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

Technical field of chloro-pyrazine carboxamide derivatives that can be used to treat diseases caused by insufficient mucosal hydration

[0001] The present invention relates to novel compounds useful as sodium channel blockers, which include 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-( 4-(3-(Bis((2S, 3R, 4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl) (Cai-1-yl)butyl)methanyl)-6-chloropyrazine-2-carboxamide and related compounds and pharmaceutically acceptable salts thereof, compositions containing said compounds, treatment methods and uses of said compounds And the method for preparing the compound.

Background technique

[0002] A variety of "innate defenses", namely protection mechanisms, have evolved on the mucosal surface at the interface between the environment and the body. The main form of these innate defenses is to clean these surfaces with liquids. Generally speaking, the amount of liquid layer on the mucosal surface reflects the balance between epithelial fluid secretion and epithelial fluid absorption. Epithelial fluid secretion usually reflects anions (C1 and C1 and C1 and C1) coupled with water (and cation counter-ion). /Or HCO₃) Secretion, epithelial fluid absorption is usually reflected with water and counter anions (C1 and/or HC0₃) Coupling absorption. Many diseases of the mucosal surface are caused by too little protective fluid on these mucosal surfaces, and the latter is caused by the imbalance between secretion (too little) and absorption (relatively too much). The salt transport process that characterizes the defects of these mucosal dysfunctions exists in the epithelial layer of the mucosal surface.

[0003] One way to replenish the protective liquid layer on the mucosal surface is to "rebalance" the system by blocking channels and liquid absorption. The epithelial protein that mediates the rate-limiting step of fluid absorption is the epithelial Na* channel ("ENaC"). ENaC is located on the top surface of the epithelium, the mucosal surface-environment interface. Ideally, in order to inhibit ENaC-mediated Na⁺For fluid absorption, amiloride-like ENaC blockers must be delivered to the mucosal surface and maintained at that site to achieve maximum therapeutic benefit.

[0004] ENaC blockers have been reported to be used for various diseases that are improved by improving mucosal hydration. In particular, ENaC blockers have been reported to be used in the treatment of respiratory diseases, such as chronic bronchitis (CB), cystic fibrosis (CF) and COPD, which are reflected in the body's inability to normally recover from the lungs. Partially clears mucus, which eventually leads to chronic airway infections. See Evidence for airway surface dehydration as the initiating event in CF airway disease, RC Boucher, Journal of Internal Medicine, Vol. 261, No. 1, January 2007, page 5T6; and Cystic fibrosis: a disease of vulnerab. airway surface dehydration, RC Boucher, Trends in Molecular Medicine, Volume 13, Issue 6, June 2007, pp. 231-240.

[0005] Data show that the initial problem of both chronic bronchitis and cystic fibrosis is the inability to clear the mucus on the airway surface. The inability to clean the mucus reflects the imbalance in the amount of mucus that is the airway surface liquid (ASL) on the airway surface. This imbalance causes a relative decrease in ASL, which leads to mucus concentration, periciliary liquid (PCL) lubrication activity, mucus adhesion to the airway surface, and the inability to clear mucus to the oral cavity through ciliary activity. The reduction in mucus clearance leads to long-term bacterial colonization of mucus that adheres to the airway surface. The long-term retention of bacteria, the inability of local antimicrobial substances to kill bacteria trapped in mucus on a long-term basis, and the subsequent chronic inflammatory response to this type of surface infection are manifested as chronic bronchitis and cystic fibrosis.

[0006] There is currently a huge unmet medical need for products that specifically treat various diseases (including chronic bronchitis, COPD, cystic fibrosis, etc.) that are alleviated by improving mucosal hydration. Current treatments for chronic bronchitis, COPD, and cystic fibrosis focus on treating the symptoms and/or late effects of these diseases. However, none of these treatments effectively solve the fundamental problem of inability to remove mucus from the lungs.

[0007] RC Boucher in US 6,264,975 describes the use of pyrazinoylguanidine sodium channel blockers to hydrate the mucosal surface with the well-known diuretics amiloride and benzamil (benzamil) And phenamil is the representative. However, considering the limited weight of drugs that can be breathed into the lungs; (2) rapid absorption, and thus an undesirable short half-life on the mucosal surface; and (3) free dissociation from ENaC, these compounds are relatively weak effect. There is a need for more effective drugs with a longer half-life on the mucosal surface.

[0008] Too little protective surface fluid on other mucosal surfaces is a common pathophysiology of many diseases. For example, in xerostomia (xerostomia), the oral cavity lacks fluid because the parotid, sublingual, and submandibular glands cannot secrete fluid, and continuous Na* (ENaC) transport mediates the absorption of fluid from the oral cavity. Keratoconiunctivitis sira (dry eye) is caused by the inability of the lacrimal glands to secrete fluid with continuous and dependent fluid absorption on the surface of the conjunctiva. In sinusitis, there is an imbalance between mucin secretion and relative ASL consumption. The inability to secrete C1 (and fluid) in the proximal small intestine, together with the increased absorption of (and fluid) in the terminal ileum, leads to distal intestinal obstruction syndrome (DIOS)_oIn elderly patients, excessive Na* (and volume) absorption in the descending colon produces constipation and diverticulitis _o

[0009] The published literature includes several patent applications and granted patents by Par ion Sciences Inc. for pyrazinyl muscle analogues as sodium channel blockers. Examples of these publications include PCT Publication No.WO2003/070182, WO2003/070184, WO2004/073629, WO2005/025496, WO2005/016879, WO2005/018644, WO2006/022935, WO2006/023573, WO2006/023617, WO2007/018640, W02007/146869, W02008/031028, W02008/031048, and U.S. Patent Nos. 6858614, 6858615, 6903105, 7064129, 7186833, 7189719, 7192958, 7192959, 7192960, 7241766, 7247636, 7247637, 7317013,

7332496、7368447、7368450、7368451、7375102、7388013、7399766、7410968、7807834、

7842697 And 7868010.

[0010] There is still a need for new sodium channel blocking compounds with enhanced potency and effectiveness on mucosal tissues. There is still a need for new sodium channel blocking compounds that provide a therapeutic effect but minimize or eliminate the occurrence or development of hyperkalemia in the recipient.

[0011] Summary of the invention

[0012] The present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0013]

<img file="CN105073717A_D0001.tif" />

<img file="CN105073717A_D0002.tif" />

[0014] where:

[0015] Ar is selected from:

[0016]

<img file="CN105073717A_D0003.tif" />

<img file="CN105073717A_D0004.tif" />

<img file="CN105073717A_D0005.tif" />

[0017] n is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

[0018] W is selected from hydrogen, C plant. 8 alkyl groups and polyvinylated alkyl groups having 3 to 8 carbon atoms;

[0019] R²Is hydrogen or a polyalkylated alkyl group having 3 to 8 carbon atoms;

[0020] W and R are independently hydrogen or C_rC₃alkyl.

[0021] The present invention also provides solvates and hydrates of the compound of formula (I), individual stereoisomers, including optical isomers (enantiomers and diastereomers) and geometric isomers (cis Formula/trans isomers), mixtures of stereoisomers and tautomers, or pharmaceutically acceptable salts thereof, and pharmaceutical compositions containing the compounds or pharmaceutically acceptable salts thereof, which are useful in treatment methods Uses and preparation methods.

[0022] The present invention also provides the compound 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S, 3R, 4R, 5R) -2, 3,4, 5,6-Pentylhexyl) amino) propyl) phenylamino) -3-oxopropyl) acetylene-1-yl) butyl) methylene Yl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salts, as well as its optical isomers (enantiomers and diastereomers) and geometric isomers (cis/trans isomers) , Mixtures of stereoisomers and tautomers, and pharmaceutical compositions containing the compounds or pharmaceutically acceptable salts thereof, their use in therapeutic methods and their preparation methods.

[0023] Brief description of the drawings

[0024] By referring to the information in this article in conjunction with the following drawings, a more comprehensive understanding of the present invention and its many advantages can be more easily obtained:

[0025] FIG. 1 is a graph showing the effect of compound 33 on sheep MCC 4 hours after administration.

[0026] FIG. 2 is a graph showing the effect of compound 123 on sheep MCC 4 hours after administration.

[0027] FIG. 3 is a graph showing the effect of compound 48 on sheep MCC 4 hours after administration.

[0028] FIG. 4 is a graph showing the effect of compound 33 on sheep MCC 8 hours after administration.

[0029] FIG. 5 is a graph showing the effect of compound 152 on sheep MCC 8 hours after administration.

[0030] FIG. 6 is a graph showing the enhancement effect of compound 33 on sheep MCC 8 hours after administration of hypertonic saline.

[0031] FIG. 7 is a graph comparing the effect of Example 1 on sheep MCC at 4 hours after administration.

[0032] FIG. 8 is a graph comparing the effect of Example 1 on the plasma potassium level of sheep.

9 is a graph comparing the activity of Comparative Example 1 and Compound 33 on sheep MCC 4 hours after administration.

10 is a graph comparing the effects of Comparative Example 1 and Compound 33 on sheep plasma Q levels.

11 is a graph comparing the activity of Comparative Example 1 and Compound 123 on sheep MCC 4 hours after administration.

12 is a graph comparing the effects of Comparative Example 1 and Compound 123 on sheep plasma Q levels.

[0037] FIG. 13 is a graph comparing the activity of Comparative Example 1 and Compound 48 on sheep MCC at 4 hours after administration.

14 is a graph comparing the effects of Comparative Example 1 and Compound 48 on sheep plasma Q levels.

[0039] Detailed description of the invention

[0040] As used herein, the following terms are the specified definitions.

[0041] "The compound of the present invention" means a compound of formula I or a salt thereof, especially a pharmaceutically acceptable salt thereof.

[0042] "Compound of Formula I" means a compound having the structural formula designated as Formula I herein. The compound of formula I includes solvates and hydrates (ie, adducts of the compound of formula I with a solvent). In those embodiments where the compound of formula I contains one or more chiral centers, the term is intended to encompass each individual stereoisomer, including optical isomers (enantiomers and diastereomers) and Geometric isomers (cis/trans isomers) and mixtures of stereoisomers. In addition, the compound of formula I also includes tautomers of the shown formula.

[0043] Throughout and in the examples, standard IUPAC naming principles are used to name the compounds, which may include naming the compounds using the ChemDraw Ultra 11.0 software program sold by Cambridge So ft Corp. /PerkinElmer.

[0044] In some chemical structure representations, when it is not depicted that the carbon atoms have sufficient connection variables to obtain tetravalent, it is assumed that the remaining carbon substituents that need to provide tetravalent are hydrogen. Similarly, in some chemical structures, the bond is drawn without specifying the end group. According to the convention in the art, such a bond represents a methyl group (Me, -CHJ.

[0045] In one embodiment, the compound of formula (1) is 3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(bis (2,3,4,5,6-Pentaylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methionyl)-6-chloro Pyrazin-2-carboxamide or its pharmaceutically acceptable salt:

[0046]

H0

<img file="CN105073717A_D0006.tif" />

<img file="CN105073717A_D0007.tif" />

[0047] In another embodiment, the compound of formula (1) is 3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-( Bis(2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahydrozea-1-yl ) Butyl) Methyl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salt:

[0048]

OH OH

<img file="CN105073717A_D0008.tif" />

^g 1'η₂ν ν νη_{2 ο}

<img file="CN105073717A_D0009.tif" />

Cl

[0049] In yet another embodiment, the compound of formula (1) is 3,5-diamino-N-(Ν-(4-(6-(2-amino-3-(4-(3-( Bis(2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Ze-2-yl)butyl)methionyl)-6- Chlorpyrazine-2-carboxamide or its pharmaceutically acceptable salt:

<img file="CN105073717A_D0010.tif" />

[0051] In another embodiment, the compound of formula (1) is 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-(4- (3-(Bis((2S, 3R, 4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Cai- 1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salt:

[0052]

<img file="CN105073717A_D0011.tif" />

<img file="CN105073717A_D0012.tif" />

[0053] Three separate embodiments respectively include compounds of formula (II), formula (IID and formula (IV) or pharmaceutically acceptable salts thereof:

[0054]

<img file="CN105073717A_D0013.tif" />

<img file="CN105073717A_D0014.tif" />

[0055] Where:

[0056] n is an integer selected from 0, 1, 2, 3, 4, 5 or 6;

[0057] W is selected from hydrogen, C plant. 8 alkyl groups and polyvinylated alkyl groups having 3 to 8 carbon atoms;

[0058] R²Is hydrogen or a polyhydroxylated alkyl group having 3 to 8 carbon atoms;

[0059] W and R are independently hydrogen or C_rC₃alkyl.

[0060] In each compound group independently represented by formula (I), (II), (IID and (IV) or a pharmaceutically acceptable salt thereof, there are additional embodiments, in which:

[0061] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0062] W is selected from hydrogen, C plant. 8 alkyl groups and polyvinylated alkyl groups having 3 to 8 carbon atoms;

[0063] R²Is hydrogen or a polyalkylated alkyl group having 3 to 8 carbon atoms;

[0064] W and R are independently hydrogen or C_rC₃alkyl.

[0065] In each compound group independently represented by formula (I), (II), (IID and (IV) or a pharmaceutically acceptable salt thereof, there are additional embodiments, in which:

[0066] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0067] M is selected from hydrogen and Ci-C₈alkyl;

[0068] R²Is hydrogen or a polyalkylated alkyl group having 3 to 8 carbon atoms;

[0069] W and R are independently hydrogen or C_rC₃alkyl.

[0070] In each compound group independently represented by formulas (I), (II), (IID and (IV) or a pharmaceutically acceptable salt thereof, there is another embodiment, wherein:

[0071] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0072] M is selected from hydrogen and Ci-Cg alkyl;

[0073] F is hydrogen;

[0074] W and R are independently hydrogen or C_rC₃alkyl.

[0075] In each compound group independently represented by formula (I), (II), (IID and (IV) or a pharmaceutically acceptable salt thereof, there is yet another embodiment, wherein:

[0076] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0077] W and R²Each independently is a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0078] W and R are independently hydrogen or C_rC₃alkyl.

[0079] In each compound group independently represented by formulas (I), (II), (IID and (IV) or a pharmaceutically acceptable salt thereof, there is another embodiment, wherein:

[0080] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0081] W and R²Each independently is a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0082] W and R° are hydrogen.

[0083] In each compound group or pharmaceutically acceptable salt thereof independently represented by formula (1), (II), (IID and (IV), there is another embodiment in which:

[0084] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0085] W and R²Each independently is a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0086] W and R 4 are each independently C_rC₃alkyl.

[0087] In each compound group or pharmaceutically acceptable salt thereof independently represented by formula (1), (II), (IID and (IV), there are additional embodiments, in which:

[0088] n is an integer selected from 1, 2, 3, 4, 5 or 6;

[0089] W and R²Each independently is a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0090] 0 is hydrogen;

[0091] R is the C plant. 3 alkyl.

[0092] The polyalkylated alkyl groups of the present invention are those in which 3 to 8 carbon atoms are substituted with two or more alkyl chains. Examples of polyvinylated 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-pentanol, heptane-1,2,3,4,5,6-hexanol and octane-1,2,3 ,4, 5,6,7-Heptanol.

[0093] One embodiment in each compound group described herein is where the polyvinylated alkyl group has the formula -CH₂-(CHR⁵）_n-H, where n is an integer selected from 2, 3, 4, 5, 6 or 7 and in each case independently H or OH, provided that at least two R § groups are OH.

[0094] Another embodiment in each compound group described herein is where the polyvinylated alkyl group has the formula -CH₂-CH0H-(CHR⁶）_n-H those compounds, where m is an integer selected from 1, 2, 3, 4, 5 or 6 and in each case independently H or OH, provided that at least one R &amp; group is OH

[0095] Yet another embodiment in each compound group described herein includes wherein the polyvinylated alkyl group has the formula -CH₂-(CH0H)_n-CH₂Compounds of OH, wherein n is an integer selected from 1, 2, 3, 4, 5, or 6. Another embodiment in each compound group described herein includes compounds wherein n is an integer selected from 2, 3, 4, or 5. Another embodiment in each group includes compounds wherein n is an integer selected from 3, 4, or 5.

[0096] In another embodiment of each compound group described herein, the formula -CH₂-(CH0H)_n-CH₂0H

The chain represented is 2, 3, 4, 5, 6-Pentyl hexane, which has the following formula:

[0097]

<img file="CN105073717A_D0015.tif" />

[0098] In yet another embodiment of each of the compound groups described herein, by formula one CH₂-(CH0H)_n-CH₂The chain represented by 0H is a chain of the following formula:

[0099]

OH OH

<img file="CN105073717A_D0016.tif" />

OH OH

[0100] Three additional independent embodiments respectively include compounds of formula (V), formula (VI) and formula (VII) or pharmaceutically acceptable salts thereof:

[0101]

<img file="CN105073717A_D0017.tif" />

<img file="CN105073717A_D0018.tif" />

<img file="CN105073717A_D0019.tif" />

[0103] n is an integer selected from 1, 2, 3, 4, 5 or 6; and

[0104] W and R are independently hydrogen or C_rC₃alkyl.

In each of the embodiments represented by formulas (V), (VI) and (VII), there are additional embodiments or pharmaceutically acceptable salts thereof, wherein n is selected from 1, 2, 3, 4, An integer of 5 or 6; and W and R° are each hydrogen. In each of the embodiments represented by formulas (V), (VI) and (VII), there is another embodiment or a pharmaceutically acceptable salt thereof, wherein n is selected from 1, 2, 3, 4, 5, or 6. And R is an integer of St alkyl.

[0106] In each of the embodiments described herein, there are additional embodiments in which n is an integer selected from 1, 2, or 3. In each of the embodiments described herein, there are additional embodiments in which n is an integer selected from 4, 5, or 6. In each of the embodiments described herein, there are six additional independent embodiments where n is an integer of 1, 2, 3, 4, 5, and 6, respectively.

[0107] The compounds herein, including those of formula (I), (Ia), (II), (III), (IV), (V), (VI) and (VII), may be free bases or salts, especially those of The form of medicinal salt. For a review of pharmaceutically acceptable salts, see Berge et al., J. Pharma Sci. (1977) 66: 1-19.

[0108] Pharmaceutically acceptable salts formed by inorganic or organic acids include, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogensulfate, nitrate, sulfamate, phosphate, Hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinic acid Salt, pyruvate, citrate, ascorbate, palmitate, salicylate, stearate, phthalate, alginate, polyglutamate, oxalate, Oxaloacetate, saccharate, benzoate, alkyl or aryl sulfonate (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or sulfonate Acid salt) and isothionate; complexes formed with amino acids such as lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine Acid, leucine, etc. The compound of the present invention may also be in the form of a salt formed from an anion of an element such as chloride, bromide, or iodide.

[0109] For therapeutic use, the salt of the active ingredient of the compound of formula I is pharmaceutically acceptable, that is, it is a salt derived from a pharmaceutically acceptable acid. However, it can also be seen that salts of non-pharmaceutically acceptable acids are used, for example, in the preparation or purification of pharmaceutically acceptable compounds. For example, trifluoroacetic acid can be used for such applications. All salts, whether derived from pharmaceutically acceptable acids or not, are within the scope of the present invention.

[0110] The term chiral refers to a molecule that has the property of not overlapping with the mirror image partner, and the term "achiral" refers to a molecule that can overlap with its mirror image partner.

[0111] The term "stereoisomers" refers to compounds that have the same chemical composition but differ in the arrangement of atoms or groups in space. "Diastereomers" refer to stereoisomers that have two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated under high-resolution analytical operations such as electrophoresis and chromatography. "Enantiomers" refer to two stereoisomers of a compound that do not overlap with each other's mirror images.

[0112] The definitions and rules of stereochemistry used herein generally follow SP Parker, Ed., McGraw-H, 1 Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and W. en, S., Stereochemistry of Organic Compounds(1994) John Wiley&amp;Sons, Inc., New Yorko

[0113] Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. In the description of optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to its chiral center. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can react in a chemical reaction

Or it occurs when there is no stereoselectivity or stereospecificity in the process. The term "racemic mixture" or "racemate" refers to an equimolar mixture of two enantiomeric materials.

[0114] The term "tautomer" refers to a type of stereoisomer in which the migration of a hydrogen atom results in two or more structures. The compounds of formula I may exist in different tautomeric forms. Those skilled in the art should recognize that squins, amides, muscles, glands, sulfur glands, heterocycles, etc. can exist in tautomeric forms. By way of example and not limitation, the compounds of formula I may exist in multiple tautomeric forms as shown below:

[0115]

<img file="CN105073717A_D0020.tif" />

<img file="CN105073717A_D0021.tif" />

[0116] All the tautomeric forms of squint, amide, muscle, gland, sulphur gland, heterocycle, etc. of all embodiments of Formula I are within the scope of the present invention. Tautomers exist in equilibrium, so those skilled in the art should understand that the description of a single tautomer in the provided formula means all possible tautomers.

[0117] It should be noted that all enantiomers, diastereomers and racemate mixtures, tautomers, polymorphs, pseudopolymorphs and their possible compounds within the scope of formula I Pharmaceutical salts are all encompassed in the present invention. All mixtures of these enantiomers and diastereomers, including mixtures enriched in enantiomers and mixtures enriched in diastereomers, are within the scope of the present invention. An enantiomerically enriched mixture is a mixture of enantiomers in which the ratio of the designated enantiomer to the alternative enantiomer is greater than 50:50. More particularly, the enantiomerically enriched mixture contains at least about 75% of the designated enantiomer, and preferably at least about 85% of the designated enantiomer. In one embodiment, the enantiomerically enriched mixture is substantially free of other enantiomers. Similarly, a diastereomer-enriched mixture is a mixture of diastereomers in which the amount of a designated diastereomer is greater than the amount of each alternative diastereomer. More particularly, the diastereomer-enriched mixture contains at least about 75% of the designated diastereomer, and preferably at least about 85% of the designated diastereomer. In one embodiment, the diastereomer-enriched mixture is substantially free of all other diastereomers. Those skilled in the art will understand that the term "substantially free" means that there are less than 5% of other diastereomers, preferably less than 1%, more preferably less than 0. 1%. In other embodiments, no other diastereomers are present or the amount of any other diastereomers present is below the detection level. Stereoisomers can be separated by techniques known in the art including high performance liquid chromatography (HPLC) and crystallization of chiral salts.

[0118] A single stereoisomer (for example, an enantiomer substantially free of its stereoisomer) can be obtained by resolving a racemic mixture using, for example, a method of forming diastereomers with an optically active resolving agent ( Stereochemistry of

Carbon Compounds, (1962) by EL Eliel, McGraw Hill; Lochmuller, C. Η., (1975)

J. Chromatogr., 113: (3) 283-302). The racemic mixture of the chiral compound of the present invention can be separated and separated by any suitable method, including: (1) forming an ionic diastereomeric salt with the chiral compound and separating it by fractional crystallization or other methods, (2) Form diastereomeric compounds with chiral derivatization reagents, separate diastereomers and convert them into pure stereoisomers, and (3) directly separate substantially pure or enriched stereoisomers under chiral conditions.

[0119] In one embodiment, the present invention provides 3,5-diamino-N-(N-(4-(4-((S)-2-amino-3-( 4-(3-(Bis((2S, 3R, 4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl) An enantiomerically enriched mixture or composition of (Cai-1-yl)butyl)methyl)-6-chloropyrazine-2-carboxamide or a pharmaceutically acceptable salt thereof.

[0120] Other embodiments include formulas (1), (Ia). (II), (III), (IV), (V), (VI) contained in each of their respective mixtures as major isomers, respectively An enantiomerically enriched mixture or composition of the compound of (VII) or its pharmaceutically acceptable salt.

[0121] In another embodiment, the present invention provides 3,5-diamino-N-(N-(4-(4-((S)-2-amino- 3-(4-(3-(Bis((2S, 3R, 4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxo (Propyl) Choi-1-yl) Butyl) Methyl)-6-chloropyrazine-2-carboxamide or an enantiomerically enriched mixture or composition of a pharmaceutically acceptable salt thereof.

[0122] Four other embodiments include formulas (1), (Ia), (II), (III), (IV), (V), which are contained in each of their respective mixtures substantially free of other isomers, respectively. ), (VI) and (VII) compounds or their pharmaceutically acceptable salts are enantiomerically enriched mixtures or compositions.

[0123] This document also provides each compound or group of compounds described herein, including formula (I), (Ia). (II), (III), (IV), (V), (VI) and ( Those of VII) or their use in salts, which are used as drugs.

[0124] The compounds of formula I and their pharmaceutically acceptable salts may exist as different polymorphs or pseudopolymorphs. As used herein, crystalline polymorphs means the ability of crystalline compounds to exist in different crystal structures. Crystalline polymorphs can be caused by differences in crystalline packing (packing polymorphs) or packing differences between different conformers of the same molecule (conformation polymorphs). As used herein, crystalline pseudopolymorphs also include the ability of hydrates or solvates of compounds to exist in different crystal structures. The pseudopolymorphs of the present invention may exist due to differences in crystal packing (packing pseudopolymorphs) or packing differences between different conformational isomers of the same molecule (conformational pseudopolymorphs). The present invention includes all polymorphs and pseudopolymorphs of the compound of formula I and pharmaceutically acceptable salts thereof.

[0125] The compound of formula I and its pharmaceutically acceptable salts may also exist as amorphous solids. As used herein, an amorphous solid is a solid that has no long-range order of atomic positions in the solid. This definition also applies when the crystal size is 2 nanometers or less. Additives containing solvents can be used to produce the amorphous form of the present invention. The present invention, which includes all the pharmaceutical compositions, treatment methods, combination products, and uses described herein, includes all amorphous forms of the compound of formula I and pharmaceutically acceptable salts thereof.

[0126] Purpose

[0127] The compounds of the present invention exhibit activity as sodium channel blockers. Without being limited to any specific theory, it is believed that the compounds of the present invention can act in vivo by blocking the epithelial sodium channels existing on the mucosal surface, thereby reducing the absorption of water on the mucosal surface. This effect increases the volume of protective liquid on the mucosal surface and rebalances the system.

[0128] Therefore, the compounds of the present invention can be used as drugs, especially for the treatment of clinical conditions suitable for sodium channel blockers. Such conditions include pulmonary conditions in people in need, such as reversible or irreversible airway obstruction.

Related diseases, chronic obstructive pulmonary disease (COPD) (including acute exacerbations of COPD), asthma, bronchiectasis (including bronchiectasis caused by diseases other than cystic fibrosis), acute bronchitis, chronic bronchitis, after viral infection Cough (post-viral cough), cystic fibrosis, emphysema, pneumonia, panbronchiolitis, transplantation-related bronchiolitis (including lung transplantation and bone marrow transplantation-related bronchitis). The compounds of the present invention can also be used to treat ventilator-associated tracheobronchitis and/or prevent ventilator-associated pneumonia in ventilated patients. The present invention includes methods for treating each of the conditions described herein in a mammal in need thereof (preferably a human in need), each method comprising administering to said mammal a pharmaceutically effective amount of the present The compound of the invention or a pharmaceutically acceptable salt thereof. It also provides (a) a method for slowing down the deterioration of COPD in mammals in need; (b) a method for slowing down the deterioration of CF in mammals in need; (c) breastfeeding in need Method for improving lung function (FEV1) in animals; (d) Method for improving lung function (FEV1) in mammals with COPD; (e) Method for improving lung function (FEV1) in mammals with CF ; (F) Methods to slow down airway infections in mammals in need.

[0129] A method for stimulating, enhancing or improving mucociliary clearance in a mammal is also provided, the method comprising administering a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof to a mammal in need thereof. Mucociliary clearance should be understood to include the natural mucociliary action involved in the transfer or removal of mucus in the airway, including the self-clearing mechanism of the bronchi. Therefore, a method for improving mucus clearance in the airway of a mammal in need is also provided.

[0130] In addition, sodium channel blockers may be suitable for the treatment of diseases that are improved by increasing the mucosal hydration of mucosal surfaces other than the lung mucosal surface. Examples of such conditions include dry mouth (xerostomia), dry skin, vaginal dryness, sinusitis, sinusitis, nasal dehydration (including nasal dehydration due to the application of dry oxygen), dry eyes, Sjogren's disease (Sjogren, s disease), otitis media, primary ciliary dyskinesia&gt; distal intestinal obstruction syndrome, esophagitis, constipation and chronic diverticulitis. The compounds of the present invention can also be used to promote hydration of the eye or cornea.

The compounds of the present invention can also be used in methods for obtaining sputum samples from humans. The method can be carried out as follows: administer an effective amount of the compound of the present invention to at least one lung of a patient, and then induce and collect a sputum sample from the person.

[0132] Therefore, in one aspect, the present invention provides a method for treating conditions suitable for sodium channel blockers in mammals (eg, humans).

[0133] In other embodiments, each of the methods described herein provided by the present invention has the additional benefit of minimizing or eliminating hyperkalemia in the recipient of the method. Also provided are some embodiments including each of the methods described herein in which an improvement in the therapeutic index is achieved.

[0134] The term "treatment" as used herein refers to reversing, alleviating, inhibiting the progression of a disease or condition or one or more symptoms of these diseases or conditions, or preventing them.

[0135] All the therapeutic methods described herein are performed by administering an effective amount of a compound of the present invention, a compound of formula I, or a pharmaceutically acceptable salt thereof, to a subject in need of treatment (usually a mammal, and preferably a human).

[0136] In one embodiment, the present invention provides a method for treating conditions improved by increasing mucosal hydration in mammals (especially humans) in need thereof. In one embodiment, the present invention provides methods for treating diseases related to reversible or irreversible airway obstruction in mammals (especially humans) in need thereof. In a specific embodiment, the present invention provides a method for treating chronic obstructive pulmonary disease (COPD) in mammals (especially humans) in need thereof. In a specific embodiment, the present invention provides a method for reducing the frequency, severity, or duration of acute exacerbations of COPD in mammals (especially humans) in need thereof, or for treating acute exacerbations of COPD One or more symptoms. In one embodiment, the present invention provides

There is a need for a method of treating asthma in mammals (especially humans). In one embodiment, the present invention provides a method for treating bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis) in mammals (especially humans) in need thereof. In one embodiment, the present invention provides methods for treating bronchitis (including acute bronchitis and chronic bronchitis) in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating cough following a viral infection in a mammal in need thereof, particularly a human. In one embodiment, the present invention provides a method for treating cystic fibrosis in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating emphysema in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating pneumonia in a mammal (especially a human) in need thereof. In one embodiment, the present invention provides a method for the treatment of panbronchiolitis in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating transplantation-associated bronchiolitis (including lung transplantation and bone marrow transplantation-associated bronchiolitis) in mammals (especially humans) in need thereof. In one embodiment, the present invention provides for use in this need Methods for the treatment of ventilator-associated tracheobronchitis and/or prevention of ventilator-associated pneumonia among people with ventilator.

[0137] The present invention provides specific methods for the treatment of diseases selected from the group consisting of: reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), asthma, Bronchiectasis (including bronchiectasis caused by diseases other than cystic fibrosis), acute bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, panbronchiolitis, transplant-related diseases For bronchitis and ventilator-associated tracheobronchitis, each method includes administering to the person an effective amount of a compound of formula 1(a) or a pharmaceutically acceptable salt thereof. In other embodiments of each method of treatment, the pharmaceutically acceptable salt form is the hydrochloride or vatyl zeoate of the compound of formula (la). In other embodiments of each treatment method, the free base of the compound of formula (la) is used.

[0138] In one embodiment, the present invention provides a method for treating dry mouth (xerostomia) in a mammal (especially a human) in need thereof. In one embodiment, the present invention provides a method for treating dry skin in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating vaginal dryness in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating sinusitis, sinusitis, or nasal dehydration (including nasal dehydration due to the administration of dry oxygen) in a mammal (especially a human) in need thereof. In one embodiment, the present invention provides a method for treating dry eye or Sjogren's disease or promoting eye or corneal hydration in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating otitis media in a mammal (especially a human) in need thereof. In one embodiment, the present invention provides a method for treating primary ciliary dyskinesia in mammals (especially humans) in need thereof. In one embodiment, the present invention provides a method for treating distal intestinal obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a mammal (especially a human) in need thereof.

[0139] There is also provided a compound of the present invention for use in drug therapy, particularly for the treatment of conditions suitable for sodium channel blockers in mammals (such as humans). All the therapeutic uses described herein are carried out by administering an effective amount of the compound of the present invention to a subject in need of treatment. In one embodiment, the compounds of the present invention are provided for use in the treatment of pulmonary disorders, such as diseases associated with reversible or irreversible airway obstruction, in mammals (especially humans) in need thereof. In a specific embodiment, a compound of the present invention is provided for use in the treatment of chronic obstructive pulmonary disease (COPD) in mammals (especially humans) in need thereof. In one embodiment, the compound of the present invention is provided for use in reducing the frequency, severity or duration of acute exacerbations of COPD or for the treatment of acute exacerbations of COPD in mammals (especially humans) in need thereof One or more symptoms. In one embodiment, the compound of the present invention is provided

It is used to treat asthma in mammals (especially humans) in need. In one embodiment, a compound is provided for use in the treatment of bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis) or bronchitis (including acute bronchitis) in mammals (especially humans) in need thereof Inflammation and chronic bronchitis). In one embodiment, there is provided a compound for use in the treatment of cough following a viral infection in a mammal in need thereof, particularly a human. In one embodiment, there is provided a compound for use in the treatment of cystic fibrosis in a mammal (especially a human) in need thereof. In one embodiment, there is provided a compound of the present invention for use in the treatment of emphysema in mammals (especially humans) in need thereof. In one embodiment, there is provided a compound of the present invention for use in the treatment of pneumonia in mammals (especially humans) in need thereof. In one embodiment, the compound of the present invention is provided for use in the treatment of panbronchiolitis or transplantation-associated bronchiolitis (including lung transplantation and bone marrow transplantation-associated bronchiolitis in mammals (especially humans) in need thereof ). In one embodiment, there is provided a compound of the present invention for use in a method of treating ventilator-associated tracheobronchitis or preventing ventilator-associated pneumonia in a person who needs a ventilator. [0140] In one embodiment, a compound of the present invention is provided for use in mammals in need thereof (particularly It is human) that treats diseases that are alleviated by increasing the hydration of the mucosa on the mucosal surface. In one embodiment, a compound is provided for use in the treatment of dry mouth (xerostomia) in mammals (especially humans) in need thereof. In one embodiment, a compound is provided for use in the treatment of dry skin in mammals, particularly humans, in need thereof. In one embodiment, compounds are provided for use in the treatment of vaginal dryness in mammals, particularly humans, in need thereof. In one embodiment, the compound of the present invention is provided for use in a method of treating sinusitis, sinusitis or nasal dehydration (including nasal dehydration due to the administration of dry oxygen) in a mammal (especially human) in need thereof . In one embodiment, a compound of the present invention is provided for use in treating dry eye or Sjogren's disease or promoting eye or corneal hydration in mammals (especially humans) in need thereof. In one embodiment, there is provided a compound of the present invention for use in the treatment of otitis media in mammals (especially humans) in need thereof. In one embodiment, there is provided a compound of the present invention for use in the treatment of primary ciliary dyskinesia in mammals (especially humans) in need thereof. In one embodiment, there is provided a compound of the present invention for use in the treatment of distal intestinal obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a mammal (especially human) in need thereof.

[0141] The present invention also provides the use of the compounds of the present invention in the manufacture of drugs for the treatment of disorders suitable for sodium channel blockers in mammals (such as humans). In one embodiment, there is provided the use of the compound of the present invention in the preparation of a medicament for the following purposes: treatment of diseases related to reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), acute exacerbation of COPD, asthma, Bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis), bronchitis (including acute bronchitis and chronic bronchitis), cough after viral infection, cystic fibrosis, emphysema, pneumonia, panbronchiole Inflammation, transplant-associated bronchiolitis (including bronchiolitis associated with lung transplantation and bone marrow transplantation), ventilator-associated tracheobronchiolitis, or prevention of ventilator-associated pneumonia.

[0142] In a specific embodiment, there is provided the use of the compound of the present invention in the manufacture of drugs for the following: treatment of diseases alleviated by increasing the hydration of the mucosa on the mucosal surface, treatment of dry mouth (xerostomia), dry skin , Vaginal dryness, sinusitis, sinusitis, nasal dehydration (including nasal dehydration due to the application of dry oxygen), treatment of dry eye, Sjogren's disease, promotion of eye or corneal hydration, treatment of otitis media, primary ciliary dyskinesia, Distal ileus syndrome, esophagitis, constipation, or chronic diverticulitis.

[0143] As used herein, the terms "effective amount", "pharmaceutically effective amount", "effective dose" and "pharmaceutical effective dose" refer to cells that are sufficient to cause, for example, the cells sought by researchers or clinicians in an administered subject. The amount of the compound of the present invention that responds to the biology or medicine of a culture, tissue, system, or mammal (including human). The term also includes within its scope

Effectively strengthen the amount of normal physiological functions. In one embodiment, an effective amount means that when such a composition is administered by inhalation, it provides a desired drug level in the airway and lung secretions and tissues or blood stream of the subject to be treated to obtain the desired physiological response or The amount required for the desired biological effect. For example, the effective amount of the compound of the present invention for treating disorders suitable for sodium channel blockers is sufficient to treat a particular disorder in the subject to be administered. In one embodiment, the effective amount is an amount of the compound of the invention sufficient to treat COPD or cystic fibrosis in a human.

The precise and effective amount of the compound of the present invention depends on a variety of factors, including but not limited to: the species, age and weight of the subject to be treated, the precise condition to be treated and its severity, and the bioavailability of the specific compound being administered , Potency and other properties, the nature of the formulation, the route of administration and the delivery device, and ultimately determined by the attending physician or veterinarian. Further guidance on appropriate dosages can be found by considering the routine administration of other sodium channel blockers (such as amiloride), but also by appropriately considering any difference in efficacy between amiloride and the compound of the invention.

[0145] The pharmaceutically effective dose for topical application to the airway surface of a 70 kg human subject may be about 10 ng to about 10 mg_oIn another embodiment, the pharmaceutically effective dose may be from about 0.1 ug to about 1000 μg_oGenerally speaking, the daily dose for topical application to the airway surface will be sufficient to obtain a dissolved concentration of the active agent on the airway surface of about 10 SO* or 10 ⁷To about 10, 10 3, 10 ²Or 10 [mol/liter, more preferably an amount of about 10 9 to about 10 mol/liter. The choice of the specific dosage for the patient will be determined by the attending physician, clinician or veterinarian of ordinary skill in the art based on a variety of factors including those described above. In a specific embodiment, the dose of the compound of the invention for the treatment of a 70 kg human will be from about 10 nanograms (ng) to about 10 mg_oIn another embodiment, the effective dose will be from about 0.1 μg to about 1,000 μg_oIn one embodiment, the dose of the compound of the invention for the treatment of a 70 kg human will be from about 0.5 ug to about 0.5 mg_oIn yet another embodiment, the dosage will be from about 0.5 ug to about 60 Jie_oIn another embodiment, the pharmaceutically effective dose will be about 1 Pg to about 10 μg_oIn another embodiment, the pharmaceutically effective dose will be from about 5 Pg to about 50 μg_oAnother embodiment has an effective dose of about 10 μg to about 40 μg. In two additional embodiments, the pharmaceutically effective dose will be about 15 ug to about 50 μg, and about 15 ug to about 30 μg, respectively_oIt should be understood that in each of these dosage ranges, all increased dosages within the range are included. For example, the range of 0.5 g to 50 g includes the following individual doses: 0.5 μg&gt;0.6ug&gt;0.7ug&gt;0.8ug&gt;0.9ug&gt;1.0ug&gt;l.lug&gt;1.2ug&gt;1.3 ug&gt;1.4ug&gt;1.5ug&gt;1.6ug&gt;

1. 7 μ g&gt;1.8 μ g&gt;1.9 μ g&gt;2.0 μ g&gt;2.1 μ g&gt;2.2 μ g&gt;2.3 μ g&gt;2.4 μ g&gt;2.5 μ g&gt;2.6 μ g&gt;2.7 μ g&gt;

2. 8 μ g&gt;2.9 μ g&gt;3.0 μ g&gt;3.1 μ g&gt;3.2 μ g&gt;3.3 μ g&gt;3.4 μ g&gt;3.5 μ g&gt;3.6 μ g&gt;3.7 μ g&gt;3.8 μ g&gt;

3. 9 μ g&gt;4.0 μ g&gt;4.1 μ g&gt;4.2 μ g&gt;4.3 μ g&gt;4.4 μ g&gt;4.5 μ g&gt;4.6 μ g&gt;4.7 μ g&gt;4.8 μ g&gt;4.9 μ g&gt;

5. 0 μ g&gt;5.1 μ g&gt;5.2 μ g&gt;5.3 μ g&gt;5.4 μ g&gt;5. 5 μ g&gt;5.6 μ g&gt;5.7 μ g&gt;5.8 μ g&gt;5.9 μ g&gt;6.0 μ g&gt;

6. 1 μ g&gt;6.2 μ g&gt;6.3 μ g&gt;6.4 μ g&gt;6.5 μ g&gt;6.6 μ g&gt;6.7 μ g&gt;6.8 μ g&gt;6.9 μ g&gt;7.0 μ g&gt;7.1 μ g&gt;

7. 2 μ g&gt;7.3 μ g&gt;7.4 μ g&gt;7.5 μ g&gt;7.6 μ g&gt;7.7 μ g&gt;7.8 μ g&gt;7.9 μ g&gt;8.0 μ g&gt;8.1 μ g&gt;8. 2 μ g&gt;

8. 3 μ g&gt;8.4 μ g&gt;8.5 μ g&gt;8.6 μ g&gt;8.7 μ g&gt;8.8 μ g&gt;8.9 μ g&gt;9.0 μ g&gt;9.1 μ g&gt;9.2 μ g&gt;9.3 μ g&gt;

9. 4 μ g&gt;9.5 μ g&gt;9. 6 μ g&gt;9.7 Pg&gt;9.8ug&gt;9. 9ug&gt;10.0ug&gt;10.1 μ g&gt; 10. 2 μ g&gt; 10. 3 μ g&gt;

10. 4ug&gt;10.5ug&gt;10. 6ug&gt;10.7ug&gt;10.8ug&gt;10.9ug&gt;11.0ug&gt;ll.lug&gt;11.2ug&gt; 11.3ug&gt;11.4ug&gt;11.5ug&gt;11.6ug&gt;11.7ug&gt;11.8ug&gt;11.9 ug&gt;12.0ug&gt;12. lug&gt; 12.2ug&gt;12.3ug&gt;12.4ug&gt;12.5ug&gt;12.6ug&gt;12.7ug&gt;12.8ug&gt;12.9ug&gt;13.0ug&gt;

13. lug&gt;13.2ug&gt;13.3ug&gt;13.4ug&gt;13.5ug&gt;13.6ug&gt;13.7ug&gt;13.8ug&gt;13.9ug&gt;

14. 0ug&gt;14. lug&gt;14. 2ug&gt;14. 3ug&gt;14.4ug&gt;14.5ug&gt;14. 6ug&gt;14. 7ug&gt;14.8ug&gt; 14. 9ug&gt;15.0ug&gt;15. lug&gt;15.2ug&gt;15.3ug&gt; 15.4ug&gt;15.5ug&gt;15.6ug&gt;15.7ug&gt; 15.8ug&gt;15.9ug&gt;16.0ug&gt;16. lug&gt;16.2ug&gt;16.3ug&gt;16.4ug&gt;16.5ug&gt;16.6ug&gt;

16.7ug&gt;16.8ug&gt;16.9ug&gt;17.0ug&gt;17. lug&gt;17.2ug&gt;17.3ug&gt;17.4ug&gt;17.5ug&gt; 17.6ug&gt;17.7ug&gt;17.8ug&gt;17.9ug&gt;18.0ug&gt;18. lug&gt;18.2ug&gt; 18.3ug&gt;18.4ug&gt; 18.5ug&gt;18.6ug&gt;18.7ug&gt;18.8ug&gt;18.9ug&gt;19.0ug&gt;19. lug&gt;19.2ug&gt;19.3ug&gt; 19.4ug&gt;19.5ug&gt;19.6ug&gt;19.7ug&gt;19.8ug&gt;19.9 ug&gt;20.0ug&gt;20.lug&gt;20.2ug&gt; 20.3ug&gt;20. 4ug&gt;20.5ug&gt;20.6ug&gt;20.7ug&gt;20.8ug&gt;20. 9ug&gt;21.0ug&gt;21.1ug&gt; 21.2ug&gt;21.3ug&gt;21.4 ug&gt;21.5ug&gt;21.6ug&gt;21.7ug&gt;21.8ug&gt;21.9ug&gt;22.0ug&gt;

twenty two. 1 μ g&gt;22.2 μ g&gt;22.3 μ g&gt;22.4 μ g&gt;22.5 μ g&gt;22.6 μ g&gt;22.7 μ g&gt;22.8 μ g&gt;22.9 μ g&gt;

twenty three. 0 μ g&gt; 23. lug&gt; 23.2ug&gt; 23.3ug&gt; 23.4ug&gt; 23.5ug&gt; 23.6ug&gt; 23.7ug&gt; 23.8ug&gt; 23.9 μ g&gt; 24.0 μ g&gt; 24.1 μ g&gt; 24.2 μ g&gt; 24.3 μ g&gt; 24.4 μ g&gt; 24.5 μ g&gt; 24.6 μ g&gt; 24.7 μ g&gt; 24.8 ug&gt; 24.9 ug&gt; 25.0 ug&gt; 25. lug&gt;25.2ug&gt;25.3ug&gt;25.4ug&gt;25.5ug&gt;25.6ug&gt; 25.7ug&gt;25.8ug&gt;25.9ug&gt;26.0ug&gt;26. lug&gt;26. 2ug&gt;26. 3ug&gt;26.4ug&gt;26 . 5ug&gt; 26.6ug&gt;26. 7ug&gt;26.8ug&gt;26.9ug&gt;27.0ug&gt;27. lug&gt;27.2ug&gt;27.3ug&gt;27.4ug&gt; 27.5ug&gt;27.6ug&gt;27.7ug&gt;27.8ug&gt;27.9ug&gt;28.0ug &gt;28.lug&gt;28.2ug&gt;28.3ug&gt; 28.4ug&gt;28.5ug&gt;28.6ug&gt;28.7ug&gt;28.8ug&gt;28.9ug&gt;29.0ug&gt;29. lug&gt;29. 2ug&gt;

29. 3ug&gt;29.4ug&gt;29.5ug&gt;29.6ug&gt;29.7ug&gt;29.8ug&gt;29.9ug&gt;30.0ug&gt;30.lug&gt;

30. 2ug&gt;30. 3ug&gt;30.4ug&gt;30. 5ug&gt;30.6ug&gt;30. 7ug&gt;30.8ug&gt;30.9ug&gt;31.0ug&gt; 31.1ug&gt;31.2ug&gt;31.3ug&gt;31.4ug&gt;31.5ug&gt;31.6ug&gt;31.7ug &gt;31.8ug&gt;31.9ug&gt; 32.0 μ g&gt; 32.lug&gt;32. 2ug&gt;32.3ug&gt;32.4ug&gt;32.5ug&gt;32.6ug&gt;32.7ug&gt;32.8ug&gt; 32.9ug&gt;33.0ug&gt; 33. lug&gt;33.2ug&gt;33.3ug&gt;33.4ug&gt;33.5ug&gt;33.6ug&gt;33.7ug&gt; 33.8ug&gt;33.9ug&gt;34. 0ug&gt;34. lug&gt;34.2ug&gt;34.3ug&gt;34. 4ug&gt; 34.5ug&gt;34. 6ug&gt; 34.7ug&gt;34.8ug&gt;34.9ug&gt;35.0ug&gt;35. lug&gt;35.2ug&gt;35.3ug&gt;35.4ug&gt;35. 5ug&gt; 35.6ug&gt;35.7ug&gt;35.8ug &gt;35.9ug&gt;36.0ug&gt;36.lug&gt;36. 2ug&gt;36.3ug&gt;36.4ug&gt; 36.5ug&gt;36. 6ug&gt;36. 7ug&gt;36.8ug&gt;36.9ug&gt;37.0ug&gt;37.lug&gt;37.2ug&gt;37.3ug&gt; 37.4ug&gt;37.5ug&gt;37.6ug&gt;37.7ug&gt;37.8ug&gt;37.9ug&gt;38.0ug&gt;38 .lug&gt;38.2ug&gt;

38. 3ug&gt;38. 4ug&gt;38.5ug&gt;38.6ug&gt;38.7ug&gt;38.8ug&gt;38. 9ug&gt;39.0ug&gt;39.lug&gt;

39. 2ug&gt;39.3ug&gt;39.4ug&gt;39.5ug&gt;39.6ug&gt;39. 7ug&gt;39.8ug&gt;39.9ug&gt;40.0ug&gt;

40. 1 μ g&gt;40.2 μ g&gt;40.3 μ g&gt;40.4 μ g&gt;40.5 μ g&gt;40.6 μ g&gt;40.7 μ g&gt;40.8 μ g&gt;40.9 μ g&gt;41.0ug&gt;41.lug &gt;41.2ug&gt;41.3ug&gt;41.4ug&gt;41.5ug&gt;41.6ug&gt;41.7ug&gt;41.8ug&gt;

41. 9 μ g&gt;42.0 μ g&gt;42.1 μ g&gt;42.2 μ g&gt;42.3 μ g&gt;42.4 μ g&gt;42.5 μ g&gt;42.6 μ g&gt;42.7 μ g&gt; 42.8ug&gt;42.9ug&gt;43.0ug&gt;43. lug&gt;43.2ug&gt;43.3ug&gt;43.4ug&gt;43.5ug&gt;43.6ug&gt;

43. 7 μ g&gt;43.8 μ g&gt;43.9 μ g&gt;44.0 μ g&gt;44.1 μ g&gt;44.2 μ g&gt;44.3 μ g&gt;44.4 μ g&gt;44.5 μ g&gt;

44. 6 μ g&gt;44.7 μ g&gt;44.8 μ g&gt;44.9ug&gt;45.0ug&gt;45. lug&gt;45. 2ug&gt;45.3ug&gt;45.4ug&gt; 45.5ug&gt;45.6ug&gt;45.7ug&gt;45.8ug&gt;45.9 ug&gt;46.0ug&gt;46.lug&gt;46.2ug&gt;46.3ug&gt;46.4ug&gt;46.5ug&gt;46.6ug&gt;46.7ug&gt;46.8ug&gt;46.9ug&gt;47.0ug&gt;47.lug&gt;47.2ug&gt; 47.3ug&gt;47.4ug&gt; 47.5ug&gt;47.6ug&gt;47.7ug&gt;47.8ug&gt;47.9ug&gt;48.0ug&gt;48.lug&gt;48.2ug&gt;48. 3ug&gt;48.4ug&gt;48.5ug&gt;48.6ug&gt;48.7ug&gt;48.8ug&gt;38.9ug&gt;49.0ug&gt; 49.1 μ g&gt;49.2 μ g&gt;49.3 μ g&gt;49.4 μ g&gt;49.5 μ g&gt;49.6 μ g&gt;49.7 μ g&gt;49.8 μ g&gt;39. 9 g and 50 μ g_o

[0146] If the compound is administered by a different route, the aforementioned recommendations can be adjusted by using conventional dosing algorithms

the amount. Based on the foregoing description and common general knowledge in the art, those skilled in the art can determine the appropriate dosage for administration via other routes.

