Chloro-pyrazine carboxamide derivatives with epithelial sodium channel blocking activity

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, including 3,5-diamino-N-(Ν-(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 the compounds, treatment methods and uses of the compounds, and The method used to prepare the compound.

Background technique

[0002] A variety of "innate defenses", that is, 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 fluid layer on the mucosal surface reflects the balance between epithelial fluid secretion and epithelial fluid absorption. Epithelial fluid secretion usually reflects anions (Cl" coupled with water (and cation counter-ion). And/or HCO3J secretion, epithelial fluid absorption is usually reflected with water and counter anions (C "and/or HC0₃-) Coupled Na+ absorption. Many diseases of the mucosal surface are caused by too little protective fluid on these mucosal surfaces, and the latter is caused by an 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 Na+ channels and liquid absorption. The epithelial protein that mediates the rate-limiting step of Na+ and 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+ and fluid absorption, amiloride-like ENaC blockers must be delivered to the mucosal surface and maintained at the site for 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 bodys inability to recover from the lungs normally. Partially clears mucus, which eventually leads to chronic airway infections. See Evidence for airway surface dehydration as the initiating event in CF airway disease, RCBoucher, Journal of Internal Medicine, Volume 261, Issue 1, January 2007, pages 5-16; and Cystic f ibrosis: a disease of vulnerability to airway surface dehydration, RCBoucher, 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 results in a relative decrease in ASL, which leads to mucus concentration, pericliary liquid (PCL) lubrication activity, mucus adhesion to the airway surface, and the inability to clear mucus to the oral cavity through cilia 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.

CN 105073717 Β

[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] RCBoucher in US6,264,975 describes the use of pyrazinoylguanidine sodium channel blockers to hydrate the mucosal surface, with the well-known diuretics amiloride, benzamil (benzami 1) and phenalamine Mir (phenam order) as a representative. However, considering the limited weight of drugs that can be inhaled 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. 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 lack of fluid in the oral cavity is because the parotid, sublingual, and submandibular glands cannot secrete fluid, and persistent 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 under conditions of continuous Na+-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 C (and fluid) in the proximal small intestine, together with the increased absorption of Na* (and fluid) in the terminal ileum, leads to distal intestinal obstruction syndrome (DIOS). In elderly patients , Excessive Na* (and volume) absorption in the descending colon produces constipation and diverticulitis.

[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 Nos. W02003/070182, W02003/070184, W02004/073629, W02005/025496, W02005/016879, W02005/018644, W02006/022935, W02006/023573, W02006/023617, W02007/018640, W02007/146869, W02008/031028, W02008/ 031048, and U.S. Patent Nos. 6858614, 6858615, 6903105, 7064129, 7168833, 7189719,

7192958、7192959、7192960、7241766、7247636、7247637、7317013、7332496、7368447、

7368450, 7368451, 7375102, 738013, 7397666, 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="CN105073717B_D0001.tif" />

R_t

<img file="CN105073717B_D0002.tif" />

<img file="CN105073717B_D0003.tif" />

Μ ΗΗ₂ (I)

[0014] where:

[0015] Ar is selected from:

CN 105073717 Β

<img file="CN105073717B_D0004.tif" />

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

[0018] P is selected from the group consisting of hydrogen, C-C8 alkyl and polyalkylated alkyl having 3 to 8 carbon atoms;

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

[0020] Furnace and M are each independently hydrogen or C-C3 alkyl.

[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-(Ν-(4-(4-((S)-2-amino-3-(4-(3-(bis((2S, 3R,4R, 5R) -2,3,4,5,6-Pentylhexyl)amino)propyl)phenylamino)-3-oxopropyl)cae-1-yl)butyl)methionine 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] Figure 1 is a graph showing the effect of compound 33 on sheep MCC 4 hours after administration.

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

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

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

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

[0030] Figure 6 is a graph showing the enhancing 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.

[0033] FIG. 9 is a graph comparing the activity of Comparative Example 1 and Compound 33 on sheep MCC at 4 hours after administration.

[0034] FIG. 10 is a graph comparing the effects of Comparative Example 1 and Compound 33 on sheep plasma K+ levels.

[0035] FIG. 11 is a graph comparing the activity of Comparative Example 1 and Compound 123 on sheep MCC at 4 hours after administration.

[0036] Figure 12 is a graph comparing the effects of Comparative Example 1 and Compound 123 on sheep plasma K+ 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.

[0038] Figure 14 is a graph comparing the effects of Comparative Example 1 and Compound 48 on sheep plasma K+ levels.

[0039] Detailed description of the invention

CN 105073717 Β

[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 compounds, which may include naming compounds using the ChemDraw Ultra 11.0 software program sold by CambridgeSoft 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, -CH3).

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

[0046]

<img file="CN105073717B_D0005.tif" />

N C1

[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-Pentaylhexyl)amino)propyl)phenylamino)-3-oxopropyl)-5,6,7,8-tetrahydrozea-1-yl) Butyl) Methyl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salt:

[0048]

<img file="CN105073717B_D0006.tif" />

<img file="CN105073717B_D0007.tif" />

<img file="CN105073717B_D0008.tif" />

[0049] In yet another embodiment, the compound of formula (1) is 3,5-diamino-N-(N-(4-(6(2-amino-3-(4-(3-(bis (2,3,4,5,6-Pentylhexyl) amino) propyl) phenylamino) -3-oxopropyl) Cai-2 yl) butyl) methyl sulfonyl) -6-chloropyridine Pyrazine-2-carboxamide or its pharmaceutically acceptable salt:

CN 105073717 Β

[0050]

<img file="CN105073717B_D0009.tif" />

ΝΗ Ο

<img file="CN105073717B_D0010.tif" />

[0051] In another embodiment, the compound of formula (1) is 3,5-diamino-N-(Ν-(4-(4-(

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) Methyl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salt:

[0052]

<img file="CN105073717B_D0011.tif" />

<img file="CN105073717B_D0012.tif" />

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

<img file="CN105073717B_D0013.tif" />

<img file="CN105073717B_D0014.tif" />

<img file="CN105073717B_D0015.tif" />

<img file="CN105073717B_D0016.tif" />

Ν Dan

<img file="CN105073717B_D0017.tif" />

*;⁵

<img file="CN105073717B_D0018.tif" />

CN 105073717 Β

[0055] Where:

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

[0057] P is selected from hydrogen, C1-C8 alkyl and polyalkylated alkyl having 3 to 8 carbon atoms;

[0058] is hydrogen or a polyalkylated alkyl group having 3 to 8 carbon atoms;

[0059] M and M are each independently hydrogen or C1-C3 alkyl.

[0060] In each compound group or pharmaceutically acceptable salt thereof independently represented by formula (1), (II), (III) and (IV), there are additional embodiments, in which:

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

[0062] P is selected from the group consisting of hydrogen, C-C8 alkyl and polyalkylated alkyl having 3 to 8 carbon atoms;

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

[0064] M and M are each independently hydrogen or a C-C3 alkyl group.

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

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

[0067] P is selected from hydrogen and C1-C8 alkyl;

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

[0069] M and M are each independently hydrogen or C-C3 alkyl.

[0070] In each compound group or pharmaceutically acceptable salt thereof independently represented by formula (1), (II), (III) and (IV), there is another embodiment in which:

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

[0072] P is selected from hydrogen and C1-C8 alkyl;

[0073] R? is hydrogen;

[0074] M and M are each independently hydrogen or a C-C3 alkyl group.

[0075] In each compound group independently represented by formulas (I), (II), (III) 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] P and F are each independently a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0078] M and M are each independently hydrogen or C-C3 alkyl.

[0079] In each of the compound groups independently represented by formula (1), (II), (III) and (IV) or a pharmaceutically acceptable salt thereof, there is another embodiment, in which:

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

[0081] P and F are each independently a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0082] Less and shochu is hydrogen.

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

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

[0085] P and F are each independently a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0086] R3 and M are each independently a Ci-Cs alkyl group.

[0087] In each of the compound groups independently represented by formula (1), (II), (III) and (IV) or a pharmaceutically acceptable salt thereof, there is

CN 105073717 Β

Another embodiment, in which:

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

[0089] P and F are each independently a polyvinylated alkyl group having 3 to 8 carbon atoms;

[0090] The furnace is hydrogen;

[0091] M is C1-C3 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 T, 2,2-triol, butane T, 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) Those compounds of nH, 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 of the H groups are OH.

[0094] Another embodiment in each compound group described herein is where the polyvinylated alkyl group has the formula -CH2-CHOH-(CHR⁶), wherein 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 H group is OH.

[0095] Yet another embodiment in each of the compound groups described herein includes wherein the polyvinylated alkyl group has the formula -Gamma 2-(CHOH) _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- (CHOH) _n-CH₂The chain represented by 0H is 2,3,4,5,6-Pentylhexane, which has the following formula:

<img file="CN105073717B_D0019.tif" />

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

<img file="CN105073717B_D0020.tif" />

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

CN 105073717 Β

<img file="CN105073717B_D0021.tif" />

<img file="CN105073717B_D0022.tif" />

<img file="CN105073717B_D0023.tif" />

<img file="CN105073717B_D0024.tif" />

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

[0104] 2 and M are each independently hydrogen or C1-C3 alkyl.

[0105] 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 2 and R⁴Each is 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 2 and M are each a C1-C3 alkyl group.

[0106] In each of the embodiments described herein, there are additional embodiments where 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 (1), (la), (II), (HI), (IV), (V), (VI) and (VII), may be free bases or salts, especially those of formula (1), (la), (II), (HI), (IV), (V), (VI) and (VII) 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 from 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 Amino acid, leucine

CN 105073717 Β

Sour 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 the 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-Hi Π Dictionary of Chemical Terms (1984) McGraw-Hi 11 Book Company, New York; and Eliel, E. and W. ,S.,Stereochemistry of Organic Compounds (1994) John Wiley&amp;Sons, Inc._?New York_o

[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 can also be called an enantiomer, and a mixture of such isomers is usually called an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or 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:

CN 105073717 Β

<img file="CN105073717B_D0025.tif" />

ΝΗ

[0115]

<img file="CN105073717B_D0026.tif" />

<img file="CN105073717B_D0027.tif" />

[0116] All tautomeric forms of squam, amide, muscle, gland, sulphur gland, heterocycle, etc. of all embodiments of Formula I are within the scope of the present invention. Tautomers exist in an equilibrium state, 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 enantiomerically-enriched mixtures and diastereomer-enriched mixtures, 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 less than 5% of other diastereomers are present, 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. The 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 (^/z Stereo chemistry of Carbon Compounds, (1962) by EL Eliel, McGraw Hill; Lochmuller, CH, (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) with the hand The derivatization reagent forms diastereomeric compounds, separates the diastereomers and converts them into pure stereoisomers, and (3) directly separates 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)phenyl

CN 105073717 Β

Amino)-3-oxopropyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide or its pharmaceutically acceptable salt enantiomerically enriched mixture or combination Things.

[0120] Other embodiments include formulas (1), (la), (II), (IID, (IV), (V), (VI), and (VI), respectively, contained in each of their respective mixtures as major isomers Enantiomerically enriched mixtures or compositions of the compound of (VII) or its pharmaceutically acceptable salt.

[0121] In another embodiment, the present invention provides 3,5-diamino-N-(Να-(4-(-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 or an enantiomerically enriched mixture or composition of a pharmaceutically acceptable salt thereof.

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

[0123] Also provided herein are each compound or group of compounds described herein, including formulas (I), (la), (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 compounds of formula I and their 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 lung conditions in people in need, such as diseases related to reversible or irreversible airway obstruction, 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, post-viral cough after viral infection, cystic fibrosis, emphysema, pneumonia, panbronchiolitis, Transplant-related bronchiolitis (including bronchitis related to lung transplantation and bone marrow transplantation). 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. also

CN 105073717 Β

Provides (a) a method for slowing down COPD deterioration in mammals in need; a method for slowing down CF deterioration in mammals in need; (c) improvement in mammals in need Method of lung function (FEV1); (d) Method of improving lung function (FEV1) in mammals with COPD; (e) Method of improving lung function (FEV1) in mammals with CF; (f) ) A method to slow down airway infections in mammals in need.

[0129] There is also provided a method for stimulating, enhancing or improving mucociliary clearance in a mammal, the method comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound of formula (D or a pharmaceutically acceptable salt thereof. Mucosal Ciliary 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 bronchus. Therefore, it also provides a mechanism for improving mucus clearance in the airway of mammals in need. method.

[0130] In addition, sodium channel blockers may be suitable for the treatment of conditions 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, Sjogrens disease (Sjogrens disease) 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.

[0131] 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 methods for the treatment of conditions improved by increased 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 a method for treating asthma in a mammal (especially a human) in need thereof. 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. On one implementer In this case, 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 for the treatment of pneumonia in mammals (especially humans) in need

CN 105073717 Β

Methods. In one embodiment, the present invention provides a method for treating panbronchiolitis in a mammal (especially a human) in need thereof. In one embodiment, the present invention provides methods for the treatment of 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 methods for the treatment of ventilator-associated tracheobronchitis and/or prevention of ventilator-associated pneumonia in persons who use ventilator in need thereof.

[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 tracheobronchiolitis, each method includes administering to the human an effective amount of a compound of formula 1(a) or a pharmaceutically acceptable salt thereof. In other embodiments of each treatment method, 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 administration of dry oxygen) in mammals (especially humans) 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 (eg, 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, there is provided a compound of the present invention for use in the treatment of asthma in mammals (especially humans) in need thereof. In one embodiment, a compound is provided for use in the treatment of bronchiectasis (including bronchiectasis caused by conditions other than cystic fibrosis) or bronchitis (including acute bronchitis) in mammals (especially humans) in need thereof Inflammation and chronic bronchitis). In one embodiment, mentioning A compound is provided for use in the treatment of cough after viral infection in mammals in need thereof, especially humans. 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 breastfeeding in need thereof

CN 105073717 Β

Treatment of panbronchiolitis or transplantation-related bronchiolitis (including lung transplantation and bone marrow transplantation-related bronchiolitis) in animals (especially human). 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, the compounds of the present invention are provided for use in the treatment of conditions alleviated by increased mucosal hydration of the mucosal surface in mammals (especially humans) in need thereof. In one embodiment, there is provided a compound 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, transplantation-related bronchiolitis (including lung transplantation and bone marrow transplantation-related bronchiolitis), ventilator-related tracheobronchiolitis, or prevention of ventilator-related 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 caused by the application of dry oxygen), treatment of dry eye, Sjogrens disease, promotion of eye or corneal hydration, treatment of otitis media, primary ciliary dyskinesia, Distal ileus syndrome, esophagitis, constipation, or chronic diverticulitis.

[0143] The terms "effective amount", "pharmaceutically effective amount", "effective dose" and "pharmaceutically effective dose" as used herein 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 an amount effective to enhance normal physiological functions. In one embodiment, an effective amount means that when such a composition is administered by inhalation, it provides a desired level of drug in the secretions and tissues of the airway and lungs of the subject or the blood stream 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.

[0144] 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, properties of the formulation, route of administration and 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), and

CN 105073717 Β

And any difference in potency between amiloride and the compound of the present invention should be properly considered.

[0145] The pharmaceutically effective dose for topical application to the airway surface of a 70-blood human subject may be from about 10 ng to about. In another embodiment, the pharmaceutically effective dose may be from about 0.1 g to about 1000 ug_oGenerally speaking, the daily dose administered locally to the airway surface will be sufficient to obtain a dissolved concentration of the active agent on the airway surface of about ιο-\ιο-⁸Or KT? to about 10-\10-\10-²Or 10-1 mol/L, more preferably about 1 (Γ⁹To about 10-4 moles/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 used to treat a 70 kg human will be from about 10 nanograms (ng) to about. In another embodiment, the effective dose will be from about 0.1 g to about 1,000 ug_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 dose will be from about 0.5 ug to about 60 μg. In another embodiment, the pharmaceutically effective dose will be about 1 ug to about. In another embodiment, the pharmaceutically effective dose will be about 5 ug to about 60 μg. 50μ "Another embodiment has an effective dose of about 40 ug. In two other embodiments, the pharmaceutically effective dose will be about 15 ug to about 50 ug, about 15 ug to about It should be understood that in these dosage ranges In each, all increased doses within the range are included. For example, the range of 0.5μg to 50ug includes the following individual doses: 0.5ug&gt;0.6ug&gt;0.7ug&gt;0.8ug&gt;0.9ug&gt;1.0ug&gt;l. lug, 1.2ug&gt; 1.3ug&gt;1.4ug&gt;1.5ug&gt;1.6ug&gt;1.7ug&gt;1.8ug&gt;1.9ug&gt;2.0ug&gt;2.1ug&gt; 2.2ug&gt; 2.3ug&gt; 2.4ug&gt; 2.5μ g&gt; 2.6ug&gt; 2.7 ug&gt; 2.8ug&gt; 2.9ug&gt; 3.Oug&gt; 3.lug&gt;3.2ug&gt;3.3ug&gt;3.4ug&gt;3.5ug&gt;3.6ug&gt;3.7ug&gt;3.8 g, 3.9ug, 4.0ug, 4.lug&gt;4.2ug &gt;4.3ug&gt;4.4ug&gt;4.5ug&gt;4.6ug&gt;4.7ug&gt;4.8ug&gt;4.9ug&gt;5.0ug&gt; 5.lug, 5.2ug, 5.3ug, 5.4ug, 5.5ug, 5.6ug, 5.7ug, 5.8ug, 5.9ug, 6.Oug, 6.lug, 6.2ug, 6.3μg&gt;6.4ug&gt;6.5ug&gt;6.6ug&gt;6.7ug&gt;6.8ug&gt;6.9ug&gt;7.0ug&gt;7.lug&gt;7.2ug&gt;7.3ug&gt;7.4 ug&gt;7.5ug&gt;7.6 i^Jg&gt;7.7ug&gt;7.8ug&gt;7.9ug&gt;8.0ug&gt;8.lug&gt;8.2ug&gt;8.3ug&gt;8.4ug&gt;8.5ug&gt;8.6ug&gt;8.7ug&gt;8.8ug&gt; 8.9ug&gt;9.0ug&gt;9.lug&gt;9.2 ug&gt;9.3ug&gt;9.4ug&gt;9.5ug&gt;9.6ug&gt;9.7ug&gt;9.8ug&gt;9.9ug&gt;10.0ug&gt;10.1 ug&gt;10.2ug&gt;10.3ug&gt;10.4ug&gt;10.5ug&gt;10.6ug&gt;10.7ug&gt;10.8ug&gt; 10.9ug&gt;ll.Oug&gt;ll. lug, 11.2 ug&gt;11.3ug&gt;11.4ug&gt;ll.5ug&gt;11.6ug&gt;ll.7ug&gt;11.8ug&gt;11.9ug&gt;12.Oug&gt;12.lug&gt;12.2ug&gt;12.3 Pg&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;13.4 ug&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;14.5 ug&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;15.6 Pg&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;16.7 Pg&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;17.8 Pg&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;18.9 Pg&gt;19.0ug&gt;19.lug&gt;19.2ug&gt;19.3ug&gt;19.4 ug&gt;19.5ug&gt;19.6ug&gt;19.7ug&gt;19.8ug&gt;19.9ug&gt;20.0 Ug&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;21.1 Ug&gt;21.2ug&gt;21.3ug&gt;21.4ug&gt;21.5ug&gt;21.6ug&gt;21.7ug&gt;21.8ug&gt;21.9ug&gt;22.0ug&gt;22.1ug&gt;22.2 Pg&gt;22.3ug&gt;22.4ug&gt;22.5ug&gt;22.6ug&gt;22.7 ug&gt;22.8ug&gt;22.9ug&gt;23.0ug&gt;23.lug&gt;23.2ug&gt;23.3 Pg&gt;23.4ug&gt;23.5ug&gt;23.6ug&gt;23.7ug&gt;23.8ug&gt;23.9ug&gt;24.0ug&gt;24.lug&gt; 24.2ug&gt; 24.3 ug &gt;24.4 Ug&gt;24.5ug&gt;24.6ug&gt; 24.7ug&gt; 24.8ug&gt; 24.9ug&gt; 25.0ug&gt;25.lug&gt;25.2ug&gt;25.3ug&gt;25.4ug&gt;25.5 Ug&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;26.6 Pg&gt; 26.7ug&gt; 26.8ug&gt; 26.9ug&gt; 27.Oug&gt; 27.lug&gt;27.2ug&gt;27.3ug&gt;27.4ug&gt;27.5ug&gt;27.6ug&gt;27.7 Ug&gt; 27.8ug&gt; 27.9ug&gt; 28.Oug&gt; 28.lug&gt;28.2ug&gt;28.3ug&gt;28.4ug&gt;28.5ug&gt;28.6ug&gt;28.7ug&gt;28.8 Ug&gt; 28.9ug&gt; 29.Oug&gt; 29.lug&gt;29.2ug&gt;29.3ug&gt;29.4ug&gt;29.5 ug&gt;29.6ug&gt;29.7ug&gt;29.8ug&gt;29.9

CN 105073717 Β

ug&gt;30.0ug&gt;30.lug, 30.2ug&gt;30.3ug&gt;30.4ug&gt;30.5ug&gt;30.6ug&gt;30.7ug&gt;30.8ug&gt;30.9pg, 31.0 Pg, 31.lyg, 31.2yg, 31.3yg, 31.4yg, 31.5 yg, 31.6yg, 31.7yg, 31.8yg, 31.9yg, 32.0yg, 32.1 Pg, 32.2pg, 32.3pg, 32.4pg, 32.5pg, 32.6pg, 32.7pg, 32.8pg, 32.9pg, 33.0pg, 33.lug , 33.2 Pg, 33.3ug, 33.4ug, 33.5ug, 33.6ug, 33.7ug, 33.8ug, 33.9ug, 34.Oug, 34.lug, 34.2ug&gt;34.3 Pg, 34.4ug, 34.5ug&gt;34.6ug, 34.7yg , 34.8ug, 34.9ug&gt;35.Oug, 35.lug, 35.2ug&gt;35.3ug&gt;35.4 Pg, 35.5pg, 35.6pg, 35.7pg, 35.8pg, 35.9pg, 36.0pg, 36. lug, 36.2pg, 36.3pg, 36.4pg, 36.5 Pg, 36.6pg, 36.7pg, 36.8pg, 36.9pg, 37.0pg, 37.lyg, 37.2yg, 37.3yg, 37.4yg, 37.5yg, 37.6 Pg, 37.7pg , 37.8pg, 37.9pg, 38.0pg, 38.lug, 38.2pg, 38.3pg, 38.4pg, 38.5pg, 38.6pg, 38.7 Pg, 38.8pg, 38.9pg, 39.0pg, 39.lug, 39.2pg, 39.3pg , 39.4pg, 39.5pg, 39.6pg, 39.7pg, 39.8 Pg, 39.9ug&gt;40.0ug&gt;40.lug, 40.2ug&gt;40.3ug&gt;40.4ug&gt;40.5ug&gt;40.6ug&gt;40.7ug&gt;40.8ug&gt;40.9 Pg, 41.0 song, 41.lug, 41.2ug, 41.3ug, 41.4 song, 41.5 song, 41.6 song, 41.7 song, 41.8ug, 41.9 song, 42.0 Pg, 42. lug, 42.2ug&gt;42.3ug&gt;42.4ug, 42.5ug, 42.6ug, 42.7yg, 42.8ug, 42.9ug, 43.Oug&gt;43.1 Pg, 43.2ug, 43.3ug, 43.4ug, 43.5ug, 43.6ug, 43.7yg , 43.8ug, 43.9ug, 44.Oug, 44.lug&gt;44.2 Pg&gt;44.3ug, 44.4ug, 44.5ug, 44.6ug, 44.7ug, 44.8ug, 44.9ug, 45.0ug, 45.lug, 45.2ug&gt; 45.3 Pg&gt;45.4pg, 45.5pg, 45.6pg, 45.7pg, 45.8pg, 45.9pg, 46.0ug&gt;46.lug, 46.2pg, 46.3pg, 46.4 Pg, 46.5ug, 46.6ug, 46.7yg, 46.8ug, 46.9 ug, 47.Oug, 47.lug, 47.2ug, 47.3ug, 47.4ug&gt;47.5 Pg&gt;47.6pg, 47.7pg, 47.8pg, 47.9pg, 48.0ug&gt;48. lug, 48.2pg, 48.3ug&gt;48.4ug&gt;48.5pg, 48.6 Pg, 48.7pg, 48.8pg, 38.9pg, 49.0ug&gt;49.lug, 49.2pg, 49.3pg, 49.4pg, 49.5pg, 49.6pg, 49.7 Pg , 49.8ug, 39.9ug and 50ug.

[0146] If the compound is administered by a different route, the aforementioned recommended dosage can be adjusted by using a conventional dosage algorithm. 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 a compound of the present invention may require the delivery of a single dosage form or multiple unit dosages, which may 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 present 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 carried out the 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 American civilian populations to spores of Bacillus anthracis revealed a gap in the country's overall preparations for bioterrorism. In addition, the report detailed that these attacks revealed

CN 105073717 Β

There is an unmet demand for vaccines and immunotherapy, drugs and biological agents used to cure 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 having potential for bioterrorism, 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 each bioterrorism threat 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 includes administering an effective amount of a compound of formula (D) to an individual in need of preventive treatment of infection by an airborne pathogen. 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 (D) is administered to the lungs of people with symptoms of the disease, wherein effective amounts of sodium channel blockers and osmolytes are sufficient to reduce the risk of infection in humans. A specific example of an airborne pathogen is anthrax.

[0154] In another aspect, there is provided a post-exposure preventive treatment or therapeutic treatment method for the treatment of airborne pathogen infections, which comprises administering to the lungs of individuals in need of treatment against airborne pathogen infections An effective amount of the compound of formula (1). 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. usually, The pathogen may 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 lists compiled by the Centers for Disease Control and Prevention (CDC). As established by the CDC, Class 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 (severe acute respiratory syndrome). respiratory syndrome, SARS) _o

[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 radioactive materials from nuclear attacks such as radiological dispersal devices (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

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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 in recipients in need (including those in need), so The method includes administering to the human an effective amount of a compound of formula (D 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] Studies have 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 removed by BAL in a reasonable order. 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, inhaled insoluble in adult dogs¹⁴⁴Ce-FAP particles. Give two groups of dogs known to cause radiation pneumonitis and pulmonary fibrosis]⁴⁴Ce 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 treatment]⁴⁴Ce level (approximately IMBq/kg), but these animals are not treated. Allow all animals to spend their lifespan (which is up to 16 years). Because of the initial lungs between the dogs in each group]⁴⁴The Ce content is different, so 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 1.5MBq/kg to 2.5MBq/kg, the average survival time is 370±65 days_oFor the treated dogs, the average survival was 1270±240d, which is a statistically significant difference. Accept 0.6MBq/kg to 1.4MBq¹⁴⁴In the third group of Ce lung content, the average survival time was 1800±230, which was 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

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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 which (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, mitigating and/or treating definite health effects on the respiratory tract and/or other body organs caused by inhalable aerosols containing radionuclides include The individual administers an effective amount of the sodium channel blocker of formula (D or a pharmaceutically acceptable salt thereof. In a feature of this aspect, the sodium channel blocker is administered together with an osmotic agent. This feature is further described The osmotic agent is hypertonic saline (HS). In another feature, the sodium channel blocker and the osmotic agent are administered together with the ion transport modifier. Further to this feature, the ion transport modifier can be selected from B -Agonists, CFTR potentiators, purinergic receptor agonists, lubiprostone and protease inhibitors. In another feature of this aspect, the radionuclide is selected from the group consisting of -60, Xinse T37, Iron T92, Radium-226, Phosphorus-32, Saw-89 and Saw-90, Iodine T25, Pliers-201, Lead-210, Mu-234, Uranium-238, Brazing, Drilling-58 , Rong-51, shrink and copper. In another feature, the radionuclide comes from a radioactive disposal device. In another feature, the sodium channel blocker or its pharmaceutically acceptable salt is an aerosol of inhalable particles inhaled by the individual Agent suspension for administration. 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 contains 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 (I), (la), (II), (IID, (IV), (V), (VI) and (VII) The compound or a pharmaceutically acceptable salt thereof, exists alone or in combination with a pharmaceutically acceptable excipient, diluent or carrier.

[0166] In some embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of formula (I), (la), (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), (la), (II), (IID, (IV) in 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 an embodiment, it is 2.8% w/v.

[0167] A kit is also provided, which comprises: i) a pharmaceutically effective amount of formula (1), (la), (II), (IID, (IV), (V),

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(VI) and (VII) compounds or their pharmaceutically acceptable salts; ii) one or more pharmaceutically acceptable excipients, carriers or diluents; iii) for administering group i) to subjects in need The compound and the excipient, carrier or diluent of group ii); and iv) container. 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) formulas (1), (la), (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 (I), (la), (II), (IID, (IV), Solutions of the compounds of (V), (VI) and (VII) or 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) formulas (1), (la), (II), (III), (IV), (V), (VI) 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 diluent 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), (la), (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 administers 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), (la), (II), (IID, (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 to about 1,000 ug; b) about 0.5 to about 0.5mg; and c) about 0.5μg to about 50ug_o

[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] A pharmaceutically acceptable excipient, diluent, or carrier must be acceptable in terms of compatibility with the other ingredients of the formulation and not harmful to the recipient. Generally, the 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 Williams &amp; Wilkins; 21st edition (May 1, 2005). Preferred

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In particular, pharmaceutically acceptable excipients, diluents or carriers comply with FDA's Generally Regarded As Safe (GRAS).

[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 formulation can be presented in a unit dosage form or in a bulk form (for example, in the case of metering the formulation 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 containing particles for delivery using a nebulizer, a pressurized metered dose inhaler (MDI), a soft mist inhaler or a dry powder inhaler (DPI). The aerosol formulation used in the method of the present invention may be a liquid (e.g., 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 generally polydisperse, that is, they are composed of many particles of different sizes. The particle size distribution is usually described by Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD). For the best drug delivered to the intrabronchial space, the MMAD is about 1pm to about 10um, preferably about 1pm to about 5pm, and the GSD is less than 3, particularly preferably less than about 2. When inhaled into the lungs, aerosols with MMAD greater than 10 wn 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. In order to obtain these particle sizes in powder formulations, conventional techniques such as micronization or spray drying can be used 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. The desired fraction can be separated by air classification or sieving. In one embodiment, the particles are crystalline. 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 lung 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. Store

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Examples of library DPI include, but are not limited to, AstraZeneca's Turbohalo® and Vectura's ClickHaler®_o[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. During inhalation, 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 cyclone 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, US Published Patent Application No. 2006/0237010_oA paper published by the University of Groningen clarified that using this technology, a 60 mg dose of pure micronized colistin sulfomethate can be effectively delivered as a respirable dry powder.

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

[0186] Examples of suitable dry powder inhalers and dry powder dispersion devices for delivering the compounds and compositions according to 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. US5,458,135, US4,805,811 and U.S. Published Patent Application No. 2006/0237010_o

[0187] In one embodiment, the pharmaceutical formulation according to the present invention is a dry powder for inhalation, which is formulated for delivery through a Diskus®-type device. The Diskus® device includes an elongated band formed from a substrate, The elongated belt has a plurality of recesses spaced along its length and a cover sheet hermetically but releasably closed thereon to define a plurality of containers, each of which has a predetermined amount of active ingredient inhalable In the formulation, the active ingredient 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)_oThe TwincerTM inhaler contains a foil laminate blister, which has one or more recesses and a dense but peelable closure on it

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The cover sheet thus defines multiple 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 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. WO2009/015286 or WO2007/114881. 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 US7,108,159_oIn 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 to about 5 wn, and a GSD of less than about 2.

[0191] Liquid aerosol formulations for delivery to the intrabronchial space or lungs by inhalation may, for example, be formulated as an aqueous solution or suspension or aerosol delivered by 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 tank (for example, an aluminum can) with a valve (for example, a metering valve), and is installed in an actuator with 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: U.S. 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 invention as a dry powder, wherein the emitted particles have an MMAD of about 1 to about 5 wn, and a GSD of 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 formulations for nebulization are formulated by dissolving or reconstituted solid particle formulations, 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.

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[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 a chlorine concentration&gt; 30 mM, is at a pH of 4.5 to 7.4, preferably 5.0 to 5.5, and has an osmolarity of about 800 m0sm/kg to 1600 m0sm/kg. The pH can be controlled by titration with common acids (e.g., hydrochloric acid or sulfuric acid) or bases (e.g., sodium hydroxide) or by using buffers. Commonly used buffers include citrate buffers (for example, citric acid/sodium citrate buffer), acetate buffers (for example, acetic acid/sodium acetate buffer), and phosphate buffers. The strength of the buffer can be 2 mM to 50 mM.

[0196] Usable acetate, phosphate, and citrate buffers include sodium acetate, sodium acetate trihydrate, sodium acetate, potassium acetate, sodium phosphate, sodium phosphate dibasic&gt; sodium 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 salt (such as its citrate or sodium citrate salt), the salt of lactic acid and lactic acid (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 nebulizers (pneumatic jet nebulizers), exhaust or breath-enhanced jet nebulizers or ultrasonic nebulizers (including Static or vibrating perforated plate atomizer). Commercial nebulizers include Aeroneb® 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, the aerosol having an MMAD of about 1 to about 5 wn and a GSD of less than 2 . For best effectiveness and avoid upper respiratory tract and systemic side effects, the MMAD of the aerosol should not be greater than about 5 wn, and the GSD should not be greater than about 2. If the aerosol

With MMAD greater than about 5 wn or GSD 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 lwn, a large proportion of the particles may remain suspended in the inhaled air and thus may be expelled during exhalation.

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

CN 105073717 Β

[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 may 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 formulations 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, requiring 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 diapers can be prepared in dosage unit form to give a given amount containing a predetermined 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 may 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 for 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 wn to 500 wn can be used to ensure stay 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 make

CN 105073717 Β

Formulated with 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 comprises a compound of the present invention in an amount sufficient for the compound to obtain ιο-\ιο- on the airway surface⁸Or ΙΟ to about 1 (Γ⁴, 1 (Γ?, 1 (Γ? or 10 "mol / liter, more preferably 1 (Γ? to about 10 insect mol / liter dissolved concentration.

[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, 10" on the airway surface 8 or 10 "7 to about 1 () 7, 10 "3, 10 "2 or 10" mole/liter, more preferably 10" 9 to about mole/liter dissolved concentration.

