Targeting compounds and uses thereof

Abstract

The invention provides a targeting compound capable of being specifically bound to integrin alpha v beta 6. The compound is the compound shown in the formula I. The invention further provides a preparation method of the targeting compound. The targeting compounds can be used for the transport delivery of active molecules to cells or tissues expressing integrin [alpha] v [beta] 6. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine. Specifically, the present invention relates to a targeting compound and its use. Background Art

[0002] Integrin αvβ6 is a heterodimer composed of αv and β6 integrin subunits, and each subunit consists of a large extracellular head, a flexible leg, a transmembrane segment, and a short cytoplasmic tail. Integrin αvβ6 is a receptor for the latency-associated peptide (LAP) of TGF-β and extracellular matrix (ECM) proteins such as fibronectin, vitronectin, and tenascin. Integrin αvβ6 is often upregulated in diseases such as cancer and fibrosis, and is estimated to be upregulated in about one-third of all solid cancers. Its expression is associated with disease progression and poor prognosis. Multiple studies have shown that the severity of many cancers such as lung cancer, breast cancer, colon cancer, gastric cancer, and ovarian cancer is related to the expression of αvβ6. Integrin αvβ6 is attractive as a tumor marker and potential therapeutic target, especially considering its role in the expression of matrix metalloproteinases (MMPs) and the activation of TGF-β1.

[0003] Compounds targeting integrin αvβ6 are suitable for conjugation with active molecules to transport and deliver the active molecules in vivo to cells expressing integrin αvβ6. For a specific molecule to be transported, such as a therapeutic oligonucleotide-based compound (e.g., antisense oligonucleotide or RNAi agent), a targeting ligand targeting integrin αvβ6 can be conjugated with the oligonucleotide-based compound to deliver the therapeutic agent to cells and / or tissues expressing integrin αvβ6 and promote the entry of the therapeutic agent into cells 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 confirmed to be associated with type II and non-type II inflammatory responses in preclinical animal models. Currently, the small nucleic acid compound ARO-RAGE targeting RAGE is in the clinical phase I study 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 has low expression in other tissues and organs. Therefore, a targeting ligand targeting integrin αvβ6 and a compound capable of silencing the expression of RAGE protein are suitable for evaluating the delivery ability of the targeting ligand. Summary of the Invention

[0005] The object of the present invention is to provide a targeting compound, which is a targeting ligand capable of targeting integrin αvβ6, has an affinity for integrin αvβ6, and can specifically bind to integrin αvβ6. The targeting compound can bind to 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 can exert its function in these cells or tissues.

[0006] The targeting compound of the present invention is the compound shown in Formula I or its tautomer or stereoisomer. The targeting compound contains a linker, and an active molecule is further connected through the linker. A scaffold may further exist between the targeting compound and the active molecule, and the scaffold has a structure of monodentate, bidentate, tridentate or tetradentate. The active molecule is connected to one, two, three or four of the targeting compounds through the scaffold.

[0007] In the first aspect of the present invention, there is provided a compound shown in Formula I or its tautomer or stereoisomer:

[0008]

[0009] wherein, ring A is a 5-membered heteroaryl ring;

[0010] ring B is a 6-10 membered aryl ring, a 5-6 membered heteroaryl ring;

[0011] ring C is a 6-10 membered aryl ring, a 5-10 membered heteroaryl ring, a 5-10 membered heterocycloalkyl;

[0012] L1 is a C1-C6 alkylene group, a C1-C6 haloalkylene group;

[0013] 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 ;

[0014] R2 and R3 are each independently selected from: H, halogen, -NH2, -CN, -OH, -SF5, -COOH, C 1-6 alkyl, C 1-6 alkoxy, -CONH2, -CONR11 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 ;

[0015] Wherein, R 11 、 R 12 、 R 13 、 R 14 、 R 15 are each independently C 1-6 alkyl;

[0016] Said 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;

[0017] Said ring B or ring C is optionally connected with a linker T;

[0018] Said linker T is used to directly or indirectly connect an active molecule;

[0019] When there are multiple R1s, said R1s are identical or different substituents;

[0020] When there are multiple R2s, said R2s are identical or different substituents;

[0021] When there are multiple R3s, said R3s are identical or different substituents;

[0022] m is 1, 2 or 3; n is 1 or 2; p is 1 or 2.

[0023] It should be noted that the above "R1, R2, R3 are optionally substituted by substituents" means that the hydrogen in the group containing hydrogen in R1, R2, R3 (such as C 1-6 alkyl) is optionally substituted by a substituent group.

[0024] In an alternative embodiment of the present invention, ring B is a 6- to 10-membered aromatic ring.

[0025] In an alternative embodiment of the present invention, ring B is a 6- to 8-membered aromatic ring.

[0026] In a preferred embodiment, the structural fragment has the structure

[0027] In a preferred embodiment, the group Among them, the definitions of R2, R3, ring C, n, and p are as described in the first aspect.

[0028] In a preferred embodiment, R2 is H.

[0029] In a preferred embodiment, ring C is a 5- to 10-membered heteroaryl ring.

[0030] In a preferred embodiment, ring C is a 5- to 8-membered heteroaryl ring.

[0031] In a preferred embodiment, ring C is a 5- or 6-membered heteroaryl ring, wherein the heteroatoms are selected from N, O, and S, and when there are multiple heteroatoms, the heteroatoms are the same or different.

[0032] In a preferred embodiment, ring C is a 5-membered N-containing heteroaryl ring.

[0033] In a preferred embodiment, ring C is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole, pyridine, pyridazine, pyrimidine, and pyrazine.

[0034] In a preferred embodiment, ring C is pyrazole.

[0035] In a preferred embodiment, ring C is

[0036] In a preferred embodiment, R3 is halogen, -NH2, -CN, -OH, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl.

[0037] In a preferred embodiment, R3 is methyl.

[0038] In a preferred embodiment, L1 is a C1-C6 alkylene group.

[0039] In a preferred embodiment, L1 is -CH2CH2CH2-.

[0040] In a preferred embodiment, the compound represented by formula I has the following structure:

[0041]

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

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

[0044]

[0045] Among them, the definitions of ring A, R1, and m are as described in the first aspect.

[0046] In a preferred embodiment, ring A is a 5-membered heteroaryl; the definition of R1 is as described in the first aspect; m is 0, 1, or 2.

[0047] In a preferred embodiment, the structural fragment

[0048] In a preferred embodiment, ring A is a 5-membered heteroaromatic ring, wherein the heteroatoms are selected from N, O, and S, and when there are multiple heteroatoms, the heteroatoms are the same or different.

[0049] In a preferred embodiment, ring A is a 5-membered N-containing heteroaromatic ring.

[0050] In a preferred embodiment, ring A is selected from furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, and tetrazole.

[0051] In a preferred embodiment, ring A is imidazole or triazole.

[0052] In a preferred embodiment, ring A is

[0053] In a preferred embodiment, wherein, R 1a 、R 1b are each independently R1.

[0054] 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 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.

[0055] In a preferred embodiment, R1 is H, -F, -Cl, -CN, -SF5, -COOH, -CF3, -CHF2, -COCH3, -CONH2, -S(O)2CH3.

[0056] In a preferred embodiment, R1 is H, -F, -Cl, -CN.

[0057] In a preferred embodiment, selected from

[0058] In a preferred embodiment, the compound of formula I has one linker T.

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

[0060] In a preferred embodiment, the linker T comprises 2 - 20 polyethylene glycol units.

[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, the linker T is where t is 1 - 10.

[0064] In a preferred embodiment, t as described above in any one is 1, 2, 3, 4, 5, 6.

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

[0066] In a preferred embodiment, the linker T is

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

[0068]

[0069] In a preferred embodiment, the compound of formula I has one or more structures selected from the following:

[0070]

[0071]

[0072] In a preferred embodiment, t is 1 - 10.

[0073] In a preferred embodiment, t is 1, 2, 3, 4, 5, 6.

[0074] In a preferred embodiment, the compound has one or more of the following structures:

[0075]

[0076] wherein, Denotes a connection point; each R1 is independently defined as described in the first aspect.

[0077] In a preferred embodiment, each R1 is independently H, -F, -Cl, -CN.

[0078] In a preferred embodiment, the compound of formula I or its tautomer or stereoisomer has the following structure:

[0079]

[0080]

[0081] In a preferred embodiment, the compound of formula I or its tautomer or stereoisomer has the following structure:

[0082]

[0083]

[0084] In a preferred embodiment, the compound of formula I (connected to linker T) has one or more structures selected from the following:

[0085]

[0086]

[0087]

[0088]

[0089]

[0090] wherein t is 3, 4, or 5; Denotes a connection point.

[0091] In a preferred embodiment, the compound of formula I (connected to linker T) has one or more structures selected from the following:

[0092]

[0093]

[0094]

[0095]

[0096] wherein, Denotes a connection point.

[0097] In a second aspect of the present invention, there is provided a conjugate which comprises a compound of formula I or a tautomer or stereoisomer thereof as described in the first aspect, and an active molecule G; wherein, the compound of formula I contains a linker T, and the active molecule G is linked to the linker T in the compound of formula I.