[0147] Delivery of an effective amount of the compound of the present invention may require the delivery of a single dosage form or multiple unit dosages, which can be delivered simultaneously or separately at a time within a specified period (eg, 24 hours). The dose of the compound of the present invention (alone or in the form of a composition containing the compound of the present invention) can be administered from 1 to 10 times a day. Generally speaking, the compound of the invention (alone or in the form of a composition containing the compound of the invention) will be administered 4, 3, 2 or 1 times a day (24 hours).

[0148] The compounds of formula (I) of the present invention can also be used to treat airborne infections. Examples of airborne infections include, for example, RSV. The compound of formula (1) of the invention can also be used to treat anthrax infection. The present invention relates to the use of the compound of formula (1) of the present invention for prevention, post-exposure prevention, prophylactic or therapeutic treatment of diseases or disorders caused by pathogens. In a preferred embodiment, the present invention relates to the use of a compound of formula (D) for the prevention, post-exposure prophylaxis, prophylactic or therapeutic treatment of diseases or disorders caused by pathogens useful in bioterrorism.

[0149] In recent years, a number of research projects and biodefense measures have been carried out to solve problems related to the use of biological agents in terrorist acts. These measures are aimed at solving problems related to bioterrorism or the use of microorganisms or biotoxins to kill, spread fear, and disrupt society. For example, the National Institute of Allergy and Infectious Diseases (National Institute of Allergy and Infectious Diseases, NIAID) has conducted a Strategic Plan for Biodefense Research (Strategic Plan for Biodefense Research), which lists the And plans needed for research in a wide range of areas where infectious diseases appear and reappear. According to the plan, the intentional exposure of the civilian population of the United States to spores of Bacillus anthracis (Bac delus anthracis) revealed a gap in the country's overall preparations for bioterrorism. In addition, reports detailing these attacks revealed an unmet need for tests for rapid diagnosis, vaccines and immunotherapy for prevention, and drugs and biological agents for curing diseases caused by bioterrorist activities.

[0150] The focus of a variety of research work is mostly on the biology of pathogens with the same potential as bioterrorism substances, studying the host's response to these substances, developing vaccines against infectious diseases, and evaluating currently available and under research Therapeutic agents for these substances, and the development of diagnostics for identifying the signs and symptoms of threatening substances. These efforts are commendable, but considering the large number of pathogens that have been identified as potential bioterrorist activities, these efforts have not yet been able to provide a satisfactory response to all possible bioterrorism threats. In addition, many pathogens that have been identified as potentially dangerous as bioterrorist substances do not provide sufficient economic motivation for the development of treatment or preventive measures in industry. In addition, even if preventive measures (such as vaccines) are available for every pathogen that can be used for bioterrorism, the cost of administering all these vaccines to the general population is prohibitive.

[0151] Until convenient and effective treatments for every threat of bioterrorism are available, there is still a strong demand for preventive, preventive, or therapeutic treatments that can prevent or reduce the risk of pathogen infection.

[0152] The present invention provides such preventive treatment methods. In one aspect, a preventive treatment method is provided, which comprises administering an effective amount of a compound of formula (I) to an individual in need of preventive treatment of infection by airborne pathogens. A specific example of an airborne pathogen is anthrax.

[0153] In another aspect, there is provided a preventive treatment method for reducing the risk of infection by air-borne pathogens that can cause disease in humans, the method comprising: An effective amount of the compound of formula (I) is administered to the lungs of a person with symptoms of the disease, wherein the effective amount of sodium channel blocker and osmotic agent are sufficient to reduce the risk of infection in the person. A specific example of an airborne pathogen is anthrax.

[0154] In another aspect, there is provided a post-exposure prophylaxis for the treatment of airborne pathogen infections.

A treatment or therapeutic treatment method, which comprises administering an effective amount of a compound of formula (I) to the lungs of an individual in need of such treatment against airborne pathogen infection. Pathogens can be prevented by the post-exposure prophylactic, rescue, and therapeutic treatment methods of the present invention, which include any pathogen that can enter the body through the mouth, nose, or nasal airway to further enter the lungs. Generally speaking, the pathogen can be a pathogen that exists naturally or is spread through atomized air. The pathogen may be naturally occurring or may be introduced into the environment deliberately by nebulization or introduced into the environment by other methods. Many pathogens that cannot spread naturally in the air have been or can be atomized for bioterrorism. The pathogens that can be treated with the present invention include, but are not limited to, the pathogens of the A, B, and C priority levels listed by NIAID. These categories usually correspond to the Centers for Disease Control and Prevention (Centers for Disease Control and Prevention, CDC) compiled list. As established by the CDC, category A pathogens are those that are easy to spread or spread from person to person, cause high mortality, and may have a significant impact on public health. Next in priority are category B pathogens, and it includes those that are moderately easy to spread and cause moderate morbidity and low mortality. Category C consists of emerging pathogens that can be adapted for mass transmission in the future because of their availability, ease of production and distribution, and the potential for high morbidity and mortality. Specific examples of these pathogens are anthrax and plague. Other pathogens that can prevent or reduce the risk of infection include influenza virus, rhinovirus, adenovirus, and respiratory syncytial virus. Another pathogen that can be prevented is the coronavirus, which is believed to cause severe acute respiratory syndrome (SARS).

[0155] The present invention also relates to a sodium channel blocker of formula I or a pharmaceutically acceptable salt thereof for use in the prevention, reduction and/or treatment of exposure to radioactive materials, especially those containing from a nuclear attack such as a radiological dispersal device (radiological dispersal device). , RDD) explosions or accidents such as nuclear power plant disasters inhalable aerosols of radionuclides and the use of deterministic health effects on the respiratory tract. Therefore, this article provides for the prevention, alleviation and/or treatment of respiratory tract and/or other body organs caused by inhalable aerosols containing radionuclides in recipients in need (including those in need) A method for determining the health effects of a method comprising administering to the human an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

[0156] Major issues related to consequence management planning for exposing members of the public to inhalable aerosols containing radionuclides from nuclear attacks such as radioactive dispersal device (RDD) explosions or accidents such as nuclear power plant disasters How to prevent, reduce or treat the potential deterministic health effects on the respiratory tract (mainly the lungs). It is necessary to prepare drugs, technology, procedures, and trained personnel to manage and deal with such highly contaminated individuals.

[0157] Research has been conducted to determine methods to prevent, reduce, or treat potential damage to the respiratory tract and various organs in the body caused by internally deposited radionuclides. So far, most of the research attention has been focused on strategies designed to mitigate the health effects of internally deposited radionuclides by promoting their excretion or removal. These strategies have focused on soluble chemical forms that can reach the bloodstream and be deposited at remote systemic sites specific for a given radionuclide. Such a method does not work when the deposited radionuclide is in a relatively insoluble form. Studies have shown that many, if not most, physical forms of the deposited radionuclides from RDDs will be relatively insoluble forms.

[0158] The only method known to effectively reduce the lung radiation dose from inhaled insoluble radioactive aerosol is bronchoalveolar lavage or BAL. This technique is modified from a method that has been used to treat patients with alveolar protein deposits, and it has proven to be a safe and reproducible method, even when performed over a long period of time. However, there are some changes in the method. The basic method used for BAL anesthetize the subject and then slowly introduce isotonic saline into a single lung lobe until the functional residual capacity is reached. Then add additional volume and drain by gravity.

[0159] The results of studies using BAL in animals indicate that about 40% of the deep lung content can be divided by BAL in a reasonable order.

go with. In some studies, there are considerable differences in the amount of radionuclides recovered between animals. The reason for the discrepancy is currently unknown.

[0160] In addition, based on studies in animals, it is believed that BAL treatment significantly reduces the radiation dose, thereby reducing the health effects of inhalation of insoluble radionuclides. In this study, adult dogs were made to breathe insoluble¹⁴⁴Ce-FAP particles. Give two groups of dogs known to cause radiation pneumonitis and pulmonary fibrosis¹⁴⁴Ce lung content (approximately 2MBq/kg body weight), one group was given 10 unilateral lavage treatments between 2 days and 56 days after exposure, and the other group was not treated. The third group was exposed to levels similar to those in the BAL treatment group after treatment¹⁴⁴Ce level (about IMBq/kg), but these animals are not treated. Allow all animals to spend their lifespan (which is up to 16 years). Since the initial lung content of dogs in each group is different, the dose rate and cumulative dose of each group overlap. However, it can be seen from the survival curve that BAL has a significant effect on reducing the risk of pneumonia/fibrosis. In untreated dogs with lung content ranging from 1.5 MBq/kg to 2.5 MBq/kg, the average survival time is 370 ± 65 days_oFor the treated dogs, the average survival was 1270±240 days, which is a statistically significant difference. Accept 0.6MBq/kg to 1.4MBq¹⁴⁴In the third group of Ce lung content, the average survival time is 1800±230, which is not statistically different from the treated group. Equally important for prolonging survival, dogs in the high-dose untreated group died of definite effects on the lungs (pneumonia/fibrosis), while dogs in the treated group did not. Instead, most of the treated dogs had lung tumors (angiosarcoma or carcinoma) as in the low-dose untreated group. Therefore, the dose reduction caused by BAL treatment appears to have a biological effect on the lung that can be predicted based on the radiation dose received by the lung.

[0161] Based on these results, it is believed that further reduction of residual radiation dose by any method or combination of methods that enhances the removal of particles from the lungs can further reduce the possibility of causing health effects on the lungs. However, the BAL method has many disadvantages. BAL is a highly invasive method, which must be performed by a trained pulmonologist in a professional medical center. Therefore, the BAL method is expensive. Considering the shortcomings of BAL, it is not an easy and immediately available treatment option for people who need to accelerate the removal of radioactive particles, such as in the event of a nuclear attack. In the case of a nuclear attack or nuclear accident, immediate and relatively easy-to-administer treatment is required for people who have been exposed or are at risk of exposure. Sodium channel blockers administered as inhaled aerosols have been shown to restore airway surface hydration. This hydration of the airway surface helps remove accumulated mucus secretions and related particulate matter from the lungs. Therefore, without being bound by any particular theory, it is believed that sodium channel blockers can be used to accelerate the removal of radioactive particles from airway channels.

[0162] As discussed above, after a radiation attack (eg, a dirty bomb), the greatest threat to the lungs is caused by the inhalation and retention of insoluble radioactive particles. Due to the retention of radioactive particles, the cumulative exposure to the lungs is significantly increased, eventually leading to pulmonary fibrosis/pneumonia and possible death. Insoluble particles cannot be systematically removed by chelating agents because these particles do not dissolve. So far, the physical removal of particulate matter through BAL is the only treatment that has been shown to be effective in reducing radiation-induced lung disease. As discussed above, BAL is not a realistic treatment option for reducing the effect of radioactive particles that have been inhaled into the body. Therefore, it is desirable to provide a treatment plan that effectively helps to remove radioactive particles from the airway passage, and it (unlike BAL) is relatively simple for the user and can be scaled up to large-scale radiation exposure. In addition, it is also hoped that the treatment plan will be easily available to many people in a relatively short period of time.

[0163] In one aspect of the present invention, methods for preventing, reducing and/or treating definite health effects on the respiratory tract and/or other body organs caused by inhalable aerosols containing radionuclides include Administer an effective amount of the sodium channel blocker of formula (I) or a pharmaceutically acceptable salt thereof. In a feature of this aspect, the sodium channel blocker is administered with an osmotic agent. Further to this feature, the osmotic agent is hypertonic saline (HS). In another feature, sodium channel blockers and osmotic agents are administered together with ion transport modifiers. For this feature further, the ion transport modulator can be selected from B-agonists, CFTR potentiators, purinergic receptor agonists (purinergic receptor agonists).

agonist), lubiprostone and protease inhibitors. In another feature of this aspect, the radionuclide is selected from the group consisting of drill-60, octane-137, iron-192, radium-226, phosphorus-32, saw-89 and saw-90, iodine-125, armor-201 , Lead-210, mu-234, uranium-238, brazing, drill-58, yong-51, shrinkage and copper. In another feature, the radionuclide comes from a radioactive disposal facility. In another feature, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered as an aerosol suspension of inhalable particles inhaled by the individual. In another feature, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to the radionuclide.

Composition

[0165] Although the compound of the present invention may be administered alone, in some embodiments, it is preferably presented in the form of a composition and particularly a pharmaceutical composition (formulation). Therefore, in another aspect, the present invention provides a composition, particularly a pharmaceutical composition (such as an inhalable pharmaceutical composition), which comprises as an active ingredient a pharmaceutically effective amount of the compound of the present invention, alone or in combination, and a pharmaceutically acceptable Excipients, diluents and carriers. The term "active ingredient" as used herein refers to any compound of the present invention or a combination of two or more compounds of the present invention in a pharmaceutical composition. A specific embodiment is also provided, wherein the pharmaceutical composition comprises a pharmaceutically effective amount of formula (D, (Ia), (II), (III), (IV), (V), (VI) and (VII) The compound of or a pharmaceutically acceptable salt thereof exists alone or in combination with a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of formula (I), (Ia), (II), (III), (IV), (V), alone or in combination, in a diluent. ), (VI) and (VII) compounds or pharmaceutically acceptable salts thereof. In a separate embodiment, the pharmaceutical composition comprises a pharmaceutically effective amount of formula (I), (Ia), (ID, (IID, (IV), (IV), (ID, (IID, (IV), hypertonic saline, sterile water, and hypertonic saline, respectively). (V), (VI) and (VII) compounds or pharmaceutically acceptable salts thereof, wherein the salt water concentration may be as described herein. In one embodiment, the salt water concentration is 0.17% w/v, and in another In one embodiment, it is 2.8% w/v_o[0167] A kit is also provided, which comprises: i) a pharmaceutically effective amount of formula (1), (Ia), (II), (III), (IV), (V), (VI) and (VII) ) Compound or a pharmaceutically acceptable salt thereof; ii) one or more pharmaceutically acceptable excipients, carriers or diluents; iii) for administering the compound of group i) and group ii) to a subject in need thereof Instructions for excipients, carriers or diluents; and iv) containers. The subject in need includes any subject in need of the treatment methods described herein, and particularly includes human subjects in need. Another embodiment also includes an atomizing device selected from: atomizers, including vibrating screen atomizers and jet atomizers; dry powder inhalers, including active and passive dry powder inhalers; and metered-dose inhalers, including Pressurized metered-dose inhalers, dry powder metered-dose inhalers and soft mist metered-dose inhalers.

[0168] In one embodiment, the kit includes i) Formula (I), (Ia), (II), (III), (iv), (V), (VI), and (VII) compound or a pharmaceutically acceptable salt thereof; ii) a diluent of about 1 mL to about 5 mL per dose; iii) for administering the compound of group i) and the diluent of group ii) to a subject in need ; And iv) container. In another embodiment, the diluent is about 1 mL to about 5 mL of the saline solution described herein per dose. In another embodiment, the diluent is about 1 mL to about 5 mL of hypotonic saline per dose. In another embodiment, the diluent is about 1 mL to about 5 mL of hypertonic saline per dose. In yet another embodiment, the diluent is about 1 mL to about 5 mL of sterile water per dose.

[0169] A kit is also provided, which includes i) a pharmaceutically effective amount of formula (1), (Ia), (II), (III), (IV), ( V), (VI) and (VII) compounds or solutions of their pharmaceutically acceptable salts; iii) for administering the solution of group i) to a subject in need thereof; and iii) a container.

[0170] A kit is also provided, which includes i) Formula (I), (Ia), (II), (III), (IV), (V), (VI) comprising about 10 ng to about 10 mg And a solution of the compound of (VII) or a pharmaceutically acceptable salt thereof; dissolved in a pharmaceutically acceptable diluent; iii) for administering the solution of group i) to a subject in need thereof; and iii) a container. In another embodiment, the dilute

The release agent is about 1 mL to about 5 mL of the saline solution described herein per dose.

[0171] Another embodiment includes a kit comprising i) a pharmaceutically effective amount of formula (I), (Ia). (II), (III), (IV), (V), (VI) and ( VII) compound or a pharmaceutically acceptable salt thereof; in a dry powder formulation suitable for inhalation ii) optionally, one or more pharmaceutically acceptable excipients or carriers suitable for inhalation; iii) The subject in need is administered the compound of group i) and the excipient and carrier of group ii); and; iv) a container. In another embodiment, the kit further includes a dry powder inhaler adapted to deliver a dry powder formulation to a recipient. In other embodiments, the dry powder inhaler may be a single-dose inhaler or a multi-dose inhaler.

[0172] Other embodiments of each kit described herein include those below, wherein each dose of formula (1), (Ia). (II), (III), (IV), (V) The concentration of the compounds of (VI) and (VII) or their pharmaceutically acceptable salts is one of the effective dose ranges described herein, including a) about 0.1 ug to about 1,000 Jg; b) about 0 . 5 Jie g to about 0.5 mg; and c) about 0.5 Jie g to about 50 μ go

[0173] For each of the kits described above, there are additional embodiments in which the diluent is hypertonic saline at the concentration described herein. In another embodiment, for each kit, the diluent is hypotonic saline at the concentration described herein. In another embodiment, for each kit, the diluent is sterile water suitable for inhalation.

[0174] The pharmaceutically acceptable excipient, diluent or carrier must be acceptable in terms of compatibility with the other ingredients of the formulation and harmless to its recipient. Generally, pharmaceutically acceptable excipients, diluents or carriers used in pharmaceutical formulations are "non-toxic", meaning that they are considered safe to be consumed in the amount delivered in the formulation, and "inert" means It does not have a measurable reaction with the active ingredient or has no undesirable influence on the therapeutic effect of the active ingredient. Pharmaceutically acceptable excipients, diluents, and carriers are conventional in the art, and can be selected according to the desired route of administration using conventional techniques. See Remington's, Pharmaceutical Sciences, Lippincott W subscribes to liams&amp;W subscribes to kins; 21st edition (May 1, 2005). Preferably, the pharmaceutically acceptable excipients, diluents or carriers comply with the General Regarded As Safe (GRAS) according to the FDA.

[0175] Pharmaceutical compositions according to the present invention include those suitable for administration via the following routes: oral administration; parenteral administration, including subcutaneous, intradermal, intramuscular, intravenous and intraarticular; topical administration, including topical administration to Skin, eyes, ears, etc.; vaginal or rectal administration; and administration to the respiratory tract, including the nasal cavity and sinuses, oral cavity and extrathoracic airways, and lungs, including the use of aerosols, which can be applied through various types of dry powder inhalers, pressurized Delivery by metered-dose inhaler, soft mist inhaler, nebulizer or insufflator. The most suitable route of administration may depend on several factors, including the patient being treated and the condition or disease.

[0176] The preparation can be presented in a unit dosage form or in a bulk form (for example, in the case of metering the preparation through an inhaler), and can be prepared by any known method in the pharmaceutical field. Generally speaking, the method includes the step of combining the active ingredient with a carrier, diluent or excipient and optionally one or more accessory ingredients. Generally, the formulation is prepared by making the active ingredient and one or more liquid carriers, diluents or excipients or finely divided solid carriers, diluents or excipients or both uniform and compact Associate and then, if necessary, shape the product into the desired formulation.

[0177] In a preferred embodiment, the composition is an inhalable pharmaceutical composition, which is suitable for inhalation and delivery to the intrabronchial space. Generally speaking, such compositions are in the form of an aerosol that contains particles for delivery using a nebulizer, pressurized metered dose inhaler (MDI), soft mist inhaler or dry powder inhaler (DPI). The aerosol formulation used in the method of the present invention may be a liquid (for example, a solution) suitable for administration via a nebulizer, soft mist inhaler or MDI or a dry powder suitable for administration via MDI or DPI.

[0178] Aerosols used to administer drugs to the respiratory tract are usually polydisperse, that is, they are composed of many particles of different sizes. The particle size distribution usually passes through the mass median aerodynamic diameter (Mass Median Aerodynamic

Diameter, MMAD) and geometric standard deviation (Geometric Standard Deviation, GSD) description. For the best drug delivered to the intrabronchial space, the MMAD is about 1 pm to about 10 pm, preferably about 1 pm to about 5 um, and the GSD is less than 3, particularly preferably less than about 2. When inhaled into the lungs, the MMAD is greater than 10 um aerosols are usually too large. Aerosols with a GSD greater than about 3 are not preferred for pulmonary delivery because they deliver a large proportion of the drug into the oral cavity. To obtain these particle sizes in powder formulations, conventional Techniques such as micronization or spray drying to reduce the particle size of the active ingredient. Non-limiting examples of other methods or techniques that can be used to produce respirable particles include spray drying, precipitation, supercritical fluids, and freeze drying. They can be classified by air or sieved. The desired fraction is separated by fractionation. In one embodiment, the particles are crystals. For fluid formulations, the particle size is determined by selecting a specific model of nebulizer, soft mist inhaler or MDI.

[0179] The aerosol particle size distribution was measured using a device known in the art. For example, a multi-stage Anderson cascade impactor or other suitable methods, such as those specifically cited in Chapter 601 of the United States Pharmacopoeia, are devices characterized as emitting aerosols from metered-dose inhalers and dry powder inhalers.

[0180] The dry powder composition for local delivery to the lungs by inhalation can be formulated without excipients or carriers, and only contains the active ingredient in the form of a dry powder having a particle size suitable for inhalation. The dry powder composition may also comprise a mixture of the active ingredient and a suitable powder base (carrier/diluent/excipient material) (e.g., monosaccharide, disaccharide or polysaccharide). For dry powder formulations, lactose is usually the preferred excipient. When a solid excipient such as lactose is used, generally the particle size of the excipient will be much larger than the active ingredient to aid dispersion of the formulation in the inhaler.

[0181] Non-limiting examples of dry powder inhalers include reservoir multi-dose inhalers&gt;pre-metered multi-dose inhalers&gt;capsule-based inhalers and Single-dose disposable inhaler. A reservoir type inhaler contains many (for example, 60) doses in one container. Before inhalation, the patient activates the inhaler so that the inhaler measures a dose of medicine from the reservoir and prepares it for inhalation. Examples of reservoir DPI include, but are not limited to, AstraZeneca's Turbohaler® and Vectura's

ClickHaler®.,

[0182] In a multi-dose inhaler, each single dose is manufactured in a separate container, and the inhaler is activated before inhalation so that a new dose of drug is released from its container and prepared for inhalation. Examples of multi-dose DPI inhalers include, but are not limited to, Diskus® by gsk, Gyrohaler® by Vectura, and Prohaler® by Valois_oDuring the inhalation process, the patient's inspiratory air flow accelerates the powder out of the device and into the oral cavity. For capsule inhalers, the formulation is in a capsule and stored outside the inhaler. The patient places the capsule in the inhaler, activates the inhaler (punctures the capsule), and then inhales. Examples include RotohalerTM (GlaxoSmithKline)&gt; SpinhalerTM (Novartis)&gt; HandiHalerTM (IB), TurboSpinTM (PH&amp;T)_OFor single-dose disposable inhalers, the patient activates the inhaler to prepare it for inhalation, inhales and then discards the inhaler and packaging. Examples include TwincerTM (U Groningen)&gt; OneDoseTM (GFE) and Manta InhalerTM (Manta Devices)_o

[0183] Generally speaking, dry powder inhalers use the turbulent nature of the powder path to disperse the excipient-drug assembly and deposit the active ingredient particles in the lungs. However, some dry powder inhalers use cyclonic dispersion chambers to produce particles of the desired inhalable size. In the cyclonic dispersion chamber, the medicine enters the coin-shaped dispersion chamber tangentially, causing its air path and medicine to move along the outer annular wall. As the drug formulation moves along the annular wall, it bounces around and causes the aggregate to be broken up by the impact force. The air path spirals toward the center of the chamber and leaves vertically. Particles of sufficiently small aerodynamic size can leave the chamber along the air path. In fact, the dispersion chamber works like a small jet mill. Depending on the characteristics of the formulation, large lactose particles can be added to the formulation to aid dispersion (by collision with API particles).

[0184] The TwincerTM single-dose disposable inhaler that appears to operate using a coin-shaped cyclone dispersing chamber is called an "air classifier. See Rijksuni ver site it Groningen's U.S. Published Patent Application No. 2006/0237010_oA paper published by the University of Groningen states that using this technology, a 60 mg dose of pure micronized colistin sulfomethate can be effectively delivered as a dry powder inhalable.

[0185] In some preferred embodiments, a dry powder inhaler is used to deliver the aerosol formulation as a dry powder, wherein the MMAD of the particles ejected from the inhaler is about 1 μm to about 5 μm, and the GSD is about less than 2.

Examples of suitable dry powder inhalers and dry powder dispersion devices for delivering the compounds and compositions of the present invention include, but are not limited to, those disclosed in the following patent documents: US7520278, US7322354, US7246617, US7231920, US7219665, US7207330, US6880555, US5, 522, 385, US6845772, US6637431, US6329034. US5, 45 &amp; 135, US4, 805, 811 and US Published Patent Application No. 2006/0237010.

[0187] In one embodiment, the pharmaceutical formulation according to the invention is a dry powder for inhalation, which is formulated for delivery through a Diskus®-type device. The Disku$® device includes an elongated belt formed from a substrate, the elongated belt having a plurality of recesses spaced along its length and a cover sheet hermetically but releasably closed thereon to define a plurality of containers, Each container has an inhalable preparation containing a predetermined amount of active ingredient, which is present alone or mixed with one or more carriers or excipients (for example, lactose) and/or other therapeutically active agents. Preferably, the belt is flexible enough to be wound into a roll. The cover sheet and the base sheet preferably have leading end portions that are not closed to each other and at least one leading end portion is configured to be connected to the winding device. In addition, preferably, the airtight seal between the base and the cover sheet covers the entire width thereof. In order to prepare a dose for inhalation, the cover sheet may preferably be peeled off from the first end of the base sheet in the axial direction of the base sheet.

[0188] In one embodiment, the pharmaceutical formulation according to the present invention is a dry powder for inhalation, which is formulated for delivery using a single-dose disposable inhaler (especially TwincerTM inhaler). The Twincer torture inhaler includes a foil laminate blister, which has one or more recesses and a cover sheet that is tightly but releasably closed, thereby defining a plurality of containers . Each container has an inhalable preparation containing a predetermined amount of active ingredient, which is present alone or mixed with one or more carriers or excipients (for example, lactose). The cover sheet will preferably have a leading end portion configured to protrude from the main body of the inhaler. The patient will operate the device to administer the aerosol formulation as follows: 1) remove the outer packaging packaged on the outside, 2) pull out the foil tab to uncover the drug in the blister, and 3) inhale the drug from the blister.

[0189] In another embodiment, the pharmaceutical formulation according to the present invention is a dry powder for inhalation, wherein the dry powder is formulated into microparticles as described in NexBio's PCT Publication No. W02009/015286 or W02007/114881. Such microparticles are usually formed as follows: adding a counter ion to a solution containing the compound of the present invention in a solvent, adding an antisolvent to the solution; gradually cooling the solution to a temperature lower than about 25°C to form a solution containing A composition containing microparticles of the compound. Then, the microparticles containing the compound can be separated from the solution by any suitable means, such as precipitation, filtration or lyophilization. Suitable counterions, solvents and antisolvents for preparing microparticles of the compounds of the present invention are described in WO2009/015286.

[0190] In another embodiment, a metered-dose inhaler is used to deliver the pharmaceutical composition according to the invention as a dry powder. Non-limiting examples of metered-dose inhalers and devices include those disclosed in the following patent documents: US5,261,538, US5,544,647, US5,622,163, US4,955,371, US3,565,070, US3,361306 And US6, 116, 234 and US7, 10&amp; 159. In a preferred embodiment, a metered dose inhaler is used to deliver the pharmaceutical composition of the present invention as a dry powder, wherein the emitted particles have an MMAD of about 1 μm to about 5 μm, and a GSD of less than about 2.

[0191] Liquid aerosol formulations for delivery to the intrabronchial space or lungs by inhalation can be formulated, for example, by pressurizing

The aqueous solution or suspension or aerosol for delivery is packaged in a pressurized package, such as a soft mist inhaler, nebulizer, or metered-dose inhaler using a suitable liquefied propellant. This aerosol composition suitable for inhalation may be a suspension or solution, which usually contains the active ingredient, and a pharmaceutically acceptable carrier or diluent (for example, water (distilled water or sterile water), saline, high Saline or ethanol) and optionally one or more other therapeutically active agents.

[0192] Aerosol compositions for delivery via a pressurized metered-dose inhaler usually also contain a pharmaceutically acceptable propellant. Examples of such propellants include fluorocarbons or hydrogen-containing chlorofluorocarbon billets or mixtures thereof, especially hydrofluorocarbon billets, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1 ,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3, -heptafluoro-n-propane or mixtures thereof. The aerosol composition may be free of excipients or may optionally contain additional formulation excipients known in the art, such as surfactants, such as oleic acid or lecithin, and co-solvents, such as ethanol. The pressurized formulation is usually stored in a can (for example, an aluminum can) sealed with a valve (for example, a metering valve), and is installed in an actuator having a mouthpiece.

[0193] In another embodiment, a metered-dose inhaler is used to deliver the pharmaceutical composition according to the invention as a liquid. Non-limiting examples of metered-dose inhalers and devices include those disclosed in the following patent documents: US Patent Nos. 6, 253, 762, 6, 413, 497, 7, 601, 336, 7, 481, 995, 6, 743 , 413 and 7, 105, 152. In a preferred embodiment, a metered-dose inhaler is used to deliver the compound of the present invention as a dry powder, wherein the MMAD of the emitted particles is from about 1 μm to about 5 μm, and the GSD is less than about 2.

[0194] In one embodiment, the aerosol formulation is suitable for atomization by jet atomizers or ultrasonic atomizers (including static and vibrating perforated plate atomizers). Liquid aerosol preparations for nebulization are formulated by dissolving or reconstituting solid particle preparations, or formulated with an aqueous carrier and adding reagents such as acids or bases, buffer salts, and isotonicity regulators. It can be sterilized by in-process techniques such as filtration or terminal treatments such as heating in an autoclave or gamma radiation. It can also be presented in an unsterilized form.

[0195] Patients may be sensitive to the pH, osmolarity, and ion content of the nebulized solution. Therefore, these parameters should be adjusted to be compatible with the active ingredient and tolerated by the patient. The most preferred solution or suspension of the active ingredient contains chlorine concentration&gt; 30 mM, at pH 4.5 to 7.4, preferably 5.0 to 5.5, and the osmolality concentration is about 800mOsm/kg to 1600mOsm/kg_oThe pH can be controlled by titration with common acids (for example, hydrochloric acid or sulfuric acid) or bases (for example, sodium hydroxide) or by using buffers. Commonly used buffers include citrate buffers (such as citric acid/sodium citrate buffer), acetate buffers (such as acetic acid/sodium acetate buffer) and phosphate buffers. The buffer strength can be 2mM to 50mM_o

[0196] Usable acetate, phosphate and citrate buffers include sodium acetate, sodium acetate trihydrate, saddle acetate, potassium acetate, sodium phosphate, sodium phosphate dibasic&gt; disodium hydrogen phosphate (Disodium hydrogen phosphate), potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium citrate and potassium citrate. Other buffers that can be used include sodium hydroxide, potassium hydroxide, saddle hydroxide, aminomethylpropanol, tromethamine, tetravyl propyl ethylene diamine, citric acid, acetic acid, vat based triple acid or Its salts (such as its citrate or sodium citrate), lactic acid and lactic acid salts (including sodium lactate, potassium lactate, lithium lactate, calcium lactate, magnesium lactate, lactate, aluminum lactate, zinc lactate, silver lactate, copper lactate, Iron lactate, manganese lactic acid, saddle lactic acid), monoethanolamine, diethanolamine, triethanolamine, diisopropanolamine and combinations thereof.

[0197] Such a formulation can be administered using a commercially available atomizer or other sprayer that can break the formulation into particles or droplets suitable for deposition in the respiratory tract. Non-limiting examples of nebulizers that can be used for the aerosol delivery of the composition of the present invention include: pneumatic jet nebulizer&gt; exhaust or breath-enhanced jet nebulizer or ultrasonic nebulizer (including Static or vibrating perforated plate atomizer). Commercial nebulizers include AeiOHeb® Go nebulizer (Aerogen) and eFlow nebulizer (Pari Pharma).

[0198] Jet atomizers use high-speed airflow to jet upward through a water column to produce droplets. Particles that are not suitable for inhalation impinge on the wall or aerodynamic baffle. Exhaust or breath-enhanced nebulizers work in essentially the same way as jet nebulizers, except that the inhaled air passes through the main droplet generation area to increase the output rate of the nebulizer when inhaled by the patient.

[0199] In the ultrasonic nebulizer, the vibration of the piezoelectric crystal creates surface instability in the drug reservoir, resulting in the formation of droplets. In the perforated plate atomizer, the pressure field generated by sound energy drives the liquid through the mesh, where it is broken into droplets by Rayleigh breakup. Acoustic energy can be applied through a vibrating arm or a vibrating plate driven by a piezoelectric crystal or the vibrating mesh itself. Non-limiting examples of sprayers include any single-stream or dual-stream sprayer or nozzle that produces droplets of suitable size. Single-stream sprayers work by driving liquid through one or more holes, where the sprayed liquid is broken into droplets. The dual-flow nebulizer works by driving both gas and liquid through one or more holes or by causing the ejected gas to collide with another ejected liquid or gas.

[0200] The choice of a nebulizer for nebulizing an aerosol formulation is important for the application of the active ingredient. Different atomizers have different efficiencies based on their design and operating principles, and are sensitive to the physical and chemical properties of the preparation. For example, two formulations with different surface tensions may have different particle size distributions. In addition, formulation properties such as pH, osmolality concentration, and osmotic ion content can affect drug tolerance, so some preferred embodiments meet certain ranges of these properties.

[0201] In a preferred embodiment, a suitable nebulizer is used to deliver the preparation for nebulization as an aerosol to the intrabronchial space, and the aerosol has an MMAD of about 5 μm to about 5 μm and a GSD of less than 2 . For best efficacy and avoid upper respiratory tract and systemic side effects, the MMAD of the aerosol should not be greater than about 5 um, and the GSD should not be greater than about 2. If the MMAD of the aerosol is greater than about 5 mm or the GSD is greater than about 2, a large percentage of the dose can be deposited in the upper respiratory tract, reducing the amount of drug delivered to the desired site in the lower respiratory tract. If the MMAD of the aerosol is less than about 1 um, a large proportion of the particles may remain suspended in the inhaled air, which may be expelled during exhalation.

[0202] The compounds of the present invention can also be administered by transbronchoscopic lavage.

[0203] Formulations suitable for oral administration may exist as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of active ingredient; as powders or granules; as A solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be provided in sachet, bolus, syrup or paste.

[0204] Tablets can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (eg, powder or granules), optionally mixed with binders, lubricants, inert diluents, surfactants or dispersants, in a suitable machine. Molded tablets can be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored, and may be formulated so as to provide slow or controlled release of the active ingredient therein.

[0205] Formulations for topical administration in the mouth (for example, buccal or sublingual) include: lozenges, which contain active ingredients in a flavoring base such as sucrose and gum arabic or tragacanth; and lozenges, which Active ingredients contained in a base such as gelatin and glycerin or sucrose and acacia.

[0206] Preparations for parenteral administration include: aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the preparations isotonic with the blood of the intended recipient; aqueous and non-aqueous Aqueous sterile suspensions, which may contain suspending agents and thickening agents. The preparations can be presented in single-dose or multi-dose containers, such as sealed retorts and vials, and can be stored under lyophilized (freeze-dried) conditions, with only the addition of a sterile liquid carrier, such as saline or water for injection, just before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

[0207] Oral fluids such as solutions, syrups, and drugs can be prepared in dosage unit form to give a predetermined

A given amount of active ingredient. Syrups can be prepared by dissolving the active ingredient in a suitable flavored aqueous solution, while the drug is prepared using a pharmaceutically acceptable alcohol carrier. Suspensions can be formulated by dispersing the active ingredient in a pharmaceutically acceptable carrier. Oral liquid compositions can also incorporate solubilizers and emulsifiers, such as ethoxylated isostearyl alcohol and polyoxyethylene sorbitol, preservatives, flavor additives such as peppermint oil or natural sweeteners or other artificial sweetenersAgent etc.

[0208] Liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles can also be used as delivery means of the compounds of the present invention. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, and phosphatidylcholine.

[0209] Pharmaceutical compositions for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. Compositions designed to treat the eye or other external tissues (such as the mouth and skin) can be applied as a topical ointment or cream. When formulated as an ointment, the active ingredient can be used with paraffin wax or an ointment base that is miscible with water. Alternatively, the active ingredient can be formulated into a cream with an oil-in-water cream base or a water-in-oil base.

[0210] Other compositions designed for topical application to 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 an aqueous solvent (including saline).

[0211] Compositions designed for nasal administration include aerosols, solutions, suspensions, sprays, mists, and drops. Aerosol formulations for nasal administration can be formulated in a very similar manner to aerosol formulations for inhalation, provided that non-inhalable sized particles are preferred in formulations for nasal administration. Generally speaking, particles with a size of about 5 microns can be used until small drops are visible. Therefore, for nasal administration, a particle size range of 10 um to 500 um can be used to ensure that it stays in the nasal cavity.

[0212] Transdermal patches can also be used, which are designed to maintain long-term contact with the patient's epidermis and promote the absorption of active ingredients therethrough.

[0213] Compositions for vaginal or rectal administration include ointments, creams, suppositories, and enemas, all of which can be formulated using conventional techniques.

[0214] In another aspect, the present invention provides a method for promoting mucosal surface hydration or restoring mucosal defense in a person in need thereof, which comprises administering to said person a pharmaceutical composition comprising a compound of the present invention, wherein said The compound is administered in an effective amount. In a preferred embodiment, the method comprises administering a pharmaceutical composition as an inhalable composition, which contains a compound of the present invention in an amount sufficient to obtain 10% of the compound on the airway surface. 7 to about 10 4, 10 3, 10²Or 10 1 mol/liter, more preferably a dissolved concentration of 109 to about 10 mol/liter.

[0215] In another aspect, the present invention provides a method for treating any of the following diseases or preventing ventilator-associated pneumonia in a person in need thereof, which comprises administering to said person a drug comprising a compound of the present invention A composition, wherein the compound is administered in an effective amount: reversible or irreversible airway obstruction related diseases, chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis (including bronchiectasis caused by diseases other than cystic fibrosis), acute Bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, panbronchiolitis, transplant-associated bronchiolitis, and ventilator-associated tracheobronchiolitis. In a preferred embodiment, the method comprises administering a pharmaceutical composition as an inhalable composition, which contains a compound of the present invention in an amount sufficient for the compound to obtain 10 9, 108, or 10 on the airway surface. 10 7 to about 10 \10 \10²Or 10 1 mol/liter, more preferably a dissolved concentration of 109 to about 104 mol/liter.

[0216] In another aspect, the present invention provides a method of treating any of the following diseases in a person in need thereof: dry mouth (xerostomia), dry skin, vaginal dryness, sinusitis, sinusitis, or nasal dehydration (Including nasal dehydration caused by the application of dry oxygen), dry eye or Sjogren's disease, promote eye or corneal hydration, treat distal intestinal obstruction syndrome, treat otitis media, primary fibrosis

For hair dyskinesia, distal intestinal obstruction syndrome, esophagitis, constipation, and chronic diverticulitis, the method comprises administering to the human a pharmaceutical composition comprising a compound of the present invention, wherein the compound is administered in an effective amount.

[0217] Preferred unit dose formulations of the compounds of the present invention are those that contain an effective amount of the active ingredient or an appropriate fraction thereof.

[0218] It should be understood that, in addition to the ingredients specifically mentioned above, taking into account the types of formulations in question, the formulations of the present invention may include other conventional agents in the art, for example, those suitable for oral administration may include Flavoring agent.

[0219] According to the specific condition to be treated and the desired route of administration, the composition of the present invention can be formulated as immediate release, controlled release or sustained release as needed. For example, a controlled release formulation for oral administration may be desirable to treat constipation, so as to deliver as much active agent to the colon as possible. Such formulations and suitable excipients are well known in the pharmaceutical art. Since the free base of the compound is generally less soluble in aqueous solution than the salt, a composition comprising the free base of the compound of formula I can be used to provide a more sustained release of the active agent delivered to the lungs by inhalation. The active agent present in the lung in the form of particles that cannot be dissolved in the solution cannot induce a physiological response, but acts as a reservoir for the biocompatible drug that gradually dissolves into the solution. In another example, the formulation may use both the free base form and the salt form of the compound of the present invention to provide both immediate and sustained release of the active ingredient for dissolution into mucus secretions such as the nose.

[0220] Combination

[0221] The compounds of the present invention can be formulated and/or used in combination with other therapeutically active agents. Other therapeutically active agents that can be formulated or used in combination with the compounds of the present invention include, but are not limited to, osmotic agents, anti-inflammatory agents, anticholinergic agents, agonists (including selective B 2^_Agonists), P2Y2 receptor agonists, peroxisome proliferator-activated receptor (PPAR) delta agonists, other epithelial sodium channel blockers (ENaC receptor blockers), capsules Cystic fibrosis transmembrane conductance regulator (CFTR) regulators, kinase inhibitors, anti-infectives, antihistamines, non-antibiotic anti-inflammatory macrolides, elastase and protease inhibitors, And mucus or mucin modifiers, such as surfactants. In addition, for cardiovascular indications, the compounds of the present invention can be used in combination with beta blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular agents.

[0222] Therefore, in another aspect, the present invention provides a composition comprising an effective amount of a compound of the present invention and one or more other therapeutically active agents selected from the group consisting of osmotic agents, anti-inflammatory agents , Anticholinergic agents, agonists (including selective B 2-agonists), P2Y2 receptor agonists, PPAR δ agonists, ENaC receptor blockers, cystic fibrosis transmembrane conductance regulator (CFTR) Modulators, kinase inhibitors, anti-infectives, antihistamines, non-antibiotic anti-inflammatory macrolides, elastase and protease inhibitors, and mucus or mucin modifiers, such as surfactants. Therefore, in another aspect, the present invention provides a composition comprising an effective amount of a compound of the present invention and one or more other therapeutically active agents selected from the group consisting of B blockers, ACE inhibitors. HMGCoA Reductase inhibitors and calcium channel blockers. The use of the compound of the present invention in combination with one or more additional therapeutically active agents (especially osmotic agents) can reduce the dose of the compound of the present invention required to fully hydrate the mucosal surface, thereby reducing systemic (e.g., renal Middle) Possibility of undesirable side effects of blocking sodium channels.

[0223] The "osmotic agent" according to the present invention is a molecule or compound with osmotic activity. "Osmotic active" molecules and compounds are membrane-impermeable (ie, substantially non-absorbable) on the surface of the airway or lung epithelium. The terms "airway surface" and "lung surface" as used herein include lung airway surfaces, such as bronchi and bronchioles, alveolar surfaces, and nasal and sinus surfaces. Suitable osmotic agents include ionic osmotic agents (ie, salts) and non-ionic osmotic agents (ie, sugars, sugar alcohols, and organic osmotic agents). Generally speaking, osmotic agents (ionic and non-ionic) used in combination with the compounds of the present invention

The two) are preferably osmotic agents that do not promote or actually prevent or delay the growth of bacteria. Osmotic agents suitable for use in the present invention may be in the form of racemates or in the form of enantiomers, diastereomers, tautomers, polymorphs and pseudopolymorphs.

[0224] Examples of ion tonicity agents that can be used in the present invention include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably, either (or both) of the anions and cations have osmotic activity for their application to the airway surface and will not be actively transported quickly. Such compounds include, but are not limited to, the anions and cations contained in the FDA-approved commercially available salts, see, for example, Remington: The Science and Practice of Pharmacy&gt; Vol. II&gt; page 1457 (19th edition, 1995), and such Any combination known in the art can be used.

[0225] Specific examples of pharmaceutically acceptable osmotic active anions include, but are not limited to: acetate, benzenesulfonate, benzoate, bicarbonate, hydrogen tartrate, bromide ion, calcium edetate, camphor Sulfonate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate, ethanedisulfonate (1,2-ethanedisulfonate), etonate (lauryl sulfate), Ethylsulfonate (1,2-ethane disulfonate), fumarate, gluceptate, gluconate, glutamate, glycollylarsan order (paracetamol) Shen acid radical (p-glycollamidophenylarsonate), hexylresorcinate (hexylresorcinate), hybamin (Ν, Ν'-bis(dehydrorosinyl) ethylenediamine), hydrogen bromide, hydrochloride, vat carboxylate, iodide, vat ethyl sulfonate, lactate, lacturonate, malate, maleate, Mandelate (mandelate), methanesulfonate, methyl bromide anion (methylbromide), methyl nitrate, methyl sulfate, mucate, sulfonate, nitrate, nitrite (nitrte), catabolite (en Borate), pantothenate, phosphate or hydrogen phosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, horseradish, tartrate, naphtha chloride Teoclate (8-chloronaphtha), triethiodide, bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide and phosphate.

[0226] Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to: organic cations, such as Mexin (N, N,-dijasylethylenediamine), chloroprocaine, choline, diethanolamine, ethyl Diamine, meglumine (N-methyl D-glucosamine), procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylsulfonate , N-methyl D-glycerol, etc.; and metal cations, such as aluminum ion, calcium ion, lithium ion, magnesium ion, potassium ion, sodium ion, zinc ion, iron ion, saddle ion, etc. Preferred organic cations include 3-carbon, 4-carbon, 5-carbon and 6-carbon organic cations. Preferred cations include sodium ion, potassium ion, choline ion, lithium ion, meglumine ion, D-lysine ion, saddle ion, magnesium ion and calcium ion.

[0227] Specific examples of ionotonizers that can be administered in combination with the compounds of the present invention include, but are not limited to: sodium chloride (especially hypertonic saline), potassium chloride, choline chloride, choline iodide, chlorine Lithium, meglumine chloride, L-lysine chloride, D-lysine chloride, saddle chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride, Potassium bromide and a combination of any two or more of the foregoing substances. In one embodiment, the invention provides a combination of a compound of the invention and two different osmotically active salts. When different salts are used, one of the anions or cations in the different salts may be the same. Hypertonic saline is the preferred ionotonizer for use in combination with the compounds of the present invention.

