Compounds targeting αvβ6 and use thereof

Abstract

The present invention provides compounds targeting αvβ6. The compounds are compounds shown as formula I in the present invention and can be used for delivering active molecules to cells or tissue expressing integrin αvβ6.

Description

Compounds targeting αvβ6 and their uses Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to a compound targeting αvβ6 and uses thereof. Background Art

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

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

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

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

[0006] The targeting compound of the present invention is a compound shown in Formula I, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof. The targeting compound is further connected to a linker, through which an active molecule is further connected. A scaffold may further be provided between the targeting compound and the active molecule, wherein the scaffold is a monodentate, bidentate, tridentate or quadridentate structure. The active molecule is connected to one, two, three or four of the targeting compounds via the scaffold.

[0007] In the first aspect of the present invention, there is provided a compound of formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs:

[0008] Wherein, L1 is empty, -CH2- or

[0009] L2 is -(CH2) m -, 3- to 10-membered cycloalkylene or 3- to 10-membered heterocycloalkylene, m is 1, 2, 3, 4, 5, or 6;

[0010] Said L2 is optionally substituted by C1-C3 alkyl or halogen;

[0011] A is selected from:

[0012] Ra is a 6-10 membered aromatic ring or a 5-10 membered heteroaromatic ring; said Ra is optionally substituted by R2;

[0013] Rb is dioxane;

[0014] R1, R2, R3, R4, and R5 are each independently H, halogen, -OH, -NH2, -COOH, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -CON(C1-C6 alkyl)2, -CON(C1-C6 haloalkyl)2, -CO-C1-C6 alkyl, -CO-C1-C6 haloalkyl;

[0015] Said A is optionally connected to a linker T;

[0016] And the compound satisfies one or more of the following conditions i)-iii):

[0017] i) L1 is A is selected from:

[0018] ii) L1 is empty, -CH2-, A is

[0019] iii), L1 is empty, -CH2- or A is And Ra is a 5-6 membered heteroaromatic ring.

[0020] In an optional embodiment of the present application, the compound represented by Formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs:

[0021] Wherein, L1 is -CH2- or

[0022] L2 is -(CH2) m -, m is 1, 2, 3, 4, 5, 6;

[0023] Said L2 is optionally substituted by C1-C3 alkyl or halogen;

[0024] A is selected from:

[0025] Ra is a 6-10 membered aromatic ring or a 5-10 membered heteroaromatic ring; said Ra is optionally substituted by R2;

[0026] Rb is dioxane;

[0027] R1, R2, R3, R4, and R5 are each independently H, halogen, -OH, -NH2, -COOH, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -CON(C1-C6 alkyl)2, -CON(C1-C6 haloalkyl)2, -CO-C1-C6 alkyl, -CO-C1-C6 haloalkyl;

[0028] Said A is optionally connected to a linker T;

[0029] And the compound satisfies one or more of the following conditions i)-iii):

[0030] i) L1 is A is selected from:

[0031] ii) L1 is empty, -CH2-, A is

[0032] iii), L1 is empty, -CH2- or A is And Ra is a 5-6 membered heteroaromatic ring.

[0033] In a preferred embodiment, R1 is C1-C6 alkyl or C1-C6 haloalkyl.

[0034] In a preferred embodiment, R1 is C1-C3 alkyl or C1-C3 haloalkyl.

[0035] In a preferred embodiment, for

[0036] In a preferred embodiment, With structure

[0037] In a preferred embodiment, It is a Z configuration.

[0038] In a preferred embodiment, when L1 is When A is Said A is optionally connected to a linker T.

[0039] In a preferred embodiment, Ra is a naphthalene ring.

[0040] In a preferred embodiment, R3 is H.

[0041] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -、 3- to 10-membered cycloalkyl or 3- to 10-membered heterocycloalkyl, wherein m is 1, 2, 3, 4, 5, or 6.

[0042] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -、 3- to 10-membered cycloalkyl or 3- to 10-membered heterocycloalkyl, wherein m is 2, 3, or 4.

[0043] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -、 or a 3- to 6-membered cycloalkyl group, wherein m is 2, 3, or 4.

[0044] In a preferred embodiment, -L1-L2- is selected from -(CH2)3-, or a 4- to 5-membered cycloalkyl group.

[0045] In a preferred embodiment, when L1 is empty, L2 is a 3- to 10-membered cycloalkylene group or a 3- to 10-membered heterocycloalkylene group.

[0046] In a preferred embodiment, when L1 is empty, L2 is a 3- to 6-membered cycloalkylene group or a 3- to 6-membered heterocycloalkylene group.

[0047] In a preferred embodiment, when L1 is empty, L2 is a 3- to 6-membered cycloalkylene group.

[0048] In a preferred embodiment, when L1 is empty, L2 is

[0049] In a preferred embodiment, when L1 is empty, A is selected from:

[0050] In a preferred embodiment, when L1 is empty, L2 is A is selected from:

[0051] In a preferred embodiment, when L1 is empty and L2 is a 3-10 membered cycloalkylene group or a 3-10 membered heterocycloalkylene group, A is selected from:

[0052] Wherein, Ra is a 5-6 membered heteroaromatic ring; Rb is dioxane; and the definitions of R3, R4 and R5 are as shown in the first aspect of the present invention.

[0053] In a preferred embodiment, when L1 is empty and L2 is a 3-10 membered cycloalkylene group or a 3-10 membered heterocycloalkylene group, A is selected from:

[0054] Wherein, Ra is a 5-6 membered heteroaromatic ring; R3, R4, and R5 are defined as shown in the first aspect of the present invention.

[0055] In a preferred embodiment, when L1 is empty and L2 is a 3-10 membered cycloalkylene group or a 3-10 membered heterocycloalkylene group, A is selected from:

[0056] Wherein, Ra is a 5-6 membered heteroaromatic ring; R3 is H; R4 and R5 are hydroxyl or C1-C6 alkoxy.

[0057] In a preferred embodiment, when L1 is empty and L2 is a 3-10 membered cycloalkylene group or a 3-10 membered heterocycloalkylene group, A is selected from:

[0058] Wherein, Ra is a 5-6 membered heteroaromatic ring; R3 is H; and R4 and R5 are methoxy, ethoxy or propoxy.

[0059] In a preferred embodiment, when L1 is -CH2-, L2 is -(CH2) m -, A is selected from: wherein Ra is a 5-6 membered heteroaromatic ring; Rb is dioxane; and R3, R4, and R5 are as defined above.

[0060] In a preferred embodiment, R3 is H.

[0061] In a preferred embodiment, R4 and R5 are hydroxy or C1-C6 alkoxy.

[0062] In a preferred embodiment, R4 and R5 are methoxy, ethoxy or propoxy.

[0063] In a preferred embodiment, the compound of formula I has structure Ia:

[0064] Wherein, Ra and R3 are defined as above; Optionally, a linker T is attached.

[0065] In a preferred embodiment, in structure Ia, R3 is H.

[0066] In a preferred embodiment, in structure Ia, Ra is a 5-6 membered heteroaromatic ring optionally substituted by R2; said R2 is C1-C3 alkyl or C1-C3 haloalkyl.

[0067] In a preferred embodiment, in structure Ia, R2 is methyl.

[0068] In a preferred embodiment, in structure Ia, when When the connector T is connected and R3 is H, With structure

[0069] In a preferred embodiment, in structure Ia, when When the connector T is connected and R3 is H, With structure

[0070] In a preferred embodiment, in structure Ia, Ra is a 5-6 membered heteroaromatic ring optionally substituted by R2.

[0071] In a preferred embodiment, in structure Ia, Ra is a 5-membered N-containing heteroaromatic ring optionally substituted by R2.

[0072] In a preferred embodiment, in structure Ia, Ra is the following substituent optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole.

[0073] In a preferred embodiment, in structure Ia, Ra is methylpyrazole, such as

[0074] In a preferred embodiment, the compound of formula I has structure Ib:

[0075] Among them, R b , R4, and R5 are as defined above.

[0076] In a preferred embodiment, in structure Ib, R4 and R5 are OH or -OCH3.

[0077] In a preferred embodiment, in structure Ib, Rb is 1,3-dioxane, and Rb is optionally connected to a linker T.

[0078] In a preferred embodiment, in structure Ib, Rb is

[0079] In a preferred embodiment, the compound of formula I has structure Ic:

[0080] Here, Ra and R3 are as defined above.

[0081] In a preferred embodiment, in structure Ic, Ra is a naphthalene ring, and said Ra is optionally connected to a linker T;

[0082] In a preferred embodiment, in structure Ic, R3 is H.

[0083] In a preferred embodiment, in structure Ic, With structure

[0084] In a preferred embodiment, in structure Ic, With structure

[0085] In a preferred embodiment, the compound of formula I has structure Id:

[0086] Wherein, Ra and R3 are defined as described in the first aspect; Optionally, a linker T is attached.

[0087] In a preferred embodiment, in structure Id, when The structure is connected with a joint T.

[0088] In a preferred embodiment, in Structure Id, The benzene ring in the structure is connected with a linker T.

[0089] In a preferred embodiment, in structure Id, R3 is H.

[0090] In a preferred embodiment, in structure Id, when When the connector T is connected and R3 is H, With structure

[0091] In a preferred embodiment, in structure Id, when the benzene ring is connected to a linker T and R3 is H, With structure

[0092] In a preferred embodiment, in structure Id, when When the connector T is connected and R3 is H, With structure

[0093] In a preferred embodiment, in structure Id, when the benzene ring is connected to a linker T and R3 is H, With structure

[0094] In a preferred embodiment, in structure Id, Ra is a 5-6 membered heteroaromatic ring optionally substituted by R2.

[0095] In a preferred embodiment, in structure Id, Ra is a 5-membered N-containing heteroaromatic ring optionally substituted by R2.

[0096] In a preferred embodiment, in structure Id, Ra is the following substituent optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole.

[0097] In a preferred embodiment, in structure Id, Ra is methylpyrazole, such as

[0098] In a preferred embodiment, when said A is connected to a linker T, With structure

[0099] In a preferred embodiment, when said A is connected to a linker T, With structure

[0100] In a preferred embodiment, when R3 is H, for

[0101] In a preferred embodiment, when R3 is H, for

[0102] In a preferred embodiment, for

[0103] In a preferred embodiment, when said A is connected to a linker T, With structure

[0104] In a preferred embodiment, for

[0105] In a preferred embodiment, for

[0106] In a preferred embodiment, for

[0107] In a preferred embodiment, when A is connected to a linker T, the compound represented by formula I has the structure:

[0108] In a preferred embodiment, the linker T comprises a polyethylene glycol unit.

[0109] In a preferred embodiment, the linker T comprises 2-20 polyethylene glycol units.

[0110] In a preferred embodiment, the linker T is Where t is 1-10.

[0111] In a preferred embodiment, the linker T is Where t is 1-10.

[0112] In a preferred embodiment, t is 1, 2, 3, 4, or 5.

[0113] In a preferred embodiment, the linker T is

[0114] In a preferred embodiment, the linker T is

[0115] In a preferred embodiment, the compound of formula I has the structure Ie or If (attached with a linker T):

[0116] wherein T and R3 are as defined above; and q is selected from 0, 1 or 2.

[0117] In a preferred embodiment, in structure Ie, R3 is hydrogen.

[0118] In a preferred embodiment, in structure Ie, q is selected from 0.

[0119] In a preferred embodiment, in structure Ie, q is selected from 1.

[0120] In a preferred embodiment, in structure If, ​​the linker T is

[0121] In a preferred embodiment, in structure If, ​​the linker T is

[0122] In a preferred embodiment, the compound of Formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, includes the following structure:

[0123] in, Indicates a connection point.