[0098] It should be noted that when the compound of formula I is linked to other molecules (such as in the conjugate described in the second aspect), the compound of formula I always refers to the compound of formula I containing the linker T. When N3 is contained in the linker T of the compound of formula I, the compound of formula I is a precursor before the preparation of the conjugate; when N3 is not contained in the linker T of the compound of formula I but contains then, in the compound of formula I is linked to the active molecule G.

[0099] In a preferred embodiment, the conjugate further comprises a scaffold; wherein, the active molecule G is linked to the linker T in the compound of formula I through the scaffold.

[0100] As used herein, the term "scaffold" refers to a structure that connects one molecule or a part of a molecule to another molecule or another part of a molecule. For example, in the present application, the active molecule G can be conjugated to one or more compounds of formula I via a scaffold, and the scaffold includes at least one attachment point for each ligand (the compound of formula I or its tautomer or stereoisomer) and at least one attachment point for each active molecule G.

[0101] In a preferred embodiment, the conjugate has one or more of the following characteristics:

[0102] aa) The active molecule G is an active pharmaceutical ingredient or a prodrug thereof;

[0103] 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;

[0104] cc) The active molecule G is a natural or modified oligonucleotide;

[0105] dd) The active molecule G is an ASO, siRNA, miRNA;

[0106] ee) The scaffold is a monodentate, bidentate, tridentate, or tetradentate structure;

[0107] (ff) The active molecule G is linked to one, two, three or four compounds of formula I via the scaffold.

[0108] Oligonucleotide As used herein, the term "natural or modified oligonucleotide" refers to an oligonucleotide composed of natural nucleic acids, or an oligonucleotide containing modified nucleic acids. The term "natural or modified polynucleotide" refers to a polynucleotide composed of natural nucleic acids, or a polynucleotide containing modified nucleic acids.

[0109] In a preferred embodiment, the active molecule G is linked to ring B and / or ring C of the compound of formula I. In a preferred embodiment, the active molecule G is linked to one, two, three or four compounds of formula I via the scaffold.

[0110] In a preferred embodiment, the conjugate is

[0111]

[0112]

[0113] wherein formula I is the compound of formula I shown, and G is the active molecule G;

[0114] v is from 1 to 10.

[0115] In a preferred embodiment, t is 1, 2, 3, 4, 5, 6.

[0116] In a preferred embodiment, t is 4.

[0117] In a preferred embodiment, v is 4, 5, 6, 7, 8.

[0118] In a preferred embodiment, the active molecule G is a natural or modified oligonucleotide. In a preferred embodiment, the active molecule G is an ASO, siRNA, miRNA. In a preferred embodiment, the structure of the conjugate is as follows:

[0119]

[0120]

[0121] For the specific structures of IIb-1 to IIb-28, see the structures marked above in the first aspect of the invention content. In a preferred embodiment, the conjugate includes the following structures

[0122]

[0123]

[0124] wherein, Indicates the junction with the oligonucleotide.

[0125] In a third aspect of the present invention, there is provided a composition comprising a compound of formula I as described in the first aspect or a tautomer or stereoisomer thereof, or a conjugate as described in the second aspect.

[0126] In a preferred embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0127] In a fourth aspect of the present invention, there is provided the use of a compound of formula I as described in the first aspect or a tautomer or stereoisomer thereof, a conjugate as described in the second aspect, or a composition as described in the third aspect in the preparation of a medicament.

[0128] In a preferred embodiment, the medicament is used to deliver the active molecule G to cells.

[0129] In a preferred embodiment, the cells are cells expressing αvβ6 integrin.

[0130] 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 (cancers).

[0131] In a preferred embodiment, the medicament is used for:

[0132] i) inhibiting the expression of a target gene in the cells; or

[0133] ii) preparing a medicament for inhibiting the expression of a target gene in the cells; or

[0134] iii) preparing a medicament for treating lung diseases.

[0135] In a fifth aspect of the present invention, there is provided a method for delivering an active molecule to cells or treating lung diseases, the method comprising administering to a patient a compound of formula I as described in the first aspect or a tautomer or stereoisomer thereof, a conjugate as described in the second aspect, or a composition as described in the third aspect.

[0136] In a preferred embodiment, the cells are the cells described in the fourth aspect.

[0137] In a preferred embodiment, the active molecule is the active molecule G.

[0138] In a preferred embodiment, the active molecule G is an ASO, siRNA, miRNA.

[0139] In the sixth aspect of the present invention, a compound is provided, which comprises any of the following structures:

[0140]

[0141] Wherein, represents a connection point.

[0142] linker

[0143] As disclosed herein, the targeting compound further has a linker, and an active molecule is further connected through the linker. In some embodiments, the linker is linker T described in the present application. In some embodiments, the linker may be a structure containing polyethylene glycol (PEG) units. The linker may contain 2-20 polyethylene glycol unit structures.

[0144] In some embodiments, the linker is represents connection sites respectively connected to the targeting compound and the active molecule.

[0145] In some embodiments, at one end where the active molecule is connected, the linker has a reactive group, and is directly conjugated to the active molecule through the reactive group, or conjugated to the active molecule after being connected to the scaffold. In some embodiments, the reactive group is an azide or an alkynyl-containing group.

[0146] In some embodiments, the linker is

[0147] In some embodiments, t is 1-10.

[0148] In some embodiments, preferably, t in any of the above is 1, 2, 3, 4, 5, 6, 7.

[0149] There is no particular limitation on the connection site of the linker to the targeting compound. In some embodiments, the linker is connected to ring B or ring C of the targeting compound (the compound shown by formula I in the present invention).

[0150] In some embodiments, the compound of formula I has the structure:

[0151]

[0152] Targeting compound and scaffold

[0153] As disclosed herein, in some embodiments, one or more targeting compounds (compounds of Formula I as described in the present invention) can be linked to one or more active molecules to be transported. 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 "divalent" 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.

[0154] As disclosed herein, in some embodiments, the scaffold and the active molecule are linked to the targeting compound via a linker. In some embodiments, the scaffold has a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the active molecule is linked to one targeting compound via a scaffold having a monodentate structure. In some embodiments, the active molecule is linked to two targeting compounds via a scaffold having a bidentate structure. In some embodiments, the active molecule is linked to three targeting compounds via a scaffold having a tridentate structure. In some embodiments, the active molecule is linked to four targeting compounds via a scaffold having a tetradentate structure.

[0155] 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 alkynyl-containing moieties (such as alkynyl, cyclooctyne). In some embodiments, the alkynyl-containing moieties include, but are not limited to: alkynyl maleimide, alkynyl NHS ester.

[0156] In some embodiments, the scaffold can 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.

[0157] In some embodiments, exemplary scaffolds having a typical tridentate structure are, for example:

[0158]

[0159]

[0160] The scaffold includes an amine-reactive group, an amide bond, three PEG2 units, and an alkyne. The amine-reactive group can be conjugated via amide formation to a primary amine (such as a terminal amine group (e.g., NH2-C6)) on the active molecule to be transported. The alkyne can be conjugated with an azide-modified linker to form a triazole structure, which is then connected to the targeting compound. In this scaffold, the amine-reactive group is p-nitrophenol (also known as 4-nitrophenol) ester.

[0161] In some embodiments, the amine-reactive group is not particularly limited. The amine-reactive group can also be, for example:

[0162]

[0163] In some embodiments, the scaffold can be synthesized as a phosphoramidite compound, which allows 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:

[0164]

[0165] Wherein, represents an oligonucleotide, such as siRNA.

[0166] Conjugate

[0167] The present invention provides a conjugate having the structure As disclosed above herein, in some embodiments, one or more αvβ6 integrin ligands (such as the compound of formula I described in the present invention) can be linked to one or more active molecules to be transported.

[0168] In some embodiments, the active molecule G can be coupled to the scaffold via phosphoramidite synthesis. The scaffold can have a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the azide group on the targeting compound (such as ) can react with the alkyne (such as benzocyclooctyne, propargyl) of the scaffold (such as a click chemical reaction) to form a triazole structure, which is then connected to the targeting compound. For example, the structure:

[0169]

[0170] Wherein represents the connection point with the active molecule (such as an oligonucleotide).

[0171] The active molecule described in the present application can be linked to the targeting compound to form a conjugate. In some embodiments, the conjugate is the conjugate described in the third aspect.

[0172] Active molecule

[0173] As used herein, the "active molecule" can be linked to the targeting compound and then transported to cells and / or tissues expressing integrin αvβ6. The transported "active molecule" (or transported molecule) is any molecule that will have a desired effect on cells containing the αvβ6 integrin receptor when separated from the αvβ6 integrin ligand described herein. The transported active molecule can be, but is not limited to, a pharmaceutical ingredient, a pharmaceutical product, a prodrug, a substance with known therapeutic benefits, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a native or modified nucleic acid, an oligonucleotide or polynucleotide, a peptide, a polymer, a polyamine, a protein, a nucleic acid 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 αvβ6 integrin ligands to target the transported active molecules to cells expressing αvβ6 integrin. In some embodiments, the active molecule is active molecule G of the present invention.

[0174] oligonucleotide

[0175] As used in this application, the term "oligonucleotide" refers to a short chain of nucleotides (including nucleotides within deoxyribonucleic acid DNA or ribonucleic acid RNA) consisting of less than 50 bases. The oligonucleotides of this application can be in single-stranded or double-stranded structures, and the specific types are not limited and are all within the scope of protection of this application. Exemplarily, when in 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 substrate; when in a single-stranded structure, the RNAi agent includes but is not limited to antisense oligonucleotide (ASO).

