Targeting compound and use thereof

Abstract

The present invention provides a targeting compound capable of specifically binding to an integrin αvβ6, wherein the compound is the compound as shown in formula (I) of the present invention. The targeting compound is used for transferring and delivering active molecules to a cell or tissue expressing the integrin αvβ6.

Description

Targeted compounds and their uses Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to a targeting compound and uses thereof. Background Art

[0002] Integrin αvβ6 is a heterodimer composed of αv and β6 integrin subunits, each of which consists of a large extracellular head, a flexible leg, a transmembrane segment, and a short cytoplasmic tail. Integrin αvβ6 is a receptor for the delayed-associated peptide (LAP) of TGF-β and the extracellular matrix (ECM) proteins fibronectin, vitronectin, and tenascin. Integrin αvβ6 is frequently upregulated in diseases such as cancer and fibrosis, being upregulated in an estimated one-third of all solid cancers, and its expression correlates with disease progression and poor prognosis. Multiple studies have shown that the severity of many cancers, including lung, breast, colon, gastric, and ovarian cancers, is associated with αvβ6 expression. Integrin αvβ6 is attractive as a tumor marker and potential therapeutic target, particularly given its role in matrix metalloproteinase (MMP) expression and TGF-β1 activation.

[0003] Compounds targeting integrin αvβ6 are suitable for conjugation with active molecules to transport and deliver the active molecules to cells expressing integrin αvβ6 in vivo. For specific molecules to be transported, such as therapeutic oligonucleotide-based compounds (e.g., antisense oligonucleotides or RNAi agents), targeting ligands targeting integrin αvβ6 can be conjugated with oligonucleotide-based compounds to deliver the therapeutic agent to cells and / or tissues expressing integrin αvβ6 and promote the entry of the therapeutic agent into the cell through receptor-mediated endocytosis, pinocytosis or other means. Therefore, there is a need in the art to develop targeting ligands targeting integrin αvβ6.

[0004] The receptor for advanced glycation end products (RAGE) is a new pattern recognition receptor that is widely involved in the pathological processes of many diseases, such as Alzheimer's disease, pneumonia, tumors and diabetes. In particular, this receptor has been shown to be associated with type II and non-type II inflammatory responses in preclinical animal models. Currently, ARO-RAGE, a small nucleic acid compound targeting RAGE, is in Phase I clinical research for the treatment of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. According to genomic analysis, this receptor is highly specifically expressed on the surface of alveolar cells and is less expressed in other tissues and organs. Therefore, targeting ligands targeting integrin αvβ6 and compounds that can silence RAGE protein expression are suitable for evaluating the delivery ability of targeting ligands. Summary of the Invention

[0005] The present invention aims to provide a targeting compound, which is a targeting ligand capable of targeting integrin αvβ6. The targeting compound has affinity for integrin αvβ6 and can specifically bind to integrin αvβ6. The targeting compound can be combined with an active molecule to promote the transport and delivery of the active molecule (such as an antisense oligonucleotide) to cells or tissues expressing integrin αvβ6, so that the active molecule exerts its effect in these cells or tissues.

[0006] The targeting compound of the present invention is a compound represented by Formula I or a tautomer or stereoisomer thereof. The targeting compound contains a linker, through which an active molecule is further connected. A scaffold may further be provided between the targeting compound and the active molecule, wherein the scaffold has a monodentate, bidentate, tridentate, or tetradentate structure. The active molecule is connected to one, two, three, or four of the targeting compounds via the scaffold.

[0007] In the first aspect of the present invention, there is provided a compound represented by formula I or its tautomers or stereoisomers:

[0008] wherein Ring A is a 5-membered heteroaromatic ring;

[0009] Ring B is a 6-10 membered aromatic ring or a 5-6 membered heteroaromatic ring;

[0010] Ring C is a 6-10 membered aromatic ring, a 5-10 membered heteroaromatic ring, or a 5-10 membered heterocycloalkyl ring;

[0011] L1 is C1-C6 alkylene, C1-C6 haloalkylene;

[0012] R1 is selected from: H, halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 Alkyl, C 1-6 Halogenated alkyl, C 1-6 Alkoxy, -CONH2, -CONR 11 R 12 、-C(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -COOR 13 、-NH-C 1-6 Alkyl, -NR 14 R 15 ;

[0013] R2, R3 are each independently selected from: H, halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 Alkyl, C 1-6 Alkoxy, -CONH2, -CONR 11 R 12、-C(O)-C 1-6 Alkyl, -S(O)2-C 1-6 Alkyl, -COOR 13 、-NH-C 1-6 Alkyl, -NR 14 R 15 ;

[0014] Among them, R 11 、R 12 、R 13 、R 14 、R 15 Each independently is C 1-6 alkyl;

[0015] The R1, R2, R3 are optionally substituted by 1, 2 or 3 identical or different substituents selected from the following: halogen, NH2, CN, OH, SF5, COOH, C 1-6 Alkyl, C 1-6 alkoxy;

[0016] The ring B or ring C is optionally connected to a linker T;

[0017] The linker T is used to directly or indirectly connect the active molecule;

[0018] When there are multiple R1s, the R1s are the same or different substituents;

[0019] When R2 is multiple, the R2 are the same or different substituents;

[0020] When R3 is multiple, the R3 are the same or different substituents;

[0021] m is 1, 2, or 3;

[0022] n is 1 or 2;

[0023] p is 1 or 2.

[0024] It should be noted that the above “R1, R2, R3 are optionally substituted by a substituent” means that R1, R2, R3 contain a hydrogen group (e.g., C 1-6 The hydrogen in the alkyl group may be substituted with a substituent.

[0025] In an alternative embodiment of the present invention, Ring B is a 6-10 membered aromatic ring.

[0026] In an alternative embodiment of the present invention, Ring B is a 6-8 membered aromatic ring.

[0027] In a preferred embodiment, the structural fragment With structure

[0028] In a preferred embodiment, the group for Wherein, the definitions of R2, R3, ring C, n, and p are as described in the first aspect.

[0029] In a preferred embodiment, R2 is H.

[0030] In a preferred embodiment, Ring C is a 5-10 membered heteroaromatic ring.

[0031] In a preferred embodiment, Ring C is a 5-8 membered heteroaromatic ring.

[0032] In a preferred embodiment, ring C is a 5- or 6-membered heteroaromatic ring, wherein the heteroatom is selected from N, O, and S. When there are multiple heteroatoms, the heteroatoms are the same or different.

[0033] In a preferred embodiment, Ring C is a 5-membered N-containing heteroaromatic ring.

[0034] In a preferred embodiment, ring C is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine.

[0035] In a preferred embodiment, Ring C is pyrazole.

[0036] In a preferred embodiment, Ring C is

[0037] In a preferred embodiment, R3 is halogen, -NH2, -CN, -OH, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkyl.

[0038] In a preferred embodiment, R3 is methyl.

[0039] In a preferred embodiment, L1 is C1-C6 alkylene.

[0040] In a preferred embodiment, L1 is -CH2CH2CH2-.

[0041] In a preferred embodiment, the compound represented by formula I has the following structure:

[0042] Wherein, the definitions of ring A, R1, and m are as described in the first aspect.

[0043] In a preferred embodiment, when a linker T is connected, the compound represented by formula I has the following structure:

[0044] Wherein, the definitions of ring A, R1, and m are as described in the first aspect.

[0045] In a preferred embodiment, ring A is a 5-membered heteroaryl group; R1 is as defined in the first aspect; and m is 0, 1 or 2.

[0046] In a preferred embodiment, the structural fragment for

[0047] In a preferred embodiment, ring A is a 5-membered heteroaromatic ring, wherein the heteroatom is selected from N, O, and S. When there are multiple heteroatoms, the heteroatoms are the same or different.

[0048] In a preferred embodiment, Ring A is a 5-membered N-containing heteroaromatic ring.

[0049] In a preferred embodiment, ring A is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole.

[0050] In a preferred embodiment, ring A is imidazole or triazole.

[0051] In a preferred embodiment, Ring A is

[0052] In a preferred embodiment, for Among them, R 1a 、R 1b are each independently R1.

[0053] In a preferred embodiment, R1 is halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 Alkyl, -CONH2, -C(O)-C1-6 alkyl, -S(O)2-C 1-6 Alkyl; the C 1-6 Alkyl, -C(O)-C 1-6 Alkyl, -S(O)2-C 1-6 The alkyl group is optionally substituted with 1, 2 or 3 substituents which are the same or different and are selected from the group consisting of halogen, -NH2, -CN, -OH, -SF5, C 1-3 Alkyl, C 1-3 Alkoxy.

[0054] In a preferred embodiment, R1 is H, -F, -Cl, -CN, -SF5, -COOH, -CF3, -CHF2, -COCH3, -CONH2, -S(O)2CH3.

[0055] In a preferred embodiment, R1 is H, -F, -Cl, or -CN.

[0056] In a preferred embodiment, Selected from

[0057] In a preferred embodiment, the compound of formula I has one linker T.

[0058] In a preferred embodiment, the linker T comprises a polyethylene glycol unit.

[0059] In a preferred embodiment, the linker T comprises 2-20 polyethylene glycol units.

[0060] In a preferred embodiment, the linker T is Where t is 1-10.

[0061] In a preferred embodiment, the linker T is Where t is 1-10.

[0062] In a preferred embodiment, the linker T is Where t is 1-10.

[0063] In a preferred embodiment, any of the above t is 1, 2, 3, 4, 5, or 6.

[0064] In a preferred embodiment, the linker T is

[0065] In a preferred embodiment, the linker T is

[0066] In a preferred embodiment, the compound represented by formula I has one or more structures selected from the following:

[0067] In a preferred embodiment, the compound represented by formula I has one or more structures selected from the following:

[0068] In a preferred embodiment, t is 1-10.

[0069] In a preferred embodiment, t is 1, 2, 3, 4, 5, or 6.

[0070] In a preferred embodiment, the compound has one or more of the following structures:

[0071] in, Represents a connection point; each R1 is independently defined as described in the first aspect.

[0072] In a preferred embodiment, each R1 is independently H, -F, -Cl, or -CN.

[0073] In a preferred embodiment, the compound represented by Formula I or its tautomers or stereoisomers has the following structure:

[0074] In a preferred embodiment, the compound represented by Formula I or its tautomers or stereoisomers has the following structure:

[0075] In a preferred embodiment, the compound of formula I (connected with a linker T) has one or more structures selected from the following:

[0076] Wherein, t is 3, 4, or 5; Indicates a connection point.

[0077] In a preferred embodiment, the compound of formula I (connected with a linker T) has one or more structures selected from the following:

[0078] in, Indicates a connection point.

[0079] In a second aspect, the present invention provides a conjugate comprising a compound represented by formula I as described in the first aspect, or its tautomers or stereoisomers, and an active molecule G; wherein the compound represented by formula I contains a linker T, and the active molecule G is connected to the linker T in the compound represented by formula I.