[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 Sjogrens disease, promote eye or corneal hydration, treat distal intestinal obstruction syndrome, treat otitis media, primary ciliary dyskinesia, and distal intestinal obstruction. Symptoms, 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 containing 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 being 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 solutions 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 that exists 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 biocompatible drugs that gradually dissolve 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, beta-agonists (including selective beta-agonists), P2Y2 receptor agonists Agents, peroxisome proliferator-activated receptor (PPAR) 6 agonists, other epithelial sodium channel blockers (ENaC receptor blockers), cystic fibrosis transmembrane conductance regulator protein (Cystic fibrosis transmembrane conductance regulator, CFTR) regulators, kinase inhibitors, anti-infectives, antihistamines, non-antibiotic anti-inflammatory macrolides, bombs

CN 105073717 Β

Sexual proteases and protease inhibitors, as well as 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 iso-agonists), P2Y2 receptor agonists, PPAR6 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 effects (e.g., in the kidneys). Middle) Possibility of undesirable side effects of blocking sodium channels.

[0223] The "osmotic agent" according to the present invention is a molecule or compound having osmotic activity. "Osmotically 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, the compounds used in combination with osmolytes of the present invention (from both the child and nonionic) is preferably not actually promote or prevent or delay the growth of bacteria osmolyte. 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 acid (paraacetaminophen) P-glycollamidophenylarsonate (p-glycollamidophenylarsonate), hexylresorcinate (hexylresorcinate), Hybamine (Ν,Ν'-bis(dehydrorosinyl) ethylenediamine), hydrobromide, hydrochloride, Weng Cai formic acid Root, iodide, vat ethyl sulfonate, lactate, lacturonate, malate, maleate, mandelate, methanesulfonate, methyl bromide anion (methylbromide), methyl nitrate, methyl Sulphuric acid Root, mucinate, zesylate, nitrate, nitrite (nitrte), catabolite (emboate), pantothenate, phosphate or hydrogen phosphate, polygalacturonate, salicylate, stearate , Subacetate, succinate, sulfate, succinate, tartrate, teoclate (8-chloronaphtha), triethiodide, bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide and phosphate.

CN 105073717 Β

[0226] Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to, organic cations, such as Junxing (N, N'ethylenediamine), chloroprocaine, choline, diethanolamine, and ethylenediamine. Amine, 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, saddleium chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride, bromine Potassium and a combination of any two or more of the foregoing. In one embodiment, the invention provides a combination of a compound of the invention and two different osmotically active salts. When different salts are used, one of the anions or cations 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 (for example, glycerol, diacetone), 4-carbon sugars (for example, 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 -Carbon sugars (for example, D and L forms of maltose, allose, glucose, mannose, gulose, idose, galactose and talose, and D and L forms of allose-hepta Kulose, allose-heptulose (hepulose), glucose-heptulose, mannose-heptulose, gulose-heptulose, idose-heptulose, galactose-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 used to control the intracellular osmolality (osmolality) in the kidney. See, for example, J. S. Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995). Organic 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-inosito 1 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, certain 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 present 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 osmolytes amino acids by metabolic steps such as transamination. example

CN 105073717 Β

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 alters 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., et al., J. Med. Chem. 19: 113T17 (1976) ; Bodor, N., et al., J. Pharm. Sci. 67: 1045T050 (1978); Bodor, N., et al., 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 (especially 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 CF or COPD_oRecent findings indicate that although the relative ratio of HC03 "conductivity/C" conductivity in a single CFTR channel activated by cAMP and ATP is 0.1 to .2, the ratio in the sweat duct is actually 0 depending on the stimulation conditions. To almost 1.0. That is, the combination of cAMP+cGMP+α-ketoglutarate can produce CFTR HCOs with conductivity almost equal to Cl"^-Conductivity (Quiton et al. Physiology, Volume 22, Issue 3, 212-225, June 2007). In addition, formulations containing 7% and &gt;7% of the hypertonic saline of bicarbonate anions 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 airway surface liquid layer ( 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 would 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 the salt concentration is greater than normal saline (normal saline, NS), that is, greater than 9g/L or 0.9% w/v, the salt concentration of hypotonic saline is lower than normal saline, for example, about 1g/ L or 0.1% w/v to about 8g/L or 0.8% w/v. The salt concentration of the hypertonic salt solution that can be used in the formulations and treatment methods described herein can 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 1 g/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 20g/L (2% w/v) o

[0236] Salt 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 (1 %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) &gt; 110g/L (11 %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), 2lOg/L (21 %w/

CN 105073717 Β

ν), 220g/L (22%w/v) and 230g/L (23%w/v). It is also possible to use each salt concentration between these listed concentrations/percentages, such as 1.7g/L (0.17%w/v), 1.25g/L (1.25%w/v), 1.5g/L (1.5%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] Hypotonic saline solutions of specific usable concentrations include those of about 0.12 g/L (0.012% w/v) to about 8.5 g/L (0.85% w/v). Any concentration in this range can be used, for example, based on w/v, 0.05%, 0.1%, 0.15%, 0.2%, 0.225% (1/4NS), 0.25%, 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 osmotic agent (or precursor) with another chemical group that alters 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. et al., J. Med. Chem. 19: 113-117 ( 1976); Bodor, N., et al., J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N., et al., J. Med. Chem. 26: 313-318 (1983); Bodor, N., et al. , J. Pharm. Sci. 75: 29-35 (1986), each 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-fluid anti-inflammatory agents (NSAIDs), particularly 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, 6α,9α-difluoro 17α-[ (2-soranyl dialyl)oxy]-11B- Urnyl-16α_methyl-3-oxo-andro-1,4-diene-17B-thioacid S-fluoromethyl ester, 6α, 9α-difluoro-1 IB-urnyl-16α _Methyl-3-oxo-17α_propionyloxy-andro-1,4-diene T7B thioacid S-(2-oxo-tetrahydro-soran-3S-yl) ester, Beclomethasone ester (for example, 17-propionate or 17,21-dipropionate, fluoromethyl ester, triamcinolone acetonide, rofluronide or any combination or subset thereof. For use with the compound of the present invention The preferred corticosteroids formulated or used together are selected from ciclesonide, desisobutyryl ciclesonide, budesonide, mometasone, fluticasone propionate and fluticasone furoate or any combination or subset thereof.

[0241] The NSAIDs used in the present invention include, but are not limited to, cromolyn sodium, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g., nicotine, aminophenrin, PDE4 inhibitor, mixed PDE3/PDE4 inhibitor or mixed PDE4/PDE7 inhibitor), leukotriene antagonist, leukotriene synthesis inhibitor (for example, 5L0 and FLAP inhibitor), nitric oxide synthase (iNOS) inhibitor, protease inhibitor Agents (for example, tryptase inhibitors, neutrophil elastase inhibitors and metalloproteinase inhibitors, B2-integrin antagonists and adenosine receptor agonists or antagonists (for example, adenosine 2a agonists), cell Factor antagonists (e.g., chemokine antagonists) or cytokine synthesis inhibitors (e.g., prostaglandin D2 (CRTh2) receptor antagonists). Examples of leukotriene modulators suitable for administration by the method of the invention include Rust, Zileuton, and Zalust.

[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 Fenqun, Jie bilamilast, ibudilast, apremilast (apremilast), 2-[4-[6,7-diethoxy-2,3-bis (diameter

CN 105073717 Β

Methyl) T-Caiyl]-2-Pyridanyl]-4-(3-Pyridanyl)-1 (2H)-Diaza Zeaphenone Biphenyl (T2585), N-(3,5-Di chlorine-

4-Pyridanyl)-1-[(4-Fluorophenyl)methyl]-5-Vonyl-α-oxo-1H-Jie Ding-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-bisulfanyl] dialyl]amino]methyl]-3-fluorophenoxy]- (2R)-propionic acid (CP-671305), N-(4,6-dimethyl-2-ketyl)-4-[4,5,6,7-tetrahydro-2-(4-methyl Oxy-3-methylphenyl)-

5-(4-Methyl-1-piperazinyl)-1H-injido-1-yl]-benzenesulfonamide, (2E)-2-butenedioate (YM-393059), 9[ (2-Fluorophenyl)methyl]-N-methyl-2-(trifluoromethyl)-9H-Zyin-6-amine (NCS-613), N-(2,5-dichloro-3 -Pyridanyl)-8-methoxy-5-P1P#formamide (D-4418), N- [(3R) -9-amino-3,4,6,7-tetrahydro-4-oxo -1 -Phenylpyrrolo[3,2,1-] [1,4]benzodiazepine-3-yl]-3Η-cast a glancein-6-amine (PD-168787), 3-[ [3-(Cyclopentyloxy)-4-methoxyphenyl]methyl]-N-ethyl-8-(1-methylethyl)-3Η-cast a glancein-6-amine hydrochloride ( V-11294A) Small (3,5-dichloro-1-oxo-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quineline carboxamide (Sch351591) , 5[3-(cyclopentyloxy)-4-methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S, 5S) -2-piperidone (HT-0712), 5(2-((1R,4R) -4-amino-1-(3-(cyclopentyloxy)-4-methoxyphenyl) (Cyclohexyl) Ethyl)-Ctromethan-2-amine, cis-[4-oxy-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexyl-1 -Alcohol] and 4-[6,7-diethoxy-2,3-bis(vinylmethyl)-1-naphthyl]-1-(2-methoxyethyl)-2 (1H)- Pyridone (T-440) and any combination or subset thereof.

[0243] Leukotriene antagonists and inhibitors of leukotriene synthesis include zafirukast, 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, acropine and its various forms of salt (e.g., anhydrous atropine, atropine sulfate, atropine oxide or atropine HC1, methylatropine nitrate, homatropine hydrobromide, homatropine methyl bromide, chrysopine hydrobromide, chrysopine sulphate, scoprine hydrobromide, dong Chrystine methyl bromide), or any combination or subset of them.

[0245] Other anticholinergic agents used in combination formulation or use include: mettyline, bromprofen, methyl bromide (anisotropine methyl bromide) or Valpin 50, aclidinium bromide (aclidinium bromide) ), Robinul (Robinul), Isopropyl iodide, Mepentyl bromide, tridihexethyl chloride (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 (brimide compound), oxitropium (brimide compound) and cetopine (brimide compound) or Any combination or subset thereof.

[0247] Examples of B-agonists to be formulated or used in combination with the compounds of the present invention include, but are not limited to, salmeterol, R-salmeterol and its xic acid salt, 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, and are described in, for example, U.S. Patent No. 6,264,975, columns 9-10, and U.S. Patent Nos. 5,656,256 and 5,292,498.

CN 105073717 Β

[0249] P2Y2 agonists that can be administered by the method of the present invention include P2Y2 receptor agonists, such as ATP.UTP.UTP-.γ.-S and dinucleotide P2Y2 receptor agonists (such as denufosol (denufosol)). ) Or diquafosol) or a pharmaceutically acceptable salt thereof. The P2Y2 receptor agonist is usually included in an amount effective to stimulate the secretion of chloride and water on the airway surface (especially the nasal airway surface). Suitable P2Y2 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. 6,348,589, U.S. Patent No. 6,818,629, 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, all of which are incorporated herein by reference.

[0250] The combination therapies and formulations herein may include adenosine 2b (A2b) agonists, and also include BAY60-6583.NECA (N ethyl formamide 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 : 667-98 (September 2006 December); 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 Nos. 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 No. 6,858,614, US Patent No. 6,858,615, 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 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. 7,368,447, U.S. Patent No. 7,368,450, U.S. Patent No. 7,368,451, 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, U.S. Patent No. 7,868,010, U.S. 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. The protease inhibitors that can be used in the combinations 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 combinations herein may comprise one or more suitable nucleic acids (or polynucleic acids), including but not limited to antisense oligonuclei

CN 105073717 Β

Acid, siRNA, miRNA, miRNA analogs, antagomir, ribozymes, aptamers and bait oligonucleotides. See, for example, U.S. Patent Application Publication No. 20100316628. Generally, such nucleic acids can range from 17 or 19 nucleotides up to 23, 25, or 27 nucleotides long 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 long and up to 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 A1, US 2009/0253736 A1, US 2010/0227888 A1, Patent No. 7,645,789 ,US 2009/0246820 A1, US 2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US 2010/0168094 Al and authorized patents: 7,553,855; US 7,772,259 B2, US 7,405,233 B2, US 2009/0203752, US 7,499,570.

[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 mucus viscosity and improve clearance in vivo. 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 improvements in mucus rheology and tend to improve lung function and reduce acute exacerbation of the lungs-however, most clinical data indicate that in 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 as a reaction state of negatively charged thiolates. Therefore, clinically, NAC is administered at a very high concentration. However, it is expected that current aerosol devices 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) commonly used.

[0260] In non-clinical studies, administered by inhalation]⁴C-labeled NAC appears to be 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 well suited 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 di palm thick acyl phosphatidyl choline (DPPC), PF, palmitic acid, palm thick acyl-oil

CN 105073717 Β

Acylphosphatidylglycerol, surface-active protein (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 minced 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 of products that indicate active agents include ExOSUrF Neonatal (brown plant 1 choline (colfoscer order palmitate))&gt; Pumactant^ (DPPC and egg phosphatidylcholine), κl-4 surfactant, Wnticute (1 u su 1 ptide, r sρ-c surfactant), Alveofact® (bovactant), CurO$lirf® (P°ractant alfa) JnfosurF (calfactant), NewfacteiT' (modified bovine surfactant)'Surface, NatsurfTM (non-ionic alcohol ethoxy The surface of the chemical substance is ι biochemical) and Survanta (beractant). Examples of detergents include, but are not limited to, Tween-80 and triton-X 100.

[0264] Any suitable expectorant may be used, including but not limited to guaifenesin (see, for example, U.S. Patent No. 7,345,051). Any suitable deoxyribonuclease can be used, including but not limited to Dornase Alpha. (see, for example, U.S. 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. Additional anti-infective agents that can be used herein include aminoglycosines, dapatocins, fluoroquinolones, ketolides, carbapenems, cephalosporins, erythromycin, Linezolin, penicillins, azithromycin, clindamycin, serotones, tetracyclines, and vancomycin.

[0267] Examples of usable carbapenem antibiotics are imipenem, panipenem, meropenem, biapenem, MK826 (L-749,345), DAT131, ER-35786, lenapem South, S-4661, CS-834 (prodrug of R-95867), KR21056 (prodrug of KR-21012), L-084 (prodrug of LJC11036) and CXA-10L·

[0268] Antihistamines (ie, HI-receptor antagonists) for use in combination with the compounds of the present invention 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, citric acid Trepinamine rafter and Antapyrine; Alkylamines, such as pheniramine, chlorpheniramine, brompheniramine, dexchlorpheniramine, triprolidine, and atorvastatin; Pyridoxines, such as mesapiline, piperazines, such as vulcanizine HC1, pyrrolizine, cyclizine HC1, cyclizine lactate, meclizine HC1, and cetirizine HC1; pipidines, such as Aspirin, levocabastine HC1, loratadine, decarbonized ethoxyloratadine, terfenadine, and fexofenadine HC 1; tricyclic and tetracyclic rings, such as isopropyl Oxazine, chlorpromethazine, isobutyrazine, and azastatine; and azalastine HC1, or any combination or subset thereof.

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-l-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 having 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 combined with the treatment using the compound of the present invention

CN 105073717 Β

The treatment is compatible. 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 compounds of the invention are used in combination with one or more osmotic agents, particularly hypertonic 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.

[0273] 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 embodiment, 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>meaning</td>

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

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

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

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

<td>[0282]</td><td>DIPEA</td><td>Ν,Ν-Diisopropylethylamine</td>

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

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

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

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

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

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

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

<td>[0290]</td><td>HATU</td><td>2- (1H-7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyladenohexafluorophosphate</td>

<td>[0291]</td><td>HPLC</td><td>High performance liquid chromatography</td>

<td>[0292]</td><td>iPrOH</td><td>Isopropanol</td>

<td>[0293]</td><td>it or IT</td><td>Intratracheal</td>

CN 105073717 Β

<td>[0294]</td><td>Me</td>

<td>[0295]</td><td>MeOH</td>

<td>[0296]</td><td>AcOH</td>

<td>[0297]</td><td>m/z or m/e</td>

<td>[0298]</td><td>MH⁺</td>

<td>[0299]</td><td>MH^-</td>

<td>[0300]</td><td>MIC</td>

<td>[0301]</td><td>MS or ms</td>

<td>[0302]</td><td>Rt or rt</td>

<td>[0303]</td><td>Rf</td>

<td>[0304]</td><td>t-Bu</td>

<td>[0305]</td><td>THF</td>

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

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

<td>[0308]</td><td>δ</td>

<td>[0309]</td><td>Cbz</td>

<td>[0310]</td><td>AUC</td>

<td>[0311]</td><td>MTBE</td>

<td>[0312]</td><td>tR</td>

<td>[0313]</td><td>GC-MS</td>

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

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

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

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

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

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

<td>[0320]</td><td>Boc</td>

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

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

<td>[0323]</td><td>DIPEA</td>

<td>[0324]</td><td>PhaP</td>

Methyl methanol acetic acid mass-to-charge ratio mass plus 1 mass minus 1 minimum inhibitory concentration mass spectrometry room temperature retention factor tert-butyltetrahydrofuran thin layer chromatography meaning relative to the parts per million of the low magnetic field of tetramethylsilane Base, i.e. -(C0) 0-benzyl curve or area under the peak methyl tert-butyl sulfide retention time gas chromatography-mass spectrometry weight percentage hour minutes megahertz trifluoroacetic acid ultraviolet tert-butoxy dial azodiac acid two Isopropyl acetic acid

Ν,Ν-diisopropylethylamine or Htinig base triphenylphosphonium

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

<img file="CN105073717B_D0028.tif" />

<img file="CN105073717B_D0029.tif" />

CN 105073717 Β

[0328] These methods are described in, for example, E. J. Cragoe, The Synthesis of Ami lor ide and Its Analogs (Chapter 3), Andioride and Its Analogs, pages 25-36. Other methods of preparing amiloride analogs are described, for example, in U.S. Patent No. 3,318,813, see in particular 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 described in PCT Publication Nos. Described in 2007/018640, all belong to Par ion Sciences, Inc_o

[0329] Methyl Ν'-3,5-diamino6-chloropyrazine-2-carbonylcarbamimido thioate (methyl Ν'-3,5-diamino6-chloropyrazine-2-carbonylcarbamimido thioate) ( 2) For the preparation, please refer to WO 2009/074575_oGenerally 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, for example at 70 °C, with an amine of formula 3 and a base (such as triethylamine (TEA) or diisopropylethylamine). Amine (DIPEA)). 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&gt; Chem-Impex International Inc. and TCI chemical industry Co. Ltd_o With Bruker AC 400 (^XH NMR at 400 MHz and i NMR at 100 MHz) and Bruker AC 300 (^XH NMR at 300 MHz and scoldNMR at 75 MHz) to obtain NMR spectra. The proton spectrum refers to tetramethylsilane as the internal standard, and the carbon spectrum refers to CDC1₃&gt;CDsOD or DMSO-de (unless otherwise specified, purchased from Aldrich or Cambridge Isotope Laboratories). Use a Combif lash system (Comb if lash Rf, Teledyne Isco) equipped with a silica gel column (Redi Sep. Rf, Teledyne Isco) or a reverse phase column (high efficiency C18 Gold column) for flash chromatography. ESI mass spectra were obtained with Shimadzu LCMS-2010EV mass spectrometer. The Shimadzu Prominence HPLC system was used to detect by Waters XTerra MS C18 5um 4.6X 150mm analytical column at 220nm (unless otherwise specified) to obtain HPLC analysis. The flow rate is 1.0 mL/min, and the following time program is used:

<img file="CN105073717B_D0030.tif" />

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

CN 105073717 Β

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 (minutes)</td><td>Percentage A (out(), with 0.05% NHXOOH and 0.1% HCOOH)</td><td>Percentage B (CHsCN/water 80:20%, with 0.05% NH₄COOH and 0.1% HCOOH)</td>

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

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

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

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

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

1. (S)-2-Amino-3-(4-(4-(3-(3-(3,5-diamino-6-chloropyrazine-2-propelyl) quaternyl) butyl) Preparation of the hydrochloride (16) of propionic acid (Cai-1-yl)

[0338] Scheme 2

CN 105073717 Β

[0339]

<img file="CN105073717B_D0031.tif" />

<img file="CN105073717B_D0032.tif" />

<img file="CN105073717B_D0033.tif" />

<img file="CN105073717B_D0034.tif" />

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

[0341] A solution of 4-hydroxyl-carbaldehyde (10.0 g, 58.1 mmol) in anhydrous THF (200 mL) was cooled to 0° C., and imidazole (12.0 g, 174 mmol) and tert-butyl were added sequentially. Tertbutyldimethyls yl chloride (TBSC1) (13. lg, 87.1 mmol). 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 NHKl (100 mL), water (100 mL) and brine (100 mL) and washed with Na₂S0₄dry. 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 light yellow solid:

[0342] ^XH NMR (300MHz, CDC13): δ10.22 (s, ΙΗ), 9.30 (d, J = 8.10Hz, ΙΗ), 8.27 (d, J =

8.1Hz,lH) ,7.86 (d,J = 7.8Hz, 1H) ,7.69 (ddd, J = 8.4,7.0,1.3Hz, 1H) ,7.57 (ddd,J = 8.4,

CN 105073717 Β

7.0,1.3Hz,lH) ,6.95(d,J = 7.5Hz,lH), 1.10 (s,9H) ,0.36 (s,6H)

[0343] Preparation of (Z)-2-(tert-butoxy dialyl)amino-3-[l-(tert-butyldimethylsilyloxy) azee-4-yl]methyl acrylate (4);

[0344] To (MeO) ₂P (0) CH (NHBoc) C0₂DBU (10.1 mL, 67.3 mmol) was added to a dry CH2CI2 (100 mL) solution of Me, 3 (23.0 g, 52.7 mmol), and the mixture was stirred at 0 °C for 30 minutes. Slowly add 1 (14.8g, 51.74mmol) of dry CH via a syringe₂C1₂ (60 mL) solution and the reaction mixture was heated to room temperature for 16 h. After the solvent was removed under reduced pressure, the residue was dissolved in CH2CI2 (500 mL), washed quickly with saturated aqueous NHKl (2×150 mL) and brine (200 mL) and dried over Na2S04. The solvent was evaporated and the crude product was purified by flash chromatography on silica gel (20% EtOAc/hexane with 1% NEt3) to give 4 (20.0 g, 85%) as a yellow solid:

[0345] ^XH NMR (300MHz,CDCI3): 68.23 (dd, J = 8.6,2.1Hz, 1H) ,7.93 (dd,J = 8.6,2.1Hz, 1H) ,7.67 (s,lH) ,7.57 (d, J = 8.4 Hz, 1H) ,7.53-7.47 (m,2H) ,6.85 (d, J = 7.8Hz, 1H) ,6.05 (brs,lH) ,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 (1 atm, 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): δ8.23 (d, J = 8.2Hz, 1Η), 7.99 (d, J = 8.2Hz, 1Η), 7.577.44 (m,2H), 7.10 (d, J = 8.2 Hz, 1H) ,6.77 (d, J = 8.2Hz, 1H) ,5.07-4.94 (brs, 1H) ,4.744.61 (m,lH) ,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-(4-½ ylcae-1-yl)propionate (6);

[0350] To a solution of 5 (17.0 g, 37.0 mmol) in anhydrous THF (200 mL) at 0° C. was added tetrabutyl fluoride (48.1 mL, 48.1 mmol). The resulting solution was stirred for 15 minutes and quenched with saturated aqueous NHK1 (150 mL). After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (500mL), quickly wash with saturated aqueous water (2X150mL) and brine (200mL) and pass Na₂S0₄dry. 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): 58.23 (d, J = 8.2Hz, 1H), 7.98 (d, J = 8.2Hz, 1H), 7.577.44 (m,2H), 7.07 (d, J = 8. OHz , 1H), 6.68 (d, J = 7.6Hz, 1H), 6.55 (brs, 1H) ,5.14-4.85 (brs,lH) ,4.77-4.51 (m,lH) ,3.78-3.31 (m,5H), 1.40 (s,6H) ,1.10 (s,3H).

[0352] Preparation of Compound 7 and Compound 8;

[0353] Using isocratic system IPA/heptane (7.5%, with 0.4% DEA), CHIRALPAK AD column 5 cm I.DX 50 cm L, particles 20 J were used to separate the enantiomers. 8.0 g 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%) .

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

[0355] At 0° C., to a solution of compound 8 (1.22 g, 3.53 mmol) in Vitro (20 mL) was added triflate (0.9 mL, 5.30 mmol), and the reaction mixture was kept at room temperature Stir for 2h. After concentration, the reaction mixture is in CH₂Partition between Ch (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 Na2S04 and concentrated to give compound 9 (1.51g, 89%) as a brown oil:

CN 105073717 Β

[0356] ^XH NMR (400MHz, CDCls): δ8.19-8.07 (m,2H), 7.69-7.64 (m,2H), 7.38 (d,J = 8.1Hz, 1H), 7.28 (d,J = 7.9Hz,lH ), 5.12-5.06 (brs,lH), 4.78-4.67 (m,lH), 3.68-3.46 (m,5H), 1.39 (s,8H), 1.25 (s,lH).

[0357] (S) -3-{4-[4-(Benzyloxy dialylamino)butyl T-alkynyl] Cai T-yl} ~2~ (tert-butoxy dialylamino) methyl propionate (11) Preparation

At room temperature, TEA (1.27mL, 12.6mmol) was added to a solution of compound 9 (1.50g, 3.14mmol) in anhydrous CHsCN (60mL), 10% in hexane (t-Bu) ₃P (1.27 mL, 0.62 mmol), but-3- alkynyl carbamate (10,948 mg, 4.7 lmmol) and Cui (30 mg, 0.16 mmol). The resulting mixture was degassed with nitrogen for 10 minutes and Pd(PPh3)4 (363 mg, 0.31 mmol) 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 was purified by column chromatography (silica gel, 60:40 ethyl acetate/hexane) to give compound 11 (1.30g, 78%) as a brown oil:

[0359] ^XH NMR (400MHz,CDCI3): 68.33 (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,lH) ,5.13(s,2H) ,5.07-4.99, 4.74-4.65 ,3.59 (s,3H) ,3.913.42 (m,2H) ,3.53 (d, J = 6.2Hz,2H) ,2.79 (t, J = 6.4Hz,2H) ,1.39 (s,8H) ,1.25(s,lH).

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) 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 give amine salt 12 (820mg, 95%) as a white solid:

[0362] ^XH NMR (300MHz, CDsOD): 58.17-8.05 (m,2H) ,7.62-7.48 (m,2H), 7.27 (brs, 2H), 4.47 (t,J = 7.4Hz,lH) ,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) Preparation of methyl citrulline] butyl} Cai-1-yl) propionate (14);

[0364] At room temperature, to the amine salt 125mg, 1.77mmol 1) and 3,5-diamino-6-chloropyrazine-2-pyridylmethyl thioacid methyl ester (13,1.1g, 2.83 A solution of EtOH (6.0 mL) in mmol) was added to DIPEA (2.50 mL, 14.2 mmol). 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 purified by column chromatography (silica gel, 80: 18: 2CHCI3/CH3OH/NH4OH) to give muscle 14 (870mg, 80%) as a yellow solid:

[0365] ^XH NMR (400MHz,CDsOD): δ8.17-8.07 (m,2H), 7.58-7.48 (m,2H), 7.26 (q,J = 7.4Hz, 2H), 4.56-3.68 (m,lH), 3.75 -3.68 ,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-pyridine) Guarinyl) butyl) Cai-1-yl) propionic acid (15);

To a solution of methyl ester 14 (510mg, 0.83mmol) in a mixture of THF (3mL), methanol (3mL) and water (1mL) was added solid LiOH (120mg, 4.99mmol) and the reaction mixture was stirred at room temperature for 2h . 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. DCM (2X

50mL) extraction. Combine the organic layers and pass Na₂S0₄Dry, filter and concentrate to give compound 15 as a white solid (375mg,

CN 105073717 Β

76%):

[0368] ^XH NMR (300MHz, DMSO-ch): 58.22-8.02 (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 ,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-(3,5-diamino-6-chloropyrazine-2-pyridyl)inosyl)butyl)na-1 -Preparation of the HC1 salt of propionic acid (16);

[0370] 4N HC1 (8.0 mL) in dioxane was added to 15 (258 mg, 0.43 mmol), then water (4.0 mL) was added 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] ^XH NMR (400MHz, DMSO-ch): δ10.54 (brs, ΙΗ), 9.33 (t, J = 5.92Hz, ΙΗ), 9.03-8.80 (m,2H), 8.60 (brs,3H), 8.17 (ddd ,J = 10.1,7.6,4.5Hz,2H) ,7.59 (ddd,J = 9.2,6.7,4.5Hz, 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-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)benzene Base ammonia

(Yl)-3-oxopropyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide (23)

[0373] Scheme 3

CN 105073717 Β

[0374]

<img file="CN105073717B_D0035.tif" />

NaOH

Mei ill THE I io()

<img file="CN105073717B_D0036.tif" />

<img file="CN105073717B_D0037.tif" />

<img file="CN105073717B_D0038.tif" />

2()

<img file="CN105073717B_D0039.tif" />

N HC1 aqueous solution, dioxane

<img file="CN105073717B_D0040.tif" />

[0375] (S) -3-{4-[4-(Benzyloxy dialylamino) butyl T-alkynyl] Cai T-yl} ~2~ (tert-butoxy dialyl amino) methyl propionate (17) Preparation;

To a solution of methyl ester 11 (1.71g, 3.22mmol) in a mixture of THF (21mL), methanol (21mL) and water (7.0mL) was added solid NaOH (1.29g, 32.3mmol) and The reaction mixture was stirred at room temperature for 3h. 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 part₂C1₂ (2 X 50mL) extraction. Combine the organic layers, pass Na₂S0₄Dried, filtered and concentrated to give compound 17 (1.55g, 93%) as a brown solid:

[0377] ^XH NMR (400MHz,DMSO-cU): 68.32 (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 ,5.12(s,2H) ,5.07-4.93 ,4.70-4.54 (m,lH) ,3.77-3.62(m,lH) ,3.57-3.35 (m,2H) ,2.84-2.68 (m,2H), 1.37(s,9H).

CN 105073717 Β

Preparation of Compound 19;

To containing compound 18 (100mg, 0.56mmol) in THF (2.5mL), sequentially add DEPBT (218mg, 0.72mmol), 17 (289mg, 0.56mmol) and DIPEA (0.3mL, 1.68mmol) and at room temperature Stir for 16h. After the solvent was removed under reduced pressure, the residue was dissolved in CH2CI2 (100 mL), washed quickly with saturated aqueous NaHCOs (2 X 50 mL) and brine (50 mL) and subjected to NaHCOs (50 mL).₂S0₄dry. The solvent was evaporated and the crude product was purified by silica gel flash chromatography (8% methanol/CH2CI2) to give amide 19 (250mg, 66%) as a yellow solid:

[0380] ^XH NMR (400MHz, CDC13): 68.34 (dd, J = 8.3,1.4Hz, 1H), 8.21 (d, J = 8.3Hz, 1H), 7.61-7.47 (m,4H) ,7.39-7.27 (m,5H ) ,7.16 (d, J = 8.3Hz,2H) ,7.05 (d,J = 8.3Hz,2H) ,5.365.19 (m,2H) ,5.12(s,2H) ,4.36-4.53 ,3.66-3.42 ( m,4H), 2.79 (t, J = 6.6Hz, 2H),

2.57 (t,J = 7.5Hz,2H), 2.40 (t, J = 7.5Hz, 2H), 2.32 (s,6H), 1.86T. 75 (m, 2H), 1.39 (s, 9H).

[0381] Preparation of Compound 20;

A suspension of 19 (210 mg, 0.3 lmmol) and 10% Pd/C (150 mg) in a mixture of MeOH (3.0 mL) and AcOH (0.3 mL) was degassed and hydrogenated at room temperature Conditions (latm) 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 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%) as a white solid :

[0383] ^XH NMR (300MHz,CD3OD): 68.24 (dd, J = 8.1,2.1Hz, 1H), 8.08 (dd, J = 8.2,1.5Hz, 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) ,3.663.55 (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) ,1.84-1.61 (m,6H), 1.36 (s,7H) ,1.10(s,2H).

[0384] Preparation of 22;

[0385] At room temperature, to amine 20 (122mg, 0.22mmol) and 3,5-diamino-6-chloropyrazine-2-pyrimylmethyl thiomethionate (21,139mg, 0.35mmol) DIPEA (0.3ImL, 1.76mmo 1) was added to the EtOH (4.0 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 purified by column chromatography (silica gel, 80:18:2CHCI3/CH3OH/NH4OH) to give muscle 22 (111 mg, 66%) as a yellow solid:

[0386] ^XH NMR (400MHz, CDsOD): 68.23 (dd, J = 7.5,2.4Hz, 1H), 8.10 (d, J = 8.1Hz, 1H), 7.57-7.48 (m,2H) ,7.29 (d, J = 7.3 Hz ,2H) ,7.24 (d, J = 8.0Hz,2H) ,7.13-7.05 (m,2H) ,4.53 (t,J = 8.0Hz,lH) ,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 23-3,5-diamino-N-(N-(4-(4-(2-amino-3-(4-(3-(dimethylamino)propyl)phenylamino) Preparation of the HC1 salt of -3-oxopropyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide

[0388] 4N HC1 (3.0 mL) in dioxane was added to 22 (100 mg, 0.13 mmol), then water (1.0 mL) was added 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 give compound 22 (65mg, 65%) as a yellow solid:

[0389] ^XH NMR (400MHz, DMSO-ch) 10.50 (s,lH), 10.48 (s,lH), 10.46-10.40, 9.26 (t,J = 4.9Hz,lH), 9.01-8.74 (m,2H), 8.61 ( brs,lH) ,8.35 (dd, J = 6.6,3.4Hz, 1H) ,8.13 (dd,J = 6.5,3.3Hz,lH), 7.58 (ddd, J = 9.9,6.6,3.6Hz, 2H), 7.42 (brs, 1H) ,7.40(d,J =

CN 105073717 Β

7.3Hz, 2H) ,7.34(d,J = 7.3Hz,lH), 7.28 (d, J = 7.3Hz, 1H) ,7.16 (d, J = 8.6Hz,2H) ,4.294.20 ,3.64-3.49 ( m,2H) ,3.12-3.03 (m,2H) ,3.02-2.94 (m,2H) ,2.72 (s,3H) ,2.70 (s,

3H), 2.56 (t,J = 8.1Hz,2H), 1.97-1.88 (m,2H), 1.79-1.61 (m,4H).

