3,5-Diamino-6-chloro-N-(N-(4-(4-(2-(hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

Description: (The description is published in the applicant's editorial office)

The present invention relates to novel compounds, including in particular 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl (2,3,4,5,6-pentahydroxyhexyl) amino)). ethoxy) phenyl) butyl) carbamimidoyl) -pyrazine-2-carboxamide and its pharmaceutically acceptable salt forms, useful as sodium channel blockers, their compositions, therapeutic methods and their applications and processes for their preparation.

Prerequisites for creating the invention

The mucosal surfaces at the interface between the environment and the body have evolved a series of "inherent protections", for example, protective mechanisms. The main form of such inherent protection is to clean these surfaces with liquid. Usually, the amount of fluid layer on the mucosal surface reflects the balance between the secretion of the epithelial fluid, which often reflects the secretion of anions (Cl 'and / or НСОз) coupled with water (and a cation counterion), and the absorption of the epithelial fluid, which often absorbs N / A⁺, coupled with water and against anion (Cl 'and / or НСОз') · Many diseases of the mucosal surfaces are caused by the protective fluid too little on these mucosal surfaces created by an imbalance between secretion (too little) and absorption (relatively too much) . Defective salt transfer processes that characterize these mucosal membrane dysfunctions are found in the epithelial layer of the mucosal surface.

The approach of restoring the protective liquid layer on the mucosal surfaces is to “re-balance” the system by blocking the Na channel.⁺ and the absorption of the liquid. Epithelial protein that mediates the factor that reduces the rate of Na uptake⁺ and the fluid is the epithelial channel of Na⁺ ( "ENaC"). ENaC is positioned on the apical surface of the epithelium, that is, the mucosal interface surface-environment. Ideally, to inhibit the absorption of liquid and Na⁺ mediated by ENaC, an ENaC blocker from the amiloride class will be administered to the mucosal surface and maintained at this site for maximum therapeutic benefit.

The use of ENaC blockers has been reported for a variety of diseases that are enhanced by increased hydration of the mucous membrane. In particular, the use of ENaC blockers in the treatment of respiratory diseases, such as chronic bronchitis (ВС), cystic fibrosis (FC) and ВРОС, which reflects the body's inability to purge mucus normally from the lungs and ultimately results in chronic infection of the respiratory tract, has been reported (RC Boucher. Evidence for airway surface dehydration as the initiating event in CF airway disease. Journal of Internal Medicine, 2007, vol. 261, no. 1, pp. 5-16; RC Boucher. Cystic fibrosis: a disease of vulnerability to airway surface dehydration. Trends in Molecular Medicine, 2007, vol. 13, ed. 6, pp. 231-240).

The data indicate that the problem that causes both chronic bronchitis and cystic fibrosis is the inability to purge the mucus from the airway surfaces. The inability to purge the mucus reflects a lump of waste in the quantities of mucus as airway surface fluid (ASL) on the airway surfaces. This imbalance results in a relative reduction of ASL which results in mucus concentration, reduced lubricant activity of the perilous fluid (PCL), adhesion of the mucus to the surface of the airways and inability to purge the mucus through the ciliary activity in the mouth. Reduction of mucus clearance results in chronic bacterial colonization of the mucus adherent to the airway surfaces. The chronic retention of bacteria, the inability of local antimicrobial substances to kill the bacteria trapped by mucus on a chronic basis and the chronic inflammatory response consistent with this type of surface infection, is manifested in chronic bronchitis and cystic fibrosis.

There is currently a large, unmet medical need for products to specifically treat the variety of diseases that are enhanced by increased hydration of the mucous membrane, including chronic bronchitis, ВРОС and cystic fibrosis, among others. Current therapies for chronic bronchitis, ВРОС and cystic fibrosis are focused on treating the symptoms and / or delayed effects of these diseases. However, none of these therapies effectively addresses the fundamental problem of inability to clear mucus from the lungs.

RC Boucher, describes the use of sodium channel blockers in the pyrazinoilguanidine class for hydrating the mucosal surfaces characterized by the well-known diuretics amiloride, benzamyl and phenamyl. However, these compounds are relatively impotent, considering the limited mass of drug that can be

MD 4574 Cl 2019.01.31 inhaled into the lungs; (2) rapidly absorbed and thus having an undesirable short half-life on the mucosal surface, and (3) are freely dissociable from EnaC [1]. More powerful drugs are needed, with prolonged half-lives on the mucosal surface.

Too little liquid of protective surface on other mucosal surfaces is the common pathophysiology of a number of diseases. For example, in xerostomia (dry mouth) is the oral cavity depleted of fluid due to the inability of the sublingual and submandibular parotid glands to secrete? liquid despite the continuous absorption of the liquid mediated by the Na transport⁺ (ENaC) in the oral cavity. Is keratoconjunctivitis sicca (dry keratitis) caused by disability? tear glands to secrete? liquid despite continuous absorption of the Na-dependent liquid⁺ on the conjunctival surfaces. In rhinosinusitis, there is an imbalance between mucin secretion and relative ASL exhaustion. Incapacity? to secret? Cl '(and fluid) in the proximal small intestine, combined with increased Na uptake⁺ (and fluid) in the terminal ileum leads to distal intestinal obstruction syndrome (SOID). In older patients, excessive absorption of Na⁺(and volume) in the descending colon causes constipation and diverticulitis.

The published literature includes the number of patent applications and patents granted by Parion Sciences Inc., aimed at pyrazinoilguanidine analogues as sodium channel blockers. Examples of such publications include PCT publication numbers: WO 2003070182 A2 2003.08.28, WO 2003070184 A2 2003.08.28, WO 2004073629 A2 2004.09.02, WO 2005025496 A2 2005.03.24, WO 2005016879 A2 2005.02.24, WO

2005018644 To 2005.03.03, WO 2006022935 To 2006.03.02, WO 2006023573 A2

2006.03.02, WO 2006023617 A2 2006.03.02, WO 2007018640 To 2007.02.15, WO

2007146869 To 2007.12.21, WO 2008031028 A2 2008.03.13, WO 2008031048 A2

2008.03.13 and patent numbers US 685 8614 B2 2005.02.22, US 6858615 B2 2005.02.22, US 6903105 B2 2005.06.07, US 7064129 B2 2006.06.20, US 7186833B2

2007.03.06, US 7189719 B2 2007.03.13, US 7192958 B2 2007.03.20, US 7192959B2

2007.03.20, US 7192960 B2 2007.03.20, US 7241766 B2 2007.07.10, US 7247636B2

2007.07.24, US 7247637 B2 2007.07.24, US 7317013 B2 2008.01.08, US 7332496B2

2008.02.19, US 7368447 B2 2008.05.06, US 7368450 B2 2008.05.06, US 7368451B2

2008.05.06, US 7375102 B2 2008.05.20, US 7388013 B2 2008.06.17, US 7399766B2

2008.07.15, US 7410968 B2 2008.08.12, US 7807834 B2 2010.10.05, US 7842697B2

2010.11.30, US 7868010 B2 2011.01.11

There is also a need for new compounds to block sodium channels with increased potency and efficiency on mucosal tissues. There is also a need for new compounds to block sodium channels that provide a therapeutic effect, but minimize or eliminate the onset or progression of hyperkalemia in recipients.

The essence of the invention

The present invention relates to 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, of the formula:

OH OH

<img file="MD4574C1_D0001.tif" />

or a pharmaceutically acceptable salt thereof. The invention also relates to solvates and hydrates, individual stereoisomers, including optical isomers (enantiomers and diaster eoisomers) and geometric isomers (cis- / trans -isomerism), mixtures of 3,5-diamino-6 stereoisomers and tautomers. -chloro-N- (N- (4- (4- (2- (hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, or acceptable salt. pharmaceutical composition thereof, as well as pharmaceutical compositions containing the compound, or о a pharmaceutically acceptable salt thereof, its use in treatment methods, as well as methods for its preparation.

MD 4574 Cl 2019.01.31

Brief description of the drawings

A fuller appreciation of the invention and many of its advantages can be easily obtained by reference to the information included here with the following figures:

Fig. 1 is a representative diagram of the concentration-effect dependence of the Compound (la) on the short-circuit current by the canine bronchial epithelial cells (CBE).

Fig. 2 is a diagram of the dose-effect dependence of Compound (la) on mucociliary clearance in sheep (CMC) at 4h after administration.

Fig. 3 is a diagram of the effect of the Compound (la) and the hypertonic physiological solution (SH) on the CMC in sheep at 4h after administration.

Fig. 4 is a diagram of the effect of Compound (la) and SH on CMC in sheep at 8h after administration.

Fig. 5 is a diagram of the biocare effect of the sodium channel of Compound (Ia) on the surface fluid retention at 0-8 h in the in vitro CBE cell model.

Fig. 6 represents a histogram of the effect of Compound (Ia) on the retention of the surface fluid at 24 hours in the in vitro CBE model.

Fig. 7 represents a diagram of the effect of ENaC blockers Compound at and Comparative Example I on CMC in sheep at 8 hours.

Fig. 8 represents a diagram of the effect of ENAC blockers on Compound and Comparative Example 1 on potassium levels in sheep plasma.

Fig. 9 is a diagram comparing the activity of Comparative Example 4 and Compound over CMC in sheep at 4h after administration.

Fig. 10 is a о diagram comparing the effect on K levels⁺ in ovine plasma of Comparative Example 4 and Compound at.

Detailed description of the invention

As used herein, the following terms are defmifi as indicated.

"A compound of the invention" means a compound of Formula I or salt, in particular a pharmaceutically acceptable salt thereof.

"A compound of Formula I" means a compound having the structural formula herein designated as Formula I. Compounds of Formula I include solvates and hydrographs (i.e., adducts of a compound of Formula I with a solvent). In those embodiments wherein a compound of Formula I includes one or more chiral centers, the phrase is intended to encompass each individual stereoisomer, including optical isomers (enantiomers and diastereoisomers) and geometric isomers (cis- / trans -isomerism) and mixtures. of stereoisomers. In addition, the compounds of Formula I also include tautomers of the Formula (s) described.

Throughout the description and examples, the compounds are named using the principles of standard IUPAC designation, where possible, including the use of ChemDraw Ultra 11.0 software for compound names, sold by CambridgeSoft Corp./PerkinElmer.

In some chemical structural representations in which the carbon atoms do not have a sufficient number of attached variables described to produce о valence of four, the remaining carbon substitutes that must provide о valence of four are assumed to be hydrogen. Similarly, in some chemical structures in which the bond is formed without specifying the terminated group, this bond indicates the methyl group (Me, -SO), as is conventional in the art.

In a preferred embodiment, the compound of Formula (I) is 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) - 2,3,4,5,6pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, having the formula:

OH OH

NH О

<img file="MD4574C1_D0002.tif" />

H₃C or о its pharmaceutically acceptable salt.

MD 4574 Cl 2019.01.31 в

The compounds of Formula I may be in the form of a free base or a salt, especially a pharmaceutically acceptable salt. For a review of pharmaceutically acceptable salts see Berge et al. Pharmaceutical salts. Journal of Pharmaceutical Sciences, 1977, vol. 66, pp. 1-19.

Pharmaceutically acceptable salts consisting of inorganic or organic acids! include, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, sulfamate, phosphate, hydrogen phosphate, acetate, trifluoracetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, tanate, ascorbate palmitate, salicylate, stearate, phthalate, alginate, polyglutamate, oxalate, oxaloacetate, sugar, benzoate, alkyl or aryl sulfonates (for example, methanesulfonate, ethanesulfonate, benzolsulfonate, p-toluenesulfonate or naphthalenesulfonate) and isionate; amino acid complexes such as lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like. The compounds according to the invention may also be in the form of salts consisting of elemental anions, such as chlorine, bromine or iodine.

For therapeutic use, the salts of the active ingredients of the compounds of Formula I will be pharmaceutically acceptable, ie they will be salts derived from a pharmaceutically acceptable acid. However, salts of non-pharmaceutically acceptable acids can also be used, for example, in the preparation or purification of a pharmaceutically acceptable compound. Trifluoroacetate salts, for example, can thus be used. All salts, whether or not they are derived from a pharmaceutically acceptable acid, are included in the scope of the present invention.

The term "chiral" refers to molecules that have the non-overlapping property of the mirror image partner, while the term "achiral" refers to molecules that are superimposed on the mirror image partner.

The term "stereoisomers" refers to compounds that have the same chemical structure, but differ in terms of the arrangement of atoms or groups in space. "Diastereomer" refers to a stereoisomer with two or more chirality centers and whose molecules are not mirror images of another. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties and reactivities. Mixtures of diastereomers can be separated according to high-resolution analytical procedures, such as electrophoresis and chromatography. "Enantiomers" refers to two stereoisomers of a compound that are non-taxable mirror images of another.

The definitions and stereochemical conventions used here correspond generally (SP Parker. McGraw-Hill Dictionary of Chemical Terms. 1984, McGraw-Hill Book Company, New York; Eliel E. and Wilen S. Stereochemistry of Organic Compounds. 1994, John Wiley &amp; Sons, Inc., New York).

Many organic compounds! they exist in optically active forms, that is, they have the ability to rotate the plane of polarized plane light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule relative to its chiral center (ii). A specific stereoisomer can also be considered as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is considered to be a racemic or racemic mixture, which may occur if there was no stereoselection or stereospecificity in a chemical reaction or chemical process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species.

The term "tautomers" refers to a type of stereoisomer in which the migration of a hydrogen atom results in two or more structures. Compounds of Formula I may exist in different tautomeric forms. One skilled in the art will recognize that amidines, amides, ureas, guanidines, thiourea, heterocycles and the like may exist in tautomeric forms. By way of example and not by limitation, compounds of Formula I may exist in different tautomeric forms as shown below:

MD 4574 Cl 2019.01.31

<img file="MD4574C1_D0003.tif" />

<img file="MD4574C1_D0004.tif" />

All possible tautomer forms of amidines, amides, ureas, guanidines, thiols, heterocycles and the like of all embodiments of Formula I are within the scope of the present invention. Tautomers exist in equilibrium and, thus, the description of a single tautomer in the formulas presented will be congratulated by those skilled in the art because they refer equally to the possible tautomers tofu.

It should be noted that tofi enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs, pseudopolymorphs of the compounds of Formula I and pharmaceutically acceptable salts thereof are encompassed by the present invention. All mixtures of such enantiomers and diastereomers, including enantiomerically enriched mixtures and diastereomeric enriched mixtures are within the scope of the present invention. Enantiomerically enriched mixtures are mixtures of enantiomers in which the ratio of the enantiomer specified to the alternative enantiomer is greater than 50:50. Specifically, an enantiomerically enriched mixture confines at least about 75% of the specified enantiomer and preferably at least about 85% of the specified enantiomer. In one embodiment, the enantiomerically enriched mixture is substantially free of another enantiomer. Similarly, diastereomeric enriched mixtures are mixtures of diastereomers, wherein the amount of specified diastereomer is greater than the amount of each alternate diastereomer. More specifically, a diastereomeric enriched mixture confines at least about 75% of the specified diastereomer and preferably at least about 85% of the specified diastereomer. In one embodiment, the diastereomeric enriched mixture is substantially free of any alpha diastereomers. The term "substantially free of" will be understood by those skilled in the art as indicating о less than 5% presence of alpha diastereomers, preferably less than 1%, more preferably less than 0.1%. In other embodiments, the diastereomers will not be present or the number of any other diastereomers present will be below the detection level. Stereoisomers can be separated by techniques known in the art, including high performance liquid chromatography (HPLC) and crystallization of chiral salts.

A single stereoisomer, for example an enantiomer, substantially free of its stereoisomer can be obtained by separating the racemic mixture using a method such as diastereomers formation, using optically active decomposition agents (Eliel EL et al. Stereochemistry of Carbon Compounds. 1962, 1975 , J. Chromatogr., P. 283302). Racemic mixtures of chiral compounds of the invention can be separated and isolated by any suitable method, including: (1) formation of ionic, diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with derivative reagents. chirals, separation of diastereomers and conversion to pure stereoisomers, and (3) separation of substantially pure or enriched stereoisomers directly into chiral condices.

For illustrative purposes, specific examples of enantiomers of the compound of Formula (I) within the scope of the present invention include, but are not limited to:

MD 4574 Cl 2019.01.31

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

OH OH

<img file="MD4574C1_D0005.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5S) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide OH OH чхА ^ / Ιχ ^ ζΟΙ

OH OH

<img file="MD4574C1_D0006.tif" />

<img file="MD4574C1_D0007.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0008.tif" />

<img file="MD4574C1_D0009.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

OH OH

<img file="MD4574C1_D0010.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5S) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

<img file="MD4574C1_D0011.tif" />

О

<img file="MD4574C1_D0012.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4R, 5S) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0013.tif" />

<img file="MD4574C1_D0014.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4S, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0015.tif" />

О

<img file="MD4574C1_D0016.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3R, 4S, 5S) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0017.tif" />

ОН ОН

<img file="MD4574C1_D0018.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3S, 4R, 5R) -2,3,4,5,6 pentahydroxyhexyl) amino)) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

OH OH

<img file="MD4574C1_D0019.tif" />

<img file="MD4574C1_D0020.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0021.tif" />

<img file="MD4574C1_D0022.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3S, 4S, 5S) -2,3,4,5,6-pentalihydroxyxyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0023.tif" />

<img file="MD4574C1_D0024.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5S) -2,3,4,5,6-pentahydroxyhexyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

<img file="MD4574C1_D0025.tif" />

OH OH

<img file="MD4574C1_D0026.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentalihydroxylyl) amino) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

<img file="MD4574C1_D0027.tif" />

<img file="MD4574C1_D0028.tif" />

In one embodiment, the present invention refers! to an enantiomerically enriched mixture or composition containing 5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2.3), 4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidooyl) -pyrazin2-carboxamide, or acceptable salt! its pharmaceutical, as the predominant isomer.

Other embodiments include enantiomeric enriched mixtures or compositions containing, respectively, compounds of Formulas (Ia), (Ib), (Ic), (Id), (I), (If), (Ig), (Ih ), (Ii), (Ij), (Ik) and (II), or о is acceptable! pharmaceuticals, as the predominant isomer in each of their respective mixtures.

In another embodiment, the present invention refers! to an enantiomeric mixture or enrichment or о composition by confining 5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2.3 , 4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidooyl) -pyrazine-2-carboxamide, or acceptable salt! its pharmaceutical, substantially free! of alpha isomers.

Four other embodiments include enantiomeric enriched mixtures or compositions containing, respectively, compounds of Formulas (Ia), (Ib), (Ic), (Id), (I), (If), (Ig), ( Ih), (Ii), (Ij), (Ik) and (II), or о is acceptable! pharmaceutically thereof, substantially free of alpha isomers in each of their mixtures.

A compound of Formula I and their pharmaceutically acceptable salts may exist as different polymorphs or pseudopolymorphs. As used herein, crystalline polymorphism means! the ability of a crystalline compound to exist in different crystalline structures. Crystalline polymorphism may result from differences in crystal packing (packing polymorphism) or differences in packing between different conformations of the same molecule (conformational polymorphism). As used herein, crystalline pseudopolymorphism also includes the ability of a hydrate or solvate of a compound to exist in different crystalline structures. Pseudopolymorphs of the present invention may exist as a result of differences in crystal packing (packing pseudopolymorphism) or as a result of packing differences between different conformations of the same molecule (conformational pseudopolymorphism). The present invention comprises tofi polymorphs and pseudopolymorphs of compounds of Formula I and pharmaceutically acceptable salts thereof.

A compound of formula I and their pharmaceutically acceptable salts may also exist as an amorphous solid. As used here, an amorphous solid is a solid that does not exist! extended ordering! of the positions of atoms in the solid. This! defmifie is also applied when the crystal size is two nanometers or less. Additives, including solvents, can be used to create the amorphous forms of the present invention. The present invention, which includes all pharmaceutical compositions, methods of treatment, combined products and their uses described herein, encompasses all amorphous forms of the compounds of Formula I and their pharmaceutically acceptable salts.

Use! Ri

The compounds of the invention manifest! activity as sodium channel blockers. F! R! to be connected to a certain one! theory, it is believed that! The compounds of the invention can function in vivo by blocking the epithelial sodium channels present in the mucosal surfaces and thereby reducing the water absorption from the mucosal surfaces. This effect increases the volume of protective fluids on the mucosal surfaces and re-balances! the system.

As a result, the compounds of the invention are useful as drugs, especially for the treatment of clinical conditions for which a sodium channel blocker may be indicated. These conditions include lung disorders, such as diseases associated with reversible obstruction! or irreversible! respiratory acids, obstructive bronchopneumopathy! chronic! (ВРОС), including exacerbations ВРОС, asthma, bronchiectasis (including bronchiectasis from conditions other than cystic fibrosis!), Acute bronchitis! , chronic bronchitis !, post-viral cough !, fibrosis

MD 4574 Cl 2019.01.31 cyst, emphysema, pneumonia, panbronchiolitis and bronchiolitis associated with transplantation, including bronchiolitis associated with lung and marrow transplantation, in a man who needs it. The compounds of the invention may also be useful for treating ventilator-associated tracheobronchitis and / or prevention of ventilator-associated pneumonia in ventilatory patients. The present invention comprises methods for treating each of these conditions in a mammal in need thereof, preferably in a human in need thereof, each method including administering to the mammal a pharmaceutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt. it. There are also provided (a) a method for reducing ВРОС exacerbations in a mammal in need thereof; (b) о method for reducing FC exacerbations in a mammal in need thereof; (с) о method of improving lung function (FEV1) in a mammal in need thereof; (d) о method of improving lung function (FEV1) in a mammal suffering from ВРОС; (e) о method of improving lung function (FEV1) in a mammal suffering from CF; (f) a method of reducing respiratory tract infections in a mammal in need thereof.

Also provided is a method of stimulating, enhancing or enhancing mucociliary clearance in a mammal, a method including administering to a mammal in need thereof a pharmaceutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt. his. The mucociliary clearance will be understood to include the natural mucociliary action involved in the transfer or clearance of the airway mucus, including the bronchial self-cleaning mechanisms. Therefore, there is also a method for improving the clearance of mucus from the airway of a mammal.

In addition, sodium channel blockers may be indicated for the treatment of diseases that are ameliorated by increased hydration of mucous membranes on mucosal surfaces other than pulmonary mucosal surfaces. Examples of such conditions include dry mouth (xerostomia), dry skin, vaginal dryness, sinusitis, rhinosinusitis, nasal dehydration, including nasal dehydration caused by the administration of dry oxygen, dry keratitis, Sjogren's disease, middle otitis, dyskinesia, primary dyskinesia distal bowel, esophagitis, constipafia and chronic diverticulitis. The compounds of the invention may also be used to stimulate ocular or corneal hydration.