[0080] It should be noted that when the compound represented by Formula I is connected to other molecules (for example, in the conjugate described in the second aspect), the compound represented by Formula I refers to the compound represented by Formula I containing a linker T. When the linker T of the compound represented by Formula I contains N3, the compound represented by Formula I is a precursor before preparing the conjugate; when the linker T of the compound represented by Formula I does not contain N3 but contains When the compound represented by formula I Connected to the active molecule G.

[0081] In a preferred embodiment, the conjugate further comprises a scaffold; wherein the active molecule G is connected to the linker T in the compound represented by formula I via the scaffold.

[0082] As used herein, the term "scaffold" refers to a structure that connects one molecule or part of a molecule to another molecule or part of a molecule. For example, in this application, an active molecule G can be conjugated to one or more compounds of Formula I via a scaffold, wherein the scaffold includes at least one connection point for each ligand (a compound of Formula I or a tautomer or stereoisomer thereof) and at least one connection point for each active molecule G.

[0083] In a preferred embodiment, the conjugate has one or more characteristics selected from the group consisting of:

[0084] aa) the active molecule G is an active pharmaceutical ingredient or a prodrug thereof;

[0085] bb) the active molecule G is a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid, a natural or modified oligonucleotide, a natural or modified polynucleotide, a peptide, an aptamer, a polymer, a polyamine, a protein, a toxin, a vitamin, polyethylene glycol, a hapten, digoxigenin, biotin, a radioactive atom or molecule, or a fluorophore;

[0086] cc) the active molecule G is a natural or modified oligonucleotide;

[0087] dd) the active molecule G is ASO, siRNA, or miRNA;

[0088] ee) The bracket has a single-tooth, two-tooth, three-tooth, or four-tooth structure;

[0089] ff) The active molecule G is connected to one, two, three or four compounds represented by formula I through the scaffold.

[0090] Oligonucleotides As used herein, the term "natural or modified oligonucleotide" refers to an oligonucleotide consisting of a natural nucleic acid, or an oligonucleotide comprising a modified nucleic acid. The term "natural or modified polynucleotide" refers to a polynucleotide consisting of a natural nucleic acid, or a polynucleotide comprising a modified nucleic acid.

[0091] In a preferred embodiment, the active molecule G is connected to ring B and / or ring C of the compound of formula I. In a preferred embodiment, the active molecule G is connected to one, two, three or four compounds of formula I via the scaffold.

[0092] In a preferred embodiment, the conjugate is

[0093] Wherein, Formula I is the compound represented by Formula I, and G is the active molecule G;

[0094] v is 1-10.

[0095] In a preferred embodiment, t is 1, 2, 3, 4, 5, or 6.

[0096] In a preferred embodiment, t is 4.

[0097] In a preferred embodiment, v is 4, 5, 6, 7, or 8.

[0098] In a preferred embodiment, the active molecule G is a natural or modified oligonucleotide.

[0099] In a preferred embodiment, the active molecule G is ASO, siRNA, or miRNA.

[0100] In a preferred embodiment, the structure of the conjugate is as follows:

[0101] For the specific structures of IIb-1 to IIb-28, please refer to the structures marked above in the first aspect of the present invention.

[0102] In a preferred embodiment, the conjugate comprises the structure

[0103] in, Indicates the point of attachment to the oligonucleotide.

[0104] In a third aspect, the present invention provides a composition comprising the compound of formula I as described in the first aspect or its tautomers, stereoisomers, or the conjugate as described in the second aspect.

[0105] In a preferred embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0106] In a fourth aspect, the present invention provides use of the compound of formula I as described in the first aspect, or its tautomers or stereoisomers, the conjugate as described in the second aspect, or the composition as described in the third aspect in the preparation of a drug.

[0107] In a preferred embodiment, the medicament is used for:

[0108] i) delivering the active molecule G to the cell; or

[0109] ii) inhibiting the expression of a target gene in the cell; or

[0110] iii) Treatment of lung diseases.

[0111] In a preferred embodiment, the cell is a cell expressing αvβ6 integrin.

[0112] In a preferred embodiment, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells and epithelial tumors (carcinomas).

[0113] In a fifth aspect, the present invention provides the compound of formula I as described in the first aspect, or its tautomers or stereoisomers, the conjugate as described in the second aspect, or the composition as described in the third aspect, having the following uses:

[0114] i) delivering the active molecule G to the cell; and / or

[0115] ii) inhibiting the expression of a target gene in the cell; and / or

[0116] iii) treatment of lung disease; and / or

[0117] iv) preparing a medicament for inhibiting expression of a target gene in a cell; and / or

[0118] v) preparing a medicament for the treatment of lung diseases.

[0119] In a preferred embodiment, the cell is a cell expressing αvβ6 integrin.

[0120] In a preferred embodiment, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells and epithelial tumors (carcinomas).

[0121] In a sixth aspect, the present invention provides a method for delivering active molecules to cells, inhibiting the expression of target genes in cells, or treating lung diseases, the method comprising administering to a patient a compound of formula I as described in the first aspect or its tautomers, stereoisomers, the conjugate as described in the second aspect, or the composition as described in the third aspect.

[0122] In a preferred embodiment, the cell is the cell described in the fourth aspect.

[0123] In a preferred embodiment, the active molecule is the active molecule G.

[0124] In a preferred embodiment, the active molecule G is ASO, siRNA, or miRNA.

[0125] In a seventh aspect, the present invention provides a compound comprising any of the following structures:

[0126] in, Indicates a connection point.

[0127] connector

[0128] As disclosed herein, the targeting compound further comprises a linker, through which the active molecule is further connected. In some embodiments, the linker is a linker T as described herein. In some embodiments, the linker may be a linker containing a polyethylene glycol (PEG) unit structure. The linker may comprise 2-20 polyethylene glycol unit structures.

[0129] In some embodiments, the linker is Indicates the attachment sites for the targeting compound and the active molecule, respectively.

[0130] In some embodiments, the linker has a reactive group at one end connected to the active molecule, and the active molecule is directly conjugated to the linker through the reactive group, or the active molecule is conjugated to the linker after being connected to the scaffold. In some embodiments, the reactive group is an azide or an alkyne-containing group.

[0131] In some embodiments, the linker is

[0132] In some embodiments, t is 1-10.

[0133] In some embodiments, preferably, any of the above t is 1, 2, 3, 4, 5, 6, or 7.

[0134] There is no particular limitation on the connection site between the linker and the targeting compound. In some embodiments, the linker is connected to Ring B or Ring C of the targeting compound (the compound represented by Formula I of the present invention).

[0135] In some embodiments, the compound of Formula I has the structure:

[0136] Targeted compounds and scaffolds

[0137] As disclosed herein, in some embodiments, one or more targeting compounds (compounds of formula I described herein) may be connected to one or more transported active molecules. In some embodiments, only one targeting compound is conjugated to the active molecule (referred to herein as a "monodentate" or "monovalent" ligand). In some embodiments, two targeting compounds are conjugated to the active molecule (referred to herein as a "bidentate" or "bivalent" ligand). In some embodiments, three targeting compounds are conjugated to the active molecule (referred to herein as a "tridentate" or "trivalent" ligand). In some embodiments, four targeting compounds are conjugated to the active molecule (referred to herein as a "tetradentate" or "tetravalent" ligand). In some embodiments, more than four are conjugated to the active molecule.

[0138] As disclosed herein, in some embodiments, the scaffold and the active molecule are connected to the targeting compound via a linker. In some embodiments, the scaffold is a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the active molecule is connected to one targeting compound via a monodentate scaffold. In some embodiments, the active molecule is connected to two targeting compounds via a bidentate scaffold. In some embodiments, the active molecule is connected to three targeting compounds via a tridentate scaffold. In some embodiments, the active molecule is connected to four targeting compounds via a tetradentate scaffold.

[0139] Scaffolds are generally known in the art. Non-limiting examples of scaffolds that can be used with the targeting compounds disclosed herein include, but are not limited to, polymers and polyamino acids (e.g., diglutamic acid, poly-L-lysine, etc.). In some embodiments, the scaffold can include amine reactive groups, amide bonds, and alkyne-containing moieties (e.g., alkynyl, cyclooctyne). In some embodiments, alkyne-containing moieties include, but are not limited to, alkyne maleimides, alkyne NHS esters.

[0140] In some embodiments, the scaffold may include a cysteine ​​linker or group, dibenzocyclooctyne (DBCO)-PEG 1-24 -NHS, propargyl-PEG 1-24 -NHS and / or multidentate DBCO and / or propargyl moieties.

[0141] In some embodiments, a typical tridentate scaffold is, for example:

[0142] This scaffold comprises an amine-reactive group, an amide bond, three PEG2 units, and an alkyne. The amine-reactive group can be conjugated to a primary amine on the transported active molecule, such as a terminal amine (e.g., NH2-C6), through amide formation. The alkyne can be conjugated to an azide-modified linker to form a triazole structure, which can then be linked to the targeting compound. In this scaffold, the amine-reactive group is p-nitrophenol (also known as 4-nitrophenol) ester.

[0143] In some embodiments, the amine-reactive group is not particularly limited. The amine-reactive group can also be, for example:

[0144] In some embodiments, the scaffold can be synthesized as a phosphoramidite compound, which can allow the tridentate scaffold to be coupled to an oligonucleotide (such as the 5' end of the sense strand) via phosphoramidite synthesis, as shown in the following structure:

[0145] in, Represents an oligonucleotide, such as siRNA.

[0146] Conjugate

[0147] The present invention provides a conjugate having the structure As disclosed herein above, in some embodiments, one or more αvβ6 integrin ligands (such as the compounds of Formula I described herein) can be linked to one or more active molecules to be transported.

[0148] In some embodiments, the active molecule G can be coupled to the scaffold via phosphoramidite synthesis. The scaffold can be a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the azide group on the targeting compound (e.g. It can react with the alkyne of the scaffold (such as benzocyclooctyne, propargyl) (such as click chemistry reaction) to form a triazole structure, which can then be connected to the target compound. For example, the structure:

[0149] in Indicates the point of attachment to an active molecule (e.g., oligonucleotide).

[0150] The active molecule described herein can be linked to a targeting compound to form a conjugate. In some embodiments, the conjugate is the conjugate described in the third aspect.

[0151] Active molecule

[0152] As used herein, an "active molecule" can be linked to the targeting compound and thereby transported to cells and / or tissues expressing the integrin αβ. A transported "active molecule" (or transported molecule) is any molecule that, when separated from an αβ integrin ligand as described herein, will have a desired effect on cells containing an αβ integrin receptor. The transported active molecule can be, but is not limited to, a pharmaceutical ingredient, a pharmaceutical product, a prodrug, a substance with known therapeutic benefit, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid, an oligonucleotide or polynucleotide, a peptide, a polymer, a polyamine, a protein, an aptamer, a toxin, a vitamin, PEG, a hapten, digoxin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, one or more transported active molecules (e.g., the same or different transported active molecules) are linked to one or more αβ integrin ligands to target the transported active molecules to cells expressing the αβ integrin. In some embodiments, the active molecule is the active molecule G described in the present invention.

