Chloro-pyrazine carboxamide derivatives useful for the treatment of diseases favoured by insufficient mucosal hydration

The invention relates to novel compounds, including 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis (2S, 3R) 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide and the related compounds and their pharmaceutically acceptable salts, useful as sodium channel blockers, compositions containing them, their utility in therapeutic methods and their uses and methods for their preparation.

Background of the Invention [0002] Mucosal surfaces at the interface between the environment and the organism have evolved a number of protective mechanisms through evolution; "Innate defense." The main form of such innate defense is to purify these surfaces with liquid. Typically, the amount of liquid layer on the mucosal surface reflects the balance between the secretion of liquid through the epithelium, often reflecting the secretion of the anion (Cland / or HCO3^-) coupled with water (and counter-ionic cation), and absorption of liquid through the epithelium, often reflecting the absorption of Na⁺, coupled with water and a counterion anion (Cl^- and / or HCO3^-). Many mucosal surface diseases are caused by too little protective liquid on these mucosal surfaces, caused by imbalance between secretion (too little) and absorption (relatively too much). Faulty methods of transferring salts that characterize these mucosal disorders are located in the epithelial layer of the mucosal surface.

[0003] One approach to supplementing the protective liquid layer on the mucosal surfaces is "restoring the balance" of the system by blocking the Na channel.⁺ and absorption of liquids. Epithelial protein that regulates the speed limiting absorption stage of Na^{+ and} liquid is the epithelial Na channel⁺ ( "ENaC"). ENaC is located on the apical surface of the epithelium, i.e. the interfacial surface between the environment and the mucosal surface. Under ideal conditions to inhibit the absorption regulated by ENaC^{+ and} the liquid, ENaC blocker of the amiloride class would be delivered to the mucosal surface and maintained at this site to achieve maximum therapeutic benefit.

[0004] The use of ENaC blockers has been reported in many diseases that are alleviated by increased hydration of the mucosa. In particular, the use of ENaC blockers in the treatment of respiratory diseases, such as chronic bronchitis (CB), cystic fibrosis (CF) and COPD, which reflect failure of the body to normally remove mucus from the lungs and eventually lead to chronic respiratory infections is reported. See, Evidence for airway surface. dehydration as initiating event in CF airway disease, RC Boucher, Journal of Internal Medicine, volume 261, issue 1,

- 2 January 2007, pages 5-16; and Cystic fibrosis: a disease of vulnerability to airway surface dehydration, RC Boucher, Trends in Molecular Medicine, volume 13, issue 6, June 2007, pages 231-240.

[0005] The data indicate that the initial problem in both chronic bronchitis and cystic fibrosis is failure in removing mucus from the airway surfaces. Failure to remove mucus reflects an imbalance in the amount of mucus as a liquid on the surface of the airways (ASL) on the surface of the airways. This imbalance results in a relative decrease in the level of ASL that leads to the concentration of mucus, reduced peripheral fluid slippage (PCL), mucus adhesion to the airway surface, and mucosal clearance by means of cilia activity in the oral cavity. Decrease in mucus clearance leads to chronic bacterial colonization of mucus adhering to the surface of the airways. Chronic bacterial retention,

[0006] There is now a large unmet medical need for products that specifically treat a range of diseases that are relieved by increased hydration of the mucosa, including chronic bronchitis, COPD and cystic fibrosis. Current therapies for chronic bronchitis, COPD and cystic fibrosis are focused on the treatment of symptoms and / or late effects of these diseases. However, none of these therapies effectively cure the fundamental problem of failure to remove mucus from the lungs.

[0007] RC Boucher, in US 6,264,975, discloses the use of pyrazine-guanoguanidine sodium channel blockers to moisturize the mucosal surface characterized by the well-known diuretics amiloride, benzamil and fenamil. However, these compounds are relatively weak, given the limited mass of the drug that can be absorbed into the lungs; (2) rapidly absorbed, and thus exhibit an undesirably short half-life on the mucosal surface; and (3) are slowly separating from ENaC. Stronger drugs with a longer half-life on the mucosal surface are required.

[0008] Too little protective surface liquid on other mucosal surfaces is a frequent pathophysiological condition in many diseases. For example, in xerostomia (dry mouth) the oral cavity is devoid of liquid due to parotid, sublingual and submandibular insufficiency in fluid secretion despite ongoing regulation by Na transport⁺ (ENaC) absorption of liquid from the mouth. Inflammation of the cornea and conjunctiva (dry eye) is caused by the insufficiency of the lacrimal gland in secretion of fluid in the light of ongoing, Na dependent⁺ absorption of liquid on conjunctival surfaces. In inflammation of the paranasal sinuses, there is an imbalance between mucin secretion and relative ASL deficiency. Failure in the secretion of Cl- (and liquid) in the proximal part of the small intestine in connection with increased absorption of Na⁺ (and liquid) in the terminal part of the ileum leads to the distal small intestinal patency syndrome (DIOS)

- 3 intestinal obstruction syndrome). In older patients, excessive absorption of Na⁺ (and volume) in the stool causes constipation and diverticulitis.

[0009] Published articles include many patent applications and patents awarded to Parion Sciences Inc. regarding pyrazine gluguanidine analogs as sodium channel blockers. Examples of such publications include publications PCT WO2003 / 070182, WO2003 / 070184, WO2004 / 073629, WO2005 / 025496, WO2005 / 016879, WO2005 / 018644, WO2006 / 022935, WO2006 / 023573, WO2006 / 023617, WO2007 / 018640, WO2007 / 146869, WO2008 / 031028, WO2008 / 031048 and the US patents US 6858614, 6858615, 6903105, 7064129, 7186833, 7189719, 7192958, 7192959, 7192960, 7241766, 7247636, 7247637, 7317013, 7332496, 7368447, 7368450, 7368451, 7375102, 7388013, 7399766 , 7410968, 7807834, 7842697 and 7868010.

[0010] There is still a need for new sodium channel blocking compounds with increased potency and efficacy against mucosa tissues. There is also a need for new sodium channel blocking compounds that provide a therapeutic effect, but minimize or eliminate the occurrence or progression of hyperkalemia in recipients.

Brief description of the invention [0011] The invention provides compounds of Formula I:

<img file="PL2931713T3_D0001.tif" />

wherein:

Ar is selected from the group:

<img file="PL2931713T3_D0002.tif" />

n is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

R¹ is selected from hydrogen, C₁-C₈ alkyl and a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R² is hydrogen or a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ means each of them, independently, hydrogen or C₁-C₃ alkyl;

Or a pharmaceutically acceptable salt thereof.

[0012] The invention also provides solvates and hydrates, individual stereoisomers, including optical isomers (enantiomers and diastereomers) and geometric isomers (cis- / transizomers), mixtures of stereoisomers, and tautomers of compounds of Formula (I), or a pharmaceutically acceptable salt thereof, as well as pharmaceutical compositions containing the compounds, or pharmaceutically acceptable salts thereof, all of them useful in methods of treatment, and methods for their preparation.

[0013] This invention also provides the compound 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4 - 3- (bis ((2S, 3R, 4R) , 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide, or a pharmaceutically acceptable salt thereof, as well as optical isomers (enantiomers and diastereomers) and geometric isomers (cis- / trans isomerism), mixtures of stereoisomers, and their tautomers, as well as pharmaceutical compositions containing the compound, or a pharmaceutically acceptable salt thereof, all of them useful in methods of treatment, and methods their receipt.

BRIEF DESCRIPTION OF THE FIGURES [0014] A more complete picture of the meaning of the invention and its many advantages can easily be obtained by reference to the information contained in the description in connection with the following figures:

FIG. 1 is a plot of the effect of compound 33 on sheep MCC at 4 hours post dose.

FIG. 2 is a graph of the effect of compound 123 on sheep MCC at 4 hours post dose.

FIG. 3 is a plot of the effect of compound 48 on sheep MCC at 4 hours post dose.

FIG. 4 is a plot of the effect of compound 33 on sheep MCC at 8 hours post dose.

FIG. 5 is a plot of the effect of compound 152 on sheep MCC at 8 hours post dose.

FIG. 6 is a graph of increasing the effect of compound 33 on sheep MCC at 8 hours post dose using hypertonic saline.

FIG. 7 is a graph of the effect of Comparative Example I on sheep MCC at 4 hours after dosing.

FIG. 8 is a graph of the effect of Comparative Example 1 on the level of potassium in sheep plasma.

FIG. 9 is a graph comparing the activity of Comparative Example 1 and Compound 33 to sheep MCC at 4 hours post-dose.

FIG. 10 is a graph comparing the effect of Comparative Example 1 and Compound 33 on K levels⁺ in sheep's serum.

FIG. 11 is a graph comparing the activity of Comparative Example 1 and the Compound

123 on sheep MCC 4 hours after dosing.

- FIG. 12 is a graph comparing the effect of Comparative Example 1 and Compound 123 on K levels⁺ in sheep's serum.

FIG. 13 is a graph comparing the activity of Comparative Example 1 and Compound 48 to sheep MCC at 4 hours post-dose.

FIG. 14 is a graph comparing the effect of Comparative Example 1 and Compound 48 on K levels⁺ in sheep's serum.

DETAILED DESCRIPTION OF THE INVENTION [0015] The following terms are used in the description and have the definitions indicated.

[0016] "Compound of the invention" means a compound of Formula I or a salt, particularly a pharmaceutically acceptable salt thereof.

[0017] "Compound of Formula I" means a compound having the structural formula designated herein as Formula I. The compounds of Formula I include solvates and hydrates (i.e., adducts of a compound of Formula I with a solvent). In those embodiments in which the compound of Formula I comprises one or more chiral centers, the term is intended to include each individual stereoisomer, including optical isomers (enantiomers and diastereomers) and geometric isomers (cis- / trans isomerism) and mixtures of stereoisomers. . In addition, the compounds of Formula I also include tautomers of the formulas (formulas) shown.

Throughout the description and examples, the compounds are named using standard IUPAC naming rules whenever possible, including using ChemDraw Ultra 11.0 for naming compounds marketed by CambridgeSoft Corp./PerkinElmer.

[0019] In some presentations of a chemical structure where the carbon atoms do not have a sufficient number of associated variables shown, to obtain a valence of the order of four, it should be assumed that the remaining carbon substituents necessary to provide valency four are hydrogen. Similarly, in some chemical structures where the bond is drawn without specifying the end group, such a bond indicates a methyl group (Me, -CH₃), which is conventional in the art.

[0020] In one embodiment, the compound of Formula (I) is 3,5-diamino-N- (N (4- (4- (2-amino-3- (4- (3- (bis (2.3) , 4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide, having the structure:

<img file="PL2931713T3_D0003.tif" />

Or a pharmaceutically acceptable salt thereof.

[0021] In a further embodiment, the compound of Formula (I) is 3,5-diamino-N- (N (4- (4- (2-amino-3- (4- (3- (bis (2,3), 4,5,6-pentahydroksyheksylo) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrahydronaphthalen-1-yl) butyl) karbamimidoilo) -6-chloropyrazine-2-

<img file="PL2931713T3_D0004.tif" />

[0022] In a further embodiment, the compound of Formula (I) is 3,5-diamino-N- (N (4- (6- (2-amino-3- (4- (3- (bis (2,3), 4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide having the structure:

<img file="PL2931713T3_D0005.tif" />

or a pharmaceutically acceptable salt thereof.

[0023] In a further embodiment, the compound of Formula (I) is 3,5-diamino-N (N- (4- (4 - ((S) -2-amino-3- (4- (bis) ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide, the formula:

<img file="PL2931713T3_D0006.tif" />

Or a pharmaceutically acceptable salt thereof.

[0024] The three independent embodiments contain, respectively, compounds of Formula (II), Formula (III) and Formula (IV):

<img file="PL2931713T3_D0007.tif" />

wherein:

n is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

R¹ is selected from hydrogen, C₁-C₈ alkyl and a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R² is hydrogen or a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ means each of them, independently, hydrogen or C₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.

[0025] Within each group of compounds independently represented by Formulas (I), (II), (III) and (IV), there is a further embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

R¹ is selected from hydrogen, C₁-C₈ alkyl and a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R² is hydrogen or a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ each of them is independently hydrogen or C1-C3 alkyl;

Or a pharmaceutically acceptable salt thereof.

[0026] Within each group of compounds independently represented by Formulas (I), (II), (III) and (IV), there is a further embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

R¹ is selected from hydrogen and C₁-C₈ alkyl;

R² is hydrogen or a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ means each of them, independently, hydrogen or C₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.

[0027] Within each group of compounds independently represented by Formulas (I), (II), (III) and (IV), there is another embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5, or 6;

R¹ is selected from hydrogen and C₁-C₈ alkyl;

R² is hydrogen;

R^{3 and} R⁴ means each of them, independently, hydrogen or C₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.

[0028] Within each group of compounds independently represented by formulas (I), (II), (III) and (IV), there is still yet another embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

R^{1 and} R² are each independently a polyhydroxylated alkyl group having from 3 to 8 carbon atoms; R^{3 and} R⁴ means, independently, hydrogen or C₁-C₃ alkyl;

or a pharmaceutically acceptable salt thereof.

[0029] Within each group of compounds independently represented by formulas (I), (II), (III) and (IV), there is another embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

R^{1 and} R² are each independently a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ means hydrogen;

or a pharmaceutically acceptable salt thereof.

[0030] Within each group of compounds independently represented by formulas (I), (II), (III) and (IV), there is another embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

- 9th^{1 and} R² are each independently a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R^{3 and} R⁴ means, independently, C₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.

[0031] Within each group of compounds independently represented by formulas (I), (II), (III) and (IV), there is an additional embodiment, wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6;

R^{1 and} R² are each independently a polyhydroxylated alkyl group having from 3 to 8 carbon atoms;

R³ is hydrogen; and R⁴ means C₁-C₃ alkyl;

or a pharmaceutically acceptable salt thereof.

[0032] The polyhydroxylated alkyl groups of the invention are those in which an alkyl chain having 3 to 8 carbon atoms is substituted with two or more hydroxyl groups. Examples of polyhydroxylated alkyl groups are butane 1,4-diol; butane-1,2,2-triol; butane-1,1,2,3, -tetraol; pentane-1,2,3,4-tetraol; hexane-1,2,3,4,5pentaol; heptane-1,2,3,4,5,6-heksaol; and octane-1,2,3,4,5,6,7-heptaol.

[0033] One embodiment within each group of compounds described herein are those in which the polyhydroxylated alkyl group has the formula ^CH2(CHR⁵)_n-H, wherein n is an integer selected from 2, 3, 4, 5, 6 or 7, and R⁵ means independently in each case H or OH, assuming that at least two of the R groups⁵OH.

[0034] A further embodiment within each group of compounds described herein are those in which the polyhydroxylated alkyl group has the formula -CH₂-CHOH (CHR⁶)_m-H, wherein m is an integer selected from 1, 2, 3, 4, 5 or 6, and R⁶is independently in each case H or OH, assuming that at least one of the R groups⁶ means OH.

[0035] A further embodiment within each group of compounds described herein comprises compounds in which the polyhydroxylated alkyl group has the formula -CH₂(CHOH)_nCH₂OH, wherein n is an integer selected from 1, 2, 3, 4, 5, or 6. A further embodiment within each group of compounds described herein comprises compounds wherein n is an integer selected from 2, 3, 4 or 5. A further embodiment within each group contains compounds in which n is an integer selected from 3, 4 or 5.

[0036] In a further embodiment within each group of compounds described herein, a chain represented by the formula -CH₂- (CHOH)_nCH₂OH means

2,3,4,5,6-pentahydroxyhexane, of the formula:

<img file="PL2931713T3_D0008.tif" />

[0037] In a further embodiment within each group of compounds described herein, a chain represented by the formula -CH₂- (CHOH)_nCH₂OH is one of the formula:

<img file="PL2931713T3_D0009.tif" />

[0038] Three further independent embodiments include compounds of Formula (V), Formula (VI) and Formula (VII) respectively:

<img file="PL2931713T3_D0010.tif" />

wherein:

n is an integer selected from 1, 2, 3, 4, 5 or 6; and

R^{3 and} R⁴ means each of them, independently, hydrogen or C₁-C₃ alkyl;

or a pharmaceutically acceptable salt thereof.

[0039] Within each of the embodiments represented by Formulas (V), (VI) and (VII) there is a further embodiment, wherein n is an integer selected from 1,

- 11 2, 3, 4, 5, or 6; and R^{3 and} R⁴ mean, each of them, hydrogen; or a pharmaceutically acceptable salt thereof. Within each of the embodiments represented by Formulas (V), (VI) and (VII), there is another embodiment, wherein n is an integer selected from 1, 2, 3, 4, 5, or 6; and R^{3 and} R⁴ means, each of them, C₁-C₃ alkyl; or a pharmaceutically acceptable salt thereof.

[0040] Within each of the embodiments described herein, there is a further embodiment, wherein n is an integer selected from 1, 2 or 3. Within each of the embodiments described herein, there is a further embodiment, wherein is an integer selected from 4, 5 or 6. Within each of the embodiments described herein, there are six further independent embodiments, wherein n is an integer of 1, 2, 3, 4, 5 and 6, respectively.

[0041] The compounds of the description, including those of Formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), may be in the form of the free base or a salt, especially a pharmaceutically acceptable salt. for a discussion of pharmaceutically acceptable salts, see Berge et al., J. Pharma Sci. (1977) 66: 1-19.

[0042] Pharmaceutically acceptable salts formed from inorganic or organic acids include, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, nitrate, sulfamate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, tannate, ascorbyl, palmitate, salicylate, stearate, phthalate, alginate, polyglutamate, oxalate, oxalacetate, saccharinate, benzoate, alkyl aryl sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or naphthalenesulphonate) ) and isothionate; complexes formed with amino acids such as lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine and leucine.

For therapeutic use, the salts of the active ingredients of the compounds of Formula I will be pharmaceutically acceptable, i.e. they will be salts derived from a pharmaceutically acceptable acid. However, salts of acids that are not pharmaceutically acceptable may also find use, for example in the preparation or purification of a pharmaceutically acceptable compound. For example, trifluoroacetate salts may find such use. All salts, whether or not derived from a pharmaceutically acceptable acid, are within the scope of the invention.

[0044] The term "chiral" refers to molecules that have the property of not impregnating their mirror image, whereas the term "achiral" refers to molecules that are superimposable on their mirror image.

[0045] The term "stereoisomers" refers to compounds that have identical chemical composition but differ with respect to the arrangement of atoms or groups in space. "Diastereomer" refers to a stereoisomer with two or more centers of chirality, and whose molecules

- 12 are not mirror images of each other. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties and reactivity. Mixtures of diastereomers can be separated in high resolution analytical procedures, such as electrophoresis and chromatography. "Enantiomers" refer to two stereoisomers of a compound that are non-superimposable mirror images of each other.

[0046] Stereochemical definitions and conventions used according to the description generally follow SP Parker, Ed., McGRAW-HILL DICTIONARY OF CHEMICAL TERMS (1984) McGrawHill Book Company, New York; and Eliel, E. and Wilen, S., STEREOCHEMISTRY OF ORGANIC COMPOUNDS (1994) John Wiley &amp; Sons, Inc., New York.

[0047] Many organic compounds exist in optically active forms, i.e. they have the ability to rotate the plane of polarized light. When describing the optically active compound, prefixes D and L, or R and S are used to determine the absolute configuration of the molecule around its center (centers) of chirality. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate that can occur when stereoselection or stereospecificity has not occurred in a chemical reaction or method. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric forms.

[0048] The term "tautomers" refers to a type of stereoisomer in which the migration of a hydrogen atom results in the formation of two or more structures. The compounds of Formula I may exist in various tautomeric forms. The skilled person will recognize that amidines, amides, guanidines, ureas, thioureas, heterocycles and the like may exist in tautomeric forms. By way of example, compounds of Formula I may exist in various tautomeric forms as shown below:

<img file="PL2931713T3_D0011.tif" />

[0049] All potential tautomeric forms of amidines, amides, guanidines, ureas, thioureas, heterocycles and the like for all embodiments of Formula I are within the scope of the invention. The tautomers are in equilibrium and hence the representation of a single tautomer in the provided formulas will mean for a person skilled in the art reference equally to all potential tautomers.

[0050] It should be noted that all enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs, pseudopolymorphs of compounds falling within the definition of Formula I and their pharmaceutically acceptable salts are included in the invention. All mixtures of such enantiomers and diastereomers, including enantiomerically enriched mixtures and diastereomerically enriched mixtures, are within the scope of the invention. Enantiomerically enriched mixtures are mixtures of enantiomers, wherein the ratio of the specified enantiomer to the alternative enantiomer is higher than 50:50. More specifically, the enantiomerically enriched mixture contains 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 the other enantiomer. Similarly, diastereomerically enriched mixtures are mixtures of diastereomers, wherein the amount of the specific diastereomer is greater than the amount of each alternative diastereomer. More specifically, the diastereomerically enriched mixture contains at least about 75% of the specific diastereomer, and preferably at least about 85% of the specific diastereomer. In one embodiment, the diastereomerically enriched mixture is substantially free of all other diastereomers. The term "substantially free" will be understood by those skilled in the art as indicative of less than 5% presence of other diastereomers, preferably less than 1%, more preferably less than 0.1%. In other embodiments, no other diastereomers will be present, or the amount of any other diastereomer present will be below the detection level. Stereoisomers may be separated by techniques known in the art, including high performance liquid chromatography (HPLC) and the crystallization of chiral salts.

A single stereoisomer, e.g. an enantiomer, substantially free of its stereoisomer can be obtained by resolving the racemic mixture using a method such as the formation of diastereomers using optically active resolving agents ("Stereochemistry of Carbon Compounds," (1962) by EL Eliel, McGraw Hill; Lochmuller, CH, (1975) J. Chromatogr., 113: (3) 283-302). The racemic mixtures of the 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) diastereomeric compounds formation with chiral derivatizing agents, separation diastereomers and conversion to pure stereoisomers,

[0052] In one embodiment, the invention provides an enantiomerically enriched mixture or composition comprising 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroksyheksylo) amino) propyl) phenylamino) -3-oxopropyl) naphthalene-1-yl) butyl) karbamimidoilo) -6-chloropyrazine-2 carboxamide, or a pharmaceutically acceptable salt thereof, as the dominant isomer.

[0053] Other embodiments include enantiomerically enriched mixtures or compositions containing compounds of Formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), respectively. , or a pharmaceutically acceptable salt thereof, as the dominant isomer in each of their respective mixtures.

[0054] In a further embodiment, the invention provides an enantiomerically enriched mixture or a composition of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3-bis) ( (2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroksyheksylo) amino) propyl) phenylamino) -3-oxo-propyl) naphthalen-1-yl) butyl) karbamimidoilo) -6-chloropyrazine-2- carboxamide, or a pharmaceutically acceptable salt thereof, substantially free of other isomers.

[0055] Four other embodiments include enantiomerically enriched mixtures or compositions containing compounds of Formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII, respectively. ) or a pharmaceutically acceptable salt thereof, substantially free of other isomers in each of their respective mixtures.

[0056] Also provided herein are each of the compounds and groups of compounds described herein, including those of Formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), or a pharmaceutically acceptable salt thereof, for use as a medicament.

[0057] The compound of Formula I and its pharmaceutically acceptable salts may exist as different polymorphs or pseudopolymorphs. As used herein, polymorphism of crystalline forms means the ability of a crystalline compound to exist in different crystal structures. The polymorphism of crystalline forms may be the result of differences in crystal packing (packing polymorphism) or differences in packing between different conformers of the same molecule (conformational polymorphism). As used herein, the pseudopolymorphism of the crystalline forms also includes the ability of the hydrate or solvate of the compound to exist in different crystal structures. The pseudopolymorphs of the invention may occur due to differences in crystal packing (pseudopolymorphism of packing) or due to differences in packing between different conformers of the same molecule (conformational pseudopolymorphism). The invention includes all polymorphs and pseudopolymorphs of compounds of Formula I and their pharmaceutically acceptable salts.

[0058] The compound of Formula I and its pharmaceutically acceptable salts may also be present as an amorphous solid. As used herein, an amorphous solid is a solid in which there is no ordering of the long-range position of atoms in the solid. This definition also applies to a crystal with a size of two nanometers or less. Additives, including solvents, can be used to generate amorphous forms according to the invention. The invention, including all pharmaceutical compositions, methods

- treatments, combination products, and uses thereof described herein, includes all of the amorphous forms of the compounds of Formula I and their pharmaceutically acceptable salts.

APPLICATIONS [0059] The compounds of the invention show activity as sodium channel blockers. Without wishing to be bound by any particular theory, it is believed that the compounds of the invention may function in vivo by blocking the epithelial sodium channels present in the mucosal surface, thereby reducing the absorption of water through the mucosal surfaces. This effect increases the volume of protective liquids on mucosal surfaces, and restores balance to the system.

[0060] Consequently, the compounds of the invention are useful as drugs, particularly for the treatment of clinical conditions for which a sodium channel blocker may be indicated. Such conditions include lung diseases, such as diseases associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), including acute exacerbations of COPD, asthma, bronchiectasis (including bronchiectasis due to conditions other than cystic fibrosis), acute bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis, and transplant associated bronchiolitis, including those associated with lung transplantation and bone marrow bronchiolitis, in a man who requires it. The compounds of the invention may also be useful for treating tracheal and bronchitis-related inflammation and / or prevention of ventilator-associated pneumonia in ventilated patients. Disclosed herein are methods for treating any of the disorders described herein in a mammal that requires, preferably in a human that requires it, any method comprising administering to said mammal a pharmaceutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof. Also disclosed is (a) a method of reducing the number of COPD exacerbations in a mammal in need thereof; (b) a method for reducing the number of flares of CF in a mammal in need thereof; (c) a method for improving pulmonary function (FEV1) in a mammal in need thereof, (d) a method of improving pulmonary function (FEV1) in a mammal suffering COPD,

[0061] Also disclosed is a method of stimulating, enhancing or ameliorating mucosal clearance in a mammal, a method comprising administering to a mammal that requires a pharmaceutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. Mucosal clearance will be understood as including the natural activities of the epithelial epithelium associated with the transmission or clearance of mucus in the airways, including the mechanisms of bronchial self-purification. Thus, a method is also disclosed for improving mucus clearance in the mammalian airways that requires it.

[0062] In addition, sodium channel blockers may be indicated for the treatment of conditions that are alleviated by increased hydration of the mucosa on mucosal surfaces other than the surface of the lung mucosa. Examples of such conditions include dry mouth

- 16 (xerostomia), dry skin, dry vagina, sinusitis, rhinosinusitis, nasal dehydration, including nasal dehydration caused by administration of dry oxygen, dry eye, Sjogren's disease, otitis media, primary ciliary dyskinesia, distal patency syndrome small intestine, esophagitis, constipation and chronic diverticulitis. The compounds of the invention may also be used to stimulate the hydration of the eye or cornea.

[0063] The compounds of the invention may also be useful in methods for obtaining a human sputum sample. The method may be carried out by administering an effective amount of a compound of the invention to at least one lung of a patient, followed by induction and sampling of a sputum sample from a given human.

[0064] Accordingly, in one aspect, the disclosure discloses a method of treating a disease condition in a mammal, such as a human, where the use of a sodium channel blocker is indicated.

[0065] In other embodiments, the description discloses each of the methods provided herein with the added benefit of minimizing or eliminating hyperkalemia in the recipient of the method. Also disclosed are embodiments including each of the methods described herein, wherein an improvement of the therapeutic index is achieved.

[0066] The terms "treat", "treat" and "treatment" as used herein refer to reversing, alleviating, inhibiting the progress of, or preventing a disorder or disorder, or one or more symptoms of such disorder or condition.

[0067] All 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 (typically a mammal, preferably a human) who requires treatment.

[0068] In one embodiment, the disclosure discloses a method of treating a condition that is alleviated by increased hydration of the mucosa in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating a disease associated with reversible or irreversible airway obstruction in a mammal, particularly a human in need thereof. In one specific embodiment, the disclosure discloses a method of treating chronic obstructive pulmonary disease (COPD) in a mammal, particularly a human in need thereof. In one specific embodiment, the disclosure discloses a method of decreasing the incidence, severity or duration of an acute exacerbation of COPD, or treating one or more symptoms of an acute exacerbation of COPD in a mammal, particularly a human in need thereof. In one embodiment, the specification discloses a method of treating asthma in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis) in a mammal, particularly a human in need thereof. In one embodiment, the description discloses a method of treatment

17 bronchitis, including acute and chronic bronchitis in a mammal, particularly in a human that requires it. In one embodiment, the specification discloses a method of treating a cough after viral infection in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating cystic fibrosis in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating emphysema in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating pneumonia in a mammal, particularly a human in need thereof. In one embodiment, the specification discloses a method of treating bronchiolitis in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treatment associated with bronchiolitis transplantation, including lung bronchiolitis associated with a lung transplant and bone marrow bronchiolitis in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating tracheal and bronchitis-associated inflammation and / or prevention associated with respiratory-related pneumonia in the ventilated human that needs it.

In further embodiments for each treatment method, the pharmaceutically acceptable salt form is a hydrochloride salt or a hydroxy naphthoate salt of the compound of formula (1a). In a further embodiment, within each treatment method, the free base of the compound of formula (1a) is used.

[0070] In one embodiment, the description discloses a method of treating dry mouth (xerostomia) in a mammal, particularly a human, which requires it. In one embodiment, the description discloses a method of treating skin dryness in a mammal, particularly a human in need thereof. In one embodiment, the description discloses a method of treating vaginal dryness in a mammal, particularly a human in need thereof. In one embodiment, the disclosure discloses a method of treating sinusitis, rhinosinusitis, or dehydration of the nose, including nasal dehydration caused by administration of dry oxygen, in a mammal, particularly a human in need thereof. In one embodiment, the description discloses a method of treating eye dryness, or Sjogren's disease, or stimulating the hydration of the eye or cornea in a mammal, especially for a man who needs it. In one embodiment, the description discloses a method of treating otitis media in a mammal, particularly a human in need thereof. In one embodiment, the description discloses a method of treatment

- 18 primary ciliary dyskinesia, in a mammal, especially in a human that requires it. In one embodiment, the disclosure discloses a method of treating distal small intestinal patency disorder, esophagitis, constipation or chronic diverticulitis in a mammal, particularly a human in need thereof.

[0071] Also provided is a compound of the invention for use in medical therapy, particularly for use in treating a disease state in a mammal, such as a human for whom the use of a sodium channel blocker is indicated. All therapeutic uses described herein are administered by administering an effective amount of a compound of the invention to a subject in need of treatment. In one embodiment, a compound of the invention is provided for use in the treatment of a lung disease, such as a disease associated with reversible or irreversible airway obstruction in a mammal, particularly a human in need thereof. In one specific embodiment, a compound of the invention is provided for use in the treatment of chronic obstructive pulmonary disease (COPD) in a mammal, particularly a human in need thereof. In one embodiment, a compound of the invention is provided for use to reduce the incidence, severity or duration of an acute exacerbation of COPD, or to treat one or more symptoms of an acute exacerbation of COPD 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 asthma in a mammal, particularly a human in need thereof. In one embodiment, a compound is provided for use in the treatment of bronchiectasis, including bronchiectasis caused by conditions other than cystic fibrosis, or bronchitis, including acute bronchitis and chronic bronchitis, in a mammal, particularly a human, which requires . In one embodiment, a compound is provided for use in the treatment of cough following viral infection 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, particularly a human in need thereof. In one embodiment, a compound of the invention is provided for use in treating emphysema 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 pneumonia 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 bronchiolitis or transplantation of bronchiolitis, including bronchiolitis associated with a lung transplant and a bone marrow in a mammal, particularly a human who requires it. In one embodiment, a compound of the invention is provided for use in the treatment of tracheal and bronchitis-related inflammation or prevention of ventilatory-related pneumonia in the ventilated human that needs it.

[0072] In one embodiment, a compound of the invention is provided for use in treating a disease condition alleviated by increased hydration of the mucosa on the mucosal surface of a mammal, particularly a human in need thereof. In one embodiment, a compound is provided for use in the treatment of dryness

- oral cavity (xerostomia) in a mammal, especially in a human that requires it. In one embodiment, a compound is provided for use in the treatment of dry skin in a mammal, particularly a human in need thereof. In one embodiment, a compound is provided for use in treating 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 administering dry oxygen 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 ocular dryness or Sjogren's disease or to stimulate the hydration of the eye or cornea in a mammal, especially for a man who needs it. 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 treating distal small intestinal patency disorder, esophagitis, constipation or chronic diverticulitis in a mammal, particularly a human in need thereof. who needs it. 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 treating distal small intestinal patency disorder, esophagitis, constipation or chronic diverticulitis in a mammal, particularly a human in need thereof. who needs it. 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 treating distal small intestinal patency disorder, esophagitis, constipation or chronic diverticulitis in a mammal, particularly a human in need thereof.

[0073] The invention also provides the use of a compound of the invention in the manufacture of a medicament for the treatment of a disease condition in a mammal, such as a human for whom the use of a sodium channel blocker is indicated. In one embodiment, a compound of the invention is provided in the manufacture of a medicament for treating a disease associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), acute exacerbations COPD, asthma, bronchiectasis (including bronchiectasis caused by other conditions) than cystic fibrosis), bronchitis (including acute bronchitis and chronic bronchitis), cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis associated with transplantation of bronchiolitis,

[0074] In one particular embodiment, a compound of the invention is provided for the preparation of a medicament for treating a disease condition alleviated by increased hydration of the mucosa on the mucosal surface, treatment of dry mouth (xerostomia), dry skin, dry vagina, sinusitis, sinusitis paranasal, nose dehydration, including nasal drainage caused by administration of dry oxygen, treatment of eye dryness, Sjogren's disease, stimulation of eye or corneal moisturization, treatment of otitis media, primary ciliary dyskinesia, distal small intestine patency syndrome, esophagitis, constipation or chronic diverticulitis

[0075] The terms "effective amount", "pharmaceutically effective amount", "effective dose" and "pharmaceutically effective dose" as used herein refer to the amount of a compound of the invention that is sufficient for the subject to which it is administered, to induce biological or therapeutic the medical response of a tissue culture, tissue, system or mammal (including a human) sought after, for example by a researcher or clinician. The term also includes within its scope amounts effective to enhance the normal physiological function. In one embodiment, an effective amount is an amount necessary to provide the desired level of drug in airway and lung secretions and tissues, or alternatively, in the bloodstream of the subject to be treated to achieve the expected physiological response or desired biological effect, when such composition is administered by inhalation. For example, an effective amount of a compound of the invention for treating a disease state for which the use of a sodium channel blocker is indicated is sufficient in an individual to whom it is administered for the treatment of a particular disease state. In one embodiment, an effective amount is an amount of a compound of the invention that is sufficient to treat COPD 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 subject to be treated, the precise condition requiring treatment and its severity, bioavailability, potency and other properties of the particular compound being administered, the nature of the formulation, the route of administration, and the delivery device, and will ultimately depend on the decision of the attending physician or veterinarian. Further guidance for the appropriate dose can be found by considering the conventional dosage of other sodium channel blockers, such as amiloride, also including any power differences between the amiloride and the compounds of the invention.

[0077] A pharmaceutically effective dose of a compound of the invention administered topically to an individual's respiratory tract surfaces (e.g., inhalation) for treating a 70 kg human subject may range from about 10 ng to about 10 mg. In a further embodiment, the pharmaceutically effective dose can be from about 0.1 to about 1000 μg. Typically, a daily dose administered topically to the airway surfaces will mean an amount sufficient to achieve a dissolved active agent concentration on airway surfaces of about 10^-9, 10^-8 or 10^-7to around 10^-4, 10^-3, 10^-2 or 10^-1 moles / liter, more preferably from about 10^-9 to around 10^-4moles / liter. The selection of a specific dose for the patient will be determined by the attending physician, clinician or veterinarian with the usual knowledge in the given field on the basis of a number of factors, including those noted above. In one specific embodiment, a dose of a compound of the invention for treating a human with a body weight of 70 kg will range from about 10 nanograms (ng) to about 10 μg. In a further embodiment, the effective dose will be from about 0.1 mg to about 1000 μg. In one embodiment, the dose of a compound of the invention for treating a human with a body weight of 70 kg will range from about 0.5 μg to about 0.5 mg. In a further embodiment, the dose will be from about 0.5 μg to about 60 μg. IN

In a further embodiment, a pharmaceutically effective dose will range from about to about 10 μg. In a further embodiment, the pharmaceutically effective dose will be from about 5 μg to about 50 μg. A further embodiment will have an effective dose in the order of about 10 μg to about 40 μg. In two further embodiments, the pharmaceutically effective dose will suitably be from about 15 μg to about 50 gg, about 15 μg to about 30 μg. It will be understood that in each of these dose ranges, all incremental doses in the range are included. For example, the range of 0.5-50 μg includes individual doses of: 0.5 gg, 0.6 gg, 0.7 gg, 0.8 μg, 0.9 μg, 1.0 gg, 1.1 gg 1 , 2 gg, 1.3 gg, 1.4 gg, 1.5 gg, 1.6 gg, 1.7 gg, 1.8 gg, 1.9 gg, 2.0 gg, 2.1 gg,

2.2 gg, 2.3 gg 2.4 gg 2.5 gg 2.6 gg, 2.7 gg, 2.8 gg, 2.9 gg 3.0 gg 3.1 gg 3.2 gg, 3.3 gg

3.4 gg, 3.5 gg 3.6 gg 3.7 gg 3.8 gg, 3.9 gg, 4.0 gg, 4.1 gg 4.2 gg 4.3 gg 4.4 gg, 4.5 gg gg

4.6 gg, 4.7 gg 4.8 gg, 4.9 gg 5.0 gg 5.1 gg 5.2 gg, 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 6.6 gg 6.7 gg 6.8 gg, 6 , 9 gg,

7.0 gg, 7.1 gg 7.2 gg 7.3 gg 7.4 gg, 7.5 gg, 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 gg, 8.7 gg, 8.8 gg, 8.9 gg 9.0 gg 9.1 gg 9.2 gg, 9.3 gg

9.4 gg 9.5 gg, 9.6 gg 9.7 gg, 9.8 gg, 9.9 gg 10.0 gg, 10.1 gg, 10.2 gg, 10.3 gg, 10.4 gg,

10.5gg 10.6gg 10.7gg 10.8gg, 10.9gg, 11.0gg, 11.1gg 11.2gg 11.3gg, 11.4gg 11.5gg, 11.6g gg, 11.7 gg, 11.8 gg, 11.9 gg, 12.0 gg, 12.1 gg, 12.2 gg, 12.3 gg, 12.4 gg, 12.5 gg,

12.6 gg, 12.7 gg, 12.8 gg, 12.9 gg, 13.0 gg, 13.1 gg, 13.2 gg, 13.3 gg, 13.4 gg, 13.5 gg, 13, 6gg, 13.7gg, 13.8gg, 13.9gg, 14.0gg, 14.1gg, 14.2gg, 14.3gg, 14.4gg, 14.5gg, 14g, 6 gg,

14.7 gg, 14.8 gg, 14.9 gg, 15.0 gg, 15.1 gg, 15.2 gg, 15.3 gg, 15.4 gg, 15.5 gg, 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 gg, 16, 7 gg,

16.8 gg, 16.9 gg, 17.0 gg, 17.1 gg, 17.2 gg, 17.3 gg, 17.4 gg, 17.5 gg, 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 gg, 18.7 gg, 18, 8 gg,

18.9 gg, 19.0 gg, 19.1 gg, 19.2 gg, 19.3 gg, 19.4 gg, 19.5 gg, 19.6 gg, 19.7 gg, 19.8 gg, 19, 9 gg, 20.0 gg, 20.1 gg, 20.2 gg, 20.3 gg, 20.4 gg, 20.5 gg, 20.6 gg, 20.7 gg, 20.8 gg, 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, 9 gg, 23.0 gg,

23.1 gg, 23.2 gg, 23.3 gg, 23.4 gg, 23.5 gg, 23.6 gg, 23.7 gg, 23.8 gg, 23.9 gg, 24.0 gg, 24, 1 gg, 24.2 gg, 24.3 gg, 24.4 gg, 24.5 gg, 24.6 gg, 24.7 gg, 24.8 gg, 24.9 gg, 25.0 gg, 25, 1 gg,

25.2 gg, 25.3 gg, 25.4 gg, 25.5 gg, 25.6 gg, 25.7 gg, 25.8 gg, 25.9 gg, 26.0 gg, 26.1 gg, 26, 2 g, 26.3 g, 26.4 g, 26.5 g, 26.6 g, 26.7 g, 26.8 g, 26.9 g, 27.0 g, 27.1 g, 27, 2 gg,

27.3 gg, 27.4 gg, 27.5 gg, 27.6 gg, 27.7 gg, 27.8 gg, 27.9 gg, 28.0 gg, 28.1 gg, 28.2 gg, 28, 3 gg, 28.4 gg, 28.5 gg, 28.6 gg, 28.7 gg, 28.8 gg, 28.9 gg, 29.0 gg, 29.1 gg, 29.2 gg, 29, 3 gg,

29.4 gg, 29.5 gg, 29.6 gg, 29.7 gg, 29.8 gg, 29.9 gg, 30.0 gg, 30.1 gg, 30.2 gg, 30.3 gg, 30, 4 g, 30.5 g, 30.6 g, 30.7 g, 30.8 g, 30.9 g, 31.0 g, 31.1 g, 31.2 g, 31.3 g, 31, 4 gg,

31.5 gg, 31.6 gg, 31.7 gg, 31.8 gg, 31.9 gg, 32.0 gg, 32.1 gg, 32.2 gg, 32.3 gg, 32.4 gg, 32, 5 g, 32.6 gg, 32.7 gg, 32.8 gg, 32.9 gg, 33.0 gg, 33.1 gg, 33.2 gg, 33.3 gg, 33.4 gg, 33, 5 gg,

33.6 gg, 33.7 gg, 33.8 gg, 33.9 gg, 34.0 gg, 34.1 gg, 34.2 gg, 34.3 gg, 34.4 gg, 34.5 gg, 34, 6 gg, 34.7 gg, 34.8 gg, 34.9 gg, 35.0 gg, 35.1 gg, 35.2 gg, 35.3 gg, 35.4 gg, 35.5 gg, 35, 6 gg,

35.7 gg, 35.8 gg, 35.9 gg, 36.0 gg, 36.1 gg, 36.2 gg, 36.3 gg, 36.4 gg, 36.5 gg, 36.6 gg, 36, 7 gg, 36.8 gg, 36.9 gg, 37.0 gg, 37.1 gg, 37.2 gg, 37.3 gg, 37.4 gg, 37.5 gg, 37.6 gg, 37, 7 gg,

- 37.8 gg 37.9 gg 38.0 gg 38.1 gg, 38.2 gg, 38.3 gg, 38.4 gg 38.5 gg 38.6 gg, 38.7 gg 38.8 gg, 38 9 gg 39.0 gg, 39.1 gg, 39.2 gg, 39.3 gg 39.4 gg 39.5 gg, 39.6 gg 39.7 gg, 39.8 gg,

39.9 gg 40.0 gg 40.1 gg 40.2 gg, 40.3 gg, 40.4 gg, 40.5 gg 40.6 gg 40.7 gg, 40.8 gg 40.9 gg, 41.0 g gg 41.1 g, 41.2 g, 41.3 g, 41.4 g, 41.5 g, 41.6 g, 41.7 g, 41.8 g, 41.9 g, 42.0 g, 42.1 gg 42.2 gg 42.3gg 42.4gg, 42.5gg 42.6gg, 42.7gg 42.8gg 42.9gg, 43.0gg, 43.1gg, 43.2gg , 43.3 g, 43.4 g, 43.5 g, 43.6 g, 43.7 g, 43.8 g, 43.9 g, 44.0 gg,

44.1 gg, 44.2 gg, 44.3 gg, 44.4 gg, 44.5 gg, 44.6 gg, 44.7 gg, 44.8 gg, 44.9 gg, 45.0 gg, 45, 1 gg, 45.2 gg, 45.3 gg, 45.4 gg, 45.5 gg, 45.6 gg, 45.7 gg, 45.8 gg, 45.9 gg, 46.0 gg, 46, 1 gg,

46.2 gg, 46.3 gg, 46.4 gg, 46.5 gg, 46.6 gg, 46.7 gg, 46.8 gg, 46.9 gg, 47.0 gg, 47.1 gg, 47, 2 gg, 47.3 gg, 47.4 gg, 47.5 gg, 47.6 gg, 47.7 gg, 47.8 gg, 47.9 gg, 48.0 gg, 48.1 gg, 48, 2 gg,

48.3 gh, 48.4 gg, 48.5 gg, 48.6 gg, 48.7 gg, 48.8 gg, 38.9 gg, 49.0 gg, 49.1 gg, 49.2 gg, 49, 3 gg, 49.4 gg, 49.5 gg, 49.6 gg, 49.7 gg, 49.8 gg, 39.9 gg and 50 gg. [0078] The above suggested doses can be adjusted using conventional dose calculations if the compound is administered by a different route. Determining the appropriate dose for administration by other routes is well within the capabilities of those skilled in the art, in the light of the foregoing description and general knowledge of the prior art.