[0176] Additional aspects and advantages of the present invention will be given in part in the following description, will become apparent in part from the following description, or will be learned through the practice of the present invention.

[0177] Terms and Definitions

[0178] Unless otherwise specified, the definitions of groups and terms recited in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. The group definitions and compound structures after such combination and combination should fall within the scope described in the specification of this application.

[0179] Unless otherwise defined, all technical terms used herein have the same meanings as those commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. All patents, patent applications, and published materials cited herein in their entirety are incorporated herein by reference, unless otherwise indicated. If there are multiple definitions of a term in this document, the definitions in this chapter shall prevail.

[0180] It should be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and do not limit the subject matter of the present invention in any way. In this application, the singular also includes the plural unless specifically stated otherwise. It must be noted that, unless clearly stated otherwise in the text, the singular forms used in this specification and the claims include the plural forms of the indicated items. It should also be noted that, unless otherwise indicated, the terms "or" or "either...or" mean "and / or". In addition, the term "comprising" and other forms such as "including", "containing", and "having" are not restrictive.

[0181] Definitions of standard chemical terms can be found in reference works, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED.", 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 employed. Unless otherwise specifically defined, the terms used in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and the treatment of patients. For example, the instructions provided by the manufacturer for a kit can be utilized, or reactions and purifications can be carried out in a manner known to those skilled in the art or as described in the present invention. Generally, the above-mentioned techniques and methods can be implemented according to the descriptions in a number of general and more specific references cited and discussed in this specification, in accordance with conventional methods well known in the art. In this specification, groups and their substituents can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0182] When a substituent is described by a conventional chemical formula written from left to right, that substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left. For example, CH2O is equivalent to OCH2. As used herein, represents the point of attachment of a group. As used herein, "R1" and "R1" have the same meaning and can be used interchangeably. Similar definitions have the same meaning for other symbols such as R2.

[0183] The section headings used in this text are for the purpose of organizing the article only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, operating manuals, and theses, are hereby incorporated by reference in their entirety.

[0184] Except as otherwise noted, when used in the specification and claims of this application, the following terms have the meanings set forth below unless otherwise specifically indicated.

[0185] For a numerical range recited in the specification and claims of this application, when the numerical range is understood as "integers", it should be understood that the two endpoints of the range and each integer within the range are recited. For example, "integers from 0 to 5" should be understood to recite each of 0, 1, 2, 3, 4, and 5.

[0186] In this application, when used alone or as part of another substituent, the term "halogen" means fluorine, chlorine, bromine, or iodine.

[0187] As used herein, when used alone or as part of another substituent, the term "amino" means -NH2.

[0188] As used herein, when used alone or as part of another substituent, the term "nitro" means -NO2.

[0189] As used herein, when used alone or as part of another substituent, the term "cyano" means -CN.

[0190] As used herein, when used alone or as part of another substituent, the term "alkyl" means a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing no unsaturated bonds, having, for example, 1 to 6 carbon atoms, and being 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. The alkyl group may be unsubstituted or substituted by one or more suitable substituents. The alkyl group may also be an isotopic isomer of a natural abundance alkyl group enriched in carbon and / or hydrogen isotopes (i.e., deuterium or tritium).

[0191] When alone or as part of another substituent, the term "C1-C6 alkyl" is understood to represent a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. Examples of such alkyl groups include 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, etc. or their isomers. The term "C1-C5 alkyl" is understood to represent a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1, 2, 3 or 5 carbon atoms. In particular, the group has 1, 2 or 3 carbon atoms ("C1-C3 alkyl"), such as methyl, ethyl, n-propyl or isopropyl.

[0192] When alone or as part of another substituent, the term "C1-C6 alkoxy" is understood to represent a straight-chain or branched-chain saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms and an oxygen atom, or is defined as C1-C6 alkyl - O - C1-C6 alkyl as described in this specification, and the oxygen atom can be attached to any carbon atom of the straight-chain or branched-chain of the C1-C6 alkyl. It includes but is not limited to: methoxy (CH3 - O-), ethoxy (C2H5 - O-), propoxy (C3H7 - O-), butoxy (C4H9 - O-).

[0193] When alone or as part of another substituent, the term "cycloalkyl" or "carbocyclic group" refers to a cyclic alkyl group. The term "m - n membered cycloalkyl" or "C m -C n cycloalkyl" is understood to represent a saturated, unsaturated or partially saturated carbocyclic 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 monocyclic, bicyclic, tricyclic, spiro or bridged rings. Examples of unsubstituted cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and adamantyl, or bicyclic hydrocarbon groups such as decahydronaphthalene ring. The cycloalkyl group can be substituted with one or more substituents. In some embodiments, the cycloalkyl group can be a cycloalkyl group fused to an aryl or heteroaryl group.

[0194] When alone or as part of other substituents, the term "heterocycloalkyl" or "heterocyclic group" 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 "m-n membered heterocycloalkyl" or "C m -C n heterocycloalkyl" should be understood to represent a saturated, unsaturated, or partially saturated ring having m to n atoms. For example, the term "4-10 membered heterocycloalkyl" should be understood to represent 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 denote a heterocycloalkyl, the number of carbons also means including the heteroatoms. It includes monocyclic, bicyclic, tricyclic, spirocyclic, or bridged rings.

[0195] When alone or as part of other substituents, the term "heteroaryl" can be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic group" and refers to a monocyclic or polycyclic aromatic ring system in which, in certain embodiments, 1 to 3 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 furyl, imidazolyl, dihydroindolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl. The heteroaryl can optionally be fused to a benzene ring and can also be a monocyclic, bicyclic, tricyclic, spirocyclic, or bridged ring.

[0196] When alone or as part of other substituents, the term "5- to 10-membered heteroaryl" should be understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring group having 5 to 10 ring atoms and containing heteroatoms selected from N, O and S, and should be understood to have 5, 6, 7, 8, 9 or 10 ring atoms - in particular 5 or 6 or 9 or 10 carbon atoms - and which contains 1 to 5, preferably 1 to 3 - heteroatoms independently selected from N, O and S, and which, additionally in each case, may be benzo-fused. The "5- to 8-membered heteroaryl" should be understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring group having 5 to 8 ring atoms - in particular 5 or 6 carbon atoms - and containing 1 to 5 heteroatoms independently selected from N, O and S. Preferably 1 to 3 - heteroatoms independently selected from N, O and S, and which, additionally in each case, may be benzo-fused. Examples of heteroaryl include, but are not limited to: thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, etc. and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc. and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc.

[0197] When alone or as part of other substituents, the term "halo" may be used interchangeably with the term "halogen substitution". "Haloalkyl" or "halogen-substituted alkyl" means a branched or straight-chain saturated aliphatic hydrocarbon group including a specific number of carbon atoms and substituted by one or more halogens (such as -CvFw, where v = 1 to 3 and w = 1 to (2v + 1)). Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl and heptachloropropyl.

[0198] The compounds provided herein, including intermediates useful for preparing the compounds provided herein, contain reactive functional groups (such as, but not limited to, carboxyl, hydroxyl, and amino moieties), and also include their protected derivatives. "Protected derivatives" are those compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as protecting groups). Suitable carboxyl moiety protecting groups include benzyl, tert-butyl, etc., as well as isotopes, etc. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable protecting groups are well known to those of ordinary skill in the art.

[0199] In this application, "optional" or "optionally" means that the subsequent described event or condition may or may not occur, and this description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally substituted aryl" means that the aryl is either substituted or unsubstituted, and this description includes both the substituted aryl and the unsubstituted aryl.

[0200] In this application, the terms "salt" or "pharmaceutically acceptable salt" include 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, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0201] As used herein, "pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic acids or organic acids that are capable of retaining the biological effectiveness of the free base without other side effects. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic bases or organic bases that are capable of maintaining the biological effectiveness of the free acid without other side effects. In addition to pharmaceutically acceptable salts, the present invention also contemplates other salts. They can serve as intermediates in the purification of the compounds or in the preparation of other pharmaceutically acceptable salts or can be used for the identification, characterization, or purification of the compounds of the present invention.

[0202] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereoisomers, and conformational isomers.

[0203] Depending on the choice of starting materials and methods, the compounds of the present invention may exist in the form of one or a mixture of possible isomers, for example as pure enantiomers, or as a mixture of isomers such as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. When describing a compound with optical activity, 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(s) in the molecule. The prefixes D and L or (+) and (–) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (–) or L indicates that the compound is levorotatory. A compound with the prefix (+) or D is dextrorotatory.

[0204] When depicting the bonds to the chiral carbon in the formula of the present invention as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and thus both the enantiomerically pure compounds and mixtures thereof are included within the scope of this general formula. The illustration method for racemates or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62: 114 - 120. The absolute configuration of a stereocenter is represented by a wedge bond and a dashed bond.

[0205] The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom within a molecule between two positions. The compounds of the present invention may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible forms. Prototropic tautomers result from the migration of a hydrogen atom covalently bonded between two atoms. Tautomers generally exist in an equilibrium form, and attempting to isolate a single tautomer usually results in a mixture whose physical and chemical properties are consistent with those of a mixture of compounds. The position of the equilibrium depends on the chemical characteristics 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.