[0228] Non-ionic osmotic agents include sugars, sugar alcohols and organic osmotic agents. Sugars and sugar alcohols that can be used as osmotic agents in the present invention include, but are not limited to, 3-carbon sugars (e.g., glycerol, diacetone), 4-carbon sugars (e.g., D and L forms of erythreose, threose Arabinose and erythreulose), 5-carbon sugars (for example, D and L forms of ribose, arabinose, xylose, lysut, allulose, fructose, sorbose and tagatose), and 6 -Carbosaccharides (for example, D and L forms of maltose (altose), allose, glucose, mannose, gulose, idose, galactose and talose, and D and L forms of allose-heptose) Kulose, allose-heptulose (hepulose), glucose-heptulose, mannose-heptulose, gulose-heptulose, idose-heptulose, galactose

Sugar-heptulose, talose-heptulose). Other sugars that can be used in the practice of the present invention include raffinose, raffinose series oligosaccharides, and stachyose. Both the D and L forms of the reduced form of each sugar/sugar alcohol are also suitable for the present invention. For example, when reduced, glucose becomes sorbitol, which is an osmotic agent within the scope of the present invention. Therefore, sorbitol and other reduced forms of sugar/sugar alcohol (for example, mannitol, dulcitol, arabitol) are suitable osmotic agents for use in the present invention. Mannitol is the preferred non-ionic osmotic agent for use in combination with the compounds of the present invention.

[0229] "Organic osmolality agent" generally refers to a molecule that controls the intracellular osmolality in the kidney. See example Nvkou JS Handler, etc., Comp. Biochem. Physiol, 117&gt;301-306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995)_oOrganic osmotic agents include, but are not limited to, three main types of compounds: polyols (polyvinyl alcohols), methylamines, and amino acids. Suitable polyol organic osmotic agents include, but are not limited to: inositol, myo-inositol, and sorbitol. Suitable methylamine organic osmotic agents include, but are not limited to: choline, betaine, carnitine (L-, D- and DL forms), phosphorylcholine, lyso-phosphorylcholine, glycerophosphorylcholine Alkali, creatine and creatine phosphate. Suitable amino acid organic osmotic agents include, but are not limited to: D-form and L-form glycine, alanine, glutamine, glutamic acid, aspartic acid, proline and taurine. Additional organic osmotic agents suitable for use in the present invention include tihulose and sarcosine. Mammalian organic osmotic agents are preferred, and human organic osmotic agents are most preferred. However, some organic osmotic agents are derived from bacteria, yeast and marine animals, and these compounds can also be used in the present invention.

[0230] Osmotic agent precursors can be used in combination with the compounds of the invention. The term "osmotic agent precursor" as used herein refers to a compound that is converted into an osmotic agent through a metabolic step (catabolism or anabolic). Examples of osmotic agent precursors include, but are not limited to: glucose, glucose polymers, glycerol, choline, phosphatidylcholine, lysophosphatidylcholine, and inorganic phosphate, which are precursors of polyols and methylamines . The precursors of amino acid osmolytes include proteins, peptides and polyamino acids that are hydrolyzed to produce osmolyte amino acids, as well as metabolic precursors that can be converted into osmolyte amino acids by metabolic steps such as transamination. For example, the precursor of the amino acid glutamine is poly-L-glutamine, and the precursor of glutamic acid is poly-L-glutamic acid.

[0231] Chemically modified osmotic agents or osmotic agent precursors can also be used. Such chemical modification includes linking the osmotic agent (or precursor) with another chemical group that changes or enhances the effect of the osmotic agent or the osmotic agent precursor (for example, inhibits the osmotic agent molecule Degradation). Such chemical modifications have been applied to drugs or prodrugs, and are known in the art (see, for example, U.S. Patent Nos. 4,479,932 and 4,540,564; Shek, E., etc., J. Med. Chem. 19: 113-117 (1976); Bodor, N., etc., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N., etc., J. Med. Chem. 26: 313-318 ( 1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986).

[0232] Preferred osmotic agents for use in combination with the compounds of the present invention include sodium chloride (particularly hypertonic saline) and mannitol.

[0233] For 7% and &gt;7% hypertonic saline formulations, formulations containing carbonate anions may be particularly useful, especially for respiratory diseases with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction, such as The recent findings of CF or COPD indicate that although the relative ratio of HCO3 conductivity/C1 conductivity in a single CFTR channel activated by cAMP and ATP is 0.1 to .2, the ratio in sweat ducts is based on the stimulation conditions. Actually 0 to almost 1.0. That is, the combination of cAMP+cGMP+α-ketoglutarate can produce CFTR HCO3 conductivity that is almost equal to the conductivity of Cl (Quiton et al. Physiology, Vol. 22, No. 3, 212-225, June 2007). In addition, formulations containing 7% of bicarbonate anions and &gt;7% hypertonic saline can be particularly useful because they better control the pH of the airway surface liquid. First, it has been shown that airway acidification occurs in CF. (Tate et al. 2002), and the lack of CFTR-dependent bicarbonate secretion can lead to impaired responsiveness to airway disorders related to the acidification of the liquid layer of the airway surface (Coakle

Et al. 2003). Secondly, adding a bicarbonate-free HS solution to the lung surface can further dilute the bicarbonate concentration and reduce the pH or the ability to respond to airway acidification in the liquid layer of the airway surface. Therefore, adding bicarbonate anions to HS can help maintain or increase the pH of the airway surface liquid layer of CF patients.

[0234] Because of this evidence, formulations containing 7% or &gt;7% hypertonic saline of bicarbonate anions applied by the method of the present invention will be particularly useful. Formulations containing bicarbonate anions up to a concentration of 30 mM to 200 mM are particularly useful for 7% or&gt; 7% HS solutions.

[0235] Hypertonic saline should be understood as a salt concentration greater than normal saline (NS), that is, greater than 9g/L or 0.9% w/v, and the salt concentration of hypotonic saline is lower than that of normal saline, for example, about lg/L or 0.1% w/v to about 8g/L or 0.8% w/v_oThe salt concentration of the hypertonic salt solution that can be used in the formulations and treatment methods described herein may be from about 1% to about 23.4% (w/v). In one embodiment, the salt concentration of the hypertonic salt solution is from about 60 g/L (6% w/v) to about 100 g/L (10% w/v). In another embodiment, the salt concentration of the salt solution is about 70 g/L (7% w/v) to about 100 g/L (10% w/v). In other embodiments, the salt concentration of the salt solution is: a) about 0.5 g/L (0.05% w/v) to about 70 g/L (7% w/v); b) about 1g /L (0.1% w/v) to about 60g/L (6% w/v); c) about lg/L (0.1% w/v) to about 50g/L (5% w/v) ); d) about lg/L (0.1% w/v) to about 40g/L (4% w/v); e) about lg/L (0.1% w/v) to about 30g/L (3% w/v); and f) about lg/L (0.1% w/v) to about 20 g/L (2% w/v).

[0236] Saline solutions of specific concentrations that can be used in the formulations and treatment methods herein independently include those having the following salt concentrations: 1g/L (0.1% w/v), 2g/L (0.2% w/ v), 3g/L (0.3% w/v), 4g/L (0.4% w/v), 5g/L (0.5% w/v), 6g/L (0.6% w/v), 7g/L(0. 7% w/v), 8g/L(0. 8% w/v), 9g/L(0. 9% w/v), 10g/ L(l% w/v), 20g/L(2% w/v), 30g/L(3% w/v), 40g/L(4% w/v) , 50g/L(5% w/v), 60g/L(6% w/v), 70g/L(7% w/v), 80g/L(8% w/v), 90g/L(9 % w/v), 100g/L (10% w/v), 110g/L (ll% w/v), 120g/L (12% w/v), 130g/L (13% w/v), 140g/L (14% w/v), 150g/L (15% w/v), 160g/ L (16% w/v), 170g/'L (17% w/v), 180g/L (18 % w/v), 190g/L (19% w/v), 200g/L (20% w/v), 210g/L (21% w/v), 220g/L (22% w/v) and 230g/L (23% w/v). Salt concentrations between each of these listed concentrations/percentages can also be used, such as 1. 7g/L (0.17% w/v), 1.25g/L (1.25% w/v), l. 5g/L(l. 5% w/v), 25g/L(2.5% w/v), 28g/L(2. 8 % w/v), 35g/L (3.5% w/v), 45g/L (4.5% w/v) and 75g/L (7.5% w/v) brine.

[0237] Specific usable concentrations of hypotonic salt solutions include those from about 0.12 g/L (0.012% w/v) to about 5 g/L (0.85% w/v). Any concentration in this range can be used, for example based on w/v, 0.05%, 0.1%, 0.15%, 0.2%, 0.225% (l/4NS), 0.25% 25%, 0.3% (1/3NS), 0.35%, 0.4%, 0.45% (1/2NS), 0.5%, 0.55%, 0.6% (2/ 3NS), 0.65%, 0.675% (3/4NS), 0.7%, 0.75% and 0.8%.

[0238] Each range and specific concentration of saline can be used in the formulations, treatment methods, protocols, and kits described herein.

[0239] Osmotic agents or osmotic agent precursors intended for chemical modification are also within the scope of the present invention. Such chemical modification includes linking the osmolyte (or precursor) with another chemical group that changes or enhances the effect of the osmolyte or the osmolyte precursor (for example, inhibits the osmolyte molecule Degradation). Such chemical modifications have been applied to drugs or prodrugs, and are known in the art (see, for example, U.S. Patent Nos. 4,479,932 and 4,540,564; Shek, E., etc., J. Med. Chem. 19: 113-117 (1976); Bodor, N., etc., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N., etc., J. Med. Chem. 26: 313-318 ( 1983); Bodor, N. et al., J. Pharm. Sci. 75: 29-35 (1986), each of which is incorporated herein by reference.

[0240] Suitable anti-inflammatory agents for use in combination with the compounds of the present invention include corticosteroids and non-steroidal anti-inflammatory

Agents (NSAID), especially phosphodiesterase (PDE) inhibitors. 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 its esters (for example, mometasone furoate), propionic acid Fluticasone, fluticasone furoate, beclomethasone, methylprednisolone, prednisolone, dexamethasone, 6 α ,9 α -difluoro-17α-[(2-pyranyl dialyl)oxyl 11Β-Vat group-16α-Methyl-3-oxo-andron-1,4-diene T7 β-thioacid S-fluoromethyl ester, 6 α ,9α-difluoro-11 Β-Vat Group-16 α -methyl-3-oxo-17 α -propionyloxy-andro-1,4-diene-17 Beta thioacid S-(2-oxo-tetrahydro-soran -3S-base) ester, beclomethasone ester (for example, 17-propionate or 17,21-dipropionate, fluoromethyl ester, triamcinolone acetonide, rofluronide or any combination or subset thereof The preferred corticosteroid for formulation or use with the compounds of the present invention is selected from ciclesonide, desisobutyryl ciclesonide, budesonide, mometasone, fluticasone propionate and fluticasone furoate or any combination thereof Or a subset.

[0241] The NSAIDs used in the present invention include, but are not limited to, cromolyn sodium, nedocromil sodium, phosphodiesterase (PDE) inhibitors (for example, naphtha, aminophenrin, PDE4 inhibitor, mixed PDE3/PDE4 inhibitors or mixed PDE4/PDE7 inhibitors), leukotriene antagonists, leukotriene synthesis inhibitors (for example, 5L0 and FLAP inhibitors), nitric oxide synthase (iNOS) inhibitors, protease inhibitors Agents (for example, tryptase inhibitors, neutrophil elastase inhibitors and metalloproteinase inhibitors, B 2-integrin antagonists and adenosine receptor agonists or antagonists (for example, adenosine 2a agonists), Cytokine antagonists (eg, chemokine antagonists) or cytokine synthesis inhibitors (eg, prostaglandin D2 (CRTh2) receptor antagonists). Examples of leukotriene modulators suitable for administration by the method of the present invention include Montelukast, zileuton, and zalukast.

[0242] The PDE4 inhibitor, mixed PDE3/PDE4 inhibitor or mixed PDE4/PDE7 inhibitor can be any compound known to inhibit the PDE4 enzyme or found to act as a PDE4 inhibitor, and which is a selective PDE4 inhibitor (ie , The compound does not significantly inhibit other members of the PDE family). Examples of specific PDE4 inhibitors for formulation and use with the compounds of the present invention include, but are not limited to, Roflumilast, Promafentran, Aldrin, Cilomilast, Tofilast, Omilast, Procrastinate Phentran, Piramimilast, Ibumilast, apremilast, 2-[4-[6, 7~diethoxy-2, 3-bis(vnmethyl)-1- Caiji]-2-Pyridanyl]-4-(3-Pyridanyl)-1(2 specific-diazazepone biphenyl (T2585), N- (3, 5-Dichloro-4-pyrrolidinyl) fluorophenyl) methyl]-5-yl-α-oxo-1H-in Jie Do 3-acetamide (AWD-12-281), 4- [(2R) -2- [3-(cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pichuan (CDP-840), 2-[4-[[ [ [2-(1,3-benzodioxol-5-yloxy)-3-pyridyl] dialyl]amino]methyl]-3-fluorophenoxy]-( 2R)-propionic acid (CP-671305), N-(4,6-dimethyl-2-ketyl)-4-[4, 5,6,7-tetrahydro-2-(4-methoxy 3-methylphenyl)-5-(4-methyl-1-piperazinyl)-1H-Jie In Jie Dor-1-yl]-benzenesulfonamide, (2E)-2-butene two Acid salt (YM-393059), 9-[(2-fluorophenyl)methyl]shu-methyl-2-(trifluoromethyl)-9H-yanyin-6-amine (NCS-613), N -(2,5-Dichloro-3-pyruvyl)-8-methoxy-5-quineline carboxamide (D-4418), N-[(3R)-9-amino-3,4 ,6,7-Tetrahydro-4-oxo-1-phenylpyrrolo[3, 2,1-] [1,4] benzodiazepine-3-yl]-3H-pyridine-6-amine (PD-168787), 3- [[3-(cyclopentyloxy)-4 -Methoxyphenyl]methyl]-N-ethyl-8-(1-methylethyl)-3H-Zyin-6-amine hydrochloride (V-11294A), N-(3,5 -Dichloro-1-oxo-4-pyrrolidinyl)-8-methoxy-2-(trifluoromethyl)-5-quineline carboxamide (Sch351591), 5-[3-(cyclopentane Oxy)-4-methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S, 5S)-2-piperidone (HT-0712), 5-(2- ((1R, 4R)-4-amino-1-(3-(cyclopentyloxy)-4-methoxyphenyl)cyclohexyl)ethynyl)-castan-2-amine, cis- [4-oxy-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol] and 4-[6, 7-diethoxy-2, 3-Bis(Vatmethyl)-1-Czeyl]-1-(2-Methoxyethyl)-2(1-Pyridone (T-440) and any

Combination or subset.

[0243] Leukotriene antagonists and inhibitors of leukotriene synthesis include zalukast, montelukast sodium, zileuton, and plukast.

[0244] Anticholinergic agents formulated or used in combination with the compounds of the present invention include, but are not limited to, muscarinic receptor antagonists, particularly including pan-antagonists and M3 receptor antagonists. Exemplary compounds include the alkaloids of the belladonna plant, such as atropine, scotiatine, homatropine, atropine, and salts thereof (e.g., anhydrous atropine, atropine sulfate, atropine oxide, or atropine HC1, methylatropine nitrate, homatropine hydrobromide, homatropine methyl bromide, chrysopine hydrobromide, chrysopine sulphate, scoprine hydrobromide, and Chrystine methyl bromide), or any combination or subset thereof.

[0245] Other anticholinergic agents used in combination formulation or use include: mettyline, bropromethine, anisotropine methyl bromide or Valpin 50, aclidinium bromide ), Robinul, Isopropyl iodide, bromepentate, tridihexethyl chloride &gt;hexocyclium methylsulfate, cyclopentolate HC1 (cyclopentolate HC1), Bicaamide, trihexyphenidyl CC1, pirenzepine, telenzepine, and methotramine, or any combination or subset thereof.

[0246] Preferred anticholinergic agents for formulation or use in combination with the compounds of the present invention include ipratropium plating (bromo

Compounds), oxitropium (desertification compounds) and Satolan (desertification compounds) or any combination or subset thereof.

[0247] Examples of B-agonists formulated or used in combination with the compounds of the present invention include, but are not limited to, salmeterol, R-salmeterol and its xicaeate, salbutamol or R-salbutamol (free base or sulfuric acid) Salt), levalbuterol, salbutamol, formoterol (fumarate), fenoterol, procaterol, pibuterol, metaprterenol, terbutaline and its salts, and Any combination or subset.

[0248] The P2Y2 receptor agonist that can be formulated and used in combination with the compound of the present invention can be used in an amount effective to stimulate the airway surface (especially the nasal airway surface) to secrete chloride and water. Suitable P2Y2 receptor agonists are known in the art, for example in columns 9 to 10 of US Patent No. 6,264, 975, and US Patent Nos. 5, 656, 256 and 5, 292, 498 Described in.

[0249] P2Y® agonists that can be administered by the method of the present invention include P2Y® receptor agonists, such as ATP, UTP, UTP-. γ. -S, and dinucleotide P2Y® receptor agonists (such as denucleotide). Denufosol or diquafosol) or a pharmaceutically acceptable salt thereof. The P2Y receptor agonist is usually contained in an amount effective to stimulate the secretion of chloride and water on the airway surface (especially the nasal airway surface). Suitable P2Y 2 receptor agonists are described in (but not limited to) U.S. Patent No. 6,264,975, U.S. Patent No.5,656,256, U.S. Patent No.5,292,498, U.S. Patent No.6 , 34 &amp; 589, U.S. Patent No. 6, 81 &amp; 629, U.S. Patent No. 6, 977, 246, U.S. Patent No. 7, 223, 744, U.S. Patent No. 7, 531, 525 and U.S. Patent Application 2009/0306009 , Which are all incorporated herein by introduction.

[0250] The combination therapies and formulations herein may include adenosine 2b (A2b) agonists, and also include BAY60-6583, NECA (N-ethylformamide adenosine), (S)-PHPNECA, LUF-5835 and LUF- 5845. Usable A2b agonists are described in the following documents: Volpini et al., Journal of Medicinal Chemistry 45 (15): 3271-9 (2002); Volpini et al., Current Pharmaceutical Design 8(26): 2285-98 (2002); Baraldi et al. , Journal of Medicinal Chemistry 47(6): Cacciari et al., 1434-47 (2004); Mini Reviews in Medicinal Chemistry 5(12): 1053-60 (December 2005); Baraldi et al., Current Medicinal Chemistry 13(28) :3467-82 (2006); Beukers et al., Medicinal Research Reviews 26 (5): 667-98 (September 2006); Elzein et al., Bioorganic&amp;Medicinal Chemistry Letters 16(2):

302-6 (January 2006); Carotti et al., Journal of Medicinal Chemistry 49 (1): 282-99 (January 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).

[0251] Examples of other ENaC receptor blockers for formulation and use with the compounds of the present invention include, but are not limited to, amiloride and its derivatives, such as those described in the following documents: U.S. Patent No. 6858615 and PCT Publication No. W02003/070182, W02004/073629, W02005/018644, W02006/022935, W02007/018640, and W02007/146869, all belong to Par ion Sciences, Inc_o

[0252] Small molecule ENaC blockers can directly prevent sodium transport through ENaC channel pores. ENaC blockers that can be administered in the combinations herein include, but are not limited to, amiloride, benzamil, finamir, and amiloride analogs, as exemplified in the following documents: US Patent Nos. 6, 85 &amp; 614 , U.S. Patent No. 6, 85 &amp; 615, U.S. Patent No. 6, 903, 105, U.S. Patent No. 6, 995, 160, U.S. Patent No. 7, 026, 325, U.S. Patent No. 7, 030, 117, U.S. Patent No. 7, 064, 129, U.S. Patent No. 7, 186, 833, U.S. Patent No. 7, 189, 719, U.S. Patent No. 7, 192, 958, U.S. Patent No. 7, 192, 959, U.S. Patent No. 7, 241, 766, U.S. Patent No. 7, 247, 636, U.S. Patent No. 7, 247, 637, U.S. Patent No. 7, 317, 013, U.S. Patent No. 7, 332, 496, U.S. Patent No. 7, 345, 044, U.S. Patent No. 7, 36 &amp; 447, U.S. Patent No. 7, 36 &amp; 450, U.S. Patent No. 7, 36 &amp; 451, U.S. Patent No. 7, 375, 107, U.S. Patent No. 7, 399, 766, U.S. Patent No. 7, 410, 968, U.S. Patent No. 7, 820, 678, U.S. Patent No. 7 , 842, 697, US Patent No. 7, 86 &amp; 010, US Patent No. 7, 875, 619.

[0253] ENaC proteolysis that enhances the transport of sodium through ENaC is described in detail. Protease inhibitors block the activity of endogenous airway proteases, thereby preventing ENaC cleavage and activation. The proteases that cleave ENaC include furin, meprin, matriptase, trypsin, channel-associated protease (CAP), and neutrophil elastase. Protease inhibitors that can be administered in the combination herein that can inhibit the proteolytic activity of these proteases include, but are not limited to, camostat, prostasin, furin, aprotinin, leupeptin, and trypsin Inhibitor.

[0254] The combination herein may comprise one or more suitable nucleic acids (or polynucleic acids), including but not limited to antisense oligonucleotides, siRNA, miRNA, miRNA analogs, antagomir, ribozymes, aptamers And bait oligonucleotides. See, for example, U.S. Patent Application Publication No. 20100316628. Generally speaking, such nucleic acids can range from 17 or 19 nucleotides in length up to 23, 25 or 27 nucleotides in length or longer. Examples include, but are not limited to, those described in the following documents: U.S. Patent No. 7, 517, 865 and U.S. Patent Application Nos. 20100215588, 20100316628, 20110008366 and 20110104255. Generally speaking, siRNA is 17 or 19 nucleotides as long as 23, 25 or 27 nucleotides long or longer.

[0255] CFTR activity modulating compounds that can be administered in the combination of the present invention include but are not limited to those described in the following documents: US 2009/0246137 Al, US 2009/0253736 Al, US 2010/0227888 Al, Patent No. 7,645,789, US 2009/0246820 Al, US 2009/0221597 Al, US 2010/0184739 Al, US 2010/0130547 Al, US 2010/0168094 A1 and authorized patents: 7, 553, 855; US 7, 772, 259 B2, US 7,405, 233 B2, US 2009/0203752, US 7,499,570_o

[0256] Mucus or mucin modifiers that can be used in the combinations and methods herein include reducing agents, surfactants and detergents, expectorants, and deoxyribonuclease agents.

[0257] Organize mucins into high molecular weight polymers by forming covalent (disulfide) bonds and non-covalent bonds. The use of reducing agents to break covalent bonds is a well-known method to reduce the viscoelasticity of mucus in vitro and is expected to minimize the viscosity of mucus in vivo.

And improve clearance. Reducing agents are known to reduce the viscosity of mucin in vitro and are often used to help process sputum samples . Examples of reducing agents include sulfide-containing molecules or phosphonium compounds capable of reducing protein disulfide bonds, including but not limited to N-acetylcysteine, N-acystelyn, acetostein, glutathione, disulfide Thrreitol, thioredoxin-containing protein and tris (2-ethyl) lin.

[0258] N-Acetyl Cysteine (NAC) is approved for use in combination with thoracic physical therapy to loosen viscous or thickened airway mucus. Clinical studies evaluating the role of oral or inhaled NAC in CF and COPD have reported that the rheology of mucus improves and tends to improve lung function and reduce the acute exacerbation of the lung.However, most clinical data indicate that the oral or inhaled NAC When administered by inhalation, NAC is at best a slightly effective therapeutic agent for the treatment of airway mucus obstruction. A recent Cochrane review of the existing clinical literature on the use of NAC found no evidence to support the efficacy of NAC for CF. The marginal clinical benefits of NAC are reflected in:

[0259] NAC is a relatively inefficient reducing agent, which is only partially active on the airway surface. Very high concentrations of NAC (200mM or 3.26%) are required in vitro to completely reduce Muc5B (the main gel-forming airway mucin). In addition, in the pH environment of the airway surface (the range measured in the CF and COPD airways is 6.0 to 7.2), NAC only partially exists in its reaction state as a negatively charged thiolate. Therefore, clinically, NAC is administered at a very high concentration_OHowever, it is expected that the current aerosol device cannot achieve even a therapeutic concentration of 20% Mucomyst solution on the distal airway surface in a relatively short time domain (7.5 minutes to 15 minutes) that is commonly used.

In non-clinical studies, administered by inhalation¹⁴C-labeled NAC is rapidly excreted from the lungs with a half-life of

6 to 36 minutes.

[0261] NAC is administered as a high-concentration hypertonic inhalation solution (20% or 1.22 mol/L) and has been reported to cause bronchoconstriction and cough. In many cases, it is recommended that NAC be administered with a bronchodilator to improve tolerance to the agent.

[0262] Therefore, reducing agents such as NAC are not very suitable for bolus aerosol administration. However, it is expected that the delivery of the reducing agent via lung aerosol infusion will increase the efficacy while allowing the reduction of the concentration of the reducing agent in the inhaled solution (expected to improve tolerance).

[0263] Surfactants and detergents are spreading agents, which are shown to reduce the viscoelasticity of mucus and improve the ability to clear mucus. Examples of surfactants include dipalmitoylphosphatidylcholine (DPPC), PF, palmic acid, palmitoyl-oleoylphosphatidylglycerol, surface-active proteins (for example, SP-A, SP-B or SP-C) or may be of animal origin (for example, from cow or calf lung lavage or extracted from chopped pig lung) or a combination thereof. See, for example, U.S. Patent Nos. 7, 897, 577, 5, 876, 970, 5, 614, 216, 5, 100, 806 and 4, 312, 860. Examples showing active agent products include EX0SUrf^R,Neonatal (colfosceril palmitate)&gt; Pumactant* (DPPC and egg phosphatidylcholine), KL-4 surfactant, (lusulptide, rSP-C surfactant), AlvCOfiiCt^ (bovactant), Curosurf ® (poractant alfa), Infesurf® (calfactant), New'facteii':® (modified bovine surfactant), Suring®, NatsurfTM (non-ionic alcohol ethoxylate surfactant) and Survanta® (beractant) . Examples of detergents include, but are not limited to, Tween-80 and triton-X 100.

[0264] Any suitable expectorant can be used, including but not limited to guaifenesin (see, for example, US Patent No. 7, 345, 051). Any suitable deoxyribonuclease can be used, including but not limited to Dornase Alpha. (see, for example, US Patent No. 7,482,024).

[0265] Examples of kinase inhibitors include inhibitors of NFkB, P13K (phosphatidylinositol 3-kinase), p38-MAP kinase, and Rho kinase.

[0266] Anti-infective agents for formulation and use with the compounds of the present invention include antiviral agents and antibiotics. Examples of suitable antiviral agents include Tamiflu® (oseltamivir) and Relenza® (zanamivir). Examples of suitable antibiotics include, but are not limited to: aztreonam (arginine or lysine), fosfomycin, and aminoglycosides (such as tobramycin) or any combination or subset thereof. Other anti-infective agents that can be used herein include aminoglycosines, dapatomycins, fluoroquinolones, ketolides, carbapenems, cephalosporins, erythromycin, Linezamide, penicillins, azithromycin, clindamycin, oxazinones, tetracyclines, and vancomycin.

[0267] Examples of usable carbapenem antibiotics are imipenem, panipenem, meropenem, biapenem, MK-826 (L-749,345), DA-113L·ER-35786 , Lenapenem, S-4661, CS-834 (prodrug of R-95867), KR-21056 (prodrug of KR-21012), L-084 (prodrug of LJC11036) and CXA-10L·

[0268] Antihistamines (ie, H1-receptor antagonists) used in combination with the compounds of the present invention for formulation and use include but are not limited to ethanolamines, such as diphenhydramine HC1, carbisamine maleate, Doxylamine, clemastine fumarate, diphenhydramine HC1 and dimenhydrinate; ethylene diamines, such as piramine maleate (Mepilamine), trapinamine HC1, citrate Trepinamine rafter and Antapyrin; Alkylamines, such as pheniramine, chlorpheniramine, brompheniramine, dexchlorpheniramine, triprolidine, and atorvastatin; Pyridoxines, such as methapyrine, piperazines, such as vulcanizine HC1, pyrrolizine, cyclizine HC1, cyclizine lactate, meclizine HC1 and cetirizine HC1; piperazines, such as Astemizide, levocabastine HC1, loratadine, decarbohydrate ethoxyloratadine, terfenadine and fexofenadine HC1; tricyclic and tetracyclic, such as promethazine , Chlorpromethazine, isobutyrazine and azastatine; and azastatine HC1, or any combination or subset thereof.

[0269] Examples of other types of therapeutic agents suitable for use in the combinations and methods herein include: antiviral agents such as ribavirin, antifungal agents such as amphotericin, itraconazole, and voriconazole , Anti-rejection drugs, such as cyclosporin, tacrolimus and sirolimus, bronchodilators, including but not limited to anticholinergic agents such as atrovent, siRNA, gene therapy vectors, aptamers , Endothelin receptor antagonist, α-1-antitrypsin and prostacyclin.

In the above-mentioned treatment methods and uses, the compounds of the present invention can be used alone or in combination with one or more other therapeutically active agents. Generally speaking, any therapeutically active agent that has a therapeutic effect in the disease or condition treated by the compound of the present invention can be used in combination with the compound of the present invention, as long as the specific therapeutically active agent is compatible with the treatment using the compound of the present invention . Typical therapeutically active agents suitable for combination with the compounds of the present invention include the agents described above.

[0271] In a preferred embodiment, the compound of the present invention is used in combination with one or more osmotic agents, in particular osmotic saline or mannitol.

[0272] In another aspect, the present invention provides the above-mentioned treatment methods and uses, which comprise administering an effective amount of a compound of the present invention and at least one additional therapeutically active agent. The compound of the present invention and at least one additional therapeutically active agent may be used concomitantly or sequentially in any therapeutically suitable combination. The compound of the present invention and one or more additional therapeutically active agents may be administered concomitantly, these components being present in: 1) a single pharmaceutical composition, such as the above-mentioned composition, or 2) separate pharmaceutical compositions, which Each contains one or more component active ingredients. The components of the combination can be administered separately in a sequential manner, wherein the compound of the invention is administered first, followed by the other therapeutically active agent, and vice versa.

In some embodiments where the compound of the present invention is administered in combination with one or more osmotic agents, each component is preferably administered concomitantly, and may be administered in a single composition or in separate compositions . In one embodiment, the compound of the invention and one or more osmotic agents are administered concomitantly by transbronchoscope lavage. In another implementation

In the protocol, the compound of the invention and one or more osmotic agents are administered concomitantly by inhalation.

[0274] When the compound of the present invention is used in combination with another therapeutically active agent, the dose of each compound may be different from when the compound of the present invention is used alone. Those of ordinary skill in the art can easily determine the appropriate dosage. The selection of the appropriate dosage and relative administration time of the compound of the present invention, the additional therapeutically active agent, and the relative administration time to achieve the desired combined therapeutic effect depends on the expertise and discretion of the attending physician, clinician or veterinarian.

[0275] The experimental procedure is described in detail below. The present invention also provides methods for preparing the compounds of the present invention and synthetic intermediates that can be used in these methods.

[0276] Certain abbreviations and acronyms are used in the description of synthesis methods and experimental details. However, most of them are understood by those skilled in the art, and the following table contains a list of many of these abbreviations and acronyms.

<td>[0277]</td><td>abbreviation</td>

<td>[0278]</td><td>AcOH</td>

<td>[0279]</td><td>A1BN</td>

<td>[0280]</td><td>DIAD</td>

<td>[0281]</td><td>abbreviation</td>

<td>[0282]</td><td>DIPEA</td>

<td>[0283]</td><td>DCE</td>

<td>[0284]</td><td>DCM</td>

<td>[0285]</td><td>DMF</td>

<td>[0286]</td><td>Et</td>

<td>[0287]</td><td>EtOAc or EA</td>

<td>[0288]</td><td>EtOH</td>

<td>[0289]</td><td>ESI</td>

<td>[0290]</td><td>HATU</td>

Hexafluorophosphoric acid

[0291] HPLC

[0292] iPrOH

[0293] i. t or IT

[0294] Me means azobisisobutyronitrile acetate diisopropyl ester

Ν, N-diisopropylethylamine dichloroethane dichloromethane dimethyl formamide ethyl ethyl acetate ethanol electrospray ionization

2-(1Η-7-azabenzotriazole-1-yl 3-tetramethyl gland HPLC isopropanol intratracheal methyl

[0295] MeOH

[0296] AcOH Methanol Acetic Acid

[0297] m/z or m/e

[0298] MH⁺

[0299] ΜΗ

[0300] MIC

[0301] MS or ms

[0302] Rt or rt

[0303] R_f Mass to charge ratio Mass plus 1 Mass minus 1 Minimum inhibitory concentration Mass spectrometry room temperature retention factor

[0304] t-Bu tert-butyl

[0305] THF Tetrahydrofuran

<td>[0306]</td><td>TLC or tic</td><td>TLC</td>

<td>[0307]</td><td>abbreviation</td><td>meaning</td>

<td>[0308]</td><td>δ</td><td>Parts per million relative to the low magnetic field of tetramethylsilane</td>

<td>[0309]</td><td>Cbz</td><td>Benzyl group, ie-(co) 0- benzyl</td>

<td>[0310]</td><td>AUC</td><td>Area under the curve or peak</td>

<td>[0311]</td><td>MTBE</td><td>Methyl tert-butyl</td>

<td>[0312]</td><td></td><td>keep time</td>

<td>[0313]</td><td>GC-MS</td><td>Gas chromatography-mass spectrometry</td>

<td>[0314]</td><td>wt%</td><td>Weight percentage</td>

<td>[0315]</td><td>h</td><td>hour</td>

<td>[0316]</td><td>min</td><td>minute</td>

<td>[0317]</td><td>MHz</td><td>Megahertz</td>

<td>[0318]</td><td>TFA</td><td>Trifluoroacetate</td>

<td>[0319]</td><td>UV</td><td>Ultraviolet rays</td>

<td>[0320]</td><td>Boc</td><td>Tert-Butoxyl</td>

<td>[0321]</td><td>DIAD</td><td>Diisopropyl azodicarbonate</td>

<td>[0322]</td><td>AcOH</td><td>Acetic acid</td>

<td>[0323]</td><td>DIPEA</td><td>N, N-diisopropylethylamine or Hunig base</td>

<td>[0324]</td><td>Ph₃P</td><td>Triphenylphosphonium</td>

[0325] The compound of formula I can be synthesized using techniques known in the art. A representative synthesis procedure is exemplified in Scheme 1 below.

[0326] Scheme 1

[032 knife

<img file="CN105073717A_D0022.tif" />

[0328] These methods are described in, for example, EJ Cragoe, The Synthesis of Andioride and Its Analogs (Chapter 3), Andioride and Its Analogs, pages 25-36. Other methods of preparing amiloride analogs are described in, for example, US Patent Nos. 3, 31 &amp; 813, and in particular, see methods A, B, C, and D in the '813 patent. Other preparation methods that can be used in the preparation of the compounds of the present invention are in PCT Publication Nos. W02003/07182, W02005/108644, W02005/022935, US 7, 064, 129, US 6, 85 &amp; 615, US 6, 903, 105, Described in W0 2004/073629, W0 2007/146869 and W0 2007/018640, all belong to Par ion Sciences, Inc_o

[0329] 2-3,5-Diamino-6-chloropyrazine-2-Palylmethyl methyl sulfide (methyl N*-3_? The preparation of 5-diamino6-chloropyrazine-2-carbonylcarbamimido thioate) (2) can be found in WO 2009/074575_o

Generally speaking, the compounds of the present invention can be conveniently prepared by treating the compound of formula (II) with an amine of formula (III). More specifically, the compound of formula 2 is heated to a high temperature in a suitable solvent such as methanol, ethanol or tetrahydrofuran such as

Treat with amine of formula 3 and alkali (such as triethylamine (TEA) or diisopropylethylamine (DIPEA)) at 70°C. Further purification, resolution of stereoisomers, crystallization and/or preparation of salt forms can be performed using conventional techniques.

[0331] In some cases, it is obvious to those skilled in the art that the starting compound or intermediate compound in the synthesis may have other functional groups that provide alternative reaction sites. Interference with such functional groups can be avoided by using appropriate protecting groups such as amine or alcohol protecting groups, and the synthesis steps can be prioritized appropriately when applicable. Suitable protecting groups will be obvious to those skilled in the art. Methods for setting and removing such protecting groups are well known in the art, and such conventional techniques can also be used in the method of the present invention.

[0332] The following specific examples provided herein are for illustrative purposes only, and do not limit the scope of the present invention, which is defined by the claims.

[0333] Materials and Methods. All reagents and solvents were purchased from Aldrich Chemical Company, Chem-Impex International Inc. and TCI chemical industry Co. Ltd. Use Bruker AC 400 (¾ NMR at 400MHz and¹³C NMR at 100MHz) and Bruker AC 300 (^ΧΗ NMR at 300MHz and ¹³C NMR at 75MHz) Obtain NMR spectrum. The proton spectrum refers to tetramethylsilane as the internal standard, and the carbon spectrum refers to CDC1₃&gt; CD₃0D or DMS0-d₆ (Unless otherwise specified, purchased from Aldrich or Cambridge Isotope Laboratories) Use a silica gel column (Redi Sep. Rf_? Teledyne Isco) or reversed-phase column (high efficiency C18 Gold column) Combi flash system (Comb if lash Rf _? Teledyne Isco) performs flash chromatography. ESI mass spectra were obtained with Shimadzu LCMS-2010EV mass spectrometer. Use Shimadzu Prominence HPLC system to use Waters XTerra MS C18 5 μm 4. 6X 150mm analytical column to detect at 220nm (unless otherwise specified) to obtain HPLC analysis. The flow rate is 1.0 mL/min, and the following time program is used:

[0334]

<td>Hours (minutes)</td><td>Percentage A (H₂O, with 0.05% TFA)</td><td>Percent Β (CH₃CN, with &lt;0.05% TFA)</td>

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

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

<td>30.00</td><td>IQ</td><td>90</td>

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

[0335] Waters ACQUITY UPLC HSS T3 1.8um was used with Shimadzu Prominence UFLC system

2. IX 100mm analytical column is detected at 220nm (unless otherwise specified) to obtain UPLC analysis. The flow rate is 0.3mL/min, and the following time program is used:

[0336]

<td>Time can be minutes)</td><td>Percentage A (than 0, with 0.05% NH₄COOH and 0.1% HCOOH)</td><td>Percent B (CH₃CN/ Water 80: 20%, with 0.05% NH₄COOH and O. i% HCOOH)</td>

<td>1 00</td><td>90</td><td>10</td>

<td>4 00</td><td>30</td><td>70</td>

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

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

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

[0337] 1. (S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-pyridyl) cucurbital) butyl ) Preparation of the hydrochloride salt of propionic acid (16)

[0338] Scheme 2

[0339]

one

II.CC)₂C

Boel IN

<img file="CN105073717A_D0023.tif" />

TBSi!

Miwa, THF

BocHN

<img file="CN105073717A_D0024.tif" />

()11

TfQ" noise ratio

CIi₂Cl₂

BoeHN one

<img file="CN105073717A_D0025.tif" />

OTBS

II_?C()₂C

<img file="CN105073717A_D0026.tif" />

NH

<img file="CN105073717A_D0027.tif" />

Oil'

Ρ(ΟχΟΟΗ₃)₂

1 ζ people

BocHN

TBAb

ΗΧΌΛ.

Till·

BoJIN

Pd C.

Pd C, il: MeOH

Pu(PPh.h, CuI (^Bu)₃P, Et₃N

CH₃CN

BocHN, /COM

Y:

NHfbz [1Yi_ () C

<img file="CN105073717A_D0028.tif" />

OIBS

()TBS

B(kNII

Nil·· AcOH

DIPEA, 1: ()Ii

Li()li MeOH THF-IbO

HoN ^_N NH

NH.H]

A

N SCHH

NH O

II II

NHCbz

MeO

<img file="CN105073717A_D0029.tif" />

BocHN

IK)

<img file="CN105073717A_D0030.tif" />

BocHN

NH O / Π

<img file="CN105073717A_D0031.tif" />

<img file="CN105073717A_D0032.tif" />

N HC1 aqueous solution

N

H

HN

NHr

Nil () &lt;UCJ

IkN^N

<img file="CN105073717A_D0033.tif" />

NH·,

[0340] Preparation of 4-(tert-butyldimethylsilyloxy)Cai-1-carbaldehyde (2);

[0341] Anhydrous THF (200 mL) solution of 4-onyl acetylene-1-carboxaldehyde (10.0 g, 58.1 mmol) was cooled to 0° C., and imidazole (12.0 g, 174 mmol) was added sequentially And tert-butyldimethylsilyl chloride (TBSC1) (13. lg, 87.1 mmol) _o After stirring for 16 h at room temperature, the reaction mixture was filtered and the solvent was evaporated. The residue was taken up in EtOAc (500 mL), washed with saturated aqueous N&amp;C1 (100 mL), water (100 mL) and brine (100 mL) and dried over NapSOq. The solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography (2% EtOAc/hexane) to give 2 (14.8 g, 90%) as a pale yellow solid:

[0342] ^XH NMR(300MHz, CDCI3): 6 10. 22 (s, 1H), 9. 30 (d, J = &amp; 10Hz, 1H), 8. 27 (d, J = &amp; 1Hz, 1H), 7. 86 ( d, J = 7. 8Hz, 1H), 7. 69 (ddd, J = &amp; 4, 7.0, 1.3Hz, 1H), 7. 57 (ddd, J = &amp; 4,7.0, 1. 3Hz, 1H), 6.95 (d, J = 7.5Hz, 1H), 1. 10(s, 9H), 0.36(s, 6H)

(Z)-2-(tert-butoxy dialyl)amino^_Preparation of methyl 3-[1-(tert-butyldimethylsilyloxy) Cai-4-yl]acrylate (4);

[0344] To (MeO)₂P (0) CH(NHBoc) C0₂Me,3 (23.0g, 52.7mmol) of dry CH₂C1₂ (100 mL) DBU (10.1 mL, 67.3 mmol) was added to the solution, and the mixture was stirred at 0°C for 30 minutes. Slowly add 1 (14.8g, 51.74mmol) of dry CH via a syringe₂Cl₂(60 mL) solution and the reaction mixture was heated to room temperature for 16 h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(500mL), quickly washed with saturated NH£1 aqueous solution (2X150mL) and brine (200mL) and dried over Na^SOq. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexane with 1% NEts) to give 4 (20.0 g, 85%) as a yellow solid:

[0345] ^XH NMR(300MHz, CDCI3): 6 &amp; 23(dd,J = &amp;6,2.1Hz, 1H), 7.93 (dd, J = &amp;6,2.1Hz, 1H), 7.67(s, 1H) , 7.57 (d, J = 8.4Hz, 1H), 7.53-7.47 (m, 2H), 6.85 (d, J = 7.8Hz,lH),

6. 05 (brs, 1H), 3.88 (s, 3H), 1.30 (s, 9H), 1.09 (s, 9H), 0.30 (s, 6H).

Preparation of methyl 2-(tert-butoxy dialylamino)-3-(4-(tert-butyldimethylsilyloxy)cae-1-yl)propionate (5);

[0347] A suspension of 4 (17.2 g, 37.6 mmol) and 10% Pd/C (3.40 g) in EtOH (200 mL) was degassed and placed under hydrogenation conditions (1atm, balloon) at room temperature for 16 h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to give 5 (17.0 g, 99%) as a white solid:

[0348] ^XH NMR(300MHz, CDCI3): δ 8.23 (d, J = 8. 2Hz, 1H), 7. 99 (d, J = &amp; 2Hz, 1H),

7. 57-7. 44 (m, 2H), 7. 10 (d, J = &amp; 2Hz, 1H), 6. 77 (d, J = &amp; 2Hz, 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).

Preparation of methyl 2-(tert-butoxy dialylamino)-3-(4-hydroxyl-acetylene-1-yl)propionate (6);

[0350] To a solution of 5 (17.0 g, 37.0 mmol) in anhydrous THF (200 mL) was added fluorinated tetrabutyl saddle (4 &amp; 1 mL, 4 &amp; 1 mmol) at 0°C. The resulting solution was stirred for 15 minutes and saturated NH₄C1 aqueous solution (150 mL) was quenched. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(500mL), quickly washed with saturated aqueous water (2X150mL) and brine (200mL) and dried over Na^SOq. The solvent was evaporated and the crude product was purified by silica gel flash chromatography (25% EtOAc/hexane) to give rotamer 6 (14.0 g, 94%) as a yellow solid:

[0351] ^XH NMR(300MHz, CDC13): δ 8.23 (d, J = 8. 2Hz, 1H), 7.98 (d, J = 8. 2Hz, 1H), 7. 57-7.44 (m, 2H ), 7.07 (d, J = 8. ΟΗζ, 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).

[0352] Preparation of Compound 7 and Compound 8;

[0353] Using an isocratic system IPA/heptane (7.5% with 0.4% DEA), a CHIRALPAK AD column 5 cm 1.DX50 cm L, particles 20 J were used to separate the enantiomers. &amp; 0g of racemic compound 6 was purified by the column to obtain S-isomer 8 as a white solid (3.5 g, 44% yield) and R-isomer 7 as a white solid (2.2 g, 28 % ).

[0354] Preparation of (S)-2-(tert-butoxy dialylamino)-3-[4-(trifluoromethylsulfonyloxy)cae-1-yl]propionic acid methyl ester (9);

[0355] At 0° C., to a solution of compound 8 (1.22 g, 3.53 mmol) in pyruvate (20 mL) was added triflate (0.9 mL, 5.30 mmol), and reacted The mixture was stirred at room temperature for 2h. After concentration, the reaction mixture

CH₂C1₂Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂(2X50mL) extraction. The combined organic extracts were washed with brine, dried over Na^SOq and concentrated to give compound 9 (1.51 g, 89%) as a brown oil:

[0356] ^XH NMR (400MHz, CDC1₃): δ 8.19-&amp; 07 (m, 2H), 7.69-7.64 (m, 2Η), 7.38 (d, J = &amp; 1Hz, 1H), 7. 28 (d, J = 7.9Hz, 1H), 5.12-5.06 (brs, 1H), 4.78-4.67 (m, 1H), 3.68-3.46 (m, 5H), 1.39( s,8H), 1. 25 (s, 1H).

[0357] (S) -3- {4-[4-(Benoxy dialylamino) butyl T-alkynyl] Zeayl} ~2~ (tert-butoxy dialyl amino

Preparation of methyl) propionate (11)

[0358] At room temperature, the anhydrous CH of compound 9 (1.50 g, 3.14 mmol)₃Add TEA (1.27mL, 12.6mmol) to CN (60mL) solution, 10% (t-Bu) in hexane₃Ρ (1.27 mL, 0.62 mmol), but-3- alkynyl carbamate (10,948 mg, 4.71 mmol) and Cui (30 mg, 0.16 mmol). The resulting mixture was degassed with nitrogen for 10 minutes and Pd(PPh3)4 (363 mg, 0.1%) was quickly added in one portion. After degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 16 h. The reaction mixture was concentrated under vacuum and the residue Purified by column chromatography (silica gel, 60:40 ethyl acetate/hexane) to obtain compound 11 (1.30 g, 78%) as a brown oil:

[0359] ^XH NMR(400MHz, CDC1₃): 6 &amp; 33 (dd, J = 7.5, 2.2Hz, 1H), 8.07 (dd, J = 7, 5, 2.2Hz, 1H), 7.58-7.51(m, 2H), 7.52(d, J = 7.5Hz, 1H), 7.35-7.29 (m, 5H), 7.19 (d, J = 7.5Hz, 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 = 6.2Hz,2H), 2.79 (t, J = 6.4Hz, 2H), 1.39 (s, 8H), 1. 25 (s, 1H).

Preparation of Acetate (12) of (S)-3-(4-(4-Aminobutyl) Zea-1-yl)-2-(tert-butoxy dialylamino) propionic acid methyl ester (12) ;

[0361] A suspension of 11 (1.00 g, 1.88 mmol) and 10% Pd/C (200 mg) in a mixture of MeOH (20 mL) and AcOH (2 mL) was degassed and placed under hydrogenation conditions (latm ) 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 12 (820mg, 95%) as a white solid:

[0362] ^XH NMR(300MHz, CD3OD): 6 &amp; 17-&amp; 05 (m, 2H), 7. 62-7. 48 (m, 2H), 7. 27 (brs, 2H),

4. 47 (t, J = 7.4Hz, 1H), 3.75-3.51 (m, 5H), 3. 13 (t, J = 7.5Hz,2H), 2.93 (t, J = 7 66Hz, 2H), 1.93 (s, 3H), 1.88-1.65 (m, 4H), 1.34 (s, 7H), 1. 01 (s, 2H).

