Compounds targeting alpha v beta 6 and uses thereof

Abstract

The invention provides a compound targeting alpha v beta 6, and the compound is the compound shown in the formula I and can be used for delivering active molecules to cells or tissues expressing integrin alpha v beta 6. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medicine. Specifically, the present invention relates to a compound targeting αvβ6 and its use. Background Art

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

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

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

[0005] The object of the present invention is to provide a compound targeting αvβ6. The compound targeting αvβ6 (or referred to as the targeting compound) is a targeting ligand capable of targeting integrin αvβ6. The targeting compound has an 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 can exert its function in these cells or tissues.

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

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

[0008]

[0009] wherein, L1 is empty, -CH2- or

[0010] L2 is -(CH2) m -, a 3- to 10-membered sub-cycloalkyl or a 3- to 10-membered sub-heterocycloalkyl, and m is 1, 2, 3, 4, 5, 6;

[0011] The L2 is optionally substituted by a C1-C3 alkyl or a halogen;

[0012] A is selected from:

[0013] Ra is a 6- to 10-membered aromatic ring or a 5- to 10-membered heteroaromatic ring; the Ra is optionally substituted by R2;

[0014] Rb is dioxane;

[0015] R1, R2, R3, R4, R5 are each independently H, a halogen, -OH, -NH2, -COOH, -CN, a C1-C6 alkyl, a C1-C6 haloalkyl, a C1-C6 alkoxy, a C1-C6 haloalkoxy, -CON(C1-C6 alkyl)2, -CON(C1-C6 haloalkyl)2, -CO-C1-C6 alkyl, -CO-C1-C6 haloalkyl;

[0016] A is optionally linked to linker T;

[0017] And the compound satisfies one or more of the following conditions i)-iii):

[0018] i) L1 is A is selected from:

[0019] ii) L1 is empty, -CH2-, and A is

[0020] iii), L1 is empty, -CH2- or A is And Ra is a 5- to 6-membered heteroaryl ring.

[0021] In an alternative embodiment of the present application, the compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug:

[0022]

[0023] Wherein, L1 is -CH2- or

[0024] L2 is -(CH2) m -, and m is 1, 2, 3, 4, 5, 6;

[0025] The L2 is optionally substituted by C1-C3 alkyl or halogen;

[0026] A is selected from:

[0027] Ra is a 6- to 10-membered aryl ring, a 5- to 10-membered heteroaryl ring; the Ra is optionally substituted by R2;

[0028] Rb is dioxane;

[0029] 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;

[0030] The A is optionally linked to linker T;

[0031] And the compound satisfies one or more of the following conditions i)-iii):

[0032] i) L1 is A is selected from:

[0033] ii) L1 is empty, -CH2-, and A is

[0034] iii), L1 is empty, -CH2- or A is and Ra is a 5- to 6-membered heteroaryl ring.

[0035] In a preferred embodiment, R1 is C1-C6 alkyl, C1-C6 haloalkyl.

[0036] In a preferred embodiment, R1 is C1-C3 alkyl, C1-C3 haloalkyl.

[0037] In a preferred embodiment, is

[0038] In a preferred embodiment, has the structure

[0039] In a preferred embodiment, is in the Z configuration.

[0040] In a preferred embodiment, when L1 is then A is Said A is optionally linked to linker T.

[0041] In a preferred embodiment, Ra is a naphthalene ring.

[0042] In a preferred embodiment, R3 is H.

[0043] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -, 3- to 10-membered cycloalkyl or 3- to 10-membered heterocycloalkyl, where m is 1, 2, 3, 4, 5, 6.

[0044] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -, 3- to 10-membered cycloalkyl or 3- to 10-membered heterocycloalkyl, where m is 2, 3, 4.

[0045] In a preferred embodiment, -L1-L2- is selected from -(CH2) m -, or 3- to 6-membered cycloalkyl, where m is 2, 3, 4.

[0046] In a preferred embodiment, -L1-L2- is selected from -(CH2)3-, or a 4- to 5-membered cycloalkyl group.

[0047] In a preferred embodiment, when L1 is empty, L2 is a 3- to 10-membered cycloalkylidene group or a 3- to 10-membered heteroalkylidene group.

[0048] In a preferred embodiment, when L1 is empty, L2 is a 3- to 6-membered cycloalkylidene group or a 3- to 6-membered heteroalkylidene group.

[0049] In a preferred embodiment, when L1 is empty, L2 is a 3- to 6-membered cycloalkylidene group.

[0050] In a preferred embodiment, when L1 is empty, L2 is

[0051] In a preferred embodiment, when L1 is empty, A is selected from:

[0052] In a preferred embodiment, when L1 is empty, L2 is A is selected from:

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

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

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

[0056] Wherein, Ra is a 5- to 6-membered heteroaryl ring; the definitions of R3, R4, and R5 are as shown in the first aspect of the present invention.

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

[0058] Wherein, Ra is a 5- to 6-membered heteroaryl ring; R3 is H; R4 and R5 are hydroxyl groups or C1-C6 alkoxy groups.

[0059] In a preferred embodiment, when L1 is empty and L2 is a 3- to 10-membered cycloalkylidene group or a 3- to 10-membered heteroalkylidene group, A is selected from:

[0060] Wherein, Ra is a 5- to 6-membered heteroaryl ring; R3 is H; the said R4 and R5 are methoxy, ethoxy, or propoxy groups.

[0061] In a preferred embodiment, when L1 is -CH2- and L2 is -(CH2) m -, A is selected from: wherein Ra is a 5- or 6-membered heteroaryl ring; Rb is dioxane; and R3, R4, and R5 are as defined above.

[0062] In a preferred embodiment, R3 is H.

[0063] In a preferred embodiment, R4 and R5 are hydroxyl or C1-C6 alkoxy.

[0064] In a preferred embodiment, R4 and R5 are methoxy, ethoxy, or propoxy.

[0065] In a preferred embodiment, the compound of formula I has the structure Ia:

[0066]

[0067] wherein Ra and R3 are as defined above; optionally linked to linker T.

[0068] In a preferred embodiment, in structure Ia, R3 is H.

[0069] In a preferred embodiment, in structure Ia, Ra is a 5- or 6-membered heteroaryl ring optionally substituted with R2; R2 is C1-C3 alkyl or C1-C3 haloalkyl.

[0070] In a preferred embodiment, in structure Ia, R2 is methyl.

[0071] In a preferred embodiment, in structure Ia, when linked to linker T and R3 is H, it has the structure

[0072] In a preferred embodiment, in structure Ia, when linked to linker T and R3 is H, it has the structure

[0073] In a preferred embodiment, in structure Ia, Ra is a 5- or 6-membered heteroaryl ring optionally substituted with R2.

[0074] In a preferred embodiment, in structure Ia, Ra is a 5-membered N-containing heteroaryl ring optionally substituted with R2.

[0075] In a preferred embodiment, in Structure Ia, Ra is one of the following substituents optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole.

[0076] In a preferred embodiment, in Structure Ia, Ra is methylpyrazole, as

[0077] In a preferred embodiment, the compound of Formula I has Structure Ib:

[0078]

[0079] wherein, R b , R4, and R5 are as defined above.

[0080] In a preferred embodiment, in Structure Ib, R4 and R5 are OH or -OCH3.

[0081] In a preferred embodiment, in Structure Ib, Rb is 1,3-dioxane, and Rb is optionally connected to linker T.

[0082] In a preferred embodiment, in Structure Ib, Rb is

[0083] In a preferred embodiment, the compound of Formula I has Structure Ic:

[0084]

[0085] wherein, Ra and R3 are as defined above.

[0086] In a preferred embodiment, in Structure Ic, Ra is a naphthalene ring, and Ra is optionally connected to linker T;

[0087] In a preferred embodiment, in Structure Ic, R3 is H.

[0088] In a preferred embodiment, in Structure Ic, has Structure

[0089] In a preferred embodiment, in Structure Ic, has Structure

[0090] In a preferred embodiment, the compound of Formula I has Structure Id:

[0091]

[0092] wherein, Ra and R3 are as defined in the first aspect; optionally connected to linker T.

[0093] In a preferred embodiment, in Structure Id, when the structure is connected to joint T.

[0094] In a preferred embodiment, in Structure Id, the benzene ring in the structure is connected to joint T.

[0095] In a preferred embodiment, in Structure Id, R3 is H.

[0096] In a preferred embodiment, in Structure Id, when it is connected to joint T and R3 is H, it has the structure

[0097] In a preferred embodiment, in Structure Id, when the benzene ring is connected to joint T and R3 is H, it has the structure

[0098] In a preferred embodiment, in Structure Id, when it is connected to joint T and R3 is H, it has the structure

[0099] In a preferred embodiment, in Structure Id, when the benzene ring is connected to joint T and R3 is H, it has the structure

[0100] In a preferred embodiment, in Structure Id, Ra is a 5- or 6-membered heteroaryl ring optionally substituted by R2.

[0101] In a preferred embodiment, in Structure Id, Ra is a 5-membered N-containing heteroaryl ring optionally substituted by R2.

[0102] In a preferred embodiment, in Structure Id, Ra is one of the following substituents optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole.

[0103] In a preferred embodiment, in Structure Id, Ra is methylpyrazole, such as

[0104] In a preferred embodiment, when A is connected to joint T, it has the structure

[0105] In a preferred embodiment, when A is connected to joint T, it has the structure

[0106] In a preferred embodiment, when R3 is H, is

[0107] In a preferred embodiment, when R3 is H, is

[0108] In a preferred embodiment, is

[0109] In a preferred embodiment, when the A is connected to the linker T, has the structure

[0110]

[0111] In a preferred embodiment, is In a preferred embodiment, is In a preferred embodiment, is In a preferred embodiment, when the A is connected to the linker T, the compound represented by Formula I has the structure:

[0112]

[0113] In a preferred embodiment, the linker T comprises a polyethylene glycol unit.

[0114] In a preferred embodiment, the linker T comprises 2 - 20 polyethylene glycol units.

[0115] In a preferred embodiment, the linker T is wherein t is 1 - 10.

[0116] In a preferred embodiment, the linker T is wherein t is 1 - 10.

[0117] In a preferred embodiment, t is 1, 2, 3, 4, 5.

[0118] In a preferred embodiment, the linker T is

[0119] In a preferred embodiment, the linker T is

[0120] In a preferred embodiment, the compound of formula I has structure Ie or If (linked to linker T):

[0121]

[0122] wherein, the definitions of T and R3 are as described above; q is selected from 0, 1 or 2.

[0123] In a preferred embodiment, in structure Ie, R3 is hydrogen.

[0124] In a preferred embodiment, in structure Ie, q is selected from 0.

[0125] In a preferred embodiment, in structure Ie, q is selected from 1.

[0126] In a preferred embodiment, in structure If, linker T is

[0127] In a preferred embodiment, in structure If, linker T is

[0128] In a preferred embodiment, the compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, includes the following structures:

[0129]

[0130]

[0131]

[0132] wherein, represents the connection point.

