Pyridine and pyrazine derivative for the treatment of chronic obstructive pulmonary disease

[0001] The present invention relates to pyridine compounds, their preparation and use as pharmaceuticals.

[0002] Cystic fibrosis (CF) is a deadly genetic disease caused by mutations in the transmembrane conduction CF regulatory gene (CFTR), the protein kinase A (PKA) epithelial anion channel involved in the transport of salts and fluids in many organs, including the lung. Most CF mutations either reduce the number of CFTR channels on the cell surface (e.g., synthesis or processing mutations) or attenuate channel activity (e.g., gating mutations or conductivity) or both. Currently, there are no approved therapies that target directly in CFTR. The present invention discloses compounds that restore or enhance the function of a mutant and / or wild-type CFTR for the treatment of obstructive pulmonary disease.

[0003] In one aspect, the invention provides compounds or pharmaceutically acceptable salts thereof, as defined in claim 1, for use in the treatment of chronic obstructive pulmonary disease.

[0004] Various embodiments of the invention have been described. It is recognized that the features described in each embodiment may be combined with other specific features to provide additional embodiments.

[0005] Another embodiment of the invention as defined above provides compounds for use with substantially pure enantiomers with the R configuration.

[0006] Another embodiment of the invention as defined above provides compounds for use with substantially pure enantiomers with an S configuration.

[0007] Another embodiment of the invention as defined above provides compounds for use represented by 3-amino-6-methoxy- ((S) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) amide. 5-trifluoromethyl-pyridine-2-carboxylic acid or (3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid ((R) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) -amide; that any and all embodiments of the present invention may be considered in any other embodiment to describe additional embodiments of the present invention. Furthermore, all elements of the embodiment are to be combined with any and all other elements from any embodiment to describe additional embodiments. It is understood by those skilled in the art that combinations of substituents, if not possible,

[0008] A second aspect of the invention provides a compound as defined herein for use in a pharmaceutical composition for use in the treatment of chronic obstructive pulmonary disease.

[0009] A further aspect of the invention provides a compound as defined herein for use in the treatment of chronic obstructive pulmonary disease.

[0010] An embodiment of the present invention provides the use of a compound as defined herein, in free or pharmaceutically acceptable salt form, for the manufacture of a medicament for the treatment of chronic obstructive pulmonary disease.

[0011] Throughout this specification and in the following claims, unless the context otherwise provides, the word "includes", or variants such as "includes" or "includes", should be understood to imply the inclusion of a given integer or a step or group of integers or steps, but not excluding any other integer or step or group of integers or steps.

[0012] As used herein, the term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds of the present invention and that are typically not biologically or otherwise undesirable. In many cases, the compounds for use in the present invention are capable of forming acidic and / or basic salts due to the presence of amino and / or carboxyl groups or groups similar to them.

[0013] Pharmaceutically acceptable acid addition salts may be formed with inorganic acids and organic acids, e.g. acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, hydrogen sulphate / sulfate, camphorsulfonate, chloride / hydrochloride, chlorthopilylate, citrate , ethanedisulphonate, fumarate, gluaptane, gluconate, glucuronate, hippurate, iodide / hydroiodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, malate, malate, mandelate, mesilate, methylsulfate, naphthoate, napsilane, nicotinate, nitrate, octadecanoate, oleate, oxalate , palmitate, embonate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate,tosylate and trifluoroacetate salts.

[0014] Inorganic acids from which salts can be formed include, for example, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric and the like.

[0015] The organic acids from which salts can be formed include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid. , methanesulfonic acid, ethanesulfonic acid, toluenesulphonic acid and sulfosalicylic acid. [0016] Pharmaceutically acceptable base addition salts may be formed with inorganic and organic bases.

[0017] Inorganic bases from which salts can be formed include, for example, ammonium salts and metals from columns I to XII of the Periodic Table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.

[0018] Organic bases from which salts can be formed include, for example, primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Some organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine and tromethamine.

[0019] Pharmaceutically acceptable salts may be synthesized from the parent compound, basic or acidic moiety by conventional chemical methods. In general, such salts may be prepared by reacting the free acids of these compounds with a stoichiometric amount of a suitable base (such as Na, Ca, Mg or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free bases of these compounds with a stoichiometric amount of the corresponding acid. Such reactions are typically carried out in water or in an organic solvent or in a mixture of the two. In general, it is desirable when it is practically possible to use anhydrous agents, such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile. A list of additional suitable salts can be found e.g. in "Remington's Pharmaceutical Sciences", 20th ed., Mack Publishing Company, Easton, Pa., (1985); and in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0020] Furthermore, the compounds for use in the present invention, including their salts, may also be obtained in the form of their hydrates or may include other solvents used for their crystallization.

[0021] The compounds used in the invention, which contain groups capable of acting as donors and / or acceptors of hydrogen bonds, may be capable of forming co-crystals with the corresponding co-crystal-forming compounds. These co-crystals can be prepared from said compounds according to known co-crystal formation procedures. Such procedures include grinding, heating, sublimation, co-melting or contacting in a solution of said compounds with the copolymer under crystallization conditions and isolating the crystals in this way. Suitable co-crystal forming compounds include those described in WO 2004/078163.

[0022] As used herein, the term "isomers" refers to different compounds that have the same molecular formula, but differ in the arrangement and configuration of the atoms. The term "optical isomer" or "stereoisomer" as used herein refers to any of the various stereo isomeric configurations that may exist for a given compound for use in the present invention and includes geometric isomers. It is understood that the substituent may be attached at the chiral center of the carbon atom. Thus, the invention includes enantiomers, diastereomers or racemates of the compound. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images. A 1: 1 mixture of a pair of enantiomers is a "racemic" mixture. This term is used to determine the racemic mixture where appropriate. "Diastereoisomers" are stereoisomers that have at least two asymmetric atoms but which are not mirror images of one another. The absolute stereochemistry is determined according to the RS CahnIngold-Prelog system. When the compound is a pure enantiomer, the stereochemistry at any chiral carbon can be determined by either R or S. Separated compounds whose absolute configuration is unknown can be labeled as (+) or (- ) depending on the direction (clockwise or counterclockwise) that twist the plane of polarized light at the wavelength of the sodium line D. Some of the compounds described herein contain one or more asymmetric or axial centers and thus can form enantiomers, diastereoisomers and other stereoisomeric forms, which can be determined in terms of absolute stereochemistry as (R) or (S). The present invention includes all such possible isomers, including racemic mixtures, optically pure forms and intermediate mixtures. Optically active (R) and (S) isomers can be prepared using chiral synthons or chiral reagents or separated using conventional techniques. If the compound contains a double bond, the substituent may have the E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have the cis- or trans configuration. All tautomeric forms are included. The substituent may have the E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have the cis- or trans configuration. All tautomeric forms are included. The substituent may have the E or Z configuration. If the compound contains a disubstituted cycloalkyl, the cycloalkyl substituent may have the cis- or trans configuration. All tautomeric forms are included.

[0023] Any asymmetric atom (e.g., a coal or the like) of the compound (s) for use in the present invention may exist in racemic or enantiomerically enriched, for example (R) -, (S) - or (R, S) - configuration. In certain embodiments, each asymmetric atom has at least a 50% enantiomeric excess, at least a 60% enantiomeric excess, at least a 70% enantiomeric excess, at least an 80% enantiomeric excess, at least a 90% enantiomeric excess, at least a 95% enantiomeric excess , or at least 99% enantiomeric excess in the (R) or (S) configuration. Bases on atoms with unsaturated bonds may, if possible, be present in the cis- (Z) or trans- (E) form. [0024] Accordingly, the compound used in the present invention may be in the form of one of the possible isomers, rotamers, atropisomers,

Each resulting mixture of isomers can be separated on the basis of physicochemical differences of its constituents on pure or substantially pure geometric or optical isomers, diastereomers, racemates, e.g. by chromatography and / or by fractional crystallization.

[0026] Any racemates of the end products or intermediates obtained may be separated into optical antipodes by known methods, for example by separation of their diastereoisomeric salts, obtained with an optically active acid or base and the release of an optically active acidic or basic compound. In particular, a basic moiety can be used to convert the compounds of the present invention into their optical antipodes, for example by fractional crystallisation of a salt formed with an optically active acid, e.g. tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O, O'-p -toluic acid, mandelic acid, malic acid or camphor sulfonic acid. Racemic products can also be separated by chiral chromatography,

[0027] Since the compounds for use in the invention are intended for use in pharmaceutical compositions, it is understood that each of them is preferably provided in a substantially pure form, e.g. at least 60% pure, more preferably at least 75% pure and preferably at least 85%, especially at least 98% pure (% based on weight relative to mass) .None preparations of the compounds can be used to produce the purer forms used in pharmaceutical compositions; These less pure preparations of the compounds should contain at least 1%, more preferably at least 5%, and preferably from 10 to 59% of the compound.

[0028] The compounds for use in the present invention are either obtained in free form as salts or in the form of prodrug derivatives.

[0029] When both the basic group and the acid group are present in the same molecule, the compounds for use in the present invention may also form internal salts, for example zwitterionic molecules.

[0030] Any formula given herein is also intended to represent unlabelled forms as well as isotopically labeled forms of the compounds. The isotopically-labeled compounds have a structure represented by the formulas given herein, except that one or more atoms are substituted for an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as²H ³H ¹¹C ¹³C ¹⁴C ¹⁵N ¹⁸F ³¹P ³²P ³⁵S ³⁶Cl, ¹²⁵And accordingly. Various isotopically labeled compounds are included as defined in the present invention, e.g. those in which radioactive isotopes are present, such as³H ¹³C and ¹⁴C. Such isotope-labeled compounds are useful in metabolic studies (z ¹⁴C), reaction kinetics (e.g. ²H or ³H), detection or imaging techniques, such as positron emission tomography (PET) or single photon computed tomography (SPECT), including drug distribution tests or substrates in tissues, or in radioactive treatment of patients. Especially¹⁸The F or labeled compound may be particularly desirable in PET or SPECT studies. The isotopically-labeled compounds of the present invention can generally be prepared following the procedures disclosed in the schemes or in the examples and regulations described below, replacing the readily available labeled reagent with an unlabeled isotope reagent.