[0153] Oligonucleotides

[0154] In this application, the term "oligonucleotide" refers to a short chain of nucleotides (including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA) consisting of 50 or less bases. The oligonucleotides of the present application may be single-stranded or double-stranded, and the specific type is not limited and is within the scope of protection of the present application. Exemplarily, when it is a double-stranded structure, the RNAi agent consists of a sense strand and an antisense strand, and includes but is not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates; when it is a single-stranded structure, the RNAi agent is but not limited to antisense oligonucleotides (ASO).

[0155] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

[0156] Terms and Definitions

[0157] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the description of this specification.

[0158] Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by persons skilled in the art to which the claimed subject matter belongs. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated by reference in their entirety. If multiple definitions of a term are used herein, the definitions in this section shall prevail.

[0159] It should be understood that the above brief description and the detailed description below are exemplary and are only used for explanation, and do not impose any restrictions on the subject matter of the present invention. In this application, unless otherwise specifically stated, the use of the singular also includes the plural. It must be noted that unless otherwise clearly stated in the text, the singular forms used in this specification and claims include the plural forms of the things referred to. It should also be noted that unless otherwise stated, the use of "or" and "or" means "and / or". In addition, the use of the term "including" and other forms, such as "comprising", "including" and "containing" are not restrictive.

[0160] Definitions of standard chemical terms can be found in the references (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4THED." Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise indicated, conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV / VIS spectroscopy and pharmacological methods, are used. Unless otherwise specified, the terms used herein in the descriptions of analytical chemistry, synthetic organic chemistry, and pharmaceuticals and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and in the treatment of patients. For example, the manufacturer's instructions for use of the kit can be utilized, or reactions and purifications can be carried out in accordance with methods well known in the art or the description of the present invention. The above techniques and methods can generally be implemented according to conventional methods well known in the art, as described in the various general and more specific references cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0161] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents that would result from writing the formula from right to left. For example, CHO is equivalent to OCH. As used herein, Indicates the attachment site of a group. As used herein, "R1" and "R2" have the same meaning and are interchangeable. For other symbols such as R2, similar definitions have the same meaning.

[0162] The section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0163] In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings indicated below unless otherwise specifically stated.

[0164] When a numerical range is described in the specification and claims of this application and is understood as an "integer," it should be understood to include both endpoints of the range as well as every integer within the range. For example, "an integer from 0 to 5" should be understood to include every integer from 0, 1, 2, 3, 4, and 5.

[0165] As used herein, the term "halogen" by itself or as part of another substituent refers to fluorine, chlorine, bromine, or iodine.

[0166] As used herein, the term "amino" by itself or as part of another substituent refers to -NH2.

[0167] As used herein, the term "nitro" by itself or as part of another substituent refers to -NO2.

[0168] As used herein, the term "cyano," by itself or as part of another substituent, refers to -CN.

[0169] As used herein, the term "alkyl" when used alone or as part of another substituent means a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, free of unsaturated bonds, having, for example, 1 to 6 carbon atoms and connected to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl. Alkyl groups may be unsubstituted or substituted with one or more suitable substituents. Alkyl groups may also be isotopomers of naturally abundant alkyl groups enriched in isotopes of carbon and / or hydrogen (i.e., deuterium or tritium).

[0170] The term "C1-C6 alkyl" alone or as part of another substituent is understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof. The term "C1-C5 alkyl" is understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3 or 5 carbon atoms. In particular, such radicals have 1, 2 or 3 carbon atoms ("C1-C3 alkyl"), for example methyl, ethyl, n-propyl or isopropyl.

[0171] The term "C1-C6 alkoxy" when used alone or as part of other substituents shall be understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms and an oxygen atom, or as C1-C6 alkyl-O-C1-C6 alkyl, as defined herein, wherein the oxygen atom may be attached to any carbon atom of the linear or branched C1-C6 alkyl radical. These include, but are not limited to, methoxy (CH3-O-), ethoxy (C2H5-O-), propoxy (C3H7-O-), and butoxy (C4H9-O-).

[0172] The term "cycloalkyl" or "carbocyclyl" when used alone or as part of another substituent refers to a cyclic alkyl group. m -C n "Cycloalkyl" is understood to mean a saturated, unsaturated or partially saturated carbon ring having m to n atoms. For example, "3-10 membered cycloalkyl" or "C3-C 10 "Cycloalkyl" refers to a cyclic alkyl group containing 3 to 10 carbon atoms, which may contain 1 to 3 rings. The cyclic alkyl group includes a monocyclic, bicyclic, tricyclic, spirocyclic or bridged ring. Examples of unsubstituted cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and adamantyl, or a bicyclic hydrocarbon group such as a decalin ring. The cycloalkyl group may be substituted with one or more substituents. In some embodiments, the cycloalkyl group may be a cycloalkyl group fused to an aryl or heteroaryl group.

[0173] The term "heterocycloalkyl" or "heterocyclyl" when used alone or as part of another substituent refers to a cycloalkyl group in which one or more (in some embodiments, 1 to 3) carbon atoms are replaced by heteroatoms such as, but not limited to, N, O, S, and P. The term "mn-membered heterocycloalkyl" or "C m -C n "Heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having m to n atoms. For example, the term "4-10 membered heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having 4 to 10 atoms. In some embodiments, the heterocycloalkyl can be a heterocycloalkyl fused to an aryl or heteroaryl group. When a prefix such as 3-8 membered is used to represent a heterocycloalkyl, the number of carbons is also meant to include heteroatoms. Monocyclic, bicyclic, tricyclic, spirocyclic or bridged rings are included.

[0174] The term "heteroaryl" is used interchangeably with the term "heteroaromatic ring" or "heteroaromatic group" when used alone or as part of another substituent and refers to a monocyclic or polycyclic aromatic ring system. In certain embodiments, one to three atoms in the ring system are heteroatoms, i.e., elements other than carbon, including but not limited to N, O, S, or P. Examples include furanyl, imidazolyl, indolinyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl. The heteroaryl group may be optionally fused to a benzene ring and may be monocyclic, bicyclic, tricyclic, spirocyclic, or bridged.

[0175] The term "5-10 membered heteroaryl" is understood, either alone or as part of another substituent, to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5 to 10 ring atoms and containing heteroatoms selected from N, O and S, and is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, which contains 1 to 5, preferably 1 to 3, heteroatoms independently selected from N, O and S, and which may additionally in each case be benzo-fused. "5-8 membered heteroaryl" is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5 to 8 ring atoms, in particular 5 or 6 carbon atoms, which contains 1 to 5 heteroatoms independently selected from N, O and S. Preference is given to monovalent monocyclic, bicyclic or tricyclic aromatic ring radicals having 1 to 3 heteroatoms independently selected from N, O and S and, in addition, in each case may be benzo-fused. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or acinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.

[0176] The term "halo" is used interchangeably with the term "halogen-substituted" when used alone or as part of another substituent. "Haloalkyl" or "halogen-substituted alkyl" refers to saturated aliphatic hydrocarbon groups, including branched and straight-chain groups, having the specified number of carbon atoms, substituted with one or more halogens (e.g., -CvFw, where v = 1 to 3 and w = 1 to (2v+1)). Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

[0177] Compounds provided herein include intermediates that can be used to prepare compounds provided herein, which contain reactive functional groups (such as, but not limited to, carboxyl, hydroxyl, and amino moieties), and also include protected derivatives thereof."Protected derivatives" are compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as blocking groups). Suitable carboxyl moiety protecting groups include benzyl, tert-butyl, etc., and isotopes, etc. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable blocking groups are well known to those of ordinary skill in the art.

[0178] In this application, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted aryl groups and unsubstituted aryl groups.

[0179] In this application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0180] As used herein, "pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are contemplated herein. These salts may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or may be used in the identification, characterization, or purification of the compounds of the present invention.

[0181] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformational isomers.

[0182] Depending on the choice of starting materials and methods, the compounds of the present invention may exist in one of the possible isomers or in a mixture thereof, for example as pure optical isomers, or as a mixture of isomers, such as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. When describing 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 the chiral center (or centers) in the molecule. The prefixes D and L or (+) and (–) are used to designate the signs for the rotation of plane-polarized light caused by the compound, where (–) or L indicates that the compound is levorotatory. Compounds prefixed with (+) or D are dextrorotatory.

[0183] When bonds to chiral carbon atoms in formulae of the present invention are depicted as straight lines, it is understood that both the (R) and (S) configurations of the chiral carbon atoms and the enantiomerically pure compounds and mixtures thereof are encompassed within the scope of the formula. The diagrammatic representation of racemates or enantiomerically pure compounds herein is adapted from Maehr, J. Chem. Ed. 1985, 62: 114-120. Wedge-shaped bonds and dashed bonds are used to represent the absolute configuration of a stereocenter.

[0184] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture with physical and chemical properties consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0185] As used herein, a "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0186] In this application, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

[0187] As used herein, the term "prodrug" refers to a compound of the present invention that can be converted to a biologically active compound under physiological conditions or by solvolysis. Prodrugs of the present invention are prepared by modifying functional groups within the compound. These modifications can be removed by conventional procedures or in vivo to yield the parent compound. Prodrugs include compounds in which a hydroxyl or amino group within a compound of the present invention is attached to any group. When a prodrug of a compound of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free hydroxyl group or free amino group, respectively.

[0188] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0189] As used herein, the term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients enhance the handling characteristics of pharmaceutical formulations, i.e., by increasing flowability and / or adhesion, making the formulation more suitable for direct compression.

[0190] As used herein, the term "treatment" and other similar synonyms include the following meanings:

[0191] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0192] (ii) inhibiting the disease or condition, i.e., curbing its development;

[0193] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0194] (iv) Alleviate the symptoms of the disease or condition.

[0195] For each step of the reaction, the reaction temperature can be appropriately selected according to the solvent, starting materials, reagents, etc., and the reaction time can also be appropriately selected according to the reaction temperature, solvent, starting materials, reagents, etc. After the reaction of each step is completed, the target compound can be separated and purified from the reaction system according to conventional methods, such as filtration, extraction, recrystallization, washing, silica gel column chromatography, etc. If it does not affect the next step of the reaction, the target compound can also be directly entered into the next step of the reaction without separation and purification. Each step of the reaction of the present invention is preferably carried out in an inert solvent, and the inert solvent includes but is not limited to: toluene, benzene, water, methanol, ethanol, isopropanol, ethylene glycol, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, or a combination thereof. DETAILED DESCRIPTION

[0196] The present invention is further described below with reference to specific examples. It should be understood that the following description is only the most preferred embodiment of the present invention and should not be considered as limiting the scope of protection of the present invention. On the basis of a full understanding of the present invention, the experimental methods in the following examples where no specific conditions are specified are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0197] Those skilled in the art may make non-essential changes to the technical solution of the present invention, and such changes should be considered as included in

[0198] The present invention is within the scope of protection.

[0199] The following reagents were used herein:

[0200] SEM, namely (trimethylsilyl)ethoxymethyl, has the chemical formula -CH2OCH2CH2Si(CH3)3, and is an amino protecting group.

[0201] Boc, or tert-butyloxycarbonyl, is an amino protecting group.

[0202] PMB, or p-methoxybenzyl, is a protecting group for amino groups.