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

4,5,6-Pentylhexyl) amino) propyl) phenylamino) -3-oxopropyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine-2- Preparation of formamide (28)

[0391] Scheme 4

[0392]

<img file="CN105073717B_D0041.tif" />

<img file="CN105073717B_D0042.tif" />

[0393] Preparation of Compound 25;

[0394] To the THF (10mL) containing compound 24 (165mg, 0.38mmol) was added DEPBT (148mg, 0.48mmol), 17 (200mg, 0.38mmol) and DIPEA (0.2mL, 1.14mmol) in sequence and at room temperature Stir for 16h. After the solvent was removed under reduced pressure, the residue was dissolved in CH2CI2 (100 mL), washed quickly with saturated aqueous NaHCOs (2 X 50 mL) and brine (50 mL) and subjected to NaHCOs (50 mL).₂S0₄dry. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol/

CH2CI2) was purified to give amide 25 (210mg, 60%) as a yellow solid:

[0395] ^XH NMR (300MHz, CDCI3): δ8.35 (d, J = 8.2Hz, 1Η), 8.21 (d, J = 8.3Hz, 1Η), 7.637.52 (m,2H), 7.51 (d, J = 7.3 Hz, 1H) ,7.44-7.39, 7.37-7.27 (m,6H) ,7.16-7.02 (m,

3H) ,5.24-5.16(m,lH), 5.13 (s, 2H), 4.68 (ddd, J = 11.3,10.3,5.1Hz, 1H) ,4.56 (q,J =

CN 105073717 Β

7.2Hz, 1H), 4.19-4.09 (m,lH), 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) ,1.81-1.67 (m,2H) ,1.63-1.53 (m,lH) ,1.35-1.20 (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 placed under hydrogenation conditions at room temperature (latm) 4h. 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 22, which was neutralized with NaHCOs, and the crude product was purified by silica gel flash chromatography (CMA, 80: 18 (2) to obtain the free amine 26 (160 mg, 67%) as a yellow solid:

[0398] ^XH NMR (400MHz,CDsOD): 58.29 (d, J = 8.2Hz, 1H), 8.07 (d, J = 8.6Hz, 1H), 7.60 (t, J = 6.9Hz,lH), 7.55 (ddd,J = 8.2,6.9,1.ΙΗζ,ΙΗ), 7.24 (d, J = 7.1Hz, 1H), 7.18 (d, J = 7.1Hz, 1H), 7.02-7.96 (m,lH), 7.95-6.88 (m, 2H), 6.77-6.69 (m, 1H) ,5.56-5.35,

4.68(q,J = 5.1Hz,lH) ,4.61-4.53, 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 ,2.68 (t, J = 7.lHz,2H) ,2.61-2.47 (m,5H) ,2.46-2.37 (m,4H) ,1.771.63 (m,4H), 1.33 (d, J = 5.1Hz) , 3H) ,1.31-1.20 (m,8H), 1.21 (s,9H) ,0.88 (t, J = 6.7Hz, 3H).

Preparation of Compound 27;

[0400] At room temperature, to amine 26 (155mg, 0.20mmol) and 3,5-diamino-6-chloropyrazine-2-methylpyrimidine methyl thioate (21,123mg, 0.31mmol) 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 purified by silica gel column chromatography (80:18:2 CHCI3/CH3OH/NH4OH) followed by reverse phase chromatography (Gold C18) to give muscle 27 (100 mg, 51%) as a yellow solid:

[0401] ^XH NMR (400MHz, CDsOD): 68.23 (dd, J = 8.8,2.5Hz, 1Η), 8.10 (d, J = 8.2Hz, 1Η), 7.56-7.49 (m,2H), 7.29 (d,J = 7.9 Hz, 2H), 7.24 (d, J = 7.4Hz, 2Η), 7.08 (d, J = 7.9Hz, 2H), 4.67 (q, J = 5. ΙΗζ, ΙΗ), 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 ,3.25 (t, J = 7.1Hz,2H) ,3.15 -3.06 (m,2H) ,2.71 (dd,J=13.2,5.2Hz,lH) ,2.60-2.45 (m,6H) ,1.87-1.63 (m,6H), 1.48T .40 (m,6H), 1.331.26 (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)6 -Preparation of HC1 salt of chlorpyrazine-2-carboxamide

[0403] 4N HCI (3.0 mL) in water was added to 27 (80 mg, 0.08 mmol) of ethanol (0.5 mL) and the reaction mixture was stirred at 40° C. 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, DMSO-ch): δ 10.58 (brs, 1H), 10.56 (brs, 1Η), 9.70-9.58 (m, 1Η), 9.38-9.31, 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 = 8.1Hz ,2H) ,5.52-5.46 (m,lH) ,4.85-4.76 ,4.68-4.52 (m,2H) ,4.49-4.37 (m,

1H) ,4.32-4.22 ,4.05-3.97 (m, 1H) ,3.72-3.43 (m,6H) ,3.17-2.97 (m,8H) ,2.02-1.90

CN 105073717 Β

(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, CDsOD): δ8.23 (d, J = 8.3Hz, 1H), 8.17 (d, J = 8.2Hz, 1H), 7.627.53 (m,2H) ,7.41-7.36 ,7.35-7.32 ,7.31-7.25 (m,2H), 7.21-7.12 (m, 2H),

4.35-4.25 ,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), 1.91T .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-(-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

Preparation of 2-formamide (33)

[0407] Scheme 5

Sugar, N-sugar

<img file="CN105073717B_D0043.tif" />

<img file="CN105073717B_D0044.tif" />

Preparation of Compound 30:

To containing compound 29 (290mg, 0.54mmol) in THF (8.0mL), sequentially add DEPBT (210mg, 0.70mmol), 17 (311mg, 0.60mmol) and DIPEA (0.28mL, 1.62mmol) and in room temperature Stir for 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100mL), quickly wash with saturated NaHCOs aqueous solution (2 X 50mL) and brine (50mL), after NaHCOs₂S0₄dry. The solvent was evaporated and the crude product was passed through silica gel flash chromatography (8% methanol/

CN 105073717 Β

CH₂C1₂) Purification to obtain amide 30 (400mg, 72%) as a yellow solid:

[0411] ^XH NMR (400MHz, CDCls): δ8.36-8.26 (m, 1H), 8.20-8.09 (m, 1H), 8.03-7.85 (m, 1H), 7.61-7.46,7.49 (d,J = 7.2Hz, 2H) ,7.38-7.28 (m,5H) ,7.18-6.96 (m,4H) ,5.51-5.36 (m,lH) ,5.32-5.21 (m,lH) ,5.12(s,2H) ,4.67 (q, J = 5.lHz,2H) ,4.66-4.53 ,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) ,3.683.56 (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), 1.93T .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] The suspension of 30 (400mg, 0.39mmol) and 10%Pd/C (210mg) in a mixture of EtOH (54mL) and AcOH (6.0mL) was degassed and placed under hydrogenation conditions (latm) at room temperature for 4h . 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 31 (333 mg, 84%) as a yellow solid:

[0414] ^XH NMR (400MHz, CDsOD): 68.25 (dd, J = 7.5,2.5Hz, 1Η), 8.10 (d, J = 7.3Hz, 1Η), 7.60-7.51 (m,2H),7.36-7.32,7.31 (d , J = 7.2Hz, 2Η), 7.26 (d, J = 7.8Hz, 1Η), 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 = 9.5,1.8Hz, 2H) ,3.39 (t,J = 9.2Hz,3H) ,3.35-3.30 (m,2H), 3.153.08 (m, 2H), 2.92 (t, J = 8.0Hz , 2H), 2.09-2.00 (m,4H) ,2.77-2.58 (m,2H) ,1.95 (s,6H), 1.88T .60 (m,4H),1.36(s,9H),1.25 (d, J = 4.9Hz,6H).

[0415] Preparation of 32;

[0416] At room temperature, to 31 (370 mg, 0.36 mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,226 mg, 0.58 mmol) in EtOH ( 12mL) DIPEA (0.51mL, 2.88mmol) was added to the 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: 2CHC13/CH₃0H/NH₄0H) purification to obtain muscle 32 (250mg, 63%) as a yellow solid:

[0417] ^XH NMR (400MHz,CDsOD): δ8.23 (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=10.8,5.4Hz,2H) ,3.91-3.84 (m,2H ), 3.82-3.72 (m,5H), 3.48-3.43 (m,5H), 3.413.34 (m,2H), 3.13-3.10 (m,2H), 2.68-2.50 (m,8H), 1.87T. 67 (m,6H) ,1.36(s,9H),1.23 (d, J = 4.9Hz,6H).

33~3,5-Diamino-N-(N~(4-(4-(-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-chloropyrazine 2-carboxamide Preparation of HC1 salt

[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 and 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, DMSO-ch): δ 10.48 (brs,lH), 10.45-10.41 (m,lH), 9.25-9.19 (m, 1H), 8.95-8.85, 8.81-8.69, 8.64-8.46 (m, 4H) ,8.36-8.29 (m, 1H) ,8.18-8.10 (m,lH) ,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,

CN 105073717 Β

1Η), 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), 4.284.17(m,lH) ,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) ,2.03T .90 (m,2H) ,1.78-1.61 (m,4H).

[0421] ^XH NMR (400MHz, CD3OD): 58.24-8.20, 8.18-8.15 (m, 1H), 7.57 (td, J = 4.6,

1.5Hz, 2H) ,7.38 (d,J = 7.4Hz,lH), 7.33 (d, J = 7.4Hz, 1H), 7.28 (dd, J = 8.4,2.6Hz, 2H), 7.16(dd,J = 8.4,2.0Hz,2H) ,4.28 (t,J = 6.9Hz, 1H) ,4.19-4.13 (m, 1H) ,4.12-4.07 (m, 1H), 3.85-3.79 (m,2H), 3.77 (dd , J= 10.4,5.2Hz, 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,lH) ,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-(-2-amino-3-oxo-3-(4-(3-( (2S,3R,4R ,5R) -2,

3,4,5,6-Pentylhexylamino) propyl) phenylamino) propyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine-2-carboxamide (38 ) Preparation

[0423] Scheme 6

[0424]

ΒοΖ sugar

<img file="CN105073717B_D0045.tif" />

C person

OH Ο Ο

5h oh

<img file="CN105073717B_D0046.tif" />

ΝΗ Ο gg J nh₂

<img file="CN105073717B_D0047.tif" />

Ν !1(; 1 aqueous solution, I toil

<img file="CN105073717B_D0048.tif" />

<img file="CN105073717B_D0049.tif" />

CN 105073717 Β

Preparation of Compound 35

To the THF (15mL) containing compound 34 (400mg, 0.91mmol), sequentially add DEPBT (389mg, 1.30mmol), 17 (516mg, 1.00mmol) and DIPEA (0.52mL, 3.00mmol) and stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved in CH₂C1₂ (100mL), washed quickly with saturated aqueous NaHCOs (2×50mL) and brine (50mL) and passed through NaHCOs₂S0₄dry. The solvent was evaporated and the crude product was purified by silica gel flash chromatography (8% methanol/CH2CI2) to give amide 35 (700mg, 83%) as a yellow solid:

[0427] ^XH NMR (400MHz,CDCI3): 68.35 (dd, J = 8.2,1.5Hz, 1H), 8.22 (d, J = 8.1Hz, 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 = ll .4,5, 6Hz,lH) ,4.11-4.02 (m,lH) ,4.07-3.92 (m, 1H) ,3.88- 3.77, 3.73-3.67 (m, 1H), 3.643.49 (m, 5H), 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) ,l,42(s,18H), 1.32 (d, J = 5.2Hz, 3H).

[0428] Preparation of Compound 36;

[0429] A suspension of 35 (700 mg, 0.74 mmol) and 10% Pd/C (400 mg) in a mixture of EtOH (90 mL) and AcOH (10 mL) was degassed and 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 give amine salt 36 (650 mg, 95%) as a yellow solid:

[0430] ^XH NMR (400MHz, CDCI3): δ8.20 (d, J = 8.4Hz, 1Η), 7.96 (d, J = 7.3Hz, 1Η), 7.867.71 (m,lH), 7.70-7.63 (m,lH ) ,7.58-7.43 (m,2H) ,7.36-7.26 (m,2H) ,7.02-6.91 (m,2H), 4.70-4.63 ,4.61-4.54 (m, 1H) ,4.20-4.05 (m,2H) ,4.04-3.90 ,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 = 5.1Hz,3H),1.25 (s,3H) ).

[0431] Preparation of 37;

[0432] At room temperature, to 36 (650mg, 0.70mmol) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,436mg, 1.13mmol) DIPEA (0.90mL, 5.60mmol) was added to the EtOH (12mL) 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 purified by silica gel column chromatography (80: 18: 2CHCI3/CH3OH/NH4OH) to obtain muscle 37 (444 mg, 62%) as a yellow solid:

[0433] ^XH NMR (400MHz, CD3OD): 68.23 (dd, J = 7.7,2.2Hz, 1H), 8.10 (d, J = 8.1Hz, 1H), 7.57-7.47 (m,2H) ,7.33-7.21 (m,4H ) ,7.08 (d,J = 8.lHz,2H) ,4.68 (q, J = 5.0Hz, 1H) ,4.53 (t,J = 7.2Hz,lH) ,4.04 (dd, J = 10.8,5.4Hz, 1H), 4.03-3.93 (m,lH), 3.25 (ddd,J=10.3, 9.2,5.2Hz,H),3.71-3.65,3.58-3.37 (m,4H),3.27-3.20 (m,4H), 3.20-3.15 (m,1H),

3.14-3.05 (m,2H) ,2.66 (q, J = 7.5Hz, 1H) ,2.53 (t, J = 7.2Hz,2H), 1.89T .76 (m,4H) ,1.761.64(m,2H) ) ,1.36 (s,6H) ,1.42 (s,9H) ,1.25 (d, J = 5.OHz,3H) ,1.11 (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:

CN 105073717 Β

[0436] ^XH NMR (400MHz, DMSO-de): 510.50 (brs,lH) ,9.28 (t, J = 5.7Hz, 1H) ,9.02-8.87 (m, 1H) ,8.86-8.75 ,8.72-8.55 (m,4H) ,8.39-8.33 (m, 1H), 8.16-8.10 (m, 1H) ,7.61-7.55 (m,2H) ,7.45-7.40 (m,lH) ,7.40 (d, J = 7.4Hz, 2H), 7.34 (d, J = 7.3Hz, 1H) ,7.28 (d,J = 7.4Hz,lH) ,7.14(d,J = 8.5Hz,2H), 5.38 (d, J = 4.3Hz, 1H) ,4.74 (d, J = 4.9Hz, 1H) ,4.644 .51 (m,2H) ,4.49-4.35 (m,lH) ,4.30-4.20 (m,2H) ,3.94-3.86 (m,lH) ,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, CDsOD): 58.26-8.20 (m, 1H), 8.19-8.14 (m, 1H), 7.60-7.53 (m,2H), 7.38 (d. J = 7.2Hz,lH), 7.33 (d , J = 8.4Hz, 1H), 7.28 (dd, J = 8.4,2.0Hz, 2H), 7.14 (d, J = 8.4Hz ,2H), 4.29 (t,J = 8.3Hz,lH) ,4.07-4.00 ,3.83 (dd, J = 9.8,1.5Hz, 1H) ,3.77 (dd,J = 9.8,2.6Hz,lH) ,3.73-3.64 (m,5H), 3.37 (t, J = 7.2Hz, 2H), 3.21-3.11 (m,4H), 3.06-2.96 (m,2H), 2.66 (t,J = 7.7Hz,2H), 2.03-1.94 (m,2H), 1.90-1.75 (m,4H).

6. (S)-3,5-Diamino-Tian (N-(4-(4-(2-amino-3-(4-(6-(dimethylamino)hexyl)phenylamine

(Yl)-3-oxopropyl) Cai-1-yl)butyl)methanyl)-6-chloropyrazine-2-carboxamide (43)

[0439] Scheme 7

CN 105073717 Β

[0440]

CH. i_：

H; C out of C

<img file="CN105073717B_D0050.tif" />

Pd/C. Ho

ΕιΟΗ. AcOH

<img file="CN105073717B_D0051.tif" />

<img file="CN105073717B_D0052.tif" />

DIPEA. ElOH

Ο ΝΪ^ΗΙ Pay

N 9

H two

BocHN

<img file="CN105073717B_D0053.tif" />

\

H nh₂·

Nil O

N

Cl

Ν''N

Η H

HqN factory, N NH₂

<img file="CN105073717B_D0054.tif" />

TFA. CH₂d₂

H; C

<img file="CN105073717B_D0055.tif" />

N Cl

N NH₂

<img file="CN105073717B_D0056.tif" />

[0441] Preparation of Compound 40;

[0442] In an ice bath, acid 17 (880mg_?A solution of 1.70 mmol) in THF (30 mL) was cooled to 0°C. Add NMM (0.37mL_?3.40mmol), then PivCl (0.20mL_? 1.70mmol), and the reaction mixture was stirred at the same temperature for 2h. Add 39 (375mg_?l. 7Ommo 1 _?15 mL THF) 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 CH₂C1₂(3X

100mL) extraction. Combine the organic layers and pass Na₂S0₄Dry, filter and concentrate. The residue was passed through column chromatography (4% methylbenzene in chloroform)

Alcohol) was purified to give amide 40 (719mg_?59%): [M+H]⁺720_o

CN 105073717 Β

Preparation of Compound 41;

[0444] The suspension of 40 (719 mg, 1,000 mmol) and 10% Pd/C (300 mg) in a mixture of EtOH (110 mL) and Ac OH (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 41 (660 mg, 93%) as a yellow solid: [M+H]⁺589 ο

[0445] Preparation of Compound 42;

[0446] At room temperature, to amine 41 (660 mg, 0.93 mmol) and 3,5-diamino-6-chloropyrazine-2-methylpyrimidine methyl thioate (21,650 mg, 1.67 mmol) DIPEA (1.66mL, 9.3mmol) was added to the EtOH (10mL) 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 purified by silica gel column chromatography (80: 18: 2 CHCI3/CH3OH/NH4OH) to obtain muscle 42 (370 mg, 50%) as a yellow solid: [M+H]⁺801

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

[0448] TFA (10 mL) was added to 42 (370 mg, 0.46 mmol) in CH 2 Cl 2 (10 mL) 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, DMSO-ch): 510.39 (brs, 2H), 9.25 (brs, ΙΗ), 9.02-8.87 (m, ΙΗ), 8.86-8.73 (m,2H), 8.71-8.44 (m, 2H) , 8.35 (brs, 1H), 8.13 (dd, J = 6.8,3.8Hz. 1H) ,7.58 (dd,J = 6.5,3.2Hz,2H), 7.42 (brs, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.33 (d, J = 7.8Hz, 1H), 7.27 (d,J = 7.3Hz,lH) ,7.11 (d, J = 8.4Hz,2H) ,4.26-4.18 (m, 1H), 3.65-3.48 (m,2H), 3.393.32 (m,3H), 3.06 (t, J = 6.5Hz, 2H), 2.99-2.91 (m,2H), 2.69(s,6H), 1.77T. 56 (m, 6H), 1.52(t,J = 8.2Hz,2H) ,1.34-1.21 (m,4H).

[0450] ^XH NMR (400MHz,CDsOD): 58.22-8.17 (m, 1H) ,8.16-8.12 (m, 1H) ,7.58-7.51 (m,2H), 7.36 (d,J = 7.2Hz,lH) ,7.30 (d , J = 7.4Hz, 1H) ,7.19 (d,J = 8.04Hz,2H) ,7.05 (d,J = 8.3Hz, 2H) ,4.24 (t,J = 8.2Hz,lH) ,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 = 8.6Hz,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-(-2-amino-3-(4-(6-(bis((2S,3R,4R,5R) -2,3,4,

5,6-Pentylhexyl) amino) hexyl) phenylamino) -3-oxopropyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine 2-carboxamide

[0452] Scheme 8

CN 105073717 Β

[0453]

<img file="CN105073717B_D0057.tif" />

Pd/C'. Η, tlOl I. AcOI!

sugar

<img file="CN105073717B_D0058.tif" />

\!U2AcOll sugar

<img file="CN105073717B_D0059.tif" />

[0455] Preparation of Compound 45;

[0456] In an ice bath, a solution of acid 17 (900 mg, 1.74 mmol) in THF (40 mL) was cooled to 0 °C. NMM (0.38 mL, 3.48 mmol) was added, followed by PivCl (0.21 mL, 1.74 mmol), and the reaction mixture was stirred at the same temperature for 2 h. 44 (1.21 g, 1.74 mmol, 20 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 CH2CI2 (3X

CN 105073717 Β

100mL) washing. Combine the organic layers and pass Na₂S0₄Dry, filter and concentrate. 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)⁺1196o

Preparation of Compound 46;

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 (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 the amine salt 46, which was neutralized with NaHCOs, and the crude product was purified by silica gel flash chromatography (CMA, 80: 18: 2) to obtain the free amine 46 (500 mg, after two steps) as a yellow solid 27% for each step): [M+H]⁺1067„

Preparation of Compound 47;

[0460] At room temperature, to amine 46 (500mg, 0.47mmol) and 3,5-dihydro-6-chloropyrazine-2-pyridylmethyl thiomethionate (21,330mg, 0.84mmol) DIPEA (0.84mL, 94.70mmol) was added to the EtOH (20mL) 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 purified by silica gel column chromatography (80: 18: 2 CHCI3/CH3OH/NH4OH) to obtain myo 47 (325 mg, 55%) as a yellow solid: [M+H]⁺1278_O

Compound 48 3,5-diamino-N-(N-(4-(4-(-2-amino-3-(4-(6-(bis((2S, 3R, 4R, 5R) -

2.3.4.5.6- Pentayl (hexyl) amino) hexyl) phenylamino) -3-oxopropyl) acetyl-1-yl) butyl) methyl)-6-chloropyrazine-2-methan Preparation of amide 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, DMSO-ch): 510.52 (brs, 1H), 10.44 (brs, 1H), 9.28 (t, J = 5.2Hz, 1H), 9.00-8.88 (m,lH), 8.87-8.75, 8.63 (brs,2H) ,8.60-8.50 (m, 1H) ,8.39-8.33 (m,

1H) ,8.17-8.11 (m,lH) ,7.58 (dd,J = 6.5,3.3Hz,2H) ,7.47-7.35 (m,2H) ,7.36 (d,J = 8.7Hz, 2H) ,7.33 (d ,J = 6.8hz,lH) ,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) ,lH) ,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 = 8.5Hz,2H), 1.76-1.60 (m,6H), 1.58-1.47 (m,2H), 1.35-1.23 (m,4H).

[0464] ^XH NMR (400MHz,CDsOD): 58.23-8.18 (m, 1H), 8.17-8.12 (m, 1H), 7.59-7.52 (m,2H), 7.36 (d,J = 7.8Hz,lH), 7.31 (d , J = 7.2Hz, 1H) ,7.18 (d, J = 8.2Hz,2H) ,7.04 (d,J = 8.2Hz, 2H) ,4.25 (t,J = 7.8Hz,lH) ,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,lH) ,3.71-3.60 (m,8H) ,3.49-3.41 ( m,2H) ,3.40-3.33 (m,4H) ,3.32-3.30 (m,lH) ,3.25-3.19(m,lH) ,3.18-3.10 (m,2H) ,2.52 (t, J = 7.4Hz, 2H), 1.88T .69 (m,6H), 1.62-1.53 (m,2H) ,1.44-1.30 (m,4H).

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

3.4.5.6- Preparation of Pentaylhexylamino)hexyl)Phenylamino)propyl)Ze-1-yl)butyl)methionyl)-6-chloropyrazine-2-carboxamide

[0466] Scheme 9

CN 105073717 Β

<img file="CN105073717B_D0060.tif" />

<img file="CN105073717B_D0061.tif" />

[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 force [JPivCl (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 Na2S04, filtered, and concentrated. The residue was purified by column chromatography (4% methanol in chloroform) to give amide 50 (1.40 g, impure) as a pale yellow solid: (M+H)⁺1043o

[0470] Preparation of Compound 51;

[0471] A mixture of 50 (1.40g, impure) and 10% Pd/C (400m) in a mixture of EtOH (120mL) and AcOH (20mL)

CN 105073717 Β

The suspension was degassed and placed under hydrogenation conditions (latm) for 16 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 51 (1.20g, crude) used directly in the next step: [M+H]⁺913_O

[0472] Preparation of Compound 52;

[0473] At room temperature, to amine 51 (1.20g, 0.47mmol, crude) and 3,5-diamino-6-chloropyrazine-2-pyridylmethionyl methyl thioate (21,723mg, 1.86) A solution of mmo 1) in EtOH (20 mL) was added to DIPEA (2.00 mL, 11.6 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 purified by silica gel column chromatography (80:18:2CHCI3/CH3OH/NH4OH) to give Mus 52 (500mg, 24% in three steps) as a yellow solid: [M+H]⁺1125o

Compound 53 3,5-Diamino-N-(N-(4-(4-(-2-amino-3-oxo-3-(4-(6-((2S, 3R, 4R , 5R) -2,3,4,5,6-Pentaylhexylamino)hexyl)phenylamino)propyl) Cai-1-yl)butyl)methionyl)-6-chloropyrazine-2 -Preparation of HC1 salt of formamide

[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, DMSO-ch): 510.52 (brs,lH), 10.45-10.41 (m,lH), 9.31-9.24 (m, 1H), 9.02-8.89, 8.88-8.76, 8.70-8.58 (m,3H) ) ,8.57-8.46 (m,2H) ,8.40-8.31 (m,lH) ,8.17-8.10(m,lH) ,7.62-7.54 (m,2H) ,7.42 (brs,2H) ,7.36 (d, J = 8.7Hz,2H) ,7.33 (d,J = 6.7Hz,lH) ,7.27(d,J = 7.5Hz,lH) ,7.11 (d,J = 8.7Hz,2H) ,5.41-5.35 (m, 1H) ) ,4.794.72 ,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,CDsOD) :58.24-8.20 (m, 1H) ,8.19-8.14 (m, 1H) ,7.60-7.53 (m,2H), 7.38(d,J = 7.6Hz,lH) ,7.33 (d, J = 7.2Hz, 1H) , 7.24-7.18 (m, 2H) ,7.07 (d, J = 8.1Ηζ,2Η), 4.31-4.22 ,4.08-4.01 (m,lH) ,3.84 (dd, J = 4.8,1.3Hz , 1H) ,3.77 (dd, J= 10.1,

2.5Hz,lH) ,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) , 1.73-1.64 (m,2H) , 1.63T .53 (m,2H) ,1.451.31 (m,4H).

[0478] 9. 3,5-Diamino-Ν- (N- (4- (4-(-2-Amino-3- (4- (6-(Hexyl((2S,3R,4R,5R) -2,3,

4,5,6-Five Urn Base Hexyl)Amino)Hexyl)Phenylamino)-3-Oxopropyl)Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide preparation

[0479]Program 10

CN 105073717 Β

IK.KC^v

BocHN

<img file="CN105073717B_D0062.tif" />

NHCbz

<img file="CN105073717B_D0063.tif" />

'Hu

[0480]

<img file="CN105073717B_D0064.tif" />

OH OH δ OH

<img file="CN105073717B_D0065.tif" />

NHCbz.

Ν il

BocHN

Pd (J, Η.

1-iOIL Aci)H .6out3"

<img file="CN105073717B_D0066.tif" />

N ί! + Hoc! IN »6

<img file="CN105073717B_D0067.tif" />

NU₂&lt;Ac()II

O NH-ffl Mouth"NS\Pay*foot

Η:Ν people N^NH: 13

<img file="CN105073717B_D0068.tif" />

[0481]

[0482]

<img file="CN105073717B_D0069.tif" />

[0483] Towards THF (50mL)Compounds in 54 (770mg , 1.45mmo 1)Join in DEPBT (564mg, 1.88mmol),17 (752mg, 1.45mmol)andDIPEA (0.77mL,4.35mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (lOOmL)Medium, use saturationNaHCOsAqueous solution(2 X 100mL)And salt water(50mL)Wash quickly andNa₂S0₄dry. The solvent is evaporated and the crude product is passed through silica gel flash chromatography(5%Methanol/ CH2CI2)Purify and pass reverse phase chromatography(Goldcolumn)Purify to obtain the amide as a yellow solid55 (800mg,54%): [M+H]⁺1027o

[0484] Compound56Preparation;

[0485] will55 (800mg,0.78mmol)and 10%Pd/C (400mg)existEt0H(120mL)andAcOH (30mL)mixture

CN 105073717 Β

The suspension in is degassed and under hydrogenation conditions at room temperature(latm) 16h. The reaction mixture was filtered through a plug of diatomaceous earth and usedMeOHWash the stopper. The filtrate was concentrated under vacuum to obtain the amine salt as a yellow solid56(780mg,99%) : [M+H] ⁺897ο

[0486] Compound57Preparation;

[0487] At room temperature, to the amine salt56 (780mg, 0.75mmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(21,466mg,1.20mmol)ofEtOH (20mL)Add to solutionDIPEA (1.37mL,7.67mmol). In the sealed tube70°CHeat the reaction mixture2h,Cool to room temperature and concentrate under vacuum. The residue is chromatographed on a silica gel column (80:18:2 CHCI3/CH3OH/NH4OH)purification,Get the muscle as a yellow solid57 (455mg,55%) : [M+H]⁺1110_o[0488] Compound58 3,5-Diamino-Ν- (N- (4- (4- ((S) -2-Amino-3- (4- (6-(Hexyl((2S, 3R,4R,

5R) -2,3,4,5,6-Five Urn Base Hexyl)Amino)Hexyl)Phenylamino)-3-Oxopropyl)Cai-1-base)Butyl)Caramel)6-Clopiazine-2-FormamideHC1Salt preparation

[0489] In the water4N HC1 (25mL)Add to57 (455mg,0.4lmmol)Ethanol(10mL)Neutralize and stir the reaction mixture at room temperature2h. Reversed phase chromatography(Goldcolumn)Purify and lyophilize the residue to give the compound as a yellow solid58 (230mg,55%):

[0490] ^XH NMR (400MHz, DMSO-ch) : δ10.45 (brs, 1H) , 9.30 (brs , 1Η) ,9.09-8.49 (m,3H), 8.41-8.32 ,8.16-8.08 (m, 1H) ,7.62-7.52 (m,2H) ,7.42 (brs,2H) ,7.37 (t,J = 8.4Hz,

2H) ,7.32(d,J = 7.8Hz,lH) ,7.27 (d, J = 7.2Hz, 1H) ,7.10 (d, J = 8. lHz,2H) ,5.52-5.36 (m, 1H) ,4.87-4.70 ,4.63-4.51 (m,2H) ,4.47-4.38 (m, 1H) ,4.23 (t, J = 6.7Hz, 1H) ,4.033.94 ,3.71-3.66 ,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,lH) ,3.09-2.96 (m,6H) , 1.77-1.58 (m,8H) , 1.57T . 46 (m, 2H) ,1.35-

I. 21 (m,10H) ,0.86 (t, J = 6.4Hz,3H).

[0491] ^XH NMR (400MHz,CDsOD) :58.26-8.20 (m, 1H) ,8.19-8.12 (m, 1H) ,7.60-7.51 (m,2H), 7.38(d,J = 7.2Hz,lH) ,7.32 (d, J = 7.4Hz , 1H) ,7.21 (d, J = 8.3Hz,2H) ,7.06 (d,J = 8.5Hz, 2H) ,4.26 (t,J = 7.4Hz,lH) ,4.16-4.09 (m, 1H) ,3.82 (dd, J = 5.0,1.5Hz, 1H) ,3.78(dd,J =

II. 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) , 1.64T . 54 (m,2H) , 1.44T . 30 (m, 10H) ,0.92 (t, J = 6.7Hz, 3H).

[0492] 10. (S) -2-Amino-3- (6- (4- (3- (3,5-Diamino-6-Clopiazine-2-Base)Muscle base)Butyl)Cai-2base)Propionic acid(80)Preparation

[0493]Program 11

CN 105073717 Β

[0494]

<img file="CN105073717B_D0070.tif" />

11(0

<img file="CN105073717B_D0071.tif" />

64R-Me

65:R = Et

OCH.

NaOH

MeOH/THF4bO

<img file="CN105073717B_D0072.tif" />

OCH,

1. KHN1DS. THF

2. TrisylNv AcOH 3jCH₃)₄N^AcO- ,JBn

Nt

<img file="CN105073717B_D0073.tif" />

THF/H-,Ο

OCH.

iOH, H.O

BocHN

<img file="CN105073717B_D0074.tif" />

O

<img file="CN105073717B_D0075.tif" />

OCHPd/C/Ί-Ε

OH

Boe.O, NaIlCO₃

MeOH

<img file="CN105073717B_D0076.tif" />

BocHN

CH₂CI₂

OTf

NaOH

<img file="CN105073717B_D0077.tif" />

Ο

Δο()Η·Η^Ν

HO

I HBr AcOH

OH

AcCkMeOH

<img file="CN105073717B_D0078.tif" />

Ο

HBlHjN

HO

PdiPPh.^.Cul (ί-BuhP, EtjN (U;C\

OH

HO

BocHN

MvOH/THF/H₂O

NHCb2

NHCbz

<img file="CN105073717B_D0079.tif" />

H_SCO

BocHN

MICH/ h₃co

BocHN

<img file="CN105073717B_D0080.tif" />

AcOI-ί

NH°

II· CO

BqcHN

DIPEA, EtOH

Pd (\ l b (ϊ αίίπ)

EtOH/AcOH

NIHU Λ_；H

<img file="CN105073717B_D0081.tif" />

h₂n

O

<img file="CN105073717B_D0082.tif" />

H-CO

BocTlN

<img file="CN105073717B_D0083.tif" />

MK'bz

SCH^

Ν N Η H

H.N

<img file="CN105073717B_D0084.tif" />

CN 105073717 Β

<img file="CN105073717B_D0085.tif" />

[0496] Compound62Preparation;

[0497] In a nitrogen atmosphere, the appropriateWittigInner Radium Salt(ylide)Pyridylmethoxymethylenetriphenyloxane (Ph₃PCHC0₂Me,43.Og, 129mmol)Add to aldehyde59 (20.Og, 107mmol)ofCH2CI2 (200mL)Solution and stir the reaction mixture at ambient temperature16h_oTLCMonitoring the completion of the reaction(16h). Remove under reduced pressureCH2CI2,And use10%Ethyl acetate-HexaneFCCGet the corresponding trans form as a white solid-α,Β-Unsaturated ester62 (24.Og, 92%):

[0498] ^XH NMR (400MHz,CDCI3) 67.88-7.82 (m, 1H) ,8.81 (d, J= 15.8Hz, 1H) ,7.73 (d,J = 9.0Hz,lH) ,7.70 (d,J = 8.8Hz, 1H) ,7.61 (dd,J = 8.8,2.2Hz, 1H) ,7.15 (dd, J = 9.2,2.2Hz, 1H) ,7.11 (d,J = 2.2Hz,lH) ,6.49 (d, J = 15.8Hz, 1H) ,3.82 (s,3H) ,3.82 (s,3H).