The compounds of the present invention can also be used in methods of obtaining a sputum sample from a human. The method can be accomplished by administering a compound of the invention to the patient's lung, and then inducing and collecting a sputum sample from that man.

Therefore, in one aspect, the present invention relates to a method for treating a disease in a mammal, such as a human, for which a sodium channel blocker is indicated.

In other embodiments, the present invention relates to each of the methods described herein with the added advantage of minimizing or eliminating hypercalcemia in the method recipient. Also provided are embodiments of each of the methods described herein, wherein an improved therapeutic index is achieved.

The terms "treated", "treated" and "treated", as used herein, refer to reversal, mitigation, inhibition of evolution or prevention of the disorder or condition or of one or more symptoms of such disorder or condition.

All of the therapeutic methods described herein are carried out by administering an effective amount of a compound of the invention, a compound of Formula I or a pharmaceutically acceptable salt thereof, to a subject (usually a mammal and preferably a human) in need of treatment.

In one embodiment, the invention relates to a method for treating a condition that is ameliorated by increased hydration of the mucous membranes in a mammal, particularly a human, in need thereof. In one embodiment, the invention relates to a method for treating a disease associated with reversible or irreversible airway obstruction in a mammal, particularly a human, in need thereof. In one particular embodiment, the present invention relates to a method for treating chronic obstructive bronchopneumopathy (ВРОС) in a mammal, in particular a human, in need thereof. In a particular embodiment, the present invention relates to a method of reducing the frequency, severity, or duration of acute exacerbation of ВРОС or for the treatment of one or more symptoms of acute exacerbation of ВРОС to a

MD 4574 Cl 2019.01.31 mammal, in particular a human, who needs this. In one embodiment, the invention relates to a method for treating asthma in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating bronchiectasis (including bronchiectasis from conditions other than cystic fibrosis) in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for the treatment of bronchitis, including acute and chronic bronchitis in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating post-viral cough in a mammal, particularly a human, in need thereof. In one embodiment, the invention relates to a method for treating cystic fibrosis in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating emphysema in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating pneumonia in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for the treatment of panbronchiolitis in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating transplant-associated bronchiolitis, including bronchiolitis associated with lung and medullary transplants in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating ventilator-associated tracheobronchitis and / or prevention of ventilator-associated pneumonia in a ventilated man in need thereof.

The present invention relates to specific methods for treating a disease selected from the group of reversible or irreversible airway obstruction, chronic obstructive bronchopneumopathy (ВРОС), asthma, bronchiectasis (including bronchiectasis from diseases other than cystic fibrosis), bronchitis bronchitis post-viral cough, cystic fibrosis, emphysema, pneumonia, panbronchiolitis, - transplant-associated bronchiolitis and ventilator-associated tracheobronchitis or the prevention of ventilator-associated pneumonia in a person in need thereof, each method consisting in administering to the affected man an effective amount of a compound of Formula 1 (a) or о pharmaceutically acceptable salt of it. In other embodiments for each treatment method, the pharmaceutically acceptable salt form is hydrochloride or a float hydroxine of the compound of Formula (Ia). In another embodiment in each treatment method, the free base of the compound of Formula (Ia) is used.

In one embodiment, the invention relates to a method for treating dry mouth (xerostomia) in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating dry skin on a mammal, particularly a human, in need thereof. In one embodiment, the invention relates to a method for treating vaginal dryness in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating sinusitis, rhinosinusitis or nasal dehydration, including nasal dehydration caused by the administration of dry oxygen, to a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for treating dry keratitis syndrome or Sjogren's disease, or stimulating coconut or human hydration in a mammal, particularly a human, in need thereof. In one embodiment, the invention relates to a method for treating otitis media in a mammal, particularly a human, in need thereof. In one embodiment, the invention relates to a method for treating primary ciliary dyskinesia in a mammal, in particular a human, in need thereof. In one embodiment, the invention relates to a method for the treatment of distal intestinal obstruction syndrome, esophagitis, constipation, or chronic diverticulitis in a mammal, particularly a human, in need thereof.

Also provided is a compound of the invention for use in medical therapy, in particular for use in the treatment of affection in a mammal, such as a human, for which a sodium channel blocker is indicated. All therapeutic uses described herein are carried out by administering an effective amount of a compound of the invention to the subject in need of treatment. In one embodiment, a compound of the invention is provided for use in the treatment of a pulmonary disease, such as a disease associated with reversible or irreversible airway obstruction in a mammal, in particular a human, in need thereof. In one embodiment

MD 4574 Cl 2019.01.31 In particular, there is provided a compound of the invention for use in the treatment of chronic obstructive bronchopneumopathy (ВРОС) in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in reducing the frequency, severity or duration of acute exacerbation of ВРОС or for the treatment of one or more symptoms of acute exacerbation of ВРОС in a mammal, particularly a human, which requires This one. In one embodiment, a compound of the invention is provided for use in the treatment of asthma in a mammal, in particular a human, in need thereof. In one embodiment, a compound is provided for use in the treatment of bronchiectasis, including bronchiectasis from conditions other than cystic fibrosis, or bronchitis, including acute bronchitis and chronic bronchitis, in a mammal, particularly a human, in need thereof. In one embodiment, a compound is provided for use in the treatment of post-viral cough in a mammal, particularly a human, in need thereof. In one embodiment, a compound is provided for use in the treatment of cystic fibrosis in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of emphysema in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of pneumonia in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of transplant-associated panbronchiolitis or bronchiolitis, including bronchiolitis associated with lung and marrow transplantation in a mammal, particularly a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of ventilator-associated tracheobronchitis or the prevention of ventilator-associated pneumonia in a ventilated human in need thereof.

In one embodiment, a compound of the invention is provided for use in the treatment of an improved condition by increased hydration of the mucosa to the mucosal surfaces of a mammal, in particular a human, in need thereof. In one embodiment, a compound is provided for use in the treatment of dry mouth (xerostomia) in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of skin drying in a mammal, particularly a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of vaginal dryness in a mammal, particularly a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of sinusitis, rhinosinusitis or nasal dehydration, including nasal dehydration caused by administration of dry oxygen to a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of dry keratitis or Sjogren's disease, or stimulation of coconut or corneal hydration in a mammal, in particular a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of otitis media in a mammal, particularly a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of primary ciliary dyskinesia in a mammal, particularly a human, in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of distal intestinal obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a mammal, particularly a human, in need thereof.

The present invention also relates to the use of a compound of the invention in the manufacture of a medicament for the treatment of a disease in a mammal, such as a human, for which a sodium channel blocker is indicated. In one embodiment, it is envisaged to use a compound of the invention in the manufacture of a medicament for the treatment of diseases associated with reversible or irreversible airway obstruction, chronic obstructive bronchopneumopathy (ВРОС), exacerbations ВРОС, asthma, bronchiectasis (including bronchiectasis other than affec cystic fibrosis), bronchitis (including acute bronchitis and chronic bronchitis), post-viral cough, cystic fibrosis, emphysema, pan-bronchiolitis pneumonia, transplant-associated bronchiolitis (including bronchiolitis associated with lung and marrow transplantation), ventilator-associated raheobronchitis, or prevention of ventilator-associated pneumonia.

In a particular embodiment, it is envisaged to use a compound of the invention in the manufacture of a medicament for the treatment of an improved condition by

MD 4574 Cl 2019.01.31 increased hydration of the mucous membranes on the mucosal surfaces, treatment of dry mouth (xerostomia), skin dryness, vaginal dryness, sinusitis, rhinosinusitis, nasal dehydration, including nasal dehydration caused by drying, oxygen treatment, stimulation of ocular or comean hydration, treatment of otitis media, primary ciliary dyskinesia, distal intestinal obstruction syndrome, esophagitis, chronic constipation or diverticulitis

The terms "effective amount", "pharmaceutically effective amount", "effective dose" and "pharmaceutically effective dose", as used herein, refer to a quantity of a compound of the invention which is sufficient in the subject to be administered to disclose the biological or medical response of a cell, tissue, system, or mammalian (including human) culture, which is sought, for example, by a researcher or clinician. The term also includes, in its domain, effective amounts to improve normal physiological function. In one embodiment, the effective amount is the amount needed to provide a desired level of the drug in the secretions and tissues of the airways and lungs, or alternatively, in the blood flow of a subject to be treated to give a physiological response. anticipated or a desired biological effect when such composition is administered by inhalation. For example, an effective amount of a compound of the invention for the treatment of a condition for which a sodium channel blocker is indicated is sufficient in the subject to which it is administered to treat the particular condition. In one embodiment, the effective amount is the amount of a compound of the invention which is sufficient for the treatment of CVD or cystic fibrosis in a human.

The precise effective amount of the compounds of the invention will depend on a number of factors including but not limited to the species, age and weight of the treated subject, the precise affectation that requires treatment and its severity, bioavailability, potency and other properties of the specific compound administered, nature formulation, the route of administration, as well as the administration device, and will ultimately be at the discretion of the physician or veterinarian. Additional dosage guidance can be found when examining the conventional dosage of other sodium channel blockers, such as amiloride, with due regard to any efficacy differences between amiloride and compounds of the present invention.

The pharmaceutically effective dose administered topically to the surfaces of the airway of a subject (eg, by inhalation) of a compound of the invention for the treatment of a 70 kg human may range from about 10 µg to about 10 mg. In another embodiment, the pharmaceutically effective dose may be from about 0.1 to about 1000 µg. Usually, the daily dose administered topically to the airway surfaces will be in sufficient quantity to reach the dissolved concentration of the active agent on the airway surfaces at approximately IO '.⁹, 10'⁸ or IO '⁷ up to about 1 '⁴, I '³, I '² or 10¹ moles / liter, more preferably from about IO '⁹up to about 10 '⁴ moles / liter. The selection of the specific dose for a patient will be determined by the caring physician, clinician or veterinarian in the art in the art based on a number of factors, including those mentioned above. In a particular embodiment the dose of a compound of the invention for the treatment of a 70 kg human will be in the range of about 10 nanograms (pg) to about 10 mg. In another embodiment, the effective dose will be from about 0.1 µg to about 1000 µg. In one embodiment, the dose of a compound of the invention for the treatment of a 70 kg human will be in the range of about 0.5 µg to about 0.5 mg. In another embodiment, the dose will be from about 0.5 µg to about 60 µg. In another embodiment, the pharmaceutically effective dose will be from about 1 to about 10 µg. In another embodiment, the pharmaceutically effective dose will be from about 5 µg to about 50 µg. In another embodiment the effective dose will be from about 10 µg to about 40 µg. In two other examples In this embodiment, the pharmaceutically effective dose will be from about 15 pg to about 50 pg, from about 15 pg to about 30 pg, respectively. It will be understood that in each of these dose ranges, all increasing doses in the range are included. For example, the range of 0.5-50 pg includes individual doses of: 0.5 pg, 0.6 pg, 0.7 pg, 0.8 pg, 0.9 pg, 1.0 pg, 1.1 pg , 1.2 pg, 1.3 pg, 1.4 pg, 1.5 pg, 1.6 pg, 1.7 pg, 1.8 pg, 1.9 pg,

2.0 pg, 2.1 pg, 2.2 pg, 2.3 pg, 2.4 pg, 2.5 pg, 2.6 pg, 2.7 pg, 2.8 pg, 2.9 pg, 3.0 pg, 3.1 pg, 3.2 pg, 3.3 pg, 3.4 pg, 3.5 pg, 3.6 pg, 3.7 pg, 3.8 pg, 3.9 pg, 4.0 pg, 4.1 pg, 4.2 pg,

MD 4574 Cl 2019.0131

4.3 days, 4.4 days, 4.5 days, 4.6 days, 4.7 days, 4.8 days, 4.9 days, 5.0 days, 5.1 days, 5.2 days, 5.3 gg, 5.4 gg, 5.5 gg, 5.6 gg, 5.7 gg, 5.8 gg, 5.9 gg, 6.0 gg, 6.1 gg, 6.2 gg, 6.3 gg, 6.4 gg, 6.5 gg

6.6gg, 6.7gg, 6.8gg, 6.9gg, 7.0gg, 7.1gg, 7.2gg, 7.3gg, 7.4gg, 7.5gg, 7.6 gg, 7.7 gg, 7.8 gg, 7.9 gg, 8.0 gg, 8.1 gg, 8.2 gg, 8.3 gg, 8.4 gg, 8.5 gg, 8.6 days, 8.7 days, 8.8 days,

8.9gg, 9.0gg, 9.1gg, 9.2gg, 9.3gg, 9.4gg, 9.5gg, 9.6gg, 9.7gg, 9.8gg, 9.9 gg, 10.0 gg, 10.1 gg, 10.2 gg, 10.3 gg, 10.4 gg, 10.5 gg, 10.6 gg, 10.7 gg, 10.8 gg, 10.9 gg, 11.0 gg, 11.1 gg, 11.2 gg, 11.3 gg, 11.4 gg, 11.5 gg, 11.6 gg, 11.7 gg, 11.8 gg, 11, 9g, 12.0g, 12.1gg, 12.2gg, 12.3gg, 12.4gg, 12.5gg, 12.6gg, 12.7gg, 12.8gg, 12, 9gg, 13.0gg, 13.1gg, 13.2gg, 13.3gg, 13.4gg, 13.5gg, 13.6gg, 13.7gg, 13.8gg, 13, 9 days, 14.0 days, 14.1 days, 14.2 days, 14.3 days, 14.4 days, 14.5 days, 14.6 days, 14.7 days, 14.8 days, 14.9 days, 15.0 days, 15.1 days, 15.2 days, 15.3 days, 15.4 days, 15.5 days, 15.6 gg, 15.7 gg, 15.8 gg, 15.9 gg, 16.0 gg, 16.1 gg, 16.2 gg, 16.3 gg, 16.4 gg, 16.5 gg, 16.6 days, 16.7 days, 16.8 days, 16.9 days, 17.0 days, 17.1 days, 17.2 days, 17.3 days, 17.4 days, 17.5 days, 17.6 gg, 17.7 gg, 17.8 gg, 17.9 gg, 18.0 gg, 18.1 gg, 18.2 gg, 18.3 gg, 18.4 gg, 18.5 gg, 18.6 days, 18.7 days, 18.8 days, 18.9 days, 19.0 days, 19.1 days, 19.2 days, 19.3 days, 19.4 days, 19.5 days, 19.6 days, 19.7 days, 19.8 days, 19.9 days, 20.0 days, 20.1 days, 20.2gg, 20.3gg, 20.4gg, 20.5gg, 20.6gg, 20.7gg, 20.8gg, 20.9gg, 21.0gg, 21.1gg, 21.2gg, 21.3gg, 21.4gg, 21.5gg, 21.6gg, 21.7gg, 21.8gg, 21.9gg, 22.0gg, 22.1gg, 22.2gg, 22.3gg, 22.4gg, 22.5gg, 22.6gg, 22.7gg, 22.8gg, 22.9gg, 23.0gg, 23.1gg, 23.2gg, 23.3gg, 23.4gg, 23.5gg, 23.6gg, 23.7gg, 23.8gg, 23.9gg, 24.0gg, 24.1gg, 24, 2gg, 24.3gg, 24.4gg, 24.5gg, 24.6gg, 24.7gg, 24.8gg, 24.9gg, 25.0gg, 25.1gg, 25, 2 days, 25.3 days, 25.4 days, 25.5 days, 25.6 days, 25.7 days, 25.8gg, 25.9gg, 26.0gg, 26.1gg, 26.2gg, 26.3gg, 26.4gg, 26.5gg, 26.6gg, 26.7gg, 26.8gg, 26.9gg, 27.0gg, 27.1gg, 27.2gg, 27.3gg, 27.4gg, 27.5gg, 27.6gg, 27.7gg, 27.8gg, 27.9gg, 28.0gg, 28.1gg, 28.2gg, 28.3gg, 28.4gg, 28.5gg, 28.6gg, 28.7gg, 28.8gg, 28.9gg, 29.0gg, 29.1gg, 29.2gg, 29.3gg, 29.4gg, 29.5gg, 29.6gg, 29.7gg, 29.8 gg, 29.9 gg, 30.0 gg, 30.1 gg, 30.2 gg, 30.3 gg, 30.4 gg, 30.5 gg, 30.6 gg, 30.7 gg, 30.8 days, 30.9 days, 31.0 days, 31.1 days, 31.2 days, 31.3 days, 31.4gg, 31.5gg, 31.6gg, 31.7gg, 31.8gg, 31.9gg, 32.0gg, 32.1gg, 32.2gg, 32.3gg, 32.4gg, 32.5gg, 32.6gg, 32.7gg, 32.8gg, 32.9gg, 33.0gg, 33.1gg, 33.2gg, 33.3gg, 33.4gg, 33.5gg, 33.6gg, 33.7gg, 33.8gg, 33.9gg, 34.0gg, 34.1gg, 34.2gg, 34.3gg, 34.4 days, 34.5 days, 34.6 days, 34.7 days, 34.8 days, 34.9 days, 35.0 days, 35.1 days, 35.2 days, 35.3 days, 35.4gg, 35.5gg, 35.6gg, 35.7gg, 35.8gg, 35.9gg, 36.0gg, 36.1gg, 36.2gg, 36.3gg, 36.4 days, 36.5 days, 36.6 days, 36.7 days, 36.8 days, 36.9 days, 37.0 days, 37.1 days, 37.2 days, 37.3 days, 37.4 days, 37.5 days, 37.6 days, 37.7 days, 37.8 days, 37.9 days, 38.0 days, 38.1 days, 38.2 days, 38.3 days, 38.4 days, 38.5 days, 38.6 days, 38.7 days, 38.8 days, 38.9 days, 39.0 days, 39.1 days, 39.2 days, 39.3 days, 39.4 days, 39.5 days, 39.6 days, 39.7 days, 39.8 days, 39.9 days, 40.0 days, 40.1 days, 40.2 days, 40.3 days, 40.4 days, 40.5 days, 40.6 days, 40.7 days, 40.8 days, 40.9 days, 41.0 days, 41.1 days, 41.2 days, 41.3 days, 41.4 days, 41.5 days, 41.6 days 41.7 days, 41.8 days, 41 , 9 days, 42.0 days, 42.1 days, 42.2 days, 42.3 days, 42.4 days, 42.5gg, 42.6gg, 42.7gg, 42.8gg, 42.9gg, 43.0gg, 43.1gg, 43.2gg, 43.3gg, 43.4gg, 43.5gg, 43.6gg, 43.7gg, 43.8gg, 43.9gg, 44.0gg, 44.1gg, 44.2gg, 44.3gg, 44.4gg, 44.5gg, 44.6gg, 44.7gg, 44.8gg, 44.9gg, 45.0gg, 45.1gg, 45.2gg, 45.3gg, 45.4gg, 45.5gg, 45.6gg, 45.7gg, 45.8gg, 45.9gg, 46.0gg, 46.1gg, 46.2gg, 46.3gg, 46.4gg, 46.5gg, 46.6gg, 46.7gg, 46.8gg, 46.9gg, 47.0gg, 47.1gg, 47.2gg, 47.3gg, 47.4gg, 47.5 gg, 47.6 gg, 47.7 gg, 47.8 gg, 47.9 gg, 48.0 gg, 48.1 days, 48.2 days, 48.3 days, 48.4 days, 48.5 days, 48.6 days, 48.7 days, 48.8 days, 38.9 days, 49.0 days, 49, 1 day, 49.2 days, 49.3 days, 49.4 days, 49.5 days, 49.6 days, 49.7 days, 49.8 days, 39.9 days and 50 bg ·

The doses suggested above can be adjusted using conventional dose calculations, if the compound is administered in a different way. Determining an appropriate dose for administration by other means is within the competence of those skilled in the art, in light of the above description and general knowledge in the field.

Administration of an effective amount of a compound of the invention may result in the administration of a single drug form or multiple single doses that may be administered concurrently or separately over time for a designated period, such as 24 hours. The dose of a compound of the invention (alone or in the form of a confining composition) may be administered one to ten times daily. Typically, a compound of the invention (alone or in the form of a confining composition) will be administered four, three, twice, or once daily (24 hours).

The compounds of Formula (I) of the present invention are also useful for treating airborne infections. Examples of airborne infections include, for example,

MD 4574 Cl 2019.01.31

RSV. The compounds of Formula (I) of the present invention are also useful for treating an anthrax infection. The present invention relates to the use of compounds of Formula (I) of the present invention for the prophylactic, prophylactic post-exposure, preventive or therapeutic treatment of diseases or disorders caused by pathogens. In a preferred embodiment, the present invention relates to the use of compounds of Formula (I) for prophylactic, prophylactic post-exposure, preventive or therapeutic treatment of diseases or conditions caused by pathogens that may be used in bioterrorism.

In recent years, a variety of research programs and measures of biological protectionism have been elaborated that fine-tuned the problems related to the use of biological agents in acts of terrorism. These measures are intended to solve problems related to bioterrorism or the use of microorganisms or biological toxins to kill people, spread fear and disrupt society. For example, the National Institute of Allergic and Infectious Diseases (NIAID) has developed a Strategic Plan for Biological Protection Research that presents plans to meet the research needs in the field of bioterrorism and emerging and re-emerging infectious diseases. According to the plan, the deliberate exposure of the US civilian population to Bacillus anthracis spores revealed a gap in the nation's preparedness against bioterrorism. Moreover, the report details that these attacks have uncovered an unmet need for tests to quickly diagnose vaccines and immunotherapies to prevent, and biologic drugs to cure the disease caused by bioterrorism agents.

The major focus of the various research efforts was placed on studying the biology of pathogens identified as potentially dangerous as bioterrorism agents, studying the response of the host to these agents, developing vaccines against infectious diseases, evaluating currently available and currently undergoing therapies. against these agents, as well as developing the diagnosis to identify the signs and symptoms of dangerous agents. Such efforts are commendable, but, given the large number of pathogens that have been identified as potentially available for bioterrorism, these efforts have not yet been able to provide satisfactory responses to all possible bioterrorism threats. In addition, many of the pathogens identified as potentially dangerous as bioterrorism agents do not provide adequate economic incentives for the development of therapeutic or preventive measures by the Industry. Moreover, even if preventive measures such as vaccines were available for each pathogen that can be used in bioterrorism, the costs of administering all these vaccines to the population generated are prohibitive.

As long as convenient and effective treatments are available against any threat of bioterrorism, there is a great need for preventive, prophylactic or therapeutic treatments that can prevent or reduce the risk of infection with pathogens.