[0124] In a preferred embodiment, the compound of Formula I, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, includes the following structure:

[0125] in, Indicates a connection point.

[0126] In a second aspect, the present invention provides a compound II, its tautomers, stereoisomers or salts thereof, including a compound represented by formula I as described in any one of the first aspects, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, and an active molecule G; wherein the active molecule G is connected to the linker T in the compound represented by formula I.

[0127] In a preferred embodiment, the active molecule G is an active pharmaceutical ingredient or a prodrug thereof.

[0128] In a preferred embodiment, the active molecule G is an antibody, immunoglobulin, label or marker, lipid, natural or modified nucleic acid, natural or modified nucleic acid oligonucleotide, natural or modified nucleic acid polynucleotide, peptide, nucleic acid aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, biotin, radioactive atom or molecule, or fluorophore.

[0129] In a preferred embodiment, the active molecule G is a natural or modified nucleic acid oligonucleotide.

[0130] As used herein, the terms "natural or modified nucleic acid oligonucleotide," "oligonucleotide comprising a natural or modified nucleic acid," or "natural or modified oligonucleotide" are synonymous and refer to oligonucleotides composed of natural nucleic acids or oligonucleotides comprising modified nucleic acids. The term "natural or modified nucleic acid polynucleotide" refers to polynucleotides composed of natural nucleic acids or polynucleotides comprising modified nucleic acids.

[0131] In a preferred embodiment, the active molecule G is ASO, siRNA, or miRNA.

[0132] In a third aspect, the present invention provides a conjugate comprising a compound of formula I as described in any one of the first aspects, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, a scaffold and an active molecule G; wherein the scaffold is connected to the linker T in the compound of formula I, and the active molecule G is connected to the compound of formula I through the scaffold.

[0133] In a preferred embodiment, the stent has a single-tooth, two-tooth, three-tooth, or four-tooth structure.

[0134] In a preferred embodiment, the active molecule G is connected to one, two, three or four compounds of formula I via the scaffold.

[0135] In a preferred embodiment, the conjugate has the following structure:

[0136] Wherein, Formula I is the compound represented by Formula I, and G is the active molecule G;

[0137] t is 1-10; preferably, t is 1, 2, 3, 4, 5, more preferably, t is 2, 3;

[0138] v is 1-10; preferably, v is 4, 5, 6, 7, or 8.

[0139] In a preferred embodiment, the active molecule G is a natural or modified nucleic acid oligonucleotide. In a preferred embodiment, the active molecule G is ASO, siRNA, or miRNA.

[0140] In a preferred embodiment, the conjugate has the following structure:

[0141] in, Indicates the point of attachment to the oligonucleotide.

[0142] In a fourth aspect, the present invention provides a composition comprising a compound of formula I as described in any one of the first aspects, its tautomers, stereoisomers, hydrates, solvates, pharmaceutically acceptable salts or prodrugs, compound II as described in any one of the second aspects, or a conjugate as described in any one of the third aspects.

[0143] In a preferred embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0144] In a fifth aspect, the present invention provides a compound of formula I as described in any one of the first aspects, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof, a compound II as described in any one of the second aspects, a conjugate as described in any one of the third aspects, or a composition as described in the fourth aspect, having the following uses, the uses being selected from:

[0145] delivering the active molecule G to the cell; and / or

[0146] Inhibiting the expression of target genes in epithelial cells; and / or

[0147] Treating lung disease; and / or

[0148] preparing a medicament for inhibiting expression of a target gene in a cell; and / or

[0149] Preparation of a medicament for treating lung disease.

[0150] In a preferred embodiment, the cell is an αvβ6 integrin-expressing cell or an epithelial cell.

[0151] In a preferred embodiment, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells and epithelial tumors (carcinomas).

[0152] In a sixth aspect, the present invention provides a compound of formula I as described in any one of the first aspects, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt, or prodrug thereof, compound II as described in any one of the second aspects, a conjugate as described in any one of the third aspects, or a composition as described in the fourth aspect, for use in:

[0153] delivering the active molecule G to the cell; and / or

[0154] Inhibiting the expression of target genes in epithelial cells; and / or

[0155] Treating lung disease; and / or

[0156] preparing a medicament for inhibiting expression of a target gene in a cell; and / or

[0157] Preparation of a medicament for treating lung disease.

[0158] In a preferred embodiment, the cell is an αvβ6 integrin-expressing cell or an epithelial cell.

[0159] In a preferred embodiment, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells and epithelial tumors (carcinomas).

[0160] In the seventh aspect, the present invention provides a method for delivering active molecules to cells, inhibiting the expression of target genes in cells, or treating lung diseases, which method comprises administering to a patient the compound II as described in the second aspect, the conjugate as described in the third aspect, or the composition as described in the fourth aspect.

[0161] In a preferred embodiment, the cell is the cell described in the fifth aspect.

[0162] connector

[0163] As disclosed herein, in some embodiments, the targeting compound further comprises a linker (linker T) to further connect the active molecule. In some embodiments, the linker can be a structure containing a polyethylene glycol (PEG) group. The polyethylene glycol linker can comprise 2-20 polyethylene glycol units.

[0164] In some embodiments, the linker is

[0165] In some embodiments, the linker T is

[0166] Indicates the attachment sites for the targeting compound and the active molecule, respectively.

[0167] In some embodiments, wherein t is an integer from 1 to 10; for example, t is 1, 2, 3, 4, 5, 6, or 7.

[0168] In some embodiments, the linker has a reactive group at one end connected to the active molecule, and the active molecule is directly conjugated to the linker through the reactive group, or the active molecule is conjugated to the linker after being connected to the scaffold. In some embodiments, the reactive group is an azide or an alkyne-containing group.

[0169] There is no particular limitation on the connection site between the linker and the targeting compound. In some embodiments, A in Formula I is connected to a linker T.

[0170] In some embodiments, the structure of the compound of Formula I connected with a linker T comprises:

[0171] Multidentate targeting compounds and scaffolds

[0172] As disclosed herein, in some embodiments, one or more compounds that target αβ (targeting compounds) can be linked to one or more active molecules that are 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 "bivalent" ligand). In some embodiments, three targeting compounds are conjugated to the active molecule (referred to herein as a "tridentate" or "trivalent" ligand). In some embodiments, four targeting compounds are conjugated to the active molecule (referred to herein as a "tetradentate" or "tetravalent" ligand). In some embodiments, more than four are conjugated to the active molecule.

[0173] As disclosed herein, in some embodiments, the scaffold and the active molecule are connected to the targeting compound via a linker. In some embodiments, the scaffold is a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the active molecule is connected to one targeting compound via a monodentate scaffold. In some embodiments, the active molecule is connected to two targeting compounds via a bidentate scaffold. In some embodiments, the active molecule is connected to three targeting compounds via a tridentate scaffold. In some embodiments, the active molecule is connected to four targeting compounds via a tetradentate scaffold.

[0174] Scaffolds are generally known in the art. Non-limiting examples of scaffolds that can be used with the targeting compounds disclosed herein include, but are not limited to, polymers and polyamino acids (e.g., diglutamic acid, poly-L-lysine, etc.). In some embodiments, the scaffold can include amine reactive groups, amide bonds, and alkyne-containing moieties (e.g., alkynyl, cyclooctyne). In some embodiments, the cyclooctyne-containing moiety contains moieties such as dibenzocyclooctyne (DBCO), bicyclo[6.1.0]non-4-yne. In some embodiments, the alkyne-containing moiety includes, but is not limited to, alkyne maleimide, alkyne NHS ester. In some embodiments, the scaffold can include a cysteine ​​linker or group, DBCO-PEG 1-24 -NHS, propargyl-PEG 1-24 -NHS and / or multidentate DBCO and / or propargyl moieties.

[0175] In some embodiments, a typical tridentate scaffold is, for example:

[0176] This scaffold comprises an amine-reactive group, an amide bond, three PEG2 units, and an alkyne. The amine-reactive group can be conjugated to a primary amine on the transported active molecule, such as a terminal amine (e.g., NH2-C6), through amide formation. The alkyne can be conjugated to an azide-modified linker to form a triazole structure, which can then be linked to the targeting compound. In this scaffold, the amine-reactive group is p-nitrophenol (also known as 4-nitrophenol) ester.

[0177] In some embodiments, the amine-reactive group is not particularly limited. The amine-reactive group can also be, for example:

[0178] In some embodiments, the scaffold can be synthesized as a phosphoramidite compound, which can allow the tridentate scaffold to be coupled to an oligonucleotide (such as the 5' end of the sense strand of an siRNA) via phosphoramidite synthesis, as shown in the following structure:

[0179] in, Indicates siRNA.

[0180] Conjugate

[0181] The active molecule described herein can be connected to a targeting compound to form a conjugate. In some embodiments, the active molecule is an oligonucleotide, which can be coupled to the scaffold by phosphoramidite synthesis. The scaffold can be a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the azide group on the targeting compound can react (e.g., click chemistry reaction) with the alkyne (e.g., benzocyclooctyne, propargyl) of the scaffold to form a triazole structure, which is then connected to the targeting compound.

[0182] In some embodiments, the conjugate is the conjugate of the third aspect. The conjugate has a tridentate structure, and the conjugate is

[0183] Wherein, Formula I is the compound represented by Formula I, and G is the active molecule G;

[0184] t is 1-10; preferably, t is 1, 2, 3, 4, 5;

[0185] v is 1-10; preferably, v is 4, 5, 6, 7, or 8.

[0186] In a preferred embodiment, the active molecule G is a natural or modified oligonucleotide.

[0187] Active molecule

[0188] As used herein, an "active molecule" can be linked to the targeting compound and thereby transported to cells and / or tissues expressing the integrin αβ. A transported "active molecule" (or transported molecule) is any molecule that, when separated from an αβ integrin ligand as described herein, will have a desired effect on cells containing an αβ integrin receptor. The transported active molecule can be, but is not limited to, a pharmaceutical ingredient, a pharmaceutical product, a prodrug, a substance with known therapeutic benefit, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide, a peptide, a polymer, a polyamine, a protein, an aptamer, a toxin, a vitamin, PEG, a hapten, digoxin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, one or more transported active molecules (e.g., the same or different transported active molecules) are linked to one or more αβ integrin ligands to target the transported active molecules to cells expressing the αβ integrin. In some embodiments, the active molecule G is a natural or modified oligonucleotide.

[0189] Oligonucleotides

[0190] In the present application, the term "oligonucleotide" refers to a short chain of nucleotides (including nucleotides in deoxyribonucleic acid DNA or ribonucleic acid RNA) consisting of 50 or less bases. The oligonucleotides of the present application may be single-stranded or double-stranded, and the specific type is not limited and is within the scope of protection of the present application. Exemplarily, when it is a double-stranded structure, it consists of a sense strand and an antisense strand, and includes but is not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA) and dicer substrates; when it is a single-stranded structure, RNAi agents include but are not limited to antisense oligonucleotides (ASOs).

[0191] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

[0192] Terms and Definitions

[0193] Unless otherwise indicated, the definitions of groups and terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, definitions in tables, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with one another. The resulting group definitions and compound structures shall fall within the scope of the description of this specification.

[0194] Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by persons skilled in the art to which the claimed subject matter belongs. Unless otherwise indicated, all patents, patent applications, and publications cited herein are incorporated by reference in their entirety. If multiple definitions of a term are used herein, the definitions in this section shall prevail.

[0195] It should be understood that the above brief description and the detailed description below are exemplary and are only used for explanation, and do not impose any restrictions on the subject matter of the present invention. In this application, unless otherwise specifically stated, the use of the singular also includes the plural. It must be noted that unless otherwise clearly stated in the text, the singular forms used in this specification and claims include the plural forms of the things referred to. It should also be noted that unless otherwise stated, the use of "or" and "or" means "and / or". In addition, the use of the term "including" and other forms, such as "comprising", "including" and "containing" are not restrictive.