[0203] Example 1: Preparation of Compound I-1

[0204] The synthetic route is as follows:

[0205] Step 1: Synthesis of methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (01B)

[0206] (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoic acid (1.20 g, 3.49 mmol) was dissolved in anhydrous DMF (10 mL) at room temperature. Potassium carbonate (726 mg, 5.25 mmol) and iodomethane (1.03 g, 7.23 mmol) were added sequentially at 0°C. After complete conversion of the starting material was monitored by TLC, the mixture was diluted with 20 mL of water and extracted with tert-butyl methyl ether (10 mL x 3). The combined organic phases were washed once with saturated NaHCO₃ solution (10 mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to yield methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (01B) (1.21 g, 97.2% yield).

[0207] LC-MS,M / Z(ESI):358.0,360.0[M+H] + .

[0208] Step 2: Synthesis of methyl (S)-3-((tert-butyloxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01C)

[0209] To a 100 mL round-bottom flask was added methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (1.21 g, 3.39 mmol), 1-methyl-1H-pyrazol-5-ylboronic acid (643 mg, 5.11 mmol), Pd(PPh3)2Cl2 (249 mg, 0.355 mmol), Na2CO3 (1.08 g, 10.2 mmol), DME (10 mL), and H2O (2.5 mL). The mixture was sonicated, the atmosphere was replaced with nitrogen three times, and the mixture was stirred at 100°C. After complete conversion of the starting material as monitored by TLC, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 3:1) gave (S)-methyl 3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01C) (360 mg, 29.7% yield).

[0210] LC-MS, M / Z(ESI):360.2[M+H] + .

[0211] Step 3: Synthesis of methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01D)

[0212] To a 100 mL single-necked flask, (S)-methyl 3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (600 mg, 1.67 mmol), dichloromethane (20 mL), and trifluoroacetic acid (3.81 g, 33.4 mmol) were added sequentially and stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in dichloromethane (20 mL), washed sequentially with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01D) (430 mg, 99.3% yield).

[0213] LC-MS, M / Z(ESI):260.13[M+H] + .

[0214] Step 4: Synthesis of methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetylamino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01E)

[0215] To a 100 mL single-necked flask, (S)-methyl 3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (400 mg, 1.54 mmol), DMF (10 mL), BOC-glycine (298 mg, 1.70 mmol), TBTU (740 mg, 2.30 mmol), and DIPEA (598 mg, 4.63 mmol) were added sequentially. The mixture was stirred at room temperature for 16 h under nitrogen. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave (S)-methyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01E) (600 mg, 93.4% yield).

[0216] LC-MS, M / Z(ESI):417.27[M+H] + .

[0217] Step 5: Synthesis of (S)-3-(2-aminoacetamide)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01F)

[0218] To a 100 mL single-necked flask, (S)-methyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (600 mg, 1.44 mmol), dichloromethane (10 mL), and trifluoroacetic acid (3.29 g, 28.85 mmol) were added sequentially and stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to obtain a crude product. The crude product was dissolved in dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01F) (460 mg).

[0219] LC-MS, M / Z(ESI):317.32[M+H] + .

[0220] Step 6: Synthesis of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01H)

[0221] To a 250 mL single-necked flask, 1H-imidazole-4-carbonitrile (6.00 g, 64.5 mmol), acetone (60 mL), and potassium carbonate (17.82 g, 128.9 mmol) were added sequentially and cooled to 0°C in an ice bath. SEMCl (11.82 g, 70.90 mmol) was added, and the mixture was stirred in an ice bath for 20 min, followed by stirring at room temperature for 16 h. The reaction mixture was diluted with ethyl acetate (200 mL) and washed with water (200 mL). The resulting organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-1:1) afforded 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (8.00 g, 55.6% yield).

[0222] LC-MS, M / Z(ESI):224.11[M+H] + .

[0223] Step 7: Synthesis of 2-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01I)

[0224] To a 250 mL single-necked flask were added 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (8.00 g, 35.8 mmol), carbon tetrachloride (100 mL), NBS (7.01 g, 39.4 mmol), and AIBN (590 mg, 3.59 mmol) in sequence, and the mixture was stirred at 60°C for 6 h. After cooling to room temperature, the reaction solution was diluted with ethyl acetate (200 mL), washed sequentially with saturated sodium bicarbonate solution (200 mL) and saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-1:1) afforded 2-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01I) (4.50 g, 41.6% yield).

[0225] LC-MS, M / Z(ESI):302.02[M+H] + .

[0226] Step 8: Synthesis of 2-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01J)

[0227] To a 100 mL single-necked flask were added 2-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (3.00 g, 9.93 mmol), 4-methoxybenzylamine (2.04 g, 14.9 mmol), Pd2(dba)3 (454 mg, 0.496 mmol), Xantphos (574 mg, 0.992 mmol), cesium carbonate (9.70 g, 29.8 mmol), and 1,4-dioxane (30 mL) in sequence, and the mixture was stirred at 100°C for 16 h. The reaction mixture was filtered and concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave 2-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01J) (850 mg, 23.9% yield).

[0228] 1 H NMR (400MHz, DMSO-d6) δ7.75 (s, 1H), 7.25 (d, J = 8.6Hz, 2H), 6.90–6.84 (m, 3H), 5.19 (s, 2H), 4.32(d,J=5.9Hz,2H),3.72(s,3H),3.49–3.45(m,2H),0.82(d,J=8.0Hz,2H),-0.04(s,9H).

[0229] LC-MS, M / Z(ESI):359.47[M+H] + .

[0230] Step 9: Synthesis of ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoate (01K)

[0231] To a 100 mL single-necked flask, 2-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (850 mg, 2.37 mmol) and DMF (10 mL) were added sequentially. Under nitrogen protection, the temperature was lowered to -10°C, NaH (60%, 142 mg, 3.55 mmol) was added, and the mixture was stirred at this temperature for 0.5 h. Ethyl 4-bromobutyrate (694 mg, 3.56 mmol) was added, and the mixture was stirred at -10°C for 0.5 h, then transferred to room temperature and stirred for 2 h. Saturated aqueous ammonium chloride solution was added, followed by 10 mL of water, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoate (200 mg, 17.8% yield).

[0232] 1 H NMR (400MHz, DMSO-d6) δ8.05(s,1H),7.15(d,J=8.6Hz,2H),6.85(d,J=8.7Hz,2H),5.21(s,2H),4.17(s,2H),4.00–3.94(m,2H),3.71(s,3H) ),3.51–3.47(m,2H),3.04–2.99(m,2H),2.21(t,J=7.4Hz,2H),1.66-1.61(m,2H),1.12(t,J=7.1Hz,3H),0.84–0.78(m,2H),-0.06(s,9H).

[0233] LC-MS, M / Z(ESI):473.51[M+H] + .

[0234] Step 10: Synthesis of 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoic acid (01L)

[0235] To a 100 mL single-necked flask, ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoate (200 mg, 0.423 mmol) and THF (9 mL) were added sequentially. The mixture was dissolved in an aqueous solution (3 mL) of lithium hydroxide monohydrate (23.1 mg, 0.550 mmol) and stirred at room temperature for 6 h. The THF was removed by concentration under reduced pressure, and water (10 mL) was added. The pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL x 3). The combined organic phase was washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoic acid (01L) (100 mg, 53.2% yield).

[0236] LC-MS, M / Z(ESI):445.22[M+H] + .

[0237] Step 11: Synthesis of methyl (S)-3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01M)

[0238] To a 100 mL single-necked flask were added 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoic acid (100 mg, 0.225 mmol), DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (71.2 mg, 0.225 mmol), TBTU (108 mg, 0.321 mmol), and DIPEA (87.2 mg, 0.675 mmol). The mixture was stirred at room temperature under nitrogen for 16 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave (S)-methyl 3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (90.0 mg, 53.9% yield).

[0239] LC-MS, M / Z(ESI):743.36[M+H] + .

[0240] Step 12: Synthesis of (S)-3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (01N)

[0241] To a 100 mL single-necked flask, (S)-methyl 3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (90.0 mg, 0.121 mmol), THF (6 mL), and an aqueous solution (2 mL) of lithium hydroxide monohydrate (6.6 mg, 0.16 mmol) were added sequentially, and the mixture was stirred at room temperature for 6 h. The THF was removed by concentration under reduced pressure, and water (5 mL) was added. The pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was extracted with ethyl acetate (5 mL x 3). The combined organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give (S)-3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (70.0 mg, 79.3% yield).

[0242] LC-MS, M / Z(ESI):729.35[M+H] + .

[0243] Step 13: Synthesis of (S)-3-(2-(4-((4-cyano-1H-imidazol-2-yl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-1)

[0244] To a 100 mL single-necked bottle, (S)-3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (70.0 mg, 96.0 μmol), dichloromethane (8 mL) and trifluoroacetic acid (438 mg, 3.84 mmol) were added sequentially and stirred at room temperature for 6 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified by reverse phase chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% aqueous ammonia, B = acetonitrile; gradient: 30%-100%) to give (S)-3-(2-(4-((4-cyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-1) (11 mg, 24% yield).

[0245] 1H NMR (400MHz, DMSO-d6) δ8.44(d,J=8.3Hz,1H),8.09(t,J=5.8Hz,1H),7.72(s, 1H),7.45–7.42(m,2H),7.41(d,J=7.4Hz,1H),7.39–7.33(m,2H),6.38(d,J=1 .8Hz,1H),5.23(q,J=7.4Hz,1H),3.82(s,3H),3.69(d,J=5.8Hz,2H),3.12(t, J=7.0Hz,2H),2.73(d,J=7.2Hz,2H),2.16(t,J=7.4Hz,2H),1.73–1.66(m,2H).

[0246] LC-MS, M / Z(ESI):479.3[M+H] + .

[0247] Example 2: Preparation of Compound I-2

[0248] The synthetic route is as follows:

[0249] Step 1: Synthesis of ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (02B)

[0250] To a 100 mL single-necked flask, tert-butyl (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)carbamate (2.80 g, 15.3 mmol) and DMF (30 mL) were added. Under nitrogen protection, the mixture was cooled to 0°C in an ice bath, and NaH (1.07 g, 26.8 mmol, 60%) was added. After stirring at 0°C for 0.5 h, ethyl 4-bromobutyrate (7.47 g, 38.3 mmol) and potassium iodide (2.54 g, 15.3 mmol) were added. The mixture was stirred at 0°C for 1 h, then transferred to room temperature and stirred for 16 h. The reaction was quenched by the addition of saturated ammonium chloride, and water (30 mL) was added, followed by extraction with ethyl acetate (60 mL x 3). The combined organic phases were washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-2:3) gave the product ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (02B) (2.80 g, 61.5% yield) as a colorless oil.

[0251] LC-MS, M / Z(ESI):428.35[M+H] + .