The present invention relates to such methods of prophylactic treatment. In one aspect, a prophylactic treatment method is provided which includes administering an effective prophylactic amount of compounds of Formula (I) to an individual requiring prophylactic treatment against infection with one or more airborne pathogens. A particular example of an airborne pathogen is anthrax.

In another embodiment, the prophylactic treatment method is provided to reduce the risk of infection with an airborne pathogen, which may cause disease in humans, the method mentioned including administering an effective amount of compounds of Formula (I) to the lungs. to the man who shows a laughter of infection with the pathogen transmitted through the air, but is asymptomatic for the disease, wherein the effective amount of a sodium channel blocker and osmolite is sufficient to reduce the risk of human infection. A particular example of an airborne pathogen is anthrax.

In another embodiment, post-exposure prophylactic treatment or therapeutic treatment is provided for the treatment of infection with an airborne pathogen including the administration of an effective amount of compounds of Formula (I) to the lungs of an individual in need of such treatment against infection with an airborne pathogen. The pathogens that need to be protected by the therapeutic, prophylactic post-exposure and urgent treatment methods of the invention include any pathogens that can enter the body through the mouth, nose or nasal airways, thus passing into the lungs. Typically, pathogens will be airborne pathogens either way

MD 4574 Cl 2019.01.31 natural or by aerosolization. Pathogens may occur naturally or may be introduced into the intended environment after aerosolization or another method of introducing pathogens into the environment. Many pathogens that are not naturally transmitted through the air have been or can be aerosolized for use in bioterrorism. The pathogens for which the treatment according to the invention may be useful include, but are not limited to, the priority pathogens of category A, В and C established by NIAID. These categories generally correspond to the lists compiled by the Center for Disease Control and Prevention (CDC). As established by the CDC, Category A agents are those that can be easily spread or transmitted from person to person, causing high mortality, with the potential to have a major impact on public health. Agents of category В are the following as a priority and include those that are moderately widespread and cause moderate morbidity and reduced mortality. Category C consists of emerging pathogens that could be designed for mass dissemination in the future, because of their availability, productivity and ease of dissemination and the potential for high morbidity and mortality. Particular examples of these pathogens are anthrax and plague. Additional pathogens to be protected or reduced risk of infection with these include influenza viruses, rhinoviruses, adenoviruses and respiratory syncytial viruses and the like. An additional pathogen that must be protected is coronavirus that is thought to cause severe acute respiratory syndrome (SARS).

The present invention relates to the use of sodium channel blockers of Formula I, or their pharmaceutically acceptable salt, for the prevention, mitigation and / or treatment of deterministic health effects on the respiratory tract caused by exposure to radiological materials, in particular breathable aerosols containing radionuclides. from nuclear attacks, such as detonation of radiological dispersal devices (RDDs), or accidents, such as nuclear station disasters. As such, there is provided a method for preventing, mitigating and / or treating deterministic health effects on the respiratory tract and / or other bodily organs caused by respirable aerosols containing radionuclides in a container in need thereof, including in a person in need thereof. , said method, including administering to said human an effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.

The major concern associated with management planning for exposure of members of the public to breathable aerosols containing radionuclides from nuclear attacks, such as detonation of radiological dispersal devices (RDDs), or accidents, such as nuclear station disasters, is how to prevent, reduce or treatment of the potential deterministic effects on the health of the respiratory tract, first of all, the lung. It is necessary to have medicines, techniques and procedures, as well as trained personnel trained to manage and treat such highly contaminated persons internally.

Research has been conducted to determine how to prevent, alleviate or treat the potential damage to the respiratory tract and the various organs in the body, which is caused by radionuclides deposited in the body. So far, most of the research attention has been focused on strategies aimed at mitigating the health effects of radionuclides deposited in the body, by accelerating their excretion or elimination. These strategies focused on soluble chemical forms, which are capable of reaching the bloodstream and are stored in remote systemic locations specific to a particular radio element. These approaches will not work in cases where the deposited radionuclide is in a relatively insoluble form. Studies have shown that many, if not most, of the physico-chemical forms of radionuclides dispersed from RDDs will be in a relatively insoluble form.

The only known method of effectively reducing the lung radiation dose from inhaled insoluble radioactive aerosols is bronchoalveolar lavage or LBA. This method, which has been adapted from that already used for the treatment of patients with alveolar proteinosis, has proven to be a safe, repeatable procedure, even when extended for an extended period of time. Although there are variations in the process, the basic method for LBA is to anesthetize the subject, followed by the slow introduction of the isotonic solution into a single lobe of the lung until functional residual capacity is reached. The extra volumes are then added and drained by gravity.

MD 4574 Cl 2019.01.31

The results of studies using animal LBA indicate that approximately 40% of the deep lung content can be removed by a reasonable LBA sequence. In some studies, there was considerable variability among animals in the restored amount of radionuclides. The reasons for the variability are not currently understood.

Further, based on an animal study, it is considered that a significant dose reduction from LBA therapy results in attenuation of health effects due to inhalation of insoluble radionuclides. In the study, adult dogs inhaled insoluble particles¹⁴⁴What FAP. Two groups of dogs were given pulmonary content¹⁴⁴What is known to produce radiation pneumonitis and pulmonary fibrosis (approximately 2 MBq / kg body weight), with one group treated with 10 unilateral washes between 2 and 56 days after exposure, the other untreated. A third group was exposed to a level of¹⁴⁴Which is comparable to that seen in the BAL-treated group after treatment (about 1 MBq / kg), but these animals were not treated. All animals were allowed to live their life span, which extended to 16 years. Because there is variability in the initial lung content of¹⁴⁴What among the dogs in each group, dose rates and cumulative doses for each group overlap. However, the effect of LBA in reducing the risk of pneumonitis / fibrosis was evident from the survival curves. In untreated dogs with a lung content of 1.5-2.5 MBq / kg, the average survival time was 370 ± 65 d. For treated dogs, the average survival was 1270 ± 240 d, which was statistically significant different. Third group, which received pulmonary confinement ¹⁴⁴What of 0.6-1.4 MBq had an average survival time of 1800 ± 230, which was not statistically different from the treated group. Equally important for increased survival, dogs in the high-dose untreated group died from deterministic effects on the lungs (pneumonitis / fibrosis), while the treated dogs did not die. In contrast, treated dogs, unlike dogs in the low-dose group, had mostly lung tumors (hemangiosarcoma or carcinoma). Therefore, the dose reduction resulting from the treatment of BAL seems to have produced biological effects in the lungs that were predictable based on the radiation doses received by the lungs.

Based on these results, it is considered that decreasing the residual radiological dose further by any method or combination of methods for increasing clearance of the lung particles would further reduce the likelihood of effects on lung health. However, LBA is a procedure that has many disadvantages. LBA is an extremely invasive procedure, which must be performed in specialized medical centers by trained pulmonologists. As such, the LBA procedure is costly. Given the disadvantages of LBA, this is not a treatment option that would be readily and immediately available to people who require the accelerated removal of radioactive particles, for example, in the event of a nuclear attack. In the event of a nuclear attack or a nuclear accident, immediate and relatively easy treatment for people who have been exposed or are at risk of being exposed is required. Blockers of sodium channels administered as an inhalation aerosol have been shown to restore the hydration of the airway surfaces. О Such hydration of the airway surfaces helps to eliminate accumulated mucus secretions and associated particles from the lungs. As such, without being bound by any theory, it is believed that sodium channel blockers can be used to accelerate the removal of radioactive particles from the airways.

As discussed above, the highest rise for lungs following a radiological attack, such as a radioactive bomb, results from inhaling and refining insoluble radioactive particles. As a result of the retention of the radioactive particles, the cumulative exposure at the pulmonary level is significantly increased, ultimately leading to pulmonary fibrosis / pneumonitis and eventually death. The insoluble particles cannot be systemically eliminated by the chelating agents, because these particles are not in solution. So far, physical removal of particles by LBA is the only therapeutic scheme proven to be effective in alleviating radiation-induced lung disease. As discussed above, LBA is not a realistic treatment solution for reducing the effects of radioactive particles that have been inhaled into the body. As such, it is desirable to provide a treatment scheme that effectively helps eliminate radioactive particles from the airway passages and which, unlike LBA, is relatively easy to administer and scalable in a radiation exposure scenario. wide scale. In addition,

MD 4574 Cl 2019.01.31 it is also desirable that the treatment scheme be readily available! to a number of people in a period! of relatively short time!

In one aspect of the present invention, the method! prevention, mitigation and / or treatment of deterministic effects on the respiratory tract and / or other bodily organs caused by respirable aerosols containing radionuclides includes the administration of an effective amount of sodium channel blocker with Formula I or о is acceptable! its pharmaceutical to an individual in need! This one. In a feature! of this aspect, the sodium channel blocker is administered together! with an osmolite. Regarding this! characteristic !, the osmolite is a hypertonic solution! (SH). In another! characteristic !, the sodium channel blocker and the osmolite are administered in combination with a modulator of ion transport. About this! Characteristically, the ion transport modulator can be selected from the stand group of β-agonists, CFTR potentiators, purinergic receptor agonists, lubiprostones and protease inhibitors. In another! characteristic! of this aspect, radionuclides are selected from the group consisting of Colbalt-60, Cesium-137, Iridium-192, Radium-226, Phosphorus-32, Stronfiu-89 and 90, Iod-125, Thallium-201, Lead-210, Thorium -234, Uranium-238, Plutonium, Cobalt-58, Chromium-51, Americium and Curium. In another! characteristic !, the radionuclides are from a radioactive elimination device! In another! characteristic !, the sodium channel blocker or the acceptable salt! Its pharmaceutical formulation is administered in an aerosol suspension of respirable particles inhaled by the individual. In a feature! extra !, the sodium channel blocker or о is acceptable! its pharmaceutical is administered after! it is exposed to radionuclides.

Compozifii

Although it is possible that a compound of the invention s! be administered alone, in some embodiments it is preferable to! be present in the form of a composition, in particular a pharmaceutical composition (formulation). Thus, in another aspect, the invention refers! in the compositions and especially the pharmaceutical compositions (such as о pharmaceutical composition! inhalable!) which confines о effective amount! Pharmaceutical composition of an invention as an active ingredient and a pharmaceutically acceptable excipient, diluent or carrier. The term "active ingredient", as used here, refers! to any compound of the invention or a combination of two or more sulfur compounds of the invention in a pharmaceutical composition! Also provided are specific embodiments m which о pharmaceutical composition! confine о quantity efficiently! pharmaceutically of a compound of Formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or о is acceptable! pharmaceutical agent thereof, independently or in combination, and a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical composition! confine о quantity efficiently! pharmaceutically of a compound of Formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ш), (Ii), (Ij), (Ik) and (II) or о is acceptable! pharmaceutically thereof, independently or in combination, in a diluent. In different embodiments, the pharmaceutical composition! confine о quantity efficiently! pharmaceutically of a compound of Formulas (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or о is acceptable! its pharmaceutical, in hypertonic solution! sterile! and saline solution! hypertonic !, respectively, in which the concentration of saline solution! can be as described here. In one embodiment, the concentration of the saline solution is 0.17% m / v and in another example it is 2.8% m / v.

There is also a set containing: i) о effective quantity! pharmaceutically of a compound of Formula (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or о is acceptable! its pharmaceutical; ii) one or more pharmaceutically acceptable excipients, carriers or diluents; iii) instructions for administering the compound in group i) and excipients, carriers or diluents in group ii) to a subject that needs! This one; and iv) a container. A topic I need! this includes any topic I need! the treatment methods described here, including in particular a human subject that I need! This one. Other embodiments also include an aerosolizing device selected from the group of a nebulizer, including vibrating nebulizers with mesh! and jet nebulizers, a dry powder inhaler !, including dry powder inhalers! active! and passive !, and a dose inhaler! m! sourat !, including inhaled inhalers, with dose! ml measured! dry powder! and soft spray.

In one embodiment, a set bound i) from about 10 pg up! to about 10 mg of a compound of Formula (I), (la), (lb), (Ic), (Id), (le), (If), (Ig), (Ш),

MD 4574 Cl 2019.01.31 (li), (Ij), (Ik) and (II) or о its pharmaceutically acceptable salt, per dose; ii) from about 1 to about 5 mL of diluent per dose; iii) instructions for administering the compound of group i) and diluent of group ii) to a subject in need thereof; and iv) a container. In another embodiment, the diluent is from about 1 to about 5 mL of saline, as described herein, per dose. In another embodiment, the diluent is from about 1 to about 5 mL of hypotonic saline solution per dose. In another embodiment, the diluent is from about 1 to about 5 mL of hypertonic saline solution per dose. In another embodiment, the diluent is from about 1 to about 5 mL of sterile water per dose.

Also provided is a set containing: i) a solution which confines о a pharmaceutically effective amount of a compound of Formula (I), (la), (lb), (Ic), (Id), (le), ( If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II), or the pharmaceutically acceptable salt thereof, dissolved in a pharmaceutically acceptable diluent; iii) instructions for administering the solution in group i) to a subject who needs it; and iii) a container.

Also provided is a set containing: i) о solution containing from 10 µg to 10 mg of a compound of Formula (I), (to), (lb), (Ic), (Id), ( le), (If), (Ig), (Ih), (Ii), (lj), (Ik) and (II), or a pharmaceutically acceptable salt thereof, dissolved in a pharmaceutically acceptable diluent; iii) instructions for administering the solution in group i) to a subject who needs it; and iii) a container. In another embodiment, the diluent is from about 1 to about 5 mL of saline, as described herein, per dose.

Another embodiment includes a set containing: i) a pharmaceutically effective amount of a compound of Formula (I), (la), (lb), (Ic), (Id), (le), (If), ( Ig), (Ш), (Ii), (Ij), (lk) and (II) or о a pharmaceutically acceptable salt thereof, in a dry powder formulation suitable for inhalation; ii) optionally, one or more pharmaceutically acceptable excipients or carriers suitable for inhalation; iii) instructions for administering the compound in group i) and excipients or carriers in group ii) to a subject in need thereof; and iv) a container. In another embodiment, the set also confines a dry powder inhaler suitable for administering the dry powder formulation to a container. The dry powder inhaler may, in the additional embodiments, be a single dose inhaler or a multiple dose inhaler.

Other embodiments of each of the kits described herein include those in which the concentration of the compound of Formula (I), (la), (lb), (Ic), (Id), (le), (If), (Ig) , (Ih), (Ii), (Ij), (Ik) and (II) or its pharmaceutically acceptable salt, per dose, is one of the most effective dose ranges described here, including a) from about 0 , 1 pg to about 1000 pg; b) from about 0.5 µg to about 0.5 mg; and c) from about 0.5 pg to about 50 pg.

For each of the sets described above, there is an additional embodiment in which the diluent is hypertonic solution with the concentrations described herein. In another embodiment for each set the diluent is a hypotonic solution with the concentrations described herein. In another embodiment for each set, the diluent is sterile water suitable for inhalation.

The pharmaceutically acceptable excipient (s), diluent (s) or carrier (s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not be harmful to the recipient. Generally, the pharmaceutically acceptable excipient (s), diluent (s) or carrier (s) used in the pharmaceutical formulation are "non-toxic", which means that he / she is / are considered harmless / harmless for consumption in the quantity delivered in formulations and "inert" which means that he / she does not react appreciably with or results in an undesirable effect on the therapeutic activity of the active ingredient (s). Pharmaceutically acceptable excipients, diluents and carriers are conventional in the art and can be selected using conventional techniques, based on the desired mode of administration (Remington's, Pharmaceutical Sciences, Lippincott Williams &amp; Wilkins, Ed. 21, 2005). Preferably, pharmaceutically acceptable excipients, diluents and carriers are generally considered to be harmless (CRCFI) according to the FDA.

Pharmaceutical compositions according to the invention include those suitable for oral administration, parenteral administration, including subcutaneous administration,

MD 4574 Cl 2019.01.31 intradermal !, intramuscular !, intravenous! and intra-articular !, topic !, including local administration! skin, eyes, ears, etc .; vaginal administration! or rectal !, and administration to the respiratory tract, including the nasal cavities and sinuses, the oral and extrathoracic respiratory cells, and the palms, including by using aerosols that can be administered through different types of dry powder inhalers !, hermetically sealed inhalers! m! sado !, easily disintegrable aerosol inhalers, nebulizers or insufflators. The most appropriate! The route of administration may depend on several factors, including the patient and the condition or disorder treated !.

The forms can be presented in the form! medicamentoas! unified! or form! medicamentoas! unwrapped! as, for example, in the following formulations! can be dosed by an inhaler and can be prepared by any of the methods well known in the pharmaceutical field. Generally, the methods include the step of bringing the active ingredient together with the carrier, diluent or excipient and optionally one or more additional ingredients. In general, the formulations are prepared by bringing them evenly! and intimate! in combination of the active ingredient with one or more carriers, diluents or liquid excipients or carriers, finely divided solid carriers, diluents or excipients, or both, and then, if! it is necessary, modeling the product in the desired formulation!

In a preferred embodiment, the composition is a pharmaceutical composition! Inhalable! which is appropriate! for inhalation and administration to the endobronchial space. Usually this one! composition is in the form! spray containing particles for administration with a nebulizer, inhaler dosed hermetically! ml measured! (IDM), easily disintegrated aerosol inhaler or dry powder inhaler! (IPU). The aerosol formulation used! In the methods of the present invention there may be a liquid (for example, solution) suitable for administration by a nebulizer, easily disintegrable aerosol inhaler or IDM, or о powder! dry! suitable! for administration by an IDM or IPU.

Aerosols used to administer respiratory tract drugs are typically polydisperse, that is! they are made of particles of different sizes. Particle size distribution is usually described! by Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD). For optimal administration! of the drug at the endobronchial space MMAD is in the range of about 1 to! at about 10 pm and preferably from about 1 until! at about 5 pm and GSD is lower! of 3, preferably! smaller! about 2. Aerosols with an MMAD greater than 10 pm are generally too large when inhaled to reach the plume. Aerosols with о GSD greater than about 3 are not preferred for pulmonary administration! because I administer! a high percentage of the drug in the oral cavity! To obtain these particle sizes in the powder formulation! The active ingredient particles can be small in size using conventional techniques, such as micronization or spray drying. Non-limiting examples of other processes or techniques that can be used to produce breathable particles include spray drying, precipitation, supercritical fluid and lyophilization. The fraction wanted! it can be separated! by sorting with air or sifting. In one embodiment, the particles will be crystalline. For liquid formulations, the particle size is determined! by selecting a specific nebulizer, easily disintegrable aerosol inhaler or IDM model.

The size distribution of the aerosol particles is determined! with the help of devices well known in the art. For example, a cascading impactor! Anderson with several steps or another! method! adequate !, such as those specifically mentioned in the US Pharmacopoeia Chapter that characterizes! the devices for aerosols emitted from the measured dose! and dry powder inhalers!

Dry powder compositions! for topical administration! at inhalation can be formulated lightly by inhalation! excipient or carrier and including only active ingredients in dry powder instead! having a suitable particle size! for inhalation. Dry powder compositions can also contain a mixture of active ingredient and о base! adequate powder! (carrier! diluent / excipient) such as mono-, di- or polysaccharides (eg lactose or starch). Lactose is usually the preferred excipient for dry powder formulations. When a solid excipient such as lactose is used, generally the particle size of the excipient will be much larger than the active ingredient to contribute to the dispersion of the formulation in the inhaler.

MD 4574 Cl 2019.01.31

Non-limiting examples of dry powder inhalers include multi-dose tank inhalers, pre-measured multi-dose inhalers, capsule-based inhalers, and single-use single-dose inhalers. A tank inhaler confines a large number of doses (eg 60) in a single container. Before inhalation, the patient starts up the inhaler which causes the inhaler to measure a dose of drug from the reservoir and to prepare it for inhalation. Examples of reservoir IPUs include, but are not limited to Turbohaler® by AstraZeneca and ClickHaler® by Vehicle.

In a pre-measured multi-dose inhaler, each individual dose was manufactured in a separate container, and commissioning the inhaler before inhalation causes a new dose of drug to be released from its container and ready for inhalation. Examples of multi-dose IPU inhalers include, but are not limited to, Diskus® by GSK, Gyrohaler® by Vectura and Prohaler® by Valois. During inhalation, the patient's inspiratory flow accelerates the output of the powder from the device and entry into the oral cavity. For a capsule inhaler, the formulation is in a capsule and stored on the outside of the inhaler. The patient places the capsule in the inhaler, starts the inhaler (pierces the capsule), then inhales. Examples include Rotohaler ™ (GlaxoSmithKline), Spinhaler ™ (Novartis), HandiHaler ™ (IB), TurboSpin ™ (PH&amp;T). Regarding single dose single-use inhalers, the patient starts the inhaler to prepare it for inhalation, inhales, then tightens the inhaler and packaging. Examples include Twincer ™ (U Groningen), OneDose ™ (GFE) and Manta Inhaler ™ (Manta Devices).

Generally, dry powder inhalers use the characteristics of the turbulent flow path of the powder to lump the excipient-drug aggregates to disperse, and the active ingredient particles to be stored in the lungs. However, certain dry powder inhalers use a cyclone dispersion chamber to produce particles of the desired breathable size. In a cyclone dispersion chamber, the drug enters a tangenfial coin dispersion chamber so that the air duct and the drug travel along the outer circular wall. As the drug formulation moves along this circular wall it spreads around and the agglomerates are decomposed separately from the impact forks. The air duct spirals towards the center of the chamber, exiting vertically. Particles that have small enough aerodynamic dimensions can follow the air duct and exit the chamber. In fact, the dispersion chamber works as a small jet mill.

The single-dose, single-use Twincer ™ inhaler appears to work with a о-cyclone dispersion chamber in the form of a coin referred to as an "air classifier" (US 2006237010 Al 2006.10.26). Papers published by the University of Groningen stated that a dose of 60 mg of purely micronized colistin sulfometate could be effectively administered in the form of inhalable dry powder using this technology.

In preferred embodiments, the aerosol formulation is administered as a dry powder by means of a dry powder inhaler in which the particles emitted from the inhaler have an MM AD in the range of about 1 gm to about 5 gm and о GSD approximately more. less than 2.

