Untitled record

Full description

Background of the invention

The present invention relates to new 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- 5

Carboxamide and related compounds and their pharmaceutically accepted salts are useful in the form of sodium channel blockers, their composition, therapeutic methods, uses, and preparation processes.

Mucosal surfaces at the interface between the environment and the body have developed a number of “innate 10 defences,” that is, protective mechanisms. The initial picture is the defense

The fungal infection mentioned in cleaning those surfaces using liquid. Typically, the amount of liquid layer on the mucosal surface reflects the balance between surfaces with liquid, typically anion (Cl- and/or HCO3-) secretion (and the corresponding anion cation), and absorption of epithelial cell liquid surfaces with liquid. liquid, reversing Na+ absorption,

15 Combined with water and the corresponding anion (Cl- and/or HCO3-). Many diseases caused by mucous surfaces are caused by the presence of too little protective fluid on said mucosal surfaces due to an imbalance between secretion (too little) and absorption (relatively too much). Defective salt transport processes that characterize mucosal dysfunction reside in the epithelial layer of the mucosal surface.

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One way to replenish the protective fluid layer on mucosal surfaces is to “re-balance

balance "system by blocking the Na+ channel and liquid absorption. The epithelial protein that stimulates the Na+ restriction step and liquid absorption is represented in the epithelial Na+ channel ("ENaC").

5 ENaC placed the apical surface of the epithelium, i.e. the mucosal surface-environmental interface. Typically, to inhibit ENaC-induced Na+ and fluid absorption, an amiloride-type ENaC inhibitor is delivered to the mucosal surface and maintained in that position for maximum therapeutic benefit.

10 The use of ENaC blockers has been suggested for a range of diseases that occur with increased mucosal hydration. Specifically, ENaC inhibitors are proposed for the treatment of respiratory diseases such as chronic bronchitis (CB chronic bronchitis), cystic fibrosis (CF), and COPD, which reflects the body's inability to eliminate mucus normally. From lungs

15 It eventually leads to chronic infection of the airways. See:

Evidence for airway surface dehydration as the initiating event in CF airway disease, R. C. Boucher, Journal of Internal Medicine, Vol. 261, Issue 1, January 2007, pages 5-16; and Cystic fibrosis: a disease of vulnerability to airway surface dehydration, R. C. Boucher, Trends in Molecular Medicine, Vol. 13, Issue 6, June 2007, pages 231-240 20

Data indicate that the primary problem in both chronic bronchitis and cystic fibrosis is a failure to clear mucus from airway surfaces. Failure to eliminate mucus reflects an imbalance in the amounts of mucus as airway surface liquid (ASL) on the surfaces of the airways.

25 Airways. The aforementioned imbalance leads to a relative decrease in ASL, which leads to...

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Concentration of mucus, reduced lubricating activity of pericilial fluid (PCL) activity, adhesion of mucus to the surface of the airways, and failure to remove mucus through ciliary activity to the mouth. Decreased ability to remove mucus leads to the formation of bacterial colonies Chronic bacterial colonization of mucus

5 Adherent to airway surfaces. Chronic retention of bacteria, the inability of local antimicrobial substances to kill mucus entrapped bacteria on a chronic basis, subsequent to a chronic inflammatory response of the aforementioned type following surface injury, is evident in chronic bronchitis chronic bronchitis and fibrosis

10 Cystic fibrosis.

There is currently a significant unmet medical need for products that treat a range of ailments that are improved by increased mucus hydrolysis, including COPD, chronic bronchitis, cystic fibrosis, and more. Current treatments for chronic bronchitis, COPD and cystic fibrosis focus on treating the following symptoms and/or effects:

15 That mother has land. However, none of the treatments effectively address the underlying problem of clearing mucus from the lungs.

RC Boucher, in US Patent No. 6,264,975, describes the use of the sodium channel blocker pyrazinoylguanidine sodium to hydrolyze mucosal surfaces embolized by the well-known diuretics amiloride, Ben-Izmil, and phenamyl. However, these compounds are latent

20 Relatively speaking, taking into account the restricted mass of the drug that can be inhaled into the lung; (2) which is done

They are rapidly absorbed and thus exhibit a short, undesired half-life at the mucosal surface; and (3) can be freely separated from ENaC. More latent drugs that have longer half-lives on the mucosal surface are needed.

The presence of a small amount of surface protective fluid on other mucosal surfaces is known in physics

25 Pathogenicity of several Earth mothers. For example, in dry mouth and fluid depletion

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From the oral cavity due to parotopathy Sublingual glands Submandibular glands secrete fluid despite continuous Na+ transport (ENaC) induced by absorption of fluid from the oral cavity. Keratoconjunctivitis sicca (dry eye) results from failure of the lacrimal glands Liquid rice at the surface of the liquid absorption process of Na+ continuous 5 harmonic surfaces In rhinosinusitis, there is an imbalance between mucin secretion and related ASL depletion. Lack of secretion of Cl- (and fluid) near the small intestine, combined with increased absorption of Na+ (and fluid) at the end of the ileum results in

in . DIOS (distal intestinal obstruction syndrome).

In elderly patients, increased Na+ absorption (and volume) in the descending 10 colon leads to constipation and diverticulitis.

Published patents include a number of patent applications and patents granted to Parion Sciences Inc., relating to pyrazinoylguanidine analogues as sodium channel blockers. Examples of such bulletins include PCT bulletin numbers for International Application 070182/2003, International Application 070184/2003, International Application 073629/2004, International Application

15 025496/2005, international application 016879/2005, international application 018644/2005, application

International Application 022935/2006, International Application 023573/2006, International Application 023617/2006, International Application 018640/2007, International Application 146869/2007, International Application

031028/2008, International Application 031048/2008, and US Patent Numbers: 6858614, 6858615, 6903105, 7064129, 7186833, 7189719, 7192958, 7192959, 20 7192960, 7241766, 7247636, 7247637, 7317013, 7332496, 7368447,

7368450, 7368451, 7375102, 7388013, 7399766, 7410968, 7807834, 7842697, and 7868010.

There is a need for new sodium channel blocking compounds that have enhanced and effective mucus-texturing ability. There is still a need for new sodium channel blocking compounds that provide a therapeutic effect.

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With the reduction or elimination of the initiation or progression of hyperkalemia in

recipients

General description of the invention

This invention provides compounds of formula I:

5

where:

<img file="SA5673B1_D0001.tif" />

Ar be selected from a set:

<img file="SA5673B1_D0002.tif" />

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

10

R1 consists of a C1-C8 alkyl, a hydrogen, and a polyhydroxylated alkyl group with 3 to 8 carbon atoms;

R2 is a hydrogen or polyhydroxylated alkyl group with 3 to 8 carbons;

R3 and R4 are each, independently, a hydrogen or C1-C3 alkyl;

15 Or acceptable salt from it.

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This invention also provides solutes and hydrates, individual stereoisomers, including optical isomers (enantiomers and diastereomers) and geometric isomers (cis-/trans-isomerism), mixtures of stereoisomers, and compounds A class of tautomers of 5 compounds with formula (I), or a pharmaceutically acceptable salt thereof, in addition to pharmaceutical compositions that include the compounds, or pharmaceutically acceptable salts thereof, for use in processing methods, and methods for their preparation.

This invention also presents the compound:

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- 10

chloropyrazine-2-carboxamide

<p dir="rtl">Use of in</p>

<p dir="rtl">15 Processing methods and preparation methods.</p>

Brief explanation of the drawings

The invention is fully explained and many advantages of the invention are obtained by reference to the information set forth herein in conjunction with the following figures:

Figure 1 is a plot of the effect of compound 33 on sheep MCC at 4 h postdose.

20 Figure 2 is a plot of the effect of compound 123 on sheep MCC at 4 h postdose.

Figure 3 is a plot of the effect of compound 48 on sheep MCC at 4 h postdose.

Figure 4 is a plot of the effect of compound 33 on sheep MCC at 8 h postdose.

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

Figure 6 is a diagram of the enhancement of compound 33 on sheep MCC at 8 hours post-dose with hypertonic saline.

Figure 7 is a plot of the effect of Comparative Example I on MCC of sheep at 4 h post

5 Dosage.

Figure 8 is a chart of the effect of Comparative Example 1 on sheep plasma potassium levels

sheep plasma potassium levels

Figure 9 is a chart comparing the activity of Comparator Example 1 and Compound 33 on sheep MCC at 4 h postdose.

10 Figure 10 is a chart comparing the effect on K+ levels of sheep plasma of Comparative Example 1 and Compound 33.

Figure 11 is a chart comparing the activity of Comparator Example 1 and Compound 123 on sheep MCC at 4 hours post-dose.

Figure 12 is a chart comparing the effect on K+ levels of sheep plasma Comparative Example 1 15 and compound 123.

Figure 13 is a chart comparing the activity of Comparator Example 1 and Compound 48 on sheep MCC at 4 hours post-dose.

Figure 14 is a chart comparing the effect on K+ levels of sheep plasma of Comparative Example 1 and Compound 48.

20 Detailed description:

As used in this document, the following expressions are defined as referenced in this document.

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The expression “compound of the invention” means a compound having formula I or a salt, specifically a pharmaceutically acceptable salt thereof.

The expression “compound with formula I” means a compound with the structural formula designated in this document as formula I.

Compounds with formula I include solutes and hydrates (that is, the products of adding a compound with formula I to...

5 (solvent). In those embodiments where a compound of formula I includes one or more chiral centers, the expression is intended to include all of the individual isomers, including the optical isomers.

Optical isomers (diastereomers and enantiomers)

Geometric mart (formation of cis-/trans-isomerism) and mixtures of enantiomers. In addition to

Therefore, compounds with formula I also include tautomers of formula(s)

10 Explained.

By description and examples, compounds are named using standard IUPAC nomenclature principles, if applicable, including the use of ChemDraw Ultra 11.0 compound naming software, sold by CambridgeSoft Corp./PerkinElmer.

In some chemical structural models, in the case where the carbon atom does not have a sufficient number of attached variants that form quaternary valence, it is assumed that the carbon substitution groups are

The residue that provides quaternary valence is a hydrogen group. Likewise, in some structural formulas when ligands are depicted without specifying the final group, that group is referred to as the CH3-,Me(methyl) group, as is appropriate in the art.

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- 20)

pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-

1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

By structural composition:

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<img file="SA5673B1_D0003.tif" />

Or acceptable salt from it.

In another 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)-5,6,7,8- 5 tetrahydronaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-

carboxamide, by structural structure:

<img file="SA5673B1_D0004.tif" />

Or acceptable salt from it.

10 In an additional 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

By structural composition:

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<img file="SA5673B1_D0005.tif" />

Or acceptable salt from it.

In another embodiment, the compound of formula (I) is:

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, with the formula:

<img file="SA5673B1_D0006.tif" />

Or acceptable salt from it.

10

The three independent forms include compounds of formula (II), formula (III), and formula (IV), respectively:

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<img file="SA5673B1_D0007.tif" />

<img file="SA5673B1_D0008.tif" />

where:

5 n is an integer selected from 0,

1, 2, 3, 4, 5, or 6;

R1 is a selectivity of alkyl C1-C8, hydrogen, and its polyhydroxylated alkyl group.

3 to 8 carbon atoms;

R2 is a hydrogen or polyhydroxylated alkyl group with 3 to 8 carbons;

R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

10 Or acceptable salt from it.

Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is an additional model where:

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

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R1 consists of a C1-C8 alkyl, a hydrogen, and a polyhydroxylated alkyl group from 3 to 8 carbons;

R2 is a hydrogen or polyhydroxylated alkyl group with 3 to 8 carbons;

R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

5 Or acceptable salt from it.

Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is an additional model where:

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

R1 is hydrogen- and alkyl-selective C1-C8;

10 R2 is a hydrogen or polyhydroxylated alkyl group with 3 to 8 carbons;

R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

Or acceptable salt from it.

Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is another model where:

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

R1 is hydrogen- and alkyl-selective C1-C8;

R2 is hydrogen;

R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

Or acceptable salt from it.

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Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is also another form where:

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

R1 and R2 are each, independently, a polyhydroxylated alkyl group with 3 5 to 8 carbons;

R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

Or acceptable salt from it.

Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is also another form where:

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

R1 and R2 are each, independently, a polyhydroxylated alkyl group with 3 to 8 carbons;

R3 and R4 are hydrogen;

Or acceptable salt from it.

15 Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is another model where:

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

R1 and R2 are each, independently, a polyhydroxylated alkyl group with 3 to 8 carbons;

20 R3 and R4 are each, independently, alkyl C1-C3;

Or acceptable salt from it.

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Within each group of compounds represented independently by formulas (III), (II), (I), and (IV), there is an additional model where:

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

R1 and R2 are each, independently, a polyhydroxylated alkyl group with 3 5 to 8 carbons;

R3 is hydrogen; And

R4 is alkyl C1-C3;

Or acceptable salt from it.

The polyhydroxyl-treated alkyl groups of the invention are those in which the alkyl chain has 3 to 8 carbon atoms substituted by two or more

Hydroxyl groups. Examples of polyhydroxyl-treated 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-1,2,3,4,5-pentaol; heptane-1,2,3,4,5,6-hexaol; And the. octane-1,2,3,4,5,6,7-heptaol

15 One embodiment within each group of compounds described herein is those whose polyhydroxyl alkyl group has the formula CH2-(CHR5)nH–, where n is an integer selected from 2, 3, 4, 5, 6, or 7, and R5 is independently in each case an H or an OH, provided that at least two of the R5 groups are OH.

Another form within each group of compounds described herein is those compounds

20 whose polyhydroxyl-treated alkyl group has the formula -CH2-CHOH– CHR6)mH), where m is an integer chosen from 1, 2, 3, 4, 5, or 6, and R6 is independently in each case an H or OH, provided that at least one of the R6 groups is an OH.

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An additional embodiment within each group of compounds described herein includes compounds whose polyhydroxyl-treated alkyl group has the formula -CH2-(CHOH)n–CH2OH, where n is an integer selected from 1, 2, 3, 4, 5, or 6. Another embodiment within each group of compounds described herein includes compounds for which n is an integer 5 selected from 2, 3, 4, or 5. Another embodiment within each group includes compounds for which n is an integer selected From 3, 4, or 5.

In another embodiment within each group of compounds described herein, the chain represented by the formula CH2-(CHOH)n-CH2OH is 2,3,4,5,6-pentahydroxy

hexane, with the formula:

<img file="SA5673B1_D0009.tif" />

10 .

In a further embodiment within each group of compounds described herein, the series represented by the formula CH2-(CHOH)n-CH2OH– has the formula:

<img file="SA5673B1_D0010.tif" />

Three additional independent forms include compounds of formula (V), formula (VI), and formula

15 (VII), respectively:

<img file="SA5673B1_D0011.tif" />

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<img file="SA5673B1_D0012.tif" />

where:

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

5 R3 and R4 are each, independently, hydrogen or alkyl C1-C3;

Or acceptable salt from it.

Within each model represented by the formulas (VI), (V), and (VII) there is an additional model where n is an integer chosen from 1, 2, 3, 4, 5, or 6; and R3 and R4 are each a hydrogen; or a salt. It is pharmaceutically acceptable. Within each form represented by the formulas (VI), (V), and (VII), there is another form where 10 n is an integer selected from 1, 2, 3, 4, 5, or 6; and R3 and R4 are each an expression. for C1-C3 alkyl; or a pharmaceutically acceptable salt thereof.

Within each of the embodiments described in this document, there is an additional embodiment where n is an integer selected from 1, 2, or 3. Within each of the embodiments described in this document, there is an additional embodiment where n is an integer selected from 4, 5, or 6. Within each of the models described in this 15 document, there are six additional independent models where n is an integer, respectively, 1, 2, 3, 4, 5, and 6.

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The compounds in this document, which include those in the formula (IV), (III), (II), (Ia), (I), (VI), (V), and (VII), may be in the form of a free base or Salt, specifically pharmaceutically acceptable salt.

Berge et al., J. Pharma Sci. (1977) For pharmaceutically acceptable salts see 66:1-19.

5 Pharmaceutical acceptable salts include, for example, organic or inorganic acids

hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, sulfamate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, tannate, ascorbate, palmitate, salicylate , stearate, 10 phthalate, alginate, polyglutamate, oxalate, oxaloacetate, saccharate, methanesulfonate, ethanesulfonate, such as (benzoate, alkyl or aryl sulfonates)

f)benzenesulfonate, p-toluenesulfonate or naphthalenesulfonate

isothionate; And complexes formed with amino acids such as lysine and arginine

And glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine 15

Like that. The compounds of the invention may also be in the form of salts formed from elemental anions such as chlorine, bromine or iodine.

For therapeutic use, the salts of the active ingredients of compounds having Formula I are pharmaceutically acceptable, i.e. they are salts derived from a pharmaceutically acceptable acid. However, salts may be used

20 Acids that are not pharmaceutically acceptable, for example, in the process of preparing or purifying a pharmaceutically acceptable compound. Trifluoroacetate salts, for example, can also be used. All salts, whether derived from a pharmaceutically acceptable acid, are within the scope of the present invention.

The expression "chiral" refers to molecules that are unable to combine in their mirror image pattern, while the expression "chiral" refers to molecules that are compounded in their mirror image pattern.

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The term “stereoisomers” refers to compounds that have an identical chemical conformation, but differ in the arrangement of atoms or groups in the space. The term "diastereomer" refers to an enantiomer with two or more chiral centers in which molecules are non-identical images of each other. Diastereomers have 5 different physical properties, such as melting points and boiling points.

<p dir="rtl">, spectral properties, and reactivities rates. Mixtures of dimers can be separated using high-precision analytical procedures such as electrophoresis and chromatography. The term enantiomers refers to two enantiomers of a compound that have identical, non-combined images of each other.</p>

<p>SP Parker, Ed., McGraw-Hill 10 Stereochemistry definitions and terms follow Reference Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds</p>

(1994) John Wiley &amp; Sons, Inc., New York

Many organic compounds exist in optically 15 active forms, that is, they have the ability to rotate the plane of polar light products. When describing a photoactive compound, the prefixes D and L or R and S are used to indicate the absolute shape of the part around its chiral center. Specific enantiomers can also be referred to as enantiomers, and the aforementioned isomeric mixture is referred to as enantiomers. A 50:50 mixture of enantiomers is referred to as a racemic mixture, which occurs in the absence of 20 stereoselection or stereospecificity in the chemical reaction or process. The expression "graphic mixture" refers to

"Arcimat" refers to a stoichiometric mixture of two types of enantiomers.

The term "selective compounds" indicates that a type of enantiomer in which the transfer of a hydrogen leads to the existence of two or more structures. Compounds with formula I can exist as different tautomers. Those skilled in the art recognize that amidines, amides, guanidines, ureas, thioureas, heterocycles, etc.

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Similar can be found in the form of synthetic compounds. For example, but not limited to, compounds with formula I can be found in many forms of tautomers, as explained below.

5 All forms of possible atomic compounds fall into the following compounds :

Heterocyclic rings and the like, amidines, amides, guanidines, ureas, thioureas

This is for all embodiments having formula I within the scope of the present invention. The nucleotide compounds exist in equilibrium, so the single nucleotide compound is depicted in the formulas provided by those skilled in the art and refers equally to all possible nucleotide compounds.

10 It is shown that enantiomers, dimers, formal mixtures, atomic compounds, polymorphs, and pseudopolymorphs within the range of compounds having formula I and pharmaceutically acceptable salts are included in the present invention. All mixtures of enantiomers and homodimers mentioned, including isomorphically rich mixtures and isotropically rich mixtures, are within the scope of the present invention. Enantiomerically rich mixtures are mixtures of 15 enantiomers where the ratio of identified enantiomers to alternative enantiomers is greater than 50:50. More specifically, a isomorphically rich mixture contains at least about 75% of the identified enantiomer, and

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Preferred by at least 85% of the identified conformer. In one embodiment, the enantiomer-rich mixture is largely free of other enantiomers. Likewise, mixtures rich in diastereomers are mixtures of diastereomers in which the proportion of the specific diastereomer is large compared to the amount of the substituent diastereomer. More specifically, the homodimer-rich mixture 5 comprises at least about 75% of the identified homodimer, and preferably about 85%

At least one of the specified stereograms. In one embodiment, the diastereomeric-rich mixture is substantially free of all other diastereomers. Those skilled in the art will recognize that “substantially free of” refers to the presence of less than 5% of other dimers, preferably less than 1%, and often preferably less than 0.1%. In other embodiments there are no other dimers 10 or the amount of other dimers is less than the detectable level. Enantiomers can be separated by techniques known to those skilled in the art, including high performance liquid chromatography (HPLC).

Chromatography and the crystallization process of chiral salts.

Single enantiomers and enantiomers that are largely free of their enantiomers 15 can be obtained by formal mixture analysis using a method such as the formation of enantiomers

"Stereochemistry of Carbon Compounds," (Using Optically Active Dissolving Agents (1962) by EL Eliel, McGraw Hill; Lochmuller, CH, (1975) J.

Chromatogr 283-302 (3):113). Formal mixtures of the chiral compounds of the invention may be separated and isolated using any suitable method, which includes: (1) the formation of anionic substances, 20 dimer salts with the chiral compounds and separation by the crystallization process Partial or other methods, (2) formation of diastereomers using chiral derivatization agents, separation and conversion of diastereomers, and conversion to pure enantiomers, and (3) separation of pure and highly enriched dimers directly under chiral conditions.

In one embodiment, the present invention provides a conformationally rich mixture or composition comprising:

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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, or a pharmaceutically acceptable salt thereof, as the isomer

<p dir="rtl">5 prevailing.</p>

Other embodiments include conformationally rich mixtures or compositions comprising, respectively, compounds of the formulas (VI), (V), (IV), (III), (II), (Ia), (I), and (VII), or a pharmaceutically acceptable salt thereof, in the form of the dominant isomer in each of their respective mixtures.

In another embodiment, the present invention provides a conformationally rich mixture or composition:

including 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3- 10)

(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

<p dir="rtl">, or a pharmaceutically acceptable salt thereof, largely free of other isomers.</p>

<p dir="rtl">15 The other four models include conformationally rich mixtures or compositions that include, respectively, compounds of the formulas (VI), (V), (IV), (III), (II), (Ia), (I), and (VII). , or a pharmaceutically acceptable salt thereof, are substantially free of other isomers in each of their respective mixtures.</p>

Each of the compounds and groups of compounds described herein are also presented, including those in the formulas (VI), (V), (IV), (III), (II), (Ia), (I), and (Ia). VII), or a pharmaceutically acceptable salt thereof, for use as a medicine.

A compound with formula I and its pharmaceutically acceptable salts may exist in a different polymorph form or in a false polymorph form. As used herein, pseudopolymorphs mean the ability of a crystal compound to assume different crystal structures. can produce

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Pseudo-crystalline polymorphism consists of differences in crystal packing (packing polymorphism) or differences in packing between different conformations of the same molecule (combinatorial polymorphisms). As used herein, pseudo-crystalline polymorphisms also include the ability of hydrates or solutes of a compound to Existing in the form of different crystal structures, multiple images can exist

<p dir="rtl">5 The pseudopolymorphism of the present invention is the result of differences in crystal packing (packing pseudopolymorphism) or differences in packing between different conformational materials of the same molecule (conformational pseudopolymorphism). Polymorphic polymorphs of compounds with formula I and its pharmaceutically acceptable salts.</p>

<p dir="rtl">10 A compound with formula I and its pharmaceutically acceptable salts may also exist in the form of a non-crystalline solid. As used herein, a non-crystalline material is a solid in which the long range positions of the atoms in the solid are no longer arranged. The aforementioned definition also applies when the crystal size is 2 nm or less. Additives, including solvents, can be used to create non-crystalline forms of the present invention. The present invention includes compositions</p>

<p dir="rtl">15 Pharmaceuticals, treatment methods, combination products, and their uses mentioned in this document, on all counts</p>

Non-crystalline forms of compounds with formula I and their pharmaceutically acceptable salts.

Uses

The compounds of the invention demonstrate the effectiveness of sodium channel blockers. Without specifically restricting the theory, it is shown that the compounds of the invention work in living organisms by obstructing epithelial sodium channels located on mucosal surfaces, which works to reduce the absorption of water by the mucous surfaces. The aforementioned effect leads to an increase in the volume of protective fluids on the mucous surfaces, and the system is rebalanced.

Therefore, the compounds of the invention are useful as therapeutic agents, specifically for the treatment of clinical conditions for which a sodium channel blocker is indicated. These conditions include lung conditions such as diseases

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Accompanied by recurrent and non-recurrent airway obstruction, chronic obstructive pulmonary disease (COPD), including exacerbations of COPD, asthma, bronchiectasis (including bronchiectasis resulting from conditions other than cystic fibrosis), inflammation Acute people

<p dir="rtl">5 bronchitis, chronic bronchitis, post-viral cough, cystic fibrosis, emphysema, pneumonia, inflammatory lung disease panbronchiolitis, and transplant-associated bronchiolitis, including bronchitis</p>

lung- and bone marrow-transplant associated

<p dir="rtl">10 bronchiolitis, in a patient requiring treatment. Compounds of the invention may be useful in the treatment of rejuvenator-associated tracheobronchitis and/or the prevention of refrigerant-associated pneumonia in patients due to rejuvenation. The present invention includes methods for treating each of these conditions described herein in a mammal requiring treatment, and preferably in a human being requiring treatment, each method comprising administering to said mammalian organism a therapeutically effective amount of a compound.</p>

<p dir="rtl">15 The present invention, or pharmaceutically acceptable salts thereof. (a) A method for reducing cases is also provided</p>

Exacerbation of COPD in a mammal requires treatment; (b) A method for reducing exacerbations of CF in a mammal requiring treatment; (c) A method for improving lung function (FEV1) in a mammal requiring treatment; (d) A method for improving lung function (FEV1) in a mammal with COPD (e) How to improve lung function (FEV1) in a mammal with CF,

<p dir="rtl">20 (g) A method of reducing the incidence of airway infections in a mammal requiring treatment.</p>

Also provided is a method for stimulating, enhancing or improving mucus ciliary clearance in a mammal, the method comprising administering to a mammal in need of treatment a therapeutically effective amount of a compound having formula (I), or pharmaceutically acceptable salts thereof. It is clarified that mucus clearance involves mucin ciliary processes The natural processes involved in transmission and filtration in the airways, including mechanisms of...

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Self-filtration of bronchial tubes. Thus, a method is provided to improve mucus clearance in the airways of a mammal in need of treatment.

In addition, the use of sodium channel blockers may be suggested to treat conditions that have improved increased mucosal hydration on mucosal surfaces other than those

<p dir="rtl">5 Pulmonary mucosa. Examples of these conditions include dry mouth, dry skin, vaginal dryness, sinusitis, nasal dehydration, nasal dryness, including dry oxygen, Dry eye, Sjogren's disease, otitis media, primary ciliary dysfunction</p>

<p dir="rtl">10 dyskinesia, distal intestinal obstruction syndrome, esophagitis, constipation, and chronic diverticulitis. The compounds of the invention can also be used to promote ocular or corneal hydration</p>

hydration

The compounds of the present invention are also useful in methods for obtaining a nasopharyngeal sample from human organism 15. The method may be performed by administering an effective amount of the compound of the invention into at least one lung of the patient, and collecting a nasopharyngeal sample from the human.

Thus, in one aspect, the present invention provides a method for treating a condition in a mammalian organism, such as a human being, for which the use of a sodium channel blocker is described.

In other embodiments, the present invention provides each of the methods described herein with additional features 20 of reducing or eliminating hyperleukemia in a person subject to the method. It is also provided

Other embodiments include each of the methods mentioned herein where the therapeutic parameter is optimized.

The expression “treat” or “treat,” as used herein, refers to reversing, alleviating, inhibiting the development of, or preventing the disorder or condition or one or more of the symptoms of said disorder or condition.

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All therapeutic methods described herein are performed by administering an effective amount of the compound of the invention, a compound having Formula I or pharmaceutically acceptable salts thereof, to a patient (typically a mammal but preferably a human) in need of treatment.

In one embodiment the invention provides a method for treating a condition improved by increasing the hydrolysis of mucus in an organism

<p dir="rtl">5 A mammal, specifically a human being, in need of treatment. In one embodiment the invention provides a method for treating a disease associated with recurrent and non-reversible airway obstruction in a mammal, specifically a human, in need of treatment. In one specific embodiment the present invention provides a method for treating a disease</p>

In chronic obstructive pulmonary disease (COPD).

A mammal, specifically a human being, in need of treatment. In one particular embodiment the present invention provides

<p dir="rtl">10 A method to reduce the frequency, severity or duration of acute exacerbation symptoms of COPD or to treat one or more symptoms of acute exacerbation of COPD in a mammal, specifically a human being in need of treatment. In one embodiment the invention provides a method for treating asthma in a mammal, specifically a human, in need of treatment. In one embodiment the invention provides a method for treating bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis).</p>

<p dir="rtl">15 In a mammal, specifically a human, in need of treatment. In one embodiment the invention provides a method for treating bronchitis, including acute and chronic bronchitis in a mammalian organism, specifically a human organism, in need of treatment. In one embodiment the invention provides a method for treating a cough following infection with a virus in a mammal, specifically a human being, in need of treatment. In one embodiment the invention provides a method for treating cystic fibrosis in a mammalian organism, specifically</p>

<p dir="rtl">20 A human being, in need of treatment. In one embodiment the invention provides a method for treating emphysema in a mammal, specifically a human being in need of treatment. In one embodiment the invention provides a method for treating pneumonia in a mammalian organism, specifically a human organism in need of treatment. In one embodiment the invention provides a method for treating inflammatory lung disease in a mammalian organism, specifically a human organism in need of treatment. In one embodiment the invention provides a method for treating transplant-associated bronchitis, including:</p>

<p dir="rtl">25 That's transplant-associated bronchitis in the lung and bone marrow of a mammal, specifically</p>

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A human being in need of treatment. In one embodiment the invention provides a method for treating refrigerant-associated tracheobronchitis and/or preventing refrigerant-associated pneumonia in a human subject due to refrigerant in need of treatment.

The invention provides specific methods for treating a disease selected from a group of airway obstructions

<p dir="rtl">5 Recurrent and irreversible, chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis), acute bronchitis, chronic bronchitis, post-viral cough, cystic fibrosis, emphysema , pneumonia, inflammatory lung disease, transplant-associated bronchitis, and ventilator-associated tracheobronchitis or prevention of ventilator-associated pneumonia in a human subject requiring treatment,</p>

<p dir="rtl">10 Each method includes administering to said human being an effective amount of a compound having formula 1(a), or pharmaceutically acceptable salts thereof. In further embodiments of each treatment method, the form of the pharmaceutically acceptable salt is a hydrochloride salt or a hydroxynaphthoate salt of a compound having the formula (1). 1a). In another embodiment within each treatment method, the free base of a compound with the formula (a1) is used.</p>

In one embodiment the invention provides a method for treating xerostomia (dry mouth) in a mammal, in particular

<p dir="rtl">15 Identification: A human being in need of treatment. In one embodiment the invention provides a method for treating dry skin in a mammal, specifically a human being in need of treatment. In one embodiment the invention provides a method for treating vaginal dryness in a mammal, specifically a human being in need of treatment. In one embodiment the invention provides a method for treating sinusitis, rhinosinusitis, or nasal dryness, including nasal dryness resulting from the administration of dry oxygen, in a mammalian organism, in particular</p>

<p dir="rtl">20 Identification: A human being in need of treatment. In one embodiment the invention provides a method for treating dry eye, Sjögren's disease, or enhancing eye and corneal lubrication in a mammalian organism, specifically a human organism in need of treatment. In one embodiment the invention provides a method for treating otitis media in a mammalian organism, specifically a human organism in need of treatment. In one embodiment, the invention provides a method for treating primary ciliary dyskinesia, in a mammalian organism, in particular</p>

<p dir="rtl">25 Identification: A human being in need of treatment. In one embodiment the invention provides a method for treating an occlusion syndrome</p>

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Distal intestinal obstruction syndrome, esophagitis, constipation, or chronic diverticulitis in a mammal, specifically a human, needs to be treated.

The compound of the invention is also provided for use in medical treatment, specifically for use in

<p dir="rtl">5 Treat a condition in a mammal, such as a human, for which the use of a sodium channel blocker has been described. All therapeutic uses mentioned herein are accomplished by administering an effective amount of the inventive compound to a patient in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of a lung injury such as a disease associated with recurrent and non-regressive airway obstruction in a mammalian organism, specifically a human organism, in need of treatment. In one of the specified models</p>

<p dir="rtl">10 The compound of the invention is provided for use in the treatment of chronic obstructive pulmonary disease (COPD) in a mammalian organism, specifically a human organism in need of treatment. In one embodiment, the compound of the invention is provided for use in reducing the frequency, severity or duration of acute worsening symptoms of COPD Or to treat one or more symptoms of an acute exacerbation of COPD in a mammalian organism, specifically a human being, in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of asthma in a mammalian organism</p>

<p dir="rtl">15 Mammals, specifically human beings, need to be treated. In one embodiment a compound is provided for use in the treatment of bronchiectasis, including bronchiectasis resulting from conditions other than cystic fibrosis, or bronchitis, including acute bronchitis and chronic bronchitis, in a mammalian organism, specifically a human organism, Need treatment. In one embodiment a compound is provided for use in the treatment of post-viral cough, in a mammalian organism, specifically a</p>

<p dir="rtl">20 Human, needs treatment. In one embodiment a compound is provided for use in the treatment of cystic fibrosis in a mammalian organism, specifically a human organism in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of emphysema in a mammal, specifically a human, in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of pneumonia in a mammalian organism, specifically a human organism, in need of treatment. In one embodiment the compound of the invention is provided</p>

<p dir="rtl">25 For use in the treatment of inflammatory lung disease or transplant-associated bronchitis, including</p>

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Transplant-associated bronchitis of the lung and bone marrow in a mammal, specifically a human, needs to be treated. In one embodiment the compound of the invention is provided for use in treating airflow-associated tracheobronchitis or preventing airflow-associated pneumonia in a human subject due to airflow in need of treatment.

