (10N) was added to adjust the pH to 12. The aqueous layer was extracted with ethyl acetate. (2 x 150 ml) and dried (magnesium sulfate), and then the solvent was removed under reduced pressure to give ester piperidin-4-yl acid Biphenyl-2-ylcarbamic (155 g, 100%). HPLC (10-70) R t = 2.52; MS m / z: [M + H] + calculated for C 18 H 20 N 2 O 2: 297.15; Found: 297.3.

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Example 2 Acid 3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionic

To an ester solution piperidin-4-yl acid biphenyl-2-ylcarbamic (50 g, 67.6 mmol) in dichloromethane (500 ml) acrylic acid (15.05, 100 mmol). The resulting mixture was heated at 50 ° C under reflux for 2 hours and then the solvent was removed. Methanol (600 ml) was added and this mixture was heated at 75 ° C for 2 hours and then cooled to room temperature to form a thick paste. The solid is collected by filtration, washed with methanol (50 ml) and dried on air to provide acid 3- (4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionic (61 g, 96% purity) as white powder.

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Example 3 N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} -formamide acetic acid salt

He passed TO

N - {5 - [( R ) -2-benzylamino-1- (tert-butyldimethylsthyloxy) ethyl] -2-benzyloxyphenyl} formamide

To a round bottom flask and three 500 ml necks was added N- {2-benzyloxy-5 - [( R ) -2-bromo-1- (tert-butyldimethylsilyloxy) ethyl] phenyl) formamide (100 g, 215 mmol) and N-methyl-2-pyrrolidone (300 ml). Benzyl amine (69.4 ml, 648 mol) was added and the reaction mixture was purged with nitrogen. The reaction mixture was then heated to 90 ° C and stirred for approximately 8 hours. The reaction mixture was then cooled to room temperature and water (1.5 L) and ethyl acetate (1.5 L) were added. The layers were separated and the organic layer was washed with water (500 ml), a 1: 1 mixture of water and saturated brine (500 ml total), and again with water (500 ml). The organic layer was then dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure to provide N - {5 - [( R ) -2-benzylainino-1- (tert-butyldimethylsilyloxy) ethyl] -2-benzyloxyphenyl} formamide raw (100 g, 90% yield, 75-80% purity) as a thick orange-brown oil.

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He passed B

N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} formamide acetic acid salt

N - {5 - [( R ) -2-benzylamino-1- (tert-butyldimethylsilyloxy) ethyl] -2-benzyloxyphenyl} formamide (100 g, 194 mmol) was dissolved in methanol (1 L) and acetic acid (25 ml, 291 mmol). The resulting mixture was purged with dry nitrogen and then palladium hydroxide in carbon (20 g, 20% by weight, approximately 50% water) was added. Hydrogen was bubbled into the reaction mixture with stirring at room temperature for approximately 10 hours. The mixture was then purged with dry nitrogen and the mixture was filtered with celite. The filtrate was concentrated on a rotary evaporator and ethyl acetate (600 ml) was added to the residue. This mixture was stirred for approximately 2 hours, at which time a thick yellow paste developed. The paste was filtered and the precipitate was air dried to provide N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} formamide acetic acid salt (48 g , 98% purity) as a yellow-white solid. LCMS (10-70) R t = 3.62; [M + H +] found
311.3.

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Example 4 Biphenyl acid 1- [2- (4 - {[( R ) -2- {3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} 2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl ester -2-ilcarbamic

He passed TO

2,5-Dimethyl-4-nitrobenzoate of methyl

To a stirred acid solution 2,5-dimethyl-4-nitrobenioic (480 mg, 2.4 mmol) in dry methanol (8.2 ml) at 0 ° C under nitrogen Dry thionyl chloride (0.538 ml, 7.38 mmol) was added. The resulting mixture was allowed to warm to room temperature and stirred for about 7 hours. More thionyl chloride was added (0.300 ml) and stirring was continued at room temperature for the night. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate. This solution was washed with bicarbonate. saturated aqueous sodium, dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide 2,5-dimethyl-4-nitrobenzoate of methyl (578 mg) as a pale yellow solid. HPLC (10-70) R t = 4.61; 1 H NMR (300 MHz, CDCl 3) δ 2.57 (s, 3H), 2.61 (s, 3H), 3.94 (s, 3H), 7.82 (s, 1H), 7.87 (s, 1H).

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He passed B

4-Amino-2,5-dimethylbenzoate of methyl

To a stirred solution of 2,5-dimethyl-4-nitrobenzoate of methyl (523 mg, 2.5 mmol) in a 9: 1 mixture of methanol and water (25 ml total) at 0 ° C was added ammonium chloride (401 mg, 7.5 mmol). Portions of zinc (1.63 g, 25 mmol) and the mixture were added The resulting was stirred at room temperature overnight. The reaction mixture was filtered through celite and the support of Celite was washed with methanol. The filtrate was concentrated under pressure. reduced and the resulting residue was dissolved in ethyl acetate. This solution was washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium sulfate and concentrated under pressure reduced to provide 4-amino-2,5-dimethylbenzoate of methyl (450 mg) as a yellow oil. 1 H NMR (300 MHz, CDCl 3) δ 2.14 (s, 3H), 2.53 (s, 3H), 3.83 (s, 3H), 3.85 (br s, 2H), 6.48 (s, 1H), 7.72 (s, 1H).

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He passed C

4- {3- [4- (Biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionylamino} -2,5-dimethylbenzoate of methyl

To a stirred acid solution 3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionic (670 mg, 1.82 mmol) and 4-amino-2,5-dimethylbenzoate methyl (390 mg, 2.18 mmol) in dichloromethane (3.6 ml) and diisopropylethylamine (0.413 ml) was added hexafluorophosphate O- (7-azabenzotriazol-1-yl) -1,3,3-tetramethyluronium (HATU) (829 mg, 2.18 mmol). The resulting mixture was stirred at room temperature overnight. Then the mixture was washed with saturated aqueous sodium bicarbonate, dried over anhydrous sodium, filtered and concentrated under reduced pressure. The residue it was purified by chromatography on silica gel eluting with dichloromethane containing 3% to 5% methanol, to provide 4- {3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionylamino} -2,5-dimethylbenzoate of methyl (568 mg, 59% yield). LCMS (10-70) R t = 4.55; [M + H +] found 530.4.

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He passed D

Ester 1- [2- (4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of the acid biphenyl-2-yl-carbamic

To a stirred solution of 1M hydride of lithium aluminum in THF (1.52 ml, 1.52 mmol) at 0 ° C was added 4- {3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionylamino} -2,5-dimethylbenzoate of methyl (400 mg, 0.76 mmol). The resulting mixture was stirred at 0 ° C. for 30 minutes and then a 1: 1 mixture of 1M of aqueous sodium hydroxide (5 ml) and water (5 ml) and stirring is He continued for 2 hours. Dichloromethane and the organic layer were added separated, dried over sodium sulfate and the solvent was removed at reduced pressure The residue was purified by chromatography on silica gel eluting with dichloromethane containing 5% methanol to provide ester 1- [2- (4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl) -ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid. LCMS (10-70) R t = 3.94; [M + H +] found 502.5.

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He passed AND

Ester of 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

To a solution of 1- [2- (4-hydroxymethyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl ester of biphenyl-2-ylcarbamic acid (151 mg, 0.3 mmol) in dichloromethane (3 ml) at 0 ° C, dimethyl sulfoxide (128 µL, 1.8 mmol) and diisopropylethylamine (157 µL, 0.9 mmol) were added. After 15 minutes, the sulfide trioxide and pyridine complex (143 mg, 0.9 mmol) was added and stirring was continued at 0 ° C for 1 hour. Water was added to quench the reaction and the layers were separated. The organic layer was dried over anhydrous sodium sulfate, filtered and the solvent removed under reduced pressure to give biphenyl acid 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester -2-ilcarbamic (150 mg, 100% yield), which was used without further purification. [M + H +] found
500.4

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He passed F

1- [2- (4 - {[( R ) -2- (tert-Butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidine- ester 4-Ilic Biphenyl-2-ylcarbamic acid

A solution of 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl ester of biphenyl-2-ylcarbamic acid (150 mg, 0.30 mmol) and N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} formamide (112 mg, 0.36 mmol) in a 1: 1 mixture of dichloromethane and methanol (3.0 ml total) at room temperature for 30 minutes. Sodium triacetoxyborohydride (191 mg, 0.9 mmol) was added and the resulting mixture was stirred at room temperature overnight. Acetic acid was added to quench the reaction and the mixture was concentrated under reduced pressure to give ester 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) -2- (3-formylamino-4- Hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl of biphenyl-2-ylcarbamic acid, which was used without further purification. LCMS (10-70) R t = 4.55; [M + H +] found 794.6.

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He passed G

1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester of biphenyl acid -2-ilcarbamic

To a suspension of 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl ester ] Biphenyl-2-ylcarbamic acid piperidine-4-yl (238 mg, 0.30 mmol) in dichloromethane (3.0 ml) was added triethylamino trihydrofluoride (147 µL, 0.90 mmol). This mixture was stirred at room temperature overnight and then the mixture was concentrated under reduced pressure. The residue was purified by preparative RP-HPLC (gradient: 2 to 50% acetonitrile in water with 0.05% TFA). The appropriate fractions were collected and lyophilized to give 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl ester ] piperidin-4-yl of biphenyl-2-ylcarbamic acid as ditrifluoroacetate salt (50 mg, 97% pure). LPLC (2-90) R t = 2.76; [M + H +] found 680.8.