[0079] Providing an effective amount of a compound of the invention may comprise providing a single or multiple unit dosage form that can be delivered simultaneously or separately over a period of time, such as 24 hours. The dose of a compound of the invention (alone or in the form of a composition comprising this) may be administered one to ten times daily. Typically, a compound of the invention (alone or in the form of a composition comprising thereof) will be administered four, three, two, or once a day (24 hours).

[0080] The compounds of Formula (I) of the invention are also useful for the treatment of infections transmitted by air. Examples of infections transmitted by air include, for example, RSV. The compounds of Formula (I) according to the invention are also useful for the treatment of anthrax infections. The invention relates to the use of the compounds of Formula (I) according to the invention for the prophylactic, post-prophylactic, prophylactic or therapeutic treatment of diseases or pathological conditions caused by pathogens. In a preferred embodiment, the invention relates to the use of compounds of Formula (I) for the prophylactic, post-prophylactic, prophylactic or therapeutic treatment of diseases or conditions caused by pathogens that can be used in bioterrorism.

[0081] In recent years, a number of research programs and biological defense measures have been implemented to deal with concerns about the use of biological agents in terrorist acts. These measures are intended to address bioterrorism or the use of micro-organisms or biological toxins to kill people, spread fever and disrupt society. For example, the National Institute of Allergy and Infectious Diseaes (NIAID) has developed Strategic Plan for Biodefense Research which outlines plans for addressing research needs in a wide area of ​​bioterrorism

- 23 and emerging and emerging infectious diseases. According to the plan, the intentional exposure of the civilian population of the United States to the spores of Bacillus anthracis has revealed a gap in the general readiness of the nation for bioterrorist threats. In addition, the report discusses in detail that these attacks have identified an unmet need for rapid diagnosis tests, vaccines and immunotherapy for prevention, and biological medicines for the treatment of disease caused by bioterrorist agents.

[0082] Many of the various research papers have been directed to biology analysis of pathogens identified as potentially dangerous as bioterrorist agents, examining host responses to such factors, developing vaccines against infectious diseases, evaluating therapeutics currently available and being developed against such agents, and developing diagnostics to identify the symptoms of the subject and subjective threatening factors. Such efforts are commendable, but given the large number of pathogens that have been identified as potentially available for bioterrorist purposes, these efforts have not yet yielded satisfactory responses to all possible bioterrorist threats. Additionally, many of the pathogens identified as potentially dangerous as bioterrorist agents do not provide adequate economic incentives for the development of therapeutic or preventive measures in industry. In addition, even if preventive measures such as vaccines will be available to any pathogen that can be used as part of bioterrorism, the cost of administering all such vaccines to the general population is prohibitive.

[0083] Until a convenient and effective treatment directed against each bioterrorist threat arises, there is a strong need for preventive, prophylactic or therapeutic therapies that can prevent or reduce the risk of infection with a pathogenic agent.

[0084] Disclosed herein are methods of prophylactic treatment. In one aspect, a prophylactic treatment method is provided, comprising administering a prophylactically effective amount of the compounds of Formula (I) to a subject in need of prophylactic treatment against infection with one or more airborne pathogens. A particular example of an airborne pathogen is anthrax.

[0085] In a further aspect, a prophylactic treatment is disclosed for reducing the risk of infection by an airborne pathogen that can cause disease in a human, the method comprises administering an effective amount of the compounds of Formula (I) to human lungs that may be exposed to infection. airborne pathogen, but does not cause disease symptoms, where the effective amount of sodium channel blocker and osmolyte is sufficient to reduce the risk of infection in humans. A particular example of an airborne pathogen is anthrax.

[0086] In a further aspect, prophylactic treatment after exposure or a method of therapeutic treatment is disclosed for the treatment of infections caused by a pathogenic pathway.

- air, comprising administering an effective amount of the compounds of Formula (I) to the lungs of an individual in need of such treatment against infection caused by airborne pathogens. Pathogens against which one can protect itself by means of prophylactic treatment after exposure, rescue and therapeutic treatment according to the invention include any pathogens that can enter the body through the mouth, nose or nasal airways, thereby passing into the lungs. Typically, pathogens will be pathogens carried by air, naturally occurring or by aerosol production. Pathogens may be naturally occurring or may be introduced into the environment deliberately after aerosol formation or other way of introducing pathogens into the environment. Many pathogens, which are not naturally transmitted in the air has been or can be subjected to aerosol production for use in bioterrorism. The pathogens against which the treatment of the invention may be useful include, but are not limited to, categories A, B and C of priority pathogens as given in the NIAID. These categories correspond generally to the lists compiled by the Centers for disease prevention and prevention (CDC). As established by the CDC, category A measures are those that can be easily distributed or transferred between people, causing high mortality, with the potential to create a high public health risk. Category B measures are further important and include those that are moderately easy to spread and cause moderate morbidity and low mortality. Category C consists of emerging pathogens, which can be modified for massive proliferation in the future, due to their availability, ease of production and spread, and the potential to achieve high morbidity and mortality. Particular examples of these pathogens are anthrax and plague. Additional pathogens that may or may not be protected against them include influenza viruses, rhinoviruses, adenoviruses, and respiratory syncytial viruses, and the like. A further pathogen against which one can protect is a coronavirus that is believed to cause acute severe respiratory distress syndrome (SARS). Particular examples of these pathogens are anthrax and plague. Additional pathogens that may or may not be protected against them include influenza viruses, rhinoviruses, adenoviruses, and respiratory syncytial viruses, and the like. A further pathogen against which one can protect is a coronavirus that is believed to cause acute severe respiratory distress syndrome (SARS). Particular examples of these pathogens are anthrax and plague. Additional pathogens that may or may not be protected against them include influenza viruses, rhinoviruses, adenoviruses, and respiratory syncytial viruses, and the like. A further pathogen against which one can protect is a coronavirus that is believed to cause acute severe respiratory distress syndrome (SARS).

[0087] Also described herein is the use of sodium channel blockers of Formula I, or a pharmaceutically acceptable salt thereof, for the prevention, alleviation and / or treatment of deterministic health effects on the respiratory tract caused by exposure to radioactive materials, particularly respirable aerosols containing radionuclides from Nuclear attacks, such as detonation of radiological equipment scattering materials (RDD), or accidents, such as nuclear plant disasters. As such, the description provides a method for the prevention, alleviation and / or treatment of deterministic health effects on the respiratory tract and / or other organs of the body caused by respirable aerosols containing radionuclides in a recipient who requires it, including a human in need thereof,

[0088] The main problem associated with the planning of effects management in the context of the exposure of members of society to respirable aerosols containing radionuclides from nuclear attacks, such as the detonation of radiological equipment scattering materials

- 25 (RDD), or accidents, such as a nuclear plant disaster, is how to prevent, mitigate or treat potential deterministic health effects on the respiratory tract, especially the lungs. It is necessary to have medicines, techniques and procedures, and trained personnel prepared to deal with and treat such highly internally contaminated persons.

[0089] Studies have been carried out to determine how to prevent, alleviate or treat potential damage to the airways and various organs in the body that are caused by radionuclides deposited inside the body. So far, most of the attention has been focused on strategies designed to mitigate the effects of intraocular radionuclides on health by accelerating their expulsion or removal. These strategies focused on soluble chemical forms that are able to reach the circulatory system and are deposited on distant system-specific locations specific for a given radioactive element. Such approaches will not work in cases where the embedded radionuclide occurs in a relatively insoluble form. Studies have shown that many,

[0090] The only method known to effectively reduce the radiation dose to the lung from inhaled insoluble radioactive aerosols is bronchoalveolar lavage or BAL. This technique, which has been adapted on the basis of that already used for the treatment of patients with follicular proteinosis, has proved to be a safe, repeatable procedure, even if performed over a prolonged period. Although there are variants of the procedure, the BAL primary method involves anesthesia of the subject, followed by the slow introduction of isotonic saline into a single lobe of the lung until the functional residual volume is reached. Additional volumes are then added and emptied by gravity.

[0091] The results of studies using BAL used in animals indicate that about 40% of the deep lung contents can be removed by the rational BAL sequence. In some studies, significant variability was found among animals as to the amount of radionuclide recovered. Reasons for variability are currently unknown.

[0092] Furthermore, based on an animal study, it is believed that a significant dose reduction in BAL therapy results in alleviation of health effects due to the inhalation of insoluble radionuclides. In the study, adult dogs inhaled insoluble particles¹⁴⁴Ce-FAP. Two groups of dogs were given lung contents from¹⁴⁴Ce known to cause lung inflammation due to radiation and pulmonary fibrosis (approximately 2 MBq / kg body weight), with one group treated with one-sided rinsing between 2 and 56 days after exposure and the other group untreated. The third group was exposed at the level¹⁴⁴Ce comparable to that observed in the BAL treated group after treatment (approximately 1 MBq / kg), but these animals were untreated. All animals were allowed to live their life to the end, which covered up to 16 years. Due to the variability in the initial lung content as to¹⁴⁴Ce

- 26 among dogs in each group, dose levels and total doses for each group overlap. Nevertheless, the effect of BAL on reducing the risk of pneumonia / fibrosis was evident from survival curves. In untreated dogs with a lung content of 1.5-2.5 MBq / kg, the average time of survival was 370 ± 65 d. In treated dogs, the mean survival time was 1270 ± 240 d, which was statistically significantly different. The third group that received the lung content of the order¹⁴⁴Ce at 0.61,4 MBq had an average survival time of 1800 ± 230, which was not statistically different from that for the treatment group. What is equally important for prolonged survival, dogs in the untreated group died of a deterministic impact on the lung (pneumonitis / fibrosis), as opposed to treated dogs. Instead, treated dogs, like dogs in the untreated group, with a low dose, mainly suffered from lung tumors (angiosarcoma or cancer). Thus, the reduction in dose under the treatment of BAL seems to produce biological effects in the lungs that were predictable on the basis of doses of radiation received by the lungs.

[0093] Based on these results, it is contemplated that further reducing the residual radiological dose by any method or combination of methods for enhancing the clearance of particles from the lungs will further reduce the likelihood of an effect on lung health. However, BAL is a procedure that has many disadvantages. BAL is a highly invasive procedure that should be performed in specialized medical centers, with the participation of trained pulmonologists. As such, the BAL procedure is expensive. Given the disadvantages of BAL, this is not a treatment option that will be readily and immediately available to people requiring accelerated removal of radioactive particles, for example, in the case of a nuclear attack. In the event of a nuclear attack or nuclear accident, immediate and relatively easy-to-administer treatment for people is necessary, have been exposed or are at risk of exposure. It has been shown that sodium channel blockers administered as an inhalation aerosol restore the hydration of the airway surface. This hydration of the airway surface helps in cleaning the accumulated secreted mucus and associated suspended solid particles from the lung. As such, without wishing to be bound by any particular theory, it is believed that sodium channel blockers can be used to accelerate the removal of radioactive particles from the airways.

[0094] As discussed above, the greatest risk for lung after a radiological attack, such as a dirty bomb, is the inhalation and persistence of insoluble radioactive particles. As a result of the persistence of radioactive particles, total lung exposure is significantly elevated, ultimately leading to pulmonary fibrosis / pneumonia and potentially death. Insoluble particles can not be removed systemically with chelating agents because these particles are not present in the solution. So far, physical removal of suspended particles through BAL is the only therapeutic regimen that has been shown to be effective in alleviating radiation-induced lung disease. As discussed above, BAL is not a realistic solution to treatment in reducing the effects of radioactive particles, which have been inhaled in the body. As such, it is desirable to provide a scheme

- a therapeutic that will effectively help in the removal of radioactive particles from the airways and which, unlike BAL, is relatively simple to administer and allows a scale change in the distribution of large-scale radiation exposure. Furthermore, it is also desirable that the therapeutic regimen be readily available to many people in a relatively short period of time.

[0095] In aspect of the disclosure, a method of preventing, alleviating and / or treating deterministic health effects on the respiratory tract and / or other body organs caused by respirable aerosols comprising radionuclides comprises administering an effective amount of a sodium channel blocker of Formula I or a pharmaceutically acceptable salt thereof to a subject. who needs it. In the description of this aspect, the sodium channel blocker is administered together with an osmolyte. With further reference to this function, the osmolite is a hypertonic saline solution (HS). In the following description, the sodium channel blocker and osmolyte are administered together with the ion transport modulator. With further reference to this function, the ion transport modulator can be selected from the group consisting of β-agonists, CFTR enhancers, purinergic receptor agonists, oriprostones and protease inhibitors. With further reference to this aspect, radionuclides are selected from the group consisting of cobalt-60, cesium-137, iridium-192, radium-226, phosphorus-32, Strontium-89 and 90, iodine-125, talu-201, lead -210, thorium-234, uranium-238, plutonium, cobalt-58, chromium-51, americium and kiuru. In a further description of the functions, the radionuclides are derived from a device for the utilization of radioactive materials. In yet another description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that is inhaled by a given subject. In the additional description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides. cesium-137, iridium-192, radium-226, phosphorus-32, Strontium-89 and 90, iodine-125, talu-201, lead-210, thorium-234, uranium-238, plutonium, cobalt-58, chromium 51, america and kiuru. In a further description of the functions, the radionuclides are derived from a device for the utilization of radioactive materials. In yet another description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that is inhaled by a given subject. In the additional description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides. cesium-137, iridium-192, radium-226, phosphorus-32, Strontium-89 and 90, iodine-125, talu-201, lead-210, thorium-234, uranium-238, plutonium, cobalt-58, chromium 51, america and kiuru. In a further description of the functions, the radionuclides are derived from a device for the utilization of radioactive materials. In yet another description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that is inhaled by a given subject. In the additional description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides. In yet another description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that is inhaled by a given subject. In the additional description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides. In yet another description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that is inhaled by a given subject. In the additional description of the function, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides.

COMPOSITIONS [0096] While it is possible for a compound of the invention to be administered alone, it is preferred in some embodiments to exist in the form of a composition, particularly a pharmaceutical composition (formulation). Thus, in a further aspect, the invention provides compositions, and particularly pharmaceutical compositions (such as an inhalable pharmaceutical composition) comprising a pharmaceutically effective amount of a compound of the invention as an active ingredient, and a pharmaceutically acceptable excipient, diluent or carrier. The term "active ingredient" as used herein refers to any compound of the invention or a combination of two or more compounds of the invention in a pharmaceutical composition. Specific embodiments are also provided,

[0097] In certain embodiments, a pharmaceutical composition comprises a pharmaceutically effective amount of a compound of formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), or a compound thereof. a pharmaceutically acceptable salt, independently or in combination, in

- 28 thinner. In separate embodiments, the pharmaceutical composition contains a pharmaceutically effective amount of a compound of formulas (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), or a pharmaceutically acceptable salt thereof. , in hypertonic saline, sterile water, and hypertonic saline, respectively, wherein the saline concentration can be as described herein. In one embodiment, the concentration of physiological saline is 0.17% w / v, and in another it is 2.8% w / v.

[0098] Also provided is a kit comprising i) a pharmaceutically effective amount of a compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), or its pharmaceutically acceptable salt; ii) one or more pharmaceutically acceptable excipients, carriers or diluents; iii) instructions for administration of a compound of group i) and excipients, carriers or diluents of group ii) to a subject in need thereof; and; iv) container. A subject in need thereof includes any individual that requires the use of the treatment methods set forth herein, especially a human who requires it. Further embodiments also include an aerosol device selected from a group of nebulizers, including rotating membrane nebulisers and jet nebulizers, a dry powder inhaler,

[0099] In one embodiment, the kit comprises i) from about 10 ng to about 10 mg of a compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII) or a pharmaceutically acceptable salt thereof, per dose; ii) from about 1 to about 5 ml of diluent per dose; iii) instructions for administering a compound of group i) and a diluent of group ii) to a subject in need thereof; and; iv) container. In a further embodiment, the diluent has from about 1 to about 5 ml of physiological saline, as described herein, per dose. In a further embodiment, the diluent has from about 1 to about 5 ml of hypotonic saline per dose. In a further embodiment, the diluent has from about 1 to about 5 ml of hypertonic saline per dose.

[0100] Also provided is a kit comprising i) a solution containing a pharmaceutically effective amount of a compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII) or its pharmaceutically acceptable salt; dissolved in a pharmaceutically acceptable diluent; (iii) instructions for administering a solution from the group i) to the individual in need thereof; and iii) container.

[0101] Also provided is a kit comprising i) a solution containing from about 10 ng to about 10 mg of a compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII) or a pharmaceutically acceptable salt thereof; dissolved in a pharmaceutically acceptable diluent; (iii) instructions for administering a solution from the group i) to the individual in need thereof; and iii) container. In a further embodiment, the diluent has from about to about 5 ml of physiological saline as described herein per dose.

[0102] A further embodiment includes a kit comprising i) a pharmaceutically effective amount of a compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII) , or 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 a compound of group i) and excipients or carriers from the group ii) to a subject in need thereof; and; iv) container. In a further embodiment, the kit also includes a dry powder inhaler suitable for providing a recipient dry powder formulation. A dry powder inhaler can mean, in additional embodiments, a single-dose inhaler or a multi-dose inhaler.

[0103] Further embodiments of each of the kits described herein include those in which the concentration of the compound of Formula (I), (Ia), (II), (III), (IV), (V), (VI) and ( VII) or a pharmaceutically acceptable salt thereof, per dose, is one of the ranges of effective doses described herein, including a) from about 0.1 μg to about 1000 μg; b) from about 0.5 μg to about 0.5 mg; and c) from about 0.5 μg to about 50 μg.

[0104] For each of the kits described above, there is an additional embodiment in which diluent means hypertonic saline at the concentrations described herein. In a further embodiment, for each kit, the diluent is hypotonic saline at the concentrations described herein. In a further embodiment for each kit, the diluent is a sterile water suitable for inhalation.

[0105] Pharmaceutically acceptable excipients, diluents or carriers must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In general, the pharmaceutically acceptable excipients, diluents or carriers used in the pharmaceutical formulation are "non-toxic" which means that they are considered safe to consume in the amount provided in the formulation and "inert", meaning that they do not react appreciably or lead to an undesirable effect on the therapeutic activity of the active ingredient. Pharmaceutically acceptable excipients, diluents and carriers are conventional in the art and can be selected using conventional techniques based on the desired route of administration. See REMINGTON'S, PHARMACEUTICAL SCIENCES, Lippincott Williams &amp; Wilkins; Issue 21. (May 1, 2005). Preferably, pharmaceutically acceptable excipients, diluents or carriers are generally recognized as safe (GRAS) according to the FDA.

[0106] The pharmaceutical compositions of the invention include those suitable for oral administration; parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous and intraarterial administration; topical administration, including topical administration to the skin, eyes, ears, etc .; vaginal or rectal administration; and administration to the airways, including nasal cavities and sinuses, orally and outside the chest to the respiratory tract, and lungs, including through the use of aerosols that can be delivered by various types of dry powder inhalers, pressurized inhalers with a dispenser, soft inhalers fog, nebulisers, or

- 30 insufflators. The most appropriate route of administration may depend on several factors, including the patient and the medical condition or disorder to be treated.

[0107] The formulations may be in the form of unit dosage forms or bulk forms, as for example in the case of a dosage formulation by an inhaler, and may be prepared by any of the methods well known in the art of pharmacy. In general, the methods include the step of associating the active ingredient with a carrier, diluent or excipient, and optionally one or more accessory ingredients. In general, the formulations are obtained by uniformly and intimately associating the active ingredient with one or more liquid carriers, diluents or excipients or finely divided solid carriers, diluents or excipients, or both, and then, if necessary, shaping the product to the desired form. formulation.

[0108] In one preferred embodiment, the composition is an inhalable pharmaceutical composition that is suitable for inhalation and delivery to the endobronchial space. Typically, such a composition is in the form of an aerosol comprising particles to be delivered using a nebulizer, a pressurized inhaler with a dispenser (MDI), a soft mist inhaler or a dry powder inhaler (DPI). The aerosol formulation used in the methods of the invention can be a liquid (e.g., solution) suitable for administration by a nebulizer, a soft mist inhaler, or MDI, or a dry powder suitable for administration by MDI or DPI.

[0109] Aerosols used to administer medications to the respiratory tract are typically polydisperse; which means that they consist of particles of many different sizes. The particle size distribution is typically described by mean aerodynamic diameter (MMAD) and geometric standard deviation (GSD). For optimal delivery of the drug to the endobronchial space, the MMAD ranges from about 1 to about 10 gm and preferably from about 1 to about 5 gm, and the GSD is less than about 3, and preferably less than about 2. Aerosols having an MMAD above 10 gm are generally too large when inhaled to reach the lungs. Aerosols with GSD greater than about 3 are not preferred for delivery to the lungs, as they provide a high proportion of the drug to the oral cavity. To achieve these particle sizes in the powder formulation, the active ingredient particles can be reduced in size using conventional techniques such as micronization or spray drying. Non-limiting examples of other methods or techniques that can be used to produce respirable particles include spray drying, precipitation, supercritical fluid, and lyophilization. The desired fraction can be separated by means of air classification or sieving. In one embodiment, the particles will be crystalline. In liquid formulations, particle size is determined by selecting a particular model of nebulizer, soft mist inhaler or MDI. precipitation, supercritical fluid and freeze-drying. The desired fraction can be separated by means of air classification or sieving. In one embodiment, the particles will be crystalline. In liquid formulations, particle size is determined by selecting a particular model of nebulizer, soft mist inhaler or MDI. precipitation, supercritical fluid and freeze-drying. The desired fraction can be separated by means of air classification or sieving. In one embodiment, the particles will be crystalline. In liquid formulations, particle size is determined by selecting a particular model of nebulizer, soft mist inhaler or MDI.

[0110] Particle size distributions in an aerosol are determined using devices well known in the art. For example, a multi-stage Anderson cascade inactivator or other suitable method such as those specifically cited in the American Pharmacopoeia, chapter

601 as for devices characterizing aerosols emitted from inhalers with a dispenser and dry powder inhalers.

[0111] Dry powder compositions for topical delivery to inhaled lung may be formulated without vehicle or carrier and instead contain only active ingredients in the form of a dry powder having a suitable particle size for inhalation. The dry powder compositions may also contain a blend of the active ingredient and a suitable powder base (carrier / diluent / excipient substance) such as mono-, di- or polysaccharides (e.g., lactose or starch). Lactose is typically a preferred dry powder formulation. When a solid excipient such as lactose is used, generally the size of the excipient particles will be much larger than the active ingredient to aid dispersion of the formulation in the inhaler.

[0112] Examples of dry powder inhalers include multi-dose reservoir inhalers, multi-dose metered dose inhalers, capsule-based inhalers and disposable mono-dose inhalers. A reservoir inhaler contains a large number of doses (e.g., 60) in one container. Before inhalation, the patient starts the inhaler, which causes the inhaler to measure one dose of medicine from the reservoir and prepare it for inhalation. Examples of DPIs with a tank include Turbohaler® from AstraZeneca and ClickHaler® from Vectura.

[0113] In multidose dose inhalers, each individual dose is prepared in a separate container, and actuation of the inhaler before inhalation releases a new dose of drug from the container and preparation for inhalation. Examples of multi-dose DPIs include, but are not limited to, Diskus® from GSK, Gyrohaler® from Vectura and Prohaler® from Valois. During inhalation, the inspiratory flow of the patient accelerates the release of the powder from the device and into the mouth. Regarding the inhaler for capsules, the formulation is in the form of a capsule and is stored outside the inhaler. The patient places the capsule in the inhaler, activates the inhaler (pierces the capsule) and then inhales. Examples include Rotohaler ™ (GlaxoSmithKline), Spinhalator ™ (Novartis), HandiHaler ™ (IB), TurboSpin ™ (PH &amp; T). In single-dose, single-dose inhalers, the patient starts the inhaler to prepare for inhalation, inhales, then disposes of the inhaler and the package. Examples include Twincer ™ (U Groningen), OneDose ™ (GFE), and Manta Inhaler ™ (Manta Devices).

[0114] In general, dry powder inhalers use the flow characteristics of a turbulent powder path to cause dispersion of the excipient-drug aggregates, and the active ingredient particles are deposited in the lungs. However, some dry powder inhalers use a cyclone dispersion chamber to produce particles of the desired respirable size. In the cyclone dispersion chamber, the drug moves tangentially into the dispersion chamber in the shape of a coin, so that the air and drug path moves along the outer circular wall. As the drug formulation moves along this circular wall, it bounces around and the agglomerates are broken down by impact forces. The air path moves spirally towards the center of the chamber, going up vertically. particles which have a small aerodynamic size can follow the air path and leave the chamber. Consequently, the dispersion chamber acts as a small jet mill. Depending on the formulation's specifications, large lactose particles can be added to the formulation to help dispersion by colliding with API particles.

[0115] A single-dose Twincer ™ single dose inhaler appears to work using a coin-shaped cyclone dispersion chamber, referred to as an "air classifier". Look. US patent application US 2006/0237010 to Rijksuniversiteit Groningen. The publications published by the University of Groningen reported that it was possible to effectively administer a 60 mg dose of pure micronized colistin sulphomethane as a dry powder inhalable using this technology.

[0116] In preferred embodiments, the aerosol formulation is provided as a dry powder using a dry powder inhaler, wherein the particles emitted from the inhaler have an MMAD in the range of about 1 μm to about 5 μm and GSD about less than 2.

[0117] Examples of suitable dry powder inhalers and dry powder dispersions for use in providing compounds and compositions of the invention include those disclosed in US7520278; US7322354; US7246617; US7231920; US7219665; US7207330; US6880555; US5,522,385; US6845772; US6637431; US6329034;

US5,458,135; US4,805,811; and US patent application US 2006/0237010.

[0118] In one embodiment, the pharmaceutical formulation of the invention is a dry powder for inhalation that is formulated for delivery by a Diskus® device. The Diskus® device comprises an elongated strip made of a basic sheet having a plurality of recesses arranged along its length and a cover sheet sealed therein, but allowing opening to define a plurality of containers, each container having an inhalable formulation containing a predetermined quantity of active ingredient , alone or in admixture with one or more carriers or excipients (e.g., lactose) and / or other therapeutically active agents. Preferably, the strip is flexible enough to roll into a roll. The cover sheet and the base sheet will preferably have leading end portions that are not sealed relative to each other, and at least one of the leading end portions are designed to be attached to the means for winding. Also, preferably, the hermetic seal between the base and cover sheets extends over their entire width. To prepare the dose for inhalation, the cover sheet can advantageously be folded away from the base sheet in the longitudinal direction starting from the first end of the base sheet. preferably, the hermetic seal between the base and cover sheets extends over their entire width. To prepare the dose for inhalation, the cover sheet can advantageously be folded away from the base sheet in the longitudinal direction starting from the first end of the base sheet. preferably, the hermetic seal between the base and cover sheets extends over their entire width. To prepare the dose for inhalation, the cover sheet can advantageously be folded away from the base sheet in the longitudinal direction starting from the first end of the base sheet.

[0119] In one embodiment, the pharmaceutical formulation of the invention is a dry powder for inhalation that is formulated for delivery using a single-dose single-use inhaler, in particular a Twincer ™ inhaler. The Twincer ™ inhaler contains a blister of a film laminate, with one or more depressions, and a cover sheet hermetically sealed, but allowing opening, to define multiple containers. Each container has an inhalable formulation therein containing a predetermined quantity of the active ingredient, alone or mixed with one or more carriers or excipients (e.g., lactose). The cover sheet will preferably have a leading end portion that is designed to protrude from the inhaler body. The patient will use the device and the same

- administer the aerosol formulation by 1) removing the outer wrapping, 2) pulling out the foil tab to reveal the drug in the blister and 3) inhaling the medicine from the blister.

[0120] In a further embodiment, the pharmaceutical formulation of the invention is a dry powder for inhalation, wherein the dry powder is formulated into microparticles as described in PCT WO2009 / 015286 or WO2007 / 114881 both for NexBio. Such microparticles are generally formed by adding the counterion to a solution containing the compound of the invention in a solvent, adding an anti-solvent to the solution; and gradually cooling the solution to a temperature below about 25 ° C to form a composition containing microparticles containing the compound. The microparticles containing the compound can then be separated from the solution by any suitable means, such as deposition, filtration or lyophilization. Suitable counterions,

[0121] In a further embodiment, the pharmaceutical composition of the invention is provided as a dry powder using a metered dose inhaler. Non-limiting examples of metered dose inhalers and devices containing them are disclosed in

US5,261,538; US5,544,647; US5,622,163; US4,955,371; US3,565,070; US3,361, 306 and US6,116,234 and US7,108,159. In a preferred embodiment, the compound of the invention is provided as a dry powder using a metered dose inhaler, with the emitted particles having an MMAD that is in the range of about 1 gm to about 5 gm and a GSD that is less than about 2.

[0122] Liquid aerosol formulations for delivering to the endobronchial or pulmonary space, by inhalation, can for example be formulated as aqueous solutions or suspensions or as aerosols delivered from pressure packages, such as metered dose inhalers, using suitable liquefied propellants, soft mist inhalers or nebulizers. Such aerosol compositions suitable for inhalation may be a suspension or solution and generally contain the active ingredient together with a pharmaceutically acceptable carrier or diluent (e.g., water (distilled or sterile), physiological saline, hypertonic saline, or ethanol) and optionally one or a greater number of other therapeutically active agents.

[0123] Aerosol compositions for delivery by means of pressurized metered dose inhalers typically further comprise a pharmaceutically acceptable propellant. Examples of such propellants include hydrofluorocarbons or hydrogen-containing chlorofluorocarbons or mixtures thereof, especially hydrofluoroalkanes, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethab, in particular 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3, -heptafluoro-n-propane or a mixture thereof. The aerosol composition may be exempt from the excipient or may optionally contain an additional excipient formulation well known in the art, such as surfactants e.g. oleic acid or lecithin, and co-solvents e.g. ethanol. The pressure formulations will generally be contained in a canister (e.g., an aluminum canister) with a closed valve (e.g.

[0124] In a further embodiment, the pharmaceutical composition of the invention is provided as a liquid using a metered dose inhaler. Non-limiting examples of metered dose inhalers and devices containing them include those disclosed in US Patent Nos. 6,253,762, 6,413,497, 7,601,336, 7,481,995, 6,743,413, and 7,105,152. In a preferred embodiment, a compound of the invention is provided as a dry powder using a metered dose inhaler, with the emitted particles having an MMAD that is in the range of about 1 gm to about 5 gm and a GSD that is less than about 2.

[0125] In one embodiment, the aerosol formulation is suitable for producing an aerosol by a jet nebulizer, or ultrasonic nebulizer, including static and vibrating nebulisers with a porous plate. Liquid nebulizer aerosol formulations may be generated by solubilizing or reconstituting particulate formulations, or may be formulated with an aqueous vehicle, with the addition of agents such as acid or bases, buffered salts, and isotonicity adjusting agents. They can be sterilized using techniques within the framework of the method, such as filtration, or end-processes such as autoclaving or gamma radiation. They can also be presented in a non-sterile form.

[0126] Patients may be sensitive to the pH, osmolality and ionic content of the nebulized solution. Thus, these parameters should be adjusted to be compatible with the active ingredient and tolerated by patients. The most preferred solution or suspension of the active ingredient will contain a chloride concentration&gt; 30 mM at pH 4.5-7.4, preferably 5.0-5.5, and osmolality of about 800-1600mOsm / kg. The pH of the solution can be controlled by titration with common acids (hydrochloric acid or sulfuric acid, for example) or bases (sodium hydroxide, for example) or using buffers. Commonly used buffers include citrate buffers, such as citric acid / sodium citrate buffers, acetate buffers such as acetic acid / sodium acetate buffers, and phosphate buffers.

[0127] Useful acetate, phosphate and citrate buffers include sodium acetate, sodium acetate trihydrate, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate dibasic dihydrate, disodium hydrogenphosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium citrate and potassium citrate. Other buffers which may be used include sodium hydroxide, potassium hydroxide, ammonium hydroxide, aminomethylpropanol, tromethamine, tetrahydroxypropylethylenediamine, citric acid, acetic acid, hydroxytrocarboxylic cokes, or their salts such as citrate or its citrate salt, 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, lactate lactate, manganese lactate, ammonium lactate, monoethanolamine,

[0128] Such formulations may be administered using commercially available nebulizers or other atomisers that can cleave the formulation into particles or droplets suitable

- 35 for embedding in the airways. Non-limiting examples of nebulizers that can be used to deliver an aerosol of the composition of the invention include pneumatic jet nebulizers, ventilated or breath-activated jet nebulisers, or ultrasonic nebulisers, including static or vibrating nebulisers with a porous plate. Commercially available nebulisers include the Aeroneb® Go nebulizer (Aerogen) and the eFlow nebulizer (Pari Pharma).

[0129] The jet nebulizer uses a high velocity air stream traveling through a water column to generate droplets. Particles unsuitable for inhalation collide with walls or aerodynamic baffles. A ventilated or breath-actuated nebulizer operates essentially the same as a jet nebulizer, except that the inhaled air passes through the basal droplet generation area to increase the nebulizer output rate while the patient inhales.

[0130] In the ultrasonic nebulizer, the vibration of the piezoelectric crystal creates surface instabilities in the drug reservoir, which causes the formation of droplets. In nebulisers with a porous plate, pressure fields generated by sound energy force liquid through the pores of the mesh, where it is broken down into droplets by Rayleigh scattering. Sound energy can be provided by the vibration of a tube or plate driven by a piezoelectric crystal, or by the vibration of the mesh itself. Non-limiting examples of atomizers include any single or double fluid atomizers or nozzles that generate droplets of the appropriate size. A single fluid atomizer operates by forcing the liquid to flow through one or more openings where the liquid stream is broken down into droplets.

[0131] The choice of a nebulizer that generates an aerosol from an aerosol formulation is important when administering the active ingredient (s). Different nebulizers have varying yields in relation to their construction and principle of action, and are sensitive to the physical and chemical properties of the formulation. For example, two formulations with different surface tensions may have different particle size distributions. In addition, formulation properties such as pH, osmolality, and permeation ion content can affect drug tolerance, so preferred embodiments are compatible with certain ranges of these properties.

[0132] In a preferred embodiment, the nebulizer formulation is delivered to the endobronchial space as an aerosol having a MMAD between about 1 Pm and about Pm and GSD less than 2 using a suitable nebulizer. For optimal efficacy and avoidance of side effects in the upper respiratory system and systemically, the aerosol should not have an MMAD greater than about 5 μm and have a GSD greater than about 2. If the aerosol has an MMAD greater than about 5 μm or GSD greater than about 2, a high dose rate It can settle in the upper respiratory tract, reducing the amount of drug delivered to the desired location in the lower roads

- 36 breaths. If the MMAD of the aerosol is less than about 1 gm, then a large proportion of particles may remain suspended in the inhaled air and may then be exhaled when exhaled.

[0133] The compounds of the invention may also be administered via transbronchoscopic rinsing.

[0134] Formulations suitable for oral administration may be formulated into discrete units, such as capsules, cachets or tablets, each containing a predetermined quantity of active compound, as a powder or granules or as a solution or suspension in an aqueous liquid, in a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a sachet, intravenous injection, medicine mixed with honey or syrup or paste.

[0135] The tablet may be made by compression or molding, optionally with one or more additives. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient, in a free flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or provided with a dividing line and may be formulated so as to provide slow or controlled release of the active ingredient therefrom.

Formulations for topical administration in the mouth, e.g. bucally or sublingually, include lozenges comprising the active ingredient in a flavor base, such as sucrose and acacia or gum tragacanth, and lozenges comprising the active ingredient in a base such as gelatin and glycerin or sucrose and arabic gum.

[0137] Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be in the form of unit dose containers or multiple doses, e.g. sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, e.g. saline, water for injection, immediately before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders,

[0138] Oral fluids, such as solutions, syrups and elixirs, may be prepared in unit dosage form such that the amount contains a predetermined quantity of active ingredient. Syrups can be obtained by dissolving the active ingredient in a suitably flavored aqueous solution, while the elixirs are prepared from

Using a pharmaceutically acceptable alcohol excipient. The suspensions may be formulated by dispersing the active ingredient in a pharmaceutically acceptable excipient. Solubilizers and emulsifiers, such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavors, such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be included in liquid oral compositions.

[0139] Systems for delivering liposomes, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles may also be used as delivery forms for compounds of the invention. Liposomes can be produced from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

[0140] Pharmaceutical compositions for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Compositions designed to treat eyes or other external tissues, e.g. mouth and skin, can be applied as a topical ointment or cream. When formulated into an ointment, active ingredients may be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

[0141] Other compositions designed for topical administration to the eyes or ears include eye and ear drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, such as, for example, an aqueous solvent, including physiological saline.

[0142] Compositions designed for nasal administration include aerosols, solutions, suspensions, nebulisers, mists and drops. Aerosol formulations for nasal administration may be formulated using substantially the same solutions as aerosol inhalation formulations, provided that the non-responsive particles are preferred in nasal formulations. Typically, particles of about 5 microns in size can be used up to the size of visible droplets. Hence, for nasal administration, a particle size in the range of 10-500 μm can be used to provide retention in the nasal cavity.

[0143] Transdermal patches that are designed to remain in contact with the patient's epidermis for an extended period of time and stimulate the absorption of the active ingredient through the epidermis can also be used.

[0144] Compositions for vaginal or rectal administration include ointments, creams, suppositories and enemas, all of which may be formulated using conventional techniques.

[0145] In a further aspect, the invention provides a method of stimulating hydration of the mucosal surface or restoring mucosal defense in a human that requires it, comprising administering to a human a pharmaceutical composition comprising a compound of the invention wherein the compound is administered in an effective amount. In one

In a preferred embodiment, the method comprises administering a pharmaceutical composition as an inhalable composition comprising an amount of a compound of the invention that is sufficient to achieve a dissolved concentration of compound on surfaces of the airways of about 10^-9, 10^-8 or 10^-7 to around 10^-4.

10^-3, 10^-2 or 10^-1 moles / liter, more preferably from about 10^-9 to around 10^-4 moles / liter.

[0146] In a further aspect, the invention provides a method of treating any of: a disease associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (POCnP), asthma, bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis), acute inflammation bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis associated with transplantation bronchiolitis, and respiratory-related tracheitis and bronchitis or the prevention of respiratory-related pneumonia in a human which requires it, comprising administering to a human a pharmaceutical composition comprising a compound of the invention wherein the compound is administered in an effective amount.In one preferred embodiment, the method comprises administering a pharmaceutical composition as an inhalable composition comprising an amount of a compound of the invention that is sufficient to achieve a dissolved concentration of compound on airway surfaces of about 10^-9, 10^-8 or 10^-7 to around 10^-4.

10^-3, 10^-2 or 10^-1 moles / liter, more preferably from about 10^-9 to around 10^-4 moles / liter.

[0147] In a further aspect, the invention provides a method of treating any of: dry mouth (xerostomia), dry skin, dry vagina, sinusitis, rhinosinusitis, or nasal dehydration, including nasal dehydration caused by administration of dry oxygen, dry eye or Sjogren's disease, stimulation of eye or corneal moisturization, treatment of distal small intestinal patency syndrome, treatment of otitis media, primary ciliary dyskinesia, distal small intestinal patency syndrome, esophagitis, constipation or chronic diverticulitis that requires it, including administering to a human a pharmaceutical composition comprising a compound of the invention wherein the compound is administered in an effective amount.

[0148] A preferred unit dosage form of the formulations for compounds of the invention means those containing an effective amount of the active ingredient or a suitable fraction thereof.

[0149] It should be understood that in addition to the ingredients specifically mentioned above, the formulations of the invention may contain other agents conventional in the art with respect to the type of formulation in question, for example those suitable for oral administration may contain flavoring agents.

[0150] The compositions of the invention may be formulated for immediate, controlled or sustained release as desired for the particular disease state to be treated and the desired route of administration. For example, formulation

Controlled release, for oral administration may be desirable for the treatment of constipation, to maximize the delivery of the active agent to the large intestine. Such formulations and suitable excipients for this are well known in the state of the art pharmaceutical field. Since the free base of the compound is generally less soluble in aqueous solutions than the salt, the compositions containing the free base of the compound of Formula I can be used to provide a more prolonged release of the active agent delivered via inhalation to the lungs. The active agent present in the lungs in the form of particles, which are not dissolved in the solution, is not available for inducing a physiological response, but serves as a composition of a bioavailable drug that gradually dissolves in solution. As another example,

COMBINATIONS [0151] The compounds of the invention may be formulated and / or used in combination with other therapeutically active agents. Examples of other therapeutically active agents that may be formulated or used in combination with the compounds of the invention include, but are not limited to, osmolites, anti-inflammatory agents, anticholinergic agents, β-agonists (including selective e2 agonists), P2Y2 receptor agonists, peroxisomal proliferator-activated receptor proliferators (PPAR) delta, other epithelial sodium channel blockers (ENaC receptor blockers), modulators of transmembrane transmission regulators in cystic fibrosis (CFTR), kinase inhibitors, anti-infective agents, antihistamines, non-antibiotic anti-inflammatory macrolides, elastase and protease inhibitors, and mucus or mucin modifying agents . such as surfactants. In addition, for the cardiovascular indications, the compounds of the invention may be used in combination with beta blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents.

[0152] The invention thus provides, in a further aspect, a composition comprising an effective amount of a compound of the invention and one or more other therapeutically active agents selected from osmolytes, anti-inflammatory agents, anticholinergic agents, β-agonists (including selective e2 agonists), P2Y2 receptor agonists, PPAR delta agonists, ENaC receptor blockers, modulators of transmembrane transmission regulators in cystic fibrosis (CFTR), kinase inhibitors, anti-infective agents, antihistamines, non-antibiotic anti-inflammatory macrolides, elastase and protease inhibitors, and mucin or mucin modifying agents, such as surfactants. The invention thus provides, in a further aspect, a composition comprising an effective amount of a compound of the invention and one or more other therapeutically active agents selected from beta blockers, ACE inhibitors, HMGCoA reductase inhibitors, and calcium channel blockers. The use of the compounds of the invention in combination with one or more others

Therapeutically active agents (particularly osmolytes) may reduce the dose of a compound of the invention that is required for sufficiently moisturizing the mucosal surface, thereby reducing the potential for undesirable side effects attributable to systemic blockade of sodium channels, such as in the kidneys, for example.

[0153] "Osmolytes" according to the invention are molecules or compounds which are osmotically active. "Osmotically active" molecules and compounds mean those with an impermeable membrane (i.e., substantially non-resorbable) on the surface of the airway or lung epithelia. The terms "airway surface" and "lung surface" as used herein refer to pulmonary surfaces of the airways such as bronchi and bronchioles, surfaces of alveoli, and nasal surfaces and sinuses. Suitable osmolites include ionic osmolites (i.e., salts), and non-ionic osmolites (i.e., sugars, sugar alcohols, and organic osmolytes). In general, osmolites (both ionic and non-ionic) used in combination with the compounds of the invention are preferably osmolites that do not stimulate or actually inhibit or retard bacterial growth.

[0154] Examples of the ionic osmolytes useful in the invention include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably, any (or both) of the anion and cation are osmotically active and are not subject to rapid active transport relative to the surfaces of the airways to which they are administered. Such compounds include, but are not limited to, anions and cations that are contained in FDA approved commercially available salts, see, e.g., Remington: Science and Practice of Pharmacy, Volume II, page 1457 (19th edition 1995), and can be used in any combinations as is known in the art.

[0155] Specific examples of pharmaceutically acceptable osmotically active anions include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, hydrogen tartrate, bromide, calcium edetate, camphor sulphate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate (1,2-ethanedisulphonate) ), estolate (lauryl sulfate), esylate (1,2-ethanedisulphonate), fumarate, gluceptane, gluconate, glutamate, glycolylsanilane (p-glycolamidophenyleneate), hexylresorcinate, hydrabamine (N, N'di (dehydroabiethyl) ethylenediamine), hydrobromide, hydrochloride, hydroxynftoane, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methylsulphonate, muconate, naphthalenesulphonate, nitrate, nitrite, palmate (pantonate), pantothenate, phosphate or diphosphate, polygalacturonate,salicylate, stearate, podoctane, succinate, sulfate, tannate, tartrate, theocoate (8-chloroteophilate), triethyliodide, bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide and phosphate.

[0156] Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to, organic cations such as benzathine (N, N'-dibenzylethylenediamine), chloroprocaine, 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; and metal cations such as aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron, ammonium and the like. Preferred organic cations include organic 3-carbon, 4-carbon, 5-carbon and 6-carbon cations. Preferred cations include sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, magnesium and calcium.

[0157] Specific examples of ionic osmolytes that may be used in combination with a compound of the invention include, but are not limited to, sodium chloride (especially hypertonic saline), potassium chloride, choline chloride, choline iodide, lithium chloride, meglumine chloride, L-lysine chloride, D-lysine chloride, ammonium chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride, potassium bromide, and combinations of any two or more of the foregoing. In one embodiment, the invention provides a combination of a compound of the invention and two different osmotically active salts. When different salts are used, one of the anions or cations may be the same among the different salts.

