Integrin αvβ6 specific ligand compound, conjugate, pharmaceutical composition and use thereof

Abstract

Provided are a compound capable of specifically binding to integrin αvβ6, a conjugate and pharmaceutical composition containing a compound-based ligand group, and a method for preparing the compound, conjugate and pharmaceutical composition and the use thereof. The compound has a structure as shown in formula (1). The provided conjugate can specifically deliver a functional group to cells and / or tissues expressing integrin αvβ6, thereby achieving the objective of diagnosing or treating diseases.

Description

Integrin αvβ6-specific ligand compound, conjugate, pharmaceutical composition and use thereof TECHNICAL FIELD

[0001] The present disclosure relates to a ligand compound capable of specifically binding to integrin αvβ6, a conjugate containing a ligand group based on the compound, and a pharmaceutical composition, as well as a preparation method and use of the compound, the conjugate and the pharmaceutical composition. BACKGROUND

[0002] Integrin αvβ6 is a transmembrane heterodimer composed of α and β subunits combined by non-covalent bonds, which is expressed on the surface of various cells, especially epithelial cells and muscle cells, wherein the epithelial cells include alveolar epithelial cells, secretory epithelial cells, ciliated epithelial cells, corneal and conjunctival epithelial cells, dermal epithelial cells, bile duct epithelial cells, intestinal epithelial cells, duct epithelial cells, glandular epithelial cells or epithelial tumor cells; the muscle cells include various skeletal muscle cells. When the tissue is damaged or inflamed, the expression of integrin αvβ6 increases, which can promote the proliferation and migration of epithelial cells to the damaged site, and help the reconstruction of epithelial tissue. On the other hand, integrin αvβ6 can also regulate the expression of matrix metalloproteinase (MMP) and activate TGF-β1. Increasing evidence, mainly from in vitro studies, shows that integrin αvβ6 can promote cancer development. Therefore, further research on integrin αvβ6 can provide new ideas for the diagnosis and treatment of various epithelial cell and muscle cell related diseases, such as pulmonary fibrosis diseases or malignant tumors and related diseases.

[0003] As a receptor that can be expressed in various cell types, integrin αvβ6 can deliver different molecules into target cells by specific binding of specific ligands to integrin αvβ6, thereby achieving the purpose of diagnosis or treatment of different diseases or symptoms. SUMMARY

[0004] The inventors of the present disclosure provide a ligand compound capable of specifically binding to integrin αvβ6, a conjugate containing a ligand group based on the compound, and a pharmaceutical composition, as well as a preparation method and use of the compound, the conjugate and the pharmaceutical composition. The conjugate and / or the pharmaceutical composition provided by the present disclosure can specifically target cells or tissues expressing integrin αvβ6, and play a role in diagnosis, treatment and / or prevention of related diseases, symptoms and / or disorders. Therefore, the inventors make the following invention.

[0005] In one aspect, the present disclosure provides a compound having the structure shown in formula (1):

[0006] wherein:

[0007] A1, A2, A3, and A4 are each CR 11 Or N, and at least one of A1 and A3 is CR 11 At least one of A2 and A4 is CR 11 Each R 11 Each element is independently selected from H, OH, SH, halogens, and C1-C. 10 One of the straight-chain or branched hydrocarbon groups;

[0008] R 21 R 22 and R 23 Whether the two are the same or different, each is independently selected from one of O, S and NH;

[0009] R 31 Each R 32 and R 33 Whether the groups are the same or different, each group is independently selected from one of H, C3-C6 cycloalkyl, C3-C6 substituted cycloalkyl, C1-C6 straight-chain or branched hydrocarbon groups and C1-C6 straight-chain or branched substituted hydrocarbon groups;

[0010] R 41 R 42 R 51 R 52 and each R 53 Whether the elements are the same or different, they are each independently selected from H, OH, SH, NH2, halogens, and C3-C. 10 cycloalkyl, C3-C 10 Substituted cycloalkyl, C1-C 10 Straight-chain or branched hydrocarbon groups and C1-C 10 One of the straight-chain or branched substituted hydrocarbon groups;

[0011] R 10 R 61 R 62 R 63 R 64 R 65 and R 66 Whether they are the same or different, each is independently R. p Or R q ;R p Selected from hydrogen, hydroxyl, halogen, C4-C 20 Polyoxyethyl, C4-C 20 Substituted polyoxyethyl, C3-C 10 cycloalkyl, C3-C 10 Substituted cycloalkyl, C1-C 20 Straight-chain or branched hydrocarbon groups and C1-C 20 One of the straight-chain or branched substituted hydrocarbon groups, R qone selected from the group consisting of active functional groups or protected active functional groups;

[0012] Y is selected from the group consisting of OR k , N(R k )2, and SR k , wherein each R k is independently H or a protecting group;

[0013] m is an integer selected from 1-10, and n is an integer selected from 0-2.

[0014] In another aspect, the present disclosure also provides a conjugate comprising at least one ligand group and at least one functional group, the ligand group being formed by removing one hydrogen atom and / or one functional group from a compound of the present disclosure, each of the ligand group and the functional group being linked by a covalent bond or through a linker.