<p dir="rtl">5 In one embodiment the compound of the invention is provided for use in treating a condition ameliorated by increased mucus hydrolysis of the mucosal surfaces of a mammalian organism, specifically a human organism, in need of treatment. In one embodiment a compound is provided for use in the treatment of xerostomia (xerostomia) in a mammal, specifically a human being, in need of treatment. In one embodiment a compound is provided for use in the treatment of dry skin in a mammalian organism, specifically a human being, in need of treatment. For treatment, in one embodiment</p>

<p dir="rtl">10 Formulated for use in the treatment of vaginal dryness in a mammal, specifically a human being in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of sinusitis, nasal dehydration, or nasal dryness, including nasal dryness resulting from the administration of dry oxygen in a mammalian organism, specifically a human organism, in need of treatment. In one embodiment the compound of the invention is provided for use in...</p>

<p dir="rtl">15 Treating dry eye, Sjogren's disease or promoting ocular or corneal hydration in a mammal, specifically a human, is in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of otitis media in a mammalian organism, specifically a human organism, in need of treatment. In one embodiment the compound of the invention is provided for use in the treatment of primary cilia dysfunction</p>

<p dir="rtl">20 Primary ciliary dyskinesia in a mammal, specifically a human, needs treatment. In one embodiment the compound of the invention is provided for use in the treatment of distal intestinal obstruction syndrome, esophagitis, constipation, or chronic diverticulitis in a mammalian organism, specifically a human organism, in need of treatment.</p>

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The present invention also provides for the use of the compound of the invention in the manufacture of a drug to treat a condition in a mammalian organism, such as a human being, for which the use of a sodium channel blocker is described. In one embodiment the compound of the invention is provided for use in the manufacture of a drug for the treatment of diseases associated with recurrent and non-recurrent obstructive airways disease, chronic obstructive pulmonary disease.

<p dir="rtl">5 COPD disease, acute worsening symptoms of COPD, asthma, bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis), bronchitis (including acute bronchitis and chronic bronchitis), cough post-viral infection, cystic fibrosis, emphysema, pneumonia, inflammatory lung disease, transplant-associated bronchitis (including transplant-associated bronchitis)</p>

<p dir="rtl">10 Lung and bone marrow bone marrow-transplant associated bronchiolitis, ventilator-associated tracheobronchitis or preventing ventilator-associated pneumonia.</p>

In one specific embodiment the compound of the invention is provided for use in the manufacture of a drug to treat a condition improved by increased mucus hydration at mucosal surfaces, the treatment of dry mouth,

<p dir="rtl">15 dry skin, vaginal dryness, sinusitis, nasal dehydration, nasal dryness, including dry oxygen, treatment of dry eye, Sjogren's disease, promoting eye hydration and cornea, treatment of otitis media, primary ciliary dyskinesia, distal bowel obstruction syndrome</p>

<p dir="rtl">20 Distal intestinal obstruction syndrome, esophagitis, constipation, or</p>

chronic diverticulitis

The expressions “effective amount”, “pharmaceutical effective amount”, “effective dose”, and “pharmaceutical effective dose” as used herein, refer to the amount of the compound of the invention which is sufficient for administration to a patient, to demonstrate a biological and medical response to the cell culture, Tissue, organ, or mammary organism) including

<p dir="rtl">25 (human being) to be treated, for example by a researcher or doctor. It also includes the expression in</p>

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Its range, effective amount to promote normal physiological function. In one embodiment, the effective amount is the amount required to deliver the desired level of drug into the excretory processes and tissues of the airways and lungs, or alternatively, into the bloodstream of the treated patient to obtain the desired prior physiological response or biological effect when said composition is administered by

<p dir="rtl">5 Inhalation. For example, an effective amount of the inventive compound to treat a condition for which a sodium channel blocker is prescribed would be sufficient to administer to a patient to treat a specific condition. In one embodiment the effective amount is the amount of the compound of the invention that is sufficient to treat COPD or cystic fibrosis in a human subject.</p>

The exact effective amount of the compounds of the invention depends on several factors, including, but not limited to, the type,

<p dir="rtl">10 The age and weight of the patient to be treated, the specific condition requiring treatment and its severity, the bioavailability, potency, and other properties of the compound being administered, the nature of the formulation, the method of administration, and the means of delivery, are ultimately determined by the medical practitioner or veterinarian. Guidance can be found regarding the appropriate dosage, taking into account the conventional dosage of sodium channel blockers, such as amiloride, taking into account</p>

<p dir="rtl">15 Consider the differences in potency between the amiloride compounds of the present invention.</p>

The pharmaceutically effective dose administered topically to the airway surfaces of the case (i.e., by inhalation) of the inventive compound for treating a human subject weighing 70 kg is in the range from about 10 nanograms to about 10 mg. In another embodiment, the Typically, the effective dosage ranges from about 0.1 to about 1000 micrograms

<p dir="rtl">20 The daily dose administered topically to the airway surfaces in an amount sufficient to achieve the dissolved concentration of the active agent on the airway surfaces ranges from about 10-9, 10-8, or 10-7 to about 10-4, 10- 3, 10-2, or 10-1 moles/L, preferably from about 10-9 to about 10-4 moles/L. The specific dose selection for the patient will be determined by the medical practitioner, physician or veterinarian of ordinary skill</p>

<p dir="rtl">25 in the field based on several factors including those mentioned previously. In one of the specified embodiments it is</p>

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The dose of the compound of the invention to treat a patient weighing 70 kg ranges from about 10 nanograms to about 10 mg. In another embodiment, the effective dose can range from about 0.1 micrograms to about 1,000 micrograms. In one embodiment, the dose of the compound of the invention for treating a 70 kg human subject falls in the range from about 0.5 micrograms to about 0.5 mg. In another embodiment the

<p dir="rtl">5 The dose ranges from about 0.5 micrograms to about 60 micrograms. In another embodiment, it goes away</p>

The pharmaceutically effective dose ranges from about 1 to about 10 micrograms. In another embodiment, the pharmaceutically effective dose ranges from about 5 micrograms to about 50 micrograms. In another embodiment, the effective dose ranges from about 10 micrograms to about 40 micrograms. Typically, the pharmaceutically effective dose ranges from about 15 micrograms to about 50 micrograms from about 15 micrograms.

<p dir="rtl">10 µC to about 30 µC, respectively. It is shown that in each of the dose ranges mentioned, all increased doses are included. For example, the 0.5-50 mcg range includes single doses of: 0.5 mcg, 0.6 mcg, 0.7 mcg, 0.8 mcg, 0.9 mcg, 1.0 mcg, 1.1 mcg. 1.2micro-arm, 1.3micro-arm, 1.4micro-arm, 1.5micro-arm, 1.6micro-arm, 1.7micro-arm, 1.8micro-arm, 1.9micro-arm,</p>

<p dir="rtl">15 2.0µg arm, 2.1µg arm, 2.2µg arm, 2.3µg arm, 2.4µg arm, 2.5µg arm, 2.6µg arm, 2.7µg arm, 2.8µg arm, 2.9µg arm 3.0 micrograms,</p>

3.1micro-arm, 3.2micro-arm, 3.3micro-arm, 3.4micro-arm, 3.5micro-arm, 3.6micro-arm, 3.7micro-arm, 3.8micro-arm, 3.9micro-arm, 4.0micro-arm, 4.1 micrograms,

4.2µg arm, 4.3µg arm, 4.4µg arm, 4.5µg arm, 4.6µg arm, 4.7µg arm

20 G arm, 4.8 micro g arm, 4.9 micro g arm, 5.0 micro g arm, 5.1 micro g arm, 5.2 micro g arm,

5.3micro-arm, 5.4micro-arm, 5.5micro-arm, 5.6micro-arm, 5.7micro-arm, 5.8micro-arm, 5.9micro-arm, 6.0micro-arm, 6.1micro-arm, 6.2micro-arm, 6.3 micrograms,

6.4µg arm, 6.5µg arm, 6.6µg arm, 6.7µg arm, 6.8µg arm, 6.9µg arm, 7.0µg arm, 7.1µg arm, 7.2µg arm, 7.3µg arm, 7.4 micrograms,

25 7.5µg arm, 7.6µg arm, 7.7µg arm, 7.8µg arm, 7.9µg arm, 8.0µg arm, 8.1µg arm, 8.2µg arm, 8.3µg arm, 8.4µg arm 8.5micrograms,

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8.6µg arm, 8.7µg arm, 8.8µg arm, 8.9µg arm, 9.0µg arm, 9.1µg arm, 9.2µg arm, 9.3µg arm, 9.4µg arm, 9.5µg arm 9.6 micrograms,

9.7 µg arm, 9.8 µg arm, 9.9 µg arm,

10.0 micrograms, 10.1 micrograms, 10.2 micrograms, 10.3 micrograms, 10.4 micrograms,

5 10.5 micrograms, 10.6 micrograms, 10.7 micrograms, 10.8 micrograms, 10.9 micrograms,

11.0micro-arm, 11.1micro-arm, 11.2micro-arm, 11.3micro-arm, 11.4micro-arm,

11.5micro-arm, 11.6micro-arm, 11.7micro-arm, 11.8micro-arm, 11.9micro-arm,

12.0µg arm, 12.1µg arm, 12.2µg arm, 12.3µg arm, 12.4µg arm,

12.5micro-arm, 12.6micro-arm, 12.7micro-arm, 12.8micro-arm, 12.9micro-arm,

10 13.0µg arm, 13.1µg arm, 13.2µg arm, 13.3µg arm, 13.4µg arm,

13.5micro-arm, 13.6micro-arm, 13.7micro-arm, 13.8micro-arm, 13.9micro-arm,

14.0µg arm, 14.1µg arm, 14.2µg arm, 14.3µg arm, 14.4µg arm,

14.5micro-arm, 14.6micro-arm, 14.7micro-arm, 14.8micro-arm, 14.9micro-arm,

15.0µg arm, 15.1µg arm, 15.2µg arm, 15.3µg arm, 15.4µg arm,

15 15.5micro-arm, 15.6micro-arm, 15.7micro-arm, 15.8micro-arm, 15.9micro-arm,

16.0µg arm, 16.1µg arm, 16.2µg arm, 16.3µg arm, 16.4µg arm,

16.5µg arm, 16.6µg arm, 16.7µg arm, 16.8µg arm, 16.9µg arm,

17.0µg arm, 17.1µg arm, 17.2µg arm, 17.3µg arm, 17.4µg arm,

17.5µg arm, 17.6µg arm, 17.7µg arm, 17.8µg arm, 17.9µg arm,

20 18.0µg arm, 18.1µg arm, 18.2µg arm, 18.3µg arm, 18.4µg arm,

18.5µg arm, 18.6µg arm, 18.7µg arm, 18.8µg arm, 18.9µg arm,

19.0µg arm, 19.1µg arm, 19.2µg arm, 19.3µg arm, 19.4µg arm,

19.5µg arm, 19.6µg arm, 19.7µg arm, 19.8µg arm, 19.9µg arm,

20.0µg arm, 20.1µg arm, 20.2µg arm, 20.3µg arm, 20.4µg arm,

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20.5µg arm, 20.6µg arm, 20.7µg arm, 20.8µg arm, 20.9µg arm,

21.0µg arm, 21.1µg arm, 21.2µg arm, 21.3µg arm, 21.4µg arm,

21.5micro-arm, 21.6micro-arm, 21.7micro-arm, 21.8micro-arm, 21.9micro-arm,

22.0µg arm, 22.1µg arm, 22.2µg arm, 22.3µg arm, 22.4µg arm,

5 22.5micro-arm, 22.6micro-arm, 22.7micro-arm, 22.8micro-arm, 22.9micro-arm,

23.0µg arm, 23.1µg arm, 23.2µg arm, 23.3µg arm, 23.4µg arm,

23.5µg arm, 23.6µg arm, 23.7µg arm, 23.8µg arm, 23.9µg arm,

24.0µg arm, 24.1µg arm, 24.2µg arm, 24.3µg arm, 24.4µg arm,

24.5µg arm, 24.6µg arm, 24.7µg arm, 24.8µg arm, 24.9µg arm,

10 25.0µg arm, 25.1µg arm, 25.2µg arm, 25.3µg arm, 25.4µg arm,

25.5micro-arm, 25.6micro-arm, 25.7micro-arm, 25.8micro-arm, 25.9micro-arm,

26.0µg arm, 26.1µg arm, 26.2µg arm, 26.3µg arm, 26.4µg arm,

26.5micro-arm, 26.6micro-arm, 26.7micro-arm, 26.8micro-arm, 26.9micro-arm,

27.0µg arm, 27.1µg arm, 27.2µg arm, 27.3µg arm, 27.4µg arm,

15 27.5µg arm, 27.6µg arm, 27.7µg arm, 27.8µg arm, 27.9µg arm,

28.0µg arm, 28.1µg arm, 28.2µg arm, 28.3µg arm, 28.4µg arm,

28.5µg arm, 28.6µg arm, 28.7µg arm, 28.8µg arm, 28.9µg arm,

29.0µg arm, 29.1µg arm, 29.2µg arm, 29.3µg arm, 29.4µg arm,

29.5µg arm, 29.6µg arm, 29.7µg arm, 29.8µg arm, 29.9µg arm,

20 30.0µg arm, 30.1µg arm, 30.2µg arm, 30.3µg arm, 30.4µg arm,

30.5µg arm, 30.6µg arm, 30.7µg arm, 30.8µg arm, 30.9µg arm,

31.0µg arm, 31.1µg arm, 31.2µg arm, 31.3µg arm, 31.4µg arm,

31.5µg arm, 31.6µg arm, 31.7µg arm, 31.8µg arm, 31.9µg arm,

32.0µg arm, 32.1µg arm, 32.2µg arm, 32.3µg arm, 32.4µg arm,

25 32.5µg arm, 32.6µg arm, 32.7µg arm, 32.8µg arm, 32.9µg arm,

33.0µg arm, 33.1µg arm, 33.2µg arm, 33.3µg arm, 33.4µg arm,

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33.5µg arm, 33.6µg arm, 33.7µg arm, 33.8µg arm, 33.9µg arm,

34.0µg arm, 34.1µg arm, 34.2µg arm, 34.3µg arm, 34.4µg arm,

34.5µg arm, 34.6µg arm, 34.7µg arm, 34.8µg arm, 34.9µg arm,

35.0µg arm, 35.1µg arm, 35.2µg arm, 35.3µg arm, 35.4µg arm,

5 35.5µg arm, 35.6µg arm, 35.7µg arm, 35.8µg arm, 35.9µg arm,

36.0µg arm, 36.1µg arm, 36.2µg arm, 36.3µg arm, 36.4µg arm,

36.5µg arm, 36.6µg arm, 36.7µg arm, 36.8µg arm, 36.9µg arm,

37.0µg arm, 37.1µg arm, 37.2µg arm, 37.3µg arm, 37.4µg arm,

37.5µg arm, 37.6µg arm, 37.7µg arm, 37.8µg arm, 37.9µg arm,

10 38.0µg arm, 38.1µg arm, 38.2µg arm, 38.3µg arm, 38.4µg arm,

38.5µg arm, 38.6µg arm, 38.7µg arm, 38.8µg arm, 38.9µg arm,

39.0µg arm, 39.1µg arm, 39.2µg arm, 39.3µg arm, 39.4µg arm,

39.5µg arm, 39.6µg arm, 39.7µg arm, 39.8µg arm, 39.9µg arm,

40.0µg arm, 40.1µg arm, 40.2µg arm, 40.3µg arm, 40.4µg arm,

15 40.5µg arm, 40.6µg arm, 40.7µg arm, 40.8µg arm, 40.9µg arm,

41.0µg arm, 41.1µg arm, 41.2µg arm, 41.3µg arm, 41.4µg arm,

41.5µg arm, 41.6µg arm, 41.7µg arm, 41.8µg arm, 41.9µg arm,

42.0µg arm, 42.1µg arm, 42.2µg arm, 42.3µg arm, 42.4µg arm,

42.5micro-arm, 42.6micro-arm, 42.7micro-arm, 42.8micro-arm, 42.9micro-arm,

20 43.0µg arm, 43.1µg arm, 43.2µg arm, 43.3µg arm, 43.4µg arm,

43.5µg arm, 43.6µg arm, 43.7µg arm, 43.8µg arm, 43.9µg arm,

44.0µg arm, 44.1µg arm, 44.2µg arm, 44.3µg arm, 44.4µg arm,

44.5µg arm, 44.6µg arm, 44.7µg arm, 44.8µg arm, 44.9µg arm,

45.0µg arm, 45.1µg arm, 45.2µg arm, 45.3µg arm, 45.4µg arm,

25 45.5micro-arm, 45.6micro-arm, 45.7micro-arm, 45.8micro-arm, 45.9micro-arm,

46.0µg arm, 46.1µg arm, 46.2µg arm, 46.3µg arm, 46.4µg arm,

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46.5µg arm, 46.6µg arm, 46.7µg arm, 46.8µg arm, 46.9µg arm,

47.0µg arm, 47.1µg arm, 47.2µg arm, 47.3µg arm, 47.4µg arm,

47.5µg arm, 47.6µg arm, 47.7µg arm, 47.8µg arm, 47.9µg arm,

48.0µg arm, 48.1µg arm, 48.2µg arm, 48.3µg arm, 48.4µg arm,

5 48.5µg arm, 48.6µg arm, 48.7µg arm, 48.8µg arm, 38.9µg arm, 49.0µg arm, 49.1µg arm, 49.2µg arm, 49.3µg arm, 49.4µg arm

49.5µg arm, 49.6µg arm, 49.7µg arm, 49.8µg arm, 39.9µg arm,

And 50 micrograms.

10 Previous suggested doses may be adjusted using conventional dose calculations if the compound is administered by a different route. The appropriate dose for administration is determined by other methods in the field according to previous description and general knowledge in the field.

The delivery of an effective amount of the compound of the invention can result in the delivery of a single-dose form or a multiple-dose form which is delivered simultaneously or separately over a period of time exceeding the allotted period, such as 24 hours. The compound of the invention may be dosed (alone or in combination).

15 Comprising the same compound) from one to 10 marts per day. Typically, the compound of the invention will be administered (alone or as a composition comprising it) four, three, twice, or once per day (24 hours).

Compounds of formula (I) of the present invention are useful for treating airborne infections. Examples of airborne infections include, for example, RSV.

20 Compounds having formula (I) of the present invention are useful in treating any anthrax infection

Malicious. The present invention relates to the use of compounds of formula (I) The present invention relates to the use of compounds of formula (I) for prophylaxis, post-exposure prophylaxis, preventive or curative treatment of diseases or conditions resulting from pathogens in preferred embodiments, the present invention relates to the use of compounds of formula (I) for prophylaxis, Post-exposure prophylaxis, preventive or curative treatment against diseases or conditions resulting from etiologies

25 disease, which has been used in bioterrorism.

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In recent years, several research programs and biodefense standards have been activated to deal with matters relating to the use of biological agents in terrorism. The aforementioned criteria relate to bioterrorism or the use of microorganisms or biological toxins to kill individuals, spread fear, and disrupt society. For example, the National Institute of Allergy and Infectious Diseases developed

5 Diseases (NIAID) A plan to conduct biodefense research that outlines plans related to needs

Research on a broad spectrum of bioterrorism and integration of re-emerging infectious diseases. According to the plan, the exposure of a civilian group in the United States of America to Bacillus anthracis spores shows a gap in the overall preparedness of nations against the phenomenon of bioterrorism. Moreover, the report explains that these attacks expose the unmet need for rapid tests, vaccines, and therapeutics.

10 Immunological agents to prevent, and drugs and biological materials to treat disease resulting from bioterrorism agents.

Further efforts also include many research efforts to study the biology of pathogens that are presumed to be dangerous, such as bioterrorism agents, study the host response to the aforementioned agents, develop vaccines against infectious diseases, evaluate the therapeutic agents currently available and subject to investigation against the aforementioned agents, and develop Diagnostics to identify signs and symptoms of threatening factors. Count those

15 The efforts are benign, but given the large number of potential bioterrorism pathogens available, these efforts have not been able to provide satisfactory responses to all potential bioterrorism risks. In addition, many potentially dangerous pathogens such as bioterrorism agents do not provide sufficient economic incentives for the development of therapeutic or preventive standards in the field. Moreover, even if preventive standards such as vaccines for each pathogen are available, which can...

<p dir="rtl">20 For use in bioterrorism, the cost of administering all vaccines to a general population is a constraint.</p>

Until appropriate and effective treatments are available against the risk of bioterrorism, there is still a great need for preventive and curative treatments that can prevent or reduce the incidence of infection resulting from the pathogens causing the disease.

The present invention provides preventive treatment methods. In one aspect, a preventive treatment method is provided including:

<p dir="rtl">25 To give an effective prophylactic amount of compounds having formula (I) to a patient in need of prophylactic treatment against</p>

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Infections from one or more airborne pathogens. A specific example of an airborne pathogen is anthrax.

In another aspect, a preventive treatment method is provided to reduce the risk of infection resulting from an airborne pathogen that causes illness in a human being. The aforementioned method includes administering an

<p dir="rtl">5 A specific example of a pathogen is effective for compounds of formula I in the lungs of a human subject who is at risk of becoming infected with an airborne pathogen and is asymptomatic, where the effective amount of sodium channel blocker resulting from osmotic breakdown is sufficient to reduce the risk of human infection. A specific example of a pathogen is Airborne is anthrax.</p>

In another aspect, post-exposure prophylaxis or a therapeutic method for treating infection with an airborne pathogen includes administering an effective amount of compounds having formula (I) into the lung of an individual in need of said treatment against infections with an airborne pathogen. It includes pathogens that Immunization is carried out through post-exposure prophylaxis, rescue methods and treatment of any pathogen that enters the body through the mouth, nasal tract, and then reaches the lungs. Typically, the pathogens are airborne pathogens15, occurring naturally or by spraying. Pathogens can occur naturally or can be introduced into the media spontaneously after spraying or other methods of introducing pathogens into the media. Many pathogens can be naturally airborne or aerosolized for use in bioterrorism. Pathogens for which the remedy of the present invention is useful may include, but are not limited to, types B, A and C of

<p dir="rtl">20 Priority pathogens as previously described by NIAID. These categories generally correspond to lists approved by the Centers for Disease Control and...</p>

Prevention (CDC). As approved by the CDC, Category A agents are easily spread or transmitted from person to person, increasing the number of deaths, with the potential for public health impacts. Category B agents are close in priority and include those that are moderately easy Spread and can

<p dir="rtl">25 It causes moderate disease and reduces the death rate. Category C consists of the appearance of a pathogen</p>

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Modern viruses that can be genetically modified to spread a mass in the future due to their availability, ease of production and spread, and high probability of disease and death. Specific examples of pathogens are anthrax and plague. Additional pathogens that can be prevented or reduced risk include influenza viruses, rhinoviruses, adenoviruses, respiratory cell viruses, and the like. Another pathogen that must be prevented is the coronavirus, which causes severe acute respiratory syndrome (SARS).

The present invention also relates to the use of sodium channel blockers of Formula I, or pharmaceutically acceptable salts thereof, to prevent, soothe, and/or treat serious health effects on the respiratory tract resulting from radioactive materials, specifically spray agents that can Inhaled radionuclides resulting from nuclear attacks

nuclear attacks, such as the explosion of radiological dispersal devices (RDD), or accidents, such as nuclear power unit disasters. Therefore, a method is provided herein to prevent, soothe, and/or treat serious health effects on the respiratory tract and/or other body organs resulting from inhaled aerosols containing radionuclides.

<p dir="rtl">15 radionuclides in a recipient in need, including in a human subject in need of treatment, said method comprising administering to said human subject an effective quantity of a compound having formula (I), or pharmaceutically acceptable salts thereof.</p>

A key concern is planning for subsequent exposure of group members to radionuclides, including inhaled aerosols, resulting from nuclear attacks, such as a device explosion.

<p dir="rtl">20 Radiological dispersal devices (RDD), or accidents, such as nuclear power plant disasters, in how to prevent, mitigate and treat potential serious health effects on the respiratory tract, primarily in the lungs. It is necessary to have drugs, techniques, procedures, and trained people equipped to control and treat internally infected individuals.</p>

Research has been conducted to determine ways to prevent, mitigate or treat potential injury

<p dir="rtl">25 The respiratory pathway and many parts of the body resulting from the deposition of radionuclides. To our time</p>

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Currently, most research has focused on ways to mitigate the health effects resulting from internal deposition of radionuclides by accelerating their excretion or removal. These methods focused on soluble chemical forms that have the ability to reach the bloodstream and are deposited at distant systemic sites specific to a specific radioactive element. These methods do not work in cases where the deposited radionuclide 5 is in a relatively insoluble form. Studies have shown that it is most, if not,

All the physicochemical profiles of the precipitated radionuclides obtained as RDDs are in a relatively insoluble form.

A known method to effectively reduce the radiation dose to the lung from inhaled insoluble radioactive aerosols is bronchoalveolar lavage.

<p dir="rtl">10 bronchoalveolar lavage or BAL. The technique, which was adapted from techniques already used to treat patients with alveolar proteinosis, appeared to be a safe, repeatable procedure when performed over an extended period of time. Although there are variations in procedures, the basic method for BAL is to anesthetize the patient, followed by slow infusion of isotonic saline into a single lobe of the lung until residual capacity for function is reached. An additional 15 volumes are then added and dried through gravity cavitation.</p>

Results from animal BAL studies indicate that approximately 40% of deep lung content is removed by the acceptable succession of BALs. In some studies, there is significant variation between animals in terms of the amount of radionuclide. The reasons for the change are not currently clear.

Moreover, on the basis of an animal study, it appears that reducing the high dose of BAL treatment leads to attenuation of the health effects resulting from the inhalation of radionuclides.

Insoluble radionuclides. In the study, adult dogs inhaled insoluble 144Ce-FAP particles. Two groups of dogs are given 144Ce lung contents that cause radiation-induced pneumonia and lung fibrosis (about 2 minimum batch amount/kg body mass), with one group treated with 10-sided lavage between 2 and 56 days post-exposure The group was exposed to the infection and the untreated group

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The third infection level is below 144Ce compared to the level seen in the group treated with post-treatment BAL (about 1 minimum batch/kg), but these organisms are untreated. These animals are allowed to survive for the duration of their lives, which extends to 16 years. As the initial lung content of 144Ce among the dogs in each group, the dose rates overlap the cumulative doses in each group. However, the effect of BAL in reducing the risk of pneumonia/fibrosis is evident from the survival curves. In the untreated CAPS group with lung contents of 1.5-2.5 minimum batch volume/kg, median survival was 370 ± 65 days. For the dogs that were done

treated, the mean survival is 1270±240 days, which is statistically significantly different. The third group, which receives lung contents of 144Ce of 0.6-1.4, has the lowest boost amount.

10

15

20

25

The mean survival time was 1800±230, which was statistically significantly different from the treated group. Importantly, compared to the increased survival time, dogs in the group not treated with high doses die from serious lung effects (pneumonitis/fibrosis) while untreated dogs survive. Alternatively, dogs that have been treated, such as dogs in The group not treated with low doses developed lung cancer (hemangiosarcoma or carcinoma). Thus, the dose reduction resulting from treatment with BAL appears to produce biological effects in the lung that would be expected on the basis of radiation doses received by the lungs.

received

On the basis of these results, it is clear that further reducing residual radiation dose using any method or combination of methods to enhance particle clearance from the lung can potentially reduce lung health effects. However, BAL is a procedure that has several drawbacks. BAL is a highly invasive procedure that should be performed in specialized medical centers by trained pulmonologists. Therefore, BAL is expensive. Based on the disadvantages of BAL, it is not the treatment of choice that is readily available and accessible to thousands of individuals who require accelerated removal of radioactive particles, for example, in the event of a nuclear attack. In the event of a nuclear attack or

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In nuclear accidents, there is a need for direct and easy treatment of thousands of individuals at risk of injury. Sodium channel blockers administered via inhaled aerosol restore rehydration of airway surfaces. Hydration of these surfaces of the airways helps in eliminating the accumulation of mucus secretions and related particulate matter.

<p dir="rtl">5 From the lung. Therefore, without being limited to theory specifically, it is clear that sodium channel blockers can be used to accelerate the removal of radioactive particles from the airways.</p>

airway passages

As previously explained, significant lung hazards following a radiation attack, such as contaminant explosion, result from inhalation and retention of insoluble radioactive particles. As a result of detention

<p dir="rtl">10 With radioactive particles, cumulative lung injury is greatly increased, eventually leading to lung fibrosis/pneumonitis and possible death. Insoluble particles are not completely eliminated by chelating agents due to the absence of those particles in solution. To date, physical removal of particulate matter via the BAL method is a treatment method that appears to be effective for remission of radiation-induced lung disease. As explained previously, BAL is not an actual mitigation treatment</p>

<p dir="rtl">15 Radioactive particles inhaled into the body. Therefore, it is preferable to provide a treatment that helps eliminate radioactive particles from the airways. Unlike BAL, the treatment is simple to apply and is graduated according to a large-scale radiation injury scenario. In addition, it is also required that the treatment method be available to a number of individuals in short periods of time.</p>

<p dir="rtl">20 In one aspect of the present invention, a method of preventing, soothing, and/or treating serious health effects on the respiratory tract and/or other body organs resulting from inhaled aerosols containing radionuclides includes the step of administering an effective amount of a sodium channel blocker. It has Formula I or pharmaceutically acceptable salts from it to an individual in need of treatment. In one aspect of this aspect, a sodium channel blocker is given in combination with the product of osmotic glycolysis.</p>

<p dir="rtl">25 Taking into account the mentioned characteristic, the product of osmotic decomposition is excessive brine</p>

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Hypertonic saline (HS) stress. In another aspect, the sodium channel blocker and the product of osmotic decomposition are given in combination with the ion transport rate. Taking also into account that aspect, the anion transport rate can be chosen from the group consisting of β-agonists β CFTR potentiators, purine receptor cofactors

5 purinergic receptor agonists, lubiprostones, and protease inhibitors. In another aspect of this aspect, radionuclides were selected from the group consisting of:

Colbalt-60, Cesium-137, Iridium-192, Radium-226, Phospohrus-32,

Strontium-89 and 90, Iodine-125, Thallium-201, Lead-210, Thorium-

234, Uranium-238, Plutonium, Cobalt-58, Chromium-51, Americium, and 10

Curium. In another aspect, radionuclides are obtained from a radiodeposition device. In yet another aspect, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered as an aerosol suspension for particles that can be inhaled by an individual. In an additional feature, a sodium channel blocker or pharmaceutically acceptable salts thereof are administered prior to exposure to the radionuclide.

15

Installations

20

25

Whereas it is possible to give the compound of the invention alone, in some embodiments it is preferable to provide it in the form of a composition, specifically a pharmaceutical composition (formulation). Hence, in another aspect, the invention provides compositions, specifically pharmaceutical compositions (e.g. Inhalable pharmaceutical compositions) comprising a therapeutically effective amount of the inventive compound in the form of an active ingredient, a pharmaceutically acceptable excipient, a diluent or a carrier. The term “active ingredient” as used herein means a compound of the invention or a combination of two or more compounds of the invention in a pharmaceutical composition. Specific embodiments are also provided where the pharmaceutical composition includes a therapeutically effective amount of a compound having the formulas (VI), (V), (IV), (III), (II), (Ia), (I), and (VII). or pharmaceutically acceptable salts thereof, alone or in combination, and a pharmaceutically acceptable excipient, diluent or carrier.

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In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound having the formulas (I), (VI), (V), (IV), (III), (II), (Ia), and (VII), or salts pharmaceutically acceptable thereof, alone or in combination, in a diluent. In separate embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a compound having the formulas (VI), (V), (IV), (III), (II), (Ia), (I), and (VII), or salts 5 pharmaceutically acceptable, in hypertonic saline, sterile water, and hypertonic saline, respectively, wherein the concentration of the saline solution as described herein in one embodiment is 0.17% w/v and in one embodiment Another 2.8% w/v.

A set is also provided that includes (1) a therapeutically effective quantity of a compound with the formula (Ia), (I), (VI), (V), (IV), (III), (II), and (VII). or pharmaceutically acceptable salts thereof; (2) one or more

of pharmaceutically acceptable excipients, carriers, or diluents; (3) Instructions for administering the Group (1) compound and excipients, carriers, or diluents of Group (2) to a patient requiring treatment; and (4) a container. Patients requiring treatment is any patient requiring treatment using the treatment methods described in this document. It specifically includes 15 human beings in need of treatment. Furthermore, embodiments also include a spray device selected from a range of atomizing devices, including a vibrating mesh atomizer and jet atomization devices, a dry powder inhaler, including both active and passive dry powder inhalers, and a single-dose inhaler. Metered dose inhalers, including pressurized inhalers, dry powder, and soft metered dose inhalers

mis metered dose inhalers 20

In one embodiment the group includes (1) from about 10 nanograms to about 10 mg of a compound having the formula (VI), (V), (IV), (III), (II), (Ia), (I), (VII), or pharmaceutically acceptable salts thereof, per dose; (2) from about 1 to about 5 mL of diluent per dose; (3) instructions for administering the Group (1) compound and the Group (2) diluent to a patient requiring treatment ; 25 and (4) containers. In another embodiment, the diluent ranges from about 1 to about 5 mL of solution.

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Saline, as described herein, per dose. In another embodiment, the diluent ranges from about 1 to about 5 mL of hypotonic saline per dose. In another embodiment, the diluent ranges from about 1 to about 5 mL of hypertonic saline per dose. In yet another embodiment, the diluent ranges from about 1 to about 5 ml

5 of sterile water for each dose.

A kit is also provided that includes (1) a solution containing a therapeutically effective amount of a compound with the formula (VI), (V), (IV), (III), (II), (Ia), (I), and (VII). , or pharmaceutically acceptable salts thereof; dissolved in a pharmaceutically acceptable diluent; (3) instructions for administering the solution to (1) a patient in need of treatment; and (3) a container.