[0158] Non-ionic osmolytes include sugars, sugar alcohols, and organic osmolytes. Sugars and sugar alcohols useful as osmolytes in the invention include, but are not limited to, 3-carbon sugars (e.g., glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both D and L forms of erythrose, trerose, and erythrulose); 5-carbon sugars (e.g., both D and L forms of ribose, arabinose, xylose, lysosose, psicose, fructose, sorbose, and tagatose); and 6-carbon sugars (e.g., both forms D and L of altose, allose, glucose, mannose, gulose, idose, galactose, and talose, and form D and L of alloheptulose, allo-hepulose, glucose-heptulose, manno-heptulose, gulo -heptulose, ido-heptulose, galacto-heptulose, talo-heptulose). Additional sugars useful in the practice of the invention include raffinose, a series of refined oligosaccharides, and stachyose. Both forms D and L of the deduced form of each sugar / sugar alcohol are also suitable in the invention. For example, glucose, when reduced, becomes sorbitol; an osmolyte within the scope of the invention. Accordingly, sorbitol and other reduced sugar / sugar alcohol forms (e.g., mannitol, dulcitol, arabitol) are suitable osmolytes for use in the invention. Mannitol is a preferred nonionic osmolyte for use in combination with the compounds of the invention.

[0159] "Organic osmolytes" are generally used to refer to molecules that control intracellular osmolality in the kidney. See, e.g., JS Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995). Organic osmolytes include, but are not limited to, three major classes of compounds: polyols (polyhydric alcohols), methylamines and amino acids. Suitable polyol organic osmolytes include, but are not limited to, inositol, myo-inositol, and sorbitol. Suitable methylamino organic osmolites include, but are not limited to, choline, betaine, carnitine (L-, D- and DL-form), phosphorylcholine, lyso-phosphorylcholine, glycerophosphorylcholine, creatinine, and creatinine phosphate. Suitable amino acid organic osmolytes include, but are not limited to, D- and L-glycine, alanine, glutamine,

- 42 glutamate, aspartate, proline and tautyna. Additional organic osmolytes suitable for use in the invention include tihulose and sarcosine. Organic osmolytes are preferred, and human organic osmolites are most preferred. However, some organic osmolytes are of bacterial, yeast and marine origin, and these compounds can also be used in the invention.

[0160] Osmolyte precursors can be used in combination with the compounds of the invention. "Osmolyte precursor" as used herein refers to a compound that is converted to osmolyte in a metabolic, catabolic or anabolic step. Examples of osmolyte precursors include, but are not limited to, glucose, glucose polymers, glycerol, choline, phosphatidylcholine, lyso-phosphatidylcholine and inorganic phosphates, which are precursors of polyols and methylamines. Precursors of amino acid osmolytes include proteins, peptides, and polyamino acids that are hydrolyzed to yield osmolytic amino acids, and metabolic precursors that can be converted to osmolytic amino acids by a metabolic step, such as transamination. For example, the precursor of the amino acid glutamine is poly-L-glutamine, and the glutamate precursor is poly-L-glutamic acid.

[0161] Chemically modified osmolytes or osmolyte precursors may also be used. Such chemical modifications involve the combination of an osmolyte (or precursor) with an additional chemical group that alters or enhances the action of the osmolyte or osmolyte precursor (e.g., inhibits the degradation of the osmolyte molecule). Such chemical modifications have been used with drugs or prodrugs, and they are known in the art. (see, for example, US Patents 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19: 113-117 (1976); Bodor, N. et al., J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N. et al., J. Med Chem. 26: 313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75: 29- 35 (1986).

[0162] Preferred osmolytes for use in combination with the compounds of the invention include sodium chloride, a defined hypertonic saline solution, and mannitol.

[0163] With respect to the formulation of 7% and&gt; 7% hypertonic saline physiological solution, formulations containing bicarbonate anions can be particularly useful, especially in respiratory disorders with dysfunction of the transmembrane conductivity regulator in cystic fibrosis (CFTR), such as CF or POChP. Recent observations indicate that, despite the relative ratio of HCO conductivity^-/ Clis between 0.1 and 2 for single cAMP and ATP activated CFTR channels, the sweat channel ratio can be substantially from 0 to almost 1.0, depending on the stimulation conditions. That is, the combination of cAMP + cGMP + u-ketoglutarate^{can give}CFTR HCO's leadership₃^- almost equal to the conduct of Cl^- (Quiton et al. ^{Physiology, vol} 22, No. 3, 212 - 225, June 2007). In addition, formulations of 7% and&gt; 7% hypertonic saline solution containing bicarbonate anions can be particularly useful in connection with better control of pH in the liquid on the surface of the airways. first, it has been shown that there is acidification of the airways in CF (Tate et al. 2002) and that absent CFTR-dependent secretion of bicarbonate may lead to disruption of the ability to respond to conditions in the airways associated with acidification of the liquid on the airway surface layer (Coakley

- 43 et al. 2003). Second, the addition of a HS solution without bicarbonate to the lung surface can further dilute the bicarbonate concentration, and potentially reduce the pH or ability to respond to acidification of the airways in the liquid layer on the surface of the airways. Thus, the addition of bicarbonate anions to HS may help to maintain or improve the pH of the liquid on the airway surface layer in patients with CF.

[0164] Because of this evidence, the incorporation of a bicarbonate anion into the 7% or&gt; 7% formulation of the hypertonic saline solution administered by the method of the invention will be particularly useful. Formulations containing up to 30 to 200 mM concentrations of bicarbonate anions are particularly interesting as solutions of 7% or&gt; 7% HS.

[0165] The hypertonic saline solution is intended to have a salt concentration greater than that of normal saline (NS) saline, i.e. greater than 9 g / L or 0.9% w / v, and the hypotonic saline solution has salt concentration less than that for a normal physiological saline solution, such as from about 1 g or L / 0.1% w / v. up to about 8 g / L or 0.8% w / v. The hypertonic saline solution useful in the formulations and methods of treatment herein may have a salt concentration of about 1% to about 23.4% (w / v). In one embodiment, the hypertonic saline solution has a salt concentration of about 60 g / L (6% w / v) to about 100 g / L (10% w / v). In another embodiment, the saline solution has a salt concentration of about 70 g / L (7% w / v) to about 100 g / L (10% w / v). ). In further embodiments, the saline solution has salt concentrations of a) from about 0.5 g / L (0.05% w / v) to about 70 g / L (7% w / v); b) from about 1 g / L (0.1% w / v) to about 60 g / L (6% w / v); c) from about 1 g / L (0.1% w / v) to about 50 g / L (5% w / v); d) from about 1 g / L (0.1% w / v) to about 40 g / L (4% w / v); e) from about 1 g / L (0.1% w / v) to about 30 g / L (3% w / v); if) from about 1 g / L (0.1% w / v) to about 20 g / L (2% w / v). e) from about 1 g / L (0.1% w / v) to about 30 g / L (3% w / v); if) from about 1 g / L (0.1% w / v) to about 20 g / L (2% w / v). e) from about 1 g / L (0.1% w / v) to about 30 g / L (3% w / v); if) from about 1 g / L (0.1% w / v) to about 20 g / L (2% w / v).

200 g / l (20% w / v), 210 g / l (21% w / v), 220 g / l (22% w / v), and 230 g / l (23% wag./obj.). Concentrations of physiological saline between each of the specified concentrations / percentages can also be used, such as saline solutions having 1.7 g / L (0.17% w / v), 1.25 g / L (1.25 g / L). % w / v), 1.5 g / L (1.5%)

- 44 w / v), 25 g / l (2.5% w / v), 28 g / l (2.8% w / v), 35 g / l (3.5%) weight / volume), 45 g / l (4.5% w / v), and 75 g / l (7.5% w / v).

[0167] Specific useful concentrations of hypotonic saline solutions include those having from about 0.12 g / L (0.012% w / v) to about 8.5 g / L (0.85% w / v). Any concentration in this range can be used, such as, based on weight / volume. 0.05%, 0.1%,

0.15%, 0.2%, 0.225% (1/4 NS), 0.25%, 0.3% (1/3 NS), 0.35%, 0.4%, 0.45% ( 1/2 NS), 0.5%, 0.55%, 0.6% (2/3 NS), 0.65%, 0.675% (3/4 NS), 0.7%, 0.75% , and 0.8%.

[0168] Each of the ranges and specific physiological saline concentrations described herein can be used with the formulations, treatment methods, schedules, and kits described herein.

[0169] Also within the scope of the invention, chemically modified osmolites or osmolyte precursors are considered. Such chemical modifications involve the combination of an osmolyte (or precursor) with an additional chemical group that alters or enhances the action of the osmolyte or osmolyte precursor (e.g., inhibits the degradation of the osmolyte molecule). Such chemical modifications have been used with drugs or prodrugs, and they are known in the art. (See, for example, US Patents 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19: 113-117 (1976); Bodor, N. et al., J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N. et al., J. Med Chem. 26: 313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75: 29- 35 (1986), each included in the description by reference.

[0170] Suitable anti-inflammatory agents for use in combination with the compounds of the invention include corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs), especially phosphodiesterase (PDE) inhibitors. Examples of corticosteroids for use in the invention include oral or inhaled corticosteroids or their prodrugs. Specific examples include, but are not limited to, ciclesonide, desisobutyryl-ciclesonide, budesonide, flunizolide, mometasone and their esters (e.g., mometasone furoate), fluticasone propionate, fluticasone furoate, beclomethasone, methylprednisolone, prednisolone, dexamethasone, 6a, 9a-difluoro- 17a - [( 2-furanylcarbonyl) oxy] -β, 3-hydroxy-16a-methyl-3-oxo-androstene-1,4-dieno-17e-carbothioic acid, S-fluoromethyl ester, 6α, 9α-difluoro-1-hydroxy-16-methyl-3-oxo-17a -propionyloxy-androstine-1,4-dieno-β-carotin,

[0171] NSAIDs for use in the invention include, but are not limited to, sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, mixed PDE3 / PDE4 inhibitors or mixed PDE4 / PDE7 inhibitors), leukotriene antagonists, leukotriene synthesis inhibitors (e.g., LO and FLAP inhibitors), nitric oxide synthase inhibitors (iNOS), protease inhibitors (e.g., tryptase inhibitors, neutrophil elastase inhibitors, and metalloprotease inhibitors) of e2-integrin antagonists and agonists or

Adenosine receptor antagonists (e.g., adenosine 2a agonists), cytokine antagonists (e.g.

chemokine antagonists) or inhibitors of cytokine synthesis (e.g., prostaglandin D2 receptor antagonists (CRTh2)). Examples of leukotriene modifiers suitable for administration by the method of the invention include montelukast, zileuton and zafirlukast.

[0172] A PDE4 inhibitor, a mixed PDE3 / PDE4 inhibitor, or a mixed PDE4 / PDE7 inhibitor can be any compound that is known to inhibit the PDE4 enzyme or which is found to act as a PDE4 inhibitor, and which are selective PDE4 inhibitors (i.e. compounds that do not significantly inhibit other members of the PDE family). Examples of specific PDE4 inhibitors for formulation and use in combination with the compounds of the invention include, but are not limited to, roflumilast, pumafenthrine, arofylin, cilomilast, tofimilast, oglemilast, tolafenthrin, piclamilast, ibudilast, apremilast, 2 [4- [6,7-diethoxy-2,3 -bis (hydroxymethyl) -1-naphthalenyl] -2-pyridinyl] -4- (3-pyridinyl) -1 (2H) -phthalazinone (T 2585), N- (3,5-dichloro-4-pyridinyl) -1- [ (4-fluorophenyl) methyl] -5-hydroxy-α-oxo-1H-indole-3-acetamide (AWD-12-281,

[0173] Leukotriene antagonists and leukotriene synthesis inhibitors include zafirlukast, montelukast sodium, zileuton, and pranlukast.

[0174] Anticholinergic agents for formulation or use in combination with the compounds of the invention include, but are not limited to, muscarinic receptor antagonists, particularly general antagonists and M-receptor antagonists.₃. Exemplary compounds include wolfberry plant alkaloids such as atropine, scopolamine, homatropine, hyoscyamine, and various forms, including their salts (e.g., anhydrous atropine, atropine sulfate, atropine oxide or HCl, methylatropine nitrate, homatropin hydrobromide, methyl homatropine bromide, hydrosamine hydrosulfide, hyoscyamine sulphate, scopolamine hydrobromide, scopolamine methyl bromide), or any combination or subset thereof.

[0175] Additional anticholinergic agents for formulation and use in combination are metantelin, propantheline bromide, anisotropin methyl bromide or Valpin 50, aclidinium bromide, glycopyrrolate (Robinul), isopropamide iodide, mepenzolate bromide, tridihexetyl chloride, hexocyclic methyl sulphate, cyclopentolate HCl, tropicamide, trihexyphenidyl CCl, pyrenzepine, telenzepin, and metoctramine, or any combinations or subsets thereof.

[0176] Preferred anticholinergic agents for formulation and use in combination with the compounds of the invention include ipratropium (bromide), oxitropium (bromide) and tiotropium (bromide), or any combinations or subsets thereof.

[0177] 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, and xinafoate, their salts, albuterol or R-albuterol (free base or sulfate), levalbuterol, salbutamol, formoterol (fumarate) ), fenoterol, prokaterol, pirbuterol, metaprterenol, terbutaline and their salts, and any combinations or subsets thereof.

[0178] The P2Y2 receptor agonists for formulation and use in combination with the compounds of the invention can be used in an amount effective to stimulate the secretion of chloride and water through the surfaces of the airways, particularly through nasal airways. Suitable P2Y2 receptor agonists are known in the art and described, for example, in columns 9-10 of US Patent No. 6,264,975, as well as US Patents US 5,656,256 and 5,292,498.

[0179] P2Y agonists₂ which can be administered by the methods of the invention include P2Y receptor agonists₂ such as ATP, UTP, UTP-.gamma.-S and P2Y agonist dinucleotide₂ (e.g., denufosol or diquafosol) or a pharmaceutically acceptable salt thereof. P2Y receptor agonists₂ they are typically enclosed in an amount effective to stimulate the secretion of chloride and water through the surfaces of the airways, particularly the nasal surfaces of the airways. Suitable P2Y receptor agonists₂ are described in, but not limited to, U.S. Patent No. 6,264,975, U.S. Patent No. 5,656,256, U.S. Patent No. 5,292,498, U.S. Patent No. 6,348,589, U.S. Patent 6,818,629, U.S. Patent No. 6,977,246, U.S. Patent 7,223,744, U.S. Patent No. 7,,511,525, and U.S.A. .2009 / 0306009, each of which is incorporated herein by reference.

[0180] Combination therapies and formulations as herein described may include adenosine 2b (A2b) agonists, including BAY 60-6583, NECA (N-ethylcarboxamide adenosine), (S) PHPNECA, LUF-5835 and LUF-5845. A2b agonists that can be used are described in

Volpini et al., Journal of Medicinal Chemistry 45 (15): 3271-9 (2002); Volpini et al., Current Pharmaceutical Design 8 (26): 2285-98 (2002); Baraldi et al., Journal of Medicinal Chemistry 47 (6): Cacciari et al., 1434-47 (2004); Mini Reviews in Medicinal Chemistry 5 (12): 1053-60 (Dec. 2005); Baraldi et al., Current Medicinal Chemistry 13 (28): 3467-82 (2006); Beukers et al., Medicinal Research Reviews 26 (5): 667-98 (Sept. 2006); Elzein et al., Bioorganic &amp; Medicinal Chemistry Letters 16 (2): 302-6 (Jan. 2006); Carotti, et al.

- 47 Journal of Medicinal Chemistry 49 (1): 282-99 (Jan. 2006); Tabrizi et al., Bioorgonic &amp; Medicinal Chemistry 16 (5): 2419-30 (March 2008); and Stefanachi, et al., Bioorgonic &amp; Medicinal Chemistry 16 (6): 2852-69 (March 2008).

[0181] 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 its derivatives, such as those described in US Patent 6,858,615, and PCT Publication WO2003 / 070182, WO2004 / 073629, WO2005 / 018644 , WO2006 / 022935, WO2007 / 018640, and WO2007 / 146869, all for Parion Sciences, Inc.

[0182] The small molecule ENaC blockers are directly capable of preventing sodium transport through ENaC channel pores. An ENaC blocker that can be administered in combinations as described herein includes, but is not limited to, amiloride, benzamil, phenamil, and amiloride analogs as detailed in US Patent No. 6,858,614, US 6,858,615, US 6,903,105, US 6,995,160, US 7,266,325 , US Patent No. 7,030,117, US 7,064,612, US 7,186,833, US 7,189,719, US 7,192,958, US 7,192,959, US 7,241,766, US 7,247,636, US 7,247,637, US 7,317,013 , US 7,332,496,

[0183] Proteolysis of ENaC is well described as increasing sodium transport through ENaC. The protease inhibitor blocks the activity of endogenous respiratory proteases, thereby preventing clearance and activation of ENaC. Prostheses that cleave ENaC include furin, meprin, matryptase, trypsin, channel-associated proteases (CAPs), and neutrophil elastases. Protease inhibitors that can inhibit the proteolytic activity of these proteases that can be administered in combinations herein include, but are not limited to, kamostat, protazin, furin, aprotinin, leupeptin, and trypsin inhibitors.

[0184] Combinations herein may comprise one or more suitable nucleic acids (or a polynucleic acid), including, but not limited to, antisense oligonucleotides, siRNA, miRNA, mimetic miRNA, antagomir, ribozyme, aptamer, and decoy oligonucleotide nucleic acids. See, e.g., US Pat. No. 20100316628. In general, such nucleic acids may 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 described in US 7,517,865 and US patent applications US 20100215588; 20100316628; 20110008366; and 20110104255. Generally, siRNAs have from 17 or 19 nucleotides in length, up to 23, 25 or 27 nucleotides in length, or more.

[0185] Compounds that modulate CFTR activity that can be administered in combinations of the invention include, but are not limited to, those described in US 2009/0246137 A1, US

2009/0253736 A1, US 2010/0227888 A1, US 7,645,789, US 2009/0246820 A1, US 2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US 2010/0168094 A1 and the issued patent: 7,553,855; US 7,772,259 B2, US 7,405,233 B2, US 2009/0203752, US 7,499,570.

[0186] Mucin or mucin modifiers useful in the combinations and methods herein include reducing agents, surfactants and detergents, expectorants, and deoxyribonuclease agents.

[0187] Mucin proteins are organized into high molecular weight polymers by forming covalent (disulphide) and non-covalent bonds. Breaking covalent bonds with reducing agents is a well-established way of reducing viscoelastic mucus properties in vitro and it is predicted that this minimizes mucus adhesion and improves clearance in vivo. Reducing agents are well known for reducing mucus viscosity in vitro and are widely used as an aid in the processing of sputum samples. Examples of reducing agents include sulphide or phosphine-containing molecules capable of reducing disulphide bonds in the protein, including, but not limited to, N-acetylcysteine, N-acystellins, carbocysteine, glutathione, dithiothreitol, thioredoxin-containing proteins, and tris (2-carboxyethyl) phosphine.

[0188] N-acetylcysteine ​​(NAC) is approved for use in combination with chest physiotherapy to loosen viscous or thickened mucus in the airways. In clinical trials assessing the effect of oral or inhaled NAC in CF and COPD, it was reported to improve rheumatic mucosal properties and trends to improve lung function and reduce the number of pulmonary exacerbations⁹. However, the prevailing clinical data indicate that NAC is at most a marginal effective therapeutic agent for the treatment of airway obstruction caused by mucus when administered orally or inhaled. A recent review of Cochrane's existing clinical literature on the use of NAC has not found any evidence of the effectiveness of NAC in CF. The marginal clinical benefit of NAC reflects:

NAC is a relatively ineffective reducing agent that is only partially active on the surface of the airways. Very high concentrations of NAC (200 mM or 3.26%) are required for the full reduction of Muc5B, the mucin-forming major airway gel, in vitro. In addition, the pH of the airway surface environment (measured in the pH range 6.0 to 7.2 in the airways with CF and

COPD), NAC only occurs partially in its reactive stage as a negatively charged thiolate. Hence, in clinical settings, NAC is administered at very high concentrations. However, it is anticipated that current aerosol delivery devices will not be able to achieve therapeutic concentrations of up to 20% of the Mucomyst solution on the distal airway surfaces within the relatively short periods (7.5-15 minutes) typically used.

- [0189] In non-clinical studies, ¹⁴C-labeled NAC, administered by inhalation, shows rapid elimination from the lungs, with a half-life in the range of 6 to 36 minutes.

[0190] NAC is administered as a highly concentrated, hypertonic solution for inhalation (20% or 1.22 molar) and reported to cause bronchospasm and coughing. In many cases, it is recommended that NAC be administered with a bronchodilator to improve the tolerance of the agent.

[0191] Hence, reducing agents such as NAC are not well suited for administering a bolus aerosol. However, it is envisaged that delivery of reducing agents in the infusion of aerosol administered to the lungs will increase efficacy, while also allowing reducing the concentration of the reducing agent in the solution for inhalation (predicted increase in tolerance).

[0192] Surfactants and detergents are spreading agents that have been shown to reduce the viscoelasticity of mucus, improving mucus clearance. Examples of surfactants include dipalmitoylphosphatidylcholine (DPPC), PF, palmitic acid, palmitoyl-oleophilatidylglycerol, surfactant-related proteins (e.g., SP-A, B or C), or those of animal origin (e.g., from lung or calf lung or extractable lung lungs) from a crushed pig's lungs) or combinations thereof. See. e.g. US patents US 7,897,577; 5,876,970; 5,614,216; 5,100,806; and 4,312,860. Examples of surfactant products include Exosurf^® Neonatal (colfosceril palmitate), Pumactant^® (DPPC and phosphatidylglycerol from eggs), surfactant KL-4, Venticute^®(lusulptide, rSP-C surfactant), Alveofact^® (bovactant), Curosurf^® (poractant alfa), Infasurf^®(calfactant), Newfacten^® (modified bovine surfactant), Surface^®, Natsurf ™ (non-ionic alcohol ethoxylated surfactant) and Survanta^® (Beractant). Examples of detergents include, but are not limited to, Tween-80 and Triton-X 100.

[0193] Any suitable expectorant can be used, including, but not limited to, guaifenazine (see, e.g., US Patent 7,345,051). Any suitable deoxyribonuclease can be used, including but not limited to Dornase Alpha. (see, e.g., U.S. Patent No. 7,482,024).

[0194] Examples of kinase inhibitors include NFkB inhibitors, PI3K (phosphatidylinositol-3-kinase), p38-MAP kinases, and Rho kinases.

[0195] Anti-infective agents for formulation and use in combination with the compounds of the invention include antiviral agents and antibiotics. Examples of suitable antiviral agents include Tamiflu® (oseltamivir) and Relenza® (zanamivir). Examples of suitable antibiotics include, but are not limited to, aztreonam (arginine or lysine), fosfomycin, and aminoglycosides such as tobramycin, or any combinations or subsets thereof. Additional anti-infective agents that can be used herein include aminoglycosides, daptomycin, fluoroquinolones, ketolides, carbapenems, cephalosporins, erythromycin, linezolid, penicillins, azithromycin, clindamycin, oxazolidinones, tetracklins, and vancomycin.

[0196] Examples of useful carbapenamic antibiotics are impenam, panipenam, meropenam, biapenam, MK-826 (L-749,345), DA-1131, ER-35786, lenapenam, S-4661, CS834 (pro-drug R-95867), KR -21056 (prodrug KR-21012), L-084 (LJC prodrug 11036) and ceftolozane (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 dimenhydrinate; ethylenediamines, such as pyrimine maleate (metpyramine), tripelennamine HCl, tripelennamine citrate, and antazoline; alkylamines, such as phenyramine, chlorpheniramine, brompeniramine, deoxylorfeniramine, triprolidine and acacotine; pyridines, such as metapyrilene, piperazines, such as hydroxyl HCl, hydroxyzine pamoate, cyclizine HCl, cyclizine lactate, hemoglobin HCl and cetirizine HCl; piperidines, such as astemizole, levocabastine HCl, loratadine, descarboethoxyloratadine, terfenadine, and fexofenadine HCl; tri-and tetracyclic compounds, such as promethazine, chlorprometazine, trimeprazine and azatadine; and azelastine HCl, or any combinations or subsets thereof.

[0198] Examples of other classes of therapeutic agents suitable for use in the combinations and methods herein include antiviral agents such as ribavirin, antifungal agents such as amphotericin, itraconazole and voriconazole, anti-graft rejection agents such as cyclosporine, tacrolimus and sirolimus, agents bronchodilators, including, but not limited to, anticholinergic agents such as atrovent, siRNAs, gene therapy vectors, aptamers, endothelin receptor antagonists, alpha-1-antitrypsin and prostacyclin.

[0199] In the above-described treatment methods and uses, a compound of the invention can be used alone or in combination with one or more other therapeutically active agents. Typically, any therapeutically active agent that has a therapeutic effect in a disease or a disease subject to treatment with a compound of the invention may be used in combination with the compounds of the invention, provided that the particular therapeutically active agent is compatible with therapy using the compound of the invention. Typical therapeutically active agents that are suitable for use in combination with the compounds of the invention include those described above.

[0200] In one preferred embodiment, the compounds of the invention are used in combination with one or more osmolytes, particularly hypertonic saline or mannitol.

[0201] In a further aspect, the disclosure discloses methods of treatment and use as described above, which comprises administering an effective amount of a compound of the invention and at least one other therapeutically active agent. The compounds of the invention and at least one additional therapeutically active agent may be used in combination either simultaneously or sequentially, in any therapeutically suitable combination. Administration of a compound of the invention with one or more other therapeutically

The active agents can be administered by administering simultaneously in the form of 1) a unitary pharmaceutical composition, such as the compositions described above, or 2) separate pharmaceutical compositions each containing one or more active ingredient ingredients. The components of the combination may be administered separately sequentially, wherein the compound of the invention is administered first and the second therapeutically active agent is administered second or vice versa.

[0202] When a compound of the invention is administered in combination with one or more osmolytes, the administration of each component is preferably concurrent, and may involve a unit composition or separate compositions. In one embodiment, the compound of the invention and one or more osmolytes are administered simultaneously by transbronchoscopic lavage. In a further embodiment, the compound of the invention and one or more osmolytes are administered simultaneously, by inhalation.

[0203] When a compound of the invention is used in combination with another therapeutically active agent, the dose of each compound may differ from that when the compound of the invention is used alone. Suitable doses will be readily determined by those skilled in the art. A suitable dose of a compound of the invention, another therapeutically active agent (s), and relative times of administration will be selected to achieve the desired overall therapeutic effect, and lie within the skill limit and depend on the discretion of the attending physician, clinician or veterinarian.

[0204] Experimental Procedures The invention also provides methods for the preparation of compounds of the invention and the synthesis of intermediates useful in such methods as described in detail below.

[0205] Some abbreviations and acronyms are used in describing synthetic methods and details about experiments. Although most of them will be understood by those skilled in the art, the following table lists many of these abbreviations and acronyms.

<td>Abbreviation</td><td>Importance</td>

<td>AcOH</td><td>Acetic acid</td>

<td>AIBN</td><td>azobisisobutyronitrile</td>

<td>DIAD</td><td>Diisopropyl azodicarboxylate</td>

<td>DIPEA</td><td>N, N-diisopropylethylamine</td>

<td>DCE</td><td>dichloroethane</td>

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

<td>DMF</td><td>dimethylformamide</td>

<td>et</td><td>Ethyl</td>

<td>EtOAc or EA</td><td>Ethyl acetate</td>

<td>EtOH</td><td>Ethanol</td>

<td>ESI</td><td>Ionization with electrospray</td>

<td>HATU</td><td>2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3 methyltronic tetrarofluorophosphate hexafluorophosphate</td>

<td>Abbreviation</td><td>Importance</td>

<td>HPLC</td><td>High performance liquid chromatography</td>

<td>iPrOH</td><td>Isopropyl alcohol</td>

<td>it or IT</td><td>Dotchawiczy</td>

<td>Me</td><td>Methyl</td>

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

<td>AcOH</td><td>Acetic acid</td>

<td>m / z or m / e</td><td>The ratio of mass to load</td>

<td>MH +</td><td>Weight plus 1</td>

<td>MH</td><td>Weight minus 1</td>

<td>MIC</td><td>Minimum inhibitor concentration</td>

<td>MS or ms</td><td>Mass spectrum</td>

<td>rt or rt</td><td>room temperature</td>

<td>^Rf</td><td>Delay coefficient, R value</td>

<td>t-Bu</td><td>tert-butyl</td>

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

<td>TLC or tic</td><td>Thin layer chromatography</td>

<td>δ</td><td>parts per million below tetramethylsilane</td>

<td>cbz</td><td>Benzyloxycarbonyl, i.e. - (CO) O-benzyl</td>

<td>AUC</td><td>Area under the curve or peak</td>

<td>MTBE</td><td>T-butyl methyl ether</td>

<td>^tR</td><td>Retention time</td>

<td>GC-MS</td><td>Gas chromatography-mass spectrometry</td>

<td>wt%</td><td>Weight percent</td>

<td>h</td><td>Hours</td>

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

<td>MHz</td><td>megahertz</td>

<td>TFA</td><td>Trifluoroacetic acid</td>

<td>UV</td><td>ultraviolet</td>

<td>Boc</td><td>tert-butyloxycarbonyl</td>

<td>DIAD</td><td>Diisopropyl azodicarboxylate</td>

<td>AcOH</td><td>Acetic acid</td>

<td>DIPEA</td><td>N, N-diisopropylethylamine or Iiiniga base</td>

<td>Ph 3 P</td><td>triphenylphosphine</td>

[0206] The compounds of Formula I may be synthesized using techniques known in the art. A representative synthesis procedure is illustrated in Scheme 1 below.

<img file="PL2931713T3_D0012.tif" />

[0207] These procedures are described in, for example, EJ Cragoe, "The Synthesis of Amiloride and Its Analogs" (Chapter 3) in Amiloride and Its Analogs, pp. 25-36. Other methods for the preparation of amiloride analogs are described in, for example, US Pat. No. 3,318,813 to Cragoe, particularly with respect to methods A, B, C and D from the '813 patent. Still other methods that can be adapted for the preparation of the compounds of the invention are described in PCT WO2003 / 07182, WO2005 / 108644, WO2005 / 022935, US 7,064,129, US 6,858,615, US 6,903,105, WO 2004/073629, WO 2007/146869, and WO 2007. / 018640, all awarded to Parion Sciences, Inc.

[0208] The preparation of N'-3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (2) can be observed in WO 2009/074575.

[0209] In general, compounds of the invention may be conveniently prepared by treating a compound of Formula II with an amine of Formula III. More specifically, compounds of formula 2 are treated with an amine of Formula 3 in a suitable solvent such as methanol, ethanol or tetrahydrofuran, and a base such as triethylamine (TEA) or diisopropylethylamine (DIPEA), with heating to an elevated temperature, e.g. 70 ° C. Further purification, separation of stereoisomers, crystallization and / or obtaining the salt form can be carried out using conventional techniques.

[0210] As will be apparent to those skilled in the art, in some cases, the initial compounds or intermediates in the synthesis may have other functional groups that provide alternative reactive sites. Disruptions from such functional groups can be avoided by the use of suitable protecting groups, such as amino or alcohol protecting groups, and where appropriate, appropriately prioritizing the synthesis steps. Suitable protecting groups will be apparent to those skilled in the art. The methods are well known in the art for incorporating and removing such groups

- protective ones, and such conventional techniques can also be used in the methods of the invention.

[0211] The following specific examples are provided herein for purposes of illustration. 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 a Bruker AC 400 device (¹1 H NMR at 400 MHz and ¹³C NMR at 100 MHz) or Bruker AC 300 (¹1 H NMR at 300 MHz and ¹³C NMR at 75 MHz). Proton spectra were referenced to tetramethylsilane as an internal standard, and carbon spectra were referenced to CDCl₃.

CD₃OD or DMSO-d₆ (purchased from Aldrich or Cambridge Isotope Laboratories, unless otherwise stated). Flash chromatography was carried out using the Combiflash system (Combiflash Rf, Teledyne Isco) with a column loaded with silica gel (Redi Sep. Rf, Teledyne Isco) or a reverse-phase column (high-performance C18 column)

Gold). ESI mass spectra were obtained on a Shimadz LCMS2010 EV mass spectrophotometer. HPLC analyzes were performed using a Waters XTerra MS C18 5μm 4.6 x 150mm analytical column with detection at 220 nm (unless indicated otherwise) using the Shimadzu Prominence HPLC system. The following time program was used with a flow rate of 1.0 ml per minute:

<td>Time (min)</td><td>Percent A (H₂About with 0.05% TFA)</td><td>Percent B (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 a Waters ACQUITY UPLC HSS T3 analytical column of 1.8Lim 2.1x100mm with detection at 220 nm (unless otherwise stated) using the 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>Percent A (HgO with 0.05% NH₄COOH and 0.1% HCOOH)</td><td>Percent B (CH₃CN / 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>thirty</td><td>70</td>

<td>5.00</td><td>thirty</td><td>70</td>

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

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

1. Preparation of (S) -2-amino-3- (4- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) naphthalen-1-yl) hydrochloride salt propane (16) [0212]

<img file="PL2931713T3_D0013.tif" />

[0213] Preparation of 4- (tert-butyldimethylsilyloxy) naphthalene-1-carbaldehyde (2);

A solution of 4-hydroxynaphthalene-1-carbaldehyde (1) (10.0 g, 58.1 mmol) in anhydrous THF (200 ml) was cooled to 0 ° C, and imidazole (12.0 g, 174 mmol) was added sequentially. and chloride

56 tert-butyldimethylsilyl (TBSCl) (13.1 g, 87.1 mmol). After finishing the 16-hour period of stirring at room temperature, the reaction mixture was filtered and the solvent was evaporated. The residue was taken in EtOAc (500 mL), washed with saturated aqueous NH 4 Cl (100 mL), water (100 mL) and brine (100 mL), and dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue was purified by flash chromatography on silica gel (2% EtOAc / hexane) to afford 2 (14.8 g, 90%) as a pale yellow solid:¹H NMR (300 MHz, CDCl₃): δ 10.22 (s, 1H),

9.30 (d, J = 8.10 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7, 69 (ddd, J =

8.4, 7.0, 1.3 Hz, 1H), 7.57 (ddd, J = 8.4, 7.0, 1.3 Hz, 1H), 6.95 (d, J = 7, 5 Hz, 1H), 1.10 (s,

9H), 0.36 (s, 6H)

Preparation of methyl (Z) -2- (tert-butyloxycarbonyl) amino-3- [1- (tert-butyldimethylsilyloxy) naphthalen-4-yl] acrylate (4);

[0214] To a solution (MeOhP (O) CH (NHBoc) CO₂Me, 3 (23.0 g, 52.7 mmol) in anhydrous

CH₂cl₂ (100 ml) was charged with DBU (10.1 ml, 67.3 mmol) and the mixture was stirred for 30 min at 0 ° C. Solution 1 (14.8 g, 51.74 mmol) in anhydrous CH₂cl₂ (60 ml) was added slowly with a syringe and the reaction mixture was warmed to room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (500 ml), washed rapidly with saturated aqueous NH₄Cl (2 x 150 mL) and brine (200 mL), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (20% EtOAc / hexane with 1% NEt₃), yielding 4 (20.0 g, 85%) as a yellow solid: ¹1 H NMR (300 MHz, CDCl 3): δ 8.23 ​​(dd, J = 8.6, 2.1 Hz, 1H), 7.93 (dd, J = 8.6, 2.1 Hz, 1H), 7.67 (s, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.53-7.47 (m, 2H), 6.85 (d, J = 7.8 Hz) , 1H), 6.05 (brs, 1H), 3.88 (s, 3H), 1.30 (s, 9H), 1.09 (s, 9H), 0.30 (s, 6H).

Preparation of methyl 2- (tert-butoxycarbonylamino) -3- (4- (tert-butyldimethylsilyloxy) naphthalen-1-yl) propanoate (5);

[0215] Suspension 4 (17.2 g, 37.6 mmol) and 10% Pd / C (3.40 g) in EtOH (200 mL) was degassed and subjected to hydrogenation conditions (1 atm, balloon) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was rinsed with MeOH. The filtrate was concentrated in vacuo to give 5 (17.0 g,^{99%) in the form}white solid: ¹H NMR (300 MHz, CDCl3): δ 8.23 ​​(d, J = 8.2 Hz, 1H), 7.99 (d, J =

8.2 Hz, 1H), 7.57-7.44 (m, 2H), 7.10 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 5.07- 4.94 (br s, 1H), 4.74- 4.61 (m, 1H), 3.66 (s, 3H), 3.55-3.17 (m, 2H) , 1.40 (s, 9H), 1.18 (s, 9H), 0.30 (s, 6H).

Preparation of methyl 2- (tert-butoxycarbonylamino) -3- (4-hydroxynaphthalen-1-yl) propanoate (6);

[0216] To a solution of 5 (17.0 g, 37.0 mmol) in anhydrous THF (200 ml) at 0 ° C was introduced tetrabutylammonium fluoride (48.1 ml, 48.1 mmol). The resulting solution was stirred for 15 min and quenched with saturated aqueous NH₄Cl (150 ml). After removing

- solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (500 ml), washed rapidly with saturated aqueous solution (2 x 150 ml) and brine (200 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (25% EtOAc / hexane) to yield rotamer 6 (14.0 g, 94%) as a yellow solid:¹H NMR (300 MHz, CDCl3): δ 8.23 ​​(d, J = 8.2 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.57-7.44 ( m, 2H), 7.07 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 7.6 Hz, 1H), 6.55 (brs, 1H), 5.14- 4.85 (br s, 1H), 4.77-4.51 (m, 1H), 3.78-3.31 (m, 5H), 1.40 (s, 6H), 1.10 (s, 3H).

Preparation of compounds 7 and 8;

[0217] A CHIRALPAK AD 5 cm ID x 50 cm L column was used, 20μ particles for the resolution of enantiomers using an IPA / heptane isocratic system (7.5% with 0.4% DEA). 8.0 g of racemic compound 6 was purified on a column to give S-isomer 8 (3.5 g, 44% yield) as a white solid and R-isomer 7 (2.2 g, 28%) as a white solid.

Preparation of methyl (S) -2- (tert-butoxycarbonylamino) -3- [4- (trifluoromethylsulfonyloxy) naphthalen-1-yl] propanoate (9);

[0218] To a solution of compound 8 (1.22 g, 3.53 mmol) pyridine (20 ml) was introduced triflate (0.9 ml, 5.30 mmol) at 0 ° C, and the reaction mixture was stirred at room temperature. for 2 hours After concentration, the reaction mixture was partitioned between CH₂cl₂ (100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 50 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated to give compound 9 (1.51 g, 89%) as a brown oil: ¹H NMR (400 MHz, CDCl3): δ 8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H). ), 7.28 (d, J = 7.9 Hz, 1H), 5.12-5.06 (br s, 1H), 4.78- 4.67 (m, 1H), 3.68-3, 46 (m, 5H), 1.39 (s, 8H), 1.25 (s, 1H).

[0219] Preparation of methyl (S) -3- {4- [4- (benzyloxycarbonylamino) but-1-ynyl] naphthalen-1-yl} -2- (tert-butoxycarbonylamino) propanoate (11); To a solution of compound 9 (1.50 g, 3.14 mmol) in anhydrous CH₃CN (60 ml) was charged with TEA (1.27 ml, 12.6 mmol), 10% (t-Bu)₃P in hexanes (1.27 mL, 0.62 mmol), benzyl but-3-ylcarbamate (10, 948 mg, 4.71 mmol), and CuI (30 mg, 0.16 mmol) at room temperature. The resulting mixture was degassed with argon for 10 min and Pd was added quickly (PPh₃)₄ (363 mg, 0.31 mmol) in one portion. After degassing with argon for 5 min, the resulting mixture was heated under reflux for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 60:40 ethyl acetate / hexanes) to yield compound 11 (1.30 g, 78%) as a brown oil:¹1 H NMR (400 MHz, CDCl 3): δ 8.33 (dd, J = 7.5, 2.2 Hz, 1H), 8.07 (dd, J = 7.5, 2.2 Hz, 1H), 7.58-7.51 (m, 2H), 7.52 (d, J = 7.5 Hz, 1H), 7.35-7.29 (m, 5H), 7.19 (d, J = 7.5 Hz, 1H), 5.166, 12 (m, 1H), 5.13 (s, 2H), 5.07- 4.99 (m, 1H), 4.74-4.65 (m, 1H); ), 3.59 (s, 3H), 3.91-3.42 (m, 2H), 3.53 (d, J = 6.2 Hz, 2H), 2.79 (t, J = 6, 4 Hz, 2H), 1.39 (s, 8H), 1.25 (s, 1H).

Preparation of the methyl (S) -3- (4- (4-aminobutyl) naphthalen-1-yl) -2- (tert-butoxycarbonylamino) propanoate (12) salt;

[0220] Suspension 11 (1.00 g, 1.88 mmol) and 10% Pd / C (200 mg) in a mixture of MeOH (20 ml) and AcOH (2 ml) were degassed and hydrogenated (1 atm) for 16 minutes. h.

in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 12 (820 mg,^95%)in the form of a white solid: ¹H NMR (300 MHz, CD₃OD): δ 8.17-8.05 (m, 2H), 7.62^{7.48 (m,} 2H), 7.27 (br s, 2H), 4.47 (t, J = 7.4 Hz, 1H), 3.75-3.51 (m, 5H), 3.13 (t, J = 7). , 5 Hz, 2H), 2.93 (t, J = 7.66 Hz, 2H), 1.93 (s, 3H), 1.88-1.65 (m, 4H), 1.34 (s , 7H), 1.01 (s, 2H).

[0221] Preparation of (S) -2- (tert-butoxycarbonylamino) -3- (4- {4- [3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino] butyl} naphthalen-1-yl) methyl propanoate (14);

To a solution of amine salt 12 (815 mg, 1.77 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 1.1 g, 2.83 mmol) in EtOH (6.0 mL) was charged DIPEA (2.50 mL, 14.2 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 14 (870 mg, 80%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD): δ 8.17-8.07 (m, 2H), 7.58-7.48 (m, 2H), 7.26 (q, J = 7.4 Hz, 2H), 4.56- 3.68 (m, 1 H), 3.75-3.68 (m, 1H), 3.64 (s, 2H), 3.583.43 (m, 2H), 3.13 (t, J = 6, 7 Hz, 2H), 2.98 (q, J = 7.2 Hz, 2H), 1.86-1.70 (m, 4H), 1.33 (s, 7H), 0.98 (s, 2H).

[0222] (S) -2- (tert-Butoxycarbonylamino) -3- (4- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) naphthalen-1-yl) acid) propane (15); To a solution of methyl ester 14 (510 mg, 0.83 mmol) in a mixture of THF (3 mL), methanol (3 mL) and water (1 mL) was introduced solid LiOH (120 mg, 4.99 mmol) and the reaction mixture was stirred at room temperature for 2 hours When the TLC of the reaction mixture showed the reaction was complete, the pH of the reaction mixture was adjusted to 9-10 by addition of 1 N

HCl (aqueous), and the organic solvent was removed. The pH of the aqueous portion was adjusted to 5-6 and the resulting precipitate was extracted with dichloromethane. The aqueous portion was extracted with DCM (2 x 50 mL). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated to give compound 15 (375 mg, 76%) as a white solid: ¹H NMR (300 MHz, DMSO-d₆):

δ 8.22-8.02 (m, 2H), 7.59-7.47 (m, 2H), 7.34-7.22 (m, 2H), 6.82 (brs, 2H), 4 19- 4.06 (m, 1H), 3.59-3.46 (m, 1H), 3.25-3.13 (m, 2H), 3.09- 2.94 (m, 10H) , 1.80-1.55 (m, 4H), 1.28 (s, 7H), 0.93 (s, 2H).

[0223] Preparation of (S) -2-amino-3- (4- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) naphthalen-1-yl) propanoic acid HCl salt (16); 4 N HCl in dioxane (8.0 mL) was added to 15 (258 mg, 0.43 mmol) followed by water (4.0 mL) and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed and the residue was lyophilized to yield compound 16 (250 mg, 99%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.54 (brs, 1H), 9.33 (t, J = 5.92 Hz, 1H),

59-93-8.80 (m, 2H), 8.60 (br s, 3H), 8.17 (ddd, J = 10.1, 7.6, 4.5 Hz, 2H), 7, 59 (ddd, J = 9.2, 6.7, 4.5 Hz, 2H), 7.46-7.36 (m, 2H), 7.34 (dd, J = 9.9, 7.5 Hz, 2H), 4.13-4.02 (m, 1H), 3.753.44 (m, 3H), 3.43-3.33 (m, 2H), 3.09 (t, J = 6, 4 Hz, 2H), 1.81-1.62 (m, 4H).

2. Preparation of (S) -3,5-Diamino-N- (N- (4- (4- (2-amino-3- (4- (3- (dimethylamino) propyl) phenylamino) -3-oxopropyl) naphthalene -1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (23) [0224]

<img file="PL2931713T3_D0014.tif" />

Preparation of methyl (S) -3- {4- [4- (benzyloxycarbonylamino) but-1-ynyl] naphthalen-1-yl} -2 (tert-butoxycarbonylamino) propanoate (17);

[0225] To a solution of methyl ester 11 (1.71 g, 3.22 mmol) in a mixture of THF (21 mL), methanol (21 mL) and water (7.0 mL) was added solid NaOH (1.29 g, 32). , 3 mmol), and the reaction mixture was stirred at room temperature for 3 hours. When the TLC of the reaction mixture showed the reaction was complete, the pH of the reaction mixture was adjusted to 9-10 by the addition of 1 N HCl (aqueous) and the organic solvent was removed. The pH of the aqueous portion was adjusted to 5-6 and the resulting precipitate was extracted with dichloromethane. The aqueous portion was extracted with CH₂cl₂ (2 x 50 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated to give compound 17 (1.55 g, 93%) as a brown solid: ¹1 H NMR (400 MHz, DMSO-d6): δ 8.32 (d, J = 7.4 Hz, 1H), 8.13-8.05 (m, 1H), 7.58-7.48 (m , 4H), 7.38-7.29 (m, 5H), 5.21-5.15 (m, 1H), 5.12 (s, 2H), 5.07- 4.93 (m, 1H); ), 4.70-4.54 (m, 1H), 3.77-3.62 (m, 1H), 3.57-3.35 (m, 2H), 2.84-2.68 (m , 2H), 1.37 (s, 9H).