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Example 5 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl) -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester of biphenyl acid -2-ilcarbamic

He passed TO

Dibenzyl- (4-iodo-2,5-dimethylphenyl) amine

To a 2 liter round bottom bottle equipped with a top stirrer, temperature control and a funnel was added 4-iodo-2,5-dimethylaniline (100.0 g, 0.405 mol) (from Spectra Group Limited, Inc., Millbury, OH). Ethanol (1 L) and solid potassium carbonate (160 g, 1,159) were added mol) and then pure benzyl bromide (140 ml, 1,179 mol) was added in one serving The resulting mixture was stirred at 30 ° C for approximately 18 hours, at which time the HPLC shows a Conversion higher than 98%. Then the mixture was cooled to room temperature and hexanes (1 L) were added. This mixture is stirred for 15 minutes and then filtered with a paper filter to remove solids and the filter cake was washed with hexanes (200 ml). Using a rotary evaporator, the volume of the filtrate was reduced to approximately 500 ml and acid was added concentrated hydrochloric (30 ml). The remaining solvent was removed then using a rotary evaporator. To the resulting residue hexanes (500 ml) were added and this mixture was stirred for approximately 30 minutes, at which time a paste formed fluid The paste was filtered and the filter cake was washed with hexanes (200 ml) and dried to provide hydrochloride of dibencil- (4-iodine-2,5- dimethylphenyl) amine (115 g, 62% yield, 97.5% of purity) as a greenish solid.

Dibenzyl- (4-iodo-2,5-dimethylphenyl) amine hydrochloride was transferred to a 3 I bottle and toluene (1 L) and 1 M aqueous sodium hydroxide (1 L) were added. The resulting mixture was stirred for 1 hour and then the layers were separated. The organic layer was washed with dilute brine (500 ml) and the solvent was removed by rotary evaporation to provide dibenzyl- (4-iodo-2,5-dimethylphenyl) amine (80 g) as a semi-solid thick oil. (Alternatively, dichloromethane can be used instead of toluene in this step). 1 H NMR (300 MHz, DMSO- d 6) δ 2.05 (s, 3H), 2.19 (s, 3H), 3.90 (s, 4H), 6.91 (s, 1H), 7.05-7.20 (M, 10H), 7.42 (s, 1H); [M + H +] found 428.

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He passed B

Hydrochloride 4-dibenzylamino-2,5-dimethylbenzaldehyde

To a round bottom jar and 3 1 l necks equipped with a top stirrer, temperature control and a addition funnel was added dibenzyl- (4-iodo-2,5-dimethylphenyl) amine (15 g, 35 mmol). Toluene (300 ml) was added and the resulting mixture stirred for about 15 minutes. The reaction flask purged with dry nitrogen and cooled to about -20 ° C and added 1.6 M of n-butyllithium in hexanes (33 ml, 53 mmol) dropwise through the addition funnel. During the In addition, the internal temperature of the reaction mixture was maintained below -10 ° C. When the addition was completed, the mixture The resulting was stirred at about -15 ° C for 15 minutes. Then N, N-dimethylformamide (10 ml, 129) was added mmol) drop by drop while maintaining the internal temperature of reaction below 0 ° C. Then the resulting mixture was stirred to between -20ºC and 0ºC for approximately 1 hour. Then it was added 1 M aqueous hydrochloric acid (200 ml) over a period of 5 minutes and the resulting mixture was stirred for 15 minutes. So the layers were separated and the organic layer was washed with brine diluted (100 ml). The organic layer was then dried over sulfate of anhydrous sodium was filtered and the solvent was removed under reduced pressure to provide hydrochloride of 4-dibenzylamino-2,5-dimethylbenzaldehyde (11.5 g, 90% yield, 95% purity) as a thick oil which solidified after rest. The product contained approximately 3 to 5% of the secondary product of des-iodine 1 H NMR (300 MHz, CDCl 3) δ 2.42 (s, 3H), 2.50 (s, 3H), 4.25 (s, 4H), 6.82 (s, 1H), 7.10-7.30 (10H, m), 7.62 (1H, s), 10.15 (1H, s); MS [M + H +] found 330.3.

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He passed C

4- [1,3] Dioxolan-2-yl-2,5-dimethylphenylamine

A 500 ml round bottom bottle will be added hydrochloride of 4-dibenzylamino-2,5-dimethylbenzaldehyde (11.5 g, 31.4 mmol) and toluene (150 ml) and the resulting mixture is stirred until the salt dissolved completely. The jar of The reaction was then purged with dry nitrogen for 5 minutes. Be added ethylene glycol (5.25 ml, 94.2 mmol) and acid p-toluenesulfonic acid (760 mg, 6.2 mmol) and the mixture resulting heated at 60 ° C to 80 ° C for approximately 20 hours. Then the solvent was removed slowly (during approximately 40 minutes) at 40 ° C in a rotary evaporator. Be added toluene (100 ml) to the residue and the solvent was removed again slowly at 40 ° C in a rotary evaporator. This process is repeated using another aliquot of toluene (100 ml) and the mixture was evaporated to dry. Ethyl acetate (150 ml) was added and saturated aqueous sodium bicarbonate (100 ml) to the residue and layers They separated. The organic layer was washed with brine (50 ml) and then dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to provide dibenzyl- (4- [1,3] dioxolan-2-yl-2,5-dimethyl-phenyl) amine raw (11.4 g).

The crude dibenzyl- (4- [1,3] dioxolan-2-yl-2,5-dimethyl-phenyl) amine was dissolved in a 2: 1 mixture of ethanol and water (150 ml total) and the resulting mixture was purged with dry nitrogen for 5 minutes. Palladium on carbon (2.3 g, 10% by weight containing approximately 50% water) and solid sodium bicarbonate (1.0 g) were added and the resulting mixture was hydrogenated at about 1 atm of hydrogen at between 25 ° C and 30 ° C for approximately 8 hours The mixture was filtered with celite and the filtrate was concentrated on a rotary evaporator to provide crude 4- [1,3] dioxolan-2-yl-2,5-dimethylphenylamine (5.6 g, 92% yield) as an oil. thick. 1 H NMR (300 MHz, DMSO- d 6) δ 2.05 (s, 3H), 2.22 (s, 3H), 3.7-3.9 (m, 4H), 3 , 95 (s, 4H), 5.59 (s, 1H), 6.72 (s, 1H), 7.0-7.25
(m, 11H).

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He passed D

N- (4- [1,3] Dioxolan-2-yl-2,5-dimethylphenyl) acrylamide

A 500 ml round bottom bottle will be added 4- [1,3] dioxolan-2-yl-2,5-dimethylphenylamine crude (5.6 g, 29 mmol), dichloromethane (100 ml) and diisopropylethylamine (7.6 ml, 43.5 mmol). The resulting mixture is stirred at room temperature until the ingredients are dissolved and then the mixture was cooled to 0 ° C. Then it was added acryloyl chloride (2.35 ml, 29 mmol) dropwise during a 5 minute period. The reaction mixture was stirred at 0 ° C and 5 ° C for 1 hour and then water (50 ml) was added and continued stirring for approximately 30 minutes, at which time solids formed. The mixture was filtered to collect the solid. The filtrate layers were separated and the organic layer was separated. concentrated under reduced pressure to dry. Dichloromethane was added (50 ml) to the residue and this mixture was stirred until it developed a fluid paste The paste was filtered (using the same funnel as was used to collect the fine solids above) and the cake from filtration was washed with dichloromethane (10 ml) and dried to provide N- (4- [1,3] dioxolan-2-yl-2,5-dimethylphenyl) acrylamide (3.1 g, 97% purity) as a white solid at whitish

The filtrate above was evaporated to dryness and methanol (10 ml) was added to the residue. This mixture was stirred for 15 minutes and then the precipitate was collected by filtration, washed with methanol (5 ml) and dried to give a second production of N - (4- [1,3] dioxolan-2-yl-2 , 5-dimethylphenyl) acrylamide (0.8 g, 95% purity). 1 H NMR (300 MHz, CD 3 OD) δ 2.10 (s, 3H), 2.23 (s, 3H), 3.85-4.10 (m, 4H), 5.60 -6.40 (M, 3H), 5.59 (s, 1H), 7.18 (s, 1H), 7.23 (s, 1H).

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He passed AND

Ester 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of the acid biphenyl-2-yl-carbamic

To a 50 ml round bottom bottle was added piperidin-4-yl ester of biphenyl-2-ylcarbamic acid (1.2 g, 4.04 mmol) and N - (4- [1,3] dioxolan-2 -yl-2,5-dimethylphenyl) acrylamide (1.0 g, 4.04 mmol). Ethanol (10 ml) and dichloromethane (10 ml) were added to form a paste. The reaction mixture was heated at 45 ° C to 50 ° C for approximately 18 hours and cooled to room temperature. 1M aqueous hydrochloric acid (10 ml) was added and the resulting mixture was vigorously stirred for approximately 3 hours. Dichloromethane (10 ml) was added and the resulting mixture was stirred for approximately 5 minutes. The layers were then separated and the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated on a rotary evaporator to provide 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidine ester hydrochloride Crude biphenyl-2-ylcarbamic acid 4-yl (1.9 g). 1 H NMR (300 MHz, DMSO- d 6) δ 1.2-1.4 (m, 2H), 1.58-1.75 (m, 2H), 2.0- 2.17 (m, 2H), 2.19 (s, 3H), 2.38 (s, 3H), 2.41-2.50 (m, 4H), 2.5-2.75 (m, 2H), 4.31-4.42 (m, 1H), 7.10-7.35 (m, 9H), 7.55 (s, 1H), 7.75 (s, 1H), 8.59 (s, 1H), 9.82 (s, 1H), 9.98 (s, 1H); MS [M + H +] found 500.2.

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He passed F

1- [2- (4 - {[( R ) -2- (tert-Butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl) -2,5-dimethylphenyl-carbamoyl) ethyl] piperidine ester -4-yl of biphenyl-2-ylcarbamic acid

To a round bottom flask of 2 I was added 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester hydrochloride (38 g, 70 mmol) and N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} formamide acetic acid salt (33.6 g, 91 mmol). Dichloromethane (500 ml) and methanol (500 ml) were added and the resulting mixture was stirred at room temperature under dry nitrogen for approximately 3 hours. The reaction mixture was then cooled to 0 ° C to 5 ° C and solid sodium triacetoxyborohydride (44.5 g, 381 mmol) was added portionwise over a period of 10 minutes. The reaction mixture was slowly heated from 0 ° C to room temperature for a period of about 2 hours and then cooled to 0 ° C. Saturated aqueous sodium bicarbonate (500 ml) and dichloromethane (500 ml) were added. This mixture was stirred thoroughly and then the layers were separated. The organic layer was washed with brine (500 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give ester 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) ) Biphenyl-2-ylcarbamic acid -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl as a yellow solid.