<p dir="rtl">10 A kit is also provided that includes (1) a solution containing from about 10 nanograms to about 10 mg of a compound with the formula (VI), (V), (IV), (III), (II), (Ia), (I). (VII), or pharmaceutically acceptable salts thereof; dissolved in a pharmaceutically acceptable diluent; (3) instructions for administering the solution to (1) a patient in need of treatment; and (3) a container. In another embodiment, the diluent ranges from Approximately 1 to approximately 5 mL of saline, as stated herein, per dose.</p>

<p dir="rtl">15 Another embodiment includes a group comprising (1) a therapeutically effective amount of a compound having the formula (I), (VI), (V), (IV), (III), (II), (Ia) and (VII), or pharmaceutically acceptable salts thereof; in a dry powder form suitable for inhalation (2) Optionally, one or more pharmaceutically acceptable excipients or carriers suitable for inhalation; (3) Instructions for administration of the compound of the group (1) and the excipients or carriers of the group (2) (to a patient in need of treatment; and; (4) a container.</p>

<p dir="rtl">20 In another embodiment, the assembly also includes a dry powder inhalation device suitable for delivering the dry powder formulation to a recipient. The dry powder inhalation device may, in additional embodiments, be a single-dose or multi-dose inhalation device.</p>

Other embodiments of each of the groups mentioned herein include those in which the concentration of a compound having the formula (VI), (V), (IV), (III), (II), (Ia), (I), and (VII) is located. or pharmaceutically acceptable salts thereof,

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per dose, in the effective dose ranges listed herein, including (a) from about 0.1 mcg to about 1,000 mcg; (b) from about 0.5 mcg to about 0.5 mg; and (c) from about 0.5 mcg to about 0.5 mg. About 0.5 micrograms to about 50 micrograms.

Each of the previously mentioned groups has an additional form in which the diluent is the diluent

<p dir="rtl">5 It is a hypertonic saline solution from Turkey mentioned in this document. In another embodiment for each group, the diluent is a hypotonic saline solution of the Turkish azt mentioned herein. In another embodiment of each assembly, the diluent is sterile water suitable for inhalation.</p>

10 Pharmaceutical acceptable excipient(s), diluent(s) or carrier(s) must be acceptable in terms of their compatibility with other components of the formulation that do not present a risk to the recipient of these substances. Generally, the pharmaceutically acceptable excipient(s), diluent(s) or carrier(s) used in a pharmaceutical formulation are “non-toxic” meaning they are safe to use in the amounts delivered in the formulation and are “inert” meaning they are not Interact to a significant extent with or produce an effect on the therapeutic efficacy of the active ingredient(s).

<p dir="rtl">15 Pharmaceutical acceptable excipients, diluents and carriers are conventional in the art and can be selected using conventional techniques, on the basis of the desired route of administration. See, Remington's</p>

Pharmaceutical Sciences, Lippincott Williams &amp; Wilkins; 21st Ed (May 1,

(2005). Preferably, the pharmaceutically acceptable excipient(s), diluent(s) or carrier(s) shall be Generally Regarded As Safe (GRAS).

FDA 20

Pharmaceutical compositions according to the invention include those suitable for oral administration; For administration by injection, including subcutaneously, into the skin, intramuscularly, into a vein and into joints; Topical administration, including topical administration to the skin, eye, ear, etc.; Vaginal or rectal administration; Administration via the respiratory route, including the nasal cavity, sinuses, oral airways

<p dir="rtl">25 Outside the mouth, and in the lung, including using aerosols delivered by species</p>

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Various types of dry powder inhalers, high-pressure metered dose inhalers, wet inhalers, nebulizers, or puffs. The appropriate method of administration can depend on several factors including the patient and the condition or disorder being treated.

<p dir="rtl">5 The formula can be presented in the form of a unit dosage or in mass form, for example in the case of formulas intended to be measured by inhalation means, and it can be prepared by any of the methods well known in the field of pharmacy. Generally, the method includes the step of combining the active ingredient with a carrier, diluent or excipient and optionally one or more additional ingredients. Generally, the formula is prepared by systematically or vigorously combining the active ingredient with one or</p>

<p dir="rtl">10 More liquid carriers, excipient diluents or excipients or finely divided solid carriers, diluents or excipients, or both, and then, if necessary, form the product into the desired formulation.</p>

In one preferred embodiment, the composition is an inhalable pharmaceutical composition that is suitable for inhalation and delivery into an endotracheal space. Typically, this is

<p dir="rtl">15 The composition is an aerosol containing particles for delivery using a nebulizer, metered dose inhaler (MDI), nebulizer, or dry powder inhaler (DPI). The aerosol formulation used in the methods of the present invention can be a liquid (e.g., solution) suitable for administration via a nebulizer, nebulizer inhaler, or MDI, or a suitable dry powder</p>

20 To give via MDI or DPI.

Aerosols used for respiratory drug administration are typically polydisperse; As it consists of particles of different sizes. Typically the particle size distribution is described by the Mass Median Aerodynamic Diameter (MMAD).

Geometric Standard (GSD) and Aerodynamic Diameter

25 Deviation. For optimal intrabronchial drug delivery, the MMAD has a range of approx

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1 to about 10 µM and preferably from about 1 to about 5 µM, and a GSD of less than 3, preferably less than about 2. Aerosols with a MMAD greater than 10 µM are usually too large when inhaled to reach the lung. Aerosols with a GSD greater than about 3 are not preferred for delivery as they deliver a large proportion of the drug to the oral cavity. To achieve the desired particle sizes in a powder formulation, the particle size on the active ingredient can be reduced using conventional techniques such as crushing or spray drying. Non-restrictive examples of other processes or techniques that can be used to obtain respirable particles include spray drying, sedimentation, supercritical fluid, and freeze-drying. The required portion can be separated by grading or air sieving. In one

10 Models, the particles are in crystalline form. For crystalline formulations, particle size is determined by selection of a specific model of nebulization unit, nebulization inhaler, or MDI.

Aerosol particle size distributions are determined using equipment well known in the art. For example, a multi-stage cascade device or other appropriate methods such as those specified in the chapters of the American Pharmacology Encyclopedia appear as distinct devices from the metered dose and dry powder inhaler 15.

Dry powder formulations may be formed for topical delivery to the lung by inhalation without the use of excipients or carriers, alternatively including the active ingredients in a dry powder form that have a particle suitable for inhalation. Dry powder formulations may also include a combination of the active ingredient and a suitable powder base (carrier 20 / diluent / excipient) such as mono-, di- or poly-saccharides (e.g.

lactose or starch). Typically lactose is a preferred excipient for dry powder formulations. When a solid excipient such as lactose is used, the particle size of the excipient is generally larger than the active ingredient to aid in dispersion of the formulation in the inhaler.

Examples of non-captive dry powder inhalers include a 25-dose multi-dose inhaler, multiple pre-measured dose devices, and capsule-based inhalers.

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and single-dose inhalers. An inhaler reservoir contains a large number of doses (such as 60) in a single container. Before inhalation, the patient turns on the inhaler, causing the inhaler to measure a single dose of medication obtained from the reservoir and prepare it for inhalation. Examples of inhaler reservoirs include DPIs for example

5 Exclusively Turbohaler® by AstraZeneca and ClickHaler® by Vectura.

In a pre-measured multi-dose nebulizer, each single dose is prepared in a separate container, and turning on the nebulizer before inhalation releases a new dose of drug from its container and prepares it for inhalation. Examples of multidose DPI sensors include but are not limited to SKUS® acquired from GSK,

10 Gyrohaler® obtained from Vectura, and Prohaler® obtained from Valois. During inhalation, the patient's respiratory flow accelerates the exit of the powder from the device and into the oral cavity. For a capsule inhaler, the formula is contained in a capsule and stored outside the inhaler. The patient pops the capsule into the inhaler, turns on the inhaler (and pierces the capsule), and then inhales. Examples include the RotohalerTM

(IB) HandiHalerTM, (Novartis) SpinhalerTM, (GlaxoSmithKline) 15

TurboSpinTM (PH&amp;T). With single-use inhalers, the patient turns on the inhaler to prepare it for inhalation, inhales, then disposes of the inhaler and packaging. Examples include TwincerTM (U Groningen), OneDoseTM (U GroningenTM).

(Manta Devices) and Manta InhalerTM (GFE).

20 In general, a dry powder inhaler uses the turbulent flow properties of the powder path

To disperse the excipient-drug accumulation products and deposit the active ingredient particles in the lung. However, a specific dry powder inhaler uses a vortex dispersion chamber to produce particles of the desired shape. In a vortex dispersion chamber, the drug enters the circular dispersion chamber tangentially so that the flow path and the drug move along the outer chamber wall. As the drug formula moves along the wall

25 The aforementioned round bounces off and the agglomeration product is broken away by the impact forces. The air path turns towards

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The center of the orthogonally projecting chamber. Particles that have sufficiently small dynamic sizes can follow the flow path and exit the chamber. When applied, the dispersion chamber acts like a small jet mill. Based on the properties of the formulations, macroformulated lactose particles can be added to assist in dispersion through impaction with API particles.

5 The TwincerTM single-dose inhaler appears to operate using a circular vortex dispersion chamber referred to as an “air classifier” See US Patent Publication No. 2006/0237010 of Rijksuniversiteit Groningen. Research published by the University of Groningen has shown that a 60 mg dose of pure powdered colistin sulfomethate can be effectively delivered as an inhalable dry powder (Technique 10).

In preferred embodiments, the aerosol formulation is delivered in dry powder form using a dry powder inhalation device where particles are emitted from the inhalation device comprising an MMAD in a range from about 1 µm to about 5 µm and a GSD of less than about 2.

Suitable examples of a dry powder inhaler and dry powder dispenser 15 for use in the delivery of compounds and compositions according to the present invention include but are not limited to those disclosed in US Patent No. 7,520,278; US Patent 7,322,354; US Patent 7246617; US Patent 7231920; US Patent 7,219,665; Innocence

American 7207330; US Patent 6880555; US Patent 5,522,385; US Patent 6,845,772; US Patent 6,637,431; US Patent 6,329,034; Innocence

20 American 5,458,135; US Patent 4,805,811; And the published US patent bulletin

0237010/2006.

In one embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation which is shaped for delivery by a Diskus®-type device. The Diskus® device comprises an elongated strip formed from a core foil with a set of recesses spaced along its length and a sanitary covering foil

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They can be peel-sealed to identify a set of containers, and each container contains within an inhalable formulation containing a predetermined amount of the active ingredient alone or in mixture with one or more carriers, excipients (e.g., lactose) and/or other therapeutic active agents. Preferably, the tape is flexible enough to be folded into a reel.

<p dir="rtl">5 Preferably the top sheet and the base sheet shall have end portions which are not tight to each other and at least one of the guide end portions shall be formed to secure them to the winding means. Preferably, the sanitary seal between the base and top sheets shall preferably extend the full length of their width. To prepare the inhalation dose, the top foil can preferably be peeled off from the base foil in the longitudinal direction from the first end of the base foil.</p>

<p dir="rtl">10 In one embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation that is shaped for delivery using a single-dose disposable inhaler, specifically a TwincerTM inhaler. The TwincerTM nebulizer includes an aluminum foil strip with one or more recesses and a sanitary cover foil which is removably sealed to identify a group of containers. Each container includes a reusable formula</p>

<p dir="rtl">15 For inhalation includes a pre-determined amount of the active ingredient(s) alone or in a mixture with one or more carriers or excipients (e.g., lactose). The top wafer preferably includes percussion guide portions that are shaped to protrude from the body of the inhaler. The patient is able Turn on the device and then administer the aerosol formulation by (1) removing the outer packaging, (2) removing the ammonium strip to expose the drug in the strip, and (3) inhaling the drug from the strip.</p>

<p dir="rtl">20 In another embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation wherein the dry powder is formed into fine particles as described in PCT Publication ISBN 015286/2009 or ISBN 114881/2007, both for NexBio. These microparticles are generally formed by adding any corresponding ion to a solution containing the compound of the invention in a solvent, adding an antisolvent to the solution; Gradually cooling the solution to a temperature</p>

<p dir="rtl">25 Less than about 25 m, to form a composition including fine particles comprising the compound. can then</p>

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Separating the fine particles comprising the compound from the solution by any suitable means such as sedimentation, filtration or freeze-drying. Suitable corresponding ions, solvents and antisolvents for preparing fine particles of the compounds of the invention are described in the international application.

015286/2009.

5 In another embodiment, the pharmaceutical composition according to the invention is delivered in dry powder form using a metered dose inhaler. Non-restricted examples of metered dose inhalers and devices include those disclosed in US Pat. 5,261,538; US Patent 5,544,647; US Patent 5,622,163; US Patent 4,955,371; US Patent 3, 565,070; U.S. Patent 3,361,306, U.S. Patent 6,116,234, and U.S. Patent 6,116,234.

10 7,108,159. In preferred embodiments, the compound of the invention is delivered in dry powder form

Using a metered dose inhaler, the particles emitted have an MMAD ranging from about 1 micrometer to about 5 micrometers and less than about 2.

Liquid aerosol formulations can, for example, be formed for delivery into the bronchoalveolar space or lung by inhalation, as aqueous solutions or suspensions, or as aerosols delivered from

15 High-pressure fillings, such as metered-dose inhalers, with appropriate liquid propellants, wet inhalers, or nebulizers. Aerosol formulations suitable for inhalation may be a suspension or solution and generally include the active ingredient(s) in combination with a pharmaceutically acceptable carrier or diluent (e.g., water (distilled or sterile), saline, hypertonic saline, Or ethanol (optionally one

20 or more other therapeutic active agents.

Aerosol formulations for delivery by high-pressure metered dose inhalers typically also include a pharmaceutically acceptable propellant. Examples of said propellants include fluorocarbon, fluorocarbon containing hydrogen, or mixtures thereof, specifically hydrofluoroalkanes, e.g., dichlorodifluoromethane,

25 dichlorotetrafluoroethane, trichlorofluoromethane, specifically -1,1,1,2

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1,1,1,2,3,3,3,-heptafluoro-n-propane, tetrafluoroethane or mixtures thereof. The aerosol formulation may be excipient-free or optionally the formulation may include additional excipients well known in the art such as surfactants such as oleic acid or lecithin and co-solvents such as ethanol. Formulas are usually held high

5 Pressure in a can (e.g., aluminum can) closed by a valve (e.g., metering valve) and mounted on an actuator with a mouthpiece.

In another embodiment, the pharmaceutical composition according to the invention is delivered in liquid form using a metered dose inhaler. Non-restricted examples of metered dose inhalers and devices include those disclosed in US Pat. No. 6,253,762,

10 6,413,497, 7,601,336, 7,481,995, 6,743,413, and 7,105,152. In models

Preferably, the compound of the invention is delivered in dry powder form using a metered dose inhaler wherein the emitted particles have an MMAD in the range from about 1 µm to about 5 µm and a GSD of less than about 2.

In one embodiment the aerosol formulation is suitable for spraying by jet atomization or wave atomization

15 Ultrasonic including fixed and vibrating porous dishes and atomization devices. Liquid aerosol formulations for atomization may be formed by dissolution to reconstitute the solid particle formulation or may be formed with an aqueous excipient by adding agents such as acid or alkali, brine salts, and isotonic adjusting agents. They can be sterilized by techniques such as filtration, or downstream processes such as heating in an autocalf or gamma ray. It can also be served in

20 Unsterilized image.

Patients may develop sensitivity to the pH, osmosis, and ionic content of the nebulization solution. Therefore, these variables must be adjusted to match the active ingredient and so that the patient is able to tolerate them. The preferred solution or suspension of the active ingredient will have a chloride concentration of &lt;30 mM at pH 4.5-7.4, preferably 5.0-5.5, and an osmosis of

25 From about 800-1600 mOsm/kg. The pH of the solution can be controlled

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By titration with known acids (hydrochloric acid or sulfuric acid, for example) or bases (sodium hydroxide, for example) or by the use of buffer solutions. The buffer solutions typically used include buffer solutions of citrate, such as citric acid/sodium buffer solutions Of citrate, buffered solutions of acetate, e.g

5 acetic acid/sodium buffered solutions of acetate, and buffered solutions of... The resistance of the buffer solution can range from about 2 mM to 50 mM.

Useful examples include phosphate, acetate, and buffer solutions of sodium citrate.

potassium, ammonium acetate, sodium acetate trihydrate, acetate

sodium phosphate, sodium phosphate, dibasic acetate, sodium

potassium dihydrogen, disodium hydrogen phosphate, dibasic phosphate 10

Sodium citrate, phosphate, and potassium citrate. Other buffer solutions that may be used include:

sodium hydroxide, potassium hydroxide, ammonium hydroxide, aminomethylpropanol, tromethamine, tetrahydroxypropyl ethylenediamine,

citric acid, acetic acid, hydroxytricarboxylic acid 15

Or a salt thereof, such as salts of citrate or sodium citrate thereof, lactic acid, and salts of lactic acid including:

sodium lactate, potassium lactate, lithium lactate, calcium lactate, magnesium lactate, barium lactate, aluminum lactate, zinc lactate, silver

lactate, copper lactate, iron lactate, manganese lactate, ammonium 20 lactate, monoethanolamine, diethanolamine, triethanolamine,

diisopropanolamine

, in addition to combinations thereof, and the like.

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These formulations may be administered using commercially available nebulization devices or another nebulization device capable of breaking the formulation into particles or droplets suitable for deposition into the respiratory tract. Non-restrictive examples of nebulization means that may be used for aerosol delivery of the composition of the invention include airjet nebulization means, air or breath-assisted nebulization means, or nebulization means

<p dir="rtl">5 Ultrasonic, including atomization methods with fixed or rotating porous dishes. Includes and means</p>

Commercially available nebulizers are Aeroneb® Go nebulizer (Aerogen) and eFlow nebulizer (Pari (Pharma).

A jet atomizer uses a high-velocity stream of air blown through a water column to produce droplets. Particles unsuitable for inhalation affect walls or aerodynamic barriers.

<p dir="rtl">10 The air-assisted nebulizer, or breather, operates essentially the same as a jet nebulizer except that inhaled air passes through the primary droplet generation area to increase the nebulizer's output rate as the patient inhales.</p>

In the ultrasonic sputtering process, piezoelectric crystal vibration causes surface variations in the drug reservoir such that droplets are formed. In atomization media with porous dishes,

<p dir="rtl">15 The generation of pressure fields by a liquid forces sound energy through the pores of the lattice where it breaks into droplets by the Rayleigh refraction process. The sound energy may be supplied by a vibrating horn or plate guided by a piezoelectric crystal, or by the vibrating grid itself. Examples of non-restricting sprayers include any single or double liquid sprayer or nozzle that produces droplets of the appropriate size. The single fluid spray works by forcing liquid through one or more nozzles.</p>

<p dir="rtl">20 The process of jetting the liquid causes it to disperse into droplets. Dual-fluidic atomizers operate by either gas or liquid forces through one or more nozzles, or by the impingement of the liquid jet by another liquid or gas jet.</p>

Choosing a nebulization medium that atomizes the aerosol formulation is essential in delivering the active ingredient(s). Different nebulization media have different efficiency rates based on their design

<p dir="rtl">25 The operating principles are sensitive to the physical and chemical properties of the formulas. For example, be</p>

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The two formulas having different surface tensions have different particle size distributions. In addition, formulation properties such as pH, osmosis, and permanent anion content can affect drug tolerability, and preferred models correspond to the specified ranges for those properties.

5 In a preferred embodiment, the nebulization formulation is delivered into the endotracheal space as an aerosol with a MMAD ranging from about 1 micrometer to about 5 micrometer and less than 2 GSD using an appropriate nebulization medium. To be optimally effective and to avoid upper respiratory and systemic side effects, the aerosol must not have a MMAD of more than about 5 microM and must not contain a GSD of more than about 2.

10 About 5 micrometers or a GSD exceeding about 2, a large percentage of the dose is deposited in the upper airways, which reduces the amount of drug that is delivered to the lower respiratory tract. If the MMAD of the aerosol is less than about 1 micrometer, a large amount of particles remain suspended in the inhaled air and can be exhaled during exhalation.

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

15 Formulations suitable for oral administration may be provided in separate units such as capsules, blister packs or tablets, each containing a pre-determined amount of the active ingredient; In the form of powder or granules; In the form of a solution or suspension in an aqueous or non-aqueous liquid; Or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient is also available in the form of a sachet, bolus, lozenge or paste.

20 The tablet may be formed by compression or forging, optionally with one or more additional components. Compressed tablets may be prepared by pressing in a suitable machine the active ingredient into a free-flowing form as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active agent or dispersing agent. Molded tablets can be prepared by molding in a suitable machine a mixture of powdered compound moistened with a substance

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Inert liquid diluent. The tablets may optionally be coated or scored and may be shaped to provide a controlled or slow release of the active ingredient.

Formulations for topical administration in the mouth, for example buccal or sublingual, include emulsions, comprising the active ingredient in a flavored base such as sucrose, gum arabic or tartar gum, tablets comprising the active ingredient in a base such as gelatin and glycerin or sucrose and gum arabic acacia.

Parenteral formulations for administration include sterile aqueous and non-aqueous injection solutions containing antioxidants, buffer solutions, bacterial stabilizing agents and degradation products that make the formulation isotonic with the recipient's blood; and aqueous and non-aqueous sterile suspensions which may include suspension agents and thickening agents. Formulations can be provided in single-dose or multi-dose containers, e.g. ampoules and sealed vials, and can be stored in a freeze-drying medium requiring only the addition of a sterile liquid carrier, e.g. saline or water - for injections, Immediately before use. Solutions for concurrent injection and suspensions may be prepared from sterile powders, granules and tablets of the type mentioned above.

15 Oral fluids such as solutions, syrups and elixirs can be prepared as a unit dosage such that the specified quantity includes a pre-determined amount of the active ingredient. Drinks can be prepared by dissolving the active ingredient in any suitable flavored aqueous solution, while elixirs are prepared by using a pharmaceutically acceptable alcohol excipient. Suspensions may be formed by dispersing the active ingredient in a pharmaceutically acceptable excipient. Factors may also be included

20 Solubilizing agents such as isostearyl ethoxylate alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavoring agents such as peppermint oil, natural sweeteners, saccharin or other artificial sweeteners, and the like in oral liquid formulations.

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Liposome delivery systems such as small monolayer vesicles, large monolayer vesicles and multilayer vesicles can also be used as delivery vehicles for the compounds of the invention. Liposomes can be formed from a variety of phospholipids such as:

phosphatidylcholines, stearylamine, cholesterol

5 Pharmaceutical compositions for topical administration may be formed into the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Formulations intended for the treatment of the eye or other external tissues, for example the mouth and skin, can be applied as a topical ointment or cream. When forming the formulation into an ointment, the active ingredient may be used with a parvin or water-miscible ointment base. Alternatively, the active ingredient can be formed into a cream with

10 Oil-in-water cream base or water-in-oil base.

Other formulations intended for topical administration into the eye or ear include eye drops and ear drops in which the active ingredient is dissolved or suspended in a suitable carrier, such as for example an aqueous solvent, including saline.

Compositions for nasal administration include aerosols, solutions, suspensions, aerosols, sprays

15 And art cat. Sprayable nasal formulations can be formed in the same manner as other spray formulations

Sprayable inhalants provided that the particle size of non-inhalables in nasal administration formulations is preferred. Typically, particles of about 5 microns in size can be used, which grow to the size of visible droplets. Therefore, intranasal administration, with a particle size of 10-500 µm, can be used to ensure retention in the nasal cavity.

20 Transdermal pads can also be used, which are designed to be in contact with the patient's skin for an extended period of time and to enhance absorption of the active ingredient.

Formulations for vaginal or rectal administration include ointments, creams, suppositories and enemas, all of which are formed using traditional techniques.

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In another aspect, the invention provides a method for enhancing the hydration of mucosal surfaces or restoring mucosal defense in a human subject in need of treatment, comprising administering to the human subject a pharmaceutical composition comprising the compound of the invention, wherein said compound is administered in an effective quantity. In one preferred embodiment, the method comprises administering the pharmaceutical composition in the form of an inhalable composition comprising an amount of

5 The compound of the invention, which is sufficient to obtain the dissolved composition of the compound on the airway surfaces of about 10-9, 10-8, or 10-7 to about 10-4,10-3, 10-2, or 10-1 mol/ litres, preferably often from about 10-9 to about 10-4 moles/litre.

In another aspect, the invention provides a method for treating any of: a disease associated with airway obstruction

10 Recurrent and non-recurrent, chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis), acute bronchitis, chronic bronchitis, post-viral cough, cystic fibrosis, emphysema Lung, pneumonia, inflammatory lung disease, transplant-associated bronchitis, and airway-associated tracheobronchitis or prevention of airway-associated pneumonia in a human being

15 In need of treatment, it includes administering to a human being a pharmaceutical composition that includes the compound of the invention, whereby the said compound is administered in an effective quantity. In one preferred embodiment, the method comprises administering the pharmaceutical composition in the form of an inhalable composition comprising an amount of the compound of the invention sufficient to have the composition dissolved on airway surfaces of about 10-9, 10-8, or 10-10. 7 to about 10-4,10-3, 10-2, or 10

20 1 mol/L, preferably often from about 10-9 to about 10-4 mol/L.

In another aspect, the invention provides a method for treating any of dry mouth, dry skin, vaginal dryness, sinusitis, nasal dehydration, or nasal dryness, including resulting nasal dryness. When giving dry oxygen, dry eye or Sjögren's disease

25 Sjogren's disease, promotion of hydration of the eye and cornea, treatment of distal bowel obstruction syndrome

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distal intestinal obstruction syndrome, treatment of otitis media, primary ciliary dyskinesia, distal intestinal obstruction syndrome, esophagitis, constipation, or chronic diverticulitis in a human subject requiring treatment, including the administration of the organism

5 Human is a pharmaceutical composition that includes the compound of the invention, where the said compound is administered in an effective quantity.

The preferred unit dosage forms for the compounds of the invention are those that include an effective amount of the active ingredient or an appropriate portion thereof.

It is shown that in addition to the components specifically mentioned above, the formulas of the invention can include other factors conventional in the art, taking into account that the type of formula it is about

For example, those suitable for oral administration may include flavoring agents.

The compositions of the present invention may be formed for direct, controlled, or sustained release administration as required for the specific conditions being treated and the method of administration required. For example, a metered-release formulation for oral administration is required to treat constipation.

15 To make the best possible delivery of the active agent into the colon. Those formulations and suitable excipients can be well known in the field of pharmacy. Since the free base of a compound is generally less soluble in aqueous solutions than a salt, formulations containing a free base of a compound of formula I can be used to provide sustained release of the active agent delivered by inhalation into the lung. The active agent present in the lung is not in a specific form which is not

20 Soluble in solution Available to induce a physiological response, but acts as a buffer for bioavailable drug that gradually dissolves in solution. As another example, the formulation may utilize both the free base and the salt form of the inventive compound to provide both direct release and sustained release of the active ingredient to dissolve in nasal mucus secretions, for example.

Combinations

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The compounds of the invention may be formed and/or used in combination with other therapeutic active agents. Examples of other therapeutic active agents formed or used in combination with the compounds of the invention include, but are not limited to, osmotic breakdown products, anti-inflammatory agents, anticholinergic agents, β-cofactors (including

5 These include facultative β2 cofactors, P2Y2 receptor cofactors, and delta cofactors

Peroxisome proliferator-activated receptor (PPAR)

receptor, other epithelial sodium channel blockers (ENaC receptor or blockers), CFTR fibrosis transmembrane conductance regulator, kinase inhibitors

10 inhibitors, antiinfective agents, antihistamines, non-biotic anti-inflammatory macrolides, elastase and protease inhibitors, mucus or mucin modifying agents, such as substances Surfactants. In addition,

15 For cardiovascular symptoms, compounds of the invention may be used in combination with beta-agonists, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents.

The present invention thus provides, as a further aspect, a composition comprising an effective amount of the compound of the invention and one or more other therapeutic active agents selected from the products of osmotic decomposition,

20 Anti-inflammatory agents, anticholinergic agents, β-cofactors (including selective β2 cofactors), P2Y2 receptor cofactors, PPAR delta cofactors, ENaC receptor or antagonists, transmembrane conductance modulators in cystic fibrosis ( CFTR, kinase inhibitors, anti-transmissible agents, antihistamine agents, non-antibiotic macrolides, elastase inhibitors

25 and proteases, agents that transfect mucus or mucin, such as surfactants. Therefore it saves

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The present invention, as another aspect, is a composition comprising an effective amount of the compound of the invention and one or more other therapeutic 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 other therapeutic active agents (specifically osmotic degradation products) can reduce the dose of the compound of the invention required to adequately hydrate mucosal surfaces, thereby reducing the potential for unwanted side effects attributable to regimens. Obstruction of sodium channels, such as for example in the kidneys.

“Osmolytes” according to the present invention are osmotically inactive molecules or compounds. “Osmotically active” molecules and compounds are impermeable (i.e., essentially non-absorbable) membranes on the airways or pulmonary epithelial surface. The terms “airway surface” and “pulmonary surface,” as used herein, include pulmonary airway surfaces such as bronchi and bronchioles, alveolar surfaces, nasal surfaces and sinus surfaces. . Suitable osmotic degradation products include ionic osmolytes (i.e., salts) and non-ionic osmolytes (i.e., sugars, sugar alcohols, organic osmolytes). Osmotic decompositions (both ionic and non-ionic) used in combination with the compounds of the invention 20 preferably are osmotic decomposition products that do not promote, or that inhibit, bacterial growth.

The osmotic decomposition products suitable for use in the present invention shall be in the formal form or in the form of an enantiomer, dimer, monomeric compound, polymorph or pseudopolymorph.

25

Examples of useful ionic osmotic decomposition products in the present invention include any pharmaceutically acceptable anion salt and a pharmaceutically acceptable cation. Preferably, either (or both) of the anion and cation are osmotically active and not subject to rapid active transport, to airway surfaces

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airway surfaces used on them. These compounds include, but are not limited to, anions and cations contained in salts available on the market that have been approved by the FDA. See, for example, Remington: The Science and Practice of Pharmacy (1995). Vol. II, pg. 1457 (19th). Ed, can be used in any combination as is known.

5 Specific examples include, but are not limited to, pharmaceutically acceptable and osmotically active anions.

acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate, edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate), esylate (1,2) -ethanedisulfonate), fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate (p- 10

glycollamidophenylarsonate), hexylresorcinate, hydrabamine (N,N'-Di(dehydroabietyl)ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate , nitrate, nitrite, pamoate (embonate), 15 pantothenate, phosphate or diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate (8-

chlorotheophyllinate), triethiodide, bicarbonate, etc. Includes favorite anions

chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide

20 And phosphate.

Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to,

benzathine (N,N'-dibenzylethylenediamine), organic cations such as chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine), procaine, D-lysine, L-lysine, D-arginine, L-metallic and the like that; And arginine, triethylammonium, N-methyl D-glycerol 25

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Aluminum, calcium, lithium, magnesium, potassium, sodium, as cations

zinc, iron, ammonium, and the like. Preferred organic cations include 3 carbon, 4 carbon, 5 carbon and 6 carbon organic cations. Favorite cations include:

sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, 5

magnesium, and calcium.

Specific examples of ionic osmotic decomposition products that may be used in combination with the compound of the invention include, but are not limited to, sodium chloride (specifically saline

potassium chloride, choline chloride, choline, hypertonic saline iodide, lithium chloride, meglumine chloride, L-lysine chloride, D-lysine 10 chloride, ammonium chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride,

potassium bromide, and combinations of two or more of the above. In one embodiment, the present invention provides a combination of the compound of the invention and two different osmotically active salts. When using 15 different salts, it is possible to have one anions or one cations between the different salts.

The hypertonic saline solution is a preferred ionic product for use in combination with the compounds of the invention.

Non-ionic osmolytes include sugars, sugar-alcohols, and organic osmolytes.

20 Sugars and sugar alcohols useful as osmotic hydrolysis products of the present invention include, but are not limited to, 3-carbon sugars (e.g., glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both D-form, erythrose, L-threose, and erythrulose). ; sugars consisting of 5 carbons (e.g., both D and ribose, L).

; And sugars (tagatose, arabinose, xylose, lyxose, psicose, fructose, sorbose altose, allose, glucose, mannose, from L and D 25 have 6 carbons) (e.g., each of the pictures

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Allo-heptulose, allo- L and D, and the pictures, gulose, idose, galactose, and talose hepulose, gluco-heptulose, manno-heptulose, gulo-heptulose, ido- include the beneficial sugars when (heptulose, galacto-heptulose, talo-heptulose).

The present invention applies raffinose, a group of oligosaccharides, and stachyose.

5 Both forms D and L of the reduced form of sugar and sugar alcohol are suitable for the present invention. For example, glucose, upon reduction, becomes sorbitol; And the product of osmotic decomposition within the scope of the invention. Therefore, sorbitol and other reduced forms of sugars/sugar alcohols (e.g., arabitol, dulcitol, mannitol) are suitable osmotic degradation products for use in the present invention. Mannitol is a non-ionic degradation product 10 for use in combination with compounds of the invention. Take care.

In general, “products of organic osmotic decomposition” are used to refer to the molecules that control...

J. S. Handler et al., Comp. Intracellular osmosis in the kidney. See, for example Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol. 268,

(1995) F983-F996. Osmotic decomposition products include, but are not limited to, organic matter

Polyols (polyhydric alcohols), methylamines, and polyols: 15: three main classes of compounds

amino acids. Organic osmotic decomposition products include, but are not limited to, inositol

The appropriate amine for the products of osmotic decomposition includes methyl. myo-inositol, and sorbitol

Membership includes, but is not limited to:

choline, betaine, carnitine (L-, D- and DL forms), phosphorylcholine, lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatine 20

phosphate. Amino acids suitable for the products of organic osmotic decomposition include others

glycine, alanine, glutamine, glutamate, aspartate, -L and -D to name a few, images

proline and taurine. Additional organic osmotic decomposition products suitable for use in the present invention include tihulose and sarcosine. The organo-osmotic breakdown products of mammalian organisms 25 are preferable, while the human organo-osmotic breakdown products are more preferable. however,

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The identified organic osmotic decomposition products are of bacterial, yeast, or marine origin, and these compounds can be used in the present invention.