[0226] Preparation of compound 19; Compound 18 (100 mg, 0.56 mmol) in THF (2.5 mL) was followed sequentially with DEPBT (218 mg, 0.72 mmol), 17 (289 mg, 0.56 mmol) and DIPEA (0.3 mL). , 1.68 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (100 ml), washed rapidly with saturated aqueous solution ^NaHCO3 (2 x 50 ml) and brine (50 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (8% methanol / CH 2 Cl 2) to afford amide 19 (250 mg, 66%) as a yellow solid:¹1 H NMR (400 MHz, CDCl 3): δ 8.34 (dd, J =

8.3, 1.4 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.61-7.47 (m, 4H), 7.39-7.27 (m , 5H), 7.16 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 5.36-5.19 (m, 2H), 5 , 12 (s, 2H), 4.36-4.53 (m, 1H), 3.66-3.42 (m, 4H), 2.79 (t, J = 6.6 Hz, 2H), 2.57 (t, J = 7.5 Hz, 2H), 2.40 (t, J = 7.5 Hz, 2H), 2.32 (s, 6H), 1.86-1.75 (m , 2H), 1.39 (s, 9H).

[0227] Preparation of compound 20; A suspension of 19 (210 mg, 0.31 mmol) and 10% Pd / C (150 mg) in a mixture of MeOH (3.0 mL) and AcOH (0.3 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 12 hours. . in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 22 which was neutralized with triethylamine and the crude product was purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 20 (130 mg, 77%) as a white solid. :¹H NMR (300 MHz, CD3OD): δ 8.24 (dd, J = 8.1, 2.1 Hz, 1H), 8.08 (dd, J = 8.2, 1.5 Hz, 1H), 7.58-7.47 (m, 2H), 7.33-7.20 (m, 4H), 7.07-7.05 (m, 2H), 4.53 (t, J = 7.2 Hz, 1H), 3.66-3.55 (m, 2H), 3.09 (t, J = 7.5 Hz, 2H), 2.82 (t, J = 7.4 Hz, 2H), 2.57 (t, J = 7.2 Hz, 2H), 2.35 (dd, J = 10.5, 7.5 Hz, 2H), 2.24 (s, 6H), 1.84-1 61 (m, 6H), 1.36 (s, 7H), 1.10 (s, 2H).

[0228] Preparation 22; To a solution of amine (122 mg, 0.22 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 139 mg, 0.35 mmol) in EtOH (4.0 mL) was charged DIPEA (0, 31 ml, 1.76 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to

- room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl 3 / CH 3 OH / NH 4 OH) to give guanidine 22 (111 mg, 66%) as a yellow solid:¹H NMR (400 MHz, CD3OD): ^{δ 8.23} (dd, J = 7.5, 2.4 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.57-7.48 (m, 2H), 7.29 ( d, J = 7.3 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.13-7.05 (m, 2H), 4.53 (t, J = 8 , 0 Hz, 1H), 3.60-3.37 (m, 2H), 3.23 (t, J = 7.3 Hz, 2H), 3.15-3.03 (m, 2H), 2 55 (t, J = 7.3 Hz, 2H), 2.29 (dd, J = 9.7, 7.6 Hz, 2H), 2.21 (s, 6H), 1.86-1, 64 (m, 6H), 1.36 (s, 7H), 1.12 (s, 2H).

Preparation of HCl salt of compound 23 (S) -3,5-diamino-N- (N- (4- (4- (2-amino-3- (4- (3-dimethylamino) propyl) phenylamino) -3-oxopropyl) naphthalene-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0229] 4 N HCl in dioxane (3.0 ml) was added to 22 (100 mg, 0.13 mmol) followed by water (1.0 ml). ), and the reaction mixture was stirred at room temperature for 3 hours. The solvent was removed and neutralized with 1N NaOH (aq), the resulting solid was washed with water and treated again with 1 N HCl (aqueous), the water was removed and the residue was lyophilized to yield compound 22 (65 mg, 65%) as a yellow solid:¹1 H NMR (400 MHz, DMSO-d 6) 10.50 (s, 1H), 10.48 (s, 1H), 10.46-10.40 (m, 1H), 9.26 (t, J = 4) , 9 Hz, 1H), 9.01- 8.74 (m, 2H), 8.61 (br s, 1H), 8.35 (dd, J = 6.6, 3.4 Hz, 1H), 8 13 (dd, J = 6.5, 3.3 Hz, 1H), 7.58 (ddd, J = 9.9, 6.6, 3.6 Hz, 2H), 7.42 (brs, 1H); ), 7.40 (d, J = 7.3 Hz, 2H), 7.34 (d, J = 7.3 Hz, 1H), 7.28 (d, J = 7.3 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 4.29-4.2 (m, 1H), 3.643.4 (m, 2H), 3.12-3.03 (m, 2H) , 3.02- 2.94 (m, 2H), 2.72 (s, 3H), 2.70 (s, 3H), 2.56 (t, J = 8.1 Hz, 2H), 1, 97-1.88 (m, 2H), 1.79-1.61 (m, 4H).

3. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (hexyl) (2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxy-hexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (28) [0230]

Scheme 4

<img file="PL2931713T3_D0015.tif" />

[0231] To compound 24 (165 mg, 0.38 mmol) in THF (10 mL), DEPBT (148 mg, 0.48 mmol), 17 (200 mg, 0.38 mmol) were sequentially introduced, and DIPEA (0, 2 ml, 1.14 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (100 ml), washed rapidly with saturated aqueous solution ^NaHCO3 (2 x 50 ml) and brine (50 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (8% methanol / CH 2 Cl 2) to afford amide 25 (210 mg, 60%) as a yellow solid:¹1 H NMR (300 MHz, CDCl 3): δ 8.35 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.63-7.5 ( m, 2H), 7.51 (d, J = 7.3 Hz, 1H), 7.44-7.39 (m, 1H), 7.37-7.27 (m, 6H), 7,167.02 (m, 3H), 5.24-5.16 (m, 1H), 5.13 (s, 2H), 4.68 (ddd, J = 11.3, 10.3, 5.1 Hz, 1H); ), 4.56 (q, J = 7.2 Hz, 1H), 4.19-4.09 (m, 1H), 3.90-3.76 (m, 5H), 3.74-3, 68 (m, 1H), 3.63-3.46 (m, 5H), 3.45-3.24 (m, 3H), 2.80 (t, J = 6.7 Hz, 2H), 2 , 70-2.35 (m, 8H), 1.81- 1.67 (m, 2H), 1.63-1.53 ​​(m, 1H), 1.35-1.20 (m, 6H). , 1.21 (s, 9H), 1.31 (d, J = 5.1 Hz, 3H), 0.87 (t, J = 6.2 Hz, 3H).

Preparation of compound 26;

[0232] A suspension of 25 (280 mg, 0.30 mmol) and 10% Pd / C (560 mg) in a mixture of EtOH (9.0 mL) and AcOH (1.0 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 4 hours. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOI. The filtrate was concentrated in vacuo to give the amine salt 22, which was neutralized with NaICO₃and the crude product was purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 26 (160 mg, 67%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD): δ 8.29 (d, J = 8.2 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.60 (t, J = 6.9 Hz, 1H). ), 7.55 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 7.24 (d, J = 7.1 Hz, 1H), 7.18 (d, J = 7.1 Hz, 1H), 7.02-7.96 (m, 1H), 7.95-6.88 (m, 2H), 6.77-6.69 (m, 1H), 5.56 -5.35 (m, 1H), 4.68 (q, J = 5.1 Hz, 1H), 4.61-4.53 (m, 1H), 4.12 (dd, J = 10.8 , 5.4 Hz, 1H), 3.89-3.80 (m, 2H), 3.74 (t, J = 3.3 Hz, 2H), 3.46 (d, J = 3.8 Hz). , 1H), 3.39 (t, J = 10.7 Hz, 2H), 3.18-3.09 (m, 1H), 3.02- 2.92 (m, 1H), 2.68 ( t, J = 7.1 Hz, 2H), 2.61-2.47 (m, 5H), 2.46-2.37 (m, 4H), 1.77-1.63 (m, 4H). , 1.33 (d, J = 5.1 Hz, 3H), 1.31-1.20 (m, 8H), 1.21 (s, 9H), 0.88 (t, J = 6.7 Hz, 3H).

Preparation of compound 27;

[0233] A solution of amine 26 (155 mg, 0.20 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 123 mg, 0.31 mmol) in EtOH (8.0 mL) was charged with DIPEA (0.28 mL, 1.56 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CICl₃/ CH₃OH / NH₄OI) followed by reverse phase chromatography (Gold C18) to give guanidine 27 (100 mg, 51%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD): δ 8.23 ​​(dd, J = 8.8, 2.5 Hz, 1H), 8.10 (d, J = 8.2 Hz, 1H), 7.56-7.49 (m, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.24 (d, J = 7.4 Hz, 2H), 7.08 (d, J = 7.9 Hz, 2H). 4.67 (q, J = 5.1 Hz, 1H), 4.56-4.50 (m, 1H), 4.04 (dd, J = 10.8, 5.4 Hz, 1H), 3.92-3.86 (m, 1H), 3.82-3.74 (m, 2H), 3.51-3.46 (m, 1H), 3.25 (t, J = 7.1 Hz, 2H), 3.15-3.06 (m, 2H), 2.71 (dd, J = 13.2, 5.2 Hz, 1H), 2.60-2.45 (m, 6H). , 1.87-1.63 (m, 6H), 1.48-1.40 (m, 6H), 1.33-1.26 (m, 6H), 1.23 (d, J = 5, 1 Hz, 3H), 1.20 (s, 9H), 0.89 (t, J = 6.7 Hz, 3H).

[0234] Preparation of the HCl salt of 28 - 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4 - 3- (hexyl) (2S, 3R) 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) propyl) phenylamino) -3-oxo-propyl) naphthalen-1-yl) butyl) karbamimidoilo) -6-chloropyrazine-2-carboxamide; 4 N HCl in water (3.0 mL) was added to 27 (80 mg, 0.08 mmol) in ethanol (0.5 mL), and the reaction mixture was stirred at 40 ° C for 6 h. The solvent was removed, 4 N IC1 was added, and the mixture was heated at 40 ° C for a further 4 hours. The solvent was removed, water was added and the residue was lyophilized to yield compound 28 (78 mg, 99%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.58 (brs, 1I), 10.56 (brs, 1I), 9.70-9.58 (m, 1H), 9.38-9.31 (m, 1H), 9.04-8.84 (m, 2H), 8.70 (br s, 1H), 8.43-8.34 (m, 1H), 8.16-8.08 (m , 1H), 7.62-7.5 (m, 2H), 7.46-7.37 (m, 4H), 7.34 (d, J = 7.1 Hz, 1H), 7.27 ( d, J = 7.1 Hz, 1H), 7.17 (d, J = 8.1 Hz, 2H), 5.52-5.46 (m, 1H), 4.85- 4.76 (m , 1H), 4.68-4.52 (m,

64H), 4.49-4.37 (m, 1H), 4.32-4.22 (m, 1H), 4.05-3.97 (m, 1H), 3.72-3, 43 (m, 6H), 3.17-2.97 (m, 8H), 2.02-1.90 (m, 2H), 1.77-1.54 (m, 6H), 1.33- 1.21 (m, 6H), 0.86 (t, J = 6.6 Hz, 3H).

[0235] ¹H NMR (400 MHz, CD3OD): δ 8.23 ​​(d, J = 8.3 Hz, 1H), 8.17 (d, J = 8.2 Hz, 1H), 7.62-7.53 ( m, 2H), 7.41-7.36 (m, 1H), 7.35-7.32 (m, 1H), 7.31-7.25 (m, 2H), 7.21-7, 12 (m, 2H), 4.35-4.25 (m, 1H), 4.17-4.02 (m, 1H), 3.86-3.75 (m, 2H), 3.73- 3.59 (m, 6H), 3.23-3.08 (m, 9H), 2.73-2.60 (m, 2H), 2.11-1.97 (m, 2H), 1, 91-1.75 (m, 4H), 1.74-1.62 (m, 2H), 1.441.30 (m, 6H), 0.92 (t, J = 6.6 Hz, 3H).

4. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (33) [0236]

<img file="PL2931713T3_D0016.tif" />

- 65 Preparation of compound 30:

[0237] To compound 29 (290 mg, 0.54 mmol) in THF (8.0 mL), DEPBT (210 mg, 0.70 mmol), 17 (311 mg, 0.60 mmol) and DIPEA (0) were added sequentially. , 28 ml, 1.62 mmol), and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (100 mL), washed rapidly with saturated aqueous NaHCO 3 (2 x 50 mL) and brine (50 mL), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (8% methanol / CH₂^cl2) to give the amide 30 (400 mg^{, 72%) as a yellow}solid body: ¹1 H NMR (400 MHz, CDCl 3): δ 8.36-8.26 (m, 1H), 8.20-8.09 (m, 1H), 8.03.85 (m, 1H), 7.61-7 46 (m, 1H), 7.49 (d, J = 7.2 Hz, 2H), 7.38-7.28 (m, 5H), 7.18-6.96 (m, 4H), 5.51-5.36 (m, 1H), 5.32-5.21 (m, 1H), 5.12 (s, 2H), 4.67 (q, J = 5.1 Hz, 2H) , 4.66-4.53 (m, 1H), 4.11 (dd, J = 10.4, 5.2 Hz, 2H), 4.06-3.96 (m, 2H), 3.93 -3.86 (m, 2H), 3.86-3.77 (m, 2H), 3.68-3.56 (m, 2H), 3.56-3.44 (m, 6H), 3 39 (t, J = 10.4 Hz, 2H), 3.05 (q, J = 7.6 Hz, 2H), 2.96-2.88 (m, 2H), 2.79 (t, J = 6.1 Hz, 2H), 2.64-2.61 (m, 4H), 1.93-1.72 (m, 4H), 1.48-1.40 (m, 2H), 1 35 (s, 9H), 1.29 (d, J = 5.1 Hz, 6H).

Preparation of compound 31;

[0238] A suspension of 30 (400 mg, 0.39 mmol) and 10% Pd / C (210 mg) in a mixture of EtOH (54 ml) and AcOH (6.0 ml) was degassed and subjected to hydrogenation conditions (1 atm) for 4 hours. h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 31 (333 mg,^{84%) in the form} yellow ^{solid: 1}H NMR (400 MHz, CD₃OD): δ 8.25 (dd, J = 7.5, 2.5 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 7.60-7.51 (m, 2H), 7.36-7.32 (m, 1H), 7.31 (d, J = 7.2 Hz, 2H), 7.26 (d, J = 7.8 Hz, 1H), 7, 15 (d, J = 7.8 Hz, 2H), 4.70 (q, J = 4.9 Hz, 2H), 4.54 (d, J = 7.3 Hz, 1H), 4.18- 4.10 (m, 2H), 4.06 (dd, J = 10.6, 5.3 Hz, 2H), 3.87-3.82 (m, 2H), 3.81-3.68 ( m, 3H), 3.53 (dd, J = 9.5, 1.8 Hz, 2H), 3.39 (t, J = 9.2 Hz, 3H), 3.35-3.30 (m , 2H), 3.15-3.08 (m, 2H), 2.92 (t, J = 8.0 Hz, 2H), 2.09-2.00 (m, 4H), 2.77- 2.58 (m, 2H), 1.95 (s, 6H), 1.88-1.60 (m, 4H), 1.36 (s, 9H), 1.25 (d, J = 4, 9 Hz, 6H).

[0239] Preparation 32; To a solution of 31 (370 mg, 0.36 mmol) and ethyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 226 mg, 0.58 mmol) in EtOH (12 mL) was charged with DIPEA (0.51 mL). 2.88 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 32 (250 mg, 63%) as a yellow solid: ¹H NMR (400 MHz, CD3OD): ^{δ 8.23 ​​(d,} J = 8.6 Hz, 1H), 8.13-8.03 (m, 1H), 7.54-7.49 (m, 2H), 7.30-7.20 (m, 4H), 7 13-7.04 (m, 2H), 4.67 (q, J = 4.9 Hz, 2H), 4.54-4.49 (m, 1H), 4.03 (dd, J = 10 , 8, 5.4 Hz, 2H), 3.91-3.84 (m, 2H), 3.82-3.72 (m, 5H), 3.48-3.43 (m, 5H), 3.41-3.34 (m, 2H), 3.113, 10 (m, 2H), 2.68-2.50 (m, 8H), 1.87-1.67 (m, 6H), 1, 36 (s, 9H), 1.23 (d, J = 4.9 Hz, 6H).

Preparation of HCl salt 33 - 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis (2S, 3R, 4R) 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0240] 4 N HCl in water (6.0 mL) was added to 32 (200 mg, 0.18 mmol) in ethanol (2.0 mL) and the reaction mixture was stirred at 40 ° C for 8 h. The solvent was removed, 4N HCl was added, and the mixture was heated at 40 ° C for a further 6 hours. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 33 (138 mg, 59%) as a yellow solid:¹H NMR (400 MHz, DMSO-d₆): δ 10.48 (br s, 1H), 10.45-10.41 (m, 1H), 9.25-9.19 (m, 1H), 8.95-8.85 (m, 1H) , 8.81- 8.69 (m, 1H), 8.64- 8.46 (m, 4 H), 8.36-8.29 (m, 1H), 8.18-8.10 (m, 1H), 7.62-7.55 (m, 2H), 7.46-7.38 (m, 4H), 7.34 (d, J = 7.5 Hz, 1H), 7.28 (d , J = 7.3 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 5.48-5.39 (m, 2H), 4.87- 4.75 (m, 2H), 4.68-4.33 (m, 4H), 4.28-4.17 (m, 1H), 4.05-3.93 (m, 2H), 3.72-3.65 ( m, 2H), 3.623.53 (m, 4H), 3.52-3.35 (m, 8H), 3.27-3.13 (m, 6H), 3.3,10- 3.00 ( m, 2H), 2.62-2.48 (m, 4H), 2.03-1.90 (m, 2H), 1.78-1.61 (m, 4H).

[0241] ¹1 H NMR (400 MHz, CD 3 OD): δ 8.24-8.20 (m, 1H), 8.18-8.15 (m, 1H), 7.57 (td, J = 4.6, 1, 5 Hz, 2H), 7.38 (d, J = 7.4 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.28 (dd, J = 8.4, 2.6 Hz, 2H), 7.16 (dd, J = 8.4, 2.0 Hz, 2H), 4.28 (t, J = 6.9 Hz, 1H), 4.19-4, 13 (m, 1H), 4.12-4.07 (m, 1H), 3.85-3.79 (m, 2H), 3.77 (dd, J = 10.4, 5.2 Hz, 2H), 3.73-3.60 (m, 6H), 3.49-3.45 (m, 2H), 3.42-3.34 (m, 6H), 3.26-3.23 ( m, 1H), 3.19-3.13 (m, 2H), 3.14-3.11 (m, 1H), 2.74-2.59 (m, 2H), 2.14-2, 00 (m, 2H), 1.90-1.72 (m, 4H).

5. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3-oxo-3- (4- (3 - ((2S, 3R, 4R, 5R) ) 2,3,4,5,6-pentahydroxyhexylamino) propyl) phenylamino) propyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (38) [0242]

Diagram 6

<img file="PL2931713T3_D0017.tif" />

Preparation of compound 35;

[0243] To compound 34 (400 mg, 0.91 mmol) in THF (15 mL) sequentially DEPBT (389 mg, 1.30 mmol), 17 (516 mg, 1.00 mmol) were introduced, and DIPEA (0, 52 ml, 3.00 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (100 mL), washed rapidly with saturated aqueous NaHCO 3 (2 x 50 mL) and brine (50 mL), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (8% methanol / CH₂^cl2), yielding amide 35 (700 mg^{, 83%) in the form of yellow}solid body: ¹1 H NMR (400 MHz, CDCl 3): δ 8.35 (dd, J = 8.2, 1.5 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.64- 7.35 (m, 4H), 7.38-7.26 (m, 5H), 7.06 (d, J = 7.8 Hz, 2H), 7.17-7.09 (m, 2H) , 5.21-5.13 (m, 2H), 5.12 (s, 2H), 4.69 (q, J = 5.1 Hz, 1H), 4.55 (q, J = 7.25 Hz, 1H), 4.15 (dd, J = 11.4, 5.6 Hz, 1H), 4.11-4.02 (m, 1H), 4.07- 3.92 (m, 1H). , 3.88-3.77 (m, 1H), 3.73-3.67 (m, 1H), 3.64-3.49 (m, 5H), 3.41 (d, J = 10, 6 Hz, 2H), 3.37-3.30 (m, 2H), 3.29-3.20 (m, 3H), 2.80.

- 68 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 7.8 Hz, 2H), 1.90-1.76 (m, 3H), 1.42 (s, 18H), 1.32 (d, J = 5.2 Hz, 3H).

Preparation of compound 36;

[0244] A suspension of 35 (700 mg, 0.74 mmol) and 10% Pd / C (400 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 36 (650 mg,^95%)in the form of a yellow solid: ¹H NMR (400 MHz, CDCl₃): δ 8.20 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.3 Hz, 1H), 7.86-7.71 (m, 1H), 7, 70-7.63 (m, 1H), 7.58-7.43 (m, 2H), 7.36-7.26 (m, 2H), 7.02-6.91 (m, 2H), 4.70-4.63 (m, 1H), 4.61-4.54 (m, 1H), 4.20-4.05 (m, 2H), 4.04- 3.90 (m, 1H); ), 3.89-3.68 (m, 3H), 3.67-3.46 (m, 3H), 3.45-3.27 (m, 5H), 3.29-3.21 (m , 4H), 3.129.91 (m, 4H), 2.90-2.76 (m, 2H), 2.48 (d, J = 7.3 Hz, 2H), 2.08 (s, 6H) , 1.86-1.61 (m, 6H), 1.41 (s, 15H), 1.32 (d, J = 5.1 Hz, 3H), 1.25 (s, 3H).

Preparation 37;

[0245] To a solution of 36 (650 mg, 0.70 mmol) and 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 436 mg, 1.13 mmol) in EtOH (12 mL) was charged with DIPEA (0, 90 mL, 5.60 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 37 (444 mg, 62%) as a yellow solid: ¹H NMR (400 MHz, CD3OD): δ 8.23 ​​(dd, J = 7.7, 2.2 Hz, 1H), 8.10 (d, J = 8.1 Hz, 1H), 7.57- 7.47 (m, 2H), 7.33-7.21 (m, 4H), 7.08 (d, J = 8.1 Hz, 2H), 4.68 (q, J = 5.0 Hz); , 1H), 4.53 (t, J = 7.2 Hz, 1H), 4.04 (dd, J = 10.8, 5.4 Hz, 1H), 4.03-3.93 (m, 1 H), 3.25 (ddd, J = 10.3, 9.2, 5.2 Hz, H), 3.71-3.65 (m, 1H), 3.58-3.37 (m, 4H), 3.27-3.20 (m, 4H), 3.20-3.15 (m, 1H), 3.14-3.05 (m, 2H), 2.66 (q, J = 7.5 Hz, 1H), 2.53 (t, J = 7.2 Hz, 2H), 1.89-1.76 (m, 4H), 1.76-1.64 (m, 2H), 1.36 (s, 6H), 1.42 (s, 9H), 1.25 (d, J = 5.0 Hz, 3H), 1.11 (s, 3H).

Preparation of HCl 38 salt;

[0246] 4 N HCl in water (6.0 mL) was added to 37 (240 mg, 0.23 mmol) in ethanol (3.0 mL) and the reaction mixture was stirred at 40 ° C for 8 h. The solvent was removed, 4N HCl was added, and the mixture was heated at 40 ° C for a further 8 hours. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 38 (251 mg, 64%) as a yellow solid:¹H NMR (400 MHz, DMSO-d₆): δ 10.50 (br s, 1H), 9.28 (t, J = 5.7 Hz, 1H), 9.02-8.87 (m, 1H), 8.86-8.75 (m , 1H), 8.72-8.55 (m, 4H), 8.39-8.33 (m, 1H), 8.16-8.10 (m, 1H), 7.61-7.55 (m, 2H), 7.45-7.40 (m, 1H), 7.40 (d, J = 7.4 Hz, 2H), 7.34 (d, J = 7.3 Hz, 1H) , 7.28 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 5.38 (d, J = 4.3 Hz, 1H), 4 , 74 (d, J = 4.9 Hz, 1H), 4.64-4.51 (m, 2H), 4.49-4.35 (m, 1H), 4.30-4.2 (m) , 2H), 3.94-3.86 (m, 1H), 3.70-3.64 (m, 1H), 3.63-3.52 (m, 3H), 3.51-3.34 (m, 6H), 3.15-2.98 (m, 3H), 2.98-2.81 (m, 3H), 2.58 (t, J = 7.6 Hz, 2H), 1, 96-1.85 (m, 2H), 1.79-1.61 (m, 4H).

- 69 [0247] ¹H NMR (400 MHz, CD₃OD): δ 8.26-8.20 (m, 1H), 8.19-8.14 (m, 1H), 7.60-7.53 (m, 2H), 7.38 (d, J) = 7.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.28 (dd, J = 8.4, 2.0 Hz, 2H), 7.14 (d); , J = 8.4 Hz, 2H), 4.29 (t, J = 8.3 Hz, 1H), 4.07-4.00 (m, 1H), 3.83 (dd, J = 9, 8, 1.5 Hz, 1H), 3.77 (dd, J = 9.8, 2.6 Hz, 1H), 3.73-3.64 (m, 5H), 3.37 (t, J = 7.2 Hz, 2H), 3.21-3.11 (m, 4H), 3.06-2.96 (m, 2H), 2.66 (t, J = 7.7 Hz, 2H) 2.03-1.94 (m, 2H), 1.90-1.75 (m, 4H).

6. Preparation of (S) -3,5-Diamino-N- (N- (4- (4- (2-amino-3- (4- (6 (dimethylamino) hexyl) phenylamino) -3-oxopropyl) naphthalene 1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (43) [0248]

<img file="PL2931713T3_D0018.tif" />

- Compound 40;

[0249] A solution of acid 17 (880 mg, 1.70 mmol) in THF (30 ml) was cooled to 0 ° C in an ice bath. NMM (0.37 mL, 3.40 mmol) was added followed by PivCl (0.20 mL, 1.70 mmol) and the reaction mixture was stirred at the same temperature for 2 hours. 39 (375 mg, 1.70 mmol, 15 mL of THF) were added, and the reaction mixture was stirred at the same temperature for a further 10 min. The reaction mixture was brought to room temperature and stirred for 16 hours. The organic solvent was removed. Water was added to the residue and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. ^{The residue was purified by column chromatography} (4% methanol in chloroform) to yield amide 40 (719 mg, 59%) as a slightly yellow solid: [M + H]⁺ 720.

Preparation of compound 41;

[0250] A suspension of 40 (719 mg, 1.00 mmol) and 10% Pd / C (300 mg) in a mixture of EtOH (110 mL) and AcOH (20 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 41 as a yellow solid (660 mg, 93%).^: [M + H]⁺ 589.

Preparation of compound 42;

To a solution of amine 41 (660 mg, 0.93 mmol) and 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 650 mg, 1.67 mmol) in EtOH (10 mL) was charged DIPEA (1 66 ml, 9.3 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 42 (370 mg, 50%) as a yellow solid: [M + H]⁺ 801.

Preparation of HCl salt of 43 (S) -3,5-diamino-N- (N- (4- (4- (2-amino-3- (4- (6 (dimethylamino) hexyl) phenylamino) -3-oxopropyl) naphthalene-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0252] TFA (10 ml) was added to 42 (370 mg, 0.46 mmol) in CH₂cl₂ (10 mL) and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, 1N HCl was added, and the solvent was removed. The mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 43 (290 mg, 92%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.39 (brs, 2H), 9.25 (brs, 1H), 9.02-8.87 (m, 1H), 8.86-8.73. (m, 2 H), 8.718,44 (m, 2 H), 8.35 (br s, 1H), 8.13 (dd, J = 6.8, 3.8 Hz, 1H), 7.58 (dd, J = 6.5, 3.2 Hz, 2H), 7.42 (br s, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 7.3 Hz, 1H), 7.11 (d, J = 8.4 Hz, 2H), 4.26-4.18 (m, 1H), 3.65-3.48 (m, 2H), 3.39-3.32 (m, 3H), 3.06 (t, J = 6.5 Hz, 2H), 2.99-2.91 ( m, 2H), 2.69 (s, 6H), 1.77-1.56 (m, 6H), 1.52 (t, J = 8.2 Hz, 2H), 1.34-1.21 (m, 4H).

- 71 [0253] ¹H NMR (400 MHz, CD3OD): δ 8.22-8.17 (m, 1H), 8.16-8.12 (m, 1H), 7.58-7.5 (m, 2H), 7 36 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 7.4 Hz, 1H), 7.19 (d, J = 8.04 Hz, 2H), 7.05 (d, J =

8.3 Hz, 2H), 4.24 (t, J = 8.2 Hz, 1H), 3.70-3.58 (m, 2H), 3.33 (t, J = 6.9 Hz, 2H). , 3.14 (t, J =

7.4 Hz, 2H), 3.09-3.03 (m, 2H), 2.84 (s, 6H), 2.53 (t, J = 8.6 Hz, 2H), 1.88-1, 73 (m, 4H), 1.72-1.63 (m, 2H), 1.61-1.52 (m, 2H), 1.41-1.32 (m, 4H).

7. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (6- (bis ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) hexyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0254]

Scheme 8

<img file="PL2931713T3_D0019.tif" />

<img file="PL2931713T3_D0020.tif" />

Preparation of compound 45;

[0255] A solution of acid 17 (900 mg, 1.74 mmol) in THF (40 mL) was cooled to 0 ° C in an ice bath. NMM (0.38 mL, 3.48 mmol) was added followed by PivCl (0.21 mL, 1.74 mmol) and the reaction mixture was stirred at the same temperature for 2 hours. 44 (1.21 g, 1.74 mmol, 20 mL THF) were added, and the reaction mixture was stirred at the same temperature for a further 10 min. The reaction mixture was brought to room temperature and stirred for 16 hours. The organic solvent was removed. Water was added to the residue and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. ^{The residue was purified by column chromatography} (4% methanol in chloroform) to give amide 45 (2.00 g, impure) as a slightly yellow solid: [M + H]⁺ 1196.

Preparation of compound 46;

[0256] Suspension 45 (2.00 g, contaminated) and 10% Pd / C (400 mg) in a mixture of EtOH (120 mL) and AcOH (20 mL) were degassed and hydrogenated (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 46, which was neutralized with NaHCO₃and the crude product was purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 46 as a yellow solid (500 mg, 27% in two steps): [M + H]⁺ 1067.

Preparation of compound 47;

[0257] To a solution of amine 46 (500 mg, 0.47 mmol) and methyl 3,5-diamino-6-chloropyrazine-2carbonylcarbamimidothioate (21, 330 mg, 0.84 mmol) in EtOH (20 mL) was charged DIPEA (0 , 84 mL, 94.70 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified using

- Silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 47 (325 mg, 55%) as a yellow solid: [M + I]⁺ 1278.

Preparation of HCl salt of the compound 48 - 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (6-bis (2S, 3R, 4R, 5R) ) -2,3,4,5,6-pentahydroxyhexyl) amino) hexyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0258] 4 N HCl in water ( 20 ml) was added to 47 (325 mg, 0.25 mmol) in EtOH (2.0 ml) and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 48 (165 mg, 60%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.52 (brs, 1I), 10.44 (brs, 1H), 9.28 (t, J = 5.2 Hz, 1H), 9.00- 8.88 (m, 1 H), 8.87-8.75 (m, 1 H), 8.63 (br s, 2 H), 8.60-8.50 (m, 1H), 8.39-8, 33 (m, 1H), 8.17-8.11 (m, 1H), 7.58 (dd, J = 6.5, 3.3 Hz, 2H), 7.47-7.35 (m, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 6.8Hz, 1H), 7.27 (d, J = 3.6Hz, 1H). , 7.11 (d, J = 8.8 Hz, 2H), 3.72-3.66 (m, 3H), 3.60 (d, J = 3.6 Hz, 1H), 3.57 ( d, J = 2.8 Hz, 1H), 3.53-3.46 (m, 3H), 3.45-3.38 (m, 3H), 3.37-3.27 (m, 4H) , 3.26-3.12 (m, 4H), 3.06 (t, J = 8.5 Hz, 2H), 1.76-1.60 (m, 6H), 1.58-1.47 (m, 2H), 1.35-1.23 (m, 4H).

[0259] ¹1 H NMR (400 MHz, CD 3 OD): δ 8.23-8.18 (m, 1H), 8.17-8.12 (m, 1H), 7.59-7.5 (m, 2H), 7 36 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 4.25 (t, J = 7.8 Hz, 1H), 4.18-4.10 (m, 2H), 3.83-3.79 ( m, 2H), 3.77 (d, J = 3.1 Hz, 1H), 3.74 (d, J = 3.5 Hz, 1H), 3.71-3.60 (m, 8H), 3.49-3.41 (m, 2H), 3.40-3.33 (m, 4H), 3.32-3.30 (m, 1H), 3.25-3.19 (m, 1H); ), 3.18-3. 10 (m, 2H), 2.52 (t, J = 7.4 Hz, 2H), 1.88-1.69 (m, 6H), 1.62-1, 53 (m, 2H), 1.44-1.30 (m, 4H).

8. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3-oxo-3- (4- (6 - ((2S, 3R, 4R, 5R) ) 2,3,4,5,6-pentahydroxyhexylamino) hexyl) phenylamino) propyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0260]

Scheme 9

<img file="PL2931713T3_D0021.tif" />

Preparation of compound 50;

[0261] A solution of acid 17 (950 mg, 1.84 mmol) in THF (30 mL) was cooled to 0 ° C in an ice bath. NMM (0.40 mL, 3.68 mmol) was added followed by PivCl (0.23 mL, 1.84 mmol) and the reaction mixture was stirred at the same temperature for 2 hours. 49 (800 mg, 1.47 mmol, 10 mL THF) was added, and the reaction mixture was stirred at the same temperature for a further 10 min. The reaction mixture was brought to room temperature and stirred for 16 hours. The organic solvent was removed. To the residue

- water was added and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. The residue was purified using^{column chromatography} (4% methanol in chloroform) to give amide 50 (1.40 g, impure) as a slightly yellow solid: [M + H]⁺ 1043.

Preparation of compound 51;

[0262] A suspension of 50 (1.40 g, contaminated) and 10% Pd / C (400 mg) in a mixture of EtOH (120 ml) and AcOH (20 ml) was degassed and subjected to hydrogenation conditions (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 51 directly used in the next step (1.20 g, crude): [M + H]⁺ 913.

Preparation of compound 52;

[0263] To a solution of amine 51 (1.20 g, 0.47 mmol, crude) and 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate methyl (21, 723 mg, 1.86 mmol) in EtOH (20 mL) DIPEA (2.00 mL, 11.6 mmol) was introduced at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl3 / CH3OH / NH4OH) to give guanidine 52 (500 mg, 24% in three steps) as a yellow solid: [M + H] + 1125.

Preparation of HCl salt of 53 - 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3-oxo-3- (4 (6 - ((2S, 3R, 4R) , 5R) -2,3,4,5,6-pentahydroxyhexylamino) hexyl) phenylamino) propyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0264] 4 N HCl in water (25 ml) was added to 52 (500 mg, 0.44 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 53 (170 mg, 41%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.52 (brs, 1H), 10.45-10.41 (m, 1H), 9.31-9.24 (m, 1H), 9.02 -8.89 (m, 1H), 8.88-8.76 (m, 1H), 8.70-8.5 (m, 3H), 8.57- 8.46 (m, 2H), 8 , 40-8.31 (m, 1H), 8.17-8.10 (m, 1H), 7.62-7.54 (m, 2H), 7.42 (br s, 2H), 7.36 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 6.7 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.11 (d , J = 8.7 Hz, 2H), 5.415,35 (m, 1H), 4.79- 4.72 (m, 1H), 4.62- 4.53 (m, 2H), 4.47- 4.38 (m, 1H), 4.29-4. 19 (m, 1H), 3.94-3.87 (m, 1H), 3.63-3.52 (m, 3H), 3, 50-3.39 (m, 3H), 3.38-3.32 (m, 2H), 3.12-2.96 (m, 3H), 2.97-2.90 (m, 1H), 2.89-2.80 (m, 2H), 1.77-1.56 (m, 6H), 1.54-1.45 (m, 2H), 1.35-1.20 (m, 4H); ).

[0265] ¹1 H NMR (400 MHz, CD 3 OD): δ 8.24-8.20 (m, 1H), 8.19-8.14 (m, 1H), 7.60-7.53 (m,

2H), 7.38 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.24-7.18 (m, 2H), 7, 07 (d, J = 8.1 Hz,

2H), 4.31-4.22 (m, 1H), 4.08-4.01 (m, 1H), 3.84 (dd, J = 4.8, 1.3 Hz, 1H), 3 77 (dd, J = 10.1,

2.5 Hz, 1H), 3.71-3.62 (m, 5H), 3.36 (t, J = 7.2 Hz, 2H), 3.19-3.12 (m, 4H), 3.03-2.96 (m,

- 76 2H), 2.55 (t, J = 7.7 Hz, 2H), 1.90-1.74 (m, 4H), 1.73-1.64 (m, 2H), 1.63 -1.53 ​​(m, 2H), 1.451.31 (m, 4H).

9. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (6- (hexyl) ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxy-hexyl) amino) hexyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0266]

Diagram 10

<img file="PL2931713T3_D0022.tif" />

[0267] Preparation of compound 55; Compound 54 (770 mg, 1.45 mmol) in THF (50 mL) was followed sequentially with DEPBT (564 mg, 1.88 mmol), 17 (752 mg, 1.45 mmol) and DIPEA (0.77 mL, 4). 35 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (100 mL), washed rapidly with saturated aqueous NaHCO 3 (2 x 100 mL) and brine (50 mL), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (5% methanol / CH₂cl₂) and using

- reverse phase chromatography (Gold column) to give amide 55 as a yellow solid (800 mg, 54%): [M + H]⁺ 1027.

Preparation of compound 56;

[0268] A suspension of 55 (800 mg, 0.78 mmol) and 10% Pd / C (400 mg) in a mixture of EtOH (120 mL) and AcOH (30 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 56 as a yellow solid (780 mg, 99%): [M + H]⁺ 897.

Preparation of compound 57;

[0269] To a solution of the amine salt 56 (780 mg, 0.75 mmol) and methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate (21, 466 mg, 1.20 mmol) in EtOH (20 mL) was charged DIPEA ( 1.37 mL, 7.67 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 57 (455 mg, 55%) as a yellow solid: [M + H]⁺ 1110.

Preparation of HCl salt of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (6) hexyl ((2S, 3R, 4R, 5R) compound 58 -2,3,4,5,6-pentahydroxyhexyl) amino) hexyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide [0381] 4 N HCl in water (25) ml) was added to 57 (455 mg, 0.41 mmol) in ethanol (10 ml), and the reaction mixture was stirred at room temperature for 2 hours. The mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to afford compound 58 (230 mg, 55%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6): δ 10.45 (br s, 1H), 9.30 (brs, 1H), 9.09-8.4 (m, 3H), 8.41-8.38 (m, 1 H), 8.16-8.08 (m, 1H), 7.62-7.5 (m, 2H), 7.42 (br s, 2H), 7.37 (t, J = 8 , 4 Hz, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 7.10 (d, J = 8.1 Hz, 2H), 5.52-5.36 (m, 1H), 4.87- 4.70 (m, 1H), 4.63-4.51 (m, 2H), 4.47-4, 38 (m, 1 H), 4.23 (t, J = 6.7 Hz, 1H), 4.033,94 (m, 1H), 3.71-3.66 (m, 1H), 3.65-3. 52 (m, 2H), 3.50-3.34 (m, 5H), 3.21 (d, J = 3.2 Hz, 1H), 3.12 (d, J = 3.2 Hz, 1H), 3.09-2.96 (m, 6H), 1.77-1.58 (m, 8H), 1.57-1.46 (m, 2H), 1.351.21 (m, 10 H). ), 0.86 (t, J = 6.4 Hz, 3H).

[0271] 1H NMR (400 MHz, CD₃OD): δ 8.26-8.20 (m, 1H), 8.19-8.12 (m, 1H), 7.60-7.5 (m, 2H), 7.38 (d, J) = 7.2 Hz, 1H), 7.32 (d, J = 7.4 Hz, 1H), 7.21 (d, J = 8.3 Hz, 2H), 7.06 (d, J =

8.5 Hz, 2H), 4.26 (t, J = 7.4 Hz, 1H), 4.16- 4.09 (m, 1H), 3.82 (dd, J = 5.0, 1 , 5 Hz, 1H), 3.78 (dd, J = 11.3, 3.2 Hz, 1H), 3.72-3.61 (m, 6H), 3.35 (t, J = 6, 7 Hz, 2H), 3.24-3.11 (m, 7H), 2.54 (t, J = 7.4 Hz, 2H), 1.90-1.67 (m, 8H), 1, 64-1.54 (m, 2H), 1.44-1.30 (m, 10H), 0.92 (t, J = 6.7Hz, 3H).

10. Preparation of (S) -2-amino-3- (6- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) naphthalen-2-yl) propane (80) [0272]

- 79 Scheme 11

<img file="PL2931713T3_D0023.tif" />

<img file="PL2931713T3_D0024.tif" />

Preparation of compound 62;

[0273] Stable Wittig Wettig carbomethoxymethylene triphenylphosphate (Ph₃PCHCO₂Me, 43.0 g, 129 mmol) was added to a solution of aldehyde 59 (20.0 g, 107 mmol) in CH₂cl₂ (200 ml) under a nitrogen atmosphere, and the reaction mixture was stirred for 16 hours. at ambient temperature. The completion of the reaction was monitored by TLC (16 h). CH₂cl₂ was removed under reduced pressure and FCC using 10% ethyl acetate-hexanes gave the corresponding trans-α, e-unsaturated ester 62 (24.0 g, 92%) as a white solid: ¹1 H NMR (400 MHz, CDCl 3) δ 7.88-7.82 (m, 1H), 8.81 (d, J = 15.8 Hz, 1H), 7.73 (d, J = 9.0 Hz , 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.61 (dd, J = 8.8, 2.2 Hz, 1H), 7.15 (dd, J = 9, 2, 2.2 Hz, 1H), 7.11 (d, J = 2.2 Hz, 1H), 6.49 (d, J = 15.8 Hz, 1H), 3.82 (s, 3H). , 3.82 (s, 3H).

Preparation of compound 62 (additional pathway);

[0274] To trimethyl phosphonoacetate (55.6 ml, 381 mmol) in 250 ml of anhydrous CH₂cl₂ DBU (48.8 ml, 322 mmol) was added to 0 ° C and the mixture was stirred for 15 min. Aldehyde 59 (40.0 g, 215 mmol) in 50 ml of CH₂cl₂ added dropwise. The temperature of the reaction mixture was brought to room temperature and the resulting reaction mixture was stirred at rt for 16 h and then quenched with 100 mL of water. The mixture was separated and the aqueous layer was extracted with CH₂cl₂ (3 x 150 ml). The combined organic fractions were washed with brine, dried (Na 2 SO 4), filtered, concentrated and the residue was purified by silica gel column chromatography (10: 1 hexanes / ethyl acetate) to afford the desired trans-α, unsaturated ester 62 (48.0 g). , 92%) as a white solid.