[0206] In the present application, a "pharmaceutical composition" refers to a preparation of a compound of the present invention with a medium commonly accepted in the art for delivering a bioactive compound to a mammal (such as a human). This medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate the administration to an organism, facilitate the absorption of the active ingredient, and thereby exert its biological activity.

[0207] In the present application, a "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier that has been approved by the relevant government regulatory authorities for use in humans or livestock.

[0208] In the present application, the term "prodrug" refers to a compound that can be converted into the bioactive compound of the present invention under physiological conditions or by solvolysis. The prodrugs of the present invention are prepared by modifying the functional groups in the compound, and such modification can be removed by conventional operations or in vivo to obtain the parent compound. Prodrugs include compounds formed by connecting a hydroxyl group or an amino group in the compound of the present invention to any group. When the prodrug of the compound of the present invention is administered to a mammalian individual, the prodrug is cleaved to form a free hydroxyl group and a free amino group respectively.

[0209] The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compounds can be labeled with radioactive isotopes such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). All isotopic compositions of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0210] In the present application, the term "excipient" refers to a pharmaceutically inert ingredient. Non-limiting examples of the types of "vehicles" include binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of pharmaceutical formulations, i.e., make the formulations more suitable for direct compression by increasing fluidity and / or adhesiveness.

[0211] As used herein, the terms "treat" and other similar synonyms include the following meanings:

[0212] (i) Prevent the occurrence of a disease or disorder in a mammal, especially when such a mammal is susceptible to the disease or disorder but has not been diagnosed as having the disease or disorder;

[0213] (ii) Inhibit a disease or disorder, i.e., curb its development;

[0214] (iii) Alleviate a disease or disorder, i.e., cause the condition of the disease or disorder to subside; or

[0215] (iv) Reduce the symptoms caused by the disease or disorder.

[0216] For the reactions of each step, 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, purified, etc. from the reaction system by common methods, such as filtration, extraction, recrystallization, washing, silica gel column chromatography and other methods. Without affecting the next reaction, the target compound can also directly enter the next 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 implementation manners

[0217] The following further illustrates the present invention in conjunction with specific embodiments. It should be understood that the following description is only the most preferred implementation manner of the present invention, and should not be considered as a limitation on the protection scope of the present invention. On the basis of fully understanding the present invention, for the experimental methods without specific conditions noted in the following embodiments, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Those skilled in the art can make non-essential changes to the technical solutions of the present invention, and such changes should be regarded as being included in the protection scope of the present invention.

[0218] The following reagents as used herein:

[0219] SEM, namely (trimethylsilyl)ethoxymethyl, with the chemical formula of —CH2OCH2CH2Si(CH3)3, is an amino protecting group.

[0220] Boc, namely tert-butoxycarbonyl, is an amino protecting group.

[0221] PMB, namely p-methoxybenzyl, is a protecting group for amino.

[0222] Example 1: Preparation of Compound I-1

[0223] The synthetic route is as follows:

[0224]

[0225] First step: Synthesis of methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (01B)

[0226]

[0227] At room temperature, dissolve (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoic acid (1.20 g, 3.49 mmol) in anhydrous DMF (10 mL). Add potassium carbonate (726 mg, 5.25 mmol) and methyl iodide (1.03 g, 7.23 mmol) sequentially at 0 °C. After monitoring by TLC that the raw materials were completely converted, add 20 mL of water for dilution and extract with tert-butyl methyl ether (10 mL × 3). Wash the combined organic phases once with saturated NaHCO3 solution (10 mL), separate the organic phase, dry it over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (01B) (1.21 g, yield 97.2%).

[0228] LC-MS, M / Z(ESI): 358.0, 360.0 [M+H] + .

[0229] Step 2: Synthesis of methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01C)

[0230]

[0231] Add 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) to a 100 mL round-bottom flask. After the mixture is ultrasonically shaken, displace nitrogen three times and stir at 100 °C. After monitoring by TLC that the raw materials were completely converted, filter the reaction solution and concentrate the filtrate under reduced pressure to obtain the crude product. Purify by column chromatography (petroleum ether / ethyl acetate (V / V) = 3:1) to obtain methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01C) (360 mg, yield 29.7%).

[0232] LC-MS, M / Z(ESI): 360.2 [M+H] + .

[0233] Step 3: Synthesis of methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01D)

[0234]

[0235] To a 100 mL single-necked flask, methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (600 mg, 1.67 mmol), dichloromethane (20 mL), and trifluoroacetic acid (3.81 g, 33.4 mmol) were added successively, and the mixture was 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 successively 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)propionate (01D) (430 mg, yield 99.3%).

[0236] LC-MS, M / Z(ESI): 260.13[M+H] + .

[0237] Step 4: Synthesis of methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01E)

[0238]

[0239] To a 100 mL single-necked flask, methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (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 successively. Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1) to obtain methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01E) (600 mg, yield 93.4%).

[0240] LC-MS, M / Z(ESI): 417.27[M+H] + .

[0241] Step 5: Synthesis of methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01F)

[0242]

[0243] To a 100 mL single-necked flask, methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (600 mg, 1.44 mmol), dichloromethane (10 mL), and trifluoroacetic acid (3.29 g, 28.85 mmol) were successively added, and the mixture was 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 successively 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)propionate (01F) (460 mg).

[0244] LC-MS, M / Z(ESI): 317.32[M+H] + .

[0245] Step 6: Synthesis of 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01H)

[0246]

[0247] 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 successively added, and the temperature was cooled to 0 °C in an ice bath. SEMCl (11.82 g, 70.90 mmol) was added, and the mixture was stirred in the ice bath for 20 min and then at room temperature for 16 h. The reaction solution was diluted with ethyl acetate (200 mL) and washed with water (200 mL). The obtained organic phase was 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 1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (8.00 g, yield 55.6%).

[0248] LC-MS, M / Z(ESI): 224.11[M+H] + .

[0249] Step 7: Synthesis of 2-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01I)

[0250]

[0251] 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) were successively added to a 250 mL single-necked flask and stirred at 60 °C for 6 h. After cooling to room temperature, ethyl acetate (200 mL) was added to dilute the reaction solution, which was then washed successively 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) gave 2-bromo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01I) (4.50 g, yield 41.6%).

[0252] LC-MS, M / Z(ESI): 302.02[M + H] + .

[0253] Step 8: Synthesis of 2-((4-Methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (01J)

[0254]

[0255] 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) were successively added to a 100 mL single-necked flask and stirred at 100 °C for 16 h. The reaction solution was filtered and concentrated to obtain the 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, yield 23.9%).

[0256] 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).

[0257] LC-MS, M / Z(ESI): 359.47 [M+H] + .

[0258] Step 9: Synthesis of Ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoate (01K)

[0259]

[0260] Add 2-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole-4-carbonitrile (850 mg, 2.37 mmol) and DMF (10 mL) into a 100 mL single-necked flask in sequence. Under nitrogen protection, cool down to -10 °C, add NaH (60%, 142 mg, 3.55 mmol), and stir at this temperature for 0.5 h. Add ethyl 4-bromobutyrate (694 mg, 3.56 mmol), stir at -10 °C for 0.5 h, then transfer to room temperature and stir for 2 h. Add saturated ammonium chloride aqueous solution, then add 10 mL of water, and extract with ethyl acetate (20 mL × 3). The combined organic phases are washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purify by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 1:1) to obtain ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butanoate (200 mg, yield 17.8%).

[0261] 1 H NMR (400 MHz, DMSO-d6) δ 8.05 (s, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.7 Hz, 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 = 7.4 Hz, 2H), 1.66 - 1.61 (m, 2H), 1.12 (t, J = 7.1 Hz, 3H), 0.84–0.78 (m, 2H), -0.06 (s, 9H).

[0262] LC-MS, M / Z(ESI): 473.51 [M+H] + .

[0263] Step 10: Synthesis of 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyric acid (01L)

[0264]

[0265] Ethyl 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyrate (200 mg, 0.423 mmol) and THF (9 mL) were successively added to a 100 mL single-necked flask, 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. THF was removed by concentration under reduced pressure. Water (10 mL) was added, the pH was adjusted to 6 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (10 mL × 3). The combined organic phases were washed with saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 4-((4-cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyric acid (01L) (100 mg, yield 53.2%).

[0266] LC-MS, M / Z (ESI): 445.22 [M+H] + .

[0267] Step 11: Synthesis of methyl (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)propionate (01M)

[0268]

[0269] 4-((4-Cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyric acid (100 mg, 0.225 mmol), DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (71.2 mg, 0.225 mmol), TBTU (108 mg, 0.321 mmol) and DIPEA (87.2 mg, 0.675 mmol) were successively added to a 100 mL single-necked flask. Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phases were successively washed with saturated aqueous sodium bicarbonate solution (20 mL) and 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 - 0:1) gave methyl (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)propionate (90.0 mg, yield 53.9%).

[0270] LC-MS, M / Z(ESI): 743.36[M+H] + .

[0271] Step 12: Synthesis of (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)propionic acid (01N)

[0272]

[0273] To a 100 mL single-necked flask, (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)propionic acid methyl ester (90.0 mg, 0.121 mmol), THF (6 mL), and an aqueous solution of lithium hydroxide monohydrate (6.6 mg, 0.16 mmol) (2 mL) were added successively, and the mixture was stirred at room temperature for 6 h. THF was removed by concentration under reduced pressure. Water (5 mL) was added, and the pH was adjusted to 6 with 1N hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain (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)propionic acid (70.0 mg, yield 79.3%).