[0499]Compound62Preparation(Extra route);

[0500] Cool down to0°CTrimethyl phosphonoacetate(55.6mL,38lmmol)of250mLAnhydrousCH2CI2Add inDBU (48.8mL, 322mmol)And stir the mixture15minute. Add drop by drop50mL CH2CI2Aldehydes in59 (40.0g, 215mmol). The temperature of the reaction mixture was returned to room temperature and the resulting reaction mixture was stirred at room temperature16h,And use100mLThe water is quenched. Distribute the mixture and use the aqueous layerCH₂Cl₂(3X150mL)extraction. Combined organics with brine, dry(Na₂S0₄)Wash, filter, concentrate and pass the residue through silica gel column chromatography(10: 1Hexane/Ethyl acetate)Purification to obtain the desired trans form as a white solid-α,Β-Unsaturated ester62 (48.0g,92%).

[0501] Compound64Preparation;

[0502] At room temperature, the compound62 (48.0g,196mmol)and 10%Pd/C (10g)ofEtOAc/THF (600mL/ 75mL)Suspension is under hydrogenation condition(latm) 16h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate under vacuum,Obtained as a white solid64 (46.5g, 96 %):

[0503] ^XH NMR(400MHz,CDC13) 67.67 (d,J = 9.4Hz,2H) , 7.57-7.54 (m, 1H) ,7.29(dd,J = 8.6,1.8Hz,lH) ,7.12 (dd,J = 8.8,2.5Ηζ,1Η) ,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] Compound66Preparation;

[0505] Methyl ester64 (46.5g, 19lmmo 1)ofTHF/MeOH/H₂O (500mL/500mL/150mL)Add to solution NaOH (45.6g,114mmo 1)And stir the reaction mixture at room temperature2h. Remove the solvent andpHValue useIN HC1Adjust the aqueous solution to1;A white solid precipitated. The solid was filtered, washed with water and dried under vacuum to obtain the acid as a white solid66 (42.5g,97%):

CN 105073717 Β

[0506] ^XH NMR (400MHz, DMSO-ch) 512.14 (brs, 1H) ,7.73 (dd, J = 9.5,2.3Hz , 2H) ,7.647.61 (m,lH) ,7.35(dd,J = 8.5,1.5Hz,lH) ,7.26(d,J = 2.8Hz,lH) ,7.129(dd,J = 9.1, 2.5Hz,lH) ,3.85 (s,3H) ,2.94 (t, J = 7.6Hz,2H) ,2.60 (t, J = 7.6Hz,2H).

[0507] Compound67Preparation;

[0508] exist-78°CDown, to the compound60 (39.3g,222mmol)AnhydrousTHF (500mL)Add n-butyl lithium dropwise to the solution(11 OmL,In cyclohexane2MSolution)And stir the reaction mixturelh,Get the compound61The solution. To the compound66 (42.5g,185mmol)AnhydrousTHF(lOOOmL)To another solution ofNMM (26.3mL,240mmo 1)And in-78°CAdd drop by dropPivCl (27.3mL,222mmol). Stir the reaction mixture at the same temperature1Minutes and then-78°CAdd compound slowly66The preparation solution. Stir the reaction mixture additionally10Minutes before returning to 0°CAnd stirlh,Then stir at room temperature30Minutes, with saturationNHKlQuench and concentrate to removeTHF,And inCH₂C1₂(lOOOmL)with water(lOOOmL)Distribution between. Separate the water layer and useCH2CI2 (2 X lOOOmL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate. The residue passes through column chromatography(Silica gel,CH2C12)Purify to obtain the compound as a white solid67 (45.0g,63%):

[0509] ^XH NMR (400MHz, CDC13) 57.68 (d,J = 8.6Hz,2H) ,7.64-7.61 (m,lH) ,7.31 (dd,J = 8.5,1.8Hz,lH) ,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,lH) ,3.90(s,3H) ,3.46-3.21 (m,3H) ,3.20-3.08 (m, 2H) ,2.74 (dd,J = 13.6,9.4Hz,lH).

[0510] Compound68Preparation;

[0511] exist-78°CDown, to the compound67 (45.0g,116mmol)AnhydrousTHF (700mL)Add dropwise to the solution KHMDS(34.6g,174mmol). The resulting mixture is stirred30Minutes later,Add triisopropylbenzenesulfonyl azide(trisyl azide) (53.6g, 174mmol)And stir the reaction mixture5minute. Then, slowly add acetic acid at the same temperature(69.6mL, 1158mmol), Then add tetramethyl acetic acid saddle(30.9g,232mmol). Heat the reaction mixture to24°C ,Stir16h,Saturated withNaHCOs (300mL)Quench and concentrate to removeTHFAnd useCH₂C1₂ (2 X 500mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate. The residue passes through column chromatography(Silica gel,10:90 EtOAc/Hexane, followed byDCM)purification,Obtain the compound as a yellow solid68 (31.0g,62%):

[0512] ^XH NMR (400MHz,CDCI3) 67.70 (d,J = 9.1Ηζ,2Η) ,7.68-7.65 ,7.40(dd,J =

8.6,1.8Hz,lH) ,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,lH) ,5.36 (dd, J = 9.0,6.OHz, 1H) ,4.58-4.50 (m, 1H), 4.11 (dd,J = 9.1,2.6Hz,lH) ,3.90(s,3H) ,3.91 (t,J = 8.6Hz,lH) ,3.34(dd,J=13.8, 6.5Hz,lH) ,3.30 (dd,J=13.0,3.5Hz,lH) ,3.19 (dd, J= 13.4,8.6Hz , 1H) ,2.81 (dd,J = 13.4,9.5Hz,lH).

[0513] Compound69Preparation;

[0514] exist0°CDown, to the compound68 (31.0g,72. lmmol)ofTHF/H2O (300mL/100mL)Add dropwise to the solutionH2O2 (49mL,433mmol), Then addLiOH (6.04g, 144mmol). Stir the reaction mixture at the same temperature 10Minutes, then stir at room temperaturelhr,Then use saturationNa₂SO₃ (200mL)Quench and concentrate under reduced pressure to removeTHFAnd use CH2CI2 (500mL)washing. For water layerIN HC1Aqueous solution acidification and useCH₂C1₂ (2X500mL)extraction. The combined organic extractsNa2S04Dry, concentrate and useMTBEWashing to obtain the compound as an off-white solid69 (15.0g,82%):

[0515] ^XH NMR (400MHz,MeOD-ds) δ7.70 (t, J = 8.4Ηζ,2Η) ,7.66-7.63 (m, 1Η) ,7.35(dd,J = 8.6,1.7Hz,lH) ,7.19(d,J = 2.8Hz,lH) , 7.10 (dd, J = 9.1,2.6Hz , 1H) , 4.25 (dd, J = 8.6,

CN 105073717 Β

5.3Ηζ,1Η) ,3.88 (s,3H) ,3.29 (dd, J = 13.9,5.1Hz, 1H) ,3.10 (dd, J = 14.3,8.6Hz, 1H).

[0516] Compound70Preparation;

[0517] At room temperature, the compound69 (15.0g, 55. lmmo 1)and 10%Pd/C (3.50g)ofAcOH/H₂O (300mL/ lOOmL)Suspension is under hydrogenation condition(latm) 3h. The reaction mixture was filtered through diatomaceous earth and usedAcOH/IfeOWash, then useMeOHwashing. Concentrate the filtrate under vacuum,Acetate is obtained as a yellow solid70 (14.0g,83%):

[0518] ^XH NMR (400MHz,DMSO-de,TFA) δ8.38-8.18 (m,3Η) ,7.78 (dd, J=ll .4,8.1Ηζ,2Η), 7.75-7.70 ,7.41 (dd, J = 8.6,1.6Hz, 1Η) ,7.29 (d, J = 2.3Hz, 1H) ,7.18 (dd,J = 8.8,

2.4Hz,lH) ,4.33-4.23 ,3.89 (s,3H) ,3.33 (dq,J = 14.5,5.9Hz,2H) ,1.92(s,3H).

[0519] Compound71Preparation;

[0520] To the compound at room temperature70 (14.0g,45.9mmol)Acetic acid(140mL)Add hydrobromic acid dropwise to the solution (140mL)And reflux the reaction mixture3h. The reaction mixture was cooled to room temperature and concentrated. Crude brown residue71 (12.4g,87%)Used directly in the next step without any purification:

[0521] ^XH NMR (400MHz,DMSO-ch) 513.83 (brs,lH) ,9.71 (brs,lH) ,8.41 (brs,lH) ,8.25 (brs,2H) ,7.67 (dd,J=13.8,8.7Hz,2H) ,7.64-7.61 (m,lH) ,7.29 (dd, J = 8.6,1.7Hz, 1H), 7.13-7.05 (m,2H) ,4.29-4.19 (m, 1H) ,3.20 (t,J = 5.5Hz,2H).

[0522] Compound72Preparation;

[0523] exist0°CNext, the acetyl chloride(38.4mL,540mmol)Add to anhydrous methanol(400mL)And then add the compound71 (24.0g, 77.2mmo 1). Reflux the reaction mixture4hAnd concentrated. Leftovers inCH₂C1₂ (500mL)With saturation NaHCOs (300mL)Distribution between. Separate the water layer and useCH2CI2 (2 X 300mL)extraction. The combined organic extractsNazSOq Dry and concentrate,Obtain the compound as a white solid72 (16.6g,88%):

[0524] ^XH NMR (400MHz, DMSO-ch) 69.62 (brs,lH) , 7.67 (d, J = 9.4Hz , 1H) , 7.58 (d, J = 8.8Hz,lH) ,7.53 (s,lH) ,7.22 (dd,J = 8.2,1.4Hz, 1H) ,7.09-7.06 ,7.04 (dd,J = 8.8,

2.6Hz,lH) ,3.67 (t,J = 6.5Hz,lH) ,3.57 (s,3H) ,2.97 (dd, J= 13.5,6.1Hz, 1H) ,2.86 (dd,J = 13.2,7.4Hz,lH) ,1.90 (brs,2H).

[0525] Compound73Preparation;

[0526] exist0°CDown, to the compound72 (16.6g,67.8mmol)ofMeOH/H₂O (360mL/120mL)Solution addition NaHCOs (22.8g, 27lmmo 1)andB0C2O (17.7g,81.3mmo 1). Warm the resulting mixture to room temperature and stirlh. The reaction mixture is inCH2CI2 (200mL)with water(200mL)Distribution between. Separate the water layer and useCH2CI2 (2 X 400mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate. use20%Ethyl acetate-Hexane is subsequently used CH2CI2ofFCCObtain the compound as a white solid73 (17.0g,73%):

[0527] ^XH NMR (400MHz,CDCls) δ7.60 (d, J = 9.5Hz, 1H) ,7.51 (d, J = 8.2Ηζ, 1Η) ,7.49-7.43 (m,lH) ,7.15(d,J = 8.2Hz,lH) ,7.09-6.99 (m,2H) ,6.31 (brs, 1H) ,5.15-4.84 (m, 1H) ,4.734.46 ,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).

[0528] Compound74Preparation

[0529] exist0°CDown, to the compound73 (7.0g, 20.3mmol)ofCH2CI2 (300mL)Solution addition(16.5mL, 203mmo 1)And triflate(5.1 ImL,30.4mmol)And stir at the same temperaturelh,Then stir at room temperature2h. After concentration, the reaction mixtureCH₂C1₂ (300mL)with water(200mL)Distribution between. Separate the water layer and useCH₂C1₂(2X300mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dried and concentrated to obtain a brown oil

CN 105073717 Β

Compound74(8.80g,91%)(Depend onNMRSure to exist). by usingLC-MSMonitor the reaction and the product composition consists ofLM-MSData to determine:

[0530] ^XH NMR (400MHz,CDCI3) 7.85 (d,J = 9.2Ηζ,1Η) ,7.80 (d, J = 8.7Hz, 1H) ,7.71 (d,J = 2.7Hz,lH) ,7.66-7.63 (m,lH) , 7.37 (ddd, J= 10.0,7.3,2.0Hz , 2H) , 5.12-5.03 (m, 1H), 4.73-4.61 (m,lH) ,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] Compound75Preparation;

[0532] At room temperature, the compound74 (16.5g, 34.6mmol)He Ding-3-Alkynyl carbamate(17,10.4g, 51,9mmol)AnhydrousCHsCN (450mL)Degas with nitrogen10Minutes, then add at room temperatureTEA(19.3mL, 138mmol), In hexane 10% (t-Bu) ₃P (13.9mL,6.91mmol)andCui (0.33g, 1.72mmol). Degas the resulting mixture with nitrogen10Minutes and add quickly all at oncePd (PPh3)4 (3.99g, 3.45mmol). Degassing with nitrogen 5Minutes later,Reflux the resulting mixture18h. Concentrate the reaction mixture under vacuum and pass through the column(Silica gel,75:25Hexane/EA)Purification residue,Obtain the compound as a brown solid75 (14. lg,77%):

[0533] ^XH NMR (400MHz, CDCI3) 67.86 (brs,lH) , 7.68 (t, J = 7.8Hz , 2H) , 7.53 (brs, 1H), 7.41 (dd,J = 8.5,1.6Hz,lH) ,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,lH) ,4.70-4.59 , 3.69 (s, 3H) , 3.46 (q, J = 6.7Hz , 2H) ,3.27 (dd,J=14.1,5.9Hz,lH) ,3.16 (dd, J= 13.2,6.2Hz, 1H) ,2.67 (t, J = 6.6Hz ,2H) ,1.38 (s, 9H).

[0534] Compound76Preparation;

[0535] Methyl ester75 (12. lg,22.8mmol)ofTHF/MeOH/H₂O (150mL/150mL/50mL)Solution additionNaOH (4.56g,114mmol)And the reaction mixture was stirred at room temperature2h. willpHValue useIN HC1Adjust the aqueous solution to9And remove the organic solvent. Remnants ofpHValue adjusted to5to6,And the suspension is inCH2CI2 (500mL)with water(200mL)Distribution between. Separate the water layer and useCH₂Cl₂ (2X400mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate,Obtain the compound as a brown solid76 (10.50g,89%):

[0536] ^XH NMR (400MHz,CD3OD) 67.83 (s,lH) ,7.73-7.61 (m,3H) ,7.44-7.19 (m,7H) ,5.10 (s,2H) ,4.42-4.34 (m,lH) ,3.41-3.32 (m,3H) , 3.06 (dd, J= 14.3,9.3Hz, 1H) , 2.64 (t,J = 7.OHz,2H) ,1.31 (s,7H) ,1.21 (s,2H).

[0537] Compound 77 Preparation;SG-SJL-B-27

[0538] will75 (2.0g,3.77mmo 1)and 10%Pd/C (500mg)existEtOH (90mL)andAcOH(lOmL)The suspension in the mixture is degassed and then under hydrogenation conditions at room temperature(latm) 16h. The reaction mixture was filtered through a plug of diatomaceous earth and usedMeOHWash the stopper. The filtrate was concentrated under vacuum to obtain the amine salt as a white solid77(1.60mg,93%): [0539] ^XH NMR (400MHz, CD3OD) 7.73 (d,J = 8.5,1H) , 7.72 (d, J = 8.7Hz, 1H) ,7.62 ((brs, 2H) ,7.73 (ddd,J=10.0,8.7,2.7Hz,2H) ,4.44 (dd, J = 8.8,5.6Hz, 1H) ,3.68 (s,3H) ,3.25 (dd,J=14.0,6.6Hz,lH) ,3.04 (dd, J= 13.5,9.2Hz, 1H) ,2.93 (t, J = 7.4Hz,2H) ,2.83 (t, J = 7.4Hz,2H) ,1.96 (s,6H) , 1.85-1.75 (m,2H) , 1.74-1.68 (m,2H) , 1.33 (s,7H) , 1.26 (s,2H). [0540] Compound 78 Preparation;SG-SJL-B-30

[0541 ] At room temperature, to the amine salt77 (1.60g, 3.47mmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,2.16g, 5.56mmo 1)ofEtOH (40mL)Add to solutionDIPEA (6.20mL, 34.70mmol). In a sealed tube70 °CHeat the reaction mixturelh,It was then cooled to room temperature and concentrated in vacuo. The residue passes through column chromatography

CN 105073717 Β

(Silica gel80:18:2CHC13/CH₃0H/NH₄0H)Purified to obtain muscle as a yellow solid78 (1.24g,59%):

[0542] ^XH NMR(400MHz,CD₃OD)57.71 (dd, J = 8.4,2.8Hz,2H) ,7.60 (brs,2H) ,7.34(dd,J = 8.5,1.9Hz,lH) ,7.30 (dd, J = 8.7,1.7Hz , 1H) ,4.45 (dd,J = 8.9,5.7Hz, 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,lH) ,2.82(t,J = 7.2Hz,2H) , 1.86T .77 (m,2H) ,1.73-1.63 (m,2H) , 1.32 (s,7H) , 1.23 (s, 2H).

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

[0544] Methyl ester 78(1.24g,2. OOmmol)exist THF (25mL), Methanol(25mL)And water(lOmL)Add solids to the solution in the mixtureNaOH (324mg,8.00mmo 1)And the reaction mixture was stirred at room temperaturelh. Reaction mixtureTLCShow the completion of the reaction, and then the reaction mixturepHBy addingIN HC1 (Aqueous solution)resumedpH 9to10And remove the organic solvent. The water partpHAdjusted topH 5to6Precipitation occurred, which was extracted with dichloromethane. For water-based partCH2CI2 (2 X 50mL)extraction. Combine the organic layers, afterNa₂S0₄dry,Filter and concentrate. Dry yellow solid compound under vacuum (79,1.10g,92%):

[0545] ^XH NMR ((400MHz,CDsOD) 7.70 (t, J = 9.4,2Η) ,7.61 (d, J = 5.3Hz,2H) ,7.33(dd,J = 8.4,1.4Hz,2H) ,4.38 (dd,J = 8.4,5.1Hz, 1H) ,3.05 (dd,J=14.1,9.1Hz, 1H) ,2.84 (t,J = 6.9Hz,2H) ,3.35-3.34(m,3H) , 1.88T . 79 (m,2H) ,1.76-1.67 (m,2H) , 1.32 (s,7H) , 1.21 (s, 2H).

[0546] Compound80--2-Amino-3- (6- (4- (3- (3,5-Diamino-6-Clopiazine-2-Base)Muscle base)Butyl) Cai-2-base)Preparation of propionic acid hydrochloride

[0547] Put the dioxane in4N HC1 (25mL)Add toEtOH (5.OmL)middle79 (1.10g, 1.83mmo 1)And stir the reaction mixture at room temperature2h. Remove solvent and pass through reversed phase column(GoIdcolumn)Purify and lyophilize the residue to give the compound as a yellow solid80 (700mg,67%):

[0548] ^XH NMR (400MHz,DMS0-d6) 10.48 (s,lH) ,9.24 (brs, 1H) ,8.99-8.86 (m,lH) ,8.848.70 (m,lH) ,8.38 (brs,3H) ,7.80 (t, J = 9.2Hz ,2H) ,7.73 (s, 1H) ,7.69 (s,lH) ,7.45-7.35 (m,4H) ,4.25 (dd,J=11.4,5.9Hz,lH) ,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, CDsOD) 7.82 (d, J = 8.5Hz, 1H) ,7.78 (d, J = 8.7Hz , 1H) ,7.73 (s, 1H) ,7.68 (s,lH) ,7.40 (ddd, J= 10.5,8.6,1.6Hz ,2H) ,4.33 (dd, J = 7.7,5.2Hz, 1H) ,3.46 (dd,J=14.9,6.0Hz,lH) ,3.37 (t, J = 7.5Hz , 2H) ,3.33-3.29 (m,lH) , 2.87 (t, J = 7.7Hz, 2H) ,1.90-1.80 (m,2H) ,1.79-1.71 (m,2H).

[0550] 11. (S) -3,5-Diamino-6-chlorine-Ν- (Ν- (4- (6- (2,3-Diamino-3-Oxopropyl)Cai-2-base)Ding

base)Caramel)Pyrazine-2-Formamide(84)Preparation

[0551]Program 12

CN 105073717 Β

ο

11(people

BocIIN

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NIKbz f-BCF. NMM middle7 Ν NH3

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II?N

BocilN

NUCbz

Pd/C, IL (1 atm)

Ο

[0552]

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N UCIAqueous solution

II₂N

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bomb ¥ ·21ΚΊ

NpeopleNPeople openΝ] η ^Η Λ Λ π₂ν ν νπ₂ Compound81Preparation; In an ice bath, the acid76 (2.0g,3.87mmol)ofTHF (80mL)The solution is cooled to0°C ,Add toNMM

[0553]

[0554] (0.63mL,5.03mmol), Then add drop by dropi-BCF (0.63mL,5.80mmol)And stir the reaction mixture at the same temperature2h. Add drop by dropNH3(7.0NOf methanol,5.52mL,38.7mmol)And stir the reaction mixture at the same temperature.2h. Then the reaction mixture was returned to room temperature and stirred16h. Remove the organic solvent. Add water to this residue and useCH2CI2 (3X 100mL)extraction. Combine the organic layers, afterNa₂S0₄Dry, filter and concentrate. The residue passes through column chromatography (In chloroform3%Methanol)Purified to obtain the amide as a pale yellow solid81(1.75g,88%):

[0555] ^XH NMR (400MHz,CDsOD) 7.83 (s, 1Η) ,7.69 (d,J = 8.1Ηζ,2Η) ,7.66 (s,lH) ,7.39 (dt, J = 8.8,1.9Hz,2H) ,7.35-7.21 (m,5H) ,5.09 (s,2H) ,4.40 (dd, J = 9.6,5.8Hz, 1H) ,3.37 (t,J = 6.9Hz,2H) ,3.27 (dd, J= 13.8,5.2Hz, 1H) , 2.97 (dd, J= 13.7,9.4Hz , 1H) , 2.63 (t,J = 7.0Hz,2H) ,1.27 (s,7H) ,1.21 (s,2H).

CN 105073717 Β

[0556] Compound82Preparation;

[0557] will81 (1.75mg, 3.39mmo 1)and 10 % Pd/C (600mg)existEtOH (11 OmL)andAcOH (15mL)The suspension in the mixture is degassed and then under hydrogenation conditions at room temperature(latm) 12h. The reaction mixture was filtered through a plug of diatomaceous earth and usedMeOHWash the stopper. The filtrate was concentrated in vacuo to obtain the amine salt as a white solid82(1.40g,93%): [0558] ^XH NMR (400MHz, CD3OD) 7.72 (dd,J = 8.3,5.6Hz, 2H) ,7.66 (s,lH) ,7.61 (s, 1H), 7.38 (dd,J = 8.6,1.3Hz,lH) ,7.34 (dd, J = 8.5,1.5Hz , 1H) ,4.38 (dd, J = 9.0,5.OHz , 1H), 3.27 (dd,J = 13.8,5.0Hz,lH) ,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).

[0559] Compound83Preparation;

[0560] At room temperature, to the amine salt82 (1.40g, 3.15mmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,1.96g,5.04mmol)ofEtOH(40mL)Add to solutionDIPEA (5.64mL,31.5mmol). In the sealed tube, in70°CHeat the reaction mixture2h,It was then cooled to room temperature and concentrated in vacuo. The residue passes through column chromatography(Silica gel,80:18:2CHC13/CH₃0H/NH₄0H)Purified to obtain muscle as a yellow solid83 (1.15g,61%):

[0561] ^XH NMR (400MHz, CD3OD) 67.70 (d,J = 8.3Hz, 2H) , 7.64 (s, 1H) , 7.60 (s, 1H) , 7.34 (dt,J = 8.9,1.9Hz,2H) ,4.38 (dd,J = 9.0,5.5Hz, 1H) ,3.28-3.20 (m,3H) ,2.96 (dd,J = 9.6, 14. ΙΗζ,ΙΗ) ,2.81 (t, J = 7.4Hz , 2H) , 1.85T . 76 (m, 2H) ,1.70-1.61 (m,2H) ,1.27(s,7H), 1.20 (s, 2H).

[0562] Compound(S) -3,5-Diamino-6-Chlorinated-Ν- (Ν- (4- (6- (2,3-Diamino-3-Oxopropyl)Cai-2base)Butyl)Caramel)Pyrazine-2-Formamide(84)ofHC1Salt preparation

[0563] Put the dioxane in4Ν HC1 (25mL)Add toEtOH (6.OmL)middle83 (1.15g, 1.92mmo 1)And stir the reaction mixture at room temperature2h. Remove solvent and pass through reversed phase column(Goldcolumn)Purify and freeze-dry the residue, Obtain the compound as a yellow solid840mg,28%):

[0564] ^XH NMR (400MHz,DMSO-de) 10.56 (s,lH) ,9.38 (t,J = 5.5Hz, 1H) ,9.06-8.83 (m,2H), 8.31 (brs,3H) ,8.02 (s,lH) ,7.79 (t,J = 8.6Hz,2H) ,7.73 (s,lH) ,7.69 (s,lH) ,7.51 (s,lH), 7.46-7.36 (m,4H) ,4.06 (dd,J= 11.5,6.0Hz, 1H) ,3.37 (q,J = 6.4Hz,2H) ,3.27 (dd,J = 6.6, 1.4Hz,lH) ,3.18(dd,J=13.7,6.9Hz,lH) ,2.79 (t, J = 7.1Hz, 2H) , 1.79T . 69 (m, 2H) ,1.641.54(m,2H).

[0565] ^XH NMR (400MHz, CDsOD) 7.82 (d, J = 8.4Hz , 1H) , 7.79 (d, J = 8.7Hz , 1H) ,7.74 (s, 1H) ,7.68 (s,lH) ,7.41 (td,J = 8.1,1.6Hz,2H) ,4.18 (dd,J = 8.1,6.3Hz, 1H) ,3.42-3.34 (m, 3H) ,3.21 (dd,J=14.1,8.0Hz,lH) ,2.86 (t, J = 7.4Hz,2H) ,1.91-1.80 (m,2H) ,1.79-1.71 (m,2H).

[0566] 12. 3,5-Diamino-Ν- (N^_ (4^_ (6^_ ((S) -2-Amino-3- (4^_ (3^_ (Hexyl((2S, 3R, 4R, 5R) -2,

3,4,5,6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(89)Preparation

[0567]Program 13

CN 105073717 Β

<img file="CN105073717B_D0090.tif" />

HO

BocHN

DEPBT/DCPEA Pay THF

<img file="CN105073717B_D0091.tif" />

NHCbz

<img file="CN105073717B_D0092.tif" />

[0568]

OH OH

6,,δ διι NS

Ph . Dish,n

<img file="CN105073717B_D0093.tif" />

<img file="CN105073717B_D0094.tif" />

H OH

<img file="CN105073717B_D0095.tif" />

N

OH ΟΠ OH

Ο

ΝΗπ

CMComment

<img file="CN105073717B_D0096.tif" />

BocHN

Pd/C, H,

PtOWAcOH

NHCbz

<img file="CN105073717B_D0097.tif" />

forN

O NH*HI

H

NHo

SCII3

DIPEA. BtOH

<img file="CN105073717B_D0098.tif" />

BccHN

NH people

<img file="CN105073717B_D0099.tif" />

Ο

N H

N[IC1Aqueous solution

EtOH

<img file="CN105073717B_D0100.tif" />

I o -JL

N NH₂

[0569]

[0570] Compound86Preparation; toTHF (50mL)Compounds in85 (1 . 10g,2.32mmol)Add in orderDEPBT (766mg, 2.56mmol) &gt;76 (1.00g,1.97mmol)andDIPEA (1.OmL,5.9lmmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH₂C1₂ (lOOmL)Medium, with saturated aqueous water(2 X 100mL)And salt water(50mL) Quickly wash and cureNa₂S0₄dry. The solvent is evaporated and the crude product is passed through silica gel flash chromatography(5%Methanol/CH2CI2)Purification to obtain the amide as a yellow solid product86 (1.19g,57%):

[0571] ^XH NMR (400MHz,CD3OD) :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 = 8.3Hz , 2H) ,5.52(s,lH) ,5.10(s,2H) ,4.51 (t,J = 7.8Hz,lH) ,4.31-4.25(m,lH) ,4.24 (dd, J= 11.0,5.4Hz, IH) ,4.01-3.91 (m,2H) ,3.88 (dd,J = 5.5,2. ΙΗζ,ΙΗ) ,3.76 (dd,J = 9.3,2. ΙΗζ,ΙΗ) ,3.61 (t,J= 10.6Hz, IH) ,3.37 (t,J = 6.9Hz,2H) ,3.12-3.00 (m,lH) ,2.74 (dd, J= 13.2,5.3Hz, IH) ,2.64 (t, J = 7.1Hz,2H) ,2.57-

2.37 (m,7H) , 1.74T .64 (m,2H) ,1.31 (s,9H) , 1.29-1.16 (m,8H) ,0.86 (t, J = 6.9Hz,3H).

CN 105073717 Β

[0572] Compound87Preparation;

[0573] will86 (1.19g,mixture)and 10%Pd/C (220mg)existEtOH (11 OmL)andAcOH(15mL)The suspension in the mixture is degassed and then under hydrogenation conditions at room temperature(latm) 3h. The reaction mixture was filtered through a plug of diatomaceous earth and usedMeOHWash the stopper. Concentrate the filtrate under vacuum to obtain the amine salt87,Then it usesNaHCOsNeutralize and pass the crude product through silica gel flash chromatography(CMA, 80 : 18 : 2)purification,Free amine is obtained as a yellow solid87 (550mg, 58% ,After two steps):

[0574] ^XH NMR (400MHz,CD3OD) 7.71 (t, J = 8.4Hz,2H) ,7.62 (d, J=1.8Hz, 1H) ,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=15.6,8.4Hz,lH) ,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,lH) ,3.63 (t, J= 10.4Hz, 1H) ,3.27-3.20 ,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) , 1.95T .86 (m,2H) ,1.851.75 (m,2H) , 1.74-1.65 (m,2H) ,1.57-1.47 (m,2H) , 1.39T . 19 (m,7H) ,1.33(s,9H) ,0.88 (t, J = 6.9Hz,3H).

[0575] 88Preparation;

[0576] At room temperature, to amine87 (550mg, 0.65mmol)and3,5-Diamino-6-Clopiazine-2-Methyl thiomethionate(21,400mg,1.04mmol)ofEtOH (20mL)Add to solutionDIPEA (1.15mL,6.44mmol). In the sealed tube70 °CHeat the reaction mixture2h,It was then cooled to room temperature and concentrated in vacuo. The residue is chromatographed on a silica gel column(80: 18: 2CHCI3/CH3OH/NH4OH)Purification, followed by reverse phase column(Gold C18)Purified to obtain muscle as a yellow solid88 (333mg,48%):

[0577] ^XH NMR (400MHz,CD3OD) 7.69 (dd,J = 8.6,3.5Hz,2H) ,7.66 (s,lH) ,7.60 (s, 1H), 7.48-7.44 (m, 2H) , 7.35 (ddd, J = 10.4,8.6,1.6Hz , 2H) , 7.33-7.28 (m, 5H) , 7.04 (d, J = 8.3Hz ,2H) ,5.52 (s,lH) ,4.52-4.55 ,4.24 (dd, J= 10.6,5.4Hz , 1H) ,4.00-3.91 (m,

2H) ,3.88 (dd,J = 5.4,2.0Hz, 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= 13.5,8.3Hz , 1H) , 2.82 (t, J = 7 . OHz , 2H) ,2.77 (dd,J = 13.9,5.6Hz,lH) ,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-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine2-Formamide compoundHC1Salt(89)Preparation;

[0579] In the water4N HC1 (20mL)Add to88 (333mg,0.31mmol)Ethanol(10mL)Neutralize and stir the reaction mixture at room temperature2h. Pass through reversed phase column(Goldcolumn)Purify and freeze-dry the residue,Obtain the compound as a yellow solid89 (210mg,68%):

[0580] ^XH NMR (400MHz,DMSO-de) 10.94 (brs, 1H) ,9.29 (brs, 1H) ,9.02-8.77 (m,2H) ,8.648.17 (m,2H) ,7.80-7.73 (m,3H) , 7.68 (s, 1H) , 7.52 (d, J = 8.8Hz , 2H) , 7.47 (dd, J = 8.2,

Ι.ΟΗζ,ΙΗ) ,7.44-7.36 (m,3H) , 7.19 (d, J = 8.6Hz , 2H) ,5.52-5.41 (m,lH) ,4.86-4.71 (m,

1H) ,4.60 (d,J = 5.4Hz,lH) ,4.59-4.53 (m, 1H) ,4.42 (t, J = 5.8Hz, 1H) ,4.38 (t,J = 7.0Hz,

1H) ,4.03-3.95 ,3.71-3.66 (m, 1H) ,3.62-3.55 (m, 1H) ,3.53-3.34 (m,5H) ,3.27 (d,J = 7.7Hz,lH) ,3.23(d,J = 7.4Hz,lH) ,3.16-2.99 (m,5H) ,2.78 (t, J = 7.4Hz,2H) ,2.58 (t,J = 7.9Hz,2H) ,2.01-1.90 (m,2H) , 1.78T .68 (m,2H) , 1.66T . 54 (m,4H) ,1.32-1.21 (m,6H),

CN 105073717 Β

0.85(t,J = 6.6Hz,3H).

[0581] ^XH NMR (400MHz , CDsOD) 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= 14.0,6.6Hz , 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) ,2.102.00 (m,2H) ,1.89-1.80 (m,2H) , 1.79T .72 (m,2H) , 1.71T .63 (m,2H) , 1.40T . 30 (m,6H),

0.91 (t,J = 6.6Hz,3H).

[0582] 13. 3,5-Diamino-N- (N~ (4- (6- ((S) -2-Amino-3- (4- (3-(pair((2S, 3R,4R, 5R) -2,3,

4,5,6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(94)Preparation

[0583]Program 14

Ο

<img file="CN105073717B_D0101.tif" />

NHCbz

[0584]

<img file="CN105073717B_D0102.tif" />

Ο ΝΗ·ΗΙ Λ.

Η

<img file="CN105073717B_D0103.tif" />

scn₃ threw upNpeople

<img file="CN105073717B_D0104.tif" />

uo.

<img file="CN105073717B_D0105.tif" />

η

Boel IN ^Η0&lt;

NH Ο

A

Ν

II

<img file="CN105073717B_D0106.tif" />

Ν C1

<img file="CN105073717B_D0107.tif" />

Ν

Η

HrN Ν ΝΗ.