Preparation of compound 64;

[0275] A suspension of compound 62 (48.0 g, 196 mmol) and 10% Pd / C (10 g) in EtOAc / THF (600 mL / 75 mL) was subjected to hydrogenation conditions (1 atm) for 16 h. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to afford 64 (46.5 g, 96%) as a white solid:¹1 H NMR (400 MHz, CDCl 3) δ 7.67 (d, J = 9.4 Hz, 2H), 7.57-7.54 (m, 1H), 7.29 (dd, J = 8.6, 1.8 Hz,

- 81 1H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H), 7.11-7.09 (m, 1H), 3.90 (s, 3H), 3.66 (s, 3H), 3.07 (t, J =

7.7 Hz, 2H), 2.70 (t, J = 7.7 Hz, 2H).

Preparation of compound 66;

[0276] To a solution of methyl ester 64 (46.5 g, 191 mmol) in THF / MeOH / H₂O (500 ml / 500 ml / 150 ml) was added NaOI (45.6 g, 114 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed and the pI value was adjusted to 1 with 1 N aqueous phase. ICl; a white precipitate precipitated. The solid was filtered, washed with water and dried under vacuum to give acid 66 (42.5 g, 97%) as a white solid:¹1 H NMR (400 MHz, DMSO-d6) δ 12.14 (br s, 1 L), 7.73 (dd, J = 9.5, 2.3 Hz, 2H), 7.64-7.61 (m, 1 H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 7.26 (d, J = 2.8 Hz, 1H), 7.12 9 (dd, J = 9, 1

2.5 Hz, 1H), 3.85 (s, 3H), 2.94 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 7.6 Hz, 2H).

Preparation of compound 67;

[0277] To a solution of compound 60 (39.3 g, 222 mmol) in anhydrous THF (500 mL) was added n-butyllithium (110 mL, 2M solution in cyclohexane) dropwise at -78 ° C, and the reaction mixture was stirred for 1 hour to obtain a solution of compound 61. To a further solution of compound 66 (42.5 g, 185 mmol) in anhydrous THF (1000 mL) was added NMM (26.3 mL, 240 mmol) and PivCl (27.3 mL, 222 mmol) dropwise in -78 ° C. The reaction mixture was stirred for 1 min at the same temperature, and then a solution of 66 was added, added slowly at -78 ° C. The reaction mixture was stirred for a further time^{10 min, a}then brought to 0 ° C and stirred for 1 hour, then at room temperature for 30 min, quenched with saturated NH 4 Cl, concentrated to remove TIF, and partitioned between CI 2 Cl 2 (1000 mL) and water (1000 mL). The aqueous layer was separated and extracted with CH 2 Cl 2 (2 x 1000 mL). Connected^extracts the organic was dried over Na2SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, CH2Cl2) to obtain compound 67 (45.0 g, 63%) as a white solid:¹1 H NMR (400 MIz, CDCl 3) δ 7.68 (d, J = 8.6 Hz, 2H), 7.64-7.61 (m, 1H), 7.31 (dd, J = 8.5,

1.8 Hz, 1H), 7.33-7.24 (m, 4H), 7.17-7.12 (m, 2H), 7.11-7.09 (m, 1H), 4.69-4 61 (m, 1 H), 4.15 (d, J = 2.4 Hz, 1H), 4.13 (s, 1H), 3.90 (s, 3H), 3.46-3.21 ( m, 3H), 3.20-3.08 (m, 2H), 2.74 (dd, J = 13.6, 9.4 Hz, 1H).

Preparation of compound 68;

[0278] To a solution of compound 67 (45.0 g, 116 mmol) in anhydrous THF (700 mL) was added KHMDS (34.6 g, 174 mmol) in portions at -78 ° C. After stirring the resulting mixture for 30 min, triisopropylbenzenesulfonyl azide (53.6 g, 174 mmol) was added and the reaction mixture was stirred for 5 min. Acetic acid (69.6 mL, 1158 mmol) was then added slowly followed by tetramethylammonium acetate (30.9 g, 232 mmol) at the same temperature. The reaction mixture was allowed to warm to 24 ° C, stirred for 16 h, quenched with saturated NaHCO₃ (300 ml), concentrated to remove THF and extracted with CH₂cl₂ (2 x 500 ml). The combined organic extracts were dried over Na₂SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, 10:90 EtOAc / hexane followed by DCM) to obtain compound 68 (31.0 g, 62%).

- 82 in the form of a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 7.70 (d, J = 9.1 Hz, 2H), 7.687.65 (m, 1H), 7.40 (dd, J = 8.6, 1.8 Hz, 1H), 7.36 -7.23 (m, 3H), 7.20 (d, J = 1.8 Hz, 1H), 7.19-7.17 (m, 1H), 7.13 (dd, J = 9.0 , 2.6 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 5.36 (dd, J = 9.0, 6.0 Hz, 1H), 4.58-4 , 50 (m, 1H), 4.11 (dd, J = 9.1, 2.6 Hz, 1H), 3.90 (s, 3H), 3.91 (t, J = 8.6 Hz, 1 H), 3.34 (dd, J = 13.8, 6.5 Hz, 1H), 3.30 (dd, J = 13.0, 3.5 Hz, 1H), 3.19 (dd, J = 13.4,

8.6 Hz, 1H), 2.81 (dd, J = 13.4, 9.5 Hz, 1H).

Preparation of compound 69;

[0279] To a solution of compound 68 (31.0 g, 72.1 mmol) in THF / H₂O (300 ml / 100 ml) was added H₂ABOUT₂ (49 mL, 433 mmol) followed by LiOH (6.04 g, 144 mmol) in portions at 0 ° C.

The reaction mixture was stirred for 10 min at the same temperature, then at room temperature for 1 h, and then quenched with saturated NaOH.₂SO₃ (200 mL), concentrated under reduced pressure to remove THF and washed with CH₂cl₂ (500 ml). The aqueous layer was acidified with 1 N aqueous. HCl and extracted with CH₂cl₂ (2 x 500 ml). The combined organic extracts were dried over Na₂SO₄, concentrated and washed with MTBE to afford compound 69 (15.0 g, 82%) as an off-white solid: ¹H NMR (400 MHz, MeOD-d₃) δ 7.70 (t, J = 8.4 Hz, 2H), 7.66-7.63 (m, 1H), 7.35 (dd, J = 8.6, 1.7 Hz, 1H) , 7.19 (d, J = 2.8 Hz, 1H), 7.10 (dd, J = 9.1, 2.6 Hz, 1H), 4.25 (dd, J = 8.6, 5). , 3 Hz, 1H), 3.88 (s, 3H), 3.29 (dd, J = 13.9, 5.1 Hz, 1H), 3.10 (dd, J = 14.3, 8, 6 Hz, 1H).

Preparation of compound 70;

[0280] A suspension of Compound 69 (15.0 g, 55.1 mmol) and 10% Pd / C (3.50 g) in AcOH / H₂O (300 ml / 100 ml) were subjected to hydrogenation conditions (1 atm) for 3 hours. in room temperature. The reaction mixture was filtered through Celite and washed with AcOH / H2O, followed by MeOH. The filtrate was concentrated in vacuo to give the acetic salt 70 (14.0 g, 83%) as a yellow solid:¹H NMR (400 MHz, DMSO-_d6, TFA) δ 8.38-8.18 (m, 3H), 7.78 (dd, J = 11.4, 8.1 Hz, 2H), 7.75-7.70 (m, 1H), 7.41 (dd, J = 8.6, 1.6 Hz, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.18 (dd, J = 8.8, 2, 4 Hz, 1H), 4.33-4.23 (m, 1H), 3.89 (s, 3H), 3.33 (dq, J = 14.5,

5.9 Hz, 2H), 1.92 (s, 3H).

Preparation of compound 71;

[0281] To a solution of compound 70 (14.0 g, 45.9 mmol) in acetic acid (140 ml) was added hydrobromic acid (140 ml) dropwise at room temperature, and the reaction mixture was heated under reflux for 3 h. The reaction mixture was cooled to room temperature and concentrated. The crude brown residue 71 (12.4 g, 87%) was used directly in the next step without any purification:¹1 H NMR (400 MHz, DMSO-d6) δ 13.83 (br s, 1H), 9.71 (br s, 1H), 8.41 (br s, 1H), 8.25 (br s, 2H), 7.67 (dd, J = 13.8,

8.7 Hz, 2H), 7.64-7.61 (m, 1H), 7.29 (dd, J = 8.6, 1.7 Hz, 1H), 7.13-7.05 (m, 2H); ), 4.29-4. 19 (m, 1H), 3.20 (t, J = 5.5 Hz, 2H).

Preparation of compound 72;

[0282] Acetyl chloride (38.4 mL, 540 mmol) was added to anhydrous methanol (400 mL) at 0 ° C, followed by addition of compound 71 (24.0 g, 77.2 mmol). The reaction mixture

- 83 was heated under reflux for 4 hours. and concentrated. The residue was partitioned between CH₂cl₂ (500 ml) and saturated NaHCO₃ (300 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 300 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated to give compound 72 (16.6 g, 88%) as a white solid: ¹1 H NMR (400 MHz, DMSO-d6) δ 9.62 (br s, 1H), 7.67 (d, J = 9.4 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H). ), 7.53 (s, 1H), 7.22 (dd, J = 8.2, 1.4 Hz, 1H), 7.09-7.06 (m, 1H), 7.04 (dd, J = 8.8, 2.6 Hz, 1H), 3.67 (t, J = 6.5 Hz, 1H), 3.57 (s, 3H), 2.97 (dd, J = 13.5). , 6.1 Hz, 1H), 2.86 (dd, J = 13.2, 7.4 Hz, 1H), 1.90 (brs, 2H).

Preparation of compound 73;

[0283] To a solution of compound 72 (16.6 g, 67.8 mmol) in MeOH / H₂O (360 ml / 120 ml) was added NaHCO₃ (22.8 g, 271 mmol) and Boc2O (17.7 g, 81.3 mmol) at 0 ° C. The resulting mixture was allowed to warm to room temperature and stirred for 1 hour. The reaction mixture was partitioned between CH₂cl₂ (200 ml) and water (200 ml). The aqueous layer was separated and extracted with CH 2 Cl 2 (2 x 400 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated. FCC using 20% ​​ethylhexane acetate followed by CH₂cl₂ gave compound 73 (17.0 g, 73%) as a white solid:

¹1 H NMR (400 MHz, CDCl 3) δ 7.60 (d, J = 9.5 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.49-7.43 (m , 1 H), 7.15 (d, J = 8.2 Hz, 1H), 7.09-6.99 (m, 2H), 6.31 (br s, 1 H), 5.15-4.84 ( m, 1 H), 4.734,46 (m, 1H), 3.71 (s, 3H), 3.23 (dd, J = 13.7, 5.3 Hz, 1H), 3.14 (dd, J = 13.7, 5.5 Hz, 1H), 1.39 (s, 9H).

Preparation of compound 74;

[0284] To a solution of compound 73 (7.0 g, 20.3 mmol) in CH₂cl₂ (300 ml) was added pyridine (16.5 ml, 203 mmol), triflate (5.11 ml, 30.4 mmol) at 0 ° C and stirred at the same temperature for 1 hour and then at room temperature. for 2 hours After concentration, the reaction mixture was partitioned between CH₂cl₂ (300 ml) and water (200 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 300 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to give compound 74 (8.80 g, 91%) as a brown oil (presence of pyridine as confirmed by NMR). The reaction was monitored by LC-MS and product formation was confirmed using LM-MS data:¹1 H NMR (400 MHz, CDCl 3) 7.85 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 8.7 Hz, 1H), 7.71 (d, J = 2, 7 Hz, 1H), 7.66-7.63 (m, 1H), 7.37 (ddd, J = 10.0, 7.3, 2.0 Hz, 2H), 5.1255.03 (m, 1H); ), 4.73-4.61 (m, 1H), 3.72 (s, 3H), 3.32 (dd, J = 13.3, 5.3 Hz, 1H), 3.20 (dd, J = 13.3, 6.2 Hz, 1H), 1.38 (s, 9H).

Preparation of compound 75;

Compound 74 (16.5 g, 34.6 mmol) and benzyl but-3-ylcarbamate (17, 10.4 g, 51.9 mmol) on anhydrous CH₃CN (450 ml) was degassed with argon for 10 min at

room temperature, followed by TEA (19.3 ml, 138 mmol), 10% (t-Bu)₃P in hexanes (13.9 mL, 6.91 mmol) and CuI (0.33 g, 1.72 mmol) at room temperature. The resulting mixture was degassed with argon for 10 min and Pd (PPh3) was added quickly.₄ (3.99 g,

84.45 mmol) in one portion. After the completion of degassing with argon for 5 min, the resulting mixture was heated under reflux for 18 h. The reaction mixture was concentrated in vacuo and the residue was purified on a column (silica gel, 75:25 hexanes / EA) to give compound 75 (14.1 g, 77%) as a brown solid:¹1 H NMR (400 MHz, CDCl 3) δ 7.86 (br s, 1H), 7.68 (t, J = 7.8 Hz, 2H), 7.53 (br s, 1H), 7.41 (dd, J = 8.5, 1.6 Hz, 1H), 7.38-7.28 (m, 5H), 7.27-7.22 (m, 1H), 5.26-5.17 (m, 1H); ), 5.13 (s, 2H), 5.06- 4.99 (m, 1 H), 4.70-9.5 (m, 1H), 3.69 (s, 3H), 3.46 ( q, J = 6.7 Hz, 2H), 3.27 (dd, J = 14.1, 5.9 Hz, 1H), 3.16 (dd, J = 13.2, 6.2 Hz, 1H); ), 2.67 (t, J = 6.6 Hz, 2H), 1.38 (s, 9H).

Preparation of compound 76;

[0286] To a solution of methyl ester 75 (12.1 g, 22.8 mmol) in THF / MeOH / H₂O (150 ml / 150 ml / 50 ml) was added NaOH (4.56 g, 114 mmol) and the reaction mixture was stirred at room temperature for 2 hours. The pH was adjusted to 9 1 N aqueous. HCl, and the organic solvent was removed. The pH of the residue was adjusted to 5-6 and the suspension was partitioned between CH₂cl₂ (500 ml) and water (200 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 400 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated to give compound 76 (10.50 g, 89%) as a brown solid: ¹H NMR (400 MHz, CD3OD) δ 7.83 (s, 1H), 7.73-7.61 (m, 3H), 7.44-7.19 (m, 7H),

5.10 (s, 2H), 4.42-4.34 (m, 1H), 3.41-3.32 (m, 3H), 3.06 (dd, J = 14.3, 9.3 Hz, 1H), 2.64 (t, J = 7.0 Hz, 2H), 1.31 (s, 7H), 1.21 (s, 2H).

Preparation of compound 77; SG-SJL-B-27 [0287] A suspension of 75 (2.0 g, 3.77 mmol) and 10% Pd / C (500 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed, and then subjected to hydrogenation conditions (1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 77 (1.60 mg, 93%) as a white solid:¹H NMR (400 MHz, CD₃OD) 7.73 (d, J =

8.5, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.62 ((brs, 2H), 7.73 (ddd, J = 10.0, 8.7, 2). , 7 Hz, 2H), 4.44 (dd, J = 8.8, 5.6 Hz, 1H), 3.68 (s, 3H), 3.25 (dd, J = 14.0, 6, 6 Hz, 1H), 3.04 (dd, J = 13.5, 9.2 Hz, 1H), 2.93 (t, J = 7.4 Hz, 2H), 2.83 (t, J = 7.4 Hz, 2H), 1.96 (s, 6H), 1.85-1.75 (m, 2H), 1.74-1.68 (m, 2H), 1.33 (s, 7H); ), 1.26 (s, 2H).

Preparation of compound 78; SG-SJL-B-30 [0288] To a solution of the amine salt 77 (1.60 g, 3.47 mmol) and methyl 3,5-diamino-6-chloropyrazine-2carbonylcarbamimidothioate (13, 2.16 g, 5.56 mmol) in EtOH (40 mL) was added DIPEA (6.20 mL, 34.70 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 1 h, then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 78 (1.24 g, 59%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.71 (dd, J =

8.4, 2.8 Hz, 2H), 7.60 (br s, 2H), 7.34 (dd, J = 8.5, 1.9 Hz, 1H), 7.30 (dd, J = 8 , 7, 1.7 Hz, 1H),

4.45 (dd, J = 8.9, 5.7 Hz, 1H), 3.68 (s, 3H), 3.28-3.26 (m, 1H), 3.25 (t, J = 2.4 Hz, 1H), 3.22

- 85 (d, J = 5.9 Hz, 1H), 3.04 (dd, J = 14.0, 9.2 Hz, 1H), 2.82 (t, J = 7.2 Hz, 2H) , 1.86-1.77 (m, 2H), 1.73-1.63 (m, 2H), 1.32 (s, 7H), 1.23 (s, 2H).

Preparation of compound 79; SG-SJL-B-32 [0289] To a solution of methyl ester 78 (1.24 g, 2.00 mmol) in a mixture of THF (25 mL), methanol (25 mL) and water (10 mL) was added solid NaOH (324). mg, 8.00 mmol) and the reaction mixture was stirred at room temperature for 1 hour. When the TLC of the reaction mixture showed the reaction was complete, the pH of the reaction mixture was adjusted to pH 9-10 by the addition of 1 N HCl (aqueous) and the organic solvent was removed. The pH of the aqueous portion was adjusted to pH 5-6, a precipitate formed, and it was extracted with dichloromethane. The aqueous portion was extracted with CH₂cl₂ (2X 50 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. ^{Relationship of a yellow solid} (79, 1.10 g, 92%) were dried under vacuum: ¹1 H NMR (400 MHz, CD3OD) 7.70 (t, J = 9.4, 2H),

7.61 (d, J = 5.3 Hz, 2H), 7.33 (dd, J = 8.4, 1.4 Hz, 2H), 4.38 (dd, J = 8.4, 5, 1 Hz, 1H), 3.05 (dd, J = 14.1, 9.1 Hz, 1H), 2.84 (t, J = 6.9 Hz, 2H), 3.35-3.34 ( m, 3H), 1.88-1.79 (m, 2H), 1.76- 1.67 (m, 2H), 1.32 (s, 7H), 1.21 (s, 2H).

Preparation of Compound 80 - (S) -2-Amino-3- (6- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) naphthalen-2-yl) propanoic acid hydrochloride salt [0290] 4 N HCl in dioxane (25 mL) was added to 79 (1.10 g, 1.83 mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, purified by reversed phase column (Gold column) and the residue was lyophilized to yield compound 80 (700 mg, 67%) as a yellow solid:¹1 H NMR (400 MHz, DMSO-d6) 10.48 (s, 1H), 9.24 (br s, 1H), 8.99-8.86 (m, 1H), 8.84-8.70 (m) , 1 H), 8.38 (br s, 3H), 7.80 (t, J = 9.2 Hz, 2H), 7.73 (s, 1H), 7.69 (s, 1H), 7.45 -7.35 (m, 4H), 4.25 (dd, J = 11.4, 5.9 Hz, 1H), 3.34 (q, J = 6.6 Hz, 2H), 3.27 ( d, J = 6.9 Hz, 2H), 2.79 (t, J = 7.70 Hz, 2H), 1.79-1.67 (m, 2H), 1.65-1.54 (m , 2H).

[0291] ¹1 H NMR (400 MHz, CD 3 OD) 7.82 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 8.7 Hz, 1H), 7.73 (s, 1H), 7.68 (s, 1H), 7.40 (ddd, J = 10.5, 8.6, 1.6 Hz, 2H), 4.33 (dd, J = 7.7, 5.2 Hz, 1H), 3.46 (dd, J = 14.9, 6.0 Hz, 1H), 3.37 (t, J = 7.5 Hz, 2H), 3.33-3.29 (m, 1H); ), 2.87 (t, J = 7.7 Hz, 2H), 1.90-1.80 (m, 2H), 1.79-1.71 (m, 2H).

11. Preparation of (S) -3,5-Diamino-6-chloro-N- (N- (4- (6- (2,3-diamino-3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) pyrazine-2 -carboxamide (84) [0292]

Diagram 12

<img file="PL2931713T3_D0025.tif" />

Preparation of compound 81;

[0293] A solution of acid 76 (2.0 g, 3.87 mmol) in THF (80 mL) was cooled to 0 ° C in an ice bath, NMM (0.63 mL, 5.03 mmol) was added, and then with i-BCF (0.63 ml),

5.80 mmol) and the reaction mixture was stirred at the same temperature for 2 hours.

- 87 NH was added₃ (7.0 N in methanol, 5.52 mL, 38.7 mmol) dropwise, and the reaction mixture was stirred at the same temperature for a further 2 hours. The reaction mixture was then brought to room temperature and stirred for 16 hours. The organic solvent was removed. Water was added to this residue and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. The residue was purified by column chromatography (3% methanol in chloroform) to give amide 81 (1.75 g, 88%) as a slightly yellow solid:¹1 H NMR (400 MHz, CD 3 OD) 7.83 (s, 1H), 7.69 (d, J = 8.1 Hz, 2H), 7.66 (s, 1H), 7.39 (dt, J = 8.8, 1.9 Hz, 2H), 7.35-7.21 (m, 5H), 5.09 (s, 2H), 4.40 (dd, J = 9.6, 5.8 Hz). , 1H), 3.37 (t, J = 6.9 Hz, 2H), 3.27 (dd, J = 13.8, 5.2 Hz, 1H), 2.97 (dd, J = 13, 7, 9.4 Hz, 1H), 2.63 (t, J = 7.0 Hz, 2H), 1.27 (s, 7H), 1.21 (s, 2H).

Preparation of compound 82;

[0294] A suspension of 81 (1.75 mg, 3.39 mmol) and 10% Pd / C (600 mg) in a mixture of EtOH (110 mL) and AcOH (15 mL) was degassed and then subjected to hydrogenation conditions (1 atm). for 12 hours in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 82 as a white solid (1.40 g, 93%):¹H NMR (400 MHz, CD₃OD) 7.72 (dd, J = 8.3,

5.6 Hz, 2H), 7.66 (s, 1H), 7.61 (s, 1H), 7.38 (dd, J = 8.6, 1.3 Hz, 1H), 7.34 ( dd, J = 8.5, 1.5 Hz, 1H), 4.38 (dd, J = 9.0, 5.0 Hz, 1H), 3.27 (dd, J = 13.8, 5, 0 Hz, 1H), 2.93 (t, J = 7.9 Hz, 2H), 2.83 (t, J = 7.5 Hz, 2H), 3.01 - 2.95 (m, 1H) , 1.96 (s, 3H), 1.86-1.75 (m, 2H), 1.74-1.64 (m, 2H), 1.29 (s, 7H), 1.23 (s , 2H).

Preparation of compound 83;

[0295] To a solution of the amine salt 82 (1.40 g, 3.15 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 1.96 g, 5.04 mmol) in EtOH (40 mL) ) DIPEA (5.64 mL, 31.5 mmol) was added at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 83 (1.15 g, 61%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) δ 7.70 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.60 (s, 1H), 7.34 (dt, J = 8.9, 1). , 9 Hz, 2H), 4.38 (dd, J = 9.0, 5.5 Hz, 1H), 3.28-3.20 (m, 3H), 2.96 (dd, J = 9, 6, 14.1 Hz, 1H), 2.81 (t, J = 7.4 Hz, 2H), 1.851.76 (m, 2H), 1.70-1.61 (m, 2H), 1, 27 (s, 7H), 1.20 (s, 2H).

Preparation of the compound HCl (S) -3,5-diamino-6-chloro-N- (N- (4- (6- (2,3-diamino-3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) pyrazine 2-carboxamide (84) [0296] 4 N HCl in dioxane (25 ml) was added to 83 (1.15 g, 1.92 mmol) in EtOH (6.0 ml) and the reaction mixture was stirred at room temperature for 2 hours. h. The solvent was removed, purified by reversed phase column (Gold column) and the residue was lyophilized to yield compound 84 (310 mg, 28%) as a yellow solid:¹H NMR (400 MHz, DMSO-d6) 10.56 (s, 1H), 9.38 (t, J = 5.5 Hz, 1H), 9.06-8.83 (m, 2H), 8, 31 (br s, 3 H), 8.02 (s, 1H), 7.79 (t, J = 8.6 Hz, 2H), 7.73 (s, 1H), 7.69 (s, 1H), 7.51 (s, 1H), 7.46-7.36

88 (m, 4H), 4.06 (dd, J = 11.5, 6.0 Hz, 1H), 3.37 (q, J = 6.4 Hz, 2H), 3.27 (dd, J = 6.6, 1.4 Hz, 1H), 3.18 (dd, J = 13.7, 6.9 Hz, 1H), 2.79 (t, J = 7.1 Hz, 2H), 1.79-1.69 (m, 2H), 1.64-1.54 (m, 2H).

[0297] ¹1 H NMR (400 MHz, CD 3 OD) 7.82 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.7 Hz, 1H), 7.74 (s, 1H), 7 68 (s, 1H), 7.41 (td, J = 8.1, 1.6 Hz, 2H), 4.18 (dd, J = 8.1, 6.3 Hz, 1H), 3, 42-3.34 (m, 3H), 3.21 (dd, J = 14.1, 8.0 Hz, 1H), 2.86 (t, J = 7.4 Hz, 2H), 1.91 -1.80 (m, 2H), 1.791.71 (m, 2H).

12. Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3- (4- (3- (hexyl) ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (89)

Scheme 13 [0298]

<img file="PL2931713T3_D0026.tif" />

Preparation of compound 86;

[0299] To compound 85 (1.10 g, 2.32 mmol) in THF (50 mL) DEPBT (766 mg, 2.56 mmol), 76 (1.00 g, 1.97 mmol) and DIPEA were added sequentially. (1.0 mL, 5.91 mmol) and stirred at rt for 16 h. After removing the solvent under reduced pressure, the residue was dissolved in Cl₂cl₂ (100 mL), washed rapidly with saturated aqueous solution (2 x 100 mL) and brine (50 mL), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (5% methanol / CH₂cl₂) to give the amide 86 as a yellow solid product (1.19 g, 57%):

- 90 ¹H NMR (400 MHz, CD3OD): 7.82 (d, J = 5.8 Hz, 2H), 7.73-7.61 (m, 4H), 7.51-7.43 (m, 2H) , 7.39-7.19 (m, 10H), 7.05 (d, J = 8.3 Hz, 2H), 5.52 (s, 1H), 5.10 (s, 2H), 4, 51 (t, J = 7.8 Hz, 1H), 4.31-4.25 (m, 1H), 4.24 (dd, J = 11.0, 5.4 Hz, 1H), 4.01 -3.91 (m, 2 H), 3.88 (dd, J = 5.5, 2.1 Hz, 1H), 3.76 (dd, J = 9.3, 2.1 Hz, 1H), 3.61 (t, J = 10.6 Hz, 1H), 3.37 (t, J = 6.9 Hz, 2H), 3.12-3.00 (m, 1H), 2.74 (dd) , J = 13.2, 5.3 Hz, 1H), 2.64 (t, J = 7.1 Hz, 2H), 2.57-2.37 (m, 7H), 1.74-1, 64 (m, 2H), 1.31 (s, 9H), 1.29-1.16 (m, 8H), 0.86 (t, J = 6.9 Hz, 3H).

Preparation of compound 87;

[0300] A suspension of 86 (1.19 g, mixture) and 10% Pd / C (220 mg) in a mixture of EtOH (110 mL) and AcOH (15 mL) was degassed and then subjected to hydrogenation conditions (1 atm) for 3 hours. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 87, which was then neutralized with NaHCO_{3 a} the crude product was purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 87 as a yellow solid (550 mg, 58%, in two steps):

¹1 H NMR (400 MHz, CD 3 OD) 7.71 (t, J = 8.4 Hz, 2H), 7.62 (d, J = 1.8 Hz, 1H), 7.49-7.45 (m, 3H), 7.40 (d, J = 8.2 Hz, 2H), 7.36-7.28 (m, 5H), 7.09 (d, J = 8.2 Hz, 2H), 5, 55 (s, 1 H), 4.51 (dd, J = 15.6, 8.4 Hz, 1H), 4.25 (dd, J = 10.6, 5.4 Hz, 1H), 4.17 -4.03 (m, 2H), 3.98-3.90 (m, 2H), 3.81-3.74 (m, 1H), 3.63 (t, J = 10.4 Hz, 1H). ), 3.27-3.20 (m, 1H), 3.09-2.98 (m, 5H), 2.93 (t, J = 7.6 Hz, 2H), 2.83 (t, J = 6.8 Hz, 2H), 2.61-2.54 (m, 2H), 1.95-1.86 (m, 2H), 1.851.75 (m, 2H), 1.74-1 65 (m, 2H), 1.57-1.47 (m, 2H), 1.39-1.19 (m, 7H), 1.33 (s, 9H), 0.88 (t, J = 6.9 Hz, 3H).

Preparation 88;

[0301] To a solution of amine 87 (550 mg, 0.65 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 400 mg, 1.04 mmol) in EtOH (20 mL) was added

DIPEA (1.15 mL, 6.44 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, then cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) followed by reverse phase columns (Gold C18) to give guanidine 88 (333 mg, 48%) as a yellow solid: ¹1 H NMR (400 MHz, CD 3 OD) 7.69 (dd, J = 8.6, 3.5 Hz, 2H), 7.66 (s, 1H), 7.60 (s, 1H), 7.48- 7.44 (m, 2H), 7.35 (ddd, J = 10.4, 8.6, 1.6 Hz, 2H), 7.33-7.28 (m, 5H), 7.04 ( d, J = 8.3 Hz, 2H), 5.52 (s, 1H), 4.52-4.55 (m, 1H), 4.24 (dd, J = 10.6, 5.4 Hz) , 1H), 4.00-3.91 (m, 2H), 3.88 (dd, J = 5.4, 2.0 Hz, 1H), 3.75 (dd, J = 9.6, 2). , 2 Hz, 1H), 3.60 (t, J =

10.6 Hz, 2H), 3.28-3.23 (m, 3H), 3.06 (dd, J = 13.5, 8.3 Hz, 1H), 2.82 (t, J = 7). , 0 Hz, 2H),

2.77 (dd, J = 13.9, 5.6 Hz, 1H), 2.59-2.40 (m, 7H), 1.86-1.76 (m, 2H), 1.74- 1.68 (m, 4H), 1.42-1.60 (m, 7H), 1.33 (s, 9H), 0.86 (t, J = 7.1 Hz, 3H).

Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3- (4- (3) hexyl (2S, 3R, 4R, 5R) HCl salt -2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide compound (89);

[0302] 4 N HCl in water (20 ml) was added to 88 (333 mg, 0.31 mmol) in ethanol (10 ml) and the reaction mixture was stirred at rt for 2 h. Purified by a reverse phase column (Gold column) and the residue was lyophilized to give compound 89 (210 mg, 68%) as a yellow solid:¹H NMR (400 MHz, DMSO-d₆) 10.94 (br s, 1 H), 9.29 (br s, 1 H), 9.02- 8.77 (m, 2 H), 8.64-8.17 (m, 2 H), 7.80-7. 73 (m, 3H), 7.68 (s, 1H), 7.52 (d, J = 8.8 Hz, 2H), 7.47 (dd, J = 8.2, 1.0 Hz, 1H), 7.44-7.36 (m, 3H), 7.19 (d, J = 8.6 Hz, 2H), 5.52-5.4 (m, 1H), 4.86- 71 (m, 1H), 4.60 (d, J = 5.4 Hz, 1H), 4.59-4.53 (m, 1H), 4.42 (t, J = 5.8 Hz, 1H), 4.38 (t, J = 7.0 Hz, 1H), 4.03-3.95 (m, 1H), 3.71-3.66 (m, 1H), 3.62-3. 55 (m, 1H), 3.53-3.34 (m, 5H), 3.27 (d, J = 7.7 Hz, 1H), 3.23 (d, J = 7.4 Hz, 1H), 3.16-2.99 (m, 5H), 2.78 (t, J = 7.4 Hz, 2H), 2.58 (t, J = 7.9 Hz, 2H), 2, 01-1.90 (m, 2H), 1.78-1.68 (m, 2H), 1.66-1.54 (m, 4H), 1.32-1.21 (m, 6H), 0.85 (t, J = 6.6 Hz, 3H).

[0303] ¹1 H NMR (400 MHz, CD 3 OD) 7.79 (d, J = 8.5 Hz, 1H), 7.77-7.73 (m, 2H), 7.67 (s, 1H), 7.47- 7.37 (m, 4H), 7.21 (d, J = 8.5 Hz, 2H), 4.30 (dd, J = 7.7, 6.7 Hz, 1H), 4.12-4 , 05 (m, 1 H), 3.82-3.74 (m, 1H), 3.71-3.61 (m, 2H), 3.49 (dd, J = 14.0, 6.6 Hz). , 1H), 3.47 (t, J = 6.9 Hz, 2H), 3.33-3.27 (m, 3H), 3.26-3.13 (m, 4H), 2.86 ( t, J = 7.6 Hz, 2H), 2.73-2.64 (m, 2H), 2.102.00 (m, 2H), 1.89-1.80 (m, 2H), 1.79 -1.72 (m, 2H), 1.71-1.63 (m, 2H), 1.40-1.30 (m, 6H), 0.91 (t, J = 6.6 Hz, 3 H). ).

13. Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (94) [0304]

Scheme 14

<img file="PL2931713T3_D0027.tif" />

[0305] To compound 90 (484 mg, 0.91 mmol) in THF (30 mL) DEPBT (300 mg, 1.00 mmol), 19 (400 g, 0.77 mmol) and DIPEA (0.40) were added sequentially. ml, 2.31 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH 2 Cl 2 (100 mL), washed rapidly with saturated aqueous solution (2 x 100 mL) and brine (50 mL), and dried over Na₂SO₄. ^Solventevaporation and the crude product was purified by flash gel chromatography

- silica (5% methanol / CH₂cl₂) to give the amide 91 as a yellow solid product (600 mg, 76%, impure). The creation of the product was confirmed with

LCMS.

Preparation of compound 92;

[0306] A suspension of 91 (600 mg, 0.59 mmol) and 10% Pd / C (200 mg) in a mixture of EtOH (90 ml) and AcOI (10 ml) was degassed and then subjected to hydrogenation conditions (1 atm) for 16 hours. h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOI. The filtrate was concentrated in vacuo to give an amine salt 92 which was then neutralized with NaICO_{3 a} the crude product is purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 36 as a yellow solid (350 mg, 66%, impure): ¹1 H NMR (400 MHz, CD 3 OD) 7.71 (d, J = 8.1 Hz, 2H), 7.68 (s, 1H), 7.61 (s, 2H), 7.43-7.37 ( m, 2H), 7.34 (dd, J = 8.3, 1.3 Hz, 1H), 7.16 (d, J = 8.2 Hz, 2H), 4.69 (q, J = 5 , 1 Hz, 2H), 4.50 (t, J = 7.1 Hz, 1H), 4.13-4.06 (m, 2H), 4.05 (dd, J = 11.0, 5, 6 Hz, 2H), 3.83 (dd, J = 4.8, 2.1 Hz, 2H), 3.81-3.73 (m, 2H), 3.51 (dd, J = 9.5 , 2.3 Hz, 2H), 3.38 (t, J = 10.8 Hz, 2H), 3.13-3.03 (m, 6H), 2.93 (t, J = 7.6 Hz). , 2H), 2.82 (t, J = 7.2 Hz, 2H), 2.74-2.57 (m, 2H), 2.04-1.95 (m, 2H), 1.84- 1.75 (m, 3H), 1.74-1.63 (m, 3H), 1.33 (s, 9H), 1.25 (d, J = 5.1 Hz, 6H).

Preparation 93;

[0307] A solution of amine 92 (350 mg, 0.38 mmol) and 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 242 mg, 0.62 mmol) in EtOH (10 mL) was charged with DIPEA (0 67 ml, 3.80 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, then cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CICl₃/ CH₃OH / NH₄OH) followed by reverse phase columns (Gold C18) to afford guanidine 93 (170 mg, 20% in three steps) as a yellow solid: 1H NMR (400 MHz, CD)₃OD) 7.71 (d, J = 8.2

Hz, 2H), 7.68 (s, 1H), 7.62 (s, 1H), 7.43 (d, J = 8.2 Hz, 2H), 7.26 (ddd, J = 10.6 , 8.6, 1.3 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 4.70 (q, J = 0.5 Hz, 2H), 4.49 (t , J = 7.8 Hz, 1H), 4.22-4.09 (m, 2H), 4.06 (dd, J = 10.4, 5.1 Hz, 2H), 3.89-3, 81 (m, 2H), 3.80-3.71 (m, 2H), 3.60-3.49 (m, 2H), 3.43-3.32 (m, 8H), 3.31- 3.23 (m, 2H), 3.10-2.98 (m, 2H), 2.85 (t, J = 6.9 Hz, 2H), 2.772.61 (m, 2H), 2.12 -2.02 (m, 2H), 1.89-1.79 (m, 2H), 1.78-1.68 (m, 2H), 1.31 (s, 9H), 1.25 (d); , J = 5.1 Hz, 6H).

Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3- (4- (3- (bis (2S, 3R, 4R, 5R) 2 HCl salt) , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (94) [0308] 4 N HCl in water ( 20 ml) was added to 93 (170 mg, 0.15 mmol) in ethanol (5.0 ml) and the reaction mixture was stirred at 40 ° C for 2 hours. The solvent was removed, 4N IC1 was added again and heated at 40 ° C for a further 2 hours. This addition was repeated two consecutive times. The solvent was removed and purified by a reverse phase column (Gold column) and the residue was lyophilized to yield compound 94 (80 mg,

- 94 50%) in the form of a yellow solid: ¹H NMR (400 MHz, DMSO-d₆) 10.74 (br s, 1H), 9.288.9 (m, 1H), 9.03-8.60 (m, 2H), 8.58-8.04 (m, 1H), 7.81-7. 73 (m, 3H), 7.68 (s, 1H), 7.50 (d, J = 8.4 Hz, 2H), 7.48-7.34 (m, 4H), 7.19 ( d, J = 9.0 Hz, 2H), 5.39-5.35 (m, 1H), 4.87- 4.63 (m, 1H), 4.62- 4.47 (m, 3H). 4.45-4.35 (m, 2H), 4.32-4.23 (m, 1H), 4.01-3.85 (m, 1H), 3.67 (d, J =

4.6 Hz, 1H), 3.62-3.55 (m, 2H), 3.53-3.38 (m, 5H), 3.37-3.29 (m, 2H), 3.24 -3.09 (m, 2H),

2.78 (t, J = 7.2 Hz, 2H), 2.62-2.53 (m, 2H), 2.01-1.86 (m, 2H), 1.79-1.68 ( m, 2H), 1.64-1.55 (m, 2H).

[0309] ¹1 H NMR (400 MHz, CD 3 OD) 7.78 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.73 (s, 1H), 7 66 (s, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8, 7 Hz, 2H), 4.16 (t, J = 7.0 Hz, 1H), 4.13-4.05 (m, 2H), 3.81 (dd, J = 4.7, 1.9 Hz, 2H), 3.77 (dd, J = 10.6, 3.0 Hz, 2H), 3.72-3.61 (m, 6H), 3.44-3.30 (m, 10 H). ), 2.86 (t, J = 7.0 Hz, 2H), 2.76-2.61 (m, 2H), 2.11-2. 01 (m, 2H), 1.89-1, 80 (m, 2H), 1.79-1.72 (m, 2H).

14. Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3-oxo-3- (4- (3 - ((2S, 3R, 4R, 5R) ) 2,3,4,5,6-pentahydroxyhexylamino) propyl) phenylamino) propyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (99) [0310]

Scheme 15

<img file="PL2931713T3_D0028.tif" />

Preparation of compound 96;

[0311] A solution of acid 19 (1.17 g, 2.27 mmol) in THF (60 mL) was cooled to 0 ° C in an ice bath, NMM (0.30 mL, 2.95 mmol) was added, and then PivCl (0.30 mL, 2.49 mmol), and the reaction mixture was stirred at the same temperature for 2 hours. 34 (1.0 g, 2.27 mmol, 10 mL THF) of aniline 171 was added and the reaction mixture was stirred at the same temperature for a further 10 min. The reaction mixture was then brought to room temperature and stirred for 16 hours. The organic solvent was removed. Water was added to this residue and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. ^{The residue was purified using}^{column chromatography} (4% methanol in chloroform) to afford amide 96 (1.40 g, 66%, impure) as a slightly yellow solid. The formation of the product was confirmed by LCMS.

- 96 Preparation of compound 97;

[0312] A suspension of 96 (1.40 g, 1.50 mmol) and 10% Pd / C (300 mg) in a mixture of EtOH (120 mL) and AcOH (12 mL) was degassed and then subjected to hydrogenation conditions (1 atm). for 16 hours in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 97, which was then neutralized with NaHCO_{3 a} the crude product was purified by flash chromatography on silica gel (CMA, 80: 18: 2) to give the free amine 97 as a yellow solid (550 mg, 30%, in two steps):

¹1 H NMR (400 MHz, CD 3 OD) 7.74-7.65 (m, 3H), 7.59 (s, 1H), 7.41-7.29 (m, 4H), 7.11 (d, J) = 8.4 Hz, 2H), 4.69 (q, J = 4.9 Hz, 1H), 4.50 (t, J = 7.9 Hz, 1H), 4.04 (dd, J = 10 , 4, 5.2 Hz, 1H), 4.02-3.94 (m, 1H), 3.79-3.71 (m, 1H), 3.70-3.63 (m, 1H), 3.54-3.39 (m, 3H), 3.26- (dd, J = 13.6, 6.8 Hz, 1H), 3.07 (dd, J = 13.1, 8.3 Hz , 1H), 2.79 (t, J = 7.5 Hz, 2H), 2.75-2.67 (m, 2H), 2.55 (t, J = 7.3 Hz, 2H), 1 , 91-1.80 (m, 2H), 1.79-1.69 (m, 2H), 1.62-1.52 (m, 2H), 1.501.37 (m, 12H), 1.33 (s, 9H), 1.25 (d, J = 4.9 Hz, 3H).

Preparation of compound 98;

[0313] To a solution of amine 97 (550 mg, 0.68 mmol) and methyl 3,5-diamino-6-chloropyrazine-2carbonylcarbamimidothioate (13, 423 mg, 0.62 mmol) in EtOH (20 mL) was added DIPEA (1 , 21 mL, 6.80 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, then cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) followed by reverse phase columns (Gold C18) to give guanidine 98 (500 mg, 72%) as a yellow solid: ¹H NMR (400 MHz, CD₃OD) 7.73-7.64 (m, 3H), 7.61 (s, 1H), 7.407.30 (m, 4H), 7.11 (d, J = 8.5 Hz, 2H), 4 , 68 (d, J = 4.9 Hz, 1H), 4.49 (t, J = 7.2 Hz, 1H), 4.04 (dd, J = 10.9, 5.5 Hz, 1H) , 4.02- 3.93 (m, 1H), 3.78-3.70 (m, 1H), 3.69-3.64 (m, 1H), 3.543.38 (m, 4H), 3 , 30-3.20 (m, 2H), 3.15-3.01 (m, 1H), 2.83 (t, J = 7.4 Hz, 2H), 2.54 (t, J = 7 , 3 Hz, 2H), 1.90-1.78 (m, 4H), 1.73-1.64 (m, 2H), 1.53-1.37 (m, 12H), 1.32 ( s, 9H), 1.25 (d, J = 4.9 Hz, 3H).

Preparation of 3,5-diamino-N- (N- (4- (6 - ((S) -2-amino-3-oxo-3- (4- (3 ((2S, 3R, 4R, 5R) HCl salt) -2,3,4,5,6-pentahydroxyhexylamino) propyl) phenylamino) propyl) naphthalen-2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (99) [0314] 4 N HCl in water (20 ml) was added to 98 (500 mg, 0.15 mmol) in ethanol (5.0 ml) and the reaction mixture was stirred at 40 ° C for 2 hours. The solvent was removed, 4N HCl was added again and heated at 40 ° C for a further 2 hours. This addition was repeated two consecutive times. The solvent was removed, purified by a reverse phase column (Gold column) and the residue was lyophilized to yield compound 99 (206 mg,

50%) in the form of a yellow solid: ¹H NMR (400 MHz, DMSO-d₆) 11.0 (br s, 1H), 9.34 (br s, 1H), 9.09-8.25 (m, 6H), 7.82-7.73 (m, 2H), 7.68 (s , 1H), 7.53 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 9.2 Hz, 1H), 7.41 (s, 2H), 7.39 (d , J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H), 5.39 (d, J = 3.7 Hz, 1H), 4.80-4.70 (m, 1 H), 4.62 (d, J = 4.3 Hz, 1H), 4.60-4.54 (m, 1H), 4.46- 4.36 (m,

- 97 2H), 3.96-3.88 (m, 1H), 3.71-3.65 (m, 1H), 3.62-3.54 (m, 1H), 3.51-3, 35 (m, 5H), 3.09 (d, J = 13.3 Hz, 1H), 2.94 (d, J = 10.9 Hz, 1H), 2.87 (t, J = 9.1 Hz, 2H), 2.78 (t, J = 6.7 Hz, 2H), 2.60 (t, J = 7.7 Hz, 2H), 2.00-1.86 (m, 2H), 1.85- 1.67 (m, 2H), 1.65-1.53 ​​(m, 2H).

[0315] ¹1 H NMR (400 MHz, CD 3 OD) 7.80 (d, J = 9.5 Hz, 1H), 7.78-7.73 (m, 2H), 7.68 (s, 1H), 7.45- 7.37 (m, 4H), 7.19 (d, J = 7.1 Hz, 2H), 4.31 (t, J = 6.3 Hz, 1H), 4.09-4.00 (m , 1 H), 3.87-3.81 (m, 1 H), 3.78 (d, J = 11.4 Hz, 1H), 3.73-3.61 (m, 3 H), 3.46 ( dd, J = 13.6, 6.3 Hz, 1H), 3.37 (t, J = 6.8 Hz, 2H), 3.30-3.25 (m, 1H), 3.22-3 , 12 (m, 2H), 3.03 (t, J = 7.9 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H), 2.69 (t, J = 7, 4 Hz, 2H), 2.07-1.95 (m, 2H), 1.91-1.81 (m, 2H), 1.801.69 (m, 2H).