[0031] In addition, substitution with heavier isotopes, in particular deuterium (i.e. ²H or D) may provide certain therapeutic advantages resulting from greater metabolic stability, e.g. an increased in vivo half-life or reduced dosage requirements or an improvement of the therapeutic index. It is understood that deuterium in this context is considered a substituent in the compound. The concentration of such a heavier isotope, especially deuterium, can be determined by an isotopic enriching agent. The term "isotopic enrichment agent" as used in this text means the relationship between isotopic prevalence and the natural prevalence of a particular isotope. If a substituent in a compound of the present invention is designated by deuterium, such compound has an isotopic enrichment factor for each deuterium atom of at least 3500 (52.5% incorporation of deuterium in each designated deuterium atom), at least 4,000 (60% incorporation of deuterium), at least 4,500 (67.5% incorporation of deuterium), at least 5,000 (75% deuterium incorporation), at least 5,500 (82,5% incorporation of deuterium), at least 6,000 (90% incorporation of deuterium), at least 633.3 (95% incorporation of deuterium), at least 6466,7 (97% incorporation of deuterium), at least 6600 (99% incorporation of deuterium) or at least 6633,3 (99.5% incorporation of deuterium) ). [0032] Radiolabelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents instead of the previously unlabeled reagent. at least 5,000 (75% incorporation of deuterium), at least 5,500 (82,5% incorporation of deuterium), at least 6,000 (90% incorporation of deuterium), at least 6333,3 (95% incorporation of deuterium), at least 6466,7 ( 97% incorporation of deuterium), at least 6600 (99% incorporation of deuterium) or at least 6633,3 (99.5% incorporation of deuterium). [0032] Radiolabelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents instead of the previously unlabeled reagent. at least 5,000 (75% incorporation of deuterium), at least 5,500 (82,5% incorporation of deuterium), at least 6,000 (90% incorporation of deuterium), at least 6333,3 (95% incorporation of deuterium), at least 6466,7 ( 97% incorporation of deuterium), at least 6600 (99% incorporation of deuterium) or at least 6633,3 (99.5% incorporation of deuterium). [0032] Radiolabelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents instead of the previously unlabeled reagent. at least 6600 (99% incorporation of deuterium) or at least 6633,3 (99.5% incorporation of deuterium). [0032] Radiolabelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents instead of the previously unlabeled reagent. at least 6600 (99% incorporation of deuterium) or at least 6633,3 (99.5% incorporation of deuterium). [0032] Radiolabelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods analogous to those described in the accompanying examples and preparations, using the appropriate isotopically labeled reagents instead of the previously unlabeled reagent.

[0033] Pharmaceutically acceptable solvates of the invention include compounds wherein the crystallization solvent may be isotopically substituted, e.g. D2O, d6-acetone, d6DMSO.

Synthesis. In general, the compounds may be synthesized by the routes described in Scheme 1, 2 and 3 and examples. Schemes 2 and 5 are provided as a reference and groups A, R¹, R², R³, R⁴, R⁵ and R⁶, are referred to as corresponding compounds for use in the present invention.

[0035] The pyridinyl moiety can be synthesized according to the general scheme 1 shown below.

Scheme 1

<img file="PL2845593T3_D0001.tif" />

or

<img file="PL2845593T3_D0002.tif" />

[0036] The pyrazine moiety can be synthesized according to the general scheme shown below.

Scheme 2

<img file="PL2845593T3_D0003.tif" />

or

<img file="PL2845593T3_D0004.tif" />

[0037] The right side of the moiety is typically added by the amide forming reaction as shown below on general scheme 3.

<img file="PL2845593T3_D0005.tif" />

[0038] HATU (2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium ethanaminium hexafluorophosphate) is a peptide coupling agent. The skilled person will know that other coupling factors could probably work. The halogen group in the above schemes may be replaced by other groups by selecting the appropriate nucleophile and catalyst. The protection of the NH 2 aryl group may be necessary and is represented by P. Schemes 4-7 below are some representative examples.

Scheme 4

<img file="PL2845593T3_D0006.tif" />

Scheme 5

<img file="PL2845593T3_D0007.tif" />

Diagram 6

<img file="PL2845593T3_D0008.tif" />

Scheme 7

<img file="PL2845593T3_D0009.tif" />

[0039] The skilled person will appreciate that the general synthetic pathways detailed above show typical reactions that convert the starting materials as needed. Specific reaction conditions are not given, but they are well known to those skilled in the art and the respective considered conditions are within the ordinary general knowledge of those skilled in the art.

[0040] The starting materials are either commercially available compounds or are known compounds and can be prepared thanks to the procedures described in the field of organic chemistry.

[0041] The compounds in free form may be converted into a salt form and vice versa, in a conventional manner understood by one of ordinary skill in the art. The compounds in free or salt form can be obtained in the form of hydrates or solvates containing the solvent used for crystallization. The compounds can be recovered from reaction mixtures and purified in a conventional manner. Isomers, such as stereoisomers, can be obtained in a conventional manner, e.g. by fractional crystallization or asymmetric synthesis from correspondingly asymmetrically substituted, e.g. optically active, starting materials.

[0042] The compounds may be prepared, e.g. using the reactions and techniques described below and in the examples. The reactions can be carried out in a solvent suitable for the reagents used and the materials used and suitable for effective transformation. A specialist in the field of organic synthesis will be aware that the functionality present on the molecule should be consistent with the proposed transformations. This will sometimes require evaluation to modify the order of the synthetic steps or to select one particular process scheme relative to another to obtain the desired compound of the invention.

[0043] Various substituents on synthetic intermediates and final products shown in the following reaction schemes can exist in their fully formulated forms, with suitable protecting groups, if necessary, within the meaning of the person skilled in the art, or in precursor forms that can be further developed to final forms by methods known to those skilled in the art. The substituents can also be added at various stages of the synthesis sequence or at the completion of the synthesis sequence. In many cases, commonly used functional group manipulations can be used to convert one intermediate into another intermediate, or one compound for use in the present invention in another compound for use in the present invention. Examples of such manipulations are the conversion of an ester or a ketone into an alcohol; ester conversion into ketone; transformations between esters, acids and amides; alkylation, acylation and sulfonylation of alcohols and amines; and many others. Substituents can also be added using conventional reactions, such as alkylation, acylation, halogenation or oxidation. Such manipulations are well known in the art, and many literature references discuss procedures and methods for such manipulations. Some references, which give examples and references to the basic literature on organic synthesis in terms of many manipulations of functional groups, as well as other transformations commonly used in the field of organic synthesis, are March'sOrganic Chemistry, 5th edition, Wiley and Chichester, ed. (2001); Comprehensive Organic Transformations, Larock, eds., VCH (1989); Comprehensive Organic Functional Group Transformations, Katritzky et al. (Publisher of the series), Pergamon (1995); and Comprehensive OrganicSynthesis, Trost and Fleming (series publishers), Pergamon (1991). It should also be appreciated that another important consideration in the design of any synthetic path in the art is the reasonable choice of the protecting group used to protect the reactive functional group present in the compounds described in this invention. Multiple protecting groups can be selected within the same molecule, so that each of these protecting groups can either be removed without removing other protecting groups in the same molecule, or several protecting groups can be removed using the same reaction step, depending on the desired result. .

Pharmacological Activity [0044] Considering their modulation of CFTR activity, the compounds described in the present invention in free or pharmaceutically acceptable salt form, hereinafter referred to alternatively as "agents of the invention", are useful in treating conditions that respond to modulation of CFTR activity, especially those benefiting from hydration of the mucosa, such as cystic fibrosis.

[0045] Diseases mediated by modulation of CFTR activity include diseases associated with the regulation of fluid volume through epithelial membranes. For example, the volume of airway surface fluid is a key regulator of mucociliary clearance and lung health. Modulation of CFTR activity will stimulate the accumulation of fluid on the mucosal side of the airway epithelium, thereby promoting mucus clearance and preventing the accumulation of mucus and phlegm in the respiratory tissues (including the airways of the lungs). Such diseases include respiratory diseases such as cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, chronic obstructive pulmonary disease (COPD), asthma, respiratory infections (acute and chronic, in viral and bacterial), and lung cancer. Diseases,

In addition, modulation of CFTR activity in the kidneys can be used to stimulate diuresis and thereby induce the effect of reducing the pressure.

[0046] The therapy according to the invention may be symptomatic or prophylactic.

[0047] Asthma includes congenital (non-allergic) asthma and extrinsic asthma (allergic), benign asthma, moderate asthma, severe asthma, bronchial asthma, post-stroke asthma, occupational asthma and asthma induced after bacterial infection. Therapy for asthma is also understood to include the treatment of subjects, e.g. younger than 4 or 5 years old, showing symptoms of wheezing and being diagnosed or diagnosed as "wheezing infants", an established category of patients with special medical care and now often identified as first-line asthmatics. or early phase. (For convenience, these individual asthma conditions are referred to as "wheezing syndrome in infants".) Preventive efficacy in the treatment of asthma will be demonstrated by the lower frequency or severity of the symptomatic attack, e.g. acute asthmatic attack or bronchoconstriction, improvement in lung function or improvement of airway hyperresponsiveness. This may be further demonstrated by the lower need for other, symptomatic therapy, i.e. therapy for the purpose or intention of limiting or eliminating symptomatic attack when it occurs, e.g. anti-inflammatory (e.g., corticosteroid) or bronchodilatory. The preventive advantage in asthma can be seen, in particular, in subjects prone to morning dipping. A "morning crisis" is a known asthmatic syndrome, typical of a significant percentage of asthmatics, and is characterized by an asthma attack, e.g. between about 4 to 6 am, i.e., at a time normally significantly distant from any previously administered symptomatic asthma therapy. improvement of lung function or improvement of airway hyperresponsiveness. This may be further demonstrated by the lower need for other, symptomatic therapy, i.e. therapy for the purpose or intention of limiting or eliminating symptomatic attack when it occurs, e.g. anti-inflammatory (e.g., corticosteroid) or bronchodilatory. The preventive advantage in asthma can be seen, in particular, in subjects prone to morning dipping. A "morning crisis" is a known asthmatic syndrome, typical of a significant percentage of asthmatics, and is characterized by an asthma attack, e.g. between about 4 to 6 am, i.e., at a time normally significantly distant from any previously administered symptomatic asthma therapy. improvement of lung function or improvement of airway hyperresponsiveness. This may be further demonstrated by the lower need for other, symptomatic therapy, i.e. therapy for the purpose or intention of limiting or eliminating symptomatic attack when it occurs, e.g. anti-inflammatory (e.g., corticosteroid) or bronchodilatory. The preventive advantage in asthma can be seen, in particular, in subjects prone to morning dipping. A "morning crisis" is a known asthmatic syndrome, typical of a significant percentage of asthmatics, and is characterized by an asthma attack, e.g. between about 4 to 6 am, i.e., at a time normally significantly distant from any previously administered symptomatic asthma therapy. therapy for the purpose or intended to limit or remove symptomatic attack when it occurs, e.g. anti-inflammatory (e.g., corticosteroid) or bronchodilatory. The preventive advantage in asthma can be seen, in particular, in subjects prone to morning dipping. A "morning crisis" is a known asthmatic syndrome, typical of a significant percentage of asthmatics, and is characterized by an asthma attack, e.g. between about 4 to 6 am, i.e., at a time normally significantly distant from any previously administered symptomatic asthma therapy. therapy for the purpose or intended to limit or remove symptomatic attack when it occurs, e.g. anti-inflammatory (e.g., corticosteroid) or bronchodilatory. The preventive advantage in asthma can be seen, in particular, in subjects prone to morning dipping. A "morning crisis" is a known asthmatic syndrome, typical of a significant percentage of asthmatics, and is characterized by an asthma attack, e.g. between about 4 to 6 am, i.e., at a time normally significantly distant from any previously administered symptomatic asthma therapy.