Substances producing the decomposition product may be used in combination with the compounds of the invention. The term “decomposition product” as used herein refers to a compound that is converted into a degradation product

5 By a metabolic step, either a catabolic or anabolic step. Examples of substances that produce an output include:

glucose, glucose polymers, glycerol, choline, hydrolysis, including but not limited to phosphate and phosphatidylcholine compounds, lyso-phosphatidylcholine

Organic, which are substances that produce polyols and methylamines. Substances that produce amino acids for osmotic breakdown products include proteins, peptides, and polyaminos.

10 acids, which are hydrolyzed to produce amino acid degradation products, and metabolic products, which are converted into amino acid degradation products by a metabolic step such as osmolyte amino acids. For example, the substance producing amino acid glutamine is poly-L-glutamine, and the substance producing glutamate is poly-L-glutamic acid.

15 Chemically modified osmotic decomposition products or materials that produce the decomposition product can also be used. Chemical modifications involve attaching an additional chemical group to the catalytic converter (or substance) that alters or enhances the effect of the catalytic converter (or substance producing the catalytic converter) (e.g., inhibiting the breakdown of the catalytic converter molecule). The chemical modifications mentioned have been used using drugs or drugs. known primaries in the field (see, for example, patents).

20 US No. 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)

Osmotic decomposition products preferred for use in combination with the compounds of the invention include sodium

25 chloride, specifically hypertonic saline, and mannitol

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For the formation of 7% and &lt;7% hypertonic saline, formulations including bicarbonate anions can be particularly useful, particularly those for apnea associated with impaired transmembrane conduction rates in fibrosis.

C such as cystic fibrosis transmembrane conductance regulator (CFTR).

5 For the duration of COPD. Recent findings indicate that, although the relative ratio of HCO3 conduction/Cl − conductance is between 0.1 and .2 for single cAMP- and ATP-activated CFTR channels, the ratio in the sweat channel can actually range from 0 to nearly 1.0, although Basis of Stimulation Conditions In other words, the combination of cAMP + cGMP + α-ketogaloate leads to conduction

Cl–(Quiton et al. Physiology, Vol. 22, No. 3, equal to the CFTR HCO3– conduction

10 2007 212-225, June). Furthermore, the formulas of 7% and &lt;7% solution can be

Hypertonic saline containing bicarbonate anions is beneficial precisely because of its good pH control at the surface of the airways. Initially, the airways appear to be converted to an acidic state in Tate et al. CF (2002) Absent CFTR, which depends on bicarbonate secretion, can lead to impaired ability to

15 Response to airway conditions associated with the acidification process of the liquid layer of the airway surface (Coakley et al. 2003). Second, adding a HS solution without bicarbonate to the lung surface can also dilute the bicarbonate concentration, and potentially reduce the pH or It depends on the response to convert the airways to an acidic state within the liquid layer of the airway surface, so the addition of bicarbonate anions HS helps

20 Maintaining or improving the pH of the liquid layer of the airway surface in patients

CF

As a result, inclusion of the bicarbonate anion in the formulation of 7% or &lt;7% hypertonic saline administered by the method of the present invention is particularly beneficial. Formulas that contain up to 30 to 200 mM of Turkish AZT

25 Bicarbonate anions are especially important for 7% solutions or &lt; 7% HS solutions.

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It turns out that hypertonic brine contains a salt concentration greater than that of normal brine, i.e. more than 9 g/L or 0.9% w/v, and hypotonic brine contains a salt concentration lower than that of normal brine, i.e. from about 1 g or L/0.1% w/v to about 8 g/L or 0.8% w/v. Salt solutions are hypertonic

5 Useful formulas and processing methods described herein have a salt concentration ranging from about 1% to about 23.4% (w/v). In one embodiment the hypertonic brine has a salt concentration ranging from about 60 g/L (6% w/v). ) to about 100 g/L (10% w/v). In another embodiment, the brine has a brine concentration ranging from about 70 g/L (7% w/v) to about 100 g/L (10% Weight/volume). In additional embodiments, includes

10 Brine is a solution with a salt concentration ranging 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

(d) From 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

15 g/L (3% w/v); f) from about 1 g/L (0.1% w/v) to about 20 g/L (2% w/v).

The specified concentrations of useful brines in the formulas and processing methods mentioned herein include, separately, those solutions with brine concentrations of 1 g/L (0.1% w/v), 2 g/L (0.2% w/v) , 3 g/L (0.3% w/v), 4 g/L (0.4%

20 w/v), 5 g/L (0.5% w/v), 6 g/L (0.6% w/v), 7 g/L (0.7% w/v), 8 g/L (0.8% w/v). vol), 9 g/L (0.9% w/v), 10 g/L (1% w/v), 20 g/L (2% w/v), 30 g/L (3% w/v) , 40 g/L (4% w/v), 50 g/L (5% w/v), 60 g/L (6% w/v), 70 g/L (7% w/v), 80 g/L (8% w/v), 90 g/L (9%

25 w/v), 100 g/L (10% w/v), 110 g/L (11% w/v), 120 g/

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l (12% w/v), 130 g/l (13% w/v), 140 g/l (14% w/v), 150 g/l (15% w/v), 160 g/l ( 16% w/v), 170 g/L (17% w/v), 180 g/L (18% w/v), 190 g/L (19% w/v), 200 g/L (20% w/v), 210 g/l (21% w/v), 220 g/l (22% w/v),

5 And 230 g/L (23% w/v). It is also possible to use brine concentrations that range between the concentrations/ratios mentioned, such as a brine solution that includes 1.7 g/L (0.17% w/v), 1.25 g/L (1.25% w/v), 1.5 g/L (1.5% w/v), 25 g/L (2.5% w/v), 28 g/L (2.8% w/v), 35 g/L (3.5 % w/v), 45 g/L (4.5% w/v), and 75 g/L (7.5% w/v).

10 Specifically useful concentrations of hypotonic brines include solutions with concentrations ranging from about 0.12 g/L (0.012% w/v) to about 8.5 g/L (0.85% w/v). Any concentration can be used in this Range, such as, based on a weight/volume basis,

(NS 3/1) 0.25%, 0.3% (NS 4/1) 0.225%, 0.15%, 0.15%, 0.1%, 0.65%, 0.05% (NS 3/2) 0.55%, 0.6% (NS 2/1) 0.5%, 0.45%, 0.35%

15 0.675% (4/3), NS, 0.7%, 0.75%, and 0.8%.

Each of the ranges and concentrations specified for brine given herein may be used with the formulas, processing methods, systems, and combinations given herein.

Within the scope of the invention, chemically modified osmotic decomposition products or substances producing the decomposition product are also targeted. Chemical modifications involve binding to the product of osmotic decomposition (or substance).

20 Producer) an additional chemical group that alters or enhances the effect of the osmolyte or the substance producing the osmotic product (e.g., inhibiting the degradation of the osmotic molecule). Chemical modifications are used with drugs or prodrugs and are known in the art. (See, for example Example, US Patents Nos. 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 25

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1983), where all are included); Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986)

For reference in this document.

Anti-inflammatory agents suitable for use in combination with the compounds of the patent include corticosteroids and nonsteroidal anti-inflammatory drugs

5 Non-steroidal anti-inflammatory drugs (NSAIDs), specifically inhibitors

Particularly phosphodiesterase (PDE). Examples of corticosteroids used in the present invention include oral or inhaled corticosteroids or prodrugs thereof. Specific examples include, but are not limited to, ciclesonide

desisobutyryl-ciclesonide, budesonide, flunisolide, mometasone and

10 esters (such as fluticasone propionate, mometasone furoate).

fluticasone furoate, beclomethasone, methyl prednisolone, prednisolone, -difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-

hydroxy- -methyl-3-oxo-androsta-1,4-diene-17β-carbothioic acid

S- -difluoro-11β-hydroxy- -methyl-3-oxo-

-propionyloxy-androsta-1,4-diene-17β-carbothioic acid S-(2-oxo- 15

tetrahydro-furan-3S-yl) ester, beclomethasone esters (eg, the 17-propionate ester or the 17,21-dipropionate ester, fluoromethyl ester, triamcinolone acetonide, rofleponide, or any combination or subset thereof. Preferred corticosteroids for formulation or use in combination

With the compounds of the invention are selected from ciclesonide, 20 desisobutyryl-ciclesonide, budesonide, mometasone, fluticasone

propionate, and fluticasone furoate, or any combination or subset thereof. The preferred corticosteroid has been selected for formulation or use in combination with the compounds of the invention of fluticasone propionate, fluticasone furoate, or any combination or subcombination.

25 So.

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NSAIDs for use in the present invention include but are not limited to sodium nedocromil sodium, cromoglycate, phosphodiesterase (PDE) inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, mixed PDE3/PDE4 inhibitors or mixed PDE7/PDE4 inhibitors), leukotrienes 5 leukotriene antagonists, inhibitors of leukotriene synthesis

synthesis (e.g., 5-LO and FLAP inhibitors), nitric oxide synthase (iNOS) inhibitors, protease inhibitors (e.g., tryptase inhibitors, nucleophilic elastase inhibitors, neutrophil elastase inhibitors) metalloprotease inhibitors (integrin-β2 antagonists). And the factors

10 Cofactors and adenosine antagonists (e.g., adenosine a2 cofactors), cytokine antagonists (e.g., chemokine antagonists) or cytokine synthesis inhibitors (e.g.,

D2 prostaglandin receptor antagonists (CRTh2). Examples of leukotriene modifiers suitable for administration via the method of the invention include montelukast, zileuton and zafirlukast.

A PDE4 inhibitor, a combined PDE4/PDE3 inhibitor or a combined PDE7/PDE4 inhibitor 15 may be any compound which inhibits the PDE4 enzyme or which acts as a PDE4 inhibitor, which

It is a selective PDE4 inhibitor (i.e., compounds that do not significantly inhibit members of the PDE family). Specific examples of PDE4 inhibitors for composition or use in combination with compounds of the present invention include but are not limited to:

roflumilast, pumafentrine, arofylline, cilomilast, tofimilast, oglemilast, tolafentrine, piclamilast, ibudilast, apremilast, 2-[4-[6,7-diethoxy-2,3- 20

bis(hydroxymethyl)-1-naphthalenyl]-2-pyridinyl]-4-(3-pyridinyl)-1(2H)-phthalazinone (T2585), N-(3,5-dichloro-4-pyridinyl)-1-[ (4-

fluorophenyl)methyl]-5-hydroxy- -oxo-1H-indole-3-acetamide (AWD-

12-281, 4-[(2R)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-

phenylethyl]-pyridine (CDP-840), 2-[4-[[[[2-(1,3-benzodioxol-5-yloxy)- 25

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3-pyridinyl]carbonyl]amino]methyl]-3-fluorophenoxy]-(2R)-propanoic acid (CP-671305), N-(4,6-dimethyl-2-pyrimidinyl)-4-[4,5,6 ,7-tetrahydro-2-(4-methoxy-3-methylphenyl)-5-(4-methyl-1-piperazinyl)-1H-indol-1-

yl]-benzenesulfonamide, (2E)-2-butenedioate (YM-393059), 9-[(2-

fluorophenyl)methyl]-N-methyl-2-(trifluoromethyl)-9H-purin-6-amine 5

(NCS-613), N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-

quinolinecarboxamide (D-4418), N-[(3R)-9-amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrrolo[3,2,1-][1,4]benzodiazepin- 3-yl]-3H-purin-6-amine (PD-168787), 3-[[3-(cyclopentyloxy)-4-methoxyphenyl]methyl]-N-ethyl-

8-(1-methylethyl)-3H-purin-6-amine hydrochloride (V-11294A), N- 10

(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-

quinolinecarboxamide (Sch351591), 5-[3-(cyclopentyloxy)-4-

methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S,5S)- 2-piperidinone (

HT-0712), 5-(2-((1R,4R)-4-amino-1-(3-(cyclopentyloxy)-4-

methyoxyphenyl)cyclohexyl)ethynyl)-pyrimidine-2-amine,cis-[4-cyano- 15

4-(3-cyclopropylmethoxy-4-difluoromethoxy phenyl)cyclohexan-1-ol], and 4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-1-(2-

440-methoxyethyl)-2(1H)-pyridinone (T), and any combination or subset thereof.

The processes involved in the synthesis of Leukotriene antagonists and inhibitors

20 leukotriene containing: zileuton, montelukast sodium, zafirlukast, and

pranlukast

The anticholinergic agents of the formulation or for use in combination with compounds of the invention include but are not limited to muscarinic receptor antagonists, specifically including systemic antagonists and M3 receptor antagonists. Examples of alkaloid compounds include belladonna plants,

And multiple forms including, atropine, scopolamine, homatropine, hyoscyamine 25 such as

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These include salts (for example, anhydrous atropine, atropine sulfate, atropine oxide or methylatropine nitrate, HCl,

hyoscyamine, homatropine methyl bromide, homatropine hydrobromide, scopolamine hydrobromide, hyoscyamine sulfate, hydrobromide

5 scopolamine methyl bromide, or any combination or subgroup thereof.

Additional anticholinergics for the formulation or for use in combination with:

methaneline, propantheline bromide, anisotropine methyl bromide or

Valpin 50, aclidinium bromide, glycopyrrolate (Robinul), isopropamide iodide, mepenzolate bromide, tridihexethyl chloride, hexocyclium methylsulfate, cyclopentolate HCl, tropicamide, trihexyphenidyl CCl, 10 pirenzepine, telenzepine, and methoctramine

Or any combination or subset thereof.

The preferred anticholinergic action of the formulation and use in combination with the compounds of the invention includes ipratropium (bromide), oxitropium (bromide) and tiotropium (bromide), or any 15 combinations or subsets thereof.

Examples of β cofactors for forming a formulation or use in combination with compounds of the invention include but are not limited to R-salmeterol, salmeterol, xinafoate salts thereof, albuterol or R-albuterol (free base or sulfate), and

levalbuterol, salbutamol, formoterol (fumarate), fenoterol, procaterol,

20 pirbuterol, metaprterenol, terbutaline and salts thereof, and any combination or subset thereof.

The P2Y2 receptor cofactors may be used to form the formula or used in combination with the compounds of the invention in an amount effective for stimulating the secretion of chloride and water by airway surfaces.

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airway surfaces, specifically nasal airway surfaces. Suitable P2Y2 receptor cofactors are known in the art and are described, for example, in columns 9-10 in US Patents 6,264,975, and US Patents 5,656,256 and 5,292,498.

5 The P2Y2 cofactors that can be used by the methods of the invention include P2Y2 receptor cofactors such as ATP, UTP, gamma.-S.-UTP and a dinucleotide P2Y2 receptor cofactor (such as denufosol or diquafosol) or pharmaceutically acceptable salts thereof. Typically the agent is included P2Y2 receptor cofactors in an effective amount to stimulate chloride and water secretion by airway surfaces, specifically 10 nasal airway surfaces are described

Appropriate P2Y2, without limitation, in US Patent No. 6,264,975, US Patent No. 5,656,256, US Patent No. 5,292,498, US Patent No. 6,348,589, US Patent No. 6,818,629, US Patent No. 6,977,246, US Patent No. 7,223,744, US Patent No. 7,531,525, US Patent Application No. 15 0306009/2009, each of which is incorporated by reference in its entirety herein. document.

Combination therapies and formulations mentioned in this document may include cofactors 2b adenosine (A2b), also including ethylcarboxamidoadenosine (ethylcarboxamidoadenosine), 6583-60-60 N-NECA (BAY) S)-PHPNECA, 5835-LUF, and 5845-LUF.

Volpini et al., Journal of Used by A2b Cofactors are described Medicinal Chemistry 45(15): 3271–9 (2002); Volpini et al., Current 20

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., 25

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Bioorganic &amp; Medicinal Chemistry Letters 16(2): 302–6 (Jan. 2006); Carotti, et al., Journal of Medicinal Chemistry 49 (1): 282–99 (Jan.

2006); Tabrizi et al., Bioorganic &amp; Medicinal Chemistry 16(5): 2419–30 (March 2008); and Stefanachi, et al., Bioorganic &amp; Medicinal Chemistry

16(6):2852–69 (March 2008))5

Examples of other ENaC receptors and hindrances for formulation formation or use in combination with compounds of the invention include, but are not limited to, amiloride and derivatives thereof of those compounds mentioned in US Patent No. 6,858,615, PCT Publication, International Application No. 070182/2003, International Application 073629/2004, International Application 018644/2005, International Application 022935/2006,

10 International Application 018640/2007 and International Application 146869/2007, all for Parion.

Sciences, Inc

Small molecules of ENaC blockers are able to directly block sodium transport to the pores of the ENaC channel. ENaC inhibitor, which may be administered in the combinations described herein, includes, but is not limited to, phenamil, benzamil, amiloride, and amiloride analogues as indicated.

15 Illustration by US Patent No. 6,858,614, US Patent No. 6,858,615, Patent

US Patent No. 6,903,105, US Patent No. 6,995,160, US Patent No. 7,026,325, US Patent No. 7,030,117, US Patent No. 7,064,129, US Patent No. 7 186,833, US Patent No. 7,189,719, US Patent No. 7,192,958, US Patent No. 7,192,959, US Patent No. 7,241,766, Patent

20 US Patent No. 7,247,636, US Patent No. 7,247,637, US Patent No. 7,317,013, US Patent No. 7,332,496, US Patent No. 7,345,044, US Patent No. 7,368,447, US Patent No. 7,368,450, US Patent No. 7,368,451, US Patent No. 7,375,107, US Patent No. 7,399,766, US Patent No. 7,410,968 , US Patent No. 7,820,678, US Patent No.

25 7,842,697, US Patent No. 7,868,010, US Patent No. 7,875,619.

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ENaC proteolysis is well described to increase sodium transport through ENaC. The protease inhibits or inactivates endogenous airway proteases, preventing ENaC cleavage and activation. The proteases that function to cleave ENaC include furin, meprin, matriptase, trypsin, canalicular-associated proteases (CAPs), and aliphatic elastases.

<p dir="rtl">5 Nucleus. Protease inhibitors capable of inhibiting the proteolytic activity of protease enzymes that may be given in the combinations described herein include, but are not limited to, camostat prostasin, furin, aprotinin, leupeptin, and trypsin inhibitors.</p>

The combinations described herein may include one or more suitable nucleic acids (or polynucleic acid), including but not limited to

<p dir="rtl">10 Oligonucleotides that inhibit the expression of sense, miRNA, siRNA, miRNA mimetic, aptamer, antagomir, and attract decoy oligonucleotide nucleic acids. See, for example, US Patent Application Publication No. 20100316628. In general, said nucleic acids can be obtained from 17 or 19 nucleotides in length, to as high as 23, 25 or 27 nucleotides in length, or</p>

<p dir="rtl">15 more. Examples include, but are not limited to, those cited in US Patent No. 7,517,865 and US Patent Publication 20100215588; 20100316628;</p>

20110008366; And 20110104255. In general, siRNAs are obtained from 17 or 19 nucleotides in length, increasing to 23, 25 or 27 nucleotides in length, or more.

<p dir="rtl">20 Compounds that modify CFTR activity that can be administered in combinations of the present invention include, but are not limited to, compounds described in US Patent 0246137/2009-1A, US Patent 0253736/2009-1A, US Patent 0227888/2010-1A, Patent No.</p>

7,645,789, US Patent 0246820/2009-1a, US Patent 0221597/2009-1a, US Patent 0184739/2010-1a, US Patent 0130547/2010-1a, Patent

25 US 0168094/2010-1A and Published Patent No. 7,553,855; American patent

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7,772,259-2B, US Patent 7,405,233-2B, US Patent

0203752/2009, US Patent 7,499,570.

Mucus modifying agents or mucins useful in the combinations and methods described herein include reducing agents, surfactants, detergents, expectorants, and 5-deoxyribonuclease agents.

10

Mucin proteins are organized according to high molecular weight polymerases through the formation of covalent (disulfide) and non-covalent bonds. The distribution of covalent bonds with reducing agents is a well-known method of reducing the viscoelastic properties of mucus in the organism and would be expected to act on Reducing mucus adhesion and improving organism clearance Reducing agents are well known in reducing mucus viscosity in the laboratory and are commonly used as adjuncts to processed sputum samples. Examples of reducing agents include sulfide-containing molecules or phosphines capable of cleaving protein di-sulfide bonds including, but not limited to:

N-acetyl cysteine, N-acystelyn, carbocysteine, glutathione, dithiothreitol, thioredoxin containing proteins, and tris (2-carboxyethyl) phosphine.

15 N-acetyl cysteine (NAC) has been approved for use in combination with chest physiotherapy to loosen viscous or thickened airway mucus. Clinical studies evaluating the effects of oral or inhaled NAC in CF and COPD have shown improvement in the rheological properties of mucus and trends in improved function. Lung and pulmonary exacerbations9 However, the preponderance of clinical data suggests that NAC is best used as a marginal therapeutic agent for the treatment of NSAIDs.

20 Airway mucus when covering through the mouth or by inhalation. The recent Cochrane review of current clinical areas on the use of NAC has not provided evidence of the effectiveness of NAC for CF. The marginal clinical benefit of NAC is reflected in:

NAC is a relatively inactive reducing agent that is only partially active on the surface of the airways. Very high concentrations of NAC are 200 mM (or 3.26%).

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It is absolutely required to reduce MuC5B, the major airway mucin reducing gel, in vitro. Moreover, at the average pH of the airway surface (measured in the pH range of 6.0 to 7.2 in CF and COPD airways), NAC exists only partially in the reactive state as a negatively charged thiolate compound. Thus, in vitro, it is given However, current aerosol devices would be expected to be unable to detect NAC 5 at very high concentrations

The therapeutic efficacy of 20% Mucomyst solution was achieved on distal airway surfaces in the relatively short time scales (7.5-15 minutes) typically used.

In nonclinical studies, NAC labeled 14C, administered by inhalation,10 shows rapid elimination from the lung with a half-life ranging from 6 to 36 minutes.

NAC is given in high concentration, hypertonic inhalation solution (20% or 1.22 M) causes bronchoconstriction and cough. In several cases, it is suggested to give NAC with a bronchodilator or to improve the tolerance of the said agent.

Therefore, reducing agents such as NAC are not well suited for bolus or spray administration. 15 However, delivery of reducing agents by pulmonary aerosol infusion is expected to increase efficacy, and allow the concentration of the reducing agent in the inhalation solution to be reduced (expected to increase tolerability).

Surfactants and detergents are diffusion agents that reduce mucus viscosity and improve mucus clearance. Examples include surfactants

palmitoyl-, palmitic acid, PF, dipalmitoylphosphatidylcholine

20 oleoylphosphatidylglycerol, surfactant-binding proteins (such as B, SP-A, or C), or can be derived from animals (such as obtained from lung lavage of cow or calf or minced pig lung) extracted from minced pig lung or combinations thereof. See, for example, US Patents Nos. 7,897,577; 5,614,216; and 4,312,860

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Surfactant products Colfosceril palmitate (Exosurf® Neonatal), Pumactant® (DPPC) and KL-4 surfactant eggs (phosphatidylglycerol), Rsp-C®, Venticute (surfactant lusulptide),

(calfactant) Infasurf®, (poractant alfa) Curosurf®, (bovactant) Alveofact®

5 Newfacten® (modified bovine surfactant), SurfaceTM®, NatsurfTM® (nonionic ethoxylate alcohol surfactant) and Survanta® (survanta). Examples of detergents include, but are not limited to, 80-Tween and 100 triton-X.

Any suitable repellent may be used, including but not limited to guaifenesin (see, for example, US Patent No. 7,345,051). Any suitable deoxyribonuclease may be used, including but not limited to 10 Exclusively Dornase alpha.

(See, for example, US Patent No. 7,482,024.)

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

Agents forming the formula or use in combination with compounds of the invention include antiviral agents 15 and antibiotics. Examples of suitable antiviral agents include Tamiflu®

Suitable examples of antibiotics include (oseltamivir) and zanamivir (Relenza®). Suitable examples of antibiotics include but are not limited to aztreonam (arginine or lysine), fosfomycin, aminoglycosides such as tobramycin, or any combination or subset thereof. Antiinfective agents include antiinfective agents Additional information used in this document:

Aminoglycosides, Daptomycin, Fluoroquinolones, Ketolides, Carbapenems, 20 Cephalosporins, Erythromycin, Linezolid, Penicillins, Azithromycin,

Clindamycin, Oxazolidinones, Tetracyclines, and Vancomycin

Useful examples of carbapenam antibiotics are panipenam,

(749,345-L) MK-826, DA-1131, ER-35786, biapenam, meropenam

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Lenapenam, 4661-CS-834S (initial drug of 95867-KR-21056, R (initial drug of 21012-L-084), Ceftolozane (initial drug of 11036-LJC) and CXA-101).

Antihistamine agents include antihistamines (i.e., 1H-receptor antagonists) to form the formulation or use in combination with compounds of the invention including but not limited to ethanolamines 5 such as:

diphenhydramine HCl, carbinoxamine maleate, doxylamine, clemastine fumarate, diphenylhydramine HCl and dimenhydrinate; ethylenediamines

such as pyrilamine maleate (metpyramine), tripelennamine HCl, tripelennamine citrate, and antazoline; alkylamines such as pheniramine, chloropheniramine, bromopheniramine, dexchlorpheniramine, triprolidine 10 and acrivastine; pyridines such as methapyrilene, piperazines such as

hydroxyzine HCl, hydroxyzine pamoate, cyclizine HCl, cyclizine lactate,

meclizine HCl and cetirizine HCl; piperidines such as astemisole, levocabastine HCl, loratadine, descarboethoxyloratadine, terfenadine, and

fexofenadine HCl; tri- and tetracyclics such as promethazine, 15

chlorpromethazine trimeprazine and azatadine; and azelastine HCl, or any combination or subset thereof.

Examples of other classes of other therapeutic agents suitable for use in the combinations and methods described herein include antiviral agents such as ribavirin, antifungal agents such as intraconazol 20, amphotericin and voriconazol, anti-rejection drugs such as tacrolimus, cyclosporine and sirolimus, bronchodilator agents including bronchodilators. Including, but not limited to, anticholinergic agents such as Atrovent siRNAs, Atrovent, gene therapy vectors, AptamiArt. aptamers, endothelin-receptor antagonists, 25 alpha-1-antitrypsin antagonists and prostacyclins.

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In accordance with previously used treatment methods, the inventive compound may be used alone, or in combination with one or more other therapeutic active agents. Typically, any therapeutically active agent that has a therapeutic effect on the disease or condition treated using the compound of the invention may be used in combination with compounds of the invention, provided that the therapeutically active agent is

Specifically compatible with a treatment using the compound of the invention. Typical therapeutically active agents suitable for use in combination with compounds of the invention include the aforementioned agents.

In one preferred embodiment, the compounds of the invention are used in combination with one or more osmotic decomposition products, namely hypertonic saline or

mannitol

10 In another aspect, the invention provides methods of treatment and uses as previously described, which include administering an effective amount of the compound of the invention and at least one of the therapeutically active agents. maybe

15

The use of the compounds of the invention and at least one of the additional therapeutic active agents in a simultaneous combination or sequentially in any therapeutically appropriate combination. Administration of the compound of the invention with one or more other therapeutic active agents may be simultaneous administration in 1) a single pharmaceutical composition, such as the aforementioned compositions, or 2) a separate pharmaceutical composition including one or more active ingredients. The components of the combination may be administered separately or in a sequential manner, whereby the compound of the invention is administered initially and the active therapeutic agent is administered secondarily, or vice versa.

In embodiments in which the compound of the invention is administered in combination with one or more osmotic breakdown products, each component is administered simultaneously, or may be in the form of a single or

20 separate. In one embodiment, the compound of the invention and one or more osmotic degradation products are administered concomitantly in bronchoalveolar lavage. In another embodiment, the compound of the inventor and one or more osmotic degradation products are administered simultaneously by inhalation.

When the inventive compound is used in combination with therapeutically active agents, the dose of each compound may differ from the dose when the inventive compound is used alone. Doses are determined

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This can be easily done by those skilled in the field. The appropriate dose of the inventive compound, of the therapeutically active agents, and the relative timing of administration to achieve combined therapeutic effects are selected and are within the knowledge of the medical practitioner or veterinarian.

Experimental Procedures: The present invention also provides processes for preparing the compounds of the invention and relates to synthetic intermediates useful in those processes, as described in detail below.

Specific abbreviations and expressions have been used in describing the synthetic processes and experimental details. Although most of these are self-explanatory for those skilled in the art, the following table includes a list of many of these abbreviations.

<tr><td><p dir="rtl">the meaning</p><p>Acetic acid</p></td><td><p dir="rtl">Abbreviation</p><p>AcOH</p></td></tr><tr><td><p>Azobisisobutyrolnitrile</p><p>Diisopropyl azidocarboxylate</p><p>N,N-diisopropylethylamine</p></td><td><p>AIBN</p><p>DIAD</p><p>DIPEA</p></td></tr><tr><td><p>dichloroethane</p></td><td><p>DCE</p></td></tr><tr><td><p>dichloromethane</p></td><td><p>DCM</p></td></tr><tr><td><p>dimethylformamide</p></td><td><p>DMF</p></td></tr><tr><td><p>Ethyl</p></td><td><p>Et</p></td></tr><tr><td><p>ethyl acetate</p><p>Ethanol</p></td><td><p>Or EtOAc</p><p>EA</p><p>EtOH</p></td></tr>

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<tr><td><p dir="rtl">the meaning</p><p>Acetic acid</p></td><td><p dir="rtl">Abbreviation</p><p>AcOH</p></td></tr><tr><td><p dir="rtl">Electrospray ionization</p></td><td><p>ESI</p></td></tr><tr><td><p>2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate</p></td><td><p>HATU</p></td></tr><tr><td><p dir="rtl">Chromotog RV is a high-performance liquid</p></td><td><p>HPLC</p></td></tr><tr><td><p>Isopropyl alcohol</p></td><td><p>iPrOH</p></td></tr><tr><td><p dir="rtl">Inside the Kasbah</p></td><td><p dir="rtl">it or IT</p></td></tr><tr><td><p>methyl</p></td><td><p>Me</p></td></tr><tr><td><p>methanol</p></td><td><p>MeOH</p></td></tr><tr><td><p dir="rtl">Mass-to-charge ratio</p></td><td><p>m/e or m/z</p></td></tr><tr><td><p dir="rtl">Increased mass 1</p></td><td><p>+MH</p></td></tr><tr><td><p dir="rtl">Missing mass 1</p></td><td><p>-MH</p></td></tr><tr><td><p dir="rtl">Minimum inhibitory concentration</p></td><td><p>MIC</p></td></tr><tr><td><p dir="rtl">Mass spectrum</p></td><td><p>ms or MS</p></td></tr><tr><td><p dir="rtl">Room temperature</p></td><td><p dir="rtl">rt or rt</p></td></tr><tr><td><p dir="rtl">Delay factor</p></td><td><p>Rf</p></td></tr><tr><td><p>tert-butyl</p></td><td><p>t-Bu</p></td></tr><tr><td><p>tetrahydrofuran</p></td><td><p>THF</p></td></tr>

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<tr><td><p dir="rtl">the meaning</p><p>Acetic acid</p></td><td><p dir="rtl">Abbreviation</p><p>AcOH</p></td></tr><tr><td><p dir="rtl">Thin-layer chromoautog arv</p></td><td><p>tlc or TLC</p></td></tr><tr><td><p dir="rtl">A ppm subfield of tetramethylsilane</p></td><td><p>δ</p></td></tr><tr><td><p>Benzyloxycarbonyl, i.e. -(CO)O-benzyl</p></td><td><p>Cbz</p></td></tr><tr><td><p dir="rtl">Area under the curve or peak</p></td><td><p>AUC</p></td></tr><tr><td><p>Methyl tertiary butyl ether</p></td><td><p>MTBE</p></td></tr><tr><td><p dir="rtl">Detention time</p></td><td><p>tR</p></td></tr><tr><td><p dir="rtl">Gas chromatography mass spectrometry</p></td><td><p>GC-MS</p></td></tr><tr><td><p dir="rtl">Percentage by weight</p></td><td><p>%wt</p></td></tr><tr><td><p dir="rtl">hour</p></td><td><p>h</p></td></tr><tr><td><p dir="rtl">minutes</p></td><td><p>min</p></td></tr><tr><td><p dir="rtl">MHz</p></td><td><p>MHz</p></td></tr><tr><td><p>Trifluoroacetic acid</p></td><td><p>TFA</p></td></tr><tr><td><p dir="rtl">Ultraviolet</p></td><td><p>UV</p></td></tr><tr><td><p>tert-butyloxycarbonyl</p></td><td><p>Boc</p></td></tr><tr><td><p>Diisopropyl azodicarboxylate</p></td><td><p>DIAD</p></td></tr><tr><td><p>Acetic acid</p></td><td><p>AcOH</p></td></tr>

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<tr><td><p dir="rtl">the meaning</p><p>Acetic acid</p></td><td><p dir="rtl">Abbreviation</p><p>AcOH</p></td></tr><tr><td><p>Hünig's or N,N-diisopropylethylamine base</p><p>Triphenylphosine</p></td><td><p>DIPEA</p><p>Ph3P</p></td></tr>

Compounds with formula I can be synthesized using techniques known in the art. A typical synthetic procedure is illustrated in the following Scheme 1.

Scheme 1

10

15

These procedures are described, for example, in E. J. Cragoe, “The Synthesis of

Amiloride and Its Analogs” (Chap 3) in Amiloride and Its Analogs, pp. 25

36.Other processes for preparing amiloride analogues are described, for example, in US Patent No. 3,318,813, for Cragoe, specifically at methods C, B, A, and D of patient 813.' Other processes adapted for preparing the compounds of the invention are also described in PCT Publication, International Application No. 07182/2003, International Application No. 108644/2005, International Application 022935/2005, US Patent 7,064,129, US Patent 6,858,615, US Patent 6,903,105, International Application 073629/2004, International Application 146869/2007, and International Application 018640/2007, All assigned to Parion Sciences, Inc

The method for preparing 3,5N'-methyl-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate(2) can be found in International Application 074575/2009.