[0133] In a preferred embodiment, the compound of formula I, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, includes the following structures:

[0134]

[0135]

[0136]

[0137]

[0138] wherein, represents the connection point.

[0139] In a second aspect of the present invention, there is provided a compound II, a tautomer, stereoisomer or salt thereof, including the compound represented by formula I as described in any one of the first aspect, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, and an active molecule G; wherein the active molecule G is linked to the linker T in the compound represented by formula I.

[0140] In a preferred embodiment, the active molecule G is an active pharmaceutical ingredient or a prodrug thereof.

[0141] 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.

[0142] In a preferred embodiment, the active molecule G is a natural or modified nucleic acid oligonucleotide.

[0143] As used herein, the terms "natural or modified nucleic acid oligonucleotide", "oligonucleotide containing natural or modified nucleic acid", or "natural or modified oligonucleotide" are synonymous and all refer to an oligonucleotide composed of natural nucleic acids or an oligonucleotide containing modified nucleic acids. The term "natural or modified nucleic acid polynucleotide" refers to a polynucleotide composed of natural nucleic acids or a polynucleotide containing modified nucleic acids.

[0144] In a preferred embodiment, the active molecule G is an ASO, siRNA, miRNA.

[0145] In a third aspect of the present invention, there is provided a conjugate, including the compound represented by formula I as described in any one of the first aspect, a tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug thereof, a scaffold and an active molecule G; wherein the scaffold is linked to the linker T in the compound represented by formula I, and the active molecule G is linked to the compound represented by formula I through the scaffold.

[0146] In a preferred embodiment, the scaffold has a monodentate, bidentate, tridentate, or tetradentate structure.

[0147] In a preferred embodiment, the active molecule G is linked to one, two, three, or four compounds represented by formula I through the scaffold.

[0148] In a preferred embodiment, the conjugate has the following structure:

[0149]

[0150]

[0151] Among them, Formula I is the compound shown in Formula I, and G is the active molecule G;

[0152] t is from 1 to 10; preferably, t is 1, 2, 3, 4, 5, and more preferably, t is 2, 3;

[0153] v is from 1 to 10; preferably, v is 4, 5, 6, 7, 8.

[0154] 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 an ASO, siRNA, miRNA.

[0155] In a preferred embodiment, the conjugate has the following structure:

[0156]

[0157]

[0158] Among them, represents the connection point with the oligonucleotide.

[0159] In the fourth aspect of the present invention, there is provided a composition comprising the compound shown in Formula I according to any one of the first aspect, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, the compound II according to any one of the second aspect, or the conjugate according to any one of the third aspect.

[0160] In a preferred embodiment, the composition further comprises a pharmaceutically acceptable excipient.

[0161] In the fifth aspect of the present invention, there is provided the use of the compound shown in Formula I according to any one of the first aspect, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, the compound II according to any one of the second aspect, the conjugate according to any one of the third aspect, or the composition according to the fourth aspect in the preparation of a drug.

[0162] In a preferred embodiment, the drug is used to deliver the active molecule G to cells.

[0163] In a preferred embodiment, the drug is used for:

[0164] inhibiting the expression of a target gene in epithelial cells; and / or

[0165] preparing a drug for inhibiting the expression of a target gene in cells; and / or

[0166] preparing a drug for treating lung diseases.

[0167] In a preferred embodiment, the cell is a cell expressing αvβ6 integrin or an epithelial cell.

[0168] In a preferred embodiment, the cells are type I and type II alveolar epithelial cells, goblet cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, ductal epithelial cells, glandular epithelial cells, and epithelial tumors (cancers).

[0169] In a sixth aspect of the present invention, there is provided a method for delivering an active molecule to a cell or treating a lung disease, the method comprising administering to a patient a compound II as described in the second aspect, a conjugate as described in the third aspect, or a composition as described in the fourth aspect.

[0170] In a preferred embodiment, the cell is the cell as described in the fifth aspect.

[0171] Linker

[0172] As disclosed herein, in some embodiments, the targeting compound further has a linker (linker T), and the active molecule is further linked through the linker. In some embodiments, the linker may be a structure containing a polyethylene glycol (PEG) group. The polyethylene glycol linker may comprise 2 - 20 polyethylene glycol units.

[0173] In some embodiments, the linker is

[0174] In some embodiments, the linker T is

[0175] representing the attachment sites respectively connected to the targeting compound and the active molecule.

[0176] In some embodiments, where t is an integer from 1 to 10; for example, t is 1, 2, 3, 4, 5, 6, 7.

[0177] In some embodiments, at one end of the linker connecting the active molecule, the linker has a reactive group, and is directly conjugated to the active molecule through the reactive group, or conjugated to the active molecule after being connected to a scaffold. In some embodiments, the reactive group is an azide or an alkynyl-containing group.

[0178] There is no particular limitation on the attachment site of the linker to the targeting compound. In some embodiments, A in formula I is connected to linker T.

[0179] In some embodiments, the structure of the compound shown in formula I connected to linker T includes:

[0180]

[0181] Multidentate targeting compounds and scaffolds

[0182] As disclosed herein, in some embodiments, one or more compounds that target αvβ6 (targeting compounds) can be linked to one or more transported active molecules. In some embodiments, only one targeting compound is conjugated to the active molecule (referred to herein as a "monodentate" or "monovalent" ligand). In some embodiments, two targeting compounds are conjugated to the active molecule (referred to herein as a "bidentate" or "divalent" ligand). In some embodiments, three targeting compounds are conjugated to the active molecule (referred to herein as a "tridentate" or "trivalent" ligand). In some embodiments, four targeting compounds are conjugated to the active molecule (referred to herein as a "tetradentate" or "tetravalent" ligand). In some embodiments, more than four are conjugated to the active molecule.

[0183] As disclosed herein, in some embodiments, the scaffold and the active molecule are linked to the targeting compound via a linker. In some embodiments, the scaffold is a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the active molecule is linked to one targeting compound via a scaffold having a monodentate structure. In some embodiments, the active molecule is linked to two targeting compounds via a scaffold having a bidentate structure. In some embodiments, the active molecule is linked to three targeting compounds via a scaffold having a tridentate structure. In some embodiments, the active molecule is linked to four targeting compounds via a scaffold having a tetradentate structure.

[0184] Scaffolds are generally known in the art. Non-limiting examples of scaffolds that can be used with the targeting compounds disclosed herein include, but are not limited to, polymers and polyamino acids (e.g., diglutamic acid, poly-L-lysine, etc.). In some embodiments, the scaffold can include amine-reactive groups, amide bonds, and alkynyl-containing moieties (such as alkynyl, cyclooctyne). In some embodiments, the cyclooctyne-containing moiety contains moieties such as dibenzocyclooctyne (DBCO), bicyclo[6.1.0]nonyne-4-yne. In some embodiments, the alkynyl-containing moiety includes, but is not limited to: alkynyl maleimide, alkynyl 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.

[0185] In some embodiments, a typical scaffold having a tridentate structure, for example:

[0186]

[0187] The scaffold includes an amine-reactive group, an amide bond, three PEG2 units, and an alkyne. The amine-reactive group can be conjugated to a primary amine (such as a terminal amine group (e.g., NH2-C6)) on the active molecule to be transported through amide formation. The alkyne can be conjugated to an azide-modified linker to form a triazole structure, which is then linked to the targeting compound. In this scaffold, the amine-reactive group is p-nitrophenol (also known as 4-nitrophenol) ester.

[0188] In some embodiments, the amine-reactive group is not particularly limited. The amine-reactive group can also be, for example:

[0189]

[0190] In some embodiments, the scaffold can be synthesized as a phosphoramidite compound, which allows the tridentate scaffold to be coupled to an oligonucleotide (such as the 5'-end of the sense strand of siRNA) through phosphoramidite synthesis, as shown in the following structure:

[0191] Wherein, represents siRNA.

[0192] Conjugate

[0193] The active molecule described in this application can be linked to a targeting compound to form a conjugate. In some embodiments, the active molecule is an oligonucleotide, and the oligonucleotide can be coupled to the scaffold through phosphoramidite synthesis. The scaffold can have a monodentate, bidentate, tridentate, or tetradentate structure. In some embodiments, the azide group on the targeting compound can react with the alkyne (such as benzocyclooctyne, propargyl) of the scaffold (such as click chemical reaction) to form a triazole structure, which is then linked to the targeting compound.

[0194] In some embodiments, the conjugate is the conjugate described in the third aspect. The conjugate has a tridentate structure, and the conjugate is

[0195] Wherein, formula I is the compound shown in formula I, and G is the active molecule G;

[0196] t is 1-10; preferably, t is 1, 2, 3, 4, 5;

[0197] v is 1-10; preferably, v is 4, 5, 6, 7, 8.

[0198] In a preferred embodiment, the active molecule G is a natural or modified oligonucleotide.

[0199] Active molecule

[0200] As used herein, the "active molecule" can be linked to the targeting compound and then transported to cells and / or tissues expressing integrin αvβ6. The transported "active molecule" (or transported molecule) is any molecule that will have a desired effect on cells containing the αvβ6 integrin receptor when separated from the αvβ6 integrin ligand described herein. The transported active molecule can be, but is not limited to, a pharmaceutical ingredient, a pharmaceutical product, a prodrug, a substance with known therapeutic benefits, a small molecule, an antibody, an antibody fragment, an immunoglobulin, a monoclonal antibody, a label or marker, a lipid, a natural or modified nucleic acid or polynucleotide, a peptide, a polymer, a polyamine, a protein, a nucleic acid aptamer, a toxin, a vitamin, PEG, a hapten, digoxin, biotin, a radioactive atom or molecule, or a fluorophore. In some embodiments, one or more transported active molecules (e.g., the same or different transported active molecules) are linked to one or more αvβ6 integrin ligands to target the transported active molecules to cells expressing αvβ6 integrin. In some embodiments, the active molecule G is a natural or modified oligonucleotide.

[0201] oligonucleotide

[0202] As used herein, the term "oligonucleotide" refers to a short chain of nucleotides (including nucleotides within deoxyribonucleic acid DNA or ribonucleic acid RNA) composed of less than 50 bases. The oligonucleotides of the present application can be in single-stranded or double-stranded structures, and the specific types are not limited and are all within the scope of protection of the present application. Exemplarily, when in a double-stranded structure, it is composed of a sense strand and an antisense strand, and includes but is not limited to: short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrate; when in a single-stranded structure, the RNAi agent includes but is not limited to antisense oligonucleotide (ASO).

[0203] Additional aspects and advantages of the present invention will be given in part in the following description, will become apparent in part from the following description, or will be learned through the practice of the present invention.

[0204] Terms and Definitions

[0205] Unless otherwise specified, the definitions of groups and terms recited in the specification and claims of the present application, including their definitions as examples, exemplary definitions, preferred definitions, definitions recited in tables, definitions of specific compounds in examples, etc., can be combined and combined with each other arbitrarily. The group definitions and compound structures after such combination and combination should fall within the scope described in the specification of the present application.