[0274] LC-MS, M / Z (ESI): 729.35 [M+H] + .

[0275] Step 13: Synthesis of (S)-3-(2-(4-((4-cyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-1)

[0276]

[0277] To a 100 mL single-necked flask, (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)propionic acid (70.0 mg, 96.0 μmol), dichloromethane (8 mL), and trifluoroacetic acid (438 mg, 3.84 mmol) were added successively, and the mixture was stirred at room temperature for 6 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% ammonia water, B = acetonitrile; gradient: 30% - 100%) to obtain (S)-3-(2-(4-((4-cyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-1) (11 mg, yield 24%).

[0278] 1 1H NMR (400 MHz, DMSO-d6) δ 8.44 (d, J = 8.3 Hz, 1H), 8.09 (t, J = 5.8 Hz, 1H), 7.72 (s, 1H), 7.45–7.42 (m, 2H), 7.41 (d, J = 7.4 Hz, 1H), 7.39–7.33 (m, 2H), 6.38 (d, J = 1.8 Hz, 1H), 5.23 (q, J = 7.4 Hz, 1H), 3.82 (s, 3H), 3.69 (d, J = 5.8 Hz, 2H), 3.12 (t, J = 7.0 Hz, 2H), 2.73 (d, J = 7.2 Hz, 2H), 2.16 (t, J = 7.4 Hz, 2H), 1.73–1.66 (m, 2H).

[0279] LC-MS, M / Z (ESI): 479.3 [M+H] + .

[0280] Example 2: Preparation of Compound I-2

[0281] The synthetic route is as follows:

[0282]

[0283]

[0284] The first step: Synthesis of ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (02B)

[0285]

[0286] Add tert-butyl (1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)carbamate (2.80 g, 15.3 mmol) and DMF (30 mL) to a 100 mL single-necked flask. Under nitrogen protection, cool the mixture to 0 °C in an ice bath, add NaH (1.07 g, 26.8 mmol, 60%), stir at 0 °C for 0.5 h, then add ethyl 4-bromobutyrate (7.47 g, 38.3 mmol) and potassium iodide (2.54 g, 15.3 mmol), stir at 0 °C for 1 h, transfer to room temperature and stir for 16 h. Quench the reaction with saturated ammonium chloride, add water (30 mL), and extract with ethyl acetate (60 mL × 3). Wash the combined organic phases with saturated brine (100 mL), dry over anhydrous sodium sulfate, filter, and concentrate to obtain the crude product. Purify by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 2:3) to obtain ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (02B) as a colorless oil (2.80 g, yield 61.5%).

[0287] LC-MS, M / Z(ESI): 428.35[M+H] + .

[0288] Step 2: Synthesis of 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyric acid (02C)

[0289]

[0290] Add ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (215 mg, 0.503 mmol), THF / H2O (4 mL, v:v = 1:1), and lithium hydroxide monohydrate (63 mg, 1.5 mmol) to a 100 mL single-necked flask in sequence, and stir at room temperature for 3 h. After the reaction is completed, add 1N HCl (2 mL) and extract with ethyl acetate (20 mL). Dry the combined organic phases over anhydrous sodium sulfate, filter, and concentrate to obtain 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyric acid (02C) (200 mg, yield 99.6%).

[0291] LC-MS, M / Z(ESI): 400.35[M+H] + .

[0292] Step 3: Synthesis of Methyl (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)propionate (02D)

[0293]

[0294] To a 100 mL single-necked flask, sequentially add 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)propionate (01F) (158 mg, 0.499 mmol), TBTU (176 mg, 0.548 mmol) and DIPEA (194 mg, 1.50 mmol), and stir at room temperature for 16 h. Add water (10 mL), and extract with ethyl acetate (20 mL × 3). The combined organic phases are washed successively with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. Purify by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1) to obtain Methyl (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)propionate (02D) (230 mg, yield 66.0%).

[0295] LC-MS, M / Z(ESI): 698.44[M+H] + .

[0296] Step 4: Synthesis of (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)propionic Acid (02E)

[0297]

[0298] To a 100 mL single-necked flask, (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)methyl propionate (230 mg, 0.330 mmol), THF / H2O (4 mL, v:v = 1:1), and lithium hydroxide monohydrate (42 mg, 1.0 mmol) were added successively, 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 obtain (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)propionic acid (02E) (225 mg, yield 99.8%).

[0299] LC-MS, M / Z(ESI): 684.45[M+H] + .

[0300] Step 5: Synthesis of (3S)-3-((N-(4-((1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-2)

[0301]

[0302] To a 100 mL single-necked flask, (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)propionic acid (225 mg, 0.329 mmol), DCM (5 mL), and TFA (2 mL) were added successively, 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 purified by reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% ammonia water, B = acetonitrile; gradient: 30% - 100%) to obtain (3S)-3-((N-(4-((1H-imidazol-2-yl)amino)butanoyl)glycyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-2) (73 mg, yield 49%).

[0303] 11H NMR (400 MHz, DMSO-d6) δ 9.47 (d, J = 8.0 Hz, 1H), 8.69 (s, 1H), 8.46 (t, J = 6.0 Hz, 1H), 7.45–7.39 (m, 2H), 7.39–7.30 (m, 3H), 6.74 (s, 2H), 6.36 (d, J = 2.0 Hz, 1H), 5.04 (m, 1H), 3.80 (m, 5H), 3.55 (m, 2H), 2.65–2.52 (m, 2H), 2.37 (t, J = 6.8 Hz, 2H), 1.85 (m, 2H).

[0304] LC-MS, M / Z (ESI): 454.31 [M+H] + .

[0305] Example 3: Preparation of Compound I-3

[0306] The synthetic route is as follows:

[0307]

[0308] The first step: Synthesis of methyl 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanoate (03C)

[0309]

[0310] To a 100 mL single-necked flask were successively added 2-amino-4,5-imidazoledicarbonitrile (150 mg, 1.13 mmol), methanol (5 mL), methyl 4-oxobutanoate (131 mg, 1.13 mmol) and acetic acid (135 mg, 2.25 mmol). Under nitrogen protection, 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 with saturated ammonium chloride solution, water (5 mL) was added, and the organic solvent was removed by reduced pressure concentration. The residue was extracted with ethyl acetate (10 mL × 3), 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%).

[0311] 1 1H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 7.29 (t, J = 5.5 Hz, 1H), 3.55 (s, 3H), 3.12 (q, J = 6.6 Hz, 2H), 2.32 (t, J = 7.4 Hz, 2H), 1.75–1.68 (m, 2H).

[0312] LC-MS, M / Z (ESI): 234.23 [M+H] + .

[0313] Step 2: Synthesis of 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyric acid (03D)

[0314]

[0315] To a 100 mL single-necked flask, add successively methyl 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyrate (50 mg, 0.21 mmol), tetrahydrofuran (5 mL), and lithium hydroxide monohydrate (11.7 mg, 0.279 mmol) dissolved in water (0.5 mL). Stir at room temperature for 16 h. Concentrate under reduced pressure to remove the organic solvent, adjust the pH to 7 with 1N hydrochloric acid and concentrate under reduced pressure to obtain the product 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyric acid (03D), which is directly used in the next reaction.

[0316] LC-MS, M / Z (ESI): 220.21 [M+H] + .

[0317] Step 3: Synthesis of methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (03E)

[0318]

[0319] To a 100 mL single-necked flask, add successively methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (3.2 g, 7.7 mmol), methanol (20 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 9.61 mL). Stir at room temperature for 2 h. Concentrate the reaction solution under reduced pressure to obtain methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (03E) (2.6 g, yield 96%).

[0320] LC-MS, M / Z (ESI): 317.32 [M+H] + .

[0321] Step 4: Synthesis of methyl (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (03F)

[0322]

[0323] To a 100 mL single-necked flask were successively added 4-((4,5-dicyano-1H-imidazol-2-yl)amino)butyric acid obtained in the second step, DMF (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (91 mg, 0.26 mmol), TBTU (103 mg, 0.321 mmol) and DIPEA (111 mg, 0.859 mmol). Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (10 mL) and ethyl acetate (20 mL×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 (dichloromethane / methanol (V / V)=1:0 - 9:1) gave methyl (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (03D) (35 mg, two-step yield 32%).

[0324] LC-MS, M / Z(ESI): 518.26 [M+H] + .

[0325] Step 5: Synthesis of (S)-3-(2-(4-((4,5-dicyano-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-3)

[0326]

[0327] 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)propionate (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 successively, and the mixture was stirred at room temperature for 3 h. The organic solvent was removed by concentration under reduced pressure, and the pH was adjusted to 6 with 1 N hydrochloric acid and concentrated under reduced pressure to obtain the crude product. After reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); 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)propionic acid (I-3) (3.2 mg, yield 9.4%) was obtained.

[0328] 1 H NMR (400 MHz, DMSO-d6) δ 8.54 (d, J = 8.3 Hz, 1H), 8.14–8.11 (m, 1H), 7.44–7.32 (m, 5H), 6.37 (d, J = 1.7 Hz, 1H), 5.18 (q, J = 7.2 Hz, 1H), 3.81 (s, 3H), 3.67–3.63 (m, 2H), 3.08–3.05 (m, 2H), 2.69 (d, J = 6.8 Hz, 2H), 2.16 (t, J = 7.4 Hz, 2H), 1.71–1.64 (m, 2H).