In general, the compounds of the invention can be conveniently prepared by treating a compound of formula II with an amine of formula III. More specifically, compounds with formula 2 are processed using

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An amine with formula 3 in a suitable solvent such as ethanol, methanol, or tetrahydrofuran, and a base such as triethylamine (TEA), or di-isoproylethylamine (DIP EA), with heating to an elevated temperature, e.g., 70°C.

In addition, the process of purification, dissolution of solids, crystallization and/or method of preparing images can be carried out.

5 Salt using traditional techniques.

As will be evident to those skilled in the art, in specific cases, initiating and intermediate compounds in the synthesis may include functional groups that provide alternative reactive sites. Interference with the mentioned functional groups can be avoided by using appropriate protecting groups, such as amine groups or protective alcohols, when applicable, and prioritizing the synthesis steps. 10 It is clear to those skilled in the art that the appropriate protection packages are available. The methods are well known in the field for addition

Or remove the oblique protection groups and the conventional techniques mentioned can be used in the processes of the present invention as well.

The following specific examples are provided herein for the purposes of illustrating and limiting the invention, which is described in the claims.

15 Materials and Methods: All reagents and solvents were obtained from Aldrich Chemical Corp

Chem-Impex International Inc. and TCI chemical industry Co. Ltd

Obtain NMR spectra on either Bruker AC (400 1H NMR at 400 MHz and 13C NMR at 100 MHz) or Bruker AC (300 1H NMR at 300 MHz and 13C NMR at 75 MHz). The proton spectrum is denoted by

20 tetramethylsilane is used as an internal standard and a carbon spectrum is indicated by the expression CDCl3, CD3OD, or DMSO-d6 (obtained by Aldrich or Cambridge Isotope Laboratories, unless otherwise specified).

Redi) Combiflash System Packed with Silica Gel Column (Combiflash Rf, Teledyne Isco)

Sep. Rf, Teledyne Isco) or reverse phase column (High Performance C18 Gold column).

25 ESI mass spectra were acquired on a 2010-Shimadzu LCMS mass spectrometer

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EV. HPLC degradation products were obtained using a 5 Waters XTerra MS C18 µm 150x4.6 mM analytical column that can be detected at 220 nm (unless otherwise specified) on a Shimadzu Prominence HPLC system. The following time program is used which has Flow rate 1.0 ml per minute:

<tr><td><p dir="rtl">B ratio</p><p>0.05% with CH3CN)</p><p>)TFA</p></td><td><p dir="rtl">A ratio</p><p>(TFA 0.05% with H2O)</p></td><td><p dir="rtl">time</p><p dir="rtl">)minute(</p></td></tr><tr><td><p>10</p></td><td><p>90</p></td><td><p>2.50</p></td></tr><tr><td><p>90</p></td><td><p>10</p></td><td><p>20.00</p></td></tr><tr><td><p>90</p></td><td><p>10</p></td><td><p>30.00</p></td></tr><tr><td><p>10</p></td><td><p>90</p></td><td><p>32.50</p></td></tr>

5 UPLC degradation products were obtained using a Waters ACQUITY UPLC HSS 1.8 T3 100x2.1 mM analytical column that can be detected at 220 nm (unless otherwise specified) on a Shimadzu Prominence UFLC system. The following flow rate schedule is used It is 0.3 ml per minute:

<tr><td><p dir="rtl">B ratio</p><p dir="rtl">)/CH3CN water 20:80%</p><p>NH4COOH %0.05 with</p><p>(HCOOH 0.1% and</p></td><td><p dir="rtl">A ratio</p><p>NH4COOH 0.05% with H2O)</p><p>(HCOOH 0.1% and</p></td><td><p dir="rtl">time</p><p dir="rtl">)minute(</p></td></tr><tr><td><p>10</p></td><td><p>90</p></td><td><p>1.00</p></td></tr><tr><td><p>70</p></td><td><p>30</p></td><td><p>4.00</p></td></tr>

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<tr><td><p>70</p></td><td><p>30</p></td><td><p>5.00</p></td></tr><tr><td><p>10</p></td><td><p>90</p></td><td><p>5.50</p></td></tr><tr><td><p>10</p></td><td><p>90</p></td><td><p>6.50</p></td></tr>

1. Preparing salt:

S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-(

carbonyl)guanidino)butyl)naphthalen-1-yl)propanoic acid

)16(

5

Scheme 2

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<img file="SA5673B1_D0013.tif" />

<img file="SA5673B1_D0014.tif" />

<img file="SA5673B1_D0015.tif" />

<img file="SA5673B1_D0016.tif" />

<img file="SA5673B1_D0017.tif" />

<img file="SA5673B1_D0018.tif" />

<img file="SA5673B1_D0019.tif" />

<img file="SA5673B1_D0020.tif" />

<img file="SA5673B1_D0021.tif" />

Preparation of 2(4-(tert-Butyldimethylsilyloxy)naphthalene-1-carbaldehyde); a solution of 10(1)4-hydroxynaphthalene-1-carbaldehyde g, 58.1 mmol) in dry THF (200 mL) was cooled to 0 °C , imidazole (12.0 g, 174 mmol) and tert-butyldimethylsilyl chloride (13.1) g (TBSCl), 87.1 mmol).

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Add sequentially. After stirring at room temperature for 16 hours, the reaction mixture was filtered and the solvent was evaporated. The residue was drawn out in 500 ml EtOAc, washed with a saturated aqueous volume of 100 ml NH4Cl, water (100 ml), and 100 ml brine, and dried over Na2SO4. The solvent was removed under reduced pressure and the residue purified. by

5 Flash chromatography on silica gel (2% hexane/EtOAc), to obtain

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) 10

to prepare :

(Z)-Methyl 2-(tert-Butyloxycarbonyl)amino-3-[1-(tert-)

4(;(butyldimethylsilyloxy)naphthalen-4-yl]acrylate

A solution of 3 (MeO)2P(O)CH(NHBoc)CO2Me, 23.0 g, 52.7 mmol, was charged

15 in dry CH2Cl2 (100 ml) with 10.1 ml DBU (67.3 mmol), and the mixture was stirred for 30 minutes at 0°C. A solution of 1 (14.8 g, 51.74 mmol) in dry CH2Cl2 (60 ml) was added slowly Using a syringe, the reaction mixture was warmed to room temperature over 16 hours. After the solvent was removed under low pressure, the residue was dissolved in (500 ml) CH2Cl2, and washed quickly with a saturated amount of NH4Cl (2).

20 × 150 ml (200 ml brine), and dried over Na2SO4. The solvent was evaporated and the product was purified.

The crude oil was chromatographed and flashed on silica gel (20% hexane/EtOAc) with 1% NEt3, to obtain 4 (20.0 g, 85%) in the form of a yellow solid:

1H NMR (300 MHz, CDCl3): 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

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(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).

Methyl 2-(tert-butoxycarbonylamino)-3-(4-(tert-) preparation

5(;(butyldimethylsilyloxy)naphthalen-1-yl)propanoate

5 A suspension of 4 (17.2 g, 37.6 mmol) and 10% Pd/C (3.40 g) in EtOH (200 mL) was degassed and exposed to hydrogenation conditions (1 atmosphere, color) for 16 hours at room temperature. The reaction mixture was passed through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain 5 (17.0 g, 99%) as a white solid:

1H NMR (300 MHz, CDCl3): δ 8.23 (d, J = 8.2 Hz, 1H), 7.99 (d, J = 8.2 10

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 (brs, 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).

Methyl 2-(tert-butoxycarbonylamino)-3-(4-hydroxynaphthalen-1- preparation

15 6(yl)propanoate(;

A solution of 5 (17.0 g, 37.0 mmol) was charged in dry THF (200 mL) at 0 °C with butylammonium fluoride tartar (48.1 mL, 48.1 mmol). The resulting solution was stirred for 15 minutes and quenched with saturated aqueous (150 ml) of NH4Cl. After the solvent was removed under reduced pressure, the residue was dissolved in 500 (500 ml) of CH2Cl2, washed at 20 ml with a saturated volume of water (2 x 150 ml) and brine (200 ml), and dried. above

Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (25% hexane/EtOAc), to obtain Rotamer 6 (14.0 g, 94%) as a yellow solid:

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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 (brs, 1H), 4.77–4.51 (m, 1H), 3.78–

3.31 (m, 5H), 1.40 (s, 6H), 1.10 (s, 3H).

5 Preparation of compounds 7 and 8;

CHIRALPAK AD Column 5 cm inner diameter x 50 cm length, a 20 micro particle was used to separate the enantiomers using an isomeric IPA/heptane system (7.5% using 0.4% DEA). 8.0 g of enantiomer 6 was purified by the column to obtain the isomer 8 S (3.5 g, 44% yield) as a white solid and 10 R(7 R) isomer 2.2 g, 28%) as a white solid.

(S)-methyl 2-(tert-butoxycarbonylamino)-3-[4- Preparation

9(((trifluoromethylsulfonyloxy)naphthalen-1-yl]propanoate

A solution of compound 8 (1.22 g, 3.53 mmol) in pyridine (20 ml) was charged with

triflate (0.9 ml, 5.30 mmol) at 0°C, and the reaction mixture was stirred at

15 At room temperature for two hours. After concentration, the reaction mixture was divided between (100 ml) CH2Cl2 and water

(50 ml). The aqueous layer was separated and extracted with 50 x 2 (CH2Cl2 ml). The combined organic extraction products were washed with brine, dried over Na2SO4, and concentrated to obtain 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 (brs, 1H), 20

4.78–4.67 (m, 1H), 3.68–3.46 (m, 5H), 1.39 (s, 8H), 1.25 (s, 1H).

S)-methyl 3-{4-[4-(benzyloxycarbonylamino)but-1-( Preparation

11(ynyl]naphthalen-1-yl}-2-(tert-butoxycarbonylamino)propanoate(;

Charging a solution of compound 9 (1.50 g, 3.14 mmol) in a non-aqueous amount of CH3CN

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(60 ml) with TEA (1.27 ml, 12.6 mmol), 10% t-Bu(3P) in hexanes (1.27 ml, 0.62 mmol), benzyl but-3-ynylcarbamate (10, 948 mg, 4.71 mmol) (30 mg CuI, 0.16 mmol) at room temperature. The resulting mixture was eluted with Argon for 10 minutes and 363 (4) Pd(PPh3 mg, 0.31 mmol) was quickly charged onto

5 lump sum. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 16 hours. The reaction mixture was concentrated under vacuum and the remaining material was purified using a chromatography column (silica gel hexanes/ethyl acetate 40:60, silica gel) to obtain compound 11 (1.30 g, 78%) in the form of a brown oil:

1H NMR (400 MHz, CDCl3): 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), 7.35–10

7.29 (m, 5H), 7.19 (d, J = 7.5 Hz, 1H), 5.16–5.12 (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).

(S)-methyl 3-(4-(4-aminobutyl)naphthalen-1-yl)-2-(tert- 15 Preparation of 12(;(butoxycarbonylamino)propanoate) salt

A suspension of 11 (1.00 g, 1.88 mmol) and 10% (200 mg Pd/C) in a mixture of (20 MeOH ml) and AcOH (2 ml) was degassed and exposed to atmospheric hydrogenation conditions (1) for 16 hours at At room temperature, the reaction mixture was filtered through a Celite plug

20 The socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain 12 amine salt (820 mg, 95%) as a white solid:

1H NMR (300 MHz, CD3OD): δ 8.17–8.05 (m, 2H), 7.62–7.48 (m, 2H),

7.27 (brs, 2H), 4.47 (t, J = 7.4 Hz, 1H), 3.75–3.51 (m, 5H), 3.13 (t, J =

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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).

to prepare :

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

(14) with 2.50 ml DIPEA, 14.2 mmol) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours,

10 Cool to room temperature, and concentrate under vacuum. The remaining material was purified by a chromatography column (silica gel CH3OH/NH4OH/CHCl3 80:18:2, silica gel) to obtain 14 (870 mg) guanidine, 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, 1H), 3.75–3.68 (m, 1H), 3.64

(s, 2H), 3.58–3.43 (m, 2H), 3.13 (t, J = 6.7 Hz, 2H), 2.98 (q, J = 7.2 Hz, 15 2H), 1.86–1.70 (m, 4H), 1.33 (s, 7H), 0.98 (s, 2H).

(S)-2-(tert-butoxycarbonylamino)-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-1-yl)propanoic

acid (15); a solution of 14 (510 mg methyl ester, 0.83 mmol) was charged into a mixture

20 of THF (3 ml), methanol (3 ml), and water (1 ml) with 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 indicated that the reaction was complete, the reaction mixture was stirred at room temperature for 2 hours. The pH of the reaction mixture was converted to 9-10 by adding 1 p Hydrochloric acid (aqueous) and the organic solvent was removed. The pH of the aqueous portion was adjusted to 5-6, and the resulting precipitate was extracted using

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dichloromethane. The aqueous fraction was extracted with 50 × 2 DCM ml. The organic layers were collected, dried over Na2SO4, filtered, and concentrated to obtain 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 5

(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).

(S)-2-amino-3-(4-(4-(3-(3,5-diamino-) from Hydrochloric acid Preparation of 6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-1-yl)propanoic salt

10 (16 ml) Hydrochloric acid was added to 15 (258) dioxane (8.0 ml).

mg, 0.43 mmol) followed by water (4.0 ml) and the reaction mixture was stirred at room temperature for 3 hours. The solvent was removed and the remaining material was freeze-dried to obtain compound 16 (250 mg, 99%) as a solid color yellow:

1H NMR (400 MHz, DMSO-d6): δ 10.54 (brs, 1H), 9.33 (t, J = 5.92 Hz,

1H), 9.03–8.80 (m, 2H), 8.60 (brs, 3H), 8.17 (ddd, J = 10.1, 7.6, 4.5 15

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), 3.75–3.44 (m, 3H), 3.43–

3.33 (m, 2H), 3.09 (t, J = 6.4 Hz, 2H), 1.81–1.62 (m, 4H).

2. to prepare :

(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3- 20)

(dimethylamino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-)23(yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

Scheme 3

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<img file="SA5673B1_D0022.tif" />

<img file="SA5673B1_D0023.tif" />

<img file="SA5673B1_D0024.tif" />

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

A solution of 11 (1.71 g methyl ester, 3.22 mmol) was charged in a mixture of THF

5 (21 ml), methanol (21 ml), water (7.0 ml) with solid NaOH (1.29 g, 32.3 g).

mmol) and the reaction mixture was stirred at room temperature for 3 hours. When the TLC of the reaction mixture showed that the reaction was complete, the pH of the reaction mixture was converted to 9-10

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Adding 1 p Hydrochloric acid (aqueous) and the organic solvent was removed. The pH of the aqueous fraction was adjusted to 5-6, and the resulting precipitate was extracted using dichloromethane. The aqueous fraction was extracted using 50 x 2 (CH2Cl2 ml). The layers were assembled. organic matter, drying over Na2SO4, filtration, and concentration to obtain compound 5 17 (1.55 g, 93%) as a brown solid:

1H 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).

10 Preparation of compound 19; Compound 18 (100 mg, 0.56 mmol) was charged in THF (2.5 ml) with 218 mg DEPBT (218 mg, 0.72 mmol), 17 (289 mg, 0.56 mmol), and DIPEA (0.3 ml, 1.68 mmol) sequentially and stirred. At room temperature for 16 hours after the solvent was removed under low pressure, the residue was dissolved in (100 CH2Cl2 mL), and washed quickly with saturated water of (50 x 2 NaHCO3 mL) and brine (50 mL).

15 Drying over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (CH2Cl2/methanol 8%), to obtain 19 amide (250 mg, 66%) in the form of a yellow solid:

1H NMR (400 MHz, CDCl3): 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), 20

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).

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Preparation of compound 20; A suspension of 19 (210 mg, 0.31 mmol) and 10% Pd/C (150 mg) in a mixture of 3.0 MeOH (3.0 ml) and AcOH (0.3 ml) was degassed and exposed to hydrogenation conditions (1 atm) for 12 hours at Room temperature. The reaction mixture was filtered through a Celite socket, the socket was washed with MeOH, the filtrate was concentrated under vacuum to obtain 5 amine salt, which was neutralized with triethylamine, and the crude product was purified by

Flash chromatography on silica gel (CMA 80:18:2) to obtain free amine 20 (130 mg, 77%) in the form of 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 10

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).

preparation 22; A solution of 20 amine (122 mg, 0.22 mmol) and 3,5-methyl 15 (21, 139 diamino-6-chloropyrazine-2-carbonylcarbamimidothioate mg) was charged,

0.35 mmol (4.0 ml) EtOH with 0.31 ml DIPEA (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 under Discharging. The remaining material was purified using a chromatographic column (silica gel CH3OH/NH4OH/CHCl3 2:18:80, silica gel) to obtain 20 (22) guanidine (111 mg, 66%) in the form of 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

٥٦٧٣

-٩٩-

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 Hydrochloric acid salt from compound 23:

(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenylamino)-3-oxopropyl)naphthalen-1- 5 yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

4 p Hydrochloric acid in 3.0 ml 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 1 p NaOH (aqueous), the solid was washed

10 The resultant mixture was obtained using water and treated again with 1 p Hydrochloric acid (aqueous), the water was removed, and the remaining material was freeze-dried to obtain compound 22 (65 mg, 65%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6) 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 (brs, 1H), 8.35 (dd, J = 6.6, 3.4 Hz, 1H), 8.13 (dd, J = 6.5, 3.3 Hz, 1H), 7.58 15

(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), 4.29–4.20 (m, 1H), 3.64–3.49 (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). 20

3. to prepare :

of 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R)-2), 3,4,5,6-

٥٦٧٣

١٠٠-

pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-)28(1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

Scheme 4

<img file="SA5673B1_D0025.tif" />

<img file="SA5673B1_D0026.tif" />

5 Preparation of compound 25;

Compound 24 (165 mg, 0.38 mmol) were charged in THF (10 ml) with DEPBT (148 mg, 0.48 mmol), 17 (200 mg, 0.38 mmol), and DIPEA (0.2 ml, 1.14 mmol). After the solvent was removed under low pressure, the residue was dissolved in 100 ml (CH2Cl2) and washed rapidly with sequential stirring at room temperature for 16 hours.

10 Saturated aqueous volume of 50 x 2 NaHCO3 mL and 50 brine mL, and dried over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on a silica gel.

٥٦٧٣

-١٠١-

CH2Cl2/ methanol (8% silica gel), to obtain 25 (210 mg amide, 60%) in the form of a yellow solid:

1H NMR (300 MHz, CDCl3): δ 8.35 (d, J = 8.2 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.63–7.52 (m, 2H), 7.51 (d, J = 7.3 Hz, 1H), 7.44–7.39 (m,

1H), 7.37–7.27 (m, 6H), 7.16–7.02 (m, 3H), 5.24–5.16 (m, 1H), 5.13 (s, 5

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). 10

Preparation of compound 26;

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 exposed to hydrogenation conditions (1 atmosphere) for 4 hours at Room temperature. The reaction mixture was filtered through a Celite socket and washed

15 Socket using MeOH. The filtrate was concentrated under vacuum to obtain amine salt 22, neutralized with NaHCO3, and the crude product was purified by flash chromatography on silica gel (CMA, 80:18:2) to obtain free amine 26 (160 mg, 67%). (In the form of 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, 20 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),

٥٦٧٣

-١٠٢-

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).

5 Preparation of compound 27;

A solution of 26 amine (155 mg, 0.20 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (21, 123 mg, 0.31 mmol) in EtOH (8.0 ml) was charged with 0.28 ml DIPEA. (1.56 mmol) at room temperature. The reaction mixture was heated at 70 °C in a sealed tube for 2 hours, cooled to 10 °C at room temperature, and concentrated under vacuum. The remaining material was purified by an Arf gel chromatography column.

Silica gel (CH3OH/NH4OH/CHCl3 80:18:2) followed by reverse phase chromatography (Gold C18) to obtain 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 15 = 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, 20 3H), 1.20 (s, 9H), 0.89 (t, J = 6.7 Hz, 3H).

Preparation of Hydrochloric acid salt from compound 28:

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-

٥٦٧٣

-١٠٣-

oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

; 4 p Hydrochloric acid 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 hours.

<p dir="rtl">5 The solvent was removed, an additional amount of 4 p HCl was added, and the mixture was heated at 40 °C for another 4 hours. The solvent was removed, water was added, and the remainder was freeze-dried to obtain compound 28 (78 mg, 99%) as a yellow solid:</p>

1H NMR (400 MHz, DMSO-d6): δ 10.58 (brs, 1H), 10.56 (brs, 1H), 9.70–9.58 (m, 1H), 9.38–9.31 (m, 1H), 9.04–8.84 (m, 2H), 8.70 (brs, 1H), 8.43–8.34(m, 1H), 8.16–8.08 (m, 1H), 7.62–7.52 (m, 2H), 7.46–10

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, 2H), 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). 15

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 20

(m, 2H), 1.44–1.30 (m, 6H), 0.92 (t, J = 6.6 Hz, 3H)

<p dir="rtl">4 to prepare :</p>

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)

٥٦٧٣

-١٠٤-

phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-

(33) chloropyrazine-2-carboxamide

Scheme 5

<img file="SA5673B1_D0027.tif" />

<img file="SA5673B1_D0028.tif" />

<img file="SA5673B1_D0029.tif" />

<img file="SA5673B1_D0030.tif" />

<img file="SA5673B1_D0031.tif" />

<img file="SA5673B1_D0032.tif" />

<img file="SA5673B1_D0033.tif" />

<img file="SA5673B1_D0034.tif" />

<img file="SA5673B1_D0035.tif" />

<img file="SA5673B1_D0036.tif" />

<img file="SA5673B1_D0037.tif" />

<img file="SA5673B1_D0038.tif" />

<p dir="rtl">5 Preparation of compound 30:</p>

Compound 29 (290 mg, 0.54 mmol) were charged in THF (8.0 ml) with DEPBT (210 mg, 0.70 mmol), 17 (311 mg, 0.60 mmol), and DIPEA (0.28 ml, 1.62 mmol). After the solvent was removed under low pressure, the residue was dissolved in 100 ml (CH2Cl2) and washed rapidly with sequential stirring at room temperature for 16 hours.

10 Saturated aqueous volume of 50 x 2 NaHCO3 mL and 50 brine mL, and dried over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on a silica gel.

٥٦٧٣

-١٠٥-

CH2Cl2/ methanol (8% silica gel), to obtain 30 (400 mg amide, 72%) in the form of a yellow solid:

1H NMR (400 MHz, CDCl3): δ 8.36–8.26 (m, 1H), 8.20–8.09 (m, 1H), 8.03–7.85 (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, 5

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), 10

1.35 (s, 9H), 1.29 (d, J = 5.1 Hz, 6H).

Preparation of compound 31;

A suspension of 30 (400 mg, 0.39 mmol) and 10% Pd/C (210 mg) was degassed in a mixture

of EtOH (54 ml) and AcOH (6.0 ml) and exposed to hydrogenation conditions (1 atmosphere) for 4

15 Hours at room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 31 (333 mg, 84%) as a yellow solid:

1H 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 = 20

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,

٥٦٧٣

-١٠٦-

2H), 1.95 (s, 6H), 1.88–1.60 (m, 4H), 1.36 (s, 9H), 1.25 (d, J = 4.9 Hz,

6H).

preparation 32; A solution of 31 (370 mg, 0.36 mmol) and 21, 3.5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate methyl 226 mg was charged,

5 0.58 mmol (12 ml EtOH) with 0.51 ml DIPEA, 2.88 mmol) at

The room temperature. The reaction mixture was heated at 70 °C in a sealed tube for 2 h, cooled to room temperature, and concentrated under vacuum. The remaining material was purified using a silica gel chromatography column (CH3OH/NH4OH/CHCl3 (80:18:2) silica gel) to obtain 32 (250 mg guanidine, 63%) in the form of a yellow solid:

1H NMR (400 MHz, CD3OD): δ 8.23 (d, J = 8.6 Hz, 1H), 8.13–8.03 (m, 10

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), 3.13–3.10 (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). 15

Prepare Hydrochloric acid salt from:

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 20

4 p Hydrochloric acid 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 hours. The solvent was removed, an additional amount of 4 p HCl, and the mixture was heated at 40°C for another 6 hours. The solvent was removed, and the mixture was purified by phase chromatography

٥٦٧٣

-١٠٧-

Reverse (Gold column), and the remaining material was freeze-dried to obtain compound 33 (138 mg, 59%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6): δ 10.48 (brs, 1H), 10.45–10.41 (m, 1H), 9.25–9.19 (m, 1H), 8.95–8.85 (brs, 1H), 8.81–8.69 ( m, 1H), 8.64–

8.46 (m, 4H), 8.36–8.29 (m, 1H), 8.18–8.10 (m, 1H), 7.62–7.55 (m, 5

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), 3.62–3.53 (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 10

(m, 4H).

1H NMR (400 MHz, CD3OD): 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), 3.85–15

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).

20 5. Preparation:

diamino-N-(N-(4-(4-((S)-2-amino-3-oxo-3-(4-(3--3,5((2S,3R,4R,5R)-2) ,3,4,5,6-

pentahydroxyhexylamino)propyl)phenylamino)propyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

٥٦٧٣

-١٠٨-

)38(

Scheme 6

<img file="SA5673B1_D0039.tif" />

<img file="SA5673B1_D0040.tif" />

Preparation of compound 35;

5 Compound 34 (400 mg, 0.91 mmol) was charged in THF (15 ml) with DEPBT (389 mg, 1.30 mmol), 17 (516 mg, 1.00 mmol), and DIPEA (0.52 ml, 3.00 mmol) After the solvent was removed under low pressure, the residue was dissolved in 100 (100 CH2Cl2 mL) and washed quickly with saturated water of (50 x 2 NaHCO3 mL) and 50 brine (50 mL). (, and drying above

10 Na2SO4. The solvent was evaporated and the crude product was purified by Arf chromatography and flashing on silica gel

٥٦٧٣

-١٠٩-

CH2Cl2/ methanol (8% silica gel), to obtain 35 (700 mg amide, 83%) in the form of a yellow solid:

1H NMR (400 MHz, CDCl3): 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

= 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 (t, J = 6.2 Hz, 2H), 2.52 (t, J = 7.8

Hz, 2H), 1.90–1.76 (m, 3H), 1.42 (s, 18 H), 1.32 (d, J = 5.2 Hz, 3H). 10

Preparation of compound 36;

A suspension of 35 (700 mg, 0.74 mmol) and 10% Pd/C (400 mg) in a mixture of (90 EtOH ml) and 10 (10 ml AcOH ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 16 hours at °C. The reaction mixture was filtered through a Celite socket and the socket was washed

15 Using MeOH. The filtrate was concentrated under vacuum to obtain amine salt 36 (650 mg, 95%) as 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), 20

3.67–3.46 (m, 3H), 3.45–3.27 (m, 5H), 3.29–3.21 (m, 4H), 3.11–2.91

(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).

٥٦٧٣

-١١٠-

preparation 37;

A solution of 36 (650 mg, 0.70 mmol) was charged

(21) methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

436 mg, 1.13 mmol (in 12 ml EtOH) with 0.90 ml DIPEA, 5.60 mmol)

5 At room temperature. The reaction mixture was heated at 70 °C in a sealed tube for 2 h, cooled to room temperature, and concentrated under vacuum. The remaining material was purified using a silica gel chromatography column (CH3OH/NH4OH/CHCl3 (80:18:2) silica gel) to obtain 37 (444 mg) guanidine, 62%, in the form of 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 10

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, 1H), 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 15

(s, 9H), 1.25 (d, J = 5.0 Hz, 3H), 1.11 (s, 3H).

Prepare Hydrochloric acid salt of 38;

4 p Hydrochloric acid 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 hours.

20 The solvent was removed, an additional amount of 4 p HCl was added, and the mixture was heated at 40 °C for another 8 hours. The solvent was removed, the mixture was purified by reverse-phase chromatography (Gold column), and the remaining material was freeze-dried to obtain compound 38 (251 mg, 64%) as a yellow solid:

٥٦٧٣

-١١١-

1H NMR (400 MHz, DMSO-d6): δ 10.50 (brs, 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 = 5

4.9 Hz, 1H), 4.64–4.51 (m, 2H), 4.49–4.35 (m, 1H), 4.30–4.20 (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).

1H NMR (400 MHz, CD3OD): δ 8.26–8.20 (m, 1H), 8.19–8.14 (m, 1H), 10

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 15

(m, 2H), 1.90–1.75 (m, 4H).

6. to prepare :

of (S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1 -

yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide 20

)43(

Scheme 7

٥٦٧٣

-١١٢-

<img file="SA5673B1_D0041.tif" />

<img file="SA5673B1_D0042.tif" />

<img file="SA5673B1_D0043.tif" />

Preparation of compound 40;

A solution of acid 17 (880 mg, 1.70 mmol) in THF (30 ml) was cooled to 0 °C in an ice bath. (0.37 ml NMM, 3.40 mmol) was added, followed by (0.20 ml PivCl, 1.70 mmol). The reaction mixture was stirred at the same temperature for two hours

٥٦٧٣

-١١٣-

Add 39 (375 mg, 1.70 mmol, 15 mL THF) and the reaction mixture was stirred at the same temperature for another 10 minutes. The reaction mixture was transferred to room temperature and stirred for 16 hours. The organic solvent was removed. The The remaining material was mixed with water and extracted using 100 x 3 CH2Cl2 ml. The organic layers were collected, dried over Na2SO4, and filtered.

5 And focus. The remaining material was purified using a chromatography column (4% methanol in...

chloroform (to obtain 719 mg (40 amide, 59%) as a light yellow solid: 720 + [M + H).

Preparation of compound 41;

A suspension of 40 (719 mg, 1.00 mmol) and 10% Pd/C (300 mg) was degassed in a mixture

10 of EtOH (110 ml) and AcOH (20 ml) and exposed to hydrogenation conditions (1 atm) for 16 hours at room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain Amine salt 41 is a yellow solid (660 mg, 93%): 589 + [M + H].

Preparation of compound 42;

15 A solution of amine 41 (660 mg, 0.93 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (21, 650 mg, 1.67 mmol) in EtOH (10 ml) was charged with 1.66 ml DIPEA. (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 under vacuum. The residue was purified by an Arf gel chromatography column.

20 Silica CH3OH/NH4OH/CHCl3 2:18:80 (silica gel) to obtain guanidine

42 (370 mg, 50%) as a yellow solid: 801 + [M + H].

Preparation of Hydrochloric acid salt from compound 43:

٥٦٧٣

-١١٤-

(S)-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1- yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

TFA (10 ml) was added to 42 (370 mg, 0.46 mmol) in CH2Cl2 (10 ml)

5 The reaction mixture was stirred at room temperature for two hours. The solvent was removed, an additional amount of 1 V HCl was added, and the solvent was removed. The mixture was purified by reverse phase chromatography (Gold column) and the remaining material was freeze-dried to obtain compound 43 (290 mg, 92%) in the form of 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, 2H), 8.71–8.44 (m, 2H), 8.35 (brs, 1H), 10

8.13 (dd, J = 6.8, 3.8 Hz, 1H), 7.58 (dd, J = 6.5, 3.2 Hz, 2H), 7.42 (brs,

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, 15

4H).

1H NMR (400 MHz, CD3OD): δ 8.22–8.17 (m, 1H), 8.16–8.12 (m, 1H), 7.58–7.51 (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, 20

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. to prepare :

٥٦٧٣

-١١٥-

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

5 Scheme 8

<img file="SA5673B1_D0044.tif" />

<img file="SA5673B1_D0045.tif" />

٥٦٧٣

-١١٦-

<img file="SA5673B1_D0046.tif" />

Preparation of compound 45;

A solution of acid 17 (900 mg, 1.74 mmol) in (40 ml THF) was cooled to 0°C in an ice bath. 0.38 (0.38 ml NMM, 3.48 mmol) was added, followed by (0.21 ml) PivCl.

5 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 another 10 minutes. The reaction mixture was brought to room temperature and stirred for 16 hours. The remaining organic solvent was removed with water and extracted with 100 x 3 CH2Cl2 ml. The organic layers were collected, dried over Na2SO4, and filtered.

10 And focus. The remaining material was purified using a chromatography column (4% methanol in chloroform) to obtain 45 (2.00 g) amide, uncontaminated) as a light yellow solid: 1196 + [M + H].

Preparation of compound 46;

A suspension of 45 (2.00 g, uncontaminated) and 10% Pd/C (400 mg) was degassed in a mixture of

15 EtOH (120 ml) and AcOH (20 ml) and exposed to hydrogenation conditions (1 atmosphere) for 16 hours

At room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 46, which was done

٥٦٧٣

-١١٧-

It was neutralized using NaHCO3, and the crude product was purified by Arf chromatography and flashed on silica gel (CMA, 80:18:2) to obtain free amine 46 in the form of a yellow solid (500 mg, 27% in two steps): 1067 +] M+H].

Preparation of compound 47;

5 A solution of amine 46 (500 mg, 0.47 mmol) and 6-methyl 3,5-diamino was charged

330 (21) chloropyrazine-2-carbonylcarbamimidothioate mg, 0.84 mmol)

in 20 ml EtOH with 0.84 ml DIPEA, 94.70 mmol at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, cooled to room temperature, and concentrated under vacuum. The material was purified The remainder was analyzed by Arf-Gel chromatography column

10 Silica CH3OH/NH4OH/CHCl3 2:18:80 (silica gel) to obtain guanidine

47 (325 mg, 55%) as a yellow solid: 1278 +[M + H].

Preparation of Hydrochloric acid salt 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- 15

carboxamide

Add 4 p Hydrochloric acid in water (20 ml) to 47 (325 mg, 0.25 ml).

(mol) in 2.0 ml (EtOH) and the reaction mixture was stirred at room temperature for two hours.

The solvent was removed, the mixture was purified by reversed phase chromatography (Gold column), and dried.