[0206] Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. All patents, patent applications, and published materials cited herein in their entirety are incorporated herein by reference unless otherwise indicated. If there are multiple definitions of a term in this document, the definitions in this chapter shall prevail.

[0207] It should be understood that the foregoing summary and the following detailed description are exemplary and explanatory only and do not limit the subject matter of the present invention in any way. In this application, the singular form also includes the plural unless specifically stated otherwise. It must be noted that, unless clearly stated otherwise in the text, the singular forms used in this specification and the claims include the plural forms of the indicated items. It should also be noted that, unless otherwise indicated, the terms "or" and "or" mean "and / or". In addition, the terms "comprising" and other forms, such as "including", "containing", and "having" are not restrictive.

[0208] Definitions of standard chemical terms can be found in reference works (including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED.", Vols. A (2000) and B (2001), Plenum Press, New York). Unless otherwise indicated, conventional methods within the skill of the art are employed, such as mass spectrometry, NMR, IR, and UV / VIS spectroscopy and pharmacological methods. Unless a specific definition is provided, the terms used in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry herein are known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, as well as the treatment of patients. For example, the instructions provided by the manufacturer for a kit can be utilized, or reactions and purifications can be carried out in a manner known in the art or as described in the present invention. Generally, the above-mentioned techniques and methods can be implemented according to the descriptions in a number of general and more specific documents cited and discussed in this specification, in accordance with conventional methods well-known in the art. In this specification, groups and their substituents can be selected by one of ordinary skill in the art to provide stable structural moieties and compounds.

[0209] When a substituent is described by a conventional chemical formula written from left to right, the substituent also includes the chemically equivalent substituent obtained when the structural formula is written from right to left. For example, CH2O is equivalent to OCH2. As used herein, represents the point of attachment of a group. As used herein, "R1" and "R1" have the same meaning and can be substituted for each other. Similar definitions have the same meaning for other symbols such as R2.

[0210] The chapter headings used in this document are for the sole purpose of organizing the article and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals of operation, and theses, are hereby incorporated by reference in their entirety.

[0211] Except as otherwise provided above, when used in the specification and claims of this application, unless otherwise specifically indicated, the following terms have the meanings set forth below.

[0212] For a numerical range recited in the specification and claims of this application, when the numerical range is understood as "integers", it should be understood that the two endpoints of the range and each integer within the range are recited. For example, "integers from 0 to 5" should be understood to recite each of 0, 1, 2, 3, 4, and 5.

[0213] In this application, when used alone or as part of other substituents, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0214] As used herein, when used alone or as part of other substituents, the term "alkyl" means a straight-chain or branched hydrocarbon chain group consisting only of carbon and hydrogen atoms, containing no unsaturated bonds, having, for example, 1 to 6 carbon atoms, and connected to the rest of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl. The alkyl group may be unsubstituted or substituted with one or more suitable substituents. The alkyl group may also be an isotopic isomer of a naturally occurring abundance alkyl group enriched in carbon and / or hydrogen isotopes (i.e., deuterium or tritium).

[0215] When used alone or as part of other substituents, the term "C1-C6 alkyl" should be understood to represent a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, or 6 carbon atoms. The alkyl group is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, etc. or their isomers. The term "C1-C5 alkyl" should be understood to represent a straight-chain or branched saturated monovalent hydrocarbon group having 1, 2, 3, or 5 carbon atoms. In particular, the group has 1, 2, or 3 carbon atoms ("C1-C3 alkyl"), such as methyl, ethyl, n-propyl, or isopropyl.

[0216] When alone or as part of another substituent, the term "C1-C6 alkoxy" is understood to mean a straight-chain or branched-chain saturated monovalent hydrocarbyl group having 1, 2, 3, 4, 5, or 6 carbon atoms and an oxygen atom, or is defined as C1-C6 alkyl-O-C1-C6 alkyl as described in this specification, and the oxygen atom can be attached to any carbon atom of the straight-chain or branched-chain C1-C6 alkyl. It includes, but is not limited to: methoxy (CH3-O-), ethoxy (C2H5-O-), propoxy (C3H7-O-), butoxy (C4H9-O-).

[0217] When alone or as part of another substituent, the term "cycloalkyl" or "carbocyclic group" refers to a cyclic alkyl group. The term "m-n membered cycloalkyl" or "C m -C n cycloalkyl" is understood to mean a saturated, unsaturated, or partially saturated carbocyclic ring having m to n atoms. For example, "3-10 membered cycloalkyl" or "C3-C 10 cycloalkyl" refers to a cyclic alkyl group containing 3 to 10 carbon atoms, which may contain 1 to 3 rings. The said cyclic alkyl groups include monocyclic, bicyclic, tricyclic, spirocyclic, or bridged-ring groups. Examples of unsubstituted cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl, or bicyclic hydrocarbon groups such as the decahydronaphthalene ring. The cycloalkyl group can be substituted with one or more substituents. In some embodiments, the cycloalkyl group can be a cycloalkyl group fused to an aryl or heteroaryl group.

[0218] When alone or as part of another substituent, the term "heterocycloalkyl" or "heterocyclic group" refers to a cycloalkyl group in which one or more (in some embodiments 1 to 3) carbon atoms are replaced by heteroatoms such as, but not limited to, N, O, S, and P. The term "m-n membered heterocycloalkyl" or "C m -C n heterocycloalkyl" is understood to mean a saturated, unsaturated, or partially saturated ring having m to n atoms. For example, the term "4-10 membered heterocycloalkyl" is understood to mean a saturated, unsaturated, or partially saturated ring having 4 to 10 atoms. In some embodiments, the heterocycloalkyl group can be a heterocycloalkyl group fused to an aryl or heteroaryl group. When a prefix such as 3-8 is used to denote a heterocycloalkyl group, the number of carbons also means including the heteroatoms.

[0219] When alone or as part of another substituent, the term "aromatic ring" refers to an aryl ring structure, including compounds having one or more ring structures, such as monocyclic, bicyclic, tricyclic spiro or bridged ring compounds, and benzo-fused carbocyclic moieties, wherein at least one ring system is aromatic. The aryl group is generally, but not necessarily, linked to the parent molecule through the aromatic ring of the aryl group. The term "aromatic ring" can be used interchangeably with the terms "aromatic ring group" or "aryl ring group". Examples of aryl groups can include phenyl, indenyl, naphthyl, and anthracenyl. The said aryl group is optionally substituted with one or more substituents described in the present invention. Representative aryl groups include phenyl, anthracenyl, fluorenyl, indenyl, phenanthryl, and naphthyl.

[0220] When alone or as part of another substituent, the term "heteroaryl" can be used interchangeably with the terms "heteroaromatic ring" or "heteroaromatic group", and refers to a monocyclic or polycyclic aromatic ring system, in certain embodiments, 1 to 3 atoms in the ring system are heteroatoms, i.e., elements other than carbon, including but not limited to N, O, S, or P. For example, furyl, imidazolyl, dihydroindolyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl, pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl, and isoquinolinyl. The heteroaryl can be optionally fused with a benzene ring and can also be monocyclic, bicyclic, tricyclic, spiro or bridged.

[0221] When used alone or as part of other substituents, the term "5- to 10-membered heteroaryl" shall be understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring group having 5 to 10 ring atoms and containing heteroatoms selected from N, O and S, and shall be understood to have 5, 6, 7, 8, 9 or 10 ring atoms - especially 5 or 6 or 9 or 10 carbon atoms - and containing 1 to 5, preferably 1 to 3 - heteroatoms independently selected from N, O and S, and being, additionally in each case, benzo-fused. The "5- to 8-membered heteroaryl" shall be understood to mean a monovalent monocyclic, bicyclic or tricyclic aromatic ring group having 5 to 8 ring atoms - especially 5 or 6 carbon atoms - and containing 1 to 5 heteroatoms independently selected from N, O and S. Preferably 1 to 3 - heteroatoms independently selected from N, O and S, and being, additionally in each case, benzo-fused. Examples of heteroaryl include, but are not limited to: thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, etc. and their benzo derivatives, such as benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl, isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, etc., and their benzo derivatives, such as quinolinyl, quinazolinyl, isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc. and their benzo derivatives; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, etc.

[0222] When used alone or as part of other substituents, the term "halo" may be used interchangeably with the term "halogen substitution". "Haloalkyl" or "halogen-substituted alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group including a specific number of carbon atoms and substituted by one or more halogens (such as -CvFw, where v = 1 to 3 and w = 1 to (2v + 1)). Examples of haloalkyl include, but are not limited to, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl and heptachloropropyl.

[0223] The compounds provided herein, including the intermediates useful for preparing the compounds provided herein, which contain reactive functional groups (such as but not limited to carboxyl, hydroxyl, and amino moieties), also include their protected derivatives. "Protected derivatives" are those compounds in which one or more reactive sites are blocked by one or more protecting groups (also referred to as protecting groups). Suitable carboxyl moiety protecting groups include benzyl, tert-butyl, etc., as well as isotopes, etc. Suitable amino and amido protecting groups include acetyl, trifluoroacetyl, tert-butoxycarbonyl, benzyloxycarbonyl, etc. Suitable hydroxyl protecting groups include benzyl, etc. Other suitable protecting groups are well known to those of ordinary skill in the art.

[0224] In this application, "optional" or "optionally" means that the subsequent described event or condition may or may not occur, and this description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally substituted aryl" means that the aryl is substituted or unsubstituted, and this description includes both the substituted aryl and the unsubstituted aryl.

[0225] In this application, the terms "salt" or "pharmaceutically acceptable salt" include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0226] "Pharmaceutically acceptable acid addition salts" refer to salts formed with inorganic acids or organic acids that can retain the biological effectiveness of the free base without other side effects. "Pharmaceutically acceptable base addition salts" refer to salts formed with inorganic bases or organic bases that can maintain the biological effectiveness of the free acid without other side effects. In addition to pharmaceutically acceptable salts, the present invention also contemplates other salts. They can serve as intermediates in the purification of the compounds or in the preparation of other pharmaceutically acceptable salts or can be used for the identification, characterization, or purification of the compounds of the present invention.

[0227] The term "stereoisomer" refers to isomers produced by the different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers, diastereoisomers, and conformational isomers.

[0228] Depending on the choice of starting materials and methods, the compounds of the present invention may exist in the form of one or a mixture of possible isomers, for example as pure enantiomers, or as a mixture of isomers, such as a racemic and diastereoisomer 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 denote the absolute configuration of the molecule with respect to the chiral center(s) in the molecule. The prefixes D and L or (+) and (–) are symbols used to specify the rotation of plane-polarized light caused by the compound, where (–) or L indicates that the compound is levorotatory. Compounds with the prefix (+) or D are dextrorotatory.