[0329] LC-MS, M / Z (ESI): 504.41 [M+H] + .

[0330] Example 4: Preparation of Compound I-4

[0331] The synthetic route is as follows:

[0332]

[0333] First step: Synthesis of ethyl 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (04A)

[0334]

[0335] Ethyl 4-((tert-butoxycarbonyl)(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (400 mg, 0.935 mmol), DMF (10 mL), and N-chlorosuccinimide (125 mg, 0.936 mmol) were successively added to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 2 h. The reaction solution was heated to 40 °C and stirred for another 16 h. Water (10 mL) and ethyl acetate (20 mL × 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)butyrate (04A) (280 mg, yield 64.8%).

[0336] LC-MS, M / Z(ESI): 462.3, 464.3[M+H] + .

[0337] Step 2: Synthesis of 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyric acid (04B)

[0338]

[0339] Ethyl 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyrate (70 mg, 0.15 mmol), THF (5 mL), and lithium hydroxide monohydrate (19 mg, 0.45 mmol) dissolved in water (0.5 mL) were successively added to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 16 h. The organic solvents were removed by concentration under reduced pressure. Water (5 mL) was added, and the pH was adjusted to 6 with 1N hydrochloric acid. 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 obtain 4-((tert-butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyric acid (04B) (66 mg, yield 100%).

[0340] LC-MS, M / Z(ESI): 434.3, 436.3[M+H] + .

[0341] Step 3: Synthesis of Methyl (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-triazaoctadec-16-enoate (04C)

[0342]

[0343] 4-((tert-Butoxycarbonyl)(4-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)amino)butyric acid (66 mg, 0.15 mmol), DMF (10 mL), Methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (54 mg, 0.15 mmol), TBTU (73 mg, 0.23 mmol) and DIPEA (79 mg, 0.61 mmol) were successively added to a 100 mL single-necked flask. Under nitrogen protection, 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 × 3). The combined organic phases were successively washed 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 (dichloromethane / methanol (V / V) = 1:0 - 9:1) gave Methyl (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-triazaoctadec-16-enoate (04C) (60 mg, yield 54%).

[0344] LC-MS, M / Z(ESI): 732.3, 734.3 [M+H] + .

[0345] 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-triazaoctadec-16-enoic acid (04D)

[0346]

[0347] To a 100 mL single-necked flask were successively added methyl (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-triazapentadec-16-enoate (60 mg, 0.082 mmol), THF (6 mL), and lithium hydroxide monohydrate (10 mg, 0.24 mmol) dissolved in water (1 mL). The mixture was stirred at room temperature for 6 h. The organic solvent was removed by concentration under reduced pressure. Water (5 mL) was added, and the pH was adjusted to 6 with 1 N hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL × 3). The combined organic phases were 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-triazapentadec-16-enoic acid (04D) (45 mg, yield 76%).

[0348] LC-MS, M / Z (ESI): 718.4, 720.4 [M+H] + .

[0349] Step 5: Synthesis of (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)

[0350]

[0351] To a 100 mL single-necked flask were successively added (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-triazapentadec-16-enoic acid (45 mg, 0.063 mmol), dichloromethane (4 mL), and trifluoroacetic acid (1 mL). The mixture was stirred at room temperature for 16 h. The crude product was obtained by concentration under reduced pressure and purified by 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, yield 11%).

[0352] 1 1H NMR (400 MHz, DMSO-d6) δ 10.05 (s, J = 8.3 Hz, 1H), 8.44 (d, J = 8.3 Hz, 1H), 8.11 (t, J = 5.7 Hz, 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 = 7.5 Hz, 1H), 3.84 (s, 3H), 3.70 (d, J = 5.7 Hz, 2H), 3.04 (q, J = 6.3 Hz, 2H), 2.75 (d, J = 7.2 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.72–1.65 (m, 2H).

[0353] LC-MS, M / Z (ESI): 488.2, 490.2 [M+H] + .

[0354] Example 5: Preparation of Compound I-5

[0355]

[0356] First step: 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)

[0357]

[0358] To a 100 mL single-necked flask were successively added methyl (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)propionate (300 mg, 0.404 mmol), THF (3 mL), an aqueous solution of lithium hydroxide monohydrate (84.8 mg, 2.02 mmol) (3 mL), and the mixture was stirred at room temperature for 6 h. THF was removed by concentration under reduced pressure. Water (5 mL) was added, and the pH was adjusted to 6 with 1 N hydrochloric acid. The mixture was extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain (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)propionic acid (180 mg, yield 59.7%).

[0359] LC-MS, M / Z (ESI): 747.36 [M+H] + 。

[0360] Step 2: Synthesis of (S)-3-(2-(4-((4-formamido-1H-imidazol-2-yl)amino)butanamido)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionic acid (I-5)

[0361]

[0362] To a 100 mL single-necked flask, (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) were added successively, and the mixture was stirred at room temperature for 30 min. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% ammonia water, B = acetonitrile; gradient: 30% - 100%) to obtain (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, yield 54%).

[0363] 1 H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.4 Hz, 1H), 8.19 (q, J = 6.4 Hz, 1H), 7.47–7.43 (m, 2H), 7.41 (t, J = 6.8 Hz, 1H), 7.39–7.35 (m, 2H), 7.13 (s, 1H), 6.39 (d, J = 2.0 Hz, 1H), 6.25 (s, 1H), 5.21 (q, J = 7.2 Hz, 1H), 3.83 (s, 3H), 3.76–3.62 (m, 2H), 3.13 (d, J = 6.0 Hz, 2H), 2.72 (d, J = 7.2 Hz, 2H), 2.21 (t, J = 7.2 Hz, 2H), 1.76–1.68 (m, 2H).

[0364] LC-MS, M / Z (ESI): 497.22 [M + H] + 。

[0365] Example 6: Preparation of Compounds II-1A & II-1B

[0366] The synthetic route is as follows:

[0367]

[0368] First step: Synthesis of ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (06B)

[0369]

[0370] Ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate (14.00 g, 37.01 mmol), dichloromethane (150 mL) and di-tert-butyl dicarbonate (12.12 g, 55.53 mmol) were successively added to a 500 mL single-necked flask and stirred at room temperature for 3 h. The reaction mixture was washed with water (150 mL), and the combined organic phases were 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 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (06B) (14.00 g, yield 79.07%).

[0371] LC-MS, M / Z(ESI): 478.12, 480.11 [M+H] + .

[0372] Step 2: Synthesis of ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (06C)

[0373]

[0374] Ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (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 successively added to a 250 mL single-necked flask and stirred at 100 °C for 16 h. After cooling to room temperature, the filtrate was filtered and 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)propionate (06C) (14.00 g, yield 99.75%).

[0375] LC-MS, M / Z(ESI): 480.24 [M+H] + .

[0376] Step 3: Synthesis of ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (06D)

[0377]

[0378] Ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (06D) (6.00 g, yield 86.5%) was obtained by adding ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (8.00 g, 16.7 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 41.7 mL) to a 250 mL single-necked flask in sequence and stirring at room temperature for 2 h. The reaction solution was concentrated under reduced pressure.

[0379] LC-MS, M / Z (ESI): 380.19 [M+H] + .

[0380] Step 4: Synthesis of ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (06E)

[0381]

[0382] At room temperature, ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (1.0 g, 2.4 mmol) was dissolved in anhydrous DMF (10 mL), and then TBTU (0.93 g, 2.9 mmol) was added. DIPEA (1.02 g, 7.89 mmol) was added at 0 °C, and after returning to room temperature and stirring for half an hour, N-(tert-butoxycarbonyl)glycine (0.46 g, 2.6 mmol) was added and stirred. After the reaction was monitored by TLC to be complete, saturated NH4Cl solution (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed once with saturated NaHCO3 solution (50 mL), dried over anhydrous sodium sulfate, filtered by suction, and concentrated under reduced pressure. The obtained residue was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:1 - 1:0) to obtain ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (06E) (0.80 g, yield 62%).

[0383] LC-MS, M / Z (ESI): 537.6 [M+H] + .

[0384] Step 5: Synthesis of Ethyl 3-((N-(tert-Butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (06F)

[0385]

[0386] At room temperature, dissolve ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (0.80 g, 1.5 mmol) in methanol (10 mL), add 10% wet palladium on carbon (100 mg), replace hydrogen three times, and then stir the reaction under a hydrogen atmosphere at room temperature. After monitoring by TLC that the raw materials are completely converted, filter the reaction solution by suction, and concentrate the filtrate under reduced pressure to obtain ethyl 3-((N-(tert-butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (06F) (0.66 g, yield 99%).

[0387] LC-MS, M / Z(ESI): 447.5 [M+H] + .

[0388] Step 6: Synthesis of Ethyl 3-(4-((14-Azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (06G)

[0389]

[0390] At room temperature, dissolve ethyl 3-((N-(tert-butoxycarbonyl)glycyl)amino)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (0.66 g, 1.5 mmol) in anhydrous DMF (5 mL), add potassium carbonate (0.51 g, 3.7 mmol) and azido-pentaethylene glycol-p-toluenesulfonate (0.80 g, 1.9 mmol), and stir at 80 °C for 6 hours. After monitoring by TLC that the raw materials are completely converted, cool to room temperature, add saturated NaHCO3 solution (10 mL), and extract with ethyl acetate (10 mL × 3). Wash the combined organic phase once with saturated NaCl solution (20 mL), dry over anhydrous sodium sulfate, filter by suction, and concentrate under reduced pressure to obtain ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (06G) (0.97 g, yield 95%).