20 The remaining material was frozen to obtain compound 48 (165 mg, 60%) in the form of a solid

In yellow:

1H NMR (400 MHz, DMSO-d6): δ 10.52 (brs, 1H), 10.44 (brs, 1H), 9.28 (t, J = 5.2 Hz, 1H), 9.00–8.88 (m, 1H), 8.87–8.75 (m, 1H), 8.63 (brs, 2H), 8.60–8.50 (m, 1H), 8.39–8.33 (m, 1H), 8.17–8.11 (m, 1H), 7.58

٥٦٧٣

-١١٨-

(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 = 6.8 Hz, 1H), 7.27 (d, J = 3.6 Hz, 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 5

(m, 2H), 1.35–1.23 (m, 4H).

1H NMR (400 MHz, CD3OD): δ 8.23–8.18 (m, 1H), 8.17–8.12 (m, 1H), 7.59–7.52 (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), 10

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).

15 8. Preparation:

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

20 Scheme 9

٥٦٧٣

-١١٩-

<img file="SA5673B1_D0047.tif" />

<img file="SA5673B1_D0048.tif" />

<img file="SA5673B1_D0049.tif" />

<img file="SA5673B1_D0050.tif" />

<img file="SA5673B1_D0051.tif" />

<img file="SA5673B1_D0052.tif" />

<img file="SA5673B1_D0053.tif" />

<img file="SA5673B1_D0054.tif" />

<img file="SA5673B1_D0055.tif" />

Preparation of compound 50;

A solution of acid 17 (950 mg, 1.84 mmol) in THF (30 ml) was cooled to 0°C in an ice bath. 0.40 (0.40 ml) NMM, 3.68 mmol) was added, followed by (0.23 ml) PivCl.

5 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) were added and the reaction mixture was stirred at the same temperature for another 10 minutes. Bring the reaction mixture to room temperature and stir for a while

٥٦٧٣

-١٢٠-

16 hour. The organic solvent has been removed. The residue was charged with water and extracted with 100 x 3 (CH2Cl2 ml). The organic layers were collected, dried over Na2SO4, filtrated, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to obtain 50 amide (1.40). g, uncontaminated) as a light yellow solid: 1043 +[.[M + H

Preparation of compound 51;

A suspension of 50 (1.40 g, uncontaminated) and 10% Pd/C (400 mg) in a mixture of (120 EtOH) and AcOH (20 ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 16 hours at temperature The reaction mixture was filtered through a Celite socket and the socket 10 was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 51, used directly in the next step (1.20 g, crude): 913 + [M + H].

Preparation of compound 52;

A solution of amine 51 (1.20 g, 0.47 mmol, crude) and methyl 3,5-diamino 723, 21, 6-chloropyrazine-2-carbonylcarbamimidothioate mg, 1.86 mg, was charged.

15 (20 mol) in 20 ml EtOH with 2.00 ml DIPEA, 11.6 mmol) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, cooled to room temperature, and concentrated under vacuum. The remaining material was purified using a chromatography column using silica gel (CH3OH/NH4OH/CHCl3 (80:18:2) to obtain guanidine 52 (500 mg, 24% in three steps) in the form of a yellow solid: +[M + H]

20 1125.

Preparation of Hydrochloric acid salt from compound 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-

٥٦٧٣

-١٢١-

pentahydroxyhexylamino)hexyl)phenylamino)propyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

4% Hydrochloric acid in water (25 ml) was added to 52 (500 mg, 0.44 mmol) in 5.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours. 5 The solvent was removed, the mixture was purified by chromatography. Draw in reverse phase (Gold column), and the material is dried

The remainder was frozen to obtain compound 53 (170 mg, 41%) in the form of 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 (brs, 1H), 8.88–8.76 ( m, 1H), 8.70–

8.58 (m, 3H), 8.57–8.46 (m, 2H), 8.40–8.31 (m, 1H), 8.17–8.10 (m, 10

1H), 7.62–7.54 (m, 2H), 7.42 (brs, 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), 5.41–5.35 (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 15

(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).

1H NMR (400 MHz, CD3OD): 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– 20

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, 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), 1.45–1.31 (m, 4H).

٥٦٧٣

-١٢٢-

9. to prepare :

3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(hexyl((2S,3R,4R,5R))-

2,3,4,5,6-pentahydroxyhexyl)amino)hexyl)phenylamino)-3-

oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-

10 Scheme carboxamide 5

<img file="SA5673B1_D0056.tif" />

<img file="SA5673B1_D0057.tif" />

<img file="SA5673B1_D0058.tif" />

٥٦٧٣

-١٢٣-

Preparation of compound 55; The compound was charged 54 (770 mg, 1.45 mmol) in THF (50 ml) with 564 mg DEPBT (564 mg, 1.88 mmol), 17 (752 mg, 1.45 mmol), and DIPEA (0.77 ml, 4.35 mmol) sequentially and stirred. At room temperature for 16 hours after the solvent was removed under low pressure, the remaining material was dissolved in (100 ml) CH2Cl2.

5 Washing quickly using a saturated amount of water (100 × 2 ml NaHCO3) and 50 brine (50 ml), and drying over Na2SO4. The solvent was evaporated and the crude product was purified by Arf chromatography and flashed on a silica gel (CH2Cl2/methanol 5% silica gel) and by Arf chromatography in a phase. Reverse (Gold column), to obtain 55 amide as a yellow solid (800 mg, 54%): 1027 +[M + H].

10 Preparation of compound 56;

A suspension of 55 (800 mg, 0.78 mmol) and 10% Pd/C (400 mg) in a mixture of (120 EtOH) and AcOH (30 ml) was degassed and exposed to hydrogenation conditions (1 atm) for 16 hours at °C. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine 56 as a yellow solid (780 mg, 99%): 897 + [M + H]. .

Preparation of compound 57;

A solution of 56 amine salt (780 mg, 0.75 mmol) and 21)-methyl 3,5-diamino 466-2-carbonylcarbamimidothioate mg, 1.20 mmol) in EtOH (20 ml) was charged with 1.37 DIPEA. ml, 7.67 mmol) at room temperature.

20 The reaction mixture was heated at 70 °C in a sealed tube for 2 h, cooled to room temperature, and concentrated under vacuum. The remaining material was purified by Arff silica gel chromatography column (CH3OH/NH4OH/CHCl3 (80:18:2) silica gel) to obtain guanidine 57 (455 mg, 55%) in the form of a yellow solid: 1110 + [M + H].

Preparation of Hydrochloric acid salt from compound 58:

٥٦٧٣

-١٢٤-

3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(hexyl((2S,3R,4R,5R))-2,3) ,4,5,6-pentahydroxyhexyl)amino)hexyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

5 4 p Hydrochloric acid in water (25 ml) was added to 57 (455 mg, 0.41 mmol) in 10 ethanol (10 ml) and the reaction mixture was stirred at room temperature for 2 hours. The mixture was purified by reverse phase chromatography (column Gold) and the remaining material was freeze-dried to obtain compound 58 (230 mg, 55%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6): δ 10.45 (brs, 1H), 9.30 (brs, 1H), 9.09–

8.49 (m, 3H), 8.41–8.32 (m, 1H), 8.16–8.08 (m, 1H), 7.62–7.52 (m, 10

2H), 7.42 (brs, 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, 1H), 4.23 (t, J =

6.7 Hz, 1H), 4.03–3.94 (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), 15

3.09–2.96 (m, 6H), 1.77–1.58 (m, 8H), 1.57–1.46 (m, 2H), 1.35–1.21 (m, 10H), 0.86 (t, J = 6.4 Hz, 3H).

1H NMR (400 MHz, CD3OD): δ 8.26–8.20 (m, 1H), 8.19–8.12 (m, 1H), 7.60–7.51 (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, 20 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, 10 H), 0.92 (t, J = 6.7 Hz, 3H).

٥٦٧٣

-١٢٥-

10. to prepare :

5

(S)-2-amino-3-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-2-yl)propanoic acid

)80(

Scheme 11

<img file="SA5673B1_D0059.tif" />

<img file="SA5673B1_D0060.tif" />

<img file="SA5673B1_D0061.tif" />

<img file="SA5673B1_D0062.tif" />

<img file="SA5673B1_D0063.tif" />

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Preparation of compound 62;

carbomthoxymethylenetriphenylphosphorane 5 produced stable hydration

(43.0 g Ph3PCHCO2Me, 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. Monitor the completion of TLC Reaction (16 hours). CH2Cl2 was removed under low pressure and FCC using 10% ethyl acetate-hexanes

10 To obtain unsaturated ester (24.0 g, α-trans, 62 g, 92%) in the form of a white solid:

1H NMR (400 MHz, CDCl3) 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 =

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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 method);

To 55.6 ml (381 mmol) Trimethyl phosphonoacetate (381 mmol) in 250 ml

5 Nonaqueous CH2Cl2 was cooled to 0 °C. 48.8 ml DBU (322 mmol) was added and the mixture was stirred for 15 minutes. Aldehyde 59 (40.0 g, 215 mmol) was added in 50 ml CH2Cl2 dropwise. The temperature of the reaction mixture became At room temperature, the resulting reaction mixture was stirred at room temperature for 16 hours, and then quenched with 100 ml of water. The mixture was divided, and the aqueous layer was extracted using 150 x 3 CH2Cl2 ml.

10 The collected organic materials were washed using brine, drying (Na2SO4), filtration, and concentration, and the remaining material was purified by chromatography using a 10:1 silica gel hexanes column (silica gel / ethyl acetate) to obtain an unsaturated ester of 62 (48.0), -, - trans. gm, 92%) as a white solid.

Preparation of compound 64;

15 A suspension of compound 62 (48.0 g, 196 mmol) and 10% Pd/C (10 g) in EtOAc/THF (600 ml/75 ml) was exposed to hydrogenation conditions (1 atm) for 16 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in the blank to obtain 64 (46.5 g, 96%) as a white solid:

1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 9.4 Hz, 2H), 7.57-7.54 (m, 1H), 7.29(dd, J = 8.6, 1.8 Hz, 1H), 7.12 (dd, J = 8.8, 2.5 Hz, 1H), 7.11- 20

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;

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To a solution of 64 (46.5 g) methyl ester (191 mmol) in THF/MeOH/H2O (500 ml/500 ml/150 ml) NaOH (45.6 g, 114 mmol) was added and the reaction mixture was stirred at room temperature. For two hours, the solvent was removed and the pH value was adjusted to 1 using 1 p of aqueous Hydrochloric acid; a white solid was precipitated. The solid was filtered, washed with water and dried under vacuum to obtain 66 (42.5 g, 97%) acid. (In the form of a white solid:

1H NMR (400 MHz, DMSO-d6) δ 12.14 (brs, 1H), 7.73 (dd, J = 9.5, 2.3 Hz, 2H), 7.64-7.61 (m, 1H), 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). 10

Preparation of compound 67;

To a solution of compound 60 (39.3 g, 222 mmol) in dry THF (500 ml) n-butyl lithium (110 ml), a 2 M solution in cyclohexane (110 ml) was added by distillation at -78 °C and the reaction mixture was stirred for 1 hour to obtain Solution of compound 61. To

15 Another solution of compound 66 (42.5 g, 185 mmol) in dry THF (1000 ml) was added (26.3 ml NMM, 240 mmol) and PivCl (27.3 ml, 222 mmol) by distillation at -78 °C. The reaction mixture was stirred for 1 minute at the same temperature, after which the prepared solution of compound 66 was added slowly at -78 °C. The reaction mixture was stirred for another 10 minutes, then transferred to 0 °C and stirred for 1 hour followed

20 At room temperature for 30 minutes, quenching with saturated NH4Cl, concentrating to remove THF, and partitioning between (1000 CH2Cl2) (1000 ml) and water (1000 ml). The aqueous layer was separated and extracted with CH2Cl2 (1000 x 2 ml). The extraction products were dried The combined organic matter was concentrated over Na2SO4 and the remaining substance was purified using a chromatography column (silica gel, CH2Cl2) to obtain compound 67 (45.0 g, 63%) in the form of a white solid:

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1H NMR (400 MHz, CDCl3) 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, 1H), 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). 5

Preparation of compound 68;

To a solution of compound 67 (45.0 g, 116 mmol) in dry THF (700 ml) KHMDS (34.6 g, 174 mmol) was added in batches at -78 °C. After stirring the resulting mixture for 30 minutes, 53.6 g was added. (10 trisyl azide g, 174 mmol) The reaction mixture was stirred for 5 minutes. Then (69.6 ml Acetic Acid, 1158 mmol) was added, followed by 30.9 (30.9 ml tetramethylammonium acetate, 232 mmol) slowly at the same temperature. The reaction mixture was left to warm to 24°C, stirred for 16 hours, quenched with saturated NaHCO3 (300 ml), concentrated for THF removal and extracted with CH2Cl2 (2 × 500 ml). The combined organic extraction products were dried over Na2SO4 and concentrated.

15 The remaining material was purified by a rack chromatography column (silica gel EtOAc 90:10, silica gel/hexane followed by DCM) to obtain compound 68 (31.0 g, 62%) in the form of a yellow solid:

1H NMR (400 MHz, CDCl3) δ 7.70 (d, J = 9.1 Hz, 2H), 7.68-7.65 (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 = 20

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, 1H), 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).

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Preparation of compound 69;

To a solution of compound 68 (31.0 g, 72.1 mmol) in THF/H2O (300 ml/100 ml) H2O2 (49 ml, 433 mmol) was added followed by 6.04 (6.04 g LiOH, 144 mmol) in batches at 0 °C. The reaction mixture was stirred for 10 minutes at the same temperature

5 Next, heat at room temperature for one hour and then quench with saturated Na2SO3.

(200 ml), and concentrated under low pressure THF removal and washing with 500 CH2Cl2 (500 ml). The aqueous layer was acidified with 1 aqueous Hydrochloric acid and extracted with 2 x 500 CH2Cl2 (500 ml). The combined organic extraction products were dried over Na2SO4, and concentrated Washing with MTBE obtained the compound 69 (15.0 g, 82%) 10 in the form of a tan-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).

15 Preparation of compound 70;

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) was exposed to hydrogenation conditions (1 atm) for 3 hours at room temperature. Filtering the reaction mixture through Celite and washing with AcOH/H2O followed by MeOH. The filtrate was concentrated in the blank to obtain acetic salt 70 (14.0 g, 83%) in the form of 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).

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Preparation of compound 71;

To a solution of compound 70 (14.0 g, 45.9 mmol) in Acetic Acid (140 ml) hydrobromic acid (140 ml) was added dropwise at room temperature and the reaction mixture was refluxed for 3 hours. The reaction mixture was cooled to h Chamber temperature and concentration. The remaining crude brown 71 (12.4 g, 87%) was used directly for the next step without any purification:

1H NMR (400 MHz, DMSO-d6) δ 13.83 (brs, 1H), 9.71 (brs, 1H), 8.41 (brs, 1H), 8.25 (brs, 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).

10 Preparation of compound 72;

Acetyl chloride (38.4 ml, 540 mmol) was added to dry methanol (400 ml) at 0°C and then compound 71 (24.0 g, 77.2 mmol) was added. The reaction mixture was refluxed for 4 hours and concentrated. The material was aliquoted The remaining 500 ml CH2Cl2 (500 ml) and saturated NaHCO3 (300 ml). The aqueous layer was separated and extracted using 300 ml x 15 CH2Cl2 (2). The combined organic extraction products were dried over Na2SO4 and concentrated to obtain

Compound 72 (16.6 g, 88%) is a white solid:

1H NMR (400 MHz, DMSO-d6) δ 9.62 (brs, 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 = 20

13.2, 7.4 Hz, 1H), 1.90 (brs, 2H).

Preparation of compound 73;

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To a solution of compound 72 (16.6 g, 67.8 mmol) in MeOH/H2O (360 ml/120 ml) were added 22.8 g NaHCO3 (271 mmol) and Boc2O (17.7 g, 81.3 mmol) at 0 °C The resulting mixture was left to warm to room temperature and stirred for one hour. The reaction mixture was divided between (200 ml) CH2Cl2 and water. The layer was separated.

5 aqueous and extraction using 400 ml Solid white:

1H NMR (400 MHz, CDCl3) δ 7.60 (d, J = 9.5 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.49-7.43 (m, 1H), 7.15 (d, J = 8.2 Hz , 1H), 7.09-6.99 (m, 10

2H), 6.31 (brs, 1H), 5.15-4.84 (m, 1H), 4.73-4.46 (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;

15 To a solution of the compound 7.0 (73 g, 20.3 mmol) in 300 ml CH2Cl2, 16.5 ml pyridine, 203 mmol, 5.11 triflate ml, 30.4 mmol were added at 0°C and stirring at the same temperature. Heat for 1 hour followed by room temperature for 2 hours. After concentration, the reaction mixture was divided between (300 CH2Cl2) and water (200 ml). The aqueous layer was separated and extracted with 300 × 2 CH2Cl2 ml. The products were washed.

20 The combined organics were extracted using brine, dried over Na2SO4 and concentrated to obtain compound 74 (8.80 g, 91%) as a brown oil (pyridine present as confirmed by NMR). The reaction was monitored using LC-MS, and product formation was confirmed by - LM

:MS data

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1H NMR (400 MHz, CDCl3) 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), 5.12-5.03 (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). 5

Preparation of compound 75;

Compound 74 (16.5 g, 34.6 mmol) and benzyl but-3-ynylcarbamate (17, 10.4 g, 51.9 mmol) were degassed in a non-aqueous amount of CH3CN (450 ml) with Argon for 10 minutes at rt, then added 19.3 (TEA ml, 138 mmol(, 10% -t)

10 Bu(3P) in 13.9 g hexanes, 6.91 mmol, and 0.33 g CuI, 1.72 mmol at room temperature. The resulting mixture was dehydrated with Argon for 10 minutes and 3.99 g Pd(PPh3) was added. (3.45 mmol) rapidly in one batch. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 18 hours. The reaction mixture was concentrated in vacuum and the residue was purified by a silica gel column (EA/hexanes 75:25). , silica gel

15 To obtain compound 75 (14.1 g, 77%) as a brown solid:

1H NMR (400 MHz, CDCl3) δ 7.86 (brs, 1H), 7.68 (t, J = 7.8 Hz, 2H), 7.53 (brs, 1H), 7.41 (dd, J = 8.5, 1.6 Hz, 1H), 7.38 -7.28 (m, 5H), 7.277.22 (m, 1H), 5.26-5.17 (m, 1H), 5.13 (s, 2H), 5.06-4.99 (m, 1H),

4.70-4.59 (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, 20

2H), 1.38 (s, 9H).

Preparation of compound 76;

To a solution of 75 (12.1 g) methyl ester (22.8 mmol) in THF/MeOH/H2O (150 ml/150 ml/50 ml) NaOH (4.56 g, 114 mmol) was added and stirred.

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The reaction mixture was kept at room temperature for two hours. The pH value was adjusted to 9 using 1 p of aqueous Hydrochloric acid and the organic solvent was removed. The pH value of the residue was adjusted to 5-6, and the suspension was divided between 500 CH2Cl2 (500 ml) and water (200 ml). The aqueous layer was separated and extracted using 5 x 2 CH2Cl2 (400 ml). The combined organic extraction products were dried over Na2SO4 and concentrated To obtain 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), 7.447.19 (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). 10

Preparation of compound 77; 27-SG-SJL-B

A suspension of 75 (2.0 g, 3.77 mmol) and 10% Pd/C (500 mg) was degassed in a mixture of (90 ml) EtOH and 10 (10 ml AcOH) and then exposed to hydrogenation conditions (1 atmosphere) for 16 hours at Room temperature. The reaction mixture was filtered through a Celite socket and washed

15 Socket using MeOH. The filtrate was concentrated in the blank to obtain 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), 20

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; 30-SG-SJL-B

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To a solution of amine salt 77 (1.60 g, 3.47 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (13, 2.16 g, 5.56 mmol) in EtOH (40 ml) DIPEA (6.20 ml) was added. 5 ml, 34.70 mmol) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 1 hour, then cooled to room temperature, and concentrated in blank. The residue was purified by column chromatography.

(Silica gel CH3OH/NH4OH/CHCl3 2:18:80, silica gel) to obtain 1.24 (78 guanidine g, 59%) in the form of a yellow solid:

1H NMR (400 MHz, CD3OD) δ 7.71 (dd, J = 8.4, 2.8 Hz, 2H), 7.60 (brs, 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 = 10

2.4 Hz, 1H), 3.22 (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; 32-SG-SJL-B

15 A solution of 78 (1.24 g) methyl ester (2.00 mmol) in a mixture of THF (25 ml), methanol (25 ml) and water (10 ml). Solid NaOH (324 mg, 8.00 mmol) was added and the reaction mixture was stirred at a temperature At room temperature for one hour, TLC of the reaction mixture showed that the reaction was complete, then the pH of the reaction mixture was converted to pH 10-9 by adding 1 p Hydrochloric acid (aqueous) and the organic solvent was removed. The pH was adjusted.

20 Dilute the aqueous portion to pH 5-6, form a precipitate, and extract using dichloromethane. The aqueous fraction was extracted with 50 x 2 ml (CH2Cl2). The organic layers were combined, dried over Na2SO4, filtrated, and concentrated. A yellow colored solid compound (79, 1.10 g, 92%) was dried under vacuum:

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1H 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).

5 Preparation of compound 80:

Hydrochloride salt of (S)-2-amino-3-(6-(4-(3-(3,5-diamino-6-)

chloropyrazine-2-carbonyl)guanidino)butyl)naphthalen-2-yl)propanoic acid

4 p Hydrochloric acid in 25 dioxane ml was added to 79 (1.10 g, 1.83 10 mmol) in 5.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours.

The solvent was removed, purified using a reverse phase column (Gold column), and the remaining material was freeze-dried to obtain compound 80 (700 mg, 67%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6)10.48 (s, 1H), 9.24 (brs, 1H), 8.99-8.86

(m, 1H), 8.84-8.70 (m, 1H), 8.38 (brs, 3H), 7.80 (t, J = 9.2 Hz, 2H), 15

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).

1H NMR ((400 MHz, CD3OD) 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, 20 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. to prepare :

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(S)-3,5-diamino-6-chloro-N-(N-(4-(6-(2,3-diamino-3-)

oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)pyrazine-2-carboxamide

)84(

Scheme 12

<img file="SA5673B1_D0069.tif" />

<img file="SA5673B1_D0070.tif" />

<img file="SA5673B1_D0071.tif" />

<img file="SA5673B1_D0072.tif" />

<img file="SA5673B1_D0073.tif" />

Preparation of compound 81;

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A solution of acid 76 (2.0 g, 3.87 mmol) in THF (80 ml) was cooled to 0°C in an ice bath, 0.63 NMM (0.63 ml, 5.03 mmol) was added followed by distillation -i (0.63 ml BCF, 5.80 mmol) and the reaction mixture was stirred at the same temperature for two hours. Methanol (7.0 ml, 38.7 mmol) was added by distillation.

5 The reaction mixture was stirred at the same temperature for another two hours. The reaction mixture was then brought to room temperature and stirred for 16 hours. The organic solvent has been removed. To this residue water was added and extracted with 100 x 3 CH2Cl2 ml. The organic layers were collected, dried over Na2SO4, filtrated, and concentrated. The residue was purified by a column chromatogram (3% methanol in chloroform) to obtain 1.75 (81). amide g,

10 88% as a light yellow solid:

1H NMR (400 MHz, CD3OD) 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). 15

Preparation of compound 82;

A suspension of 81 (1.75 mg, 3.39 mmol) and 10% Pd/C (600 mg) was degassed in a mixture of (110 EtOH) and AcOH (15 ml) and then exposed to hydrogenation conditions (1 atmosphere) for 12 hours at Room temperature. The reaction mixture was filtered through a Celite socket

20 The socket was washed with MeOH. The filtrate was concentrated in a vacuum to obtain an 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),

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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;

5 To a solution of 82 amine salt (1.40 g, 3.15 mmol) and 13-chloropyrazine-2-carbonylcarbamimidothioate (1.96 g, 5.04 mmol) in EtOH (40 ml) DIPEA (5.64 ml) was added. ml, 31.5 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 blank. The residue was purified by column

10 Chromatogarve (CH3OH/NH4OH/CHCl3 80:18:2, silica gel) to obtain 83 (1.15 g guanidine, 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), 1.85-1.76 (m, 2H), 1.70-1.61 (m, 2H), 1.27 (s, 7H), 1.20 (s, 15

2H).

Prepare the compound Hydrochloric acid salt from:

(S)-3,5-diamino-6-chloro-N-(N-(4-(6-(2,3-diamino-3-oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)pyrazine-2- carboxamide

)84( 20

4 p Hydrochloric acid in 25 ml dioxane was added to 1.15 g (83 g, 1.92 mmol) in 6.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed and purified by a reverse-phase column. reverse phase column

٥٦٧٣

-١٤٠-

Gold) and the remaining material was freeze-dried to obtain compound 84 (310 mg, 28%) in the form of 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 (brs, 3H), 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 (m, 4H), 4.06 5

(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).

1H NMR (400 MHz, CD3OD) 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 10

(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), 1.79-1.71(m, 2H).

12. to prepare :

3,5-diamino-N-(N-(4-(6-((S)-2-amino-3-(4-(3-(hexyl((2S,3R,4R,5R))- 15)

2,3,4,5,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

)89(

20 Scheme 13

٥٦٧٣

-١٤١-

^C6^H13 _N

sugar

OH OH

(R) (S)

<img file="SA5673B1_D0074.tif" />

sugar = (R) (R)

OO OH

Ph

<img file="SA5673B1_D0075.tif" />

DEPBT/DIPEA/THF

O

NH2

C6H13_N

sugar

<img file="SA5673B1_D0076.tif" />

BocHN

86

Pd/C,H₂EtOH/AcOH

NHCbz

C6H13_N

sugar

<img file="SA5673B1_D0077.tif" />

BocHN

N

O NH•HI 87

Cl

HN

SCH₃

NH₂

DIPEA, EtOH

N

^C6^H13 _N

<img file="SA5673B1_D0078.tif" />

Preparation of compound 86;

To the 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 (1.0 ml, 5.91 mmol) were added sequentially. After the solvent was removed under low pressure, the residue was dissolved in 100% CH2Cl2 and washed quickly with a saturated amount of water (50X 2ml) and stirred at room temperature for 16 hours. Drying over Na2SO4 was done

٥٦٧٣

-١٤٢-

Evaporate the solvent and purify the crude product by flash chromatography on silica gel (5% CH2Cl2 / methanol), to obtain amide 86 as a yellow solid product (1.19 g, 57%):

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

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, 2H), 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), 10

1.31 (s, 9H), 1.29-1.16 (m, 8H), 0.86 (t, J = 6.9 Hz, 3H).

Preparation of compound 87;

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 exposed to hydrogenation conditions (1 atmosphere) for 3 hours at 15°C. At room temperature, the reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated in a vacuum to obtain the amine salt 87, which was neutralized with NaHCO3, and the fermented product was purified by flash chromatography on silica gel (CMA, 80:18:2), to obtain free amine 87 in the form of a yellow solid. (550 mg, 58%, in two steps):

1H NMR (400 MHz, CD3OD) 7.71 (t, J = 8.4 Hz, 2H), 7.62 (d, J = 1.8 20

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 , 1H), 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),

٥٦٧٣

-١٤٣-

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) , 1.85-1.75 (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).

5 preparation 88;

To a solution of amine 87 (550 mg, 0.65 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (21, 400 mg, 1.04 mmol) 3,5-diamino-6-methyl (20 ml) in EtOH (1.15 ml DIPEA) was added. 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 a blank. The remaining material was purified by chromatography.

Silica gel column CH3OH/NH4OH/CHCl3 silica gel (80:18:2) followed by a column

88 guanidine to obtain (Gold C18) reverse phase column

(333 mg, 48%) as a yellow solid:

1H NMR (400 MHz, CD3OD) 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, 15 2H), 7.33-7.28 (m, 5H), 7.04 (d, J = 8.3 Hz, 2H), 5.52 (s, 1H), 4.524.55 (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), 20

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).

Prepare Hydrochloric acid salt from:

٥٦٧٣

-١٤٤-

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-

compound carboxamide (89);

5 4 p Hydrochloric acid in water (20 ml) was added to 88 (333 mg, 0.31 mmol) in 10 ethanol (10 ml) and the reaction mixture was stirred at room temperature for 2 hours. Purification was done using a reverse phase column (reverse phase column). Gold column) and the remaining material was freeze-dried to obtain compound 89 (210 mg, 68%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6) 10.94 (brs, 1H), 9.29 (brs, 1H), 9.02- 10

8.77 (m, 2H), 8.64-8.17 (m, 2H), 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), 5.52-5.41 (m, 1H), 4.86-4.71 (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, 15

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).

1H NMR (400 MHz, CD3OD) 7.79 (d, J = 8.5 Hz, 1H), 7.77-7.73 (m, 20

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 , 1H), 3.82-3.74 (m, 1H), 3.713.61 (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-

٥٦٧٣

-١٤٥-

2.64 (m, 2H), 2.10-2.00 (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, 3H).

13. to prepare :

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- 5 oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-(94) carboxamide

Scheme 14

٥٦٧٣

-١٤٦-

<img file="SA5673B1_D0079.tif" />

<img file="SA5673B1_D0080.tif" />

Preparation of compound 91;

To the compound 90 (484 mg, 0.91 mmol) in THF (30 ml) were added DEPBT (300 mg, 1.00 mmol), 19 (400 g, 0.77 mmol) and 0.40 ml DIPEA, 2.31

5 (mmol) sequentially and stirring at room temperature for 16 hours. After the solvent was removed under reduced pressure, the residue was dissolved in 100 (100 mL) of CH2Cl2, and quickly washed with a saturated volume of water (2 x 100 mL) and 50 brine ml), and drying over Na2SO4. Done

٥٦٧٣

-١٤٧-

Evaporate the solvent and purify the crude product by flash chromatography on silica gel (5% CH2Cl2 / methanol), to obtain amide 91 as a yellow solid product (600 mg, 76%, uncontaminated). The composition of the product was confirmed by LCMS.

Preparation of compound 92;

5 A suspension of 91 (600 mg, 0.59 mmol) and 10% Pd/C (200 mg) was degassed in a mixture of (90 EtOH ml) and 10 (10 ml AcOH) and then exposed to hydrogenation conditions (1 atmosphere) for 16 hours at Room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated in a vacuum to obtain the amine salt 92, which was neutralized with NaHCO3. The fermented product was purified by flash chromatography on silica gel (silica gel, 10:18:80, 2 CMA) to obtain the free amine 36 in the form of a colored solid. Yellow (350 mg, 66%, uncontaminated):

1H NMR (400 MHz, CD3OD) 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, 15

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).

20 preparation 93;

To a solution of amine 92 (350 mg, 0.38 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (13, 242 mg, 0.62 mmol) in EtOH (10 ml) DIPEA (0.67 ml) was added. (3.80 mmol) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, then

٥٦٧٣

-١٤٨-

Cooled to room temperature, and concentrated in a blank. The remaining material was purified by chromatography using a silica gel column (CH3OH/NH4OH/CHCl3 (80:18:2)), followed by a silica gel column.

93 guanidine to obtain (Gold C18) reverse phase column

(170 mg, 20% in three steps) as a yellow solid:

1H NMR (400 MHz, CD3OD) 7.71 (d, J = 8.2 Hz, 2H), 7.68 (s, 1H), 5

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), 2.77- 10

2.61 (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).

Prepare Hydrochloric acid salt from:

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- 15 oxopropyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-

(94) carboxamide

4 p Hydrochloric acid 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.

20 Remove the solvent again, add 4 p Hydrochloric acid, and heat at 40°C for

Another two hours. This addition was repeated several times. The solvent was removed and purified using a reverse phase column (Gold column), and the remaining material was freeze-dried to obtain compound 94 (80 mg, 50%) as a yellow solid:

٥٦٧٣

-١٤٩-

1H NMR (400 MHz, DMSO-d6) 10.74 (brs, 1H), 9.28-9.19 (m, 1H), 9.03-8.60 (m, 2H), 8.58-8.04 (brs, 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, 5 1H), 3.62-3.55 (m, 2H), 3.53-3.38 (m, 5H), 3.37-3.29 (m, 2H), 3.243.09 (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).

1H NMR (400 MHz, CD3OD) 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 10 = 8.4 Hz, 2H), 7.21 (d, J = 8.7 Hz, 2H), 4.16 (t, J = 7.0 Hz, 1H), 4.134.05 (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, 10H) , 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). 15

14. to prepare :

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-

(99(yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide 20

Scheme 15

٥٦٧٣

-١٥٠-

<img file="SA5673B1_D0081.tif" />

<img file="SA5673B1_D0082.tif" />

<img file="SA5673B1_D0083.tif" />

Compound preparation 96;

A solution of acid 19 (1.17 g, 2.27 mmol) in THF (60 ml) was cooled to 0 °C in an ice bath, (0.30 ml NMM, 2.95 mmol) was added, followed by (0.30 ml PivCl, 2.49 mmol) The reaction mixture was stirred at the same temperature for two hours. 34 (1.0 g, 2.27 mmol, 10 ml THF) of aniline 171 were added, and the reaction mixture was stirred at the same temperature for another 10 minutes. Then the reaction mixture was transferred to the mixture. At room temperature and stirring for 16 hours, the organic solvent was removed, and water was added

٥٦٧٣

-١٥١-

and extraction with 100 × 3 CH2Cl2 ml). The organic layers were combined, dried over Na2SO4, filtered, and concentrated. The remainder was purified by column chromatography (4% methanol in chloroform) to obtain 96(1.40 g) amide, 66%, untreated. Contaminant) as a light yellow solid. Product composition confirmed by LCMS

5 Compound preparation 97;

A suspension of 96 (1.40 g, 1.50 mmol) and 10% Pd/C (300 mg) was degassed in a mixture of (120 EtOH ml) and AcOH (12 ml) and then exposed to hydrogenation conditions (1 atmosphere) for 16 hours at Room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated in the blank to obtain amine salt 97

10 It is neutralized using NaHCO3, and the fermented product is purified by flash chromatography on silica gel (CMA, 80:18:2), to obtain free amine 97 in the form of a yellow solid (550 mg, 30%, in two steps):

1H NMR (400 MHz, CD3OD) 7.74-7.65 (m, 3H), 7.59 (s, 1H), 7.417.29 (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), 15

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), 1.50-1.37 (m, 12H), 1.33 (s,

9H), 1.25 (d, J = 4.9 Hz, 3H). 20

preparation 98;

To a solution of amine 97 (550 mg, 0.68 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (13, 6-methyl 3,5-diamino 423 mg, 0.62 mmol) in EtOH (20 ml) DIPEA (1.21 ml) was added. 6.80 mmol) at temperature

٥٦٧٣

-١٥٢-

the room. The reaction mixture was heated at 70 °C in a sealed tube for 2 hours, then cooled to room temperature, and concentrated in a vacuum. The remaining material was purified by chromatography using a silica gel column (CH3OH/NH4OH/CHCl3 (80:18:2)), followed by a silica gel column.