Examples of suitable dry powder inhalers and devices for dispersing dry powder for use in the administration of compounds and compositions according to the present invention include, but are not limited to those described in US 2006237010 Al 2006.10.26, US 7520278 B2 2009.04.21, US 7322354 B2 2008.01.29, US 7246617 Bl

2007.07.24, US 7231920 B2 2007.06.19, US 7219665 Bl 2007.05.22, US 7207330 Bl

2007.04.24, US 6880555 Bl 2005.04.19, US 5522385 A 1996.06.04, US 6845772 B2

2005.01.25, US 6637431 B2 2003.10.28, US 6329034 Bl 2001.12.11, US 5458135 A

1995.10.17 and US 4805811 A 1989.02.21. In one embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation, which is formulated for administration by a Diskus® device. The Diskus® device encloses an elongated band consisting of a base plate having a plurality of distal cavities along its length and a plate with a hermetically sealed but removable lid to define a plurality of containers, each having a container inside. inhalable which confers a predetermined amount of active ingredient either alone or in combination with one or more carrier moieties or excipients (eg, lactose) and / or therapeutically active agents. Preferably, the tape is flexible enough to be wrapped in a roll.

MD 4574 Cl 2019.01.31

The cover plate and the base plate will preferably have front ends that are not sealed together and at least one of the front ends is constructed to be attached to a winding means. Also, preferably, the sealing groove between the cover plate and the base plate extends across their width. In order to prepare the dose for inhalation, the lid plate may preferably be detached from the base plate in a longitudinal direction from a first end of the base plate.

In one embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation, which is formulated for administration using a single dose single dose inhaler and in particular the Twincer ™ inhaler. The Twincer ™ inhaler encloses a tin foil blister with one or more cavities and a hermetically sealed but removable lid plate to define a plurality of containers. Each container has an inhalable formulation containing a predetermined amount of the active ingredient (s) either alone or in combination with one or more carrier moieties or excipients (eg, lactose). The lid plate will preferably have a front end which is prominently constructed from the body of the inhaler. The patient will operate the device and thereby administer the aerosol formulation by 1) removing the outer shell of the package, 2) pulling the staniol foil to thaw the blister and 3) inhaling the blister.

In another embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation wherein the dry powder is formulated into microparticles as described in WO 2009015286 A2 2009.01.29 and WO 2007114881 Al 2007.10.11. These microparticles are generally formed by adding a counterion to the solution which confines a compound of the invention in a solvent, adding an antisolvent to the solution; and the gradual cooling of the solution to a temperature below about 25 ° C, to form a composition that confines microparticles that confine the compound. The microparticles confining the compound can then be separated from the solution by any suitable means, such as sedimentation, filtration or lyophilization. Adequate contractions, solvents and antisolves for the preparation of the microparticles of the compounds of the invention are described in WO 2009015286 A2 2009.01.29.

In another embodiment, the pharmaceutical composition according to the invention is administered as a dry powder using a metered dose inhaler. Non-limiting examples of metered dose devices and inhalers include those disclosed in US 5261538 A 1993.11.16, US 5544647 A 1996.08.13, US 5622163 A 1997.04.22, US 4955371 A 1990.09.11, US 3565070 A 1971.02.23, US 3361306 A 1968.01.02, US 6116234 A 2000.09.12 and US 7108159 A 2000.09.12. In a preferred embodiment, a compound of the invention is administered in the form of a dry powder, using a metered dose inhaler wherein the emitted particles have an MMAD that is in the range of about 1 µm to about 5 µm and о GSD which is less than about 2.

Liquid aerosol formulations for administration to the endobronchial sphincter or lung by inhalation may, for example, be formulated as aqueous solutions or suspensions or as aerosols administered from pressure packs, such as metered dose inhalers, with the use of proper liquefaction propulsion, easily disintegrable aerosol inhalers or nebulizers. These aerosol compositions suitable for inhalation may be either suspension or solution and generally contain the active ingredient (s) together with an acceptable pharmaceutical carrier or diluent (eg, water (distilled or sterile), saline, solution hypertonic saline or ethanol) and optionally one or more therapeutically active agents.

Aerosol compositions for administration by metered dose inhalers typically contain a pharmaceutically acceptable propellant. Examples of such propellants include fluorocarbon or chlorofluorocarbon which contain hydrogen or mixtures thereof, in particular hydrofluoroalkanes, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1,1,1,2tetrafluoroethane, 1,1,1,2, 3,3,3, -heptafluoro-n-propane or a mixture thereof. The aerosol composition may be excipient-free or may optionally confine additional formulation excipients well known in the art, such as surfactants for example, oleic acid or lecithin and co-solvents for example, ethanol. The pressure formulations will generally be kept in a canister (for example, an aluminum canister).

MD 4574 Cl 2019.01.31 closed with о valve (for example, о metering valve) and mounted in an actuator provided with a nozzle.

In another embodiment, the pharmaceutical composition according to the invention is administered in liquid form using a metered dose inhaler. Non-limiting examples of metered dose devices and inhalers include those described in US 6253762 Bl 2001.07.03, US 6413497 Bl 2002.07.02, US 7601336 B2 2009.10.13, US 7481995 B2 2009.01.27, US 6743413 Bl 2004.06.01 and US 7105152 Bl 2006.09.12. In a preferred embodiment, a compound of the invention is administered in the form of a dry powder, using a metered dose inhaler wherein the emitted particles have an MMAD that is in the range of about 1 µm to about 5 µm and о GSD which is smaller. about 2.

In one embodiment, the aerosol formulation is suitable for aerosolization of a jet nebulizer or ultrasonic nebulizer including vibrating and static porous plate nebulizers. Liquid aerosol formulations for nebulization may be generated by solubilizing or reconstituting a solid particle formulation or may be formulated with an aqueous vehicle with the addition of agents such as acids or bases, buffer salts and isotonicity-adjusting agents. They can be sterilized by processing techniques, such as filtration, or termination processes such as heating in an autoclave or irradiation range. They can also be presented in a non-sterile form.

Patients may be sensitive to the pH, osmolarity and ionic content of a nebulized solution. Therefore, these parameters need to be adjusted to be compatible with the active ingredient and tolerable to patients. The most preferred active ingredient solution or suspension will contain о chloride concentrations of&gt; 30 mM at pH 4.5-7.4, preferably 5.0-5.5, and о osmolarity of about 800-1600m0sm / kg. The pH of the solution can be controlled either by titration with common acids (hydrochloric acid or sulfuric acid, for example) or bases (sodium hydroxide, for example) or by using buffers. Commonly used buffers include citrate buffers, such as sodium citrate / citric acid buffers, acetate buffers, such as sodium acetate / acetic acid buffers and phosphate buffers. Buffer concentrations can range from 2 mM to 50 mM.

Useful acetate, phosphate and citrate buffers include sodium acetate, sodium acetate trihydrate, ammonium acetate, potassium acetate, sodium phosphate, dibasic sodium phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium phosphate potassium, sodium citrate and potassium citrate. Other buffer solutions that may be used include sodium hydroxide, potassium hydroxide, ammonium hydroxide, aminomethylpropanol, tromethamine, tetrahydroxypropylethylenediamine, citric acid, acetic acid, hydroxycitricarboxylic acid or its salt, such as citrate salt or its sodium citrate, lactic acid, and lactic acid salts, including sodium lactate, potassium lactate, lithium lactate, calcium lactate, magnesium lactate, barium lactate, aluminum lactate, zinc lactate, silver lactate, copper lactate, iron lactate, manganese lactate, ammonium lactate, monoethanolamine, di ethanol amine, triethanolamine, diisopropanolamine, and combinations thereof, and the like.

These formulations may be administered using commercially available nebulizers or other atomizers which may break down the formulation into particles or droplets suitable for deposition in the respiratory tract. Non-limiting examples of nebulizers that may be used for administering an aerosol composition according to the invention include pneumatic jet nebulizers, ventilated or improved breathing nebulizers, or ultrasonic nebulizers, including static or vibrating plate nebulizers. Commercially available nebulizers include Aeroneb® Go nebulizer (Aerogen) and eFlow nebulizer (Pari Pharma).

A jet nebulizer uses a high-speed airflow blown through a column of water to generate drops. Inappropriate particles for inhalation influence aerodynamic drifts or baffles. A ventilated or improved respiratory nebulizer functions essentially as a jet nebulizer except that inhaled air passes through the primary droplet generation area to increase nebulizer productivity while the patient inhales.

In an ultrasonic nebulizer, the vibration of a piezoelectric crystal creates superficial instabilities in the drug reservoir that produces drop formation. In the porous plate nebulizers the pressure fields generated by the sonic energy shed the liquid through the pores of the mesh where it decomposes into droplets by decomposition.

MD 4574 Cl 2019.01.31

Rayleigh. The sonic energy can be fumigated by a vibrating combo or plate driven by a piezoelectric crystal, or by the mesh itself by vibration. Non-limiting examples of atomizers include any single-component or two-component liquid atomizer or nozzle that produces droplets of appropriate size. A single component liquid atomizer works by forcing a liquid through one or more holes, where the liquid stream breaks down into droplets. Two-component liquid atomizers work either by forcing both a gas and a liquid through one or more holes or by pushing a liquid jet against another jet or liquid or gas.

Choosing the aerosolizing nebulizer aerosol formulation is important in the administration of the active ingredient (s). Different nebulizers have different yields based on their construction and operating principle and are sensitive to the physical and chemical properties of the formulation. For example, two formulations with different surface voltages may have different particle size distributions. In addition, the properties of the formulation such as pH, osmolality and permeant ion content can affect the tolerability of the drug, so preferred embodiments respect certain ranges of these properties.

In a preferred embodiment, the nebulization formulation is administered to the endobronchial sphincter as an aerosol having an MMAD of between about 1 µm and about 5 µm and о GSD less than 2 using a suitable nebulizer. To be very effective and to avoid superior systemic and respiratory side effects, the aerosol should not have an MMAD greater than about 5 pm and should not have о GSD greater than about 2. If an aerosol has an MMAD greater than about 5 µm or a GSD greater than about 2 a large percentage of the dose may be deposited in the upper respiratory tract reducing the amount of drug administered to the desired site m the lower respiratory tract. If the aerosol MMAD is less than about 1 µm, then a large percentage of particles may remain suspended in the inhaled air and may then be exhaled during expiration.

The compounds of the invention may also be administered by transbronchoscopic lavage.

Formulations suitable for oral administration may be presented as discrete units such as capsules, capsules or tablets, each containing a predetermined amount of active ingredient; in the form of powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be presented as an envelope, bowl, excipient or paste.

The tablet can be obtained by compression or tuming, optionally with one or more additional ingredients. The compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a powdered form, such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant. Turned tablets can be manufactured by pouring them into a suitable machine of a mixture of a powdery compound moistened with an inert liquid diluent. The tablets can be optionally coated or labeled and can be formulated to ensure the slow or controlled effect of the active ingredient in them.

Formulations for topical administration to the mouth, for example buccal or sublingually, include tablets that contain the active ingredient in a flavored base, such as sucrose and acacia or tragacanth, and pills that contain the active ingredient in a base, such as gelatin and glycerin or sucrose and acacia.

Formulations for parenteral administration include sterile aqueous and non-aqueous injectable sterile solutions that may contain antioxidants, buffers, bacteriostats and dissolved substances that make the isotonic formulation with the blood of the container; and sterile aqueous and non-aqueous suspensions which may include suspending agents and thickening agents. The formulations may be presented in single-dose or multi-dose containers, for example sealed vials and vials, and may be stored in a freeze-dried state that requires only the addition of the sterile liquid carrier, for example, saline solution or water for injections, immediately before delivery. use. Extemporaneous injectable solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

Oral fluids such as solutions, syrups and elixirs can be prepared in a unified drug form so that the given amount is confined to the predetermined amount of active ingredient. The syrups may be prepared by dissolving the active ingredient in a suitable flavored aqueous solution, while the elixirs are prepared by the use of a pharmaceutically acceptable alcohol carrier. Suspensions can be formulated by dispersion

MD 4574 Cl 2019.01.31 to the active ingredient in a pharmaceutically acceptable vehicle. Solubilizing agents and emulsifiers, such as ethoxylation isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavoring additions such as peppermint oil or natural sweeteners or saccharin or artificial sweeteners !, and such alcohols may also be incorporated into composts. oral fluids.

Liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles can also be used as administration means for the compounds of the invention. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholine.

The pharmaceutical compositions for topical administration may be formulated as ointments, creams, suspensions, slurries, powders, solutions, pastes, gels, sprays, aerosols or oils. Compositions intended for the treatment of eyes or other extraneous tissues, for example mouth and skin, can be applied as an ointment or cream. When is the formula! As an ointment, the active ingredient can be used with either a paraffmic base or a water soluble ointment. Alternatively, the active ingredient may be the formula! in a cream with a creamy oil-in-water base or a water-in-oil base.

Other compositions intended for topical administration to the eyes or ears include eye drops or ear drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, such as, for example, an aqueous solvent, including saline.

The compositions intended for nasal administration include aerosols, solvents, suspensions, sprays, aerosols and drops. The aerosolable formulations for nasal administration can be formulated in the same way as the aerosolable formulations for inhalation, provided that non-breathable particles will be preferred in the nasal administration formulations. Usually particles of about 5 microns up to the size of visible droplets can be used. Thus, for nasal administration, a particle size in the range of 10-500 microns can be used to ensure retention in the nasal cavity.

Transdermal patches may also be used, which are designed to remain in contact with the patient's epidermis for an extended period of time and to stimulate the absorption of the active ingredient through it.

Compositions for vaginal or rectal administration include ointments, creams, suppositories and enema, all of which can be formulated using conventional techniques.

In another aspect, the invention relates to a method of stimulating the hydration of the mucosal surfaces or of restoring the protection of the mucous membranes to a person in need thereof, which includes administering to man a pharmaceutical composition which confines a compound of the invention, wherein the compound mentioned is administered in ο effective amount. In a preferred embodiment, the method includes administering the pharmaceutical composition as an inhalable composition containing an amount of a compound of the invention which is sufficient to reach the dissolved concentration of the compound on the airway surfaces, from about IO '.⁹, 10'⁸ or IO '⁷ up to about 10 '⁴, I '³, I '² or 10¹ moles / liter, more preferably from about IO '⁹ up to about 1 O '⁴ moles / liter.

In another aspect, the invention refers to a method of treating any of: о disease associated with reversible or irreversible airway obstruction, chronic obstructive bronchopneumopathy (ВРОС), asthma, bronchiectasis (including bronchiectasis from conditions other than cystic fibrosis), acute bronchitis, chronic bronchitis, post-viral cystic fibrosis , panbronchiolitis, transplant-associated bronchiolitis and ventilator-associated tracheobronchitis or the prevention of ventilator-associated pneumonia in a person in need of it, which includes administering to the human body a pharmaceutical composition which confines a compound of the invention, wherein the mentioned compound is administered in an effective amount. In a preferred embodiment, the method includes administering the pharmaceutical composition as an inhalable composition containing an amount of a compound of the invention which is sufficient to reach the dissolved concentration of the compound on the airway surfaces at about 10 '⁹, 10‘⁸ or 10 '⁷ up to about 10 '⁴, 10‘³, 10‘² or 10¹ moles / liter, more preferably from about 10 '⁹ up to about 1 '⁴ moles / liter.

In another embodiment, the invention relates to a method of treating any of the dry mouth (xerostomia), skin dryness, vaginal dryness, sinusitis, rhinosinusitis or nasal dehydration, including nasal dehydration caused by the administration of dry oxygen or dry eye. , stimulation of ocular or coma hydration, treatment of distal intestinal obstruction syndrome,

MD 4574 Cl 2019.01.31 treatment of otitis media, primary ciliary dyskinesia, distal intestinal obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a man in need of it, which includes administering to man a pharmaceutical composition that confines a compound of the invention, m which the aforementioned compound is administered in an effective amount.

Preferred unified drug formulations for the compounds of the invention are those which contain an effective amount of active ingredient or a suitable fraction thereof.

It should be appreciated that, in addition to the ingredients mentioned in particular above, the formulations of this invention may include other conventional agents in the art having regard to the type of formulation concerned, for example those suitable for oral administration may include flavoring agents.

The compositions of the present invention may be formulated for immediate, controlled or prolonged action after wishful thinking for the specific condition to be treated and the desired route of administration. For example, controlled-action formulations for oral administration may be desirable for the treatment of constipation to maximize the release of the active agent in the colon. Such formulations and excipients suitable for this are well known in the pharmaceutical field. Since the free base of the compound is generally more soluble in aqueous solutions than the salt, the compositions containing the free base of a compound of Formula I may be used to provide a longer action of the active agent administered by inhalation to the lungs. An active agent present in the lungs in the form of particles, which has not dissolved m solution, is not available to induce a physiological response, but serves as a bioavailable drug store that gradually dissolves m solution. As another example, the formulation may include both the free base and the salt form of a compound of the invention to provide both immediate and prolonged action of the active ingredient for dissolving the mucus secretions of for example, the nose.

Combinafii

The compounds of the invention may be formulated and / or used in combination with therapeutically active agents. Examples of therapeutically active agents that may be formulated or used in combination with the compounds of the invention include, but are not limited to, osmolifes, anti-inflammatory agents, anticholinergic agents, selective β-agonists (including β2-agonists), P2Y2 receptor agonists, delta of peroxisome proliferator activafi receptors (RAPPs), alpha blockers of epithelial sodium channels (ENAC receptor blockers), modulators of the cystic fibrosis transmembrane conductance regulator (CFTR), kinase inhibitors, anti-infectious agents, antihistamines, non-antibiotic anti-inflammatory macrolides, protease and elastase inhibitors, and mucus or mucin-modifying agents, such as surfactants. In addition, for cardiovascular indications, the compounds of the invention can be used in combination with beta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents!

The present invention relates, as a further aspect, to a composition comprising an effective amount of a compound of the invention and one or more therapeutically active agents selected from osmolifes, anti-inflammatory agents, anticholinergic agents, β-agonists (including β2 ^ οηφ! selective), P2Y2 receptor agonists, PPAR delta agonists, ENAC receptor blockers, modulators of the transmembrane conductance regulator in cystic fibrosis (CFTR), kinase inhibitors, anti-infectious agents, antihistamines, non-antibiotic anti-inflammatory macrolides, protease and elastase inhibitors, and mucus or mucin-modifying agents, such as surfactants. The present invention relates, as a further aspect, to о composition that confines о effective amount of a compound of the invention and one or more therapeutically active agents of beta-blockers, ACE inhibitors, HMGCoA reductase inhibitors and channel blockers of calcium. The use of compounds of the invention in combination with one or more sulfur therapeutically active agents (especially osmolifices) may reduce the dose of the compound of the invention which is required for sufficient hydration of mucosal surfaces, thereby reducing the potential for undesirable side effects that may be attributed to systemic blockade. sodium channels, such as, for example, the kidneys.

"Osmoliphs" according to the present invention are molecules or compounds that are osmotically active. The "osmotic active" molecules and compounds are membrane-impermeable (ie, essentially non-absorbable) on the respiratory tract or pulmonary epithelial surface. The terms "airway surface" and "pulmonary surface", as used herein, encompass the surfaces of the pulmonary airways, such as the bronchi and bronchioles, the surfaces

MD 4574 Cl 2019.01.31 alveolar and nasal and sinus surfaces. Suitable osmoliphs include ionic osmoliphs (eg, salts) and nonionic osmoliphs (eg sugars, alcohol sugars and organic osmoliphs). In general, osmolifes (both ionic and nonionic) used in combination with the compounds of the invention are preferable! osmolifs that do not promote !, or actually discourage! or delay! bacteria development. Osmolifices suitable for use in the present invention may be in the form! racemic! or in the form of an enantiomer, diastereomer, tautomer, polymorph or pseudopolymorph.

Examples of osmoliphons useful in the present invention include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably, the salt of either the anion or the cation (or both) is osmotically active! and is not subject! to the active rapid transport, in relation to the respiratory tract surfaces to which they are administered. These compounds include, but are not limited to, anions and cations that are confined to FDA approved commercially available salts (Remington. Science and Practice of Pharmacy. 1995, ed. 19, vol. II, p. 1457), and may be used in any combination as known in the art.

Specific examples of pharmaceutically acceptable osmotically active anions include, but are not limited to, acetate, benzolsulfonate, benzoate, bicarbonate, bicarbonate, bromide, calcium edetate, camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate, edisilate (1,2-ethanesulphonate), estolate (lauryl sulphate), esylate (1,2-ethanesulphonate), fumarate, gluceptate, gluconate, glutamate, glycolylarsanylate (p-glycolamidophenylarsonate), hexylresorcinate, hydrabamine! (N, N'-di (dehydroabietyl) ethylenediamine!), Hydrobromide !, hydrochloride !, hydroxinaftoate, iodide !, isetionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide!, Methylnitrate, methylsulfate, mucate, naps nitrate, nitrite, pamoate (powdery mildew), pantothenate, phosphate or diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulphate, tanate, tartrate, teoclate (8-chloroteophilinate), triethiodide!, bicarbonate, etc. Preferred anions include chloride !, sulfate, nitrate, gluconate, iodide !, bicarbonate, bromide! and phosphate.

Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to, organic cations such as benzathine (N, N'-dibenzylethylenediamine), chlorprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine), procaine. , D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium, N methyl D glycerol and the like, as well as metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron. , such as ammonium and alfalfa. Preferred organic cations include 3-carbon, 4-carbon, 5-carbon and 6-carbon organic cations. Preferred cations include sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, magnesium and calcium.

Specific examples of ionic osmolifics that may be used in combination with a compound of the invention include, but are not limited to, chloride! sodium (in particular the saline solution! hypertonic!), chloride! of potassium, chloride! of choline !, iodide !, chloride! of lithium, chloride! of meglumin !, chloride! of L-lysine !, chloride! of D-lysine !, chloride! of ammonium, potassium sulfate, potassium nitrate, potassium gluconate, iodide! of potassium, chloride! happy, chloride! feroas !, bromide! of potassium, as well as combinations of any two! or more of the above. In one embodiment, the present invention refers! to the combination of a compound of the invention and two! different osmotic active salts. When different salts are used, one anion or cation may be the same among different salts. Hypertonic solution! is a preferred ionic osmolite for use in combination with the compounds of the invention.

Non-ionic osmoliphs include sugars, sugar-alcohols and organic osmoliphs. The sugars and sugars-alcohols useful as osmolifices in the present invention include, but are not limited to, 3-carbon sugars (eg, glycerol, dihydroxyacetone); 4-carbon sugars (for example, both D and L forms of erythrocytes !, threesome! and erythrocytes!), 5-carbon sugars (for example, both D and L forms of ribose !, arabinose !, xylose !, lixose !, psychosis !, fructose !, sorbose! and tagatoz!); and 6-carbon sugars (for example, both D and L forms of barley !, allose !, glucose !, mannose !, gulose !, idose !, galactose! and thallium!), as well as forms D and L of hello-heptulose! , alo-hepulose !, gluco-heptulose !, mano-heptulose !, gulo-heptulose !, IDOheptulose !, galacto-heptulose !, talo-heptulose!). The additional sugars useful in the practice of the present invention include rafmosis, oligosaccharides from the series of rafmosis and stachiosis. Both D and L forms of the reduced form of each sugar / sugar are also suitable for the present invention. For example, glucose, when reduced!, Becomes sorbitol; an osmolite in the scope of the invention. Therefore, sorbitol and other reduced forms of sugar / sugar-alcohol (for example, mannitol, dulcitol,

MD 4574 Cl 2019.01.31 arabitol) are suitable osmolites for use in the present invention. Mannitol is a nonionic osmoly preferred for use in combination with the compounds of the invention.