[0252] Step 2: Synthesis of 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (02C)

[0253] To a 100 mL single-necked flask, ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (215 mg, 0.503 mmol), THF / H2O (4 mL, v:v=1:1), and lithium hydroxide monohydrate (63 mg, 1.5 mmol) were added sequentially and stirred at room temperature for 3 h. After the reaction was completed, 1N HCl (2 mL) and ethyl acetate (20 mL) were added for extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (02C) (200 mg, 99.6% yield).

[0254] LC-MS, M / Z(ESI):400.35[M+H] + .

[0255] Step 3: Synthesis of methyl (3S)-3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyryl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (02D)

[0256] To a 100 mL single-necked flask were added 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (02C) (200 mg, 0.501 mmol), DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01F) (158 mg, 0.499 mmol), TBTU (176 mg, 0.548 mmol), and DIPEA (194 mg, 1.50 mmol) in sequence, and the mixture was stirred at room temperature for 16 h. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give (3S)-methyl 3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (02D) (230 mg, 66.0% yield).

[0257] LC-MS, M / Z(ESI):698.44[M+H] + .

[0258] Step 4: Synthesis of (3S)-3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyryl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (02E)

[0259] To a 100 mL single-necked bottle, (3S)-methyl 3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (230 mg, 0.330 mmol), THF / H2O (4 mL, v:v=1:1), and lithium hydroxide monohydrate (42 mg, 1.0 mmol) were added sequentially, and the mixture was stirred at room temperature for 3 h. 1N HCl (1 mL) and ethyl acetate (20 mL) were added for extraction. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give (3S)-3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (02E) (225 mg, 99.8% yield).

[0260] LC-MS, M / Z(ESI):684.45[M+H] + .

[0261] Step 5: Synthesis of (3S)-3-((N-(4-((1H-imidazol-2-yl)amino)butyryl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-2)

[0262] To a 100 mL single-necked bottle was added (3S)-3-((N-(4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (225 mg, 0.329 mmol), DCM (5 mL) and TFA (2 mL) in sequence, and the mixture was stirred at room temperature for 2 days. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified by reverse phase chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% aqueous ammonia, B = acetonitrile; gradient: 30%-100%) to give (3S)-3-((N-(4-((1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-2) (73 mg, 49% yield).

[0263] 1H NMR (400MHz, DMSO-d6) δ9.47(d,J=8.0Hz,1H),8.69(s,1H),8.46(t,J=6.0Hz,1H),7.45–7.39(m,2H),7.39–7.30(m,3H),6.74 (s,2H),6.36(d,J=2.0Hz,1H),5.04(m,1H),3.80(m,5H),3.55(m,2H),2.65–2.52(m,2H),2.37(t,J=6.8Hz,2H),1.85(m,2H).

[0264] LC-MS, M / Z(ESI):454.31[M+H] + .

[0265] Example 3: Preparation of Compound I-3

[0266] The synthetic route is as follows:

[0267] Step 1: Synthesis of methyl 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyrate (03C)

[0268] To a 100 mL single-necked flask, 2-amino-4,5-imidazole dicarbonitrile (150 mg, 1.13 mmol), methanol (5 mL), methyl 4-oxobutanoate (131 mg, 1.13 mmol), and acetic acid (135 mg, 2.25 mmol) were added sequentially. Under nitrogen, the mixture was stirred at room temperature for 16 h. Sodium cyanoborohydride (212 mg, 3.37 mmol) was added, and the mixture was stirred at room temperature for 3 h. The reaction was quenched by the addition of saturated ammonium chloride solution, and water (5 mL) was added. The organic solvent was removed by concentration under reduced pressure. Ethyl acetate (10 mL x 3) was added for extraction, and the combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave methyl 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanoate (03B) (100 g, yield 38.0%).

[0269] 1 H NMR (400MHz, DMSO-d6) δ12.47(s,1H),7.29(t,J=5.5Hz,1H),3.55(s,3H),3.12(q,J=6.6Hz,2H),2.32(t,J=7.4Hz,2H),1.75–1.68(m,2H).

[0270] LC-MS, M / Z(ESI):234.23[M+H]+ .

[0271] Step 2: Synthesis of 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyric acid (03D)

[0272] To a 100 mL single-necked flask, methyl 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanoate (50 mg, 0.21 mmol), tetrahydrofuran (5 mL), and lithium hydroxide monohydrate (11.7 mg, 0.279 mmol) dissolved in water (0.5 mL) were added sequentially and stirred at room temperature for 16 h. The organic solvent was removed by concentration under reduced pressure, and the pH was adjusted to 7 with 1N hydrochloric acid. The product, 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanoic acid (03D), was obtained and used directly in the next reaction.

[0273] LC-MS, M / Z(ESI):220.21[M+H] + .

[0274] Step 3: Synthesis of (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid methyl ester hydrochloride (03E)

[0275] To a 100 mL single-necked flask, (S)-methyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (3.2 g, 7.7 mmol), methanol (20 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 9.61 mL) were added sequentially and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (03E) (2.6 g, 96% yield).

[0276] LC-MS, M / Z(ESI):317.32[M+H] + .

[0277] Step 4: Synthesis of methyl (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (03F)

[0278] To a 100 mL single-necked flask was added 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyric acid obtained in step 2, DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (91 mg, 0.26 mmol), TBTU (103 mg, 0.321 mmol), and DIPEA (111 mg, 0.859 mmol). The mixture was stirred at room temperature for 16 h under nitrogen. Water (10 mL) and ethyl acetate (20 mL x 3) were added for extraction. The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to yield the crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) gave (S)-methyl 3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (03D) (35 mg, 32% yield over two steps).

[0279] LC-MS, M / Z(ESI):518.26[M+H] + .

[0280] Step 5: Synthesis of (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-3)

[0281] To a 100 mL single-necked flask, (S)-methyl 3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (35 mg, 0.068 mmol), tetrahydrofuran (5 mL), and lithium hydroxide monohydrate (3.7 mg, 0.088 mmol) dissolved in water (0.5 mL) were added sequentially and stirred at room temperature for 3 h. The organic solvent was removed by concentration under reduced pressure, the pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was concentrated under reduced pressure to give the crude product. After reverse phase preparation (column: YMC-Triart Prep C18 (30mm×40cm, 7μm); solvent: A=0.1% formic acid, B=acetonitrile; gradient: 42%–90%), (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5)-yl)phenyl)propanoic acid (I-3) (3.2 mg, yield 9.4%) was obtained.

[0282] 1H NMR (400MHz, DMSO-d6) δ8.54(d,J=8.3Hz,1H),8.14–8.11(m,1H),7.44–7.32(m,5H),6.37(d,J=1.7Hz,1H),5.18(q,J=7.2H z,1H),3.81(s,3H),3.67–3.63(m,2H),3.08–3.05(m,2H),2.69(d,J=6.8Hz,2H),2.16(t,J=7.4Hz,2H),1.71–1.64(m,2H).

[0283] LC-MS, M / Z(ESI):504.41[M+H] + .

[0284] Example 4: Preparation of Compound I-4

[0285] The synthetic route is as follows:

[0286] Step 1: Synthesis of ethyl 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (04A)

[0287] To a 100 mL single-necked flask, ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (400 mg, 0.935 mmol), DMF (10 mL), and N-chlorosuccinimide (125 mg, 0.936 mmol) were added sequentially and stirred at room temperature for 2 h. The reaction solution was heated to 40°C and stirred for an additional 16 h. Water (10 mL) and ethyl acetate (20 mL x 3) were added for extraction. The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave ethyl 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (04A) (280 mg, 64.8% yield).

[0288] LC-MS,M / Z(ESI):462.3,464.3[M+H] + .

[0289] Step 2: Synthesis of 4-((tert-Butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (04B)

[0290] To a 100 mL single-necked flask were added ethyl 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoate (70 mg, 0.15 mmol), THF (5 mL), and lithium hydroxide monohydrate (19 mg, 0.45 mmol) dissolved in water (0.5 mL), and stirred at room temperature for 16 h. The organic solvent was removed by concentration under reduced pressure, and water (5 mL) was added. The pH was adjusted to 6 with 1N hydrochloric acid, and ethyl acetate (10 mL × 3) was added for extraction. The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (04B) (66 mg, 100% yield).

[0291] LC-MS,M / Z(ESI):434.3,436.3[M+H] + .

[0292] Step 3: Synthesis of (S)-5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl)-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleic acid methyl ester (04C)

[0293] To a 100 mL single-necked flask were added 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butanoic acid (66 mg, 0.15 mmol), DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (54 mg, 0.15 mmol), TBTU (73 mg, 0.23 mmol), and DIPEA (79 mg, 0.61 mmol). The mixture was stirred at room temperature for 16 h under nitrogen. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by column chromatography (dichloromethane / methanol (V / V) = 1:0-9:1) to give (S)-5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl)-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleic acid methyl ester (04C) (60 mg, 54% yield).

[0294] LC-MS,M / Z(ESI):732.3,734.3[M+H] + .

[0295] Step 4: Synthesis of (S)-5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleic acid (04D)

[0296] To a 100 mL single-necked bottle, (S)-methyl 5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl)-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleate (60 mg, 0.082 mmol), THF (6 mL), and lithium hydroxide monohydrate (10 mg, 0.24 mmol) dissolved in water (1 mL) were added sequentially and stirred at room temperature for 6 h. The organic solvent was removed by concentration under reduced pressure, and water (5 mL) was added. The pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was extracted with ethyl acetate (5 mL x 3). The combined organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give (S)-5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleic acid (04D) (45 mg, 76% yield).

[0297] LC-MS,M / Z(ESI):718.4,720.4[M+H] + .

[0298] Step 5: Synthesis of (S)-3-(2-(4-((4-chloro-1H-imidazol-2-yl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl))phenyl)propanoic acid (I-4)

[0299] To a 100 mL single-necked bottle, (S)-5-(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2,2-dimethyl-14-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecane-16-oleic acid (45 mg, 0.063 mmol), dichloromethane (4 mL), and trifluoroacetic acid (1 mL) were added sequentially and stirred at room temperature for 16 h. The crude product was concentrated under reduced pressure and subjected to reverse phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); solvent: A = 0.1% formic acid, B = acetonitrile; gradient: 42%–98%) to give (S)-3-(2-(4-((4-chloro-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl))phenyl)propanoic acid (I-4) (3.4 mg, 11% yield).

[0300] 1 H NMR (400MHz, DMSO-d6) δ10.05 (s, J=8.3Hz, 1H), 8.44 (d, J=8.3Hz, 1H), 8.11 (t,J=5.7Hz,1H),7.50–7.34(m,5H),6.54(s,1H),6.44–6.37(m,1H),5.97(s ,1H),5.25(q,J=7.5Hz,1H),3.84(s,3H),3.70(d,J=5.7Hz,2H),3.04(q,J= 6.3Hz,2H),2.75(d,J=7.2Hz,2H),2.17(t,J=7.4Hz,2H),1.72–1.65(m,2H).

[0301] LC-MS,M / Z(ESI):488.2,490.2[M+H] + .