98 guanidine to obtain (Gold C18) reverse phase column

5 (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), 3.54-3.38 (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 10

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).

Prepare Hydrochloric acid salt from:

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) 15 propyl)naphthalen-2-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-

(99) carboxamide

4 p Hydrochloric acid 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.

20 After removing the solvent again, 4 p Hydrochloric acid was added and heated at 40°C

For another two hours. This addition was repeated several times. The purified solvent was removed by a reverse phase column (Gold column) and the remaining material was freeze-dried to obtain compound 99 (206 mg, 50%) as a yellow solid:

٥٦٧٣

-١٥٣-

1H NMR (400 MHz, DMSO-d6) 11.0 (brs, 1H), 9.34 (brs, 1H), 9.098.25 (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, 1H), 4.62 (d, J = 4.3 Hz, 1H), 4.60-4.54 (m, 1H), 4.46-4.36 ( m, 2H), 3.96-3.88 (m, 5

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).

1H NMR (400 MHz, CD3OD) 7.80 (d, J = 9.5 Hz, 1H), 7.78-7.73 (m, 10

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, 1H) ), 3.87-3.81 (m, 1H), 3.78 (d, J = 11.4 Hz, 1H), 3.73-3.61 (m, 3H), 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- 15

1.95(m, 2H), 1.91-1.81(m, 2H), 1.80-1.69(m, 2H).

15. Preparation of (3,5-(S-diamino-2)-6(-4)-N(-N-amino-3-(4-)3-(dimethylamino)propyl)phenylamino)-3-oxo Propyl(naphthalene-2-yl(butyl(carbamimidoyl(-6-chloropyarzine-2-carboxy103) amide)

20 Scheme 16

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<img file="SA5673B1_D0084.tif" />

<img file="SA5673B1_D0085.tif" />

<img file="SA5673B1_D0086.tif" />

Preparation of compound 100;

A solution of acid 19 (1.75 g, 3.39 mmol) in THF (70 ml) was cooled to 0°C in an ice bath, 0.74 ml NMM, 6.78 mmol) was added, followed by 0.41 ml PivCl.

5 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) were added and the reaction mixture was stirred at the same temperature for another 10 minutes. Then The reaction mixture was brought to room temperature and stirred

٥٦٧٣

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For 16 hours. The organic solvent has been removed. To this residue water was added and extracted with 100 x 3 CH2Cl2 ml (100). The organic layers were collected, dried over Na2SO4, filtrated, and concentrated. The residue was purified by a column chromatogram (4% methanol in chloroform) to obtain 100 (1.60). amide g, 71%) as a light yellow solid:

1H NMR (400 MHz, CDCl3) 7.87 (s, 1H), 7.71 (d, J = 8.5 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), 7.40-7.29 (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 10 = 7.9 Hz, 2H), 2.26 (ddt, J = 11.5 , 9.3, 2.5 Hz, 2H), 2.21 (s, 6H), 2.22

2.19 (m, 1H), 1.78-1.69 (m, 3H), 1.39 (s, 9H).

Compound Preparation 101;

A suspension of 100 (1.60 g, 2.30 mmol) and 10% Pd/C (400 mg) in 15 mixtures of (130 ml) EtOH and 20 (20 ml) AcOH was degassed and then exposed to hydrogenation conditions (1

Atmospheric) for 16 hours at room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated in a blank to obtain 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), 20

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,

٥٦٧٣

-١٥٦-

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;

To a solution of amine 101 (1.60 g, 2.30 mmol) and chloropyrazine-2-carbonylcarbamimidothioate (21, 1.60 g, 4.14 mmol)

In 25 ml EtOH (4.1 ml DIPEA, 23.0 mmol) was added at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, then cooled to room temperature, and concentrated in blank. The remaining material was purified by silica gel column chromatography (CH3OH/NH4OH/CHCl3 (80:18:2 silica gel)) to obtain guanidine 10 102 (645 mg, 37% and 640 mg, 37% contamination) in the form of a yellow solid:

1H NMR (400 MHz, CD3OD) 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, 15

9H).

Prepare Hydrochloric acid salt from:

of (S)-3,5-diamino-N-(N-(4-(6-(2-amino-3-(4-(3-(dimethylamino)propyl)phenylamino)-3-oxopropyl)naphthalen-2 -

yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide 20

)103(

47 (545 mg, 0.71 mmol) TFA (10 ml) in CH2Cl2 (15 ml) was added and the reaction mixture was stirred at room temperature for 1 hour. The solvent was removed again.

٥٦٧٣

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1 p Hydrochloric acid was added, the solvent was removed, and purified using a reverse phase column (Gold column), and the remaining material was freeze-dried to obtain compound 48 (206 mg, 50%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6) 11.02 (brs, 1H), 10.81-10.58 (m, 1H), 10.53 (s, 1H), 9.32 (s, 1H), 9.04-8.72 (m, 2H), 8.50 (brs, 3h), 7.82- 5

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, 1H), 3.74-3.45 (m, 1H), 3.27 (dd, J = 14.7, 8.3 Hz, 1H), 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). 10

1H NMR (400 MHz, CD3OD) 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). 15

16. to prepare :

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-

(123) chloropyrazine-2-carboxamide 20

Scheme 17

٥٦٧٣

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<img file="SA5673B1_D0087.tif" />

<img file="SA5673B1_D0088.tif" />

<img file="SA5673B1_D0089.tif" />

<img file="SA5673B1_D0090.tif" />

<img file="SA5673B1_D0091.tif" />

<img file="SA5673B1_D0092.tif" />

<img file="SA5673B1_D0093.tif" />

<img file="SA5673B1_D0094.tif" />

<img file="SA5673B1_D0095.tif" />

<img file="SA5673B1_D0096.tif" />

<img file="SA5673B1_D0097.tif" />

<img file="SA5673B1_D0098.tif" />

<img file="SA5673B1_D0099.tif" />

<img file="SA5673B1_D0100.tif" />

Chart 17 (continued)

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<img file="SA5673B1_D0101.tif" />

<img file="SA5673B1_D0102.tif" />

Compound preparation 105;

A solution of 104 (100 g, 0.675 mmol) in dry THF (800 mL) was charged with

(32.0 mg NaOH, 0.809 mmol) and 102 (dimethylsulfate mg, 0.809 mmol)

5 By distillation at 0°C. The reaction mixture was stirred for two hours at room temperature. The THF was removed under low pressure, and the mixture was divided between 1.0 L CH2Cl2 (1.0 L) and water. The aqueous layer was separated and extracted with 1.0 × 2 CH2Cl2 (1.0 L). The combined organic extraction products were dried over Na2SO4 and concentrated. The material was purified The remainder was separated using a chromatography column (100% silica gel, CH2Cl2, silica gel) to obtain compound 105.

10 (108.0 g, 90%) as a yellow liquid:

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1H NMR (400 MHz, DMSO-d6): δ 7.06 (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).

Compound preparation 106;

5 A solution of dry DMF (71.45 mL, 0.923 mmol) was charged with 57.40 mL POCl3, 0.616 mmol) by distillation under a nitrogen atmosphere at 0 °C. The reaction mixture was stirred for 30 minutes at 0 °C. A solution of 105 (50.0 g, 0.308 mmol) in 1,2-dichloromethane dry (500 mL) was added to the reaction mixture by distillation under a nitrogen atmosphere at 0 °C. After the addition was complete, the reaction mixture was heated at 80 °C

10 Celsius for 6 hours. The reaction mixture was quenched with cold H2O and divided between (1.0 L CH2Cl2) and water (1.0 L). The aqueous layer was separated and extracted with 1.0 × 2 CH2Cl2 (1.0 L). The combined organic extraction products were dried over Na2SO4 and concentrated. The residue was purified by column Chromatogruff (EA 5% hexane, silica gel) to obtain compound 106 (35.0 g, 61%) in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6): δ 10.10 (s, 1H), 7.65 (d, J = 7.81, 1H), 15

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).

Compound preparation 107;

A solution of 55.0 ml Trimethyl phosphonoacetate (0.378 mmol) was charged in

20 100 ml nonaqueous CH2Cl2 cooled to 0°C with 58.0 g DBU (58.0 g, 0.380 ml

mol) and the mixture was stirred for 15 minutes. Aldehyde 106 (16.0 g, 0.084 mmol) was added in 50 mL CH2Cl2 dropwise. The reaction mixture was brought to room temperature, stirred for 16 hours, and quenched with 100 mL of water. The mixture was divided, and the aqueous layer was extracted using 150 x 3 CH2Cl2 ml. The collected organic materials were washed using brine,

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Drying (Na2SO4), filtration, and concentration, and the remaining material was purified using a chromatography column. Ethyl acetate/hexanes 1:10 (silica gel) to obtain an unsaturated cis ester and 15.0) 107 β, α - trans g, 72%. (In the form of a white solid:

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 5

(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

A suspension of 107 (33.0 g, 0.134 mmol) and 10% Pd/C (15 g, 10 (0.127) in 300 EtOH ml) was exposed to hydrogenation conditions (1 atmosphere) for 3 hours at room temperature. The mixture was filtered Reaction through Celite and washing with MeOH The filtrate was concentrated under vacuum to obtain 108 (28.0 g, 90%) as a white solid:

1H NMR (400 MHz, CDCl3): 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). 15

Compound preparation 109;

A solution of 108 (28.0 g) methyl ester, 0.106 mmol (200 ml/200 ml/60 ml) THF/MeOH/H2O (25.0 g, 0.625 mmol) was charged and the reaction mixture was stirred at room temperature. For 3 hours, the solvent was removed and the pH was adjusted to 1 using 1 p of aqueous Hydrochloric acid; a white solid was precipitated, filtered, washed with water, and dried under vacuum to obtain acid 109 (25.5 g, 92%). White solid body:

٥٦٧٣

-١٦٢-

1H NMR (400 MHz, CDCl3): 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), 1.80–1.73 (m, 4H).

Preparation of compound 110

5 A solution of 60 (13.70 g, 77.31 mmol) in dry THF (200 ml) with n-butyl lithium (45.07 ml, 90.08 mmol, 2 M solution in cyclohexane) was charged by distillation at -78 °C, and the reaction mixture was stirred For one hour to obtain a solution of 61 lithium salt, another solution of 109 (15.0 g, 64.37 mmol) was charged in dry THF (200 ml) with 9.30 NMM (83.64 mmol) and 10.30 ml PivCl.

10 83.64 mmol) by distillation at -78 °C. The reaction mixture was stirred for 30 minutes

Heating to -20°C for one hour, the prepared solution of lithium salt was added slowly at -78°C. The reaction mixture was stirred for another 10 minutes, transferred to 0°C and stirred for 1 hour, transferred to room temperature and stirred for 30 minutes, quenched with saturated NH4Cl, THF buffer concentration, and divided between 300 CH2Cl2

15 ml) and water (100 ml). The aqueous layer was separated and extracted using CH2Cl2 (150 ml). The combined organic extraction products were dried over Na2SO4 and concentrated. The remaining material was purified by a chromatography column (CH2Cl2, silica gel) to obtain compound 110 (15.0 g, 60%) as a white solid.

Compound preparation 111;

20 A solution of 110 (15.0 g, 38.14 mmol) in dry THF (250 ml) was charged with 13.70 g KHMDS, 68.67 mmol) in batches at -78 °C. After stirring the resulting mixture for 30 minutes, 19.0 (19.0 g) was added. trisyl azide (g, 61.40 mmol) and the reaction mixture was stirred for 5 minutes. Acetic acid (15.0 ml, 228 mmol) and tetramethylammonium acetate (30.9 g, 76.28 mmol) were added slowly at the same temperature

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The heat. The reaction mixture was warmed to 24 °C, stirred for 16 h, quenched with saturated NaHCO3 (100 ml), THF buffer concentration, and extracted with 300 ml CH2Cl2. The combined organic extraction products were dried over Na2SO4 and concentrated. The residue was purified by Arf chromatography column (EtOAc/hexanes 10:90, silica gel)

5 This is followed by DCM) to obtain compound 111 (8.80 g, 54%) as a yellow solid:

1H NMR (400 MHz, CDCl3): 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 10

(m, 2H), 2.63 (m, 2H), 1.78–1.75 (m, 5H), 1.58 (m, 2H).

Preparation of compound 112;

A solution of 111 (31.0 g, 72.1 mmol) was charged in THF/H2O (300 ml/100 ml)

With 49 (49 ml H2O2, 433 mmol) followed by 6.04 (6.04 g, 144 mmol) LiOH

15 Batches at 0°C. The reaction mixture was stirred for 10 minutes at 0 °C and at room temperature for 1 hour, quenched with saturated Na2SO3 (200 mL), concentrated under low pressure to remove THF, and washed with CH2Cl2 (500 mL). 1 p of aqueous Hydrochloric acid and extraction with 500 x 2 mL CH2Cl2. The combined organic extraction products were dried over Na2SO4, concentrated, and washed.

20 Using MTBE to obtain compound 112 (15.0 g, 82%) as a solid color

White to black:

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 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).

٥٦٧٣

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Compound preparation 113;

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 exposed to hydrogenation conditions (1 atm) for 3 hours at room temperature. The reaction mixture was mixed through Celite and washing with AcOH/H2O followed

5 MeOH. The filtrate was concentrated under vacuum to obtain 113 (14.0 g) acetic salt, 83%, in the form of a yellow solid.

Compound preparation 114;

A solution of 113 (11.0 g, 44.1 mmol) in Acetic Acid (120 ml) with 120 hydrobromic acid (120 ml) was charged by distillation at room temperature and the reaction mixture was refluxed for

10 3 hours. The reaction mixture was cooled to room temperature and concentrated. The remaining material was used

Brown crude 114 (8.90 g, 80%) directly to the next step without any purification:

1H NMR (400 MHz, CDCl3): 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).

15 Compound preparation 115;

Acetyl chloride (17.0 ml, 243 mmol) was added to dry methanol (300 ml) at 0°C and 114 (8.90 g, 28.2 mmol) were added. The reaction mixture was refluxed for 4 hours and concentrated. The remainder was divided between 200 (CH2Cl2 ml) and saturated NaHCO3 (100 ml). The aqueous layer was separated and extracted using CH2Cl2

20 (200 ml). The collected organic extraction products were dried over Na2SO4 and concentrated to obtain

On compound 115 (7.30 g, 90%) as a white solid:

٥٦٧٣

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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), 2.70–2.67

(m, 2H), 2.63 (t, J = 6.16 Hz, 2H).

Compound preparation 116;

5 A solution of 115 (7.30 g, 25.60 mmol) in (100) MeOH/H2O ml/60 ml) was charged with 12.0 (12.0 g) NaHCO3 (145 mmol) and 10.0 (10.0 g) Boc2O (45.8 mmol) at 0°C. The resulting mixture was warmed to room temperature and stirred for 1 hour. The reaction mixture was divided between (100 mL CH2Cl2) and water (50 mL). The aqueous layer was separated and extracted with 100 (CH2Cl2 mL). The combined organic extraction products were washed.

10 Using brine, drying over Na2SO4, and concentrating. Chromatography with a flash column using 20% hexanes / ethyl acetate followed by CH2Cl2 to obtain compound 116 (7.1 g, 81%) in the form of 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, 1H), 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, 15

1H), 2.86 (m, 2H), 2.63 (m, 2H), 1.80–1.78 (m, 4H), 1.39 (s, 9H).

Compound preparation 117;

A solution of 116 (7.0 g, 20.05 mmol) in 80 (80 ml) CH2Cl2 with pyridine (100 ml) and triflate (4.64 ml, 24.0 mmol) was charged at 0°C, stirred for 1 hour, and stirred at 20°C. At room temperature for two hours after concentration, the reaction mixture was divided between

CH2Cl2 (150 ml) and water (70 ml). The aqueous layer was separated and extracted with CH2Cl2 (100 ml). The combined organic extraction products were washed with brine, dried over Na2SO4, and concentrated to obtain compound 117 (8.00 g, 83%) as Brown oil:

٥٦٧٣

-١٦٦-

1H NMR (400 MHz, CD3OD): δ 8.81 (d, J = 4.63 Hz, 5H), 8.56–8.51 (m, 2H), 8.02–7.99 (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).

5 Compound preparation 118;

Compound 117 (8.0 g, 16.6 mmol) and benzyl but-3-ynylcarbamate (10, 5.00 g, 24.9 mmol) in a non-aqueous amount of CH3CN (100 ml) were degassed with Argon for 10 minutes at room temperature and charged Using 9.34 ml TEA, 66.50 mmol, 10% t-Bu(3P) in 7.0 ml hexanes, 3.32 mmol, and 0.16 g CuI,

10 0.84 mmol). The resulting mixture was eluted with Argon for 10 minutes and Pd was added

2.00(4) g (PPh3, 1.73 mmol) rapidly in a single batch. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 16 hours. The reaction mixture was concentrated under vacuum and the residue was purified by a gel chromatography column. Silica 25:75, silica gel ethyl acetate / hexanes) to obtain compound 118 (4.50 g, 52%) in the form

15 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). 20

Compound preparation 119;

A solution of 118 (4.50 g) methyl ester (8.42 mmol) in THF/MeOH/H2O (30 ml/30 ml/10 ml) was charged with (3.60 g NaOH, 90 mmol) and the reaction mixture was stirred at room temperature for 3 hours. The pH value has been adjusted

٥٦٧٣

-١٦٧-

to 9 using 1 V of aqueous Hydrochloric acid and the organic solvent was removed. The pH value of the residue was adjusted to 5-6, and the suspension was divided between (100 ml CH2Cl2) and water (50 ml). The aqueous layer was separated and extracted with 100 (100 ml CH2Cl2). The combined organic extraction products were dried over Na2SO4 and concentrated to obtain On compound 5 119 (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 ), 1.73–10

1.71 (m, 4H), 1.31 (s, 9H).

Preparation of compound 120;

The compound was charged 119 (800 mg, 1.53 mmol) in THF (30 ml) with DEPBT (845 mg, 2.56 mmol), 24 (700 mg, 2.33 mmol), and DIPEA (1.0 ml, 4.65 mmol). (sequentially and stirring at room temperature for 16 hours. After the solvent was removed under low pressure, the residue was dissolved in (50 ml) CH2Cl2), and washed quickly with a saturated amount of water (50 ml) and brine (50 ml). , and drying over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on 6% silica gel (CH2Cl2/methanol), to obtain 120 (1.0 g) amide as a 20 yellow solid:

1H NMR (400 MHz, CDCl3): δ 7.46–7.44 (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, 1H), 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 =

٥٦٧٣

-١٦٨-

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;

5 A suspension of 120 (1.00 g, 1.01 mmol) and 10% Pd/C (600 mg) in a mixture of (50 ml) EtOH and 2 (AcOH ml) was degassed and exposed to hydrogenation conditions (1 atm) for 12 hours at °C. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 121 in the form of a white solid (700 mg, 80%):

1H NMR (400 MHz, CDCl3): δ 7.49–7.41 (m, 2H), 7.34–7.30 (m, 5H), 10

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). 15

Preparation of compound 122;

A solution of amine salt 121 (700 mg, 0.81 mmol) and methyl 3,5-diamino-680 mg (13, 6-carbonylcarbamimidothioate, 1.75 mmol) in EtOH (20 ml) was charged with 1.60 DIPEA. ml, 9.26 mmol) at room temperature.

20 The reaction mixture was heated at 70 °C in a sealed tube for 2 h, cooled to room temperature, and concentrated under vacuum. The remaining material was purified using an Arf chromatography column (silica gel CH3OH/NH4OH/CHCl3 80:18:2, silica gel) to obtain 122 (380 g guanidine, 48%) in the form of a yellow solid:

٥٦٧٣

-١٦٩-

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 5

Hz, 3H).

Prepare Hydrochloric acid salt from:

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- 10

chloropyrazine-2-carboxamide (compound 123);

4 p Hydrochloric acid in 15 dioxane ml was added to 122 (350 g, 0.35 mmol) in 5.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by chromatography. elution in reverse phase (Gold column), and the remaining material was freeze-dried to obtain 110 mg (45%) of compound 123 in the form of a yellow solid:

1H NMR (400 MHz, DMSO-d6): δ 10.16 (s, 1H), 9.16 (brs, 1H), 8.51– 8.34 (brs, 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 (brs, 1H), 4.42 (m, 1H), 4.53 (d, J = 5.3 Hz, 2H), 4.42 (m, 1H) , 4.01 (m, 1H), 3.93 (m, 1H), 3.60 (m, 1H), 3.50–3.38 (m, 20

4H), 3.08–3.03 (m, 6H), 2.72 (brs, 2H), 2.66–2.65 (m, 2H), 2.57 (m, 2H), 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).

٥٦٧٣

-١٧٠-

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). 5

17. to prepare :

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-(127) chloropyrazine-2-carboxamide 10

Scheme 18

<img file="SA5673B1_D0103.tif" />

<img file="SA5673B1_D0104.tif" />

<img file="SA5673B1_D0105.tif" />

<img file="SA5673B1_D0106.tif" />

<img file="SA5673B1_D0107.tif" />

<img file="SA5673B1_D0108.tif" />

<img file="SA5673B1_D0109.tif" />

<img file="SA5673B1_D0110.tif" />

<img file="SA5673B1_D0111.tif" />

<img file="SA5673B1_D0112.tif" />

<img file="SA5673B1_D0113.tif" />

<img file="SA5673B1_D0114.tif" />

٥٦٧٣

-١٧١-

Compound preparation 124;

The compound was charged 119 (1.0 g, 1.92 mmol) in THF (30 ml) with 845 mg DEPBT (845 mg, 2.82 mmol), 29 (1.25 g, 1.91 mmol), and DIPEA (1.0 ml, 5.73 mmol). Sequentially stirring at room temperature for 16 hours after the solvent was removed in the shade

5 Low pressure, the residue was dissolved in 50 ml CH2Cl2, washed quickly with saturated aqueous water (50 ml) and 50 ml brine, and dried over Na2SO4. The solvent was evaporated and the crude product was purified by chromatography and flashing on a % silica gel. 5) Silica gel CH2Cl2/methanol), to obtain 124[900 mg amide (mixture)] in the form of a yellow solid.

10 Preparation of compound 125;

A suspension of 124 [900 mg (mixture), 0.77 mmol] and 10% Pd/C (600 mg) in a mixture of (50 EtOH ml) and AcOH (1.5 ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 12 hours at room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain 125 (15 800 mg) crude oil in the form of colorless oil.

Compound preparation 126;

A solution of 125 (800 mg) ore was shipped

(13) methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

400 1.02 mg (1.02 mmol) in 40 ml EtOH (1.10 ml, 6.38 mmol) DIPEA (20) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, cooled to room temperature , and concentration under vacuum. The remaining substance was purified using a chromatograph column (silica gel CH3OH/NH4OH/CHCl3 2:18:80, silica gel) to obtain 126 (285 mg) guanidine, 12% in 3 steps) in the form of a yellow solid:

٥٦٧٣

-١٧٢-

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), 5

3.06–3.00 (m, 1H), 2.91–2.86 (m, 1H), 2.76 (m, 2H), 2.71–2.68 (m, 4H), 2.61–2.55 (m, 4H), 2.44–2.35 (m, 4H ), 1.74–1.60 (m, 10H), 1.38

(s, 9H).

Prepare Hydrochloric acid salt from:

3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R))- 10

2,3,4,5,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahydronaphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

(Complex 127)

15 4 p Hydrochloric acid in 10 dioxane ml was added to 126 (1.15 g, 0.23 mmol) in 3.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by chromatography. eluted in reverse phase (Gold column), and the remaining material was freeze-dried to obtain 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 20 = 7.6 Hz, 2H), 6.96 (d, J = 7.1, 1H), 6.89 (d, J = 7.4, 1H), 5.44 (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), 3.43–3.39 (m, 4H), 3.25–3.22 (m, 4H), 3.04

٥٦٧٣

-١٧٣-

(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).

1H NMR (400 MHz, DO): δ 7.10 (d, J = 8.30 Hz, 2H), 7.02–6.90 (m, 5

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).

10 18. Preparation:

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-tetrahydronaphthalen-1-yl)butylcarbamoyl)-6-chloropyrazine-2-(131) carboxamide

15 Scheme 19

٥٦٧٣

-١٧٤-

<img file="SA5673B1_D0115.tif" />

<img file="SA5673B1_D0116.tif" />

Compound preparation 128;

The compound was charged 119 (1.00 g, 1.92 mmol) in THF (30 ml) with 862 mg DEPBT (862 mg, 2.88 mmol), 34 (1.50 g, 2.98 mmol), and 1.0 ml DIPEA, 5.76 mmol.

5 mol) sequentially and stirring at room temperature for 16 hours. After the solvent was removed under reduced pressure, the residue was dissolved in 50 (50 ml) CH2Cl2, and washed quickly with a saturated volume of water (30 ml) and 20 ml brine. (and drying over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on 6% silica gel CH2Cl2/ methanol), to obtain 128 (780 mg amide, 42%) in the form of

10 Yellow solid:

1H NMR (400 MHz, CDCl3): δ 7.49 (m, 3H), 7.31–7.29 (m, 10H), 7.00–

7.08 (m, 3H), 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–

٥٦٧٣

-١٧٥-

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).

5 Compound preparation 129;

A suspension of 128 (780 mg, 0.776 mmol) and 10% Pd/C (300 mg) in a mixture of 30 EtOH (30 ml) and AcOH (1.0 ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 12 hours at Room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum to obtain 129 10 amine salt (720 mg, 85%) as 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), 4.34–4.33 (m, 1H), 4.25–4.21 (m, 1H), 4.03–4.02 (m, 1H), 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), 15

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;

20 A solution of amine salt 129 (720 mg, 0.77 mmol) was charged and...

(13) methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

456 mg, 1.17 mmol (20 ml EtOH) with 1.12 ml DIPEA (6.24 mmol) at room temperature. The reaction mixture was heated at 70 °C in an airtight tube for 2 hours, cooled to room temperature, Concentration under vacuum and the remaining material was purified by

٥٦٧٣

-١٧٦-

Chromatograph column (silica gel CH3OH/NH4OH/CHCl3 80:18:2, silica gel) to obtain 130 (380 mg) guanidine, 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, 5

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 ), 3.07–3.02 (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). 10

Prepare Hydrochloric acid salt from:

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-tetrahydronaphthalen-1-yl)butylcarbamoyl)-6-chloropyrazine-2-

carboxamide 15

)131(؛

4 p Hydrochloric acid in 25 ml dioxane was added to 130 (350 mg, 0.35 mmol) in 5.0 ml EtOH and the reaction mixture was stirred at room temperature for 2 hours. The solvent was removed, the mixture was purified by chromatography. The remaining material was eluted in reverse phase (Gold column) and the remaining material was freeze-dried to obtain compound 131 (125 mg, 48%) in the form of a solid.

In yellow:

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 =

٥٦٧٣

-١٧٧-

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), 1.77–1.73 (m, 5H), 1.69–

1.65 (m, 3H).

1H 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), 5

6.93 (d, J = 6.6 Hz, 1H), 5.43 (d, J = 3.8 Hz, 1H), 4.79 (d, J = 5.38 1H), 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),

<p>2.62 (t, J = 6.6 Hz, 2H), 1.95–1.94 (m, 2H), 1.76–1.15 (m, 8H). 10</p>

<p dir="rtl">19. to prepare :</p>

(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-

(135) carboxamide 15

Scheme 20

٥٦٧٣

-١٧٨-

<img file="SA5673B1_D0117.tif" />

<img file="SA5673B1_D0118.tif" />

Preparation of compound 132;

The compound was charged 119 (700 mg, 1.34 mmol) in THF (30 ml) with 600 (600 mg DEPBT, 2.00 mmol), 18 (360 mg, 1.51 mmol), and 0.80 ml DIPEA, 4.03

5 (mmol) sequentially and stirring at room temperature for 16 hours. After the solvent was removed under reduced pressure, the residue was dissolved in 50 ml (50 ml) CH2Cl2, and washed quickly with a saturated amount of water (50 ml) and 50 brine. ml), and dried over Na2SO4. The solvent was evaporated and the crude product was purified by flash chromatography on 6% silica gel (CH2Cl2/ methanol), to obtain 132[800 mg amide (mixture)] in the form of a 10-colored 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

٥٦٧٣

-١٧٩-

(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).

Compound preparation 133;

5 A suspension of 132 [800 mg (mixture), 1.01 mmol] and 10% Pd/C (350 mg) in a mixture of (30 EtOH ml) and AcOH (1 ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 12 hours At room temperature, the reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum and purified by a chromatograph column (silica gel CH3OH/NH4OH/CHCl3 2:18:80, silica gel) to obtain 10. Compound 233 (500 mg, 67% in 2 steps) in the form of a yellow solid:

1H 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), 2.60–2.55 (m, 4H), 2.35–2.31 (m, 2H), 1.82 (s, 6H), 1.58–1.57 (m, 4H), 1.40 (s, 9H). 15

Compound preparation 134;

A solution of amine salt 133 (500 mg, 0.90 mmol) and methyl 3,5-diamino 530-6-chloropyrazine-2-carbonylcarbamimidothioate (13 mg, 1.36 mmol) in EtOH (20 ml) was charged with 1.30 DIPEA. ml, 7.25 mmol) at room temperature. 20 The reaction mixture was heated at 70°C in a sealed tube for 2 hours, cooled to room temperature, and concentrated under vacuum. The remaining material was purified using a chromatography column (silica gel CH3OH/NH4OH/CHCl3 80:18:2, silica gel) to obtain 134 (285 mg) guanidine, 42%, in the form of a yellow solid:

٥٦٧٣

-١٨٠-

1H NMR (400 MHz, DMSO-d6): δ 7.29 (d, J = 7.5 Hz, 2H), 7.10 (d, J =

<p>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 (m, 2H), 2.70 (m, 2H), 2.61–2.54 (m, 4H), 2.35–2.31 (m, 2H), 2.22 (s, 6H), 1.80–1.72 (m, 5H), 1.69–1.62</p>

(m, 4H), 1.39 (s, 9H). 5

Prepare Hydrochloric acid salt from:

(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-

10 compound carboxamide 135;

4 p Hydrochloric acid in 10 dioxane ml was added to 134 (380 g, 0.35 mmol) in 5.0 ml EtOH 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 Gold column), and the remaining material was freeze-dried to obtain compound 135 (125 mg, 49%) as a yellow solid:

1H NMR (400 MHz, DMSO-d6): δ 10.69 (brs, 1H), 10.54–10.50 (d, J = 16.7 Hz, 2H), 9.32 (t, J = 4.8 Hz, 1H), 8.96 (brs, 1H ), 8.86 (brs, 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 20

(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).

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,

٥٦٧٣

-١٨١-

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).

<p dir="rtl">20. to prepare :</p>

(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2- 5)

carbonyl)guanidino)butyl)-5,6,7,8-tetrahydronaphthalen-1-yl)propanoic

acid

)139(

Scheme 21

<img file="SA5673B1_D0119.tif" />

<img file="SA5673B1_D0120.tif" />

<img file="SA5673B1_D0121.tif" />

<img file="SA5673B1_D0122.tif" />

<img file="SA5673B1_D0123.tif" />

10

Compound preparation 136;

٥٦٧٣

-١٨٢-

A suspension of 118 (800 mg, 1.49 mmol) and 10% Pd/C (350 mg) in a mixture of (50 EtOH ml) and AcOH (1.0 ml) was degassed and exposed to hydrogenation conditions (1 atmosphere) for 12 hours at Room temperature. The reaction mixture was filtered through a Celite socket and the socket was washed with MeOH. The filtrate was concentrated under vacuum and purified by a column

5 Chromatograph (CH3OH/NH4OH/CHCl3 2:18:80, silica gel) to obtain compound 136 (700 mg, 93%) in the form of a yellow solid.

Compound preparation 137;

A solution of amine salt 136 (700 mg, 1.50 mmol) was charged and...

(13) methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

10 880 mg, 2.26 mmol (in 30 ml EtOH) with 2.15 ml DIPEA, 12.03 ml

mol) at room temperature. The reaction mixture was heated at 70°C in an airtight tube for 2 hours, cooled to room temperature, and concentrated under vacuum. The residue was purified by a chromatography column (silica gel 2:18:80). , silica gel

CH3OH/NH4OH/CHCl3) to obtain 137 (560 mg guanidine, 60%)

15 Yellow solid form:

1H NMR (400 MHz, CD3OD); 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).

20 Compound preparation 138;

A solution of 137 (560 mg) methyl ester, 0.907 mmol (30 ml/30 ml/10 ml) THF/MeOH/H2O (30 ml) with 3.60 g NaOH, 7.25 mmol was charged and the reaction mixture was stirred at room temperature. For 3 hours, the pH value was adjusted to 9 using 1 V of hydrochloric acid and the organic solvent was removed

٥٦٧٣

-١٨٣-

The pH value of the residue was adjusted to 5-6, and the suspension was divided between CH2Cl2 (100 ml) and water (50 ml). The aqueous layer was separated and extracted with 100 ml (100 ml CH2Cl2). The combined organic extraction products were dried over Na2SO4 and concentrated to obtain Compound 138 (420 mg, 78%) is a brown solid:

1H NMR (400 MHz, DMSO-d6); δ 6.93 (d, J = 6.7 Hz, 1H), 6.84 (d, J = 5

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).