"Organic osmoliphs" are generally used to refer to molecules that control intracellular osmolality in the kidney (JS Handler et al. Comp. Biochem. Physiol. 1997, vol. 117, pp. 301-306; Burg. Molecular basis of osmotic regulation, American Journal of Physiology, 1995, vol. 268, pp. F983-F996). Organic osmoliphs include, but are not limited to, three major classes of compounds: polyols (polyalcohols), methylamines and amino acids. Suitable organic polyol osmoliphs include, but are not limited to, inositol, myo-inositol and sorbitol. Suitable methylamine organic osmoliphs include, but are not limited to, choline, betaine, camitin (L-, D- and DL-forms), phosphorylcholine, lysophosphorylcholine, glycerophosphorylcholine, creatine, phosphate and creatine. Suitable organic amino acid osmoliphs include, but are not limited to, D- and L- forms of glycine, alanine, glutamine, glutamate, aspartate, proline and taurine. Additional organic osmoliphs suitable for use in the present invention include thiulose and sarcozin. Organic mammal osmoliphs are preferred, human organic osmoliphs being the most preferred. However, certain organic osmolifices are of bacterial, yeast, marine and animal origin, and these compounds may also be used in the present invention.

Osmolific precursors may be used in combination with the compounds of the invention, A "osmolite precursor", as used herein, refers to a compound that is transformed into an osmolite by a metabolic step, either catabolic or anabolic. Precursor examples! of osmolifes include, but are not limited to, glucose, glucose polymers, glycerol, choline, phosphatidylcholine, lysate-phosphatidylcholine, and inorganic phosphates. of polyols and methylamines. The precursors of the osmolific amino acids include proteins, peptides and polyamino acids, which are hydrolyzed to produce osmolific amino acids, and the metabolic precursors! which can be converted to osmolific amino acids by a metabolic step, such as transamination. For example, a glutamine precursor of the amino acid is poly-L-glutamine, and a precursor of glutamate is poly-Lglutamic acid.

Chemically modified osmoliphs or osmoliph precursors may also be used. These chemical modifications involve binding of the osmolite (or precursor) to a further chemical group which modifies or enhances the effect of the osmolite or osmolite precursor (for example, inhibits the degradation of osmolite molecules). These chemical modifications have been used with drugs or prodrugs and are known in the art (US 4479932 A 1984.10.30; US 4540564 A 1985.09.10; Ephraim Shek et al. Improved delivery through biological membranes.) 3. Delivery of N-methylpyridinium-2carbaldoxime chloride through the blood-brain barrier in its dihydropyridine pro-drug form Journal of Medicinal Chemistry, 1976, vol. 19, pp. 113-117; Nicholas Bodor et al. Controlled delivery of theophylline: Chemistry of 7-acyl- and 7,7'-acylditheophylline derivatives. Jomal of Pharmaceutical Sciences, 1978, vol. 67, pp. 1045-1050; Nicholas Bodor et al. Improved delivery through biological membranes. 11. The redox chemical drug-delivery system and its use for brain-specific delivery of phenylethyl amine. Journal of Medicinal Chemistry, 1983, vol. 26, pp. 313-318; Tits. 2002).

Preferred osmoliphs for use in combination with the compounds of the invention include sodium chloride, in particular hypertonic solution and mannitol.

For the formulation of the 7% and&gt; 7% hypertonic solution, the formulations containing bicarbonate anions may be particularly useful, especially for respiratory disorders with dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), such as FC or ВРОС. Recent findings have shown that although the relative ratio between HCO conductance³/ CF conductance is between 0.1 and 0.2 for single CFTR channels activated by cAMP and ATP, the ratio in the sweat channel can range from about 0 to about 1.0, in the stimulation conditions. That is, the combination of cAMP + cGMP + α-ketoglutarate can produce CFTR НСОз 'conductance almost equal to that of conductance C1' (Paul M. Quinton. Cystic Fibrosis: Lessons from the Sweat Gland. Physiology, 2007, vol. 22, p. 212 -225). Moreover, the formulations of the 7% and&gt; 7% hypertonic solution containing bicarbonate anions may be particularly useful due to better pH control in the airway fluid. First, it has been shown that this acidification of the airways occurs in the FC. 2002) and that CFTR-dependent bicarbonate secretion is absent may lead to impaired responsiveness to airway conditions associated with acidification of the airway surface fluid layer (Coakley.

MD 4574 Cl 2019.01.31

2003). Second, the addition of the SH solution without bicarbonate to the surface of the lung may further dilute the concentrations of bicarbonate and possibly reduce the pH or the ability to respond to the acidification of the airways in the surface fluid layer of the airways. Therefore, the addition of bicarbonate anions to SH may contribute to lowering or improving the pH of the airway surface fluid layer in CF patients.

Due to this sample, the inclusion of the bicarbonate anion in the formulation of the 7% or&gt; 7% hypertonic solution administered by the method of the present invention would be particularly useful. Formulations containing up to 30 to 200 mM concentrations of bicarbonate anions are of particular interest for 7% or&gt; 7% SH solutions.

The hypertonic saline solution is found to have о higher salt concentrations than that of the normal saline solution (SN), ie greater than 9 g / L or 0.9% m / v, and the hypotonic saline solution has о higher salt concentrations. smaller than that of normal saline, such as from about 1 g or L / 0.1% m / v to about 8 g / L or 0.8% m / v. The hypertonic saline solutions useful in the formulations and treatment methods of the present invention may have salt concentrations from about 1% to about 23.4% (m / v). In one embodiment, the hypertonic saline has salt concentrations of from about 60 g / L (6% m / v) to about 100 g / L (10% m / v). In another embodiment, the saline solution has о salt concentrations from about 70 g / L (7% m / v) to about 100 g / L (10% m / v). In another embodiment, the saline solution has salt concentrations of a) from about 0.5 g / L (0.05% m / v) to about 70 g / L (7% m / v); b) from about 1 g / L (0.1% m / v) to about 60 g / L (6% m / v); c) from about 1 g / L (0.1% m / v) to about 50 g / L (5% m / v); d) from about 1 g / L (0.1% m / v) to about 40 g / L (4% m / v); e) from about 1 g / L (0.1% m / v) to about 30 g / L (3% m / v) and f) from about 1 g / L (0.1% m / v) ) up to about 20 g / L (2% m / v).

Specific concentrations of saline solutions useful in the formulations and methods of treatment of the present invention include, independently, those having salt concentrations of 1 g / L (0.1% m / v), 2 g / L (0, 2% m / v), 3 g / L (0.3% m / v), 4 g / L (0.4% m / v), 5 g / L (0.5% m / v), 6 g / L (0.6% m / v), 7 g / L (0.7% m / v), 8 g / L (0.8% m / v), 9 g / L (0.9% m / v), 10 g / L (1% m / v), 20 g / L (2% m / v), 30 g / L (3% m / v), 40 g / L (4% m / v) v), 50 g / L (5% m / v), 60 g / L (6% m / v), 70 g / L (7% m / v), 80 g / L (8% m / v) , 90 g / L (9% m / v), 100 g / L (10% m / v), 110 g / L (11% m / v), 120 g / L (12% m / v), 130 g / L (13% m / v), 140 g / L (14% m / v), 150 g / L (15% m / v), 160 g / L (16% m / v), 170 g / L (17% m / v), 180 g / L ( 18% m / v), 190 g / L (19% m / v), 200 g / L (20% m / v), 210 g / L (21% m / v), 220 g / L (22% m / v) and 230 g / L (23% m / v). Saline solution concentrations from each of these listed concentrations / percentages may also be used, such as 1.7 g / L (0.17% m / v), 1.25 g / L (1.25%) saline m / v), 1.5 g / L (1.5% m / v), 25 g / L (2.5% m / v), 28 g / L (2.8% m / v), 35 g / L (3.5% m / v), 45 g / L (4.5% m / v) and 75 g / L (7.5% m / v).

Useful specific concentrations of hypotonic saline include those from about 0.12 g / L (0.012% m / v) to about 8.5 g / L (0.85% m / v). Any concentrates in this range can be used, such as, on the basis of m / v, 0.05%, 0.1%, 0.15%, 0.2%, 0.225% (1/4 SN), 0 , 25%, 0.3% (1/3 SN), 0.35%, 0.4%, 0.45% (1/2 SN), 0.5%, 0.55%, 0.6% (2/3 SN), 0.65%, 0.675% (3/4 SN), 0.7%, 0.75% and 0.8%.

Each of the specific ranges and concentrations of the saline solution described herein may be used with the formulations, treatment methods, schemes and sets described herein.

Also, destinations within the scope of this invention are osmolifiers or precursors of chemically modified osmolifes. These chemical modifications involve the binding to the osmolite (or precursor) of an additional chemical group which modifies or enhances the effect of the osmolite or osmolite precursor (for example, inhibits the degradation of the osmolite molecule). These chemical modifications have been used with drugs or prodrugs and are known in the art (US 4479932 A 1984.10.30; US 4540564 A 1985.09.10; Ephraim Shek et al. Improved delivery through biological membranes.) 3. Delivery of N-methylpyridinium-2 -carbaldoxime chloride through the blood-brain barrier in its dihydropyridine pro-drug form Journal of Medicinal Chemistry, 1976, vol 19, p.113-117; Nicholas Bodor et al. Controlled delivery of theophylline: Chemistry of

7-acyl- and 7,7'-acylditheophylline derivatives. Jomal of Pharmaceutical Sciences, 1978, vol. 67, pp. 1045-1050; Nicholas Bodor et al. Improved delivery through biological membranes. ILA redox chemical drug delivery system and its use for brain-specific

MD 4574 Cl 2019.01.31 delivery of phenylethylamine. Journal of Medicinal Chemistry, 1983, vol. 26, p. 313318), each incorporated herein by reference.

Suitable anti-inflammatory agents for use in combination with the compounds of the invention include corticosteroids and nonsteroidal anti-inflammatory drugs (MNSAIs), in particular phosphodiesterase (PDE) inhibitors. Examples of corticosteroids for use in the present invention include oral or inhaled corticosteroids or prodrugs thereof. Specific examples include, but are not limited to, cyclesonide, desisobutyrylcyclesonide, budesonide, flunisolid, momethasone and their esters (eg, momethasone furoate), fluticasone propionate, fluticasone furoate, beclomethasone, methyl prednisolone, prednisolone, prednisolone, prednisolone 6ct, 9ct-difluoro1-S-fluoromethyl acid at - [(2-furanylcarbonyl) oxy] -11 β-hydroxy-16ct-methyl-3-oxo-androsta-1,4-dien-17 β-carboxylic acid, 6ct, 9ct-difluoro-1β-hydroxy16ct-methyl-3-oxo-17ct-propionyloxy-androsta-1,4-dien-17β ^ ester S- (2-oxo-tetrahydro-furan-3S-yl) ? τΙχ) 6οκ, beclometazone esters (for example, 17-propionate ester or 17,21-dipropionate ester, fluoromethyl ester, triamcinolone acetonide, rofleponid) or any combination or subset thereof. Preferred corticosteroids for formulation or use in combination with the compounds of the invention are selected from cyclesonide, desizoobutyryl-cyclesonide, budesonide, momethasone, fluticasone propionate and fluticasone furoate, or any combination or subset thereof.

MNSAIs for use in the present invention include, but are not limited to, sodium chromoglycate, sodium nedocromyl, phosphodiesterase (PDE) inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, PDE3 / PDE4 mixed inhibitors or PDE4 / PDE4 mixed inhibitors ), leukotriene antagonists, leukotriene synthesis inhibitors (e.g., 5 LO and FLAP inhibitors), nitric oxide (iNOS) synthesis inhibitors, protease inhibitors (e.g., tryptase inhibitors, neutrophil elastase inhibitors and metalloprotease inhibitors), β2-integrin antagonists and adenosine receptor agonists or antagonists (eg, adenosine 2a agonists), cytokine antagonists (eg, chemokine antagonists) or cytokine inhibitors for example, prostaglandin D2 (CRTh2), receptor antagonists). Examples of leukotriene modifiers suitable for administration by the method of the present invention include montelukast, dayuton, and zafirlukast.

The PDE4 inhibitor, the PDE3 / PDE4 mixed inhibitor or the PDE4 / PDE7 mixed inhibitor can be any compound that is known to inhibit the PDE4 enzyme or which is found to act as an inhibitor of PDE4, and which are selective inhibitors of PDE4 (e.g., compounds that are PDE4). does not significantly inhibit other members of the PDE family). Examples of specific PDE4 inhibitors for the preparation and use in combination with the compounds of the present invention include, but are not limited to, roflumilast, pumafentrin, arophylline, cilomilast, tofimilast, oglemilast, tolafentrin, piclamilast, ibudilast, apremilast, 2- [6,7-diethoxy-2,3-bis (hydroxymethyl) -l-naphthalenyl] -2-pyridinyl] -4- (3-pyridinyl) -l (2H) -phthalazinone (T2585), N- (3,5-dichloro -4-pyridinyl) -l - [(4-fluorophenyl) methyl] -5-hydroxy-ct-oxo-1H-indole-3-acetamide (A WD-12-281, 4- [(2R) -2- [3 - (cyclopentyloxy) -4-methoxyphenyl] -2-enyl-ethyl] pyridine (CDP-840), 2- [4 - [[[[2- (1,3 -benzodioxol-5-yloxy) -3-pyridinyl] carbonyl] amino] methyl] -3-fluorophenoxy] - (2R) -propanoic (CP-671305), N- (4,6-dimethyl-2-pyrimidinyl) -4- [4, 5,6,7-tetrahydro-2- (4-methoxy-3-methylphenyl) -5- (4-methyl-lpiperazinyl) -1H-indol-1-yl] -benzolsulfonamide, (2E) -2-butenedioate (YM -393059), 9 - [(2-fluorophenyl) methyl] -N-methyl-2- (trifluoromethyl) -9H-purine-6-amine (NCS-613), N - (2,5-Dichloro-3-pyridinyl) -8-methoxy-5-quinolinecarboxamide (D-4418), N - [(3R) -9-amino-3,4,6,7-tetrahydro-4-oxo-1 phenylpyrrolo [3,2, 1 -] [1,4] benzodiazepin-3-yl] -3H-purine-6-amine (PD168787), 3 - [[3- (cyclopentyloxy) -4-methoxyphenyl] methyl] hydrochloride -N-ethyl-8- (methylethyl) -3H-purine-6-amine (V 1294A), N- (3,5-dichloro-1-oxido-4-pyridinyl) -8-methoxy-2 (trifluoromethyl) - 5-quinolinecarboxamide (Sch3 51591), 5- [3- (cyclopentyloxy) -4-methoxyphenyl] -3 - [(3-methylphenyl) methyl] - (3S, 5S) -2-piperidinone (HT-0712), 5- (2 - ((R, 4R) -4amino-1- (3- (cyclopentyloxy) -4-methoxyphenyl) cyclohexyl) ethynyl) -pyrimidin-2-amine, cis- [4-cyano-4- (phenyl-3-cyclopropylmethoxy-4-difluoromethoxy) cyclohexane-l -ol] and 4- [6,7-diethoxy2,3-bis (hydroxymethyl) -l-naphthalenyl] -l- (2-methoxyethyl) -2 (1H) -pyridinone (T-440), as well as any combination or subset of them.

Leukotriene antagonists and their synthesis inhibitors include zafirlukast, sodium montelukast, dayuton and pranlukast.

MD 4574 Cl 2019.01.31

Anticholinergics for formulation or use in combination with the compounds of the invention include, but are not limited to, muscarinic receptor antagonists, including in particular pan antagonists and M3 receptor antagonists. Examples of compounds include belladonna plant alkaloids, such as atropine, scopolamine, homatropin, hyoscyamine, and various forms, including salts thereof (eg, anhydrous atropine, atropine sulfate, atropine oxide or HCI, methylatropine nitrate, hydrobromide hydrobromide). homatropin, homatropin methyl bromide, hyoscyamine hydrobromide, hyoscyamine sulfate, scopolamine hydrochloride, scopolamine methyl bromide), or any combination thereof or subset thereof.

Additional anticholinergics for formulation and use in combination with metanthelin, propantheline bromide, anisotropin methyl bromide or Valpin 50, aclidinium bromide, glycopyrrolate (Robin), isopropamide iodide, mepenzolate bromide, tridihexetyl chloride, methyldiophenyl acetate chloride , tropicamide, trihexiphenidyl CC1, pirenzepine, telenzepine and metoctramine, or any combination thereof or subset thereof.

Preferred anticholinergics for preparation and use in combination with the compounds of the invention include ipratropium (bromide), oxitropium (bromide) and tiotropium (bromide) or any combination or subset thereof.

Examples of β-agonists for formulation and use in combination with the compounds of the invention include, but are not limited to, salmeterol, R-salmeterol, xinafoate and their salts, albuterol or R-albuterol (free base or sulfate), levalbuterol, salbutamol, formoterol (fumarate), phenoterol, procaterol, pirbuterol, metaprterenol, terbutaline and their salts, as well as any combination or subset thereof.

P2Y2 receptor agonists for the preparation and use in combination with the compounds of the invention can be used in an effective amount to stimulate chloride and water secretion by airway surfaces, especially nasal airway surfaces. Suitable P2Y2 receptor agonists are known in the art and are described, for example, in columns 9-10 of US 6264975 Bl 2001.07.24 [1] and also (US 5656256 A 1997.08.12, US 5292498 A 1994.03.08) .

P2Y2 agonists that can be administered by the methods of this invention include P2Y2 receptor agonists, such as ATP, UTP, UTP-.gamma.-S, and dinucleotide P2Y2 receptor agonists (eg, denufosol or diquafosol) or a pharmaceutically acceptable salt. them. The P2Y2 receptor agonist is usually included in an effective amount to stimulate chloride and water secretion by the airway surfaces, especially the nasal airway surfaces. Suitable P2Y2 receptor agonists are described in, but are not limited to, US 6264975 Bl 2001.07.24, US 5656256 A 1997.08.12, US 5292498 A 1994.03.08, US 6348589 Bl 2002.02.19, US 6818629 B2 2004.11.16, US 6977246 B2 2005.12.20, US 7223744 B2 2007.05.29, US 7531525 B2 2009.05.12, US 2009306009 Al 2009.12.10, each of which is incorporated herein by reference.

The combined therapies and formulations of the present invention may include adenosine 2b agonists (A2b), also including BAY 60-6583, NECA (Netilcarboxamidoadenosine), (S) -PHPNECA, LUF-5835 and LUF-845. A2b agonists that may be uses are described (Rosaria Volpini et al. N⁶-Alkyl-2-alkynyl Derivatives of Adenosine as Potent and Selective Agonists at the Human Adenosine Аз Receptor and a Starting Point for Searching A₂b Ligands. Journal of Medicinal Chemistry, 2002, vol. 45, no. 15, pp. 3271-3279; Rosaria Volpini et al. Purine Nucleosides Bearing 1-Alkynyl Chains as Adenosine Receptor Agonists. Current Pharmaceutical Design, 2002, vol. 8, no. 26, pp. 2285-2298; Pier Giovanni Baraldi et al. Design, Synthesis, and Biological Evaluation of New 8-Heterocyclic Xanthine Derivatives as Highly Potent and Selective Human A2B Adenosine Receptor Antagonists. Journal of Medicinal Chemistry, 2004, vol. 47, no. 6, p. 1434-1447; Mini Reviews in Medicinal Chemistry, 2005, vol. 5, no. 12, pp. 1053-1060; Giovanni Baraldi Pier et al. Ligands for A2B Adenosine Receptor Subtype. Current Medicinal Chemistry, 2006, vol. 13, no. 28, pp. 3467-82; Margot W. Beukers et al. Structure-affinity relationships of adenosine A₂b receptor ligands. Medicinal Research Reviews, 2006, vol. 26, no. 5, pp. 667-698; ElfatihElzein et al. Novel l, 3-dipropyl-8- (lheteroarylmethyl-lH-pyrazol-4-yl) -xanthine derivatives as high affinity and selective A₂b adenosine receptor antagonists. Bioorganic &amp; Medicinal Chemistry Letters, 2006, vol. 16, no. 2, pp. 302-306; Angelo Carotti et al. Design, Synthesis, and Structure-Activity Relationships of 1-, 3-, 8-, and 9-Substituted-9-deazaxanthines at the Human A₂b Adenosine Receptor. Journal of Medicinal Chemistry, 2006, vol. 49, no. 1, pp. 282-299;

MD 4574 Cl 2019.01.31

Mojgan AghazadehTabrizi et al. l, 3-Dipropyl-8- (l-phenylacetamide-lH-pyrazol-3-yl) xanthine derivatives as highly potent and selective human A2B adenosine receptor antagonists. Bioorganic &amp; Medicinal Chemistry, 2008, vol. 16, no. 5, pp. 2419 -2430; AngelaStefanachi et al. 1-, 3- and 8-substituted-9-deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor. Bioorganic &amp; Medicinal Chemistry, 2008, vol. 16, no. 6, pp. 2852-2869).

Examples of other ENaC receptor blockers for formulation and use in combination with the compounds of the invention include, but are not limited to, amiloride and derivatives thereof such as those described in US 6858615 B2 2005.02.22, WO 2003070182 A2 2003.08.28 [2 ], WO 2004073629 A2 2004.09.02 [3], WO 2005018644 To 2005.03.03, WO 2006022935 To 2006.03.02, WO 2007018640 To 2007.02.15, WO 2007146869 To 2007.12.21 [4], all from Parion Sciences, Inc.