[0196] Definitions of standard chemical terms can be found in the references (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4THED." Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise indicated, conventional methods within the skill of the art, such as mass spectrometry, NMR, IR and UV / VIS spectroscopy and pharmacological methods, are used. Unless otherwise specified, the terms used herein in the descriptions of analytical chemistry, synthetic organic chemistry, and pharmaceuticals and medicinal chemistry are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and in the treatment of patients. For example, the manufacturer's instructions for use of the kit can be utilized, or reactions and purifications can be carried out in accordance with methods well known in the art or the description of the present invention. The above techniques and methods can generally be implemented according to conventional methods well known in the art, as described in the various general and more specific references cited and discussed in this specification. In this specification, groups and substituents thereof can be selected by those skilled in the art to provide stable structural moieties and compounds.

[0197] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes chemically equivalent substituents that would result from writing the formula from right to left. For example, CHO is equivalent to OCH. As used herein, Indicates the attachment site of a group. As used herein, "R1" and "R2" have the same meaning and are interchangeable. For other symbols such as R2, similar definitions have the same meaning.

[0198] The section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are incorporated herein by reference in their entirety.

[0199] In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings indicated below unless otherwise specifically stated.

[0200] When a numerical range is described in the specification and claims of this application and is understood as an "integer," it should be understood to include both endpoints of the range as well as every integer within the range. For example, "an integer from 0 to 5" should be understood to include every integer from 0, 1, 2, 3, 4, and 5.

[0201] As used herein, the term "halogen" by itself or as part of another substituent refers to fluorine, chlorine, bromine, or iodine.

[0202] As used herein, the term "alkyl" when used alone or as part of another substituent means a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, free of unsaturated bonds, having, for example, 1 to 6 carbon atoms and connected to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl. Alkyl groups may be unsubstituted or substituted with one or more suitable substituents. Alkyl groups may also be isotopomers of naturally abundant alkyl groups enriched in isotopes of carbon and / or hydrogen (i.e., deuterium or tritium).

[0203] The term "C1-C6 alkyl" alone or as part of another substituent is understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof. The term "C1-C5 alkyl" is understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3 or 5 carbon atoms. In particular, such radicals have 1, 2 or 3 carbon atoms ("C1-C3 alkyl"), for example methyl, ethyl, n-propyl or isopropyl.

[0204] The term "C1-C6 alkoxy" when used alone or as part of other substituents shall be understood to mean a linear or branched saturated monovalent hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms and an oxygen atom, or as C1-C6 alkyl-O-C1-C6 alkyl, as defined herein, wherein the oxygen atom may be attached to any carbon atom of the linear or branched C1-C6 alkyl radical. These include, but are not limited to, methoxy (CH3-O-), ethoxy (C2H5-O-), propoxy (C3H7-O-), and butoxy (C4H9-O-).

[0205] The term "cycloalkyl" or "carbocyclyl" when used alone or as part of another substituent refers to a cyclic alkyl group. m -C n "Cycloalkyl" is understood to mean a saturated, unsaturated or partially saturated carbon ring having m to n atoms. For example, "3-10 membered cycloalkyl" or "C3-C 10"Cycloalkyl" refers to a cyclic alkyl group containing 3 to 10 carbon atoms, which may contain 1 to 3 rings. The cyclic alkyl group includes a monocyclic, bicyclic, tricyclic, spirocyclic or bridged ring. Examples of unsubstituted cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and adamantyl, or a bicyclic hydrocarbon group such as a decalin ring. The cycloalkyl group may be substituted with one or more substituents. In some embodiments, the cycloalkyl group may be a cycloalkyl group fused to an aryl or heteroaryl group.

[0206] The term "heterocycloalkyl" or "heterocyclyl" when used alone or as part of another substituent refers to a cycloalkyl group in which one or more (in some embodiments, 1 to 3) carbon atoms are replaced by heteroatoms such as, but not limited to, N, O, S, and P. The term "mn-membered heterocycloalkyl" or "C m -C n "Heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having from m to n atoms. For example, the term "4-10 membered heterocycloalkyl" is understood to mean a saturated, unsaturated or partially saturated ring having from 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 indicate a heterocycloalkyl, the number of carbons is also meant to include the heteroatoms.

[0207] When used alone or as part of another substituent, the term "aromatic ring" refers to an aromatic ring structure, including compounds having one or more ring structures, such as monocyclic, bicyclic, tricyclic spirocyclic or bridged ring compounds, as well as benzo-fused carbocyclic moieties, in which at least one ring system is aromatic. The aryl group is typically, but not necessarily, attached to the parent molecule via the aromatic ring of the aryl group. The term "aromatic ring" can be used interchangeably with the terms "aromatic ring radical" or "aromatic ring radical." Examples of aryl groups include phenyl, indenyl, naphthyl, and anthracenyl. The aryl group is optionally substituted with one or more substituents described herein. Representative aryl groups include phenyl, anthracenyl, fluorenyl, indenyl, phenanthrenyl, and naphthyl.

[0208] The term "heteroaryl" is used interchangeably with the term "heteroaromatic ring" or "heteroaromatic group" when used alone or as part of another substituent and refers to a monocyclic or polycyclic aromatic ring system. In certain embodiments, one to three atoms in the ring system are heteroatoms, i.e., elements other than carbon, including but not limited to N, O, S, or P. Examples include furanyl, imidazolyl, indolinyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl. The heteroaryl group may be optionally fused to a benzene ring and may be monocyclic, bicyclic, tricyclic, spirocyclic, or bridged.

[0209] The term "5-10 membered heteroaryl" is understood, either alone or as part of another substituent, to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5 to 10 ring atoms and containing heteroatoms selected from N, O and S, and is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 carbon atoms, which contains 1 to 5, preferably 1 to 3, heteroatoms independently selected from N, O and S, and which may additionally in each case be benzo-fused. "5-8 membered heteroaryl" is understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring radical having 5 to 8 ring atoms, in particular 5 or 6 carbon atoms, which contains 1 to 5 heteroatoms independently selected from N, O and S. Preference is given to monovalent monocyclic, bicyclic or tricyclic aromatic ring radicals having 1 to 3 heteroatoms independently selected from N, O and S and, in addition, in each case may be benzo-fused. Examples of heteroaryl groups include, but are not limited to, thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl, isoquinolyl, and the like; or acinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and the like.

[0210] The term "halo" is used interchangeably with the term "halogen-substituted" when used alone or as part of another substituent. "Haloalkyl" or "halogen-substituted alkyl" refers to saturated aliphatic hydrocarbon groups, including branched and straight-chain groups, having the specified number of carbon atoms, substituted with one or more halogens (e.g., -CvFw, where v = 1 to 3 and w = 1 to (2v+1)). Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl.

[0211] Compounds provided herein include intermediates that can be used to prepare compounds provided herein, which contain reactive functional groups (such as, but not limited to, carboxyl, hydroxyl, and amino moieties), and also include protected derivatives thereof."Protected derivatives" are compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as blocking groups). Suitable carboxyl moiety protecting groups include benzyl, tert-butyl, etc., and isotopes, etc. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, tert-butyloxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable blocking groups are well known to those of ordinary skill in the art.

[0212] In this application, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both instances where the event or circumstance occurs and instances where it does not occur. For example, "optionally substituted aryl" means that the aryl group is substituted or unsubstituted, and the description includes both substituted aryl groups and unsubstituted aryl groups.

[0213] In this application, the term "salt" or "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0214] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic or organic acids that retain the biological effectiveness of the free base without other side effects. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic or organic bases that retain the biological effectiveness of the free acid without other side effects. In addition to pharmaceutically acceptable salts, other salts are contemplated by the present invention. These salts may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically acceptable salts, or may be used in the identification, characterization, or purification of the compounds of the present invention.

[0215] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereomers and conformational isomers.

[0216] Depending on the choice of starting materials and methods, the compounds of the present invention may exist in one of the possible isomers or in a mixture thereof, for example as pure optical isomers, or as a mixture of isomers, such as a racemic and diastereomeric mixture, depending on the number of asymmetric carbon atoms. When describing optically active compounds, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to the chiral center (or centers) in the molecule. The prefixes D and L or (+) and (–) are used to designate the signs for the rotation of plane-polarized light caused by the compound, where (–) or L indicates that the compound is levorotatory. Compounds prefixed with (+) or D are dextrorotatory.

[0217] When bonds to chiral carbon atoms in formulae of the present invention are depicted as straight lines, it is understood that both the (R) and (S) configurations of the chiral carbon atoms and the enantiomerically pure compounds and mixtures thereof are encompassed within the scope of the formula. The diagrammatic representation of racemates or enantiomerically pure compounds herein is adapted from Maehr, J. Chem. Ed. 1985, 62: 114-120. Wedge-shaped bonds and dashed bonds are used to represent the absolute configuration of a stereocenter.

[0218] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom between two positions in a molecule. Compounds of the present invention may exhibit tautomerism. Tautomeric compounds can exist as two or more interconvertible species. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture with physical and chemical properties consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones, such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0219] As used herein, a "pharmaceutical composition" refers to a formulation of a compound of the present invention and a medium generally accepted in the art for delivering a biologically active compound to a mammal (e.g., a human). The medium includes a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0220] In this application, "pharmaceutically acceptable carrier" includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the relevant governmental regulatory authorities as acceptable for human or livestock use.

[0221] As used herein, the term "prodrug" refers to a compound of the present invention that can be converted to a biologically active compound under physiological conditions or by solvolysis. Prodrugs of the present invention are prepared by modifying functional groups within the compound. These modifications can be removed by conventional procedures or in vivo to yield the parent compound. Prodrugs include compounds in which a hydroxyl or amino group within a compound of the present invention is attached to any group. When a prodrug of a compound of the present invention is administered to a mammalian subject, the prodrug is cleaved to form a free hydroxyl group or free amino group, respectively.

[0222] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as deuterium ( 2 H), tritium ( 3 H), iodine-125( 125 I) or C-14( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0223] As used herein, the term "excipient" refers to a pharmaceutically acceptable inert ingredient. Examples of the term "excipient" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, and diluents. Excipients enhance the handling characteristics of pharmaceutical formulations, i.e., by increasing flowability and / or adhesion, making the formulation more suitable for direct compression.

[0224] As used herein, the term "treatment" and other similar synonyms include the following meanings:

[0225] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0226] (ii) inhibiting the disease or condition, i.e., curbing its development;

[0227] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0228] (iv) Alleviate the symptoms of the disease or condition.

[0229] For each step of the reaction, the reaction temperature can be appropriately selected according to the solvent, starting materials, reagents, etc., and the reaction time can also be appropriately selected according to the reaction temperature, solvent, starting materials, reagents, etc. After the reaction of each step is completed, the target compound can be separated and purified from the reaction system according to conventional methods, such as filtration, extraction, recrystallization, washing, silica gel column chromatography, etc. If it does not affect the next step of the reaction, the target compound can also be directly entered into the next step of the reaction without separation and purification. Each step of the reaction of the present invention is preferably carried out in an inert solvent, and the inert solvent includes but is not limited to: toluene, benzene, water, methanol, ethanol, isopropanol, ethylene glycol, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, or a combination thereof. DETAILED DESCRIPTION

[0230] The present invention will be further described below in conjunction with specific examples. It should be understood that the following description is only the most preferred embodiment of the present invention and should not be considered as limiting the scope of protection of the present invention. Based on a full understanding of the present invention, the experimental methods in the following examples that do not specify specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturer. Those skilled in the art may make non-essential changes to the technical solutions of the present invention, and such changes should be considered as included in the scope of protection of the present invention.