[0049] Chronic obstructive pulmonary disease includes chronic bronchitis or related dyspnoea, emphysema, as well as exacerbation of airway hyperactivity as a consequence of other drug therapy, in particular other inhaled drug therapies. The invention can also be used to treat bronchitis of any type or genesis, including, e.g., acute bronchitis, caused by irritant effects of peanut, catarrhal, krupowego, chronic or tuberculous.

[0050] Dry eye disease is characterized by a decrease in tear production and an abnormal lipid, protein and mucin film profile of tears. There are many causes of the dry eye, some of which include age, laser eye treatments, arthritis, drug intake, chemical / thermal burns, allergies and diseases such as cystic fibrosis and Sjogren's syndrome. Increasing anion secretion by CFTR will strengthen fluid transport from corneal endothelial cells and secretory glands that surround the eye, increasing corneal hydration. This will help relieve the symptoms associated with dry eye disease. [0051] Sjogren's syndrome is an autoimmune disease in which the immune system attacks the moisture-producing glands throughout the body, including the eye, mouth, skin, and tissue of the system.breathing rate, in the liver, vagina and intestines. Symptoms include dry eye, dry mouth and dry vagina, as well as lung disease. The disease is also associated with rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and polymyositis / dermatomyositis. Faulty transmission of proteins is considered to be the cause of a disease whose treatment options are limited. CFTR activity modulators can hydrate different organs affected by the disease and help relieve the accompanying symptoms.

[0052] The suitability of modulators of CFTR activity as treatment of a disease benefiting from hydration of the mucosa can be tested by determining the movement of chloride ions in a suitable cell-based assay. For example, single cells or confluent epithelia that show endogenous expression or reconstructed for overexpression of CFTR can be used to assess channel function using electrophysiological techniques or ion flow studies. See, methods described in: Hirsh et al., J Pharm ExpTher (2004); Moody et al., Am J Physiol Cell Physiol (2005).

[0053] Modulators of CFTR activity, including those described in the present invention are also useful as co-therapeutic agents for use in combination with other drug substances, such as anti-inflammatory drug, bronchodilatory, antihistamine or anti-tussive substances, particularly in the treatment of cystic fibrosis or obstructive or inflammatory respiratory diseases, such as those mentioned previously, e.g. as potentiators of the therapeutic activity of such drugs or as reducing the necessary dose or potential side effects of such drugs.

[0054] The compounds described in the present invention can be mixed with other drug substances in a solid pharmaceutical composition or they can be administered separately, before, concurrently with or after other drug substances.

[0055] Accordingly, the invention relates to a combination of a CFTR activity modulator compound described herein with osmotic agents (hypertonic saline, dextran, mannitol, xylitol), HA inhibitors, anti-inflammatory, bronchodilatory, antihistamine, anti-cough, antibiotic and / or DNase drug substances, wherein the CFTR activity modulator and other drug substances may be in the same or different pharmaceutical composition. [0056] Suitable antibiotics include macrolide antibiotics, e.g. tobramycin (TOBI ™). [0057] Suitable DNase drug substances include dornase alfa (Pulmozyme ™), a highly purified solution of recombinant human deoxyribonuclease I (rhDNase) that selectively cleaves DNA. Dornase alfa is used to treat cystic fibrosis.

[0058] Other useful combinations of modulators of CFTR activity with anti-inflammatory drugs are those with chemokine receptor antagonists, e.g. CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR -8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists, such as SC-351125, SCH-55700 and SCH-D antagonists from Schering-Plow; Takeda antagonists, such as N chloride [[4 [

[6,7-dihydro-2- (4-methyl-phenyl) -5H-benzo-cyclohepten-8-yl] carbonyl] amino] phenyl] -methyl] tetrahydro-N, N-dimethyl-2H-pyran-4-amine -ium (TAK-770); and CCR-5 antagonists described in USP 6,166,037 (especially claims 18 and 19), WO 00/66558 (especially claim 8), WO 00/66559 (particularly claim 9), WO 04/018425 and WO 04/026873.

[0059] Suitable anti-inflammatory drugs include steroids, in particular, glucocorticosteroids, such as budesonide, dipropyl dipropyl ester, flutonazone propionate, ciclesonide or furumianmometasone or steroids described in WO 02/88167, WO 02/12266, WO 02/100879, WO 02/00679 (especially those of examples 3, 11, 14, 17, 19, 26, 34, 37, 39, 51, 60, 67, 72, 73, 90, 99 and 101), WO 03/35668, WO 03/48181, WO 03 62259, WO 03/64445, WO 03/72592, WO 04/39827 and WO 04/66920; non-steroidal glucocorticoid receptor agonists, such as those described in DE 10261874, WO 00/00531, WO 02/10143, WO 03/82280, WO 03/82787, WO 03/86294, WO 03/104195, WO 03/101932, WO 04 / 05229, WO 04/18429, WO 04/19935 and WO 04/26248; LTD4 antagonists, such as montelukast and zafirlukast; PDE4 inhibitors, such as cilomilast (Ariflo® GlaxoSmithKline), Roflumilast (Taurus Gulden), V-11294A (Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plow), Arofylline (AlmirallProdesfarma), PD189659 / PD168787 (Parke-Davis), AWD-12281 (Asta Medica), CDC-801 (Celgene), SeICID (TM) CC -10004 (Celgene), VM554 / UM565 (Vernalis), T-440 (Tanabe), KW-4490 (KyowaHakkoKogyo), and those disclosed in WO 92/19594, WO 93/19749, WO 93/19750, WO 93/19751 , WO 98/18796, WO 99/16766, WO 01/13953, WO 03/104204, WO 03/104205, WO 03/39544, WO 04/000814, WO 04/000839, WO 04/005258, WO 04/018450 , WO 04/018451, WO 04/018457, WO 04/018465, WO 04/018431, WO 04/018449, WO 04/018450, WO 04/018451, WO 04/018457, WO 04/018465, WO 04/019944 , WO 04/019945, WO 04/045607 and WO 04/037805; antagonists of the A2B adenosine receptor, such as those described in WO 02/42298; and beta-2 adrenoceptor agonists, such as albuterol (salbutamol), metaproterenol, terbutaline, salmeterolfenoterol, prokaterol,

03/99764, WO 04/16578, WO 04/22547, WO 04/32921, WO 04/33412, WO 04/37768, WO

04/37773, WO 04/37807, WO 04/39762, WO 04/39766, WO 04/45618, WO 04/46083, WO

04/80964, WO 04/108765 and WO 04/108676.

[0060] Suitable bronchodilators include anticholinergic or antimuscarinic agents, in particular, ipratropium bromide, oxitropium bromide, tiotropium salts and CHF 4226 (Chiesi), and glycopyrrolate, but also those described in EP 424021, USP 3,714,357, USP 5,171,744, WO 01 / 04118, WO 02/00652, WO 02/51841, WO 02/53564, WO 03/00840, WO 03/33495, WO 03/53966, WO 03/87094, WO 04/018422 and WO 04/05285.

[0061] Suitable dual-effect anti-inflammatory and bronchodilator drugs include a dual beta-2 adrenoceptor / muscarinic antagonist such as those disclosed in USP 2004/0167167, WO 04/74246 and WO 04/74812.

[0062] Suitable antihistamine drug substances include cetirizine hydrochloride, acetaminophen, fumaranklemastine, prometasin, loratidine, desloratidine, difenohydramine and fexofenadine hydrochloride, actinin, astemizole, azelastine, ebastine, epinastine, mizolastine and tefenadine, as well as those disclosed in JP 2004107299, WO 03 / 099807 and WO 04/026841.

In another aspect, the present invention provides a compound described herein, in free form or in the form of a pharmaceutically acceptable salt, for use in the manufacture of a medicament for treating COPD.

[0064] The agents of the invention may be administered by any suitable route, e.g. orally, e.g. in the form of a tablet or capsule; parenterally, e.g., intravenously; by inhalation, e.g. in the therapy of a disease with airway spasm; nasally, e.g. in the treatment of allergic rhinitis; locally on the skin; or to rectally. The compounds described herein may be provided as a pharmaceutical composition comprising the compound in free form or in the form of a pharmaceutically acceptable salt, optionally together with a pharmaceutically acceptable diluent or carrier therefor. The composition may contain a co-therapeutic agent such as an anti-inflammatory, bronchodilatory, antihistamine or anti-tussive drug as described hereinbefore. Such compositions may be prepared using conventional diluents or excipients and methods known in the galenical technique. Thus, oral dosage forms can include tablets and capsules. Formulations for topical administration may take the form of creams, ointments, gels or transdermal delivery systems, e.g. patches. Compositions for inhalation may include aerosol or other sprayable formulations or dry powder formulations.