The crude product (30 g) was dissolved in dichloromethane containing 2% methanol (150 ml total) and loaded onto a column of silica gel (300 g) that had been loaded and equilibrated with dichloromethane containing 2% methanol and hydroxide 0.5% ammonium. The product was eluted from the column using dichloromethane containing 2% methanol and 0.5% ammonium hydroxide (1 L); dichloromethane containing 4% methanol and 0.5% ammonium hydroxide (1 L) and dichloromethane containing 5% methanol and 0.5% ammonium hydroxide (approximately 3 L). Fractions (200 ml) were collected, and those fractions having a purity greater than 90% were combined and concentrated under reduced pressure to provide 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) ester] Biphenyl-2-ylcarbamic acid -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl (21.6 g, 96.5% purity) Like a yellowish solid. MS [M + H +] found 794.6.

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He passed G

1- [2- (4-1 {( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylaminomethyl} -2,5-dimethylphenyl-carbamoyl) ethylpiperidine-4-yl ester hydrofluoride salt

To a 1 l round bottom flask was added 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) 2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} - ester Biphenyl-2-ylcarbamic acid 2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl (21.5 g, 27.1 mmol) and dichloromethane (200 ml). The resulting mixture was stirred at room temperature until the ingredients dissolved and then triethylamine trihydrofluoride (8.85 ml, 54.2 mmol) was added and the resulting mixture was stirred at 25 ° C for approximately 48 hours. The solvent was removed on a rotary evaporator to provide a thick paste. Dichloromethane (100 ml) and ethyl acetate (200 ml) were added to the paste and the resulting mixture was stirred for 30 minutes. The resulting paste was slowly filtered under dry nitrogen and the filter cake was washed with a 1: 2 mixture of dichloromethane and ethyl acetate (100 ml total), dried under nitrogen for 2 hours and then dried under vacuum during night to provide 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenyl-carbamoyl) ethyl] piperidine-4 ester - Biphenyl-2-ylcarbamic acid as a hydrochloride salt (25 g, 96.9% pure) that was a hard clay-like solid. MS [M + H +] found 680.8.

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He passed H

1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidine-4-yl acid ester biphenyl-2-ylcarbamic

The ester hydrofluoride salt 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} 2,5-dimethylphenylcarbamoyl) ethyl] piperidine was purified 4-Ilic biphenyl-2-ylcarbamic acid (25 g) on a 6-inch reverse phase column (solid phase Microsorb) in three equal series using a 10% to 50% mixture of acetonitrile in water containing trifluoroacetic acid at 1% as mobile phase. Fractions with a purity greater than 99% were combined and then diluted with a volume of water. The resulting mixture was cooled to 0 ° C and solid sodium bicarbonate was added until the pH of the mixture was approximately 7.5 to 8.0. In about 5 minutes a white paste developed. The paste was stirred for 30 minutes and then filtered. The filter cake was washed with water (500 ml), air dried for about 4 hours and then dried under vacuum overnight to provide ester 1- [2- (4 - {[( R ) -2- ( Biphenyl-2-ylcarbamic acid 3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl (12 g, 99 +% purity), as a base semi-crystalline free.

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Example 6 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} 2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester of biphenyl- 2-ilcarbamic

He passed TO

Ester 1- [2- (4 [1,3] dioxolan-2-yl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

To a 500 ml round bottom bottle was added biphenyl-2-ylcarbamic acid piperidin-4-yl ester (17.0 g, 58 mmol) and N - (4- [1,3] dioxolan-2-yl -2,5-dimethylphenyl) acrylamide (13.1 g, 52.9 mmol). Ethanol (150 ml) and dichloromethane (150 ml) were added to form a paste. The reaction mixture was heated at 50 ° C to 55 ° C for approximately 24 hours and cooled to room temperature. Most of the solvent was removed on a rotary evaporator to provide a thick paste. Ethanol (reagent grade) was added to form a total volume of approximately 200 ml and the resulting mixture was heated to 80 ° C and then slowly cooled to room temperature. The resulting thick white paste was filtered, washed with ethanol (20 ml) and dried in vacuo to provide 1- [2- (4- [1,3] dioxolan-2-yl-2,5-dimethylphenylcarbamoyl) ethyl] piperidine ester -4-yl of biphenyl-2-ylcarbamic acid (23.8 g, approximately 98% pure) as a white solid.

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He passed B

Ester of 1- [2- (4-formyl-2,5-dimethylphenyl-carbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

To a 500 ml round bottom flask was added 1- [2- (4- [1,3] dioxolan-2-yl-2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester of biphenyl-2 acid -ylcarbamic (15 g, 27.6 mmol) and acetonitrile (150 ml) to form a paste. 2M aqueous hydrochloric acid (75 ml) was added and the resulting mixture was stirred at 30 ° C for 1 hour. The mixture was then cooled to room temperature and ethyl acetate (150 ml) was added. 2 M of aqueous sodium hydroxide (75 ml) was added, the pH was verified and then another 2 M of sodium hydroxide was added until the pH of the solution was in the range of 9 to 10. The layers were separated. and the organic layer was washed with dilute brine (75 ml; 1: 1 brine / water), dried over anhydrous sodium sulfate and the solvent was removed on a rotary evaporator to give ester 1- [2- (4-formyl- 2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl (12.5 g, approximately 98% pure). If desired, the purity of this intermediate can be increased by forming a paste with ethanol (3 volumes of ethanol), heating the paste at 80 ° C, and then slowly cooling to room temperature and insulating by
filtration.

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He passed C

1- [2- (4 - {[( R ) -2- (tert-Butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylimino] methyl} -2,5-dimethyl-phenylcarbamoyl) ethyl] piperidine ester -4-yl of biphenyl-2-ylcarbamic acid

To a 250 ml round bottom bottle was added 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl ester of biphenyl-2-ylcarbamic acid (7.1 g, 14 , 2 mmol) and N - {5 - [( R ) -2-amino-1- (tert-butyldimethylsilyloxy) ethyl] -2-hydroxyphenyl} formamide acetic acid salt (5.8 g, 15.6 mmol) . Methanol (100 ml) was added to form a paste and this mixture was stirred at between 15 ° C and 50 ° C under nitrogen for 1 hour. The mixture was then cooled to room temperature and toluene (50 ml) was added and the solvent was removed on a rotary evaporator at a temperature ranging between 35 ° C and 45 ° C. Toluene (50 ml) was added to the residue and the solvent was removed to provide ester 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) Ethylimino] methyl} -2,5-dimethyl-phenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid (12 g) as a yellow-orange solid.

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He passed D

Ester 1- [2- (4 - ([( R ) -2- (tert-Butyldimethylsilanyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4 - biphenyl-2-ylcarbamic acid

To a hydrogenation flask was added 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) 2- (3-formylamino-4-hydroxyphenyl) ethylimino] methyl} -2,5- ester Biphenyl-2-ylcarbamic acid dimethyl-phenylcarbamoyl) ethyl] piperidin-4-yl (4.6 g) and 2-methyltetrahydrofuran (50 ml). The resulting mixture was stirred until the solid dissolved (approximately 5 min.) And then the mixture was purged with nitrogen. Platinum on carbon (920 mg, 5% by weight, active carbon support) was added and the mixture was hydrogenated at 50 psi (Parr stirrer) for 6 hours. The mixture was filtered with celite (5 g), and the celite was washed with 2-methyltetrahydrofuran (10 ml). To the filtrate was added a thiopropyl modified silica gel (20% solution weight, Silicycle) and this mixture was stirred at between 25 ° C and 30 ° C for 3 hours. Then the mixture was filtered with celite and concentrated to remove the solvent. The residue was dissolved in methanol (5 ml per gram of residue) and then the resulting solution was slowly added to a vigorously stirred 1: 1 mixture of aqueous sodium bicarbonate and water (40 ml per gram of residue). The resulting whitish paste was stirred for 20 minutes and then filtered. The filter cake was washed with water (20 volumes), air dried for 3 hours and then dried under vacuum at room temperature overnight to provide ester 1- [2- (4 - {[( R ) -2 - (tert-Butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl of biphenyl-2-ylcarbamic acid (80% recovery, approximately 96% purity).

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He passed AND

Ester L-tartrate salt 1-12- (4-1 [( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl) -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4 - biphenyl-2-ylcarbamic acid

To a 200 ml round bottom bottle was added 1- [2- (4 - {[( R ) -2- (tert-butyldimethylsilyloxy) -2- (3-formylamino-4-hydroxyphenyl) ethylamino] -methyl ester } -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid (3.8 g, 4.8 mmol) and 2-methyltetrahydrofuran (400 ml). The resulting mixture was stirred at room temperature until the ingredients dissolved (approximately 15 min.) And then triethylamine trihydrofluoride (0.94 ml, 5.76 mmol) was added and the resulting mixture was stirred at 25 ° C for approximately 24 hours. . To this mixture was added a 1: 1 mixture of saturated aqueous sodium bicarbonate and water (40 ml) and 2-methyltetrahydrofuran and the resulting mixture was stirred until the solid dissolved (solution pH of about 8). The layers were separated and the organic layer was washed with brine (30 ml), dried over anhydrous sodium sulfate, filtered and concentrated on a rotary evaporator. The residue was dissolved in 2-methyltetrahydrofuran (50 ml) and cold L-tartaric acid (650 mg) was added. The resulting mixture was heated at 25 ° C to 30 ° C for 18 hours and then filtered with a paper filter. The filter cake was washed with 2-methyltetrahydrofuran (10 ml), isopropanol (10 ml) and immediately placed in vacuo to provide salt of ester L-tartaric acid 1- [2- (4 - {[( R ) - Biphenyl-2-ylcarbamic acid 2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenyl-carbamoyl) ethyl] piperidin-4-yl (3.7 g,> 97%
purity).

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He passed F

1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester of biphenyl acid -2-ilcarbamic

To a 250 ml round bottom flask was added L-tartaric acid ester salt 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl Biphenyl-2-ylcarbamic acid (3.5 g) and methanol (35 ml) -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl, and the resulting mixture was stirred for 15 min. A 1: 1 mixture of saturated aqueous sodium bicarbonate and water (70 ml) was added over a period of 5 min and stirring was continued for 2 hours. The resulting whitish plast was filtered and the filter cake was washed with water (20 ml), air dried for 2 hours and then dried under vacuum overnight to provide ester 1- [2- (4 - {[( R ) -2- (3-Formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl of biphenyl-2-ylcarbamic acid (2.3 g) as a semi-crystalline free base.