(S)-2-(tert-butoxy dialylamino)-3-(4-{4-[3-(3,5-diamino-6-chloropyrazine-2-pyridine) Μ

Preparation of methyl] butyl} Cai-1-yl) propionate (14);

[0364] At room temperature, to amine salt 12 (815 mg, 1.77 mmol) and 3,5-diamino-6-chloropyrazine-2-methylmethionine methyl thioate (13,1.1g) , 2.83mmol) in EtOH (6.0mL) solution was added DIPEA (2.50mL, 14.2mmol). The reaction mixture was heated in a sealed tube at 70°C for 2h, cooled to room temperature and concentrated under vacuum. The residue was passed through column chromatography (silica gel, 80: 18: 2CHCl₃/CH₃0H/NH₄0H) Purification to obtain muscle 140mg, 80% as a yellow solid):

[0365] ^XH NMR (400MHz, CD₃0D): 6 &amp; 17-&amp; 07 (m, 2H), 7. 58-7. 48 (m, 2H), 7. 26 (q, J = 7. 4Hz, 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 . 7Hz, 2H), 2. 98 (q, J = 7.2Hz, 2H), 1. 86-1. 70 (m, 4H), 1. 33 (s, 7H), 0.98 (s, 2H) ).

(S)-2-(tert-butoxy dialylamino)-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine~2~ dialyl) Μ

(Yl) butyl) acetyl-1-yl) propionic acid (15);

[0367] To a solution of methyl ester 14 (510 mg, 0.83 mmol) in a mixture of THF (3 mL), methanol (3 mL) and water (1 mL) was added solid LiOH (120 mg, 4.99 mmol) and the reaction mixture was left at room temperature Stir for 2h. When the TLC of the reaction mixture

When the reaction was shown to be complete, the pH of the reaction mixture was restored to 9 to 10 by adding IN HC1 (aqueous solution) and the organic solvent was removed. The pH of the aqueous portion was adjusted to 5 to 6 and the resulting precipitate was extracted with dichloromethane. The aqueous portion was extracted with DCM (2×50 mL). Merge the organic layer, menstruation is wonderful. Dry, filter and concentrate to obtain compound 15 (375 mg, 76%) as a white solid:

[0368] ^XH NMR (300MHz, DMS0-d₆): δ &amp; 22-8. 02 (m, 2H), 7. 59-7. 47 (m, 2H), 7. 34-7. 22 (m, 2H), 6. 82 (brs, 2H), 4.19-4.06 (m, 1H), 3.59-3.46 (m, 1H), 3.25-3.13 (m, 2H), 3.09-2.94 (m, 10H ), 1.80-1.55 (m, 4H), 1.28 (s, 7H), 0.93 (s, 2H).

(S)-2-Amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazinepyridinyl) cucurbital) butyl) Chua-1- The preparation of the HCl salt (16) of yl) propionic acid;

[0370] 4N HC1 (8.0 mL) in dioxane was added to 15 (258 mg, 0.43 mmol), followed by water (4.0 mL) and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed and the residue was lyophilized to give compound 16 (250 mg, 99%) as a yellow solid:

[0371] P NMR (400MHz, DMS0-d₆): δ 10. 54 (brs, 1H), 9.33 (t, J = 5.92Hz,lH),

9. 03-&amp; 80(m,2H) ,8.60(brs,3H) ,8.17(ddd, J = 10. 1,7. 6, 4.5Hz, 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. 5Hz, 2H), 4. 13 -4.02 (m, 1H),

3. 75-3.44(m,3H), 3.43-3.33 (m, 2H), 3.09 (t, J = 6.4Hz,2H), 1.81-1.62(m,4H ).

2. (S)-3,5-Diamino-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyl) (Amino)-3-oxopropyl)Cai-1-yl)butyl)methanyl)-6-chloropyrazine-2-carboxamide (23) Preparation [0373] Scheme 3

[0374]

<img file="CN105073717A_D0034.tif" />

MeOH Till·/Π.Ο

NaOH

<img file="CN105073717A_D0035.tif" />

<img file="CN105073717A_D0036.tif" />

<img file="CN105073717A_D0037.tif" />

<img file="CN105073717A_D0038.tif" />

<img file="CN105073717A_D0039.tif" />

<img file="CN105073717A_D0040.tif" />

Ο

Π Γ.

<img file="CN105073717A_D0041.tif" />

Ci

Nil·

Ν lie 1 aqueous solution, dioxane

<img file="CN105073717A_D0042.tif" />

[0375] (S) -3-{4-[4-(Benoxy dialylamino) butyl T-alkynyl] Chua~1~ group} ~2~ (tert-butoxy dialyl amino

Preparation of methyl) propionate (17);

[0376] To a solution of methyl ester 11 (1.71 g, 3.22 mmol) in a mixture of THF (21 mL), methanol (21 mL) and water (7.0 mL) was added solid NaOH (1.29 g, 32.3 mmol) and the reaction mixture was stirred at room temperature for 3 h. When the TLC of the reaction mixture showed that the reaction was complete, the pH of the reaction mixture was restored to 9 to 10 by adding IN HC1 (aqueous solution) and the organic solvent was removed. The pH of the aqueous portion was adjusted to 5 to 6 and the resulting precipitate was extracted with dichloromethane. CH for water-based part₂Cl₂(2X50mL) extraction. Combine the organic layers, menstrual blood claws. . Dry, filter and concentrate to obtain compound 17 (1.55 g, 93%) as a brown solid:

[0377] P NMR (400MHz, DMS0-d₆): Δ 8. 32 (d, J = 7. 4Hz, 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, 1Η), 3.57-3.35 (m, 2H), 2.84-2.68 (m, 2H), 1. 37 (s, 9H).

Preparation of Compound 19;

To the THF (2.5mL) containing compound 18 (100mg, 0.56mmol) was added DEPBT (218mg, 0.72mmol), 17 (289mg, 0.56mmol) and DIPEA (0.3mL, 1. 68mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100 mL), quickly washed with saturated aqueous NaHCA (2 X 50 mL) and brine (50 mL) and dried over N^SOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol/CH₂C1₂) Was purified to obtain amide 19 (250 mg, 66%) as a yellow solid:

[0380] ^XH NMR (400MHz, CDC13): δ &amp; 34 (dd, J = &amp; 3,1.4Hz, 1H), 8.21 (d, J = &amp; 3Hz, 1H), 7.61-7.47 (m, 4H) ,7.39-7.27 (m, 5H), 7.16(d, J = &amp; 3Hz,2H) ,7.05(d, J = &amp; 3Hz,2H),

5. 36-5. 19 (m, 2H), 5. 12 (s, 2H), 4. 36-4. 53 (m, 1H), 3. 66-3. 42 (m, 4H), 2. 79 ( t, J = 6. 6Hz, 2H), 2. 57 (t, J = 7.5 Hz, 2H), 2. 40 (t, J = 7.5 Hz, 2H), 2. 32 (s, 6H), 1. 86T. 75 (m, 2H),

1. 39(s,9H).

[0381] Preparation of Compound 20;

The suspension of 19 (210mg, 0.1% and 10% Pd/C (150mg) in a mixture of MeOH (3.0mL) and AcOH (0.3mL) was degassed and placed under hydrogenation conditions at room temperature ( latm) 12h_oThe reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 22, which was neutralized with triethylamine and the crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain the free amine 20 (130 mg, 77% ):

[0383] ^XH NMR(300MHz, CD₃0D): 6 &amp; 24(dd,J = &amp; 1,2. 1Hz, 1H), 8. 08 (dd, J = &amp; 2,1.5Hz, 1H), 7. 58-7.47 (m, 2H) , 7. 33-7. 20 (m, 4H), 7. 07-7. 05 (m, 2H), 4. 53 (t, J = 7. 2Hz, 1H),

3. 66-3. 55 (m, 2H), 3. 09 (t, J = 7.5Hz, 2H), 2.82 (t, J = 7.4Hz, 2H), 2. 57 (t, J = 7 . 2Hz, 2H), 2. 35 (dd, J = 10. 5, 7. 5Hz, 2H), 2. 24 (s, 6H), 1. 84T. 61 (m, 6H), 1. 36 (s , 7H), 1. 10 (s, 2H).

[0384] Preparation of 22;

[0385] At room temperature, to amine 20 (122 mg, 0.22 mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,139 mg, 0.35 mmol) ) In EtOH (4.0 mL) solution was added DIPEA (0.3I mL, 1.76 mmol). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was passed through column chromatography (silica gel, 80: 18: 2CHCl₃/CH₃0H/NH₄OH) was purified to obtain M 22 (111 mg, 66%) as a yellow solid: ^XH NMR(400MHz, CD3OD): δ &amp; 23(dd, J = 7.5, 2.4Hz, 1H), &amp; 10 (d, J = &amp; 1Hz, 1H), 7. 57-7.48(m, 2H), 7.29 (d, J = 7.3Hz, 2H), 7. 24 (d, J = 8. ΟΗζ, 2H), 7. 13-7.05 (m, 2H),

4. 53(t, J = 8. ΟΗζ, 1H), 3.60-3.37(m, 2H), 3.23(t, J = 7.3Hz, 2H), 3.15-3.03 (m , 2H),

2. 55(t, J = 7.3Hz, 2H), 2. 29(dd. J = 9. 7, 7. 6Hz, 2H), 2. 21 (s, 6H), 1. 86T. 64 (m, 6H ),

1. 36(s,7H), 1.12(s,2H).

Compound 23(S)-3,5-diaminos-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyl (Amino)-3-oxopropyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide HC1 salt preparation

[0388] 4N HC1 (3.0 mL) in dioxane 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 h. The solvent was removed and neutralized with IN NaOH (aqueous solution), the resulting solid was washed with water and treated with IN HC1 (aqueous solution) again, the water was removed and the residue was lyophilized to obtain as a yellow solid

Compound 22 (65mg, 65%):

[0389] P NMR (400MHz, DMS0-d₆) 10. 50 (s, 1H), 10. 48 (s, 1H), 10. 46-10. 40 (m, 1H), 9. 26 (t, J = 4.9Hz, 1H), 9. 01 -&amp; 74 (m, 2H), 8. 61 (brs, 1H), 8. 35 (dd, J = 6.6,3.4Hz,lH), &amp; 13 (dd, J = 6.5,3.3Hz, 1H), 7.58(ddd, J = 9. 9, 6. 6, 3.6Hz, 2H), 7.42 (brs, 1H), 7.40 (d, J = 7.3Hz, 2H), 7. 34 (d, J = 7.3Hz, 1H), 7. 28 (d, J = 7.3Hz, 1H), 7. 16 (d, J = &amp; 6Hz, 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 = &amp; 1Hz, 2H), 1.97-1.88 (m, 2H), 1.79-1.61 (m, 4H).

3. 3, 5-Diamino (N~ (4- (4- ((S) ~2~ Amino-3-(4-(3-(hexyl ((2S, 3R, 4R, 5R)- 2,

3,4,5,6-Pentaylhexyl)amino)propyl)phenylamino)-3-oxopropyl) Cai-1-yl)butyl)methan

Yl)-6-chloropyrazine-2-carboxamide (28) Preparation Scheme 4

[0391]

[0392]

<img file="CN105073717A_D0043.tif" />

<img file="CN105073717A_D0044.tif" />

<img file="CN105073717A_D0045.tif" />

[0393] Preparation of Compound 25;

[0394] To the THF (10 mL) containing compound 24 (165 mg, 0.38 mmol) was sequentially added DEPBT (148 mg, 0.48 mmol), 17 (200 mg, 0.38 mmol) and DIPEA (0.2 mL, 1.14 mmol) ) And stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100 mL), quickly washed with saturated NaHCC^ aqueous solution (2 X 50 mL) and brine (50 mL) and dried over N^SOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol

/CH₂C1₂) Was purified to obtain amide 25 (210 mg, 60%) as a yellow solid:

[0395] ^XH NMR(300MHz, CDC1₃): δ 8.35 (d, J = &amp; 2Hz,lH), &amp; 21 (d, J = &amp; 3Ηζ,1Η), 7. 63-7.52 (m, 2H), 7. 51 (d, J = 7.3Hz, 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, 2H), 4. 68 (ddd, J = 11. 3, 10. 3, 5.1 Hz, 1H), 4. 56 (q, J = 7.2Hz, 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 = 6.7Hz, 2H), 2. 70-2.35 (m, 8H ), 1.81T.67(m,2H),1.63T.53(m, 1H),1.35T.20 (m,6H), 1.21 (s,9H), 1.31 (d, J = 5.1Hz, 3H ), 0.87 (t, J = 6.2Hz, 3H).

Preparation of Compound 26;

A suspension of a mixture of 25 (280 mg, 0.30 mmol) and 10% Pd/C (560 mg) in EtOH (9.0 mL) and AcOH (1.0 mL) was degassed and hydrogenated at room temperature Condition (latm) 4h_oThe reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 22, which was used NaHC.₃Neutralized, and the crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain free amine 26 (160 mg, 67%) as a yellow solid:

[0398] ^XH NMR(400MHz, CD₃0D): δ 8.29 (d, J = &amp; 2Hz,lH), &amp;07(d, J = &amp; 6Hz,lH), 7.60(t, J = 6.9Hz, 1H), 7.55(ddd , J = &amp; 2, 6. 9, 1. 1Hz, 1H), 7. 24 (d, J = 7.1Hz, 1H), 7. 18 (d, J = 7.1Hz, 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 = 5.1Hz, 1H) ,4.61-4.53 (m, 1H), 4.12 (dd, J = 10. 8 ,5.4Hz, 1H),3.89-3.80(m,2H),3.74(t, J = 3.3Hz, 2H), 3.46 (d, J = 3.8Hz, 1H) , 3. 39 (t, J =

10. 7Hz, 2H), 3. 18-3.09 (m, 1H), 3. 02-2.92 (m, 1H), 2. 68 (t, J = 7. 1Ηζ, 2Η), 2. 61- 2.47 (m, 5H), 2.46-2.37(m,4H), 1.77-1.63(m,4H), 1.33 (d, J = 5.1Ηζ,3Η), 1. 31-1. 20(m,8H),

1. 21 (s, 9H), 0.88 (t, J = 6.7 Hz, 3H).

Preparation of Compound 27;

[0400] At room temperature, to amine 26 (155 mg, 0.20 mmol) and 3,5-diamino-6-chloropyrazine-2-methylpyridine methyl thioate (21,123 mg, 0.31 mmol) DIPEA (0.28 mL, 1.56 mmol) was added to the EtOH (8.0 mL) solution. The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was subjected to silica gel column chromatography (80: 18: 2 CHCl₃/CH₃0H/NH₄OH) Purification followed by reverse phase chromatography (Gold C18) to obtain muscle 27 (100 mg, 51%) as a yellow solid:

[0401] ^XH NMR(400MHz, CD₃0D) :6 &amp; 23 (dd, J = &amp; 8, 2. 5Hz, 1H), 8. 10 (d, J = &amp; 2Hz, 1H), 7. 56-7. 49(m, 2H), 7. 29 (d, J = 7.9Hz,2H), 7.24 (d, J = 7.4Hz,2H), 7.08 (d, J =7.9Hz,2H), 4.67(q, J = 5. 1Hz, 1H), 4. 56-4. 50 (m, 1H), 4. 04 (dd, J = 10. 8, 5. 4Hz, 1H), 3. 92-3. 86 (m, 1H), 3. 82-3. 74 (m, 2H), 3. 51-3. 46 (m, 1H), 3. 25 (t, J = 7.1Hz, 2H),

3. 15-3.06(m,2H), 2.71 (dd, J = 13. 2,5.2Hz, 1H), 2.60-2.45(m,6H), 1.87-1.63 (m,6H), 1.48T.40(m,6H),1.33T.26(m,6H),1.23(d, J = 5.1Hz, 3H), 1.20 (s, 9H), 0. 89 (t, J =

6. 7Hz, 3H).

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-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methyl)- Preparation of HC1 salt of 6-chloropyrazine-2-carboxamide

[0403] 4N HC1 (3.0 mL) in water was added to 27 (80 mg, 0.08 mmol) of ethanol (0.5 mL) and heated at 40° C.

The reaction mixture was stirred for 6 h. The solvent was removed, additional 4N HC1 was added and the mixture was heated at 40°C for another 4h. The solvent was removed, water was added and the residue was lyophilized to give compound 28 (78 mg, 99%) as a yellow solid:

[0404] ^XH NMR(400MHz, DMS0-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) ,7.46-7.37(m,4H) ,7.34(d, J = 7.1Hz, 1H), 7.27 (d, J = 7.1Hz, 1H) , 7. 17 (d, J = &amp; 1Hz, 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 = 6.6Hz, 3H).

[0405] ^XH NMR(400MHz, CD₃0D): δ 8.23 (d, J = &amp; 3Hz,lH), &amp;17(d, J = &amp; 2Hz,lH),

7. 62-7.53 (m, 2H), 7.41-7.36 (m, 1H), 7.35-7.32 (m, 1H), 7.31-7.25 (m, 2H), 7. 21-7. 12 (m,

2H), 4.35-4.25 (m, 1H), 4.17-4.02 (m, 1H), 3.86-3.75 (m, 2H), 3.73-3.59 ( m, 6H),

3. 23-3.08(m,9H), 2.73-2.60(m,2H), 2.11-1.97(m,2H), 1.91-1.75(m,4H), 1. 74-1. 62 (m,

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

4. 3,5-Diamino ^_N~ (N~ (4- (4- ((S) ~2~ Amino-3-(4-(3-(bis((2S, 3R, 4R, 5R)-2,

3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)choen-1-yl)butyl)methionyl)-6-chloropyrazine- Preparation of 2-formamide (33)

[0407] Scheme 5

[0408]

<img file="CN105073717A_D0046.tif" />

<img file="CN105073717A_D0047.tif" />

addiction

<img file="CN105073717A_D0048.tif" />

ΗΟ"

HO (V)

<img file="CN105073717A_D0049.tif" />

Ν

NH Ο

JL PeoplePep^δ Λ X

Η,Ν Ν NIL

<img file="CN105073717A_D0050.tif" />

.λΟΉ pieces, ΌΗ ^(R) ΟΗ battle)ι ("ΟΗ

ΗΟ'

<img file="CN105073717A_D0051.tif" />

ΗΟ

N HC1 aqueous solution, EtOH δ

<img file="CN105073717A_D0052.tif" />

NH Ο

Η g

<img file="CN105073717A_D0053.tif" />

Preparation of Compound 30:

[0410] To the THF (8.0 mL) containing compound 29 (290 mg, 0.54 mmol) was added DEPBT (210 mg, 0.70 mmol), 17 (311 mg, 0.60 mmol) and DIPEA (0.28 mL, 1. 62mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100 mL), quickly washed with saturated NaHCC^ aqueous solution (2 X 50 mL) and brine (50 mL), and dried over Na^SOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol/CH₂C1₂) Purification to obtain amide 30 (400 mg, 72%) as a yellow solid:

[0411] ^XH NMR (400MHz, CDC1₃): δ &amp; 36-8. 26 (m, 1H), &amp; 20-8. 09 (m, 1H), &amp; 03-7.85 (m, 1H), 7. 61-7.46 (m, 1H ), 7.49 (d, J = 7.2Hz,2H), 7.38-7.28 (m, 5H), 7.18-6.96 (m, 4H),

5. 51-5. 36 (m, 1H), 5. 32-5. 21 (m, 1H), 5. 12 (s, 2H), 4. 67 (q, J = 5.1 Hz, 2H), 4. 66-4.53 (m, 1H), 4.11 (dd, J = 10.4,5.2Hz,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. 4Hz, 2H), 3. 05 (q, J = 7.6Hz, 2H), 2.96-2.88 (m, 2H), 2.79 (t, J = 6. lHz, 2H), 2.64-2.61 (m,4H), 1.93-1.72(m,4H), 1.48T.40(m,2H),1.35(s,9H), 1.29(d, J = 5.lHz, 6H).

[0412] Preparation of Compound 31;

[0413] Mix 30 (400 mg, 0.39 mmol) and 10% Pd/C (210 mg) in EtOH (54 mL) and AcOH (6.0 mL)

The suspension in the product was degassed and placed under hydrogenation conditions (latm) for 4 h at room temperature. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 31 (333 mg, 84%) as a yellow solid: [0414]^XH NMR(400MHz, CD₃0D): δ &amp; 25(dd, J = 7.5,2.5Hz, 1H) ,&amp; 10 (d, J = 7.3Hz, 1H), 7.60-7.51(m,2H),7 . 36-7. 32(m, 1H), 7. 31(d, J = 7.2Hz, 2H), 7. 26 (d, J = 7.8Hz,lH), 7. 15 (d, J = 7.8Hz, 2H), 4.70 (q, J = 4.9Hz, 2H), 4.54 (d, J = 7.3Hz, 1H), 4.18-4. 10 (m, 2H), 4. 06 (dd, J = 10. 6, 5. 3Hz, 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. 153. 08 (m, 2H), 2.92 (t, J = &amp; ΟΗζ, 2H), 2. 09-2. 00 (m, 4H), 2. 77-2.58 (m, 2H), 1. 95 (s, 6H), 1.88-1.60 (m, 4H), 1.36 (s, 9H), 1.25 (d, J = 4.9Hz,6H).

[0415] Preparation of 32;

[0416] At room temperature, add 31 (370 mg, 0.36 mmol) and 3,5-diamino-6-chloropyrazine-2-pyrimylmethyl thiomethionate (21,226 mg, 0.58 mmol) DIPEA (0.51mL, 2.88mmol) was added to the EtOH (12mL) solution. The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was passed through silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) purification to obtain muscle 32 (250mg, 63%) as a yellow solid: [0417] ^XH NMR(400MHz, CD3OD): 6 8. 23 (d, J = 8.6Hz, 1H), 8. 13-8. 03 (m, 1H),

7. 54-7.49 (m, 2H), 7. 30-7.20 (m, 4H), 7. 13-7.04 (m, 2H), 4.67 (q, J = 4.9Hz, 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 = 4.9Hz,6H).

[0418] 33-3, 5-diamino (N~ (4~ (4~ ((S) ~2~ amino-3- (4- (3-(bis((2S, 3R, 4R, 5R) ~ 2,

3,4,5,6-Pentylhexyl) amino) propyl) phenylamino) -3-oxopropyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine- Preparation of HC1 salt of 2-formamide

[0419] 4N HC1 (6.0 mL) in water was added to 32 (200 mg, 0.18 mmol) in ethanol (2.0 mL) and the reaction mixture was stirred at 40° C. for 8 h. The solvent was removed, additional 4N HC1 was added and the mixture was heated at 40°C for another 6h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to give compound 33 (138 mg, 59%) as a yellow solid:

[0420] ^XH NMR(400MHz, DMS0-d₆): δ 10. 48 (brs, 1Η), 10. 45-10.41 (m, 1H), 9.25-9.19 (m, 1H), 8.95-8.85 (m, 1H) , 8.81-8.69 (m, 1H), 8.64-8.46 (m, 4H), 8.36-8.29 (m, 1H),

8. 18-8. 10 (m, 1H), 7.62-7.55 (m, 2H), 7.46-7.38 (m, 4H), 7.34 (d, J = 7.5Hz, 1H ), 7.28 (d, J = 7.3Hz, 1H), 7.18(d, J = 8.7Hz, 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 (m, 4H).

[0421] ^XH NMR(400MHz, CD3OD): 6 &amp; 24-&amp; 20 (m, 1H), 8. 18-&amp; 15 (m, 1H), 7. 57 (td, J =

4. 6,1. 5Hz, 2H), 7.38 (d, J = 7.4Hz, 1H), 7. 33 (d, J = 7.4Hz, 1H), 7. 28 (dd, J = &amp; 4,

2. 6Hz,2H), 7.16(dd, J = &amp; 4, 2. ΟΗζ, 2H), 4.28 (t, J = 6.9Hz,lH), 4.19-4.13 (m, 1H) , 4. 12-4. 07 (m, 1H), 3. 85-3. 79 (m, 2H), 3. 77 (dd, J = 10. 4, 5.2 Hz, 2H), 3. 73- 3. 60 (m, 6H),

3. 49-3. 45 (m, 2H), 3. 42-3. 34 (m, 6H), 3. 26-3. 23 (m, 1H), 3. 19-3. 13 (m, 2H), 3.14-3.11 (m, 1H), 2.74-2.59 (m, 2H), 2.14-2.00 (m, 2H), 1.90-1.72 (m, 4H ).

5. 3,5-Diamino-N-(N-(4-(4-(4-((S)-2-amino-3-oxo-3-(4-(3-((2S, 3R , 4R,

5R)-2,3,4,5,6-Pentaylhexylamino)propyl)phenylamino)propyl) Cai-1-yl)butyl)methyl)-6-chloropyrazine-2 -Preparation of formamide (38)

[0423] Scheme 6

[0424]

BocN sugar

<img file="CN105073717A_D0054.tif" />

<img file="CN105073717A_D0055.tif" />

<img file="CN105073717A_D0056.tif" />

OH 0H

<img file="CN105073717A_D0057.tif" />

N UC I aqueous solution, I'1( )11

<img file="CN105073717A_D0058.tif" />

Preparation of Compound 35

[0426] To contain compound 34 (400mg, 0.

THF (15mL) was added DEPBT (389mg,

1. 30mmol), 17 (516mg, 1.OOmmol) and DIPEA (0.52mL, 3.OOmmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100 mL), washed quickly with a saturated aqueous NaHCC solution (2×50 mL) and brine (50 mL) and dried over NaHSOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol/CH₂C1₂) Purification to obtain amide 35 (700 mg, 83%) as a yellow solid:

[0427] ^XH NMR(400MHz, CDC1₃): 6 &amp; 35 (dd, J = &amp; 2,1.5Hz,lH), &amp; 22(d, J = &amp; 1Hz,

1H), 7.64-7.35(m,4H), 7.38-7.26 (m, 5H), 7.06 (d, J = 7.8Hz,2H), 7.17-7. 09(m,2H),

5. 21-5. 13(m,2H), 5.12(s,2H), 4.69 (q, J = 5.1Hz, 1H), 4.55 (q, J = 7.25Hz, 1H),

4. 15 (dd, J = 11.4,5,6Hz, 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, 5Η), 3.41 (d, J = 10.6Hz,2H), 3.37-3. 30 (m, 2H),

3. 29-3.20(m,3H), 2.80 (t, J = 6.2Hz,2H), 2.52 (t, J = 7.8Hz,2H), 1.90-1.76 (m , 3H), 1, 42 (s, 18H), 1. 32 (d, J = 5.2Hz, 3H).

[0428] Preparation of Compound 36;

The suspension of 35 (700 mg, 0.74 mmol) and 10% Pd/C (400 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and placed under hydrogenation conditions (1 atm) at room temperature 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 36 (650 mg, 95%) as a yellow solid: [0430]^XH NMR(400MHz, CDC1₃): δ 8.20 (d, J = &amp; 4Hz, 1H), 7.96 (d, J = 7.3Hz,lH), 7. 86-7.71 (m, 1H), 7. 70-7 . 63 (m, 1H), 7. 58-7. 43 (m, 2H), 7. 36-7. 26 (m, 2H), 7. 02-6. 91 (m, 2H), 4. 70 -4. 63 (m, 1H), 4. 61-4. 54 (m, 1H), 4. 20-4. 05 (m, 2H), 4. 04-3. 90 (m, 1H), 3 . 89-3. 68 (m, 3H), 3. 67-3. 46 (m, 3H), 3. 45-3. 27 (m, 5H), 3. 29-3. 21 (m, 4H) , 3. 11-2. 91 (m, 4H), 2. 90-2.76 (m, 2H), 2. 48 (d, J = 7.3Hz, 2H), 2. 08 (s, 6H) , 1.86-1.61 (m, 6H), 1.41 (s, 15H), 1.32 (d, J = 5.1Ηζ, 3Η), 1.25 (s, 3H).

[0431] Preparation of 37;

[0432] At room temperature, add 36 (650 mg, 0.70 mmol) and 3,5-diamino-6-chloropyrazine to methyl 2-pyridylmethyl thioate (21,436 mg, 1.13 mmol) DIPEA (0.90 mL, 5.60 mmol) was added to the EtOH (12 mL) solution. The reaction mixture was heated in a sealed tube at 70°C for 2h, cooled to room temperature and concentrated under vacuum. The residue was passed through silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) purified to obtain muscle 37 (444mg, 62%) as a yellow solid:

[0433] ^XH NMR(400MHz, CD₃0D): 6 &amp; 23 (dd, J = 7. 7, 2. 2Hz, 1H), &amp; 10 (d, J = &amp; 1Hz, 1H), 7.57-7.47(m,2H), 7. 33-7. 21 (m, 4H), 7. 08 (d, J = 8.1 Hz, 2H), 4. 68 (q, J = 5.0 Hz, 1H), 4. 53 (t, J = 7 . 2Hz, 1H), 4. 04 (dd, J = 10. 8, 5. 4Hz, 1H), 4. 03-3.93 (m, 1H), 3. 25 (ddd, J = 10. 3, 9. 2,5.2Hz, Η), 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 .2Hz,2H), 1.89T.76(m,4H),1.76T.64(m,2H),1.36(s,6H),1.42(s,9H),1.25(d, J = 5. 0Hz,3H), 1.ll(s,3H).

[0434] Preparation of the HC1 salt of 38;

[0435] 4N HC1 (6.0 mL) in water was added to 37 (240 mg, 0.23 mmol) in ethanol (3.0 mL) and the reaction mixture was stirred at 40° C. for 8 h. The solvent was removed, additional 4N HC1 was added and the mixture was heated at 40°C for another 8h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to give compound 38 (251 mg, 64%) as a yellow solid:

[0436] P NMR (400MHz, DMSO-cQ: 6 10. 50 (brs, 1H), 9.28 (t, J = 5.7 Hz, 1H),

9. 02-&amp; 87 (m, 1H), &amp; 86-&amp; 75 (m, 1H), &amp; 72-&amp; 55 (m, 4H), &amp; 39-&amp; 33 (m, 1H), &amp; 16-&amp; 10 (m , 1H), 7.61-7.55(m,2H), 7.45-7.40(m, 1H), 7.40(d, J = 7.4Hz, 2H), 7.34 (d , J = 7.3Hz, 1H), 7.28 (d, J = 7.4Hz, 1H), 7. 14 (d, J = &amp; 5Hz, 2H), 5.38 (d, J = 4.3Hz , 1H), 4.74 (d, J =4.9Hz, 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. 6Hz, 2H), 1.96-1.85 (m, 2H), 1.79-1.61 (m, 4H).

[0437] ^XH NMR (400MHz, CD₃0D): δ &amp; 26-8. 20 (m, 1H), &amp; 19-&amp; 14 (m, 1H), 7. 60-7. 53 (m, 2H), 7.38 (d. J = 7. 2Hz, 1H), 7.33(d, J = 8.4Hz, 1H), 7.28 (dd, J = &amp; 4,2.0Hz,2H),

7. 14 (d, J = 8.4Ηζ, 2Η), 4.29 (t, J = 8.3Hz, 1Η), 4.07-4.00 (m, 1Η), 3.83 (dd, J =

9. 8,1.5Hz, 1Η), 3.77 (dd, J = 9. 8,2.6Hz, 1Η), 3.73-3.64 (m, 5Η), 3.37 (t, J = 7 . 2Hz, 2Η), 3.21-3.11 (m, 4H), 3. 06-2.96 (m, 2Η), 2.66 (t, J = 7.7Hz, 2H), 2.03 -1.94(m,2H), 1.90-1.75(m,4H).

6.-3,5-Diamino-N-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylamino) Preparation of -3-oxopropyl) acetyl-1-yl) butyl) methionyl)-6-chloropyrazine-2-carboxamide (43) [0439] Scheme 7

[0440]

<img file="CN105073717A_D0059.tif" />

Pd/C. Η,

CH., Yan

-"C

3.

Η.

<img file="CN105073717A_D0060.tif" />

Η, ct-n

<img file="CN105073717A_D0061.tif" />

ΝΗ₂·2ΑοΟΗ

<img file="CN105073717A_D0062.tif" />

ο

Λ Ν Η

Ο

<img file="CN105073717A_D0063.tif" />

[0441] Preparation of Compound 40;

[0442] In an ice bath, a solution of acid 17 (880 mg, 1.70 mmol) in THF (30 mL) was cooled to 0 °C. NMM (0.37 mL, 3.40 mmol) was added, followed by PivCl (0.20 mL, 1.70 mmol), and the reaction mixture was stirred at the same temperature for 2 h. 39 (375 mg, 1.70 mmol, 15 mL THF) was added and the reaction mixture was stirred at the same temperature for another 10 minutes. The reaction mixture was returned to room temperature and stirred for 16 h. Remove the organic solvent. Add water to the residue and use CH₂Cl₂(3X100mL) extraction. The organic layers were combined, dried over NagSOq, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to obtain amide 40 (719 mg, 59%) as a pale yellow solid: [M+H]⁺720_o

Preparation of Compound 41;

The suspension of 40 (719mg, 1.OOmmol) and 10% Pd/C (300mg) in a mixture of EtOH (110mL) and AcOH (20mL) was degassed and placed under hydrogenation conditions (latm) at room temperature for 16h . The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to give amine salt 41 (660 mg, 93%) as a yellow solid: [M+H]⁺589_O

[0445] Preparation of Compound 42;

At room temperature, to amine 41 (660mg, 0.93mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,650mg, 1.67mmol) DIPEA (1.66 mL, 9.3 mmol) was added to the EtOH (10 mL) solution. The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was subjected to silica gel column chromatography (80: 18: 2 CHCl₃/CH₃0H/NH₄0H) Purification to obtain muscle 42 (0 mg, 50%) as a yellow solid: [M+H] ⁺801

Compound 43(S)-3,5-diamino-(N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylamino )-3-oxopropyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide HC1 salt preparation

[0448] Add TFA (10 mL) to CH₂Cl₂(10 mL) in 42 (370 mg, 0.46 mmol) and the reaction mixture was stirred at room temperature for 2 h. Remove the solvent and add IN HC1 to remove the solvent. The mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to give compound 43 (290 mg, 92%) as a yellow solid:

[0449] ^XH NMR(400MHz, DMS0-d₆): δ 10. 39 (brs, 2Η), 9. 25 (brs, 1H), 9.02-8.87 (m, 1H), 8.86-8.73 (m, 2H), 8.71 -&amp; 44(m,2H) ,8.35 (brs, 1H) ,&amp; 13 (dd, J = 6. 8, 3. 8Hz. 1H), 7. 58 (dd, J = 6. 5, 3. 2Hz, 2H), 7. 42 (brs, 2H), 7. 35 (d, J = &amp; 6Hz, 2H), 7. 33 (d, J = 7. 8Hz, 1H), 7. 27 (d, J = 7. 3Hz, 1H), 7. 11 (d, J = &amp; 4Hz, 2H), 4. 26-4. 18 (m, 1H), 3. 65-3. 48 (m, 2H), 3. 39-3. 32 (m, 3H), 3. 06 (t, J = 6. 5Hz, 2H), 2. 99-2.91 (m, 2H), 2. 69 (s, 6H), 1. 77-1.56 (m, 6H), 1.52 (t, J = 8.2Hz, 2H), 1.34-1.21 (m, 4H).

[0450] ^XH NMR (400MHz, CD₃0D): δ &amp; 22-8. 17 (m, 1H), &amp; 16-8. 12 (m, 1H), 7. 58-7. 51 (m, 2H), 7. 36 (d, J = 7 . 2Hz, 1H), 7. 30 (d, J = 7.4Hz, 1H), 7. 19 (d, J = &amp; 04Hz,2H), 7. 05 (d, J =&amp; 3Hz,2H) ,4 . 24 (t, J = &amp; 2Hz, 1H), 3. 70-3. 58 (m, 2H), 3. 33 (t, J = 6.9Hz, 2H), 3. 14 (t, J = 7 . 4Hz,2H) ,3.09-3.03 (m, 2H), 2.84(s,6H) ,2.53(t, J = &amp; 6Hz,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. 3,5-Diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(bis((2S, 3R, 4R, 5R)-2,

3,4,5,6-Pentylhexyl)amino)hexyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methionyl)-6-chloropyrazine-2 -Formamide preparation

[0452] Scheme 8

[0453]

<img file="CN105073717A_D0064.tif" />

Pd C. hl· LlOt 1. AcOI ί

<img file="CN105073717A_D0065.tif" />

<img file="CN105073717A_D0066.tif" />

\II₂*2Ac()1I

[0454]

<img file="CN105073717A_D0067.tif" />

OH

<img file="CN105073717A_D0068.tif" />

5)

HO's

11(/'

OH ^iR}_λΟΗ

<img file="CN105073717A_D0069.tif" />

Ν ϊ_| _

Boel l\ aqueous solution,

<img file="CN105073717A_D0070.tif" />

HO

NH O n^An^Y^NV^{C1 HH} Λ Λ

HN \ \11_?

LtOH

<img file="CN105073717A_D0071.tif" />

Μ Η O *3HC1 Ba Kuang Phu Ya N Cl Obituaries n^nh₂

[0455]

[0456] A solution of THF (40 mL) was cooled to 0°C. Add to

Η(", ⁽^-ΌΗ

R) η Preparation of compound 45; In an ice bath, the acid 17 (900mg, 1.74mmol)

NMM (0.38 mL, 3.48 mmol), then PivCl (0.21 mL, 1.74 mmol) was added, and the reaction mixture was stirred at the same temperature for 2 h. Add 44 (1.21g, 1.74mmol, 20mL THF) and stir the reaction mixture at the same temperature.

10 minutes away. The reaction mixture was returned to room temperature and stirred for 16 h. Remove the organic solvent. Add water to the residue and use CH₂Cl₂(3X100mL) Wash. The organic layers were combined, dried over NagSOq, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to give amide 45 (2.00 g, impure) as a pale yellow solid: [M+H]⁺[0457] Preparation of Compound 46;

[0458] A suspension of 45 (2.00 g, impure) and 10% Pd/C (400 mg) in a mixture of EtOH (120 mL) and AcOH (20 mL) was degassed and placed under hydrogenation conditions (latm ) 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 46, which was used NaHC.₃Neutralized, and the crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain free amine 46 (500 mg, 27% after two steps) as a yellow solid: (M+H)⁺1067o

Preparation of Compound 47;

[0460] At room temperature, to amine 46 (500mg, 0.47mmol) and 3,5-dihydro-6-chloropyrazine-2-propenylmethyl thioacid methyl ester (21,330mg, 0.84mmol) DIPEA (0.84mL, 94.70mmol) was added to the Et0H (20mL) solution of ). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was subjected to silica gel column chromatography (80: 18: 2 CHCl₃/CH₃0H/NH₄0H) purified to obtain muscle 47 (325mg, 55%) as a yellow solid: [M+H]⁺1278_O

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-Pentylhexyl)amino)hexyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methionyl)-6- Preparation of chlorpyrazine-2-carboxamide HC1 salt

[0462] 4N HC1 (20m) in water was added to 47 (325 mg, 0.25 mmol) in EtOH (2.0 mL) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to give compound 48 (165 mg, 60%) as a yellow solid:

[0463] ^XH NMR(400MHz, DMS0-d₆): δ 10. 52 (brs, 1Η), 10. 44 (brs, 1H), 9. 28 (t, J = 5.2Hz, 1H), 9. 00-&amp; 88 (m, 1H), &amp; 87 -&amp; 75 (m, 1H), &amp; 63 (brs, 2H), &amp; 60-&amp; 50 (m, 1H), &amp; 39-&amp; 33 (m, 1H) ,8. 17-&amp; 11 (m, 1H) ,7.58(dd, J = 6.5,3.3Hz, 2H), 7.47-7.35 (m, 2H), 7.36 (d, J = &amp; 7Hz,2H), 7.33 (d, J = 6.8hz, 1H), 7. 27 (d, J = 3.6Hz, 1H), 7. 11 (d, J = 8.8Hz, 2H), 3. 72-3. 66 ( m,3H), 3.60 (d, J = 3.6Hz, 1H), 3. 57 (d, J = 2. 8Hz, 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 = &amp; 5Hz, 2H), 1.76-1.60 (m, 6H), 1.58-1.47 (m, 2H), 1.35-1.23 (m, 4H).

[0464] ^XH NMR (400MHz, CD₃0D): δ &amp; 23-8. 18 (m, 1H), &amp; 17-8. 12 (m, 1H), 7. 59-7.52 (m, 2H), 7.36 (d, J = 7 . 8Hz, 1H), 7. 31 (d, J = 7. 2Hz,lH), 7. 18 (d, J = &amp; 2Hz, 2H), 7. 04 (d, J =&amp; 2Hz, 2H) ,4 . 25 (t, J = 7. 8Hz, 1H), 4. 18-4. 10 (m, 2H), 3. 83-3. 79 (m, 2H), 3. 77 (d, J = 3. 1Hz, 1H), 3.74 (d, J = 3.5Hz, 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 = 7.4Hz, 2H), 1.88-1.69 (m, 6H), 1.62-1.53 (m, 2H), 1.44-1. 30 (m, 4H).

8. 3,5-Diamino-N-(N-(4-(4-(4-((S)-2-amino-3-oxo-3-(4-(6-((2S, 3R , 4R,

5R)-2,3,4,5,6-Pentaylhexylamino)hexyl)phenylamino)propyl) Cai-1-yl)butyl)methyl)-6-chloropyrazine-2- Formamide preparation

[0466] Scheme 9

[0467]

<img file="CN105073717A_D0072.tif" />

<img file="CN105073717A_D0073.tif" />

II .2Ac()II

<img file="CN105073717A_D0074.tif" />

Cl

HJ '

ILN^N Nil;

OH ί)Π

<img file="CN105073717A_D0075.tif" />

ΟΠ ()11

<img file="CN105073717A_D0076.tif" />

O aqueous solution, 2(Sichuan

Ν (S

<img file="CN105073717A_D0077.tif" />

NH.

<img file="CN105073717A_D0078.tif" />

o

N Cl

<img file="CN105073717A_D0079.tif" />

N Nil

[0468] Preparation of Compound 50;

[0469] In an ice bath, a solution of acid 17 (950 mg, 1.84 mmol) in THF (30 mL) was cooled to 0 °C. NMM (0.40 mL, 3.68 mmol) was added, followed by PivCl (0.23 mL, 1.84 mmol) and the reaction mixture was stirred at the same temperature for 2 h. 49 (800 mg, 1.47 mmol, 10 mL THF) was added and the reaction mixture was stirred at the same temperature for another 10 minutes. The reaction mixture was returned to room temperature and stirred for 16 h. Remove the organic solvent, add water to the residue and use CH₂Cl₂(3X100mL) extraction. The organic layers were combined, dried over NagSOq, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to give amide 50 as a pale yellow solid (1.40 g, impure): [M+H]⁺1043o

[0470] Preparation of Compound 51;

[0471] Mix 50 (1.40g, impure) and 10% Pd/C (400m) in Et0H (120mL) and Ac0H (20mL)

The suspension in the product was degassed and placed under hydrogenation conditions (latm) at room temperature for 16 h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 51 (1.20g, crude) used directly in the next step: [M+H]⁺913_O

[0472] Preparation of Compound 52;

[0473] At room temperature, amine 51 (1.20 g, 0.47 mmol, crude) and methyl 3,5-diamino-6-chloropyrazine-2-pyrimylmethyl thioate (21,723 mg , 1.86mmol) of Et0H (20mL) solution was added DIPEA (2.00mL, 11.6mmol) _oThe reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was passed through silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) purified to obtain muscle 52 as a yellow solid (500mg, 24% after three steps): [M+H]⁺1125o

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-Pentylhexylamino)hexyl)phenylamino)propyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine Preparation of HC1 salt of -2-carboxamide

[0475] 4N HC1 (25 mL) in water was added to 52 (500 mg, 0.44 mmol) of EtOH (5.0 mL) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to give compound 53 (170 mg, 41%) as a yellow solid:

[0476] ^XH NMR(400MHz, DMS0-d₆): δ 10. 52 (brs, 1Η), 10. 45-10. 41 (m, 1H), 9. 31-9. 24 (m, 1H), 9. 02-8.89 (m, 1H) , 8.88-8.76 (m, 1H), 8.70-8.58 (m, 3H), 8.57-8.46 (m, 2H), &amp; 40-&amp; 31 (m, 1H) , 8. 17-8. 10 (m, 1H), 7. 62-7. 54 (m, 2H), 7. 42 (brs, 2H), 7. 36 (d, J = &amp; 7Hz, 2H), 7. 33 (d, J = 6.7Hz, 1H), 7. 27 (d, J = 7.5Hz, 1H), 7. 11 (d, J = 8.7Hz, 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 (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).

[0477] ^XH NMR (400MHz, CD₃0D): δ &amp; 24-8. 20 (m, 1H), &amp; 19-&amp; 14 (m, 1H), 7. 60-7. 53 (m, 2H), 7.38 (d, J = 7. 6Hz, 1H), 7.33 (d, J = 7.2Hz, 1H), 7.24-7.18 (m, 2H), 7.07 (d, J = 8. lHz, 2H), 4. 31-4. 22(m, 1H), 4. 08-4. 01 (m, 1H), 3. 84 (dd, J = 4. 8, 1. 3Hz, 1H), 3. 77 (dd, J = 10. 1,2. 5Hz, 1H), 3. 71-3. 62 (m, 5H), 3. 36 (t, J = 7.2Hz, 2H), 3. 19-3. 12 (m, 4H) ,3.03-2.96(m,2H) ,2.55(t, J = 7.7Hz, 2H), 1.90T. 74 (m, 4H), 1.73T. 64 (m, 2H),

1. 63-1.53 (m, 2H), 1.45-1.31 (m, 4H).

9. 3,5-Diamino-N-(N-(4-(4-((S)-2-amino-3-(4-(6-(hexyl((2S, 3R, 4R, 5R) -2,

3,4,5,6-Pentylhexyl)amino)hexyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methionyl)-6-chloropyrazine-2 -Formamide preparation

[0479] Scheme 10

[0480]

<img file="CN105073717A_D0080.tif" />

<img file="CN105073717A_D0081.tif" />

[0481] Sugar

<img file="CN105073717A_D0082.tif" />

[0482] Preparation of Compound 55;

[0483] To compound 54 (770 mg, 1.45 mmol) in THF (50 mL), DEPBT (564 mg, 1.88 mmol), 17 (752 mg, 1.45 mmol) and DIPEA (0.77 mL, 4.35 mmol) were sequentially added And stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100 mL), quickly washed with saturated NaHCC^ aqueous solution (2 X 100 mL) and brine (50 mL) and dried over Na^SOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (5% methanol/CH₂C1₂) Was purified and purified by reverse phase chromatography (Gold column) to obtain amide 55 (800 mg, 54%) as a yellow solid: [M+H]⁺1027_o

[0484] Preparation of Compound 56;

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

[0486] Preparation of Compound 57;

[0487] At room temperature, the amine salt 56 (780 mg, 0.75 mmol) and 3,5-diamino-6-chloropyrazine-2-methylmethionine methyl thioate (21,466 mg, 1. DIPEA (1.37 mL, 7.67 mmol) was added to a solution of 20 mmol) in EtOH (20 mL). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. The residue was subjected to silica gel column chromatography (80: 18: 2 CHCl₃/CH₃0H/NH₄0H) purified to obtain muscle 57 (455mg, 55%) as a yellow solid: [M+H]⁺1110 ο

Compound 58 3,5-Diamino-N-(N-(4-(4-((S) ~2~ amino-3-(4-(6-(hexyl((2S, 3R, 4R, 5R)-2,3,4,5,6-Pentylhexyl)amino)hexyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methyl)-6- Preparation of chlorpyrazine-2-carboxamide HC1 salt

[0489] 4N HC1 (25 mL) in water was added to 57 (455 mg, 0.41 mmol) of ethanol (10 mL) and the reaction mixture was stirred at room temperature for 2 h. The mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to give compound 58 (230 mg, 55%) as a yellow solid:

[0490] ^XH NMR (400MHz, DMS0-d₆): δ 10. 45 (brs, 1H), 9. 30 (brs, 1H), 9. 09-&amp; 49(m,3H), &amp; 41-&amp; 32 (m, 1H), 8. 16-8. 08 (m, 1H), 7. 62-7. 52 (m, 2H), 7. 42 (brs, 2H), 7. 37 (t, J = &amp; 4Hz, 2H), 7. 32(d, J = 7. 8Hz, 1H), 7. 27 (d, J = 7.2Hz, 1H), 7. 10 (d, J = 8. lHz, 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. 7Hz, 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 = 3.2Hz, 1H), 3.12(d, J = 3.2Hz, 1H), 3. 09-2.96 (m, 6H), 1. 77T. 58 (m, 8H),

1. 57-1.46 (m, 2H), 1.35-1.21 (m, 10H), 0.86 (t, J = 6.4Hz, 3H).

[0491] ^XH NMR (400MHz, CD₃0D): δ &amp; 26-8. 20 (m, 1H), &amp; 19-8. 12 (m, 1H), 7. 60-7. 51 (m, 2H), 7. 38 (d, J = 7 . 2Hz, 1H), 7. 32 (d, J = 7.4Hz, 1H), 7. 21 (d, J = &amp; 3Hz,2H), 7. 06 (d, J =&amp; 5Hz,2H) ,4 . 26 (t, J = 7. 4Hz, 1H), 4. 16-4. 09 (m, 1H), 3. 82 (dd, J = 5.0, 1.5 Hz, 1H), 3. 78 ( dd, J = 11.3,3.2Hz,lH),3.72-3.61 (m, 6H), 3.35 (t, J = 6.7Hz,2H), 3.24-3.11 (m , 7H), 2.54 (t, J = 7.4Hz,2H), 1.90-1.67(m,8H), 1.64-1.54(m,2H), 1.44-1 . 30 (m, 10H), 0.92 (t, J = 6.7Hz, 3H).