[0229] When the bonds to the chiral carbon in the formula of the present invention are depicted as straight lines, it should be understood that both the (R) and (S) configurations of the chiral carbon and thus both the enantiomerically pure compounds and mixtures thereof are included within the scope of this general formula. The graphical representation of racemates or enantiomerically pure compounds herein is from Maehr, J. Chem. Ed. 1985, 62:114 - 120. The absolute configuration of a stereocenter is represented by wedge and dashed bonds.

[0230] The term "tautomer" refers to functional group isomers resulting from the rapid movement of a particular atom within a molecule between two positions. The compounds of the present invention may exhibit tautomerism. Tautomers of a compound may exist in two or more interconvertible forms. Prototropic tautomers result from the migration of a covalently bonded hydrogen atom between two atoms. Tautomers generally exist in an equilibrium form, and attempting to isolate a single tautomer usually results in a mixture whose physical and chemical properties are consistent with those of a mixture of compounds. The position of the equilibrium depends on the chemical characteristics within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; while in phenols, the enol form predominates. The present invention encompasses all tautomeric forms of the compounds.

[0231] In the present application, a "pharmaceutical composition" refers to a preparation of a compound of the present invention with a medium commonly accepted in the art for delivering a bioactive compound to a mammal (such as a human). The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to facilitate administration to an organism, promote absorption of the active ingredient, and thereby exert its biological activity.

[0232] In the present application, "pharmaceutically acceptable carrier" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, or emulsifying agent that has been approved by the relevant government regulatory authorities for use in humans or livestock.

[0233] In the present application, the term "prodrug" refers to a compound that can be converted into the bioactive compound of the present invention under physiological conditions or by solvolysis. The prodrugs of the present invention are prepared by modifying the functional groups in the compound, and the modification can be removed by conventional operations or in vivo to obtain the parent compound. Prodrugs include compounds formed by connecting a hydroxyl group or an amino group in the compound of the present invention to any group. When the prodrug of the compound of the present invention is administered to a mammalian individual, the prodrug is cleaved to form a free hydroxyl group and a free amino group, respectively.

[0234] The compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound. For example, the compounds can be labeled with radioactive isotopes such as deuterium ( 2 H), tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C). All isotopic compositions of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

[0235] In the present application, the term "excipient" refers to a pharmaceutically inert ingredient. Non-limiting examples of the types of "formulants" include binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like. Excipients can enhance the handling characteristics of pharmaceutical formulations, i.e., make the formulations more suitable for direct compression by increasing fluidity and / or adhesiveness.

[0236] As used herein, the terms "treat" and other similar synonyms include the following meanings:

[0237] (i) Prevent the occurrence of a disease or disorder in a mammal, particularly when such a mammal is susceptible to the disease or disorder but has not been diagnosed as having the disease or disorder;

[0238] (ii) Inhibit a disease or disorder, i.e., arrest its development;

[0239] (iii) Alleviate a disease or disorder, i.e., cause the condition of the disease or disorder to subside; or

[0240] (iv) Reduce the symptoms caused by the disease or disorder.

[0241] For the reactions of each step, the reaction temperature can be appropriately selected according to solvents, starting materials, reagents, etc., and the reaction time can also be appropriately selected according to reaction temperature, solvents, starting materials, reagents, etc. After the reaction of each step is completed, the target compound can be separated, purified, etc. from the reaction system by common methods, such as filtration, extraction, recrystallization, washing, silica gel column chromatography and other methods. Without affecting the next reaction, the target compound can also directly enter the next reaction without separation and purification. Each step of the reaction of the present invention is preferably carried out in an inert solvent, and the inert solvents include but are not limited to: toluene, benzene, water, methanol, ethanol, isopropanol, ethylene glycol, N-methylpyrrolidone, dimethyl sulfoxide, tetrahydrofuran, dichloromethane, chloroform, 1,2-dichloroethane, acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide, dioxane, or a combination thereof. Detailed implementation mode

[0242] The following further illustrates the present invention with specific examples. It should be understood that the following description is only the most preferred implementation mode of the present invention and should not be considered as a limitation on the protection scope of the present invention. On the basis of fully understanding the present invention, for the experimental methods without specific conditions noted in the following examples, they are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. Those skilled in the art can make non-essential changes to the technical solutions of the present invention, and such changes should be regarded as being included in the protection scope of the present invention.

[0243] Example 1: Preparation of Compound I-1

[0244] The synthesis route is as follows:

[0245]

[0246] First step: Synthesis of methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01B)

[0247]

[0248] To a 100 mL round-bottom flask, add methyl (S)-3-(3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (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). After the mixture is sonicated, nitrogen is displaced three times and the mixture is stirred at 100 °C. After monitoring by TLC that the raw materials are completely converted, the reaction solution is filtered and the filtrate is concentrated under reduced pressure to obtain a crude product. The crude product is purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 3:1) to obtain methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01B) (360 mg, yield 29.7%).

[0249] LC-MS, M / Z(ESI): 360.2[M+H] + .

[0250] Step 2: Synthesis of methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (01C)

[0251]

[0252] Dissolve methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (360 mg, 1.00 mmol) in a 1,4-dioxane solution of hydrogen chloride (4 M, 5 mL) at room temperature and stir at room temperature. After monitoring by TLC that the raw materials are completely converted, the reaction solution is concentrated under reduced pressure to obtain methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (01C) (296 mg).

[0253] LC-MS, M / Z(ESI): 260.1[M+H] + .

[0254] Step 3: Synthesis of methyl (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (01E)

[0255]

[0256] At room temperature, dissolve compound ((4-methylpyridin-2-ylamino)butanoyl)glycine (252 mg, 1.00 mmol) in anhydrous DMF (5 mL), add tetramethylurea tetrafluoroborate (TBTU, 385 mg, 1.20 mmol), and add N,N-diisopropylethylamine (DIPEA, 1.48 g, 1.15 mmol) at 0 °C. After returning to room temperature and stirring for half an hour, add compound methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (296 mg, 1.00 mmol), and stir the reaction at room temperature. After monitoring the completion of the reaction by TLC, add saturated NH4Cl solution (10 mL) to the reaction solution, extract with ethyl acetate (10 mL × 3), combine the organic phases and wash once with saturated NaHCO3 solution (10 mL). Separate the organic phase, dry it over anhydrous sodium sulfate, filter and concentrate to obtain compound methyl (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (01E) (300 mg, yield 60.7%).

[0257] LC-MS, M / Z (ESI): 493.3 [M+H] + .

[0258] Step 4: Synthesis of (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionic acid (I-1)

[0259]

[0260] At room temperature, dissolve methyl (S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (320 mg, 0.540 mmol) in THF / H2O (6 mL, v:v = 1:1), and then add LiOH·H2O (68.0 mg, 1.62 mmol). The mixture was stirred at room temperature for 3 hours and concentrated under reduced pressure. To the residue, add a 1,4-dioxane solution of hydrogen chloride (4 M, 2.7 mL) and stir for half an hour. After monitoring the completion of the reaction by TLC, the organic solvent was removed by concentration under reduced pressure. The residue was purified by reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm, mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 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)propionic acid (I-1) (210 mg, yield 81.3%).

[0261] LC-MS, M / Z(ESI): 479.2[M+H] + .

[0262] 1 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.4 Hz, 1H), 8.08 (t, J = 6.0 Hz, 1H), 7.77 (d, J = 5.2 Hz, 1H), 7.44 (d, J = 2.0 Hz, 2H), 7.42–7.32 (m, 3H), 6.37 (d, J = 2.0 Hz, 2H), 6.26 (d, J = 5.2 Hz, 1H), 6.22 (s, 1H), 5.22 (q, J = 7.2 Hz, 1H), 3.82 (s, 3H), 3.68 (d, J = 6.0 Hz, 2H), 3.15 (q, J = 6.8 Hz, 2H), 2.73 (d, J = 7.2 Hz, 2H), 2.16 (t, J = 7.2 Hz, 2H), 2.10 (s, 3H), 1.70 (quint, J = 7.2 Hz, 2H).

[0263] Example 2: Preparation of Compound I-2

[0264] The synthetic route is as follows:

[0265]

[0266] First step: Synthesis of methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04C)

[0267]

[0268] To a 100 mL single-necked flask were successively added methyl (S)-3-((tert-butoxycarbonyl)amino)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (01B, 600 mg, 1.67 mmol), dichloromethane (20 mL), and trifluoroacetic acid (3.81 g, 33.4 mmol). The mixture was stirred at room temperature for 4 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was dissolved in dichloromethane (20 mL), washed successively with saturated sodium bicarbonate (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04C) (430 mg, yield 99.3%).

[0269] LC-MS, M / Z(ESI): 260.13 [M+H] + 。

[0270] Step 2: Synthesis of methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04D)

[0271]

[0272] To a 100 mL single-necked flask were successively added methyl (S)-3-amino-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (400 mg, 1.54 mmol), DMF (10 mL), BOC-glycine (298 mg, 1.70 mmol), TBTU (740 mg, 2.30 mmol), and DIPEA (598 mg, 4.63 mmol). Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (10 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic phases were washed successively with saturated sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate (V / V)=1:0 - 0:1) to obtain methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04D) (600 mg, yield 93.4%).

[0273] LC-MS, M / Z(ESI): 417.27 [M+H] + 。

[0274] Step 3: Synthesis of Methyl (S)-3-(2-Aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04E)

[0275]

[0276] To a 100 mL single-necked flask, add successively methyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (600 mg, 1.44 mmol), dichloromethane (10 mL), and trifluoroacetic acid (3.29 g, 28.9 mmol). Stir at room temperature for 4 h. Concentrate the reaction solution under reduced pressure to obtain a crude product. Dissolve it in dichloromethane (10 mL), wash it successively with saturated sodium bicarbonate solution (10 mL) and saturated brine (10 mL), dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (04E) (460 mg, yield about 100%).

[0277] LC-MS, M / Z(ESI): 317.32 [M+H] + 。

[0278] Step 4: Synthesis of Methyl 3-((4-Methylpyridin-2-yl)amino)cyclobutane-1-carboxylate (04G)

[0279]

[0280] To a 100 mL single-necked flask, add successively 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 triacetoxyborohydride (11.8 g, 55.7 mmol). Stir at room temperature for 12 h, then add sodium cyanoborohydride (3.50 g, 55.7 mmol) and stir at room temperature for 6 h. Concentrate the reaction solution under reduced pressure to obtain a crude product. Add ethyl acetate (100 mL) and dilute hydrochloric acid (1N, 100 mL), stir and separate the layers. Add ethyl acetate (100 mL×2) to the aqueous phase and wash twice. Adjust the pH of the obtained aqueous phase to 10 with 1N aqueous sodium hydroxide solution, then extract three times with ethyl acetate (100 mL×3). Combine the organic phases, dry over anhydrous sodium sulfate, filter and concentrate to obtain a crude product. Purify by column chromatography to obtain methyl 3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carboxylate (04G) (300 mg, yield 7.36%).