[0391] LC-MS, M / Z(ESI): 692.8 [M+H] + .

[0392] Step 7: Synthesis of Ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycylamino)propionate Hydrochloride (06H)

[0393]

[0394] At room temperature, dissolve ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(tert-butoxycarbonyl)glycyl)amino)propionate (0.97 g, 1.4 mmol) in a 1,4-dioxane solution of hydrogen chloride (4 M, 7 mL), and stir for half an hour. After monitoring by TLC that the raw materials were completely converted, concentrate under reduced pressure to obtain the crude product. Add ethyl acetate, stir, filter, collect the filter cake, and dry in vacuo to obtain ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycylamino)propionate hydrochloride (06H) (0.88 g, yield 100%).

[0395] LC-MS, M / Z(ESI): 592.6 [M+H] + .

[0396] Step 8: Synthesis of Ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-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)propionate (06I)

[0397]

[0398] 4-((4-Cyano-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)(4-methoxybenzyl)amino)butyric acid (01L) (0.88 g, 2.0 mmol), DMF (20 mL), ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(glycylamino)propionate hydrochloride (1.5 g, 2.4 mmol), TBTU (0.77 g, 2.4 mmol) and DIPEA (0.77 g, 6.0 mmol) were successively added to a 100 mL single-necked flask. Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed successively with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 95:5) gave ethyl 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-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)propionate (06I) (1.5 g, yield 74%).

[0399] LC-MS, M / Z (ESI): 1018.5 [M+H] + .

[0400] Step 9: Synthesis of 3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yloxy)-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)propionic acid (06J)

[0401]

[0402] To a 100 mL single-necked flask were successively added 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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 ethyl ester (1.0 g, 0.98 mmol), THF / H2O (v:v = 1:1, 5 mL), and lithium hydroxide monohydrate (50 mg, 1.2 mmol), and the mixture was stirred at room temperature for 3 h. THF was removed by concentration under reduced pressure, the residue was adjusted to pH 6 with 1 N hydrochloric acid, and the mixture was extracted with ethyl acetate (5 mL × 3). The combined organic phases were washed with saturated brine (5 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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, yield 75%).

[0403] LC-MS, M / Z(ESI): 990.5[M+H] + .

[0404] Step 10: Synthesis of 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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)

[0405]

[0406] To a 100 mL single-necked flask were successively added 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-1-yloxy)-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), 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 preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 10% - 95%) to obtain 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-1-yloxy)-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, yield 95.0%).

[0407] 1 H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 8.44 (s, 1H), 8.07 (s, 1H), 7.49 (s, 1H), 7.42 (d, J = 6.0 Hz, 1H), 7.37–7.31 (m, 1H), 7.20 (d, J = 2.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 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 = 4.4 Hz, 2H), 3.12–3.02 (m, 2H), 2.68 (d, J = 7.2 Hz, 2H), 2.17 (t, J = 7.2 Hz, 2H), 1.72–1.63 (m, 2H).

[0408] LC-MS, M / Z (ESI): 740.3 [M + H] + .

[0409] Step 11: Synthesis of (R)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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) and (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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)

[0410]

[0411] 3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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 resolved 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 obtain the compound (R)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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 I.D., 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 the compound (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecan-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 I.D., 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).

[0412] II-1A: 11H NMR (400 MHz, DMSO-d6) δ 12.50–11.15 (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 = 2.8 Hz, 1H), 7.08 (d, J = 8.4 Hz, 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 = 7.2 Hz, 2H), 2.17 (t, J = 7.2 Hz, 2H), 1.80–1.60 (m, 2H).

[0413] II-1B: 1 1H NMR (400 MHz, DMSO-d6) δ 12.4–11.4 (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 = 2.8 Hz, 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 = 7.2 Hz, 2H), 2.17 (t, J = 7.2 Hz, 2H), 1.80–1.60 (m, 2H).

[0414] Example 7: Preparation of Compound III-1

[0415] The synthetic route is as follows:

[0416]

[0417] The first step: 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)

[0418]

[0419] To a 100 mL single-necked flask, 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)heptanediamide (500 mg, 0.803 mmol), DCM (5 mL), triethylamine (0.5 mL), and dihydro-4,4-dimethyl-2H-pyran-2,6(3H)-dione (137 mg, 0.964 mmol) were added successively, and the mixture was stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 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 (500 mg, yield 81.4%).

[0420] 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-diaza-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1)

[0421]

[0422] To a 100 mL single-necked flask, 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 (500 mg, 0.654 mmol), DCM (5 mL), 4-nitrophenol (109 mg, 0.784 mmol), and EDCI (150 mg, 0.782 mmol) were added successively, and the mixture was stirred at room temperature for 12 h. The mixture was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 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-diaza-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1) (268 mg, yield 46.3%).

[0423] 11H NMR (400 MHz, DMSO-d6) δ 8.27 (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).

[0424] LC-MS, M / Z (ESI): 887.3 [M+H] + .

[0425] Example 8: Preparation of Compound III-2

[0426] The synthetic route is as follows:

[0427]

[0428]

[0429] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azapentadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriaconta-1,32-diyne-17-yl)carbamate (07B)

[0430] 4-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo-3-(perfluorophenoxy)propyl)heptanedioic acid bis(perfluorophenyl) ester (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 successively added to a 100 mL single-necked flask and stirred at room temperature for 3 h. The crude product was obtained by concentration 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-azapentadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriaconta-1,32-diyne-17-yl)carbamate (3.0 g, yield 99%).

[0431] Step 2: Synthesis of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (07C)

[0432] To a 100 mL single-necked flask, (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-diazadotriaconta-1,32-diyne-17-yl)carbamate (3.0 g, 3.1 mmol), DMF (30 mL) and triethylamine (20 mL) were added successively, and the mixture was stirred at room temperature for 36 h. The crude product was obtained by concentration under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to obtain 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (2.0 g, yield 86%).

[0433] Step 3: Synthesis of 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 (07D)

[0434] To a 100 mL single-necked flask, 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (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 successively, and the mixture was stirred at room temperature for 3 h. The crude product was obtained by concentration under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 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 (2.0 g, yield 84%).

[0435] Step 4: Synthesis of 21,21 - dimethyl - 14,19 - dioxo - 17,17 - bis(3 - oxo - 7,10,13 - trioxa - 4 - azapentadec - 15 - yn - 1 - yl) - 4,7,10 - trioxa - 13,18 - diaza - 1 - yn - 23 - oleic acid pentafluorophenyl ester (III - 2)

[0436] Add 21,21 - dimethyl - 14,19 - dioxo - 17,17 - bis(3 - oxo - 7,10,13 - trioxa - 4 - azapentadec - 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) into a 100 mL single - necked flask in sequence, and stir at room temperature for 12 h. Concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography (ethyl acetate / acetonitrile (V / V)=1:1) to obtain 21,21 - dimethyl - 14,19 - dioxo - 17,17 - bis(3 - oxo - 7,10,13 - trioxa - 4 - azapentadec - 15 - yn - 1 - yl) - 4,7,10 - trioxa - 13,18 - diaza - 1 - yn - 23 - oleic acid pentafluorophenyl ester (III - 2) (110 mg, yield 18.6%).

[0437] 1 H NMR (400 MHz, CDCl3) δ 7.39 (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), 3.58–3.54 (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).

[0438] Preparation Example 1: Design and Synthesis of siRNA Conjugates

[0439] Taking C1 in Table 1 as an example, the synthesis steps of the siRNA conjugate with the conjugate group linked to the 5'-end of the sense strand of siRNA are as follows:

[0440] 1) Synthesis of single - stranded oligoribonucleotide: Synthesize oligoribonucleotide according to the phosphoramidite solid - phase synthesis technique. On the general controlled - pore glass CPG Synthesis was carried out thereon. All 2'-modified nucleotide phosphoramidites and auxiliary reagents are commercially available reagents (Shanghai Zhaowei Technology Development Co., Ltd.). All phosphoramidites were dissolved in anhydrous acetonitrile and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT, Suzhou Keloma Biotechnology Co., Ltd.) as the activator was 5 minutes. A solution of 50 mM 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) was used to generate the phosphorothioate bond, and the reaction time was 3 minutes. All sequences were completed after the DMT group was removed finally.

[0441] 2) Cleavage and deprotection of oligoribonucleotides bound to CPG: After the solid-phase synthesis was terminated, the protecting groups were removed by treating with an acetonitrile solution containing 20% diethylamine for 30 minutes. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia water. The combined solution was concentrated to obtain a solid mixture.

[0442] 3) Purification of single-stranded oligoribonucleotides: The oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was a 10 mM sodium perchlorate solution containing 20% acetonitrile, 20 mM Tris, 1 mM EDTA, pH 7.4; and buffer B was a 500 mM sodium perchlorate solution containing 20% acetonitrile, 20 mM Tris, 1 mM EDTA, pH 7.4. The target product was separated, and part of the product was desalted using a reverse-phase C18 column.