10. (S)-2-Amino-3-(6-(4-(3-(3,5-diamino-6-chloropyrazine-2-pyridyl) cucurbityl)butyl ) Preparation of 2-yl)propionic acid (80)

[0493] Scheme 11

[0494]

<img file="CN105073717A_D0083.tif" />

<img file="CN105073717A_D0084.tif" />

64R \1c

65: R = Et

NaOH

MeOH/Tm'/HO

<img file="CN105073717A_D0085.tif" />

Η

<img file="CN105073717A_D0086.tif" />

k KHMDS. THF

2. TrisylN₃. AcOH

3. {CH3)₄NAcO·

<img file="CN105073717A_D0087.tif" />

<img file="CN105073717A_D0088.tif" />

<img file="CN105073717A_D0089.tif" />

N Cl

<img file="CN105073717A_D0090.tif" />

N NH₂

[0495]

<img file="CN105073717A_D0091.tif" />

[0496] Preparation of Compound 62;

[0497] In a nitrogen atmosphere, the appropriate Wittig inner radium salt (ylide) dialyl methoxymethylene triphenyl oxane (Ph₃PCHC0₂Me, 43.0g, 129mmol) was added to the CH of aldehyde 59 (20.0g, 107mmol)₂C1₂(200 mL) solution and the reaction mixture was stirred at ambient temperature for 16 h. The completion of the reaction was monitored by TLC (16h). Remove CH under reduced pressure₂C1₂, And use the FCC of 10% ethyl acetate-hexane to obtain the corresponding trans-α, β-unsaturated ester 62 (24.0 g, 92%) as a white solid:

[0498] ^XH NMR(400MHz, CDC1₃) 6 7.88-7.82 (m, 1H), 8.81 (d, J = 15.8Hz, 1H), 7. 73 (d, J = 9. ΟΗζ, 1H), 7. 70 (d , J = &amp; 8Hz, 1H), 7. 61 (dd, J = &amp; 8, 2. 2Hz, 1H), 7. 15 (dd, J = 9. 2,

2. 2Hz, 1H), 7.11 (d, J = 2.2Hz, 1H), 6.49 (d, J = 15. 8Hz, 1H), 3.82(s,3H), 3.82(s, 3H).

Preparation of compound 62 (additional route);

[0500] To 250 mL anhydrous CH of trimethyl phosphonoacetate (55.6 mL, 381 mmol) cooled to 0°C₂C1₂^ DBU (4 &amp; 8 mL, 322 mmol) was added and the mixture was stirred for 15 minutes. Add 50mL CH dropwise₂C1₂^ofaldehyde 59 (40.0g, 215mmol). The temperature of the reaction mixture was returned to room temperature and the resulting reaction mixture was stirred at room temperature for 16 h, and quenched with 100 mL of water. Distribute the mixture and use CH₂Cl₂(3X150mL) extraction. The combined organics are brine, dried (Na₂S0₄) Was washed, filtered, concentrated and the residue was purified by silica gel column chromatography (10:1 hexane/ethyl acetate) to obtain the desired trans-α,β-unsaturated ester 62 (48.0 g, 92% ).

[0501] Preparation of Compound 64;

[0502] In the chamber at a temperature, Compound 62 (4 &amp; 0g, 196mmol) and 10% Pd / C (10g) in EtOAc / THF (600mL / 75mL) in a suspension hydrogenation conditions (latm) 16h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated under vacuum to give 64 (46.5 g, 96%) as a white solid:

[0503] ^XH NMR(400MHz, CDC13) δ 7.67 (d, J = 9.4Hz, 2H), 7. 57-7.54 (m, 1H), 7. 29 (dd, J =&amp; 6,1.8Hz , 1H), 7. 12 (dd, J = &amp; 8,2.5Hz, 1H), 7. 11-7.09 (m, 1H), 3. 90(s,3H), 3.66 (s, 3H), 3. 07 (t, J = 7.7Hz, 2H), 2. 70 (t, J = 7.7Hz. 2H).

[0504] Preparation of Compound 66;

To methyl ester 64 (46.5g, 191mmol) in THF/MeOH/H₂0 (500mL/500mL/150mL) solution was added NaOH (45.6g, 114mmol) and the reaction mixture was stirred at room temperature for 2h. The solvent was removed and the pH was adjusted to 1 with IN HC1 aqueous solution; a white solid precipitated. The solid was filtered, washed with water and dried under vacuum to give acid 66 (42.5 g, 97%) as a white solid:

[0506] ^XH NMR(400MHz, DMSO-cU 6 12. 14(brs, 1H), 7.73(dd, J = 9. 5, 2.3Hz, 2H),

7. 64-7. 61 (m, 1H), 7. 35 (dd, J = &amp; 5,1.5Hz, 1H), 7. 26 (d, J = 2. 8Hz, 1H), 7. 129 (dd, J = 9. 1,2. 5Hz, 1H), 3.85 (s, 3H), 2.94 (t, J = 7.6Hz, 2H), 2.60 (t, J = 7.6Hz, 2H ).

Preparation of Compound 67;

At -78° C., to a solution of compound 60 (39.3 g, 222 mmol) in anhydrous THF (500 mL) was added dropwise n-butyllithium (110 mL, 2M solution in cyclohexane) and reacted The mixture was stirred for 1 h to obtain a solution of compound 61. To another solution of compound 66 (42.5g, 185mmol) in anhydrous THF (1000mL) was added NMM (26.3mL, 240mmol) and PivCl (27.3mL, 222mmol) was added dropwise at -78°C . The reaction mixture was stirred at the same temperature for 1 minute, and then the preparation solution of compound 66 was slowly added at -78°C. The reaction mixture was stirred for another 10 minutes, then returned to 0°C and stirred for 1 h, then stirred at room temperature for 30 minutes, quenched with saturated NH1, concentrated to remove THF, and in CH₂C1₂ (1000 mL) and water (1000 mL). Separate the water layer and use CH₂C1₂ (2 X 1000mL) extraction. The combined organic extracts were dried over N^SOq and concentrated. The residue was passed through column chromatography (silica gel, CH₂C1₂) Was purified to obtain compound 67 (45.0 g, 63%) as a white solid:

[0509] ^XH NMR(400MHz, CDC13) δ 7.68 (d, J = &amp; 6Hz, 2H), 7. 64-7.61 (m, 1H), 7. 31 (dd, J =&amp; 5,1.8Ηζ,1Η ), 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 = 2.4Hz, lH), 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).

[0510] Preparation of Compound 68;

[0511] To a solution of compound 67 (45.0 g, 116 mmol) in anhydrous THF (700 mL) at -78° C., KHMDS (34.6 g, 174 mmol) was added dropwise. After the resulting mixture was stirred for 30 minutes, trisyl azide (53.6 g, 174 mmol) was added and the reaction mixture was stirred for 5 minutes. Then, acetic acid (69.6 mL, 1158 mmol) was slowly added at the same temperature, followed by tetramethyl acetic acid saddle (30.9 g, 232 mmol). The reaction mixture was heated to 24 °C, stirred for 16h, and saturated NaHC0₃(300mL) quenched, concentrated to remove THF and used CH₂C1₂(2X500mL) Extraction. The combined organic extracts were dried over Na^SOq and concentrated. The residue was purified by column chromatography (silica gel, 10:90 EtOAc/hexane, followed by DCM) to give compound 68 (31.0 g, 62%) as a yellow solid:

[0512] ^XH NMR (400MHz, CDC1₃) δ 7.70 (d, J = 9. 1Hz, 2H), 7. 68-7.65 (m, 1H), 7. 40 (dd, J =&amp; 6,1.8Hz, 1H), 7. 36-7. 23 (m, 3H), 7. 20 (d, J = 1. 8Hz, 1H), 7. 19-7. 17 (m, 1H), 7. 13 (dd, J = 9. 0 ,2.6Hz, 1H), 7. 10 (d, J = 2.4Hz, 1H), 5.36 (dd, J = 9. 0, 6. ΟΗζ, 1H), 4.58-4.50 ( m, 1H), 4. ll(dd, J = 9. 1,2. 6Hz,lH), 3.90(s, 3H), 3.91(t, J = 8.6Hz, 1H), 3. 34 (dd, J =

13. 8,6. 5Hz, 1H), 3. 30 (dd, J = 13. 0, 3.5 Hz, 1H), 3. 19 (dd, J = 13. 4, &amp; 6Hz, 1H), 2. 81 ( dd, J = 13. 4, 9.5 Hz, 1H).

[0513] Preparation of Compound 69;

[0514] At 0° C., to compound 68 (31.0 g, 72.1 mmol) in THF/H₂0 (300mL/100mL) Add H dropwise to the solution₂0₂ (49 mL, 433 mmol), followed by LiOH (6.04 g, 144 mmol). The reaction mixture was stirred at the same temperature for 10 minutes, then at room temperature for 1hr, and then saturated Na₂S0₃ (200mL) quenched, concentrated under reduced pressure to remove THF and used CH₂Cl₂(500mL) Wash. The water layer was acidified with IN HC1 aqueous solution and CH₂C1₂(2X500mL) extraction. The combined organic extracts were dried with blood claw 04, concentrated and washed with MTBE to obtain compound 69 (15.0 g, 82%) as an off-white solid:

[0515] ^XH NMR(400MHz, MeOD-d₃) δ 7. 70 (t, J = 8.4Hz, 2H), 7. 66-7.63 (m, 1H), 7. 35 (dd,

J = &amp; 6,1.7Hz, 1H), 7. 19(d, J = 2. 8Hz, 1H), 7. 10 (dd, J = 9. 1,2. 6Hz, 1H), 4. 25 ( dd, J = &amp; 6,5.3Hz, 1H) ,3.88(s,3H), 3.29 (dd, J = 13. 9,5.1Hz, 1H), 3. 10 (dd, J = 14. 3,&amp; 6Hz, 1H).

[0516] Preparation of Compound 70;

[0517] At room temperature, compound 69 (15.0 g, 55.1 mmol) and 10% Pd/C (3.50 g) of AcOH/H₂0 (300mL/100mL) suspension was left under hydrogenation conditions (latm) for 3h. The reaction mixture was filtered through Celite and washed with AcOH/O0, followed by MeOH. The filtrate was concentrated under vacuum to obtain acetate 70 (14.0 g, 83%) as a yellow solid:

[0518] ^XH NMR (400MHz, DMS0-d₆, TFA) 6 &amp; 38-8. 18 (m, 3H), 7. 78 (dd, J = 11.4, &amp; 1Hz, 2H), 7. 75-7. 70 (m, 1H), 7. 41 (dd , J = &amp; 6,1.6Hz, 1H), 7. 29 (d, J = 2.3Hz, 1H), 7. 18 (dd, J =&amp; 8,2.4Hz, 1H) ,4.33- 4.23 (m, 1H), 3.89(s,3H), 3.33(dq, J = 14.5,5.9Hz,2H), 1.92 (s, 3H).

[0519] Preparation of Compound 71;

[0520] To a solution of compound 70 (14.0 g, 45.9 mmol) in acetic acid (140 mL) was added dropwise hydrogen bromide (140 mL) at room temperature and the reaction mixture was refluxed for 3 h. The reaction mixture was cooled to room temperature and concentrated. The crude brown residue 71 (12.4 g, 87%) was used directly in the next step without any purification:

[0521] P NMR (400MHz, DMS0-d₆) δ 13. 83 (brs, 1H), 9. 71 (brs, 1H), 8.41 (brs, 1H), &amp; 25 (brs, 2H), 7. 67 (dd, J = 13. 8,&amp; 7Hz,2H), 7.64-7.61 (m, 1H), 7.29(dd, J = &amp; 6,1.7Hz, 1H), 7.13-7.05(m,2H),4 . 29-4. 19 (m, 1H), 3. 20 (t, J = 5.5Hz, 2H).

[0522] Preparation of Compound 72;

[0523] At 0° C., acetyl chloride (3 &amp; 4 mL, 540 mmol) was added to anhydrous methanol (400 mL), and then compound 71 (24.0 g, 77.2 mmol) was added_oThe reaction mixture was refluxed for 4h and concentrated. Residue in CH₂C1₂(500mL) and saturated NaHC0₃(300mL). Separate the water layer and use CH₂C1₂ (2X 300mL) extraction. The combined organic extracts were subjected to Na₂S0₄T was dried and concentrated to give compound 72 (16.6 g, 88%) as a white solid:

[0524] ^XH NMR (400MHz, DMS0-d₆) δ 9. 62 (brs, 1Η), 7. 67 (d, J = 9.4Hz, 1H), 7. 58 (d, J = 8. 8Hz, 1H), 7.53(s, 1H), 7. 22(dd, J = &amp; 2,1.4Hz, 1H), 7. 09-7. 06 (m, 1H), 7. 04 (dd, J =&amp; 8,2.6Hz, 1H), 3 . 67 (t, J = 6. 5Hz, 1H), 3. 57(s, 3H), 2. 97 (dd, J = 13. 5,6.1Hz, 1H),

2. 86 (dd, J = 13. 2, 7.4Hz, 1H), 1.90 (brs, 2H).

[0525] Preparation of Compound 73;

[0526] At 0° C., the MeOH/H of compound 72 (16.6 g, 67.8 mmol)₂0(360mL/120mL) solution add NaHC0₃(22.8g, 271mmol) and Boc₂0 (17.7 g, 81.3 mmol). The resulting mixture was heated to room temperature and stirred for 1 h_oReaction mixture in CH₂Cl₂Partition between (200 mL) and water (200 mL). Separate the water layer and use CH₂C1₂ (2 X 400mL) extraction. The combined organic extracts were washed with brine, dried over Nahoq and concentrated. Use 20% ethyl acetate-hexane and then use the FCC of Pit to obtain compound 73 (17.0 g, 73%) as a white solid:

[0527] ^XH NMR (400MHz, CDC1₃) δ 7.60 (d, J = 9. 5Hz, 1H), 7. 51 (d, J = &amp; 2Hz,lH), 7.49-7.43 (m, 1H), 7. 15 (d, J = &amp; 2Hz, 1H), 7.09-6.99 (m, 2H), 6.31 (brs, 1H), 5.15-4.84 (m, 1H),4.73-4.46 3.71 (s,3H), 3.23 (dd, J = 13. 7, 5. 3Hz, 1H), 3. 14 (dd, J = 13. 7,

5. 5Hz, 1H), 1.39(s,9H).

Preparation of Compound 74

[0529] At 0 ° C, to the CH of compound 73 (7.0 g, 20.3 mmol)₂C1₂ (300 mL) The solution was added with pyruvate (16.5 mL, 203 mmol) and triflate (5.11 mL, 30.4 mmol) and stirred at the same temperature for 1 h, and then at room temperature for 2 h. After concentration, the reaction mixture is in CH₂C1₂Partition between (300 mL) and water (200 mL). Separate the water layer and use CH₂Cl₂(2X300mL) extraction. The combined organic extracts were washed with brine and passed through the blood paw. . After drying and concentration, compound 74 (8.80 g, 91%) was obtained as a brown oil (the presence of noise was confirmed by NMR). The reaction was monitored by using LC-MS and the product composition was determined from the LM-MS data:

[0530] ^XH NMR(400MHz, CDCI3) 7. 85 (d, J = 9. 2Hz, 1H), 7. 80 (d, J = 8.7Hz, 1H), 7. 71 (d, J = 2.7Hz, 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. 3Hz, 1H), 3. 20 (dd, J = 13. 3 ,6.2Hz, 1H), 1.38(s,9H).

[0531] Preparation of Compound 75;

[0532] At room temperature, compound 74 (16.5 g, 34.6 mmol) and but-3- alkynyl carbamate benzyl ester (17, 10.4 g, 51.9 mmol) in anhydrous CH₃CN (450mL) was degassed with nitrogen for 10 minutes, then TEA (19.3mL, 138mmo 1), 10% in hexane (t-Bu) were added at room temperature ₃P (13.9mL, 6. and Cui (0.33g, 1.72mmol). The resulting mixture was degassed with nitrogen for 10 minutes and Pd(PPh₃)₄(3.99g, 3.45mmol). After degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 18h. The reaction mixture was concentrated under vacuum and the residue was purified by a column (silica gel, 75:25 hexane/EA) to give compound 75 (14.lg, 77%) as a brown solid:

[0533] ^XH NMR(400MHz, CDC1₃) 6 7. 86 (brs, 1Η), 7. 68 (t, J = 7. 8Hz, 2H), 7.53 (brs, 1H), 7.41 (dd, J = &amp; 5,1.6Hz, 1H), 7.38-7.28 (m, 5H), 7.27-7.22 (m, 1H), 5.26-5.17 (m, 1H),

5. 13(s,2H) ,5.06-4.99 (m, 1H) ,4.70-4.59 (m, 1H), 3.69(s,3H) ,3.46 (q, J = 6. 7Hz, 2H),

3. 27 (dd, J = 14. 1,5.9Hz, 1H), 3. 16 (dd, J = 13. 2,6.2Hz, 1H), 2. 67 (t, J = 6. 6Hz, 2H) ,

1. 38(s,9H).

Preparation of Compound 76;

[0535] To formaldehyde 75 (12. lg, 22.8 mmol) in THF/MeOH/H₂0 (150mL/150mL/50mL) NaOH (4.56g, 114mmol) was added to the solution and the reaction mixture was stirred at room temperature for 2h. The pH value was adjusted to 9 with IN HC1 aqueous solution and the organic solvent was removed. Adjust the pH value of the residue to 5 to 6, and the suspension in CH₂C1₂ Partition between (500mL) and water (200mL). Separate the water layer and use CH₂Cl₂(2X400mL) extraction. The combined organic extracts were passed through the blood claws. . Dry and concentrate, obtain compound 76 (10.50g, 89%) as brown solid:

[0536] ^XH NMR(400MHz, CD3OD) 6 7.83 (s, 1H), 7.73-7.61 (m, 3H), 7.44-7.19 (m, 7H), 5. 10 (s, 2H) ), 4. 42-4. 34 (m, 1Η), 3. 41-3. 32 (m, 3H), 3. 06 (dd, J = 14. 3, 9.3 Hz, 1H), 2. 64 (t, J = 7. 0Hz, 2H), 1. 31 (s, 7H), 1. 21 (s, 2H).

Preparation of Compound 77; SG-SJL-B-27

A suspension of 75 (2.0 g, 3.77 mmol) and 10% Pd/C (500 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and then under hydrogenation conditions at room temperature (latm)16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 77 (1. 60 mg, 93%) as a white solid:

[0539] ^XH NMR (400MHz, CD₃OD) 7. 73 (d, J = &amp; 5,1H), 7. 72 (d, J = &amp; 7Hz, 1H), 7. 62 ((brs, 2H), 7.73 (ddd, J = 10.0, &amp; 7, 2. 7Hz, 2H), 4. 44 (dd, J = &amp; 8, 5. 6Hz, 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 = 7.4Ηζ, 2Η), 1.96 (s, 6H), 1.85-1.75 (m, 2H), 1.74-1.68 (m, 2H), 1. 33(s,7H),

1. 26(s,2H).

Preparation of Compound 78; SG-SJL-B-30

[0541] At room temperature, to the amine salt 77 (1.60g, 3.47mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethyl thioacid methyl ester (13,2 16g, 5.56mmol) in EtOH (40mL) solution was added DIPEA (6.20mL, 34.70mmol). The reaction mixture was heated in a sealed tube at 70°C for 1 h, then cooled to room temperature and concentrated in vacuo. The residue was passed through column chromatography (silica gel 80: 18: 2CHCl₃/CH₃0H/NH₄OH) was purified to obtain M 78 (1.24 g, 59%) as a yellow solid:

[0542] ^XH NMR(400MHz, CD₃0D) δ 7. 71 (dd, J = &amp; 4, 2. 8Hz, 2H), 7. 60 (brs, 2H), 7. 34 (dd, J = &amp; 5,1.9Hz, 1H), 7. 30 (dd, J = &amp; 7,1.7Hz, 1H), 4.45 (dd, J = &amp; 9,5.7Hz, 1H), 3. 68 (s, 3H), 3. 28-3. 26 (m, 1H), 3. 25 (t, J = 2.4Hz, 1H), 3. 22 (d, J = 5.9Hz, 1H), 3. 04 (dd, J =

14. 0,9.2Hz, 1H), 2.82 (t, J = 7.2Hz,2H), 1.86-1.77 (m, 2H), 1.73-1.63 (m, 2H), 1. 32 (s, 7H), 1. 23 (s, 2H).

[0543] Preparation of Compound 79; SG-SJL-B-32

[0544] To a solution of methyl ester 78 (1.24 g, 2.00 mmol) in a mixture of THF (25 mL), methanol (25 mL) and water (10 mL) was added solid NaOH (324 mg, 8.0 mmol) and the reaction mixture was heated Stir at room temperature for 1 h. The TLC of the reaction mixture showed the completion of the reaction, and then the pH of the reaction mixture was restored to pH 9 to 10 by adding IN HC1 (aqueous solution) and the organic solvent was removed. The pH of the aqueous portion was adjusted to pH 5 to 6 and precipitation occurred, and it was extracted with dichloromethane. CH for water-based part₂Cl₂(2X50mL) extraction. Merge the organic layer, menstruation is wonderful. Dry, filter and concentrate. Dry yellow solid compound (79,1.10g, 92%) under vacuum:

[0545] ^XH NMR((400MHz, CD₃0D) 7. 70 (t, J = 9. 4, 2H), 7. 61 (d, J = 5. 3Hz, 2H), 7. 33 (dd, J =&amp; 4,1.4Hz, 2H), 4. 38 (dd, J = &amp; 4,5.1Hz, 1H), 3. 05 (dd, J = 14. 1,9.1Hz, 1H), 2.84(t, J = 6.9Hz, 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).

Compound 80-(S)-2-amino-3-(6-(4-(3-(3-(3,5-diaminochloropyrazine dialyl) guanyl) butyl) Chua-2- (Base) preparation of propionic acid hydrochloride

[0547] 4N HC1 (25 mL) in dioxane was added to 79 (1. 10 g, 1.83 mmol) in EtOH (5.0 mL) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, purified by a reverse phase column (Gold column) and the residue was lyophilized to give compound 80 (700 mg, 67%) as a yellow solid:

[0548] P NMR (400MHz, DMS0-d6) 10. 48 (s, 1H), 9.24 (brs, 1H), 8.99-8.86 (m, 1H), &amp; 84-&amp; 70 (m , 1H), &amp; 38 (brs, 3H), 7. 80 (t, J = 9. 2Hz, 2H), 7. 73 (s, 1H), 7. 69 (s, 1H), 7. 45-7 . 35(m,4H) ,4. 25 (dd, J = 11.4,5.9Hz, 1H), 3.34 (q, J = 6.6Hz,2H), 3.27 (d, J =

6. 9Hz,2H), 2.79 (t, J = 7.70Hz,2H), 1.79-1.67 (m, 2H), 1.65-1.54(m,2H).

[0549] ^XH NMR((400MHz, CD3OD) 7.82 (d, J = &amp; 5Hz, 1H), 7.78 (d, J = &amp; 7Hz, 1H), 7.73 (s, 1H), 7.68(s , 1H), 7.40 (ddd, J = 10.5, &amp; 6, 1.6 Hz, 2H), 4. 33 (dd, J = 7. 7, 5.2 Hz, 1H),

3. 46 (dd, J = 14. 9, 6. ΟΗζ, 1H), 3. 37 (t, J = 7.5Hz, 2H), 3.33-3.29 (m, 1H), 2.87 (t , J =

7. 7Hz, 2H), 1.90-1.80 (m, 2H), 1.79-1.71 (m, 2H).

11. (S)-3,5-Diamino-6-Chloro-(N-(4-(6-(2,3-Diamino-3-oxopropyl) Cai-2-yl ) Butyl) methyl) pyrazine-2-carboxamide (84) preparation

[0551] Scheme 12

[0552]

<img file="CN105073717A_D0092.tif" />

<img file="CN105073717A_D0093.tif" />

II N

<img file="CN105073717A_D0094.tif" />

BocHN

<img file="CN105073717A_D0095.tif" />

NHc

Nil Ο

Ν Ν II II

<img file="CN105073717A_D0096.tif" />

[0553] Preparation of Compound 81;

[0554] In an ice bath, the acid 76 (2.0g, 3.87mmol) in THF (80mL) solution was cooled to 0 °C, added

NMM (0.63 mL, 5.03 mmol), then i-BCF (0.63 mL, 5.80 mmol) was added dropwise and the reaction mixture was stirred at the same temperature for 2 h. Add ΝΗ dropwise₃(7.0N methanol, 5.52mL, 3&amp;7mmol) and the reaction mixture was stirred at the same temperature for another 2h. The reaction mixture was then returned to room temperature and stirred for 16 h. Remove the organic solvent. Add water to this residue and use CH₂Cl₂(3X100mL) extraction. The organic layers were combined, dried over NagSOq, filtered, and concentrated. The residue was purified by column chromatography (3% methanol in chloroform) to give amide 81 (1.75 g, 88%) as a pale yellow solid:

[0555] ^XH NMR (400MHz, CD₃OD) 7.83 (s, 1H), 7.69 (d, J = &amp; 1Ηζ, 2Η), 7.66 (s, 1H), 7.39 (dt, J = &amp; 8,1.9Hz, 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 = 6.9Hz, 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 = 7.0Hz, 2H), 1. 27 (s, 7H), 1. 21 (s, 2H).

Preparation of Compound 82;

A suspension of 81 (1.75 mg, 3.39 mmol) and 10% Pd/C (600 mg) in a mixture of EtOH (110 mL) and AcOH (15 mL) was degassed and then under hydrogenation conditions at room temperature ( latm) 12h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated in vacuo to obtain the amine salt 82 (1. 40 g, 93%) as a white solid:

[0558] ^XH NMR(400MHz, CD₃0D) 7. 72 (dd, J = &amp; 3, 5. 6Hz, 2H), 7. 66 (s, 1H), 7. 61 (s, 1H), 7. 38 (dd, J = &amp; 6,1 . 3Hz, 1H), 7. 34 (dd, J = &amp; 5,1.5Hz, 1H), 4.38 (dd, J = 9. 0,5.0Hz, 1H), 3. 27(dd, J = 13. 8, 5. ΟΗζ, 1H), 2.93 (t, J = 7.9Hz, 2H), 2.83 (t, J = 7.5Hz,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;

[0560] At room temperature, to amine salt 82 (1.40g, 3.15mmol) and 3,5-diamino-6-chloropyrazine-2-methylmethionine methyl thioate (13,1 96g, 5.04mmol) in Et0H (40mL) solution was added DIPEA (5.64mL, 31.5mmol). In a sealed tube, the reaction mixture was heated at 70 °C for 2 h, then cooled to room temperature and concentrated in vacuo. The residue was passed through column chromatography (silica gel, 80: 18: 2CHCl₃/CH₃0H/NH₄(OH) purification to obtain muscle 83 (1.15 g, 61%) as a yellow solid:

[0561] ^XH NMR (400MHz, CD₃0D) δ 7. 70 (d, J = 8. 3Hz, 2H), 7. 64 (s, 1H), 7. 60 (s, 1H), 7. 34 (dt, J = &amp; 9,1.9 Hz ,2H), 4.38 (dd, J = 9. 0, 5.5Hz, 1H), 3.28-3.20 (m, 3H), 2.96 (dd, J =9.6, 14. 1Hz, 1H), 2.81 (t, J = 7.4Hz,2H), 1.85-1.76 (m, 2H), 1.70-1.61(m,2H), 1.27 ( s, 7H), 1. 20(s, 2H).

[0562] Compound (S)-3,5-Diamino-6-chloro-Qian (Qian (4-(6-3-diamino-3-oxopropyl) Cai-2-yl)butyl) Preparation of the HC1 salt of methyl)pyrazine-2-carboxamide (84)

[0563] 4N HC1 (25 mL) in dioxane was added to 83 (1.15 g, 1.92 mmol) in EtOH (6.0 mL) and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, purified by a reverse phase column (Gold column) and the residue was lyophilized to give compound 84 (310 mg, 28%) as a yellow solid:

[0564] ^XH NMR(400MHz, DMS0-d₆) 10. 56 (s, 1H), 9. 38 (t, J = 5. 5Hz, 1H), 9. 06-8. 83 (m, 2H), &amp; 31(brs,3H), &amp; 02 (s , 1H) ,7.79 (t, J = &amp; 6Hz,2H) ,7.73 (s, 1H), 7.69 (s, 1H), 7.51 (s, 1H) ,7.46-7 36(m,4H) ,4.06 (dd, J = 11. 5,6. ΟΗζ, 1H), 3.37 (q, J = 6.4Hz,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. 1Ηζ, 2Η), 1. 79-1 . 69 (m, 2H), 1. 64-1. 54 (m, 2H).

[0565] ^XH NMR(400MHz, CD₃0D) 7.82 (d, J = 8.4Hz, 1H), 7.79 (d, J = 8.7Hz,lH), 7.74(s,lH), 7.68(s,lH), 7.41(td, J = &amp;1,1.6Hz, 2H), 4. 18 (dd, J = &amp; 1,6.3Hz,lH), 3.42-3.34(m,3H), 3. 21(dd, J = 14. l,&amp;0 Hz, 1H), 2.86 (t, J = 7.4Hz, 2H), 1.91-1.80 (m, 2H), 1.79-1. 71(m, 2H).

12. 3,5-diamino (N~(4-(6-((S)~2~amino-3-(4-(3-(hexyl((2S,3R,4R,

5R)-2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Ze-2-yl)butyl)methyl)-6 -Preparation of chlorpyrazine-2-carboxamide (89)

[0567] Scheme 13

[0568]

<img file="CN105073717A_D0097.tif" />

<img file="CN105073717A_D0098.tif" />

<img file="CN105073717A_D0099.tif" />

Preparation of Compound 86;

[0570] To compound 85 (1.10g, 2.32mmol) in THF (50mL) was sequentially added DEPBT (766mg,

2. 56mmol), 76 (1.00g, 1.97mmol) and DIPEA (1.0mL, 5.91mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂(100 mL), quickly washed with saturated aqueous water (2×100 mL) and brine (50 mL) and dried over NazSOq. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (5% methanol/CH₂C1₂) Was purified to obtain amide 86 (1.19 g, 57%) as a yellow solid product:

[0571] ^XH NMR(400MHz, CD₃0D): 7. 82 (d, J = 5. 8Hz, 2H), 7. 73-7. 61 (m, 4H), 7. 51-7. 43 (m,

2H), 7. 39-7. 19 (m, 10H), 7. 05 (d, J = &amp; 3Hz, 2H), 5. 52 (s, 1H), 5. 10 (s, 2H), 4. 51 (t, J =

7. 8Hz, 1H), 4. 31-4. 25 (m, 1H), 4.24 (dd, J = 11. 0, 5. 4Hz, 1H), 4.01-3. 91 (m, 2H), 3. 88 (dd,

J = 5. 5, 2. 1Hz, 1H), 3. 76 (dd, J = 9.3, 2. 1Hz, 1H), 3. 61 (t, J = 10. 6Hz, 1H), 3. 37 (t, J

=6.9Hz,2H) ,3.12-3.00 (m, 1H), 2.74 (dd, J = 13. 2,5.3Hz, 1H), 2.64 (t, J = 7. 1Ηζ,2Η),

2. 57-2.37(m,7H),1.74T.64(m,2H),1.31(s,9H),1.29T.16 (m, 8H), 0.86 (t, J = 6.9Hz, 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 under hydrogenation conditions (1 atm) at room temperature 3h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain the amine salt 87, which was then used NaHC0₃The neutralized and crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain the free amine 87 (550 mg, 58%, after two steps) as a yellow solid:

[0574] ^XH NMR(400MHz, CD₃0D) 7. 71 (t, J = 8.4Hz, 2H), 7.62 (d, J = 1.8Hz,lH), 7.49-7.45(m,3H), 7.40(d, J = 8. 2Hz, 2H), 7. 36-7. 28 (m, 5H), 7. 09 (d, J = 8. 2Hz, 2H),

5. 55 (s, 1H) ,4.51 (dd, J = 15. 6,&amp; 4Hz, 1H), 4.25 (dd, J = 10. 6,5.4Hz, 1H) ,4.17-4. 03 (m,

2H), 3.98-3.90(m,2H),3.81-3.74 (m, 1H), 3.63 (t, J = 10.4Hz, 1H), 3.27-3. 20 (m, 1H),

3. 09-2.98(m,5H), 2.93(t, J = 7.6Hz, 2H), 2.83 (t, J = 6.8Hz, 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 = 6.9Hz,3H).

[0575] Preparation of 88;

[0576] At room temperature, to amine 87 (550mg, 0.65mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,400mg, 1.04mmol) DIPEA (1.15 mL, 6.44 mmol) was added to the Et0H (20 mL) solution of ). The reaction mixture was heated in a sealed tube at 70°C for 2h, then cooled to room temperature and concentrated in vacuo. The residue was passed through silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄OH) purification, followed by reverse phase column (Gold C18) purification, to obtain Arc 88 (333 mg, 48%) as a yellow solid:

[057 knife ^XH NMR(400MHz, CD₃0D) 7. 69 (dd, J = &amp; 6, 3.5Hz, 2H), 7.66 (s, 1H), 7.60 (s, 1H), 7.48-7.44 (m, 2H) , 7. 35 (ddd, J = 10. 4, &amp; 6, 1.6Hz, 2H), 7. 33-7. 28 (m, 5H), 7. 04 (d, J = &amp; 3Hz, 2H), 5. 52 (s, 1H), 4. 52-4. 55 (m, 1H), 4. 24 (dd, J = 10. 6, 5. 4Hz, 1H), 4. 00-3.91 (m , 2H), 3. 88 (dd, J = 5. 4, 2. ΟΗζ, 1H), 3. 75 (dd, J = 9. 6, 2. 2Hz, 1H), 3. 60 (t, J = 10. 6Hz, 2H), 3. 28-3. 23 (m, 3H), 3. 06 (dd, J = 13. 5, &amp; 3Hz, 1H), 2.82 (t, J = 7.0 Hz, 2H), 2. 77 (dd, J = 13. 9, 5. 6Hz, 1H), 2. 59-2. 40 (m, 7H), 1. 86-1.76 (m, 2H), 1.74-1.68 (m, 4H), 1.42-1.60 (m, 7H), 1.33(s,9H), 0.86 ( t, J = 7. lHz, 3H).

[0578] 3,5-Diamino-N- (N~ (4- (6- ((S) ~2~ amino-3- (4- (3-(hexyl ((2S, 3R, 4R, 5R) ~2,

3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Cze-2-yl)butyl)methionyl)-6-chloropyrazine- Preparation of HC1 salt (89) of 2-formamide compound;

[0579] 4N HC1 (20 mL) in water was added to 88 (333 mg, 0.31 mmol) of ethanol (10 mL) and the reaction mixture was stirred at room temperature for 2 h. Purified by a reverse phase column (Gold column) and lyophilized the residue to give compound 89 (2) 0 mg, 68% as a yellow solid):

[0580] ^XH NMR(400MHz, DMS0-d₆) 10. 94 (brs, 1H), 9. 29 (brs, 1H), 9. 02-8. 77 (m, 2H), &amp; 64-&amp; 17 (m, 2H), 7. 80-7. 73 (m, 3H), 7. 68 (s, 1H), 7. 52 (d, J = 8.8Hz, 2H), 7. 47 (dd, J = &amp; 2, 1. ΟΗζ, 1H), 7. 44-7.36(m,3H) ,7.19(d, J = &amp; 6Hz, 2H), 5.52-5.41 (m, 1H),

4. 86-4.71 (m, 1H), 4.60 (d, J = 5. 4Hz, 1H), 4.59-4.53 (m, 1H), 4.42 (t, J = 5. 8Hz) ,

1H), 4.38 (t, J = 7. ΟΗζ, 1Η), 4.03-3.95 (m, 1H), 3.71-3.66 (m, 1H), 3.62-3. 55 (m, 1H),

3. 53-3.34(m,5H), 3.27(d, J = 7.7Hz, 1H), 3.23 (d, J = 7.4Hz, 1H), 3.16-2.99 (m , 5H),

2. 78(t, J = 7.4Hz, 2H) ,2.58(t, J = 7.9Hz, 2H), 2. 01T. 90 (m, 2H), 1.78T. 68 (m, 2H),

1. 66-1.54 (m, 4H), 1.32-1.21 (m, 6H), 0.85 (t, J = 6. 6Hz, 3H).

[0581] ^XH NMR(400MHz, CD₃0D) 7. 79 (d, J = 8.5Hz, 1H), 7.77-7.73 (m, 2H), 7.67 (s, 1H), 7.47-7.37(m,4H ), 7.21(d, J = 8. 5Hz, 2H), 4. 30 (dd, J = 7. 7,6.7Hz, 1H), 4. 12-4. 05 (m, 1H), 3 . 82-3. 74 (m, lH), 3.71-3. 61 (m, 2H), 3. 49 (dd, J = 14. 0, 6.6 Hz, 1H), 3. 47 (t, J =

6. 9Hz,2H), 3.33-3.27 (m, 3H), 3.26-3.13(m,4H), 2.86 (t, J = 7.6Hz,2H), 2.73- 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 = 6.6Hz, 3H).

13. 3, 5-Diamino (N~ (4- (6- ((S) ~2~ Amino-3-(4-(3-(bis((2S, 3R, 4R, 5R)- 2,

3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Cze-2-yl)butyl)methionyl)-6-chloropyrazine- Preparation of 2-formamide (94)

Scheme 14

[0584]

<img file="CN105073717A_D0100.tif" />

Ο

NH4-II

1)) PLA, LiOII

<img file="CN105073717A_D0101.tif" />

Η()

II*

<img file="CN105073717A_D0102.tif" />

Oil ^Ζ/ί )Π

S) η θ¹³ υ

Yan Ya

BocHN ν

<img file="CN105073717A_D0103.tif" />

Ν

Π ο

N^Cl

I

N Nil·

N

II

<img file="CN105073717A_D0104.tif" />

(Out

Ν

<img file="CN105073717A_D0105.tif" />

, QI

Ό)ίΙ no

4Ν I1C1 aqueous solution

ΙίιΟΙϊ

Ο

Ν

<img file="CN105073717A_D0106.tif" />

Nil·

NH People II II

<img file="CN105073717A_D0107.tif" />

Ο

N,'Cl

N NH.

Preparation of Compound 91

[0586] To compound 90 (484mg, 0.1%) in THF (30mL) was added DEPBT (300mg,

1. OOmmol), 19 (400g, 0.77mmol) and DIPEA (0.40mL, 2.31mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂(100 mL), quickly washed with saturated aqueous solution (2×100 mL) and brine (50 mL), and dried over NazSOq. Evaporate the solvent and pass through silica gel (5% methanol/CH₂C1₂) The crude product was purified by flash chromatography to obtain amide 91 (600 mg, 76%, impure) as a yellow solid product. The formation of the product was confirmed by LCMS.

Preparation of Compound 92

[0588] A suspension of 91 (600 mg, 0.59 mmol) and 10% Pd/C (200 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed at room temperature and then placed under hydrogenation conditions (latm) stirring for 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated in vacuo to obtain the amine salt 92, and then NaHC0₃Neutralize and purify the crude product by silica gel flash chromatography (CMA, 80: 18: 2) to obtain a yellow solid

Lysamine 36 (350mg, 66%, impure):

[0589] ^XH NMR(400MHz, CD₃0D) 7. 71 (d, J = 8.1Hz, 2H), 7. 68 (s, 1H), 7. 61 (s, 2H),

7. 43-7. 37(m,2H), 7.34(dd, J = &amp; 3,1.3Hz, 1H), 7.16 (d, J = 8. 2Hz, 2H), 4.69 (q, J =

5. lHz, 2H), 4.50 (t, J = 7.1Hz, 1H), 4. 13-4. 06 (m, 2H), 4. 05 (dd, J = 11. 0, 5. 6Hz, 2H ),

3. 83 (dd, J = 4.8, 2. 1Hz, 2H), 3. 81-3. 73 (m, 2H), 3. 51 (dd, J = 9.5, 2.3 Hz, 2H), 3. 38 (t, J = 10. 8Hz, 2H), 3. 13-3. 03 (m, 6H), 2.93 (t, J = 7.6 Hz, 2H), 2.82 (t, J = 7. 2Hz, 2H),

2. 74-2.57 (m, 2H), 2.04-195 (m, 2H), 1.84-1.75 (m, 3H), 1.74-1.63 (m, 3H), 1. 33 (s, 9H),

1. 25 (d, J = 5. lHz, 6H).

Preparation of Compound 93:

[0591] To amine 92 (350 mg, 0.38 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-pyrimidine sulfoate (13,242 mg, 0.62 mmol) at room temperature DIPEA (0.67 mL, 3.80 mmol) was added to the EtOH (10 mL) solution. The reaction mixture was heated in a sealed tube at 70 °C for 2 h, then cooled to room temperature and concentrated in vacuo. Then by silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) The residue was purified by a reverse phase column (Gold C18) to obtain muscle 93 as a yellow solid (170 mg, 20% yield in three steps):

[0592] ^XH NMR(400MHz, CD₃0D) 7. 71 (d, J = &amp; 2Hz, 2H), 7. 68 (s, 1H), 7. 62 (s, 1H), 7. 43 (d, J = &amp; 2Hz, 2H), 7. 26(ddd, J = 10. 6,&amp; 6,1.3Hz,2H) ,7.18 (d, J = &amp; 2Hz,2H) ,4.70 (q, J =0.5Hz,2H) ,4 . 49 (t, J = 7.8Hz, 1H) ,4.22-4.09(m,2H) ,4.06 (dd, J = 10.4,5.1Ηζ,2Η),

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 = 6.9Hz, 2H), 2. 77-2.61 (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. lHz, 6H).

[0593] 3, 5~ Diamino ^_N~ (N~ (4- (6- ((S) ~2~ amino ^_3- (4- (3-(Double((2S, 3R, 4R, 5R) -2,3,4,

5,6-Pentyl (hexyl) amino) propyl) phenyl amino) -3-oxopropyl) ze-2-yl) butyl) methyl)-6-chloropyrazine-2-carboxamide (94) Preparation of the hydrochloride

[0594] 4N HCl in water (20 mL) was added to 93 (170 mg, 0.15 mmol) in ethanol (5.0 mL) and the reaction mixture was stirred at 40° C. for 2 h_oThe solvent was removed, 4NHC1 was added again and heated at 40°C for another 2h. Repeat this addition 2 more times. The solvent was removed, and the residue was purified by a reverse phase column (Gold column) and lyophilized to obtain compound 94 (80 mg, 50%) as a yellow solid:

[0595] ^XH NMR (400MHz, DMS0-d₆) 10. 74 (brs, 1H), 9. 28-9. 19 (m, 1H), 9. 03-&amp; 60(m, 2H), &amp; 58-&amp; 04 (m, 1H), 7. 81- 7. 73 (m, 3H), 7. 68 (s, 1H), 7. 50 (d, J = &amp; 4Hz, 2H), 7. 48-7. 34 (m, 4H), 7. 19 (d , J = 9. 0Hz, 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. 6Hz, 1H ), 3. 62-3. 55 (m, 2H), 3. 53-3. 38 (m, 5H), 3. 37-3. 29 (m, 2H), 3. 24-3. 09 (m , 2H), 2.78 (t, J = 7.2Hz,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).

[0596] ^XH NMR(400MHz, CD₃0D) 7. 78 (d, J = &amp; 4Hz, 1H), 7. 75 (d, J = 7.8Hz, 1H), 7.73 (s, 1H), 7.66 (s, 1H), 7 . 42 (d, J = &amp; 8Hz,2H), 7. 39 (d, J = &amp; 4Hz, 2H), 7. 21 (d, J = &amp; 7Hz, 2H), 4. 16(t, J = 7 0Hz,lH),4.13-4.05(m,2H),381(dd, J = 4. 7,1.9Hz, 2H). 3.77 (dd. J = 10. 6, 3. 0Hz,2H),3.72-3.61 (m,6H),3.44-3.30(m, 10H), 2.86(t, J = 7.0Hz,2H),

2. 76-2.61 (m, 2H), 2.11-2.01 (m, 2H), 1.89-1.80 (m, 2H), 1.79-1.72 (m, 2H).

14. 3,5-Diamino-N-(N-(4-(6-((S)-2-amino-3-oxo-3-(4-(3-((2S, 3R , 4R,

5R)-2,3,4,5,6-Pentaylhexylamino)propyl)phenylamino)propyl) Cai-2-yl)butyl)methyl)-6-chloropyrazine-2 -Preparation of formamide (99)

[0598] Scheme 15

[0599] Sugar II

OH OH

Ph

Ο

<img file="CN105073717A_D0108.tif" />

Roc,

PivCl, NMM i!()

BocliN

NHCbz grumble,,Ν sugar

<img file="CN105073717A_D0109.tif" />

O

171XII

<img file="CN105073717A_D0110.tif" />

Β(κίίΝ

NHCbz

<img file="CN105073717A_D0111.tif" />

O

PdCJl· MOIkAcOH

<img file="CN105073717A_D0112.tif" />

mouth..

Bod IN

Nil:

<img file="CN105073717A_D0113.tif" />

[0600]

The solution was cooled to 0°C, and the preparation of compound 96 was added. Acid 19 (1.17g, 2.27mmol) in THF (60mL) was added in an ice bath

NMM (0.30 mL, 2.95 mmol), then PivCl (0.30 mL, 2.49 mmol) was added and the reaction mixture was stirred at the same temperature for 2 h. 34 (1.0 g, 2.27 mmol, 10 mL THF) aniline 171 was added, and the reaction mixture was stirred for another 10 minutes at the same temperature. The reaction mixture was then stirred at room temperature for 16 h. Remove the organic solvent. Add water to this residue and use CH₂Cl₂(3X100mL) extraction. The organic layers were combined, dried over Na^SOq, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to obtain amide 96 (1.40 g, 66%, impure) as a pale yellow solid. The formation of the product was confirmed by LCMS.

Preparation of Compound 97

[0603] A suspension of 96 (1.40 g, 1.50 mmol) and 10% Pd/C (300 mg) in EtOH (120 mL) and AcOH (12 mL) was degassed at room temperature and then placed under hydrogenation conditions (latm) next 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated in vacuo to obtain the amine salt 97, and then NaHC0₃The amine salt 97 was neutralized, and the crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain the free amine 97 as a yellow solid (550 mg, 30% yield in two steps):

[0604] ^XH NMR(400MHz, CD3OD) 7.74-7.65 (m, 3H), 7.59 (s, 1H), 7.41-7.29 (m, 4H), 7. 11 (d, J = &amp; 4Hz,2H) ,4.69 (q, J = 4.9Hz, 1H) ,4.50 (t, J = 7.9Hz, 1H) ,4.04 (dd, J = 10. 4,

5. 2Hz, 1H), 4.02-3.94 (m, 1H), 3.79-3.71 (m, 1H), 3.70-3.63 (m, 1H), 3.54-3. 39(m,3H),

3. 26-(dd, J = 13. 6,6.8Hz, 1H) ,3.07(dd, J = 13.1, &amp; 3Hz, 1H), 2.79 (t, J = 7.5Hz, 2H) ,2 .75-2.67(m,2H) ,2.55 (t, J = 7.3Hz,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 = 4.9Hz,3H).

Preparation of Compound 98:

[0606] To amine 97 (550 mg, 0.68 mmol) and 3,5-diamino-6-chloropyrazine-2-methylpyrimidine methyl thioate (13,423 mg, 0.62 mmol) at room temperature DIPEA (1.21 mL, 6.80 mmol) was added to the Et0H (20 mL) solution. The reaction mixture was heated in a sealed tube at 70 °C for 2 h, then cooled to room temperature and concentrated in vacuo. Then by silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) Purify the residue and pass it on a reverse phase column (Gold C18) to obtain Myo 98 (500 mg, 72%) as a yellow solid:

[0607] ^XH NMR(400MHz, CD3OD) 7.73-7.64 (m, 3H), 7.61 (s, 1H), 7.40-7.30 (m, 4H), 7. 11 (d, J = &amp; 5Hz,2H), 4.68 (d, J = 4.9Hz, 1H), 4.49 (t, J = 7.2Hz, 1H), 4.04 (dd, J = 10. 9, 5. 5Hz, 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 = 7.4Hz, 2H), 2. 54 (t, J = 7.3Hz,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 = 4.9Hz, 3H).