[0281] Step 5: Synthesis of 3-[(4-Methylpyridin-2-yl)amino]cyclobutane-1-carboxylic Acid (04H)

[0282]

[0283] 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 successively added to a 100 mL single-necked flask and stirred at room temperature for 12 h. The mixture was concentrated to obtain the crude product of 3-[(4-methylpyridin-2-yl)amino]cyclobutane-1-carboxylic acid (04H), which was directly used in the next reaction.

[0284] Step 6: Synthesis of methyl (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (04I)

[0285]

[0286] 3-((4-Methylpyridin-2-yl)amino)cyclobutane-1-carboxylic acid (04H), dichloromethane (10 mL), methyl (S)-3-(2-aminoacetamido)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (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) were successively added to a 100 mL single-necked flask and stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure to obtain the crude product, and ethyl acetate and water were added and stirred. After liquid separation, the organic phase was collected, and the aqueous phase was extracted twice with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The product methyl (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (04I) (200 mg, two-step yield 29.1%) was obtained by column chromatography purification.

[0287] Step 7: Synthesis of methyl (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (I-2)

[0288]

[0289] To a 100 mL single-necked flask, (3S)-3-(3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)-methyl propionate (200 mg, 0.396 mmol), tetrahydrofuran (5 mL), water (1 mL), and lithium hydroxide (19.0 mg, 0.793 mmol) were added successively, 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 purified by reverse-phase preparation 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, yield 49%).

[0290] LC-MS, M / Z(ESI): 491.2[M+H] + 。

[0291] Example 3: Preparation of Compounds II-1 & II-1A & II-1B

[0292] The synthetic route is as follows:

[0293]

[0294]

[0295] First step: Synthesis of ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (02B)

[0296]

[0297] To a 500 mL single-necked flask, ethyl 3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate (14.00 g, 37.01 mmol), dichloromethane (150 mL), and di-tert-butyl dicarbonate (12.12 g, 55.53 mmol) were added successively, and the mixture was 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 a crude product. The product ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (02B) (14.00 g, yield 79.07%) was obtained by column chromatography purification (petroleum ether / ethyl acetate (V / V) = 1:0 - 1:1).

[0298] LC-MS, M / Z(ESI): 478.12, 480.11[M+H] + 。

[0299] Step 2: Synthesis of Ethyl 3-(4-(Benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (02C)

[0300]

[0301] Add ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (14.00 g, 29.27 mmol), 1-methyl-1H-pyrazole-5-boronic acid pinacol ester (12.18 g, 58.54 mmol), Pd2(dba)3 (1.07 g, 1.17 mmol), tricyclohexylphosphine (657 mg, 2.34 mmol), dipotassium hydrogen phosphate (10.20 g, 58.56 mmol), 1,4-dioxane (100 mL) and water (30 mL) into a 250 mL single-necked flask in sequence, and stir at 100 °C for 16 h. After cooling to room temperature, filter and concentrate the filtrate to obtain the crude product. Purify by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 1:1) to obtain the product ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (02C) (14.00 g, yield 99.75%). LC-MS, M / Z(ESI): 480.24 [M+H] + 。

[0302] Step 3: Synthesis of Ethyl 3-Amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate Hydrochloride (02D)

[0303]

[0304] Add ethyl 3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (8.00 g, 16.7 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 41.7 mL) into a 250 mL single-necked flask in sequence, and stir at room temperature for 2 h. Concentrate the reaction solution under reduced pressure to obtain the product ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (02D) (6.00 g, yield 86.6%).

[0305] LC-MS, M / Z(ESI): 380.19 [M+H] + 。

[0306] Step 4: Synthesis of Ethyl 14-(4-(Benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxa-3-oxa-5,10,13-triazapentadecane-16-carboxylate (02F)

[0307]

[0308] Ethyl 3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate 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) were successively added to a 250 mL single-necked flask. Under nitrogen protection, the mixture was stirred at room temperature for 16 h. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (200 mL × 3). The combined organic phases were successively washed with saturated sodium bicarbonate solution (100 mL) and saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1) gave ethyl 14-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxa-3-oxa-5,10,13-triazapentadecane-16-carboxylate (02F) (6.00 g, yield 87.5%). LC-MS, M / Z (ESI): 713.36 [M+H] + 。

[0309] Step 5: Synthesis of Ethyl 14-(4-Hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-2,2-dimethyl-5-(4-methylpyridin-2-yl)-4,9,12-trioxa-3-oxa-5,10,13-triazapentadecane-16-carboxylate (02G)

[0310]

[0311] Under nitrogen protection, 1-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-triazapentadecane-16-carboxylate (6.00 g, 8.42 mmol), methanol (100 mL) and 10% Pd / C (600 mg) were successively added to a 500 mL single-necked flask. The single-necked flask was replaced with a hydrogen atmosphere and stirred at room temperature for 16 h. The reaction mixture was filtered through diatomaceous earth, the filter cake was washed with methanol, and the combined filtrates were concentrated under reduced pressure to obtain 1-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-triazapentadecane-16-carboxylate (02G) (5.00 g, yield 95.4%). LC-MS, M / Z (ESI): 623.31 [M+H] + .

[0312] Step 6: Synthesis of 1-ethyl 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-triazapentadecane-16-carboxylate (02H)

[0313]

[0314] 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-triazaoctadec-16-oate (1.50 g, 2.41 mmol), DMF (15 mL), potassium carbonate (670 mg, 4.85 mmol) and azido-pentaethylene glycol-p-toluenesulfonate (1.51 g, 3.62 mmol) were successively added to a 250 mL single-necked flask, and the mixture was stirred at 80 °C for 16 h. After the reaction was completed, water (15 mL) was added, and the mixture was extracted with ethyl acetate (30 mL × 3). The combined organic phases were washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain a crude product. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1) gave ethyl 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-triazaoctadec-16-oate (02H) (1.80 g, yield 86.1%).

[0315] LC-MS, M / Z(ESI): 868.45[M+H] + 。

[0316] 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-triazaoctadec-16-oic acid (02I)

[0317]

[0318] To a 100 mL single-necked flask, ethyl 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-triazapentadec-16-oate (1.80 g, 2.07 mmol), THF (20 mL), and an aqueous solution of lithium hydroxide (149 mg, 6.22 mmol) in water (10 mL) were added successively, and the mixture was stirred at room temperature for 16 h. The reaction solution was concentrated under reduced pressure to remove THF, water (10 mL) and 1N hydrochloric acid were added to adjust the pH to 7, and the product ethyl 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-triazapentadec-16-oate (02I) (1.74 g, yield 99.9%) was obtained by concentration under reduced pressure.

[0319] LC-MS, M / Z (ESI): 840.42 [M+H] + 。

[0320] 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)butanamido)acetamido)propanoic acid (II-1)

[0321]

[0322] 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-triazahexadec-16-oic acid (1.74 g, 2.07 mmol) and a 1,4-dioxane solution of hydrogen chloride (4 M, 5.18 mL) were successively added to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain a crude product, and 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, yield 42.4%) was obtained by reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm, mobile phase: A = 0.1% ammonia water, B = acetonitrile; gradient: 30% - 100%).

[0323] 1 H NMR (400 MHz, DMSO-d6) δ 9.82 (d, J = 5.6 Hz, 1H), 8.29 (t, J = 6.0 Hz, 1H), 7.75 (d, J = 5.2 Hz, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.30 (dd, J = 8.6, 2.2 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 6.98 (d, J = 8.6 Hz, 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).

[0324] LC-MS, M / Z (ESI): 740.7 [M+H] + 。

[0325] 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)

[0326]

[0327] 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 resolved by SFC (column: (S,S)WHELK-O1 (250 mm × 30 mm, 10 μm, mobile phase: A = CO2, B = isopropanol + acetonitrile (0.1% ammonia water); gradient: 75%) to obtain (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).

[0328] II-1A: 11H NMR (400 MHz, DMSO-d6) δ 9.82 (d, J = 5.6 Hz, 1H), 8.30 (t, J = 6.0 Hz, 1H), 7.75 (d, J = 5.2 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 7.30 (dd, J = 8.6, 2.2 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 6.99 (d, J = 8.6 Hz, 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).

[0329] LC-MS, M / Z (ESI): 740.5 [M+H] + 。

[0330] II-1B: 1 1H NMR (400 MHz, DMSO-d6) δ 9.83 (d, J = 5.6 Hz, 1H), 8.29 (t, J = 6.0 Hz, 1H), 7.76 (d, J = 5.2 Hz, 1H), 7.37 (d, J = 1.8 Hz, 1H), 7.31 (dd, J = 8.6, 2.2 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 6.98 (d, J = 8.6 Hz, 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).

[0331] LC-MS, M / Z (ESI): 740.5 [M+H] + 。

[0332] Example 4: Preparation of Compounds II-2 & II-2A & II-2B

[0333] The synthetic route is as follows:

[0334]

[0335] Step 1: Synthesis of Ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propionate (03B)

[0336]

[0337] Add compound 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) into a 100 mL round-bottom flask, and stir the reaction at 80 °C. After monitoring by TLC that the raw materials are completely converted, concentrate the reaction solution under reduced pressure to obtain compound ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propionate (03B) (1.56 g, yield 40.0%).

[0338] LC-MS, M / Z (ESI): 378.1, 380.1 [M+H] + 。

[0339] Step 2: Synthesis of Ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (03C)

[0340]

[0341] At room temperature, dissolve compound ethyl 3-amino-3-(3-(benzyloxy)-5-bromophenyl)propionate (1.56 g, 4.12 mmol) in water / 1,4-dioxane (40 mL, v:v = 1:1), add triethylamine (1.25 g, 12.4 mmol) and di-tert-butyl dicarbonate (1.08 g, 4.95 mmol), and stir the reaction at room temperature. After monitoring by TLC that the raw materials are completely converted, concentrate under reduced pressure to remove the organic solvent, and extract with ethyl acetate (20 mL×3). The combined organic phases are washed once with saturated NaCl solution (20 mL), dried over anhydrous sodium sulfate, filtered by suction, concentrated under reduced pressure, and the residue is purified by column chromatography (petroleum ether / ethyl acetate (V / V) = 85:15) to obtain compound ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (03C) (1.82 g, yield 92.3%).

[0342] LC-MS, M / Z (ESI): 478.1, 480.1 [M+H] + 。

[0343] Step 3: Synthesis of Ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (03D)

[0344]

[0345] To a 100 mL round-bottom flask, add ethyl 3-(3-(benzyloxy)-5-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (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 oscillation, replace nitrogen three times and stir the reaction at 100 °C. After monitoring by TLC that the raw materials are completely converted, filter the reaction solution, concentrate the filtrate under reduced pressure, and purify the residue by column chromatography (petroleum ether / ethyl acetate (V / V) = 7:3) to obtain ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (03D) (1.82 g, yield 99.8%).