When the composition comprises an aerosol formulation, it preferably contains, e.g. propellane, hydrofluoroalkanic (HFA), such as HFA134a or HFA227 or a mixture thereof, and may contain one or more co-solvents known in the art, such as ethanol (up to 20% by weight ), and / or one or more surfactants, such as oleic acid or trioleinium sorbitan, and / or one or more bulking agents, such as lactose. When the composition contains a dry powder formulation, it preferably contains, e.g. a compound with a particle diameter of up to 10 microns, optionally together with a diluent or carrier, such as lactose, with the desired particle size distribution and a compound that helps protect against deterioration of product performance due to moisture , e.g. magnesium stearate. When the composition contains a nebulized preparation,

[0066] The compound used in the present invention may include:

(a) a compound in inhalable form, e.g. in an aerosol or other composition capable of being atomised or in particulate, e.g. micronized inhalation form;

(b) an inhaled drug containing the compound in inhalable form;

(c) a pharmaceutical product containing the compound in inhalable form together with an inhalation device; and (d) an inhalation device containing the compound in inhalable form.

[0067] The dosage of the compounds used in practicing the present invention will, of course, vary depending on, e.g. the particular condition to be treated, the desired effect and the mode of administration. In general, suitable daily doses for administration by inhalation are in the order of 0.005-10 mg, while for oral administration, suitable daily doses are in the order of 0.05-100 mg.

Pharmaceutical use and testing [0068] The compounds for use in the present invention and their pharmaceutically acceptable salts, hereinafter referred to alternatively as "agents of the invention", are useful as pharmaceuticals. In particular, the compounds are suitable modulators of CFTR activity and can be tested by the following tests.

Membrane potential assay [0069] The CFTR activity can be quantified by measuring the membrane potential. Means for measuring membrane potential in a biological system can use a variety of methods, including membrane electrophysiological and optical fluorescence testing. [0070] The membrane potential optical assay utilizes a negatively charged potentiometric dye, such as FLIPR membrane potential dye (FMP) (see Baxter DF, Kirk M, Garcia AF, Raimondi A, Holmqvist MH, Flint KK, Bojanic D, Distefano PS, Curtis R , Xie Y. 'A novelmembranepotential-sensitivefluorescentdyeimprovescell-basedtests for ionchannels.' J BiomolScreen. 2002 Feb; 7 (1): 79-85), which outside the cell is bound to the quenching agent. During depolarisation, the negatively charged dye cells are re-distributed to the intracellular compartment, separating from the membrane non-transmittant quenching agent, resulting in an increase in fluorescence. This change in fluorescence is proportional to changes in membrane potential, which may be due to CFTR activity. Fluorescence changes can be monitored in real time by a suitably equipped fluorescence detector, such as a FLIPR (fluorimetric plate imaging reader) in 96 or 384 microtiter microtiter plates.

Cell culture:

[0071] Chinese hamster ovary (CHO) cells with durable expression of the ΔF508-CFTR channel were used for membrane potential experiments. The cells were maintained at 37 ° C in 5% v / v CO2 at 100% humidity in modified Eagles medium (MEM) modified with 8% v / v fetal calf serum, 100 μg / ml methotrexate and 100U / ml penicillin / streptomycin. Cells were grown at 225 cm² tissue culture flasks. For membrane potential assays, cells were seeded into 96 well plates at 40,000 cells per well, allowed to adhere, and then maintained at 26 ° C for 48 hours to facilitate insertion of the channel.

Amplifier test:

[0072] The membrane potential screening assay used an extracellular solution containing a low concentration of chloride ions (~ 5 mM) in combination with a double addition protocol. A buffer with or without a test compound was added for the first time, then forskolin (1-20 μΜ) was added 5 minutes later - this protocol promotes maximal chloride outflow in response to the activation of ΔF508-CFTR. A discharge of chloride ions through the ΔF508-CFTR leads to membrane depolarization, which is optically monitored by the FMP dye.

solutions:

[0073] Extracellular with low chloride concentration (mM): 120 Na-gluconate, 1.2 CaCl2, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 10.0 D-glucose, 20.0 HEPES, pH 7.4 with NaOH FMP dye: made in accordance with the manufacturer's instructions in the extracellular solution with low chloride concentration, as listed above, at 10x final concentration and stored as 1 ml aliquots at -20 ° C.

IonWorks Quattro test:

[0074] The activity of CFTR can also be quantified electrophysiologically using the whole cell configuration by flake technique (Hamill and other Pflugers Acrhive 1981). This test directly measures the currents associated with the flow of chlorides through CFTR channels either simultaneously or maintaining or fine-tuning the transmembrane voltage. This assay may use either single glass micropipettes or parallel planar kits for measuring CFTR activity from native or recombinant cell systems. The currents measured using parallel planar sets can be quantified using a suitably equipped instrument such as IonWorks Quattro (Molecular Devices) or Qpatch (Sophion).

Cell culture:

[0075] Chinese hamster ovary (CHO) cells with permanent ΔF508-CFTR channel expression were used for the IonWorks Quattro experiments. Cells were maintained at 37 ° C in 5% v / v CO2 at 100% humidity in D-MEM supplemented with 10% (v / v) FCS, 100 U / ml Penicillin / Streptomycin, 1% (v / v) NEAA, 1 mg / ml Zeocin and 500 μg / ml Higromycin B. Cells for experiments were grown in 225 cm tissue culture flasks² until reaching confluence, and then grown at 26 ° C for 48-72 hours to facilitate insertion of the canal. The cells were removed from the flask and resuspended either in the extracellular solution for measurements for immediate experiments or alternatively in growth medium supplemented with 10% v / v DMSO and frozen to -80 ° C as 1-2 ml aliquots for use at a later date.

Amplifier test:

[0076] Cells, density 1.5-3 million per ml, were placed in the Quattro system, added to the planar flake set and closed, allowed to stabilize for 5-10 min. After assessment of closure resistance (typically&gt; 50 ΜΩ), access to the entire cell was achieved through perforation of 100 μg / ml amphotericin B. Baseline currents were measured by a scan prior to compound addition, obtained by applying a voltage increase from -100 to +100 mV. Then, either buffer or test compound diluted in extracellular solution supplemented with 20 μΜ forskolin, was added to each of the 384 flutes of the planar flake set. After the incubation step (5-20 minutes), the currents were measured after adding the compound again by applying an increase in voltage from -100 to +100 mV.

solutions:

[0077] Extracellular solution (ECS): 145 mM NaCl, 4 mM CsCl, 5 mM D-glucose, 10 mM TES, 1 mM CaCl2, 1 mM MgCl2, pH 7.4 NaOH intracellular buffer (ICS): 113 mM L acid -Asparagine, 113 mM CsOH, 27 mM CsCl, 1 mM NaCl, 1 mM MgCl2, 1 mM EGTA, 10 mM TES. pH 7.2 with CsOH. The filter was sterilized before use.

Ion transport test:

[0078] Another method of measuring the CFTR function is to measure current in the chamber of the short circuit Ussing. The developed or native epithelial cells are grown into a confluent single layer on a semipermeable filter and layered between two plexiglass blocks. The flow of chloride ions through CFTR on one side of the epithelium to the other can be quantified by measuring the current flow while maintaining the trans-epithelial potential at 0 mV. This is achieved thanks to KCl-filled agar electrodes on both terminals of a single cell layer and current flow measurement.

Cell culture:

[0079] FRF88-CFTR stably expressed FRT cells were cultured in modified Fon Coon medium supplemented with 32 mM NaHCO3, 10% v / v fetal bovine serum, 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 30 μg / ml hygromycin B as growth medium. For experiments with the Ussing chamber, the cells were grown as polarized epithelia on permeable Snapwell insert supports (500,000 cells / contribution in growth medium) and cultured for 7 to 9 days. The cartridges were fed with fresh medium modified by F-12 Coon growth every 48 hours and 24 hours before the experiment with the Ussing chamber. To increase the expression of the CFTR ΔF508 protein on the cell surface, the plates were incubated at 27 ° C for 48 hours. before experimenting with the Ussing chamber.

Amplifier test:

[0080] Fischer Rat Thyroid epithelial cells (FRT) with sustained expression of human ΔF508-CFTR were used as monolayer cultures on permeable supports. The current Cl was measuredusing current techniques with short circuit, with imposed gradient Cl^- basolateral to apical, in the Ussing chambers. For measuring constant currents Cl^-, FRT cells were cultured for 48 hours. at 27 ° C to facilitate the introduction of ΔF508 CFTR into the plasma membrane. Studies with the Ussing chamber were similarly carried out at 27 ° C. Under these conditions, the effect of the combined addition of test compounds to the ΔF508 CFTR currents could be assessed both with end points of strength and efficacy. Compounds were added to both the apical and basolateral side after the addition of 10μΜ forskolin. The efficacy of the compounds was compared to known enhancers such as genistein.

solutions:

[0081]

Basolateral Ringer's solution (mM): 126 NaCl, 24 NaHCO3, 0.38 KH2PO4, 2.13 K2HPO4, 1 MgSO4, 1 CaCl2 and 10 glucose.

Apical Ringer's solution (mM): 140 Na-gluconate, 1 MgSO4, 2 CaCl2, 1 HCl, 10 glucose and 24 NaHCO3.

[0082] The compounds may also be tested for their ability to stimulate the introduction of the ΔF508 CFTR into the cell membrane using the above tests. For these tests the protocols were identical except that the cells were not grown at low temperature (26 or 27 ° C) but instead they were incubated with the test compounds for 12-24 hrs. before the test. [0083] The compounds of the examples, herein below, generally have EC 50 values ​​in the measurement data described above, below 10 μΜ. Table 1 provides a list of representative compounds with their EC50 value.

Table 1

<td>Example No.</td><td>EC50 μΜ</td>

<td>2 (reference)</td><td>0.015</td>

<td>3 (reference)</td><td>0,055</td>

<td>4</td><td>0.076</td>

<td>5</td><td>0.05</td>

<td>6</td><td>0.426</td>

<td>7</td><td>0,040</td>

<td>8</td><td>0,060</td>

<td>16</td><td>0,008</td>

<td>17</td><td>0,010</td>

[0084] The invention is illustrated by the following examples.

Examples General conditions:

[0085] Mass spectra were run on LC-MS systems using electrospray ionization. These were either the Agilent 1100 HPLC / Micromass Platform Mass Spectrometer or the Waters Acquity UPLC with the SQD Mass Spectrometer.

[M + H] + refers to monoisotopic molecular masses.