Small molecule ENaC blockers are capable of preventing the direct transfer of sodium through the pores of the ENaC channel. The ENaC blocker that can be administered in combinations of the present invention includes, but is not limited to, amiloride, benzamyl, phenamyl, and amiloride analogues as exemplified by U.S. Patent Nos. 685 8614 B2 2005.02.22, US 6858615 B2 2005.02.22, US 6903105 B2 2005.06.07, US 7064129 B2 2006.06.20, US

7186833 B2 2007.03.06, US 7189719 B2 2007.03.13, US 7192958 B2 2007.03.20, US

7192959 B2 2007.03.20, US 7241766 B2 2007.07.10, US 7247636 B2 2007.07.24, US

7247637 B2 2007.07.24, US 7317013 B2 2008.01.08, US 7332496 B2 2008.02.19, US

7368447 B2 2008.05.06, US 7368450 B2 2008.05.06, US 7368451 B2 2008.05.06, US

7399766 B2 2008.07.15, US 7410968 B2 2008.08.12, US 7842697 B2 2010.11.30, US

7868010 B2 2011.01.11, US 6995160 B2 2006.02.07, US 7026325 B2 2006.04.11, US

7030117 B2 2006.04.18, US 7345044 B2 2008.03.18, US 7375107 B2 2008.05.20, US

7820678 B2 2010.10.26, US 7875619 B2 2011.01.25.

ENaC proteolysis is well described to increase sodium transfer through ENaC. The protease inhibitor blocks the activity of endogenous airway proteases, thus preventing cleavage and activation of ENaC. Proteases that cleave ENaC include furin, meprin, matriptase, trypsin, channel-associated proteases (CAPs) and neutrophil elastases. Protease inhibitors that may inhibit the proteolytic activity of these proteases that may be administered in the combinations of the present invention include, but are not limited to, camostat, prostase, furin, aprotinin, leupeptin, and trypsin inhibitors.

The combinations of the present invention may include one or more suitable nucleic acids (or polynucleic acids), including, but not limited to, antisense oligonucleotides, siRNA, miRNA, mimic miRNA, antagomyr, ribozyme, aptamer, and nucleic acid oligonucleotides. 2010316628 To 2010.12.16). Generally, such nucleic acids can be from 17 or 19 nucleotides in length, up to 23, 25 or 27 nucleotides in length, or more. Examples include, but are not limited to, those disclosed in US Patents 2010,316,628 Al 2010.12.16, US 7517865 B2 2009.04.14, US 2010215588 ^ Al 2010.08.26, US 2011008366 Al 2011.01.13 and US 2011104255 Al 2011.05.05. Generally, siRNAs are from 17 or 19 nucleotides in length, up to 23, 25 or 27 nucleotides in length, or more.

The modulating compounds of CRTF activity that may be administered in combinations of this invention include, but are not limited to, the compounds described in US Pat.

2009246137 To 2009.10.01, US 2009253736 To 2009.10.08, US 2010227888A

2010.09.09, US 7645789 B2 2010.01.12, US 2009246820 To 2009.10.01, US

2009221597 To 2009.09.03, US 2010184739 To 2010.07.22, US 2010130547A

2010.05.27, US 2010168094 To 2010.07.01, US 7553855 B2 2009.06.30, US 7772259 B2 2010.08.10, US 7405233 B2 2008.07.29, US 2009203752 To 2009.08.13, US 7499570 B2 2009.03.03.

The mucus or mucin-modifying agents useful in the combinations and methods described herein include reducing agents, surfactants and detergents, expectorants and deoxyribonuclear agents.

Mucin proteins are organized into high molecular weight polymers by forming noncovalent and covalent bonds (disulfide). Destruction of covalent bonds with reducing agents is a well-established method for reducing the vascoelastic properties of mucus in vitro and is expected to minimize mucus adhesion and improve clearance in vivo. Reducing agents are well known to decrease mucosal viscosity in vitro and are commonly used as a processing aid

MD 4574 Cl 2019.01.31 sputum samples (source 8 http://www.drugs.coin./mmx/mucomy st.html # citecOO 100716). Examples of reducing agents include molecules containing sulfide or phosphine that may reduce the di-sulfuric binding of the protein, including, but not limited to, N-acetyl cysteine, N-acistelin, carbocysteine, glutathione, dithiothreitol, thioredoxin-bound proteins , and tris (2-carboxyethyl) phosphine.

N-acetyl cysteine ​​(NAC) is approved for use in combination with thoracic physiotherapy for mucus secreted by viscous or thickened respiratory tract (source 12 https://www.dru£s.com/mmx/mucomvst.html#citec00100716) . Clinical studies evaluating the effects of oral or inhaled NAC m FC and ВРОС have reported improvements in the rheological properties of mucus and tendencies toward improvements in lung function and decreases in pulmonary obstruction (source 9 httn: //www.dru£s.com/mmx/ mucomvst.html # citec00100716). However, the preponderance of clinical data suggests that NAC is at best an effective marginal therapeutic agent for treating mucus obstruction secreted by the airways when given orally or by inhalation. Cochrane's recent analysis of existing clinical literature on the use of CNA found no evidence to support the effectiveness of CNA for FC (source 10 http: //www.drugs.conVmmx/mucomyst.html#citec00100716). The marginal clinical benefit of NAC reflects:

NAC is a relatively inefficient reducing agent that is only partially active on the airway surface. Extremely high concentrations of NAC (200 mM or 3.26%) are required to completely reduce Muc5B, a major gelanitizing mucin in the airway, in vitro. Moreover, in the pH environment of the airway surface (measured in the pH range 6.0-7.2 in the airways FC and ВРОС) (source 11 http: //www.drugs.conVmmx/mucomyst.html#citec00100716 ), NAC exists only partially in its reactive state as a negatively charged thiolate. Thus, in the clinic, NAC is administered in very high concentrations. However, it is predicted that current aerosol devices will not be able to reach therapeutic concentrations even with a 20% Mucomyst solution on the distal airway surfaces in the relatively short time ranges (7.5-15 minutes) typically used.

In nonclinical studies, NAC (source 14 http: //vvww.drugs.eom/mmx/mucomyst.html#citec00100716) C-labeled, administered by inhalation, has a rapid elimination of lungs with a half-life ranging from 6 to at 36 minutes (source 12 http: //www.drugs.eom/mmx/mucomyst.html#citec00100716).

NAC is administered as a highly concentrated inhaled hypertonic solution (20% or 1.22 molar) and has been reported to cause bronchoconstriction and cough. In many cases, it is recommended that NAC be administered with a bronchodilator to improve the tolerability of this agent.

Thus, reducing agents, such as NAC, are not suitable for the administration of bolus aerosols. However, it is anticipated that the administration of reducing agents by aerosol pulmonary infusion would increase efficacy, while allowing a reduction of the concentration of reducing agent in the inhalation solution (expected to increase tolerance).

Surfactants and detergents are spreading agents proven to reduce mucus viscoelasticity, improving mucus clearance. Examples of surfactants include dipalmitoylphosphatidylcholine (DPPC), FP, palmitic acid, palmitoyloloylophosphatidylglycerol, proteins associated with surfactants (eg, SP-A, В, or С) or may be derived from animals (eg, from cow's lung or lavage). calf or extracted from porous lung) or combinations thereof. See, for example, US Patents 78,97577 B2 2011.03.01, US 5876970 A 1999.03.02, US 5614216 A 1997.03.25, US 5100806 A 1992.03.31, US 4312860 A 1982.01.26. Examples of surfactant products include Exosurf® Neonatal (colfosceril palmitate), Pumactant® (DPPC and egg phosphatidylglycerol), KL-4 surfactant, Venticute® (lusultide, rSP-C surfactant), Alveofact® (bovactant), Curosurf® (alpha poractant), Infasurf® (calfactant), Newfacten® (modified bovine surfactant), Surface®, Natsurf ™ (nonionic ethoxylated alcohol surfactant) and Survanta® (beractant). Examples of detergents include, but are not limited to, Tween-80 and triton-X 100.

MD 4574 Cl 2019.01.31

Any suitable expectorant can be used, including but not limited to guaifenesin (US 7345051 B2 2008.03.18). Any suitable deoxyribonuclease can be used, including but not limited to Domase Alpha (US 7482024 B2 2009.01.27). Examples of kinase inhibitors include inhibitors of NFkB, PI3K (phosphatidylinositol 3-kinase), p38MAP kinase, and Rho kinase.

Anti-infectious agents for formulation and use in combination with the compounds of the invention include antiviral and antibiotic preparations. Examples of suitable antiviral drugs include Tamiflu® (oseltamivir) and Relenza® (zanamivir). Examples of suitable antibiotic preparations include, but are not limited to, aztreonam (arginine or lysine), phosphomycin and aminoglycosides such as tobramycin, or any combination or subset thereof. Additional anti-infectious agents that may be used herein include aminoglycosides, Daptomycin, Fluoroquinolones, Ketolides, Carbapenems, Cephalosporins, Erythromycin, Linesolid, Penicillins, Azithromycin, Clindamycin, Oxazolidinone, Tetracycline and Vancomycin.

Examples of useful carbapenem antibiotics are impenam, panipenam, meropenam, biapenam, MK-826 (L-749,345), DA-1131, ER-35786, lenapenam, S-4661, CS-834 (prodrug of R-95867), KR- 21056 (prodrug of KR-21012), L-084 (prodrug of LJC 11036) and Ceftolozan (CXA-101).

Antihistamines (i.e., H1 receptor antagonists) for formulation and use in combination with the compounds of the invention include, but are not limited to: ethanolamines, such as diphenhydramine HCl, carbinoxamine maleate, doxylamine, clemastine fumarate, diphenylhydramine HCl, and dimen; ethylenediamines such as pyrillamine maleate (metpiramine), tripelennamine HCl, tripelennamine citrate and antazoline; alkylamines, such as pheniramine, chlorofeniramine, bromofeniramine, dexlorophenamine, triprolidine and acrivastine; pyridines, such as metapyrylene, piperazines, such as hydroxyzine HCl, hydroxyzine pamoate, cyclicine HCl, cyclicine lactate, meclizine HCl, and cetirizine HCl; piperidines, such as astemizole, levocabastine HCI, loratadine, decarboxylethoxysatadine, terfenadine and fexofenadine HCI, tri- and tetracyclic, such as prometazine, trimeprazine, chlorprometazine and azatadine; and azelastine HCl, or any combination or subset thereof.

Examples of other classes of therapeutic agents suitable for use in combinations and methods of the present invention include antiviral drugs, such as ribavirin, antifungal agents, such as amphotericin, intraconazole, and voriconazole, anti-rejection drugs, such as cyclosporine, tacrolimus and sirrolimus. bronchodilators, including, but not limited to, anticholinergic agents, such as atrovent, RNA, genotherapeutic vectors, aptamers, endothelin receptor antagonists, alpha-l-antitrypsin and prostacyclines.

In the treatment methods and uses described above, a compound of the invention may be used alone or in combination with one or more sulfur therapeutically active agents. Typically, any therapeutic active agent, which has a therapeutic effect in the disease or condition that is treated with the compound of the invention, may be used in combination with the compounds of the invention, provided that the particular therapeutic active agent is compatible with therapy using a compound of the invention. Typically therapeutically active agents that are suitable for use in combination with the compounds of the invention include the agents described above.

In a preferred embodiment, the compounds of the invention are used in combination with one or more osmolific moieties, in particular hypertonic solution or mannitol.

In another aspect, the invention relates to methods of treatment and uses, as described above, which include administering an effective amount of a compound of the invention and at least one other therapeutic active agent. The compounds of the invention and at least one additional therapeutic active agent may be used concomitantly or sequentially in any therapeutically appropriate combination. Administration of a compound of the invention with one or more sulfur therapeutically active agents may be by co-administration in a 1) unique pharmaceutical composition, such as the compositions described above, or 2) separate pharmaceutical compositions each including one or more of the ingredients active components. The components of the combination may be administered separately in a sequential manner, wherein the compound of the invention is administered first and the other therapeutically active agent is administered second or vice versa.

In embodiments in which the compound of the invention is administered in combination with one or more osmolific moieties, the administration of each component is preferably concomitant, and may be in a single composition or separate compositions. In one example

Embodiment MD 4574 Cl 2019.01.31, the compound of the invention and one or more osmolific sulfates are co-administered by transbronchoscopic lavage. In another embodiment, the compound of the invention and one or more osmolific sulfates are co-administered by inhalation.

When a compound of the invention is used in combination with another therapeutically active agent, the dose of each compound may be different! than when the compound of the invention is used alone. The appropriate doses will be easily determined by one skilled in the art. Adequate dose! of the compound of the invention, the other therapeutically active agent (s) and the relative timing of administration will be selected to achieve the desired combined therapeutic effect, and are at the discretion and discretion of the caregiver, clinician or veterinarian.

Experimental procedures The present invention refers! also for processes for the preparation of the compounds of the invention and for synthetic intermediates used in such processes, as described in detail below.

Certain abbreviations and acronyms are used in the description of synthesis processes! and experimental details. Although most of them will be defeated by a specialist in the field, the table below confines the list! many of these abbreviations and acronyms.

<td>Abbreviating AcOH AIBN DIAD DIPEA DCE DCM DMF et EtOAc or EA EtOH ESI HATU hexafluorophosphate HPLC iPrOH it or IT</td><td>Semnificafie Acetic acid Azobisizobutirolnitril Diisopropyl azidocarboxylate N, N -diisopropylethylamine! dichloroethane dichloromethane dim ethyl starch form! Ethyl Ethyl acetate ethanol ionization by electropolishing 2- (1H-7-azabenzotriazole-1-yl) -1,1,3,3-tetramethyl uronium Liquid chromatography! with performance! high! isopropyl alcohol intratracheal</td>

<td>Me MeOH m / z or m / e MH⁺MH LITTLE MS or ms rt or rt f t-Bu THF TLC or tick Cbz AUC MTBE Ir GC-MS wt% AcOH h min</td><td>Methyl methanol the relationship between mass! and task! table plus 1 mass minus 1 Minimum inhibitory concentration! mass spectrum! room's temperature Delay factor tert-butyl tetrahydrofuran sub-layer chromatography p! rfi per million in the low fields faf! of tetramethylsilane benzyloxycarbonyl, that is! - (CO) O-benzyl Area under the curve! or peak Methyl terf-butyl ether Retention time Gas chromatography-mass spectrometry! Percentage of meal! Acetic acid hours minute</td>

<td>MHz UV TFA</td><td>megacycle Trifluoroacetic acid Ultraviolet</td>

MD 4574 Cl 2019.01.31

Tertiary butyloxycarbonyl

DIAD Diisopropyl azodicarboxylate

DIPEA Ν, Ν-diisopropylethylamine or Hunig base

РЬзР Triphenylphosine

The compounds of Formula I can be synthesized using techniques known in the art. The representative synthesis procedure is illustrated in Scheme 1 below.

Scheme 1

<img file="MD4574C1_D0029.tif" />

These procedures are described in EJ Cragoe. The Synthesis of Amiloride and Its Analogs, chap. 3, and Amiloride and Its Analogs, pp. 25-36. Other processes for the preparation of amiloride analogues are described in Cragoe, in particular the methods А, В, C and D of US patent 3318813 A 1967.05.09. However, other processes that can be adapted to prepare the compounds of the invention are described in WO 2003070182 A2 2003.08.28, WO 2004073629 A2 2004.09.02, WO 2005108644 A2 2005.11.17, WO 2005022935 A2 2005.03.10, US 7064129 B2 2006.06.20, US 6858615 B2 2005.02.22, US 6903105 B2 2005.06.07, WO 2007146869 To 2007.12.21, WO 2007018640 To 2007.02.15, all assigned to Parion Sciences, Inc.

The preparation of methyl N'-3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (2) can be seen in WO 2009074575 A2 2009.06.18.

In general, the compounds of the invention may be prepared by treating a compound of Formula 2 with о amine of Formula 3. More specifically, the compounds of Formula 2 are treated with amine of Formula 3 in a suitable solvent, such as methanol, ethanol or tetrahydrofuran. and a base, such as triethylamine (TEA) or di-isoproethylamine (DIPEA), with high temperature heating, for example, 70 ° C. Purification, resolution of stereoisomers, crystallization and / or subsequent preparation of the salt forms can be performed using conventional techniques.

As will be apparent to those skilled in the art, in some cases, intermediate or synthetic compounds may possess other functional groups that provide alternative reactive sites. Interference with these functional groups can be avoided by using appropriate protective groups, such as amine or alcohol protecting groups, and, where appropriate, prioritizing the synthesis steps accordingly. Adequate protective groups will be evident to those skilled in the art. Methods are well known in the art for the installation and removal of these protecting groups and these conventional techniques can also be used in the processes of the present invention.

The following specific examples are fumigated here for the purpose of illustration only and do not limit the scope of the invention, which is defined by the claims.

Materials and methods

All reagents and solvents were purchased from Aldrich Chemical Corp. ChemImpex International Inc. and TCI chemical industry Co. Ltd. NMR spectra were obtained on either a 400 MHz Broker AC 400 fH NMR spectrometer and¹³C MRI at 100 MHz) or a Bruker AC 300 (Ή MRI at 300 MHz and ¹³C NMR at 75 MHz). Proton spectra were reported to tetramethylsilane as an internal standard, and carbon spectra were reported to CDCI3, CD3OD or DMSO-dk (purchased from Aldrich or Cambridge Isotop Laboratories, unless otherwise specified). Rapid chromatography was performed on a Combiflash system (Combiflash Rf, Teledyne Isco) in a silica gel loaded column (Redi Sep. Rf, Teledyne Isco) or a о inverse phase column (High performance C18 Gold column). ESI mass spectra were obtained on a Shimadzu LCMS-2010 EV Mass Spectrometer mass spectrometer. HPLC analyzes were obtained using a Waters Xterra MS Cl 8 5 µm 4.6x150mm analytical column detected at 220 nm (unless otherwise specified) on a Shimadzu Prominence HPLC system. The following time program was used with a flow rate of 1.0 ml per minute:

MD 4574 Cl 2019.01.31

<td>Time (min)</td><td>Percentage A (H₂O with 0.05% TFA)</td><td>Percentage В (CH₃CN with 0.05% TFA)</td>

<td> 2.50</td><td> 90</td><td> 10</td>

<td> 20.00</td><td> 10</td><td> 90</td>

<td> 30.00</td><td> 10</td><td> 90</td>

<td> 32.50</td><td> 90</td><td> 10</td>

UPLC analyzes were obtained using о Waters Acquity UPLC HSS T3 analytical column 1.8 μπι 2.1x100 mm detected at 220 nm (unless otherwise specified) on a Shimadzu Prominence UFLC system. The following time program was used with a flow rate of 0.3 ml per minute: _____________________________________________________________

<td>Time (min)</td><td>Percentage A (H₂O with 0.05% NH4COOH and 0.1% HCOOH)</td><td>Percentage В (CFFCN / water 80: 20% with 0.05% NH4COOH and 0.1% HCOOH)</td>

<td> 1.00</td><td> 90</td><td> 10</td>

<td> 4.00</td><td> 30</td><td> 70</td>

<td> 5.00</td><td> 30</td><td> 70</td>

<td> 5.50</td><td> 90</td><td> 10</td>

<td> 6.50</td><td> 90</td><td> 10</td>

Also, (Method 2) there is provided a method of preparing compound (Ia), 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6 pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, as defined hereinbefore,

OH OH

<img file="MD4574C1_D0030.tif" />

which includes the stages of:

<img file="MD4574C1_D0031.tif" />

with a protected sugar, (4aR, 6S, 7R, 8R, 8aS) -2-phenylhexahydropyran [3,2-d] [1,3] dioxin6,7,8-trio, of formula 15:

OH

<img file="MD4574C1_D0032.tif" />

<img file="MD4574C1_D0033.tif" />

Ph 15 in the presence of a reducing agent, followed by hexane treatment to form compound 16, benzyl 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R ) -5-hydroxy-2-phenyl-3dioxan-4-yl) propyl) (hexyl) amino) ethoxy) phenyl) butylcarbamate;

MD 4574 Cl 2019.01.31

OH OH

<img file="MD4574C1_D0034.tif" />

2. subjecting compound 16 to catalytic hydrogenation, to form compound 17, (1R, 2S) -3 - ((2- (4- (4-aminobutyl) phenoxy) ethyl) (hexyl) amino) -1 - ((4R, 5R) -5-hydroxy-2-phenyl-l, 3-dioxane-4-yl) propane-l, 2-diol; and

OH OH

<img file="MD4574C1_D0035.tif" />

3. concentration of compound 17 with compound 2, methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate, in the presence of a base to form 19, 3,5-diamino-6-chloro-N- (N- (4- (4- ( 2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy-2-phenyl-3dioxan-4-yl) propyl) (hexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide; and

<img file="MD4574C1_D0036.tif" />

4. hydrolysis of compound 19 in the presence of acid, to form (at).

An alternative process includes replacing the compound of formula 16, above, with compound 27, followed by the hydrogenation, condensation and hydrolysis steps described above, to form compound (Ia).

Also, (Scheme 3) an alternative method of preparing compound (Ia), 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S) , 3R, 4R, 5R) -

2.3.4.5.6- pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, as defined hereinabove.

Scheme 2. Preparation of 3,5-Diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 37?, 47?, 57?) -

2.3.4.5.6- pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

<img file="MD4574C1_D0037.tif" />

<img file="MD4574C1_D0038.tif" />

<img file="MD4574C1_D0039.tif" />

alternative of 3,5-Diamino-6-chloro-N- (N- (4- (4- (2Scheme 3. Preparation of (hexyl ((25,3R, 4R, 5R) -2,3,4,5,6pentahydroxyhexyl ) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

OH

<img file="MD4574C1_D0040.tif" />

<img file="MD4574C1_D0041.tif" />

<img file="MD4574C1_D0042.tif" />

Examples

The invention also comprises a compound prepared by the methods herein, or a pharmaceutically acceptable salt of the compound.

Synthesis of, 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroxyhexyl)) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

Preparation of benzyl 4- (4- (3- (tert-butyloxycarbonylamino) propoxy) phenyl) butyl carbamate (Compound 13):

To the solution of benzyl 4- (4-hydroxyphenyl) butyl carbamate (11.60.0 g, 300 mmol) in dry THF (600 mL) was added N-Ethanolamine (12.38.7 g, 300 mmol), Ph3P (62.9 g, 300 mmol) and DIAD (48.6 g, 300 mmol) at 0 ° C, then the reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 15:85 EA / hexane) to give the desired compound 13 (50.0 g, 57%) as a color solid. yellow: Ή NMR (400 MHz, CDC1₃) δ 7.35 (m, 5H), 7.10 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 2H), 5.10 (s, J = 4.0 Hz, 2H), 4.0 (m, 2H), 3.5 (q, 2H), 3.2 (q, 2H), 2.55 (t, J = 8.0 Hz, 2H) ), 1.60 (m, 2H), 1.55 (m, 2H), 1.45 (s, 9H).