[0015] In yet another aspect, the present disclosure also provides a pharmaceutical composition comprising a conjugate of the present disclosure and a pharmaceutically acceptable carrier.

[0016] In yet another aspect, the present disclosure also provides use of a conjugate of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for diagnosing a disease, a symptom, and / or a disorder, wherein the functional group is a diagnostic agent group that can be used to diagnose the disease, the symptom, and / or the disorder upon delivery to a cell expressing ανβ6.

[0017] In yet another aspect, the present disclosure also provides use of a conjugate of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, a symptom, and / or a disorder, wherein the functional group is a therapeutic agent group that can be used to treat the disease, the symptom, and / or the disorder upon delivery to a cell expressing ανβ6.

[0018] In yet another aspect, the present disclosure also provides use of a conjugate of the present disclosure and / or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, a symptom, and / or a disorder, wherein the functional group is an oligonucleotide group that treats the disease, the symptom, and / or the disorder by modulating the level of the target mRNA in the cell expressing ανβ6.

[0019] In yet another aspect, the present disclosure also provides a method of diagnosing a disease, a symptom, or a disorder, comprising administering to a subject in need thereof an effective amount of a conjugate of the present disclosure and / or a pharmaceutical composition of the present disclosure, wherein the functional group is a diagnostic agent group that can be used to diagnose the disease, the symptom, and / or the disorder upon delivery to a cell expressing ανβ6.

[0020] In yet another aspect, the present disclosure also provides a method of treating a disease, a symptom or a disorder, comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure, wherein the functional group is a therapeutic group, which therapeutic group can be used to treat the disease, the symptom and / or the disorder upon delivery to the avb6-expressing cell.

[0021] In yet another aspect, the present disclosure also provides a method of treating a disease, a symptom or a disorder, comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure, wherein the functional group is an oligonucleotide group, which oligonucleotide group treats the disease, the symptom and / or the disorder by adjusting the level of the target mRNA in the avb6-expressing cell.

[0022] In yet another aspect, the present disclosure also provides a kit containing the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure.

[0023] incorporated by reference

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Advantages

[0025] In one aspect, the present disclosure provides a compound having the structure shown in the above Formula (1), which exhibits a low IC50value when binding to integrin avb6, IC50concentration range is about 0.8-0.92 nM, indicating that the compound provided by the present disclosure has good binding activity to integrin avb6, showing excellent ability to carry different functional groups and deliver into the targeted cells expressing integrin avb6, and then treat or diagnose the related diseases or symptoms. 50 50 In one aspect, the present disclosure provides a compound having the structure shown in the above Formula (1), which exhibits a low IC50value when binding to integrin avb6, IC50concentration range is about 0.8-0.92 nM, indicating that the compound provided by the present disclosure has good binding activity to integrin avb6, showing excellent ability to carry different functional groups and deliver into the targeted cells expressing integrin avb6, and then treat or diagnose the related diseases or symptoms.

[0026] ​In another aspect, the conjugates provided by the present disclosure, which contain the ligand group based on the above-mentioned compounds, have good stability and inhibitory activity on the target mRNA in the cells expressing integrin ανβ6. For example, the conjugates provided by the present disclosure all show a high inhibition rate on the expression of SOD1 mRNA in the lung of mice for a long time. For example, 7 days after the last administration, the inhibition rate of conjugate 2 on SOD1 mRNA in the lung of the test group of mice is 41%, and the inhibition rate of conjugate 3 on SOD1 mRNA in the lung of the test group of mice is 44%, showing a comparable or even higher inhibitory activity compared with the reference conjugate. It is shown that the compounds provided by the present disclosure can effectively deliver siRNA to the lung tissue and show a high efficiency in inhibiting the target mRNA. These results further show that the conjugates provided by the present disclosure can effectively deliver functional groups to the cells and / or tissues expressing integrin ανβ6, so as to achieve the purpose of treating diseases.

[0027] Therefore, it is shown that the compounds provided by the present disclosure can effectively target the cells expressing integrin ανβ6, the conjugates provided by the present disclosure can efficiently enter the cells expressing integrin ανβ6, such as lung epithelial cells, and the functional groups in the conjugates can effectively play a role in the cells, so as to diagnose, treat and / or prevent diseases or symptoms related to the cells and / or tissues expressing integrin ανβ6, and have a good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is a scatter plot of the relative expression level of SOD1 mRNA in the whole lung of C57BL / 6j mice after administration of 0.5 mg / kg (calculated as siRNA) of conjugate 2, conjugate 3 and reference conjugate, and PBS, respectively. DETAILED DESCRIPTION

[0029] The detailed description of the present disclosure is described in detail below. It should be understood that the detailed description described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0030] In the present disclosure, SOD1 mRNA refers to the mRNA having the sequence shown in Genbank accession number NM_011434.2, and SOD1 gene refers to the gene transcribing the above-mentioned SOD1 mRNA, unless otherwise specified.

[0031] DEFINITIONS

[0032] Those skilled in the art will understand that, for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is not physically or synthetically realistic.