[0302] Example 5: Preparation of Compound I-5

[0303] Step 1: Synthesis of (S)-3-(2-(4-((4-formamido-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (05A)

[0304] To a 100 mL single-necked flask, (S)-methyl 3-(2-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (300 mg, 0.404 mmol), THF (3 mL), and an aqueous solution (3 mL) of lithium hydroxide monohydrate (84.8 mg, 2.02 mmol) were added in sequence, and the mixture was stirred at room temperature for 6 h. The THF was removed by concentration under reduced pressure, and water (5 mL) was added. The pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was extracted with ethyl acetate (5 mL x 3). The combined organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give (S)-3-(2-(4-((4-formamido-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (180 mg, 59.7% yield).

[0305] LC-MS, M / Z(ESI):747.36[M+H] + .

[0306] Step 2: Synthesis of (S)-3-(2-(4-((4-formamido-1H-imidazol-2-yl)amino)butyramido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-5)

[0307] To a 100 mL single-necked bottle was added (S)-3-(2-(4-((4-formamido-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (180 mg, 0.241 mol), dichloromethane (2 mL), trifluoroacetic acid (2 mL), anisole (199 mg, 1.84 mmol) and trifluoromethanesulfonic acid (170 mg, 1.13 mmol) in sequence and stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified by reverse phase chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% aqueous ammonia, B = acetonitrile; gradient: 30%-100%) to give (S)-3-(2-(4-((4-formamido-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoic acid (I-5) (65 mg, 54% yield).

[0308] 1 H NMR(400MHz, DMSO-d6)δ8.62(d,J=8.4Hz,1H),8.19(q,J=6.4Hz,1H),7.47–7.43 (m,2H),7.41(t,J=6.8Hz,1H),7.39–7.35(m,2H),7.13(s,1H),6.39(d,J=2.0Hz ,1H),6.25(s,1H),5.21(q,J=7.2Hz,1H),3.83(s,3H),3.76–3.62(m,2H),3.13( d,J=6.0Hz,2H),2.72(d,J=7.2Hz,2H),2.21(t,J=7.2Hz,2H),1.76–1.68(m,2H).

[0309] LC-MS, M / Z(ESI):497.22[M+H] + .

[0310] Example 6: Preparation of Compounds II-1A & II-1B

[0311] The synthetic route is as follows:

[0312] Step 1: Synthesis of ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (06B)

[0313] To a 500 mL single-necked flask, ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propanoate (14.00 g, 37.01 mmol), dichloromethane (150 mL), and di-tert-butyl dicarbonate (12.12 g, 55.53 mmol) were added sequentially and stirred at room temperature for 3 h. The reaction solution was washed with water (150 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-1:1) afforded ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (06B) (14.00 g, 79.07% yield).

[0314] LC-MS,M / Z(ESI):478.12,480.11[M+H] + .

[0315] Step 2: Synthesis of ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (06C)

[0316] To a 250 mL single-necked bottle, ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (14.00 g, 29.27 mmol), 1-methyl-1H-pyrazole-5-boronic acid pinacol ester (12.18 g, 58.54 mmol), Pd2(dba)3 (1.07 g, 1.17 mmol), tricyclohexylphosphine (657 mg, 2.34 mmol), dipotassium hydrogen phosphate (10.20 g, 58.56 mmol), 1,4-dioxane (100 mL), and water (30 mL) were added in sequence and stirred at 100°C for 16 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (06C) (14.00 g, yield 99.75%).

[0317] LC-MS, M / Z(ESI):480.24[M+H] +.

[0318] Step 3: Synthesis of ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (06D)

[0319] To a 250 mL single-necked flask, ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (8.00 g, 16.7 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 41.7 mL) were added sequentially and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain the product, ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (06D) (6.00 g, 86.5% yield).

[0320] LC-MS, M / Z(ESI):380.19[M+H] + .

[0321] Step 4: Synthesis of ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propanoate (06E)

[0322] Ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (1.0 g, 2.4 mmol) was dissolved in anhydrous DMF (10 mL) at room temperature, followed by the addition of TBTU (0.93 g, 2.9 mmol). DIPEA (1.02 g, 7.89 mmol) was added at 0°C, and the mixture was allowed to return to room temperature and stirred for half an hour. N-(tert-butoxycarbonyl)glycine (0.46 g, 2.6 mmol) was then added and stirred. After completion of the reaction, as monitored by TLC, a saturated NH4Cl solution (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed once with saturated NaHCO3 solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:1-1:0) to give compound 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionic acid ethyl ester (06E) (0.80 g, yield 62%).

[0323] LC-MS, M / Z(ESI):537.6[M+H] + .

[0324] Step 5: Synthesis of ethyl 3-((N-(tert-butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (06F)

[0325] Ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propanoate (0.80 g, 1.5 mmol) was dissolved in methanol (10 mL) at room temperature. 10% wet palladium on carbon (100 mg) was added, and the hydrogen atmosphere was replaced three times. The reaction was stirred at room temperature while maintaining a hydrogen atmosphere. After complete conversion of the starting material as monitored by TLC, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to provide ethyl 3-((N-(tert-butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (06F) (0.66 g, 99% yield).

[0326] LC-MS, M / Z(ESI):447.5[M+H] + .

[0327] Step 6: Synthesis of ethyl 3-(4-((14-azido-3,6,9,12-tetraoxotetrakan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propanoate (06G)

[0328] Ethyl 3-((N-(tert-butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (0.66 g, 1.5 mmol) was dissolved in anhydrous DMF (5 mL) at room temperature. Potassium carbonate (0.51 g, 3.7 mmol) and azide-pentaethylene glycol-p-toluenesulfonate (0.80 g, 1.9 mmol) were added, and the mixture was stirred at 80°C for 6 hours. After complete conversion of the starting material, as monitored by TLC, the mixture was returned to room temperature, saturated NaHCO3 solution (10 mL) was added, and extraction was performed with ethyl acetate (10 mL x 3). The combined organic phases were washed once with saturated NaCl solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 3-(4-((14-azido-3,6,9,12-tetrahydrotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionic acid ethyl ester (06G) (0.97 g, yield 95%).

[0329] LC-MS, M / Z(ESI):692.8[M+H] + .

[0330] Step 7: Synthesis of ethyl 3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycylamino)propionate hydrochloride (06H)

[0331] Ethyl 3-(4-((14-azido-3,6,9,12-tetrahydrotetradecane-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propanoate (0.97 g, 1.4 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (7 mL) at room temperature and stirred for half an hour. After complete conversion of the starting material as monitored by TLC, the mixture was concentrated under reduced pressure to afford the crude product. Ethyl acetate was added, stirred, and the filter cake was collected by filtration and dried under vacuum to afford ethyl 3-(4-((14-azido-3,6,9,12-tetrahydrotetradecane-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycylamino)propanoate hydrochloride (06H) (0.88 g, 100% yield).

[0332] LC-MS, M / Z(ESI):592.6[M+H] + .

[0333] Step 8: Synthesis of ethyl 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoate (06I)

[0334] To a 100 mL single-necked flask were added 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoic acid (0.88 g, 2.0 mmol), DMF (20 mL), ethyl 3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycinamido)propanoate hydrochloride (1.5 g, 2.4 mmol), TBTU (0.77 g, 2.4 mmol), and DIPEA (0.77 g, 6.0 mmol). The mixture was stirred at room temperature for 16 h under nitrogen. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude product. The product was purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give ethyl 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoate (06I) (1.5 g, 74% yield).

[0335] LC-MS, M / Z(ESI):1018.5[M+H] + .

[0336] Step 9: Synthesis of 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoic acid (06J)

[0337] To a 100 mL single-necked bottle, ethyl 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoate (1.0 g, 0.98 mmol), THF / H2O (v:v = 1:1, 5 mL), and lithium hydroxide monohydrate (50 mg, 1.2 mmol) were added sequentially, and the mixture was stirred at room temperature for 3 h. The THF was removed by concentration under reduced pressure, and the pH of the residue was adjusted to 6 with 1N hydrochloric acid. Ethyl acetate (5 mL x 3) was added for extraction. The combined organic phase was washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoic acid (06J) (734 mg, 75% yield).

[0338] LC-MS, M / Z(ESI):990.5[M+H] + .

[0339] Step 10: Synthesis of 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butyryl)glycyl)amino)propanoic acid (II-1)

[0340] To a 100 mL single-necked bottle was added 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)((4-methoxyphenyl)methyl)amino)butanoyl)glycyl)amino)propanoic acid (634 mg, 0.640 mmol), dichloromethane (20 mL), trifluoroacetic acid (6 mL, 80 mmol), anisole (697 mg, 6.44 mmol), and trifluoromethanesulfonic acid (510 mg, 3.40 mmol) in sequence, and the mixture was stirred at room temperature for 3 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by reverse phase chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 10%-95%) to give 3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)propanoic acid (II-1) (450 mg, 95.0% yield).

[0341] 1 H NMR (600MHz, DMSO-d6) δ11.25(s,1H),8.44(s,1H),8.07(s,1H),7.49(s,1H),7.42(d,J=6.0Hz, 1H),7.37–7.31(m,1H),7.20(d,J=2.8Hz,1H),7.08(d,J=8.4Hz,1H),6.24(s,1H),5.17(t,J=8.0 Hz,1H),4.20–4.02(m,2H),3.71–3.58(m,7H),3.61–3.56(m,2H),3.56–3.45(m,12H),3.37(d,J =4.4Hz,2H),3.12–3.02(m,2H),2.68(d,J=7.2Hz,2H),2.17(t,J=7.2Hz,2H),1.72–1.63(m,2H).

[0342] LC-MS, M / Z(ESI):740.3[M+H] + .

[0343] Step 11: Synthesis of (R)-3-(4-((14-azido-3,6,9,12-tetraoxotetrakan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butyryl)glycyl)amino)propanoic acid (II-1A) and (S)-3-(4-((14-azido-3,6,9,12-tetraoxotetrakan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butyryl)glycyl)amino)propanoic acid (II-1B)

[0344] 3-(4-((14-azido-3,6,9,12-tetraoxotetrakan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)propanoic acid was separated by SFC (column: (S,S) WHELK-O1 (250 mm × 30 mm, 3.5 μm, mobile phase: mobile phase: A = CO2, B = =methanol (0.05% ethylenediamine); gradient: 30-60%) to give compound (R)-3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)propanoic acid (II-1A; column: (S,S) Whelk-O1 50×4.6 mm ID, 3.5 μm; mobile phase: A = CO2, B = methanol (0.05% ethylenediamine); gradient: 30-60%; flow rate: 3 mL / min; retention time: 1.471 min) and compound (S)-3-(4-((14-azido-3,6,9,12-tetraoxotetracan-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(4-((4-cyano-1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)propanoic acid (II-1B; column: (S,S) Whelk-O1 50×4.6 mm ID, 3.5 μm; mobile phase: A = CO2, B = methanol (0.05% ethylenediamine); gradient: 30-60%; flow rate: 3 mL / min; retention time: 2.159 min).