[0346] LC-MS, M / Z(ESI): 480.2[M+H] + 。

[0347] Step 4: Synthesis of ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (03E)

[0348]

[0349] At room temperature, dissolve ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (4.54 g, 9.47 mmol) in a 1,4-dioxane solution of hydrogen chloride (4 M, 47.5 mL) and stir for half an hour. After monitoring by TLC that the raw materials are completely converted, concentrate under reduced pressure to obtain the crude product. Add ethyl acetate for washing, filter by suction, and collect the filter cake to obtain ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (03E) (3.95 g, yield 100%).

[0350] LC-MS, M / Z(ESI): 380.2[M+H] + 。

[0351] Step 5: Synthesis of Ethyl 3-(3-(Benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (03F)

[0352]

[0353] At room temperature, ethyl 3-amino-3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (3.95 g, 9.50 mmol) was dissolved in anhydrous DMF (50 mL), and then TBTU (3.66 g, 11.4 mmol) was added. DIPEA (3.56 g, 27.5 mmol) was added at 0 °C, and after returning to room temperature and stirring for half an hour, ((4-methylpyridin-2-ylamino)butanoyl)glycine (2.40 g, 9.55 mmol) was added, and the reaction was stirred at room temperature. After the reaction was completed monitored by TLC, saturated NH4Cl solution (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed once with saturated NaHCO3 solution (50 mL), dried over anhydrous sodium sulfate, filtered by suction, and concentrated under reduced pressure. The residue was purified by column chromatography (dichloromethane / methanol (V / V) = 9:1) to obtain ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (03F) (4.00 g, yield 68.7%).

[0354] LC-MS, M / Z (ESI): 613.3 [M+H] + 。

[0355] Step 6: Synthesis of Ethyl 3-(3-Hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (03G)

[0356]

[0357] At room temperature, dissolve ethyl 3-(3-(benzyloxy)-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (4.00 g, 6.53 mmol) in methanol (10 mL), add 10% wet palladium on carbon (600 mg), replace hydrogen three times, and then stir the reaction under a hydrogen atmosphere at room temperature. After monitoring by TLC that the raw material was completely converted, filter the reaction solution by suction, and concentrate the filtrate under reduced pressure to obtain ethyl 3-(3-hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (03G) (3.40 g, yield 99.7%).

[0358] LC-MS, M / Z(ESI): 523.3[M+H] + 。

[0359] 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)butanamido)acetamido)propionate (03H)

[0360]

[0361] At room temperature, dissolve ethyl 3-(3-hydroxy-5-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propionate (146 mg, 0.279 mmol) in anhydrous DMF (5 mL), add potassium carbonate (97 mg, 0.70 mmol) and azido-pentaethylene glycol-p-toluenesulfonate (152 mg, 0.364 mmol), and stir at 80 °C for 6 hours. After monitoring by TLC that the raw material was completely converted, cool to room temperature, add saturated NaHCO3 solution (10 mL), and extract with ethyl acetate (10 mL×3). Combine the organic phases, wash once with saturated NaCl solution (20 mL), dry over anhydrous sodium sulfate, filter by suction, and concentrate under reduced pressure to obtain 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)propionate (03H) (200 mg, yield 93.2%).

[0362] LC-MS, M / Z(ESI): 768.4[M+H] + 。

[0363] 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)butanamido)acetamido)propanoic acid (II-2)

[0364]

[0365] 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, and LiOH·H2O (33 mg, 0.79 mmol) was added. The mixture was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and a 1,4-dioxane solution of hydrogen chloride (4 M, 1.3 mL) was added and stirred for an additional half hour. After monitoring the completion of the reaction by TLC, the organic solvents were removed by concentration under reduced pressure. The residue was purified by reverse-phase 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 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, yield 36%).

[0366] LC-MS, M / Z (ESI): 740.8 [M+H] + 。

[0367] 11H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.4 Hz, 1H), 8.11 (t, J = 6.0 Hz, 1H), 7.79 (d, J = 5.2 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.02 (s, 1H), 6.97–6.91 (m, 2H), 6.40 (d, J = 2.0 Hz, 2H), 6.29 (d, J = 5.2 Hz, 1H), 6.24 (s, 1H), 5.21 (q, J = 7.2 Hz, 1H), 4.17–4.11 (m, 2H), 3.84 (s, 3H), 3.77–3.73 (m, 2H), 3.70 (d, J = 6.0 Hz, 2H), 3.58 (dt, J = 5.2, 3.2 Hz, 4H), 3.56–3.50 (m, 10H), 3.39–3.35 (m, 2H), 3.17 (q, J = 6.8 Hz, 2H), 2.73 (d, J = 7.2 Hz, 2H), 2.19 (t, J = 7.2 Hz, 2H), 2.12 (s, 3H), 1.72 (quint, J = 7.2 Hz, 2H).

[0368] 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)butanamido)acetamido)propanoic acid (II-2A)

[0369]

[0370] (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)

[0371]

[0372] 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)propionic acid was resolved by SFC (column: (S,S)WHELK-O1 (250 mm×30 mm, 10 μm), mobile phase: mobile phase: A = CO2, B = isopropanol + acetonitrile (0.1% ammonia water); gradient: 75%) to obtain 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)propionic acid (II-2A, 24 mg; column: (S,S)Whelk-O1 50×4.6 mm I.D., 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)propionic acid (II-2B, 25 mg; column: (S,S)Whelk-O1 50×4.6 mm I.D., 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).

[0373] II-2A: 1 H 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).

[0374] II-2B: 11H NMR (400 MHz, CDCl3) δ 10.15 (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), 4.25–4.10 (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).

[0375] Example 5: Preparation of Compounds II-3A & II-3B

[0376] The synthetic route is as follows:

[0377]

[0378] First step: Synthesis of (R,E)-N-(4-(benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfinamide (05B)

[0379] To a 100 mL single-necked flask, 4-(benzyloxy)-3-bromobenzaldehyde (5.00 g, 17.2 mmol), 2-methyltetrahydrofuran (10 mL), (R)-(+)-tert-butylsulfinamide (2.19 g, 18.1 mmol) and cesium carbonate (8.39 g, 25.8 mmol) were added successively. Under nitrogen protection, the mixture was stirred at room temperature for 16 h. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The product (R,E)-N-(4-(benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfinamide (05B) (5.80 g, yield 85.6%) was obtained by column chromatography purification (petroleum ether / ethyl acetate (V / V) = 1:0 - 1:1).

[0380] LC-MS, M / Z (ESI): 394.3, 396.3 [M+H] + .

[0381] Second step: Synthesis of (S)-ethyl 3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propionate (05C)

[0382] To a 250 mL three-necked flask, zinc powder (8.29 g, 127 mmol) and tetrahydrofuran (50 mL) were added successively. Under nitrogen protection, trimethylchlorosilane (2.76 g, 25.4 mmol) was added dropwise, and the temperature was raised to 60 °C and stirred for 10 min. The reaction solution 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 temperature was raised to 60 °C and stirred for 2 h. The reaction solution was quickly cooled to 0 °C, and (R,E)-N-(4-(benzyloxy)-3-bromobenzylidene)-2-methylpropane-2-sulfinamide (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 and slowly warmed to room temperature and stirred for 10 h. The reaction was quenched by adding aqueous citric acid solution, and extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine (50 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 (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propionate (05C) (3.80 g, yield 62.1%).

[0383] 1 H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J = 1.9 Hz, 1H), 7.47 (d, J = 7.1 Hz, 2H), 7.40 (t, J = 7.3 Hz, 2H), 7.36–7.28 (m, 2H), 7.14 (d, J = 8.6 Hz, 1H), 5.56 (d, J = 5.9 Hz, 1H), 5.19 (s, 2H), 4.57 (q, J = 6.7 Hz, 1H), 3.99 (q, J = 7.1 Hz, 2H), 2.98 (dd, J = 15.4, 6.6 Hz, 1H), 2.73 (dd, J = 15.4, 7.9 Hz, 1H), 1.11–1.07 (m, 12H).

[0384] LC-MS, M / Z (ESI): 482.2, 484.2 [M+H] + 。

[0385] Step 3: Synthesis of ethyl (S)-3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate hydrochloride (05D)

[0386] Ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-(((R)-tert-butylsulfinyl)amino)propionate (3.80 g, 7.88 mmol), ethanol (10 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 3.94 mL) were successively added to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain ethyl (S)-3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate hydrochloride (05D), which was directly used in the next step.

[0387] LC-MS, M / Z (ESI): 378.2, 380.2 [M+H] + 。

[0388] Step 4: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (05E)

[0389] Ethyl (S)-3-amino-3-(4-(benzyloxy)-3-bromophenyl)propionate 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 successively added to a 100 mL single-necked flask, and the mixture was stirred at room temperature for 3 h. Water (10 mL) was added, and the organic phase was separated and concentrated under reduced pressure to obtain a crude product. The product ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (05E) (2.40 g, two-step yield 63.7%) was obtained by column chromatography purification (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1).

[0390] LC-MS, M / Z (ESI): 478.2, 480.2 [M+H] + 。

[0391] Step 5: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (05F)

[0392] To a 100 mL single-necked flask, ethyl (S)-3-(4-(benzyloxy)-3-bromophenyl)-3-((tert-butoxycarbonyl)amino)propionate (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 successively, and the mixture was stirred at 100 °C for 16 h. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. The product ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (05F) (2.30 g, yield 95.6%) was obtained by column chromatography purification (petroleum ether / ethyl acetate (V / V) = 1:0 - 1:1).

[0393] LC-MS, M / Z(ESI): 480.37[M+H] + 。

[0394] Step 6: Synthesis of ethyl (S)-3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05G)

[0395] To a 100 mL single-necked flask, ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((tert-butoxycarbonyl)amino)propionate (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 successively, and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to obtain the product ethyl (S)-3-amino-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05G) (2.0 g, yield 100%).

[0396] LC-MS, M / Z(ESI): 380.2[M+H] + 。

[0397] Step 7: Synthesis of ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate (05H)

[0398] Ethyl (S)-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 successively added to a 100 mL single-necked flask. Under nitrogen protection, 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×3). The combined organic phases were successively washed 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 ethyl (S)-3-(4-(benzyloxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoate (05H) (2.5 g, yield 97%).

[0399] LC-MS, M / Z(ESI): 537.3[M+H] + 。

[0400] Step 8: Synthesis of ethyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (05I)

[0401] 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) were successively added to a 100 mL single-necked flask under nitrogen protection. The reaction was replaced with a hydrogen atmosphere and stirred at room temperature for 16 h. The reaction solution was filtered through diatomaceous earth, the filter cake was washed with methanol and filtered, and the combined filtrates were concentrated under reduced pressure to obtain ethyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propanoate (05I) (2.05 g, yield 99%).