15. Preparation of (S) -3,5-diamino-N- (N- (4- (6- (2-amino-3- (4- (3- (dimethylamino) propyl) phenylamino) -3-oxopropyl) naphthalene 2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (103) [0316]

Scheme 16

<img file="PL2931713T3_D0029.tif" />

Preparation of compound 100;

[0317] A solution of acid 19 (1.75 g, 3.39 mmol) in THF (70 mL) was cooled to 0 ° C in an ice bath, NMM (0.74 mL, 6.78 mmol) was added, and then PivCl (0.41 mL, 3.39 mmol), and the reaction mixture was stirred at the same temperature for 2 hours. 18 (825 mg, 4.61 mmol, 10 mL THF) was added and the reaction mixture was stirred at the same temperature for a further 10 min. The reaction mixture was then brought to room temperature and stirred for 16 hours. The organic solvent was removed. Water was added to this residue and extracted with CH₂cl₂ (3 x 100 ml). The organic layers were combined, dried over Na₂SO₄, filtered and concentrated. ^{The residue was purified by column chromatography} (4% methanol in chloroform) to give amide 100 (1.60 g, 71%) as a slightly yellow solid: ¹H NMR (400 MHz, CDCl₃) 7.87 (s, 1 H), 7.71 (d, J =

- 99.8 Hz, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.65-7.62 (m, 2H), 7.42 (dd, J = 8.4 , 1.9 Hz, 1H), 7.407.29 (m, 5H), 7.22 (d, J = 8.6 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 5.21-5.10 (m, 2H), 5.13 (s, 2H), 4.51 (q, J = 7.6 Hz, 1H), 3.47 (q, J = 6.5 Hz); , 2H), 3.29 (d, J = 6.9 Hz, 2H), 2.68 (t, J = 6.7 Hz, 2H), 2.57 (t, J = 7.9 Hz, 2H); ), 2.26 (ddt, J = 11.5, 9.3, 2.5 Hz, 2H), 2.21 (s, 6H), 2.222.29 (m, 1H), 1.78-1, 69 (m, 3H), 1.39 (s, 9H).

Preparation of compound 101;

[0318] A suspension of 100 (1.60 g, 2.30 mmol) and 10% Pd / C (400 mg) in a mixture of EtOH (130 mL) and AcOH (20 mL) was degassed and then subjected to hydrogenation conditions (1 atm). for 16 hours in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 101 as a yellow solid (1.60 g, 99%):¹H NMR (400 MHz, CD₃OD) 7.71 (d, J = 8.5 Hz, 2H), 7.68 (s, 1H), 7.61 (s, 1H), 7.42 (d, J = 8.5 Hz, 2H). ), 7.39 (dd, J = 8.5, 1.3 Hz, 1H), 7.33 (dd, J = 8.5, 1.3 Hz, 1H), 7.16 (d, J = 8.6 Hz, 2H), 4.50 (t, J = 7.6 Hz, 1H), 3.28 (dd, J = 14.0, 6.3 Hz, 1H), 3.07 (dd, J = 13.3, 8.7 Hz, 1H), 3.05-2.98 (m, 2H), 2.93 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7.3 Hz, 2H), 2.78 (s, 6H), 2.65 (t, J = 7.5 Hz, 2H), 2.06-1.96 (m, 2H), 1.93 (s, 6H), 1.86-1.75 (m, 2H), 1.74-1.64 (m, 2H), 1.33 (s, 9H).

Preparation 102;

[0319] To a solution of amine 101 (1.60 g, 2.30 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (21, 1.60 g, 4.14 mmol) in EtOH (25 mL) DIPEA (4.1 mL, 23.0 mmol) was added at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, then cooled to room temperature and concentrated in vacuo. The residue was purified by silica gel column chromatography (80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 102 (645 mg, 37% and 640 mg, 37% impure) as a yellow solid: ¹1 H NMR (400 MHz, CD 3 OD) 7.70 (dd, J = 9.0, 4.3 Hz, 2H), 7.66 (s, 1H), 7.61 (s, 1H), 7.39- 7.31 (m, 4H), 7.11 (d, J = 8.4 Hz, 2H), 4.48 (t, J = 7.6 Hz, 1H), 3.30-3.22 (m , 3H), 3.06 (dd, J = 13.8, 8.9 Hz, 1H), 2.83 (t, J = 7.2 Hz, 2H), 2.57 (t, J = 7, 9 Hz, 2H), 2.32 (dd, J = 10.5, 7.6 Hz, 2H), 2.23 (s, 6H), 1.86-1.74 (m, 4H), 1, 73- 1.64 (m, 2H), 1.32 (s, 9H).

Preparation of HCl (S) -3,5-diamino-N- (N- (4- (6- (2-amino-3- (4- (3-dimethylamino) propyl) phenylamino) -3-oxopropyl) naphthalene salt 2-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (103) [0320] TFA (10 ml) was added to 47 (545 mg, 0.71 mmol) in CH₂cl₂ (15 ml) and the reaction mixture was stirred at rt for 1 h. The solvent was removed, 1N HCl was added again and the solvent was removed, purified by reverse phase column (Gold column), and the residue was lyophilized to yield compound 48 (206 mg, 50%) as a yellow solid:¹H NMR (400 MHz, DMSO-d₆) 11.02 (br s, 1H), 10.8110.58 (m, 1H), 10.53 (s, 1H), 9.32 (s, 1H), 9.04-8.72 (m, 2H); ), 8.50 (br s, 3H), 7.82-7.73 (m, 3H), 7.68 (s, 1H), 7.53 (d, J = 8.5 Hz, 2H), 7 48 (d, J = 9.2 Hz, 1H), 7.45-7.35 (m, 3H), 7.18 (d, J = 8.4 Hz, 2H), 4.45-4, 35 (m, 1 H), 3.74-3.45 (m, 1H), 3.27 (dd, J = 14.7, 8.3 Hz, 1H),

- 100-3.03-2.93 (m, 2H), 2.78 (t, J = 7.3 Hz, 2H), 2.70 (s, 6H), 2.58 (t, J = 7, 3 Hz, 2H), 2.02-1.88 (m, 2H), 1.79-1.66 (m, 2H), 1.64-1.54 (m, 2H).

[0321] ¹1 H NMR (400 MHz, CD 3 OD) 7.80 (d, J = 9.2 Hz, 1H), 7.78-7.73 (m, 2H), 7.67 (s,

1H), 7.47-7.38 (m, 4H), 7.20 (d, J = 8.9 Hz, 2H), 4.33 (t, J = 7.5 Hz, 1H), 3, 46 (dd, J = 13.6, 6.6 Hz, 1H), 3.37 (t, J = 6.8 Hz, 2H), 3.36-3.26 (m, 3H), 2.87 2.83 (m, 2H), 2.87 (s, 6H), 2.68 (t, J = 7.6 Hz, 2H), 2.07-1.97 (m, 2H), 1, 89- 1.80 (m, 2H), 1.80-1.71 (m, 2H).

16. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (hexyl) (2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (123) [0322]

Diagram 17

- 101 -

<img file="PL2931713T3_D0030.tif" />

<img file="PL2931713T3_D0031.tif" />

<img file="PL2931713T3_D0032.tif" />

Diagram 17 (continued)

- 102 -

<img file="PL2931713T3_D0033.tif" />

Preparation of compound 105;

[0323] To a solution of 104 (100 g, 0.69 mmol) in anhydrous THF (800 mL) was charged NaOH (32.0 mg, 0.809 mmol) and dimethyl sulfate (102 g, 0.809 mmol) dropwise at 0 ° C. . The reaction mixture was stirred for 2 hours. in room temperature. THF was removed under reduced pressure and the mixture was partitioned between CH₂cl₂ (1.0 L) and water (1.0 L). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 1.0 L). The combined organic extracts were dried over Na₂SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, 100% CH₂cl₂) to obtain compound 105 (108.0 g, 90%) as a yellow liquid: ¹H NMR (400 MHz, DMSO-d6): δ7.06 (t, J = 7.85 Hz, 1H), 6.71 (d, J = 7.25, 1H), 6.64 (t, J = 7.7 Hz, 1H), 3.80 (s, 3H), 2.74 (t, J = 2.75 Hz, 2H), 2.65 (t, J = 2.65 Hz, 2H), 1.81-1.71 (m, 4H).

Preparation of compound 106;

[0324] POCl was introduced into a solution of anhydrous DMF (71.45 ml, 0.923 mmol)₃ (57.40 mL, 0.616 mmol) dropwise under a nitrogen atmosphere at 0 ° C. The reaction mixture was stirred

- 103 for 30 min at 0 ° C. A solution of 105 (50.0 g, 0.308 mmol) in dry 1,2-dichloromethane (500 mL) was added to the reaction mixture dropwise under a nitrogen atmosphere at 0 ° C. After the addition was complete, the reaction mixture was heated at 80 ° C for 6 hours. The reaction mixture was quenched with cold H₂O and separated between CH₂cl₂ (1.0 L) and water (1.0 L). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 1.0 L). The combined organic extracts were dried over Na₂SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, 5% EA / hexane) to afford compound 106 (35.0 g, 61%) as a yellow solid:

¹H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 7.65 (d, J = 7.81, 1H), 6.78 (d, J = 7.47 Hz, 1H). ), 3.89 (s, 3H), 3.18 (t, J = 5.80 Hz, 2H), 2.70 (t, J = 4.64 Hz, 2H), 1.82-1.73 (m, 4H).

Preparation of compound 107;

[0325] To a solution of trimethylphosphonoacetate (55.0 mL, 0.378 mmol) in 100 mL of anhydrous CH₂cl₂ cooled to 0 ° C, DBU (58.0 g, 0.380 mmol) was introduced and the mixture was stirred for 15 min. Aldehyde 106 (16.0 g, 0.084 mmol) in 50 mL of CH₂cl₂ added dropwise. The reaction mixture was brought to room temperature, stirred for 16 h, and quenched with 100 mL of water. The mixture was separated and the aqueous layer was extracted with CH₂cl₂ (3 x 150 ml). The combined organic fractions were washed with brine, dried (Na₂SO₄), filtered, and concentrated, and the residue was purified by silica gel column chromatography (10: 1 hexanes / ethyl acetate) to obtain the unsaturated cis &amp; trans-α, β107 (15.0 g, 72%) as a white body DC: ¹H NMR (400 MHz, DMSO-d6): δ 7.83 (d, J = 14.7 Hz, 1H), 7.58 (d, J = 8.3 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.35 (d, J = 15.2 Hz, 1H), 3.80 (s, 3H), 3.70 (s, 3H), 2.76 (t, J = 5.7 Hz, 2H), 2.55 (t, J = 5.4 Hz, 2H), 1.80-1.60 (m, 4H).

Preparation of compound 108;

[0326] A suspension of 107 (33.0 g, 0.134 mmol) and 10% Pd / C (15 g, 0.127) in EtOH (300 mL) was subjected to hydrogenation conditions (1 atm) for 3 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to afford 108 (28.0 g, 90%) as a white solid:¹1 H NMR (400 MHz, CDCl 3): δ 7.65 (d, J = 7.62, 1H), 6.78 (d, J = 7.96 Hz, 1H), 4.06-4.11 (m , 1H), 3.78 (s, 3H), 2.86 (t, J = 7.79 Hz, 2H), 2.69- 2.64 (m, 4H), 2.57-2.51 ( m, 2H), 1.79-1.74 (m, 4H).

Preparation of compound 109;

[0327] To a solution of methyl ester 108 (28.0 g, 0.106 mmol) in THF / MeOH / H₂O (200 ml / 200 ml / 60 ml) was charged with NaOH (25.0 g, 0.625 mmol) and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed and the pH adjusted to 1 with 1 N aqueous HCl; a white precipitate precipitated and was filtered, washed with water and dried under vacuum to give acid 109 (25.5 g, 92%) as a white solid:¹1 H NMR (400 MHz, CDCl 3): δ 6.96 (d, J = 7.29, 1H), 6.63 (d, J = 6.86 Hz, 1H), 3.78 (s, 3H), 2.88 (t, J = 7.29 Hz, 2H), 2.69-2.66 (m, 4H), 2.63-2.59 (m, 2H), 1.801.73 (m, 4H) .

- 104 Preparation of compound 110;

[0328] To a solution of 60 (13.70 g, 77.31 mmol) in anhydrous THF (200 mL) was introduced n-butyllithium (45.07 mL, 90.08 mmol, 2M solution in cyclohexane) dropwise at -78 ° C. and the reaction mixture was stirred for 1 hour. obtaining a solution of lithium salt 61. To another solution of 109 (15.0 g, 64.37 mmol) in anhydrous THF (200 ml) was introduced NMM (9.30 ml, 83.64 mmol) and PivCl (10.30 ml, 83). , 64 mmol) dropwise at -78 ° C. The reaction mixture was stirred for 30 min and heated to -20 ° C for 1 h and the resulting lithium salt solution was added slowly at -78 ° C. The reaction mixture was stirred for another 10 min, brought to 0 ° C and stirred for 1 hour, brought to room temperature and stirred for 30 min, quenched with saturated NH₄Cl, concentrated to remove THF, and partitioned between CH₂cl₂ (300 ml) and water (100 ml). The aqueous layer was separated and extracted with CH₂cl₂ (150 ml). The combined organic extracts were dried over Na₂SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, CH2Cl2) to give compound 110 (15.0 g, 60%) as a white solid.

Preparation of compound 111;

[0329] To a solution of 110 (15.0 g, 38.14 mmol) in anhydrous THF (250 mL) was introduced

KHMDS (13.70 g, 68.67 mmol) in portions at -78 ° C. After stirring the resulting mixture for 30 min, triisopropylbenzenesulfonyl azide (19.0 g, 61.40 mmol) was added and the reaction mixture was stirred for 5 min. Acetic acid (15.0 mL, 228 mmol) and tetramethylammonium acetate (30.9 g, 76.28 mmol) were added slowly at the same temperature. The reaction mixture was heated to 24 ° C, stirred for 16 h, quenched with saturated NaHCO₃ (100 mL), concentrated to remove THF, and extracted with CH₂cl₂ (300 ml). The combined organic extracts were dried over Na₂SO_{4 and} concentrated. The residue was purified by column chromatography (silica gel, 90:10 hexanes / EtOAc and then DCM) to yield compound 111 (8.80 g, 54%) as a yellow solid:¹1 H NMR (400 MHz, CDCl 3): δ 7.36-7.30 (m, 3H), 7.23 (m, 1H), 7.20 (m, 1H), 7.16 (m, 1H), 7.01 (d, J = 7.79 Hz, 1H), 6.60 (d, J = 7.59 Hz, 2H), 5.35 (t, J = 7.99, 2H), 4.89 (s, 1H), 4.58-4.51 (m, 1H), 4.13-4.10 (m, 3H), 3.93 (t, J = 7.54, 1H), 3.77 (s, 3H), 3.33-3.27 (m, 3H), 2.71 (m, 2H), 2.63 (m, 2 H), 1.78-1.75 (m, 5H) 1.58 (m, 2H).

Preparation of compound 112; [0330] To a solution of 111 (31.0 g, 72.1 mmol) in THF / H₂O (300 ml / 100 ml) was charged with H₂ABOUT₂ (49 mL, 433 mmol) followed by LiOH (6.04 g, 144 mmol) in portions at 0 ° C. The reaction mixture was stirred for 10 min at 0 ° C and at room temperature for 1 h, quenched with saturated Na₂SO₃ (200 mL), concentrated under reduced pressure to remove THF, and washed with CH₂cl₂ (500 ml). The aqueous layer was acidified with 1 N aqueous HCl and extracted with CH₂cl₂ (2 x 500 ml). The combined organic extracts were dried over Na₂SO₄, concentrated, and washed with MTBE to afford compound 112 (15.0 g, 82%) as an off-white solid: ¹H NMR (400 MHz, CD3OD): δ 6.92 (d, J = 7.7 Hz, 1H), 6.63 (d, J = 8.0 Hz, 1H), 3.75 (s, 3H) 2.81 (t, J = 7.8

105 Hz, 2H), 2.67 (t, J = 6.0 Hz, 2H), 2.61 (t, J = 5.7 Hz, 2H), 2.49-2.47 (m, 2H). ), 1.84-1.70 (m, 6H).

Preparation of compound 113;

[0331] A suspension of 112 (15.0 g, 55.1 mmol) and 10% Pd / C (3.50 g) in AcOH / H2O (300 mL / 100 mL) was subjected to hydrogenation conditions (1 atm) for 3 hours at room temperature. The reaction mixture was filtered through Celite and washed with AcOH / H₂Oh and then MeOH. The filtrate was concentrated in vacuo to give the acetic salt 113 (14.0 g, 83%) as a yellow solid.

Preparation of compound 114;

[0332] To a solution of 113 (11.0 g, 44.1 mmol) in acetic acid (120 ml) was introduced hydrobromic acid (120 ml) dropwise at room temperature and the reaction mixture was heated under reflux for 3 hours. The reaction mixture was cooled to room temperature and concentrated. The crude brown residue 114 (8.90g, 80%) was used directly in the next step without any purification:¹1 H NMR (400 MHz, CDCl 3): δ 6.80 (d, J = 7.85, 1H), 6.57 (d, J = 7.21 Hz, 1H), 3.92-3.91 (m , 1H), 3.04-2.98 (m, 1H), 2.91-2.86 (m, 1H), 2.61 (m, 2H), 2.54-2.53 (m, 2H); ), 1.69-1.68 (m, 5H).

Preparation of compound 115;

[0333] Acetyl chloride (17.0 mL, 243 mmol) was added to anhydrous methanol (300 mL) at 0 ° C, and 114 (8.90 g, 28.2 mmol) was added. The reaction mixture was heated under reflux for 4 hours. and concentrated. The residue was partitioned between CH₂cl₂ (200 ml) and saturated NaHCO₃ (100 ml). The aqueous layer was separated and extracted with CH₂cl₂ (200 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated to afford compound 115 (7.30 g, 90%) as a white solid: ¹H NMR (400 MHz, CDCl₃): δ 6.81 (d, J = 7.51, 1H), 6.59 (d, J = 7.21 Hz, 1H), 4.12-4.11 (m, 1H), 3.75 (s, 1H), 3.31-3.30 (m, 2H), 2.702.67 (m, 2H), 2.63 (t, J = 6.16 Hz, 2H).

Preparation of compound 116;

[0334] To a solution of 115 (7.30 g, 25.60 mmol) in MeOH / H₂O (100 ml / 60 ml) was charged with NaHCO₃ (12.0 g, 145 mmol) and Boc₂O (10.0 g, 45.8 mmol) at 0 ° C. The resulting mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was partitioned between CH₂cl₂ (100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (100 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated. Flash column chromatography using 20% ​​ethyl acetate / hexanes followed by CH₂cl₂ gave compound 116 (7.1 g, 81%) as a white solid: ¹H NMR (400 MHz, CDCl₃): δ 6.77 (d, J = 7.36, 1H), 6.55 (d, J = 7.86 Hz, 1H), 4.96- 4.94 (m, 1H), 4.71 (s, 1 H), 4.96- 4.94 (m, 1H), 4.71 (s, 1H), 4.50- 4.48 (m, 1H), 3.69 (s, 3H), 3.07-3.01 (m, 1H), 2.89- 2.84 (m, 1H), 2.86 (m, 2H), 2.63 (m, 2H), 1.80-1, 78 (m, 4H), 1.39 (s, 9H).

Preparation of compound 117;

[0335] To a solution of 116 (7.0 g, 20.05 mmol) in CH₂cl₂ (80 ml) was charged with pyridine (100 ml) and triflate (4.64 ml, 24.0 mmol) at 0 ° C, stirred for 1 hour, and stirred at room temperature for 2 hours. After concentration, the reaction mixture was partitioned between CH₂cl₂ (150 ml) and water (70 ml). The aqueous layer was separated and extracted with CH₂cl₂ (100 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄, and concentrated to give compound 117 (8.00 g, 83%) as a brown oil: ¹H NMR (400 MHz, CD3OD): δ 8.81 (d, J = 4.63 Hz, 5H), 8.56-8.5 (m, 2H), 8.0227.99 (m, 4H), 7, 11 (d, J = 7.98 Hz, 1H), 7.03 (d, J = 7.98, 1H), 4.39-4.35 (m, 1H), 3.68 (s, 3H) , 3.19-3.14 (dd, 1H), 2.90-2.77 (m, 5H), 1.86-1.81 (m, 4H), 1.35 (s, 9H), 1 32-1,28 (m, 4H).

Preparation of compound 118;

[0336] Compound 117 (8.0 g, 16.6 mmol) and benzyl but-3-ynylcarbamate (10, 5.00 g, 24.9 mmol) on anhydrous CH₃CN (100 ml) was degassed with argon for 10 min at room temperature and TEA (9.34 ml, 66.50 mmol), 10% (t-Bu) 3 P in hexanes (7.0 ml, 3.32 mmol) and added. CuI (0.16 g, 0.84 mmol). The resulting mixture was degassed with argon for 10 min and Pd (PPh3) 4 (2.00 g, 1.73 mmol) was added rapidly in one portion. After degassing with argon for 5 min, the resulting mixture was heated under reflux for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 75:25 hexanes / ethyl acetate) to yield compound 118 (4.50 g, 52%) as a brown solid:¹H NMR (400 MHz, CD3OD): δ 7.36-7.34 (m, 4H), 7.33-7.29 (m, 2H), 7.16 (d, J = 7.63 Hz, 1H); ), 6.82 (d, J = 7.02 Hz, 1H), 5.12-5.08 (m, 2H), 4.95 (d, J = 7.88 Hz, 1H), 4.52 -4,51 (m, 1H), 3.67 (s, 3H), 3.48-3.34 (m, 2H), 3.10-3.05 (dd, 1H), 2.84-2 83 (m, 2H), 2.68-2.65 (m, 4H), 1.81-1.76 (m, 4H), 1.39 (s, 9H).

Preparation of compound 119;

[0337] To a solution of methyl ester 118 (4.50 g, 8.42 mmol) in THF / MeOH / H₂O (30 ml / 30 ml / 10 ml) was charged with NaOH (3.60 g, 90 mmol) and the reaction mixture was stirred at room temperature for 3 h. The pH was adjusted to 9 1 N aqueous. HCl, and the organic solvent was removed. The pH of the residue was adjusted to 5-6 and the suspension was partitioned between CH₂cl₂ (100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (100 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated to give compound 119 (3.66 g, 85%) as a brown solid: ¹H NMR (400 MHz, CD3OD): δ 7.28-7.24 (m, 5H), 7.05-7.03 (d, J = 7.67 Hz, 1H), 6.89- 6.87 (d, J = 7.55 Hz, 1H), 5.04 (brs, J = 7.02 Hz, 1H), 5.12-5.08 (m, 2H), 4.95 (d, J = 7.88 Hz, 1H), 4.52-4.51 (m, 1H), 3.67 (s, 3H), 3.48-3.34 (m, 2H), 4.27-4.26 (m, 1H), 3.38-3.30 (m, 2H), 3.15-3. 10 (m, 1H), 2.78- 2.71 (m, 4H), 2.59 (d , J = 5.95, 2H), 1.73-1.71 (m, 4H), 1.31 (s, 9H).

Preparation of compound 120;

[0338] To compound 119 (800 mg, 1.53 mmol) in THF (30 mL), DEPBT (845 mg, 2.56 mmol), 24 (700 mg, 2.33 mmol) were sequentially introduced, and DIPEA (1, 0 mL, 4.65 mmol) and stirred at

- 107 at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (50 ml), washed rapidly with saturated aqueous solution (50 ml) and brine (50 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (6% methanol / CH₂^cl2) to give amide 120 (1.0 g^{) in the form of a yellow body} DC: ¹H NMR (400 MHz, CDCl3): δ 7.46-7.4 (m, 3H), 7.36-7.30 (m, 7H), 7.17 (d, J = 7.2 Hz, 2H). ), 7.07 (d, J = 7.5 Hz, 1H), 6.99-6.92 (m, 1 H), 5.49 (s, 1H), 5.10 (s, 2H), 4 35-4,31 (m, 1H), 4.05-3.90 (m, 2H), 3.80-3.82 (m, 1H), 3.75-3.72 (m, 1H) , 3.62 (t, J = 9.9 Hz, 1H), 3.43 (t, J = 5.7 Hz, 2H), 3.18-3.16 (m, 1H), 3.01- 3.08 (m, 1H), 2.83-2.82 (m, 2H), 2.68-2.48 (m, 8H), 1.86- 1.78 (m, 3H), 1, 71- 1.62 (m, 10H), 1.44 (s, 9H), 0.87 (t, J = 6.3 Hz, 3H).

Preparation of compound 121;

[0339] A suspension of 120 (1.00 g, 1.01 mmol) and 10% Pd / C (600 mg) in a mixture of EtOH (50 mL) and AcOH (2 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 12 minutes. h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 121 as a white solid (700 mg, 80%):¹H NMR (400 MHz, CDCl3): δ 7.49-7.4 (m, 2H), 7.34-7.30 (m, 5H), 7.12-6.78 (m, 5H), 4 30-4,27 (m, 2H), 4.19-4.18 (m, 1H), 3.98-391 (m, 2H), 3.78-3.58 (m, 2H), 3 , 19-3,08 (m, 3H), 3.02- 2.89 (m, 6H), 2.75- 2.73 (m, 2H), 2.65- 2.62 (m, 3H) , 2.55-2.52 (m, 3H), 1.98-1.92 (m, 2H), 1.73-1.68 (m, 3H), 1.60-1.52 (m, 7H), 1.41 (s, 9H), 1.29-1.20 (m, 7H), 0.88-0.84 (m, 3H), 0.87 (t, J = 6.4 Hz). , 3H).

Preparation of compound 122;

[0340] To a solution of amine salt 121 (700 mg, 0.81 mmol) and methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate (13, 680 mg, 1.75 mmol) in EtOH (20 mL) was charged DIPEA ( 1.60 mL, 9.26 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 122 (380 g, 48%) as a yellow solid: ¹H NMR (400 MHz, DMSO-d₆): δ 7.43-7.39 (m, 3H), 7.33-7.31 (m, 3H), 7.05 (d, J = 6.69 Hz, 2H), 6.99-6 95 (m, 2H), 6.86 (d, J = 7.59 Hz, 1H), 5.47 (s, 1H), 4.33-4.31 (m, 1H), 4.14- 4.10 (m, 1H), 3.79-3.72 (m, 4H), 3.68-3.65 (m, 2H), 2.69-2.66 (m, 6H), 2, 56-2.53 (m, 3H), 2.45-2.36 (m, 7H), 1.70 (m, 4H), 1.56 (m, 6H), 1.32 (s, 9H) 0.86 (t, J = 7.0 Hz, 3H).

Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3) hexyl (2S, 3R, 4R, 5R) HCl salt -2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -chloropyrazine-2-carboxamide (Compound 123);

[0341] 4 N HCl in dioxane (15 mL) was added to 122 (350 g, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography

108 (Gold column) and the residue was lyophilized to give 110 mg (45%) of compound 123 as a yellow solid: ¹H NMR (400 MHz, DMSO-d₆): δ 10.16 (s, 1H), 9.16 (br s, 1H), 8.513.34 (br s, 2H), 7.41 (t, J = 8.1 Hz, 4H), 7.20 ( d, J = 8.6 Hz, 2H), 6.95-6.89 (q, 2H), 5.42 (br s, 1H), 4.42 (m, 1H), 4.53 (d, J). = 5.3 Hz, 2H), 4.42 (m, 1 H), 4.01 (m, 1H), 3.93 (m, 1H), 3.60 (m, 1H), 3.50-3. 38 (m, 4 H), 3.08-3.03 (m, 6 H), 2.72 (br s, 2 H), 2.66-2.65 (m, 2 H), 2.57 (m, 2 H). ), 1.91-1.90 (m, 2H), 1.72-1.69 (m, 4H), 1.61-1.54 (m, 6H), 1.26 (s, 6H), 0.86 (t, J = 7.0 Hz, 3H).

[0342] ¹H NMR (400 MHz, D2O): δ 7.08 (d, J = 8.4 Hz, 2H), 7.04-6.98 (q, 2H), 6.92 (d, J = 8.3 Hz, 2H), 4.06-4.02 (m, 2H), 3.78-3.69 (m, 3H), 3.62-3.54 (m, 2H), 3.25 (t, J = 5.3 Hz, 1H), 3.19-3.14 (m, 3H), 3.10-3.04 (m, 4H), 2.66-2.54 (m, 7H), 1 , 90-1.86 (m, 2H), 1.65-1.58 (m, 5H), 1.50-1.40 (m, 4H), 1.19-1.18 (m, 6H) 0.78 (t, J = 6.62).

17. Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (127) [0343]

<img file="PL2931713T3_D0034.tif" />

- 109 Preparation of compound 124;

[0344] To compound 119 (1.0 g, 1.92 mmol) in THF (30 mL), DEPBT (845 mg, 2.82 mmol), 29 (1.25 g, 1.91 mmol) were sequentially introduced, and DIPEA (1.0 mL, 5.73 mmol) and stirred at room temperature for 16 h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (50 ml), washed rapidly with saturated aqueous solution (50 ml) and brine (50 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (5% methanol / CH₂cl₂) to give amide 124 [900 mg (mixture)] as a yellow solid.

Preparation of compound 125;

[0345] Slurry 124 [900 mg (mixture), 0.77 mmol] and 10% Pd / C (600 mg) in a mixture of EtOH (50 ml) and AcOH (1.5 ml) were degassed and subjected to hydrogenation conditions (1 atm ) for 12 hours in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give crude 125 (800 mg) as a colorless oil.

Preparation of compound 126;

[0346] A solution of 125 (800 mg) and 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (13, 400 mg, 1.02 mmol) in EtOH (40 ml) was charged with DIPEA (1.10 ml, 6). 38 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 126 (285 mg, 12% in 3 steps) as a yellow solid: ¹H NMR (400 MHz, DMSO-d₆): δ 7.44-7.42 (m, 4H), 7.30-7.28 (m, 6H), 7.22 (d, J = 7.27 Hz, 2H), 6.96 (d , J = 7.11 Hz, 2H), 6.91 (d, J = 7.0 Hz, 2H), 6.86 (d, J = 7.61 Hz, 2H), 5.47 (s, 2H); ), 4.33 (m, 1H), 4.23-4.19 (m, 2H), 3.97-3.91 (m, 4H), 3.84-3.82 (m, 2H), 3.71 (d, J = 2.29 Hz, 1H), 3.69 (d, J = 2.2 Hz, 1H), 3.58 (t, J = 10.08 Hz, 2H), 3, 06-3.00 (m, 1H), 2.91-2.86 (m, 1H), 2.76 (m, 2H), 2.712,68 (m, 4H), 2.61-2.5 ( m, 4H), 2.44-2.35 (m, 4H), 1.74-1.60 (m, 10H), 1.38 (s, 9H).

Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis (2S, 3R, 4R, 5R) 2 HCl salt) , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (Compound 127 ) [0347] 4 N HCl in dioxane (10 mL) was added to 126 (1.15 g, 0.23 mmol) in EtOH (3.0 mL), and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield 62 mg (32%) of compound 127:¹H NMR (400 MHz, DMSO-d6): δ 10.39 (brs, 1H), 10.03 (brs, 1H), 8.91-8.82 (brs, 2H), 8.48 (brs, 2H) ), 7.42 (d, J = 7.6 Hz, 4H), 7.18 (d, J = 7.6 Hz, 2H), 6.96 (d, J = 7.1, 1H), 6 89 (d, J = 7.4, 1H),

- 5.144 (d, J = 10.8, 2H), 4.81 (br, 2H), 4.59 (d, J = 4.2, 2H), 4.55 (d, J = 5 , 4 Hz, 2H), 4.42 (t, J = 4.4, 2H), 4.11 (br, 1H), 4.00 (brs, 2H), 3.69-3.65 (m, 2H), 3.58 (m, 2H), 3.47 (m, 4H), 3.433.39 (m, 4H), 3.25-3.22 (m, 4H), 3.04 (d, J). = 6.3, 2H), 2.73 (m, 2H), 2.64 (m, 2H), 2.58-2.56 (m, 2H), 1.98 (m, 2H), 1, 97 (m, 2H), 1.70-1.67 (m, 4H), 1.61-1.59 (m, 2H), 1.54-1.52 (m,

2H), 1.70-1.67 (m, 4H), 1.61-1.59 (m, 2H), 1.54-1.52 (m, 2H).

[0348] 1 H NMR (400 MHz, D₂O): δ 7.10 (d, J = 8.30 Hz, 2H), 7.02-6.90 (m, 2H), 6.91 (d, J = 7.42 Hz, 2H), 4 , 07-3,92 (m, 5H), 3.77-3.70 (m, 8H), 3.62-3.55 (m, 5H), 4.07-3.95 (m, 5H) , 3.74-3.56 (m, 8H), 3.60-3.55 (m, 5H), 3.30 (d, J = 8.2 Hz, 5H), 3.20-3.16 (m, 7H), 2.60-2.51 (m, 10H), 1.97-1.95 (m, 3H), 1.61-1.59 (m, 7H), 1.49-1 45 (m, 2H).

18. Preparation of 3,5-diamino-N- (4- (4 - ((S) -2-amino-3-oxo-3- (4- (3 - ((2S, 3R, 4R, 5R) 2, 3,4,5,6-pentahydroxyhexylamino) propyl) phenylamino) propyl) -5,6,7,8-tetrahydro-naphthalen-1-yl) butylcarbamoyl) -6-chloropyrazine-2-carboxamide (131) [0349]

<img file="PL2931713T3_D0035.tif" />

Preparation of compound 128;

[0350] To compound 119 (1.00 g, 1.92 mmol) in THF (30 mL), DEPBT (862 mg, 2.88 mmol) was introduced sequentially, 34 (1.50 g, 2.98 mmol). , and DIPEA (1.0 mL, 5.76 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (50 ml), washed rapidly with saturated aqueous solution (30 ml) and brine (20 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (6% methanol / CH₂^cl2) to give amide 128 (780 mg^{, 42%) in the form}^{yellow solid: 1}1 H NMR (400 MHz, CDCl 3): δ 7.49 (m, 3 H), 7.31-7.29 (m, 10 H), 7.007.08 (m, 3 H), 6.94 (d, J = 7 , 4 Hz, 1H), 5.54 (m, 1H), 5.50-5.49 (m, 1H), 5.08 (s, 2H), 4.36 (m, 1H), 4.26 -4.22 (m, 2H), 4.05 (m, 2H), 3.95-3.91 (m, 1H), 3.80 (m, 2H), 3.64-3.59 (m , 1H), 3.52-3.48 (m, 1H), 3.14-3.06 (m, 1H), 2.94-2.89 (m, 1H), 2.79 (d, J = 16.12 Hz, 4H), 2.63 (t, J = 5.98 Hz, 1H), 2.51 (t, J = 6.9 Hz, 1H), 1.82-1.75 (m , 7H), 1.41 (s, 18H).

Preparation of compound 129;

[0351] A suspension of 128 (780 mg, 0.776 mmol) and 10% Pd / C (300 mg) in a mixture of EtOH (30 mL) and AcOH (1.0 mL) was degassed and subjected to hydrogenation conditions (1 atm) for 12 h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo to give the amine salt 129 (720 mg,^85%) in the form of a white solid: ¹H NMR (400 MHz, CDCl₃): δ 7.49-7.46 (m, 2H), 7.32-7.30 (m, 5H), 7.08-7.06 (d, J = 7.2 Hz, 1H), 6 88 (d, J = 7.4 Hz, 1H), 5.53 (s, 1H), 4.34- 4.3,3 (m, 1H), 4.25-4.21 (m, 1H), 4.03-4.02 (m, 1 H), 3.96-3.89 (m, 1H), 3.78-3.76 (m, 1H), 3.71-3.69 (m, 1H); ), 3.60 (t, J = 9.9 Hz, 1H), 3.48-3.46 (m, 1H), 3.09-3.04 (m, 1H), 2.89 (t, J = 7.3 Hz, 3H), 2.79 (m, 2H), 2.69 (m, 2H), 2.58 (t, J = 6.5 Hz, 2H), 2.51 (t, J = 6.8 Hz, 2H), 1.84-1.77 (m, 6H), 1.67-1.66 (m, 2H), 1.61-1.57 (m, 2H), 1 41 (s, 18H).

Preparation of compound 130;

[0352] A solution of the amine salt 129 (720 mg, 0.77 mmol) and methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate (13, 456 mg, 1.17 mmol) in EtOH (20 mL) was charged with DIPEA ( 1.12 ml, 6.24 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give guanidine 130 (380 mg, 45%) as a yellow solid: ¹H NMR (400 MHz, CDCl₃): δ 7.48-7.46 (m, 2H), 7.30 (t, J = 2.70 Hz, 5H), 7.08-7.06 (d, J = 7.6 Hz, 2H); ), 6.93 (d, J = 7.1 Hz, 1H), 6.88 (d, J = 7.3 Hz, 1H), 5.53 (s, 1H), 4.34 (m, 1H); ), 4.25-4.21 (m, 1H), 4.04 (m, 1H), 3.96-3.90 (m, 1H), 3.79 (m, 2H), 3.60 ( t, J = 10.0 Hz, 1H), 3.50-3.46 (m, 1H), 3.25 (t, J = 5.9 Hz, 3H), 3.0073,02 (m, 1H), 2.92-2.87 (m, 1H), 2.77 (m, 2H), 2.69-2.67 (m, 2H), 2.58 (t, J = 6.0 Hz, 2H) 2.48 (t, J = 6.8 Hz, 2H), 1.82-1.74 (m, 6H), 1.67-1.64 (m, 5H), 1.40 (s, 18H); ).

Preparation of 3,5-diamino-N- (4- (4 - ((S) -2-amino-3-oxo-3- (4- (3 ((2S, 3R, 4R, 5R) -2 HCl salt) 3,4,5,6-pentahydroxyhexylamino) propyl) phenylamino) propyl) 5,6,7,8-tetrahydronaphthalen-1-yl) butylcarbamoyl) -6-chloro-pyrazine-2-carboxamide (131);

[0353] 4 N HCl in dioxane (25 mL) was added to 130 (350 mg, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to yield compound 131 (125 mg,

48%) in the form of a yellow solid: ¹H NMR (400 MHz, CD₃OD): δ 7.35 (d, J = 7.6, 2H), 7.18 (d, J = 7.3, 2H), 6.99-6.98 (m, 2H), 4.07 -4.03 (m, 2H), 3.83 (d, J = 1.30, 1H), 3.82 (d, J = 1.40 Hz, 1H), 3.78-3.75 (m). , 1H), 3.68-3.66 (m, 3H), 3.36 (t, J = 6.3, 2H), 3.18-3.15 (m, 4H), 3.04-3 , 00 (m, 2H), 2.76 (t, J = 5.3 Hz, 2H), 2.69-2.61 (m, 5H), 2.00-1.97 (m, 2H), 1.771.73 (m, 5H), 1.69-1.65 (m, 3H).

[0354] ¹1 H NMR (400 MHz, D2O): δ 10.46 (s, 1H), 9.31 (br, 1H), 8.55 (br, 4H), 7.45 (d, J = 6.6, 4H); ), 7.20 (d, J = 7.62 Hz, 2H), 7.00 (d, J = 6.6, 1H), 6.93 (d, J = 6.6 Hz, 1H), 5 43 (d, J = 3.8 Hz, 1H), 4.79 (d, J = 5.38 1 H), 4.64-4.63 (m, 2H), 4.46 (t, J = 4.9 Hz, 1H), 4.15 (t, J = 4.6 Hz, 1H), 3.96-3.94 (m, 1H), 3.71 (m, 1H), 3.64- 3.61 (m, 1H), 3.51-3.45 (m, 3H), 2.96- 2.92 (m, 3H), 2.78-2.77 (m, 2H), 2, 68-2.65 (m, 2H), 2.62 (t, J = 6.6 Hz, 2H), 1.95-1.94 (m, 2H), 1.76-1.15 (m, 8H).

19. Preparation of (S) -3,5-Diamino-N- (N- (4- (4- (2-amino-3- (4- (3 (dimethylamino) propyl) phenylamino) -3-oxopropyl) -5 , 6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (135) [0355]

Scheme 20

- 113 -

<img file="PL2931713T3_D0036.tif" />

Preparation of compound 132;

[0356] To compound 119 (700 mg, 1.34 mmol) in THF (30 mL) sequentially DEPBT (600 mg, 2.00 mmol), 18 (360 mg, 1.51 mmol) were introduced, and DIPEA (0, 80 ml, 4.03 mmol) and stirred at room temperature for 16 hours. After removing the solvent under reduced pressure, the residue was dissolved in CH₂cl₂ (50 ml), washed rapidly with saturated aqueous solution (50 ml) and brine (50 ml), and dried over Na₂SO₄. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (6% methanol / CH₂cl₂) to give amide 132 [800 mg (mixture)] as a yellow solid product: ¹H NMR (400 MHz, DMSO-d₆): δ 8.13 (d, J = 7.54 Hz, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.89-7.85 (m, 1H), 7, 71 (t, J = 7.52 Hz, 1H), 7.64-7.59 (m, 2H), 7.44 (d, J = 7.7 Hz, 2H), 7.33-7.30 (m, 6H), 7.12-7.06 (m, 3H), 7.0 (d, J = 7.6 Hz, 1H), 5.02 (s, 2H), 2.70 (m, 4H), 2.63-2.61 (m, 5H), 2.45 (m, 5H), 1.83 (s, 6H), 1.69-1.65 (m, 3H), 1.33 (s, 9H).

Preparation of compound 133;

[0357] Suspension 132 [800 mg (mixture), 1.01 mmol] and 10% Pd / C (350 mg) in the mixture

EtOH (30 ml) and AcOH (1 ml) were degassed and subjected to hydrogenation conditions (1 atm)

- 114 for 12 hours in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo and purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to obtain compound 233 (500 mg, 67% in 2 steps) as a yellow solid: ¹1 H NMR (400 MHz,

DMSO-d6): δ 7.31 (d, J = 7.54 Hz, 2H), 7.12 (d, J = 7.1 Hz, 2H), 6.93 (d, J = 7.2 Hz). , 1H), 6.88 (d, J = 6.8 Hz, 1H), 4.32 (m, 1H), 3.08-3.03 (m, 1H), 2.91-2.86 ( m, 1H), 2.77-2.76 (m, 4H), 2.69 (m, 2H), 2.60-2.5 (m, 4H), 2.35-2.3 (m, 2H), 1.82 (s, 6H), 1.58-1.57 (m, 4H), 1.40 (s, 9H).

Preparation of compound 134;

[0358] To a solution of amine salt 133 (500 mg, 0.90 mmol) and methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate (13, 530 mg, 1.36 mmol) in EtOH (20 mL) was charged with DIPEA ( 1.30 mL, 7.25 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 80: 18: 2 CHCl 3 / CH 3 OH / NH 4 OH) to give guanidine 134 (285 mg, 42%) as a yellow solid:¹H NMR (400 MHz, DMSO-d₆): δ 7.29 (d, J = 7.5 Hz, 2H), 7.10 (d, J = 8.1 Hz, 2H), 6.94-6.87 (m, 2H), 4, 33 (m, 1H), 3.27-3.24 (m, 2H), 3.07-3.00 (m, 1H), 2.92-2.87 (m, 1H), 2.76 (mp); m, 2H), 2.70 (m, 2H), 2.61-2.54 (m, 4H), 2.352.31 (m, 2H), 2.22 (s, 6H), 1.80-1 72 (m, 5H), 1.69-1.62 (m, 4 H), 1.39 (s, 9 H).

Preparation of HCl (S) -3,5-diamino-N- (N- (4- (4- (2-amino-3- (4- (3- (dimethylamino) propyl) phenylamino) -3-oxopropyl) salt , 6,7,8-tetrahydronaphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide - Compound 135;

[0359] 4 N HCl in dioxane (10 mL) was added to 134 (380 g, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (C18 Column Gold) and the residue was lyophilized to afford compound 135 (125 mg, 49%) as a yellow solid:

¹1 H NMR (400 MHz, DMSO-d6): δ 10.69 (br s, 1H), 10.54-10.50 (d, J = 16.7 Hz, 2H), 9.32 (t, J = 4 , 8 Hz, 1H), 8.96 (br s, 1H), 8.86 (br s, 1H), 8.58 (brs, 3H), 7.42 (d, J = 8.0 Hz, 4H), 7.18 (d, J = 8.2 Hz, 2H), 6.96 (d, J = 7.3 Hz, 1H), 6.88 (d, J = 7.4 Hz, 1H), 4, 15 (t, J = 4.4 Hz, 1H), 3.36-3.32 (m, 2H), 3.09-3.06 (m, 2H), 3.00-2.95 (m, 2H), 2.74-2.73 (m, 1H), 2.22 (s, 6H), 2.64 (m, 2H), 2.57-2.56 (m, 2H), 1.94 -1.90 (m, 2H), 1.70-1.67 (m, 3H), 1.62-1.58 (m, 2H), 1.54-1.52 (m, 2H).

[0360] ¹H NMR (400 MHz, D2O): δ 7.08 (d, J = 7.7 Hz, 2H), 7.00-6.97 (q, 2H), 6.91 (d, J = 8.1 Hz, 2H), 4.12-4.08 (q, 1H), 3.25 (t, J = 5.2 Hz, 3H), 3.21-3.17 (m, 1H), 3.10. (t, J = 9.8 Hz, 1H), 3.0-2.96 (m, 2H), 2.77 (s, 6H), 2.60-2.58 (m, 5H), 2, 50-2.50 (m, 4H), 1.91-1.87 (m, 2H), 1.60-1.58 (m, 6H), 1.45-1.43 (m, 2H).