[0231] Example 1: Preparation of Compound I-1

[0232] The synthetic route is as follows:

[0233] Step 1: Synthesis of methyl (S)-3-((tert-butyloxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01B)

[0234] To a 100 mL round-bottom flask was added methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (1.21 g, 3.39 mmol), 1-methyl-1H-pyrazol-5-ylboronic acid (643 mg, 5.11 mmol), Pd(PPh3)2Cl2 (249 mg, 0.355 mmol), Na2CO3 (1.08 g, 10.2 mmol), DME (10 mL), and H2O (2.5 mL). The mixture was sonicated, the atmosphere was replaced with nitrogen three times, and the mixture was stirred at 100°C. After complete conversion of the starting material as monitored by TLC, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 3:1) to give compound (S)-methyl 3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01B) (360 mg, yield 29.7%).

[0235] LC-MS, M / Z(ESI):360.2[M+H] + .

[0236] Step 2: Synthesis of (S)-methyl 3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (01C)

[0237] Methyl (S)-3-((tert-Butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (360 mg, 1.00 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (5 mL) and stirred at room temperature. After complete conversion of the starting material as monitored by TLC, the reaction mixture was concentrated under reduced pressure to afford methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (01C) (296 mg).

[0238] LC-MS, M / Z(ESI):260.1[M+H] + .

[0239] Step 3: Synthesis of methyl (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoate (01E)

[0240] ((4-Methylpyridin-2-amino)butyryl)glycine (252 mg, 1.00 mmol) was dissolved in anhydrous DMF (5 mL) at room temperature, and tetramethyluronium tetrafluoroborate (TBTU, 385 mg, 1.20 mmol) was added. N,N-diisopropylethylamine (DIPEA, 1.48 g, 1.15 mmol) was then added at 0°C. After returning to room temperature and stirring for half an hour, (S)-methyl 3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (296 mg, 1.00 mmol) was added, and the reaction was stirred at room temperature. After completion of the reaction, as monitored by TLC, saturated NH4Cl solution (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL x 3). The organic phases were combined and washed once with saturated NaHCO3 solution (10 mL). The organic phase was separated and dried over anhydrous sodium sulfate, filtered and concentrated to give compound (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetylamino)propionic acid methyl ester (01E) (300 mg, yield 60.7%).

[0241] LC-MS, M / Z(ESI):493.3[M+H] + .

[0242] Step 4: Synthesis of (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propionic acid (I-1)

[0243] Methyl (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoate (320 mg, 0.540 mmol) was dissolved in THF / H₂O (6 mL, v:v = 1:1) at room temperature, followed by the addition of LiOH·H₂O (68.0 mg, 1.62 mmol). The mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure. To the residue was added a solution of hydrogen chloride in 1,4-dioxane (4 M, 2.7 mL) and stirred for half an hour. After completion of the reaction, the mixture was concentrated under reduced pressure to remove the organic solvent. The residual solution was subjected to reverse phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm, mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 1%-98%) to obtain compound (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (I-1) (210 mg, yield 81.3%).

[0244] LC-MS, M / Z(ESI):479.2[M+H] + .

[0245] 1 H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.4Hz,1H),8.08(t,J=6.0Hz,1H),7.77(d,J=5.2Hz,1H ),7.44(d,J=2.0Hz,2H),7.42–7.32(m,3H),6.37(d,J=2.0Hz,2H),6.26(d,J=5.2Hz,1H), 6.22(s,1H),5.22(q,J=7.2Hz,1H),3.82(s,3H),3.68(d,J=6.0Hz,2H),3.15(q,J=6.8Hz, 2H), 2.73 (d, J = 7.2Hz, 2H), 2.16 (t, J = 7.2Hz, 2H), 2.10 (s, 3H), 1.70 (quint, J = 7.2Hz, 2H).

[0246] Example 2: Preparation of Compound I-2

[0247] The synthetic route is as follows:

[0248] Step 1: Synthesis of methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04C)

[0249] To a 100 mL single-necked flask were added (S)-methyl 3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (01B, 600 mg, 1.67 mmol), dichloromethane (20 mL), and trifluoroacetic acid (3.81 g, 33.4 mmol) in sequence, and the mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to give a crude product, which was dissolved in dichloromethane (20 mL), washed sequentially with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (04C) (430 mg, 99.3% yield).

[0250] LC-MS, M / Z(ESI):260.13[M+H] + .

[0251] Step 2: Synthesis of methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetylamino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04D)

[0252] To a 100 mL single-necked flask, (S)-methyl 3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (400 mg, 1.54 mmol), DMF (10 mL), BOC-glycine (298 mg, 1.70 mmol), TBTU (740 mg, 2.30 mmol), and DIPEA (598 mg, 4.63 mmol) were added sequentially. The mixture was stirred at room temperature for 16 h under nitrogen. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave (S)-methyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (04D) (600 mg, 93.4% yield).

[0253] LC-MS, M / Z(ESI):417.27[M+H] + .

[0254] Step 3: Synthesis of methyl (S)-3-(2-aminoacetamide)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04E)

[0255] To a 100 mL single-necked flask were added (S)-methyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (600 mg, 1.44 mmol), dichloromethane (10 mL), and trifluoroacetic acid (3.29 g, 28.9 mmol) in sequence, and the mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to give a crude product, which was dissolved in dichloromethane (10 mL), washed with saturated sodium bicarbonate solution (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give (S)-methyl 3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (04E) (460 mg, approximately 100% yield).

[0256] LC-MS, M / Z(ESI):317.32[M+H] + .

[0257] Step 4: Synthesis of methyl 3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carboxylate (04G)

[0258] To a 100 mL single-necked flask, 4-methylpyridin-2-amine (2.00 g, 18.5 mmol), 1,2-dichloroethane (50 mL), methyl 3-oxocyclobutane-1-carboxylate (3.60 g, 28.1 mmol), and sodium acetate borohydride (11.8 g, 55.7 mmol) were added sequentially. The mixture was stirred at room temperature for 12 h. Then, sodium cyanoborohydride (3.50 g, 55.7 mmol) was added and stirred at room temperature for 6 h. The reaction mixture was concentrated under reduced pressure to obtain the crude product. Ethyl acetate (100 mL) and dilute hydrochloric acid (1 N, 100 mL) were added, stirred, and the mixture was separated. The aqueous phase was washed twice with ethyl acetate (100 mL x 2). The resulting aqueous phase was adjusted to pH 10 with 1 N sodium hydroxide solution and then extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography gave methyl 3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carboxylate (04G) (300 mg, yield 7.36%).

[0259] Step 5: Synthesis of 3-[(4-methylpyridin-2-yl)amino]cyclobutane-1-carboxylic acid (04H)

[0260] To a 100 mL single-necked flask, methyl 3-[(4-methylpyridin-2-yl)amino]cyclobutane-1-carboxylate (300 mg, 1.36 mmol), tetrahydrofuran (5 mL), water (1 mL), and lithium hydroxide (65.2 mg, 2.72 mmol) were added sequentially and stirred at room temperature for 12 h. The crude product, 3-[(4-methylpyridin-2-yl)amino]cyclobutane-1-carboxylic acid (04H), was obtained by concentration and used directly in the next reaction.

[0261] Step 6: Synthesis of (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycol)amino)-propionic acid methyl ester (04I)

[0262] To a 100 mL single-necked flask were added 3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carboxylic acid (04H), dichloromethane (10 mL), (S)-methyl 3-(2-aminoacetamide)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (04E) (474 ​​mg, 1.50 mmol), carbodiimide (EDCI, 313 mg, 1.63 mmol), 1-hydroxybenzotriazole (HOBt, 36.8 mg, 0.272 mmol), and triethylamine (275 mg, 2.72 mmol) in sequence, and the mixture was stirred at room temperature for 12 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, to which ethyl acetate and water were added and stirred. After separation, the organic phase was collected and extracted twice with ethyl acetate added to the aqueous phase. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The product was purified by column chromatography to obtain (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycol)amino)-propionic acid methyl ester (04I) (200 mg, two-step yield 29.1%).

[0263] Step 7: Synthesis of (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycol)amino)-propionic acid methyl ester (I-2)

[0264] To a 100 mL single-necked flask were added methyl (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)-propanoate (200 mg, 0.396 mmol), tetrahydrofuran (5 mL), water (1 mL), and lithium hydroxide (19.0 mg, 0.793 mmol) in sequence, and the mixture was stirred at room temperature for 12 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then reacted with reverse phase to obtain the product (3S)-3-[3-(1-methyl-1H-pyrazol-5-yl)phenyl]-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)-propionic acid (I-2) (96 mg, 49% yield).

[0265] LC-MS, M / Z(ESI):491.2[M+H] + .

[0266] Example 3: Preparation of Compounds II-1 & II-1A & II-1B

[0267] The synthetic route is as follows:

[0268] Step 1: Synthesis of ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (02B)

[0269] To a 500 mL single-necked flask, ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propanoate (14.00 g, 37.01 mmol), dichloromethane (150 mL), and di-tert-butyl dicarbonate (12.12 g, 55.53 mmol) were added sequentially and stirred at room temperature for 3 h. The reaction solution was washed with water (150 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-1:1) afforded the product, ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (02B) (14.00 g, 79.07% yield).

[0270] LC-MS,M / Z(ESI):478.12,480.11[M+H] + .

[0271] Step 2: Synthesis of ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (02C)

[0272] To a 250 mL single-necked bottle, ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (14.00 g, 29.27 mmol), 1-methyl-1H-pyrazole-5-boronic acid pinacol ester (12.18 g, 58.54 mmol), Pd2(dba)3 (1.07 g, 1.17 mmol), tricyclohexylphosphine (657 mg, 2.34 mmol), dipotassium hydrogen phosphate (10.20 g, 58.56 mmol), 1,4-dioxane (100 mL), and water (30 mL) were added in sequence and stirred at 100°C for 16 h. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave the product, ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (02C) (14.00 g, 99.75% yield). LC-MS, M / Z (ESI): 480.24 [M+H] + .

[0273] Step 3: Synthesis of ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (02D)

[0274] To a 250 mL single-necked flask, ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (8.00 g, 16.7 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 41.7 mL) were added sequentially and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain the product, ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (02D) (6.00 g, 86.6% yield).

[0275] LC-MS, M / Z(ESI):380.19[M+H] + .

[0276] Step 4: Synthesis of 14-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylic acid ethyl ester (02F)

[0277] To a 250 mL single-necked flask were added ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (4.00 g, 9.62 mmol), DMF (100 mL), (4-((tert-butoxycarbonyl)(4-methylpyridin-2-yl)amino)butanoyl)glycine (3.70 g, 10.5 mmol), HATU (5.21 g, 13.7 mmol), and DIPEA (4.09 g, 31.6 mmol). The mixture was stirred at room temperature for 16 h under nitrogen. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (200 mL x 3). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave ethyl 14-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylate (02F) (6.00 g, 87.5% yield). LC-MS, M / Z (ESI): 713.36 [M+H] + .

[0278] Step 5: Synthesis of 14-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylic acid ethyl ester (02G)

[0279] Under nitrogen, to a 500 mL single-necked flask were added ethyl 14-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylate (6.00 g, 8.42 mmol), methanol (100 mL), and 10% Pd / C (600 mg). The single-necked flask was replaced with a hydrogen atmosphere, and the mixture was stirred at room temperature for 16 h. The reaction mixture was filtered through celite, the filter cake was washed with methanol, and the combined filtrates were concentrated under reduced pressure to give ethyl 14-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylate (02G) (5.00 g, 95.4% yield). LC-MS, M / Z (ESI): 623.31 [M+H] + .