[0608] 3, 5~ Diamino-N- (N~ (4~ (6- ((S) ~2~ Amino-3-oxo-3- (4- (3- ((2S, 3R, 4R , 5R) -23,

4,5,6-Pentylhexylamino)propyl)phenylamino)propyl)Cy-2-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide (99) Preparation of hydrochloride

[0609] 4N HCl in water (20 mL) was added to 98 (500 mg, 0.15 mmol) in ethanol (5.0 mL) and the reaction mixture was stirred at 40° C. for 2 h. The solvent is removed, 4N HC1 is added again, and 211 is heated again at 40 . This addition is repeated two more times. The solvent was removed, and the residue was purified by a reverse phase column (Gold column) and lyophilized to obtain compound 99 (206 mg, 50%) as a yellow solid:

[0610] P NMR (400MHz, DMS0-d₆) 11. 0(brs, 1H), 9.34 (brs, 1H), 9.09-8.25(m,6H), 7.82-7.73 (m, 2H), 7. 68 (s , 1H), 7. 53 (d, J = 8. 5Hz, 2H), 7. 49 (d, J = 9. 2Hz, 1H), 7. 41 (s, 2H), 7. 39 (d, J = &amp; 2Hz,2H), 7. 17 (d, J = &amp; 2Hz,2H), 5. 39 (d, J = 3. 7Hz, 1H), 4. 80-4. 70 (m, 1H), 4 . 62 (d, J = 4.3Hz, 1H), 4.60-4.54 (m, 1H), 4.46-4.36 (m, 2H), 3.96-3.88 (m, 1H), 3. 71-3. 65 (m, 1H), 3. 62-3. 54 (m, 1H), 3. 51-3. 35 (m, 5H), 3. 09 (d, J = 13.3Hz, 1H), 2.94 (d, J = 10.9Hz, 1H), 2.87 (t, J = 9. lHz,2H), 2. 78 (t, J = 6.7Hz,2H),

2. 60 (t, J = 7.7Hz, 2H), 2.00-1.86 (m, 2H), 1.85-1.67 (m, 2H), 1.65-1.53 (m, 2H ).

[0611] ^XH NMR(400MHz, CD₃0D) 7. 80 (d, J = 9. 5Hz, 1H), 7.78-7.73 (m, 2H), 7.68 (s, 1H), 7.45-7.37(m,4H ), 7.19 (d, J = 7.1Hz, 2H), 4. 31 (t, J = 6.3Hz, 1H), 4. 09-4. 00 (m, 1H),

3. 87-3.81 (m, 1H), 3. 78 (d, J = 11. 4Hz, 1H), 3. 73-3.61 (m, 3H), 3. 46 (dd, J = 13. 6 ,6.3Hz,

1H), 3. 37(t, J = 6.8Hz, 2H), 3. 30-3. 25 (m, 1H), 3.22-3. 12(m, 2H), 3. 03 (t, J = 7.9Hz, 2H),

2. 86(t, J = 6.8Hz, 2H), 2.69(t, J = 7.4Hz, 2H), 2. 07T. 95 (m, 2H), 1. 91T. 81 (m, 2H),

1. 80-1.69(m,2H).

15. (S)-3,5-Diamino-(N-(4-(6-(2-amino-3-(4-(3-(dimethylamino)propyl)phenyl (Amino)-3-oxopropyl) Cai-2-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide (103)

[0613] Scheme 16

[0614]

<img file="CN105073717A_D0114.tif" />

<img file="CN105073717A_D0115.tif" />

<img file="CN105073717A_D0116.tif" />

®fc, N

Me

<img file="CN105073717A_D0117.tif" />

Ν

Η tfa/ch₂cl_?

<img file="CN105073717A_D0118.tif" />

Ν

If ugly

<img file="CN105073717A_D0119.tif" />

Cl

Preparation of Compound 100

[0616] A solution of acid 19 (1.75 g, 3.39 mmol) in THF (70 mL) was cooled to 0 °C in an ice bath, and

NMM (0.74mL, 6.78mmol), then PivCl (0.41mL, 3, 39mmol) was added, and the reaction mixture was stirred for 2h at the same temperature_o18 (825 mg, 4.10 mL THF) was added, and the reaction mixture was stirred for another 10 minutes at the same temperature. The reaction mixture was then brought to room temperature and stirred for 16 h. Remove the organic solvent. Add water to this residue and use CH₂Cl₂(3X100mL) extraction. The organic layers were combined, dried over Na^SOq, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to give amide 100 (1.60 g, 71%) as a light yellow solid:

[0617] ^XH NMR(400MHz, CDCI3) 7. 87 (s, 1H), 7. 71 (d, J = 8. 5Hz, 1H), 7. 67 (d, J = &amp; 5Hz, 1H), 7. 65-7 . 62 (m, 2H), 7. 42 (dd, J = &amp; 4,1.9Hz, 1H), 7. 40-7. 29 (m, 5H), 7. 22 (d, J = &amp; 6Hz, 2H), 7. 08 (d, J = 8.4 Hz, 2H), 5. 21-5. 10 (m, 2H), 5. 13 (s, 2H), 4. 51 (q, J = 7. 6Hz, 1H), 3. 47 (q, J = 6.5Hz, 2H), 3. 29 (d, J = 6.9Hz, 2H), 2. 68 (t, J = 6.7Hz, 2H), 2.57 (t, J = 7.9Hz, 2H), 2.26 (ddt, J = 11. 5,9.3, 2.5Hz, 2H), 2. 21 (s, 6H), 2. 22 -2.19 (m, 1H), 1.78T.69(m,3H), 1.39(s,9H).

Preparation of Compound 101

[0619] A suspension of 100 (1.60 g, 2.30 mmol) and 10% Pd/C (400 mg) in a mixture of EtOH (130 mL) and AcOH (20 mL) was degassed at room temperature, and then placed Hydrogenation conditions (latm) 16h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated in vacuo to obtain the amine salt 101 (1.60g, 99%) as a yellow solid:

[0620] ^XH NMR(400MHz, CD₃0D) 7. 71 (d, J = &amp; 5Hz, 2H), 7. 68 (s, 1H), 7. 61 (s, 1H), 7. 42 (d, J = &amp; 5Hz, 2H), 7. 39(dd, J = &amp; 5,1.3Hz, 1H), 7. 33 (dd, J = &amp; 5,1.3Hz, 1H), 7. 16 (d, J = &amp; 6Hz, 2H), 4. 50 (t, J = 7.6Hz, 1H), 3. 28 (dd, J = 14. 0, 6.3Hz, 1H), 3. 07 (dd, J = 13. 3, 8.7Hz, 1H) ,3.05-2.98(m,2H) ,2.93(t, J = 7.6Hz, 2H), 2.82 (t, J = 7.3Hz,2H),

2. 78(s,6H), 2.65 (t, J = 7.5Hz,2H), 2.06-1.96 (m, 2H), 1.93(s,6H), 1.86-1. 75(m,2H), 1.74T.64(m,2H),1.33(s,9H).

[0621] Preparation of Compound 102;

[0622] At room temperature to amine 101 (1.60g, 2.30mmol) and 3,5-diamino-6-chloropyrazine-2-propenylmethyl thiomethionate (21,1.60g, 4. 14mmol) in EtOH (25mL) solution was added DIPEA (4.1mL, 23.0mmo 1). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, then cooled to room temperature and concentrated in vacuo. By silica gel column chromatography (80: 18: 2CHCl₃/CH₃0H/NH₄0H) The residue was purified to obtain Muscle 102 (645 mg, 37% and 640 mg, 37% impure) as a yellow solid:

[0623] ^XH NMR(400MHz, CD₃0D) 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. ll(d, J = 8.4Hz, 2H), 4. 48 (t, J = 7.6Hz, 1H), 3. 30-3. 22 (m, 3H),

3. 06 (dd, J = 13. 8,&amp; 9Hz, 1H), 2. 83 (t, J = 7.2Hz, 2H), 2. 57 (t, J = 7.9Hz, 2H), 2. 32 ( dd, J = 10. 5, 7.6 Hz, 2H), 2. 23 (s, 6H), 1. 86-1. 74 (m, 4H), 1. 73-1. 64 (m, 2H), 1.32(s,9H).

(S)-3,5-Diamino-N-(N-(4-(6-(2-amino-3-(4-(3-(dimethylamino)propyl)phenylamino )-3-oxopropyl) Cai-2-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide (103) hydrochloride

[0625] TFA (10 mL) was added to C^Cl 47 (545 mg, 0.71 mmol) and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed, IN HC1 was added, and the solvent was removed. The residue was purified by a reverse phase column (Gold column) and lyophilized to obtain compound 48 (206 mg, 50%) as a yellow solid:

[0626] ^XH NMR(400MHz, DMS0-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), &amp; 50 (brs, 3Η), 7.82-7.73 (m, 3Η), 7. 68 (s, 1Η), 7. 53 (d, J = 8. 5Hz, 2H), 7.48 (d, J = 9.2Hz, 1H), 7. 45-7. 35 (m, 3H), 7. 18 (d, J = &amp; 4Hz,2H),

4. 45-4. 35 (m, 1H), 3.74-3. 45 (m, 1H), 3. 27 (dd, J = 14. 7,8.3Hz, 1H), 3. 03-2.93 (m, 2H), 2.78(t, J = 7.3Hz, 2H), 2.70(s,6H), 2.58(t, J = 7.3Hz, 2H), 2. 02T. 88 (m, 2H),

1. 79-1.66 (m, 2H), 1.64-1.54 (m, 2H).

[0627] ^XH NMR(400MHz, CD₃0D) 7. 80 (d, J = 9.2Hz, 1H), 7.78-7.73 (m, 2H), 7.67 (s, 1H), 7.47-7.38(m,4H ), 7. 20 (d, J = &amp; 9Hz, 2H), 4. 33 (t, J = 7.5 Hz, 1H), 3. 46 (dd, J = 13. 6,

6. 6Hz, 1H), 3.37 (t, J = 6.8Hz, 2H), 3.36-3.26 (m, 3H), 2.87-2.83 (m, 2H), 2.87( s,6H),

2. 68 (t, J = 7.6Hz, 2H), 2.07-1.97 (m, 2H), 1.89-1.80 (m, 2H), 1.80-1.71 (m, 2H ).

16. 3,5-Diamino-N-(N-(4-(4-((S)~2~ Amino-3-(4-(3-(hexyl((2S,3R,4R,

5R)-2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetraylcae-1 -Yl)butyl)methanyl)-6-chloropyrazine-2-carboxamide (123)

[0629] Scheme 17

[0630]

(Chuan

<img file="CN105073717A_D0120.tif" />

Dimethyl Sulfate

NaOH, acetone

<img file="CN105073717A_D0121.tif" />

(y^Q in

1. KHMDS, THF

2. TrisylN_3i AcOH

3. (CH₃)₄N^{AeO-

<img file="CN105073717A_D0122.tif" />

POCh, DMF

Ι,ΖΜΧΈ

Bn

OCH

<img file="CN105073717A_D0123.tif" />

DBU, CH.Cb

CHO °γ^ΚΗ

O 8

1. BuLi. THF

2. PivCl.NMM, THF

<img file="CN105073717A_D0124.tif" />

MeOH/THF/H&gt;O

1B9

<img file="CN105073717A_D0125.tif" />

OCH?

<img file="CN105073717A_D0126.tif" />

PcC,ii_?

AcOH/lI.O

OH

OCH;

<img file="CN105073717A_D0127.tif" />

, NH0IRr

O

OH

IP, NH2 o

H2

<img file="CN105073717A_D0128.tif" />

<img file="CN105073717A_D0129.tif" />

Μό( η ι

TIIF/ILO/MeOII

<img file="CN105073717A_D0130.tif" />

<img file="CN105073717A_D0131.tif" />

AcCl

[0631] Scheme 17 (continued)

[0632] PayCOM r one

P(O)(OCH₃h

<img file="CN105073717A_D0132.tif" />

107

OC.'H₃lpd/C, H₂. EtOH

NaOH

<img file="CN105073717A_D0133.tif" />

och₃ m

OH

MeOH

<img file="CN105073717A_D0134.tif" />

_t ^NH₂«HC1

O OCH₃

H5

Boc₂0, NaHCO^

MeOH (Chuan

114

<img file="CN105073717A_D0135.tif" />

<img file="CN105073717A_D0136.tif" />

<img file="CN105073717A_D0137.tif" />

<img file="CN105073717A_D0138.tif" />

<img file="CN105073717A_D0139.tif" />

<img file="CN105073717A_D0140.tif" />

Preparation of Compound 105

To a solution of 104 (100g, 0.675mmol) in anhydrous THF (800mL) at 0°C was added NaOH (32.0mg, 0.809mmol) and dimethyl sulfate (102g, 0.809mmol) dropwise . Stir the reaction mixture for 2h at room temperature_oRemove THF under reduced pressure, and make the mixture in CH₂C1₂(1.OL) and water (1.0L). Separate the water layer and use CH₂C1₂(2X1.OL) extraction. The combined organic extracts were dried and concentrated. Column chromatography (silica gel, 100% CH₂C1₂) The residue was purified to obtain compound 105 (108.0 g, 90%) as a yellow liquid:

[0635] ^XH NMR(400MHz, DMS0-d₆): Δ 7. 06 (t, J = 7.85Hz, 1H), 6. 71 (d, J = 7. 25, 1H),

6. 64 (t, J = 7.7Hz, 1H), 3.80(s,3H), 2.74 (t, J = 2.75Hz,2H), 2.65 (t, J = 2.65Hz,2H ),

1. 81-1.71 (m, 4H).

Preparation of Compound 106

[0637] Add POC1 dropwise to a solution of anhydrous DMF (71.45mL, 0.923mmol) under a nitrogen atmosphere at 0°C₃(57.40mL, 0.616mmol)_oThe reaction mixture was stirred at 0°C for 30 minutes. A solution of 105 (50.0 mg, 0.38 mmol) in anhydrous 1,2-dichloromethane (500 mL) was added to the reaction mixture at 0° C. under a nitrogen atmosphere. After the addition is complete, the reaction mixture is heated to 80°C for 6h_oUse cold H₂0 quenched, and make it in CH₂C1₂(1.OL) and water (1.0L). Separate the water layer and use CH₂C1₂(2X1.OL) extraction. The combined organic extracts were dried over NahOq and concentrated. The residue was purified by column chromatography (silica gel, 5% EA/hexane) to give the product

Be the compound 106 (35.0g, 61%) of yellow solid:

[0638] ^XH NMR(400MHz, DMS0-d₆): δ 10. 10 (s, 1Η), 7. 65 (d, J = 7. 81, 1H), 6. 78 (d, J =

7. 47Hz, 1H), 3.89(s,3H), 3.18(t, J = 5.80Hz,2H), 2.70(t, J = 4.64Hz,2H), 1.82-1. 73 (m, 4H).

Preparation of Compound 107

[0640] Add trimethyl phosphonoylacetate (55.0 mL, 0.378 mmol) cooled to 0°C in 100 mL anhydrous CH₂C1₂DBU (58.0 g, 0.380 mmol) was added to the solution in and the mixture was stirred for 15 minutes. A solution of aldehyde 106 (16.0 g, 0.084 mmol) in 50 mL C^Cl? was added dropwise. The reaction mixture was warmed to room temperature, stirred for 16 h and quenched with 100 mL of water. Separate the mixture and use CH₂Cl₂(3X150 mL) The aqueous layer was extracted. The combined organics were washed with brine, dried (N^SOJ, filtered and concentrated, and the residue was purified by silica gel column chromatography (10:1 hexane/ethyl acetate) to give cis and trans α-forms as white solids. Beta unsaturated ester 107 (15.0g, 72%):

[0641] ^XH NMR (400MHz, DMS0-d₆): δ 7. 83 (d, J = 14. 7Hz, 1H), 7. 58 (d, J = &amp; 3Hz, 1H), 6. 82 (d, J = &amp; 4Hz, 1H), 6. 35 ( d, J = 15. 2Hz, 1H), 3. 80 (s, 3H), 3. 70 (s, 3H), 2. 76 (t, J =

5. 7Hz,2H), 2.55 (t, J = 5.4Hz,2H), 1.80-1.60(m,4H).

Preparation of Compound 108

[0643] A suspension of 107 (33.0 g, 0.134 mmol) and 10% Pd/C (15 g, 0.127) in EtOH (300 mL) was placed under hydrogenation conditions (latm) for 3 h at room temperature. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated under vacuum to obtain 108 (28.0 g, 90%) as a white solid:

[0644] ^XH NMR(400MHz, CDC1₃): δ 7.65 (d, J = 7.62,1H), 6.78 (d, J = 7.96Hz,lH),

4. 06-4. 11 (m, 1H), 3. 78 (s, 3H), 2. 86 (t, J = 7.79Hz, 2H), 2. 69-2.64 (m, 4H), 2. 57-2.51 (m, 2H), 1.79-1.74 (m, 4H).

Preparation of Compound 109

To methyl ester 108 (2 &amp; 0g, 0.16mmol) in THF/MeOH/H₂0 (200mL/200mL/60mL) NaOH (25.0g, 0.625mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 3h. The solvent was removed and the pH was adjusted to 1 with IN HC1 aqueous solution; a white solid was precipitated and filtered, washed with water, and dried under vacuum to obtain acid 109 (25.5 g, 92%) as a white solid:

[0647] ^XH NMR(400MHz, CDC1₃): δ 6.96 (d, J = 7.29,1H), 6.63 (d, J = 6.86Hz,lH),

3. 78 (s, 3H), 2.88 (t, J = 7.29Hz,2H), 2.69-2.66 (m, 4H), 2.63-2.59 (m, 2H), 1. 80-1.73 (m, 4H).

Preparation of Compound 110

[0649] To 60 (13.70g, 77.31mmol) in anhydrous THF (200mL) solution was added dropwise n-butyllithium (45.07mL, 90.08mmob 2M in cyclohexane at -78°C) Solution), and the reaction mixture was stirred for 1 h to obtain a solution of lithium salt 61. In another solution of 109 (15.0g, 64.37mmol) in anhydrous THF (200mL) at -78°C, NMM (9.30mL, 83.64mmol) and PivCl (10.30mL, 83. . 64mmol). The reaction mixture was stirred for 30 minutes, and the temperature was raised to -20°C for 1 h, and the prepared lithium salt solution was slowly added at -78°C. The reaction mixture was stirred for another 10 minutes, placed at 0°C and stirred for 1 h, placed at room temperature and stirred for 30 minutes, with saturated NH₄C1 is quenched, concentrated to remove THF and in CH₂Cl₂(300mL) and water (100mL) partition. Separate the water layer and use CH₂C1₂(150 mL) extraction. The combined organic extracts were dried over NazSOq and concentrated. By column chromatography (silica gel, CH₂C1₂) The residue was purified to obtain compound 110 (15.0 g, 60%) as a white solid.

Preparation of compound 111

[0651] To a solution of 110 (15.0 g, 38.14 mmol) in anhydrous THF (250 mL) was added KHMDS (13.70 g, 68.67 mmol) in batches at -78°C. The resulting mixture was stirred for 30 minutes, triisopropylbenzenesulfonyl azide (19.0 g, 61.40 mmol) was added and the reaction mixture was stirred for 5 minutes. Acetic acid (15.0 mL, 228 mmol) and tetramethyl acetate (30.9 g, 76.28 mmol) were slowly added at the same temperature. The reaction mixture was warmed to 24 °C, stirred for 16h, and saturated NaHCO was used.₃ (100mL) quenched, concentrated to remove THF, and used CH₂C1₂ (300 mL) extraction. The combined organic extracts were dried over Na^SOq and concentrated. The residue was purified by column chromatography (silica gel, 90: 10 hexane/EtOAc, then DCM was added) to give compound 111 (&amp; 80 g, 54%) as a yellow solid,

[0652] ^XH NMR (400MHz, CDC1₃): δ 7. 36-7. 30 (m, 3H), 7. 23 (m, 1H), 7. 20 (m, 1H), 7. 16 (m, 1H), 7. 01 (d, J = 7.79Hz, 1H), 6.60 (d, J = 7.59Hz,2H), 5.35(t, J = 7.99,2H),

4. 89 (s, 1H) ,4.58-4.51 (m, 1H), 4.13-4.10(m,3H) ,3.93 (t, J = 7.54, 1H), 3. 77(s,3H),

3. 33-3.27 (m, 3H), 2.71 (m, 2H), 2.63 (m, 2H), 1.78-1.75 (m, 5H), 1.58 (m, 2H) .

Preparation of Compound 112

[0654] To 111 (31.0 mmol g, 72.1 mmol) of THF/H at 0 °C₂0(300mL/100mL) add H to the solution₂0₂ (49 mL, 433 mmol), then LiOH (6.04 g, 144 mmol) was added in portions. The reaction mixture was stirred at 0 °C for 10 minutes, and at room temperature for 1 h, with saturated Na₂S0₃(200mL) quenched, concentrated under reduced pressure to remove THF, and used CH₂Cl₂(500mL) Wash. Acidify the water layer with IN HC1 water solution and use CH₂C1₂(2X500mL) extraction. The combined organic extracts were dried over NahOq, concentrated, and washed with MTBE to obtain compound 112 (15.0 g, 82%) as an off-white solid:

[0655] ^XH NMR(400MHz, CD₃0D): δ 6.92 (d, J = 7.7Hz, 1H), 6.63 (d, J = &amp; ΟΗζ,ΙΗ),

3. 75 (s, 3H), 2.81 (t, J = 7.8Hz,2H), 2.67 (t, J = 6.0Hz,2H), 2.61 (t, J = 5.7Hz,2H ),

2. 49-2.47 (m, 2H), 1.84-1.70 (m, 6H).

Preparation of Compound 113

[0657] 112 (15.0 g, 55.1 mmol) and 10% Pd/C (3.50 g) in AcOH/

H₂The suspension in 0 (300mL/100mL) was placed under hydrogenation conditions (latm) for 3h. The reaction mixture was filtered with celite and used Ac0H/H₂0 wash, followed by MeOH wash. The filtrate was concentrated under vacuum to obtain acetate 113 (14.0 g, 83%) as a yellow solid.

Preparation of compound 114

To a solution of 113 (11.0 g, 44.1 mmol) in acetic acid (120 mL) was added dropwise hydrogen bromide acid (120 mL) at room temperature, and the reaction mixture was refluxed for 3 h. The reaction mixture was cooled to room temperature and concentrated. The crude brown residue 114 (&amp;90g, 80%) was used directly in the next step without purification.

[0660] ^XH NMR(400MHz, CDC1₃): δ 6.80 (d, J = 7.85,1H), 6.57 (d, J = 7.21Hz,lH),

3. 92-3. 91 (m, 1H), 3. 04-2.98 (m, 1H), 2. 91-2.86 (m, 1H), 2.61 (m, 2H), 2. 54- 2. 53 (m, 2H),

1. 69-1.68(m,5H).

Preparation of Compound 115

[0662] Acetyl chloride (17.0 mL, 243 mmol) was added to anhydrous methanol (300 mL) at 0° C., and 114 (&amp; 90 g, 28.2 mmol) was added. The reaction mixture was refluxed for 4h and concentrated. Make the residue in CH₂C1₂(200mL) and saturated NaHC0₃(100mL). Separate the water layer and use CH₂C1₂(200 mL) extraction. The combined organic extracts were dried over N^SOq and concentrated to give compound 115 (7.30 g, 90%) as a white solid:

[0663] ^XH NMR(400MHz, CDCI3): δ 6.81 (d, J = 7.51,1H), 6.59 (d, J = 7.21Hz,lH),

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 = 6.16Hz, 2H).

Preparation of Compound 116

[0665] To 115 (7.30g, 25.60mmol) MeOH/H at 0°C₂0 (100mL/60mL) Add NaHC0 to the solution₃(12. Og, 145mmol) and Boc₂0 (10.0 g, 45.8 mmol). The resulting mixture was warmed to room temperature and stirred for 1 h. Make the reaction mixture in CH₂C1₂Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂(100 mL) extraction. The combined organic extracts were washed with brine, dried over Nahoq and concentrated. Use 20% ethyl acetate/hexane followed by flash column chromatography of C&amp;C12 to obtain compound 116 (7.1g, 81%) as a white solid:

[0666] ^XH NMR(400MHz, CDCI3): δ 6.77 (d, J = 7.36,1H), 6.55 (d, J = 7.86Hz,lH),

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, 1H), 2. 86 (m, 2H), 2 . 63 (m, 2H), 1. 80-1. 78 (m, 4H),

1. 39(s,9H).

Preparation of Compound 117

[0668] To 116 (7.0 g, 20.05 mmol) CH at 0°C₂C1₂ (80mL) The solution was added pyruvate (100mL) and triflate (4.64mL, 24.0mmol), stirred for 1h, and stirred at room temperature for 2h. After concentration, make the reaction mixture in CH₂Cl₂Partition between (150 mL) and water (70 mL). Separate the water layer and use CH₂C1₂(100 mL) extraction. The combined organic extracts were washed with brine, dried over NapSOq, and concentrated to give compound 117 (&amp; 00g, 83%) as a brown oil:

[0669] P NMR (400MHz, CD₃0D): δ 8. 81 (d, J = 4. 63Hz, 5H), 8. 56-8. 51 (m, 2H), &amp; 0 2-7. 99(m,4H), 7. ll(d , J = 7.98Hz, 1H), 7. 03 (d, J = 7.98,1H), 4. 39-4. 35 (m, 1H),

3. 68 (s, 3H), 3.19-3.14 (dd, 1H), 2.90-2.77 (m, 5H), 1.86-1.81 (m, 4H), 1.35 ( s, 9H),

1. 32-1.28(m,4H).

Preparation of Compound 118

[0671] Nitrogen will be used in anhydrous CH at room temperature₃CN (100mL) compound 117 (8.0g, 16.6mmol) and but-3-alkynyl carbamate benzyl ester (10,5.0g, 24.9mmol) degas for 10 minutes, and add TEA (9 . 34mL, 66. 50mmo 1), 10% in hexane (t-Bu) ₃P (7.0 mL, 3.32 mmol) and Cui (0.16 g, 0.84 mmol). The resulting mixture was degassed with nitrogen for 10 minutes, and Pd(PPh₃)₄(2.00g, 1.73mmol). After degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 16 h. The reaction mixture was concentrated under vacuum, and the residue was purified by column chromatography (silica gel, 75: 25 B alkane/ethyl acetate) to give compound 118 (4.50 g, 52%) as a brown solid:

[0672] ^XH NMR (400MHz, CD₃0D): δ 7. 36-7. 34 (m, 4H), 7. 33-7. 29 (m, 2H), 7. 16 (d, J =

7. 63Hz, 1H), 6.82(d, J = 7.02Hz, 1H), 5.12-5.08 (m, 2H), 4.95 (d, J = 7.88Hz, 1H),

4. 52-4. 51 (m, 1H), 3. 67 (s, 3H), 3. 48-3. 34 (m, 2H), 3. 10-3. 05 (dd, 1H), 2. 84- 2. 83 (m, 2H),

2. 68-2.65 (m, 4H), 1.81-1.76 (m, 4H), 1.39 (s, 9H).

Preparation of Compound 119

To methyl ester 118 (4.50g, &amp; 42mmol) in THF/MeOH/H₂0 (30mL/30mL/10mL) NaOH (3.60g, 90mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 3h. Adjust the pH to 9 with IN HCl aqueous solution and remove the organic solvent. Adjust the pH of the residue to 5-6, and make the suspension in CH₂C1₂Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂(100 mL) extraction. The combined organic extracts are dried and concentrated

Shrink, obtain compound 119 (3.66g, 85%) as brown solid,

[0675] ^XH NMR (400MHz, CD3OD): 6 7. 28-7. 24 (m, 5H), 7. 05-7. 03 (d, J = 7.67Hz, lH),

6. 89-6.87 (d, J = 7.55Hz, 1H), 5.04 (brs, J = 7.02Hz, 1H), 5.12-5.08 (m, 2H), 4. 95 (d , J = 7.88Hz, 1H), 4.52-4.51 (m, 1H), 3.67(s,3H), 3.48-3.34 (m, 2H), 4.27-4 . 26 (m, 1H), 3. 38-3. 30 (m, 2H), 3. 15-3. 10 (m, 1H), 2. 78-2.71 (m, 4H), 2. 59 (d, J = 5.95, 2H),

1. 73-1.71 (m, 4H), 1. 31 (s, 9H).

Preparation of Compound 120

To compound 119 (800mg, 1.53mmol) in THF (30mL), DEPBT (845mg,

2. 56mmol), 24 (700mg, 2.33mmol) and DIPEA (1.0mL, 4.65mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(50 mL), quickly washed with saturated aqueous solution (50 mL) and brine (50 mL), and dried over Nahoq. The solvent was evaporated, and flash chromatography on silica gel (6% methanol/CH₂C1₂) The crude product was purified to obtain amide 120 as a yellow solid (1.0 g):

[0678] P NMR (400MHz, CDC1₃): δ 7. 46-7. 44 (m, 3H), 7. 36-7. 30 (m, 7Η), 7. 17 (d, J = 7. 2Hz, 2H), 7.07 (d, J = 7. 5Hz, 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 = 9.9Hz, 1H), 3. 43 (t, J = 5.7Hz, 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 = 6.3Hz,3H).

Preparation of compound 121

[0680] A suspension of 120 (1.00 g, 1.01 mmol) and 10% Pd/C (600 mg) in EtOH (50 mL) and AcOH (2 mL) was degassed at room temperature and placed under hydrogenation conditions (1 atm ) Next 12h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 121 (700 mg, 80%) as a white solid

[0681] ^XH NMR (400MHz, CDCI3): 6 7. 49-7. 41 (m, 2H), 7. 34-7. 30 (m, 5H), 7. 12-6. 78 (m, 5H), 4. 30-4.27 (m,2H), 4.19-4.18 (m, 1H), 3.98-391 (m, 2H), 3.78-3.58 (m, 2H),

3. 19-3.08 (m, 3H), 3. 02-2.89 (m, 6H), 2.75-2.73 (m, 2H), 2.65-2.62 (m, 3H), 2.55-2.52 (m, 3H), 1.98-1.92 (m, 2H), 1.73-1.68 (m, 3H), 1.60-1.52 (m, 7H ), 1.41 (s, 9H), 1.29-1.20 (m, 7H), 0.88-0.84 (m, 3H), 0.87 (t, J = 6.4Hz, 3H ).

Preparation of Compound 122

[0683] To the amine salt 121 (700 mg, 0.81 mmol) and 3,5-diamino-6-chloropyrazine-2-methylpyrimidine methyl thioate (13,680 mg, 1.75 mmol) at room temperature ) DIPEA (1.60 mL, 9.26 mmol) was added to the solution in Et0H (20 mL). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature and concentrated under vacuum. By column chromatography (silica gel, 80: 18: 2 CHCl₃/CH₃0H/NH₄0H) The residue was purified to obtain muscle 122 (380g, 48%) as a yellow solid:

[0684] ^XH NMR (400MHz, DMS0-d₆): 6 7. 43-7. 39 (m, 3H), 7. 33-7. 31 (m, 3H), 7. 05 (d, J = 6. 69Hz, 2H), 6. 99-6. 95(m,2H), 6.86 (d, J = 7.59Hz, 1H), 5.47 (s, 1H) ,4.33-4.31 (m, 1H),

4. 14-4. 10 (m, 1H), 3.79-3.72 (m, 4H), 3. 68-3.65 (m, 2H), 2.69-2.66 (m, 6H), 2. 56-2. 53 (m, 3H), 2. 45-2. 36 (m, 7H), 1. 70 (m, 4H), 1. 56 (m, 6H), 1. 32 (s, 9H), 0.86 (t, J = 7.0 Hz, 3H).

[0685] 3, 5~ Diamino-N- (N~ (4~ (4~ ((S) ~2~ amino-3- (4~ (3~ (hexyl((2S, 3R, 4R, 5R) -2,3,

4,5,6-Pentylhexyl)amino)propyl)phenylamino)~3-oxopropyl)-5,6,7,8-tetrakisyl ((Ze-1-yl)butyl)methan Preparation of hydrochloride of 6-chloropyrazine-2-carboxamide (compound 123);

[0686] 4N HC1 (15 mL) in dioxane was added to 122 (350 g, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column) and the residue was lyophilized to obtain 110 mg (45%) of compound 123 as a yellow solid:

[0687] ^XH NMR(400MHz, DMS0-d₆): δ 10.16 (s, 1H), 9.16 (brs, 1H), 8.51-8.34 (brs, 2H), 7.41 (t, J = &amp; 1Ηζ, 4Η), 7. 20(d, J = 8.6Hz, 2H), 6. 95-6.89 (q, 2H), 5. 42 (brs, 1H),

4. 42 (m, 1H), 4. 53 (d, J = 5. 3Hz, 2H), 4. 42 (m, 1H), 4. 01 (m, 1H), 3. 93 (m, 1H), 3 . 60 (m, 1H), 3. 50-3. 38 (m, 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. 0Hz, 3H).

[0688] ^XH NMR(400MHz, D₂0): δ 7. 08 (d, J = 8.4Hz, 2H), 7. 04-6.98 (q, 2H), 6.92 (d, J = 8.3Hz, 2H), 4.06 -4.02(m,2H),3.78-3.69(m,3H),3.62-3.54(m,2H),3.25(t, J =

5. 3Hz, 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).

[0689] 17. 3, 5~ Diamino ^_N~ (N~ (4- (4- ((S) ~2~ amino ^_3- (4- (3-(Double((2S, 3R, 4R, 5R) -2,3,

4,5,6-Pentylhexyl)amino)propyl)phenylamino)~3-oxopropyl)-5,6,7,8-tetravinyl(Cai-1-yl)butyl)methan Preparation of Methyl)-6-Chlorpyrazine-2-Carboxamide (127)

Scheme 18

[0691]

<img file="CN105073717A_D0141.tif" />

'N sugar

Sugar sugar,

OH OH

<img file="CN105073717A_D0142.tif" />

'Ά Ύ fuse'

ΟγΟ

Ph

<img file="CN105073717A_D0143.tif" />

125

NH₂*AcOH

Ο

<img file="CN105073717A_D0144.tif" />

<img file="CN105073717A_D0145.tif" />

DIPEA. EtOH

Ο

NH nh₂)3

<img file="CN105073717A_D0146.tif" />

126

OH

HO

<img file="CN105073717A_D0147.tif" />

NH O

JL

Ν NH II II

HrN People N'NFL

<img file="CN105073717A_D0148.tif" />

Cl

<img file="CN105073717A_D0149.tif" />

<img file="CN105073717A_D0150.tif" />

OH

N .,,OH .,'OH

HO''

<img file="CN105073717A_D0151.tif" />

HO (S)

4N HC1 aqueous solution, EtOH

NH

<img file="CN105073717A_D0152.tif" />

NH₂

127

Ν N

Η H

H₂N'

<img file="CN105073717A_D0153.tif" />

Preparation of Compound 124

[0693] To compound 119 (1.0g, 1.92mmol) in THF (30mL) was sequentially added DEPBT (845mg,

2. 82mmol), 29 (1. 25g, 1.91mmol) and DIPEA (1.0mL, 5.73mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(50 mL), quickly washed with saturated aqueous solution (50 mL) and saline (50 mL), and dried with blood paw 04. The solvent was evaporated, and flash chromatography on silica gel (5% methanol/CH₂C1₂) The crude product was purified to obtain amide 124 [900 mg (mixture)] as a yellow solid.

Preparation of Compound 125

[0695] A suspension of 124 [900 mg (mixture), 0.77 mmol] and 10% Pd/C (600 mg) in a mixture of EtOH (50 mL) and AcOH (1.5 mL) was degassed at room temperature and placed Under hydrogenation conditions (latm) for 12h. The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain crude product 125 (800 mg) as a colorless oil.

Preparation of Compound 126

[0697] At room temperature, give the crude product 125 (800 mg) and 3,5-diamino-6-chloropyrazine-2-pyrimyl methyl thiomethionate (13,400 mg, 1.02 mmol) DIPEA (1. 10 mL, 6.38 mmol) was added to the Et0H (40 mL) solution. The reaction mixture was heated in a sealed tube at 70°C for 2 hours, cooled to room temperature, and concentrated under vacuum. By column chromatography (silica gel, 80: 18: 2 CHCl₃/CH₃0H/NH₄(OH) The residue was purified to obtain M 126 (285 mg, 12% yield over 3 steps) as a yellow solid:

[0698] ^XH NMR (400MHz, DMS0-d₆): 6 7. 44-7. 42 (m, 4H), 7. 30-7. 28 (m, 6H), 7. 22 (d, J =

7. 27Hz,2H), 6.96 (d, J = 7.llHz,2H), 6.91 (d, J = 7.0Hz,2H), 6.86 (d, J = 7.61Hz,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. 29Hz, 1H), 3. 69 (d, J = 2. 2Hz, 1H), 3. 58 (t, J = 10. 08Hz, 2H), 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. 74T. 60 (m, 10H), 1.38 (s, 9H).

[0699] 3, 5~ Diamino ^_N~ (N~ (4- (4- ((S) ~2~ amino ^_3- (4- (3-(Double((2S, 3R, 4R, 5R) -2,3,4,

5.6- Pentayl (hexyl) amino) propyl) phenylamino) -3-oxopropyl) -5,6,7,8-tetrakisyl (Cai-1-yl) butyl) methyl)- Preparation of the hydrochloride salt of 6-chloropyrazine-2-carboxamide (Compound 127)

[0700] 4N HC1 (10 mL) in dioxane was added to 126 (1.15 g, 0.23 mmol) in EtOH (3.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to obtain 62 mg (32%) of compound 127.

[0701] ^XH NMR (400MHz, DMS0-d₆): δ 10. 39 (brs, 1Η), 10. 03 (brs, 1H), 8.91-&amp;82(brs, 2H), 8.48(brs, 2H), 7.42(d, J = 7 . 6Hz,4H), 7.18 (d, J = 7.6Hz, 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. 4Hz, 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 (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).

[0702] ^XH NMR(400MHz, D₂0): δ 7. 10 (d, J = &amp; 30Hz, 2H), 7. 02-6.90 (m, 2H), 6.91 (d, J = 7.42Hz, 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 = &amp; 2Hz,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).

[0703] 1&amp; 3, 5~ Diamino ^_N~ (4~ (4- ((S) ~2~ Amino ^_3-oxo ^_3- (4- (3- ((2S, 3R, 4R, 5R) ~2,

3.4.5.6- Pentayl (hexylamino) propyl) phenylamino) propyl) -5,6,7,8-tetrakisyl (Cai-1-yl) butylcarbamoyl)-6-chloropyrazine Preparation of -2-carboxamide (131)

[0704] Scheme 19

[0705]

<img file="CN105073717A_D0154.tif" />

<img file="CN105073717A_D0155.tif" />

N^/Cl

Ν NH?

NH,

Ν

Preparation of compound 128

[0707] To compound 119 (1.00 g, 1.92 mmol) in THF (30 mL) was sequentially added DEPBT (862 mg,

2. 88mmol), 34 ( 1.50g, 2.98mmol) and DIPEA (1.0mL, 5.76mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(50 mL), quickly washed with saturated aqueous solution (30 mL) and brine (20 mL), and dried over Nahoq. The solvent was evaporated and flash chromatography on silica gel (6% methanol/CH₂C1₂) The crude product was purified to obtain amide 128 (780 mg, 42%) as a yellow solid:

[0708] P NMR (400MHz, CDC.: 6 7.49 (m, 3H), 7.31-7.29 (m, 10H), 7. 00-7. 08 (m, 3H), 6.94 (d, J = 7.4Hz, 1H) ,5.54 (m, 1H), 5.50-5.49 (m, 1H) ,5.08(s,2H) ,4.36 (m, 1H ), 4. 26-4. 22 (m, 2H), 4. 05 (m, 2H), 3. 95-3.91 (m, 1H), 3. 80 (m, 2H), 3. 64- 3. 59 (m, 1H),

3. 52-3. 48 (m, 1H), 3. 14-3. 06 (m, 1H), 2.94-2.89 (m, 1H), 2. 79 (d, J = 16. 12Hz, 4H ),

2. 63 (t, J = 5.98Hz, 1H), 2.51 (t, J = 6.9Hz, 1H), 1.82-1.75 (m, 7H), 1.41 (s, 18H).

Preparation of Compound 129

[0710] A suspension of 128 (780 mg, 0.776 mmol) and 10% Pd/C (300 mg) in a mixture of EtOH (30 mL) and AcOH (1.0 mL) was degassed at room temperature and placed in hydrogenation 12h under condition (latm). The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum to obtain amine salt 129 (720 mg, 85%) as a white solid

[0711] ^XH NMR (400MHz, CDC1₃): δ 7.49-7.46 (m, 2Η), 7.32-7.30 (m, 5Η), 7.08-7.06 (d, J = 7.2Hz, 1H), 6. 88(d, J = 7.4Hz, 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, 1Η), 3.78-3.76 (m, 1H), 3.71-3.69 ( m, 1H), 3.60 (t, J = 9.9Hz, 1H), 3.48-3.46(m, 1H), 3.09-3.04(m, 1H), 2.89( t, J = 7. 3Hz, 3H), 2. 79 (m, 2H), 2.69 (m, 2H), 2. 58 (t, J = 6.5 Hz, 2H), 2. 51 (t, J = 6.8Hz, 2H), 1.84T. 77 (m, 6H),

1. 67-1.66 (m, 2H), 1.61-1.57 (m, 2H), 1.41 (s, 18H).

Preparation of Compound 130

[0713] To amine salt 129 (720 mg, 0.77 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl thioate (13,456 mg, 1.17 mmol) at room temperature ) DIPEA (1.12 mL, 6.24 mmol) was added to the solution in Et0H (20 mL). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature, and concentrated under vacuum. Obtain Arc 130 (380 mg, 45%) as a yellow solid:

[0714] P NMR (400MHz, CDC.: 6 7. 48-7. 46 (m, 2H), 7. 30 (t, J = 2. 70 Hz, 5H), 7. 08-7. 06 (d, J = 7.6Hz, 2H), 6.93 (d, J = 7.1Hz, 1H), 6.88 (d, J = 7.3Hz, 1H), 5.53 (s, 1H), 4. 34 (m, 1H), 4. 25-4. 21 (m, 1H), 4. 04 (m, 1H), 3. 96-3.90 (m, 1H), 3. 79 (m, 2H) ,

3. 60 (t, J = 10. ΟΗζ, 1H), 3. 50-3.46 (m, 1H), 3. 25 (t, J = 5.9Hz, 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 = 6.0Hz,2H), 2. 48 (t, J =

6. 8Hz, 2H), 1. 82-1.74 (m, 6H), 1. 67-1. 64 (m, 5H), 1. 40 (s, 18H).

[0715] 3,5-Diamino-N- (4- (4- ((S) ~2~ amino-3-oxo-3- (4- (3- ((2S, 3R, 4R, 5R) -2,3,4,

5,6-Pentyl (hexylamino) propyl) phenylamino) propyl) -5,6,7,8-tetra-yl (Cai-1-yl) butylcarbamoyl)-6-chloropyrazine Preparation of -2-carboxamide (131) hydrochloride;

[0716] 4N HC1 (25 mL) in dioxane was added to 130 (350 mg, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h_oThe solvent was removed, the mixture was purified by reverse phase chromatography (Gold column), and the residue was lyophilized to give compound 131 (125 mg, 48%) as a yellow solid:

[0717] P NMR (400MHz, CD₃0D): 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 = 1.40Hz , 1H), 3. 78-3.75 (m, 1H), 3. 68-3.66 (m, 3H), 3. 36 (t, J = 6. 3,2H), 3. 18-3 . 15 (m, 4H),

3. 04-3. 00 (m, 2H), 2.76 (t, J = 5. 3Hz, 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).

[0718] ^XH NMR(400MHz, D2O): 6 10. 46(s, 1H), 9. 31 (br, 1H), 8. 55(br, 4H), 7. 45(d, J = 6. 6, 4H) , 7.20 (d, J = 7.62Hz, 2H), 7. 00 (d, J = 6. 6,1H), 6.93 (d, J = 6. 6Hz,lH),

5. 43 (d, J = 3. 8Hz, 1H), 4. 79 (d, J = 5.38 1H), 4. 64-4. 63 (m, 2H), 4. 46 (t, J = 4. 9Hz, 1H),

4. 15 (t, J = 4.6Hz,lH), 3.96-3.94(m, 1H), 3.71(m, 1H), 3.64-3.61(m, 1H), 3. 51-3.45(m, 3H), 2.96-2.92 (m, 3H), 2.78-2.77 (m, 2H), 2.68-2.65 (m, 2H), 2.62 (t, J = 6. 6Hz, 2H),

1. 95-1.94 (m, 2H), 1.76-1.15 (m, 8H).

19. (S)-3,5-Diaminos-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)benzene (Amino)-3-oxopropyl)-5,6,7,8-tetrakisyl (Cai-1-yl)butyl)methyl)-6-chloropyrazine-2-carboxamide (135) Preparation

[0720] Scheme 20

[0721]

<img file="CN105073717A_D0156.tif" />

Ο ΝΗ·ΗΙ

<img file="CN105073717A_D0157.tif" />

DIPEA. EtOH

<img file="CN105073717A_D0158.tif" />

<img file="CN105073717A_D0159.tif" />

<img file="CN105073717A_D0160.tif" />

Cl nh₂

Preparation of Compound 132

To compound 119 (700mg, 1.34mmol) in THF (30mL) was added DEPBT (600mg,

2. OOmmol), 18 (360mg, 1.51mmol) and DIPEA (0.80mL, 4.03mmol) and stirred at room temperature for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂Cl₂(50 mL), quickly washed with saturated aqueous solution (50 mL) and brine (50 mL), and dried over Nahoq. Evaporate the solvent and pass through flash chromatography on silica gel (6% methanol/CH₂C1₂) The crude product was purified to obtain amide 132 [800 mg (mixture)] as a yellow solid product.

[0724] ^XH NMR (400MHz, DMS0-d₆): δ &amp; 13 (d, J = 7.54Hz, 1H) ,8.03 (d, J = 7.7Hz, 1H),

7. 89-7.85(m, 1H), 7.71(t, J = 7.52Hz, 1H), 7.64-7.59 (m, 2H), 7.44 (d, J = 7. 7Hz ,2H),

7. 33-7.30(m,6H), 7.12-7.06 (m, 3H), 7. 0 (d, J = 7.6Hz, 1H), 5.02(s,2H), 2. 70(m,4H),

2. 63-2.61 (m, 5H), 2.45 (m, 5H), 1.83 (s, 6H), 1.69-1.65 (m, 3H), 1.33 (s, 9H) .

Preparation of Compound 133

[0726] A suspension of 132 [800mg (mixture), 1.01mmol] and 10% Pd/C (350mg) in a mixture of EtOH (30mL) and AcOH (1mL) was degassed at room temperature and placed in hydrogenation 12h under condition (latm). The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum and passed through column chromatography (silica gel, 80: 18: 2 CHCl₃/CH₃0H/NH₄OH) was purified to obtain compound 233 (500 mg, 67% yield over 2 steps) as a yellow solid:

[0727] ^XH NMR(400MHz, DMS0-d₆): Δ 7. 31 (d, J = 7. 54Hz, 2H), 7. 12 (d, J = 7. 1Hz, 2H), 6. 93(d, J = 7. 2Hz, 1H), 6. 88(d, J = 6.8Hz, 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).

Preparation of Compound 134

[0729] To the amine salt 133 (500mg, 0.90mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (13,530mg, 1.36mmol) at room temperature ) DIPEA (1. 30 mL, 7.25 mmol) was added to the solution in Et0H (20 mL). The reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature, and concentrated under vacuum. The residue was purified by column chromatography to obtain Mus 134 (285 mg, 42%) as a yellow solid:

[0730] ^XH NMR(400MHz, DMS0-d₆): δ 7. 29 (d, J = 7.5 Hz, 2H), 7. 10 (d, J = &amp; 1Ηζ, 2Η),

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 (m, 4H), 1.39 (s, 9H).

(S)-3,5-Diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenylamino )-3-oxopropyl)-5,6,7,8-tetraoyl (Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide (compound 135) Preparation of hydrochloride;

[0732] 4N HC1 (10 mL) in dioxane was added to 134 (380 g, 0.35 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (C18 Gold column), and the residue was lyophilized to give compound 135 (125 mg, 49%) as a yellow solid:

[0733] ^XH NMR(400MHz, DMS0-d₆): δ 10.69 (brs, 1Η), 10. 54-10.50 (d, J = 16. 7Hz, 2H), 9. 32 (t, J = 4.8Hz, 1H), 8.96 ( brs, 1H) ,8.86 (brs, 1H) ,8.58 (brs, 3H), 7.42 (d, J = &amp; 0Hz, 4H) ,7.18 (d, J = &amp; 2Hz,2H) , 6.96 (d, J = 7.3Hz, 1H), 6.88 (d, J = 7.4Hz, 1H), 4.15 (t, J = 4.4Hz, 1H), 3. 36- 3.32 (m, 2H), 3. 09-3. 06 (m, 2H), 3. 00-2.95 (m, 2H), 2.74-2.73 (m, 1H), 2. 22 (s, 6H), 2.64 (m, 2H), 257-2.56 (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).

[0734] ^XH NMR(400MHz, D₂0): δ 7. 08 (d, J = 7.7Hz, 2H), 7. 00-6.97 (q, 2H), 6.91 (d, J = 8. lHz, 2H), 4.12 -4. 08(q, 1H), 3. 25(t, J = 5.2Hz, 3H), 3. 21-3. 17 (m, 1H), 3. 10 (t, J = 9. 8Hz, 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).