[0086] NMR spectra were run on Bruker AVANCE 400 NMR open access spectrometers using ICON-NMR. The spectra were measured at 298 K and referenced to the solvent peak. [0087] Optical twist is measured at 589nm and 546nm using an Optical activity AA-1000 polarimeter at 21 ° C.

[0088] The following examples are intended to illustrate the invention and should not be considered as limiting. Temperatures are given in degrees Celsius. Unless otherwise mentioned, all evaporations are carried out under reduced pressure, preferably between about 15 mm Hg and 100 mm Hg (= 20-133 mbar). The structure of final products, intermediates and starting materials is confirmed by standard analytical methods, e.g. microanalysis and spectroscopic characteristics, e.g. MS, IR and NMR. Abbreviations used are conventional in the art. If not defined, the terms have their generally accepted meanings.

abbreviations:

[0089] app visible

ATP adenosine 5'-triphosphate

BINAP racemic 2,2'-bis (diphenylphosphino) -1,1'-binaphthyl

<td>BOC</td><td>tertiary butyl carboxy</td>

<td>year</td><td>broad (broad)</td>

<td>d</td><td>doublet</td>

<td>dd</td><td>doublet of doublets</td>

<td>DCM</td><td>dichloromethane</td>

<td>DIEA</td><td>diethylisopropylamine</td>

<td>DIPEA</td><td>diisopropylethylamine</td>

<td>DMF</td><td>N, N-dimethylformamide</td>

<td>DMSO</td><td>dimethyl sulfoxide</td>

<td>DTT</td><td>dithiothreitol</td>

<td>ESI</td><td>ionization with electrospray</td>

<td>EtOAc</td><td>ethyl acetate</td>

<td>eq</td><td>equivalent</td>

<td>h</td><td>hour (hours)</td>

<td>HATU</td><td>2- (7-aza-1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate</td>

<td>HPLC</td><td>high-pressure liquid chromatography</td>

<td>IR</td><td>infrared spectroscopy</td>

<td>LCMS</td><td>liquid chromatography and mass spectrometry</td>

<td>MeOH</td><td>methanol</td>

<td>MS</td><td>mass spectrometry</td>

<td>MW</td><td>microwave</td>

<td>m</td><td>multiplet</td>

<td>min</td><td>minutes</td>

<td>ml</td><td>milliliter (milliliters)</td>

<td>m / z</td><td>mass / load ratio</td>

<td>NMR</td><td>nuclear magnetic resonance</td>

<td>ppm</td><td>parts per million</td>

<td>PS</td><td>polymer supported</td>

<td>Rac</td><td>racemic</td>

<td>RT</td><td>room temperature</td>

<td>rt</td><td>retention time</td>

<td>s</td><td>singlet</td>

<td>SCX-2</td><td>strong cation exchange (eg Isolute® SCX-2 columns from Biotage)</td>

<td>t</td><td>triplet</td>

<td>TEA</td><td>triethylamine</td>

<td>TFA</td><td>trifluoroacetic acid</td>

<td>THF</td><td>tetrahydrofuran</td>

[0090] With respect to the examples that follow, the compounds of the preferred embodiments were synthesized using the methods described herein or other methods that are known in the art.

[0091] Various starting materials, intermediates, and compounds of preferred embodiments may be isolated and purified, if appropriate, by conventional techniques such as precipitation, filtration, crystallization, evaporation, distillation and chromatography. Unless otherwise agreed, all starting materials are obtained from market suppliers and used without further purification. Salts can be prepared from compounds by known salt-forming procedures.

[0092] It should be understood that organic compounds according to preferred embodiments may exhibit tautomerism. Since the chemical structures in this list can only mean one of the possible tautomeric forms, it should be understood that preferred embodiments include any tautomeric form with a drawn structure.

[0093] Unless otherwise indicated, analytical HPLC conditions are as follows:

<td>Method LC_v002</td><td>10min</td><td></td>

<td colspan="2">Column</td><td>Waters BEH C18 50x2,1 mm, 1.7 μιτι</td>

<td colspan="2">Temp. column.</td><td>50 ° C</td>

<td colspan="2">eluents</td><td>A: H2O, B: methanol, both contain 0.1% TFA</td>

<td colspan="2">Flow speed</td><td>0.8 ml / min</td>

<td colspan="2">Gradient</td><td>0.20 min 5% B; 5% to 95% B in 7.8 min, 1.00 min 95% B</td>

<td colspan="2"></td><td></td>

<td>Method LC_v003</td><td>10min</td><td></td>

<td colspan="2">Column</td><td>Waters BEH C18 50x2,1 mm, 1.7 μm</td>

<td colspan="2">Column temperature</td><td>50 ° C</td>

<td colspan="2">eluents</td><td>A: H2O, B: acetonitrile, both contain 0.1% TFA</td>

<td colspan="2">Flow speed</td><td>0.8 ml / min</td>

<td colspan="2">Gradient</td><td>0.20 min 5% B; 5% to 95% B at 7.80 min, 1.00 min 95% B</td>

<td colspan="2"></td><td></td>

<td>Method LC_v002</td><td>2min</td><td></td>

<td colspan="2">Column</td><td>Waters BEH C18 50x2,1 mm, 1.7 μm</td>

<td colspan="2">Column temperature</td><td>50 ° C</td>

<td colspan="2">eluents</td><td>A: H2O, B: methanol, both contain 0.1% TFA</td>

<td colspan="2">Flow speed</td><td>0.8 ml / min</td>

<td colspan="2">Gradient</td><td>0.20 min 5% B; 5% to 95% B in 1.3 min, 0.25 min 95% B</td>

<td colspan="2"></td><td></td>

<td>Method LC_v003</td><td>2min</td><td></td>

<td colspan="2">Column</td><td>Waters BEH C18 50x2,1 mm, 1.7 μm</td>

<td colspan="2">Temp. column</td><td>50 ° C</td>

<td>Method LC_v002</td><td>10min</td><td></td>

<td colspan="2">eluents</td><td>A: H2O, B: acetonitrile, both contain 0.1% TFA</td>

<td colspan="2">Flow speed</td><td>0.8 ml / min</td>

<td colspan="2">Gradient</td><td>0.20 min 5% B; 5% to 95% B in 1.30 min, 0.25 min 95% B</td>

<td colspan="2"></td><td></td>

Preparation of final compounds

Reference Example 3 (33,3-trifluoro-2-hydroxy-2-methylpropyl) -amide, 3-amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid

<img file="PL2845593T3_D0010.tif" />

[0095] 3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid (intermediate A) (397 mg, 1.392 mmol), 3-amino-1,1,1-trifluoro-2-methyl-propan hydrochloride -2-ol (250 mg, 1.392 mmol) and HATU (529 mg, 1.392 mmol) was dissolved in DMF (10 mL) and stirred at RT for 2 min. 4-Methylmorpholine (0.413 ml, 4.18 mmol) was added and stirring was continued at RT for 3 h. The reaction mixture was poured into ice / water (100 mL) and extracted with EtOAc (250 mL). The organic extract was washed with a saturated solution of NH4Cl (~ 50 mL), dried over MgSO4 and concentrated in vacuo to give a pale brown oil. The oil was dissolved in CHCl 3 (~ 3 mL) and loaded onto a 24 g ISCO column (silica), eluting with isohexane: EtOAc to give the title product; LC-MS Rt = 1.46 min;

[M + H] + 410.1, Method 2 min LC_v002. NMR 1 H (400 MHz, DMSO-d 6) δ 8.30 (NH, t), 7.72 (1H, s), 7.29 (NH 2, bs), 6.28 (OH, s), 3.68 (1 H, dd), 3.47 (1H, dd), 1.24 (3H, s). NMR 19F (400 MHz, DMSO-d6) δ -62.71 (CF3, s), - 80.48 (CF3, s).

[0096] The compound of the following table 2 was prepared by a method similar to that of example 1, from the corresponding starting compound and the amine. Single enantiomers were prepared using a chiral amine or by separation of the product using chromatography over the supercritical region. The preparation of starting compounds and the amines are described in the Intermediates section, unless they are commercially available. In some reactions, DIPEA or TEA could be used instead of 4-methylmorpholine.

Table 2

Prov.

1.25

Structure

<img file="PL2845593T3_D0011.tif" />

<td>Name</td><td>Retention time, [M + H] +, 1 H NMR</td>

<td>(3,3,3-trifluoro-2-</td><td>Rt 5.41 min;</td>

<td>hydroxy-2-</td><td>[M + H]⁺ 426;</td>

<td>methylpropyl) amide</td><td>Way</td>

<td>3-amino-6- (4-amino acid)</td><td>10minLC_v002.</td>

<td>fluorophenyl) -5-</td><td>1 H NMR: δ 8.42</td>

<td>trifluoromethyl-</td><td>(1H, m), 7.72 (1</td>

<td>pyridine-2-</td><td>H, s), 7.5 (2H, m),</td>

<td>carboxylic acid</td><td>7.3 (2H, t), 7.22 (2H, br s), 6.24 (1 H, s), 3.68 (1H, m), 3.46 (1H, m), 1.24 (3H, s)</td>

Reference Example 2 and 3 [0097] These compounds, namely:

(3-Amino-6-bromo-5-trifluoromethyl ((R) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -amide

<img file="PL2845593T3_D0012.tif" />

(3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid ((S) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) (Ex 3)

<img file="PL2845593T3_D0013.tif" />

is obtained by resolution of 3-amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) amide (Example 1) by means of chromatography. supercritical fluid under the following conditions:

Mobile phase: 12% isopropanol + 0.1% DEA / 88% CO2

Column: Chiralpak OJ-H, 250x10mm id, 5μιτι

Detection: UV at 220 nm

Flow rate: 10 ml / min

Sample concentration: 347 mg in 5 ml EtOH.

The injection volume: 50 μl

Example 2: First eluting peak: ((R) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -3-amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid.

LC-MS: R t = 4.97min [M + H] + 410.1 / 412.2 (Method 10minLC_v002).

¹H NMR (400 MHz, DMSO-d6) δ 8.30 (NH, t), 7.72 (1H, s), 7.29 (NH2, bs), 6.28 (OH, s), 3.68 (1 H, dd) 3.47 (1H, dd), 1.24 (3H, s) ¹⁹F-NMR (400 MHz, DMSO-d6) -62.70 (CF3, s), -80.47 (CF3, s)

Optical rotation [a]²¹D at 589 nm + 14.4 (c = 0.522, MeOH).