The 1 H and 13 C NMR spectra were obtained for an ester sample 1- [2- (4 - ([( R ) -2- (3-formylamino-4-hydroxyphenyl) -2 -hydroxyethylamino] methyl) -2,5-dimethylphenylcarbamoyl) -ethyl] piperidin-4-yl biphenyl-2-ylcarbamic acid (22.2 mg in approximately 0.75 ml of DMSO-d6) at room temperature using A JEOL ECX-400 NMR spectrometer:

1 H NMR (400 MHz, DMSO- d 6), major isomer, δ 9.64 (br, 1H), 9.54 (br s, 1H), 9.43 (s, 1H ), 8.67 (s, 1H), 8.26 (s, 1H), 8.03 (d, J = 1.9, 1H), 7.25-7.45 (m, 9H), sim7 , 3 (nd, 1H), 7.07 (s, 1H), 6.88 (dd, J = 8.2, 1.9, 1H), 6.79 (d, J = 8.2, 1H) , 5.15 (br, 1H), 4.53 (dd, J = 7.3, 4.7, 1H), 4.47 (m, 1H),? 3.65 and? 3.60 (pair AB , 2H), 2.68 (br m, 2H),? 2.59 (nd, 4H), 2.44 (br t, J = 6.5, 5 2H), 2.20 (s, 3H), ,12.17 (br m, 2H), 2.14 (s, 3H), 1.73 (br, 2H), 1.44 (br q, J = sim9.0, 2H).

1 H NMR (400 MHz, DMSO- d 6), minor isomer, δ 9.64 (br, 1H), 9.43 (s 1H), 9.26 (br d, J = 77.0, 1H), 8.67 (s, 1H), 8.50 (br d, J = sim7.0, 1H), 7.25 ,7.45 (m, 9H), sim7, 3 (nd, 1H), 7.07 (s, 1H),? 7.07 (nd, 1H), 6.95 (dd, J = 8.3, 1.8, 1H), 6.83 (d , J = 8.3, 1H), 5.15 (br, 1H), 4.47 (m, 1H),? 3.65 and? 3.60 (pair AB, 2H), 2.68 (br m , 2H), sim2.59 (nd, 2H), 2.44 (brt, J = 6.5, 2H), 2.20 (s, 3H), sim2.17 (br m, 2H), 2 , 14 (s, 3H), 1.73 (br, 2H), 1.44 (br q, J = 99.0, 2H).

13 C NMR (100 MHz, DMSO- d 6), major isomer, δ 170.0, 159.9, 153.9, 145.5, 139.3, 137.6, 135, 2, 135.0, 134.8, 133.4, 133.4, 130.2, 130.2, 128.6, 128.2, 127.8, 127.4, 127.2, 127.0, 126.1, 125.7, 125.6, 121.7, 118.6, 114.5, 71.4, 70.0, 57.4, 53.9, 50.3, 50.1, 33, 7, 30.7, 18.2, 17.5.

13 C NMR (100 MHz, DMSO- d 6), minor isomer, δ 170.0, 163.4, 153.9, 147.8, 139.3, 137.6, 135, 7, 135.2, 135.0, 134.8, 133.4, 133.4, 130.2, 130.2, 128.6, 128.2, 127.8, 127.4, 127.2, 127.0, 126.1, 125.7, 123.0, 119.6, 115.6, 71.0, 70.0, 57.3, 53.9, 50.3, 50.1, 33, 7, 30.7, 18.2, 17.5.

The 1 H and 13 C NMR spectra showed the presence of a major isomer (approximately 82 per percent in mol) and a minor isomer (approximately 18 percent in mol) which was believed to be the isomers resulting from the rotation restricted around the link -NH-C (O) H. It is believed that the phenyl group it is syn for carbonyl oxygen in the major and anti isomer in the minor isomer

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Example 7 Ester of 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

He passed TO

4-iodo-2,5-dimethylphenylamine

To a solution of 2,5-dimethylaniline (20 g, 165 mmol) in a mixture 1: 1 dichloromethane and methanol (400 ml) bicarbonate was added sodium (20.8 g, 250 mmol) and tetramethylammonium dichloroiodate (I) (44.7 g, 165 mmol). The resulting mixture was stirred at temperature ambient for 1 hour and then water (500 ml) was added. The layer Organic was removed and washed with 5% aqueous sodium thiosulfate (500 ml) and brine (500 ml). The organic layer was dried over sulfate of anhydrous magnesium, filtered and concentrated in vacuo to give 4-iodo-2,5-dimethylphenylamine (39.6 g, 98% yield). The product was used without further purification.

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He passed B

N - (4-iodo-2,5-dimethylphenyl) acrylamide

To a solution of 4-iodo-2,5-dimethylphenylamine (37.2 g, 151 mmol) in dichloromethane (500 ml) was added sodium bicarbonate (25.4 g, 302 mmol). The resulting mixture was cooled to 0 ° C and acryloyl chloride (12.3 ml, 151 mmol) was added slowly over a period of 25 minutes. The resulting mixture was stirred at room temperature overnight and then filtered. The volume of the filtrate was reduced to approximately 100 ml and a precipitate formed. The precipitate was filtered, dried, washed with water (1 L) and then dried again to provide N - (4-iodo-2,5-dimethylphenyl) acrylamide (42.98 g, 95% purity, 90% of performance). The product was used without further purification.

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He passed C

Ester 1- [2- (4-Iodo-2,5-dimethylphenylcarbamoyl) -ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

To a solution of N - (4-iodo-2,5-dimethylphenyl) acrylamide (32.2 g, 107 mmol) in a 6: 1 v / v mixture of N, N- dimethylformamide and isopropanol (700 ml) is added biphenyl-2-ylcarbamic acid piperidine-4-yl ester (36.3 g, 123 mmol). The resulting mixture was heated at 50 ° C for 24 hours and then at 80 ° C for 24 hours. The reaction mixture was then cooled to room temperature and concentrated in vacuo. The residue was dissolved in dichloromethane (1 L) and this solution was washed with 1N aqueous hydrochloric acid (500 ml), water (500 ml), brine (500 ml) and saturated aqueous sodium bicarbonate (500 ml). The organic layer was then dried over anhydrous magnesium sulfate and filtered. Ethanol (400 ml) was added and the resulting mixture was concentrated in vacuo to a volume of approximately 400 ml, at which time a precipitate formed. The precipitate was filtered and dried to provide biphenyl-2-ylcarbamic acid 1- [2- (4-iodo-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl ester (59.6 g, 84% purity, 79% yield), m / z: [M + H] + calculated for C 29 H 32 IN 3 O 3: 598.49; Found: 598.5.

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He passed D

Acid methyl ester 4- (3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] ethyl] propionylamino} -2,5-dimethylbenzoic acid

To an ester solution of 1- [2- (4-Iodo-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid (56 g, 94 mmol) in a 5: 1 v / v mixture of N, N-dimethylformamide and methanol (600 ml) was added diisopropylethylamine (49 ml, 281 mmol), 1,3-bis (diphenylphosphino) propane (3.9 g, 9.4 mmol) and palladium (II) acetate (2.1 g, 9.4 mmol). Mix resulting was purged with carbon monoxide and then stirred overnight at 70 ° C to 80 ° C under a monoxide atmosphere Carbon (balloon pressure). The reaction mixture was concentrated at vacuum, and the residue was dissolved in dichloromethane (500 ml). This mixture was washed with 1N hydrochloric acid (500 ml), water (500 ml) and brine (500 ml). Then the organic layer was dried over sulfate of anhydrous magnesium and concentrated in vacuo. The residue was mixed with ethanol (approximately 5: 1 v / p of ethanol and residue) and the mixture It was heated until all solid material dissolved. This solution was allowed to cool slowly to room temperature and the resulting precipitate was isolated by filtration to provide acid methyl ester 4- {3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionylamino} 2,5- dimethylbenzoic acid (47.3 g, 97% purity, 92% yield), m / z: [M + H] + calculated for C 31 H 35 N 3 O 5: 530.63; Found: 530.4.

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He passed AND

Ester 1- [2- (4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of acid biphenyl-2-yl-carbamic

An acid methyl ester solution was cooled 4- {3- [4- (biphenyl-2-ylcarbamoyloxy) piperidin-1-yl] propionylamino} -2,5-dimethylbenzoic acid (49.8 g, 93.9 mmol) in tetrahydrofuran (200 ml) at 0 ° C and it will be added lithium aluminum hydride (10.7 g, 281.7 mmol) in portions (10 x 1.07 g). The resulting mixture was stirred for 3 hours and then water (10.7 ml) was added followed by 1N hydroxide of aqueous sodium (10.7 ml) and more water (32.1 ml). This mixture was stirred. overnight and leaked. The organic layer was concentrated in vacuo and the residue was mixed with ethyl acetate (approximately 5: 1 v / p of ethyl acetate and residue). This mixture was heated until all solid material dissolved and the solution was allowed to cool to room temperature. The resulting precipitate was filtered and dried. to provide ester 1- [2- (4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl) ethyl) piperidin-4-yl of biphenyl-2-ylcarbamic acid (24.6 g, 95% purity, 47.5% yield). This material was used without further purification, m / z: [M + H] + calculated for C 30 H 35 N 3 O 4: 502.62; Found: 502.5.

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He passed F

Ester 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid

To an ester solution 1- [2- (4-Hydroxymethyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid (5.0 g, 10 mmol) in dichloromethane (200 ml) was added diisopropylethylamine (8.7 ml, 50 mmol) and dimethylsulfoxide (5.6 ml, 100 mmol) The resulting mixture was cooled to 0 ° C and the pyridine and sulfide trioxide complex (8.0 g, 50 mmol). The reaction mixture was stirred for 1 hour at 0 ° C and then added water (300 ml). The organic layer was removed and washed with 1N of aqueous hydrochloric acid (300 ml) and brine (300 ml). So The organic layer was dried over anhydrous magnesium sulfate and filter. The resulting solution containing ester 1- [2- (4-formyl-2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid se used without further purification, (m / z): [M + H] + calculated for C 30 H 33 N 3 O 4: 500.60; Found: 500.4.