MD 4574 Cl 2019.01.31

Step 2

Preparation of the benzyl hydrochloric acid salt of 4- (4- (2-aminoethoxy) phenyl) butylcarbamate (14):

Compound 13 (50.0 g, 112 mmol) was dissolved in 4 N HC1 in dioxane (250 mL) at room temperature and the solution was stirred! for 1 hour! After! it was concentrated, the residue was suspended in MTBE (500 mL) and stirred for 0.5 hours. Solid phase! was leaked! to obtain the hydrochloric acid salt 14 (40.0 g, 83%) as a white solid solution: Ή NMR (300 MHz, CD₃OD) δ 7.33 (m, 5H), 7.10 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.18 (t, 2H), 3.39 (m, 2H), 3.14 (t, J = 7.2 Hz, 2H), 2.56 (t, 7 = 7.5 Hz, 2H), 1.57 (m, 4H).

Step 3

Preparation of Benzyl 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy-2-phenyl-1, 3dioxane-4-yl) propyl ) (hexyl) amino) ethoxy) phenyl) butylcarbamate (16):

О hydrochloric acid salt solution 14 (13.5 g, 39.35 mmol) and triol 15 (10.5 g, 39.35 mmol) in MeOH (150 mL) and AcOH (18.8 g, 314.8 mmol) was agitated! at room temperature for 2 hours, cyanoborohydride! of sodium (6.1 g, 98.37 mmol) was added and the reaction mixture was stirred at room temperature overnight. Additional triol 15 (5.2 g, 19.67 mmol) was added and the reaction mixture was stirred at room temperature for 4 hours. After! what a raw material! 14 was completely consumed, hexane was added (5.9 g, 59.03 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was washed with saturated solution! from Na₂CO₃ (5.0 mL), azeotroped with MeOHl and purified by column chromatography! (silica gel, 10: 1 CILClyMeOH) to give compound 16 (12.2 g, 46% in two steps) as a gray-white solid solution: Ή NMR (300 MHz, CD)₃OD) δ 7.457.44 (m, 3H), 7.31-7.29 (m, 9H), 7.05 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 8 , 4 Hz, 2H), 5.50 (s, 1H), 5.05 (s, 2H), 4.25-4.18 (m, 2H), 4.03-3.87 (m, 6H) , 3.78-3.55 (m, 3H), 3.13-2.96 (m, 6H), 2.85-2.69 (m, 3H), 2.53 (t, J = 6, 7 Hz, 2H), 1.58-1.48 (m, 6H), 1.23 (br s, 6H), 0.86 0.7 = 6.1 Hz, 3H).

Step 4

Preparation of (1R, 2S) -3 - ((2- (4- (4-aminobutyl) phenoxy) ethyl) (hexyl) amino) -1- ((4R, 5R) -5-hydroxy-2 acetic acid salt -phenyl-1,3-dioxane-4-yl) propan-1,2-diol (17):

A suspension of carbamate 16 (12.2 g, 17.99 mmol) and 10% Pd / C (3.66 g) in EtOH / AcOH (5: 1, 120 mL) was submitted! under hydrogenation conditions (1 atm) overnight at room temperature. The reaction mixture was filtered through celite! and sprayed with EtOH. The filtrate was concentrated in vacuo to obtain the acetic salt! 17 (9.40 g, 96%) in the form! colorless oil: Ή NMR (300 MHz, CD3OD) δ 7.48-7.44 (m, 2H), 7.32-7.30 (m, 3H), 7.11 (d, 7 = 8.5 Hz, 2H), 6.84 (d, 7 = 8.5 Hz, 2H), 5.51 (s, 1H), 4.26-4.10 (m, 3H), 3.95-3.91 (m, 2H), 3.78 (dd, 7 = 1.8, 9.3 Hz, 1H), 3.60 (t, 7 = 10.4, 1H), 3.23-3.03 (m , 2H), 2.96-2.87 (m, 3H), 2.61-2.59 (m, 2H), 1.67-1.57 (m, 6H), 1.31-1,. 25 (br s, 6H), 0.89 0.7 = 6.6 Hz, 3H).

Step 5

Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - (4R, 5R) -5hydroxy- 2-phenyl-1,3-dioxan-4yl) propyl) (hexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (19)

To a solution of acetic acid salt 7 (9.40 g, 17.27 mmol) and hydroiodic acid salt of methyl 3,5-diamino-6-loropyrazine-2-carbonylcarbamimidothioate (18, 7.20 g, 27, 64 mmol) in EtOH (75 mL) was added DIPEA (17.8 g, 138.16 mmol) at room temperature. The reaction mixture was heated to 70 ° C in a tube! hermetic! for 2 hours, then cooled to room temperature, and concentrated in vacuo. The residue was purified by column chromatography! (silica gel, CH₂Cl₂/ MeOH 09: 1, 80: 18: 2 CHCL / MeOH / NFLOH) to obtain carboxamide 19 (9.20 g, 70%) as a solid solution! Yellow !: 1H NMR (300 MHz, CD3OD) δ 7.46-7.43 (m, 2H), 7.30-7.28 (m, 3H), 7.07 (d, 7 = 8.6 Hz , 2H), 6.79 (d, 7 = 8.6 Hz, 2H), 5.48 (s, 1H), 4.22 (dd, 7 = 3.9, 7.8 Hz, 1H), 4 , 06-3.88 (m, 5H), 3.75 (dd, 7 = 1.5, 6.9 Hz, 1H), 3.57 (t, 7 = 10.5 Hz, 1H), 3, 25 (t, 7 = 6.6 Hz, 2H), 2.932.83 (m, 3H), 2.68-2.56 (m, 5H), 1.70-1.64 (m, 4H), 1 , 44-1.43 (m, 2H), 1.22 (m, 6H), 0.85 0.7 = 8.1 Hz, 3H).

MD 4574 Cl 2019.01.31

Step 6

Preparation of the hydrochloric acid salt of 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5, 6pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (la):

To о carboxamide solution 19 (9.20 g, 12.16 mmol) in EtOH (30 mL) was added 4 N aqueous HCl (95 mL) at room temperature and the reaction mixture was stirred for 4 hours at room temperature. . The reaction mixture was concentrated in vacuo and the residue was purified by reverse phase column chromatography and lyophilized to give hydrochloric acid salt at (6.60 g, 81%) as a yellow hygroscopic solid solution: Д NMR (300 MHz, CD₃OD) δ 7.17 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 4.36 (br s, 2H), 4.21-4 , 19 (m, 1H), 3.84-3.61 (m, 7H), 3.46-3.30 (m, 5H), 2.64 (t, J = 6.5 Hz, 2H), 1.80-1.69 (m, 6H), 1.36 (br s, 6H), 0.91 (t, J = 6.6 Hz, 3H); ESI-MS m / z 669 [C₃oH49C1N₈0₇ + H]⁺; Anal. (СзоН4₉СШ₈0₇-2НС1-Н₂0). Calc. C 47.40, H 7.03, N 14.74; Detected. C 47.11, H 7.06, N 14.54.

Alternative synthesis of I, 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -

2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

Step 1

Preparation of Benzyl 4- (4- (3 - ((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy-2-methyl-1, 3dioxan-4-yl) propylamino) propoxy) phenyl) butylcarbamate (26):

The hydrochloric acid salt solution 14 (155 mg, 0.41 mmol) and triol 15 (84 mg, 0.41 mmol) in MeOH (5.0 mL) was stirred at room temperature for 0.5 h, then AcOH (0.036 mL, 0.6 mmol) and sodium cyanoborohydride (43 mg, 0.6 mmol) were added and the reaction mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo. The residue was washed with saturated Na solution₂CO₃ (5.0 mL), azeotroped with MeOH and purified by column chromatography (silica gel, CH₂Cl₂/ MeOH 10: 1, 10: 1: 0.1 CHCl₃/ MeOH / NH4OH) to afford carbamate 26 (163 mg, 75%) as a white gum solid solution: Д NMR (300 MHz, CD3OD) δ 7.34-7.30 (m, 5H), 7, 08-7.05 (m, 2H), 6.85-6.82 (m, 2H), 5.06 (s, 2H), 4.70-4.67 (m, IH), 4.08- 3.96 (m, 4H), 3.82-3.76 (m, 2H), 3.49-3.46 (m, IH), 3.14-3.10 (m, 2H), 3, 01-2.79 (m, 4H), 2.65-2.45 (m, 2H), 2.05-2.01 (m, 2H), 1.59-1.49 (m, 4H), 1.27 (d, J = 4.8 Hz, 3H).

Step 2

Preparation of Benzyl 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy-2-methyl-1, 3dioxane-4-yl) propyl ) (hexyl) amino) ethoxy) phenyl) butylcarbamate (27):

О carbamate solution 26 (1.02 g, 1.90 mmol), hexane (380 mg, 3.80 mmol), AcOH (0.33 mL, 5.70 mmol) and sodium cyanoborohydride (410 mg, 5, 70 mmol) in MeOH (30 mL) was stirred at room temperature for 16 hours. The solvent was removed in vacuo. The residue was washed with saturated Na solution₂CO₃ (30 mL), azeotroped with MeOH and purified by column chromatography (silica gel, CH₂Cl₂/ Mc () H 10: 1) to afford carbamate 27 (990 mg, 84%) as a white gum solid solution: Д NMR (300 MHz, CD3OD) δ 7.35-7.31 (m, 5H), 7.06 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz , 2H), 5.08 (s, 2H), 4.80 (br s, IH), 4.69-4.66 (m, IH), 4.12 (dd, J = 9.3, 2, 4 Hz, IH), 4.053.98 (m, 3H), 3.84-3.76 (m, 2H), 3.54-3.48 (m, IH), 3.38 (t, J = 10 , 5 Hz, IH), 3.20-2.96 (m, 4H), 2.83 (d, J = 6.0 Hz, 2H), 2.73-2.64 (m, 2H), 2 , 56 (t, J = 7.2 Hz, 2H), 1.63-1.50 (m, 6H), 1.32 (d, J = 5.1 Hz, 3H), 1.27-1, 24 (m, 6H), 0.87 (d, J = 6.6 Hz, 3H).

Step 3

Preparation of (1R, 2S) -3 - ((2- (4- (4Aminobutyl) phenoxy) ethyl) (hexyl) amino) -1- ((4R, 5R) -5-hydroxy-2-methyl acetic acid salt 1,3-dioxane-4-yl) propane-1,2-diol (28):

The suspension of carbamate 27 (890 mg, 1.44 mmol) and 10% Pd / C (400 mg) in MeOH / AcOH (05: 1, 60 mL) was subjected to hydrogenation conditions (1 atm) for 6 h at room temperature. The reaction mixture was filtered through celite and washed with MeOH. The filtrate was concentrated in vacuo and extracted from ether to give acetic salt 28 (782 mg, 90%) as a white gum solid solution: Д NMR (300 MHz, CD3OD) δ 7.09 (d, J = 8 , 7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.69-4.67 (m, IH), 4.00-3.85 (m, IH), 3.843 , 76 (m, 2H), 3.53-3.51 (m, IH), 3.38 (t, J = 10.5 Hz, IH), 2.98-2.59 (m, 10H), 1.96 (s, 13H), 1.67-1.47 (m, 6H), 1.40-1.27 (m, 6H), 1.26 (d, J = 5.1 Hz, 3H) , 0.88 (d, J = 6.3 Hz, 3H).

MD 4574 Cl 2019.01.31

Step 4

Preparation of 3,5-Diamino-6-chloro-N- (N- (4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5 -5-hydroxy- 2-methyl-1,3-dioxan-4yl) propyl) (hexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (20):

To о solution of acetic acid salt 28 (189 mg, 0.313 mmol) and hydroiodic acid salt of methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (18, 192 mg, 0.502 mmol) in EtOH (8 mL ) DIPEA (0.42 mL, 2.50 mmol) was added at room temperature. The reaction mixture was heated to 70 ° C in an airtight tube for 2 hours, then cooled to room temperature, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CHiCL / MeOH 09: 1, 80: 18: 2 CHCL / MeOH / NFLOH) to obtain carboxamide 20 (142 mg, 65%) as a yellow solid: Ш NMR (300 MHz, CD₃OD) δ 7.10 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 8.1 Hz, 2H), 4.66 (q, J = 5.1 Hz, IH) , 4.06-4.01 (m, 3H), 3.94-3.89 (m, IH), 3.82-3.74 (m, 2H), 3.49 (dd, J = 9, 3, 2.4 Hz, IH), 2.96-2.78 (m, 3H), 2.67-2.61 (m, 5H), 1.68-1.67 (m, 4H), 1 , 50-1.48 (m, 2H), 1.29 (br s, 6H), 1.25 (d, J = 5.1 Hz, 3H), 0.87 (t, J = 6.9 Hz , 3H).

Step 5

Preparation of the hydrochloric acid salt of 3,5-Diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5, 6pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (la)

To о carboxamide solution 20 (400 mg, 0.57 mmol) in EtOH (5 mL) was added 4 N aqueous HCl (15 mL) at room temperature and the reaction mixture was heated at 55 ° C for 24 hours. After being concentrated, the residue was dissolved in 4 N aqueous HCl (15 mL) and heated at 65 ° C for 16 hours. The reaction mixture was concentrated, extracted from EtOH / EtiO, purified by preparative TLC and lyophilized to obtain hydrochloric acid salt (Ia) (354 mg, 83%) as a yellow hygroscopic solid solution: Ή NMR (300 MHz, D₂O) δ 7.18 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 8.1 Hz, 2H), 4.30 (br s, 2H), 4.19-4 , 16 (m, 1H), 3.76-3.55 (m, 7H), 3.39-3.24 (m, 6H), 3.57 (t, J = 5.4 Hz, 2H), 1.65-1.64 (m, 6H), 1.30-1.19 (m, 6H), 0.78-0.75 (m, 3H); ESI-MS m / z 669 [C₃oH49C1N₈0₇ + H]⁺.

Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (free base a)

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) - hydrochloric acid salt of hydrochloric acid salt) -

2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (12.51 g) was dissolved in 150 mL H2O and treated, with stirring, with NaOH (0 , 1 M aqueous, 435 mL) to give a gummy precipitate. The liquid (pH ~ 11) was decanted through a filtration funnel (most of the material adhered to the walls of the vessel). The residue was treated with H2O (2 x 300 mL), stirred and decanted / filtered in a similar manner. The remaining residue was suspended in CILCN / ILO / MeOH and concentrated to give a yellowish-yellow solid solution, 9.55 g. ESI-MS m / z 669 [СзоЖСЖО? + H]⁺, purity 89% at 224 nm, 90% at 272 nm, 82% at 304 nm, 63% through MS trace. The crude product was heated with isopropanol (100 - 150 mL) at 70 ° C for 15 min, then warm filtered. The solid phases were treated similarly to isopropanol twice more, heating 30 minutes each time and allowing the mixture to cool (2 hours - O / N) before filtering. The resulting solid phases were dried to obtain 7.365 g of yellow-amber amorphous solid, for 133.1 - 135.6 ° C (11.0 mmol yield as free base). Ш NMR (400 MHz, dmso) δ 9.31-7.34 (m, 4H), 7.10 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.5 Hz , 2H), 6.61 (br, s, 3H), 4.76-4.09 (m, 5H), 3.98 (t, J = 6.1 Hz, 2H), 3.71-3, 62 (m, 2H), 3.59 (dd, J = 10.8, 3.4 Hz, IH), 3.54-3.46 (m, IH), 3.43 (dd, J = 7, 9, 1.3 Hz, IH), 3.38 (dd, J = 10.8, 5.9 Hz, IH), 3.15 (br s, 2H), 2.92-2.76 (m, 2H), 2.66 (dd, J = 13.1, 5.2 Hz, IH), 2.58-2.51 (m, 4H), 2.46 (dd, J = 13.1, 6, 5 Hz, IH), 1.66-1.45 (m, 4H), 1.45-1.31 (m, 2H), 1.31-1.09 (m, 6H), 0.90-0 , 76 (m, 3H). ¹³C NMR (01 MHz, dmso) δ 173.27, 160.96, 156.48, 154.68, 151.14, 133.70, 129.05, 119.12, 117.53, 114.14, 72 ,2. 3,

71.37, 70,60, 70,04, 65,74, 63,34, 57,39, 54,72, 52,86, 40,10, 33,72, 31,15, 28,37, 28,09,

26.38, 26.36, 22.02, 13.84. ESI-MS m / z 669 [СзоЖСМО + H]⁺.

Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2- hexyl ((2S, 3R, 4R, 5R) -2,3, 1-Hydroxy-2-naphthoate) salt 4,5,6pentahidroxihexil) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

MD 4574 Cl 2019.01.31

A mixture of 32.8 mg (0.049 mmol) of 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2, 3, 4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, 164 pL of 0.3 M solution of l-hydroxy-2-naphthoic acid in methanol (0.049 mmol of l-hydroxy-2-naphthoic acid) and about 0.33 mL of methanol was heated on the hot plate fixed at 85 ° C until the entire solid phase was dissolved. The solution was allowed to cool to ambient temperature. The solution was placed in a refrigerator (about 5 ° C) and allowed to stand overnight, during which time crystallization occurred. The liquid was decanted and the solid phase was dried in a dry air stream to give 29.5 mg (62% yield) of 3,5-diamino-6-chloro-2-naphthoate salt. N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2- carboxamide. Ή NMR (500 MHz, DMSO) 8.2 (m, 1H), 7.7 (m, 2H), 7.4 (d, 1H), 7.3 (d, 1H), 7.1 (m, 2H), 6.95 (m, 1H), 6.85 (m, 2H), 4.6-4.2 (m, 2H), 4.0 (m, 2H), 3.8-3.6 (m, 2H), 3.6-3.2 (m, 6H), 2.9-2.5 (m, 6H), 1.6 (m, 4H), 1.4 (m, 2H), 1.2 (m, 6H), 0.83 (m, 3H) ppm.

Preparation of the 3,5-diamino-6-chloro-N- (N- (4- (4- (2- hexyl ((2S, 3R, 4R, 5R) -2,3, 3-hydroxy-2-naphthoate) salt 4,5,6pentahidroxihexil) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

A mixture of 105.3 mg (0.157 mmol) of 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2, 3,4,5,6pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, 525 µL of 0.3 M solution of 1-hydroxy-2-naphthoic acid in methanol (0.158 mmol of 1 acid -hydroxy2-naphthoic) and about 1 mL of methanol was heated on the hot plate fixed at 85 ° C until the entire solid phase was dissolved. The solution was allowed to cool to ambient temperature and placed in a refrigerator (about 5 ° C). After about 20 minutes, it became cloudy and was sown. After about 2 hours, the solid phase was present. The stirring bar was added to the mixture and the solution was stirred in the refrigerator overnight, during which time it became very thick. Another 1.5 mL of methanol was added and the slurry was stirred in the refrigerator overnight. The mixture was centrifugal, the liquid was decanted and the solid phase was dried in a dry air stream to give 74 mg (55% yield) of 3,5-diamino-6 hydroxy-2-naphthoate salt. chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine -2-carboxamide.

Pharmacology of (la) 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroxyhexyl ) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

Test 1. In vitro measurement of the reversibility and activity of sodium channel biocharging

A test used to evaluate the mechanism of action and / or efficacy of the compounds of the present invention involves the determination of luminal drug inhibition of sodium epithelial currents in short-circuit current (ISC) airways using epithelial airway monolayers mounted in Ussing chambers. . The cells are obtained from freshly excised human airways, canine, sheep or rodent. This test is described in detail in Hirsh AJ et al. Pharmacological properties of N- (3,5-diamino-6-chloropyrazine-2-carbonyl) -N'-4- [4- (2,3-dihydroxypropoxy) phenyl] butylguan published in methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for CF lung disease. Journal of Pharmacology and Experimental Therapeutics, 2008, vol. 325, no. 1, pp. 77-88.

Inhibition of transcellular sodium displacement by ENaC was measured using monolayers of polarized bronchial epithelial cells mounted in a modified Ussing chamber. Primary cultures of canine or human bronchial epithelial cells cultured using the air-liquid interface were tested under voltage-setting conditions. Short-circuit current (Isc) was measured as a transepithelial sodium transporter to evaluate the efficacy.

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) ) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide was a very effective inhibitor of transcellular sodium transport and was about 60 times more active than amiloride in canine epithelial bronchial cells (CBE), and approximately 160 times in human epithelial bronchial cells (BEU) (Figure 1). In CBE

MD 4574 Cl 2019.01.31

The compound (at) had an IC50 of 13,218.0 nM and in HBE The compound (at) had an IC50 of

2,411.8 nM (Table 1).

Table 1. Inhibition of the short-circuit current by Compound (at) in canine epithelial bronchial cells and human epithelial bronchial cells (ICso nM) _____________

<td>Species</td><td>amiloride</td><td>Compound I (Parental)</td>

<td>canine</td><td> 781,5+331 (40)</td><td> 13,2±8,0 (7)*</td>

<td>human</td><td> 389+188 (22)</td><td> 2,4±1,2 (4)*</td>

Values ​​represent mean 1 DS (n) * Indicates significance (p &lt;0.05) compared to amiloride

Short-circuit current (Isc) recovery from the maximal block was used as an indirect measurement of the drug's off-rate constant. Percent recovery of Isc after complete blockage, determined after three washes of the apical surface and calculated by the formula: (Isc) restored / (Isc) pre-treatment x 100, was significantly (22 times) less reversible than amiloride in CBE and 9 , 5 times less in the HBE (Table 2), indicating that the Compound (la) produces more durable, longer biochar on ENaC.

Table 2. Reversibility of Compound (at) on short-circuit current in canine epithelial bronchial cells and human epithelial cells (%

<td>Species</td><td>amiloride</td><td>Compound</td>

<td>canine</td><td> 90,1 ±27,6 (39)</td><td> 4,1 ± 11,6 (7)*</td>

<td>human</td><td> 89,5 ± 10,7 (4)</td><td> 9,4 ± 17 (3)*</td>

Values ​​are mean ± DS (n) * Indicates significance (p &lt;0.05) compared to amiloride

Test 2. Studies of mucociliary clearance (CMC) in sheep

The animal model that has been used most often to measure changes in CMC is the sheep model. The effect of compounds for increasing mucociliary clearance (CMC) can be measured using an in vivo model described in: JR Sabater et al. Aerosolization of P2Y2-receptor agonists enhances mucociliary clearance in sheep. Journal of Applied Physiology, 1999, vol. 87, no. 6, pp. 2191-219, incorporated herein by reference.