Example 3: Second eluting peak: ((S) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -3-amino-5-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid

LC-MS Rt = 4.94 min [M + H] + 412.1 (Method 10minLC_v002).

¹H NMR (400 MHz, DMSO-d6) δ 8.30 (NH, t), 7.72 (1H, s), 7.29 (NH2, bs), 6.28 (OH, s), 3.68 (1 H, dd) 3.47 (1H, dd), 1.24 (3H, s) ¹⁹F-NMR (400 MHz, DMSO-d6) -62.70 (CF3, s), -80.48 (CF3, s).

[0098] Stereochemistry of this compound was confirmed by X-ray crystallographic analysis.

Example 4, 5 and 6 [0099] This compound, namely, 3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -amide. (Prov 4)

<img file="PL2845593T3_D0014.tif" />

made according to the following procedure:

To a solution containing 3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid (intermediate D) (4g, 16.94 mmol) and 3-amino-1,1,1-trifluoro-2-methylpropane -2-ol (intermediate R) (3.04 g, 16.94 mmol) in NMP (188 ml) was added HATU (7.73 g, 20.33 mmol) was added dropwise (2 ml portions) of DIPEA (8 , 88 ml, 50.8 mmol) within 1 hour. After stirring for an additional hour, the reaction mixture is poured into water (450 ml) and EtOAc (450 ml). The aqueous phase is acidified with 5Μ HCl (50 ml) and the layers are separated. The organic phase is washed with 2Μ NaOH (200 ml), water (4 x 200 ml), brine (2 x 100 ml), dried over MgSO4, filtered and concentrated under reduced pressure to give a brown solid. The solid is purified by chromatography on silica gel (220 g packaged silica cartridge) eluting with 0-50% EtOAc in isohexane,

¹H NMR (400 MHz, DMSO-d6) δ 8.3 (1H, t), 7.7 (1H, s), 6.7 (2H, s), 6.2 (1H, s), 3.9 (3H, s), 3.7 (1H, m), 3.5 (1H, m), 1.2 (3H, s).

LC-MS: Rt 1.24 min; MS m / z 362.4 [M + H] &lt;+&gt;; Method 2minLC_v003.

[0100] The chiral separation of the racemate by supercritical chromatography was carried out under the following conditions to give the following compounds:

Mobile phase: 12% 2-propanol + 0.1% DEA / 50% CO2

Column: Chiralcel OD-H, 250 x 10 mm id, 5 μm (2 columns connected in series) Detection: UV at 220 nm

Flow rate: 10 ml / min,

Sample concentration: 3.5 g in 30 ml EtOH,

Injection volume: 100 μl

Examples 5 and 6 are enantiomers.

Example 5: First elution peak Rt = 7.30 minutes. (3-Amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid ((S) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) -amide:

<img file="PL2845593T3_D0015.tif" />

¹H-NMR (400 MHz, DMSO-d6) δ 8.3 (1H, t), 7.6 (1H, s), 6.6 (2H, broad), 6.2 (1H, s), 3, 9 (3H, s), 3.6 (1H, m), 3.5 (1H, m), 1.3 (3H, s);

LC-MS Rt = 1.15 min, [M + H] + 362.4 (Method 2minLC_v003).

Optical rotation [a]²¹D at 589 nm -20,83 (c = 0.513, MeOH).

[0102] The stereochemistry of this compound was confirmed by X-ray crystallographic analysis. Example 6: Second elution peak Rt = 8.29 minutes ((R) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) -3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid amide

<img file="PL2845593T3_D0016.tif" />

LC-MS Rt = 1.15 min [M + H] + 362.4 (Method 2minLC_v003).

[0104] Alternatively, Example 5 can be produced according to the following method:

To a solution of 3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid (Intermediate D) (10 g, 42.3 mmol) and (S) -3-amino-1,1,1- hydrochloride trifluoro-2-methylpropan-2-ol (intermediate RA) (7.60 g, 42.3 mmol) in NMP (400 mL) is added HATU (19.3 g, 50.8 mmol) followed by dropwise addition of DIPEA (22.19 mL, 127 mmol) for ~ 1 hour. After stirring at room temperature for 30 minutes, the reaction mixture is added to EtOAc (2L), washed with 1M NaOH (2 x 1 L), water (1 L), brine (1 L), dried (MgSO4) and evaporated under reduced pressure to give a crude product in the form of a dark brown oil. Purification by chromatography on silica gel, eluting with a gradient from 1 to 25% EtOAc in isohexane gives a yellow oil. After crystallization of the oil with isohexane / DCM, the amide ((S) -3.3 is obtained,

¹1 H NMR (400 MHz, DMSO-d6) δ 8.28 (1H, t), 7.66 (1H, s), 6.67 (2H, s), 6.27 (1H, s), 3, 91 (3H, s) 3.65 (1H, m), 3.45 (1H, m), 1.24 (3H, s).

¹⁹F NMR (376 MHz, DMSO-d6) -62.58 ppm (s) -80.43 ppm (s)

Example 7:

(3-Amino-6- (4-fluorophenyl) -5-trifluoromethyl-pyridine-2-carboxylic acid ((S) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) [0105]

<img file="PL2845593T3_D0017.tif" />

[0106] A mixture containing 3 (R) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) -amide of 3 amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid (Prov 3) (100 mg, 0.244 mmol), 4-fluorophenylboronic acid (37.5 mg, 0.268 mmol) and dichloridine 1,1'-bis (diphenylphosphino) palladium (19.90 mg, 0.024 mmol) are suspended in THF (2 mL) and 1 M Cs 2 CO 3 (0.667 ml). The vial is purged with N 2, closed and heated at 160 ° C with microwave irradiation for 15 minutes. The mixture is partitioned between EtOAc (50 mL) and water (50 mL). The organic layer is separated, washed with brine (30 ml), dried (MgSO4) filtered through Celite® (filter material) and concentrated in vacuo. The crude residue is dissolved in DMSO (2 mL) and purified by LCMS using MeCN / water / 0.1% TFA as the eluent to give pure product. The product fraction obtained as a MeCN / water / 0.1% TFA solution is poured into EtOAc (50 mL) and washed with saturated NaHCO 3 solution (50 mL) to liberate the product as the free base. The organic layer is combined, dried (MgSO4) and concentrated in vacuo to give the title compound as a pale orange crystalline solid; 1H

NMR (400 MHz, DMSO-d6) δ 8.4 (1H, m), 7.7 (1H, s), 7.49 (2H, m), 7.29 (2H, t), 7.2 ( 2H, br s), 6.22 (1H, s), 3.68 (1 H, m), 3.44 (1 H, m), 1.22 (3H, s); LC-MS Rt 4.41 min [M + H] + 426 (Method 10minLC_v003).

Example 8:

(3R) -3-amino-6- (4-fluoro-phenyl) -5-trifluoromethyl-pyridine-2-carboxylic acid (R) -3,3,3-trifluoro-2-hydroxy-2-methylpropyl) -amide.

<img file="PL2845593T3_D0018.tif" />

This compound is prepared from 3-amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid ((R) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -amide. (Ex 2) in a manner analogous to that described in Example 8. 1 H-NMR (400 MHz, DMSO-d 6) δ 8.42 (1H, m), 7.7 (1H, s), 7.5 (2H,

m), 7.3 (2H, t), 7.21 (2H, br s), 6.24 (1H, s), 3.68 (1H, m), 3.44 (1H, m), 1 22 (3H, s); LC-MS Rt = 4.39 min [M + H] + 426 (Method 10minLC_v003).

Example 16 and 17 ((S) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) -3-amino-5,6-bistrifluoromethyl-pyridine-2-carboxylic acid and ((R) - 3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) carboxylic acid amide

3-amino-5,6-bis-trifluoromethyl-pyridine-2 [0109]

<img file="PL2845593T3_D0019.tif" />

Step 1: 3- (2,5-dimethylpyrrol-1-yl) -5,6 -6,6-trifluoromethyl-pyridine-2-carboxylic acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) [ 0110] To a stirred solution of 3- (2,5-dimethylpyrrol-1-yl) -5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid (intermediate M) (1.16 g, 3.29 mmol) in NMP (32 ml) 3-amino-1,1,1-trifluoro-2-methyl-propan-2-ol hydrochloride (commercially available) (591 mg 3.29 mmol) is added, followed by HATU (1.25 g, 3.29 mmol) and NEt3 (918 μΙ, 6.59 mmol) and the reaction mixture is allowed to stir at room temperature. After 1 hour a further 0.2 eq of NEt3 is added. After 15 minutes, an additional 0.4 equivalents of NEt3 and 0.2 equivalents of the amine are added. After 30 minutes, an additional 0.1 equivalent of HATU is added. After 30 min, most of the starting material is consumed. The reaction mixture is added to EtOAc (50 mL), washed with 0.1 M NaOH and the aqueous layer is back-extracted with EtOAc (2 x 50 mL). The combined organic extracts are washed with water (2 x 150 ml), brine (100 ml), dried (MgSO4) and concentrated in vacuo to give the crude product as an orange oil. The crude material is purified by chromatography on silica eluting with 0-15% EtOAc in iso-hexane to give the title product as a yellow solid; LC-MS Rt 1.32 min; MS m / z 478.2 [M + H] +; Method 2minLC_v003. Step 2: 3-Amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid (3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) amide To a stirring solution (3, 3,3-trifluoro-2-hydroxy-2-methyl-propyl) -3- (2,5-dimethylpyrrol-1-yl) -5-amide, 6-Bis-trifluoromethyl-pyridine-2-carboxylic acid (985 mg, 2.064 mmol) in 2: 1 EtOH / H2O (7.5 mL) is added hydroxylamine hydrochloride (1.43 g, 20.64 mmol) followed by NEt3 (575 ml, 4.13 mmol). The reaction mixture is heated under reflux (up to -98 ° C) for 11.5 hours and then allowed to cool to room temperature. The solvent was evaporated under reduced pressure and the resulting residue was partitioned between EtOAc (25 mL) and water (25 mL). The aqueous layer was separated, extracted with EtOAc (2 x 25 mL) and the combined organic extracts were washed with brine (50 mL), dried (MgSO4) and concentrated in vacuo. The crude material is purified by chromatography on silica gel, eluting with 0-25% EtOAc in isohexane, as a result of which the title product is obtained in the form of a pale yellow solid; LC-MS: Rt 1.24 min; MS m / z 400.0 [M + H] &lt;+&gt;; Method 2minLC_v003.