Oil 'Ν

<img file="CN105073717B_D0108.tif" />

Έ

ΟΗ ^(λ) ΟΗ (R)

4Ν[busy1Aqueous solution

ΙίίΟΙί

II()^V

<img file="CN105073717B_D0109.tif" />

HO ο

Ν

Π

<img file="CN105073717B_D0110.tif" />

ΝΠ

NH λ_μ

Ν ΐΐ

ΙΓ,Ν

<img file="CN105073717B_D0111.tif" />

Cl

NFL

CN 105073717 Β

[0585] Compound91Preparation

[0586] Xiang Zai THF (30mL)Compounds in 90 (484mg , 0.9 lmmo 1)Add in order DEPBT (300mg, 1. OOmmol),19 (400g,0.77mmol)andDIPEA (0.40mL,2.31mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (lOOmL)Medium, with saturated aqueous solution(2 X lOOmL)And salt water(50mL)Fast washing,And byNa₂SO₄dry. Evaporate the solvent and pass through the silica gel(5 %Methanol/CH2CI2)The crude product was purified by flash chromatography to obtain the amide as a yellow solid product91 (600mg,76%,Impure). passLCMSThe formation of the product was confirmed.

[0587] Compound92Preparation

[0588] At room temperature91 (600mg,0.59mmol)and 10%Pd/C (200mg)existEtOH (90mL)andAcOH (lOmL) The suspension in the mixture is degassed and then placed under hydrogenation conditions(latm)Stir down16h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. Concentrate the filtrate in vacuum,Get amine salt92,Then useNaHCOsneutralize,By silica gel flash chromatography(CMA,80: 18:2)Purify the crude product to obtain the free amine as a yellow solid36 (350mg, 66%,Impure):

[0589] ^XH NMR (400MHz ,CDsOD) 7.71 (d, J = 8.1Ηζ,2Η) ,7.68 (s,lH) ,7.61 (s,2H) ,7.43-

7.37 (m,2H) ,7.34 (dd, J = 8.3,1.3Hz, 1H) ,7.16 (d, J = 8.2Hz ,2H) ,4.69 (q, J = 5.1Ηζ,2Η), 4.50 (t,J = 7.1Hz,lH) ,4.13-4.06 (m,2H) ,4.05 (dd,J=ll .0,5.6Hz,2H) ,3.83 (dd,J = 4.8, 2.1Hz,2H) ,3.81-3.73 (m,2H) ,3.51 (dd,J = 9.5,2.3Hz,2H) ,3.38 (t, J=10.8Hz,2H) ,3.133.03 (m,6H) ,2.93 (t, J = 7.6Hz ,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 = 5.1Hz,6H).

[0590] Compound93Preparation:

[0591] To amine at room temperature92 (350mg,0.38mmo 1)and3,5-Diamino-6-Clopiazine-2-Methyl thiomethionate(13,242mg,0.62mmol)ofEtOH (1 OmL)Add to solutionDIPEA (0.67mL,3.80mmol). Place the reaction mixture in a sealed tube70°CUnder heating2h,It was then cooled to room temperature and concentrated in vacuo. Followed by silica gel column chromatography(80:18:2CHC13/CH₃0H/NH₄0H)Reversed phase column(Gold C18)Purify the residue to obtain the muscle as a yellow solid 93(170mg,Three-step yield20%):

[0592] ^XH NMR(400MHz,CD₃OD)7.71 (d, J = 8.2Hz,2H) ,7.68 (s,1H) ,7.62 (s,1H) ,7.43 (d, J = 8.2Hz,2H) ,7.26 (ddd, J= 10.6,8.6,1.3Hz , 2H) , 7.18 (d, J = 8.2Hz , 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. lHz,2H) ,3.893.81 (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 = 5.1Hz,6H).

[0593] 3,5-Diamino-N- (N~ (4~ (6- ((S) -2-Amino-3- (4- (3-(pair((2S, 3R, 4R, 5R) -2,3,4,5, 6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Chlorpyrazine-2Formamide(94)Preparation of the hydrochloride

[0594] Will be in the water(20mL)middle4N HC1Add to ethanol(5.OmL)middle93 (170mg,0.15mmol)In and in40 °CStir the reaction mixture2h. Remove the solvent and add again4NHC1And in40 °CReheat2h. Repeat this addition2Second-rate. Remove solvent and pass through reversed phase column(Goldcolumn)The residue was purified and lyophilized to give the compound as a yellow solid94 (80mg,50%):

[0595] ^XH NMR (400MHz,DMSO-ch) 10.74 (brs, 1H) ,9.28-9.19 (m, 1H) ,9.03-8.60 (m,2H), 8.58-8.04 ,7.81-7.73 (m,3H) ,7.68 (s,lH) , 7.50 (d, J = 8.4Hz , 2H) , 7.48-7.34 (m,

CN 105073717 Β

4Η) ,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) ,4.454.35 (m,2H) ,4.32-4.23 (m,lH) ,4.01-3.85 (m,lH) ,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) ,2.622.53 (m,2H) ,2.01T .86 (m,2H) , 1.79-1.68 (m,2H) , 1.64-1.55 (m,2H).

[0596] ^XH NMR (400MHz, CDsOD) 7.78 (d, J = 8.4Hz , 1H) , 7.75 (d, J = 7.8Hz , 1H) ,7.73 (s, 1H) ,7.66 (s,lH) ,7.42 (d, J = 8.8Hz ,2H) ,7.39 (d, J = 8.4Hz,2H) ,7.21 (d, J = 8.7Hz ,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.OHz ,2H) ,3.72-3.61 (m,6H) ,3.44-3.30 (m,10H) ,2.86 (t, J = 7.OHz ,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).

[0597] 14. 3,5-Diamino-Ν- (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-base)Butyl)Caramel)-6-Clopiazine-2Formamide(99)Preparation

[0598]Program 15

[0599]

<img file="CN105073717B_D0112.tif" />

<img file="CN105073717B_D0113.tif" />

<img file="CN105073717B_D0114.tif" />

CN 105073717 Β

[0600] Compound96Preparation

[0601] Put the acid in an ice bath19 (1 . 17g,2.27mmol)ofTHF (60mL)The solution is cooled to0°C ,Add toNMM (0.30mL, 2.95mmol),Add subsequentlyPivCl (0.30mL, 2.49mmo 1)And stir the reaction mixture at the same temperature2h. Add to34(1.0g,2.27mmol,10mL THF)aniline171,And stir the reaction mixture again at the same temperature 10minute. Then stir the reaction mixture at room temperature16h. Remove the organic solvent. Add water to this residue and useCH2CI2 (3X lOOmL)extraction. Combine the organic layers,Na₂S0₄Dry, filter and concentrate. Column chromatography(In chloroform4 %Methanol)Purification residue,Obtain the amide as a light yellow solid96 (1.40g,66%,Impure). passLCMSThe formation of the product was confirmed.

[0602] Compound97Preparation

[0603] At room temperature96 (1.40g, 1.50mmol)and 10%Pd/C (300mg)existEt0H(120mL)andAcOH (12mL)Degas the suspension in the system and place it under hydrogenation conditions(latm)Down16h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. Concentrate the filtrate in vacuum,Get amine salt97,Then useNaHCOsNeutralizing amine salt 97,Flash chromatography on silica gel(CMA, 80 : 18 : 2)Purify the crude product to obtain the free amine as a yellow solid97 , (550mg,Two-step yield30%):

[0604] ^XH NMR (400MHz,CD3OD) 7.74-7.65 (m,3H) ,7.59 (s,lH) ,7.41-7.29 (m,4H) ,7.11 (d, J = 8.4Hz,2H) ,4.69 (q,J = 4.9Hz,lH) ,4.50 (t, J = 7.9Hz, 1H) ,4.04 (dd, J= 10.4,5.2Hz, 1H) ,4.02-3.94 ,3.79-3.71 (m,lH) ,3.70-3.63 (m, 1H) ,3.54-3.39 (m,3H) ,3.26- (dd,

J=13.6,6.8Hz,lH) ,3.07 (dd,J= 13.1,8.3Hz, 1H) ,2.79 (t, J = 7.5Hz,2H) ,2.75-2.67 (m, 2H) ,2.55(t,J = 7.3Hz,2H) , 1.91T .80 (m,2H) , 1.79-1.69 (m,2H) , 1.62T .52 (m,2H) ,1.501.37(m,12H) ,1.33(s,9H) , 1.25 (d, J = 4.9Hz,3H).

[0605] Compound98Preparation:

[0606] To amine at room temperature97 (550mg, 0.68mmol)and3,5-Diamino-6-Clopiazine-2-Methyl thiomethionate(13,423mg,0.62mmol)ofEtOH (20mL)Add to solutionDIPEA (1.21mL,6.80mmol). Place the reaction mixture in a sealed tube70°CUnder heating2h,It was then cooled to room temperature and concentrated in vacuo. Followed by silica gel column chromatography(80:18:2CHC13/CH₃0H/NH₄0H)Purify the residue and pass it through a reverse phase column(Gold C18), Get the muscle as a yellow solid 98 (500mg,72%):

[0607] ^XH NMR (400MHz,CD3OD) 7.73-7.64 (m,3H) ,7.61 (s,lH) ,7.40-7.30 (m,4H) ,7.11 (d, J = 8.5Hz,2H) ,4.68 (d,J = 4.9Hz,lH) ,4.49 (t, J = 7.2Hz, 1H) ,4.04 (dd, J= 10.9,5.5Hz, 1H) ,4.02-3.93 ,3.78-3.70 ,3.69-3.64 (m, 1H) ,3.54-3.38 (m,4H) ,3.30-3.20 (m,2H) ,3.15-3.01 (m,lH) ,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-Ν- (N- (4- (6-(-2-Amino-3-Oxo-3- (4- (3- ((2S, 3R, 4R, 5R) -23,4,

5,6-Pentaylhexylamino)Propyl)Phenylamino)Propyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine-2-Formamide (99)Preparation of the hydrochloride

[0609] Will be in the water(20mL)middle4N HC1Add to ethanol(5.OmL)middle98 (500mg,0.15mmol)In and in40 °CStir the reaction mixture2h. Remove the solvent and add again4N HC1,exist40°CReheat2h. This addition is repeated two more times. Remove solvent and pass through reversed phase column(Goldcolumn)The residue was purified and lyophilized to give the compound as a yellow solid99 (206mg,50%):

[0610] ^XH NMR (400MHz,DMSO-de) 11.0(brs,lH) ,9.34 (brs, 1H) ,9.09-8.25 (m,6H) ,7.82

CN 105073717 Β

7.73 (m,2H) ,7.68 (s,lH) ,7.53 (d,J = 8.5Hz,2H) ,7.49 (d,J = 9.2Hz, 1H) ,7.41 (s,2H) ,7.39 (d,J = 8.2Hz,2H) ,7.17(d,J = 8.2Hz,2H) ,5.39 (d,J = 3.7Hz, 1H) ,4.80-4.70 (m, 1H) ,4.62 (d,J = 4.3Hz,lH) ,4.60-4.54 (m,lH) ,4.46-4.36 (m,2H) ,3.96-3.88 (m, 1H) ,3.71-3.65 (m, 1H) ,3.62-3.54 (m,lH) ,3.51-3.35 (m,5H) ,3.09 (d, J= 13.3Hz , 1H) ,2.94 (d,J=10.9Hz, 1H) ,2.87 (t,J = 9.1Hz,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 , CDsOD) 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. lHz,2H) ,4.31 (t,J = 6.3Hz, 1H) ,4.09-4.00 (m, 1H) ,3.873.81 (m,lH) ,3.78(d,J=11.4Hz,lH) ,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 ,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) ,1.91-1.81 (m,2H) ,1.80-1.69 (m,2H).

[0612] 15. (S) -3,5-Diamino-Ν-(N-(4-(6-(2-Amino-3-(4-(3-(Dimethylamino)Propyl)Phenylamine

base)-3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(103)Preparation

[0613]Program 16

CN 105073717 Β

<img file="CN105073717B_D0115.tif" />

[0614]

<img file="CN105073717B_D0116.tif" />

N ..W₂.

N Cl

<img file="CN105073717B_D0117.tif" />

tfa/ch₂cl₂

<img file="CN105073717B_D0118.tif" />

<img file="CN105073717B_D0119.tif" />

[0615] Compound100Preparation

[0616] Put the acid in an ice bath19 (1 .75g,3.39mmol)ofTHF (70mL)The solution is cooled to0°C ,Add toNMM (0.74mL,6.78mmol), Then addPivCl (0.41mL,3,39mmol), Stir the reaction mixture at the same temperature 2h. Add to18 (825mg,4.61mmol, 10mL THF), And stir the reaction mixture again at the same temperature10minute. Then put the reaction mixture at room temperature and stir16h. Remove the organic solvent. Add water to this residue,And useCHsCL· (3 X lOOmL)extraction. Combine the organic layers,throughNa₂S0₄Dry, filter and concentrate. Column chromatography(In chloroform4%Methanol)Purification residue,Obtain the amide as a light yellow solid100 (1.60g, 71 %):

[0617] ^XH NMR (400MHz, CDC13)7.87 (s,lH) ,7.71 (d, J = 8.5Hz , 1H) , 7.67 (d, J = 8.5Hz, 1H) ,7.65-7.62 (m,2H) ,7.42 (dd,J = 8.4,1.9Hz, 1H) ,7.40-7.29 (m,5H) ,7.22 (d,J = 8.6Hz, 2H) ,7.08(d,J = 8.4Hz,2H) ,5.21-5.10 (m,2H) ,5.13(s,2H) ,4.51 (q, J = 7.6Hz, 1H) ,3.47

CN 105073717 Β

(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).

[0618] Compound101Preparation

[0619] At room temperature 100 (1.60g,2.30mmol)and 10%Pd/C (400mg)existEt0H(130mL)andAcOH (20mL)The suspension in the mixture is degassed and then placed under hydrogenation conditions(latm) 16h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. The filtrate was concentrated in vacuo to obtain the amine salt as a yellow solid101 (1.60g,99%):

[0620] ^XH NMR(400MHz,CD₃OD)7.71 (d, J = 8.5Hz,2H) ,7.68 (s,1H) ,7.61 (s,lH) ,7.42 (d, J = 8.5Hz,2H) ,7.39(dd,J = 8.5,1.3Hz,lH) , 7.33 (dd, J = 8.5,1.3Hz , 1H) ,7.16(d,J = 8.6Hz,2H) ,4.50 (t,J = 7.6Hz,lH) ,3.28 (dd,J=14.0,6.3Hz,lH) ,3.07 (dd,J=13.3, 8.7Hz,lH) ,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) , 1.86T . 75 (m,2H) , 1.74-1.64 (m, 2H) ,1.33 (s,9H).

[0621] Compound102Preparation;

[0622] To amine at room temperature101 (1.60g, 2.30mmol)and3,5-Diamino-6-Clopiazine-2-Methyl thiomethionate(21,1.60g, 4.14mmo 1)ofEtOH (25mL)Add to solutionDIPEA (4. ImL, 23. Ommo 1). Place the reaction mixture in a sealed tube70°CUnder heating2h,It was then cooled to room temperature and concentrated in vacuo. Chromatography by silica gel column(80: 18:2CHC13/CH₃0H/NH₄0H)Purify the residue to obtain the muscle as a yellow solid102 (645mg,37%and640mg, 37%Impure):

[0623] ^XH NMR (400MHz,CD3OD) 7.70 (dd,J = 9.0,4.3Hz,2H) ,7.66 (s,lH) ,7.61 (s, 1H), 7.39-7.31 (m,4H) ,7.11 (d, J = 8.4Hz ,2H) ,4.48 (t, J = 7.6Hz, 1H) ,3.30-3.22 (m,3H) ,3.06 (dd,J=13.8,8.9Hz,lH) , 2.83 (t, J = 7.2Hz , 2H) , 2.57 (t, J = 7.9Hz , 2H) ,2.32 (dd,J = 10.5,7.6Hz,2H) ,2.23 (s,6H) , 1.86-1.74 (m,4H) , 1.73-1.64 (m,2H) ,1.32(s,9H).

[0624] (S) -3,5-Diamino-Ν- (N- (4- (6- (2-Amino-3- (4- (3-(Dimethylamino)Propyl)Phenylamino)3-Oxopropyl)Cai-2-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(103)Preparation of the hydrochloride

[0625] willTFA(lOmL)add toCH2CI2middle47 (545mg,0.71mmol)And stir the reaction mixture at room temperaturelh. Remove solvent,Add againIN HC1,And remove the solvent and pass through a reversed-phase column(Goldcolumn)Purify the residue and lyophilize the residue,Obtain the compound as a yellow solid48 (206mg,50%):

[0626] ^XH NMR (400MHz,DMSO-de) 11.02 (brs, 1H) , 10.81-10.58 (m, 1H) ,10.53 (s,lH) ,9.32 (s,lH) ,9.04-8.72 (m,2H) , 8.50 (brs, 3H) ,7.82-7.73 (m,3H) , 7.68 (s , 1H) , 7.53 (d, J = 8.5Hz ,2H) ,7.48 (d, J = 9.2Hz, 1H) ,7.45-7.35 (m,3H) ,7.18 (d, J = 8.4Hz,2H) ,4.45-4.35 (m,lH) ,3.74-3.45 (m,lH) ,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) ,2.02-1.88 (m,2H) , 1.79-1.66 (m, 2H), 1.64-1.54 (m,2H).

[0627] ^XH NMR (400MHz , CDsOD) 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 = 8.9Hz , 2H) , 4.33 (t, J = 7.5Hz , 1H) , 3.46 (dd, J= 13.6, 6.6Hz,lH) ,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).

CN 105073717 Β

[0628] 16.3,5-Diamino-Ν- (Ν- (4- (4-(-2-Amino-3- (4- (3-(Hexyl ((2S, 3R,4R, 5R) -2,

3,4,5,6-Five consumption group (hexyl) amino) propyl) phenylamino)-3-Oxopropyl)-5,6,7,8-Four consumption Kicai-1-Base) Butyl) Methyl)-6-Clopiazine-2-Formamide (123) Preparation

[0629] Program17

[0630]

ΟΜ

<img file="CN105073717B_D0120.tif" />

104 Dimethyl ketate

<img file="CN105073717B_D0121.tif" />

ΠΙ5

1.2-DCE

OCII₃¹ POC1₃, DMF

<img file="CN105073717B_D0122.tif" />

DBU_S CH₂CI₂

CHO

106 factoryJBlood

PfOKOCHs).

<img file="CN105073717B_D0123.tif" />

1(17

OCHs j Pd/C, H₂, FtOH

OCTL

<img file="CN105073717B_D0124.tif" />

O οαι;

l.KHMDS. THF

2. TrisylNg. AcOH Jade(CH₃)₄NⁱAcO^

<img file="CN105073717B_D0125.tif" />

HO

<img file="CN105073717B_D0126.tif" />

O 8

Hl

I LiOH. H₂O₂, I

<img file="CN105073717B_D0127.tif" />

Pay

Pd/C. II_y

AcOI1/H₂O

112

OCH;

<img file="CN105073717B_D0128.tif" />

,N* on

H3

OCH

1IB:, AcOH

1. BuLi, TEIF

2. PivCl, NMM, THF

<img file="CN105073717B_D0129.tif" />

McOH/THFTI_:O

AcCl

MeOH on

114

OCHi

NaOH

<img file="CN105073717B_D0130.tif" />

OH

<img file="CN105073717B_D0131.tif" />

,,NH₂«HC1

O OCH;

115

<img file="CN105073717B_D0132.tif" />

NaOH

TIlElLO/MeOIl

BocO, NaHC6 ~ MeOH

<img file="CN105073717B_D0133.tif" />

<img file="CN105073717B_D0134.tif" />

'bz

<img file="CN105073717B_D0135.tif" />

[0631]Program 17(Continued)

CN 105073717 Β

<img file="CN105073717B_D0136.tif" />

<img file="CN105073717B_D0137.tif" />

[0633] Compound105Preparation

[0634] exist0°C Towards 104 (100g,0.675mmol)AnhydrousTHF (800mL)Add dropwise to the solutionNaOH (32. Omg, 0.809mmo 1)And dimethyl sulfate(102g,0.809mmol). Stir the reaction mixture at room temperature2h. Remove under reduced pressureTHF,And make the mixture inCH₂C1₂ (1.0L)with water(1.0L)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 1.0L)extraction. The combined organic extractsNa₂S0₄Dry and concentrate. Column chromatography(Silica gel,100%CH2C12)Purification residue,Obtain the compound as a yellow liquid105 (108.0g,90%):

[0635] ^XH NMR (400MHz,DMSO-ch) : 57.06 (t, J = 7.85Hz , 1H) ,6.71 (d, J = 7.25,1H) ,6.64 (t,J = 7.7Hz,lH) ,3.80 (s,3H) , 2.74 (t, J = 2.75Hz, 2H) , 2.65 (t, J = 2.65Hz , 2H) ,1.811.71 (m,4H).

[0636] Compound106Preparation

[0637] exist0°C ,To anhydrous under nitrogen atmosphereDMF (71.45mL,0.923mmol)Add dropwise to the solutionPOCI3 (57.40mL ,0.616mmo 1). exist0 °CStir the reaction mixture30minute. exist0 °C ,Under nitrogen atmosphere,Will be in anhydrous1,2Dichloromethane(500mL)middle105 (50.0mg,0.308mmol)The solution is added to the reaction mixture. After the addition is complete, the reaction mixture is heated to80°CKeep6h. Cool out the reaction mixture with0Quench, and make it inCH₂C1₂ (1.0L)with water (1.0L)Distribution between. Separate the water layer and useCH₂Cb (2X1.0L)extraction. The combined organic extractsNa₂S0₄Dry

CN 105073717 Β

Dry and concentrate. Column chromatography(Silica gel,5%ofEA/Hexane)Purification residue,Obtain the compound as a yellow solid106 (35.0g,61%):

[0638] ^XH NMR (400MHz, DMSO-cU) : 610.10 (s , 1H) , 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).

[0639] Compound107Preparation

[0640] Cool down to0°CTrimethyl phosphonoylacetate(55.OmL,0.378mmol)existlOOmLAnhydrousCH2CI2Add to the solution inDBU(58.0g,0.380mmo 1)And stir the mixture15minute. Add aldehyde dropwise106 (16.0g, 0.084mmo 1)of50mL CH2CI2Solution. The reaction mixture was warmed to room temperature and stirred16hAnd uselOOmLThe water is quenched. Separate the mixture and useCH₂Cl₂(3X150mL)Extract the water layer. Wash the combined organics with brine and dry (NazSOd, Filtered and concentrated, and passed silica gel column chromatography(10: 1Hexane/Ethyl acetate)Purification residue,Get cis and trans forms as white solidsα-ΒUnsaturated ester107 (15.0g,72%):

[0641] ^XH NMR (400MHz,DMSO-ch) :δ7.83 (d, J= 14.7Hz, 1H) ,7.58 (d, J = 8.3Hz, 1H) ,6.82 (d,J = 8.4Hz,lH) ,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).

[0642] Compound108Preparation

[0643]At room temperature 107 (33.0g,0.134mmo 1)and 10%Pd/C (15g,0.127)existEtOH (300mL)Put the suspension in hydrogenation conditions(latm)Down3h. The reaction mixture was filtered with diatomaceous earth and usedMeOHwashing. Concentrate the filtrate under vacuum,Obtained as a white solid108 (28.0g,90%):

[0644] ^XH NMR (400MHz,CDCI3) : 67.65 (d, J = 7.62,1H) ,6.78 (d, J = 7.96Hz , 1H) ,4.064.11 (m,lH) ,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).

[0645] Compound109Preparation

[0646] Methyl ester108 (28.0g,0.106mmol)ofTHF/MeOH/H₂O (200mL/200mL/60mL)Add to solution Na0H(25.0g,0.625mmol)And stir the reaction mixture at room temperature3h. Remove the solvent and useIN HC1The aqueous solution will pHAdjusted to1; A white solid precipitated and filtered, washed with water, and dried under vacuum to obtain the acid as a white solid 109 (25.5g,92%):

[0647] ^XH NMR (400MHz,CDC13) : 66.96 (d, J = 7.29,1H) ,6.63 (d,J = 6.86Hz, 1H) ,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).

[0648] Compound110Preparation

[0649] exist-78°CTowards60 (13.70g,77.3lmmo 1)AnhydrousTHF (200mL)Add n-butyl lithium dropwise to the solution (45.07mL,90.08mmol,In cyclohexane2ΜSolution)And stir the reaction mixturelh,Get lithium salt61The solution. exist-78°CTowards109 (15.0g,64.37mmol)In anhydrousTHF (200mL)Add dropwise to another solution inNMM (9.3OmL,83.64mmol)andPivCl (10.3OmL,83.64mmol). Stir the reaction mixture30minute,And heat up to20°CKeeplh,exist-78°CSlowly add the prepared lithium salt solution. Stir the reaction mixture again10Minutes, put0°C And stirlh,Bring to room temperature and stir30Minutes, with saturationΝΗΚΙQuench and concentrate to removeTHFAnd inCH2CI2 (300mL)with water(lOOmL)Distribution between. Separate the water layer and useCH₂C1₂ (150mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate. Column chromatography(Silica gel,OC12)Purification residue,Obtain the compound as a white solid110 (15.0g, 60%) ο

CN 105073717 Β

[0650] Compound111Preparation

[0651] exist-78°C Towards 110(15.0g,38.14mmol)In anhydrous THF (250mL)Add in batches KHMDS (13.70g,68.67mmol). Stir the resulting mixture30Minutes, add triisopropylbenzenesulfonyl azide(19.0g, 61.40mmo 1)And stir the reaction mixture5minute. Slowly add acetic acid at the same temperature(15.0mL,228mmol) And tetramethylacetic acid saddle(30.9g,76.28mmol). The reaction mixture was warmed to24°C ,Stir16h,Saturated withNaHCOs (lOOmL)Quench and concentrate to removeTHF,And useCH2CI2 (300mL)extraction. The combined organic extractsNazSODry and concentrated. Column chromatography(Silica gel,90: 10Hexane/EtOAc,Then addDCM)The residue was purified to obtain the compound as a yellow solid111 (8.80g,54%),

[0652] ^XH NMR (400MHz, CDC13) : 57.36-7.30 (m,3H) ,7.23(m,lH) ,7.20 (m,lH) ,7.16 (m, 1H) ,7.01 (d,J = 7.79Hz,lH) ,6.60 (d, J = 7.59Hz,2H) ,5.35 (t, J = 7.99,2H) ,4.89 (s,lH), 4.58-4.51 (m,lH) , 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).

[0653] Compound112Preparation

[0654] exist 0°C Towards 111 (31.0mmol g, 72. lmmol)of THF/H2O (300mL/100mL)Add to solution H2O2 (49mL,433mmol),Then add in batchesLiOH(6.04g, 144mmol). exist0°CStir the reaction mixture10Minutes and stir at room temperaturelh,Saturated withNa₂SO₃ (200mL)Quench and concentrate under reduced pressure to removeTHF,And useCH₂C1₂ (500mL) washing. useIN HC1Aqueous solution to acidify the water layer and useCH2CI2 (2X500mL)extraction. The combined organic extractsNa₂S0₄Dry, concentrate and useMTBEwashing,Obtain the compound as an off-white solid112 (15.0g,82%):

[0655] ^XH NMR (400MHz,CD3OD) :δ6.92 (d, J = 7.7Hz, 1H) ,6.63 (d, J = 8.0Hz, 1H) ,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).

[0656] Compound113Preparation

[0657]At room temperature112 (15.0g, 55. lmmol)and 10 %Pd/C (3.50g)existAcOH/H₂O (300mL/lOOmL) Put the suspension in hydrogenation conditions(latm)Down3h. The reaction mixture was filtered with diatomaceous earth and usedAcOH/H₂OWash, then useMeOHwashing. Concentrate the filtrate under vacuum,Acetate is obtained as a yellow solid113 (14.0g,83%).

[0658] Compound114Preparation

[0659] At room temperature113 (11.0g,44. lmmol)Acetic acid(120mL)Add hydrobromic acid dropwise to the solution (120mL)And reflux the reaction mixture3h. The reaction mixture was cooled to room temperature and concentrated. The crude brown residue 114(8.90g,80%)It is used directly in the next step without purification.

[0660] ^XH NMR (400MHz,CDCI3) : 66.80 (d, J = 7.85,1H) ,6.57 (d, J = 7.21Hz , 1H) ,3.923.91 (m,lH) ,3.04-2.98 (m,lH) ,2.91-2.86 (m,lH) ,2.61 (m,2H) ,2.54-2.53 (m,2H) ,1.691.68 (m,5H).

[0661] Compound115Preparation

[0662] exist0°CTo anhydrous methanol(300mL)Acetyl chloride(17.0mL,243mmol)And add114(8.90g, 28.2mmol). Reflux the reaction mixture4hAnd concentrated. Make the residue inCH2CI2 (200mL)With saturationNaHCOs (100mL) Distribution between. Separate the water layer and useCH₂Cb (200mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate to obtain the compound as a white solid115(7.30g,90%):

[0663] ^XH NMR (400MHz, CDCI3) :δ6.81 (d, J = 7.51,1H) ,6.59 (d, J = 7.21Hz , 1H) ,4.124.11 (m,lH) ,3.75 (s,lH) ,3.31-3.30 (m,2H) ,2.70-2.67 (m,2H) ,2.63 (t, J = 6.16Hz,2H).

CN 105073717 Β

[0664] Compound116Preparation

[0665] exist0°C Towards 115 (7.30g,25.60mmol)ofMe0H/H₂0 (100mL/60mL)Solution additionNaHCOs (12.0g, 145mmo 1)andBoc₂0 (10.0g,45.8mmol). The resulting mixture was warmed to room temperature and stirredlh. Make the reaction mixture in CH₂C1₂ (lOOmL)with water(50mL)Distribution between. Separate the water layer and useCH2CI2 (100mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate. use20%Ethyl acetate/Hexane is subsequently usedCH₂C1₂Flash column chromatography to obtain the compound as a white solid116 (7. lg,81%):

[0666] ^XH NMR (400MHz,CDCI3) : 66.77 (d, J = 7.36,1H) ,6.55 (d, J = 7.86Hz , 1H) ,4.964.94 ,4.71 (s,lH) ,4.96-4.94 (m, 1H) ,4.71 (s,lH) ,4.50-4.48 ,3.69(s,3H),

3.07-3.01 ,2.89-2.84 (m, 1H) ,2.86 (m,2H) ,2.63 (m,2H) , 1.80T . 78 (m,4H) ,1.39 (s,

9H).

[0667] Compound117Preparation

[0668]exist0°CTowards 116(7.0g,20.05mmol)ofCH2CI2 (80mL)Vitus(lOOmL)And triflate(4.64mL,24.0mmol), Stirringlh,And stir at room temperature2h. After concentration, the reaction mixture wasCH2CI2 (150mL)with water(70mL)Distribution between. Separate the water layer and useCH₂C1₂ (lOOmL)extraction. Wash the combined organic extracts with brine,throughNa2S04dry,And concentrate,The compound is obtained as a brown oil117 (8.00g,83 %):

[0669] ^XH NMR (400MHz,CD3OD) :δ8.81 (d, J = 4.63Hz,5H) ,8.56-8.51 (m,2H) ,8.02-7.99 (m,4H) ,7.11 (d,J = 7.98Hz,lH) , 7.03 (d, J = 7.98,1H) ,4.39-4.35 ,3.68 (s,3H),

3.19-3.14(dd,lH) ,2.90-2.77 (m,5H) ,1.86-1.81 (m,4H) ,1.35 (s,9H) , 1.32-1.28 (m,4H).

[0670] Compound118Preparation

[0671] Using nitrogen at room temperature will be in anhydrousCH3CN (lOOmL)Compounds in117 (8.0g, 16.6mmol)He Ding-3Alkynyl carbamate(10,5.00g,24.9mmol)Degas10Minutes and addTEA (9.34mL,66.50mmol),In hexane 10% (t-Bu) ₃P (7. OmL, 3.32mmol)andCui (0.16g,0.84mmol). Degas the resulting mixture with nitrogen10Minutes, add quickly at oncePd (PPh3)4 (2.00g, 1.73mmol). Degas with nitrogen5Minutes later,Reflux the resulting mixture16h. The reaction mixture was concentrated under vacuum and passed through column chromatography(Silica gel,75:25Hexane/Ethyl acetate)Purification residue,Obtain the compound as a brown solid118 (4.50g, 52%):

[0672] ^XH NMR (400MHz , CDsOD) : 57.36-7.34 (m,4H) ,7.33-7.29 (m,2H) ,7.16 (d, J = 7.63Hz, 1H) ,6.82(d,J = 7.02Hz,lH) ,5.12-5.08 (m,2H) ,4.95 (d, J = 7.88Hz, 1H) ,4.524.51 (m,lH) ,3.67 (s,3H) ,3.48-3.34 (m,2H) ,3.10-3.05 (dd, 1H) ,2.84-2.83 (m,2H) ,2.682.65 (m,4H) , 1.81T .76 (m,4H) ,1.39(s,9H).

[0673] Compound119Preparation

[0674] Methyl ester 118(4.50g,8.42mmol)of THF/MeOH/H₂O (30mL/30mL/10mL)Add to solution NaOH (3.60g,90mmol), The reaction mixture was stirred at room temperature3h. useIN HC1The aqueous solution willpHValue adjusted to9And remove the organic solvent. Remnants ofpHValue adjusted to5-6,And make the suspension inCH2CI2 (lOOmL)with water(50mL)Distribution between. Separate the water layer and useCH₂C1₂ (lOOmL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate to obtain the compound as a brown solid119(3.66g,85%),

[0675] ^XH NMR (400MHz,CD3OD) :67.28-7.24 (m,5H) ,7.05-7.03 (d,J = 7.67Hz, 1H) ,6.896.87 (d,J = 7.55Hz,lH) ,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,lH) ,3.67 (s,3H) ,3.48-3.34 (m,2H) ,4.27-4.26 (m,lH) ,3.38-3.30 (m, 2H) ,3.15-3.10(m,lH) ,2.78-2.71 (m,4H) ,2.59 (d, J = 5.95,2H) ,1.73-1.71 (m,4H) ,1.31

CN 105073717 Β

(s,9H).

[0676] Compound120Preparation

[0677] Towards THF (30mL)Compounds in 119 (800mg , 1 . 53mmo 1)Add in order DEPBT (845mg, 2.56mmol),24 (700mg,2.33mmol)andDIPEA (1.OmL,4.65mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (50mL)Medium, with saturated aqueous solution(50mL)And salt water(50mL)Fast washing,And byNazSORidiculous. Evaporate solvent,And pass silica gel flash chromatography(6 %Methanol/OC12)Purified crude products,Obtain the amide as a yellow solid120 (l.Og):

[0678] ^XH NMR (400MHz,CDCls)7.46-7.44 (m,3H) ,7.36-7.30 (m,7H) ,7.17 (d,J = 7.2Hz, 2H) ,7.07 (d,J = 7.5Hz,lH) ,6.99-6.92 ,5.49(s,lH) ,5.10(s,2H) ,4.35-4.31 (m,

1H) ,4.05-3.90 (m,2H) ,3.80-3.82 ,3.75-3.72 ,3.62 (t, J = 9.9Hz, 1H) ,3.43 (t,J = 5.7Hz,2H) ,3.18-3.16(m,lH) ,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).