[0280] Step 6: Synthesis of 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylic acid ethyl ester (02H)

[0281] To a 250 mL single-necked flask, ethyl 14-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylate (1.50 g, 2.41 mmol), DMF (15 mL), potassium carbonate (670 mg, 4.85 mmol), and azide-pentaethylene glycol-p-toluenesulfonate (1.51 g, 3.62 mmol) were added sequentially. The mixture was stirred at 80°C for 16 h. After completion of the reaction, water (15 mL) was added, and the mixture was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give the product 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylic acid ethyl ester (02H) (1.80 g, yield 86.1%).

[0282] LC-MS, M / Z(ESI):868.45[M+H] + .

[0283] Step 7: Synthesis of 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-oic acid (02I)

[0284] To a 100 mL single-necked bottle, 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-carboxylic acid ethyl ester (1.80 g, 2.07 mmol), THF (20 mL) and lithium hydroxide (149 mg, 6.22 mmol) aqueous solution (10 mL) were added sequentially and stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure to remove THF, and water (10 mL) and 1N hydrochloric acid were added to adjust the pH to 7. The solution was concentrated under reduced pressure to give the product 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-oic acid (02I) (1.74 g, yield 99.9%).

[0285] LC-MS, M / Z(ESI):840.42[M+H] + .

[0286] Step 8: Synthesis of 3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoic acid (II-1)

[0287] To a 100 mL single-necked bottle, 14-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxo-3-oxa-5,10,13-triazahexadecyl-16-oic acid (1.74 g, 2.07 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 5.18 mL) were added sequentially and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was then purified by reverse phase chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm, mobile phase: A = 0.1% aqueous ammonia, B = acetonitrile; gradient: 30%-100%) to give the product 3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-1) (650 mg, 42.4% yield).

[0288] 1 H NMR (400MHz, DMSO-d6) δ9.82(d,J=5.6Hz,1H),8.29(t,J=6.0Hz,1H),7.75(d,J=5.2Hz,1H),7.37(d, J=1.8Hz,1H),7.30(dd,J=8.6,2.2Hz,1H),7.17(d,J=2.2Hz,1H),6.98(d,J=8.6Hz,2H),6.31(s,1H) ,6.22–6.21(m,2H),4.94–4.89(m,1H),4.08–4.04(m,2H),3.69–3.63(m,6H),3.58–3.47(m,12H),3. 46(s,3H),3.20–3.09(m,4H),2.36–2.31(m,2H),2.27–2.16(m,2H),2.07(s,3H),1.75–1.69(m,2H).

[0289] LC-MS, M / Z(ESI):740.7[M+H] + .

[0290] Step 9: (R)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-1A) and (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-1B)

[0291] 3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (500 mg, 0.676 mmol) was separated by SFC (column: (S,S) WHELK-O1 (250 mm×30 mm, 10 μm, mobile phase: A=CO2, B=isopropanol+acetonitrile (0.1% ammonia solution); Gradient: 75%) to give (R)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-1A, 210 mg; column: (S,S) WHELK-O1 (50 mm × 4.6 mm, 3.5 μm, mobile phase: A = CO2, B = isopropanol + acetonitrile (0.05%) DEA); gradient: 60%; flow rate: 3 mL / min; retention time: 0.554 min) and (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-1B, 205 mg; column: (S,S) WHELK-O1 (50 mm×4.6 mm, 3.5 μm, mobile phase: A=CO2, B=isopropanol+acetonitrile (0.05% DEA); gradient: 60%; flow rate: 3 mL / min; retention time: 1.451 min).

[0292] II-1A: 1H NMR (400MHz, DMSO-d6) δ9.82(d,J=5.6Hz,1H),8.30(t,J=6.0Hz,1H),7.75(d,J=5.2Hz,1H),7.38(d, J=1.8Hz,1H),7.30(dd,J=8.6,2.2Hz,1H),7.17(d,J=2.2Hz,1H),6.99(d,J=8.6Hz,2H),6.31(s,1H) ,6.23–6.21(m,2H),4.95–4.90(m,1H),4.09–4.04(m,2H),3.70–3.63(m,6H),3.60–3.47(m,12H),3. 46(s,3H),3.20–3.09(m,4H),2.36–2.31(m,2H),2.27–2.16(m,2H),2.07(s,3H),1.75–1.69(m,2H).

[0293] LC-MS, M / Z(ESI):740.5[M+H] + .

[0294] II-1B: 1 H NMR (400MHz, DMSO-d6) δ9.83(d,J=5.6Hz,1H),8.29(t,J=6.0Hz,1H),7.76(d,J=5.2Hz,1H),7.37(d, J=1.8Hz,1H),7.31(dd,J=8.6,2.2Hz,1H),7.18(d,J=2.2Hz,1H),6.98(d,J=8.6Hz,2H),6.31(s,1H) ,6.22–6.20(m,2H),4.94–4.89(m,1H),4.10–4.06(m,2H),3.69–3.63(m,6H),3.58–3.47(m,12H),3. 46(s,3H),3.21–3.10(m,4H),2.38–2.31(m,2H),2.28–2.16(m,2H),2.07(s,3H),1.75–1.69(m,2H).

[0295] LC-MS, M / Z(ESI):740.5[M+H] + .

[0296] Example 4: Preparation of Compounds II-2 & II-2A & II-2B

[0297] The synthetic route is as follows:

[0298] Step 1: Synthesis of ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propionate (03B)

[0299] To a 100 mL round-bottom flask was added 3-benzyloxy-5-bromobenzaldehyde (3.00 g, 10.3 mmol), ethyl malonate (2.72 g, 20.6 mmol), ammonium acetate (5.56 g, 72.1 mmol), and ethanol (30 mL), and the reaction was stirred at 80°C. After complete conversion of the starting material as monitored by TLC, the reaction solution was concentrated under reduced pressure to afford ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propanoate (03B) (1.56 g, 40.0% yield).

[0300] LC-MS,M / Z(ESI):378.1,380.1[M+H] + .

[0301] Step 2: Synthesis of ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (03C)

[0302] Ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propanoate (1.56 g, 4.12 mmol) was dissolved in water / 1,4-dioxane (40 mL, v:v = 1:1) at room temperature. Triethylamine (1.25 g, 12.4 mmol) and di-tert-butyl dicarbonate (1.08 g, 4.95 mmol) were added and the reaction was stirred at room temperature. After complete conversion of the starting material as monitored by TLC, the organic solvent was removed by concentration under reduced pressure, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed once with saturated NaCl solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate (v / v) = 85:15) to obtain ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (03C) (1.82 g, 92.3% yield).

[0303] LC-MS,M / Z(ESI):478.1,480.1[M+H] + .

[0304] Step 3: Synthesis of ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazole-5-)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (03D)

[0305] To a 100 mL round-bottom flask was added the compound ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (1.82 g, 3.80 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (1.58 g, 7.59 mmol), Pd2(dba)3 (140 mg, 0.153 mmol), tricyclohexylphosphine (85 mg, 0.30 mmol), dipotassium hydrogen phosphate (1.33 g, 7.64 mmol), 1,4-dioxane (10 mL) and water (5 mL). After ultrasonic vibration, nitrogen was replaced three times and the reaction was stirred at 100 °C. After complete conversion of the starting material as monitored by TLC, the reaction solution was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 7:3) to give compound 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazole-5-)phenyl)-3-((tert-butoxycarbonyl)amino)propanoic acid ethyl ester (03D) (1.82 g, yield 99.8%).

[0306] LC-MS, M / Z(ESI):480.2[M+H] + .

[0307] Step 4: Synthesis of ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (03E)

[0308] Ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (4.54 g, 9.47 mmol) was dissolved in a 4M solution of hydrogen chloride in 1,4-dioxane (47.5 mL) at room temperature and stirred for half an hour. After complete conversion of the starting material as monitored by TLC, the crude product was concentrated under reduced pressure. Ethyl acetate was added for washing, and the filter cake was collected by suction filtration to yield ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (03E) (3.95 g, 100% yield).

[0309] LC-MS, M / Z(ESI):380.2[M+H] + .

[0310] Step 5: Synthesis of ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoate (03F)

[0311] Ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (3.95 g, 9.50 mmol) was dissolved in anhydrous DMF (50 mL) at room temperature, followed by the addition of TBTU (3.66 g, 11.4 mmol). DIPEA (3.56 g, 27.5 mmol) was added at 0°C, and the mixture was allowed to return to room temperature and stirred for half an hour before the addition of ((4-methylpyridin-2-amino)butanoyl)glycine (2.40 g, 9.55 mmol). The reaction was allowed to stir at room temperature. After completion of the reaction, monitored by TLC, a saturated NH4Cl solution (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL x 3). The combined organic phase was washed once with saturated NaHCO3 solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol (V / V) = 9:1) to give the compound 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetylamino)propionic acid ethyl ester (03F) (4.00 g, yield 68.7%).

[0312] LC-MS, M / Z(ESI):613.3[M+H] + .

[0313] Step 6: Synthesis of ethyl 3-(3-hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoate (03G)

[0314] At room temperature, ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoate (4.00 g, 6.53 mmol) was dissolved in methanol (10 mL). 10% wet palladium on carbon (600 mg) was added, and the hydrogen atmosphere was replaced three times. The reaction was stirred at room temperature while maintaining a hydrogen atmosphere. After complete conversion of the starting material as monitored by TLC, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to give ethyl 3-(3-hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoate (03G) (3.40 g, 99.7% yield).

[0315] LC-MS, M / Z(ESI):523.3[M+H] + .

[0316] Step 7: Synthesis of ethyl 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoate (03H)

[0317] At room temperature, ethyl 3-(3-hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoate (146 mg, 0.279 mmol) was dissolved in anhydrous DMF (5 mL). Potassium carbonate (97 mg, 0.70 mmol) and azide-pentaethylene glycol-p-toluenesulfonate (152 mg, 0.364 mmol) were added, and the mixture was stirred at 80°C for 6 hours. After complete conversion of the starting material, as monitored by TLC, the mixture was returned to room temperature, saturated NaHCO3 solution (10 mL) was added, and extraction was performed with ethyl acetate (10 mL x 3). The combined organic phase was washed once with saturated NaCl solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetylamino)propionic acid ethyl ester (03H) (200 mg, yield 93.2%).

[0318] LC-MS, M / Z(ESI):768.4[M+H] + .

[0319] Step 8: Synthesis of 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoic acid (II-2)

[0320] Ethyl 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoate (200 mg, 0.260 mmol) was dissolved in THF / H₂O (6 mL, v:v = 1:1) at room temperature. LiOH·H₂O (33 mg, 0.79 mmol) was added and stirred at room temperature for 3 hours. The reaction mixture was concentrated under reduced pressure, and a solution of hydrogen chloride in 1,4-dioxane (4 M, 1.3 mL) was added, and stirring was continued for half an hour. After the reaction was completed as monitored by TLC, the organic solvent was removed by concentration under reduced pressure, and the residue was purified by reverse preparative chromatography (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm, mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 5%-98%) to obtain compound 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-2) (70 mg, 36% yield).

[0321] LC-MS, M / Z(ESI):740.8[M+H] + .

[0322] 1 H NMR (400MHz, DMSO-d6) δ8.42(d,J=8.4Hz,1H),8.11(t,J=6.0Hz,1H),7.79(d,J=5.2Hz,1H),7.45(d,J=2.0Hz,1H),7.02 (s,1H),6.97–6.91(m,2H),6.40(d,J=2.0Hz,2H),6.29(d,J=5.2Hz,1H),6.24(s,1H),5.21(q,J=7.2Hz,1H),4.17–4.11 (m,2H),3.84(s,3H),3.77–3.73(m,2H),3.70(d,J=6.0Hz,2H),3.58(dt,J=5.2,3.2Hz,4H),3.56–3.50(m,10H),3.39–3 .35(m,2H),3.17(q,J=6.8Hz,2H),2.73(d,J=7.2Hz,2H),2.19(t,J=7.2Hz,2H),2.12(s,3H),1.72(quint,J=7.2Hz,2H).