Prepare Hydrochloric acid salt from:

of (S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2- 10)

carbonyl)guanidino)butyl)-5,6,7,8-tetrahydronaphthalen-1-yl)propanoic

acid

compound 139;

4 p Hydrochloric acid in 10 dioxane ml was added to 138 (420 mg, 0.69 15 mmol) in 5.0 ml EtOH and the reaction mixture was stirred at room temperature for two hours.

The solvent was removed, the mixture was purified by reverse-phase chromatography (C18 Gold column), and the remaining material was freeze-dried to obtain compound 139 in the form of a yellow solid (200 mg, 49%):

1H NMR (400 MHz, DMSO-d6); δ 10.56 (brs, 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), 3.38–20

3.35 (m, 2H), 3.04 (d, J = 7.0 Hz, 2H), 2.67–2.66 (m, 4H), 2.56–2.55

(m, 2H), 1.72–1.70 (m, 4H), 1.63–1.56 (m, 4H).

٥٦٧٣

-١٨٤-

1H NMR (400 MHz, DO); δ 7.43 (brs, 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. KARLY synthesis of:

including 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3- 5)

(bis((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)propyl)

phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-

(33) chloropyrazine-2-carboxamide

Scheme 22

٥٦٧٣

-١٨٥-

<img file="SA5673B1_D0124.tif" />

<img file="SA5673B1_D0125.tif" />

<img file="SA5673B1_D0126.tif" />

<img file="SA5673B1_D0127.tif" />

<img file="SA5673B1_D0128.tif" />

<img file="SA5673B1_D0129.tif" />

<img file="SA5673B1_D0130.tif" />

<img file="SA5673B1_D0131.tif" />

<img file="SA5673B1_D0132.tif" />

<img file="SA5673B1_D0133.tif" />

<img file="SA5673B1_D0134.tif" />

Preparation of compound 141;

To a solution of 140, 10.0 naphthol (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 30

5 minute. The resulting mixture was stirred at room temperature for 4 h, and concentrated under vacuum, followed by the addition of water (200 ml) and ethyl acetate (200 ml). The aqueous layer was separated and extracted with ethyl acetate (200 × 200 ml). Organic extraction

٥٦٧٣

-١٨٦-

Assembled using brine, drying over Na2SO4 and concentrating. The remaining material was purified using an Arf chromatography column (EtOAc/hexanes 1:4, silica gel) to obtain the desired compound 141 (9.50 g, 61%) in the form of 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 5

(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;

Add 7.03 g Zinc dust, 107.6 mmol) to a round-sided flask.

10 Nitrogen-purged, flame dried. Non-aqueous DMF (50.0) added

ml) with a syringe, followed by a catalytic load of iodine (1.00 g, 3.94 mmol). The resulting mixture was observed to undergo a color change from colorless to yellow and back to colourless. Protected iodoalanine 143 (11.8 g, 35.9 mmol) was added (in one batch, followed by a catalytic load of 1.00 g iodine, 3.94 mmol) and stirring at room temperature for

15 30 minutes; Successful zinc insertion has been achieved by exothermic grinding. A solution of reagent was left behind

Organic Zinc was cooled to room temperature before 821 (821 mg Pd2dba3), SPhos (736 mg, 1.79 mmol), and 141 (8.00 aryl bromide g, 35.9 mmol) were added and the mixture was heated at 50°C for 16 hours, under positive pressure of nitrogen. The reaction mixture was left to cool to room temperature and a saturated solution of

20 NH4Cl (300 ml) and EtOAc (300 ml), and then the mixture was filtered through Celite and washed.

The aqueous layer was separated and extracted using 2 (2 x 300 ml EtOAc). The combined organic extraction products were washed with brine, dried over Na2SO4 and concentrated under vacuum. The solder product was purified by a chromatography column (EtOAc/silica gel). hexanes 1:4, silica gel (to obtain the desired compound 7) 4.60

25 gm, 37%) as a yellow solid:

٥٦٧٣

-١٨٧-

1H NMR (400 MHz, CDCl3, 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 5 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;

10 To a solution of compound 7 (7.60 g, 21.8 mmol) in CH2Cl2 (150 ml) pyridine (18.0 ml ) and Tf2O (9.19 g, 32.6 mmol) were added at 0 °C. The resulting mixture was stirred at 0°C. Room temperature for 2 hours, concentration under vacuum and partition between CH2Cl2 (100 ml) and water (50 ml). The aqueous layer was separated and extracted using 50 ml (2 x CH2Cl2). The combined organic extraction products were washed with brine, and dried over

15 Na2SO4 and concentration to obtain compound 9 (11.0 g, crude) as a brown oil. The crude product was used directly for the next step without further purification:

1H NMR (400 MHz, CDCl3, 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). 20

Preparation of compound 11;

A solution of compound 9 was degassed (11.0 g, 21.8 mmol) and -3-benzyl but

10 ynylcarbamate (6.56 g, 32.6 mmol) in a non-aqueous amount of acetonitrile (100 ml) for 10 minutes under an Argon atmosphere followed by the addition of 11.9 ml TEA (87.0 ml).

٥٦٧٣

-١٨٨-

10% t-Bu(3P) in 8.80 ml hexanes, 4.35 mmol and 207 mg CuI, 1.08 mmol at room temperature. The resulting mixture was dehydrated with Argon for another 10 minutes and 4 (4) was added. 2.51 g Pd(PPh3, 2.17 mmol) in one batch. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 16 hours. The mixture was concentrated

5 The reaction was carried out in blank and the remaining material was purified using an Arf chromatography column (silica gel, 2:3 EtOAc/hexanes) to obtain compound 11 (7.00 g, 61% in two steps) in the form of a brown oil:

1H NMR (400 MHz, CDCl3, 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), 10

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;

To a solution of 11 (7.00 g) methyl ester, 13.2 mmol (13.2 mmol) in 200 ml THF,

15 methanol (200 ml) and water (75.0 ml) added solid NaOH (16.0 g, 79.2 g).

mmol). The resulting mixture was stirred at room temperature for 1 hour until TLC indicated the reaction was complete. 1 p of hydrochloric acid was added to adjust the pH of the reaction mixture to 10. After concentration, water (100 ml) was added and the The pH was set to 5-6. The resulting precipitate was extracted using 250 x 2 (CH2Cl2) ml

20 Organic layering, drying over Na2SO4, filtration, concentration and trituration with MTBE to obtain compound 17 (5.00 g, 75%) 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,

٥٦٧٣

-١٨٩-

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;

To a solution of compound 17 (4.60 g, 8.91 mmol) in THF (160 ml), 50% T3P (50.7 ml), ethyl acetate (10.7 ml) and NMM (4.89 ml, 44.5 mmol) were added sequentially.

After stirring at room temperature for 10 minutes, amine 29 (6.11 g, 9.33 mmol) was added and the reaction mixture was stirred at room temperature for 16 hours. After the solvent was removed, the residue was dissolved in 100 (CH2Cl2). ml), washing quickly with saturated NH4Cl, saturated NaHCO3 and brine, drying over Na2SO4 and concentrating. The residue was purified

10 Using a chromatography column (MeOH/CH2Cl2 1:9, silica gel) to obtain 30 (6.60 gm amide, 64%) in the form of a tan-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), 7.377.28 (m, 11H), 7.09-6.95 (m, 4H), 5.46 (s, 2H), 5.33 (br s, 1H), 5.22 (t, J = 5.8 Hz, 1H), 5.11 (s, 2H), 4.63-4.51 (m, 1H), 4.27 (dd, J = 10.8, 15

5.4 Hz, 2H), 4.02-3.84 (m, 6H), 3.71 (t, J = 4.5 Hz, 6H impurities), 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).

Preparation of compound 31

20 A suspension of 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 with Argon using a syringe for 10 minutes and then exposing To hydrogenation conditions (1 atmosphere) for 16 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The product was concentrated

٥٦٧٣

-١٩٠-

Vacuum filtration and trituration with MTBE to obtain 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, 5

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), 2.932.81 (m, 8H), 2.54.2.39(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). 10

Preparation 32

To a solution of 31 (7.06 g, 6.18 mmol) in 50.0 ml EtOH (50.0 ml) DIPEA (8.80 ml, 49.4 mmol) was added as follows:

methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

15 (13, 3.84 g, 9.88 mmol) at room temperature. The reaction mixture was heated at 70

°C for 2 hours, cooling to room temperature and concentrating under vacuum. The remaining substance was purified twice using a chromatography column (silica gel 80:18:2, silica gel CH3OH/NH4OH/CHCl3) to obtain compound 32 (2.50 g, 33%) in the form of a yellow solid:

1H NMR (400 MHz, CD3OD, mixture of rotamers) δ 8.22 (d, J = 9.3 Hz, 20 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

٥٦٧٣

-١٩١-

(t, J = 10.8 Hz, 2H), 3.54-3.46 (m, 1H), 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), 2.57-2.47 (m, 2H), 2.46-2.34 (m, 2H), 1.841.73 (m , 2H), 1.72-1.61 (m, 4H), 1.36 (s, 7H), 1.12 (s, 2H).

5 Prepare Hydrochloric acid salt from:

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-(33) chloropyrazine-2-carboxamide

10 To a solution of 32 (2.50 g, 2.02 mmol) in 30.0 ml EtOH (30.0 ml) hydrochloric acid (80.0 ml) was added. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed, and the column was purified. Reverse phase column and freeze-drying to obtain compound 33 (1.82 g, 85%) in the form of a wet, yellow solid:

1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 10.59 (s, 1H), 9.41 (t, J

= 5.2 Hz, H), 9.01 (br s, 1H), 8.96 (br s, 1H), 8.81 (br s, 2H), 8.77 (br s, 15 2H), 8.44-8.37 (m, 1H), 8.16 -8.10 (m, 1H), 7.61-7.52 (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), 3.753.65 (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 20

(m, 2H), 1.78-1.61 (m, 4H).

1H NMR (400 MHz, CD3OD): δ 9.25 (t, J = 5.9 Hz, 0.5H), 8.26-8.21 (m, 1H), 8.17-8.12 (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,

٥٦٧٣

-١٩٢-

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), 1.91-1.73 (m, 4H).

22. to prepare :

(2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-)

(29) aminophenyl)propylazanediyl)dihexane-1,2,3,4,5-pentaol

Scheme 23

<img file="SA5673B1_D0135.tif" />

10 Preparation of compound 145

To a solution of compound 144 (8.80 g, 154.1 mmol) in CH2Cl2 (150 ml) were added 32.2 ml TEA, 231.2 mmol) and Boc2O (40.4 g, 185.3 mmol) at 0°C. Stirring of the mixture was completed. The reaction was at 0°C for 0.5 hours, left to warm to room temperature and stirred for 5 hours, then the mixture was divided between (150 ml) CH2Cl2 and water

15 (150 ml). The aqueous layer was separated and extracted using 150 x 2 CH2Cl2 (150 ml).

٥٦٧٣

-١٩٣-

The combined organic extraction products were washed with brine, dried over Na2SO4, and concentrated to obtain the desired compound 145 (22.0 g, 91%) as colorless oil.

1H NMR (400 MHz, CDCl3): 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). 5

Compound preparation 147

To a solution of compound 145 (14.0 g, 89.12 mmol) in aqueous amount of THF (150 ml) was added 0.5-9 molar BBN in 270 ml THF, 133.8 mmol) under Argon. After the reaction mixture was stirred for Two hours at room temperature, compound 146 (17.7).

10 Pd(PPh3)2Cl2 (3.12 g, 71.3 mmol), and 1 p of aqueous NaOH (150 ml) was added at room temperature. The resulting mixture was stirred for an additional one hour. After removing The solvent was divided between 200 ml EtOAc (200 ml) and water (200 ml). The aqueous layer was separated and extracted with 2 × 200 ml EtOAc. The combined organic extraction products were washed with brine, dried over Na2SO4 and concentrated under

15 unloading. The crude product was purified using a chromatography column (silica gel 1:4, silica gel EtOAc/hexanes) to obtain compound 147 (8.00 g, 43%) in the form of 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). 20

Compound preparation 148;

Compound 147 (8.00 g, 28.6) was dissolved in 4 V Hydrochloric acid in dioxane (50.0 mL) at room temperature and the solution was stirred for one hour. The reaction mixture was concentrated

٥٦٧٣

-١٩٤-

Under vacuum, the remaining material was triturated using MTBE to obtain 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). 5

Preparation of compound 150;

To a solution of compound 148 (4.00 g, 18.5 mmol) and 149 (24.8 g) triol (149 g, 92.5 mmol) in MeOH (150 ml) AcOH (11.1 ml, 185 mmol) was added and the reaction mixture was stirred at temperature Chamber for 10 minutes after adding NaCNBH3

10 g, 92.5 mmol), the solution was stirred at room temperature for 24 hours. Additional amounts of compound 149 (4.0 eq., 4.0 eq.) and NaCNBH3 (4.0 eq.) were added over 4 days. (2.0 hexanal equivalent), 2.0 AcOH (equivalent) and 2.0 NaCNBH3 equivalent). The solution was stirred again at room temperature for 1 hour. After removing the solvent, the residue was neutralized with saturated NaHCO3 and partitioned into

15 The residue was between 200 ml EtOAc and water (200 ml). The aqueous layer was separated and extracted with 2 x 300 ml (CH2Cl2). The combined organic extraction products were dried over Na2SO4 and concentrated under vacuum. The residue was purified by a gel chromatography column. Silica (MeOH/NH4OH/CHCl3 2:18:80, MeOH/CH2Cl2 1:9, silica gel) to obtain compound 150 (6.50 g, 52%) in the form of a white solid

20 To name arrrr. An additional 4.00 g of material was separated into contaminated fractions and purified by column

In reverse phase column to obtain 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),

٥٦٧٣

-١٩٥-

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).

to prepare :

(2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-aminophenyl)propylazanediyl) 5

dihexane-1,2,3,4,5-pentaol (compound 153);

A suspension of 150 (6.50 g, 9.50 mmol) and 10% Pd/C (1.30 g) in EtOH (150 ml) was degassed by bubbling with Argon using a syringe for 10 minutes and then stirring at room temperature. Under an atmosphere of hydrogen (color, 1 atmosphere) for 6

10 An hour at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in the blank to obtain 153 (6.01 g, 97%) as a tan-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), 3.98-3.89 (m, 4H), 3.83 (dd, J = 5.7, 2.3 15

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) , 2.45-2.29 (m, 2H), 1.74-1.63

(m, 2H).

23. to prepare :

3,5-diamino-N-(N-(4-(4-((R)-2-amino-3-(4-(3-(bis((2S,3R,4R,5R))- 20

2,3,4,5,6-pentahydroxyhexyl)amino)propyl)phenylamino)-3-oxopropyl)naphthalen-1-yl)butyl)carbamimidoyl)-6-chloropyrazine-2-carboxamide

٥٦٧٣

-١٩٦-

)152(

Scheme 24

<img file="SA5673B1_D0136.tif" />

<img file="SA5673B1_D0137.tif" />

<img file="SA5673B1_D0138.tif" />

<img file="SA5673B1_D0139.tif" />

<img file="SA5673B1_D0140.tif" />

<img file="SA5673B1_D0141.tif" />

<img file="SA5673B1_D0142.tif" />

<img file="SA5673B1_D0143.tif" />

<img file="SA5673B1_D0144.tif" />

<img file="SA5673B1_D0145.tif" />

<img file="SA5673B1_D0146.tif" />

<img file="SA5673B1_D0147.tif" />

<img file="SA5673B1_D0148.tif" />

Preparation of the compound 14

5 To a solution of 1 - 10.0 g naphthol, 69.4 mmol (10.0 g) in acetonitrile (70.0 ml), several portions of NBS (142, 12.3 g, 69.4 mmol) were added over 30 minutes. The resulting mixture was stirred at At room temperature for 4 hours, and concentration under vacuum, as follows

٥٦٧٣

-١٩٧-

This was done by adding water (200 ml) and 200 ethyl acetate (200 ml). The aqueous layer was separated and extracted using ethyl acetate (200 x 200 ml). The combined organic extraction products were washed with brine, dried over Na2SO4 and concentrated. The remaining material was purified by crystallization (200 ml). Heptane/EtOAc) to obtain the desired compound 14 (6.0 g, 39%) as 5 white solids.

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).

10 Preparation of compound 145

Zinc dust (4.76 g, 72.9 mmol) was added to a nitrogen-purged side-lever flask, flame dried. Non-aqueous DMF (25.0 ml) was added by syringe, followed by a catalytic load of iodine (677 mg), 2.67 mmol). It was observed that the resulting mixture undergoes a color change from colorless to yellow and back to colorless. 15 Protected iodoalanine 114 (8.00 g, 24.3 mmol) was added in one batch, followed by

Catalytic amount of iodine (677 mg, 2.67 mmol) and stirring at room temperature for 30 minutes; successful Zinc introduction was achieved by exothermic grinding. A solution of the organic Zinc reagent was allowed to cool to room temperature before 3 (556 Pd2(dba mg, 0.60 mmol), SPhos (498 mg, 1.21 mmol), 14 (5.40 g aryl bromide, 20 24.3 mmol) were added and the mixture was heated at 50°C for 16 hours, under positive pressure from

Nitrogen. The reaction mixture was left to cool to room temperature. A saturated solution of NH4Cl (300 ml) and EtOAc (300 ml) was added, and the mixture was then filtered through Celite and washed with 100 ml EtOAc. The aqueous layer was separated and extracted with 300 ml EtOAc (2 × 2). The organic extraction products were washed Collected using brine, drying over 25 Na2SO4 and concentrating under vacuum, the solder product was purified by Arf chromatography column (gel).

٥٦٧٣

-١٩٨-

Silica EtOAc/hexanes 1:4, silica gel) to obtain the desired compound 145 (3.10 g, 37%) in the form of a yellow solid.

1H NMR (400 MHz, CDCl3, 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

5.98 (brs, 0.3H), 5.59 (br s, 0.7 H), 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

10 To a solution of compound 145 (3.07 g, 8.90 mmol) in CH2Cl2 (75.0 ml) pyridine (7.25 ml, 88.9 mmol) and Tf2O (2.24 ml, 13.3 mmol) were added at 0 °C. The resulting mixture was stirred. At room temperature for 2 hours, concentrated under vacuum and divided between 100 CH2Cl2 (100 mL) and water (50 mL). The aqueous layer was separated and extracted with 2 x 50 CH2Cl2 mL. The combined organic extraction products were washed with

15 brine, drying over Na2SO4 and concentrating to obtain compound 146 (4.20 g, crude) as a brown colored oil. The crude product was used directly for the next step without further purification.

1H NMR (400 MHz, CDCl3, 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). 20

Compound preparation 147

A solution of compound 6 (4.20 g, 8.80 mmol, crude) and 3-benzyl but 7 (2.65 g, 13.2 mmol) was degassed in a non-aqueous amount of acetonitrile (50.0 mL) for 10 minutes under an Argon atmosphere. This is followed by the addition of 4.81 ml TEA (35.2 ml).

٥٦٧٣

-١٩٩-

(mol), 10% t-Bu(3P) in 3.56 mg hexanes (1.76 mmol) and CuI (84 mg, 0.44 mmol) at room temperature. The resulting mixture was stripped with Argon for another 10 minutes and 4 (4) was added. 1.01 g Pd(PPh3, 0.88 mmol) in one batch. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 18 hours. The mixture was concentrated

5 The reaction was carried out in a vacuum, and the remaining material was purified using a chromatography column (silica gel, 2:3 EtOAc/hexanes) to obtain compound 147 (3.20 g, 67% in two steps) in the form of a brown oil.

1H NMR (400 MHz, CDCl3, 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), 10

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

To a solution of 147 (3.10 g) methyl ester (5.84 mmol) in THF (60 ml), 15 (60 ml) of methanol and water (20.0 ml) solid NaOH (1.40 g, 35.09 ml) was added.

mmol). The resulting mixture was stirred at room temperature for 2 hours until TLC indicated the reaction was complete. 1 p of hydrochloric acid was added to adjust the pH of the reaction mixture to 10. After concentration, water (100 ml) was added and the number was adjusted The pH was reduced to 5-6. The resulting precipitate was extracted using 200 x 2 (CH2Cl2) ml. The layers were collected

20 organic matter, drying over Na2SO4, filtration, concentration and trituration with MTBE to obtain 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,

٥٦٧٣

٢٠٠-

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).

Compound preparation 149

To a solution of compound 148 (800 mg, 1.55 mmol) in THF (30 ml), 50% T3P (1.86 ml), ethyl acetate (50 ml) and NMM (0.85 ml, 7.75 mmol) were added sequentially.

After stirring at room temperature for 10 minutes, amine 29 (1.01 g, 1.55 mmol) was added and the reaction mixture was stirred at room temperature for 1 hour. After the solvent was removed, the residue was dissolved in 100 (CH2Cl2). ml), washing quickly with saturated NH4Cl, saturated NaHCO3 and brine, drying over Na2SO4 and concentrating. The remaining 10 was purified by a chromatography column (MeOH/CH2Cl2 silica gel 1:9, silica gel) to obtain 149 amide g (1.20 g, 67%) (In the form of a solid substance with a tan-white colour.

1H NMR (400 MHz, CDCl3): δ 8.35 (d, J = 8.0, 1.7 Hz, 1H), 8.19 (d, J = 8.5 Hz, 1H), 7.60–7.52 (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, 15 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), 3.55–3.46(m, 4H), 2.95–2.82 (m, 2H), 2.81–2.69 (m, 4H), 2.68–2.57 (m, 1H), 2.56–2.44 (m, 3H), 2.43–2.38 (m, 1H), 1.85–

1.69 (m, 2H), 1.38 (s, 9H).

20 Preparation of compound 150

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 bubbling with Argon using a syringe for 10 minutes and then exposing To hydrogenation conditions (1 atmosphere) for 16 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The product was concentrated

٥٦٧٣

-٢٠١-

Vacuum filtration and trituration using MTBE obtained 150 amine salt (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, 5

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) , 2.73–2.67 (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). 10

Preparation 151

To a solution of 150 (1.05 g, 0.92 mmol) in EtOH (15.0 ml) DIPEA (1.30 ml, 7.35 mmol) was added as follows:

methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamidothioate

15 (13, 573 mg, 1.47 mmol) at room temperature. The reaction mixture was heated at 70

°C for 2 hours, cooling to room temperature and concentrating under vacuum. The remaining substance was purified twice using a chromatography column (silica gel 80:18:2, silica gel CH3OH/NH4OH/CHCl3) to obtain compound 151 (410 mg, 36%) in the form of a yellow solid.

1H NMR (400 MHz, CD3OD, mixture of rotamers): δ 8.22 (d, J = 8.4 Hz, 20 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

٥٦٧٣

-٢٠٢-

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). 5

Synthesis:

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-(152) carboxamide 10

To a solution of 151 (480 mg, 0.42 mmol) in 5.0 ml EtOH (5.0 ml) 4 p of hydrochloric acid (25.0 ml) was added. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed, and the column was purified. Reverse phase column and freeze-drying to obtain compound 152 (300 mg, 71%) in the form of a wet, colored solid.

15 yellow.

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), 7.61–7.53 (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, 20

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).

1H NMR (400 MHz, CD3OD): 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),

٥٦٧٣

-٢٠٣-

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 ), 2.74–2.58 (m, 2H), 2.13–1.98 (m, 2H), 1.91–1.73 (m, 4H).

Calculated HRMS of 959.4418, [Na + M] C44H64ClN10O12 and detected 959.4394.

24. Preparation of the intermediate compound 18

Scheme 25

<img file="SA5673B1_D0149.tif" />

<img file="SA5673B1_D0150.tif" />

<img file="SA5673B1_D0151.tif" />

10

15

Compound preparation 155;

To a solution of compound 154 (500 mg, 9.00 mmol) in 50 ml CH2Cl2 (50 ml) TEA (1.63 ml, 11.7 mmol) and Boc2O (2.16 g, 9.90 mmol) were added at 0°C. Stirring of the mixture was completed. The reaction was at 0°C for 0.5 hours, allowed to warm to room temperature and stirred for 3 hours. Then the mixture was divided between 50 CH2Cl2 (50 ml) and water (50 ml). The aqueous layer was separated and extracted with 2 x 50 CH2Cl2 ml. The collected organic extraction products were washed with brine, dried over Na2SO4, and concentrated.

٥٦٧٣

-٢٠٤-

The remaining material was purified using a chromatography column (silica gel hexanes 3:2, silica gel /EtOAc) to obtain the desired compound 155 (1.20 g, 86%) in the form of colorless oil.

1H NMR (300 MHz, CDCl3): 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) . 5

Compound preparation 157;

A solution of compound 155 (1.00 g, 6.45 mmol) and 156 (1.30 g, 6.45 mmol) in a non-aqueous amount of THF (15 ml) was degassed for 10 minutes under an Argon atmosphere followed by the addition of TEA (3.53 ml, 25.8 ml). mmol), PPh3 (424 mg), 1.61 mmol) and CuI

10 (246 mg, 1.29 mmol) at room temperature. The resulting mixture was stripped using Argon

For another 10 minutes, 4(7.45) Pd(PPh3 g, 6.45 mmol) was added in one batch. After degassing with Argon for 5 minutes, the resulting mixture was refluxed for 16 hours. The reaction mixture was concentrated in a vacuum and the material was purified. The remaining residues were separated using a chromatography column (EtOAc/hexanes 3:2, silica gel) to obtain compound 157 (750 mg, 42%).

15 In the form of brown oil.

1H NMR (400 MHz, CDCl3): δ 8.17 (d, J = 9.2 Hz, 2H), 7.55 (d, J = 9.2 Hz, 2H), 4.79 (brs, 1H), 4.18 (d, J = 6.0 Hz, 2H), 1.47 (s, 9H).

Compound preparation 158;

Compound 157 (2.00 g, 7.24) was dissolved in 4 V Hydrochloric acid in dioxane 20 (20.0 mL) at room temperature and the solution was stirred for 2 hours. The reaction mixture was concentrated

Under vacuum, the remaining material was triturated using MTBE to obtain compound 158 (1.25 g, 82%) in the form of a brown solid.

٥٦٧٣

-٢٠٥-

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).

Compound preparation 159;

To a solution of compound 158 (100 mg, 0.47 mmol) and a solution of formaldehyde in water 5 (30%, 1.40 ml, 1.41 mmol) in MeOH (3.0 ml) AcOH (0.09 ml) was added,

(1.41 mmol) and the reaction mixture was stirred at room temperature for 30 minutes. After adding 88 mg (1.41 mmol) NaCNBH3, the solution was stirred at room temperature for 16 hours. An additional amount of formaldehyde solution in water was added (30%, 0.92 ml, 0.94 mmol), AcOH (0.09 ml, 1.41 mmol) and NaCNBH3 (88 mg, 1.41 mmol) and stirred for another 16 hours. After removing the solvent, the remaining material was neutralized with

NaHCO3 was saturated and the residue was divided between EtOAc (30 ml) and water (30 ml). The aqueous layer was separated and extracted with 40 x 2 CH2Cl2 (40 ml). The combined organic extraction products were dried over Na2SO4 and concentrated under vacuum. The residue was purified by column Chromatogarf (MeOH/CH2Cl2 1:80, 18:2, silica gel 9:1)

15 MeOH/NH4OH/CHCl3) to obtain compound 159 (50 g, 52%) in the form of a tan-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;

20 Compound 159 (100 mg, 0.49 mmol) and 10% Pd/C (40 mg) in 3.0 MeOH ml) was degassed using Argon for 10 minutes and then stirred under an atmosphere of hydrogen (colour, 1 atmosphere) for 3 hours. At room temperature, the reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in the blank and triturated with CH2Cl2/hexane to obtain 18 (48 mg, 55%) in the form of a white crystal:

٥٦٧٣

-٢٠٦-

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 the intermediate compound 29

5 Scheme 26

<img file="SA5673B1_D0152.tif" />

<img file="SA5673B1_D0153.tif" />

<img file="SA5673B1_D0154.tif" />

Compound preparation 161;

To a solution of compound 158 (4.00 g, 18.9 mmol) and 160 triol (11.7 g, 56.6 mmol) in MeOH (50 ml) AcOH (3.40 ml, 56.6 mmol) was added and stirred.

10 The reaction mixture was kept at room temperature for 30 minutes. After adding 3.55 g NaCNBH3, 56.6 mmol, stirring of the solution was completed at room temperature for 16 hours. An additional amount of compound 160 (11.7 g, 56.6 mmol) AcOH (3.40 ml, 56.6 mmol) was added. and NaCNBH3 (3.55 g, 56.6 mmol). Stirring of the solution was completed at room temperature for 16 hours. After removing the solvent, the remaining material was neutralized with

15 NaHCO3 was saturated and the residue was divided between 10 CH2Cl2 (10 ml) and water (10 ml). The aqueous layer was separated and extracted with 10 × 2 CH2Cl2 (10 ml). The combined organic extraction products were dried over Na2SO4 and concentrated under vacuum. The residue was purified by column Chromatogarf (MeOH/CH2Cl2 1:80, 18:2, silica gel 9:1)

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MeOH/NH4OH/CHCl3) to obtain compound 29 (700 mg, 7.0%) in the form of a brownish-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), 5

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;

Compound 161 (500 mg, 0.90 mmol) and 10% 10(2C/Pd(OH) (215 mg) in 230 ml EtOH) were degassed by bubbling with Argon using

syringe for 10 minutes and then stirred under an atmosphere of hydrogen (color, 1 atm) for 2 hours at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in the blank and the remaining material was purified by a chromatography column ( 15 Silica gel (MeOH/NH4OH/CHCl3 2:18:80, MeOH/CH2Cl2 1:9, gel) to obtain 15 compound 29 (264 mg, 55%) in the form of a brownish-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). 20

Preparation of the intermediate compound 24

Scheme 27

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<img file="SA5673B1_D0155.tif" />

Compound preparation 162;

To a solution of 158 (200 mg, 0.94 mmol) and 160 (194) triol (194 mg, 0.94 mmol) in MeOH (2.0 ml) AcOH (0.17 ml, 2.82 mmol) was added and

5 Stir the reaction mixture at room temperature for 30 minutes. After adding 148 mg NaCNBH3 (2.35 mmol), the solution was stirred at room temperature for 16 hours. An additional amount of compound 160 (0.2 eq., 3.0 eq.) and NaCNBH3 (1.0 eq.) were added. Stirring was completed. The solution was kept at room temperature for 16 hours. After removing the solvent, the remaining material was neutralized with saturated NaHCO3 and the remaining material was divided between...

<p dir="rtl">10 CH2Cl2 (10 ml) and water (10 ml). The aqueous layer was separated and extracted using CH2Cl2.</p>

<p dir="rtl">(2 x 10 ml). The combined organic extraction products were dried over Na2SO4 and concentrated under vacuum. The remaining material was purified by an Arf chromatography column (silica gel 1:9, silica gel MeOH/NH4OH/CHCl3 2:18:80, MeOH/CH2Cl2: 9:1). ) to obtain compound 162 (95 mg, 28%) in the form of a brownish-white solid.</p>

1H NMR (400 MHz, CD3OD): δ 8.24 (d, J = 9.1 Hz, 2H), 7.69 (d, J = 15

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).

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Compound preparation 164; To a solution of compound 162 (95 mg, 0.26 mmol) hexane 163 (52 mg, 0.51 mmol), AcOH (0.05 mg, 0.78 mmol) and NaCNBH3 (41 mg, 0.65 mmol) were added. The solution was stirred at Room temperature for 16 hours. After removing the solvent, the remaining material was neutralized with saturated NaHCO3 and the material was divided

5 The remainder was between 10 EtOAc (10 ml) and water (10 ml). The aqueous layer was separated and extracted with 10 x 2 (CH2Cl2 ml). The combined organic extraction products were dried over Na2SO4 and concentrated under vacuum. The remaining material was purified by a chromatography column (silica gel). silica MeOH/NH4OH/CHCl3 2:18:80, MeOH/CH2Cl2 1:9:9, gel) to obtain compound 164 (70 mg, 59%) in the form of a solid substance with a tan-white color.

1H NMR (400 MHz, CDCl3): δ 8.18(d, J = 8.9 Hz, 2H), 7.56(d, J = 8.9 10

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 (brs, 1H), 3.70 (brs, 1H), 3.64 (t, J = 6.2 Hz, 1H), 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–

2.55 (m, 1H), 1.75–1.43 (m, 4H), 1.34 (d, J = 5.0 Hz, 3H), 1.32–1.25 15

(m, 6H), 0.89 (t, J = 6.6 Hz, 3H).

Preparation of the compound 24

Compound 164 (1.70 g, 3.77 mmol) and 10% Pd/C (200 mg) in 40 MeOH ml were degassed using Argon for 10 minutes and then stirred under an atmosphere of hydrogen 20 (colour, 1 atmosphere) for 2 hours. At room temperature, the reaction mixture was filtered through Celite and washed with MeOH, the filtrate was concentrated in the blank, and the remaining material was purified by a chromatograph column (silica gel 18:80, silica gel 2:MeOH/CH2Cl2 9:1). NH4OH/CHCl3) to obtain compound 24 (1.20 g, 76%) in the form of a brownish-white solid.

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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, 5.5Hz, 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, 5

J = 6.6 Hz, 3H).

Preparation of intermediate compound 85

Scheme 28

<img file="SA5673B1_D0156.tif" />

10 Compound preparation 166;

To a solution of compound 148 (4.60 g, 21.3 mmol) and 165 (17.1) triol (17.1 g, 63.9 mmol) in 100 (100 MeOH ml) AcOH (12.1 ml, 63.9 mmol) was added and the reaction mixture was stirred at temperature Room for 10 minutes. After adding 4.00 g NaCNBH3, 63.9 mmol, the solution was continued stirring at room temperature for 6 hours. Then

15 163 (5.10 g hexanal, 42.6 mmol) and 2.60 g NaCNBH3 (42.6 mmol) were added. The solution was stirred again at room temperature for 2 hours. After the solvent was removed, the residue was neutralized with saturated NaHCO3 and Divide the remaining material between EtOAc