[0443] 4) Coupling of single-stranded oligoribonucleotides to the linker: 12 mg of TA14 was dissolved in DMSO, which was added to the nucleic acid amino intermediate (10 mg, dissolved in DMSO). After ultrasonic mixing, carbonate buffer solution (pH = 9) was added, and after reacting at 25 °C for 16 h, purification was carried out.

[0444] 5) Coupling of single-stranded oligoribonucleotide-TA14 conjugate to II-1A: 7 mg of nucleic acid-TA14 was dissolved in buffer salt solution, and the ligand II-1A (equivalent: 15, dissolved in DMF) was added to the above solution and vortexed; Take THPTA:CuSO4·5H2O = 5:1 (equivalents are 15 and 3 respectively), shake at 40 °C for 5 min, add the nucleic acid ligand mixture to the above reaction solution and vortex; Sodium ascorbate (equivalent of 25) was quickly added to the above solution, vortexed, and reacted at 40 °C for 1 h. After the reaction, purification was carried out to obtain the complete single-stranded oligoribonucleotide-TA14-II-1A conjugate.

[0445] 6) Annealing to produce siRNA: Dissolve the single-stranded oligoribonucleotides to be annealed in sterile RNase Free water (without RNA hydrolase) to prepare a 200 μM solution. Set up the annealing reaction system as follows. Place 10 nmol of the mixture with a total volume of 100 μL in a 95 °C water bath for 10 minutes (≥100 nmol requires 20 minutes at a higher temperature). Quickly transfer it to a 60 °C water bath and let it cool naturally. The annealed solution should not be stored at high temperatures. Complementary strands are formed by combining equimolar single-stranded oligoribonucleotide solutions to obtain various unmodified siRNAs. Among them, the nucleotide sequences of the sense and antisense strands of the siRNAs obtained in the present invention are shown in Table 1; moreover, liquid chromatography-mass spectrometry (LC-MS) is used to detect the molecular weight of the siRNAs, and the measured value of the molecular weight is compared with the theoretical value. The results show that the measured value ≈ theoretical value, indicating that the siRNAs in Table 1 are obtained. The specific structure of the ligand (II-1A) in Table 1 can be found in the description of the present invention (i.e., II-1A in the compounds or their stereoisomers, tautomers or pharmaceutically acceptable salts described in the third aspect). 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.

[0446] Table 1

[0447]

[0448]

[0449] 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 nucleotides with 2'-methoxy modification (i.e., c, g, u, and a represent that the ribose 2'-OH of the nucleotides represented by their corresponding uppercase letters is replaced by a methoxy group); f represents that the nucleotide before f is a nucleotide with 2'-fluoro modification (i.e., the 2'-OH of the nucleotide before f is replaced by a fluorine atom); s represents that there is a phosphorothioate between the two adjacent nucleotide residues on the left and right of s (i.e., the 5'-phosphate group of the nucleotide before s is replaced by a 5'-phosphorothioate group).

[0450] (TriSM6.1)(TA14)(NH2C6) is:

[0451]

[0452] Its preparation method refers to WO2022 / 216920A1.

[0453] (II-1A)(TA14)(NH2C6) is:

[0454]

[0455] (II-1B)(TA14)(NH2C6) is:

[0456]

[0457] (II-1B)(III-1)(NH2C6) is:

[0458]

[0459] (II-1B)(III-2)(NH2C6) is:

[0460]

[0461] The structures of cPrpus and cPrpas are as follows:

[0462]

[0463] Test Example 1: avβ6 Ligand ELISA Binding Experiment

[0464] Dilute the integrin avβ6 protein (source: Acro Biosystems) to 5 μg / mL with the coating solution, and add 25 μL of the protein to each well of a 384-well plate. Centrifuge at 1000 rpm for 1 minute, then let it stand overnight at 4°C. Add 100 μL of the washing solution to each well, let it stand for 5 minutes, then pat dry and wash 3 times. Add 100 μL of the blocking solution to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 90 minutes. Add 100 μL of the washing solution to each well, let it stand for 5 minutes, then pat dry and wash 3 times. Add 10 μL of the above compounds at different dilution concentrations 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 the washing solution to each well, let it stand for 5 minutes, then pat dry and wash 3 times. Add 25 μL of the fibronectin biotinylated antibody (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 the washing solution to each well, let it stand for 5 minutes, then pat dry and wash 3 times. Add 25 μL of horseradish peroxidase-labeled streptavidin to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 30 minutes. Add 100 μL of the washing solution to each well, let it stand for 5 minutes, then pat dry and wash 4 times. Add 25 μL of the ultrasensitive luminescence solution to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 5 minutes. Read the luminescence value on an enzyme-linked immunosorbent assay (ELISA) reader.

[0465] Table 2: αvβ6 integrin ligand binding activity

[0466] Compound number <![CDATA[IC 50 (nM)]]> I-1 4.42 I-2 2.11 II-1B 4.53

[0467] The results show that the compounds of the present application have high binding activity to avβ6 integrin.

[0468] Test Example 2: Silencing efficacy of tracheal administration of siRNA conjugate in rats in vivo on target expression

[0469] Referring to Patent WO2022 / 216920A1, siRNA conjugates targeting rat RAGE protein were designed and synthesized, and the compound list is shown in Table 1.

[0470] Experimental method:

[0471] 1. Administration of aerosol needle to rats and collection of serum and lung tissue samples

[0472] On day 0, whole blood of rats was taken from the jugular vein of rats to obtain serum. On day 1, after anesthetizing the rats with isoflurane, 200 μL of PBS or drug buffer was inhaled using an aerosol needle, and the administration dose was 0.1 mg / kg. Before the experimental endpoint, the rats were anesthetized with 10% chloral hydrate, blood was taken from the abdominal aorta with a negative pressure blood collection tube to obtain serum, and then the left and right lung tissues were taken. The lung tissues were quickly frozen in liquid nitrogen. All processed serum and lung tissue samples were stored at -80 °C.

[0473] 2. Homogenization of rat lung tissue and probe quantitative PCR

[0474] The lung tissue was thawed on ice and homogenized using a tissue grinder. Total RNA in the lung tissue was extracted using the Tissue RNA ExtractionKit 2.0Plus (Vazyme, R411-C3) kit. After reverse transcription (Vazyme, R333-01), the mRNA expression level of RAGE in rat lung tissue was detected by probe quantitative PCR method (Vazyme, QN211-02), and normalized with the expression level of rat GAPDH, and then compared with the expression level of the PBS control group as 100% reference.

[0475] 3. Detection of RAGE protein content in rat serum by ELISA kit

[0476] The serum was thawed on ice, and rat serum was detected according to the instructions of the ELISA detection kit Rat RAGE DuoSet ELISA (R&D systems, DY1616). According to the fitting conversion of the measured standard curve, the expression level of RAGE protein in rat serum was calculated and compared with the expression level of the PBS control group as 100% reference.

[0477] Table 3: Expression levels of RAGE protein in rat serum and RAGE mRNA in the lung

[0478]

[0479] The results showed that the siRNA conjugate significantly reduced the expression of RAGE mRNA and protein in the lungs and serum of rats. The compounds of the present application can make the siRNA silence the lung target and the expression of the corresponding secreted protein more effectively, indicating that it has a stronger ability to target the delivery of siRNA molecules to lung cells.

[0480] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to 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 with 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 to linker T; Said linker T is used to directly or indirectly connect to 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, characterized in that, The compound satisfies one or more of the following conditions: a) having a structure b) wherein R2, R3, ring C, n, and p are as defined 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 as claimed in claim 1 or its tautomer or stereoisomer, characterized in that, The compound has one or more of the following structures: Wherein, the definitions of Ring A, R1, m, and T are as described in claim 1; And / or, when connected to linker T, the compound has one or more of the following structures: Wherein, the definitions of Ring A, R1, m, and 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 where t is from 1 to 10; preferably, the 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, characterized in that, When connected to linker T, 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 of formula I or its tautomer or stereoisomer according to any one of claims 1, 3 or 5, characterized in that, The 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 , 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, characterized in that, The compound has one or more of the following structures: Among them, represents the 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 as claimed in claim 1, wherein, The compound has one or more of the following structures: wherein, t is 3, 4, 5 or 6; denotes a connection point.

9. The compound of formula I or its tautomer or stereoisomer according to claim 1, wherein, The compound includes:

10. The compound of formula I or its tautomer or stereoisomer according to claim 1, characterized in that, The compound includes: 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, Comprising 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 linked to the linker T in the compound of formula I.

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

14. The conjugate according to claim 12 or 13, wherein 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 monodentate, bidentate, tridentate, or tetradentate 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, characterized in that, The conjugate comprises the following structure wherein, formula I is the compound of formula I as described or a tautomer or stereoisomer thereof, 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 comprises the following structure Among them, represents the junction point with the oligonucleotide.

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

18. Use of a compound of formula I as described in any one of claims 1 - 10 or a tautomer or stereoisomer thereof, a conjugate as described in any one of claims 12 - 16, or a composition as described in claim 17 in the preparation of a drug.

19. The use according to claim 18, characterized in that, The drug is used for delivering the active molecule G to cells; and / or, inhibiting the expression of a target gene in cells; and / or preparing a drug for inhibiting the expression of a target gene in cells; and / or preparing a drug for 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).

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