[0323] Step 9: Synthesis of (R)-3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoic acid (II-2A)

[0324] Synthesis of (S)-3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butyramido)acetamido)propanoic acid (II-2B)

[0325] 3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid was separated by SFC (column: (S,S) WHELK-O1 (250 mm × 30 mm, 10 μm, mobile phase: mobile phase: A = CO2, B = isocyanate) Propanol + acetonitrile (0.1% ammonia water; gradient: 75%) gave compound (R)-3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-2A, 24 mg; column: (S,S) Whelk-O1 50×4.6 mm ID, 3.5 μm; mobile phase: A = CO2, B = isopropanol + acetonitrile (0.05% diethylamine); gradient: 60%; flow rate: 3 mL / min; retention time: 0.621 min) and compound (S)-3-(3-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid (II-2B, 25 mg; column: (S,S) Whelk-O1 50×4.6 mm ID, 3.5 μm; mobile phase: A = CO2, B = isopropanol + acetonitrile (0.05% diethylamine); gradient: 60%; flow rate: 3 mL / min; retention time: 1.818 min).

[0326] II-2A: 1H NMR(400MHz, CDCl3)δ10.05(s,1H),9.45(s,1H),7.55–7.40(m,2H),7.00–6.95(m,2H) ),6.80–6.65(m,2H),6.45–6.35(m,2H),6.25(s,1H),5.25–5.15(m,1H),4.30–4.05(m ,3H),3.90–3.73(m,6H),3.73–3.60(m,14H),3.45–3.15(m,4H),3.39–3.35(m,2H),2. 93–2.83(m,1H),2.75–2.65(m,1H),2.58–2.45(m,2H),2.32(s,3H),2.05(quint,2H).

[0327] II-2B: 1 H NMR (400MHz, CDCl3) δ10.15(s,1H),9.60(s,1H),7.55–7.40(m,2H),7.05–6.90(m,2H), 6.80(s,1H),6.55(s,1H),6.45–6.35(m,2H),6.25(s,1H),5.25–5.15(m,1H),4.25–4.1 0(m,3H),3.90–3.73(m,6H),3.73–3.60(m,14H),3.45–3.15(m,4H),3.39–3.35(m,2H), 2.93–2.83(m,1H),2.75–2.65(m,1H),2.58–2.45(m,2H),2.32(s,3H),2.05(quint,2H).

[0328] Example 5: Preparation of Compounds II-3A & II-3B

[0329] The synthetic route is as follows:

[0330] Step 1: Synthesis of (R,E)-N-(4-(Benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfenamide (05B)

[0331] To a 100 mL single-necked flask were added 4-(benzyloxy)-3-bromobenzaldehyde (5.00 g, 17.2 mmol), 2-methyltetrahydrofuran (10 mL), (R)-(+)-tert-butylsulfenamide (2.19 g, 18.1 mmol), and cesium carbonate (8.39 g, 25.8 mmol) in sequence. The mixture was stirred at room temperature under nitrogen for 16 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-1:1) afforded the product (R,E)-N-(4-(benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfenamide (05B) (5.80 g, 85.6% yield).

[0332] LC-MS,M / Z(ESI):394.3,396.3[M+H] + .

[0333] Step 2: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propionate (05C)

[0334] Zinc powder (8.29 g, 127 mmol) and tetrahydrofuran (50 mL) were added sequentially to a 250 mL three-necked flask. Under nitrogen, trimethylsilyl chloride (2.76 g, 25.4 mmol) was added dropwise, and the mixture was heated to 60°C and stirred for 10 min. The reaction mixture was cooled to 40°C, and ethyl bromoacetate (6.35 g, 38.0 mmol) dissolved in tetrahydrofuran (10 mL) was slowly added dropwise. After the addition was complete, the mixture was heated to 60°C and stirred for 2 h. The reaction mixture was quickly cooled to 0°C, and (R,E)-N-(4-(benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfenamide (5.00 g, 12.7 mmol) dissolved in tetrahydrofuran (10 mL) was slowly added. After the addition was complete, the mixture was stirred at 0°C for 1 h, then slowly heated to room temperature and stirred for 10 h. The reaction was quenched by adding aqueous citric acid and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0-0:1) gave (S)-ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propanoate (05C) (3.80 g, 62.1% yield).

[0335] 1H NMR (400MHz, DMSO-d6) δ7.57(d,J=1.9Hz,1H),7.47(d,J=7.1Hz,2H),7.40(t,J=7.3Hz,2H),7.36–7.28(m,2H),7.14(d,J=8.6Hz,1H),5.56(d,J=5. 9Hz,1H),5.19(s,2H),4.57(q,J=6.7Hz,1H),3.99(q,J=7.1Hz,2H),2.98( dd,J=15.4,6.6Hz,1H),2.73(dd,J=15.4,7.9Hz,1H),1.11–1.07(m,12H).

[0336] LC-MS,M / Z(ESI):482.2,484.2[M+H] + .

[0337] Step 3: Synthesis of (S)-ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate hydrochloride (05D)

[0338] To a 100 mL single-necked flask, ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propanoate (3.80 g, 7.88 mmol), ethanol (10 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 3.94 mL) were added sequentially and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain ethyl (S)-3-amino-3-(4-(benzyloxy)-3-bromophenyl)propanoate hydrochloride (05D), which was used directly in the next step.

[0339] LC-MS,M / Z(ESI):378.2,380.2[M+H] + .

[0340] Step 4: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (05E)

[0341] To a 100 mL single-necked flask, (S)-ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propanoate hydrochloride obtained in the previous step, dichloromethane (30 mL), di-tert-butyl dicarbonate (2.58 g, 11.8 mmol), and triethylamine (2.39 g, 23.6 mmol) were added in sequence and stirred at room temperature for 3 h. Water (10 mL) was added, the organic phase was separated, and the crude product was concentrated under reduced pressure. Purification by column chromatography (petroleum ether / ethyl acetate (v / v) = 1:0 to 0:1) gave the product (S)-ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (05E) (2.40 g, 63.7% yield over two steps).

[0342] LC-MS,M / Z(ESI):478.2,480.2[M+H] + .

[0343] Step 5: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (05F)

[0344] To a 100 mL single-necked flask, (S)-ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propanoate (2.40 g, 5.02 mmol), 1-methyl-1H-pyrazole-5-boronic acid pinacol ester (2.09 g, 10.0 mmol), Pd2(dba)3 (184 mg, 0.201 mmol), tricyclohexylphosphine (113 mg, 0.403 mmol), dipotassium hydrogen phosphate (1.75 g, 10.0 mmol), 1,4-dioxane (24 mL), and water (8 mL) were added sequentially and stirred at 100°C for 16 h. After cooling to room temperature, the reaction solution was filtered and the filtrate was concentrated under reduced pressure to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-1:1) gave the product (S)-ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (05F) (2.30 g, yield 95.6%).

[0345] LC-MS, M / Z(ESI):480.37[M+H] + .

[0346] Step 6: Synthesis of (S)-ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05G)

[0347] To a 100 mL single-necked flask, ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propanoate (2.30 g, 4.80 mmol), ethanol (10 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 3.6 mL) were added sequentially and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain the product, ethyl (S)-3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (05G) (2.0 g, 100% yield).

[0348] LC-MS, M / Z(ESI):380.2[M+H] + .

[0349] Step 7: Synthesis of (S)-ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate (05H)

[0350] To a 100 mL single-necked flask, (S)-ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (2.0 g, 4.8 mmol), DMF (20 mL), BOC-glycine (1.01 g, 5.77 mmol), TBTU (1.85 g, 5.76 mmol), and DIPEA (1.86 g, 14.4 mmol) were added sequentially. Under nitrogen, the mixture was stirred at room temperature for 16 h. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (40 mL x 3). The combined organic phases were washed sequentially with saturated sodium bicarbonate solution (40 mL) and saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) gave (S)-ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoate (05H) (2.5 g, 97% yield).

[0351] LC-MS, M / Z(ESI):537.3[M+H] + .

[0352] Step 8: Synthesis of ethyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (05I)

[0353] Under nitrogen protection, to a 100 mL single-necked flask were added ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoate (2.5 g, 4.7 mmol), methanol (30 mL), and 10% Pd / C (250 mg) in sequence. The reaction was replaced with a hydrogen atmosphere and stirred at room temperature for 16 h. The reaction solution was filtered through celite, the filter cake was washed with methanol and filtered, and the combined filtrates were concentrated under reduced pressure to give ethyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (05I) (2.05 g, 99% yield).

[0354] LC-MS, M / Z(ESI):447.3[M+H] + .

[0355] Step 9: Synthesis of (S)-ethyl 3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate (05J)

[0356] To a 100 mL single-necked flask, (S)-ethyl 3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (2.05 g, 4.59 mmol), DMF (20 mL), potassium carbonate (1.59 g, 11.5 mmol), and azide-pentaethylene glycol-p-toluenesulfonate (2.11 g, 5.05 mmol) were added sequentially and stirred at 80°C for 16 h. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (40 mL x 3). The combined organic phases were washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product was purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0-0:1) to give the product (S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoic acid ethyl ester (05J) (2.50 g, yield 78.7%).

[0357] LC-MS, M / Z(ESI):692.47[M+H] + .

[0358] Step 10: Synthesis of ethyl (S)-3-(2-aminoacetamido)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05K)

[0359] To a 100 mL single-necked flask were added ethyl (S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoate (2.50 g, 3.61 mmol), ethanol (10 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 3.61 mL). The mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to afford the product, ethyl (S)-3-(2-aminoacetamido)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (05K) (2.20 g, 96.9% yield).

[0360] LC-MS, M / Z(ESI):592.41[M+H] + .

[0361] Step 11: Synthesis of ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoate (05L)

[0362] To a 100 mL single-necked bottle, the purified 3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carboxylic acid (500 mg, 2.42 mmol), DMF (10 mL), ethyl (S)-3-(2-aminoacetamido)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate hydrochloride (1.83 g, 2.91 mmol), TBTU (2.34 g, 7.29 mmol), and DIPEA (940 mg, 7.27 mmol) were added sequentially and stirred at room temperature for 16 h. Water (20 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL x 3). The combined organic phases were washed sequentially with saturated aqueous sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to afford a crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 90:10) afforded ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoate (05L) (600 mg, 31.7% yield).

[0363] LC-MS, M / Z(ESI):780.4[M+H] + .

[0364] Step 12: Synthesis of (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3)

[0365] To a 100 mL single-necked bottle, ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoate (600 mg, 0.769 mmol), THF / H2O (v:v = 1:1, 2 mL), and lithium hydroxide monohydrate (97 mg, 2.3 mmol) were added sequentially and stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure to remove THF, the pH was adjusted to 6 with 1N hydrochloric acid, and the mixture was concentrated under reduced pressure to obtain the crude product. After reverse phase preparation (column: YMC-Triart Prep C18 (30mm×40cm, 7μm); mobile phase: A=0.1% formic acid, B=acetonitrile; gradient: 10%–95%), (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3) (417 mg, yield 72.1%) was obtained.

[0366] LC-MS, M / Z(ESI):752.4[M+H] + .