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Example 8 Preparation of Form I of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine- 4-Ilic Biphenyl-2-ylcarbamic acid

An ester paste 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidine- was prepared 4-yl of biphenyl-2-ylcarbamic acid (55 mg) in acetonitrile (2 ml) and stirred at room temperature for 2 days. The solvent was removed by filtration, and the solids dried at room temperature to provide Form I (50 mg). The PXRD, DSC and TGA spectra for this crystalline free base form are shown in Figures 1, 4 and 7, respectively.

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Example 9 Preparation of Form II of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine- 4-Ilic Biphenyl-2-ylcarbamic acid

An ester paste 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidine- was prepared Biphenyl-2-ylcarbamic acid 4-yl (46 mg) in isopropanol (2 ml) and stirred at room temperature for 2 days. The solvent was removed by filtration, and the solids dried at room temperature to provide Form II (43 mg). The PXRD, DSC and TGA spectra for this crystalline free base form are shown in Figures 2, 5 and 8, respectively.

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Example 10 Preparation of Form III of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine- 4-Ilic Biphenyl-2-ylcarbamic acid

An ester paste 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) -ethyl] piperidine- was prepared 4-Ilic acid biphenyl-2-ylcarbamic acid (54 mg) in ethanol (2 ml) and stirred at room temperature for 2 days. The solvent was removed by filtration, and the solids dried at room temperature to provide Form III (48 mg). The PXRD, DSC and TGA spectra for this crystalline free base form are shown in Figures 3, 6 and 9, respectively.

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Example 11 Preparation of seed crystals of Form II of the 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl ester ] Biphenyl-2-ylcarbamic acid piperidin-4-yl

1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4-yl ester was dissolved Semi-crystalline biphenyl-2-ylcarbamic acid (500 mg) in methanol (50 ml) and water was added until the cloud point was reached. The resulting mixture was stirred at 25 ° C for 3 hours and the resulting crystalline material was isolated by filtration to provide Form II of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) ) Biphenyl-2-ylcarbamic acid -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl-acid (420 mg).

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Example 12 Crystallization of Form II of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine- 4-Ilic Biphenyl-2-ylcarbamic acid

A 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) ester ester was added to a 3-necked round bottom flask equipped with a top stirrer, temperature control and addition funnel ) -2-Hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of semicrystalline biphenyl-2-ylcarbamic acid (14 g) and methanol (1.4 L). Water (500 ml) was added in one portion and then more water (200 ml) was added slowly until the cloud point was reached. Seeds 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenyl-carbamoyl) -ethyl] ester crystals were added Biphenyl-2-ylcarbamic acid piperidine-4-yl (50 mg) and the resulting mixture was stirred at 25 ° C for 3 hours, at which time a fluid paste was developed. Water (300 ml) was added over a period of 15 minutes and the resulting mixture was stirred at 25 ° C overnight. The mixture was filtered and the filter cake was washed with water (100 ml), air dried for about 2 hours and then dried under vacuum at room temperature to provide Form II of the ester 1- [2- (4- Crystalline biphenyl-2-ylcarbamic acid {12 (( R ) 2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl g, 99.6% purity).

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Example 13 X-ray powder diffraction

X-ray powder diffraction patterns were obtained with a Termo ARL X-ray diffractometer, model X'TRA (Termo ARL SA, Switzerland) using Cu Ka radiation at 1,542 A (45 kV, 40 mA) with a detector solid state Peltier. The analysis was normally performed at a scanning speed of 2 ° / min with a step size of 0.03 ° per point in a range of 2 to 40 ° at an angle of two zeta. Prior to measurement, light grinding was done by hand for 30 seconds of Form II to reduce particle size. There was no mechanical reduction in particle size in Forms I and III. The samples were carefully loaded into a custom-made quartz sample pot designed to fit the top-loading sample cup of the equipment for analysis. The equipment was calibrated weekly with a silicon metal standard, within the two zeta angle of 0.02º. Raw raw representative PXRD standards for Form I, Form II and Form III of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl of crystalline biphenyl-2-ylcarbamic acid are shown in Figures 1, 2 and 3, respectively.

Additionally, the following were obtained positions of the two zeta peaks using a function for the profile adjustment that included a background subtraction and routine attenuation:

Form I:

8.3 ± 0.3; 10.7 ± 0.3; 12.2 ± 0.3; 13.5 ± 0.3; 14.5 ± 0.3; 15.2 ± 0.3; 17.7 ± 0.3; 18.2 ± 0.3; 19.6 ± 0.3; 20.7 ± 0.3; 21.7 ± 0.3 and 23.2 ± 0.3.

Form II:

8.4 ± 0.3; 10.4 ± 0.3; 10.7 ± 0.3; 11.9 ± 0.3; 12.3 ± 0.3; 12.6 ± 0.3; 13.4 ± 0.3; 13.6 ± 0.3; 14.3 ± 0.3; 14.7 ± 0.3; 15,310.3; 16.8 ± 0.3; 17.10 ± 0.3; 17.80 ± 0.3; 18.20 ± 0.3; 18.70 ± 0.3; 19.20 ± 0.3; 19.80 ± 0.3; 20.70 ± 0.3; 21.60 ± 0.3; 22.30 ± 0.3; 22.50 ± 0.3 and 23.20 ± 0.3.

Form III:

8.5 ± 0.3; 10.5 ± 0.3; 11.3 ± 0.3; 12.1 ± 0.3; 12.8 ± 0.3; 13.6 ± 0.3; 14.2 ± 0.3; 15.5 ± 0.3; 16.6 ± 0.3; 17.1 ± 0.3; 18.1 ± 0.3; 19.2 ± 0.3; 21.0 ± 0.3; 21.8 ± 0.3; 22.6 ± 0.3; 23.3 ± 0.3 and 24.5 ± 0.3.

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Example 14 Differential scanning calorimetry

Differential scanning calorimetry (DSC) was performed using a TA Instruments model Q-100 module with a Thermal Analyst controller. Data was collected and analyzed using TA Instruments Thermal Solutions software. Exactly a sample of approximately 1 mg was weighed in an aluminum pot with a lid. The sample was evaluated using a linear heating ramp of 5 ° C / min from room temperature to approximately 300 ° C. The DSC cell was purged with dry nitrogen during use. Representative DSC traces for samples of Form I, Form II and Form III of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] Methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of crystalline biphenyl-2-ylcarbamic acid are shown in Figures 4, 5 and 6, respectively. Figure 5 demonstrates that Form II of the 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl ester ] -piperidin-4-yl of crystalline biphenyl-2-ylcarbamic acid has excellent thermal stability with a melting point at about 143 ° C.

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Example 15 Thermogravimetric analysis

Thermogravimetric analysis (TGA) was performed using a TA Instruments module model Q-500 equipped with high resolution capability. Data was collected and analyzed using TA Instruments Thermal Solutions software. A sample weighing approximately 10 mg was placed in a platinum pot and scanned with a heating rate and high resolution from room temperature to 300 ° C. The equilibrium and oven chambers were purged with nitrogen flows during use. The representative TGA traces for the samples of Form I, Form II and Form III of the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] Methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of crystalline biphenyl-2-ylcarbamic acid are shown in Figures 7, 8 and 9, respectively.

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Example 16 Dynamic moisture absorption evaluation

An evaluation of dynamic moisture absorption (DMS) (also known as moisture absorption-desorption profile) was performed for hand-ground samples of Form I, Form II and Form III of the ester 1- [2- (4 - {[( R ) -2- (3-Formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl crystalline biphenyl-2-ylcarbamic acid using a VTI atmospheric microbalance, SGA-100 system (VTI Corp., Hialeah, FL 33016). A sample size of approximately 10 mg was used and the humidity was adjusted to the ambient value at the beginning of the analysis. A typical DMS analysis consisted of three scanners: ambient at 2% relative humidity (RH), 2% RH at 90% RH, 90% RH at 5% RH at a scan rate of 5% RH / He passed. The mass was measured every two minutes and the RH was changed to the next value (± 5% RH) when the sample mass was stable at within 0.01% for 5 consecutive points. Representative traces of the DMS for Form I, Form II and Form III are shown in Figures 10, 11 and 12, respectively.

The trace of the DMS in Figure 11 demonstrates that Form II had a reversible absorption / desorption profile with a moderate hygroscopicity (<5%). Form II had an increase insignificant weight of approximately 0.3% by weight in the range of humidity from 40% RH to 75% RH. The reversible profile of moisture absorption / desorption demonstrates that Form II has a Acceptable hygroscopicity and is not deliquescent.

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Example 17 Cell culture and membrane preparation from cells expressing a muscarinic receptor M 1, M 2, M 3 and Human M4

CHO cell lines were stably expressing the cloned subtypes of the human muscarinic receptor hM1, hM2, hM3 and hM4, respectively, until almost confluence in a Ham F-12 medium supplemented with 10% FBS and 250 µg / ml of Geneticin. The cells were cultured in an incubator with 5% CO2, 37 ° C and sustained with 2 mM EDTA in dPBS. The cells were collected with a 5 minute centrifugation at 650 xg and the cell pellets were either stored frozen at -80 ° C or membranes prepared immediately for use. To prepare the membrane, the cell pellets were resuspended in a lysis buffer and homogenized with a Polytron PT2100 tissue disintegrator (Kinematica AG; 20 seconds x 2 bursts). The crude membranes were centrifuged at 40,000 xg for 15 minutes at 4 ° C. The membrane sediment was then resuspended with a resuspension buffer and homogenized again with the Polytron tissue disintegrator. The protein concentration of the membrane suspension was determined by the method described in Lowry, et al ., 1951, Journal of Biochemistry 193, 265. All frozen membranes were stored in aliquots at -80 ° C or used immediately. Aliquots of prepared hM5 receptor membranes were purchased directly from PerkinElmer, Inc. (Wellesley, MA) and stored at -80 ° C until use.