[0402] LC-MS, M / Z(ESI): 447.3[M+H] + 。

[0403] Step 9: Synthesis of ethyl (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propanoate (05J)

[0404] Ethyl (S)-3-(2-((tert-butoxycarbonyl)amino)acetamido)-3-(4-hydroxy-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate (2.05 g, 4.59 mmol), DMF (20 mL), potassium carbonate (1.59 g, 11.5 mmol), and azido-pentaethylene glycol p-toluenesulfonate (2.11 g, 5.05 mmol) were successively added to a 100 mL single-necked flask and stirred at 80 °C for 16 h. Water (20 mL) was added, and the mixture was extracted with ethyl acetate (40 mL × 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. Purification by column chromatography (petroleum ether / ethyl acetate (V / V) = 1:0 - 0:1) gave ethyl (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate (05J) (2.50 g, yield 78.7%).

[0405] LC-MS, M / Z(ESI): 692.47 [M+H] + 。

[0406] Step 10: Synthesis of ethyl (S)-3-(2-aminoacetamido)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05K)

[0407] Ethyl (S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-(2-((tert-butoxycarbonyl)amino)acetamido)propionate (2.50 g, 3.61 mmol), ethanol (10 mL), and a 1,4-dioxane solution containing hydrogen chloride (4 M, 3.61 mL) were successively added to a 100 mL single-necked flask and stirred at room temperature for 2 h. The reaction solution was concentrated under reduced pressure to obtain ethyl (S)-3-(2-aminoacetamido)-3-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (05K) (2.20 g, yield 96.9%).

[0408] LC-MS, M / Z(ESI): 592.41 [M+H] + 。

[0409] Step 11: Synthesis of ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (05L)

[0410] To a 100 mL single-necked flask, add successively 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-tetraoxatetradecyl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)propionate hydrochloride (1.83 g, 2.91 mmol), TBTU (2.34 g, 7.29 mmol) and DIPEA (940 mg, 7.27 mmol), and stir at room temperature for 16 h. Add water (20 mL) to the reaction mixture, and extract with ethyl acetate (20 mL × 3). The combined organic phases are washed successively with saturated aqueous sodium bicarbonate solution (20 mL) and saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered and concentrated to obtain the crude product. Purification by column chromatography (dichloromethane / methanol (V / V) = 90:10) gives ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (05L) (600 mg, yield 31.7%).

[0411] LC-MS, M / Z (ESI): 780.4 [M+H] + 。

[0412] Step 12: Synthesis of (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionic acid (II-3)

[0413] Ethyl (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionate (600 mg, 0.769 mmol), THF / H2O (v:v = 1:1, 2 mL), and lithium hydroxide monohydrate (97 mg, 2.3 mmol) were successively added to a 100 mL single-necked flask and stirred at room temperature for 3 h. THF was removed by concentration under reduced pressure, and the pH was adjusted to 6 with 1 N hydrochloric acid and concentrated under reduced pressure to obtain the crude product. After reverse-phase preparation (column: YMC-Triart Prep C18 (30 mm × 40 cm, 7 μm); mobile phase: A = 0.1% formic acid, B = acetonitrile; gradient: 10%–95%), (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-1-yl)oxy)-3-(1-methyl-1H-pyrazol-5-yl)phenyl)-3-((N-(3-((4-methylpyridin-2-yl)amino)cyclobutane-1-carbonyl)glycyl)amino)propionic acid (II-3) (417 mg, yield 72.1%) was obtained.

[0414] LC-MS, M / Z (ESI): 752.4 [M+H] + 。

[0415] Step 11: Synthesis of (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-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)propionic acid (II-3A) and (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-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)propionic acid (II-3B)

[0416]

[0417] (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-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 resolved 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%) to give the compound: (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-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 the compound: (3S)-3-(4-((14-azido-3,6,9,12-tetraoxatetradec-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 = CO2, B = 60% ethanol + acetonitrile (0.05% ethylenediamine); gradient: 30–60%; flow rate: 3 mL / min; retention time: 0.895 min).

[0418] II-3A: 11H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 8.4 Hz, 1H), 7.97 (t, J = 6.0 Hz, 1H), 7.79 (d, J = 5.2 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.34–7.30 (m, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.62 (d, J = 7.2 Hz, 1H), 6.31–6.27 (m, 1H), 6.22 (d, J = 2.0 Hz, 1H), 6.15 (s, 1H), 5.15 (q, J = 7.2 Hz, 1H), 4.34–4.24 (m, 1H), 4.14–4.04 (m, 2H), 3.65 (d, J = 12.0 Hz, 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).

[0419] II-3B: 1 1H NMR (400 MHz, DMSO-d6) δ 8.36 (d, J = 8.4 Hz, 1H), 7.95 (t, J = 6.0 Hz, 1H), 7.76 (d, J = 5.2 Hz, 1H), 7.39 (d, J = 2.0 Hz, 1H), 7.34–7.30 (m, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 6.31–6.27 (m, 1H), 6.22 (d, J = 2.0 Hz, 1H), 6.18 (s, 1H), 5.14 (q, J = 7.4 Hz, 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).

[0420] Example 6: Preparation of Compound III-1

[0421] The synthetic route is as follows:

[0422]

[0423] 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)

[0424] Add 4-amino-4-(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-N1,N7-bis(2-(2-((prop-2-yn-1-yl)oxy)methoxy)ethyl)heptanediamide (500 mg, 0.803 mmol), DCM (5 mL), triethylamine (0.5 mL) and dihydro-4,4-dimethyl-2H-pyranyl-2,6(3H)-dione (137 mg, 0.964 mmol) to a 100 mL single-necked flask in sequence, and stir at room temperature for 3 h. Concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diaza-1-yl-20-oic acid (500 mg, yield 81.4%).

[0425] 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)

[0426] Add 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diaza-1-yl-20-oic acid (500 mg, 0.654 mmol), DCM (5 mL), 4-nitrophenol (109 mg, 0.784 mmol) and EDCI (150 mg, 0.782 mmol) to a 100 mL single-necked flask in sequence, and stir at room temperature for 12 h. Concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 18,18-dimethyl-11,16-dioxo-14,14-bis(3-oxo-3-((2-(2-((prop-2-yn-1-yl)oxy)ethoxy)ethyl)amino)propyl)-4,7-dioxo-10,15-diaza-1-yl-20-carboxylic acid, 4-nitrophenyl ester (III-1) (268 mg, yield 46.3%).

[0427] 1 1H NMR (400 MHz, DMSO-d6) δ 8.27 (d, 2H), 7.81 (t, 3H), 7.41 (d, 2H), 7.21 (s, 1H), 4.10 (d, 6H), 3.51–3.49 (m, 12H), 3.38–3.36 (m, 9H), 3.16–3.13 (m, 6H), 2.73 (s, 2H), 2.17 (s, 2H), 2.01–1.97 (m, 6H), 1.80–1.76 (d, 6H), 1.09 (s, 6H).

[0428] LC-MS, M / Z (ESI): 887.3 [M+H] + 。

[0429] Example 7: Preparation of Compound III-2

[0430] The synthetic route is as follows:

[0431]

[0432] Step 1: Synthesis of (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azapentadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriaconta-1,32-diyne-17-yl)carbamate (07B)

[0433] To a 100 mL single-necked flask were successively added 4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(3-oxo-3-(perfluorophenoxy)propyl)heptanedioic acid bis(perfluorophenyl) ester (3.0 g, 3.1 mmol), DCM (30 mL), and 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-amine (2.1 g, 11 mmol). The mixture was stirred at room temperature for 3 h. The crude product was obtained by concentration under reduced pressure and purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to give (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azapentadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriaconta-1,32-diyne-17-yl)carbamate (3.0 g, yield 99%).

[0434] Step 2: Synthesis of 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (07C)

[0435] Into a 100 mL single-necked flask, (9H-fluoren-9-yl)methyl (14,20-dioxo-17-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10,24,27,30-hexaoxa-13,21-diazadotriaconta-1,32-diyne-17-yl)carbamate (3.0 g, 3.1 mmol), DMF (30 mL) and triethylamine (20 mL) were added successively, and the mixture was stirred at room temperature for 36 h. The mixture was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 95:5) to obtain 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (2.0 g, yield 86%).

[0436] Step 3: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-enoic acid (07D)

[0437] Into a 100 mL single-necked flask, 4-amino-4-(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-N1,N7-bis(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethyl)heptanediamide (2.0 g, 2.6 mmol), DCM (20 mL), triethylamine (2 mL) and 4,4-dimethyldihydro-2H-pyran-2,6(3H)-dione (753 mg, 5.30 mmol) were added successively, and the mixture was stirred at room temperature for 3 h. The mixture was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (dichloromethane / methanol (V / V) = 92:8) to obtain 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-enoic acid (2.0 g, yield 84%).

[0438] Step 4: Synthesis of 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid pentafluorophenyl ester (III-2)

[0439] Add 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) into a 100 mL single-necked flask in sequence, and stir at room temperature for 12 h. Concentrate under reduced pressure to obtain the crude product, and purify it by column chromatography (ethyl acetate / acetonitrile (V / V) = 1:1) to obtain 21,21-dimethyl-14,19-dioxo-17,17-bis(3-oxo-7,10,13-trioxa-4-azahexadec-15-yn-1-yl)-4,7,10-trioxa-13,18-diaza-1-yn-23-oleic acid pentafluorophenyl ester (III-2) (110 mg, yield 18.6%).

[0440] 1 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H), 6.59 (t, 3H), 4.2 (d, 6H), 3.51–3.49 (m, 12H), 3.70–3.69 (m, 12H), 3.64–3.62 (m, 12H), 3.58–3.54 (m, 6H), 3.38–3.36 (m, 6H), 3.0 (q, 3H), 2.83 (s, 2H), 2.48 (s, 2H), 2.25–2.22 (m, 6H), 2.06–2.04 (d, 6H), 1.18 (s, 6H).

[0441] Preparation Example 1: Design and synthesis of siRNA conjugate

[0442] The synthesis steps of the siRNA conjugate in which the conjugating group is linked to the 5'-end of the sense strand of siRNA are exemplified by C1 in Table 1, and the specific steps are as follows:

[0443] 1) Synthesis of single-stranded oligoribonucleotide: Synthesize oligoribonucleotide according to the phosphoramidite solid-phase synthesis technique. On the general controlled pore glass CPG Synthesis was carried out thereon. All 2'-modified nucleotide phosphoramidites and auxiliary reagents were commercially available reagents (Shanghai Zhaowei Technology Development Co., Ltd.). All phosphoramidites were dissolved in anhydrous acetonitrile and molecular sieves were added. The coupling time using 5-ethylthio-1H-tetrazole (ETT, Suzhou Kelema Biotechnology Co., Ltd.) as the activator was 5 minutes. 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) was used to generate the phosphorothioate bond, and the reaction time was 3 minutes. All sequences were completed after the DMT group was finally removed.

[0444] 2) Cleavage and deprotection of the oligoribonucleotide bound to CPG: After the solid-phase synthesis was terminated, the protecting groups were removed by treating with an acetonitrile solution containing 20% diethylamine for 30 minutes. After centrifugation, the supernatant was transferred to a new tube and the CPG was washed with ammonia water. The combined solution was concentrated to obtain a solid mixture.