[0679] Compound121Preparation

[0680] At room temperature 120 (1.00g,1.01mmo 1)and 10%Pd/C (600mg)existEtOH (50mL)andAcOH (2mL) Degas the suspension in, and place it under hydrogenation conditions(latm)Down12h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. The filtrate was concentrated under vacuum to obtain the amine salt as a white solid121 (700mg,80%)

[0681] ^XH NMR (400MHz,CDCls) :57.49-7.41 (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) , 1.29T . 20 (m,7H) ,0.88-0.84 (m,3H) ,0.87(t,J = 6.4Hz,3H).

[0682] Compound122Preparation

[0683] To amine salt at room temperature121 (700mg,0.8lmmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,680mg,1.75mmol)existEtOH (20mL)Add to the solution inDIPEA (1.60mL,9.26mmol). Place the reaction mixture in a sealed tube70°CUnder heating2h,Cool to room temperature and concentrate under vacuum. Column chromatography(Silica gel,80:18:2 CHCI3/CH3OH/NH4OH)Purify the residue to obtain the muscle as a yellow solid122 (380g,48%):

[0684] ^XH NMR (400MHz, DMSO-cU) : 67.43-7.39 (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,lH) ,4.33-4.31 (m,lH), 4.14-4.10(m,lH) ,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 = 7.0Hz,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-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(Compound123)Preparation of the hydrochloride;

[0686] Put the dioxane in4N HC1 (15mL)add toEtOH (5.OmL)middle 122 (350g,0.35mmol)In, the reaction mixture was stirred at room temperature2h. Remove solvent,Reversed phase chromatography(Goldcolumn)The mixture was purified and the residue was lyophilized to obtain11 Omg (45 %)Yellow solid compound123 :

[0687] ^XH NMR (400MHz, DMSO-ch) :δ10.16 (s,lH) , 9.16 (brs, 1Η) , 8.51-8.34 (brs, 2Η), 7.41 (t,J = 8.1Hz,4H) ,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,lH) ,4.01 (m,lH) ,3.93 (m,lH) ,3.60 (m,lH) ,3.50-3.38

CN 105073717 Β

(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, D2O) : 67.08 (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, IH) ,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-(-2-Amino-3- (4- (3-(pair((2S, 3R, 4R, 5R) -2,3,4,

5,6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(127)Preparation

[0690]Program 18

[0691] ό

sugarj

<img file="CN105073717B_D0138.tif" />

119

HO

BocHN

<img file="CN105073717B_D0139.tif" />

DEPBT, DIPEA, THF

NHCbz

124

NHCbz

OH OH

<img file="CN105073717B_D0140.tif" />

sugar='ΕΤΈ

0^-0 δΗ Ύ song 'N

<img file="CN105073717B_D0141.tif" />

Pd/C. Hr

EtOH/AcOH

<img file="CN105073717B_D0142.tif" />

125

SCH[

N _ locHN

ΝΗη·ΑοΟΗ sugar·

DIPEA, EtOH o

ΝΉ·ΗΙ.

Α

Ν

Ν 'ΝΉ,

<img file="CN105073717B_D0143.tif" />

126

OH sugar

HO ,N

Η0_λ, ®

<img file="CN105073717B_D0144.tif" />

Ν of

ΗΟ,' of

<img file="CN105073717B_D0145.tif" />

answer)OH ,_λ0Η (S)Όη

<img file="CN105073717B_D0146.tif" />

HO

NH O

Λ Λ

Ν Ν

Η ΗHow many people Η₂Ν Ν ΝΗη

4Ν HC1 Aqueous solution,ΕΙΟΗ

<img file="CN105073717B_D0147.tif" />

NH₂

127

Ν N

Η H

[0692] Compound124Preparation

[0693] Xiang Zai THF (30mL)Compounds in 119 (1.0g, 1.92mmol)Add in order DEPBT (845mg, 2.82mmol) ,29 (1.25g,1.9lmmol)andDIPEA (1. OmL,5.73mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (50mL)Medium, with saturated aqueous solution(50mL)And salt water(50mL)Fast washing,And byNazSORidiculous. Evaporate solvent,And pass silica gel flash chromatography(5 %Methanol/OC12)Purified crude products,Obtain the amide as a yellow solid124[900mg (mixture)].

CN 105073717 Β

[0694] Compound125Preparation

[0695] At room temperature 124[900mg (mixture),0.77mmo 1 ]and 10 % Pd/C (600mg)existEtOH (50mL)and AcOH (1.5mL)The suspension in the mixture is degassed and placed under hydrogenation conditions(latm)Down12h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. The filtrate was concentrated under vacuum to obtain the crude product as a colorless oil 125(800mg).

[0696] Compound126Preparation

[0697] At room temperature, to crude products125 (800mg)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,400mg, 1.02mmol)ofEtOH (40mL)Add to solutionDIPEA (1.10mL, 6.38mmo 1). Place the reaction mixture in a sealed tube70°CUnder heating2After hours, cool to room temperature and concentrate under vacuum. Column chromatography(Silica gel,80:18 :2 CHCI3/CH3OH/NH4OH)Purify the residue to obtain the muscle as a yellow solid126 (285mg,through3Step yield 12%):

[0698] ^XH NMR (400MHz, DMSO-cU) : 67.44-7.42 (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,lH) ,4.23-4.19 (m,2H) ,3.97-3.91 (m,4H) ,3.84-3.82 (m,2H) ,3.71 (d,J = 2.29Hz,lH) ,3.69(d,J = 2.2Hz,lH) , 3.58 (t, J= 10.08Hz , 2H) ,3.06-3.00 (m,lH) ,2.912.86 (m,lH) ,2.76 (m,2H) ,2.71-2.68 (m,4H) ,2.61-2.55 (m,4H) ,2.44-2.35 (m,4H) ,1.741.60(m,10H) ,1.38 (s,9H).

[0699] 3,5-Diamino-N- (N~ (4~ (4- ((S) -2-Amino-3- (4- (3-(pair((2S, 3R, 4R, 5R) -2,3,4,5, 6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(Compound127)Preparation of the hydrochloride

[0700] Put the dioxane in4N HC1 (10mL)add toEtOH (3.OmL)middle 126 (1.15g,0.23mmol)And stir the reaction mixture at room temperature2h. Remove solvent,Reversed phase chromatography(Goldcolumn)The mixture was purified and the residue was lyophilized to obtain62mg (32%)compound of127.

[0701] ^XH NMR (400MHz,DMSO-de) :δ 10.39 (brs,lH) , 10.03 (brs, ΙΗ) ,8.91-8.82 (brs,2Η), 8.48 (brs,2H) ,7.42 (d, J = 7.6Hz,4H) ,7.18 (d, J = 7.6Hz,2H) ,6.96 (d, J = 7.1, ΙΗ) ,6.89 (d,J = 7.4,lH) ,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,lH) ,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) , 1.70T . 67 (m,4H) ,1.61T.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, D2O) :δ7.10 (d, J = 8.30Hz , 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 = 8.2Hz,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] 18.3,5-Diamino-N- (4~ (4- ((S) -2-Amino-3-Oxo-3- (4- (3- ((2S, 3R,4R, 5R) -2,3,

4,5,6-Pentaylhexylamino)Propyl)Phenylamino)Propyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butylcarbamoyl)-6-Clopiazine-2-Formamide(131)Preparation

[0704]Program 19

CN 105073717 Β

[0705]

<img file="CN105073717B_D0148.tif" />

<img file="CN105073717B_D0149.tif" />

[0706] Compound128Preparation

[0707] Xiang Zai THF (30mL)Compounds in 119 (1.00g, 1.92mmol)Add in order DEPBT (862mg, 2.88mmol),34 (1.50g, 2.98mmol)andDIPEA (1. OmL, 5.76mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (50mL)Medium, with saturated aqueous solution(30mL)And salt water(20mL)Fast washing,And byNa₂S0₄dry. Evaporate the solvent and pass through silica gel flash chromatography(6 %Methanol/OfeCl2)Purify the crude product to obtain the amide as a yellow solid128 (780mg,42%):

[0708] ^XH NMR (400MHz,CDCls) 27.49 (m,3H) ,7.31-7.29 (m, 10H) ,7.00-7.08 (m,3H) ,6.94 (d,J = 7.4Hz,lH) ,5.54(m,lH) ,5.50-5.49 ,5.08 (s,2H) ,4.36(m,lH) ,4.26-4.22 (m,

2H) ,4.05 (m,2H) ,3.95-3.91 (m,lH) ,3.80 (m,2H) ,3.64-3.59 (m, 1H) ,3.52-3.48 (m, 1H), 3.14-3.06 ,2.94-2.89 ,2.79 (d, J= 16.12Hz ,4H) ,2.63 (t, J = 5.98Hz , 1H),

2.51 (t,J = 6.9Hz,lH) , 1.82-1.75 (m,7H) ,1.41 (s,18H).

[0709] Compound129Preparation

[0710] At room temperature 128 (780mg,0.776mmol)and 10%Pd/C (300mg)existEtOH (30mL)andAcOH (l.OmL)The suspension in the mixture is degassed and placed under hydrogenation conditions(latm)Down12h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. The filtrate was concentrated under vacuum to obtain the amine salt as a white solid129 (720mg,85%)

[0711] ^XH NMR (400MHz,CDCls) :57.49-7.46 (m,2H) ,7.32-7.30 (m,5H) ,7.08-7.06 (d,J =

CN 105073717 Β

7.2Hz, 1Η) ,6.88(d,J = 7.4Hz,lH) ,5.53 (s,lH) ,4.34-4.33 ,4.25-4.21 (m,lH),

4.03-4.02 ,3.96-3.89 ,3.78-3.76 ,3.71-3.69 ,3.60 (t,J =

9.9Hz,lH) ,3.48-3.46 ,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 = 6.5Hz,2H) ,2.51 (t, J = 6.8Hz , 2H) ,1.84-1.77 (m,6H) , 1.67-1.66 (m, 2H) ,1.61-1.57 (m,2H) ,1.41 (s,18H).

[0712] Compound130Preparation

[0713] To amine salt at room temperature129 (720mg,0.77mmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,456mg, 1.17mmo 1)existEtOH (20mL)Add to the solution inDIPEA (1.12mL, 6.24mmo 1). Place the reaction mixture in a sealed tube70°CUnder heating2h,Cool to room temperature,And concentrated under vacuum. Get the muscle as a yellow solid130 (380mg,45%):

[0714] ^XH NMR (400MHz,CDCI3)7.48-7.46 (m,2H) ,7.30 (t, J = 2.70Hz,5H) ,7.08-7.06 (d,J = 7.6Hz,2H) ,6.93(d,J = 7.1Hz,lH) , 6.88 (d, J = 7.3Hz , 1H) ,5.53 (s,lH) ,4.34 (m, 1H) ,4.25-4.21 (m,lH) ,4.04 (m,lH) ,3.96-3.90 ,3.79(m,2H) ,3.60 (t,J = 10.0Hz,

1H) ,3.50-3.46 ,3.25 (t, J = 5.9Hz,3H) ,3.07-3.02 (m, 1H) ,2.92-2.87 ,2.77 (m,2H) ,2.69-2.67 (m,2H) ,2.58 (t,J = 6.OHz,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-(-2-Amino-3-Oxo-3- (4- (3- ((2S, 3R,4R, 5R) -2,3,4,5,

6-Pentaylhexylamino)Propyl)Phenylamino)Propyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butylcarbamoyl)-6Clopiazine-2-Formamide(131)Preparation of the hydrochloride;

[0716] Put the dioxane in4N HC1 (25mL)add toEtOH (5.OmL)middle 130 (350mg,0.35mmol)And stir the reaction mixture at room temperature2h. Remove solvent,Reversed phase chromatography(Goldcolumn)Purify the mixture and lyophilize the residue,Obtain the compound as a yellow solid131 (125mg,48%):

[0717] ^XH NMR (400MHz,CDsOD) *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=l .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,D₂0) :610.46 (s, 1H) ,9.31 (br,lH) ,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 , 1H) , 5.43 (d, J = 3.8Hz,lH) ,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 ,3.71 (m,lH) ,3.64-3.61 (m,lH) ,3.51-3.45 (m,3H) ,2.962.92 (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 .

[0719] 19. (S) -3,5-Diamino-Ν-(N-(4-(4-(2-Amino-3-(4-(3-(Dimethylamino)Propyl)Phenylamino)-3-Oxopropyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(135)Preparation

[0720]Program 20

CN 105073717 Β

[0721]

<img file="CN105073717B_D0150.tif" />

Ο ΝΗ·ΗΙ ^C1'Y^N*i^x^N^sch₄ DIPEA. EtOH Di RenNNH2 "

<img file="CN105073717B_D0151.tif" />

<img file="CN105073717B_D0152.tif" />

[0722] Compound132Preparation

[0723] Xiang ZaiTHF (30mL)Compounds in 119 (700mg, 1.34mmol)Add in orderDEPBT (600mg,

2.OOmmo 1),18 (360mg, 1.5lmmol)andDIPEA (0.8OmL,4.03mmol)And stir at room temperature 16h. After removing the solvent under reduced pressure, the residue was dissolved inCH2CI2 (50mL)Medium, with saturated aqueous solution(50mL)And salt water(50mL)Fast washing,And byNa₂S0₄dry. Evaporate the solvent and pass through flash chromatography on silica gel(6 %Methanol/OfeCl2)Purify the crude product to obtain the amide as a yellow solid product132 [800mg (mixture)].

[0724] ^XH NMR (400MHz,DMSO-de) :δ8.13 (d,J = 7.54Hz, 1H) ,8.03 (d,J = 7.7Hz, 1H) ,7.89

7.85 (m,lH) ,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).

[0725] Compound133Preparation

[0726] At room temperature 132 [800mg (mixture),1 .Olmmol]and 10%Pd/C (350mg)existEtOH (30mL)and AcOH (ImL)The suspension in the mixture is degassed and placed under hydrogenation conditions(latm)Down12h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. Concentrate the filtrate under vacuum and pass through column chromatography(Silica gel,80:18:2 CHCI3/CH3OH/NH4OH)purification,Obtain the compound as a yellow solid233 (500mg,through2Step yield67%):

[0727] ^XH NMR (400MHz,DMSO-ch) :δ7.31 (d, J = 7.54Hz,2H) ,7.12 (d,J = 7.1Ηζ,2Η) ,6.93

100

CN 105073717 Β

(d,J = 7.2Hz,lH) ,6.88(d,J = 6.8Hz,lH) ,4.32(m,lH) ,3.08-3.03 ,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).

[0728] Compound134Preparation

[0729] To amine salt at room temperature133 (500mg,0.90mmo 1)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,530mg,1.36mmol)existEtOH (20mL)Add to the solution inDIPEA (1.30mL,7.25mmol). Place the reaction mixture in a sealed tube70°CUnder heating2h,Cool to room temperature,And concentrated under vacuum. The residue was purified by column chromatography to obtain muscle as a yellow solid134 (285mg,42%):

[0730] ^XH NMR (400MHz,DMSO-ch) :67.29 (d, J = 7.5Hz,2H) ,7.10 (d, J = 8.1Ηζ,2Η) ,6.946.87 (m,2H) ,4.33(m,lH) ,3.27-3.24 (m,2H) ,3.07-3.00 (m, 1H) ,2.92-2.87 ,2.76 (m,

2H) ,2.70 (m,2H) ,2.61-2.54 (m,4H) ,2.35-2.31 (m,2H) ,2.22 (s,6H) , 1.80T . 72 (m,5H), 1.69-1.62 (m,4H) ,1.39 (s,9H).

[0731] (S) -3,5-Diamino-Ν- (Ν- (4- (4- (2-Amino-3- (4- (3-(Dimethylamino)Propyl)Phenylamino)3-Oxopropyl)-5,6,7,8-Si Urg Ke Cai-1-base)Butyl)Caramel)-6-Clopiazine-2-Formamide(Compound135)Preparation of the hydrochloride;

[0732] Put the dioxane in4Ν HC1 (10mL)add toEtOH (5.OmL)middle 134 (380g,0.35mmol)And stir the reaction mixture at room temperature2h. Remove solvent,Reversed phase chromatography(C18 Goldcolumn)Purify the mixture and lyophilize the residue,Obtain the compound as a yellow solid135 (125mg,49%):

[0733] ^XH NMR (400MHz,DMSO-de) :510.69 (brs,lH) ,10.54-10.50 (d,J=16.7Hz,2H) ,9.32 (t,J = 4.8Hz,lH) ,8.96 (brs,lH) ,8.86 (brs,lH) ,8.58 (brs,3H) ,7.42 (d, J = 8.OHz ,4H), 7.18(d,J = 8.2Hz,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,lH) ,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) ,1.62T.58(m, 2H) ,1.54-1.52 (m,2H).

[0734] ^XH NMR (400MHz, D2O) /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,lH) , 3.25 (t, J = 5.2Hz , 3H) , 3.21-3.17 (m, 1H) , 3.10 (t, J = 9.8Hz,lH) ,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-Chlorpyrazine-2-Base)Guaqui)Butyl)-5,6,

7,8-Si Urg Ke Cai-1-base)Propionic acid(139)Preparation

[0736]Program 21

101

CN 105073717 Β

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ΝΠ_;··\ο&lt;)11 In dioxane4 N 1101

HO

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C1 ^:NH Q

Npeople

Η II :Ν :NIB

[0738] Compound136Preparation

[0739] At room temperature 118 (800mg, 1.49mmol)and 10%Pd/C (350mg)existEtOH (50mL)andAcOH (l.OmL)The suspension in the mixture is degassed and placed under hydrogenation conditions(latm)Down12h. The reaction mixture was filtered through a plug of diatomaceous earth and the plug was usedMeOHwashing. Concentrate the filtrate under vacuum and pass through column chromatography(Silica gel,80:18:2 CHCI3/CH3OH/NH4OH)purification,Obtain the compound as a yellow solid136 (700mg,93%):

[0740] Compound137Preparation

[0741] To amine salt at room temperature136 (700mg, 1.50mmol)and3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,880mg,2.26mmol)existEtOH (30mL)Add to the solution inDIPEA (2.15mL, 12.03mmol). Place the reaction mixture in a sealed tube70°CUnder heating2h,Cool to room temperature,And concentrated under vacuum. Column chromatography(Silica gel,80: 18:2 CHCI3/CH3OH/NH4OH)Purify the residue to obtain the muscle as a yellow solid137 (560mg,60%):

[0742] ^XH NMR (400MHz,CDsOD) ;66.95-6.85 (m,2H) ,4.32-4.28 (m, 1H) ,3.72-3.67 (m,2H), 3.34 (m,3H) ,3.22-3.16 (m,2H) ,3.08-3.03 ,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] Compound138Preparation

[0744] Methyl ester 137 (560mg,0.907mmol)ofTHF/MeOH/H₂OSolution(30mL/30mL/10mL)Add in NaOH (3.60g, 7.25mmol),And stir the reaction mixture at room temperature3h. useIN HC1The aqueous solution willpHValue adjusted to 9,And remove the organic solvent. Remnants ofpHValue adjusted to5-6,And make the suspension inCH2CI2 (lOOmL)with water(50mL)Of

102

CN 105073717 Β

Distribution between. Separate the water layer and useCH2CI2 (lOOmL)extraction. The combined organic extractsNazSODry and concentrate to obtain the compound as a brown solid138 (420mg,78%):

[0745] ^XH NMR (400MHz,DMSO-de) ;66.93 (d, J = 6.7Ηζ, 1Η) ,6.84 (d, J = 7.35Hz, 1H) ,6.70 (s,3H) ,3.93 (m,lH) ,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).

[0746] (S) -2-Amino-3- (4- (4- (3- (3,5-Diamino-6-Clopiazine-2-BaseΜGuaqui)Butyl)-5,6,7,8Si Urg Ke Cai-1-base)Propionic(Compound139)ofHC1Salt preparation;

[0747] Put the dioxane in4Ν HC1 (10mL)add toEtOH(5.OmL)middle 138 (420mg,0.69mmol)And stir the reaction mixture at room temperature2h. Remove solvent,Reversed phase chromatography(C18 Goldcolumn)Purify the mixture and lyophilize the residue,Obtain the compound as a yellow solid139 (200mg,49%):

[0748] ^XH NMR (400MHz,DMSO-de) ; 610.56 (brs, 1H) ,9.36 (t, J = 4.7Hz, 1H) ,8.9-8.8 (brs, 2H) ,6.98-6.93 (m,2H) ,3.95-3.92 (m,2H) ,3.38-3.35 (m,2H) ,3.04 (d,J = 7.0Hz,2H) ,2.672.66 (m,4H) ,2.56-2.55 (m,2H) , 1.72-1.70 (m,4H) , 1.63-1.56 (m,4H).

[0749] ^XH NMR (400MHz,D2O) ; 57.43 (brs,2H) ,6.94 (d, J = 7.2Hz, 1H) ,6.87 (d, J = 7.1Hz, 1H) ,3.41 (t,J = 6.0Hz,2H) ,3.28-3.26 (m,4H) , 3.11 (d, 1H) , 3.08 (m,lH) ,2.66-2.64 (m, 6H) ,1.67-1.57 (m,8H).

[0750] 21.3,5-Diamino-N- (N~ (4- (4-(-2-Amino-3- (4- (3-(pair((2S, 3R,4R, 5R) -2,3,4, 5,6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-1-base)Butyl)Caramel)-6-Clopiazine2-Formamide(33)Chiral synthesis

[0751]Program 22

103

CN 105073717 Β

[0752] on

OH

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('IH Bu Huan Substituted Succinimide (142)

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ο

NHBoc

143 uo

BocHN

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'Ν ' sugar,Ν

Zn. T₂, DMF

Pd₂ (dbah. Sphos

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NHBoc

Pd(PPh-)4- Cui

O-Bu)₃P. Et₃N

ClhCN

NHCbz

IM &lt;·.

Tf_?O.Pi noise \ Cli,ci_?

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BocHN

IK)₂C

BocHN

NaOH

MeOH/THF/H₂0

<img file="CN105073717B_D0161.tif" />

T_SP. ΝΜΜ. THF

NHCbz

NHCbz

<img file="CN105073717B_D0162.tif" />

<img file="CN105073717B_D0163.tif" />

Pd/C, Ho (1 aim)

EtOH. AcOH

N

II

BocHN

NHCbz sugar,Ν

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ΝΗ

ΝpeopleSCHbow Η '

Nhh

<img file="CN105073717B_D0165.tif" />

DiPl Λ. 1 {Oil

N

II

BocHN sugar,Ν

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11()

HO"ruler

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◎ 1

Ci

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h₂n ν

<img file="CN105073717B_D0169.tif" />

ΝΗ Ο νpeople%ν ^{11 Η} Λ Α

Η_?Ν X Nil· \

H BocHN

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W,OH OH

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HO

4N HC1 Aqueous solution,EtOH

[0753]

[0754] Compound141Is prepared in30Within minutes1-Caifen(140_?10.0g_?69.4mmo 1)Otosan(70.OmL)Add a few parts to the solutionNBS(142_?12.3g_?69.4mmol). Stir the resulting mixture at room temperature4h,Concentrate under vacuum, then add water(200mL)And ethyl acetate(200mL). Separate the aqueous layer and use ethyl acetate(2X200mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate. Column chromatography(Silica gel,4:1Hexane/EtOAc)The residue is purified to obtain the desired compound as a white solid141 (9.50g,61%):

[0755] ^XH NMR (400MHz,DMSO-ch) 610.49 (s,lH) ,8.20 (dd, J = 8.3,0.5Hz, 1H) ,8.02 (d,J =

104

CN 105073717 Β

8.3Ηζ,1Η) ,7.66 (dd,J = 8.4,1.4Ηζ,1Η) ,7.64 (d,J = 8. ΙΗζ,ΙΗ) ,7.55 (ddd, J = 8.2,7.7, Ι.ΙΗζ,ΙΗ) ,6.83(d,J = 8.2Hz,lH).

[0756] Compound7Preparation

[0757] Zinc powder(7.03g,107.6mmol)Add to a side-arm round bottom flask purged with flame-dried nitrogen. Add anhydrous via syringeDMF (50. OmL),Then add a catalytic amount of iodine(1.00g, 3.94mmol). It is observed that the resulting mixture undergoes a change from colorless to yellow and back to colorless. One-time addition of protected iodoalanine143 (11.8g, 35.9mmol), And then add a catalytic amount of iodine(1.00g, 3.94mmol),And stir at room temperature30Minutes; successful insertion of zinc is accompanied by a mild exotherm. Allow the organozinc reagent to cool to room temperature, then addPd₂dba₃ (821mg, 0.89mmol),SPhos (736mg, 1.79mmo 1)Aryl bromide141 (8.OOg,35.9mmol), And under the positive pressure of nitrogen50°CKeep heating16h. The reaction mixture was cooled to room temperature. Add saturationNHKlSolution(300mL) andEtOAc (300mL), Then filter the mixture with diatomaceous earth and useEtOAcwashing(100mL). Separate the water layer and use EtOAc (2X300mL)extraction. The combined organic extracts were washed with brine, andNazSOqDry and concentrate under vacuum. Column chromatography(Silica gel,4:1Hexane/EtOAc)Purify the crude product to obtain the desired compound as a yellow solid7(4.60g, 37%):

[0758] ^XH NMR(400MHz,CDC13,Mixture of rotamers)68.23 (d, J = 8.3Hz, 1H) ,7.99 (d,J = 8.6Ηζ,1Η) ,7.54 (t, J = 8.04Hz, 1H) ,7.48 (ddd, J = 8.3,6.9,1.3Hz, 1H) ,7.08 (d,J = 7.8Hz, 1H) ,6.70 (d,J = 7.6Hz,lH) ,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= 14.0,5.9Hz,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).

[0759] Compound9Preparation

[0760] exist0°CTo the compound7 (7.60g,21.8mmol)ofCH2CI2 (150mL)Vitus(18.OmL)and Tf₂O (9.19g,32.6mmol). Stir the resulting mixture at room temperature2h,Concentrate under vacuum and inCH2CI2 (100mL) with water(50mL)Distribution between. Separate the water layer and useCH₂C1₂ (2X50mL)extraction. Wash the combined organic extracts with brine,throughNazSOqDry and concentrate,The compound is obtained as a brown oil9 (11.0g,Crude products). The crude product is used directly in the next step without further purification.

[0761] ^XH NMR (400MHz ,CDCls,Mixture of rotamers)δ8.19-8.07 (m,2Η) ,7.69-7.64 (m, 2H) ,7.38 (d,J = 8.1Hz,lH) , 7.28 (d, J = 7.9Hz, 1H) , 5.12-5.06 (br s,lH) ,4.78-4.67 (m, 1H) ,3.68-3.46 (m,5H) ,1.39 (s,8H) ,1.25(s,lH).

[0762] Compound11Preparation

[0763] Put the compound at room temperature9(11.0g,21.8mmo 1)He Ding-3-Alkynyl carbamate10(6.56g, 32.6mmol)In Anhydrous Yiyueqing(100mL)Degassing of the solution in a nitrogen atmosphere10Minutes, then addTEA(11.9mL, 87.Ommol), In hexane 10% (t-Bu) ₃P (8.80mL,4.35mmol)andCui (207mg, 1.08mmol). Degas the resulting mixture again with nitrogen10minute,And add it all at oncePd (PPh3)4 (2.51g, 2.17mmol). Degas with nitrogen5After minutes, the resulting mixture was refluxed16h. The reaction mixture was concentrated in vacuo and used column chromatography(Silica gel,2:3Hexane/EtOAc) The residue was purified to obtain the compound as a brown oil11 (7.00g,Two-step yield61%).

[0764] ^XH NMR (400MHz,CDCI3,Mixture of rotamers)68.33 (dd, J = 8.9,1.9Hz, 1H) ,8.07 (dd,J = 9.0,1.7Hz,lH) ,7.59-7.49 (m,3H) ,7.39-7.27 (m,5H) , 7.19 (d, J = 7.3Hz , 1H), 5.24-5.16 ,5.12(s,2H) ,5.08-4.99 , 4.69 (q, J = 6.7Hz , 1H) ,3.59(s,3H),

105

CN 105073717 Β

3.57-3.40 (m,4H) ,2.79 (t, J = 6.4Hz,2H) ,1.39 (s,7.5H) ,1.11 (s,1.5H).

[0765] Compound17Preparation

[0766] Methyl esterii (7.00g,13.2mmo 1)existTHF (200mL), Methanol(200mL)And water(75.OmL)Add solids to the solutionNa0H(16.0g,79.2mmol). Stir the resulting mixture at room temperaturelh,untilTLCIt shows that the reaction is complete. Add to1NHydrochloric acid to reduce the reaction mixturepHValue to10. After concentration, add water(lOOmL)And willpHAdjusted to5-6. Use the resulting precipitateCH2CI2 (2X250mL)extraction. Combine the organic layers, afterNa₂S0₄Dry, filter, concentrate and useMTBEGrind,Obtain the compound as a white solid17 (5.00g, 75%):

[0767] ^XH NMR (400MHz,CDsOD; Mixture of rotamers)68.33 (d, J = 8.2Hz, IH) ,8.28-8.20 (m,lH) ,7.59-7.45 (m,3H) ,7.38-7.21 (m,6H) ,5.09 (s,2H) ,4.55-4.45 (m,lH) ,3.76-3.66 (m,lH) ,3.44 (t, J = 6.7Hz , 2H) ,3.28-3.20 , 2.76 (t, J = 6.7Hz , 2H) ,1.29(s,6H),

0.82 (s,3H).

[0768] Compound30Preparation

[0769] To the compound17 (4.60g,8.91mmol)ofTHF(160mL)Add to the solution one by oneT3P (In ethyl acetate 50% ,10.7mL)and NMM (4.89mL, 44.5mmol). Stir at room temperature 10 Minutes later, add amine 29 (6. llg, 9.33mmol)And stir the reaction mixture at room temperature16h. After removing the solvent, dissolve the residue inCH₂C1₂ (100mL) Medium, use saturationNH₄CL·saturationNaHCOsAnd salt water fast washing, afterNa₂S0₄Dry and concentrate. Will pass column chromatography(Silica gel,9 :1Dichloromethane/Methanol)Purification residue,Obtain the amide as an off-white solid30 (6.60g,64%)

[0770] ^XH NMR (400MHz, CDCI3) 68.33 (dd,J = 9.0,1.7Ηζ,1Η) , 8.17 (d, J = 7.3Hz , IH), 7.62-7.47 (m,4H) , 7.42 (dd, J = 7.7,4.1Hz ,4H) ,7.37-7.28 ,7.09-6.95 (m,4H),

5.46 (s,2H) ,5.33 (br s,lH) ,5.22 (t, J = 5.8Hz, IH) ,5.11 (s,2H) ,4.63-4.51 (m,lH) ,4.27 (dd,J=10.8,5.4Hz,2H) ,4.02-3.84 (m,6H) ,3.71 (t, J = 4.5Hz , 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).

[0771] Compound31Preparation

[0772] At room temperature by bubbling with nitrogen gas using a syringe30 (7.26g,6.20mmo 1)and10%Pd/C (1.50g) existEtOH/AcOH (240mL/40.0mL)Degas the suspension by bubbling10Minutes, then put under hydrogenation condition(latm) Down16h. The reaction mixture was filtered with diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in a vacuum and useMTBEGrind to obtain the amine salt as a brown solid31 (7.06g,98%):

[0773] ^XH NMR (400MHz,CD3OD,Mixture of rotamers)68.24 (dd, J = 7.2,2.OHz, 1H) ,8.09 (d,J = 7.0Hz,lH) ,7.59-7.22 (m,2H) ,7.49-7.41 (m,4H) ,7.39-7.22 (m,10H) ,6.95 (d,J = 8.5Hz,2H) ,5.51 (s,2H) ,4.55 (t, J = 7.2Hz, IH) ,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,Ηζ,2H) ,3.59-3.52 (m,lH) ,3.45-3.36 (m,lH) ,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).

[0774] Compound32Preparation

[0775] At room temperature 31 (7.06g,6.18mmol)of EtOH (50. OmL)Add to solution DIPEA (8.80mL, 49.4mmol), Then add3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,3.84g, 9 · 88mmol). Put the reaction mixture in70°Cheating2h,Cool to room temperature,And concentrated under vacuum. Column chromatography(Silica gel, 80 : 18 : 2CHCI3/CH3OH/NH4OH)Purify the residue twice,Obtain the compound as a yellow solid32 (2.50g,

106

CN 105073717 Β

33%):

[0776] ^XH NMR(400MHz,CDsOD,Mixture of rotamers)68.22 (d, J = 9.3Hz, 1H) ,8.08 (d,J = 7.8Ηζ,1Η) ,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 = 8.0Hz,2H) ,5.47 (s,2H) ,4.53 (t, J = 7.7Hz , 1H) ,4.22 (dd, J= 10.8,5.4Hz , 2H) ,3.993.89 (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,lH) ,3.47-3.38 (m,lH) ,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= 13.6,8.9,2H) ,2.57-2.47 (m,2H) ,2.462.34 (m,2H) , 1.84T . 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-(pair((2S, 3R,4R, 5R) -2,3,4,5, 6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)CaiT-base)Butyl)Caramel)-6-Chlorpyrazine-2Formamide(33)ofHC1Salt preparation

[0778] Towards32 (2.50g,2.02mmo 1)existEtOH (30.OmL)Add to the solution in4Nhydrochloric acid(80.OmL). Stir the resulting mixture at room temperature2h. Remove solvent,Purify by reverse phase column and lyophilize to obtain the compound as a yellow hygroscopic solid33 (1.82g,85%):

[0779] ^XH NMR (400MHz,DMSO-de) 510.61 (s,lH) ,10.59 (s,lH) ,9.41 (t,J = 5.2Hz,H) ,9.01 (br s,lH) ,8.96 (br s, 1H) ,8.81 (br s,2H) ,8.77 (br s,2H) ,8.44-8.37 ,8.16-8.10 (m,lH) ,7.61-7.52 (m,2H) ,7.41 (d, J = 8.6Hz , 2H) , 7.35 (d, J = 7.5Hz , 1H) , 7.27 (d,J = 7.3Hz,lH) ,7.17 (d,J = 8.5Hz,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,CD3OD) :69.25 (t, J = 5.9Hz,0.5H) ,8.26-8.21 (m,lH) ,8.17-8.12 (m,lH) ,7.60-7.54m, 2H) , 7.38 (d, J = 7.2Hz , 1H) , 7.32 (d, J = 7.2Hz , 1H) , 7.25 (d, J = 8.6Hz ,2H) ,7.15 (d,J = 8.6Hz,2H) ,4.31 (t, J = 8.1Hz, 1H) ,4.21-4.14 (m, 1H) ,4.13-4.08 (m,lH) ,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) ,2.15T .98 (m,2H) ,1.911.73 (m,4H).