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Example 18 Radioligand binding assay for receptors muscarinics

Radioligand binding assays were performed for muscarinic receptors cloned in 96-well plates in a total assay volume of 100 µl. The membranes of the CHO cells that stably express the muscarinic subtypes hM1, hM2, hM3, hM4 or hM5 were diluted in an assay buffer with the following concentrations of the specific target protein (\ mug / well): 10 \ mug for hM_ {1}, 10-15 \ mug for hM_ {2}, 10-20 \ mug for hM_ {3}, 10-20 \ mug for hM_ { 4} and 10-12? For hM_ {5} to get similar signals (cpm). The membranes were homogenized briefly using a Polytron tissue disintegrator (10 seconds) before adding them to the test plates. Saturation fixation studies were performed to determine the K D values of the radioligands using L- [ N- methyl-3 H ] scopolamine methyl chloride ([3 H] -NMS) ( TRK666, 84.0 Ci / mmol, Amersham Pharmacia Biotech, Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20 nM. Displacement assays were performed to determine the Ki values of the test compounds with [3 H] -NMS at 1 nM and eleven different concentrations of the test compounds. The test compounds were initially dissolved at a concentration of 400 µM in a dilution buffer and diluted in 5x series with dilution buffer until final concentrations ranging from 10 pM to 100 µM were reached. The order and volumes of addition to the test plates were as follows: 25 µL of radioligand, 25 µL of diluted test compound and 50 µL of membranes. The test plates were incubated for 60 minutes at 37 ° C. Fixation reactions ended with rapid filtration on GF / B glass fiber filter plates (PerkinElmer Inc.) previously treated in 1% BSA. The filter plates were washed three times with wash buffer (10 mM HEPES) to remove unbound radioactivity. The plates were air dried and 50 µL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.) was added to each well. The plates were counted in a PerkinElmer Topcount liquid scintillation counter (PerkinElmer Inc.). Binding data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the competition model of a site. The K i values for the test compounds of the observed IC 50 values and the K D value of the radioligand were calculated using the Cheng-Prusoff equation (Cheng Y; Prusoff WH (1973) Biochemical Pharmacology, 22 (23): 3099-108). The K_ {i} values were converted to pK_ {i} values to determine the geometric mean and 95% confidence intervals. These statistics were then converted back to values of K_ {i} for the presentation of data.

In this assay, a lower Ki value indicates that the test compound has a higher binding affinity for the receptor tested. It was found that the 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- ester Biphenyl-2-ylcarbamic acid (compound of the formula I) has a K i value of less than 10 nM for the subtypes of the muscarinic receptor M 1, M 2, M 3, M_ {4} and M_ {5}.

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Example 19 Cell culture and membrane preparation from cells which express the β1, β2 or adrenergic receptors human β3

[Human embryonic kidney cell lines (HEK-293) that stably expressed the β1 and β2 adrenergic receptors or Chinese hamster ovary (CHO) cell lines stably expressing the human β3 adrenergic receptors cloned to near confluence in a DMEM or Ham F-12 medium with 10% FBS in the presence of 5.00 µg / ml of Geneticin. The cell monolayer was lifted with 2 mM EDTA in PBS. The cells were pelleted by centrifugation at 1,000 rpm, and the cell pellets were either frozen frozen at -80 ° C or the membranes were prepared immediately for use. For the preparation of the membranes expressing the β1 and β2 receptors, the cell pellets were resuspended in tisis buffer (10 mM HEPES / HCl, 10 mMM EDTA, pH 7.4 a 4 ° C) and homogenized using a Dounce airtight glass homogenizer (30 strokes) on ice. For membranes expressing the β3 receptor most sensitive to protease, cell pellets were homogenized in lysis buffer (10 mM Tris / HCl, pH 7.4) supplemented with a "Complete Protease Inhibitor" tablet Cocktail Tablets "with 2 mM EDTA per 50 ml of buffer (Roche Molecular Biochemicals, Indianapolis, IN). The homogenate was centrifuged at 20,000 xg and the resulting sediment was washed once with resuspension lysis buffer and centrifugation as indicated above. The final sediment 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 ., Journal of Biochemistry 193, 265; and Bradford, Analytical Biochemistry, 1976; 72, 248-54. All frozen membranes were stored in aliquots at -80 ° C or used immediately.

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Example 20 Radioligand binding assay on receptors human β1, β2 and β3 adrenergic agents

Binding assays were performed in 96-well microtiter plates in a total assay volume of 100 µL with 10-15 µg of membrane protein containing human β1, β2 or human adrenergic receptors β 3 in the assay buffer (75 mM Tris / HCl pH 7.4 at 25 ° C, 12.5 M MgCl 2, 1 mM EDTA, 0.2% BSA). Saturation binding studies were performed to determine the K d values of the radioligand using [3 H] -dihydroalprenolol (NET-720, 100 Ci / mmol, PerkinElmer Life Sciences Inc., Boston, MA) to the β1 and β2 receptors and
[125 I] - (-) - iodocianopindolol (NEX-189, 220 Ci / mmol, PerkinElmer Life Sciences Inc., Boston, MA) in 10 or 11 different concentrations ranging from 0.01 nM to 20 nM. Displacement assays were performed for the determination of the K i values of the test compounds with [3 H] -dihydroalprenolol at 1 nM and [121 I] - (-) iodocianopindolol at 0.5 nM for 11 different concentrations of the test compound ranging from 10 pM to 10 µM. Non-specific binding was determined in the presence of 10 µM of propranolol. The assays were incubated for 1 hour at 37 ° C and then the binding reactions were terminated by rapid filtration on GF / B glass fiber plates for β 3 receptors (Packard BioScience Co., Meriden, CT) previously wetted in 0.3% polyethyleneimine. The filtration plates were washed three times with filtration buffer (75 mM Tris / HCl, pH 7.4 at 4 ° C, 12.5 mM MgCl 2, 1 mM EDTA) to eliminate unbound radioactivity. The plates were dried and 50 µl of Microscint-20 scintillation liquid (Packard BioScience Co., Meriden, CT) was added to each well and the plates were counted in a Packard Topcount liquid scintillation counter (Packard BioScience Co., Meriden , CT). Binding data were analyzed by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the competition model of a 3-parameter site. The minimum of the curve was set at the value of non-specific binding, as determined in the presence of 10 µM of propranolol. The K_ values for the test compounds were calculated from the observed IC 50 values and the K d value of the radioligand using the Cheng-Prusoff equation (Cheng Y; Prusoff WH Biochemical Pharmacology 1973, 22, 23, 3099-108).

In this assay, a lower Ki value indicates that the test compound has a higher binding affinity for the receptor tested. It was found that the 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- ester Biphenyl-2-ylcarbamic acid (compound of the formula I) has a K i value of less than 10 nM for the β 2 adrenergic receptor and K i values greater than 1000 nm for the adrenergic receptors? 1 and? 3.

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Example 21 Functional antagonism tests for subtypes of muscarinic receptor

Test TO

Agonist-mediated inhibition blockade of cAMP accumulation

In this test the potency was determined functional of a test compound as an antagonist to the hM2 receptor measuring the capacity of the test compound of block oxotremorine inhibition of cAMP accumulation forscolin mediated in CHO-K1 cells that they expressed the hM2 receptor. CAMP assays were performed with a radioimmunoassay format using the test system of Flashplate cyclase activation with 125 I-cAMP (NEN SMP004B, PerkinElmer Life Sciences Inc., Boston, MA), according to the manufacturer's instructions. The cells were rinsed once with dPBS and sustained with a Trypsin and EDTA solution (0.05% trypsin / 0.53 mM EDTA) as described in the Membrane Preparation and Culture section of cells up. Loose cells were washed twice by centrifugation at 650 x g for five minutes in 50 ml of dPBS. The cell pellet was then resuspended in 10 ml of dPBS and counted the cells with a double particle counter Coulter Z1 (Beckman Coulter, Fullerton, CA). The centrifuges were centrifuged again cells at 650 x g for five minutes and were resuspended in a Stimulation buffer until obtaining a test concentration of 1.6 x 10 6 -2.8 x 10 6 cells / ml.

The test compound was dissolved initially at a concentration of 400 µM in a dilution buffer (dPBS supplemented with 1 mg / ml BSA (0.1%)) and serially diluted 5x with dilution buffer until reaching a final molar concentration that it varied between 100 µM and 0.1 nM. Oxotremorine was diluted so Similary.

To measure oxotremorine inhibition of adenylyl cyclase activity 25 µL of forscolin (final concentration of 25 µM diluted in dPBS), 25 µL of diluted oxotremorine and 50 µL of cells to the wells of agonist test. To measure the capacity of a compound of assay to block the activity of adenylyl cyclase inhibited by oxotremorine 25 µL of forscolin and oxotremorine were added (final concentrations of 25 µM and 5 µM, respectively, diluted in dPBS), 25 µL of diluted test compound and 50 µL of cells to the rest of test wells.

The reactions were incubated for 10 minutes at 37 ° C and stopped by adding 100 µL of cooled stop buffer in ice. The plates were sealed, incubated overnight at room temperature and were counted the next morning in a liquid scintillation counter PerkinElmer Topcount (PerkinElmer Inc., Wellesley, MA). The amount of cAMP produced was calculated (pmol / well) based on the counts observed for the samples and cAMP standards, as described in the user manual of the maker. Data were analyzed by non-regression analysis. linear with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the competition equation of a nonlinear regression site. The equation was used Cheng-Prusoff to calculate the value of K_ {obs}, using the EC50 of the concentration curve of Oxotremorine-response and assay concentration of oxotremorine as the value of K_ {D} and [L], respectively.

In this test, a lower K_b value indicates that the test compound has a higher functional activity in the receptor tested. It was found that the 1- [2- (4 - {[( R -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidine-4- ester Biphenyl-2-ylcarbamic acid (compound of the formula I) has a K obs value of less than 10 nM for the blockage of oxotremorine inhibition of the accumulation of phospholin-mediated cAMP in CHO-K1 cells expressing the hM_ {2} receiver.

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Test B

[35 S] GTPγS-mediated binding of the binding the agonist

In this functional test, the functional potency of a test compound as an antagonist of hM2 receptor measuring the ability of the test compound to block the binding of [35 S] GTPγS stimulated with oxotremorine in CHOK1 cells expressing the hM2 receptor.