[0367] Step 11: Synthesis of (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((2-trans-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3A) and (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((2-cis-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3B)

[0368] (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid was separated by SFC (column: Chiralpak IG-3 (50 mm × 4.6 mm, 3 μm, mobile phase: A = CO2, B = 60% ethanol + acetonitrile (0.05% ethylenediamine); gradient: 30–60%) gave the compound: (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((2-trans-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3A, 236 mg; column: Chiralpak IG-3 (50 mm × 4.6 mm, 3 μm, mobile phase: A = CO2, B = 60% ethanol + acetonitrile (0.05% ethylenediamine); gradient: 30–60%; flow rate: 3 mL / min; retention time: 0.441 min) and compound: (3S)-3-(4-((14-azido-3,6,9,12-tetrahydrotetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((2-cis-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propanoic acid (II-3B, 250 mg; column: Chiralpak IG-3 (50 mm × 4.6 mm, 3 μm, mobile phase: A = CO 2 , B = 60% ethanol + acetonitrile (0.05% ethylenediamine); gradient: 30–60%; flow rate: 3 mL / min; retention time: 0.895 min).

[0369] II-3A: 1H NMR (400MHz, DMSO-d6) δ8.35(d,J=8.4Hz,1H),7.97(t,J=6.0Hz,1H),7.79(d,J=5.2Hz,1H),7.39(d,J=2.0Hz,1H),7.34–7 .30(m,1H),7.19(d,J=2.4Hz,1H),7.07(d,J=8.4Hz,1H),6.62(d,J=7.2Hz,1H),6.31–6.27(m,1H),6.22(d,J=2.0Hz,1H), 6.15(s,1H),5.15(q,J=7.2Hz,1H),4.34–4.24(m,1H),4.14–4.04(m,2H),3.65(d,J=12.0Hz,7H),3.57–3.53(m,2H),3.52 –3.48(m,4H),3.48–3.44(m,8H),2.97–2.88(m,1H),2.73–2.60(m,2H),2.45–2.34(m,2H),2.10(s,3H),2.04–1.93(m,2H).

[0370] II-3B: 1 H NMR (400MHz, DMSO-d6) δ8.36(d,J=8.4Hz,1H),7.95(t,J=6.0Hz,1H),7.76(d,J=5.2Hz,1H),7.39(d,J=2.0Hz,1H),7.34– 7.30(m,1H),7.18(d,J=2.4Hz,1H),7.06(d,J=8.4Hz,1H),6.60(d,J=7.6Hz,1H),6.31–6.27(m,1H),6.22(d,J=2.0Hz,1H ),6.18(s,1H),5.14(q,J=7.4Hz,1H),4.20–4.11(m,1H),4.11–4.06(m,2H),3.67–3.62(m,7H),3.57–3.53(m,2H),3.52– 3.49(m,4H),3.48–3.44(m,8H),3.37–3.32(m,2H),2.74–2.60(m,3H),2.45–2.34(m,2H),2.09(s,3H),2.01–1.90(m,2H).

[0371] Example 6: Preparation of Compound III-1

[0372] The synthetic route is as follows:

[0373] Step 1: Synthesis of 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diaza-1-yl-20-oic acid (06B)

[0374] To a 100 mL single-necked bottle were added 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)pimelane diamide (500 mg, 0.803 mmol), DCM (5 mL), triethylamine (0.5 mL) and dihydro-4,4-dimethyl-2H-pyranyl-2,6(3H)-dione ( The reaction mixture was concentrated under reduced pressure to give the crude product, which was then purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to afford 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-oic acid (500 mg, 81.4% yield).

[0375] 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)

[0376] To a 100 mL single-necked bottle, 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-oic acid (500 mg, 0.654 mmol), DCM (5 mL), p-nitrophenol (109 mg, 0.784 mmol) and EDCI (150 mg, 0.782 mmol) were added in sequence and stirred at room temperature for 12 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to give 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diazepin-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1) (268 mg, 46.3% yield).

[0377] 1 H NMR(400MHz,DMSO-d6)δ8.27(d,2H),7.81(t,3H),7.41(d,2H),7.21(s,1H),4.10(d,6H),3.51–3.49(m,12H),3. 38–3.36(m,9H),3.16–3.13(m,6H),2.73(s,2H),2.17(s,2H),2.01–1.97(m,6H),1.80–1.76(d,6H),1.09(s,6H).

[0378] LC-MS, M / Z(ESI):887.3[M+H] + .

[0379] Example 7: Preparation of Compound III-2

[0380] The synthetic route is as follows:

[0381] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (07B)

[0382] To a 100 mL single-necked bottle, bis(perfluorophenyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo-3-(perfluorophenoxy)propyl)heptanedioate (3.0 g, 3.1 mmol), DCM (30 mL) and 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (2.1 g, 11 mmol) were added in sequence and stirred at room temperature for 3 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (3.0 g, yield 99%).

[0383] Step 2: Synthesis of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)pimelamide (07C)

[0384] To a 100 mL single-necked bottle, (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriacontane-1,32-diyn-17-yl)carbamate (3.0 g, 3.1 mmol), DMF (30 mL) and triethylamine (20 mL) were added in sequence and stirred at room temperature for 36 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)pimelanediamide (2.0 g, yield 86%).

[0385] Step 3: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (07D)

[0386] To a 100 mL single-necked bottle, 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl)piperamide (2.0 g, 2.6 mmol), DCM (20 mL), triethylamine (2 mL) and 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (753 mg, 5.30 mmol) were added in sequence and stirred at room temperature for 3 h. The crude product was concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to give 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (2.0 g, yield 84%).

[0387] Step 4: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid perfluorophenyl ester (III-2)

[0388] To a 100 mL single-necked bottle, 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid (500 mg, 0.557 mmol), DCM (5 mL) and pentafluorophenyl trifluoroacetate (312 mg, 1.11 mmol) were added sequentially and stirred at room temperature for 12 h. The crude product was concentrated under reduced pressure and purified by column chromatography (ethyl acetate / acetonitrile (V / V) = 1:1) to give 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadecane-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid perfluorophenyl ester (III-2) (110 mg, yield 18.6%).

[0389] 1 H NMR(400MHz, CDCl3)δ7.39(s,1H),6.59(t,3H),4.2(d,6H),3.51–3.49(m,12H),3.70–3.69(m,12H),3.64–3.62(m,12H),3.5 8–3.54(m,6H),3.38–3.36(m,6H),3.0(q,3H),2.83(s,2H),2.48(s,2H),2.25–2.22(m,6H),2.06–2.04(d,6H),1.18(s,6H).

[0390] Preparation Example 1: Design and Synthesis of siRNA Conjugates

[0391] The synthesis steps of the siRNA conjugate in which the conjugation group is connected to the 5' end of the siRNA sense strand are as follows, taking C1 in Table 1 as an example:

[0392] 1) Synthesis of single-stranded oligoribonucleotides: Oligoribonucleotides were synthesized using phosphoramidite solid phase synthesis technology. All 2'-modified nucleotide phosphoramidites and auxiliary reagents were commercially available reagents (Shanghai Zhaowei Technology Development Co., Ltd.). All phosphoramidites were dissolved in anhydrous acetonitrile and molecular sieves were added. 5-Ethylthio-1H-tetrazole (ETT, Suzhou Kelema Biotechnology Co., Ltd.) was used as an activator, with a coupling time of 5 minutes. Phosphorothioate bonds were generated using a 50 mM solution of 3-((dimethylamino-methylene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, Shanghai Zhaowei Technology Development Co., Ltd.) in anhydrous acetonitrile / pyridine (v / v = 1 / 1) for a reaction time of 3 minutes. All sequences were synthesized after the final removal of the DMT group.

[0393] 2) Cleavage and deprotection of oligoribonucleotides bound to CPG: After solid-phase synthesis is completed, the protecting groups are removed by treatment with acetonitrile containing 20% ​​diethylamine for 30 minutes. After centrifugation, the supernatant is transferred to a new tube and the CPG is washed with aqueous ammonia. The combined solution is concentrated to obtain a solid mixture.

[0394] 3) Purification of Single-Stranded Oligoribonucleotides: Oligomers were purified by HPLC using a NanoQ anion exchange column. Buffer A consisted of 20% acetonitrile in 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4; and buffer B consisted of 20% acetonitrile in 500 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4. The desired product was isolated, and a portion of the product was desalted using a reverse-phase C18 column.

[0395] 4) Single-stranded oligoribonucleotide coupling linker: 12 mg of TA14 was dissolved in DMSO and added to the nucleic acid amino intermediate (10 mg, dissolved in DMSO). After ultrasonic mixing, carbonate buffer solution (pH = 9) was added and the mixture was reacted at 25°C for 16 h and then purified.

[0396] 5) Coupling of single-stranded oligoribonucleotide-TA14 conjugate II-1B: Dissolve 7 mg of nucleic acid-TA14 in buffered saline solution, add ligand II-1B (equivalent: 15, dissolved in DMF) to the above solution and vortex; take THPTA:CuSO4·5H2O=5:1 (equivalents are 15 and 3 respectively), shake at 40°C for 5 minutes, add the nucleic acid ligand mixture to the above reaction solution and vortex; quickly add sodium vitamin C (equivalent to 25) to the above solution, vortex, react at 40°C for 1 hour, and purify after the reaction to obtain the complete single-stranded oligoribonucleotide-TA14-II-1B conjugate.

[0397] 6) Annealing to produce siRNA: The single-stranded oligoribonucleotides to be annealed were prepared to 200 μM using sterile RNase-free water (without RNA hydrolase). The annealing reaction system was set up as follows: 10 nmol of the mixture, with a total volume of 100 μL, was placed in a 95°C water bath for 10 minutes (amounts ≥ 100 nmol require high temperature for 20 minutes). The mixture was quickly placed in a 60°C water bath and allowed to cool naturally. The annealed solution should not be stored at high temperatures. By combining equimolar single-stranded oligoribonucleotide solutions to form complementary chains, various unmodified siRNAs were obtained. The nucleotide sequences of the sense and antisense chains of the siRNAs obtained in the present invention are shown in Table 1. Furthermore, the molecular weight of the siRNAs was determined using liquid chromatography-mass spectrometry (LC-MS). The measured molecular weights were compared with the theoretical values. The results showed that the measured values ​​≈ the theoretical values, indicating that the siRNA conjugates shown in Table 1 were obtained. For the specific structure of the ligand (II-1B) in Table 1, please refer to the invention content section of this specification (i.e., II-1B in the compound described in the third aspect or its stereoisomer, tautomer or pharmaceutically acceptable salt), II-1B is derived from compound II-1B in Example 3, and III-1 and III-2 are derived from compounds III-1 and III-2 in Examples 6 and 7.

[0398] Table 1

[0399] In Table 1, lowercase letters c, g, u, a, and t are all 2'-methoxy-modified nucleotides (i.e., c, g, u, and a respectively indicate that the ribose 2'-OH of the nucleotide represented by the corresponding uppercase letter is replaced by a methoxy group); f indicates that the nucleotide represented by the letter before f is a 2'-fluoro-modified nucleotide (i.e., the 2'-OH of the nucleotide represented by the letter before f is replaced by a fluorine atom); s indicates that the residues between the two adjacent nucleotide residues on the left and right of s are phosphorothioate (i.e., the 5'-phosphate group of the nucleotide represented by the letter before s is replaced by a 5'-phosphorothioate group).

[0400] (Tri-SM6.1)(TA14)(NH2C6) is:

[0401] Its preparation method can be found in WO2022 / 216920A1.

[0402] (II-1B)(TA14)(NH2C6) is:

[0403] (II-1B)(III-1)(NH2C6) is:

[0404] (II-1B)(III-2)(NH2C6) is:

[0405] The structures of cPrpus and cPrpas are as follows:

[0406] In the test case, the I-0 structure is:

[0407] The preparation method thereof is referred to Goodman, SL et al. J. Med. Chem. 2002, 45, 1045-1051.