[0781] 22. (2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-Aminophenyl)Propylazanediyl)Dihexane-1,2,3-,4,5-Penta Alcohol(29)Preparation

[0782]Program 23

107

CN 105073717 Β

[0783]

144 (Boc)iO. TRA

145

82%

ΟΑ

<img file="CN105073717B_D0172.tif" />

PdCl.iPPh j;

N NaOHAqueous solution

60%

<img file="CN105073717B_D0173.tif" />

Ph In dioxane4Ν ilCI

93%

<img file="CN105073717B_D0174.tif" />

1. NaCABH^AcOll. MeO!!

NHexanal

<img file="CN105073717B_D0175.tif" />

14S / ΝΙΙ_?·ΙΚΊ Compound145Is prepared in0 °C To the compound 144 (8.80g, 154. lmmo 1)ofCH2CI2 (150mL)Add to solutionTEA (32.2mL,

[0784]

[0785]

231.2mmol)andB0C2O (40.4g, 185.3mmol). exist0°CContinue to stir the reaction mixture0.5h,Allow it to warm to room temperature and stir5h. Then make the mixture inCH2CI2 (150mL)with water(150mL)Distribution between. Separate the water layer and use CH₂C1₂ (2X150mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate to obtain the desired compound as a colorless oil145 (22.0g, 91 %).

[0786] ^XH NMR (400MHz,CDCI3) :65.90-5.77 (m, 1H) ,5.17 (dq, J= 17.1,1.7Hz, 1H) ,5.10 (dq,J = 10.4,1.4Hz,lH) ,4.64 (brs, 1H) ,3.74 (t,J = 5.2Hz,2H) ,1.45(s,9H).

[0787] Compound147Preparation

[0788] To the compound under nitrogen145 (14.Ommol g,89.12mmo 1)AnhydrousTHF (150mL)Add to solution9BBN (existTHFmiddle0.5M, 270mL, 133.8mmol). Stir the reaction mixture at room temperature2h,Add compound at room temperature 146 (17.7g,71.3mmol),Pd (PPhs) 2CI2 (3.12g,4.45mmol)andIN NaOHAqueous solution(150mL). Stir the resulting mixture againlh. After removing the solvent;Make the residue inEtOAc (200mL)with water(200mL)Distribution between. Separate the water layer and useEtOAc (2 X 200mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Will pass column chromatography(Silica gel,4:1Hexane/EtOAc)Purify the crude product to obtain the compound as a brown solid147 (8.00g,43%):

[0789] ^XH NMR (400MHz,CDCls) δ8.14 (d, J = 8.9Ηζ,2Η) ,7.34 (d, J = 8.9Ηζ ,2Η) ,4.56 (br s,lH) ,3.17(q,J = 6.2Hz,2H) ,2.75 (t,J = 7.7Hz,2H) , 1.89-1.79 (m,2H) ,1.44(s,9H). [0790] Compound148Preparation

[0791] Put the compound at room temperature147 (8.00g,28.6)Dissolved in dioxane4N HC1 (50.OmL)And stir the solutionlh. The reaction mixture was concentrated under vacuum and the residue was usedMTBEGrind,Obtain the compound as a brown solid 148 (4.00g,65%):

108

CN 105073717 Β

[0792] ^XH NMR (400MHz,CDsOD) δ8.19 (d, J = 8.7Hz,2H) ,7.50 (d,J = 8.7Hz,2H) ,2.98 (t, J = 7.4Hz,2H) ,2.86 (t, J = 7.6Hz,2H) ,2.07-1.97 (m,2H).

[0793] Compound150Preparation

[0794] To the compound 148 (4.00g, 18.5mmol)Triol 149 (24.8g, 92.5mmol)ofMeOH (150mL)Add to solutionAc0H(ll.lmL,185mmol)And stir the reaction mixture at room temperature10minute. Add toNaCNBHs (5 · 83g, 92.5mmol)Rear,Continue to stir the solution at room temperature24h. exist4Add additional compounds within days149 (4.0equivalent),AcOH (4.0equivalent)andNaCNBHs (4.0equivalent). Then add hexanal(2.0equivalent),AcOH (2.0equivalent)and NaCNBHs (2.0equivalent). Stir the solution again at room temperaturelh. After removing the solvent, use saturatedNaHCOsNeutralize the residue and make the residue inEtOAc (200mL)with water(200mL)Distribution between. Separate the water layer and useCH2CI2 (2 X 300mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(9:lCH₂Cl₂/MeOH,8O:18: 2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid150 (6.50g, 52%). Separation comes from the extra in the impure fraction4.00gMaterial and purified by reversed-phase column to obtain1.50g(12%)Pure compound 150 (Total7.70g,64%):

[0795] ^XH NMR (400MHz,CD3OD) 58.03 (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).

[0796] (2R,2'R,3R,3'R,4R,4'R,5S,5'S)-6,6'-(3-(4-Aminophenyl)Propylazanediyl)Dihexane-1,2,3,4,5-Penta Alcohol(Compound153)Preparation;

[0797] Inject the compound by using a syringe at room temperature150 (6.50g,9.50mmo 1)and10%Pd/C (1.30g)In ethanol(150mL)Degas the suspension by bubbling10Minutes and then in a hydrogen atmosphere(balloon,latm)Stir down6h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. The filtrate was concentrated in vacuo to obtain as an off-white solid153 (6.01g,97%):

[0798] ^XH NMR (400MHz,CDsOD) :67.49-7.42 (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 = 13.2,3.4Hz,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-(pair((2S,3R,4R,5R) -2,3,4, 5,6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)Cai-1-base)Butyl)Caramel)-6-Clopiazine2-Formamide(152)Preparation

[0800]Program 24

109

CN 105073717 Β

[0801]

<img file="CN105073717B_D0176.tif" />

OH

H.COoC

OH N-Bleached succinimide ch₃cn

<img file="CN105073717B_D0177.tif" />

Zn. b DMF Pd₂ (dba)₃. Sphos

O

NIIBoc

144

<img file="CN105073717B_D0178.tif" />

NHBoc

OCH₃ ,CH₂C1₂

NHCbz Dodin limy

<img file="CN105073717B_D0179.tif" />

BocHN

146

OTf

BocHN

NaOH / MeOH/THF/H₇O /

<img file="CN105073717B_D0180.tif" />

BocHN

OH QH sugar=tight®y

O. .5 OH

T Only NMM. THF

NHCbz

NHCbz , TangguN

Pd(PPh₃)₄. Cui

CH₃CN' Sugar

HOswell

ΗΟ₆/^λ

<img file="CN105073717B_D0181.tif" />

O (R

NH.

152 g

N

H

BocHN

N

H

BocHN

Ν Ν'

Η 3NS .λΟΗ

Ε OH

HO

<img file="CN105073717B_D0182.tif" />

Ν (Λ) OH ^,y/OH

<img file="CN105073717B_D0183.tif" />

HO'(&amp;

<img file="CN105073717B_D0184.tif" />

(S) .HO

<img file="CN105073717B_D0185.tif" />

N

H

BocHN

149

Pd/C.Pressure(1 atm)

EtOH, AcOH

<img file="CN105073717B_D0186.tif" />

Ϊ50

DIPEA. EtOH

4N HC1 Aqueous solution,EtOH

Ν N Η H

<img file="CN105073717B_D0187.tif" />

NHCbz

NH?.2AcOH

NFL

[0802]

[0803] Compound14Is prepared in30Within minutes1-Caifen(1,10.0g,69.4mmol)Yi Yueqing(70. OmL)Add a few parts to the solutionNBS (142,12.3g,69.4mmol). Stir the resulting mixture at room temperature4h ,Concentrate under vacuum, then add water (200mL)And ethyl acetate(200mL). Separate the aqueous layer and use ethyl acetate(2 X 200mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate. Through crystallization(Heptane/EtOAc)Purification residue,Obtain the desired compound as a white solid14(6.0g,39%).

[0804] ^XH NMR (400MHz,DMSO-ch) :510.49 (s,lH) ,8.20 (dd, J = 8.3,0.5Ηζ, 1Η) ,8.02 (d, J = 8.3Hz,lH) ,7.66 (dd, J = 8.4,1.4Hz, 1H) ,7.64 (d,J = 8.1Hz, 1H) ,7.55 (ddd,J = 8.2,7.7,

110

CN 105073717 Β

Ι.ΙΗζ,ΙΗ) ,6.83(d,J = 8.2Hz,lH).

[0805] Compound145Preparation

[0806] Zinc powder(4.76g, 72.9mmol)Add to flame-dried,In a side-arm round bottom flask purged with nitrogen. Add anhydrous via syringeDMF (25.0mL), Followed by the addition of a catalytic amount of iodine(677mg,2.67mmol). It is observed that the resulting mixture undergoes a change from colorless to yellow and back to colorless. One-time addition of protected iodoalanine114 (8.00g, 24.3mmol), And then add a catalytic amount of iodine(677mg, 2.67mmol),And stir at room temperature30Minutes; successful insertion of zinc is accompanied by a mild exotherm. Allow the organozinc reagent to cool to room temperature, then addPd2 (dba) 3 (556mg, 0.60mmol),SPhos (498mg, 1.2lmmo 1)Aryl bromide 14 (5.40g,24.3mmol), And under the positive pressure of nitrogen, the mixture is heated to50 °CKeep16h. The reaction mixture was cooled to room temperature. Add saturationNHaCISolution (300mL)andEtOAc (300mL), Then filter the mixture through diatomaceous earth and useEtOAcwashing(100mL). Separate the water layer and useEtOAc (2X300mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,4:1Hexane/EtOAc)Purify the crude product to obtain the desired compound as a yellow solid 145 (3.10g,37%):

[0807] ^XH NMR (400MHz,CDC13,Mixture of rotamers):68.23 (d, J = 8.3Hz, 1H) ,7.99 (d, J = 8.6Hz,lH) ,7.54(t,J = 8.04Hz,lH) ,7.48 (ddd, J = 8.3,6.9,1.3Hz , 1H) ,7.08(d,J = 7.8Hz,lH) ,6.70(d,J = 7.6Hz,lH) ,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) ,3.543. . 33 (m, 2H) , 1.39 (s, 7H) , 1.09 (s, 2H).

[0808] Compound146Preparation

[0809] exist0°C To the compound 145 (3.07g, 8.90mmo 1)ofCH2CI2 (75. OmL)Vitus(7.25mL, 88.9mmol)andTf2.(2.24mL, 13.3mmol). Stir the resulting mixture at room temperature2h,Concentrated under vacuum and at CH2CI2 (lOOmL)with water(50mL)Distribution between. Separate the water layer and useCH2CI2 (2 X 50mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate to obtain the compound as a brown oil146 (4.20g,Crude products). The crude product was used directly in the next step without further purification.

[0810] ^XH NMR (400MHz,CDCls,Mixture of rotamers):68.19-8.07 (m,2H) ,7.69-7.64 (m, 2H) ,7.38 (d,J = 8.1Hz,lH) , 7.28 (d, J = 7.9Hz, 1H) , 5.12-5.06 (br s,lH) ,4.78-4.67 (m, 1H) ,3.68-3.46 (m,5H) ,1.39 (s,8H) ,1.25(s,lH).

[0811] Compound147Preparation

[0812] In a nitrogen atmosphere, the compound6(4.20g,8.80mmol,Crude products)He Ding-3-Alkynyl carbamate7 (2.65g, 13.2mmol)In Anhydrous Yiyueqing(50 . OmL)Degas the solution in10Minutes, then add at room temperatureTEA (4.81mL,35.2mmol), In hexane 10% (t-Bu) ₃P (3.56mL , 1.76mmol)and Cui (84mg, 0.44mmol). Degas the resulting mixture again with nitrogen10Minutes and add it all at oncePd (PPh₃) 4 (1.01 g , 0.88mmol). Degas with nitrogen5After minutes, the resulting mixture was refluxed18h. The reaction mixture was concentrated in vacuo and passed through column chromatography(Silica gel,2:3Hexane/EtOAc)The residue was purified to obtain the compound as a brown oil147 (3.20g,Two-step yield67%).

[0813] ^XH NMR(400MHz,CDCI3,Mixture of rotamers):68.33 (dd, J = 8.9,1.9Hz, 1H) ,8.07 (dd,J = 9.0,1.7Hz,lH) ,7.59-7.49 (m,3H) ,7.39-7.27 (m,5H) , 7.19 (d, J = 7.3Hz , 1H), 5.24-5.16 ,5.12(s,2H) ,5.08-4.99 , 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).

111

CN 105073717 Β

[0814] Compound148Preparation

[0815] Methyl ester 147 (3.10g,5.84mmo 1)existTHF (60mL), Methanol(60mL)And water(20.OmL)Add solids to the solutionNa0H(1.40g,35.09mmol). Stir the resulting mixture at room temperature2huntilTLCIt shows that the reaction is complete. Add to1NHydrochloric acid will be the reaction mixturepHValue adjusted to10. After concentration;Add water(lOOmL)And willpHAdjusted to5-6. Use the resulting precipitateCH2CI2 (2 X 200mL)extraction. Combine organic layers,throughNazSODry, filter, concentrate and useMTBEGrind to obtain the compound as a white solid148 (3.00g, 99 %).

[0816] ^XH NMR(400MHz,CD3OD; Mixture of rotamers):68.33 (d, J = 8.2Hz, 1H) ,8.28-8.20 (m,lH) ,7.59-7.45 (m,3H) ,7.38-7.21 (m,6H) ,5.09 (s,2H) ,4.55-4.45 (m,lH) ,3.76-3.66 (m,lH) ,3.44 (t, J = 6.7Hz , 2H) ,3.28-3.20 , 2.76 (t, J = 6.7Hz , 2H) ,1.29(s,6H),

0.82 (s,3H).

[0817] Compound149Preparation

[0818] To the compound148 (800mg, 1.55mmol)ofTHFSolution(30mL)Add to the solution one by oneT3P (In ethyl acetate 50% ,1.86mL)and NMM (0.85mL, 7.75mmol). Stir at room temperature 10 Minutes later, add amine 29(1.01g, 1.55mmol)And stir the reaction mixture at room temperaturelh. After removing the solvent, dissolve the residue inCH₂C1₂ (100mL) Medium, use saturationΝΗΚΙ,saturationNaHCOsAnd salt water fast washing, afterNa₂S0₄Dry and concentrate. Column chromatography(Silica gel,9: lCH₂Cl₂/MeOH)Purification residue,Obtain the amide as an off-white solid149 (1.20g,67%).

[0819] ^XH NMR (400MHz, CDCI3) : 68.35 (d, J = 8.0,1.7Hz , 1H) ,8.19 (d, J = 8.5Hz , 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,llH) ,7.086.96 (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).

[0820] Compound150Preparation

[0821] At room temperature by using a syringe with nitrogen149 (1.15g, 1 .OOmmo 1)and10%Pd/C (230mg)exist EtOH/AcOH(80.0mL/20.0mL)Degas the suspension by bubbling10Minutes, then put under hydrogenation condition(latm)Down 16h οThe reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in a vacuum and useMTBEGrind to obtain the amine salt as a brown solid150 (1.12g,97%).

[0822] ^XH NMR(400MHz,CD3OD,Mixture of rotamers):68.25 (dd, J = 7.2,2.OHz, 1H) ,8.09 (d,J = 7.0Hz,lH) ,7.59-7.51 (m,2H) ,7.48-7.41 (m,4H) ,7.37-7.21 (m,10H) ,6.94 (d,J = 8.5Hz, 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,Ηζ,2H) ,3.60-3.51 (m,lH), 3.28-3.15 (m,2H) ,3.14-2.95 (m,4H) ,2.89 (t, J = 7.4Hz,2H) ,2.73-2.67 ,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).

[0823] Compound151Preparation

[0824] At room temperature, to 150 (1.05g,0.92mmol)ofEtOH (15.OmL)Add to solutionDIPEA (1.30mL, 7.35mmol), Then add3,5-Diamino-6-Clopiazine-2-Pyridylmethylsulfonyl methyl thioate(13,573mg, 1.47mm.1). Heat the reaction mixture to70°CKeep2h,Cool to room temperature and concentrate under vacuum. Column chromatography(Silica gel,80:18:2CHC13/CH₃0H/NH₄0H)The residue was purified twice to obtain the compound as a yellow solid151 (410mg,36%) ο

112

CN 105073717 Β

[0825] ^XH NMR (400MHz,CD30D,Mixture of rotamers):68.22 (d, J = 8.4Hz, 1H) ,8.09 (d, J = 8.2Hz,lH) ,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 = 8.1Hz , 2H) ,5.47 (s,2H) ,4.53 (t, J = 8.1Hz , 1H), 4.22(dd,J=10.8,5.4Hz,2H) ,3.99-3.89 (m,4H) ,3.84 (dd, J = 5.5,2.1Hz,2H) ,3.70 (dd,J =9. l,2.0Hz,2H) ,3.59 (t,J= 10.8Hz,2H) ,3.53-3.47 ,3.46-3.39 (m,lH) ,3.263.17(m,2H) ,3.12-3.04 (m,2H) ,2.70 (dd,J = 13.2,4.0Hz,2H) ,2.60 (dd,J = 13.0,8.2,2H), 2.57-2.49 (m,2H) ,2.47-2.33 (m,2H) , 1.84T .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-(pair((2S, 3R, 4R, 5R) -2,3,4,5, 6-Five Urn Base Hexyl)Amino)Propyl)Phenylamino)-3-Oxopropyl)CaiT-base)Butyl)Caramel)-6-Chlorpyrazine-2Formamide(152)Synthesis

[0827] Towards 151 (480mg,0.42mmo 1)existEtOH (5. OmL)Add to the solution4Nhydrochloric acid(25. OmL). Stir the resulting mixture at room temperature2h. The solvent is removed, purified by reverse phase column and lyophilized to obtain the compound as a yellow hygroscopic solid 152 (300mg,71%).

[0828] ^XH NMR (400MHz,DMSO-de) :610.57 (brs, 1Η) , 10.55 (brss, 1H) ,9.35 (t,J = 6.0Hz, 1H) ,9.04-8.84 (m,2H) ,8.81-8.66 (m,4H) ,8.42-8.36 (m, 1H) ,8.16-8.10 (m, 1H) ,7.61-7.53 (m,2H) ,7.41 (d, J = 8.6Hz,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,lH) ,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,CDsOD) :58.25-8.21 (m,lH) ,8.18-8.13 (m, 1H) ,7.59-7.53 (m,2H), 7.38(d,J = 7.3Hz,lH) ,7.32 (d, J = 7.3Hz , 1H) ,7.26 (d, J = 8.8Hz,2H) ,7.15 (d, J = 8.5Hz, 2H) ,4.30 (t,J = 7.3Hz,lH) ,4.20-4.14 (m, 1H) ,4.13-4.08 (m, 1H) ,3.84-3.80 (m,2H) ,3.793.75 (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: C44H64CIN10O12 [M+Na] + Calculated:959.4418,Actual value:959.4394.

[0831] 24.Intermediate18Preparation

[0832]Program 25

113

CN 105073717 Β

[0833] o₂n

<img file="CN105073717B_D0188.tif" />

Br

PPIh.TFA.T {Compare (¹ I

<img file="CN105073717B_D0189.tif" />

159

<img file="CN105073717B_D0190.tif" />

[0834] Compound155Preparation

[0835] exist0°C To the compound 154 (500mg,9.OOmmo 1)ofCH2CI2 (50mL)Add to solutionTEA (1.63mL, 11.7mmol)andB0C2O (2.16g, 9.90mmol). exist0°CContinue to stir the reaction mixture0.5h, Make it warm to room temperature and stir3h. Then make the mixture inCH2CI2 (50mL)with water(50mL)Distribution between. Separate the water layer and useCH2CI2 (2 X50mL)extraction. Wash the combined organic extracts with brine,throughNa2S04Dry and concentrate,Column chromatography(Silica gel,2:3 Hexane/EtOAc)The residue is purified to obtain the desired compound as a colorless oil155 (1.20g,86%).

[0836] ^XH NMR (300MHz,CDC13) :54.70 (br s,lH) ,3.91 (dd, J = 5.3,2.2Hz,2H) ,2.21 (t,J = 2.7Hz,lH) ,1.45 (s,9H).

[0837] Compound157Preparation;

[0838] In a nitrogen atmosphere, the compound 155 (1.00g,6.45mmol)and 156 (1.30g,6.45mmol)In anhydrousTHF (15mL)Degas the solution in10Minutes, then add at room temperatureTEA (3.53mL, 25.8mmol) ,PPh₃ (424mg, 1.6lmmo 1)andCui (246mg, 1.29mmol). Degas the resulting mixture again with nitrogen10Minutes and add it all at oncePd (PPh3)4 (7.45g ,6.45mmol). Degas with nitrogen5After minutes, the resulting mixture was refluxed16h. The reaction mixture was concentrated in vacuo and passed through column chromatography(Silica gel,2:3Hexane/EtOAc)The residue was purified to obtain the compound as a brown oil 157 (750mg,42%).

[0839] ^XH NMR (400MHz, CDCI3) :δ8.17 (d, J = 9.2Ηζ ,2Η) , 7.55 (d, J = 9.2Ηζ , 2Η) , 4.79 (brs,lH) ,4.18(d,J = 6.0Hz,2H) ,1.47(s,9H).

[0840] Compound158Preparation

[0841] Put the compound at room temperature157 (2.00g,7.24)Dissolved in dioxane4N HC1 (20.OmL)And stir the solution2h. The reaction mixture was concentrated under vacuum and usedMTBEGrind the residue to obtain the compound as a brown solid 158 (1.25g,82%).

[0842] ^XH NMR (300MHz,CD3OD) : 58.26 (d, J = 9.2Hz,2H) ,7.72 (d, J = 9.2Hz,2H) ,4.09 (s, 2H).

114

CN 105073717 Β

[0843] Compound159Preparation

[0844] To the compound 158 (lOOmg, 0.47mmol)And formaldehyde solution(30 % , 1.40mL, 1.4lmmol)existMeOH (3.OmL)Add to the solution inAcOH (0.09mL, 1.4lmmol), The reaction mixture was stirred at room temperature30minute. Add toNaCNBHs (88mg, 1.41mmo 1)After that, continue to stir the solution at room temperature16h. Add additional formaldehyde solution (30 % , 0.92mL,0.94mmol),AcOH (0.09mL,1.4lmmo 1)andNaCNBHs (88mg, 1.41mmol),And stir again 16h. After removing the solvent, use saturatedNaHCOsNeutralize the residue and make the residue inEtOAc (30mL)with water(30mL)Distribution between. Separate the water layer and useCH₂Cl₂(2X40mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9: lCH₂Cl₂/MeOH,80: 18: 2CHCl₃/MeOH/NH₄OH)Purification residue,Obtain the compound as an off-white oil159 (50g,52%).

[0845] ^XH NMR (300MHz,CD3OD) : δ8.17 (d, J = 9.OHz,2H) ,7.57 (d, J = 9.OHz,2H) ,3.50 (s, 2H) ,2.37 (s,6H).

[0846] Compound18Preparation

[0847] Nitrogen the compound at room temperature 159 (lOOmg,0.49mmol)and 10%Pd/C (40mg)existMeOH (3.OmL)Degassing the suspension in10Minutes and then in a hydrogen atmosphere(balloon,latm)Stir down3h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in a vacuum and useCHsCL·/Triturated with hexane to obtain white crystals 18(48mg,55%):

[0848] ^XH NMR (300MHz,CDC13) :δ6.96 (d, J = 8.3Hz,2H) ,6.60 (d, J = 8.3Hz,2H) ,3.47 (br s,2H) ,2.53 (t,J = 7.8Hz,2H) ,2.26 (dd, J = 8.7,7.2Hz , 2H) ,2.22 (s,6H) ,1.77-1.67 (m,

2H).

[0849]

[0850] Intermediate29Preparation scheme26

[0851]

<img file="CN105073717B_D0191.tif" />

Compound161Preparation;

[0852]

[08531 To the compound 158 (4.00g,18.9mmol)Triol 160 (11.7g,56.6mmol)existMeOH (50mL)Add to the solution inAcOH (3.40mL, 56.6mmo 1),Stir the reaction mixture at room temperature30minute. Add toNaCNBHs (3.55g , 56.6mmol)After that, continue to stir the solution at room temperature16h. Add additional compounds160 (11.7g, 56.6mmol),AcOH (3.40mL, 56.6mmol)andNaCNBHs (3.55g, 56.6mmol), Continue to stir the solution at room temperature16h. After removing the solvent, use saturatedNaHCOsNeutralize the residue and place the residue inCH2CI2 (10mL)with water(10mL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 10mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9: lCH₂Cl₂/MeOH, 80 : 18 : 2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid29 (700mg, 7.0 %).

115

CN 105073717 Β

[0854] ^XH NMR (300MHz,CDsOD) :58.21 (d, J = 8.8Hz,2H) ,7.66 (d,J = 8.8Hz,2H) ,4.68 (q, J =5. lHz,2H) ,4.04(dd,J=10.8,5.4Hz,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 = 5.1Hz,6H).

[0855] Compound29Preparation

[0856] Use a syringe to remove the compound with nitrogen at room temperature161 (500mg,0.90mmol)and10%ofPd (OH) ₂/C (215mg)existEtOH (230mL)Degas the suspension by bubbling10Minutes, then under a hydrogen atmosphere(balloon,latm)Stir2h οThe reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in a vacuum and pass through column chromatography(Silica gel,9: lCH₂Cl₂/MeOH,8O:18:2CHCl₃/Me0H/NH₄0H)Purification residue,Obtain the compound as an off-white solid29 (264mg,55%).

[0857] ^XH NMR (400MHz,CDsOD) :δ6.97 (d, J = 8.6Hz,2H) ,6.67 (d, J = 8.6Hz,2H) ,4.71 (q,J = 5.1Hz,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 = 9.6,2.4Hz , 2H) ,3.33-3.23 (m, 2H) , 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] Intermediate24Preparation

[0859]Program 27

[0860]

<img file="CN105073717B_D0192.tif" />

<img file="CN105073717B_D0193.tif" />

164

[0861] Compound162Preparation

[0862]To the compound 158 (200mg, 0.94mmo 1)Triol 160 (194mg, 0.94mmo 1)existMeOH (2. OmL)Add to the solutionAcOH (0.17mL,2.82mmol), The reaction mixture was stirred at room temperature30minute. Add toNaCNBHs (148mg,2.35mmol)After that, continue to stir the solution at room temperature16h. Add additional compounds160(0.2equivalent), AcOH (3.0equivalent)andNaCNBHs (1 ·0equivalent), Continue to stir the solution at room temperature16h. After removing the solvent, use saturated NaHCOsNeutralize the residue so that the residue is inCH₂C1₂ (10mL)with water(10mL)Distribution between. Separate the water layer and useCH₂C1₂(2 X 10mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9: lCH₂Cl₂/MeOH,8O:18:2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid162 (95mg,28%) ο

[0863] ^XH NMR (400MHz,CDsOD) :68.24 (d, J = 9.1Hz,2H) ,7.69 (d,J = 9. lHz,2H) ,4.70 (q, J = 5.1Hz,lH) ,4.09-4.02 (m,2H) ,4.00 (d, J = 2.1Hz , 2H) , 3.83 (dd, J = 5.1,2.3Hz , 1H), 3.81-3.71 (m,lH) , 3.53 (dd, J = 9.3,2.3Hz , 1H) , 3.38 (t, J = 11. OHz , 1H) ,3.21-3.07 (m,

116

CN 105073717 Β

2Η) ,1.25(d,J = 5.1Hz,3H).

[0864] Compound164Preparation

[0865] To the compound 162 (95mg, 0.26mmol)Hexanal 163 (52mg, 0.5lmmol)Add to the solutionAcOH (0.05mL,0.78mmol)andNaCNBHs (41mg,0.65mmol). Stir the solution at room temperature16h. After removing the solvent, use saturatedNaHCOsNeutralize the residue and make the residue inEtOAc (10mL)with water(10mL)Distribution between. Separate the water layer and use CH₂C1₂ (2 XIOmL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel9: lCH₂Cl₂/MeOH,8O:18:2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid 164 (70mg,59%).

[0866] ^XH NMR (400MHz,CDCls) :δ8.18 (d, J = 8.9Hz,2H) ,7.56 (d,J = 8.9Hz,2H) ,4.70 (q, J = 5.0Hz,lH) ,4.15 (dd,J=10.4,5.2Hz,lH) ,4.01-3.89 (m,2H) , 3.83 (dd, J = 3.8,2.7Hz, 1H) ,3.77 (brs,lH) ,3.70 (brs, 1H) ,3.64 (t, J = 6.2Hz, 1H) ,3.56 (dd, J = 9.2,4.OHz , 1H), 3.41 (t,J = 10.8Hz,lH) ,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.OHz,3H) , 1.32-1.25 (m,6H) ,0.89 (t, J = 6.6Hz,3H).

[0867] Compound24Preparation

[0868] Nitrogen the compound at room temperature 164 (1.70g,3.77mmol)and 10%Pd/C (200mg)existMeOH (40mL)Medium suspension degassing10minute,Then in a hydrogen atmosphere(balloon,latm)Stir down2h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in vacuum and pass through column chromatography(Silica gel,9: lCH₂Cl₂/MeOH,8O:18: 2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid24 (1.20g,76%).

[0869] ^XH NMR (300MHz,CDCls) *6.96 (d, J = 8.9Hz,2H) ,6.62 (d, J = 8.9Hz,2H) ,4.68 (q, J = 5.0Hz,lH) ,4.14(dd,J=11.0,5.5Hz,lH) ,3.92-3.81 (m,2H) , 3.72 (dd, J = 3.8,2.4Hz, 1H) ,3.50 (dd,J = 9.1,4.0Hz,lH) ,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. OHz, 3H) ,1.30-1.20 (m,6H) ,0.88 (t, J = 6.6Hz,

3H).

[0870] Intermediate85Preparation

[0871]Program 28

[0872]

<img file="CN105073717B_D0194.tif" />

163

<img file="CN105073717B_D0195.tif" />

<img file="CN105073717B_D0196.tif" />

[0873] Compound166Preparation

[0874] To the compound 148 (4.60g,21.3mmo 1)Triol 165 (17. lg,63.9mmol)existMeOH (100mL)Add to the solution inAcOH (12.ImL,63.9mmo 1),Stir the reaction mixture at room temperature10minute. Add toNaCNBHs

117

CN 105073717 Β

(4.00g,63.9mmol)After that, continue to stir the solution at room temperature6h. Then add hexanal163 (5.10mL,42.6mmol) andNaCNBHs (2.60g,42.6mmol). Stir the solution again at room temperature2h. After removing the solvent, use saturatedNaHCOsNeutralize the residue and make the residue inEtOAc (200mL)with water(200mL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 300mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9 : lCH₂Cl₂/MeOH,8O:18:2CHCl₃/MeOH/NH₄OH)The residue was purified to obtain the compound as an off-white solid166 (6.90g,64%) ο

[0875] ^XH NMR (400MHz,CDsOD) :δ8.12 (d, J = 8.6Hz,2H) ,7.51-7.43 (m,2H) ,7.38 (d,J = 8.6Hz , 2H) ,7.37-7.27 (m,3H) ,5.55 (s,lH) ,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.OHz,3H).

[0876] Compound85Preparation

[0877] The compound is injected with nitrogen by using a syringe166 (800mg, 1.55mmol)and10%Pd/C (300mg)exist EtOH (40mL)Degas the suspension by bubbling10minute,Then in a hydrogen atmosphere(balloon,latm)Stir at room temperature 2h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. The filtrate was concentrated in vacuo to obtain as an off-white solid85(700mg,93%).

[0878] ^XH NMR (400MHz,CDsOD) :67.52-7.42 (m,2H) ,7.38-7.25 (m,3H) ,6.88 (d,J = 8.4Hz, 2H) ,6.63(d,J = 8.4Hz,2H) ,5.53 (s,lH) ,4.24 (dd, J= 10.8,5.5Hz, 1H) ,4.05-3.84 (m,3H), 3.76 (dd,J = 9.6,1.8Hz,lH) ,3.61 (t,J= 10.8Hz, 1H) ,2.93 (dd,J=13.6,5.0Hz, 1H) ,2.79 (dd,J=13.4,9.0Hz,lH) ,2.73-2.60 (m,4H) , 2.42 (t, J = 8 . OHz , 2H) , 1.88T . 68 (m, 2H), 1.48-1.36 (m,2H) , 1.33-1.14 (m,6H) ,0.87 (t,J = 7.OHz,3H).

[0879] Intermediate34Preparation

[0880]Program 29

[0881]

[0882] Compound168Preparation

[0883] exist0°C Towards 162 (534mg, 1.45mmol)ofMeOH (30mL)Add saturation to the solutionNaHCOsAqueous solution (5.OmL)And stir10minute. Then add(Boc) 2O (350mg, 1.60mmol)And stir the reaction mixture at the same temperature3h,Bring to room temperature and stir again30minute. The mixture was concentrated and the residue was dissolved inCH₂C1₂ (lOOmL)Medium and water(lOOmL)And salt water(50mL)Wash the solution. The organic layer isNa₂S0₄Dry, filter, concentrate and pass column chromatography (Silica gel,9 : lCH₂Cl₂/MeOH,8: 2CHCl₃/MeOH)The residue was purified to obtain the compound as an off-white solid168 (435mg,64%).

[0884] ^XH NMR (400MHz,CDCls) :δ8.18 (d, J = 8.7Hz,2H) ,7.56 (d,J = 8.7Hz,2H) ,4.72 (q, J

118

CN 105073717 Β

= 5.1Ηζ,1Η) ,4.41-4.35 (m,2H) ,4.16 (dd, J= 10.8,5.5Hz , 1H) ,4.15-4.04 (m, 1H) ,3.933.83 ,3.81-3.76 ,3.66-3.53 (m,4H) ,3.40 (t, J= 11.0Hz, 1H) ,3.25-3.12 (m,

1H) ,3.08-2.96 ,1.49 (s,9H) , 1.32 (d, J = 5.1Hz,3H).

[0885] Compound34Preparation

[0886]Use a syringe to mix the compound with nitrogen 168 (80mg, 0.2 lmmo 1)and 10 % Pd/C (40mg)existEtOH (10mL) Bubbling degassing of medium suspension10minute,Then in a hydrogen atmosphere(balloon,latm)Stir at room temperature2h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in vacuum,Obtained as off-white solid34 (82mg, 89%) ο

[0887] ^XH NMR (400MHz,CDCls) : 56.96 (d, J = 8. lHz,2H) ,6.62 (d, J = 8.1Hz,2H) ,4.69 (q, J = 5.1Hz,lH) ,4.15 (dd,J=10.8,5.5Hz,lH) ,4.13-4.09 (m, 1H) ,4.01-3.93 (m, 1H) ,3.893.78 ,3.75-3.68 ,3.62-3.43 (m,4H) ,3.40 (t, J = 11.3Hz, 1H) ,3.35(dd,J =

13.5,4.0Hz,lH) ,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 = 5.1Hz,3H).