Step 3: 3-amino-5,6-bistrifluoromethyl-pyridine-2-carboxylic acid ((S) -3,3,3-trifluoro-2-hydroxy-2-methyl-propyl) and ((R) -3 3,3-trifluoro-2-hydroxy-2-methylpropyl) -3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid amide

<img file="PL2845593T3_D0020.tif" />

[0114] These compounds were prepared by separation of 3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid chiral (33,3-trifluoro-2-hydroxy-2-methylpropyl) amide;

Enantiomer 1: LC-MS Rt 1.23 min; MS m / z 400.0 [M + H] &lt;+&gt;; Method 2minLC_v003. SFC 20 Retention time 5.07 min.

Enantiomer 2: LC-MS Rt 1.23 min; MS m / z 400.0 [M + H] &lt;+&gt;; Method 2minLC_v003. SFC Retention time 5.13 min.

Preparation of intermediates. Intermediate A

3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid [0115]

<img file="PL2845593T3_D0021.tif" />

Intermediate A1: 2-bromo-3-nitro-5-trifluoromethyl-pyridine 3-Nitro-5- (trifluoromethyl) pyridin-2-ol (31.00 g, 149 mmol) is dissolved in acetonitrile (250 ml). ) and receives a dark brown solution. Phosphorous oxybrome (V) (85 g, 298 mmol) is added and the mixture is heated under reflux for 4.5 hours and then stirred at room temperature overnight. The reaction mixture is quenched by pouring into water with vigorous stirring (600 ml) containing sodium bicarbonate (110 g). The dark brown mixture is extracted with DCM (3 x 200 mL) and the organic phase is washed with water (200 mL), brine (100 mL), dried (MgSO4) and concentrated under reduced pressure to afford the title product as a brown oil.

¹H-NMR [400 MHz, CDCl 3, δH 8.87 (1H, d, J = 1.4 Hz, ArH), 8.39 (1H, d, J = 1.9 Hz, ArH). Intermediate A2: 3-nitro-5-trifluoromethyl-pyridin-2-carbonitrile 2-Bromo-3-nitro-5-trifluoromethyl-pyridine (10.00 g, 36.87 mmol) is dissolved in toluene (250 g). ml), while stirring, a light yellow solution is obtained. Tetrabutylammonium bromide (11.90 g, 36.9 mmol) is added followed by the addition of copper (I) cyanide (9.92 g, 111 mmol) and the mixture is heated at reflux for 10 hours. After cooling to room temperature, the reaction mixture is partitioned between water (750 mL) and EtOAc (750 mL). The organic portions are combined, washed with water (2 x 250 mL) and brine (100 mL), dried (MgSO4) and concentrated under reduced pressure to give the title product.¹H-NMR: [400 MHz, DMSO-d6 δH 9.55 (1H, m, ArH), 9.24 (1H, m, ArH).

Intermediate A3: 3-Amino-5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester 3-nitro-5-trifluoromethyl-pyridin-2-carbonitrile (6.5 g, 29.9 mmol) is dissolved in EtOAc ( 150 ml) to form a light yellow solution and place under a nitrogen atmosphere. 10% palladium on activated carbon (3.19 g, 2.99 mmol) is added and the reaction mixture is stirred under a hydrogen atmosphere for 18 hours. The reaction mixture is filtered and concentrated under reduced pressure. The crude residue is dissolved in concentrated HCl (45 ml) and heated under reflux for 24 hours. The reaction mixture is allowed to cool to room temperature and concentrated under reduced pressure. The solid is dissolved in MeOH (300 ml) and sulfuric acid (14.4 ml) is added. The resulting solution is heated under reflux for 48 hours. The reaction mixture is allowed to cool to room temperature and then neutralized by the addition of a 10% NaHCO3 solution (aq) (600 mL). The product is extracted with DCM (3 x 200 ml) and the combined organic phases are washed with water (200 ml), brine (50 ml), (MgSO4) and concentrated under reduced pressure. The solid obtained is purified by chromatography on silica gel: gradient elution: isohexane (500 ml), 10% ethyl acetate in isohexane (1000 ml), 20% ethyl acetate in isohexane (1500 ml) to give the title compound in the form a pale yellow solid The product is extracted with DCM (3 x 200 ml) and the combined organic phases are washed with water (200 ml), brine (50 ml), (MgSO4) and concentrated under reduced pressure. The solid obtained is purified by chromatography on silica gel: gradient elution: isohexane (500 ml), 10% ethyl acetate in isohexane (1000 ml), 20% ethyl acetate in isohexane (1500 ml) to give the title compound in the form a pale yellow solid The product is extracted with DCM (3 x 200 ml) and the combined organic phases are washed with water (200 ml), brine (50 ml), (MgSO4) and concentrated under reduced pressure. The solid obtained is purified by chromatography on silica gel: gradient elution: isohexane (500 ml), 10% ethyl acetate in isohexane (1000 ml), 20% ethyl acetate in isohexane (1500 ml) to give the title compound in the form a pale yellow solid¹H-NMR [400 MHz, DMSO-d6, δδ 8.13 (1H, d, J = 1.7 Hz, ArH), 7.60 (1H, d, J = 1.3 Hz, ArH), 7, 01 (2H, br, NH 2), 3.85 (3H, s, ArOCH 3), m / z 221.1 [M + H] +

Intermediate A4: 3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester 3-Amino-5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester (9.49 g, 43.16) mmol) is suspended in water (300 ml). Sulfuric acid (4.60 ml, 86 mmol) is added and then a solution of bromine (2.222 ml, 43.1 mmol) in acetic acid (29.6 ml, 517 mmol) is added dropwise over 30 minutes. The reaction mixture is stirred at room temperature for 18 hours. Then 100 ml of water and another 0.25 equivalents of the bromine / AcOH mixture (550 ml of bromine in 7.4 ml AcOH) are added and the reaction mixture is stirred at room temperature for an additional 90 minutes. The reaction mixture is diluted with 500 ml of water and neutralized by the addition of solid NaHCO 3 (~ 85 g). The suspension is extracted with DCM (3 x 300 ml) and the combined organic phases are washed with sat. NaHCO3 (aq) (250 mL), water (250 mL) and brine (100 mL), dried (MgSO4) and concentrated in vacuo. The crude product is recrystallized from boiling methanol (~300 ml) to give the title product as a pale orange solid, m / z 301.0 [M + H] +¹H-NMR [400 MHz, DMSO-d6 δH 7.77 (1H, s, ArH), 7.17 (2H, s, NHz), 3.86 (3H, s, ArCOzCHa).

Intermediate A: 3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid [0120] 3-Amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester (1.40 g) , 4.68 mmol) is suspended in MeOH (15 mL); Sodium hydroxide (2.0 M aqueous solution) (14.04 mL, 28.1 mmol) is added and the suspension is stirred at room temperature overnight. The mixture is concentrated under reduced pressure and the obtained residue is dissolved in water (100 ml) and then acidified by the addition of 5.0 M HCl (aqueous solution). The product is extracted with ethyl acetate (2 x 75 mL) and the combined organic extracts are washed with water (50 mL), brine (25 mL), dried (MgSO4) and concentrated under reduced pressure to give the title product as a yellow solid.¹H-NMR [400 MHz, DMSO-d6, δH 13. 24 (1H, br s, C02H), 7.74 (1H, s, ArH), 7.17H, br s, ArNH2). m / z 285.1, 287.1 [M + H] +

Intermediate D

3-Amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid

<img file="PL2845593T3_D0022.tif" />

trifluoromethyl-pyridine-2-carboxylic acid

<img file="PL2845593T3_D0023.tif" />

intermediate A4) (2 g, 6.69 mmol) is suspended in toluene (8 ml) and treated with p-toluenesulfonic acid (TsOH) (0.115 g, 0.669 mmol) and acetonyloacetone (0.941 ml, 8.03 mmol). The reaction mixture is heated at reflux for 2 hours (using a Dean-Stark apparatus) and allowed to cool to room temperature overnight. The resulting dark red / black solution is concentrated under reduced pressure to remove toluene and the crude residue is diluted with EtOAc (200 mL), washed with NaHCO 3 solution (50 mL), dried (MgSO 4) and concentrated under reduced pressure to give a brown solid. Purification of the solid by chromatography on silica gel, eluting with EtOAc / iso-hexane gives the title compound; LC-MS Rt = 5.58 min [M + H] + 377/379 (Method 10minLC_v002).¹H NMR (400 MHz, DMSO-d6) δ 8.50 (1H, s), 7.77 (2H, s), 5.83 (3H, s), 1.90 (6H, s);

¹⁹F NMR (400 MHz, DMSO-d6) δ-62.26 (CF3, s).

Intermediate D2: 3- (2,5-Dimethylpyrrol-1-yl) -6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid [0123]

<img file="PL2845593T3_D0024.tif" />

6-bromo-3- (2,5-dimethylpyrrol-1-yl) -5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester (2 g, 5.30 mmol) is dissolved in MeOH (40 ml) and treated with 2M NaOH (20 ml) to give a suspension that is stirred at room temperature for 1 hour to give a clear solution. The solvent is evaporated under reduced pressure and the residue is acidified to pH 1 with 5M HCl. The mixture is extracted with EtOAc (200 mL) and the organic extract is dried (MgSO4) and concentrated in vacuo to afford the title compound as a dark brown solid which is used in the next step without further purification; LC-MS Rt = 1.50 min [M + H] + 315.2.1 / 316.2 (Method 2minLC_v002); 1 H NMR (400 MHz, DMSO-d 6) δ 16.42-12.61 (COOH, b), 8.25 (1H, s) 5.84 (2H, s), 4.13 (3H, s), 1 97 (6H, s); 19 F NMR (400 MHz,

Intermediate D: 3-Amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid [0125] 3- (2,5-dimethylpyrrol-1-yl) -6-methoxy-5-trifluoromethyl-pyridine acid 2-carboxylic acid (2.1 g, 6.68 mmol) is dissolved in EtOH (40 mL) and water (20 mL). To this mixture is added TEA (2.79 mL, 20.05 mmol) followed by hydroxylamine hydrochloride (4.64g, 66.8 mmol). The resulting mixture is heated under reflux for 5 hours. After cooling to room temperature, the mixture is diluted with ethyl acetate (100 mL) and washed with aqueous HCl (1 M, 100 mL). The aqueous layer was re-extracted with EtOAc (100 mL) and the combined organic phases were washed with brine (100 mL), dried (MgSO4), concentrated in vacuo to give the product as an orange solid.