[0408] The structure of II-0 is:

[0409] Its preparation method refers to WO2019 / 089765A1.

[0410] Test Example 1: ɑvβ6 ligand ELISA binding assay

[0411] Dilute the integrin ɑvβ6 protein (source: Acro Biosystems) to 5 μg / mL using coating solution, and add 25 μL of protein to each well of a 384-well plate. Centrifuge at 1000 rpm for 1 minute, then incubate at 4°C overnight. Add 100 μL of wash buffer to each well, incubate at 1000 rpm for 1 minute, pat dry, and wash three times. Add 100 μL of blocking solution to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 90 minutes. Add 100 μL of wash buffer to each well, incubate at 25°C for 5 minutes, pat dry, and wash three times. Add 10 μL of compound at different dilutions to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 10 minutes. Add 10 μL of fibronectin (source: R&D) to each well, centrifuge at 1000 rpm for 1 minute, then incubate at 25°C for 60 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash three times. Add 25 μL of fibronectin biotinylated antibody (source: R&D) to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 60 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash three times. Add 25 μL of horseradish peroxidase-labeled streptavidin to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 30 minutes. Add 100 μL of washing solution to each well, let it stand for 5 minutes, pat dry, and wash four times. Add 25 μL of supersensitive luminescent solution to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 5 minutes. Read the luminescence value on a microplate reader.

[0412] Table 2: αvβ6 integrin ligand binding activity

[0413] Table 3: Binding activity of αvβ6 integrin ligand (including linker)

[0414] The results show that the compound of the present application has high binding activity with ɑvβ6 integrin.

[0415] Test Example 2: Silencing effect of target expression by siRNA conjugate administered intratracheally in rats

[0416] With reference to patent WO2022 / 216920A1, siRNA conjugates targeting rat RAGE protein were designed and synthesized. The siRNA conjugates are shown in Table 1.

[0417] On day 0, whole blood and serum were collected from the jugular vein of rats. On day 1, rats were anesthetized with isoflurane and administered 200 μL of PBS or drug buffer via an aerosol needle at a dose of 0.1 mg / kg. Before the end of the experiment, rats were anesthetized with 10% chloral hydrate, and blood and serum were collected from the abdominal aorta using a negative pressure blood collection tube. Lung tissue was then collected from both sides and snap-frozen in liquid nitrogen. All processed serum and lung tissue samples were stored at -80°C.

[0418] Lung tissue was thawed on ice and homogenized using a tissue grinder. Total RNA was extracted from the lung tissue using the Tissue RNA Extraction Kit 2.0 Plus (Novagen, R411-C3). After reverse transcription (Novagen, R333-01), the expression of RAGE mRNA in rat lung tissue was detected using a quantitative PCR probe (Novagen, QN211-02). The expression of RAGE mRNA was normalized with that of rat GAPDH and compared to the expression level in the PBS control group, which was set as 100%.

[0419] Serum was thawed on ice and assayed using the Rat RAGE DuoSet ELISA kit (R&D Systems, DY1616) according to the manufacturer's instructions. The expression level of sRAGE protein in rat serum was calculated using the standard curve fit and conversion. The expression level in the PBS control group was used as the standard for comparison. The results are shown in the table below.

[0420] Table 4: Expression of RAGE protein in rat serum and RAGE mRNA in lung

[0421] Table 5: Expression of RAGE protein in rat serum and RAGE mRNA in lung

[0422] The results showed that the siRNA conjugate significantly reduced the expression of RAGE mRNA and protein in the lungs and serum of rats. The compound of the present application can make the siRNA silencing of lung targets and the expression of corresponding secretory proteins more effective, indicating that it has a stronger ability to deliver siRNA molecules to lung cells.

[0423] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug: Among them, L1 is empty, -CH2-, or L2 is -(CH2) m -, a 3- to 10-membered subcycloalkyl or a 3- to 10-membered subheterocycloalkyl, and m is 1, 2, 3, 4, 5, 6; L2 is optionally substituted by C1-C3 alkyl or halogen; A is selected from: Ra is a 6-10 membered aromatic ring, a 5-10 membered heteroaromatic ring; Ra is optionally substituted by R2; Rb is dioxane; R1, R2, R3, R4, R5 are each independently H, halogen, -OH, -NH2, -COOH, -CN, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, C1-C6 haloalkoxy, -CON(C1-C6 alkyl)2, -CON(C1-C6 haloalkyl)2, -CO-C1-C6 alkyl, -CO-C1-C6 haloalkyl; A is optionally linked to linker T; and the compound of formula I satisfies one or more of the following conditions i)-iii): i) L1 is A is selected from: ii) L1 is empty, -CH2-, and A is iii), L1 is empty, -CH2-, or A is and Ra is a 5-6 membered heteroaromatic ring.

2. The compound represented by formula I according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, R1 is C1-C6 alkyl, C1-C6 haloalkyl; and / or For and / or Having a structure Preferably, R1 is C1-C3 alkyl, C1-C3 haloalkyl.

3. The compound represented by formula I as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, It satisfies one or more of the following conditions: a) When L1 is empty, L2 is a 3- to 10-membered cycloalkylidene or a 3- to 10-membered heterocycloalkylidene, and A is selected from: wherein, Ra is a 5-6 membered heteroaromatic ring; Rb is dioxane; the definitions of R3, R4, R5 are as described in claim 1; Preferably, when L1 is empty, L2 is and / or, A is selected from: b) When L1 is -CH2- and L2 is -(CH2) m -, A is selected from: wherein, Ra is a 5-6 membered heteroaromatic ring; Rb is dioxane; the definitions of R3, R4, R5, m are as described in claim 1; Preferably, R3 is H; R4, R5 are methoxy, ethoxy or propoxy.

4. The compound represented by Formula I according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, It has one or more of the following structures: The compound of formula I has the structure Ia: Among them, the definitions of Ra and R3 are as described in claim 1; Optionally linked to linker T; And / or, the compound of formula I has the structure Id: Among them, the definitions of Ra and R3 are as described in claim 1; Optionally linked to linker T; And / or, the compound of formula I has the structure Ie or If: wherein, the definitions of T, R3 are as described in claim 1; q is selected from 0, 1 or 2; When the compound of formula I is linked to linker T, it has structure If: wherein, the definitions of T, R3 are as described in claim 1; q is selected from 0, 1 or 2.

5. The compound represented by Formula I as described in claim 4, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, It satisfies one or more of the following conditions: c) When Connected with joint T, R3 is H, Having a structure d) Ra is a 5-6 membered heteroaromatic ring optionally substituted by R2; Preferably, the Ra is one of the following substituents optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole; alternatively, the Ra is 6. The compound represented by Formula I as described in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, The compound of formula I has the structure Ib or Ic: wherein, R b , R4, R5, Ra, and R3 are defined as described in claim 1; Preferably, R4, R5 are -OH or -OCH3; Preferably, Rb is 1,3-dioxane, and Rb is optionally linked to linker T; Preferably, Rb is or when the joint T is connected, Rb is 7. The compound represented by Formula I as described in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, When the A is connected with the joint T, has a structure and satisfies one or more of the following conditions: i) When R3 is H, Having a structure ii) When R3 is H, Having a structure iii) With structure iv) Having a structure v) Having a structure And / or, when A is attached to linker T, the compound of formula I has the structure:

8. The compound represented by Formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug according to any one of claims 1 or 4-7, characterized in that, Satisfies one or more of the following conditions: e) The linker T contains a polyethylene glycol unit; f) The linker T contains 2-20 polyethylene glycol units; g) The joint T is where t is 1-10; Or, the joint T is where t is 1-10; Preferably, t is 1, 2, 3, 4, 5; h) The joint T is Or, the joint T is 9. The compound represented by Formula I according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, It includes the following structures: Among them, Represents the connection point.

10. The compound represented by Formula I according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, It includes the following structures: Among them, Represents the connection point.

11. Compound II, its tautomer, stereoisomer or its salt, characterized in that, Comprises a compound of formula I as described in any one of claims 1-10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, and active molecule G; wherein, the active molecule G is linked to the linker T in the compound of formula I; The active molecule G satisfies one or more of the following conditions: k) The active molecule G is an active pharmaceutical ingredient or its prodrug; m) The active molecule G is an antibody, immunoglobulin, label or marker, lipid, natural or modified nucleic acid, natural or modified nucleic acid oligonucleotide, natural or modified nucleic acid polynucleotide, peptide, nucleic acid aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, biotin, radioactive atom or molecule, or fluorophore; n) The active molecule G is a natural or modified oligonucleotide; p) The active molecule G is an ASO, siRNA, or miRNA.

12. A conjugate, characterized in that, Comprising a compound of formula I as described in any one of claims 1-10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, a scaffold, and an active molecule G; Wherein the scaffold is connected to the linker T in the compound of formula I, and the active molecule G is connected to the compound of formula I through the scaffold; The active molecule G satisfies one or more of the following conditions: k) The active molecule G is an active pharmaceutical ingredient or its prodrug; m) The active molecule G is an antibody, immunoglobulin, label or marker, lipid, natural or modified nucleic acid, natural or modified nucleic acid oligonucleotide, natural or modified nucleic acid polynucleotide, peptide, nucleic acid aptamer, polymer, polyamine, protein, toxin, vitamin, polyethylene glycol, hapten, biotin, radioactive atom or molecule, or fluorophore; n) The active molecule G is a natural or modified oligonucleotide; p) The active molecule G is an ASO, siRNA, or miRNA.

13. The conjugate according to claim 12, wherein The scaffold is a monodentate, bidentate, tridentate, or tetradentate structure; And / or, the active molecule G is connected to one, two, three, or four compounds of formula I through the scaffold.

14. The conjugate according to claim 12, wherein, The conjugate has the following structure: Wherein, formula I is the compound of formula I shown, and G is the active molecule G; t is 1-10; preferably, t is 1, 2, 3, 4, 5; 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, or miRNA.

15. The conjugate according to claim 12, wherein, The conjugate has the following structure: Among them, Represents the connection point to the oligonucleotide.

16. A composition, characterized in that, Comprising a compound of formula I as described in any one of claims 1-10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, compound II as claimed in claim 11, or conjugate as claimed in any one of claims 12-15, and optionally a pharmaceutically acceptable excipient.

17. The compound of formula I as described in any one of claims 1-10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, compound II as claimed in claim 11, conjugate as claimed in any one of claims 12-15, or composition as claimed in claim 16 has the following uses, which are selected from: Delivering the active molecule G to cells; and / or Inhibiting the expression of a target gene in cells; and / or Treating lung diseases; and / or Preparing a drug for inhibiting the expression of a target gene in cells; and / or Preparing a drug for treating lung diseases.

18. The compound of formula I as described in any one of claims 1-10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, compound II as claimed in claim 11, conjugate as claimed in any one of claims 12-15, or composition as claimed in claim 16, for: Delivering the active molecule G to cells; and / or Inhibiting the expression of a target gene in cells; and / or Treating lung diseases.

19. The use according to claim 17 or 18, characterized in that, The cell is a cell expressing αvβ6 integrin or an epithelial cell; and / or, the cell is a type I and type II alveolar epithelial cell, goblet cell, secretory epithelial cell, ciliated epithelial cell, corneal and conjunctival epithelial cell, dermal epithelial cell, bile duct epithelial cell, intestinal epithelial cell, duct epithelial cell, glandular epithelial cell, and epithelial tumor (carcinoma).

20. A method for delivering an active molecule to a cell, inhibiting the expression of a target gene in the cell, or treating a lung disease, characterized in that, The method includes administering to a patient a compound represented by formula I as described in any one of claims 1-10, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, compound II as described in claim 11, conjugate as described in any one of claims 12-15, or composition as described in claim 16.

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