[0888]

[0889] Intermediate171Preparation scheme30

<img file="CN105073717B_D0197.tif" />

o₂n

<img file="CN105073717B_D0198.tif" />

Glycogen-sugarΟ,Ν

<img file="CN105073717B_D0199.tif" />

169

Boc₇O, NaHCO₃

MeOH/H^O

OH OH sugar=Wanderingw δπ

Υ

PM

O&gt;N

<img file="CN105073717B_D0200.tif" />

ΝJust sugar

Boc

H,N

170

Pd/C. Η. ,.ΕίΟΗ Sugar

Boc

<img file="CN105073717B_D0201.tif" />

To the compound 148 (6.40g, 29.6mmol)Triol 165 (11.9g, 44.5mmol)existMeOH (300mL)middle

[0891] Add to solutionAcOH (5.32mL, 88.8mmo 1),Stir the reaction mixture at room temperature30minute. Add toNaCNBHs (3.73g,59.2mmol)After that, continue to stir the solution at room temperature16h. Add additional compounds165 (11.9g, 44.5mmol),AcOH (5.32mL, 88.8mmol)andNaCNBHs (3.73g, 59.2mmol),Continue to stir the solution at room temperature 14h. Add additional compounds 165 (7.93g, 29.6mmol),AcOH (3.55mL, 59.2mmol)andNaCNBHs (2.80g,44.4mmol). Continue to stir the solution at room temperature10h. After removing the solvent, use saturatedNaHCOsNeutralize the residue so that the residue is inCH2CI2 (lOOmL)with water(lOOmL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 100mL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9:1CH₂C1₂/

MeOH,80:18: 2CHCl₃/MeOH/NH₄OH)Carry out challenging purification to get the compound150and169 (20g,mixture). Should

119

CN 105073717 Β

The mixture is used directly in the next step.

[0892] Compound170Preparation

[0893] exist0 °CTowards150and169 (20.0g,mixture)existMeOH (120mL)And water(40mL)Add saturated solution inNaHCO3 (9.99g, 118.4mmol)And stir 10minute. Add to(Boc) 2O (9.69g,44.4mmol)And stir the reaction mixture at the same temperature10Minutes at room temperature,Stir again2h. The mixture was concentrated and the residue was dissolved inCHsCb (lOOmL)Medium and water(lOOmL)And salt water(50mL)Wash the solution. The organic layer isNazSOqDry, filter, concentrate and pass column chromatography(Silica gel,9: lCH₂Cl₂/MeOH,8: 2CHCl₃/MeOH)The residue was purified to obtain the compound as an off-white solid 150 (1.50g) ίΠ170 (4.50g) _OESI-MS m/z 529[C27H32N2O9+H]⁺_o

[0894] Compound171Preparation

[0895] The compound with nitrogen 170 (4.20g,7.92mmol)and 10%Pd/C (500mg)existEtOH (lOOmL)andAcOH (lOmL)Degassing the suspension in10Minutes, then stir at room temperature under a hydrogen atmosphere(balloon,latm) 16h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashingοConcentrate the filtrate in a vacuum and useNa₂CO₃Neutralize and pass column chromatography(Silica gel,9:lCH₂Cl₂/MeOH,8:2CHCl₃/MeOH)The residue was purified to obtain the compound as an off-white solid172 (2.70g,68%).

[0896] ^XH NMR (400MHz,CDsOD) :67.52-7.44 (m,2H) ,7.36-7.29 (m,3H) ,6.89 (d,J = 8.3Hz, 2H) ,6.64 (d,J = 8.3Hz,2H) ,5.54 (s,lH) ,4.23 (dd, J= 11.9,5.9Hz, 1H) ,4.10-3.97 (m, 1H), 3.97-3.89 ,3.81-3.75 (m, 1H) ,3.74-3.69 ,3.60 (t,J= 10.9Hz, 1H) ,3.48 (dd,

J=14.1,4.6Hz,lH) ,3.28-3.22 (m,3H) ,2.41 (t, J = 7.5Hz,2H) ,1.83-1.71 (m,2H) ,1.41 (s,

9H).

[0897] Intermediate39Preparation

[0898]Program 31

[0899]

<img file="CN105073717B_D0202.tif" />

Γ72

P&lt;i(PPh₃)4.

<img file="CN105073717B_D0203.tif" />

[0900] In a nitrogen atmosphere, the compound17 (30.0g,pure12lmmo 1)and173 (14.2g, 145mmol)In Anhydrous Yiyueqing (300mL)Degas the solution in10Minutes, then add at room temperatureTEA(67mL,484mmol), In hexane10% (tBu) 3P (49. OmL, 24.2mmol)andCui (1.15g, 6.05mmol). Degas the resulting mixture again with nitrogen10Minutes and add it all at oncePd (PPh3)4 (14.0g, 12. lmmol). Degas with nitrogen5Minutes later,Heat the resulting mixture to50°Cmaintain16h. The reaction mixture was concentrated in vacuo and passed through column chromatography(Silica gel,2:3Hexane/EtOAc)The residue was purified to obtain the compound as a brown oil174 (15.0g,58%).

[0901] ^XH NMR (400MHz,CDCls) δ8.14 (d, J = 8.8Ηζ,2Η) ,7.50 (d,J = 8.8Hz,2H) ,3.71 (t,J = 6.4Ηζ,2Η) ,2.50 (t, J = 6.8Hz,2H) , 1.80-1.70 (m,4H) , 1.70-1.65 (m, 1H). [0902] Compound175Preparation

120

CN 105073717 Β

[0903] exist0°CUnder nitrogen, to the compound174 (15.0g,67.9mmol)AnhydrousCH2CI2 (50mL)Add to solution EtsN (28. OmL, 203.7mmo 1)andDMAP (4.12g, 33.9mmo 1). Stir the reaction mixture at the same temperature5Minutes later, at0°CAdd toTsCl (32.5g, 170mmol). Stir the resulting mixture again at room temperature4h. After removing the solvent; leave the residue inCH2CI2 (250mL)with water(150mL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 250mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel, hexane/ EtOAc)The residue was purified to obtain the compound as a brown oil175 (15.0g,60%).

[0904] ^XH NMR (400MHz,CDC13) : δ8.15 (d, J = 88Hz,2H) ,7.79 (d,J = 8.8Hz,2H) ,7.50 (d, J = 8.8Hz,2H) ,7.34 (d, J = 8.8Hz,2H) ,4.10 (t, J = 6.4Hz,2H) ,2.44 (t,J = 7.OHz,2H) ,2.44 (s,3H) , 1.90-1.79 (m,2H) ,1.75-1.61 (m,2H).

[0905] Compound176Preparation;

[0906] To the compound175 (5.00g, 12.9mmol,Crude products)existTHF (10mL)Add to the solution containingNHMe2 (30 %, 50. OmL)Water, then stir in a sealed tube at room temperature3h. After removing the solvent; leave the residue inCH₂C1₂(lOOmL)with water(lOOmL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X lOOmL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Pass the crude product through column chromatography(Silica gel)Purified to obtain the compound as a yellow sticky solid176 (400mg,13%).

[0907] ^XH NMR (400MHz,CDCls) :δ8.15 (d, J = 7.3Hz,2H) ,7.51 (d, J = 7.3Hz,2H) ,2.48 (t, J = 6.6Hz,2H) ,2.30 (t,J = 5.7Hz,2H) ,2.23 (s,6H) ,1.70-1.61 (m,4H).

[0908] Compound39Preparation

[0909] Use a syringe to mix the compound with nitrogen 176 (400mg, 1.62mmol)and 10%Pd/C (50mg)existEtOH (50mL) Degas the suspension by bubbling10Minutes and then in a hydrogen atmosphere(balloon,latm)Stir at room temperature16h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. The filtrate was concentrated in vacuo to obtain a brown sticky solid39 (300mg,84%) ο

[0910] ^XH NMR (400MHz,CDsOD) :δ6.91 (d, J = 7.5Hz,2H) ,6.65 (d, J = 7.5Hz,2H) ,2.47 (t,J = 7.0Hz,2H) ,2.30(dd,J = 8.4,6.5Hz,2H) ,2.23 (s,6H) ,1.60-1.52 (m,2H) ,1.51-1.41 (m, 2H) ,1.38-1.27 (m,4H).

[0911] 31Intermediate44Preparation

[0912]Program 32

121

CN 105073717 Β

<img file="CN105073717B_D0204.tif" />

[0914] Compound177Preparation

[0915] exist30°CThe compound in a sealed tube175 (6.00g, 16.Ommo 1)In contain7N NH3Methanol(150mL)Heating of the solution in5h. Temperature rises to40 °CAnd stir16h,And then rise again to60°CAnd stir4h. After removing the solvent;Make the residue inCH₂C1₂ (lOOmL)with water(lOOmL)Distribution between. Separate the water layer and useCH₂C1₂ (2X lOOmL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9: 1 CH₂Cl₂/MeOH)Purify the crude product to obtain the compound as a yellow oil177 (1.48g,43%).

[0916] ^XH NMR (400MHz,CDCls) :δ8.16 (d, J = 8.4Hz,2H) ,7.39 (d,J = 8.4Hz,2H) ,3.61 (t,J = 5.6Hz,2H) ,2.08-2.05 (m,2H) , 1.65-1.53 (m,4H).

[0917] Compound178and179Preparation

[0918] To the compound 177 (1.38g,6.33mmo 1)Triol 165 (2.03g,7.59mmol)existMeOH(lOmL)Add to the solution inAcOH (0.6mL, 9.49mmo 1)And stir the reaction mixture at room temperature30minute. Add toNaCNBHs (800mg,12.7mmol)After that, continue to stir the solution at room temperature16h. Add additional compounds165 (2.55g, 9.49mmol),AcOH (0.80mL, 12.7mmol)andNaCNBHs (1.19g, 18.9mmol),Continue to stir the solution at room temperature 16h. Add additional compounds 165 (2.55g, 9.49mmol),AcOH (0.80mL,12.7mmol)And add NaCNBHs (1.19g, 18.9mmol), Continue to stir at room temperature16h. After removing the solvent, use saturatedNaHCOsNeutralize the residue so that the residue is inCH₂C1₂ (10mL)with water(10mL)Distribution between. Separate the water layer and useCH₂C1₂ (2XIOmL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel,9:1 CH₂Cl₂/MeOH,8O: 18:2 CHCl₃/Me0H/NH₄0H)The residue was purified to obtain the compound as an off-white solid179 (2.28g,51%).

[0919] ^XH NMR (300MHz, CDsOD) : δ8.14 (d, J = 9. OHz , 2Η) , 7.54 (d, J = 9. OHz , 2H) ,7.477.44 (m,4H) ,7.34-7.30 (m,6H) ,5.48 (s,2H) ,4.24-4.19 (m,2H) ,3.99-3.94 (m,4H) ,3.863.84 (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) ,,1.61-1.51 (m,4H).

[0920] Also separated out178/179 (900mg)And use it directly in the next step(SG-GHC-G-106).

[0921] Compound44Preparation

[0922] The compound with nitrogen 179 (2.26g, 3.1 lmmo 1)and 10 %Pd/C (lOOmg)existEtOH (50mL)andAcOH

122

CN 105073717 Β

(10mL)Degas the suspension in the mixture10Minutes and then in a hydrogen atmosphere(balloon,latm)Stir at room temperature16h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in vacuum,Obtained as a brown solid 44 (1.90g,80%) ο

[0923] ^XH NMR (400MHz,CDsOD) :67.46-7.44 (m,4H) ,7.33-7.31 (m,6H) ,6.89 (d,J = 8.4Hz, 2H) ,6.65 (d,J = 8.4Hz,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).

[0924] Intermediate49Preparation

[0925]Program 33

[0926]

<img file="CN105073717B_D0205.tif" />

<img file="CN105073717B_D0206.tif" />

[0927] Compound180Preparation

[0928] exist0 °CTowards178 (900mg,Mixture, approx2. Ommol)existMeOH (20mL)And water(10mL)Add to the solution in the mixtureNaHCO3 (672mg,4.Ommol)And stir 10minute. Add to(Boc) 2O (524mg,2.40mmol), And stir the reaction mixture at the same temperaturelh,Bring to room temperature and stir again4h. The mixture was concentrated and the residue was dissolved in CH₂C1₂ (lOOmL)Medium, and the solution with water(100mL)And salt water(50mL)washing. The organic layer isNazSCkDrying, filtering,Concentrate and pass column chromatography(Silica gel,9:1 CH₂Cl₂/MeOH,8:2 CHCb/MeOH)The residue was purified to obtain the compound as an off-white solid180 (780mg,64%).

[0929] ^XH NMR (300MHz, CDsOD) : δ8.16 (d, J = 9. OHz , 2Η) , 7.55 (d, J = 9. OHz , 2H) ,7.50

7.47 (m,2H) ,7.34-7.30 (m,3H) ,5.53 (s,lH) ,4.25-4.20 (m, 1H) ,4.10(br s,lH) ,3.94-3.91 (m,lH) ,3.80-3.48 (m,4H) ,3.35-3.25 (m,3H) ,2.46 (t, J = 6.9Hz,2H) , 1.70T .49 (m,4H), 1.43 (s,9H).

[0930] Compound49Preparation

[0931] The compound is injected with nitrogen by using a syringe180 (780mg, 1.36mmol)and10%Pd/C (50mg)existEtOH (10mL)andAcOH (2.OmL)The suspension in the mixture is degassed by bubbling10Minutes and then in a hydrogen atmosphere(balloon, latm)Stir at room temperature4h. useNa2CO3Neutralize the reaction mixture, filter through diatomaceous earth and useMeOHwashing. Concentrate the filtrate in vacuum,Obtained as a white solid49 (625g,84%).

[0932] ^XH NMR (300MHz,CDsOD) :67.50-7.46 (m,2H) ,7.32-7.30 (m,3H) ,6.90 (d,J = 8.4Hz, 2H) ,6.66(d,J = 8.4Hz,2H) ,5.53 (s,lH) ,4.25-4.20 (m, 1H) ,4.04 (br s,lH) ,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).

[0933] Intermediate54Preparation

[0934]Program 34

123

CN 105073717 Β

[0935]

<img file="CN105073717B_D0207.tif" />

¹ TLA, I

9-ΙίΙΪΝ.

IMCk&lt;l»Ph₅h

IN Na0H A

CIbG. Ο»

<img file="CN105073717B_D0208.tif" />

iSJ till middle7N Nib

<img file="CN105073717B_D0209.tif" />

2.

IfM

1. NalNBiT^.. AcOTT. MeOTT

<img file="CN105073717B_D0210.tif" />

[0936] Compound182Preparation

[0937] Under nitrogen, to the compound181 (1.60g,16.00mmol)AnhydrousTHF (40mL)Add to solution9-BBN (THFmiddle0.5M, 80mL,40. Ommol). Stir the reaction mixture at room temperature2hRear,Add compound at room temperature172 (3.17g, 12.8mmol),Pd (PPhs) 2CI2 (561mg,0.80mmol)andIN NaOHAqueous solution(24mL). Stir the resulting mixture againlh. After removing the solvent;Leftovers inEtOAc (100mL)with water(100mL)Distribution between. Separate the water layer and use EtOAc (2 X 100mL)extraction. The combined organic extracts were washed with brine, andNazSOqDry and concentrate under vacuum. Column chromatography(Silica gel,4:1Hexane/EtOAc)Purify the crude product to obtain the compound as a brown solid182 (1.20g, 34%).

[0938] ^XH NMR (400MHz,CDCls) :δ8.13 (d, J = 9.0Hz,2H) ,7.31 (d, J = 9.OHz,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).

[0939] Compound183Preparation

[0940] exist0°CUnder nitrogen, to the compound182(1.20g,5.38mmo 1)AnhydrousCH2CI2 (20mL)Add to solution Et₃N (7.32mL, 53.8mmo 1). Stir the reaction mixture at the same temperature5Minutes later, at0 °CAdd methanesulfonyl chloride (0 · 62mL,8.07mmol). Stir the resulting mixture at room temperature2h. After removing the solvent; leave the residue inCH₂C1₂ (50mL) with water(50mL)Distribution between. Separate the water layer and useCH₂C1₂ (2X50mL)extraction. Wash the combined organic extracts with brine,throughNa2S0Dry and concentrate under vacuum. Crude products183 (3.00g,Crude products)Used directly in the next steps.

[0941] Compound184Preparation

[0942] exist60°CThe compound in a sealed tube183 (3.00g,5.38mmol,Crude products)In contain7N NH3Methanol (30.OmL)Heating of the solution in2h. After removing the solvent;Make the residue inCH₂C1₂ (100mL)with water(100mL)Distribution between. Separate the water layer and useCH₂C1₂ (2 X 100mL)extraction. The combined organic extracts were washed with brine, andNa₂S0₄Dry and concentrate under vacuum. Column chromatography(Silica gel)Purify the crude product to obtain the compound as a yellow oil184(390mg, Two-step yield33%).

[0943] ^XH NMR (400MHz,CDsOD) :δ8.14 (d, J = 9.0Hz,2H) ,7.42 (d,J = 9.OHz,2H) ,2.75 (t, J = 7.8Hz,2H) ,2.67(t,J = 7.3Hz,2H) ,1.72-1.63 (m,2H) ,1.53-1.46 (m,2H) ,1.42-1.35 (m,

124

CN 105073717 Β

4Η).

[0944] Compound185Preparation

[0945] To the compound 184 (620mg,2.79mmo 1)Triol 165 (938mg,3.49mmol)existMeOH (30mL)Add to the solution inAcOH (1.16mL, 27.8mmo 1),Stir the reaction mixture at room temperature10minute. Add toNaCNBHs (526mg,8.37mmol)After that, continue to stir the solution at room temperature16h. exist16hInside, add additional compounds165 (0.3 equivalent),AcOH(l0equivalent)andNaCNBHs (1.0equivalent). Then add hexanal 163 (0.96mL,8.37mmol),AcOH (1 · OOmL)andNaCNBHs (526mg, 8.37mmol). Stir the solution again at room temperature2h. After removing the solvent, use saturated NaHCOsNeutralize the residue and make the residue inEtOAc (100mL)with water(lOOmL)Distribution between. Separate the water layer and use CH₂C1₂ (2 X lOOmL)extraction. The combined organic extractsNa₂S0₄Dry and concentrate under vacuum. Column chromatography (Silica gel,9:1 CH₂Cl₂/MeOH,8O:18:2 CHCl₃/Me0H/NH₄0H)The residue was purified to give the compound as an off-white oil 185(950g,61%).

[0946] ^XH NMR (400MHz,CDCls) *8.02 (d, J = 8.7Hz,2H) ,7.48-7.42 (m,3H) ,7.37-7.34 (m, 2H) ,7.31 (d,J = 8.7Hz,2H) ,5.54 (s,lH) ,4.46-4.40 (m, 1H) ,4.30 (dd, J= 11.6,6.6Hz, 1H), 4.03 (t,J = 4.0Hz,lH) , 3.97 (dd, J= 10.5,5.4Hz, 1H) , 3.88 (dd, J = 9.4,4.0Hz, 1H) ,3.65 (t,J=10.4Hz,lH) ,3.11-3.00 (m,4H) ,2.69 (t,J = 7.8Hz,2H) ,2.00 (s,lH) ,1.70-1.55 (m, 6H) ,1.37-1.30 (m,4H) , 1.29-1.20 (m,8H) ,0.87 (t, J = 7. lHz,3H).

[0947] Compound54Preparation

[0948] The compound with nitrogen 185 (950g,1.70mmo 1)and 10%Pd/C (300mg)existEtOH (lOOmL)Degassing the suspension in10minute,Then in a hydrogen atmosphere(balloon,latm)Stir at room temperature3h. The reaction mixture was filtered through diatomaceous earth and usedMeOHwashing. Concentrate the filtrate in vacuum,Obtained as a yellow oil54 (790mg, 88 %).

[0949] ^XH NMR (400MHz,CDsOD) :67.51-7.44 (m,2H) ,7.35-7.29 (m,3H) ,6.90 (d,J = 8.5Hz, 2H) ,6.65(d,J = 8.5Hz,2H) ,5.54 (s,lH) ,4.24 (dd, J= 10.8,5.4Hz, 1H) ,4.08-4.02 (m, 1H), 4.00-3.92 (m,lH) ,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,lH) ,2.91 (dd, J= 12.1,8.1Hz , 1H) ,2.82-2.71 (m, 4H) ,2.45 (t,J = 7.5Hz,2H) , 1.59-1.42 (m,6H) , 1.37-1.13 (m, 10H) ,0.89 (t, J = 7. lHz,3H). [0950] -These assays can be used to characterize the compounds of the invention. Representative assays are discussed below.

[0951] In vitro measurement of sodium channel blocking activity and reversibility

[0952] -Species used to evaluate the mechanism of action of the compounds of the present invention and/Or the determination of potency includes use in the Uss perfusion chamber (Ussing Chamber)Short-circuit current(Isc)Measure the airway epithelial sodium current to determine intracavitary drug inhibition. Cells obtained from freshly excised human, dog, sheep or rodent airways are inoculated into porous0.4MicronSnapwellTM Inserts (CoStar)Above, in a hormone-defined medium in air-Liquid interface(air-liquid interface ,ALI)Culture under conditions, immersed in the Uss perfusion chamberKrebs Bicarbonate Ringer (KBR)Sodium transport activity(Isc). Semi-logarithm(half-log)Dosing plan to bath in the cavity(lumenal bath)Add all test compounds(1 X 10"^πΜto3 X 10cold)And recordIsc (inhibition)Cumulative changes. by1 X 10"The concentration of hoe is used as a storage solution to prepare all medicines in dimethyl inkstone and store them in-20 °CDown. Usually in parallel8Part preparation;Introduce amiloride and every two preparations/Or benzamil as a positive control. At maximum concentration(5X10-⁵M)After that, use fresh medicine-freeKBRThe solution exchange chamber is bathed three times, and each wash is about5Measured after a duration of minutesIsc. 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.

125

CN 105073717 Β

[0953] passPrism 3.0The program considers and analyzes the dosage of all compounds-Effect relationship. calculateIC50The values, maximum effective concentration and reversibility were compared with amiloride and benzamil as positive controls. 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 the table1middle.

[0954] surface1.Passing compounds in canine bronchial epithelial cells(la)Suppress short-circuit current(IC₅₀nM)

[0955]

<img file="CN105073717B_D0211.tif" />

126

CN 105073717 Β

[0956] Determination2.Mucociliary removal in sheep (Mucociliary Clearance,MCC)Research

[0957] existMCCThe most frequently used animal model for measurement is the sheep model. Can be used bySabaterWait, Journal of Applied Physiology, 1999,NS2191to2196Page (which is incorporated herein by reference) describes the in vivo model measuring the effects of compounds on enhanced mucociliary clearance (MCC).

[0958] In these studies, adult sheep were controlled and a tracheal tube was inserted through the nose. Application to sheep10-15Atomize the test sample in minutes. Then, the radioactively labeled99Tc-Sulfur colloid (TSC,3. lmg/mL; Inclusive20mCi). Administration of radiolabeled aerosol through tracheal intubation is about5minute. Then remove the cannula of the sheep, and1Hours of observation period, every5The total radioactivity count in the lungs is measured every minute. The clearance rate of radioactivity in the lungs represents theMCCrate.

[0959]The advantage of this system is that it approximates the human lung environment. The model also allows simultaneous collection by plasma and urine sampling during the testPK/PDinformation. There are also several technologies available inMCCIn the measurement, the drug concentration on the airway surface is measured. These techniques include the collection of exhaled breath condensate or the filter paper method through bronchoscopyASL.

[0960]The sheep model described above is used to evaluate the test agent pair for aerosol deliveryMCCIn vivo effects (potency/Durability). Processing includes testing by4mLTest agent or withHSCombination of test agents. In order to determine whether theHSWith compoundII-d MCCCombination, to be administered immediately after the application of the compound test agentHS. Use raindrop sprayer (Raindrop nebulizer) Atomize the test solution at a flow rate of eight liters per minute and connect it to a solenoid valve and a compressed air source (20psi) Composed of a dosimetry system. After using a raindrop sprayer for aerosol application, the deposited dose of the drug in the lungs of the sheep is estimated to be the dose8%to15%. Use raindrop sprayer, after the medicine treatment4or8Hours, administration of radioactivity TSCmake an appointment3Minutes to evaluate its effectiveness/Durability. useγThe camera is in the center of the right lung every interval5The radioactivity count is measured every minute for one hour. Use three analytical methods,1) Using linear regression to fit the first30The initial rate of clearance (slope) in minutes,2) Clear the% area under the curve within one hour, and3) The maximum clearance rate obtained in one hour.

[0961] Tested after administration4Hour,Compound33exist0.24nmol/kg (3μΜ) To sheepMCCThe role of and with the carrier (4mLSterile out0) Compared (figure1). The analysis of the effect is shown in the tableA. Compared with the vehicle control, the compound33improved MCCo

[0962] surfaceA.In the compound33Or after carrier administration4Hour of the SheepMCC

[0963]

<td>Compound33dose</td><td>Initial slope(4.0-4.511)</td><td>AUC (% Cl - h)</td><td>Maximum deduction rate</td>

<td>0.24 nmol/kg (3μΜ)</td><td>37.5* (4)</td><td>17.4* (4)</td><td>302 (4)</td>

<td>Carrier(II₂O) 4 mL.</td><td>17.2+6.8(8)</td><td>7.3+1.5 (8)</td><td>12.2+2.9(8)</td>

[0964]surfaceBAnd tableCTogether with picture2And figure3-It shows that compared with the carrier, the other compounds of the present invention similarly enhance

TMCC (See e.g. compound123and48)

[0965] surfaceB.In the compound123Or after carrier administration4Hour sheepMCC

127

CN 105073717 Β

[0966]

<td>Compound123dose</td><td>Initial slope(4.0-4.5h)</td><td>AUC (% Cl - 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* ⑵</td>

<td>Carrier (H2O)4mL</td><td>17.2 ±6.8 (8)</td><td>7.3 earth 1.5 (8)</td><td>12.2 + 2.9(8)</td>

[0967] surfaceC.In the compound48Or after carrier administration4Hour sheepMCC

[0968]

<td>Compound48dose</td><td>Initial slope(4.0-4.5h)</td><td>AlJC (% Cl - h)</td><td>Maximum clearance rate</td>

<td>0.24 nmoi/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 scholar 1.5(8)</td><td>12.2 ± 2.9(8)</td>

[0969]In order to determine whether the compound of the present invention has enhanced the duration of action, after administration8hIt was tested. surfaceDAnd tableETogether with picture4And figure5Clearly show the compound33and152Compared with the carrier, it enhances theMCCDuration of action.

[0970] surfaceD.In the compound33Or after carrier administration4Hour sheepMCC

[0971]

<td>Compound33dose</td><td>Initial slope&amp;5h)</td><td>AUC (% Cl - h)</td><td>Maximum clearance rate</td>

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

<td>Carrier (H2O)4mL</td><td>17.2 ±6.8 (8)</td><td>7.3 + 1.5 (8)</td><td>12.2 ±2.9 (8)</td>

[0972] surfaceE.In the compound152Or after carrier administration4Hour sheepMCC

[0973]

<td>Compound152dose</td><td>Initial slope (&amp;0.&amp;5h)</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 (H2O)4mL</td><td>17.2 ±6.8 (8)</td><td>7.3 scholar 1.5(8)</td><td>12.2 + 2.9(8)</td>

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[0974] to confirmHSDoes it improve the compound33ofMCCEffect, evaluated in0 · 24nmol/kgCompound33and MCCAfter combined administration8Give immediately7%ofHS (picture6). In the picture6Shown inHSImproved compound33CorrectMCCEffect.

[0975]Determination3.Clear and metabolize the airway surface drug solution through the human airway epithelium (ASL)

[0976]Assessed in human bronchial epithelium (HBE) Compounds in cells33Disappearance from the apical surface and metabolism of the airway epithelium (table3). In these experiments, the25Mof25pM ENaCThe blocking agent solution is added to the gas/Growing at the liquid interfaceHBE Apical surface of cell,And passUPLCMeasured 2hDrug concentration in the inner apical and basal side compartments.

[0977]surfaceG.Compound33The tip disappears and metabolizes

<td>Chemical agent</td><td>The initial drug mass% on the top side (maternal and metabolites,2h)</td><td>Top mass as a metabolite% ( 2h ):</td><td>The 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+18%</td><td>4%</td><td>1,1 ±0.45%</td><td>32%</td>

[0979] Values are expressed as mean soil SD

[0980] Comparative Example

[0981]Compared with known sodium channel blockers such as amiloride and the following comparative examples1Compared with the third-generation compound described in the formula of the present invention (DThe compound is more powerful and/Or from the mucosal surface, (especially the airway surface), it is not absorbed quickly. Therefore, as shown in the tableGAs evidenced by the data shown, compared with these known compounds, the formula (I) The compound has a longer half-life on the mucosal surface. Evaluated inHBEMiddle compound33The disappearance from the top surface and the metabolism of the airway epithelium, and compared with the comparative example1Made a comparison (tableH). In these experiments, the25Mof25pM ENaCThe blocking agent solution is added to the gas/ Liquid interface culturedHBEThe top surface of the cell and passUPLCMeasured2hDrug concentration in the inner apical and basal compartments. Incubate the compound of the invention on the apical surface2hRear(37°C), the compound on the top side33Most are not metabolized. In contrast, most of the comparative examples1From the top side,83%Metabolized to lower active acid, (S) -2-Amino-3-(4-(4(3-(3,5-Diamino-6-Clopiazine-2-Dial group) sarcosyl) butyl) phenoxy) propionic acid, the structure is as follows.

[0982]

<img file="CN105073717B_D0212.tif" />

NHn -L·· ' NS

<img file="CN105073717B_D0213.tif" />

[0983] surfaceH.existHBEMiddle compound33And comparative example1The tip disappears and metabolizes

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<td>Compound</td><td>On the top side of the initial drug mass% (maternal and metabolic Yang,2h)</td><td>As a metabolite, the top mass of% (2h )</td><td>The mass% of the initial top on the basal side ( 2h )</td><td>Metabolites on the basal side% ( 2h)</td>

<td>33</td><td>44.8^18%</td><td>4%</td><td>1.1±045%</td><td>32%</td>

<td>Comparative Example1</td><td>41*6±7.6%Light% maternal)</td><td>83.0±3.S%</td><td>&amp; 3±0.2(1%Maternal)</td><td>94.7±1.0%</td>

[0985] Values are expressed as mean soil SD

[0986] Comparative example 1 existW0 2003/070182 (U.S. Patent No.6,858,615;7,186,833;7,189,719; 7,192,960; and7332496) Protects, describes or discloses the structure as a sodium channel blocker with useful medicinal properties and can be prepared by the methods described therein and well-known in the art.

[0987] Comparative Example1

<img file="CN105073717B_D0214.tif" />

[0989] (S) -3,5-Diamino-6-chlorine-Ν- @-4-4- (2,3-Diamino-3-Oxopropoxy) phenyl) butyl) methyl) pyrazine)-2-Formamide

[0990] allowableUS 2005/0080093First15On the page and asW0 2008/031048NS90Compound on page2 And asW0 2008/031028NS42-43Page compound2See comparative example1compound of. In order to treat cystic fibrosis andC.0.P.DIn order to have beneficial activity, the compound must have a dose that results in enhanced mucociliary clearance (MCC) Nature. 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, with in vivoMCCThe activity is beneficial and does not cause an increase in plasma potassium at a usable dose. One evaluation model is the sheep described belowMCCModel.

[0991]As from the table1And figure7It can be seen that three different measurement methods (slope,AUCAnd maximum clearance rate) obtained in sheepMCCComparative examples in the model1ofED50For appointment240nmol/kg (3mM). At this dose (this will be the clinically active dose), the comparative example1Lead to an increase in plasma potassium (Figure8), which will cause hyperkalemia when repeated administration. Therefore, the comparative exampleINot for human use, and the compound (Ia) Produce safe and effectiveMCCAnd in this model, the benefit-risk ratio is greater than1000ο

[0992] surfaceI.After administration4Hourly carrier in sheep, comparative example1Or compound33ofMCC

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[0993]

<td>dose</td><td>Initial slope</td><td>AUC (% C1 χ h)</td><td>Maximum clearance rate</td>

<td></td><td>(4.0-4,511)</td><td></td><td></td>

<td>Comparative Example1</td><td>32.2 + 7.3* (6)</td><td>14.1 scholar 2.2* </td><td>22.9 + 2. P (6)</td>

[0994]

<td colspan="2">240 nmol/kg (3mM)</td><td colspan="2"></td>

<td>Comparative Example124 nmol/kg (300 μΜ)</td><td>14.5 + 1.3(3)</td><td>6.9 ± i.0(3)</td><td>14.6± 09(3)</td>

<td>Compound33</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]pictureIDrawn as above inMCCCompounds described in the model33And comparative example1A graph of the percentage of mucus removal over a period of time. Compound33The comparative example seen in the ratio1Low1000Provides an even higher percentage of mucus clearance at twice the dose. Therefore, the compound33Provides the greatest effect within the relevant dose range where there is no clinical increase in plasma potassium.

[0996]picture10Shown inMCCIn the study, in accepting the comparative example1In sheep plasma, a significant increase in plasma potassium level can be seen at an effective dose. In the sheep'sMCCIn the compound33And comparative example1More effective than1000Times, And the dose is as high as24nmol/kg(ED₅oDosed1000Times) when there is no increase in plasmaK. And the comparative example1In appointment3mMof ED50Blood plasmaK (picture7and8). This again shows that the compound33Has unique and unexpected advantages in efficacy and safety, as shown in the tableJAs shown in the comparative example1Compared with1000Higher kidney safety.

[0997] surfaceJ.Treatment ratio (benefit/risk)

[0998]

<td></td><td>MCCHighest sub-maximum dose</td><td>Does not increase the maximum dose of plasma potassium in sheep</td><td>Treatment ratio</td>

<td>Comparative Example1</td><td>240nmol/kg (3mM)</td><td>24nmol/kg (300μΜ)</td><td>0.1</td>

<td>33</td><td>&lt;0 · 24nmol/kg (3μΜ)</td><td>24nmol/kg (300μΜ)</td><td>&gt;100</td>

<td>ratio</td><td>&gt;1,000</td><td>1</td><td>&gt;1,000</td>

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[0999]Other compounds of the present invention have similar safety and efficacy,More than as shown in the figure11,12,13and14Known compounds exemplified in.

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