At the time of use, the thaws were thawed frozen membranes and then diluted in the assay buffer with a final concentration of the target tissue of 5 to 10 µg of protein per well. The membranes were homogenized briefly using a Polytron tissue disintegrator and then added to the test plates.

The EC90 value was determined (concentration effective for a maximum response of 90%) for the stimulation of the fixation of [35 S] GTPγS by the oxotremorine of agonist in each experiment.

To determine the capacity of a compound of [35 S] GTPγS binding inhibition assay stimulated by oxotremorine, the following was added to each well of 96-well plates: 25 µL of assay buffer with [35 S] GTPγS (0.4 nM), 25 µL of oxotremorine (EC 90) and GDP (3 µM), 25 µL of test compound diluted and 25 µL of CHO cell membranes expressing the hM_2 receiver. The plates were then incubated at 37 ° C for 60 minutes. The test plates were filtered on GF / B filters previously treated with 1% BSA using a combine 96-well PerkinElmer. The plates were rinsed with buffer ice-cold wash for 3x3 seconds and then dried air or vacuum. Scintillation fluid was added Microscint-20 (50 µL) to each well, sealed each plate and the radioactivity was counted in a Topcounter (PerkinElmer). Data were analyzed by non-regression analysis. linear with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the competition equation of a nonlinear regression site. The equation was used Cheng-Prusoff to calculate the value of K_ {obs}, using the IC_ {50} values of the curve of concentration-response for the test compound and the concentration of oxotremorine in the assay as the value of K D and the concentration of ligand [L], respectively.

In this test, a lower K_b value indicates that the test compound has a higher functional activity in the receptor tested. It was found that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4 -yl of biphenyl-2-ylcarbamic acid (compound of the formula I) has a K_b value of less than 10 nM for blocking the binding of [35 S] GTP? g stimulated with oxotremorine in CHO-K1 cells expressing the hM2 receptor.

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Test C

Agonist-mediated calcium release blockade at through FLIPR trials

In this functional test, the functional potency of a test compound as an antagonist of the hM1, hM3 and cM5 receptors measuring the capacity of the test compound to inhibit calcium increases intracellular mediated by the agonist.

CHO cells expressing from stably form the receptors in 96-well FLIPR plates the night before rehearsing. The seeded cells were washed twice with FLIPR buffer (10 mM HEPES, pH 7.4, 2 mM chloride of calcium, 2.5 mM probenecid in Hank's saline solution buffered (HBSS) without calcium or magnesium) using Cellwash (MTX Labsystems, Inc.) to eliminate the growth medium. After the washed, each well contained 50 µl of FLIPR buffer. Then incubated the cells with 50 µL / well of 4 µM of FLUO-4AM (a 2X solution was made) for 40 minutes at 37 ° C, 5% carbon dioxide. After the period of incubation of the tincture, the cells are washed twice with buffer of FLIPR leaving a final volume of 50 µL in each well.

Stimulation dependent on the dose of intracellular Ca2 + release for oxotremorine from so that the test compound could be measured with respect to the Stimulation with oxotremorine in an EC90 concentration. The cells were first incubated with dilution buffer of the compound for 20 minutes followed by oxotremorine. It generated a EC_ {90} value for oxotremorine according to the method detailed in the FLIPR data reduction and measurement section below together with the formula EC_ {F} = ((F / 100-F) ^ 1 / H) * EC 50. An oxotremorine concentration of 3 x ECF was prepared in stimulation plates so that a concentration was added EC 90 of oxotremorine to each well in the test plates of proof.

The parameters used for FLIPR were: 0.4 second exposure time, 0.5 laser power watts, 488 nm excitation wavelength and wavelength 550 nm emission. The reference value was determined by measuring the fluorescence change for 10 seconds before the addition of oxotremorine. After stimulation with oxotremorine, FLIPR continuously measured the fluorescence change every 0.5 to 1 second for 1.5 minutes to capture the maximum change of fluorescence.

The fluorescence change was expressed as the maximum fluorescence minus the reference fluorescence for each well. Initial data were analyzed with respect to the logarithm. of the concentration of the test compound by nonlinear regression with GraphPad Prism (GraphPad Software, Inc., San Diego, CA) using the included model for the sigmoid curve dose-response The values of K_ {obs} of the Prism antagonist using the EC_ {50} value of oxotremorine as the K_ {D} value and the EC_ {90} value of the oxotremorine for the concentration of ligands according to the equation of Cheng-Prusoff (Cheng & Prusoff, 1973).

In this test, a lower K_b value indicates that the test compound has a higher functional activity in the receptor tested. It was found that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4 -yl of biphenyl-2-ylcarbamic acid (compound of formula I) has a K obs value of less than 10 nM for the blockage of calcium release mediated by the agonist in CHO cells stably expressing the hM_ {1}, hM_ {3} and cM_ {5} receivers.

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Example 22 CAMP Flashpiate test on cell lines HEK-293 and CHO that express heterogeneously the human adrenergic receptors? 1,? 2 or β3

CAMP assays were performed in a format of radioimmunoassay using the activation assay system of adenylyl cyclase in Flashpiate plates with 125 I-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, MA), according to the manufacturer's instructions. For the determination of the agonist potency (EC 50) of the β1 and β2 receptors cell lines were cultured of CHO-K1 HEK stably expressing the human β1 and β2 receptors cloned to near their confluence in DMEM supplemented with 10% FBS and Geneticin (500 \ mug / ml). For the determination of the agonist power of β3 receptor, the cell line was cultured CHO-K1 stably expressing the receptors β3 or human adrenergic agents cloned to near their confluence in a Ham F-12 medium supplemented with 10% FBS and Geneticin (250 µg / ml). The cells were rinsed with PBS and separated in dPBS (saline buffered with Dulbecco phosphate, without CaCl2 or MgCl2) containing 2 mM of EDTA or Trypsin-EDTA solution (0.05% of trypsin / 0.53 mM EDTA). After counting the cells in the Counter cell counter, the cells were sedimented by centrifugation at 1,000 rpm and resuspended in a buffer of stimulation containing IBMX (PerkinElmer Kit) previously heated to room temperature for a concentration of 1.6 x 10 6 -2.8 x 10 6 cells / ml. They were used approximately 40,000 to 80,000 cells per well in this assay. Test compounds (10 mM in DMSO) were diluted in PBS containing 0.1% BSA in Beckman Biomek-2000 and it tested in 11 different concentrations ranging from 100 µM and 1 µM. The reactions were incubated for 10 min at 37 ° C and stopped by adding 100 µl of cold detection buffer containing [125 I] -AMPc (NEN SMP004, PerkinElmer Life Sciences, Boston, MA). The amount of CAMP produced (um / well) based on the observed counts for samples and cAMP standards, as described in the manufacturer's user manual. Data were analyzed by Nonlinear regression analysis with the GraphPad Prism package Software (GraphPad Software, Inc., San Diego, CA) with the equation sigmoidal Cheng-Prusoff equation was used (Cheng Y, and Prusoff WH., Biochemical Pharmacology, 1973, 22, 23, 3099-108) to calculate the values of EC_ {50}.

In this test, a lower EC50 value indicates that the test compound has a higher functional activity in the receptor tested. It was found that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4 Biphenyl-2-ylcarbamic acid -yl (compound of formula I) has an EC50 value of less than 10 nM for the β2 adrenergic receptor, an EC50 value of approximately 30 nM for the β1 adrenergic receptor and an EC50 value greater than 700 nM for the β3 adrenergic receptor.

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Example 23 Full cell Flashplate cAMP assay with a line lung epithelial cell that endogenously expresses the receptor human adrenergic? 2

In this assay the potency and intrinsic activity as an agonist of a test compound were determined using a cell line expressing endogenous levels of the β2 adrenergic receptor. Cells of a human pulmonary epithelial cell line (BEAS-2B) (ATCC CRL-9609, American Type Culture Collection, Manassas, VA) (January B, et al ., British Journal of Pharmacology, 1998, 123, 4, 701 -11) to a confluence of 75-90% in a serum-free medium (LHC-9 medium containing epinephrine and retinoic acid, Biosource International, Camarillo, CA). The day before the test, the medium was changed to LHC-8 (without epinephrine or retinoic acid, Biosource International, Camarill, CA). CAMP assays were performed in a radioimmunoassay format using the adenylyl cyclase activation assay system in Flashplate plates with125I-cAMP (NEN SMP004, PerkinElmer Life Sciences Inc., Boston, MA), according to manufacturer's instructions

On the day of the test, the cells were rinsed once with PBS, they were raised by scratching them with 5 mM EDTA in PBS and They were counted. The cells were pelleted by centrifugation at 1,000 rpm, and were resuspended in stimulation buffer preheating to 37 ° C in a final concentration of 600,000 cells / ml. They were used cells with a final concentration of 100,000 to 120,000 cells / well in this assay. The test compounds are serially diluted in assay buffer (75 mM Tris / HCl, pH 7.4 a 25 ° C, 12.5 mM MgCl 2, 1 mM EDTA, 0.2% BSA) in Beckman Biomek-2000. Test compounds were tested in the test with 11 different concentrations, ranging from 10 µm to 10 pm The reactions were incubated for 10 minutes at 37 ° C and they stopped adding 100 µl of stop buffer cooled in ice. The plates were sealed, incubated overnight at 4 ° C and they were counted the next morning in a scintillation counter TopCount (Packard BioScience Co., Meriden, CT). The amount of cAMP produced per ml of reaction based on the observed counts for samples and cAMP standards, as described in the manufacturer's user manual. Be analyzed the data by nonlinear regression analysis with the GraphPad Prism Software package (GraphPad Software, Inc., San Diego, CA) using the 4-parameter model for the curve of sigmoidal dose-response.

In this test, a lower EC50 value indicates that the test compound has a higher functional activity in the receptor tested. It was found that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] -piperidin-4 Biphenyl-2-ylcarbamic acid -yl (compound of formula I) has an EC50 value of less than 10 nM with an intrinsic activity value greater than 0.3 compared to a β-{agonist isoproterenol 2} complete (1,0).