[0735] 20. (S)-2-Amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-pyridyl) cucurbityl)butyl )-5,

Preparation of 6,7,8-tetra-urn base (Cai-1-yl) propionic acid (139)

[0736] Scheme 21

[0737]

100

<img file="CN105073717A_D0161.tif" />

<img file="CN105073717A_D0162.tif" />

ΝΗ·ΗΙ χ

SCH,

<img file="CN105073717A_D0163.tif" />

<img file="CN105073717A_D0164.tif" />

Preparation of Compound 136

[0739] A suspension of 118 (800 mg, 1.49 mmol) and 10% Pd/C (350 mg) in a mixture of EtOH (50 mL) and AcOH (1.0 mL) was degassed at room temperature and placed in hydrogenation 12h under condition (latm). The reaction mixture was filtered through a plug of celite and the plug was washed with MeOH. The filtrate was concentrated under vacuum and passed through column chromatography (silica gel,: 18: 2 CHCl₃/CH₃0H/NH₄OH) was purified to obtain compound 136 (700mg, 93%) as a yellow solid: [0740] Preparation of Compound 137

[0741] To amine salt 136 (700 mg, 1.50 mmol) and 3,5-diamino-6-chlorpyrazine-2-pyridyl methyl thioate (13,880 mg, 2.26 mmol) at room temperature ) Add DIPEA (2.15mL, 12.03mmol) to the solution in Et0H (30mL) _oThe reaction mixture was heated in a sealed tube at 70 °C for 2 h, cooled to room temperature, and concentrated under vacuum.

By column chromatography (silica gel, 80: 18: 2 CHCl₃/CH₃0H/NH₄0H) The residue was purified to give Mus 137 (560 mg, 60%) as a yellow solid:

[0742] ^XH NMR (400MHz, CD₃0D); δ 6. 95-6. 85 (m, 2Η), 4. 32-4. 28 (m, 1Η), 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. OHz,

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). [0743] Preparation of compound 138

To methyl ester 137 (560mg, 0.907mmol) in THF/MeOH/H₂0 NaOH (3.60g, 7.25mmol) was added to the solution (30mL/30mL/10mL), and the reaction mixture was stirred at room temperature for 3h. Adjust the pH value with IN HC1 aqueous solution

101

Section to 9, and remove the organic solvent. Adjust the pH of the residue to 5-6, and make the suspension in CH₂C1₂ Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂ (100 mL) extraction. The combined organic extracts are tested. Dry and concentrate, obtain compound 138 (420mg, 78%) as brown solid:

[0745] ^XH NMR (400MHz, DMS0-d₆); Δ 6. 93 (d, J = 6. 7Hz, 1H), 6. 84 (d, J = 7. 35Hz, 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).

(S) -2-Amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine~2~ dialyl) cucurbityl) butyl)- 5,

The preparation of the HC1 salt of 6,7,8-tetraonyl (Cai-1-yl) propionic acid (Compound 139);

[0747] 4N HC1 (10 mL) in dioxane was added to 138 (420 mg, 0.69 mmol) in EtOH (5.0 mL), and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed, the mixture was purified by reverse phase chromatography (C18 Gold column), and the residue was lyophilized to give compound 139 (200 mg, 49%) as a yellow solid:

[0748] P NMR (400MHz, DMSO-cQ; 6 10. 56 (brs, 1H), 9.36 (t, J = 4.7 Hz, 1H), &amp; 9-&amp; 8 (brs, 2H), 6. 98-6.93 (m, 2H), 3. 95-3.92 (m, 2H), 3. 38-3.35 (m, 2H), 3. 04 (d, J =

7. 0Hz,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).

[0749] ^XH NMR(400MHz, D₂0); δ 7. 43 (brs, 2H), 6. 94 (d, J = 7. 2Hz, 1H), 6. 87 (d, J = 7. 1Hz, 1H), 3. 41 (t, J = 6. ΟΗζ, 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).

[0750] 21. 3,5-Diamino ^_N~ (N~ (4- (4- ((S) ~2~ Amino-3- (4- (3-(Bis((2S, 3R, 4R, 5R) ~2,

3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)choen-1-yl)butyl)methionyl)-6-chloropyrazine- Chiral synthesis of 2-formamide (33)

[0751] Scheme 22

[0752]

102

<img file="CN105073717A_D0165.tif" />

HO

C'HjCN

OH N-Succinimide ¢142)

<img file="CN105073717A_D0166.tif" />

Br

141 ο

<img file="CN105073717A_D0167.tif" />

och₃

NHBoc

H3

12)? j

<img file="CN105073717A_D0168.tif" />

Zn. b- DMF

Pd₂ (dba)^, Sphos

<img file="CN105073717A_D0169.tif" />

\l ΙΒοί

NHCbz ha&lt;)₂(

Pd(PPh₃)₄. Cui (/-Bn)₃P, Et₃N (JI,(A

OCH.;

Τ4(λBia

Cil.C!.

BocW

<img file="CN105073717A_D0170.tif" />

BocMN

NaOH

MeOH/THF/FbO

<img file="CN105073717A_D0171.tif" />

sugar

NHCbz

<img file="CN105073717A_D0172.tif" />

Pd/C, tb (1 atm)

EfOK AcOH

<img file="CN105073717A_D0173.tif" />

<img file="CN105073717A_D0174.tif" />

ΝΗ₇·2ΑνΟΗ

<img file="CN105073717A_D0175.tif" />

Preparation of Compound 141

[0754] A few portions of NBS (142, 12.3 g, 69.4 mmol) were added to a solution of 1- Zeaphene (140, 10.0 g, 69.4 mmol) in ethyl gluten (70.0 mL) within 30 minutes . The resulting mixture was stirred at room temperature for 4 h, concentrated under vacuum, and then water (200 mL) and ethyl acetate (200 mL) were added. The aqueous layer was separated and extracted with ethyl acetate (2×200 mL). The combined organic extracts were washed with brine, dried over N^SOq and concentrated. The residue was purified by column chromatography (silica gel, 4: 1 hexane/EtOAc) to obtain the desired compound 141 (9.50 g, 61%) as a white solid:

[0755] ^XH NMR(400MHz, DMS0-d₆) 6 10. 49 (s, 1H), 8. 20 (dd, J = &amp; 3,0.5Hz, 1H), 8. 02 (d, J =&amp; 3Hz, 1H), 7.66 (dd, J = &amp; 4,1.4Hz, 1H), 7. 64 (d, J = &amp; 1Hz, 1H), 7. 55(ddd, J = &amp; 2,

103

7. 7,1. 1Hz, 1H), 6.83 (d, J = &amp; 2Hz, 1H).

Preparation of Compound 7

[0757] Zinc powder (7.03 g, 107.6 mmol) was added to a side arm round bottom flask purged with flame-dried nitrogen. Anhydrous DMF (50.0 mL) was added via a syringe, followed by a catalytic amount of iodine (1.00 g, 3.94 mmol). It is observed that the resulting mixture undergoes a change from colorless to yellow and back to colorless. Add protected iodoalanine 143 (11.8g, 35.9mmol) at one time, then add a catalytic amount of iodine (1.00g, 3.94mmol), and stir at room temperature for 30 minutes; successfully insert zinc Accompanied by a gentle release of heat. Allow the organozinc reagent to cool to room temperature, then add Pd₂dba₃(82lmg, 0.89mmol), SPhos (736mg, 1.79mmol) and aryl bromide 141 (8.00g, 35.9mmol), and the mixture was heated at 50°C for 16h under a positive pressure of nitrogen_oThe reaction mixture was cooled to room temperature. Add saturated NH₄C1 solution (300 mL) and EtOAc (300 mL), then the mixture was filtered through Celite and washed with EtOAc (100 mL). The aqueous layer was separated and extracted with Et0Ac (2×300 mL). The combined organic extracts were washed with brine and passed through the blood paw. . Dry and concentrate under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexane/EtOAc) to obtain the desired compound 7 (4.60 g, 37%) as a yellow solid:

[0758] ^XH NMR (400MHz, a mixture of CDC rotamers) 6 &amp; 23 (d, J = &amp; 3Hz, 1H), 7. 99 (d, J = &amp; 6Hz, 1H), 7. 54 (t, J = &amp; 04Hz, 1H), 7.48(ddd, J = &amp; 3,6.9,1.3Hz, 1H), 7. 08 (d, J =7.8Hz, 1H), 6.70(d, J = 7.6Hz, 1H), 6.57(br s, 0.2H), 6.45 (br s, 0.2H), 5.91 (br s, 0.65H), 5.05(d, J = 7.7Hz, 0.75H), 4.89 (br s, 0.25H), 4.68 (q, J = 6.8Hz, 0.7H), 4.56 (br s, 0.2H) , 3. 73 (s, 0.7H), 3. 62 (s, 2. 3H), 3. 49 (dd, J = 14. 0, 5.9 Hz, 0.8H), 3. 89 (dd, J = 14. 0, 7.2Hz, 0.7H), 3. 05 (br s, 0.2H), 1. 39 (s, 7. 5H), 1.09 (s, 2. 5H).

Preparation of Compound 9

To the CH of compound 7 (7.60 g, 21.8 mmol) at 0 °C₂C1₂ (150mL) Add Viton (1 &amp; OmL) and Tf to the solution₂0 (9.19 g, 32.6 mmol). The resulting mixture was stirred for 2h at room temperature, concentrated under vacuum and₂C1₂Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂(2X50mL) extraction. The combined organic extracts were washed with brine, dried over NahOq and concentrated to give compound 9 (11.0 g, crude product) as a brown oil. The crude product is used directly in the next step without further purification.

[0761] ^XH NMR (400MHz, CDCI3, a mixture of rotamers) δ 8.19-&amp; 07 (m, 2Η), 7.69-7.64 (m, 2H), 7.38 (d, J = 8. 1Hz, 1H), 7.28 (d, J = 7.9Hz, 1H), 5.12-5.06 (br s, 1H), 4.78-4.67 (m, 1H), 3. 68 -3. 46 (m, 5H), 1. 39 (s, 8H), 1. 25 (s, 1H).

Preparation of Compound 11

[0763] A solution of compound 9 (11.0 g, 21.8 mmol) and but-3- alkynyl carbamate benzyl ester 10 (6.56 g, 32.6 mmol) in anhydrous ethidium (100 mL) at room temperature Degas under nitrogen atmosphere for 10 minutes, then add TEA (11.9mL, 87.0mmo 1), 10% in hexane (t-Bu)₃P (8.80 mL, 4.35 mmol) and Cui (207 mg, 1.08 mmol). The resulting mixture was degassed with nitrogen for another 10 minutes, and Pd(PPh₃)₄(2.51g, 2.17mmol). After degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2:3 hexane/EtOAc) to obtain compound 11 (7.00 g, 61% yield over two steps) as a brown oil.

[0764] ^XH NMR (400MHz, CDCI3, a mixture of rotamers) 6 &amp; 33 (dd, J = &amp; 9,1.9Hz, 1Η), &amp; 07 (dd, J = 9.0, 1.7Hz, 1H), 7 . 59-7. 49 (m, 3Η), 7. 39-7. 27 (m, 5Η), 7. 19 (d, J = 7.3Hz, 1H), 5.24-5.16 (m, 1H), 5.12 (s, 2H), 5. 08-4.99 (m, 1H), 4.69 (q, J = 6.7Hz, 1H), 3.59 (s, 3H), 3 . 57-3. 40 (m, 4H), 2. 79 (t, J = 6. 4Hz, 2H), 1. 39 (s, 7. 5H), 1. 11 (s, 1. 5H).

104

Preparation of Compound 17

[0766] To a solution of methyl ester 11 (7.00 g, 13.2 mmol) in THF (200 mL), methanol (200 mL) and water (75.0 mL) was added solid NaOH (16.0 g, 79.2 mmol). The resulting mixture was stirred at room temperature for 1 h, until TLC showed that the reaction was complete. 1N hydrochloric acid was added to adjust the pH of the reaction mixture to 10. After concentration, water (100 mL) was added and the pH was adjusted to 5-6. Use CH₂Cl₂(2X250mL) extraction. The organic layers were combined, dried over Na^SOq, filtered, concentrated and triturated with MTBE to obtain compound 17 (5.00 g, 75%) as a white solid:

[0767] NMR (400MHz, CD₃0D; mixture of rotamers) 6 &amp; 33 (d, J = &amp; 2Hz, 1H), &amp; 28-8. 20 (m, 1H), 7. 59-7. 45 (m, 3H), 7. 38-7. 21 (m, 6H), 5. 09 (s, 2H), 4. 55-4. 45 (m, 1H),

3. 76-3.66(m, 1H), 3.44(t, J = 6.7Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J = 6.7Hz ,2H),

1. 29(s,6H) ,0.82(s,3H).

Preparation of Compound 30

[0769] To a solution of compound 17 (4.60 g, Ri) in THF (160 mL) was sequentially added T3P (50% in ethyl acetate, 10.7 mL) and NMM (4.89 mL, 44.5 mmol). Stir at room temperature After 10 minutes, amine 29 (6.11 g, 9.33 mmol) was added, and the reaction mixture was stirred at room temperature for 16 h. After removing the solvent, the residue was dissolved in CH₂C1₂ (100mL), quickly wash with saturated NH4CI, saturated NaHCA and brine,₂S0₄T dry and concentrate. The residue will be purified by column chromatography (silica gel, 9:1 dichloromethane/methanol) to give amide 30 (6.60g, 64%) as an off-white solid

[0770] ^XH NMR(400MHz, CDCI3) δ &amp; 33 (dd, J = 9. 0, 1.7Hz, 1H), &amp; 17 (d, J = 7.3Hz, 1H), 7. 62-7. 47 (m, 4H), 7. 42 (dd, J = 7. 7, 4. lHz, 4H), 7. 37-7. 28 (m, 11H), 7. 09-6. 95 (m, 4H), 5. 46(s,2H) ,5.33(br s, 1H), 5. 22 (t, J = 5. 8Hz, 1H), 5. 11 (s, 2H), 4. 63-4. 51 (m , 1H),

4. 27 (dd, J = 10. 8, 5. 4Hz, 2H), 4.02-3. 84 (m, 6H), 3. 71 (t, J = 4.5 Hz, 6H impurity), 3. 57 ( t, J = 10.6Hz,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

[0772] 30 (7.26 g, 6.20 mmol) and 10% Pd/C (1.50 g) were suspended in Et0H/Ac0H (240 mL/40.0 mL) at room temperature by bubbling with nitrogen gas using a syringe The liquid was bubbled and degassed for 10 minutes, and then placed under hydrogenation conditions (latm) for 16 hours. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and triturated with MTBE to obtain the amine salt 31 (7.06 g, 98%) as a brown solid:

[0773] ^XH NMR (400MHz, CD3OD, a mixture of rotamers) 6 &amp; 24 (dd, J = 7.2, 2. ΟΗζ, 1Η), &amp; 09 (d, J = 7. ΟΗζ, 1H), 7.59- 7. 22 (m, 2H), 7. 49-7. 41 (m, 4H), 7. 39-7. 22 (m, 10H), 6. 95 (d, J = &amp; 5Hz, 2H), 5 . 51(s,2H),4.55(t, J = 7. 2Hz, 1H), 4. 24 (dd, J = 10. 7,5. 4Hz, 2H),

4. 19-4. 10 (m, 2H), 3. 99-3. 88 (m, 4H), 3. 83-3. 73 (m, 8H, impurity), 3. 61 (t, J = 10. 5 , Hz, 2H),

3. 59-3.52 (m, 1H), 3.45-3.36 (m, 1H), 3.19-3.02 (m, 4H), 2.93-2.81 (m, 8H), 2.54. 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).

Preparation of Compound 32

[0775] To 31 (7.06g, 6.18mmol) in EtOH (50.0 mL) solution was added DIPEA (8.80 mL, 49.4 mmol) at room temperature, followed by 3,5-diamino-6- Chlorpyrazine-2-Pyridylmethionyl methyl thioate (13, 3.84 g, 9.88 mmol). The reaction mixture was heated at 70 °C for 2 h, cooled to room temperature, and concentrated under vacuum. By column chromatography (silica gel, 80: 18: 2CHCl₃/CH₃0H/NH₄OH) The residue was purified twice to obtain the compound as a yellow solid

105

32 (2.50g, 33%):

[0776] ^XH NMR (400MHz, CD₃0D, mixture of rotamers) 6 &amp; 22 (d, J = 9.3Hz, 1H), &amp; 08 (d, J = 7.8Hz, 1H), 7. 56-7.47 (m, 4H) , 7. 43 (dd, J = 7.4, 3. 6Hz, 4H), 7. 33-7. 14 (m, 10H), 6.94 (d, J = &amp; 0Hz, 2H), 5.47 (s,2H) ,4.53 (t, J = 7.7Hz, 1H) ,4.22 (dd, J = 10. 8,

5. 4Hz,2H) ,3.99-3.89(m,4H),3.84(dd, J = 5. 5, 2.3Hz, 2H), 3.70 (dd, J = 9.2,2.2Hz, 2H), 3.59 (t, J = 10. 8Hz, 2H), 3. 54-3.46 (m, 1H), 3. 47-3.38 (m, 1H), 3. 22 (t, J = 6.4Hz, 2H), 3.11-3.02(m,2H), 2.70(dd, J = 13. 5,4.6Hz, 2H), 2.61 (dd, J = 13.6 , &amp; 9, 2H),

2. 57-2.47 (m,2H), 2.46-2.34(m,2H), 1.84-1.73(m,2H), 1.72-1.61 (m,4H), 1. 36(s,7H),

1. 12(s,2H).

[0777] 3,5-Diamino-N- (N- (4- (4- ((S) ~2~ amino-3- (4- (3-(bis((2S, 3R, 4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)Ze-1-yl)butyl)methionyl)-6-chloro Preparation of HC1 salt of pyrazine-2-carboxamide (33)

[0778] To a solution of 32 (2.50 g, 2.02 mmol) in EtOH (30.0 mL) was added 4N hydrochloric acid (80.0 mL). Stir the resulting mixture for 2h at room temperature_oThe solvent was removed, purified by a reverse phase column and lyophilized to obtain compound 33 (1.82 g, 85%) as a yellow hygroscopic solid:

[0779] ^XH NMR(400MHz, DMS0-d₆) δ 10. 61 (s, 1H), 10. 59 (s, 1H), 9.41 (t, J = 5.2Hz, H), 9. 01 (br s,lH), &amp; 96 (br s , 1H), &amp; 81(br s,2H), &amp; 77 (br s,2H), 8. 44-&amp; 37 (m, 1H), &amp; 16-&amp; 10(m, 1H), 7. 61-7 . 52(m,2H), 7.41(d, J = 8.6Hz, 2H), 7. 35 (d, J = 7.5Hz,lH), 7. 27 (d, J = 7.3Hz, 1H), 7.17 (d, J = &amp; 5Hz, 2H), 4.28 (q, J = 7.4Hz, 1H), 4.09-3.99 (m, 2H) ,3.75-3 .65(m,3H) ,3.58(dd, J = 11. 0, 2. 6Hz, 2H), 3. 55-3. 31 (m, 10H), 3. 30-3. 13 (m, 4H), 3.32-3.00 (m, 2H), 2.63-2.53 (m, 2H), 2.05-1.92 (m, 2H), 1.78-1. 61 (m, 4H).

[0780] ^XH NMR (400MHz, CD₃0D): δ 9. 25 (t, J = 5. 9Hz, 0.5H), 8. 26-8. 21 (m, 1H), &amp; 17-&amp; 12(m, 1H), 7. 60-7 54m, 2H), 7. 38 (d, J = 7. 2Hz, 1H), 7. 32 (d, J = 7.2 Hz, 1H), 7. 25 (d, J = &amp; 6Hz, 2H), 7. 15 (d, J = &amp; 6Hz, 2H), 4. 31 (t, J = &amp; 1Hz, 1H), 4. 21-4. 14 (m, 1H), 4. 13-4. 08 (m , 1H), 3.85-3.80(m,2H), 3.79(d, J = 2.9Hz, 1H), 3.76 (d, J = 3.2Hz, 1H), 3.73 -3.62(m,8H), 3.51-3.34(m,8H), 3.15 (t, J = 6.8Hz,2H), 2.73-2.57(m,2H) ,

2. 15-1.98 (m, 2H), 1.91-1.73 (m, 4H).

22. (2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-aminophenyl)propylazane (Diyl) dihexane-1,2,3-,4, 5-pentaol (29) preparation

Scheme 23

[0783]

106

144 (Roc)_?O. TEA

9-BBN, PdCl_?(PPh₃)₂

IN NaOH water soluble roll

60%

ON

<img file="CN105073717A_D0176.tif" />

147

NHBoc

HO, pH

O &gt;-011 4N lid in dioxane

93%

<img file="CN105073717A_D0177.tif" />

y sugar it

Ο

<img file="CN105073717A_D0178.tif" />

].NaCNBHv AcO! L MeOJ I

2. Hexanal

<img file="CN105073717A_D0179.tif" />

ΝΙπ·ΙΚΊ

OH OH

0^.0 OH

Pli

[0784]

The preparation of compound 145 was changed to the CH of compound 144 (&amp; 80g, 154.1 mmol) at 0 °C₂C1₂ (150mL) TEA (32.2mL, 231.2mmol) and Boc were added to the solution₂0 (40.4g, 185.3mmol). The reaction mixture was continuously stirred at 0°C for 0.5 h, allowed to warm to room temperature and stirred for 5 h. Then make the mixture in CH₂Cl₂Partition between (150 mL) and water (150 mL). Separate the water layer and use CH₂Cl₂(2X150mL) extraction. The combined organic extracts were washed with brine and menstrualized. It was dried and concentrated to obtain the desired compound 145 (22.0 g, 91%) as a colorless oil.

[0786] NMR (400MHz, CDC1₃): 6 5. 90-5. 77 (m, 1H), 5. 17 (dq, J = 17. 1, 1. 7Hz, 1H),

5. 10(dq, J = 10. 4, 1.4Hz, 1H), 4.64 (brs, 1H), 3.74 (t, J = 5.2Hz, 2H), 1.45(s, 9H).

Preparation of compound 147

To a solution of compound 145 (14.0 mmol g, 89.12 mmol) in anhydrous THF (150 mL) under nitrogen was added

9-BBN (0.5M in THF, 270mL, 133.8mmol). The reaction mixture was stirred for 2h at room temperature, compound 146 (17.7g, 71.3mmol), Pd(PPh₃) ₂C1₂(3.12g, 4.45mmol) and 1N NaOH aqueous solution (150mL). The resulting mixture was stirred for another 1 h. After removal of the solvent; the residue was partitioned between EtOAc (200 mL) and water (200 mL).

The aqueous layer was separated and extracted with Et0Ac (2×200 mL). The combined organic extracts were washed with brine, dried over Na^Oq and concentrated under vacuum. The crude product will be purified by column chromatography (silica gel, 4: 1 hexane/EtOAc) to obtain compound 147 (&amp; 00g, 43%) as a brown solid:

[0789] ^XH NMR (400MHz, CDC1₃) 6 &amp; 14 (d, J = &amp; 9Hz, 2H), 7. 34 (d, J = &amp; 9Hz, 2H), 4. 56 (br s, 1H), 3. 17 (q, J = 6. 2Hz ,2H), 2.75 (t, J = 7.7Hz,2H), 1.89-1.79 (m, 2H), 1.44(s,9H).

Preparation of Compound 148

[0791] Compound 147 (&amp; 00g, 28.6) was dissolved in 4N HC1 (50.0 mL) in dioxane at room temperature, and the solution was stirred for 1 h_oThe reaction mixture was concentrated under vacuum and the residue was triturated with MTBE to give compound 148 (4.00 g, 65%) as a brown solid:

[0792] ^XH NMR(400MHz, CD₃0D) 6 &amp; 19 (d, J = &amp; 7Hz, 2H), 7. 50 (d, J = &amp; 7Hz, 2H), 2. 98 (t,

107

J = 7.4Ηζ, 2Η), 2.86 (t, J = 7.6Hz, 2H), 2.07-1.97 (m, 2H).

Preparation of Compound 150

To compound 148 (4.00g, 1 &amp; 5mmol) and triol 149 (24.8g, 92.5mmol) in MeOH (150mL) solution was added AcOH (11.1mL, 185mmol), and at room temperature The reaction mixture was stirred for 10 minutes. Add NaCNBH₃(5.83 g, 92.5 mmol), the solution was stirred for 24 h at room temperature. Add additional compound 149 (4.0 equivalents), AcOH (4.0 equivalents) and NaCNBH within 4 days₃(4.0 equivalent). Then add hexanal (2.0 equivalents), AcOH (2.0 equivalents) and NaCNBH₃(2.0 equivalent). The solution was stirred for another 1 h at room temperature. After removing the solvent, use saturated NaHC0₃The residue was neutralized, and the residue was partitioned between EtOAc (200 mL) and water (200 mL). Separate the water layer and use CH₂Cl₂(2X300mL) extraction. The combined organic extracts are tested. Dry and concentrate under vacuum. By column chromatography (9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 150 (6.50 g, 52%) as an off-white solid. Separate an additional 4. 00g of material from the impure fraction and purify it by a reverse phase column to obtain 1.50g (12%) of pure compound 150 (total 7.70g, 64%):

[0795] ^XH NMR (400MHz, CD₃0D) 6&amp;0 3(d,J = 8.7Hz, 2H), 7.50-7.41 (m, 4H), 7.35-7.23 (m, 8H), 5.48(s,2H) ,4.22(dd, J = 10. 6, 5. 3Hz, 2H), 3. 99-3.91 (m, 4H), 3. 85 (dd, J = 5. 5,

2. 4Hz, 2H), 3. 70 (dd, J = 9. 5, 2.4Hz, 2H), 3. 59 (t, J = 10. 6Hz, 2H), 2. 73 (dd, J = 13. 6 ,

4. 5Hz, 2H), 2.67-2.50 (m, 6H), 1.83-1.71 (m, 2H).

(2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-aminophenyl)propylazane

Preparation of dihexane-1,2,3,4,5-pentaol (compound 153);

A suspension of compound 150 (6.50 g, 9.50 mmol) and 10% Pd/C (1.30 g) in ethanol (150 mL) was degassed by bubbling at room temperature for 10 minutes by using a syringe, Then it was stirred for 6h under a hydrogen atmosphere (balloon, latm). The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 153 (6.01 g, 97%) as an off-white solid:

[0798] ^XH NMR (400MHz, CD₃0D): δ 7.49-7.42 (m, 4H), 7.35-7.26 (m, 6H), 6.82 (d, J=8.4Hz,2H), 6.60(d , J = 8. 4Hz, 2H), 5. 48 (s, 2H), 4. 22 (dd, J = 10. 8, 5. 9Hz, 2H),

3. 98-3.89(m,4H), 3.83(dd, J = 5. 7, 2. 3Hz, 2H), 3.69 (dd, J = 13. 2, 3. 4Hz, 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).

[0799] 23. 3,5-Diamino ^_N~ (N~ (4- (4- ((R) ~2~ Amino-3- (4- (3-(Bis((2S, 3R, 4R, 5R) ~2,

3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)choen-1-yl)butyl)methionyl)-6-chloropyrazine- Preparation of 2-formamide (152)

[0800] Scheme 24

[0801]

108

[0802]

[0803]

HOrC

BocHN

OH N-Hydrosuccinimide (142) CH; CN

<img file="CN105073717A_D0180.tif" />

Sugar sugar, N sugar

Pd(PPh;)₄. Cui (/-Bxi)3p. EtiN CH'CN Sugar, N

T₃P, NMM_; THt

NHCbz superior/X^ NHCbz h; co₂c

BocHN

NaOH

MeOH/THF/HG

BocHN

146

<img file="CN105073717A_D0181.tif" />

Z1L I₂. DMF Pd₂ (dba)₃. Sphos

NHCbz

<img file="CN105073717A_D0182.tif" />

Tf₂O. Ping, CH₂C1₂

<img file="CN105073717A_D0183.tif" />

<img file="CN105073717A_D0184.tif" />

R

<img file="CN105073717A_D0185.tif" />

<img file="CN105073717A_D0186.tif" />

N

H

BocHN

149

Pd/G pressure (latm)

EtOH. AcOH

<img file="CN105073717A_D0187.tif" />

15()

DIPEA. EtOH

Ν

Η

BocHN

NHCbz

NH₂*2AcOH

<img file="CN105073717A_D0188.tif" />

HO

<img file="CN105073717A_D0189.tif" />

N sch₃

Π

<img file="CN105073717A_D0190.tif" />

N ratio

NH 0

NSonN# ^HH Α Λ

Η,Ν Ν NH.

Ν

Η

BocHN (λ) OH "OH

<img file="CN105073717A_D0191.tif" />

.λΟΗ

Coh (V)OH (S)

<img file="CN105073717A_D0192.tif" />

HO

HO's

4N HC1 aqueous solution, EtOH

NH O from human Η Η Λ X

The preparation of Η, Ν N NH compound 14 gave 1- Zea phenol (1,10.0 g, 69.4 mmol) within 30 minutes

NBS (142, 12. 3 g, 69.4 mmol). The resulting mixture was stirred at room temperature for 4 h, concentrated under vacuum, and then water (200 mL) and ethyl acetate (200 mL) were added. The aqueous layer was separated and extracted with ethyl acetate (2×200 mL). The combined organic extracts were washed with brine, dried with blood and concentrated. The residue was purified by crystallization (heptane/EtOAc) to obtain the desired compound 14 (6.0 g, 39%) as a white solid.

[0804] ^XH NMR(400MHz, DMS0-d₆): δ 10. 49 (s, 1Η), 8.20 (dd, J = 8.3, 0.5 Hz, 1H), 8. 02 (d, J = &amp; 3Hz, 1H), 7.66 (dd , J = &amp; 4,1.4Hz, 1H), 7. 64(d, J = &amp; 1Hz, 1H), 7. 55(ddd, J = &amp; 2, 7. 7, 1. 1Hz, 1H) ,6 . 83 (d, J = &amp; 2Hz, 1H).

Add a few servings to the ethyl diet (70.0 mL) solution

109

Preparation of Compound 145

[0806] Zinc powder (4.76 g, 72.9 mmol) was added to a flame-dried, nitrogen-purged side-arm round bottom flask. Anhydrous DMF (25.0 mL) was added via a syringe, followed by a catalytic amount of iodine (677 mg, 2.67 mmol). It is observed that the resulting mixture undergoes a change from colorless to yellow and back to colorless. Add protected iodoalanine 114 (&amp; 00g, 24.3mmol) at one time, then add a catalytic amount of iodine (677mg, 2.67mmol), and stir at room temperature for 30 minutes; successful insertion of zinc with gentle The ground radiates heat. Allow the organozinc reagent to cool to room temperature, then add Pd₂(dba)₃(556mg, 0.60mmol), SPhos (498mg, 1.2lmmol) and aryl bromide 14 (5.40g, 24.3mmol), and under a positive pressure of nitrogen, the mixture was heated to 50°C and maintained 16h_oThe reaction mixture was cooled to room temperature. Add saturated NH₄C1 solution (300 mL) and EtOAc (300 mL), then the mixture was filtered through Celite and washed with EtOAc (100 mL). The aqueous layer was separated and extracted with Et0Ac (2×300 mL). The combined organic extracts were washed with brine and passed through the blood paw. . Dry and concentrate under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexane/EtOAc) to obtain the desired compound 145 (3.10 g, 37%) as a yellow solid:

[0807] soNMR (400MHz, CDC1₃, A mixture of rotamers): 6 &amp; 23 (d, J = &amp; 3Hz, 1H), 7. 99 (d, J = &amp; 6Hz, 1H), 7. 54 (t, J = &amp; 04Hz, 1H) , 7.48(ddd, J = &amp; 3,6.9,1.3Hz, 1H), 7. 08 (d, J =7.8Hz, 1H), 6.70(d, J = 7. 6Hz, 1H), 5.98 (brs, 0.3H), 5.59 (br s, 0.7H), 5.03 (d, J = 7.7Hz, 0.85H), 4.84 (br s, 0.15H), 4.68(q, J = 6.8Hz, 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

To the CH of compound 145 (3.07g, &amp; 90mmol) at 0°C₂C1₂ (75.0mL) in the solution, add pyridine (7.25mL, 8&amp; 9mmol) and Tf₂0 (2.24mL, 13.3mmol). The resulting mixture was stirred at room temperature for 2h, concentrated under vacuum and in CH₂C1₂Partition between (100 mL) and water (50 mL). Separate the water layer and use CH₂C1₂(2X50mL) extraction. The combined organic extracts were washed with brine, dried over N^SOq and concentrated to give compound 146 (4.20 g, crude product) as a brown oil. The crude product was used directly in the next step without further purification.

[0810] ^XH NMR (400MHz, a mixture of CDC rotamers): 6 &amp; 19-&amp; 07 (m, 2H), 7. 69-7. 64 (m, 2H), 7. 38 (d, J = 8.1 Hz , 1H), 7. 28 (d, J = 7.9Hz, 1H), 5. 12-5. 06 (br s, 1H), 4. 78-4. 67 (m, 1H), 3. 68- 3. 46 (m, 5H), 1. 39 (s, 8H), 1. 25 (s, 1H).

Preparation of Compound 147

[0812] Under a nitrogen atmosphere, compound 6 (4.20 g, 80 mmol, crude product) and but-3- alkynyl carbamate benzyl ester 7 (2.65 g, 13. 2 mmol) in anhydrous ethyl gluten (50.0 mL) Degas the solution in) for 10 minutes, then add TEA (4.81mL, 35.2mmol), 10% (t-Bu) in hexane at room temperature ₃P (3.56mL, 1.76mmol) and Cui (84mg, 0.44mmol). The resulting mixture was degassed with nitrogen for another 10 minutes, and Pd(PPh₃)₄(1. 01g, 0.88mniol) _oAfter degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 18 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2:3 hexane/EtOAc) to obtain compound 147 (3.20 g, 67% yield over two steps) as a brown oil.

[0813] soNMR (400MHz, CDC1₃, A mixture of rotamers): 6 &amp; 33 (dd, J = 8. 9,1.9 Hz, 1H), &amp; 07 (dd, J = 9. 0, 1. 7 Hz, 1Η), 7. 59- 7.49 (m, 3Η), 7.39-7.27 (m, 5Η), 7.19 (d, J = 7.3Ηζ, 1Η), 5.24-5.16 (m, 1Η), 5. 12 (s, 2Η), 5. 08-4. 99 (m, 1Η), 4.69 (q, J = 6.7Hz, 1Η), 3. 59 (s, 3Η), 3. 57- 3. 40 (m, 4Η), 2.79 (t, J = 6.4Hz, 2H), 1.39 (s, 7.5H), 1.11 (s, 1.5H).

Preparation of Compound 148

110

[0815] To a solution of methyl ester 147 (3.10 g, 5.84 mmol) in THF (60 mL), methanol (60 mL) and water (20.0 mL) was added solid NaOH (1.40 g, 35.09 mmol). The resulting mixture was stirred at room temperature for 2 h until TLC showed that the reaction was complete. 1N hydrochloric acid was added to adjust the pH of the reaction mixture to 10° after concentration; water (100 mL) was added and the pH was adjusted to 5-6. Use CH₂Cl₂(2X200mL) extraction. The organic layers were combined, dried over Na^SOq, filtered, concentrated and triturated with MTBE to obtain compound 148 (3.00 g, 99%) as a white solid.

[0816] NMR (400MHz, CD₃0D; mixture of rotamers): 6 &amp; 33 (d, J = &amp; 2Hz, 1H), &amp; 28-8. 20 (m, 1H), 7. 59-7. 45 (m, 3H), 7 . 38-7. 21 (m, 6H), 5. 09 (s, 2H), 4. 55-4. 45 (m, 1H),

3. 76-3.66(m, 1H), 3.44(t, J = 6.7Hz, 2H), 3.28-3.20 (m, 1H), 2.76 (t, J = 6.7Hz ,2H),

1. 29(s,6H) ,0.82(s,3H).

Preparation of Compound 149

To compound 148 (800mg, 1.55mmol) in THF solution (30mL) solution was added T₃P (50% in ethyl acetate, 1.86 mL) and NMM (0.85 mL, 7.75 mmol). After stirring at room temperature for 10 minutes, amine 29 (1.01g,

1. 55mmol), and the reaction mixture was stirred at room temperature for 1h. After removing the solvent, dissolve the residue in CH₂C1₂ (100mL), use saturated NH£1, saturated NaHC0₃Quickly wash with salt water, after Na ₂S0₄Dry and concentrate. Column chromatography (silica gel, 9: lCH₂Cl₂/MeOH) The residue was purified to give amide 149 (1.20 g, 67%) as an off-white solid.

[0819] ^XH NMR (400MHz, CDCI3): 6 &amp; 35(d, J = &amp;0,1. 7Hz, 1H), 8. 19 (d, J = &amp; 5Hz, 1H), 7. 60-7. 52 (m, 2H ), 7.50 (d, J = 7.3Hz, 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. 4Hz, 2H), 4. 07-4. 00 (m, 2H), 3. 99-3.91 (m, 4H), 3. 78-3. 68 (m, 3H), 3.59 (t, J = 10. 6Hz, 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).

Preparation of Compound 150

[0821] 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 mixed with air at room temperature by using a syringe Degas by bubbling for 10 minutes, and then place it under hydrogenation conditions (latm) for 16 hours. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and triturated with MTBE to obtain the amine salt 150 (1.12 g, 97%) as a brown solid.

[0822] NMR (400MHz, CD₃OD, mixture of rotamers): 6 &amp; 25 (dd, J = 7.2, 2. ΟΗζ, 1Η), &amp; 09 (d, J = 7. ΟΗζ, 1H), 7.59-7.51 (m,2H) ,7.48-7.41 (m,4H) ,7.37-7.21 (m, 10H),

6. 94(d, J = &amp; 5Hz,2H) ,5.52(s,2H) ,4.54(t, J = 7.2Hz, 1H) ,4.24(dd, J = 10.7, 5. 4Hz, 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 = 7.4Hz, 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).

Preparation of Compound 151

[0824] At room temperature, to 150 (1.05g, 0.92mmol) in EtOH (15.0mL) solution was added DIPEA (1.30mL,

7. 35mmo 1), followed by addition of 3,5-diamino-6-chloropyrazine-2-propanylmethyl thiomethionate (13,573mg, 1.47mniol) ο The reaction mixture was heated to 70°C for 2h , Cooled to room temperature and concentrated under vacuum. By column chromatography (silica gel, 80: 18: 2CHCl₃/CH₃0H/NH₄OH) The residue was purified twice to obtain compound 151 (410 mg, 36%) as a yellow solid.

111

[0825] ^XH NMR (400MHz, CD₃0D, a mixture of rotamers): &amp; &amp; 22 (d, J = &amp; 4Hz, 1H), &amp; 09 (d, J = &amp; 2Hz, 1H), 7. 56-7. 48 (m, 2H), 7. 47-7. 40 (m, 4H), 7. 33-7. 25 (m, 6H), 7. 22 (d, J = 7.5Hz, 2H), 7. 16 (d, J = 7 . 8Hz, 2H), 6.94 (d, J = &amp; 1Ηζ, 2Η), 5.47 (s, 2H), 4.53 (t, J = &amp; 1Hz, 1H), 4.22 (dd, J = 10. 8, 5. 4Hz, 2H), 3. 99-3.89 (m, 4H), 3. 84 (dd, J = 5. 5,

2. lHz,2H), 3.70(dd, J = 9. 1,2. ΟΗζ, 2H), 3.59 (t, J = 10. 8Hz, 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 . 0Hz,2H),

2. 60 (dd, J = 13. 0, &amp; 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).

[0826] 3,5-Diamino-N- (N- (4- (4- ((R) ~2~ amino-3- (4- (3-(bis((2S, 3R, 4R, 5R) -2,3,4,

5,6-Pentyl (hexyl) amino) propyl) phenylamino) -3-oxopropyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine-2-carboxamide Synthesis of (152)

[0827] To a solution of 151 (480 mg, 0.42 mmol) in EtOH (5.0 mL) was added 4N hydrochloric acid (25.0 mL). Stir the resulting mixture for 2h at room temperature_oThe solvent was removed, purified by a reverse phase column and lyophilized to obtain compound 152 (300 mg, 71%) as a yellow hygroscopic solid.

[0828] ^XH NMR(400MHz, DMS0-d₆): δ 10. 57 (brs, 1Η), 10. 55(brss, 1H), 9. 35 (t, J =

6. ΟΗζ, 1H), 9.04-8.84(m,2H), &amp; 81-&amp; 66(m,4H), &amp; 42-8.36 (m, 1H),&amp; 16-&amp; 10 (m, 1H ),

7. 61-7.53 (m, 2H), 7. 41 (d, J = &amp; 6Hz, 2H), 7. 35 (d, J = 7.5Hz, 1H), 7. 28 (d, J = 7. 8Hz, 1H), 7.17 (d, J = 9.0Hz, 2H), 4. 32-4. 23 (m, 1H), 4. 08-3. 97 (m, 2H), 3. 75- 3. 30 (m, 13H),

3. 29-3.15 (m, 4H), 3.14-2.97 (m, 2H), 2.64-2.53 (m, 2H), 2.05-1.92 (m, 2H), 1.79-1.60 (m, 4H).

[0829] ^XH NMR (400MHz, CD₃0D): δ &amp; 25-8. 21 (m, 1H), &amp; 18-8. 13 (m, 1H), 7. 59-7. 53 (m, 2H), 7. 38 (d, J = 7 . 3Hz, 1H), 7. 32 (d, J = 7.3Hz, 1H), 7. 26 (d, J = &amp; 8Hz,2H), 7. 15 (d, J =&amp; 5Hz,2H), 4 . 30 (t, J = 7.3Hz, 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. 3Hz, 2H),

2. 74-2.58 (m, 2H), 2.13-1.98 (m, 2H), 1.91-1.73 (m, 4H).

[0830] HRMS: C₄₄H₆₄C1N_1OO₁₂[M+Na]⁺ Calculated value: 959. 4418, actual value: 959. 4394_O

24. Preparation of Intermediate 18

[0832] Scheme 25

[0833]

112

<img file="CN105073717A_D0193.tif" />

Η» Pd/C

MeOH

<img file="CN105073717A_D0194.tif" />

Preparation of Compound 155

To the CH of compound 154 (500 mg, 9.0 mmol) at 0°C₂C1₂(50mL) Add TEA (1.63mL,

11. 7mmol) and Boc₂0 (2.16g, 9.90mmol). The reaction mixture was continuously stirred at 0 °C for 0.5 h, allowed to warm to room temperature and stirred for 3 h. Then make the mixture in CH₂Cl₂Partition between (50mL) and water (50mL). Separate the water layer and use CH₂Cl₂(2X50mL) extraction. The combined organic extracts were washed with brine, dried over Na^SOq, concentrated, and the residue was purified by column chromatography (silica gel, 2:3 hexane/EtOAc) to give the desired compound 155 (1. 20g, 86%) ο

[0836] ^XH NMR(300MHz, CDC1₃): δ 4. 70 (br s, 1H), 3. 91 (dd, J = 5. 3, 2. 2Hz, 2H), 2. 21 (t, J = 2.7Hz, 1H), 1. 45 (s,9H).

Preparation of Compound 157;

Under a nitrogen atmosphere, a solution of compound 155 (1.00 g, 6.45 mmol) and 156 (1.30 g, 6.45 mmol) in anhydrous THF (15 mL) was degassed for 10 minutes, and then at room temperature Add TEA (3.53mL, 25.8mmol), PPh₃(424mg, 1.61mmol) and Cui (246mg, 1.29mmol). The resulting mixture was degassed with nitrogen for another 10 minutes, and Pd(PPh₃)₄(7.45g, 6.45mmol). After degassing with nitrogen for 5 minutes, the resulting mixture was refluxed for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2:3 hexane/EtOAc) to give compound 157 (750 mg, 42%) as a brown oil.

[0839] ^XH NMR (400MHz, CDC1₃) :6&amp;17(d, J = 9. 2Hz, 2H), 7. 55 (d, J = 9. 2Hz, 2H),

4. 79 (brs, 1H), 4.18 (d, J = 6.0Hz, 2H), 1.47 (s, 9H).

Preparation of Compound 158

Compound 157 (2.00 g, 7.24) was dissolved in 4N HC1 (20.0 mL) in dioxane at room temperature, and the solution was stirred for 2 h_oThe reaction mixture was concentrated under vacuum and the residue was triturated with MTBE to give compound 158 (1.25 g, 82%) as a brown solid.

[0842] ^XH NMR(300MHz, CD₃0D): δ 8.26 (d, J = 9. 2Hz, 2H), 7. 72 (d, J = 9. 2Hz, 2H),

4. 09(s, 2H).

113

Preparation of Compound 159

To a solution of compound 158 (100mg, 0.47mmol) and aqueous formaldehyde (30%, 1.40mL, 1.41mmol) in MeOH (3.0mL) was added AcOH (0.09mL, 1.41mmol) , The reaction mixture was stirred at room temperature for 30 minutes. Add NaCNBH₃(88mg, 1.4lmmol), the solution was stirred at room temperature for 16h. Add additional aqueous formaldehyde solution (30%, 0.92mL, 0.94mmol), AcOH (0.09mL, 1. and NaCNBH₃(88mg, 1.41mmol), and stirred for another 16h. After removing the solvent, use saturated NaHC0₃The residue was neutralized and partitioned between EtOAc (30 mL) and water (30 mL). Separate the water layer and use CH₂Cl₂(2X40mL) extraction. The combined organic extracts were dried and concentrated under vacuum. By column chromatography (silica gel, 9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 159 (50 g, 52%) as an off-white oil.

[0845] ^XH NMR(300MHz, CD₃0D) :6 &amp; 17 (d, J = 9. ΟΗζ, 2H), 7. 57 (d, J = 9. 0Hz, 2H),

3. 50(s,2H), 2.37(s,6H).

Preparation of Compound 18

A suspension of compound 159 (100 mg, 0.49 mmol) and 10% Pd/C (40 mg) in MeOH (3.0 mL) was degassed with nitrogen at room temperature for 10 minutes, and then in a hydrogen atmosphere (balloon , Latm) under stirring for 3h. The reaction mixture was filtered through Celite and washed with MeOH. Concentrate the filtrate in vacuo and use CH₂C1₂/Hexane trituration to obtain 18 (48mg, 55%) as white crystals:

[0848] ^XH NMR(300MHz, CDC1₃): δ 6.96 (d, J = 8. 3Hz, 2H), 6. 60 (d, J = &amp; 3Hz, 2H),

3. 47 (br s,2H) ,2.53(t, J = 7. 8Hz, 2H), 2. 26 (dd, J = &amp; 7,7.2Hz, 2H), 2. 22 (s, 6H),

1. 77-1.67(m,2H).

Preparation of Intermediate 29

[0850] Scheme 26

[0851]

Pd(OH)-,/C,H, \ MeOH''

[0852] Preparation of Compound 161;

To a solution of compound 158 (4.00g, 1 &amp; 9mmol) and triol 160 (11.7g, 56.6mmol) in MeOH (50mL) was added AcOH (3.40mL, 56.6mmol), in The reaction mixture was stirred for 30 minutes at room temperature. Add NaCNBH₃(3.55g, 56.6mmol), the solution was stirred for 16h at room temperature. Add additional compound 160 (11.7g, 56.6mmol), AcOH (3.40mL, 56.6mmol) and NaCNBH₃ (3.55g, 56.6mmol), the solution was stirred at room temperature for 16h_oAfter removing the solvent, use saturated NaHC0₃Neutralize the residue, put the residue in CH ₂C1₂ Partition between (10mL) and water (10mL). Separate the water layer and use CH₂Cl₂(2X10mL) extraction. The combined organic extracts were dried over NapSOq and concentrated under vacuum. By column chromatography (silica gel, 9:1CH₂Cl₂/Me0H,80: 18: 2CHC1₃/

114

Me0H/NH₄0H) The residue was purified to obtain compound 29 (700 mg, 7.0%) as an off-white solid.

[0854] ^XH NMR(300MHz, CD₃0D) :6 &amp; 21 (d, J = 8. 8Hz, 2H), 7. 66 (d, J = &amp; 8Hz, 2H),

4. 68 (q, J = 5.1Ηζ, 2Η), 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. 3Hz, 2H), 3. 36 (t, J = 10. 7Hz, 2H), 2. 87 (dd, J = 13.3,4.9Hz, 2H), 2.74 (dd, J = 13.3,7.8Hz,2H), 1.25 (d, J = 5.lHz,6H).

Preparation of Compound 29

At room temperature, compound 161 (500 mg, 0.90 mmol) and 10% Pd(OH) were combined with nitrogen gas using a syringe.₂The suspension of /C (215 mg) in Et0H (230 mL) was degassed by bubbling for 10 minutes, and then stirred for 2 h under a hydrogen atmosphere (balloon, latm). The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and passed through column chromatography (silica gel, 9:1CH₂Cl₂/MeOH,8O: 18: 2CHCl₃/Me0H/NH₄0H) The residue was purified to obtain compound 29 (264 mg, 55%) as an off-white solid.