[0445] 3) Purification of single-stranded oligoribonucleotides: The oligomers were purified by HPLC using NanoQ anion exchange. Buffer A was a 10 mM sodium perchlorate solution containing 20% acetonitrile, 20 mM Tris, 1 mM EDTA, pH 7.4; and buffer B was a 500 mM sodium perchlorate solution containing 20% acetonitrile, 20 mM Tris, 1 mM EDTA, pH 7.4. The target product was separated, and some products were desalted using a reversed-phase C18 column.

[0446] 4) Coupling of the single-stranded oligoribonucleotide to the linker: 12 mg of TA14 was dissolved in DMSO, added to the nucleic acid amino intermediate (10 mg, dissolved in DMSO), sonicated and mixed evenly, then carbonate buffer solution (pH = 9) was added, and the reaction was carried out at 25 °C for 16 h followed by purification.

[0447] 5) Coupling of the single-stranded oligoribonucleotide-TA14 conjugate to II-1B: 7 mg of the nucleic acid-TA14 was dissolved in buffer salt solution, and the ligand II-1B (equivalent: 15, dissolved in DMF) was added to the above solution and vortexed; THPTA:CuSO4·5H2O = 5:1 (equivalents were 15 and 3 respectively) was taken, shaken at 40 °C for 5 min, the nucleic acid ligand mixture was added to the above reaction solution and vortexed; sodium ascorbate (equivalent of 25) was quickly added to the above solution and vortexed, and the reaction was carried out at 40 °C for 1 h. After the reaction, purification was carried out to obtain the complete single-stranded oligoribonucleotide-TA14-II-1B conjugate.

[0448] 6) Annealing to produce siRNA: Dissolve the single-stranded oligoribonucleotides to be annealed in sterile RNase Free water (RNAse-free) to prepare a 200 μM solution. Set up the annealing reaction system as follows. Place 10 nmol of the mixture with a total volume of 100 μL in a 95 °C water bath for 10 minutes (≥100 nmol requires 20 minutes at a higher temperature). Quickly transfer it to a 60 °C water bath and let it cool naturally. The annealed solution should not be stored at high temperatures. Complementary strands are formed by combining equimolar single-stranded oligoribonucleotide solutions to obtain various unmodified siRNAs. Among them, the nucleotide sequences of the sense and antisense strands of the siRNAs obtained in the present invention are shown in Table 1. Additionally, liquid chromatography-mass spectrometry (LC-MS) is used to detect the molecular weight of the siRNAs, and the measured value of the molecular weight is compared with the theoretical value. The result shows that the measured value ≈ theoretical value, indicating that the siRNA conjugates in Table 1 are obtained. The specific structure of the ligand (II-1B) in Table 1 can be found in the detailed description part of the present specification (i.e., II-1B in the compounds or their stereoisomers, tautomers, or pharmaceutically acceptable salts described in the third aspect). 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.

[0449] Table 1

[0450]

[0451] In Table 1, the lowercase letters c, g, u, a, t are all nucleotides with 2'-methoxy modification (i.e., c, g, u, a represent that the ribosyl 2'-OH of the nucleotide represented by their corresponding capital letters is replaced by a methoxy group); f represents that the nucleotide before f is a nucleotide with 2'-fluoro modification (i.e., the 2'-OH of the nucleotide before f is replaced by a fluorine atom); s represents that there is a phosphorothioate between the two adjacent nucleotide residues on the left and right of s (i.e., the 5'-phosphate group of the nucleotide before s is replaced by a 5'-phosphorothioate group).

[0452] (Tri-SM6.1)(TA14)(NH2C6) is:

[0453]

[0454] Its preparation method refers to WO2022 / 216920A1.

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

[0456]

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

[0458]

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

[0460]

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

[0462]

[0463] In the test example, the structure of I-0 is:

[0464]

[0465] Its preparation method refers to Goodman, S.L. et al. J. Med. Chem. 2002, 45, 1045 - 1051.

[0466] The structure of II-0 is:

[0467]

[0468] Its preparation method refers to WO2019 / 089765A1.

[0469] Test example 1: ɑvβ6 ligand ELISA binding experiment

[0470] Dilute integrin αvβ6 protein (source: Acro Biosystems) to 5 μg / mL with coating buffer, and add 25 μL of the protein to each well of a 384-well plate. Centrifuge at 1000 rpm for 1 minute, and then let it stand overnight at 4°C. Add 100 μL of washing buffer to each well, let it stand for 5 minutes, then pat dry, and wash 3 times. Add 100 μL of blocking solution to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 90 minutes. Add 100 μL of washing buffer to each well, let it stand for 5 minutes, then pat dry, and wash 3 times. Add 10 μL of compounds with different dilution concentrations to each well, centrifuge at 1000 rpm for 1 minute, and 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, and then incubate at 25°C for 60 minutes. Add 100 μL of washing buffer to each well, let it stand for 5 minutes, then pat dry, and wash 3 times. Add 25 μL of biotinylated fibronectin 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 buffer to each well, let it stand for 5 minutes, then pat dry, and wash 3 times. Add 25 μL of horseradish peroxidase-labeled streptavidin to each well, centrifuge at 1000 rpm for 1 minute, and then incubate at 25°C for 30 minutes. Add 100 μL of washing buffer to each well, let it stand for 5 minutes, then pat dry, and wash 4 times. Add 25 μL of ultrasensitive luminescence 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.

[0471] Table 2: αvβ6 Integrin Ligand Binding Activity

[0472] Compound number <![CDATA[IC 50 (nM)]]> I-0 4.42 I-1 1.18 I-2 4.13

[0473] Table 3: αvβ6 Integrin Ligand (with Linker) Binding Activity

[0474]

[0475]

[0476] The results show that the compounds of this application have high binding activity to αvβ6 integrin.

[0477] Test Example 2: Silencing Efficacy of Target Expression by Intratracheal Administration of siRNA Conjugates in Rats

[0478] Refer to Patent WO2022 / 216920A1 to design and synthesize siRNA conjugates against rat RAGE protein. The siRNA conjugates are shown in Table 1.

[0479] On day 0, whole blood was taken from the jugular vein of rats, and serum was collected. On day 1, after anesthetizing the rats with isoflurane, 200 μL of PBS or drug buffer was inhaled using an aerosol needle, and the administration dose was 0.1 mg / kg. Before the end of the experiment, the rats were anesthetized with 10% chloral hydrate, and blood was taken from the abdominal aorta using a negative pressure blood collection tube, and serum was collected. Then, the left and right lung tissues were taken, and the lung tissues were quickly frozen in liquid nitrogen. All processed serum and lung tissue samples were stored at -80 °C.

[0480] The lung tissues were thawed on ice and homogenized using a tissue grinder. Total RNA in the lung tissues was extracted using the Tissue RNA ExtractionKit 2.0Plus (Vazyme, R411-C3) kit. After reverse transcription (Vazyme, R333-01), the mRNA expression level of RAGE in the rat lung tissues was detected using probe quantitative PCR (Vazyme, QN211-02), and was normalized with the expression level of rat GAPDH. Then, the expression level of the PBS control group was used as a 100% benchmark for comparison.

[0481] The serum was thawed on ice, and rat serum was detected according to the instructions of the ELISA detection kit Rat RAGE DuoSet ELISA (R&D systems, DY1616). According to the conversion by fitting the measured standard curve, the expression level of sRAGE protein in the rat serum was calculated, and the expression level of the PBS control group was used as a 100% benchmark for comparison. The experimental results are shown in the following table.

[0482] Table 4: Expression levels of RAGE protein in rat serum and RAGE mRNA in the lung

[0483]

[0484] Table 5: Expression levels of RAGE protein in rat serum and RAGE mRNA in the lung

[0485]

[0486] The results showed that the siRNA conjugate significantly reduced the expression of RAGE mRNA and protein in the rat lungs and serum. The compound of the present application could make the effect of silencing the lung target and the expression of the corresponding secreted protein by siRNA stronger, indicating that it had a stronger ability to target the delivery of siRNA molecules to lung cells.

[0487] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A compound of formula I, 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 aryl ring, a 5-10 membered heteroaryl 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 a 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- or 6-membered heteroaryl ring.

2. The compound represented by Formula I as described in 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, is and / or having a structure Preferably, R1 is C1-C3 alkyl, C1-C3 haloalkyl.

3. The compound represented by formula I as described 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- to 6-membered heteroaryl ring; Rb is dioxane; and the definitions of R3, R4, and 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 heteroaryl 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 structure Ia: wherein, Ra and R3 are defined as described in claim 1; optionally connected with a connector T; and / or, the compound of formula I has structure Id: Among them, the definitions of Ra and R3 are as described in claim 1; Optionally connected with joint T; and / or, the compound of formula I has structure Ie or If: Wherein, the definitions of T, R3 are as described in claim 1; q is selected from 0, 1 or 2; and / or, when the compound of formula I is linked to a 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 according to 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 is connected to joint T, R3 is H, having the structure d) Ra is a 5-6 membered heteroaryl ring optionally substituted by R2; Preferably, the Ra is the following substituent optionally substituted by R2: furan, thiophene, pyrrole, thiazole, imidazole, pyrazole, oxazole, triazole, tetrazole; or, the Ra is 6. The compound represented by Formula I according to claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, The compound of formula I has 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 a linker T; Preferably, Rb is or when the joint T is connected, Rb is 7. The compound represented by Formula I according to 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, has the structure ii) When R3 is H, has the structure iii) having a structure iv) having a structure v) with structure and / or, when A is linked to a 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 linker T is where t is from 1 to 10; Or, the joint T is where t is from 1 to 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 as claimed in claim 1, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, characterized in that, including the following structure: 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, including the following structure: Among them, represents the connection point.

11. Compound II, its tautomer, stereoisomer or its salt, characterized in that, including 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 an 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, 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, 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, miRNA.

15. The conjugate according to claim 12, wherein, The conjugate has the following structure: Among them, represents the junction point with 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, the compound II as claimed in claim 11, or the conjugate as claimed in any one of claims 12 - 15, and optionally a pharmaceutically acceptable excipient.

17. Use of a compound of formula I as described in any one of claims 1 - 10, its tautomer, stereoisomer, hydrate, solvate, pharmaceutically acceptable salt or prodrug, the compound II as claimed in claim 11, the conjugate as claimed in any one of claims 12 - 15, or the composition as claimed in claim 16 in the preparation of a medicament.

18. The use according to claim 17, characterized in that, The medicament is used for delivering the active molecule G to cells.

19. The use according to claim 17, wherein, The medicament is used for: Inhibiting the expression of a target gene in cells; and / or Preparing a medicament for inhibiting the expression of a target gene in cells; and / or Preparing a medicament for treating lung diseases.

20. The use according to claim 18 or 19, 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).

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