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Example 24 Einthoven test to determine efficacy and duration bronchoprotective

In this test the efficacy and bronchoprotective duration of the test compounds were determined using guinea pigs. This essay is inspired by the procedures described in Einthoven (1892) Pfugers Arch. 51: 367-445; and Mohammed et al . (2000) Pulm Pharmacol Ther. 13 (6): 287-92. In this test, changes in ventilation pressure serve as a substitute measure of airway resistance. After pretreatment with a test compound, muscarinic antagonist potency was determined using dose-response curves to intravenous bronchoconstrictor methacholine in the presence of propranolol. Likewise, the bronchoprotective potency of the β2 agonist was determined using histamine. The combined bronchoprotective potency was determined using methacholine in the absence of propranolol.

The test was performed using male guinea pigs Duncan-Hartley (Harían, Indianapolis, IN), weighing between 250 and 400 g. A test compound or vehicle was dosed (it is that is, sterile water) by inhalation (IH) during a period of 10 minute time in a dosing chamber with exposure of whole body (R + S Molds, San Carlos, CA) using 5 ml of dosing solution The animals were exposed to a spray generated by an LC Star nebulizer (model 22F51, PARI Respiratory Equipment, Inc. Midlothian, VA) driven by a gas mixture Bioblend (a mixture of 2% CO2, 21% O2 and N2 at 74%) at a pressure of 22 psi. Lung function was evaluated in several time points after dosing by inhalation.

Seventy-five minutes before the start of the trial, guinea pigs were anesthetized with an intramuscular injection (IM) of a mixture of ketamine (43.7 mg / kg / xylazine (3.5 mg / kg) / acepromazine (1.05 mg / kg). One dose was administered. complementary to this mixture (50% of the initial dose) according to need. The jugular vein and the carotid artery were isolated and tubed with polyethylene catheters filled with saline solution (micro-renatane and PE-50, respectively, Beckton Dickinson, Sparks, MD). Carotid artery it was connected to a pressure transducer to allow measurement of blood pressure and jugular vein cannula was used to IV injection of methacholine or histamine. Then the trachea and intubated with a 14G needle (# NE-014, Small Parts, Miami Lakes, FL). Once the intubations are completed, the guinea pigs were ventilated using a respirator (model 683, Harvard Apparatus, Inc., MA) adjusted with a stroke volume of 1 ml / 100 g body weight but not exceeding the volume of 2.5 ml and at a speed of 100 emboladas per minute. The pressure of Ventilation (VP) was measured in the tracheal cannula using a Biopac transducer connected to a Biopac preamp (TSD 137C). Body temperature was maintained at 37 ° C using a blanket electric Before starting data collection, it was administered pentobarbital (25 mg / kg) intraperitoneally (IP) to suppress the Spontaneous breathing and get a stable reference. The changes in VP they were registered in a Biopac data collection interface Windows Reference values were collected for at least 5 minutes, after which they were administered to guinea pigs via IV non-cumulatively double doses of the bronchoconstrictor (methacholine or histamine). When methacholine was used as agent bronchoconstrictor, the animals were previously treated with propranolol (5 mg / kg, IV) to isolate antimuscarinic effects of the test compound. Propranolol was administered 30 minutes before constructing the dose-response curve to the methacholine or histamine. Changes in VP were recorded using the Acknowledgment Data Collection Software (Santa Barbara, AC). Euthanasia was performed on the animals once the study.

The change in VP was measured in cm of water. Change in VP (cm H2O) = peak pressure (after administration of the bronchoconstrictor) - the peak reference pressure. The dose-response curve to methacholine or the histamine to a four-parameter logistic equation using GraphPad Prism, version 3.00 for Windows (GraphPad Software, San Diego, California). The following equation was used:

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3

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where X is the logarithm of the dose, Y is the answer and Y starts in Min and approaches asymptotically to Max with a form sigmoidal

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The percentage inhibition of the bronchoconstrictor response to a submaximal dose of methacholine or histamine in each dose of the test compound using the following equation:% Inhibition of response = 100 - ((peak pressure (after treatment with a bronchoconstrictor) - pressure from peak reference (treated) / (peak pressure (after administering a bronchoconstrictor, water) - peak reference pressure (water) x 100). The inhibition curves were adjusted using the equation Four-parameter logistics of GraphPad software. I also know estimated the ID 50 (dose needed to produce 50% of inhibition of the response to the bronchoconstrictor) and the E max (maximum inhibition) when deemed appropriate.

The magnitude of bronchoprotection in different time points after inhalation of the test compound is used to estimate the pharmacodynamic half-life (PD T 1/2). PD T_ {1/2} was determined using a non-regression adjustment linear using an exponential degradation equation of a phase (GraphPad Prism, version 4.00): Y = Range * exp (-K * X) + Stabilization; Starts at Interval + Stabilization and degrades in Stabilization with a speed constant K. PD T_ {1/2} = 0.69 / K. The stabilization was limited to 0.

At 1.5 hours after the dose, it was discovered that the ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2 , 5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl of biphenyl-2-ylcarbamic acid (compound of formula I) has an ID 50 of less than about 50 µg / ml for both methacholine-induced bronchoconstriction and for histamine-induced bronchoconstriction.

Additionally, this compound produced a significant bronchoprotection for up to approximately 72 hours when administered as a single submaximal dose (100 µg / ml) In this test, salmeterol (3 µg / ml, IH) (an agonist of β2 adrenergic receptor) exhibited bronchoprotection significant for 6 to 14 hours; and the tiotropium (10 µg / ml) (a muscarinic receptor antagonist) exhibited a significant bronchoprotection for more than 72 hours.

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References cited in the description

This list of references cited by the applicant has been prepared solely as an aid to the reader. It is not part of the European Patent document. Although much attention has been given to compiling them, mistakes and omissions cannot be avoided, the EPO declining any responsibility in this regard .

Patent documents cited in the description

WO 2004074246 A [0002]

US 6759398 B2 [0076]

US 2007009925 W [0003]

US 6537983 B [0076]

US 79470206 P [0003]

US 20020019378 A1 [0076]

US 6750210 B2 [0076]

Non-patent literature cited in the description

Remington: The Science and Practice of Pharmacy. Lippincott Williams & White, 2000 [0050]

HC Ansel et al . Pharmaceutical Dosage Forms and Drug Delivery Systems. Lippincott Williams & White, 1999 [0050]

Eglen et al . Muscarinic Receptor Subtypes: Pharmacology and Therapuetic Potential. DN&P, 1997 , vol. 10 (8), 462-469 [0088]

Emilien et al . Current Therapeutic Uses and Potential of beta-Adrenoceptor Agonists and Antagonists. European J. Clinical Pharm ., 1998 , vol. 53 (6), 389-404 [0088]

Barnes . Chronic Obstructive Pulmonary Disease. N. Engl. J. Med , 2000 , vol. 343, 269-78 [0090]

Lowry et al. Journal of Biochemistry , 1951 , vol. 193, 265 [0152]

Cheng Y; Prusoff WH. Biochemical Pharmacology , 1973 , vol. 22 (23), 3099-108 [0153] [0156] [0174]

Lowry et al. Journal of Biological Chemistry , 1951 , vol. 193, 265 [0155]

Bradford . Analytical Biochemistry , 1976 , vol. 72, 248-54 [0155]

January B et al. British Journal of Pharmacolog and, 1998 , vol. 123 (4), 701-11 [0176]

Einthoven . Pfugers Arch ., Vol. 51, 367-445 [0179]

Mohammed et al. Pulm Pharmacol Ther ., 2000 , vol. 13 (6), 287-92 [0179]

Claims (13)

1. Crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] me-
Biphenyl-2-ylcarbamic acid til} -2,5-dimethylphenylcarbamoyl) ethyl] piperidin-4-yl. 2. Crystalline free base form of claim 1 having Form I, which is characterized by a powder x-ray diffraction pattern having diffraction peaks at 2 ta values of 17.7 ± 0.3, 18 , 2 ± 0.3, 21.7 ± 0.3 and 23.2 ± 0.3. 3. Crystalline free base form of claim 1 having Form II, which is characterized by a powder x-ray diffraction pattern having diffraction peaks at 2 ta values of 20.70 ± 0.3, 21 , 60 ± 0.3, 22.50 ± 0.3 and 23.2 ± 0.3. 4. Crystalline free base form of claim 1 having Form III, which is characterized by a powder x-ray diffraction pattern having diffraction peaks at 2 ta values of 15.50 ± 0.3, 18 , 10 ± 0.3, 19.20 ± 0.3 and 21.80 ± 0.3. 5. Crystal free base shape of the claim 1 in micronized form. 6. Pharmaceutical composition comprising a pharmaceutically acceptable support and a free base form crystal of any one of claims 1 to 5. 7. Pharmaceutical composition comprising:

(to)

the crystalline free base form of any of the claims 1 to 5;

(b)

a steroidal anti-inflammatory agent; Y

(C)

a pharmaceutically acceptable support.

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8. Isotonic saline solution prepared dissolving the crystalline free base form of any of the claims 1 to 5 in isotonic aqueous saline; where The solution has a pH in the range of 4 to 6. 9. Combination of therapeutic agents that understands:

(to)

the crystalline free base form of any of the claims 1 to 5; Y

(b)

a steroidal anti-inflammatory agent.

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10. Process to prepare the free base form crystalline of claim 1, the process comprising:

(to)

provide a solution of 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- biphenyl-2-ylcarbamic acid in a diluent;

(b)

add an antagonistic solvent to the solution of step (a) to produce a point of cloudiness

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11. Process to prepare the free base form crystal of claim 1, the process comprising:

(to)

provide a solution of 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5-dimethylphenylcarbamoyl) ethyl] piperidine-4- biphenyl-2-ylcarbamic acid in a diluent;

(b)

add an antagonistic solvent to the solution of step (a) to produce a cloud point; Y so

(C)

add a seed crystal of a crystalline free base form of ester 1- [2- (4 - {[( R ) -2- (3-formylamino-4-hydroxyphenyl) -2-hydroxyethylamino] methyl} -2,5- Biphenyl-2-ylcarbamic acid dimethylphenylcarbamoyl) ethyl] -piperidin-4-yl.

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12. Form of crystalline free base according any of claims 1 to 5 for use in therapy. 13. Use of a crystalline free base form of any one of claims 1 to 5 for the manufacture of a medicine to treat a lung disease.