20. Preparation of (S) -2-amino-3- (4- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) -5,6,7,8-tetrahydronaphthalene -1-yl) propane (139)

- 115 [0361]

<img file="PL2931713T3_D0037.tif" />

Preparation of compound 136;

[0362] Suspension 118 (800 mg, 1.49 mmol) and 10% Pd / C (350 mg) in a mixture of EtOH (50 mL) and AcOH (1.0 mL) were degassed and hydrogenated (1 atm) for 12 minutes. h. in room temperature. The reaction mixture was filtered through a Celite plug and the plug was washed with MeOH. The filtrate was concentrated in vacuo and purified by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give compound 136 (700 mg, 93%) as a yellow solid.

Preparation of compound 137;

[0363] To a solution of the amine salt 136 (700 mg, 1.50 mmol) and methyl 3,5-diamino-6-chloropyrazin-2-carbonylcarbamimidothioate (13.880 mg, 2.26 mmol) in EtOH (30 mL) was charged with DIPEA ( 2.15 ml, 12.03 mmol) at room temperature. The reaction mixture was heated at 70 ° C in a sealed tube for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified using

Column chromatography (silica gel, 80: 18: 2 ^CHCl3^{/ CH}3^{OH / NH}4^{OH) obtaining} guanidine 137 (560 mg, 60%) as a yellow solid: ¹H NMR (400 MHz, CD₃FROM); ^{δ 6.95-6.85} (m, 2H), 4.32-4.28 (m, 1H), 3.72-3.67 (m, 2H), 3.34 (m, 3H), 3.22-3.16 (m , 2H), 3.08-3.03 (m, 1H), 2.73 (m, 4H), 2.62 (t, J = 7.0 Hz, 1H), 1.81- 1.78 ( m, 4H), 1.74-1.72 (m, 2H), 1.68-1.60 (m, 2H), 1.36 (s, 9H), 1.34 (s, 5H).

Preparation of compound 138;

[0364] To a solution of methyl ester 137 (560 mg, 0.907 mmol) in THF / MeOH / H₂O (30 ml / 30 ml / 10 ml) was charged with NaOH (3.60 g, 7.25 mmol) and the reaction mixture was stirred at room temperature for 3 h. The pH was adjusted to 9 1 N aqueous. HCl, and the organic solvent was removed. The pH of the residue was adjusted to 5-6 and the suspension was partitioned between CH₂cl₂ (100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (100 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated to give compound 138 (420 mg, 78%) as a brown solid:

¹H NMR (400 MHz, DMSO-d6); δ 6.93 (d, J = 6.7 Hz, 1H), 6.84 (d, J = 7.35 Hz, 1H), 6.70 (s, 3H), 3.93 (m, 1H); , 3.16 (m, 5H), 2.98- 2.94 (m, 1H), 2.74-2.64 (m, 6H), 1.70 (m, 5H), 1.55 (m , 5H), 1.31 (s, 9H), 1.16-1.06 (m, 2H).

Preparation of H (S) -2-amino-3- (4- (4- (3- (3,5-diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) -5,6,7,8-tetrahydronaphthalene salt HCl salt -1-yl) propane - Compound

139;

[0365] 4 N HCl in dioxane (10 mL) was added to 138 (420 mg, 0.69 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 hours.

The solvent was removed, the mixture was purified by reverse phase chromatography (C18 column Gold), and the residue was lyophilized to yield compound 139 as a yellow solid (200 mg, 49%):

¹H NMR (400 MHz, DMSO-d6); δ 10.56 (br s, 1H), 9.36 (t, J = 4.7 Hz, 1H), 8.9-8.8 (brs, 2H), 6.98-6.93 (m, 2H); ), 3.95-3.92 (m, 2H), 3.38-3.35 (m, 2H), 3.04 (d, J = 7.0 Hz, 2H), 2.67-2, 66 (m, 4 H), 2.56 - 2.55 (m, 2 H), 1.72-1.70 (m, 4 H), 1.63-1.56 (m, 4 H).

[0366] ¹H NMR (400 MHz, D2O); δ 7.43 (br s, 2H), 6.94 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 7.1

Hz, 1H), 3.41 (t, J = 6.0 Hz, 2H), 3.28-3.26 (m, 4H), 3.11 (d, 1H), 3.08 (m, 1H); ), 2.66- 2.64 (m, 6H), 1.67-1.57 (m, 8H).

21. Chiral synthesis of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) 2,3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (33) [0367]

Scheme 22

- 117 -

<img file="PL2931713T3_D0038.tif" />

Preparation of compound 141;

[0368] To a solution of 1-naphthol (140, 10.0 g, 69.4 mmol) in acetonitrile (70.0 mL), several portions of NBS (142, 12.3 g, 69.4 mmol) were added over a period of 30 min. . The resulting mixture was stirred at room temperature for 4 h, concentrated in vacuo then water (200 ml) and ethyl acetate (200 ml) were added. The aqueous layer was separated and extracted with ethyl acetate (2 x 200 mL). The combined organic extracts were washed with brine, dried

- 118 ^{over Na}2^SO4 ^{and concentrated.} The residue was purified by column chromatography (silica gel, 4: 1 hexanes / EtOAc) to afford the desired compound 141 (9.50 g, 61%) as a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.49 (s, 1H), 8.20 (dd, J = 8.3, 0.5 Hz, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.66 (dd, J = 8.4, 1.4 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.55 (ddd, J = 8.2, 7.7, 1 , 1 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H).

Preparation of compound 7;

[0369] Zinc dust (7.03 g, 107.6 mmol) was added to the flame-dried, nitrogen-ejected side neck of a round bottom flask. Anhydrous DMF (50.0 mL) was added by syringe, followed by the addition of a catalytic amount of iodine (1.00 g, 3.94 mmol). The color change of the obtained mixture was observed, from colorless to yellow, and back to colorless. Protected iodoalanine 143 (11.8 g, 35.9 mmol) was added in one portion followed by a catalytic amount of iodine (1.00 g, 3.94 mmol) and stirred at room temperature for 30 min; the successful introduction of zinc was accompanied by a mild exothermic reaction. The organosyl reagent solution was allowed to cool to room temperature before the addition of Pd₂cares₃ (821 mg, 0.89 mmol), SPhos (736 mg, 1.79 mmol) and aryl bromide 141 (8.00 g, 35.9 mmol) and the mixture heated at 50 ° C for 16 h, under positive nitrogen pressure. The reaction mixture was allowed to cool to room temperature. Saturated NH solution was added₄Cl (300 mL) and EtOAc (300 mL), then the mixture was filtered through Celite and washed with EtOAc (100 mL). The aqueous layer was separated and extracted with EtOAc (2 x 300 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexanes / EtOAc) to afford the desired compound 7 (4.60 g, 37%) as a yellow solid:

¹H NMR (400 MHz, CDCl₃, a mixture of rotamers) δ 8.23 ​​(d, J = 8.3 Hz, 1H), 7.99 (d, J =

8.6 Hz, 1H), 7.54 (t, J = 8.04 Hz, 1H), 7.48 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7, 08 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 6.57 (br s, 0.2 H), 6.45 (br s, 0.2H), 5.91 (br s, 0.65 H), 5.05 (d, J = 7.7 Hz, 0.75H), 4.89 (br s, 0.25H), 4, 68 (q, J = 6.8 Hz, 0.7H), 4.56 (br s, 0.2H), 3.73 (s, 0.7H), 3.62 (s, 2.3 H) , 3.49 (dd, J = 14.0, 5.9 Hz, 0.8H), 3.89 (dd, J = 14.0, 7.2 Hz, 0.7H), 3.05 (br s, 0.2H), 1.39 (s, 7.5H), 1.09 (s, 2.5H).

Preparation of compound 9;

[0370] To a solution of compound 7 (7.60 g, 21.8 mmol) in CH₂cl₂ (150 ml) was added pyridine (18.0 ml) and Tf₂O (9.19 g, 32.6 mmol) at 0 ° C. The resulting mixture was stirred at room temperature for 2 h, concentrated in vacuo and partitioned between CH₂cl₂(100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 50 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to give compound 9 (11.0 g, crude) as a brown oil. The crude product was used directly in the next step without further purification:¹H NMR (400 MHz, CDCl₃, a mixture of rotamers) δ 8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 5.12-5.06 (br s, 1H), 4.78- 4.67 (m, 1H), 3.68-3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s, 1H).

- Compound 11;

[0371] A solution of compound 9 (11.0 g, 21.8 mmol) and benzyl 10-butylcarbamate (6.56 g,

32.6 mmol) in anhydrous acetonitrile (100 ml) was degassed for 10 min under argon followed by TEA (11.9 ml, 87.0 mmol), 10% (t-Bu).₃P in hexanes (8.80 mL, 4.35 mmol) and CuI (207 mg, 1.08 mmol) at room temperature. ^{The resulting mixture}degassed with argon for a further 10 min and Pd (PPh 3) 4 (2.51 g, 2.17 mmol) was added in ^{one serving}. After degassing with argon for 5 min, the resulting mixture was heated under reflux for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2: 3 hexanes / EtOAc) to give compound 11 (7.00 g, 61% in two steps) as a brown oil:¹1 H NMR (400 MHz, CDCl 3, mixture of rotamers) δ 8.33 (dd, J = 8.9, 1.9 Hz, 1H), 8.07 (dd, J = 9.0,

1.7 Hz, 1H), 7.59-7.49 (m, 3H), 7.39-7.27 (m, 5H), 7.19 (d, J = 7.3 Hz, 1H), 5, 24-5.16 (m, 1H), 5.12 (s, 2H), 5.08- 4.99 (m, 1H), 4.69 (q, J = 6.7 Hz, 1H), 3 59 (s, 3H), 3.57-3.40 (m, 4H), 2.79 (t, J = 6.4 Hz, 2H), 1.39 (s, 7.5 H), 1 , 11 (s, 1.5 H).

Preparation of compound 17;

[0372] To a solution of methyl ester 11 (7.00 g, 13.2 mmol) in THF (200 mL), methanol (200 mL) and water (75.0 mL) was added solid NaOH (16.0 g, 79, 2 mmol). The resulting mixture was stirred at room temperature for 1 hour. until it was shown by TLC that the reaction was complete. 1 N Hydrochloric acid was added to adjust the pH of the reaction mixture to 10. After concentration, water (100 mL) was added and the pH was adjusted to 5-6. The resulting precipitate was extracted with CH2Cl2 (2 x 250 mL). The organic layers were combined, dried over Na₂SO₄, filtered, concentrated and triturated with MTBE to afford compound 17 (5.00 g, 75%) as a white solid: ¹H NMR (400 MHz, CD₃FROM; mixture of rotamers) δ 8.33 (d, J = 8.2 Hz, 1H), 8.28-8.20 (m, 1H), 7.59-7.45 (m, 3H), 7.38- 7.21 (m, 6H), 5.09 (s, 2H), 4.55- 4.45 (m, 1H), 3.76-3.66 (m, 1H), 3.44 (t, J = 6.7 Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J = 6.7 Hz, 2H), 1.29 (s, 6H), 0, 82 (s, 3H).

Preparation of compound 30;

[0373] To a solution of compound 17 (4.60 g, 8.91 mmol) in THF (160 mL) was added sequentially with T₃P (50% in ethyl acetate, 10.7 mL) and NMM (4.89 mL, 44.5 mmol). After stirring at room temperature for 10 min, amine 29 (6.11 g, 9.33 mmol) was added and the reaction mixture was stirred at room temperature for 16 h. After removal of the solvent, the residue was dissolved in CH₂cl₂ (100 ml), washed rapidly with saturated NH₄Cl, saturated NaHCO_{3 and} with brine, dried over Na₂SO₄ and concentrated. The residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH) to give the amide 30 (6.60 g, 64%) as an off-white solid: ¹H NMR (400 MHz, CDCl₃) δ 8.33 (dd, J = 9.0, 1.7 Hz, 1H), 8.17 (d, J = 7.3 Hz, 1H), 7.62-7.47 (m, 4H) , 7.42 (dd, J = 7.7, 4.1 Hz, 4H), 7.37-7.28 (m, 11H), 7.09-6.95 (m, 4H), 5.46 (s, 2 H), 5.33 (br s, 1 H), 5.22 (t, J =

5.8 Hz, 1H), 5.11 (s, 2H), 4.63-4.51 (m, 1H), 4.27 (dd, J = 10.8, 5.4 Hz, 2H), 4, 02-3.84 (m, 6 H), 3.71 (t, J = 4.5 Hz, 6H impurity), 3.57 (t, J = 10.6 Hz, 2H), 3.54-3, 45 (m, 4H), 2.82-2.60 (m, 6H), 2.59-2.45 (m, 3H), 2.44-2.36 (m, 4H), 1.82- 1.69 (m, 2H), 1.38 (s, 9H).

- Compound 31;

[0374] Suspension 30 (7.26 g, 6.20 mmol) and 10% Pd / C (1.50 g) in EtOH / AcOH (240 mL / 40.0 mL) was degassed by bubbling through a syringe for 10 minutes. min and then subjected to hydrogenation conditions (1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and triturated with MTBE to give amine salt 31 (7.06 g, 98%) as a brown solid:¹H NMR (400 MHz, CD3OD, mixture of rotamers) δ 8.24 (dd, J = 7.2, 2.0 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1H), 7, 59-7.22 (m, 2H), 7.49-7.41 (m, 4H), 7.39-7.22 (m, 10H), 6.95 (d, J = 8.5 Hz, 2H), 5.51 (s, 2H), 4.55 (t, J = 7.2 Hz, 1H), 4.24 (dd, J = 10.7, 5.4 Hz, 2H), 4, 19-4.10 (m, 2H), 3.99-3.88 (m, 4H), 3.83-3.73 (m, 8H, impurities), 3.61 (t, J = 10.5 , Hz, 2H), 3.59-3.52 (m, 1H), 3.45-3.36 (m, 1H), 3.19-3.02 (m, 4H), 2.93-2 , 81 (m, 8H), 2.542,39 (m, 2H), 1.95 (s, 6H), 1.88- 1.80 (m, 2H), 1.80-1.65 (m, 4H), 1.36 (s, 7H), 1.09 (s, 2H).

Preparation [0375] To a solution of 31 (7.06 g, 6.18 mmol) in EtOH (50.0 mL) was added DIPEA (8.80 mL, 49.4 mmol) followed by 3,5-diamino-6. methyl chloropyrazine-2-carbonylcarbamimidothioate (13, 3.84 g, 9.88 mmol) at room temperature. The reaction mixture was heated at 70 ° C for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified twice by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give the compound ^{32 (2.50 g, 33%) in}form of a yellow solid: ¹H NMR (400 MHz, CD₃OD, mixture of rotamers) δ 8.22 (d, J = 9.3 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.56-7.47 (m, 4H) , 7.43 (dd, J = 7.4, 3.6 Hz, 4H), 7.33-7.14 (m, 10H), 6.94 (d, J = 8.0 Hz, 2H), 5.47 (s, 2H), 4.53 (t, J = 7.7 Hz, 1H), 4.22 (dd, J = 10.8, 5.4 Hz, 2H), 3.99-3 89 (m, 4H), 3.84 (dd, J = 5.5, 2.3 Hz, 2H), 3.70 (dd, J = 9.2, 2.2 Hz, 2H), 3, 59 (t, J = 10.8 Hz, 2H), 3.54-3.46 (m, 1 H), 3.47-3.38 (m, 1H), 3.22 (t, J = 6, 4 Hz, 2H), 3.11-3.02 (m, 2H), 2.70 (dd, J = 13.5, 4.6 Hz, 2H), 2.61 (dd, J = 13.6 , 8.9, 2H), 2.572.47 (m, 2H), 2.46-2.34 (m, 2H), 1.84-1.73 (m, 2H), 1.72-1.61 (m, 4H), 1.36 (s, 7H), 1.12 (s, 2H).

Preparation of 3,5-diamino-N- (N- (4- (4 - ((S) -2-amino-3- (4- (3- (bis (2S, 3R, 4R, 5R) 2 HCl salt) , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (33) [0376] To a solution of 32 (2, 50 g, 2.02 mmol) in EtOH (30.0 mL) was added 4 N hydrochloric acid (80.0 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed, purified by reversed phase column and lyophilized to yield compound 33 (1.82 g, 85%) as a yellow hygroscopic solid:¹1 H NMR (400 MHz, DMSO-d 6) δ 10.61 (s, 1H), 10.59 (s, 1H), 9.41 (t, J = 5.2 Hz,

H), 9.01 (br s, 1 H), 8.96 (br s, 1 H), 8.81 (br s, 2 H), 8.77 (br s, 2 H), 8.44-8.37 (m, 1 H), 8.168, 10 (m, 1H), 7.61-7.5 (m, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.27 (d, J = 7.3 Hz, 1H), 7.17 (d, J = 8.5 Hz, 2H), 4.28 (q, J = 7.4 Hz, 1H), 4.09-3.99 (m, 2H), 3.75-3.65

121 (m, 3H), 3.58 (dd, J = 11.0, 2.6 Hz, 2H), 3.55-3.31 (m, 10H), 3.30-3.13 (m , 4H), 3.32-3.00 (m, 2H), 2.63-2.53 (m, 2H), 2.05-1.92 (m, 2H), 1.78-1.61 (m, 4H).

[0377] 1H NMR (400 MHz, CD₃OD): δ 9.25 (t, J = 5.9 Hz, 0.5H), 8.26-8.21 (m, 1H), 8.1178.12 (m, 1H), 7.60-7, 54 m, 2H), 7.38 (d, J = 7.2 Hz, 1H), 7.32 (d, J = 7.2 Hz, 1H), 7.25 (d, J =

8.6 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 4.31 (t, J = 8.1 Hz, 1H), 4.21-4.14 (m, 1H), 4.13-4.08 (m, 1H), 3.85-3.80 (m, 2H), 3.79 (d, J = 2.9 Hz, 1H), 3.76 (d , J = 3.2 Hz, 1H), 3.73-3.62 (m, 8H), 3.51-3.34 (m, 8H), 3.15 (t, J = 6.8 Hz, 2H), 2.73-2.57 (m, 2H), 2.15-1.98 (m, 2H), 1.911,73 (m, 4H).

22. Preparation of (2R, 2'R, 3R, 3'R, 4R, 4'R, 5S, 5'S) -6,6 '- (3- (4aminophenyl) propylazanediyl) dihexane-1,2,3,4, 5-pentaol (29) [0378]

<img file="PL2931713T3_D0039.tif" />

Preparation of compound 145;

[0379] To a solution of compound 144 (8.80 g, 154.1 mmol) in CH₂cl₂ (150 ml) TEA (32.2 ml, 231.2 mmol) and Boc were added₂O (40.4 g, 185.3 mmol) at 0 ° C. Stirring of the reaction mixture was continued at 0 ° C for 0.5 h, allowed to warm to room temperature and stirred for 5 h. The mixture was then partitioned between CH₂cl₂ (150 ml) and water (150 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 150 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄, concentrated to give the desired compound 145 (22.0 g, 91%) as a colorless oil. ¹1 H NMR (400 MHz, CDCl 3): δ 5.90-5.77 (m, 1H), 5.17 (dq, J = 17.1, 1.7 Hz, 1H), 5.10 (dq, J = 10.4, 1.4 Hz, 1H), 4.64 (brs, 1H), 3.74 (t, J = 5.2 Hz, 2H), 1.45 (s, 9H).

- 122 Preparation of compound 147;

[0380] To a solution of compound 145 (14.0 g, 89.12 mmol) in anhydrous THF (150 mL) was added 9-BBN (0.5 M in THF, 270 mL, 133.8 mmol) under argon. After finishing 2 hours stirring the reaction mixture at room temperature, compound 146 (17.7 g, 71.3 mmol), Pd (PPh₃)₂cl₂ (3.12 g, 4.45 mmol) and 1 N aqueous. NaOH (150 mL) was added at room temperature. The resulting mixture was stirred for an additional 1 hour. After removal of the solvent, the residue was partitioned between EtOAc (200 mL) and water (200 mL). The aqueous layer was separated and extracted with EtOAc (2 x 200 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum.

The crude product was purified by column chromatography (silica gel, 4: 1 hexanes / EtOAc) to yield compound 147 (8.00 g, 43%) as a brown solid: ¹H

NMR (400 MHz, CDCl3) δ 8.14 (d, J = 8.9 Hz, 2H), 7.34 (d, J = 8.9 Hz, 2H), 4.56 (br s, 1H), 3.17 (q, J = 6.2 Hz, 2H), 2.75 (t, J = 7.7 Hz, 2H), 1.89-1.79 (m, 2H), 1.44 (s , 9H).

Preparation of compound 148;

[0381] Compound 147 (8.00 g, 28.6) was dissolved in 4 N HCl in dioxane (50.0 mL) at room temperature and the solution was stirred for 1 h. The reaction mixture was concentrated in vacuo and the residue triturated with MTBE to yield compound 148 (4.00 g, 65%) as a brown solid:¹H NMR (400 MHz, CD₃OD) δ 8.19 (d, J = 8.7 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 2.98 (t, J = 7.4 Hz, 2H) 2.86 (t, J = 7.6 Hz, 2H), 2.07-1.97 (m, 2H).

Preparation of compound 150;

[0382] To a solution of compound 148 (4.00 g, 18.5 mmol) and triol 149 (24.8 g, 92.5 mmol) in

MeOH (150 mL) was added AcOH (11.1 mL, 185 mmol) and the reaction mixture was stirred at room temperature for 10 min. After adding NaCNBH₃ (5.83 g, 92.5 mmol) was continued stirring the solution at room temperature for 24 h. Additional compound 149 (4.0 eq.), AcOH (4.0 eq.) And NaCNBH₃ (4.0 eq.) Was added over 4 days.

Then hexanal (2.0 eq.), AcOH (2.0 eq.) And NaCNBH were added₃ (2.0 eq.).

The solution was further stirred at room temperature for 1 hour. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} partition the residue between EtOAc (200 mL) and water (200 mL). The aqueous layer was separated and extracted

CH2Cl2 (2 x 300 ml). The combined organic extracts were dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9: 1 CH2Cl2 / MeOH, 80: 18: 2 CHCl3 / MeOH / NH4OH) to give compound 150 (6.50 g, 52%) as an off-white solid. An additional 4.00 g of material from the contaminated fractions was isolated and purified by reversed phase column to give 1.50 g (12%) of pure compound 150 (total 7.70 g, 64%):¹H NMR (400 MHz, CD₃OD) δ

8.03 (d, J = 8.7 Hz, 2H), 7.50-7.41 (m, 4H), 7.35-7.23 (m, 8H), 5.48 (s, 2H) , 4.22 (dd, J =

10.6, 5.3 Hz, 2H), 3.99-3.91 (m, 4H), 3.85 (dd, J = 5.5, 2.4 Hz, 2H), 3.70 (dd) , J = 9.5, 2.4 Hz,

2H), 3.59 (t, J = 10.6 Hz, 2H), 2.73 (dd, J = 13.6, 4.5 Hz, 2H), 2.67-2.50 (m, 6H). ), 1.83-1.71 (m, 2H).

- 123 Preparation of (2R, 2'R, 3R, 3'R, 4R, 4'R, 5S, 5'S) -6,6 '- (3- (4-aminophenyl) propylazanishiyl) dihexane-1, 2,3, 4,5-pentaol (Compound 153);

[0383] A suspension of Compound 150 (6.50 g, 9.50 mmol) and 10% Pd / C (1.30 g) in EtOH (150 mL) was degassed by bubbling an argon with a syringe for 10 min, followed by stirring in room temperature in the atmosphere of hydrogen (balloon, 1 atm) for 6 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to yield 153 (6.01 g, 97%) as an off-white solid:¹H NMR (400 MHz, CD3OD): δ 7.49-7.42 (m, 4H), 7.35-7.26 (m, 6H), 6.82 (d, J = 8.4 Hz, 2H); ), 6.60 (d, J = 8.4 Hz, 2H), 5.48 (s, 2H), 4.22 (dd, J = 10.8, 5.9 Hz, 2H), 3.983.89. (m, 4H), 3.83 (dd, J = 5.7, 2.3 Hz, 2H), 3.69 (dd, J = 13.2, 3.4 Hz, 2H), 3.62- 3.55 (m, 3H), 2.71 (dd, J = 13.2, 3.4 Hz, 2H), 2.65-2.48 (m, 3H), 2.45-2.2 ( m, 2H), 1.74-1.63 (m, 2H).

23. Preparation of 3,5-diamino-N- (N- (4- (4 - ((R) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) 2 , 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (152) [0384]

Sc hemat 24

- 124 -

<img file="PL2931713T3_D0040.tif" />

Preparation of compound 14 A few portions of NBS (142, 12.3 g, 69.4 mmol) were added to a solution of 1-naphthol (1, 10.0 g, 69.4 mmol) in acetonitrile (70.0 mL) for 30 min. The resulting mixture was stirred at room temperature for 4 h, concentrated in vacuo then water (200 ml) and ethyl acetate (200 ml) were added. The aqueous layer was separated and extracted with ethyl acetate (2 x 200 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated. The residue was purified by crystallization (heptane / EtOAc).

125 to give the desired compound 14 (6.0 g, 39%) as a white solid. ¹H NMR (400 MHz, DMSO-d6): δ 10.49 (s, 1H), 8.20 (dd, J = 8.3, 0.5 Hz, 1H), 8.02 (d, J = 8) , 3 Hz, 1H), 7.66 (dd, J = 8.4, 1.4 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.55 (ddd, J = 8.2, 7.7, 1.1 Hz, 1H),

6.83 (d, J = 8.2 Hz, 1H).

Preparation of compound 145 [0386] Zinc dust (4.76 g, 72.9 mmol) was added to the flame-dried, nitrogen-ejected side neck of a round bottom flask. Anhydrous DMF (25.0 mL) was added by syringe, followed by the addition of a catalytic amount of iodine (677 mg, 2.67 mmol). The color change of the obtained mixture was observed, from colorless to yellow, and back to colorless. Protected iodoalanine 114 (8.00 g, 24.3 mmol) was added in one portion followed by a catalytic amount of iodine (677 mg, 2.67 mmol) and stirred at room temperature for 30 min; the successful introduction of zinc was accompanied by a mild exothermic reaction. The organozinc reagent solution was allowed to cool to room temperature before addition of Pd2 (dba)₃ (556 mg, 0.60 mmol), SPhos (498 mg, 1.21 mmol), and aryl bromide 14 (5.40 g, 24.3 mmol) and the mixture heated at 50 ° C for 16 h, under positive nitrogen pressure. The reaction mixture was allowed to cool to room temperature. Saturated NH solution was added₄Cl (300 mL) and EtOAc (300 mL), then the mixture was filtered through Celite and washed with EtOAc (100 mL). The aqueous layer was separated and extracted with EtOAc (2 x 300 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexanes / EtOAc) to afford the desired compound 145 (3.10 g, 37%) as a yellow solid.

¹H NMR (400 MHz, CDCl₃, a mixture of rotamers): δ 8.23 ​​(d, J = 8.3 Hz, 1H), 7.99 (d, J =

8.6 Hz, 1H), 7.54 (t, J = 8.04 Hz, 1H), 7.48 (ddd, J = 8.3, 6.9, 1.3 Hz, 1H), 7, 08 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 5.98 (brs, 0.3H), 5.59 (br s, 0, 7H), 5.03 (d, J = 7.7 Hz, 0.85H),

4.84 (br s, 0.15H), 4.68 (q, J = 6.8 Hz, 1H), 3.76-3.68 (m, 1H), 3.62 (s, 3H), 3, 54-3.33 (m, 2H), 1.39 (s, 7H), 1.09 (s, 2H).

Preparation of compound 146 [0387] To a solution of compound 145 (3.07 g, 8.90 mmol) in CH₂cl₂ (75.0 mL) was added pyridine (7.25 mL, 88.9 mmol) and Tf₂O (2.24 ml, 13.3 mmol) at 0 ° C. The resulting mixture was stirred at room temperature for 2 h, concentrated in vacuo and partitioned between CH₂cl₂ (100 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 50 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated to give compound 146 (4.20 g, crude) as a brown oil. The crude product was used directly in the next step without further purification.¹1 H NMR (400 MHz, CDCl 3, mixture of rotamers): δ 8.19-8.07 (m, 2H), 7.69-7.64 (m, 2H), 7.38 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 7.9 Hz, 1H), 5.12-5.06 (br s, 1H), 4.78-4.67 (m, 1H), 3, 68-3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s, 1H).

Preparation of compound 147

[0388] A solution of compound 6 (4.20 g, 8.80 mmol, crude) and benzyl 7-butyl-3-ylcarbamate (2.65 g, 13.2 mmol) in anhydrous acetonitrile (50.0 mL) was degassed. for 10 min under argon, then TEA (4.81 ml, 35.2 mmol) was added, 10% (t-Bu)₃P in hexanes (3.56 mL, 1.76 mmol) and CuI (84 mg, 0.44 mmol) at room temperature. The resulting mixture was degassed with argon for a further 10 min and Pd (PPh₃)₄^{(1.01 g,}0.88 mmol) was added in one portion. After degassing with argon for 5 min, the resulting mixture was heated under reflux for 18 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2: 3 hexanes / EtOAc) to yield compound 147 (3.20 g, 67% in two steps) as a brown oil.¹H NMR (400 MHz, CDCl₃, a mixture of rotamers): δ 8.33 (dd, J = 8.9, 1.9 Hz, 1H), 8.07 (dd, J = 9.0, 1.7 Hz, 1H), 7.59- 7.49 (m, 3H), 7.39-7.27 (m, 5H), 7.19 (d, J = 7.3 Hz, 1H), 5.24-5.16 (m, 1H). , 5.12 (s, 2H), 5.08- 4.99 (m, 1H), 4.69 (q, J = 6.7 Hz, 1H), 3.59 (s, 3H), 3, 57-3.40 (m, 4H), 2.79 (t, J = 6.4 Hz, 2H), 1.39 (s, 7.5 H), 1.11 (s, 1.5 H) .

Preparation of compound 148 [0389] To a solution of methyl ester 147 (3.10 g, 5.84 mmol) in THF (60 mL), methanol (60 mL) and water (20.0 mL) was added solid NaOH (1.40 g). 35.09 mmol). The resulting mixture was stirred at room temperature for 2 hours. until it was shown by TLC that the reaction was complete. 1 N Hydrochloric acid was added to adjust the pH of the reaction mixture to 10. After concentration, water (100 mL) was added and the pH was adjusted to 5-6. The resulting precipitate was extracted with CH2Cl2 (2 x 200 mL). The organic layers were combined, dried over Na₂SO₄, filtered, concentrated and triturated with MTBE to afford compound 148 (3.00 g, 99%) as a white solid. ¹H NMR (400 MHz, CD₃OD, mixture of rotamers): δ 8.33 (d, J = 8.2 Hz, 1H), 8.28-8.20 (m, 1H), 7.59-7.45 (m, 3H), 7 38-7.21 (m, 6H), 5.09 (s, 2H), 4.55-4.45 (m, 1H), 3.76-3.66 (m, 1H), 3.44 (t, J = 6.7 Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J = 6.7 Hz, 2H), 1.29 (s, 6H) 0.82 (s, 3H).

Preparation of compound 149 [0390] To a solution of compound 148 (800 mg, 1.55 mmol) in THF (30 ml) was added in turn T₃P (50% in ethyl acetate, 1.86 ml) and NMM (0.85 ml, 7.75 mmol). After stirring at room temperature for 10 min, amine 29 (1.01 g, 1.55 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. After removal of the solvent, the residue was dissolved in CH₂cl₂ (100 ml), washed rapidly with saturated NH₄Cl, saturated NaHCO_{3 and} brine, ^{dried over Na}2^SO4 ^{and concentrated.}The residue was purified by column chromatography (silica gel, 9: 1 CH2Cl2 / MeOH) to afford amide 149 (1.20 g, 67%) as an off-white solid.

¹1 H NMR (400 MHz, CDCl 3): δ 8.35 (d, J = 8.0, 1.7 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 7.607.5 ( m, 2H), 7.50 (d, J = 7.3 Hz, 2H), 7.45-7.39 (m, 5H), 7.37-7.28 (m, 11H), 7.08 -6.96 (m,

3H), 5.47 (s, 2H), 5.33-5.17 (m, 2H), 5.12 (s, 2H), 4.59- 4.48 (m, 1H), 4.29 (dd, J = 10.8, 5.4

Hz, 2H), 4.07-4.00 (m, 2H), 3.99-3.91 (m, 4H), 3.78-3.68 (m, 3H), 3.59 (t, J = 10.6 Hz, 2H),

- 127 3.55-3.46 (m, 4H), 2.95- 2.82 (m, 2H), 2.81- 2.69 (m, 4H), 2.68-2.5 (m , 1H), 2.56-2.44 (m, 3H), 2.43-2.38 (m, 1H), 1.85-1.69 (m, 2H), 1.38 (s, 9H). ).

Preparation of compound 150 [0391] A suspension of 149 (1.15 g, 1.00 mmol) and 10% Pd / C (230 mg) in EtOH / AcOH (80.0 mL / 20.0 mL) was degassed by passing argon using syringes for 10 min and then subjected to hydrogenation conditions (1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and triturated with MTBE to give the amine salt 150 (1.12 g, 97%) as a brown solid.¹H NMR (400 MHz, CD₃OD, mixture of rotamers): δ 8.25 (dd, J = 7.2, 2.0 Hz, 1H), 8.09 (d, J = 7.0 Hz, 1H), 7.59-7.51 (m, 2H), 7.48-7.41 (m, 4H), 7.37-7.21 (m, 10H), 6.94 (d, J = 8.5 Hz, 2H), 5, 52 (s, 2H), 4.54 (t, J = 7.2 Hz, 1H), 4.24 (dd, J = 10.7, 5.4 Hz, 2H), 4.16- 4.08 (m, 2H), 3.97-3.88 (m, 4H), 3.75-3.70 (m, 2H), 3.62 (t, J =

10.5, Hz, 2H), 3.60-3.51 (m, 1H), 3.28-3.15 (m, 2H), 3.14-2.95 (m, 4H), 2, 89 (t, J = 7.4 Hz, 2H), 2.73-2.79 (m, 1H), 2.54-2.39 (m, 2H), 1.95 (s, 6H), 1 88-1,64 (m, 8H), 1.36 (s, 7.5H), 1.09 (s, 1.5H).

Preparation 151 [0392] To a solution of 150 (1.05 g, 0.92 mmol) in EtOH (15.0 mL) was added DIPEA (1.30 mL, 7.35 mmol) followed by 3,5-diamino-6- methyl chloropyrazine-2-carbonylcarbamimidothioate (13, 573 mg, 1.47 mmol) at room temperature. The reaction mixture was heated at 70 ° C for 2 hours, cooled to room temperature and concentrated in vacuo. The residue was purified twice by column chromatography (silica gel, 80: 18: 2 CHCl₃/ CH₃OH / NH₄OH) to give the compound ^{151 (410 mg, 36%) in} form of a yellow solid: ¹H NMR (400 MHz, CD3OD, mixture of rotamers): δ 8.22 (d, J = 8.4 Hz, 1H), 8.09 (d, J = 8.2 Hz, 1H), 7.56-7 48 (m, 2H), 7.47-7.40 (m, 4H), 7.33-7.25 (m, 6H), 7.22 (d, J = 7.5 Hz, 2H), 7.16 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 8.1 Hz, 2H), 5.47 (s, 2H), 4.53 (t, J = 8 , 1 Hz, 1H), 4.22 (dd, J = 10.8, 5.4 Hz, 2H), 3.99-3.89 (m, 4H), 3.84 (dd, J =

5.5, 2.1 Hz, 2H), 3.70 (dd, J = 9.1, 2.0 Hz, 2H), 3.59 (t, J = 10.8 Hz, 2H), 3, 53-3.47 (m, 1H), 3.46-3.39 (m, 1H), 3.26-3.17 (m, 2H), 3.12-3.04 (m, 2H), 2.70 (dd, J = 13.2, 4.0 Hz, 2H), 2.60 (dd, J = 13.0, 8.2, 2H), 2.57-2.49 (m, 2H); ), 2.47-2. 33 (m, 2H), 1.84-1.73 (m, 2H), 1.72-1.61 (m, 4H), 1.37 (s, 7H), 1.12 (s, 2H).

Synthesis of 3,5-diamino-N- (N- (4- (4 - ((R) -2-amino-3- (4- (3- (bis ((2S, 3R, 4R, 5R) -2, 3,4,5,6-pentahydroxyhexyl) amino) propyl) phenylamino) -3-oxopropyl) naphthalen-1-yl) butyl) carbamimidoyl) -6-chloropyrazine-2-carboxamide (152) [0393] To a solution of 151 (480 mg, 0.42 mmol) in EtOH (5.0 mL) was added 4 N hydrochloric acid (25.0 mL). The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed, purified by reversed phase column and lyophilized to yield compound 152 (300 mg, 71%) as a yellow hygroscopic solid:¹H NMR (400 MHz, DMSO-d6): δ 10.57 (brs, 1H), 10.55 (brss, 1H), 9.35 (t, J = 6.0

Hz, 1H), 9.04-8.84 (m, 2H), 8.81-8.66 (m, 4H), 8.42-8.36 (m, 1H), 8.16-8, 10 (m, 1H), 7.61- 128 7.53 (m, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.35 (d, J = 7.5 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 9.0 Hz, 2H), 4.32- 4.23 (m, 1H), 4, 08-3.97 (m, 2H), 3.75-3.30 (m, 13H), 3.29-3.15 (m, 4H), 3.14-2.97 (m, 2H), 2.64-2.53 (m, 2H), 2.05-1.92 (m, 2H), 1.79-1.60 (m, 4H).

[0394] ¹1 H NMR (400 MHz, CD 3 OD): δ 8.25-8.21 (m, 1H), 8.18-8.13 (m, 1H), 7.59-7.53 (m, 2H), 7 38 (d, J = 7.3 Hz, 1H), 7.32 (d, J = 7.3 Hz, 1H), 7.26 (d, J = 8.8 Hz, 2H), 7.15 (d, J =

8.5 Hz, 2H), 4.30 (t, J = 7.3 Hz, 1H), 4.20-4.14 (m, 1H), 4.13-4.08 (m, 1H), 3.84-3.80 (m, 2H), 3.79-3.75 (m, 2H), 3.72-3.61 (m, 8H), 3.51-3.34 (m, 8H). ), 3.15 (t, J = 7.3 Hz, 2H), 2.742,58 (m, 2H), 2.13-1.98 (m, 2H), 1.91-1.73 (m, 4H).

[0395] HRMS calculated for C₄₄H₆₄CLN₁₀ABOUT₁₂ [M + Na]⁺, 959.418 and found 959.4394.

24. Preparation of intermediate 18 [0396]

<img file="PL2931713T3_D0041.tif" />

Preparation of compound 155 [0397] To a solution of compound 154 (500 mg, 9.00 mmol) in CH₂cl₂ (50 mL) TEA (1.63 mL, 11.7 mmol) and Boc were added₂O (2.16 g, 9.90 mmol) at 0 ° C. Stirring of the reaction mixture was continued at 0 ° C for 0.5 h, allowed to warm to room temperature and stirred for 3 h. The mixture was then partitioned between CH 2 Cl 2 (50 mL) and water (50 mL). The aqueous layer was separated and extracted with CH 2 Cl 2 (2 x 50 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄, concentrated, the residue was purified by column chromatography (silica gel, 2: 3 hexanes / EtOAc) to afford the desired compound 155 (1.20 g, 86%) as a colorless

- 129 oil. ¹1 H NMR (300 MHz, CDCl 3): δ 4.70 (br s, 1H), 3.91 (dd, J = 5.3, 2.2 Hz, 2H), 2.21 (t, J = 2, 7 Hz, 1H), 1.45 (s, 9H).

Preparation of compound 157 A solution of compound 155 (1.00 g, 6.45 mmol) and 156 (1.30 g, 6.45 mmol) in anhydrous THF (15 mL) was degassed for 10 min under argon then TEA (3.53 mL, 25.8 mmol) was added, PPh₃ (424 mg, 1.61 mmol) and CuI (246 mg, 1.29 mmol) at room temperature. The resulting mixture was degassed with argon for a further 10 min and Pd (PPh₃)₄(7.45 g, 6.45 mmol) was added in one portion. After degassing with argon for 5 min, the resulting mixture was heated under reflux for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2: 3 hexanes / EtOAc) to yield compound 157 (750 mg, 42%) as a brown oil.¹1 H NMR (400 MHz, CDCl 3): δ 8.17 (d, J = 9.2 Hz, 2H), 7.55 (d, J = 9.2 Hz, 2H), 4.79 (br s, 1H) , 4.18 (d, J = 6.0 Hz, 2H), 1.47 (s, 9H).

Preparation of compound 158 [0399] Compound 157 (2.00 g, 7.24) was dissolved in 4 N HCl in dioxane (20.0 mL) at room temperature, and the solution was stirred for 2 h. The reaction mixture was concentrated in vacuo and the residue was triturated with MTBE to yield compound 158 (1.25 g, 82%) as a brown solid.¹H NMR (300 MHz, CD3OD): δ 8.26 (d, J = 9.2 Hz, 2H), 7.72 (d, J = 9.2 Hz, 2H), 4.09 (s, 2H) .

Preparation of compound 159 [0400] To a solution of compound 158 (100 mg, 0.47 mmol) and a solution of formaldehyde in water (30%, 1.40 mL, 1.41 mmol) in MeOH (3.0 mL) was added AcOH (0). , 09 mL, 1.41 mmol) and the reaction mixture was stirred at room temperature for 30 min. After adding NaCNBH₃ (88 mg, 1.41 mmol) was continued stirring the solution at room temperature for 16 hours. An additional solution of formaldehyde in water (30%, 0.92 mL, 0.94 mmol), AcOH (0.09 mL, 1.41 mmol) and NaCNBH3 (88 mg, 1.41 mmol) was added and mixed for a further 16 hours. . After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} partition between EtOAc (30 mL) and water (30 mL). The aqueous layer was separated and extracted with CH 2 Cl 2 (2 x 40 mL). The combined organic extracts were dried over Na 2 SO 4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9: 1 CH2Cl2 / MeOH, 80: 18: 2 CHCl3 / MeOH / NH4OH) to yield compound 159 (50 g, 52%) as an off-white oil.¹H NMR (300 MHz, CD3OD): ^{δ 8.17 (d,} J = 9.0 Hz, 2H), 7.57 (d, J = 9.0 Hz, 2H), 3.50 (s, 2H), 2.37 (s, 6H).

Preparation of compound 18 [0401] A suspension of compound 159 (100 mg, 0.49 mmol) and 10% Pd / C (40 mg) in MeOH (3.0 mL) was degassed with argon for 10 min, followed by stirring under a hydrogen atmosphere (balloon , atm) for 3 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and triturated with CH₂cl₂/ hexane

- 130 yielding 18 (48 mg, 55%) in the form of white crystals: ¹H NMR (300 MHz, CDCl₃): δ 6.96 (d, J = 8.3 Hz, 2H), 6.60 (d, J = 8.3 Hz, 2H), 3.47 (br s, 2H), 2.53 (t , J = 7.8 Hz, 2H), 2.26 (dd, J = 8.7, 7.2 Hz, 2H), 2.22 (s, 6H), 1.77-1.67 (m, 2H).

Preparation of intermediate 29 [0402]

Scheme 26

<img file="PL2931713T3_D0042.tif" />

Preparation of compound 161 [0403] A solution of compound 158 (4.00 g, 18.9 mmol) and triol 160 (11.7 g, 56.6 mmol) in MeOH (50 mL) was added with AcOH (3.40 mL, 56). 6 mmol) and the reaction mixture was stirred at room temperature for 30 min. After adding NaCNBH₃ (3.55 g, 56.6 mmol) was continued stirring the solution at room temperature for 16 hours. Additional compound 160 (11.7 g, 56.6 mmol), AcOH (3.40 mL, 56.6 mmol) and NaCNBH were added.₃ (3.55 g, 56.6 mmol) and stirring was continued at room temperature for 16 h. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} separated ^residue between CH₂cl₂ (10 ml) and water (10 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 10 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (silica gel, 9: 1 CH 2 Cl 2 / MeOH, 80: 18: 2 CHCl 3 / MeOH / NH 4 OH) to give compound 29 (700 mg, 7.0%) as an off-white solid.¹H NMR (300 MHz, CD3OD): ^{δ 8.21}(d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 4.68 (q, J = 5.1 Hz, 2H), 4.04 (dd) , J = 10.8, 5.4 Hz, 2H), 3.99-3.93 (m, 2H), 3.86-3.74 (m, 6H), 3.54 (dd, J = 9 , 8, 2.3 Hz, 2H), 3.36 (t, J = 10.7 Hz, 2H), 2.87 (dd, J = 13.3, 4.9 Hz, 2H), 2.74 (dd, J = 13.3, 7.8 Hz, 2H), 1.25 (d, J = 5.1 Hz, 6H).