In these studies, adult sheep were immobilized and nasally intubated with an endotracheal tube. The test aerosolized products were administered for 10-15 minutes in sheep. colloid^99mRadioactive isitope-labeled tc-sulfur (TSC, 3.1 mg / ml; containing about 20 mCi) was then administered at a specified time four or eight hours after the test product. The radioactive isotope-labeled aerosol was administered through the endotracheal tube for approximately 5 minutes. The sheep were then detubulated, and the total radioactive indices in the lungs were measured every 5 minutes for an observation period of 1 hour. The clearance rate of radioactive labeling in the lung is representative of the CMC rate in the animal. The advantage of this system is that it closely simulates the human lung environment. The model also allows the simultaneous collection of PK / PD information by selecting plasma and urine samples during the test period. There are also several techniques for measuring the concentrations of the drug on the airway surface during CMC measurements. These include collection of exhaled air condensates or perfusion method to obtain ASL by bronchoscopy.

The sheep model described above was used to evaluate the in vivo effects (efficacy / durability) of Compound (at) administered with aerosols on CMC. Treatments consisting of either 4 mL of Compound (at), Comparative Example 1, Comparative Example 4, vehicle (H₂A sterile distillate, or test agent in combination with SH were tested. To determine whether SH is combined with Compound (at) CMC, SH was administered immediately after Compound (at) was administered. The test solutions were aerosolized using a Raindrop nebulizer at a rate of eight liters per minute and connected to a dosimetric system consisting of a solenoid valve and a compressed air source (20 psi). The dose of drug stored in sheep's lungs, after aerosol administration using the Raindrop nebulizer is estimated at 8-15% of the dose. Using a Raindrop nebulizer, radioactive isotope-labeled TSC was administered over approximately 3 minutes, either 4 or 8 hours after treatment, to evaluate

MD 4574 Cl 2019.01.31 efficiency / durability. Radioactive indices were measured in a central region in the right lung at 5 min intervals for о hour with the о gamma camera. Three methods of analysis were used, 1) the initial clearance rate (tilt angle) in the first 30 min established using linear regression 2) the area under the curve for% clearance in 5 time over о hour, and 3) maximum clearance obtained in one hour.

The effects of Compound (at) at 16 pg / kg, 0.16 pg / kg and 0.016 pg / kg were tested and compared with those of the vehicle (4 mL H₂One sterile) on sheep CMC four hours after dosing (Figure 2). Effect analyzes are presented in Table 3. At all doses tested, Compound (at) intensified CMC compared to the control vehicle. The dose of 10 16 pg / kg was considered to be a maximal effect of CMC.

Table 3. CMC in sheep at 4h after dosing of Compound (at) or vehicle

<td>The dose of Compound I</td><td>Initial tilt angle (4.0-4,511)</td><td>AUC (% Cl -h)</td><td>Maximum clearance</td>

<td>16 gg / kg</td><td> 39,0±3,9* (4)</td><td> 18,6±2,2*^r (4)</td><td> 33,8, ±3,7*^f (4)</td>

<td>0.16 gg / kg</td><td> 39,1 (2)</td><td> 19(2)</td><td> 33,1 (2)</td>

<td>0.016 pg / kg</td><td> 33,3±4,4* (4)</td><td> 14,4±1,3* (4)</td><td> 25,5±1,3* (4)</td>

<td>Vehicle (H₂O) 4 mL</td><td> 17,2±6,8 (8)</td><td> 7,3+1,5 (8)</td><td> 12,2±2,9 (8)</td>

Data are reported as mean + DS (n). The study with n = 2, not included in the statistical analysis. * - Indicates the significance (p &lt;0.05) of the vehicle.^f - Indicates significance (p &lt;0.05) from 0.016 Hg / kg ·

To determine if SH increases the CMC effect of Compound (Ia), SH (6.25 mL of 10% SH; 62.5 mg stored, assuming 10% deposition) was dosed immediately after 0.016 µg / kg of Compound (Ia) and CMC was evaluated four hours after the combined dosage (Figure 12). SH increased the effect of a dose of 0.016 pg / kg of Compound (la) to a maximum effect, as seen with both 0.16 and 16 pg / kg doses of Compound (la) alone 20 (Figure 2). Therefore, a maximal effect of CMC can be achieved when SH is added at о dose (0.016 µg / kg) of Compound (IA) which produces a response below the maximum value when administered without SH.

Table 4. CMC in sheep at 4h after vehicle dosage, Compound (at) and

SH

<td>Dose</td><td>Initial tilt angle (4.0-4,511)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>Compound (0.016 gg / kg; 4 mL + SH)</td><td> 44,9 (2)</td><td> 20,7 (2)</td><td> 37,0 (2)</td>

<td>Compound (0.016 pg / kg; 4 mL)</td><td> 33,3±4,4 (4)</td><td> 14,4+1,3 (4)</td><td> 25,5+1,3 (4)</td>

<td>Vehicle H₂O (4 mL)</td><td> 17,2±6,8 (8)</td><td> 7,3+1,5 (8)</td><td> 12,2+3 (8)</td>

Data are reported as mean + DS (n). The study with n = 2, not included in the statistical analysis.

To assess both the durability of Compound (Ia) and the effect of adding SH to Compound (Ia) CMC was measured eight hours after vehicle dosing (H).₂O), 7% SH alone, 0.16, 1.6 and 16 pg / kg of Compound (to) alone or о combination of 0.16 pg / kg of Compound (to) and 7% SH (total volume (4 mL for each treatment) (Figure 4). 30 After vehicle dosing, the CMC at 4 and 8 hours was the same, indicating that the CMC rate in sheep over 4-8 hours is in a stagnant state (Figures 12 and 13). Eight hours of administration of 4 mL of 7% SH, no change in CMC was observed compared to the vehicle, indicating that the effect of SH was gone. All three dose groups (0.16, 1.6 and 16 µg / kg) of Compound (at) increased CMC in a dose-dependent manner in 35 compared to vehicle and SH, indicating that Compound (at) has a longer duration of action than SH alone (Figure 4). The combined dose of SH and Compound (at) increased the effect of a dose of 0.16 pg / kg of Compound (at) higher than that observed at the dose of 16 pg / kg, indicating о 100-fold increase in activity when SH was added to Compound (Ia) (Figure 4). The intensification of the activity of Compound (at) by SH, at a time when SH 40 has no inherent activity, clearly indicates the synergy between SH and Compound (at).

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Table 5. CMC in sheep at 8h after vehicle dosing, SH, Compound (at) or о combination of SH and Compound (at)

<td>Dose</td><td>Initial tilt angle (8.0-8.5h)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>Vehicle - H₂O (4 mL)</td><td> 17,8±5,7 (4)</td><td> 7,8±1 (4)</td><td> 14,2±0,7 (4)</td>

<td>7% SH (4 mL)</td><td> 17,8 (2)</td><td> 7,6 (2)</td><td> 14,6 (2)</td>

<td>Compound (la) (0.16 pg / kg; 4 mL)</td><td> 24,0 (2)</td><td> 10,7 (2)</td><td> 19,7 (2)</td>

<td>Compound (la) (1.6 pg / kg; 4 mL)</td><td> 24,4 (2)</td><td> 11,1(2)</td><td> 21,2 (2)</td>

<td>Compound (16 pg / kg; 4 mL)</td><td> 28,0 (2)</td><td> 13,9 (2)</td><td> 26,7 (2)</td>

<td>Compound (0.16gg / kg + 7% HS; 4mL)</td><td> 30,9±2,5 (4)</td><td> 15,3±2,2 (4)</td><td> 27,5±1,6 (4)</td>

Data are reported as mean + DS (n). Study with n = 2, not included in the statistical analysis. Test 3. d. Clearance of the drugs from the airway fluid 5 (ASL) and the metabolism through the human airway epithelium.

Disappearance of Compound (at) on apical surface and airway epithelial metabolism were evaluated in HBE (Table 6). In these experiments 25 µL of 25 µl solution of ENaC blocker was added to the apical surface of HBE cells grown at о air / liquid interface, and drug concentration in the apical and 10 basolateral compartments was measured over 2 hours by UPLC. After 2 h of incubation of Compound (at) on the apical surface (37 ° C), no metabolic detections were detected on the apical or basolateral side and neither Compound (at) was detectable on the basolateral side.

Table 9. Disappearance and apical metabolism of Compound (at) by HBE

<td>compound</td><td>% of the initial mass of the drug on the apical side (parental and metabolite, 2h)</td><td>% of apical mass as metabolites (2h)</td><td>% of initial apical mass on the basolateral side (2h)</td><td>% on the basolateral side as metabolites (2h)</td>

<td>Compound (to)</td><td> 80,7*±6,2%</td><td>no one</td><td>no one</td><td>no one</td>

Test 4. E. Hydration of the airways and blocking of the sodium channels (in vitro model)

Parion Sciences has developed experimental models for evaluating airway hydration in cell cultures (Hirsh AJ et al. Pharmacological properties of N- (3,5-diamino-6-chloropyrazine-2-carbonyl) -N'-4- [4- (2 , 3-dihydroxypropoxy) phenyl] butylguanipublicata lae methanesulfonate (552-02), a novel epithelial sodium channel blocker 20 with potential clinical efficacy for CF lung disease. Journal of Pharmacology and Experimental Therapeutics, 2008, vol. 325, no. 1, p . 77-88; Hirsh, AJ et al. Evaluation of second generation amiloride analogs as therapy for CF lung disease. Journal of Pharmacology and Experimental Therapeutics, 2004, vol. 311, no. 3, pp. 929-938).

Primary CBE cells are plated on collagen-coated porous membranes, 25 mined at the air-liquid interface to evaluate the maintenance of surface fluid volume over time. At the beginning of the experiment, each 12 mm snapwell insert was removed from the plate containing the culture medium with air-liquid interface, buffer-dried, weighed, and 50 µL of vehicle (0.1% DMSO), or ENaC blocker (10 μΜ in 0.1% DMSO) was applied to the apical surface and the mass was recorded. The inserts were immediately resuspended at о transwell plate (500 µL, Krebs Ringer Bicarbonate (KRB), pH 7.4 in the lower chamber) and placed in an incubator with 5% CO₂ at 37 ° C. To reduce the artifact due to an osmotic apical carbohydrate gradient on water loss, glucose was not included in the apical buffer. The compound (at) was tested and compared with the vehicle, and the ASL mass was monitored in series from 0-8 or 24 hours. The mass of the surface liquid was transformed into volume in pL. The data are reported as volume% inifial (100% = 50 pL).

The duration of sodium transport inhibition was indirectly determined by measuring the refined buffer after a 50 µl volume of experimental buffer was added to

MD 4574 Cl 2019.01.31 apical surface of CBE cells. Only 12,5112.1% of the vehicle (buffer) remained on the surface after 8 hours and a slight increase in surface fluid retention was observed with 10 μΜ of amiloride in the vehicle (25119.2% after 8 hours). Comparatively, Compound (la) significantly increased the apical retention of surface fluid, maintaining 88.3113% of the surface fluid over 8 hours (Figure 5).

To further test Compound (Ia), the incubation time was increased from eight to 24 hours. Amiloride was not tested for more than 24 hours because most of the action disappeared after eight hours. After 24 hours, only 11% of the vehicle buffer remained, while Compound (at) maintained 72.317.3% of surface fluid over 24 hours, о loss of only 16% compared to the 8 hour index, suggesting that the compound (at) exhibits a lasting action on fluid retention (Figure 6).

Comparative example

The present compound of formula (I) is more effective and / or absorbed more rapidly from the mucosal surfaces, especially the airway surfaces, as compared to known sodium channel blockers, such as Comparative Examples 1-5, described below. Therefore, the compound of formula (I) has a longer half-life on the mucosal surfaces compared to these compounds.

Comparative Examples 1-4 are claimed, described or disclosed in WO 2003070182 A2 2003.08.28, US 6858615 B2 2005.02.22, US 7186833 B2 2007.03.06, US 7189719 B2 2007.03.13, US 7192960 B2 2007.03.20, US 7332496 B2 2008.02.19, WO 2005044180 A2 2005.05.19, US 2005080093 To 2005.04.14, US 7745442 B2 2010.06.29, WO 2004073629 A2 2004.09.02, US 6903105 B2 2005.06.07, US 6995160 B2 2006.02.07, US 7026325 B2 2006.04 .11, US 7030117 B2 2006.04.18, US 7345044 B2 2008.03.18, US 7820678 B2 2010.10.26, US 7875619 B2 2011.01.25, WO 2005016879 A2 2005.02.24, US 7064129 B2 2006.06.20, US 7247637 B2 2007.07.24,

US 7317013 B2 2008.01.08, US 7368447 B2 2008.05.06, US 7368451 B2 2008.05.06,

US 7375107 B2 2008.05.20, US 7388013 B2 2008.06.17, US 7410968 B2 2008.08.12,

US 7868010 B2 2011.01.11, or WO 2008031028 A2 2008.03.13, US 2008090841 Al

2008.04.17, US 2009082287 Al 2009.03.26, as sodium channel blockers having useful medicinal properties and can be prepared by the methods described in the present invention

And others known in the art.

Ш Ш:

<img file="MD4574C1_D0043.tif" />

<img file="MD4574C1_D0044.tif" />

The compound in Comparative Example 1 is included in the compounds that block the sodium channel in WO 2008031028 A2 2008.03.13 [5], in which its structure can be seen on page 14.

Comparative Example 2 is 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (dihexylamino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, which is in the generic description of WO 2004073629 A2 2004.09.02 [3].

MD 4574 Cl 2019.01.31

<img file="MD4574C1_D0045.tif" />

Comparative Example 3 is 3,5-diamino-6-chloro-N- (N- (4- (4- (2 - (((2S, 3R, 4R, 5R) -5hydroxy-2,3,4,6- tetramethoxyoxy () ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, which is in the generic description of WO 2008031028 A2 2008.03.13 [5].

<img file="MD4574C1_D0046.tif" />

<img file="MD4574C1_D0047.tif" />

LExempH.c omparatiy 3

<img file="MD4574C1_D0048.tif" />

<img file="MD4574C1_D0049.tif" />

The compound in Comparative Example 4 can be seen on page 15 of US 2005080093 Al 2005.04.14 and as Compound 2 on page 90 and as Compound 2 on pages 42-43 of WO 2008031028 A2 2008.03.13 [5]. To have useful activity! In the treatment of cystic fibrosis and ВРОС, a compound must have properties that will increase the mucociliary clearance (CMC) at doses that do not increase the concentration of potassium in the plasma! Therefore, it must be avoided in this! class! of compounds, which are known to increase the concentration of potassium in the plasma! they are significantly eliminated by the kidney! To evaluate this potential, it is beneficial! AIB! CMC activity in vivo and s! does not cause increased plasma potassium concentration! the useful dose !. One model to evaluate this is the sheep CMC model described below. After! as can be seen from Table 7 below, the ED50 (AUC = 47%) for Comparative Example 1 in the sheep CMC model is approximately 3000 μΜ.

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Table 7. Vehicle CMC effect at 8 hours in sheep using 3 different measures

<td>Table 1.</td><td>Tilt angle (8-8,5h)</td><td>ASC (% Cl * h)</td><td>Clearance max (%)</td><td>ED50 approximate</td>

<td>300 μΜ (the)</td><td> 10,2(100%)</td><td> 6,2(100%)</td><td> 12,8(100%)</td><td></td>

<td>30 μΜ (the)</td><td> 6,6 (65%)</td><td> 3,3 (53%)</td><td> 7,0 (55%)</td><td>2.4 nmol / kg</td>

<td>3000µΜ (Ex. Comp. 1)</td><td> 6,1 (60%)</td><td> 2,9 (47%)</td><td> 4(31%)</td><td>240 nmol / kg</td>

<td>The maximum effect</td><td> 10,2(100%)</td><td> 6,2(100%)</td><td> 12,8(100%)</td><td></td>

As can be seen from Table 7 and Figure 7 ED50 for Comparative Example 5 in the sheep CMC model it is approximately 240 nmol / kg (3 mM) using three different measures (tilt angle, AUC and maximum clearance). At this dose, what would be a clinically active dose, Comparative Example 1 determines an increase in plasma potassium concentration which at repeated doses will lead to hyperkalemia (Figure 8). Thus, Comparative Example I is unacceptable for human use, while Compound 10 (a) produces a safe and efficient CMC with a benefit to the rise ratio greater than 1000 in this model.

To reduce the potential renal effect of the molecule, more lipophilic compounds were examined. Comparative Example 2 replacing the two hydrophilic groups in Comparative Example 1 with two lipophilic beads of equal length in Compound 15 of Comparative Example 2, which is an order of magnitude less effective than Comparative Example 1 in vitro (Table 8) and, therefore, it is not suitable to produce stable CMC in vivo. Comparative Example 3, wherein the tofi oxygen in Comparative Example 1 was refined and 5 of the methylhydroxyl groups were added to 5 of the hydroxyl groups had a similar decrease in in vitro activity. It seems, therefore, that it was not possible to produce an active and harmless renal molecule from this structural framework. Therefore, it was not expected that Compound (Ia) was found to refine in vitro activity equal to Comparative Example 1. Even more surprising and unexpected was that Compound (at) was over 100 times more effective in vivo than Comparative Example I and did not cause any increase in plasma potassium concentration at 25 effective CMC doses.

Table 8. In vitro measurement of sodium channel blocking activity

<td>compound</td><td>IC50 (nM)</td>

<td>to</td><td> 13,2</td>

<td>Comparative example 1</td><td> 11,8</td>

<td>Comparative example 2</td><td> 124,5</td>

<td>Comparative example 3</td><td> 144,1</td>

<td>Comparative example 4</td><td> 6,6</td>

Another compound that has been extensively studied is the compound of Comparative Example 4, (S) -3,5-diamino-6-chloro-N- (N- (4- (4- (2,3-diamino-330 oxopropoxy)). phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide.

Disappearance of Compound (la) from apical surface and epithelial airway metabolism were evaluated in HBE and compared with Comparative Example 4 (Table 9). In these experiments 25 µL of 25 µΜ of ENaC blocker solution was added to the apical surface of HBE cells grown at о air / liquid interface, and the concentration of 35 drugs in the apical and basolateral compartment was measured over 2 hours by UPLC. After 2 h of incubation of Compound (at) on the apical surface (37 ° C), no metabolic detectations were detected on any apical or basolateral side and neither Compound (at) was detectable on the basolateral side. On the contrary, most of the compound in Comparative Example 4 was removed from the apical side with 83% metabolized to the more active carboxylic acid 40, pufin (S) -2-amino-3- (4- (4- (3- (3, 5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) phenoxy) propanoic, the structure below.

MD 4574 Cl 2019.01.31 nh₂

<img file="MD4574C1_D0050.tif" />

Table 9. Disappearance and apical metabolism of Compound (at) by HBE

<td>compound</td><td>% of the initial mass of the drug on the apical side! (parent and metabolite, 2h)</td><td>% of apical mass as metabolites (2h)</td><td>% of initial apical mass on the basolateral side! (2h)</td><td>% on the basolateral side! as metabolites (2h)</td>

<td>Compound (to)</td><td> 80,7*±6,2%</td><td>no one</td><td>no one</td><td>no one</td>

<td>Comparative example 4</td><td> 41,617,6%</td><td> 83,0±3,5%</td><td> 8,3+0,2</td><td>94.7 ± l, 0%</td>

Values ​​represent! mean ± DS * - Indic! (p &lt;0.05) significantly different from the face! of Comparative Example 4.

Compound (la) is 10,000 times more effective in CMC in sheep than Comparative Example 4, headlight! increased concentrations of К in plasma !, while Comparative Example 4 has increased concentrations of К in plasma! at the ED50 dose of approximately 3 mM (Figures 9 and 10). This, again, I prove! the unique unexpected advantage of the Compound's effectiveness and harmlessness.

Table 10. CMC in sheep at 4h after! vehicle dose administration, Comparative Example 4 or Compound (at)

<td>Dose</td><td>Initial tilt angle (4.0-4.5h)</td><td>ASC (% Cl xh)</td><td>Maximum clearance</td>

<td>Comparative example 4 (112 pg / kg; 4 mL)</td><td> 32,2±7,3* (6)</td><td> 14,1 ±2,2* (6)</td><td> 22,9±2,1* (6)</td>

<td>Comparative Example 4 (11.2 pg / kg; 4 mL)</td><td> 14,5+1,3 (3)</td><td> 6,9±1.0 (3)</td><td> 14,6±0,9 (3)</td>

<td>Compound (0.016 pg / kg; 4 mL)</td><td> 33,3±4,4* (4)</td><td> 14,4±1,3* (4)</td><td> 25,5±1,3* (4)</td>

<td>Vehicle H₂O (4 mL)</td><td> 17,2±6.8 (8)</td><td> 7,3+1,5 (8)</td><td> 12,2±2,9 (8)</td>

It has now been proven that! renal safety! increased! of Compound (at) can be explained! 15 by marked reduction! elimination of the drug through the kidneys. If the compound can be kept away from sodium channels in the kidneys, hyperkalemia should! be reduced! significant. After! intravenous administration! in sheep, 43% of Comparative Example 1 were eliminated through the urine !, while only 5% of Compound I were eliminated from the urine !. And more dramatically! is the surprising reduction of the urinary excretion of the 20 drug when administered spray, directly in the hand. When Comparative Example 4 is administered to sheep as an inhalable aerosol, 7% of the dose! is removed! through the urine! while only 0.07% of a dose! Aerosolized! of Compound is deleted! through the urine !. Elimination reduced! 1 to the compound through the urine! (10-100 times), combined! with the requirement of significantly reducing the dose described above, it leads to о difference! unexpected! 25,000 to 1,000,000 times the risk: benefit.

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Table 11. Elimination of Compound (at) and Comparative Example 4 in the urine from the urine.

<td>Analyze</td><td>Ex. comp. 4</td><td>Compound (to)</td>

<td>Log D</td><td> 0,64</td><td> 2,2</td>

<td>IC50</td><td>6.6 ± 3.7 nM</td><td>13 ± 8 nM</td>

<td>Metabolism of human plasma</td><td>t? = 37 min</td><td>No one</td>

<td>Protein binding in human plasma</td><td> 76±2%</td><td> 97±2%</td>

<td>Elimination of dose with urine (sheep)</td><td> 7 %</td><td> 0,07 %</td>

Figure 9 graphically shows the percentage of mucus clearance over time through Compound 5 (Ia), the hydrochloric acid salt of 3,5-Diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ( (2S, 3R, 4R, 5R) 2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidooyl) pyrazine-2-carboxamide, and Comparative Example 4, as described in the CMC model above. A similar percentage of mucus clearance was provided by the Compound (at) at a dose of 7000 times lower than that observed in Comparative Example 4. Compound 10 (at) provided the maximum action within a relevant dose range. clinical.

Figure 10 illustrates the significant increase in plasma potassium levels at о effective dose seen in plasma of sheep that received Comparative Example 4, in the CMC study, above, over time. No effect on plasma potassium levels was observed at any dose tested in sheep receiving Compound (at).