[0857] ^XH NMR(400MHz, CD₃0D): δ 6. 97 (d, J = &amp; 6Hz, 2H), 6. 67 (d, J = &amp; 6Hz, 2H),

4. 71(q, J = 5. lHz, 2H), 4. 06(dd, J = 10. 6, 5. 3Hz, 2H), 4. 13-4. 05 (m, 2H), 3. 81 (dd , J = 5.0, 2. 3Hz, 2H), 3. 80-3. 72 (m, 2H), 3. 51 (dd, J = 9. 6, 2.4 Hz, 2H), 3. 33- 3. 23 (m, 2H), preparation scheme of intermediate 24 27

3. 38 (t, J = 10. 7Hz, 2H), 2.83-2.54 (m, 6H), 1.85-1.69 (m, 2H), 1. 26 (d, J = 5. lHz ,6H).

[0858]

[0859]

[0860]

<img file="CN105073717A_D0195.tif" />

<img file="CN105073717A_D0196.tif" />

<img file="CN105073717A_D0197.tif" />

Glycogen

QH_I3

160OH

NaCNBH₃. AcOH

<img file="CN105073717A_D0198.tif" />

Preparation of Compound 162

To a solution of compound 158 (200mg, 0.94mmol) and triol 160 (194mg, 0.94mmol) in MeOH (2.0mL) was added AcOH (0.17mL, 2.82mmol) at room temperature The reaction mixture was stirred for 30 minutes. Add to

NaCNBH₃(148mg, 2.35mmol), the solution was stirred for 16h at room temperature. Add additional compound 160 (0.2 equivalents), AcOH (3.0 equivalents) and NaCNBH₃(1.0 equivalent), the solution was stirred at room temperature for 16 h. After removing the solvent, use saturated NaHC0₃Neutralize the residue, make the residue in CH ₂Cl₂Partition between (10 mL) and water (10 mL). Separate the water layer and use CH₂Cl₂(2X10mL) extraction. The combined organic extracts were dried and concentrated under vacuum. By column chromatography (silica gel, 9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 162 (95 mg, 28%) as an off-white solid.

[0863] ^XH NMR(400MHz, CD₃0D): δ 8.24 (d, J = 9. 1Hz, 2H), 7. 69 (d, J = 9. lHz, 2H),

4. 70 (q, J = 5.1Hz, 1H), 4. 09-4. 02 (m, 2H), 4. 00 (d, J = 2. 1Hz, 2H), 3. 83 (dd, J = 5.1 ,

115

2. 3Ηζ, 1Η), 3.81-3.71 (m, 1H), 3.53 (dd, J = 9.3, 2. 3Hz, 1H), 3.38 (t, J = 11. ΟΗζ, 1H ),

3. 21-3.07 (m, 2H), 1. 25 (d, J = 5.lHz, 3H).

Preparation of Compound 164

To a solution of compound 162 (95mg, 0.26mmol) and hexanal 163 (52mg, 0.51mmol) was added AcOH (0.05mL, 0.78mmol) and NaCNBH₃ (41mg, 0.65mmol) _o The solution was stirred for 16 h at room temperature. After removing the solvent, use saturated NaHC0₃The residue was neutralized and partitioned between EtOAc (10 mL) and water (10 mL). Separate the water layer and use CH₂Cl₂(2X10mL) extraction. The combined organic extracts are tested. Dry and concentrate under vacuum. By column chromatography (silica gel 9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 164 (70 mg, 59%) as an off-white solid.

[0866] ^XH NMR(400MHz, CDC1₃): 6 &amp; 18 (d, J = 8. 9Hz, 2H), 7. 56 (d, J = &amp; 9Hz, 2H),

4. 70 (q, J = 5. ΟΗζ, 1H), 4. 15 (dd, J = 10. 4, 5.2 Hz, 1H), 4.01-3.89 (m, 2H), 3. 83 (dd , J =

3. 8,2.7Hz, 1H), 3. 77 (brs, 1H), 3. 70 (brs, 1H), 3. 64 (t, J = 6. 2Hz, 1H), 3. 56 (dd, J = 9. 2,

4. 0Hz,lH), 3.41(t, J = 10. 8Hz, 1H), 2.87 (dd, J = 13. 2,4.3Hz, 1H), 2.78-2.68 (m, 2H ),

2. 63-2.55 (m, 1H), 1.75-1.43 (m, 4H), 1.34 (d, J = 5.0Hz,3H), 1.32-1.25 (m, 6H ), 0.89 (t, J = 6.6 Hz, 3H).

Preparation of Compound 24

The suspension of compound 164 (1.70 g, 3.77 mmol) and 10% Pd/C (200 mg) in MeOH (40 mL) was degassed with nitrogen at room temperature for 10 minutes, and then in a hydrogen atmosphere (balloon, latm) under stirring for 2h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo and passed through column chromatography (silica gel, 9:1CH₂C1₂/ MeOH, 80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 24 (1.20 g, 76%) as an off-white solid.

[0869] ^XH NMR(300MHz, CDC1₃): δ 6. 96 (d, J = 8.9Hz, 2H), 6. 62 (d, J = &amp; 9Hz, 2H),

4. 68 (q, J = 5. ΟΗζ, 1H), 4.14 (dd, J = 11. 0, 5.5 Hz, 1H), 3. 92-3. 81 (m, 2H), 3. 72 (dd , J =

3. 8,2.4Hz, 1H), 3.50 (dd, J = 9. 1,4. ΟΗζ, 1H), 3.40 (t, J = 10. 5Hz, 1H), 2.76-2.38 (m, 10H), 1.81-1.64(m,3H), 1.48-1.36 (m, 2H), 1.33 (d, J = 5.0Hz,3H), 1.30 -1.20 (m, 6H), 0.88 (t, J = 6.6 Hz, 3H).

Preparation of Intermediate 85

[0871] Scheme 28

[0872]

116

<img file="CN105073717A_D0199.tif" />

<img file="CN105073717A_D0200.tif" />

<img file="CN105073717A_D0201.tif" />

Preparation of Compound 166

To a solution of compound 148 (4.60 g, 21.3 mmol) and triol 165 (17.1 g, 63.9 mmol) in MeOH (100 mL) was added AcOH (12.1 mL, 63.9 mmol), in The reaction mixture was stirred at room temperature for 10 minutes. Add NaCNBH₃(4.00g, 63.9mmol), the solution was stirred for 6h at room temperature. Then add hexanal 163 (5.1OmL, 42.6mmol) and NaCNBH₃(2.60g, 42.6mmol). The solution was stirred for another 2 h at room temperature. After removing the solvent, use saturated NaHC0₃The residue was neutralized, and the residue was partitioned between EtOAc (200 mL) and water (200 mL). Separate the water layer and use CH₂Cl₂(2X300mL) extraction. The combined organic extracts are tested. Dry and concentrate under vacuum. By column chromatography (silica gel, 9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 166 (6.90 g, 64%) as an off-white solid.

[0875] ^XH NMR(400MHz, CD₃0D): δ &amp;12(d,J = &amp; 6Hz, 2H), 7. 51-7.43 (m, 2H), 7.38 (d, J = &amp; 6Hz, 2H), 7.37-7.27 (m,3H) ,5.55(s, 1H), 4.24(dd, J = 11. 5, 5.5Hz, 1H), 4.18-4.01 (m, 1H) ,4.00 -3.94(m, 1H), 3.93-3.89(m, 1H), 3.77(dd, J = 9.3,1.8Hz, 1H), 3.61 (t, J = 10. 7Hz, 1H), 3.13-2.77(m,6H), 2.71(t, J = 7.5Hz, 2H), 1.99T. 85 (m, 2H), 1. 55- 1.42 (m, 2H), 1.38-1.18(m,6H), 0.87 (t, J = 7.0Hz,3H).

Preparation of Compound 85

A suspension of compound 166 (800 mg, 1.55 mmol) and 10% Pd/C (300 mg) in EtOH (40 mL) was degassed by bubbling with nitrogen using a syringe for 10 minutes, and then in a hydrogen atmosphere ( Balloon, 1atm) and stir at room temperature for 2h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 85 (700 mg, 93%) as an off-white solid.

[0878] P NMR (400MHz, CD₃0D): δ 7.52-7.42 (m, 2H), 7.38-7.25 (m, 3Η), 6.88 (d, J=8.4Hz,2H), 6.63(d , J = 8. 4Hz, 2H), 5. 53 (s, 1H), 4. 24 (dd, J = 10. 8, 5.5 Hz, 1H),

4. 05-3.84(m,3H), 3.76 (dd, J = 9. 6, 1. 8Hz, 1H), 3. 61 (t, J = 10. 8Hz, 1H), 2. 93 (dd , J = 13. 6, 5. ΟΗζ, 1H), 2.79 (dd, J = 13. 4, 9. ΟΗζ, 1H), 2.73-2.60 (m, 4H), 2. 42 ( t, J = &amp; OHz, 2H), 1.88-1.68(m,2H), 1.48-1.36 (m, 2H), 1.33-1.14(m,6H) ,0 . 87 (t, J = 7. 0Hz, 3H).

Preparation of Intermediate 34

Scheme 29

[0881]

117

<img file="CN105073717A_D0202.tif" />

EtOH HN

Pd/C, Hiro

<img file="CN105073717A_D0203.tif" />

N'sugar

The preparation of Boc compound 168 adds saturated NaHC() 3 aqueous solution in the MeOH (30mL) solution of 162 (534mg, 1.45mmol) at 0 °C

[0882]

[0883] (5.0 mL) and stirring for 10 minutes. Then add (Boc)₂0 (350mg, 1.60mmol) and the reaction mixture was stirred at the same temperature for 3h, placed at room temperature, and stirred for another 30 minutes. The mixture was concentrated and the residue was dissolved in CH₂C1₂ (100 mL), and wash the solution with water (100 mL) and brine (50 mL). The organic layer was dried over Na^SOq, filtered, concentrated and passed through column chromatography (silica gel, 9:1CH₂Cl₂/MeOH,8: 2CHCl₃/MeOH) The residue was purified to obtain compound 168 (435 mg, 64%) as an off-white solid.

[0884] ^XH NMR(400MHz, CDC1₃): 6 &amp; 18 (d, J = 8. 7Hz, 2H), 7. 56 (d, J = &amp; 7Hz, 2H),

4. 72 (q, J = 5.1Hz, 1H) ,4.41-4.35(m,2H), 4.16 (dd, J = 10.8, 5.5Hz, 1H), 4.15-4 .04 (m, 1H), 3.93-3.83 (m, 1H), 3. 81-3.76 (m, 1H), 3.66-3.53(m,4H), 3.40 (t, J = 11. ΟΗζ, 1H),

3. 25-3. 12 (m, 1H), 3. 08-2.96 (m, 1H), 1.49 (s, 9H), 1. 32 (d, J = 5. lHz, 3H).

Preparation of Compound 34

[0886] Compound 168 (80 mg, 0.1% and 10% Pd/C (40 mg) was heated with a syringe with nitrogen gas).

The suspension in EtOH (10 mL) was degassed by bubbling for 10 minutes, and then stirred at room temperature for 2 hours under a hydrogen atmosphere (balloon, latm)_oThe reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 34 (82 mg, 89%) as an off-white solid.

[0887] ^XH NMR(400MHz, CDC1₃): δ 6.96 (d, J = 8.1Hz, 2H), 6.62 (d, J = &amp; 1Ηζ,2Η),

4. 69 (q, J = 5.1Hz, 1H), 4. 15 (dd, J = 10. 8, 5.5 Hz, 1H), 4.13-4.09 (m, 1H), 4.01-3 .93 (m, 1H) ,3.89-3.78 (m, 1H), 3.75-3.68 (m, 1H), 3.62-3.43(m,4H), 3.40 (t, J = 11. 3Hz, 1H),

3. 35 (dd, J = 13. 5, 4. ΟΗζ, 1H), 3. 26 (t, J = 7.9Hz, 1H), 3. 23-3. 13 (m, 1H), 2. 48 (t , J = 7.8Hz,2H), 1.86-1.76 (m, 2H), 1.43(s,9H), 1.33 (d, J = 5.lHz,3H).

Preparation of Intermediate 171

[0889] Scheme 30

[0890]

118

<img file="CN105073717A_D0204.tif" />

BocQNaHCOs

MeOHMO

<img file="CN105073717A_D0205.tif" />

<img file="CN105073717A_D0206.tif" />

To a solution of compound 148 (6.40 g, 29.6 mmol) and triol 165 (11.9 g, 44.5 mmol) in MeOH (300 mL) was added AcOH (5.32 mL, 8 &amp; 8 mmol) at room temperature The reaction mixture was stirred for 30 minutes. Add NaCNBH₃(3.73g, 59.2mmol), the solution was stirred for 16h at room temperature. Add additional compound 165 (11.9g, 44.5mmol), AcOH (5.32mL, 8&amp; 8mmol) and NaCNBH₃(3.73g, 59.2mmol), the solution was stirred for 14h at room temperature_oAdd additional compound 165 (7.93g, 29.6mmol), AcOH (3.55mL, 59.2mmol) and NaCNBH₃(2.80g, 44.4mmol) _oStir the solution for 10h at room temperature_oAfter removing the solvent, use saturated NaHC0₃Neutralize the residue, make the residue in CH ₂C1₂Partition between (100 mL) and water (100 mL). Separate the water layer and use CH₂Cl₂(2X100mL) extraction. The combined organic extracts were passed through the blood paw. . Dry and concentrate under vacuum. By column chromatography (silica gel, 9: lCH₂Cl₂/Me0H,80: 18: 2CHCl₃/Me0H/NH₄0H) Challenging purification was performed to obtain compounds 150 and 169 (20 g, mixture). This mixture was used directly in the next step.

Preparation of Compound 170

To a solution of 150 and 169 (20.0 g, mixture) in MeOH (120 mL) and water (40 mL) was added saturated NaHCO at 0°C.₃ (9.99g, 11 &amp; 4mmol) and stir for 10 minutes. Add (Boc)₂0 (9.69 g, 44.4 mmol) and the reaction mixture was stirred at the same temperature for 10 minutes, placed at room temperature, and stirred for another 2 h. The mixture was concentrated and the residue was dissolved in CH₂C1₂ (100 mL), and wash the solution with water (100 mL) and brine (50 mL). The organic layer was dried over Na^SOq, filtered, concentrated and passed through column chromatography (silica gel, 9:1CH₂Cl₂/MeOH,8: 2CHCl₃/MeOH) The residue was purified to obtain compounds 150 (1.50 g) and 170 (4.50 g) as off-white solids. ESI-MS m/z 529[C₂₇H₃₂N₂0₉+H]⁺_oPreparation of Compound 171

[0895] A suspension of compound 170 (4.20 g, 7.92 mmol) and 10% Pd/C (500 mg) in EtOH (100 mL) and AcOH (10 mL) was degassed with nitrogen for 10 minutes, and then in hydrogen Stir at room temperature under atmosphere (balloon, latm) for 16h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo, neutralized with Chu (1) 3 and passed through column chromatography (silica gel, 9: 1CH₂Cl₂/MeOH,8: 2CHCl₃/MeOH) The residue was purified to obtain compound 172 (2.70 g, 68%) as an off-white solid.

[0896] P NMR (400MHz, CD₃0D): δ 7.52-7.44 (m, 2H), 7.36-7.29 (m, 3Η), 6.89 (d, J=8.3Hz,2H), 6.64(d , J = &amp; 3Hz, 2H), 5. 54 (s, 1H), 4. 23 (dd, J = 11. 9, 5. 9Hz, 1H),

119

4. 10-3.97 (m, 1H), 3. 97-3.89 (m, 1Η), 3. 81-3.75 (m, 1H), 3.74-3.69 (m, 1H), 3. 60 (t, J = 10. 9Hz, 1H), 3.48 (dd, J = 14. 1,4.6Hz, 1H), 3. 28-3. 22 (m, 3H), 2. 41 (t, J = 7.5Hz,2H), 1.83T.71(m,2H),1.41(s,9H).

Preparation of Intermediate 39

[0898] Scheme 31

[0899]

<img file="CN105073717A_D0207.tif" />

Under a nitrogen atmosphere, a solution of compound 17 (30.0 g, pure 121 mmol) and 173 (14.2 g, 145 mmol) in anhydrous ethidium (300 mL) was degassed for 10 minutes, and then added at room temperature TEA (67mL, 484mmo 1), 10% in hexane (t-Bu) ₃P (49.0 mL, 24.2 mmol) and Cui (1.15 g, 6.05 mmol). The resulting mixture was degassed with nitrogen for another 10 minutes, and Pd(PPh₃)₄(14.0g, 12.lmmol). After degassing with nitrogen for 5 minutes, the resulting mixture was heated to 50°C for 16 h. The reaction mixture was concentrated in vacuo and the residue was purified by column chromatography (silica gel, 2:3 hexane/EtOAc) to give compound 174 (15.0 g, 58%) as a brown oil.

[0901] ^XH NMR(400MHz, CDC1₃) 6 &amp; 14 (d, J = &amp; 8Hz, 2H), 7. 50 (d, J = &amp; 8Hz, 2H), 3. 71 (t, J = 6. 4Hz, 2H), 2. 50 (t, J = 6.8Hz, 2H), 1.80-1.70(m,4H), 1.70-1.65 (m, 1H).

Preparation of compound 175

At 0° C. under nitrogen, the anhydrous CH of compound 174 (15.0 g, 67.9 mmol)₂C1₂(50mL) Add Et to the solution₃N (2 &amp; OmL, 203.7 mmol) and DMAP (4.12 g, 33.9 mmol). After stirring the reaction mixture for 5 minutes at the same temperature, TsCl (32.5 g, 170 mmol) was added at 0°C. The resulting mixture was stirred for another 4 h at room temperature. After removing the solvent; make the residue in CH₂C1₂ Partition between (250 mL) and water (150 mL). Separate the water layer and use CH₂C1₂ (2X 250mL) extraction. The combined organic extracts were washed with brine, dried over Na^SOq and concentrated under vacuum. The residue was purified by column chromatography (silica gel, hexane/EtOAc) to obtain compound 175 (15.0 g, 60%) as a brown oil.

[0904] ^XH NMR(400MHz, CDCI3): δ &amp; 15 (d, J = 88Hz, 2Η), 7. 79 (d, J = &amp; 8Hz, 2H), 7. 50 (d, J = &amp; 8Hz, 2H) ,7 . 34 (d, J = &amp; 8Hz,2H) ,4. 10 (t, J = 6.4Hz,2H) ,2.44 (t, J = 7.0Hz,2H),

2. 44 (s, 3H), 1.90-1.79 (m, 2H), 1.75-1.61 (m, 2H).

Preparation of compound 176;

To a solution of compound 175 (5.00 g, 12.9 mmol, crude product) in THF (10 mL) was added NHMe₂(30%, 50.0 mL) of water, and then stirred in a sealed tube at room temperature for 3 h. After removing the solvent; make the residue in CH₂C1₂Partition between (100 mL) and water (100 mL). Separate the water layer and use CH₂C1₂(2X 100mL) extraction. The combined organic extracts were washed with brine, dried over Na^SOq and concentrated under vacuum. The crude product was purified by column chromatography (silica gel) to obtain compound 176 (400 mg, 13%) as a yellow viscous solid.

[0907] ^XH NMR (400MHz, CDC1₃) :6&amp;15(d, J = 7. 3Hz, 2H), 7. 51 (d, J = 7. 3Hz, 2H),

120

2. 48(t, J = 6.6Hz,2H), 2.30 (t, J = 5.7Hz,2H), 2.23(s,6H), 1.70-1.61(m,4H).

Preparation of Compound 39

A suspension of compound 176 (400 mg, 1.62 mmol) and 10% Pd/C (50 mg) in EtOH (50 mL) was degassed by bubbling for 10 minutes with a syringe with nitrogen, and then in a hydrogen atmosphere (balloon , Latm) and stirred at room temperature for 16h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to obtain 39 (300 mg, 84%) as a brown sticky solid.

[0910] ^XH NMR(400MHz, CD₃0D): δ 6.91 (d, J = 7.5Hz, 2H), 6.65 (d, J = 7.5Hz,2H), 2.47(t, J = 7. 0Hz,2H), 2 . 30(dd, J = &amp; 4,6. 5Hz, 2H), 2. 23 (s, 6H), 1. 60T. 52 (m, 2H),

1. 51-1.41 (m, 2H), 1.38-1.27 (m, 4H).

31 Preparation of Intermediate 44

[0912] Scheme 32

[0913]

<img file="CN105073717A_D0208.tif" />

Preparation of Compound 177

[0915] A solution of compound 175 (6.00 g, 16.0 mmol) in methanol (150 mL) containing 7N N2 was heated in a sealed tube at 30° C. for 5 h. The temperature was raised to 40°C and stirred for 16h, and then heated to 60°C again and stirred for 4h. After removing the solvent; make the residue in CH₂C1₂ Partition between (100 mL) and water (100 mL). Separate the water layer and use CH₂C1₂ (2X 100mL) extraction. The combined organic extracts were washed with brine, dried over Nahoq and concentrated under vacuum. By column chromatography (silica gel, 9:1 CH₂Cl₂/MeOH) The crude product was purified to obtain compound 177 (1.48 g, 43%) as a yellow oil.

[0916] ^XH NMR(400MHz, CDC1₃): 6 &amp; 16 (d, J = 8. 4Hz, 2H), 7. 39 (d, J = &amp; 4Hz, 2H),

3. 61 (t, J = 5.6Hz, 2H), 2. 08-2. 05 (m, 2H), 1. 65-1. 53 (m, 4H).

Preparation of Compound 178 and 179

To a solution of compound 177 (1.38 g, 6.33 mmol) and triol 165 (2.03 g, 7.59 mmol) in MeOH (10 mL) was added AcOH (0.6 mL, 9.49 mmol), The reaction mixture was stirred at room temperature for 30 minutes. Add NaCNBH₃(800mg, 12.7mmol), the solution was stirred at room temperature for 16h. Add additional compound 165 (2.55g, 9.49mmol), AcOH (0.80mL, 12.7mmol) and NaCNBH₃(1.19g, 1&amp; 9mmol), the solution was stirred at room temperature for 16h_oAdd additional compound 165 (2.55g, 9.49mmol), AcOH (0.8OmL, 12.7mmol)

121

And add NaCNBH₃(l. 19g, 18.9mmol), continue to stir at room temperature for 16h. After removing the solvent, use saturated NaHC0₃Neutralize the residue, make the residue in CH₂Cl₂Partition between (10 mL) and water (10 mL). Separate the water layer and use CH₂C1₂ (2X 10mL) extraction. The combined organic extracts were dried over NapSOq and concentrated under vacuum. By column chromatography (silica gel, 9:1 CH₂Cl₂/MeOH,80: 18: 2 CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 179 (2.28 g, 51%) as an off-white solid.

[0919] ^XH NMR(300MHz, CD₃0D) :6 &amp; 14 (d, J = 9. ΟΗζ, 2H), 7. 54 (d, J = 9. 0Hz, 2H), 7. 47-7. 44 (m, 4H), 7. 34- 7. 30 (m, 6H), 5.48 (s, 2H), 4. 24-4. 19 (m, 2H), 3. 99-3.94 (m, 4H),

3. 86-3.84 (m, 2H), 3. 73-3.69 (m, 2H), 3. 57 (t, J = 10. 8Hz, 4H), 3. 35-3. 25 (m, 4H ), 2.33 (d, J = 6.9Hz,2H),,1.61T.51(m,4H).

[0920] A mixture of 178/179 (900mg) was also isolated and used directly in the next step (SG-GHC-G-106) _oPreparation of Compound 44

A suspension of compound 179 (2.26 g, 3.11 mmol) and 10% Pd/C (100 mg) in a mixture of EtOH (50 mL) and AcOH (10 mL) was degassed with nitrogen for 10 minutes, and then Stir at room temperature for 16h under a hydrogen atmosphere (balloon, latm). The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to obtain 44 (1.90 g, 80%) as a brown solid.

[0923] ^XH NMR (400MHz, CD₃0D): 6 7. 46-7. 44 (m, 4H), 7. 33-7. 31 (m, 6H), 6. 89 (d, J = &amp; 4Hz, 2H), 6. 65 (d, J = &amp; 4Hz,2H), 5.51 (s,2H), 4.264.14(m,2H), 3.93-3.90(m,2H), 3.76-3.73 (m , 4H), 3. 63-3. 58 (m, 4H), 3. 35-3. 25 (m, 2H), 3. 10-3. 00 (m, 2H), 2. 41 (t, J =

7. 2Hz,2H), 1.47-145 (m,4H), 1.16-1.12(m,4H).

Preparation of Intermediate 49

[0925] Scheme 33

[0926]

<img file="CN105073717A_D0209.tif" />

Boc₂O, NaHCO.

XieOH/H^O

<img file="CN105073717A_D0210.tif" />

Preparation of Compound 180

[0928] To a solution of 178 (900 mg, mixture, about 2.0 mmol) in a mixture of MeOH (20 mL) and water (10 mL) at 0° C., NaHC() 3 (672 mg, 4.0 mmol) was added and stirred 10 minutes. Add (Boc)₂0 (524mg, 2.40mmol), and the reaction mixture was stirred at the same temperature for 1h, placed at room temperature and stirred for another 4h. The mixture was concentrated and the residue was dissolved in CH₂C1₂ (100 mL), and the solution was washed with water (100 mL) and brine (50 mL). Pass the organic layer through Na₂S0₄Dry, filter, concentrate and pass through column chromatography (silica gel, 9:1 CH₂Cl₂/MeOH,8: 2 CHCl₃/MeOH) The residue was purified to obtain compound 180 (780 mg, 64%) as an off-white solid.

[0929] ^XH NMR(300MHz, CD₃0D) :6 &amp; 16 (d, J = 9. ΟΗζ, 2H), 7. 55 (d, J = 9. 0Hz, 2H), 7. 50-7. 47 (m, 2H), 7. 34- 7. 30 (m, 3H), 5. 53 (s, 1H), 4. 25-4. 20 (m, 1H), 4. 10 (br s, 1H),

122

3. 94-3.91 (m, 1H), 3. 80-3.48 (m, 4Η), 3.35-3.25 (m, 3Η), 2.46 (t, J = 6.9Hz, 2H ),

1. 70-1.49 (m, 4H), 1.43 (s, 9H).

Preparation of Compound 49

[0931] The suspension of compound 180 (780 mg, 1.36 mmol) and 10% Pd/C (50 mg) in a mixture of EtOH (10 mL) and AcOH (2.0 mL) was drummed with nitrogen by using a syringe. The bubble was degassed for 10 minutes, and then stirred at room temperature for 4h under a hydrogen atmosphere (balloon, latm). Use Na₂C0₃The reaction mixture was neutralized, filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to obtain 49 (625 g, 84%) as a white solid.

[0932] P NMR (300MHz, CD₃0D): δ 7.50-7.46 (m, 2H), 7.32-7.30 (m, 3Η), 6.90 (d, J=8.4Hz,2H), 6.66(d , J = 8.4Hz, 2H), 5.53 (s, 1H), 4. 25-4.20 (m, 1H), 4. 04 (br s, 1H), 3. 94-3.89 ( m, 1H), 3. 77-3. 43 (m, 4H), 3. 35-3. 25 (m, 3H), 2. 45 (t, J = 7.5Hz, 2H), 1. 52- 1. 47 (m, 4H), 1. 42 (s, 9H), 1. 27-124 (m, 4H).

Preparation of Intermediate 54

[0934] Scheme 34

[0935]

<img file="CN105073717A_D0211.tif" />

Preparation of Compound 182

Under nitrogen, to a solution of compound 181 (1.60 g, 16.0 mmol) in anhydrous THF (40 mL) was added 9-BBN (0.5 M in THF, 80 mL, 40.0 mmol). After the reaction mixture was stirred for 2h at room temperature, compound 172 (3.17g, 12.8mmol), Pd(PPh₃) ₂C1₂ (561 mg, 0.80 mmol) and 1N NaOH aqueous solution (24 mL). The resulting mixture was stirred for another 1 h. After removing the solvent; the residue was partitioned between EtOAc (100 mL) and water (100 mL). The aqueous layer was separated and extracted with EtOAc (2×100 mL). The combined organic extracts were washed with brine and passed through the blood paw. . Dry and concentrate under vacuum. The crude product was purified by column chromatography (silica gel, 4: 1 hexane/EtOAc) to obtain compound 182 (1.20 g, 34%) as a brown solid.

[0938] ^XH NMR (400MHz, CDC1₃): 6&amp;13(d, J = 9. ΟΗζ, 2H), 7. 31 (d, J = 9. 0Hz,2H), 3. 64(t, J = 6. 7Hz,2H), 2. 71 (t , J = 7.8Hz,2H), 1.731.46(m,4H), 1.43-1.31(m,4H).

Preparation of Compound 183

123

[0940] At 0°C under nitrogen, to an anhydrous (20 mL) solution of compound 182 (1.20 g, 5.38 mmol) was added Et₃N (7.32 mL, 53.8 mmol). After the reaction mixture was stirred at the same temperature for 5 minutes, methanesulfonyl chloride (0.62 mL, 8.07 mmol) was added at 0°C. The resulting mixture was stirred for 2 h at room temperature. After removing the solvent; make the residue in CH₂Cl₂Partition between (50mL) and water (50mL). Separate the water layer and use CH₂C1₂(2X50mL) extraction. The combined organic extracts were washed with brine, dried over NaSO and concentrated under vacuum. Crude product 183 (3.00 g, crude product) was used directly in the next step.

Preparation of Compound 184

[0942] A solution of compound 183 (3.00 g, 5.38 mmol, crude product) in 7N methanol (30.0 mL) was heated in a sealed tube at 60° C. for 2 h. After removing the solvent; make the residue in CH₂C1₂Partition between (100 mL) and water (100 mL). Separate the water layer and use CH₂Cl₂(2X100mL) extraction. The combined organic extracts were washed with brine and subjected to Na₂S0₄Dry and concentrate under vacuum. The crude product was purified by column chromatography (silica gel) to obtain compound 184 (390 mg, 33% yield over two steps) as a yellow oil.

[0943] ^XH NMR(400MHz, CD₃0D) :6 &amp; 14 (d, J = 9. ΟΗζ, 2H), 7. 42 (d, J = 9. 0Hz, 2H),

2. 75(t, J = 7.8Hz, 2H), 2.67(t, J = 7.3Hz, 2H), 1.72T. 63 (m, 2H), 1. 53T. 46 (m, 2H),

1. 42-1.35(m,4H).

Preparation of Compound 185

To a solution of compound 184 (620mg, 2.79mmol) and triol 165 (938mg, 3.49mmol) in MeOH (30mL) was added AcOH (1.16mL, 27.8mmol), and reacted at room temperature The mixture was stirred for 10 minutes. Add NaCNBH₃(526mg, 8.37mmol), the solution was stirred at room temperature for 16h. Within 16h, add additional compound 165 (0.3 equivalent AcOH (10 equivalent) and NaCNBH₃(1.0 equivalent). Then add hexanal 163 (0.96mL, &amp; 37mmol), AcOH (1.00mL) and NaCNBH₃ (526mg, 8.37mmol) _o The solution was stirred for another 2 h at room temperature. After removing the solvent, use saturated NaHC0₃The residue was neutralized and partitioned between EtOAc (100 mL) and water (100 mL). Separate the water layer and use CH₂Cl₂(2X100mL) extraction. The combined organic extracts were dried over Na^SOq and concentrated under vacuum. By column chromatography (silica gel, 9:1 CH₂Cl₂/MeOH,80: 18: 2 CHCl₃/Me0H/NH₄OH) The residue was purified to obtain compound 185 (950 g, 61%) as an off-white oil.

[0946] P NMR (400MHz, CDC1₃): δ 8.02 (d, J = 8.7Hz, 2H), 7. 48-7. 42 (m, 3H), 7. 37-7. 34 (m, 2Η), 7. 31 (d, J = &amp; 7Hz, 2H), 5. 54 (s, 1H), 4. 46-4. 40 (m, 1H), 4. 30 (dd, J =

11. 6,6.6Hz, 1H), 4.03 (t, J = 4. ΟΗζ, 1H), 3. 97 (dd, J = 10. 5,5.4Hz, 1H), 3.88 (dd, J = 9. 4, 4. ΟΗζ, 1H), 3.65 (t, J = 10. 4Hz, 1H), 3. 11-3. 00 (m, 4H), 2. 69 (t, J = 7. 8Hz, 2H),

2. 00 (s, 1H), 1.70-1.55(m,6H), 1.37-1.30(m,4H), 1.29-1.20(m,8H) ,0.87 ( t, J = 7.1Hz, 3H).

Preparation of Compound 54

A suspension of compound 185 (950 g, 1.70 mmol) and 10% Pd/C (300 mg) in EtOH (100 mL) was degassed with nitrogen for 10 minutes, and then in a hydrogen atmosphere (balloon, latm) at room temperature Stir for 3h. The reaction mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated in vacuo to give 54 (790 mg, 88%) as a yellow oil. [0949] P NMR (400MHz, CD3OD): 67.51-7.44(m,2H), 7.35-7.29 (m,3H), 6.90(d, J=8.5Hz, 2H), 6.65(d, J = &amp; 5Hz, 2H), 5. 54 (s, 1H), 4. 24 (dd, J = 10. 8, 5. 4Hz, 1H),

4. 08-4.02 (m, 1H), 4. 00-3.92 (m, 1H), 3.91 (dd, J = 5. 6, 1. 8Hz, 1H), 3. 78 (dd, J = 9. 6, 1. 8Hz, 1H), 3. 61 (t, J = 10. 9Hz, 1H), 3. 01 (dd, J = 13. 7, 5. 4Hz, 1H), 2. 91 ( dd, J = 12. 1,

124

&amp; 1Ηζ, 1Η), 2.82-2.71 (m,4H), 2.45 (t, J = 7.5Hz, 2H), 1.59T.42 (m, 6H), 1.37-1.13 (m, 10H) ,0.89 (t, J = 7. lHz, 3H).

[0950] Some assays can be used to characterize the compounds of the invention. Representative assays are discussed below.

In vitro measurement of sodium channel blocking activity and reversibility

An assay for evaluating the mechanism of action and/or efficacy of the compounds of the present invention includes using an airway epithelial monolayer installed on the Ussing Chamber in the short-circuit current (I_sc) Measure the airway epithelial sodium current to determine intracavitary drug inhibition. Cells obtained from freshly excised human, dog, sheep or rodent airways were inoculated onto porous 0.4 micron SnapwellTM Inserts (CoStar), in a hormone-defined medium at the air-liquid interface (air-liquid Cultivation under the conditions of interface, ALI), the sodium transport activity was measured when immersed in the Krebs Bicarbonate Ringer (KBR) in the Eus perfusion chamber (lj. The half-log dose was added to the lumenal bath) Add all test compounds (1 X 10^ΠΜ to 3X10 hours), and record the cumulative change in Isc (inhibition). Take 1 X10²M concentration was used as a stock solution to prepare all drugs in dimethyl inkstone and stored at -20°C. Usually 8 preparations are performed in parallel; each two preparations introduce amiloride and/or benzamil as a positive control. In the application of the maximum concentration (5X10⁵M) After that, the cavity bath was exchanged three times with fresh drug-free KBR solution, and the resulting Isc was measured after each wash for a duration of about 5 minutes. Reversibility is defined as the percentage of sodium current returning to the baseline value after the third wash. Collect all data from the voltage clamp through the computer interface and analyze it offline.

[0953] The dose-effect relationship of all compounds was considered and analyzed by the Prism 3.0 program. The IC50 value, the maximum effective concentration and the reversibility were calculated and compared with amiloride and benzamil as a positive control. The effectiveness of representative compounds in cells freshly excised from the airway of dogs relative to the sodium channel blocking activity of amiloride is shown in Table 1.

Table 1. Inhibition of short circuit current (IC) by compound (Ia) in canine bronchial epithelial cells₅₀nM)

[0955]

125

<img file="CN105073717A_D0212.tif" />

Assay 2. Mucociliary Clearance (MCC) study in sheep

[0957] The animal model most frequently used for measurement in MCC is the sheep model. Can be used by Sabator etc.,

126

The in vivo model described in Journal of Applied Physiology, 1999, pages 2191 to 2196 (which is incorporated herein by reference) measures the effect of compounds on enhancing mucociliary clearance (MCC).

[0958] In these studies, adult sheep were controlled and a tracheal tube was inserted nasally. Spray the test sample for 10-15 minutes on the sheep. Then, the radiolabeled 99"Tc-sulfur colloid (TSC, 3.1 mg/mL; containing about 20 mCi) was administered four or eight hours after the test sample at the designated time. The radiolabeled aerosol was administered through the tracheal intubation The dose is about 5 minutes. Then the cannula of the sheep is removed, and the total radioactivity count in the lung is measured every 5 minutes during the observation period of 1 hour. The clearance rate of radioactivity in the lung represents the MCC rate in the animal.

[0959] The advantage of this system is that it approximates the human lung environment. The model also allows simultaneous collection of PK/PD information through plasma and urine sampling during the test. There are also several techniques that can measure the concentration of the drug on the airway surface in the MCC measurement. These techniques include collecting exhaled air condensate or obtaining ASL by the filter paper method through bronchoscopy_O

The above-mentioned sheep model was used to evaluate the in vivo effect (efficacy/durability) of the test agent delivered by aerosol on MCC. Treatment included testing of 4 mL of test agent or test agent in combination with HS. In order to determine whether HS has been combined with compound II-d MCC, HS is administered immediately after administration of the compound test agent. A Raindrop nebulizer was used to atomize the test solution at a flow rate of eight liters per minute and connected to a dosimetry system consisting of a solenoid valve and a compressed air source (20 psi). After using a raindrop sprayer for aerosol application, the deposited dose of the drug in the lungs of sheep is estimated to be 8% to 15% of the dose. Using a raindrop sprayer, 4 or 8 hours after the drug treatment, radioactive TSC was applied for about 3 minutes to evaluate its efficacy/durability. A gamma camera was used to measure radioactivity counts in the central area of the right lung every 5 minutes for one hour. Three analytical methods are used, 1) the initial rate of removal (slope) in the first 30 minutes using a linear regression fit, 2) the% area under the curve is removed within one hour, and 3) the maximum obtained within one hour clearance rate.

[0961] The effect of compound 33 on sheep MCC at 0.24 nmol/kg (3uM) was tested 4 hours after administration, and compared with the vehicle (4 mL sterile out of 0) (Figure 1). The analysis of the effect is shown in Table A. Compared with vehicle control, compound 33 enhanced MCCo

Table A. MCC in sheep 4 hours after administration of compound 33 or vehicle

[0963]

<td>Compound 33 dosage</td><td>Initial slope (4.0-4.5h)</td><td>AUC (% 1-h)</td><td>Maximum clearance rate</td>

<td>0.24 innol/kg (3μΜ)</td><td>37.5* (4)</td><td>17.4* (4)</td><td>30.0* (4)</td>

<td>Carrier (H2O) 4inL,</td><td>17.2±6.8(8)</td><td>7,3±1.5 (8)</td><td>12.2±2.9(8)</td>

Table B and Table C together with Figure 2 and Figure 3 show that other compounds of the invention similarly enhance MCC compared to the carrier (see, for example, compounds 123 and 48)

Table B. MCC of sheep 4 hours after administration of compound 123 or vehicle

[0966]

127

<td>Compound 123 Dong</td><td>Initial slope (4.0-4.5h)</td><td>AUC (% C1-h)</td><td>Maximum clearance rate</td>

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

<td>Carrier (Η2Ο) 4 mL</td><td>17.2±6.8(8)</td><td>7.3 ± 1.5(8)</td><td>12.2 + 2.9(8)</td>

Table C. MCC of sheep 4 hours after administration of compound 48 or vehicle

[0968]

<td>Compound 48 dose</td><td>Initial slope (4.0-4.5h)</td><td>AUG (% CI-h)</td><td>Maximum clearance rate</td>

<td>0.24 nmol/kg (3μΜ)</td><td>29.8* (2)</td><td>15.4* (2)</td><td>26.7* (2)</td>

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

In order to determine whether the compound of the present invention has enhanced the duration of action, it was tested 8 h after administration. Table D and Table E together with Figures 4 and 5 clearly show that compounds 33 and 152 enhance the duration of the effect on MCC compared to the carrier.

Table D. MCC of sheep 4 hours after administration of compound 33 or vehicle

[0971]

<td>Compound 33 dosage</td><td>Initial slope (8.0-8.5H)</td><td>AUC(% Cl- h)</td><td>Maximum clearance rate</td>

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

<td>Carrier (H2O) 4hiL</td><td>17.2 ±6.8 (8)</td><td>7.3 Soil 1.5 (8)</td><td>12.2 + 2.9(8)</td>

Table E. MCC of sheep 4 hours after administration of compound 152 or vehicle

[0973]

<td>Compound 152 dosage</td><td>Initial slope</td><td>AUC (% Cl-h)</td><td>Maximum clearance rate</td>

<td>0.24 nmol/kg (3μΜ)</td><td>37.5* (4)</td><td>17.4* (4)</td><td>30.0* (4)</td>

<td>Carrier (Η2Ο) 4 mL</td><td>17.2±6.8(8)</td><td>7.3 + 1,5(8)</td><td>12.2 ±2.9 ⑻</td>

128

In order to determine whether HS increased the MCC effect of Compound 33, it was evaluated that 7% HS was administered immediately 8 hours after the combined administration of 0.24 nmol/kg Compound 33 and MCC (Figure 6). It is shown in Figure 6 that HS improves the effect of compound 33 on MCC.

[0975] Assay 3. Clearance and metabolism of airway surface drug liquid (ASL) through human airway epithelium

[0976] The disappearance of compound 33 from the apical surface and the metabolism of airway epithelium in human bronchial epithelial (HBE) cells were evaluated (Table 3). In these experiments, 25 μL of a 25βM ENaC blocker solution was added to the apical surface of HBE cells growing at the air/liquid interface, and the drug concentration in the apical and basolateral compartments was measured by UPLC within 2 h.

Table G. Apical disappearance and metabolism of compound 33

[0978]

<td>Compound</td><td>Initial drug mass% on the top side (maternal and metabolites, 2h)</td><td>Top mass% as metabolite (2h)</td><td>Mass% of the initial top part on the basal side (2h)</td><td>Metabolites on the basal side% (2h)</td>

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

[0979] Values are expressed as mean±SD

[0980] Comparative Example

[0981] Compared with known sodium channel blockers such as amiloride and the third-generation compound as described in Comparative Example 1 below, the compound of formula (I) of the present invention is more potent and/or from the mucosal surface , (Especially the surface of the airway) is not absorbed quickly. Therefore, as evidenced by the data shown in Table G, the compound of formula (I) has a longer half-life on the mucosal surface than these known compounds. The disappearance of compound 33 from the apical surface and the metabolism of airway epithelium in HBE were evaluated and compared with Comparative Example 1 (Table H). In these experiments, 25 μL of a solution of 25 pM ENaC blocker was added to the apical surface of HBE cells cultured at the air/liquid interface, and the drug concentration in the apical and basolateral compartments was measured by UPLC within 2 h. After the compound of the present invention was incubated on the apical surface for 2 hours (37°C), most of the compound 33 was not metabolized on the apical side. In contrast, most of the comparative example 1 from the top side, 83% metabolism into the lower active acid, (S)-2-amino-3-(4-(4-(3-(3, 5-two Amino-6-chloropyrazine-2-propanyl) myoyl) butyl) phenoxy) propionic acid, the structure is as follows.

[0982]

<img file="CN105073717A_D0213.tif" />

[0983] Table H. Apparent disappearance and metabolism of Compound 33 and Comparative Example 1 in HBE

[0984]

129

<td>Compound</td><td>Initial drug mass% on the top side (maternal and metabolites, 2h)</td><td>Top mass% as metabolite (2h)</td><td>Mass% of the initial top on the basal side (2h)</td><td>Metabolites on the basal side% (2h)</td>

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

<td>Comparative Example</td><td>41・6±7・6%</td><td>83.0±3・5%</td><td>&amp; 3±0・2</td><td>94.7±1.0%</td>

<td>1</td><td>(8% of the mother)</td><td></td><td>(1% of the mother)</td><td></td>

[0985] Values are expressed as mean±SD

[0986] Comparative Example 1 In WO 2003/070182 (U.S. Patent Nos. 6,858,615; 7,186,833; 7, 189,719; 7,192,960; and 7332496), the structure is protected, described or disclosed as having Useful pharmaceutical sodium channel blockers and can be prepared by methods described therein and well known in the art.

Comparative Example 1

[0988]

<img file="CN105073717A_D0214.tif" />

[0989] (S) -3,5-Diamino-6-chloro-N-(4-4-(2,3-diamino-3-oxopropoxy)phenyl)butyl) formamide Base) pyrazine)-2-carboxamide

The compound of Comparative Example 1 can be seen on page 15 of US 2005/0080093 and as compound 2 on page 90 of WO 2008/031048 and as compound 2 on page 42-43 of WO 2008/031028. In order to have beneficial activity in the treatment of cystic fibrosis and C. 0. P. D, the compound must be based on multiple administrations without increasing plasma potassium (which ultimately leads to hyperkalemia, a serious and dangerous It has the property of enhancing mucociliary clearance (MCC) at the dosage of disease). Therefore, such compounds must be avoided, which are known to increase plasma potassium if they are significantly excreted by the kidneys. To assess this possibility, it is beneficial to have MCC activity in the body and at the available dose, it will not cause an increase in plasma potassium. One evaluation model is the sheep MCC model described below.

[0991] As can be seen from Table 1 and Figure 7, the ED50 of Comparative Example 1 in the sheep MCC model obtained using three different measurement methods (slope, AUC and maximum clearance) is about 240 nmol/kg ( 3mM). At this dose (which will be the clinically active dose), Comparative Example 1 leads to an increase in plasma potassium (Figure 8), which will lead to hyperkalemia when repeated administration. Therefore, Comparative Example I is not available for human use, and compound (Ia) produces a safe and effective MCC and the benefit-risk ratio in this model is greater than 1000.

Table 1. MCC of vehicle, comparative example 1 or compound 33 in sheep 4 hours after administration

[0993]

130

<td>dose</td><td>Initial slope (4.0-4.5H)</td><td>AUC (% (Ί χ h)</td><td>Maximum deduction rate</td>

<td>Comparative Example 1</td><td>32.2 ± 7.3* (6)</td><td>14.1 ±2.2* (6)</td><td>22.9 + 2. P (6)</td>

[0994]

<td colspan="2">240 nmol/kg (3ιηΜ)</td><td colspan="2"></td>

<td>Comparative Example 124 nmol/kg (300 μΜ)</td><td>14.5 Taxi IJ (3)</td><td>6.9 ± 1.0(3)</td><td>146 ±0.9 (3)</td>

<td>Compound 33</td><td>37.5* (4)</td><td>17.4* (4)</td><td>30 0* (4)</td>

<td>0.240 nmol/kg (30 μΜ)</td><td></td><td></td><td></td>

<td>Carrier Η₂Ο (4 mL)</td><td>17.2 ±6.8 (8)</td><td>7.3 + 1.5(8)</td><td>12.2 ±2.9 (8)</td>

[0995] FIG. 1 plots the percentage of mucus clearance of Compound 33 and Comparative Example 1 as described above in the MCC model over a period of time. Compound 33 provided an even higher percentage of mucus clearance at a dose 1000 times lower than that seen in Comparative Example 1. Therefore, compound 33 provides the maximum effect in the relevant dose range where there is no clinical increase in plasma potassium.

10 shows that in the MCC study, in sheep plasma receiving Comparative Example 1, a significant increase in plasma potassium level can be seen at an effective dose. In sheep's MCC, compound 33 is 1000 times more effective than Comparative Example 1, and the dose is as high as 24 nmol/kg (ED₅₀1000 times the dose) did not increase plasma K. However, Comparative Example 1 increased plasma K at an ED50 dose of about 3 mM (Figures 7 and 8). This again shows that Compound 33 has unique and unexpected advantages in efficacy and safety. As shown in Table J, compared with Comparative Example 1, it has a 1000-fold higher renal safety.

[0997] Table J. Treatment ratio (benefit/risk)

[0998]

<td></td><td>The highest submaximal dose of MCC</td><td>Does not increase the maximum dose of plasma potassium in sheep</td><td>Treatment ratio</td>

<td>Comparative Example 1</td><td>240nmol/kg (3mM)</td><td>24nmol/kg (300 μ Μ)</td><td>0. 1</td>

131

<td>33</td><td>&lt;0.24nmol/kg (3 μ Μ)</td><td>24nmol/kg (300 μ Μ)</td><td>&gt; 100</td>

<td>ratio</td><td>&gt; 1, 000</td><td>1</td><td>&gt; 1, 000</td>

[0999] Other compounds of the present invention have similar safety and efficacy, surpassing known compounds as exemplified in FIGS. 11, 12, 13 and 14.

132