[0345] II-1A: 1H NMR(400MHz,DMSO-d6)δ12.50–11.15(m,1H),8.50(s,1H),8.15–8.05(m,1H),7.49(s,1H) ,7.42(s,1H),7.37–7.31(m,1H),7.20(d,J=2.8Hz,1H),7.08(d,J=8.4Hz,1H),6.25(s,1H) ,5.20–5.08(m,1H),4.20–4.02(m,2H),3.71–3.58(m,7H),3.60–3.45(m,14H),3.40–3.30( m,2H),3.12–3.02(m,2H),2.68(d,J=7.2Hz,2H),2.17(t,J=7.2Hz,2H),1.80–1.60(m,2H).

[0346] II-1B: 1 H NMR (400MHz, DMSO-d6) δ12.4–11.4(m,1H),8.44(s,1H),8.15–8.05(m,1H),7.49(s,1H),7.42 (s,1H),7.37–7.31(m,1H),7.20(d,J=2.8Hz,1H),7.10–7.08(m,1H),6.24(s,1H),5.20–5.10( m,1H),4.20–4.02(m,2H),3.71–3.58(m,7H),3.61–3.56(m,2H),3.56–3.45(m,12H),3.40–3. 30(m,2H),3.12–3.02(m,2H),2.68(d,J=7.2Hz,2H),2.17(t,J=7.2Hz,2H),1.80–1.60(m,2H).

[0347] Example 7: Preparation of Compound III-1

[0348] The synthetic route is as follows:

[0349] Step 1: Synthesis of 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diaza-1-yl-20-oic acid (07B)

[0350] To a 100 mL single-necked bottle, 4-amino-4-(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-N1,N7-bis(2-(2-((prop-2-yn-1-yl)oxy)methoxy)ethyl)pimelanediamide (500 mg, 0.803 mmol), DCM (5 mL), triethylamine (0.5 mL) and dihydro-4,4-dimethyl-2H-pyranyl-2,6(3H)-dione (137 mg, 0.964 mmol) were added in sequence and stirred at room temperature for 3 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to give 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-oic acid (500 mg, 81.4% yield).

[0351] Step 2: Synthesis of 18,18-dimethyl-11,16-dioxo-14,14-bis{3-oxo-3-[(2-(2-[(prop-2-yn-1-yl)oxy]ethoxy}ethyl)amino]propyl)-4,7-dioxo-10,15-diazepine-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1)

[0352] To a 100 mL single-necked bottle, 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-oic acid (500 mg, 0.654 mmol), DCM (5 mL), p-nitrophenol (109 mg, 0.784 mmol) and EDCI (150 mg, 0.782 mmol) were added in sequence and stirred at room temperature for 12 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to give the product 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1) (268 mg, yield 46.3%).

[0353] 1H NMR(400MHz,DMSO-d6)δ8.27(d,2H),7.81(t,3H),7.41(d,2H),7.21(s,1H),4.10(d,6H),3.51–3.49(m,12H),3. 38–3.36(m,9H),3.16–3.13(m,6H),2.73(s,2H),2.17(s,2H),2.01–1.97(m,6H),1.80–1.76(d,6H),1.09(s,6H).

[0354] LC-MS, M / Z(ESI):887.3[M+H] + .

[0355] Example 8: Preparation of Compound III-2

[0356] The synthetic route is as follows:

[0357] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (07B)

[0358] To a 100 mL single-necked bottle, bis(perfluorophenyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo-3-(perfluorophenoxy)propyl)heptanedioate (3.0 g, 3.1 mmol), DCM (30 mL) and 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (2.1 g, 11 mmol) were added in sequence and stirred at room temperature for 3 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (3.0 g, yield 99%).

[0359] Step 2: Synthesis of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)pimelamide (07C)

[0360] To a 100 mL single-necked bottle, (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (3.0 g, 3.1 mmol), DMF (30 mL) and triethylamine (20 mL) were added in sequence and stirred at room temperature for 36 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)pimelanediamide (2.0 g, yield 86%).

[0361] Step 3: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (07D)

[0362] To a 100 mL single-necked bottle, 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)piperamide (2.0 g, 2.6 mmol), DCM (20 mL), triethylamine (2 mL) and 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (753 mg, 5.30 mmol) were added in sequence and stirred at room temperature for 3 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to give 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (2.0 g, yield 84%).

[0363] Step 4: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid perfluorophenyl ester (III-2)

[0364] To a 100 mL single-necked bottle, 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (500 mg, 0.557 mmol), DCM (5 mL) and pentafluorophenyl trifluoroacetate (312 mg, 1.11 mmol) were added sequentially and stirred at room temperature for 12 h. The crude product was concentrated under reduced pressure and purified by column chromatography (ethyl acetate / acetonitrile (V / V) = 1:1) to give 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid perfluorophenyl ester (III-2) (110 mg, yield 18.6%).

[0365] 1 H NMR(400MHz, CDCl3)δ7.39(s,1H),6.59(t,3H),4.2(d,6H),3.51–3.49(m,12H),3.70–3.69(m,12H),3.64–3.62(m,12H),3.5 8–3.54(m,6H),3.38–3.36(m,6H),3.0(q,3H),2.83(s,2H),2.48(s,2H),2.25–2.22(m,6H),2.06–2.04(d,6H),1.18(s,6H).

[0366] Preparation Example 1: Design and Synthesis of siRNA Conjugates

[0367] The synthesis steps of the siRNA conjugate in which the conjugation group is connected to the 5' end of the siRNA sense strand are as follows, taking C1 in Table 1 as an example:

[0368] 1) Synthesis of single-stranded oligoribonucleotides: oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. All 2'-modified nucleotide phosphoramidites and auxiliary reagents were commercially available reagents (Shanghai Zhaowei Technology Development Co., Ltd.). All phosphoramidites were dissolved in anhydrous acetonitrile and molecular sieves were added. 5-Ethylthio-1H-tetrazole (ETT, Suzhou Kelema Biotechnology Co., Ltd.) was used as an activator, with a coupling time of 5 minutes. Phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, Shanghai Zhaowei Technology Development Co., Ltd.) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for a reaction time of 3 minutes. All sequences were synthesized after the final removal of the DMT group.

[0369] 2) Cleavage and deprotection of oligoribonucleotides bound to CPG: After solid-phase synthesis is completed, the protecting groups are removed by treatment with acetonitrile containing 20% ​​diethylamine for 30 minutes. After centrifugation, the supernatant is transferred to a new tube and the CPG is washed with aqueous ammonia. The combined solution is concentrated to obtain a solid mixture.

[0370] 3) Purification of Single-Stranded Oligoribonucleotides: Oligomers were purified by HPLC using a NanoQ anion exchange column. Buffer A consisted of 20% acetonitrile in 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4; and buffer B consisted of 20% acetonitrile in 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4. The desired product was isolated, and a portion of the product was desalted using a reverse-phase C18 column.

[0371] 4) Single-stranded oligoribonucleotide coupling linker: 12 mg of TA14 was dissolved in DMSO and added to the nucleic acid amino intermediate (10 mg, dissolved in DMSO). After ultrasonic mixing, carbonate buffer solution (pH = 9) was added and the mixture was reacted at 25°C for 16 h and then purified.

[0372] 5) Coupling of single-stranded oligoribonucleotide-TA14 conjugate II-1A: Dissolve 7 mg of nucleic acid-TA14 in buffered saline solution, add ligand II-1A (equivalent: 15, dissolved in DMF) to the above solution and vortex; take THPTA:CuSO4·5H2O=5:1 (equivalents are 15 and 3 respectively), shake at 40°C for 5 minutes, add the nucleic acid ligand mixture to the above reaction solution and vortex; quickly add sodium vitamin C (equivalent to 25) to the above solution, vortex, react at 40°C for 1 hour, and purify after the reaction to obtain the complete single-stranded oligoribonucleotide-TA14-II-1A conjugate.

[0373] 6) Annealing to produce siRNA: The single-stranded oligoribonucleotides to be annealed were prepared to 200 μM using sterile RNase-free water (without RNA hydrolase). The annealing reaction system was set up as follows: 10 nmol of the mixture, with a total volume of 100 μL, was placed in a 95°C water bath for 10 minutes (amounts ≥ 100 nmol require high temperature for 20 minutes). The mixture was quickly placed in a 60°C water bath and allowed to cool naturally. The annealed solution should not be stored at high temperatures. By combining equimolar single-stranded oligoribonucleotide solutions to form complementary chains, various unmodified siRNAs were obtained. The nucleotide sequences of the sense and antisense chains of the siRNAs obtained in the present invention are shown in Table 1. Furthermore, the molecular weight of the siRNAs was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weights were compared with the theoretical values. The results showed that the measured values ​​≈ the theoretical values, indicating that the siRNAs listed in Table 1 were obtained. For the specific structure of the ligand (II-1A) in Table 1, please refer to the invention content section of this specification (i.e., II-1A in the compound described in the third aspect or its stereoisomer, tautomer or pharmaceutically acceptable salt), II-1A is derived from compound II-1A in Example 2, and III-1 and III-2 are derived from compounds III-1 and III-2 in Examples 7 and 8.

[0374] Table 1

[0375] Among them, the preparation of AD-07475 can be found in patent WO2022 / 216920A1. In Table 1, the lowercase letters c, g, u, a, and t are all 2'-methoxy-modified nucleotides (i.e., c, g, u, and a respectively indicate that the ribose 2'-OH group of the nucleotide represented by the corresponding capital letter is replaced by a methoxy group); f indicates that the nucleotide represented by the letter before f is a 2'-fluoro-modified nucleotide (i.e., the 2'-OH group of the nucleotide represented by the letter before f is replaced by a fluorine atom); s indicates that the residues between the two adjacent nucleotide residues on the left and right of s are phosphorothioate (i.e., the 5'-phosphate group of the nucleotide represented by the letter before s is replaced by a 5'-phosphorothioate group).

[0376] (TriSM6.1)(TA14)(NH2C6) is:

[0377] Its preparation method can be found in WO2022 / 216920A1.

[0378] (II-1A)(TA14)(NH2C6) is:

[0379] (II-1B)(TA14)(NH2C6) is:

[0380] (II-1B)(III-1)(NH2C6) is:

[0381] (II-1B)(III-2)(NH2C6) is:

[0382] The structures of cPrpus and cPrpas are as follows:

[0383] Test Example 1: ɑvβ6 ligand ELISA binding assay

[0384] Dilute the integrin ɑvβ6 protein (source: Acro Biosystems) to 5 μg / mL using coating solution, and add 25 μL of protein to each well of a 384-well plate. Centrifuge at 1000 rpm for 1 minute, then incubate at 4°C overnight. Add 100 μL of wash buffer to each well, incubate at 1000 rpm for 1 minute, pat dry, and wash three times. Add 100 μL of blocking solution to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 90 minutes. Add 100 μL of wash buffer to each well, incubate at 25°C for 5 minutes, pat dry, and wash three times. Add 10 μL of the above compounds at different dilutions to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 10 minutes. Add 10 μL of fibronectin (source: R&D) to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 60 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash three times. Add 25 μL of fibronectin biotinylated antibody (source: R&D) to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 60 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash three times. Add 25 μL of horseradish peroxidase-labeled streptavidin to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 30 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash four times. Add 25 μL of supersensitive luminescent solution to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 5 minutes. Read the luminescence value on a microplate reader.