LC-MS Rt = 1.0 min [M + H] + 237 (Method 2minLC_v003)

1 H NMR (400 MHz, DMSO-d 6) 8.5 (NH 2, b), 7.70 (1H, s), 3.89 (3H, s).

Reference compound G

3-Amino-6- (4-fluoro-phenyl) -5-trifluoromethyl-pyridine-2-carboxylic acid [0126]

<img file="PL2845593T3_D0025.tif" />

OH

F A mixture containing 3-amino-6-bromo-5-trifluoromethyl-pyridine-2-carboxylic acid (Intermediate A) (1 g, 3.51 mmol), 4-fluorophenylboronic acid (0.736 g, 5.26) mmol) and 1,1'-bis (diphenylphosphosio) ferrocene palladium dichloride (0.286 g, 0.351 mmol) and 1.0 M Cs2CO3 solution (3.3 mL) in THF (10 mL) is heated under reflux for 10 hours. After cooling to room temperature, the reaction mixture is partitioned between DCM (100 mL) and 1 M NaOH (2 × 100 mL). The aqueous phase is acidified with 5M HCl and the resulting milky solution is extracted with DCM (2 x 100 ml). The organic layer is separated, dried (MgSO4) and concentrated in vacuo to give the product as a crude oil. The crude material is chromatographed on a pad of silica, eluting with a gradient of DCM: MeOH, from 0% to 10% MeOH,

1H NMR (DMSO-d6, 400 MHz) δ 12.9 (1H, br s, COOH), 7.7 (1H, s, CH, Ar-H), 7.4 (2H, m, Ar-H) , 7.25 (2H, m, Ar-H), 7.1 (2H, broad s, NH 2).

Intermediate M

3- (2,5-Dimethylpyrrol-1-yl) -5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid [0128]

FO

Intermediate M1: 3- (2,5-Dimethylpyrrol-1-yl) -5,6-bistrifluoromethyl-pyridine-2-carboxylic acid A mixed mixture of KF (2.12 g, 5.62 mmol) and Cul ( 0.490 g, 8.43 mmol) is heated in a tightly closed 10.0 - 20.0 mL microwave vial under reduced pressure until a greenish appearance appears. The vial is then placed under a nitrogen atmosphere to cool. A solution of 6-bromo-3- (2,5-dimethylpyrrol-1-yl) -5-trifluoromethyl-pyridine-2-carboxylic acid methyl ester (Intermediate D) (2.64 mL, 17.65 mmol) in a 1: 1 mixture. anhydrous DMF / anhydrous NMP (14 ml) is then added, followed by TMS-CF3 (2.64 ml, 16.65 mmol). A new septum was used to seal the vial and the reaction mixture was heated using microwave irradiation at 100 ° C for 3 hours and allowed to cool. A 5M NH3 solution (50 mL) is added to the mixture and then extracted with diethyl ether (4 x 50 mL). The combined organic extracts are washed with 5M NH 3 solution (3 x 20 ml), 1M HCl (50 ml), saturated sodium bicarbonate solution (2 x 50 ml), brine (50 ml), dried (MgSO 4) and concentrated in vacuo to give brown Oil. The crude material is purified by chromatography on silica eluting with isohexane / EtOAc 0-10% to give the title compound as an orange solid; LC-MS Rt 1.37 min; MS m / z 367.1 [M + H] &lt;+&gt;; Method 2minLC_v003. it is dried (MgSO4) and concentrated in vacuo to give a brown oil. The crude material is purified by chromatography on silica eluting with isohexane / EtOAc 0-10% to give the title compound as an orange solid; LC-MS Rt 1.37 min; MS m / z 367.1 [M + H] &lt;+&gt;; Method 2minLC_v003. it is dried (MgSO4) and concentrated in vacuo to give a brown oil. The crude material is purified by chromatography on silica eluting with isohexane / EtOAc 0-10% to give the title compound as an orange solid; LC-MS Rt 1.37 min; MS m / z 367.1 [M + H] &lt;+&gt;; Method 2minLC_v003.

Intermediate M: 3- (2,5-Dimethylpyrrol-1-yl) -5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid [0130] To a stirred solution of 3- (2,5-dimethyl-pyrrole) methyl ester 1-yl) -5,6-bistrifluoromethyl-pyridine-2-carboxylic acid (1.28 g, 3.49 mmol) in methanol (25 mL) is added 1 M NaOH (7 mL, 6.99 mmol) and the reaction mixture allowed to stir at room temperature for 30 minutes. The solvent is concentrated in vacuo and water (20 ml) is added to the residue. The pH is adjusted to 4-5 by the addition of 1M HCl. The mixture is extracted with EtOAc (3 x 20 ml) and the combined organic extracts are washed with brine (30 ml), dried (MgSO4) and concentrated in vacuo and dried in a vacuum oven (50 ° C) overnight to give the crude title product as an orange substance a solid that was used without further purification; LC-MS: Rt 1.23 min; MS m / z 353, 1 [M + H] +; Method 2minLC_v003.

Intermediate R

3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride

<img file="PL2845593T3_D0026.tif" />

[0132] To LiOH (0.193 g, 8.06 mmol) in a 3-neck round bottom flask, water (25 mL), nitromethane (3.76 mL, 81 mmol) and trifluoroacetone (7.95 mL, 89 mmol) are added. Cetyltrimethylammonium chloride (3.8 g, 10.88 mmol) and MgSO4 (1.9 g, 16.12 mmol) is added and the resulting yellow solution is stirred at 20-25 ° C for 2 days. The reaction mixture is poured into diethyl ether (120 ml) and washed with water (3 x 200 ml) and brine (1x 100 ml). The organic layer is dried over MgSO 4 and concentrated in vacuo to afford the title compound as a yellow liquid. 1 H NMR (CDCl 3, 400 MHz): δ 4.7 (1H d), δ 4.5 (1H, d), δ 3.7 (1H, broad), δ 1.6 (3H, s).

Step 2: 3-amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride [0133] Pd / C is added (1g) to a 200ml glass vessel. Ethanol (50 ml, dry) is carefully added in a CO2 atmosphere. 1,1,1-trifluoro-2-methyl-3-nitropropan-2-ol (10 g, 57.8 mmol) is dissolved in ethanol (50 ml, dry) and added to a glass vessel. The reaction mixture is placed under a positive hydrogen pressure (5 bar) at room temperature and hydrogenated for 2 days. The reaction mixture is filtered through Celite® (filter material) and washed with excess ethanol. The solvent is concentrated in vacuo to give a colorless oil. The oil was dissolved in MeOH (50 mL) and treated dropwise with HCl (1M) in methanol (30 mL). The solution is allowed to stir for 30 minutes and concentrated in vacuo by azeotroping with MeCN to give the title compound as a waxy white solid;

Intermediate RA

(S) -3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride

<img file="PL2845593T3_D0027.tif" />

3,3,3-trifluoro-2-hydroxy-2 [0135] To a stirred suspension of amino-1,1,1-trifluoro-2-methylpropan-2-ol (intermediate R) (1.5 g, 8.35 mmol) in DCM (50 mL), TEA, 93.54 g, 35.0 mmol) is added, followed by addition of 2,5-dioxopyrrolidin-1-yl benzyl carbonate (1.983 g, 7.96 mmol). The mixture is stirred at room temperature for 6 hours and then diluted with water. The organic layer is separated by means of a phase separator and concentrated in vacuo. The organic phase is purified by chromatography on silica eluting with 0-70% EtOAc in isohexane to yield the title product; 1 H NMR (400 MHz, DMSO-d 6) δ 7.34 (6H, m), 5.98 (1H, s), 5.05 (2H, s), 3.31 (1H, m), 3.18 (1H, m), 1.21 (3H s). LC-MS: Rt 1.05 min; MS m / z 278.1 [M + H] +; Method 2minLC_v003.

Step 2: Separation of benzyl 3,3,3-trifluoro-2-hydroxy-2-methylpropylcarbamate enantiomers Benzyl 3,3,3-trifluoro-2-hydroxy-2-methylpropylcarbamate (1.7 g) is dissolved in 2-propanol (10 ml) and purified under the following chromatographic conditions:

Mobile phase: 10% 2-propanol / 90% CO2

Column: 2xChiralcel OJ-H, 250x10 mm ID, 5 μιτι (columns connected in series)

Detection: UV at 220 nm

Flow rate: 10 ml / min,

Sample concentration: 1.7 g in 10 ml of 2-propanol,

The injection volume: 75 μl

First eluted peak: Rt = 6.94 min (R) -benzyl 3,3,3-trifluoro-2-hydroxy-2-methylpropylcarbamate

Second elution peak: Rt = 8.04 min (S) -benzyl 3,3,3-trifluoro-2-hydroxy-2-methylpropylcarbamate (Stereochemistry confirmed by analysis of the final compound obtained according to the following steps)

Step 3: (S) -3-Amino-1,1,1-trifluoro-2-methylpropan-2-ol hydrochloride [0137] A mixture containing (S) -benzyl-3,3,3-trifluoro-2-hydroxy- The 2-methyl propyl carbamate in EtOH (165 mL) is pumped through the H-cube (hydrogenation reactor 1-2 mL / min, pressure 1 bar, RT) for 8 hours with 10% palladium on carbon. 1.25 M HCl in methanol (130 ml) is added to the mixture and the mixture is stirred for 30 minutes. The solvent is concentrated in vacuo while azeotroping with MeCN to give the title product in the form of a white powder; 1 H NMR (400 MHz, DMSO-d 6) δ 8.3 (3H, broad), 6.8 (1H, s), 3.0 (2H, s), 1.5 (3H, s).

[0138] Alternatively, the racemic mixture of 3-amino-1,1,1-trifluoro-2-methylpropan-2-ol may be separated into individual enantiomers by recrystallization from (S) -lipidic acid, L-wine acid, in isopropanol or ethanol.

Novartis AG, Switzerland Plenipotentiary:

EP 2845593B1 Z-15513/17