Preparation of compound 29 [0404] A suspension of compound 161 (500 mg, 0.90 mmol) and 10% Pd (OH)₂A / C (215 mg) in EtOH (230 ml) was degassed by bubbling an argon using a syringe for 10 min, then stirred under a hydrogen atmosphere (balloon, 1 atm) for 2 h. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and the residue was purified by column chromatography (gel

Silica, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2 CHCl 3 / MeOH / NH 4 OH) to give compound 29 (264 mg, 55%) as an off-white solid. ¹H NMR (400 MHz, CD3OD): δ 6.97 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.6 Hz, 2H), 4.71 (q, J = 5.1 Hz, 2H), 4.06 (dd, J = 10.6, 5.3 Hz, 2H), 4.13-4.05 (m, 2H), 3.81 (dd, J = 5 , 0. 2.3 Hz, 2H), 3.80-3.72 (m, 2H), 3.51 (dd, J =

9.6, 2.4 Hz, 2H), 3.33-3.23 (m, 2H), 3.38 (t, J = 10.7 Hz, 2H), 2.83-2.54 (m , 6H), 1.85-1.69 (m, 2H), 1.26 (d, J = 5.1 Hz, 6H).

Preparation of intermediate 24 [0405]

<img file="PL2931713T3_D0043.tif" />

Preparation of compound 162 [0406] A solution of compound 158 (200 mg, 0.94 mmol) and triol 160 (194 mg, 0.94 mmol) in MeOH (2.0 mL) was added with AcOH (0.17 mL, 2.82). mmol) and the reaction mixture was stirred at room temperature for 30 min. After adding NaCNBH₃ (148 mg, 2.35 mmol) was continued stirring the solution at room temperature for 16 hours. Additional compound 160 (0.2 eq.), AcOH (3.0 eq.) And NaCNBH were added₃ (1.0 eq.) And stirring was continued at room temperature for 16 hours. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} the residue was separated between CH₂cl₂ (10 ml) and water (10 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 10 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (silica gel, 9: 1 CH2Cl2 / MeOH, 80: 18: 2 CHCl3 / MeOH / NH4OH) to give compound 162 (95 mg, 28%) as an off-white solid.¹1 H NMR (400 MHz, CD 3 OD): δ 8.24 (d, J = 9.1 Hz, ^{2H), 7.69} (d, J = 9.1 Hz, 2H), 4.70 (q, J = 5.1 Hz, 1H), 4.09-4.02 (m, 2H), 4.00 (d, J = 2.1 Hz, 2H), 3.83 (dd, J = 5.1, 2.3 Hz, 1H), 3.81-3.71 (m, 1H), 3.53 (dd, J = 9 , 3, 2.3 Hz, 1H), 3.38 (t, J = 11.0 Hz, 1H), 3.21-3.07 (m, 2H), 1.25 (d, J = 5, 1 Hz, 3H).

[0407] Preparation of compound 164

To a solution of compound 162 (95 mg, 0.26 mmol) and hexanal 163 (52 mg, 0.51 mmol) was added AcOH (0.05 mL, 0.78 mmol) and NaCNBH.₃ (41 mg, 0.65mmol). ^{The solution was stirred}

- 132 ^{in room temperature} for 16 hours After removal of the solvent, the residue was neutralized with saturated NaHCO 3 and separated^residue between EtOAc (10 mL) and water (10 mL). The aqueous layer was separated and extracted with CH 2 Cl₂ (2 x 10 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2 CHCl₃/ MeOH / NH₄OH) to give compound 164 (70 mg, 59%) as an off-white solid. ¹1 H NMR (400 MHz, CDCl 3): δ 8.18 (d, J = 8.9 Hz, 2H), 7.56 (d, J = 8.9 Hz, ^2H); 4.70 (q, J = 5.0 Hz, 1H), 4.15 (dd, J = 10.4, 5.2 Hz, 1H), 4.01-3.89 (m, 2H), 3 , 83 (dd, J = 3.8, 2.7 Hz, 1H), 3.77 (br s, 1 H), 3.70 (br s, 1 H), 3.64 (t, J = 6.2 Hz, 1 H), 3.56 (dd, J = 9.2, 4.0 Hz, 1H), 3.41 (t, J = 10.8 Hz, 1H), 2.87 (dd, J = 13.2 , 4.3 Hz, 1H), 2.78-2.68 (m, 2H), 2.63-2.5 (m, 1H), 1.75-1.43 (m, 4H), 1, 34 (d, J = 5.0 Hz, 3H), 1.32-1.25 (m, 6H), 0.89 (t, J =

6.6 Hz, 3H).

Preparation of compound 24 [0408] A suspension of compound 164 (1.70 g, 3.77 mmol) and 10% Pd / C (200 mg) in MeOH (40 ml) was degassed with argon for 10 min, followed by stirring under a hydrogen atmosphere (balloon). , 1 atm) for 2 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 9: 1 CH 2 Cl 2 / MeOH, 80: 18: 2 CHCl).₃/ MeOH / NH₄OH) to give compound 24 (1.20 g, 76%) as an off-white solid. ¹H NMR (300 MHz, CDCl3): δ 6.96 (d, J = 8.9 Hz, 2H), 6.62 (d, J = 8.9 Hz, 2H),

4.68 (q, J = 5.0 Hz, 1H), 4.14 (dd, J = 11.0, 5.5 Hz, 1H), 3.92-3.81 (m, 2H), 3, 72 (dd, J = 3.8, 2.4 Hz, 1H), 3.50 (dd, J = 9.1, 4.0 Hz, 1H), 3.40 (t, J = 10.5 Hz). , 1H), 2.76-2.38 (m, 10H), 1.81- 1.64 (m, 3H), 1.48-1.36 (m, 2H), 1.33 (d, J = 5.0 Hz, 3H), 1.30-1.20 (m, 6H), 0.88 (t, J =

6.6 Hz, 3H).

Preparation of intermediate 85 [0409]

<img file="PL2931713T3_D0044.tif" />

- 133 Preparation of compound 166 [0410] To a solution of compound 148 (4.60 g, 21.3 mmol) and triol 165 (17.1 g, 63.9 mmol) in MeOH (100 mL) was added AcOH (12.1 mL). 63.9 mmol) and the reaction mixture was stirred at room temperature for 10 min. After adding NaCNBH₃ (4.00 g, 63.9 mmol) was continued stirring the solution at room temperature for 6 hours. Then hexanal 163 (5.10 mL, 42.6 mmol) and NaCNBH were added₃ (2.60 g, 42.6 mmol). ^Solutionwas further stirred at room temperature for 2 hours. After removal of the solvent, the residue was neutralized with saturated NaHCO 3 and separated^residue between EtOAc (200 mL) and water (200 mL). The aqueous layer was separated and extracted with CH 2 Cl₂ (2 x 300 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2 CHCl₃/ MeOH / NH₄OH) to give compound 166 (6.90 g, 64%) as an off-white solid. ¹H NMR (400 MHz, CD₃OD): δ 8.12 (d, J = 8.6 Hz, 2H), 7.517.43 (m, 2H), 7.38 (d, J = 8.6 Hz, 2H), 7.37-7 27 (m, 3H), 5.55 (s, 1H), 4.24 (dd, J = 11.5, 5.5 Hz, 1H), 4.18-4.01 (m, 1H), 4.00-3.94 (m, 1H), 3.93-3.89 (m, 1H), 3.77 (dd, J = 9.3, 1.8 Hz, 1H), 3.61 ( t, J = 10.7 Hz, 1H), 3.13-2.77 (m, 6H), 2.71 (t, J = 7.5 Hz, 2H), 1.99-1.85 (m , 2H), 1.55-1.42 (m, 2H), 1.38-1.18 (m, 6H), 0.87 (t, J = 7.0 Hz, 3H).

Preparation of compound 85 A suspension of compound 166 (800 mg, 1.55 mmol) and 10% Pd / C (300 mg) in EtOH (40 ml) was degassed by passing argon using a syringe for 10 min, followed by stirring at room temperature. room temperature in a hydrogen atmosphere (balloon, 1 atm) for 2 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to afford 85 (700 mg, 93%) as an off-white solid.¹H NMR (400 MHz, CD3OD): δ 7.52-7.42 (m, 2H), 7.38-7.25 (m, 3H), 6.88 (d, J = 8.4 Hz, 2H); ), 6.63 (d, J = 8.4 Hz, 2H), 5.53 (s, 1H), 4.24 (dd, J = 10.8, 5.5 Hz, 1H), 4.053.84 (m, 3H), 3.76 (dd, J = 9.6, 1.8 Hz, 1H), 3.61 (t, J = 10.8 Hz, 1H), 2.93 (dd, J = 13.6, 5.0 Hz, 1H), 2.79 (dd, J = 13.4, 9.0 Hz, 1H), 2.73-2.60 (m, 4H), 2.42 (t , J = 8.0 Hz, 2H), 1.88-1.68 (m, 2H), 1.48-1.36 (m, 2H), 1.33-1.14 (m, 6H), 0.87 (t, J = 7.0 Hz, 3H).

Preparation of intermediate 34 [0412]

<img file="PL2931713T3_D0045.tif" />

- Preparation of compound 168 [0413] A solution of saturated NaHCO was introduced into a solution of 162 (534 mg, 1.45 mmol) in MeOH (30 mL).₃ in water (5.0 ml) at 0 ° C and stirred for 10 min. Then (Boc) was added₂O (350 mg, 1.60 mmol) and the reaction mixture was stirred for 3 hours. at the same temperature, brought to room temperature, and stirred for another 30 min. The mixture was concentrated, the residue was dissolved in CH₂cl₂ (100 mL), and the solution was washed with water (100 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄, filtered, concentrated and the residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 8: 2 CHCl₃/ MeOH) to obtain compound 168 (435 mg,

64%) in the form of a greyish white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.18 (d, J = 8.7 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 4.72 (q, J = 5.1 Hz, 1H) , 4.41-4.35 (m, 2H), 4.16 (dd, J = 10.8, 5.5 Hz, 1H), 4.15-4.04 (m, 1H), 3.93 -3.83 (m, 1H), 3.81-3.76 (m, 1H), 3.66-3.53 (m, 4H), 3.40 (t, J = 11.0 Hz, 1H); ), 3.25-3.12 (m, 1H), 3.08- 2.96 (m, 1H), 1.49 (s, 9H), 1.32 (d, J = 5.1 Hz, 3H).

Preparation of compound 34 A suspension of compound 168 (80 mg, 0.21 mmol) and 10% Pd / C (40 mg) in EtOH (10 ml) was degassed by bubbling argon with a syringe for 10 min, followed by stirring under an atmosphere. hydrogen (balloon, 1 atm) for 2 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 34 (82 mg, 89%) as an off-white solid.¹H NMR (400 MHz, CDCl3): δ 6.96 (d, J = 8.1 Hz, 2H), 6.62 (d, J = 8.1 Hz, 2H), 4.69 (q, J = 5.1 Hz, 1H), 4.15 (dd, J = 10.8, 5.5 Hz, 1H), 4.13-4.09 (m, 1H), 4.01-3.93 (m , 1H), 3.89-3.78 (m, 1H), 3.75-3.68 (m, 1H), 3.62-3.43 (m, 4H), 3.40 (t, J = 11.3 Hz, 1H), 3.35 (dd, J = 13.5, 4.0 Hz, 1H), 3.26 (t, J = 7.9 Hz, 1H), 3.23-3 13 (m, 1H), 2.48 (t, J = 7.8 Hz, 2H), 1.86-1.76 (m, 2H), 1.43 (s, 9H), 1.33 ( d, J = 5.1 Hz, 3H).

Preparation of intermediate 171 [0415]

Scheme 30

- 135 -

<img file="PL2931713T3_D0046.tif" />

[0416] To a solution of compound 148 (6.40 g, 29.6 mmol) and triol 165 (11.9 g, 44.5 mmol) in MeOH (300 mL) was added AcOH (5.32 mL, 88.8 mmol). ) and the reaction mixture was stirred at room temperature for 30 min. After adding NaCNBH₃ (3.73 g, 59.2 mmol) was continued stirring the solution at room temperature for 16 h. Additional compound 165 (11.9 g, 44.5 mmol), AcOH (5.32 mL, 88.8 mmol) and NaCNBH were added.₃ (3.73 g, 59.2 mmol), stirring was continued at room temperature for 14 h. Additional compound 165 (7.93 g, 29.6 mmol), AcOH (3.55 mL, 59.2 mmol) and NaCNBH were added.₃ (2.80 g, 44.4 mmol), stirring was continued at room temperature for 10 hours. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} distributed between CH₂cl₂ (100 ml) and water (100 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 100 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. Difficult purification was carried out by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2

CHCl₃/ MeOH / NH₄OH) to give compound 150 and 169 (20 g, a mixture). The mixture was used directly in the next step.

Preparation of compound 170 [0417] To a solution of 150 and 169 (20.0 g, mixture) in MeOH (120 mL) and water (40 mL) was charged saturated NaHCO.₃ (9.99 g, 118.4 mmol) at 0 ° C and stirred for 10 min. Added (Boc)₂O (9.69 g, 44.4 mmol) and the reaction mixture was stirred for 10 min at the same temperature, brought to room temperature, and stirred for a further 2 h. The mixture was concentrated, the residue was dissolved in CH₂cl₂ (100 mL), and the solution was washed with water (100 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄, filtered, concentrated and the residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 8: 2 CHCl₃/ MeOH) to give compound 150 (1.50 g) and 170 (4.50 g) as an off-white solid. ESI-MS m / z 529 [C₂₇H₃₂N₂ABOUT₉+ H]⁺.

Preparation of compound 171

[0418] A suspension of compound 170 (4.20 g, 7.92 mmol) and 10% Pd / C (500 mg) in EtOH (100 mL) and AcOH (10 mL) was degasified with argon for 10 min, followed by stirring. in a hydrogen atmosphere (balloon, 1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo, neutralized with Na₂WHAT_{3 a} the residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 8: 2 CHCl₃/ MeOH) to give compound 172 (2.70 g,

68%) in the form of a greyish white solid. ¹H NMR (400 MHz, CD₃OD): δ 7.52-7.4 (m, 2H), 7.36-7.29 (m, 3H), 6.89 (d, J = 8.3 Hz, 2H), 6.64 (diast. d, J = 8.3 Hz, 2H), 5.54 (s, 1H), 4.23 (dd, J = 11.9, 5.9 Hz, 1H), 4.10-3.97 (m , 1H), 3.97-3.89 (m, 1H), 3.81-3.75 (m, 1H), 3.743,69 (m, 1H), 3.60 (t, J = 10.9 Hz, 1H), 3.48 (dd, J = 14.1, 4.6 Hz, 1H), 3.28-3.22 (m, 3H), 2.41 (t, J = 7.5 Hz). , 2H), 1.83-1.71 (m, 2H), 1.41 (s, 9H).

Preparation of intermediate 39 [0419]

<img file="PL2931713T3_D0047.tif" />

A solution of compound 17 (30.0 g, 121 mmol) and 173 (14.2 g, 145 mmol) in anhydrous acetonitrile (300 mL) was degassed for 10 min under argon followed by TEA (67 mL, 484). mmol), 10% (t-Bu)₃P in hexanes (49.0 mL, 24.2 mmol) and CuI (1.15 g, 6.05 mmol) at room temperature. The resulting mixture was degassed with argon for a further 10 min and Pd (PPh₃)₄ (14.0 g, 12.1 mmol) was added in one portion.

After degassing with argon for 5 min, the resulting mixture was heated at 50 ° C for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2: 3 hexanes / EtOAc) to yield 174 (15.0 g, 58%) as a brown oil.¹H NMR (400 MHz, CDCl₃) δ 8.14 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H), 3.71 (t, J = 6.4 Hz, 2H), 2.50 (t, J = 6.8 Hz, 2H), 1.80-1.70 (m, 4H), 1.70- 1.65 (m, 1H).

Preparation of compound 175 [0421] To a solution of compound 174 (15.0 g, 67.9 mmol) in anhydrous CH₂cl₂ (50 mL) Et was added₃N (28.0 mL, 203.7 mmol) and DMAP (4.12 g, 33.9 mmol) under argon at 0 ° C. The reaction mixture was stirred for 5 min at the same temperature, then TsCl (32.5 g, 170 mmol) was added at 0 ° C. The resulting mixture was stirred for

- 137 additional 4 hours in room temperature. After removal of the solvent, the residue was partitioned between CH₂cl₂ (250 ml) and water (150 ml). The aqueous layer was separated and extracted^CH2^cl2 (2 x 250 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (silica gel, hexanes / EtOAc) to yield compound 175 (15.0 g, 60%) as a brown oil.¹H NMR (400 MHz, CDCl₃): δ 8.15 (d, J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H) , 7.34 (d, J = 8.8 Hz, 2H), 4.10 (t, J = 6.4 Hz, 2H), 2.44 (t, J = 7.0 Hz, 2H), 2 44 (s, 3H), 1.90-1.79 (m, 2H), 1.75-1.61 (m, 2H).

Preparation of compound 176 A solution of compound 175 (5.00 g, 12.9 mmol, crude) in THF (10 ml) was added NHMe₂ in water (30%, 50.0 mL), and then stirred at room temperature in a sealed tube for 3 hours. After removal of the solvent, the residue was partitioned between^CH2^cl2 (100 ml) and water (100 ml). The aqueous layer was separated and extracted^CH2^cl2 (2 x 100 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel) to give compound 176 (400 mg, 13%) as a yellow sticky solid. 1H NMR (400 MHz, CDCl3): δ 8.15 (d, J = 7.3 Hz, 2H), 7.51 (d, J = 7.3 Hz, 2H), 2.48 (t, J = 6.6 Hz, 2H), 2.30 (t, J = 5.7 Hz, 2H), 2.23 (s, 6H), 1.70-1.61 (m, 4H).

Preparation of compound 39 [0423] A suspension of compound 176 (400 mg, 1.62 mmol) and 10% Pd / C (50 mg) in EtOH (50 ml) was degassed by bubbling argon with a syringe for 10 min, followed by stirring at room temperature. room temperature in a hydrogen atmosphere (balloon, 1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 39 (300 mg, 84%) as a brown viscous solid.¹H NMR (400 MHz, CD₃OD): δ 6.91 (d, J = 7.5 Hz, 2H), 6.65 (d, J = 7.5 Hz, 2H), 2.47 (t, J = 7.0 Hz, 2H); ), 2.30 (dd, J = 8.4, 6.5 Hz, 2H), 2.23 (s, 6H), 1.60-1.5 (m, 2H), 1.51-1, 41 (m, 2H), 1.38-1.27 (m, 4H).

Preparation of intermediate 44 [0424]

Scheme 32

- 138 -

<img file="PL2931713T3_D0048.tif" />

Preparation of compound 177 [0425] A solution of compound 175 (6.00 g, 16.0 mmol) in 7 N NH₃ in methanol (150 ml) was heated at 30 ° C in a sealed tube for 5 hours. The temperature was raised to 40 ° C and stirred for 16 hours, and then the temperature was raised to 60 ° C again and stirred for 4 hours. After removal of the solvent, the residue was partitioned between CH₂cl₂ (100 ml) and water (100 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 100 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH) to give compound 177 (1.48 g, 43%) as a yellow oil. ¹1 H NMR (400 MHz, CDCl 3): δ 8.16 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 3.61 (t, J = 5.6 Hz, 2H), 2.08-2.05 (m, 2H), 1.65-1.53 ​​(m, 4H).

Preparation of compounds 178 and 179 [0426] To a solution of 177 (1.38 g, 6.33 mmol) and triol 165 (2.03 g, 7.59 mmol) in MeOH (10 mL) was added AcOH (0.6 mL). , 949 mmol) and the reaction mixture was stirred at room temperature for 30 min. After adding NaCNBH₃ (800 mg, 12.7 mmol) was continued stirring the solution at room temperature for 16 hours. Additional compound 165 (2.55 g, 9.4 mmol), AcOH (0.80 mL, 12.7 mmol) and NaCNBH were added.₃ (1.19 g, 18.9 mmol) and stirring was continued at room temperature for 16 h. Additional compound 165 (2.55 g, 9.4 mmol), AcOH (0.80 mL, 12.7 mmol) and NaCNBH were added.₃ (1.19 g, 18.9 mmol), stirring was continued at room temperature for 16 hours. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} distributed between CH₂cl₂ (10 ml) and water (10 ml). The aqueous layer was separated and extracted with CH 2 Cl 2 (2 x 10 mL). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by chromatography

139 column (silica gel, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2 CHCl₃/ MeOH / NH₄OH) to give compound 179 (2.28 g, 51%) as an off-white solid. ¹H NMR (300 MHz, CD3OD): δ 8.14 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 7.47-7.4 ( m, 4H), 7.374.30 (m, 6H), 5.48 (s, 2H), 4.24-4.19 (m, 2H), 3.99-3.94 (m, 4H), 3 , 86-3,84 (m, 2H), 3,733,69 (m, 2H), 3,57 (t, J = 10.8 Hz, 4H), 3.35-3.25 (m, 4H), 2.33 (d, J = 6.9 Hz, 2H), 1.611,51 (m, 4H).

[0427] A mixture of 178/179 (900 mg) was also isolated and used directly in the next step (SG-GHC-G-106).

Preparation of compound 44 [0428] A suspension of compound 179 (2.26 g, 3.11 mmol) and 10% Pd / C (100 mg) in a mixture of EtOH (50 ml) and AcOH (10 ml) was degassed with argon for 10 min. then stirred under a hydrogen atmosphere (balloon, 1 atm) for 16 hours. in room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 44 (1.90 g, 80%) as a brown solid.¹H NMR (400 MHz, CD3OD): δ 7.46-7.44 (m, 4H), 7.33-7.31 (m, 6H), 6.89 (d, J = 8.4 Hz, 2H); ), 6.65 (d, J = 8.4 Hz, 2H), 5.51 (s, 2H), 4.26-4.14 (m, 2H), 3.93-3.90 (m, 2H), 3.76-3.73 (m, 4H), 3.63-3.58 (m, 4H), 3.35-3.25 (m, 2H), 3.10-3.00 ( m, 2H), 2.41 (t, J = 7.2 Hz, 2H), 1.47-1.45 (m, 4H), 1.161.12 (m, 4H).

Preparation of intermediate 49 [0429]

<img file="PL2931713T3_D0049.tif" />

Preparation of compound 180 [0430] A solution of 178 (900 mg, mixture, ca. 2.0 mmol) in a mixture of MeOH (20 mL) and water (10 mL) was charged with NaHCO₃ (672 mg, 4.0 mmol) at 0 ° C and stirred for 10 min. Added (Boc)₂O (524 mg, 2.40 mmol) and the reaction mixture was stirred for 1 h. at the same temperature, brought to room temperature, and stirred for a further 4 hours. The mixture was concentrated, the residue was dissolved in CH₂cl₂ (100 mL), and the solution was washed with water (100 mL) and brine (50 mL). The organic layer was dried over Na₂SO₄, filtered, concentrated and the residue was purified by column chromatography (silica gel, 9: 1 CH₂cl₂/ MeOH, 8: 2 CHCl₃/ MeOH) to give the compound

-140 180 (780 mg, 64%) as an off-white solid. ¹H NMR (300 MHz, CD₃OD): δ

8.16 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H), 7.50-7.47 (m, 2H), 7.34-7 30 (m, 3H), 5.53 (s, 1H), 4.25-4.2 (m, 1H), 4.10 (br s, 1H), 3.94-3.91 (m, 1H), 3.80-3.48 (m, 4H), 3.35-3.25 (m, 3H), 2.46 (t, J = 6.9 Hz, 2H), 1.70-1. , 49 (m, 4H), 1.43 (s, 9H).

Preparation of compound 49 [0431] A suspension of compound 180 (780 mg, 1.36 mmol) and 10% Pd / C (50 mg) in a mixture of EtOH (10 ml) and AcOH (2.0 ml) was degassed by passing argon using a syringe for 10 min, and then stirred at room temperature under a hydrogen atmosphere (balloon, 1 atm) for 4 hours. in room temperature. The reaction mixture was neutralized with Na₂WHAT₃, filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 49 (625 g, 84%) as a white solid. 1H NMR (300 MHz, CD3OD): δ7.50-7.46 (m, 2H), 7.32-7.30 (m, 3H), 6.90 (d, J = 8.4 Hz, 2H); ), 6.66 (d, J = 8.4 Hz, 2H), 5.53 (s, 1H), 4.25- 4.20 (m, 1H), 4.04 (br s, 1H), 3.94-3.89 (m, 1H), 3.773.43 (m, 4H), 3.35-3.25 (m, 3H), 2.45 (t, J = 7.5 Hz, 2H) , 1.52-1.47 (m, 4H), 1.42 (s, 9H), 1.27-1.24 (m, 4H).

Preparation of intermediate 54 [0432]

<img file="PL2931713T3_D0050.tif" />

Preparation of compound 182 [0433] To a solution of 181 (1.60 g, 16.00 mmol) in anhydrous THF (40 ml) was added 9-BBN (0.5 M in THF, 80 ml, 40.0 mmol) in the atmosphere. argon. After finishing 2 hours stirring the reaction mixture at room temperature, compound 172 (3.17 g, 12.8 mmol), Pd (PPh₃)₂cl₂ (561 mg, 0.80 mmol) and 1 N aq. NaOH (24 ml)

- 141 was added at room temperature. The resulting mixture was stirred for an additional 1 hour. After removal of the solvent, the residue was partitioned between EtOAc (100 mL) and water (100 mL). The aqueous layer was separated and extracted with EtOAc (2 x 100 mL). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexanes / EtOAc) to afford compound 182 (1.20 g, 34%) as a brown solid.¹H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 9.0 Hz, 2H), 7.31 (d, J = 9.0 Hz, 2H), 3.64 (t, J = 6.7 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H), 1.73-1.46 (m, 4H), 1.43-1.31 (m, 4H).

Preparation of compound 183 [0434] To a solution of compound 182 (1.20 g, 5.38 mmol) in anhydrous CH₂cl₂ (20 mL) Et was added₃N (7.32 ml, 53.8 mmol) under argon at 0 ° C. After 5 min of stirring the reaction mixture at the same temperature, mesyl chloride (0.62 mL, 8.07 mmol) was added at 0 ° C. The resulting mixture was stirred for an additional 2 hours. at room temperature. After removal of the solvent, the residue was partitioned between CH₂cl₂ (50 ml) and water (50 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 50 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product 183 (3.00 g, crude) was used directly in the next step.

Preparation of compound 184 [0435] A solution of compound 183 (3.00 g, 5.38 mmol, crude) in 7 N NH₃ in methanol (30.0 ml) was heated at 60 ° C in a sealed tube for 2 hours. After removal of the solvent, the residue was partitioned between CH₂cl₂ (100 ml) and water (100 ml). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 100 ml). The combined organic extracts were washed with brine, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by column chromatography (silica gel) to afford compound 184 (390 mg, 33%, in two steps) as a yellow oil.¹H NMR (400 MHz, CD3OD): δ 8.14 (d, J = 9.0 Hz, 2H), 7.42 (d, J = 9.0 Hz, 2H), 2.75 (t, J = 7.8 Hz, 2H), 2.67 (t, J = 7.3 Hz, 2H), 1.72-1.63 (m, 2H), 1.53-1.46 (m, 2H), 1.42-1.25 (m, 4H).

Preparation of compound 185 [0436] To a solution of compound 184 (620 mg, 2.79 mmol) and triol 165 (938 mg, 3.49 mmol) in MeOH (30 mL) was added AcOH (1.16 mL, 27.8 mmol). and the reaction mixture was stirred at room temperature for 10 min. After adding NaCNBH₃ (526 mg, 8.37 mmol) was continued stirring the solution at room temperature for 16 h. Additional compound 165 (0.3 eq.), AcOH (10 eq.) And NaCNBH₃ (1.0 eq.) Was added over 16 h. Then hexanal 163 (0.96 ml, 8.37 mmol), AcOH (1.00 ml) and NaCNBH3 (526 mg, 8.37 mmol) were added. The solution was further stirred at room temperature for 2 hours. After removal of the solvent, the residue was neutralized with saturated NaHCO_{3 and} separated ^residue between EtOAc (100 mL) and water (100 mL). The aqueous layer was separated and extracted with CH₂cl₂ (2 x 100 ml). The combined organic extracts were dried over Na₂SO₄ and concentrated under vacuum. The residue was purified by column chromatography (gel

- 142, silica, 9: 1 CH₂cl₂/ MeOH, 80: 18: 2 CHCl₃/ MeOH / NH₄OH) to give compound 185 (950 g, 61%) as a greyish white oil. ¹H NMR (400 MHz, CDCl₃): δ 8.02 (d, J = 8.7 Hz, 2H), 7.48-7.42 (m, 3H), 7.37-7.34 (m, 2H), 7.31 (d , J = 8.7 Hz, 2H), 5.54 (s, 1H), 4.464.40 (m, 1H), 4.30 (dd, J = 11.6, 6.6 Hz, 1H), 4 , 03 (t, J = 4.0 Hz, 1H), 3.97 (dd, J = 10.5, 5.4 Hz, 1H), 3.88 (dd, J = 9.4, 4.0 Hz, 1H), 3.65 (t, J = 10.4 Hz, 1H), 3.11-3.00 (m, 4H), 2.69 (t, J = 7.8 Hz, 2H), 2.00 (s, 1H), 1.70-1.55 (m, 6H), 1.37-1.30 (m, 4H), 1.29-1.20 (m, 8H), 0, 87 (t, J = 7.1 Hz, 3H).

Preparation of compound 54 A suspension of compound 185 (950 g, 1.70 mmol) and 10% Pd / C (300 mg) in EtOH (100 ml) was degassed with argon for 10 min then stirred under a hydrogen atmosphere (balloon, 1 atm) for 3 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to afford 54 (790 mg, 88%) as a yellow oil.¹H NMR (400 MHz, CD3OD): δ 7.51-7.4 (m, 2H), 7.35-7.29 (m, 3H), 6.90 (d, J = 8.5 Hz, 2H); ), 6.65 (d, J = 8.5 Hz, 2H), 5.54 (s, 1H), 4.24 (dd, J = 10.8, 5.4 Hz, 1H), 4.088.42 (m, 1H), 4.00-3.92 (m, 1H), 3.91 (dd, J = 5.6, 1.8 Hz, 1H), 3.78 (dd, J = 9.6 , 1.8 Hz, 1H), 3.61 (t, J = 10.9 Hz, 1H), 3.01 (dd, J = 13.7, 5.4 Hz, 1H), 2.91 (dd) , J = 12.1, 8.1 Hz, 1H), 2.82-2.71 (m, 4H), 2.45 (t, J = 7.5 Hz, 2H), 1.59-1, 42 (m, 6H), 1.37-1.13 (m, 10H), 0.89 (t, J = 7.1 Hz, 3H).

[0438] Several assays may be used to characterize compounds of the invention. Representative assays are discussed below.

In vitro measurement of sodium channel blocking activity and reversibility [0439] One of the assays used to assess the mechanism of action and / or potency of the compounds of the invention is related to the determination of drug inhibition in the lumen of the current in the airway epithelium, measured under short-circuit conditions ( AND_SC) using single layers of respiratory epithelium fixed in the Ussing chambers. Cells obtained from freshly prepared human, canine, sheep or rodent-derived respiratory tracts were inoculated into porous 0.4 micron Snapwell ™ inserts (CoStar), cultured under air-liquid interfacial (ALI) conditions in media of defined hormone composition, and determined for sodium transport activity (I_SC) with simultaneous washing in Krebs Bicarbonate Ringer bicarbonate solution (KBR) using chambers. All drug additions tested involved flushing the duct light using half-log dose addition protocols (from 1 x 10^-11 M to 3 x 10^-5 M), and the total change in ISC (inhibition) recorded. All drugs were prepared in 1 × 10 dimethyl sulfoxide stock solutions^-2 M and stored at -20 ° C. Eight preparations were typically analyzed in parallel; two analysis preparations incorporated amiloride and / or benzamil as positive controls. After administration of the maximum concentration (5 x 10^-5 M), washing the lumen of the tube was changed three times to a fresh, drug-free KBR solution, and the resulting ISC was measured after each wash, which lasted about 5 minutes. Reversibility was defined as the percentage of returns to baseline with respect to the sodium current after

- 143 third wash. All data for voltage tests in the leveling system was collected via a computer interface and analyzed off-line.

[0440] Dose-effect relationships for all compounds were analyzed, and tested using Prism 3.0. IC values₅₀, maximum effective concentrations, and reversibility were calculated and compared to amiloride and benzamyl as positive controls. The potency of sodium channel blocking activity for representative compounds relative to amiloride in freshly prepared cells from canine airways is shown in Table 1.

Table 1. Inhibition of short circuit current by Compound (Ia) in canine bronchial epithelial cells (IC50 nM)

<td>The number of the relationship</td><td>Blocking power of the sodium channel IC50 nM</td>

<td>amiloride</td><td>773</td>

<td>23</td><td>20.7</td>

<td>38</td><td>25.4</td>

<td>28</td><td>7.4</td>

<td>33</td><td>21.8</td>

<td>16</td><td>79.6</td>

<td>103</td><td>17.9</td>

<td>99</td><td>7.6</td>

<td>94</td><td>21.2</td>

<td>80</td><td>19.4</td>

<td>135</td><td>5.2</td>

<td>131</td><td>6.0</td>

<td>123</td><td>2.3</td>

<td>127</td><td>8.6</td>

<td>139</td><td>73.7</td>

<td>43</td><td>50.1</td>

<td>53</td><td>15.5</td>

<td>58</td><td>10.6</td>

<td>48</td><td>47</td>

Assay 2. Muclease clearance (MCC) studies in sheep

[0441] The animal model that was most commonly used to measure MCC changes is the sheep model. The effect of compounds on the increase in mucocyte clearance (MCC) can be measured using an in vivo model described by Sabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196, incorporated herein by reference.

[0442] In these studies, the possibilities of movement of adult sheep were limited and an endotracheal tube was introduced through the nose. Aerosolized test articles were administered for 1015 minutes to sheep. Radiolabeled^99mThe sulfur tc-colloid (TSC, 3.1 mg / ml, containing approximately 20 mCi) was then administered for a defined period of four or eight hours after the test product. A radio-labeled aerosol was administered through the endotracheal tube for approximately 5 minutes. The sheep were then extubated, and total radioactive counts were measured in the lungs every 5 minutes during the 1-hour observation period. The clearance rate of the radioactive tracer from the lung is representative of the MCC rate in the animal. The advantage of this system is that it closely mimics the human lung environment. The model also allows the simultaneous collection of PK / PD observations by collecting plasma and urine samples for the duration of the test. There are also several techniques to measure drug concentrations on the surface of the airways during MCC measurements.

[0443] The sheep model described above was used to make an in vivo evaluation of the effect (efficacy / stability) delivered as the test compound aerosol on the MCC. A treatment consisting of 4 ml of test agent or test agent in combination with HS was tested. To determine whether the combination of HS and the test agent increased MCC, HS was administered immediately after administration of the test agent. The test solutions were converted to aerosol using a Raindrop nebulizer at a flow rate of eight liters per minute and connected to a dosimetry system consisting of a solenoid valve and a compressed air source (20 psi). It was estimated that the deposited dose of the drug in sheep's lungs after aerosol administration with the Raindrop nebulizer was 8-15% of the dose. Using the Raindrop nebuliser, radio-labeled TSC was administered for approximately 3 minutes, 4 or 8 hours after drug administration to assess efficacy / persistence. Radioactive counts were measured with a gamma counter apparatus in the central right-lung region at 5-min intervals, for one hour. Three methods of analysis were used, 1) initial clearance rate (slope) for the first 30 min, with a linear regression adjustment 2) area under the curve for% clearance in time for one hour, and 3) maximum clearance achieved in one hour.

[0444] The effect of compound 33 was evaluated at 0.24 nmol / kg (3 μΜ) and compared to the vehicle (4 ml of sterile H₂O) on sheep's MCC four hours after dosing (Figure 1). The effect analyzes are shown in Table A. Compound 33 increased MCC compared to vehicle control.

Table A. MCC in sheep after 4 hours after administration of a dose of compound 33 or excipient

<td>Compound 33 Dose</td><td>Initial slope (4.0-4.5 h.)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

- 145 -

<td>0.24 nmol / kg (3 μΜ)</td><td>37.5 * (4)</td><td>17,4 * (4)</td><td>30.0 * (4)</td>

<td>Excipient (H₂AT 4 ml</td><td>17.2 ± 6.8 (8)</td><td>7.3 ± 1.5 (8)</td><td>12.2 ± 2.9 (8)</td>

[0445] Tables B and C together with Figures 2 and 3 show that other compounds of the invention similarly increase MCC as compared to the vehicle (see, e.g., Compounds 123 and 48)

Table B. MCC in sheep after 4 hours after administration of a dose of Compound 123 or excipient

<td>Relationship 123 Dose</td><td>Initial slope (4.0-4.5 h.)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>0.24 nmol / kg (3 μΜ)</td><td>29.2 * (2)</td><td>14.4 * (2)</td><td>22.8 * (2)</td>

<td>Excipient (H₂AT 4 ml</td><td>17.2 ± 6.8 (8)</td><td>7.3 ± 1.5 (8)</td><td>12.2 ± 2.9 (8)</td>

Table C. MCC in sheep after 4 hours after administration of a dose of Compound 48 or excipient

<td>Compound 48 Dose</td><td>Initial slope (4.0-4.5 h.)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>0.24 nmol / kg (3 gM)</td><td>29.8 * (2)</td><td>15,4 * (2)</td><td>26.7 * (2)</td>

<td>Excipient (H₂AT 4 ml</td><td>17.2 ± 6.8 (8)</td><td>7.3 ± 1.5 (8)</td><td>12.2 ± 2.9 (8)</td>

[0446] To determine if the compounds of the invention increased the duration of action, they were tested after 8 hours after dosing. In Tables D and E, together with Figures 4 and 5, the extended duration of action of the MCC relative to the excipient for Compounds 33 and 152 is clearly seen.

Table D. MCC in sheep after 8 hours after administration of a dose of compound 33 or excipient

<td>Compound 33 Dose</td><td>Initial slope (8.0-8.5 h.)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>0.24 nmol / kg (3 gM)</td><td>25.8 * (4)</td><td>11.7 * (4)</td><td>21.4 * (4)</td>

<td>Excipient (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>

Table E. MCC in sheep after 8 hours after administration of a dose of Compound 152 or excipient

- 146 -

<td>Compound 152 Dose</td><td>Initial slope (8.0-8.5 h.)</td><td>AUC (% Cl h)</td><td>Maximum clearance</td>

<td>0.24 nmol / kg (3 μΜ)</td><td>37.5 * (4)</td><td>17,4 * (4)</td><td>30.0 * (4)</td>

<td>Excipient (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>

[0447] To determine if HS increases the MCC effect of compound 33, 7% HS was dosed immediately after 0.24 nmol / kg of compound 33, and the MCC was evaluated eight hours after the dosing regimen (Figure 6). HS increased the effect of compound 33 on MCC, as shown in Fig. 6.

Assay 3. Clearance of drug liquids on the airway surface (ASL) and metabolism of human airway epithelium [0448] The disappearance of 33 from the apical surface and respiratory epithelial airway metabolism in human bronchial epithelial cells (HBE) were evaluated (Table 3). In these experiments, 25 μΐ of a 25 μΜ solution of ENaC blocker to the apical surface of HBE cells grown at the air / liquid interface was added, and the drug and metabolite concentrations were measured in the application and basolateral ranges for 2 hours. using UPLC.

Table G. Atrophic abstraction and metabolism of compound 33

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

<td>33</td><td>44.8 ± 18%</td><td>4%</td><td>1.1 ± 0.45%</td><td>32%</td>

<td colspan="5">Values ​​represent the mean ± SD</td>

Comparative Examples [0449] The compounds of Formula (I) are more potent and / or slower absorbed from the mucosal surface, especially of the airway surfaces, compared to known sodium channel blockers such as amiloride and third generation compounds such as Comparative Example 1 described below . Thus, the compounds of Formula (I) have a longer half-life on the mucosal surface compared to those of known compounds, as evidenced by the data shown in Table G. Disappearance of compound 33 from the apical surface and airway epithelial metabolism was estimated in HBE and compared to Example comparative 1 (Table H). In these experiments, 25 μl of a 25 μM solution of ENaC blocker to the apical surface of HBE cells grown at the air / liquid interfacial interface were added, and drug concentrations were measured in the application and basolateral ranges for 2 hours. using UPLC. After 2 hours incubation of the compounds of the invention on the apical surface (37 ° C), Compound 33 was mostly unmetabolized on the side

- 147 apikalna. In contrast, most of Comparative Example 1 was eliminated from the apical side, with 83% metabolization to less active carboxylic acid, (S) -2-amino-3- (4- (4- (3- (3,5- diamino-6-chloropyrazine-2-carbonyl) guanidino) butyl) phenoxy) propanoic, with the structure as below.

<img file="PL2931713T3_D0051.tif" />

Table H. Apical breakdown and metabolism of compound 33 against Comparative Example 1 in HBE

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

<td>33</td><td>44.8 ± 18%</td><td>4%</td><td>1.1 ± 0.45%</td><td>32%</td>

<td>Comparative example 1</td><td>41.6 ± 7.6% (8% Stock)</td><td>83.0 ± 3.5%</td><td>8.3 ± 0.2 (1% Stock)</td><td>94.7 ± 1.0%</td>

<td colspan="5">Values ​​represent the mean ± SD</td>

[0450] Comparative Example 1 is claimed, described or included in the disclosure of WO 2003/070182 (US Patents No. 6,858,615, 7,186,833, 7,189,719, 7,192,960, and 7,332,496), as sodium channel blockers having useful therapeutic properties, and which can be obtained by the methods described i.e. and others known in the art.

Comparative example 1.

[0451]

<img file="PL2931713T3_D0052.tif" />

(S) -3,5-Diamino-6-chloro-N- (N- (4- (4- (2,3-diamino-3-oxopropoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide [0452] The compound of Comparative Example 1 can be seen on page 15 of the US

2005/0080093 and as Compound 2 on page 90 of WO 2008/031048, and as Compound 2 on pages

42-43 WO 2008/031028. To provide useful activity in the treatment of cystic fibrosis and chronic obstructive pulmonary disease, the compound must have properties,

- 148 which will improve mucociliary clearance (MCC) in doses that do not increase the level of potassium in the plasma, which will ultimately lead to hyperkalemia, a serious and dangerous condition, after repeated dosing. Therefore, these compounds in this class, which are known to increase potassium in plasma, should be avoided if they are largely secreted by the kidneys. To assess this potential, it is beneficial to have MCC activity in vivo and not to increase the potassium plasma level in a useful dose. One of the models to assess this is the MCC sheep model described below.

[0453] As seen in Table I and Fig. 7 ED₅₀ for Comparative Example 1, the sheep's MCC model is approximately 240 nmol / kg (3mM) using three different measures (slope, AUC and maximum clearance). At this dose, which will mean the clinically active dose, Comparative Example 1 results in an increase in plasma potassium (Fig. 8) which, when repeatedly dosed, will lead to hyperkalemia. Hence, Comparative Example I is unacceptable for human use, while Compound (Ia) provides a safe and effective MCC with a benefit-risk ratio of greater than 1,000 in this model.

Table I. MCC in sheep after 4 hours after a dose of excipient, Comparative Example 1 or Compound 33

<td>Dose</td><td>Initial slope (4.0-4.5 hours)</td><td>AUC (% Cl xh)</td><td>Maximum clearance</td>

<td>Comparative example 1 240 nmol / kg (3 mM)</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 1 24 nmol / kg (300 μΜ)</td><td>14.5 ± 1.3 (3)</td><td>6.9 ± 1.0 (3)</td><td>14.6 ± 0.9 (3)</td>

<td>Compound 33 0.240 nmol / kg (30 gM)</td><td>37.5 * (4)</td><td>17,4 * (4)</td><td>30.0 * (4)</td>

<td>H2O excipient (4 ml)</td><td>17.2 ± 6.8 (8)</td><td>7.3 ± 1.5 (8)</td><td>12.2 ± 2.9 (8)</td>

[0454] Fig. 1 graphically depicts the percentage of mucus clearance over time, for Compound 33 and Comparative Example 1, as described in the MCC model above. Even a higher percentage of mucus clearance was provided by Compound 33 at a 1000-dose lower dose than was observed with Comparative Example 1. Therefore, Compound 33 provided maximum effect in a clinically relevant dose range, without elevating potassium levels.

[0455] Fig. 10 illustrates a significant increase in plasma potassium at the effective dose observed in sheep plasma taking Comparative Example 1 in the MCC study. Compound 33 is more than 1000-fold more potent in the context of ovine MCC than Comparative Example 1 without increasing plasma K levels at doses as high as 24 nmol / kg (1000-fold dose ED50), while Comparative Example 1 increases K-levels plasma at approximately ED50 dose^{3 mM}(Figures 7 and 8). This demonstrates once again the unique and unexpected benefit in the form of power and

- 149 ^{security of the Union} 33 as can be seen in Table J, with a therapeutic index more than 1000 times greater in the context of renal safety than Comparative Example 1.

Table J. Therapeutic ratio (Benefit / Risk)

<td></td><td>MCC The highest dose lower than the optimal one</td><td>The highest dose in sheep without increasing the level of potassium in plasma</td><td>Ratio therapeutic</td>

<td>Comparative example 1</td><td>240 nmol / kg (3 mM)</td><td>24 nmol / kg (300 μΜ)</td><td>0.1</td>

<td>33</td><td>&lt;0.24 nmol / kg (3 μΜ)</td><td>24 nmol / kg (300 μΜ)</td><td>&gt; 100</td>

<td>Ratio</td><td>&gt; 1000</td><td>1</td><td>&gt; 1000</td>

[0456] Other compounds of the invention have similar advantages in the context of safety and efficacy relative to known compounds, as detailed in Figures 11, 12, 13 and 14.

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