[0385] Table 2: αvβ6 integrin ligand binding activity

[0386] The results show that the compound of the present application has high binding activity with ɑvβ6 integrin.

[0387] Test Example 2: Silencing effect of target expression by siRNA conjugate administered intratracheally in rats

[0388] With reference to patent WO2022 / 216920A1, siRNA conjugates targeting rat RAGE protein were designed and synthesized. The compound list is shown in Table 1.

[0389] Experimental methods:

[0390] 1. Aerosol injection administration and serum and lung tissue sample collection in rats

[0391] On day 0, whole blood and serum were collected from the jugular vein of the rats. On day 1, the rats were anesthetized with isoflurane and administered 200 μL of PBS or drug buffer using an aerosol needle at a dose of 0.1 mg / kg. Before the end of the experiment, the rats were anesthetized with 10% chloral hydrate, and blood and serum were collected from the abdominal aorta using a negative pressure blood collection tube. The left and right lung tissues were then collected and snap-frozen in liquid nitrogen. All processed serum and lung tissue samples were stored at -80°C.

[0392] 2. Rat lung tissue homogenate and probe quantitative PCR

[0393] Lung tissue was thawed on ice and homogenized using a tissue grinder. Total RNA was extracted from the lung tissue using the Tissue RNA Extraction Kit 2.0 Plus (Novagen, R411-C3). After reverse transcription (Novagen, R333-01), the expression of RAGE mRNA in rat lung tissue was detected using a quantitative PCR probe (Novagen, QN211-02). The expression of RAGE mRNA was normalized with that of rat GAPDH and compared to the expression level in the PBS control group, which was set as 100%.

[0394] 3. ELISA kit detection of RAGE protein content in rat serum

[0395] Serum was thawed on ice and assayed according to the instructions of the Rat RAGE DuoSet ELISA kit (R&D systems, DY1616). The expression level of RAGE protein in rat serum was calculated based on the standard curve fitting and compared with the expression level of the PBS control group as 100%.

[0396] Table 3: Expression of RAGE protein in rat serum and RAGE mRNA in lung

[0397] The results showed that the siRNA conjugate significantly reduced the expression of RAGE mRNA and protein in the lungs and serum of rats. The compound of the present application can make the siRNA silencing of lung targets and the expression of corresponding secretory proteins more effective, indicating that it has a stronger ability to deliver siRNA molecules to lung cells.

[0398] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound of formula I or a tautomer or stereoisomer thereof: Among them, Ring A is a 5-membered heteroaryl ring; Ring B is a 6- to 10-membered aryl ring, a 5- to 6-membered heteroaryl ring; Ring C is a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring, a 5- to 10-membered heteroalkyl ring; L1 is a C1-C6 alkylene group, a C1-C6 haloalkylene group; R1 is selected from: H, halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, -CONH2, -CONR 11 R 12 , -C(O)-C 1-6 alkyl, -S(O)2-C 1-6 alkyl, -COOR 13 , -NH-C 1-6 alkyl, -NR 14 R 15 ; R2 and R3 are each independently selected from: H, halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 alkyl, C 1-6 alkoxy, -CONH2, -CONR 11 R 12 , -C(O)-C 1-6 alkyl, -S(O)2-C 1-6 alkyl, -COOR 13 , -NH-C 1-6 alkyl, -NR 14 R 15 ; Among them, R 11 , R 12 , R 13 , R 14 , R 15 are each independently C 1-6 alkyl; R1, R2, and R3 are optionally substituted by 1, 2, or 3 identical or different substituents selected from the following: halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 alkyl, C 1-6 alkoxy; Said Ring B or Ring C is optionally connected with linker T; Said linker T is used to directly or indirectly connect an active molecule; When there are multiple R1s, said R1s are the same or different substituents; When there are multiple R2s, said R2s are the same or different substituents; When there are multiple R3s, said R3s are the same or different substituents; m is 1, 2 or 3; n is 1 or 2; p is 1 or 2.

2. The compound of formula I or its tautomer or stereoisomer according to claim 1, wherein, Said compound satisfies one or more of the following conditions: a) With structure b) For Wherein, the definitions of R2, R3, Ring C, n, p are as described in claim 1; c) R2 is H; d) Ring C is a 5- to 8-membered heteroaryl ring; e) R3 is halogen, -NH2, -CN, -OH, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl; or R3 is methyl; f) L1 is a C1-C6 alkylene group; or L1 is -CH2CH2CH2-; Preferably, Ring C is a 5- or 6-membered heteroaryl ring, wherein the heteroatom is selected from N, O, S, and when there are multiple heteroatoms, said heteroatoms are the same or different; Preferably, Ring C is a 5-membered N-containing heteroaryl ring; Preferably, Ring C is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine; Preferably, Ring C is pyrazole; Preferably, ring C is 3. The compound of formula I or its tautomer or stereoisomer according to claim 1, wherein, The compound has one or more structures selected from the following: Wherein, the definitions of Ring A, R1, m, T are as described in claim 1; And / or, when connected to linker T, the compound has one or more structures selected from the following: Wherein, the definitions of Ring A, R1, m, T are as described in claim 1; Preferably, Ring A is a 5-membered heteroaryl group; and / or, m is 0, 1 or 2.

4. The compound of formula I or its tautomer or stereoisomer according to claim 1 or 3, characterized in that, Said linker T contains a polyethylene glycol unit; Or, said linker T contains 2 to 20 polyethylene glycol units; Or, the joint T is where t is from 1 to 10; preferably, the t is 1, 2, 3, 4, 5, 6; and / or, the joint T is Wherein t is 1 to 10; preferably, said t is 1, 2, 3, 4, 5, 6; Or, the joint T is and / or, the joint T is 5. The compound of formula I or its tautomer or stereoisomer as claimed in claim 1, wherein, When a linker T is attached, the compound has one or more of the following structures: Wherein, t is 1 to 10, or t is 1, 2, 3, 4, 5, 6.

6. The compound or its tautomer or stereoisomer represented by Formula I according to any one of claims 1, 3 or 5, characterized in that, Said compound satisfies one or more of the following conditions: g) Ring A is a 5-membered heteroaryl ring, wherein the heteroatom is selected from N, O, S, and when there are multiple heteroatoms, said heteroatoms are the same or different; h) Ring A is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole; i) Ring A is imidazole or triazole; j) For wherein, R 1a and R 1b are each independently R1; k) R1 is halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 alkyl, -CONH2, -C(O)-C 1-6 alkyl, -S(O)2-C 1-6 alkyl; the C 1-6 alkyl, -C(O)-C 1-6 alkyl, -S(O)2-C 1-6 alkyl is optionally substituted by 1, 2 or 3 identical or different substituents selected from the following: halogen, -NH2, -CN, -OH, -SF5, C 1-3 alkyl, C 1-3 alkoxy; m) Ring A is a 5-membered N-containing heteroaromatic ring; or Ring A is n) R1 is H, -F, -Cl, -CN, -SF5, -COOH, -CF3, -CHF2, -COCH3, -CONH2, -S(O)2CH3; or R1 is H, -F, -Cl, -CN; p) Selected from 7. The compound of formula I or its tautomer or stereoisomer according to claim 1, wherein, The compound has one or more of the following structures: Among them, Represents a connection point; Each R1 is independently defined as described in claim 1, 6 or 7; Preferably, each R1 is independently H, -F, -Cl, -CN.

8. The compound of formula I or its tautomer or stereoisomer according to claim 1, characterized in that, The compound has one or more structures selected from the following: wherein, t is 3, 4, 5 or 6; Represents a connection point.

9. The compound of formula I or its tautomer or stereoisomer as claimed in claim 1, wherein The compounds include:

10. The compound of formula I or its tautomer or stereoisomer according to claim 1, characterized in that, The compounds include: Among them, Represents a connection point.

11. A compound, characterized in that, including any of the following structures: Among them, Represents a connection point.

12. A conjugate, characterized in that, Comprises a compound of formula I as described in any one of claims 1-10 or a tautomer or stereoisomer thereof, and an active molecule G; the compound of formula I contains a linker T, and the active molecule G is connected to the linker T in the compound of formula I.

13. The conjugate according to claim 12, wherein Said conjugate further comprises a scaffold; Wherein, the active molecule G is connected to the linker T in the compound of formula I through the scaffold.

14. The conjugate according to claim 12 or 13, characterized in that, The conjugate satisfies one or more of the following conditions: aa) The active molecule G is an active pharmaceutical ingredient or a prodrug thereof; bb) The active molecule G is a small molecule, antibody, antibody fragment, immunoglobulin, monoclonal antibody, label or marker, lipid, natural or modified nucleic acid, natural or modified oligonucleotide, natural or modified polynucleotide, peptide, nucleic acid aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, digoxin, biotin, radioactive atom or molecule, or fluorophore; cc) The active molecule G is a natural or modified oligonucleotide; dd) The active molecule G is an ASO, siRNA, miRNA; ee) The scaffold is a mono-dentate, bi-dentate, tri-dentate, tetra-dentate structure; ff) The active molecule G is linked to one, two, three or four compounds of formula I through the scaffold; gg) The scaffold is the compound as described in claim 11.

15. The conjugate according to any one of claims 12 - 14, wherein, The conjugate includes the following structure Wherein, formula I is the compound of formula I or its tautomer, stereoisomer, and G is the active molecule G; v is 1-10; preferably, v is 4, 5, 6, 7, 8; Preferably, the active molecule G is a natural or modified oligonucleotide; Preferably, the active molecule G is an ASO, siRNA, miRNA.

16. The conjugate according to claim 12, wherein The conjugate includes the following structure Among them, Represents the connection point with the oligonucleotide.

17. A composition, characterized in that, Comprises the compound of formula I as described in any one of claims 1-10 or its tautomer, stereoisomer, or the conjugate as described in any one of claims 12-16, and optionally a pharmaceutically acceptable excipient.

18. The compound of formula I as described in any one of claims 1-10 or its tautomer, stereoisomer, the conjugate as described in any one of claims 12-16, or the composition as described in claim 17 has the following uses: Delivering the active molecule G to cells; and / or, Inhibiting the expression of a target gene in cells; and / or Treating lung diseases; and / or Preparing a drug for inhibiting the expression of a target gene in cells; and / or Preparing a drug for treating lung diseases.

19. The compound of formula I as described in any one of claims 1-10 or its tautomer, stereoisomer, the conjugate as described in any one of claims 12-16, or the composition as described in claim 17 is used for: Delivering the active molecule G to cells; and / or, Inhibiting the expression of a target gene in cells; and / or Treating lung diseases.

20. The use according to claim 18 or 19, characterized in that, The cells are cells expressing αvβ6 integrin or epithelial cells; and / or, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells and epithelial tumors (cancers).

21. A method for delivering an active molecule to a cell, inhibiting the expression of a target gene in the cell, or treating a lung disease, characterized in that, The method comprises administering to a patient the compound of formula I as described in any one of claims 1-10 or its tautomer, stereoisomer, the conjugate as described in any one of claims 12-16, or the composition as described in claim 17.

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