[0033] As used in the present disclosure, "hydrocarbyl" includes saturated or unsaturated "alkyl", "alkenyl", and "alkynyl". Among these, "alkyl" refers to straight and branched chains having the indicated number of carbon atoms, typically 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1-C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When reference is made to an alkyl residue having a particular number of carbons, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, iso-butyl, and t-butyl; "propyl" includes n-propyl and iso-propyl. Alkylene is a subset of alkyl, referring to the same residues as alkyl, but having two points of attachment. "Alkenyl" refers to unsaturated branched or straight chain alkyl groups having at least one carbon-carbon double bond, obtained by removing one molecule of hydrogen from adjacent carbon atoms of the parent alkyl group. The group can be in either the cis- or trans- configuration for the double bond. Typical alkenyl groups include, but are not limited to: ethenyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl; butenyl, for example but-1-en-1-yl, but-1-en-2-yl, 2-methylprop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, and but-1,3-dien-2-yl. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl, referring to the same residues as alkenyl, but having two points of attachment. "Alkynyl" refers to unsaturated branched or straight chain alkyl groups having at least one carbon-carbon triple bond, obtained by removing two molecules of hydrogen from adjacent carbon atoms of the parent alkyl group. The group can be in either the cis- or trans- configuration for the triple bond. Typical alkynyl groups include, but are not limited to: ethynyl; propynyl, such as prop-1-yn-1-yl, prop-1-yn-2-yl, prop-2-yn-1-yl (propargyl), prop-2-yn-2-yl; butynyl, for example but-1-yn-1-yl, but-1-yn-2-yl, 2-methylprop-1-yn-1-yl, but-2-yn-1-yl, but-2-yn-2-yl, but-1,3-dyn-1-yl, and but-1,3-dyn-2-yl. In certain embodiments, alkenyl groups have 2 to 20 carbon atoms, while in other embodiments, 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl, referring to the same residues as alkynyl, but having two points of attachment. "Saturated or unsaturated hydrocarbyl" refers to "alkyl", "alkenyl", "alkynyl" as described above, wherein at least one carbon of the hydrocarbyl is substituted with a substituent in a manner allowed by normal valency.

[0034] As used herein, "cycloalkyl" refers to saturated or unsaturated non-aromatic hydrocarbon ring groups having from 3 to 14 carbon atoms. Non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, and cyclohexyl. Cycloalkyl groups can include multiple spiro or fused rings. "Substituted cycloalkyl" means that a cycloalkyl group is optionally mono-, di-, tri-, tetra- or penta-substituted, at any position with substituents permitted by normal valence.

[0035] As used in the present disclosure, "aryl" refers to a radical derived by removing a hydrogen atom from a mononuclear or polynuclear aromatic hydrocarbon ring system. The mononuclear or polynuclear aromatic hydrocarbon ring system contains only carbon and six to eighteen carbon atoms, wherein one or more rings in the ring system are completely unsaturated, i.e., contain a cyclic, delocalized (4n+2) pi-electron system according to Hückel theory. Aryl groups include, but are not limited to, phenyl, fluorenyl, and naphthyl. An arylene group is a subset of aryl groups, referring to the same groups as aryl, but having two points of attachment. "Heteroaryl" refers to a radical derived from a 3- to 18-membered aromatic ring radical that contains 2 to 17 carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen, and sulfur. As used in the present disclosure, a heteroaryl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, wherein one or more rings in the ring system are completely unsaturated, i.e., contain a cyclic, delocalized (4n+2) pi-electron system according to Hückel theory. Heteroaryl groups include fused ring or bridged ring systems. The heteroatoms in a heteroaryl group can be oxidized heteroatoms. One or more nitrogen atoms, if present, can be quaternized nitrogen atoms. Heteroaryl groups are attached to the rest of the molecule through any available carbon atom. Examples of heteroaryl groups include, but are not limited to: azepinyl, acridinyl, benzimidazolyl, benzoindolyl, 1,3-benzodioxolyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[l,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H-benzo[6,7]cyclohepta[l,2-c]pyridazinyl, diphenfuranyl, dibenzothienyl, furanyl, furanonyl, furopyrrolo[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyrimidinyl, indazolyl, indazolinyl, indolizinyl, indolinyl, indolyl, indoxazinyl, indoxazonyl, indazolonyl, indazolyl, isoindolyl, isoindolinyl, isoquinolinyl, naphthoquinolinyl, oxepinyl, phenazinyl, phenarsenyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phenoxazonyl, phenyl, phthalazinyl, pteridinyl, purinyl, quinazolinyl, quinolinyl, quinoxalinyl, quinoxazolinyl, quinoxazonyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, xanthenyl, and ylidene groups.10-hexahydrocycloocta[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl, indolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl, and thiophenyl / thienyl. Heteroarylenyl is a subset of heteroaryl, referring to the same residues as heteroaryl, but with two points of attachment.

[0036] As used in the present disclosure, "heterocycle," "heterocyclic," or "heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical, containing from 2 to 12 carbon atoms and from 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless otherwise indicated, the heterocyclyl radical is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or spiro ring systems. The heteroatoms in the heterocyclyl radical can be oxidized heteroatoms. One or more nitrogen atoms, if present, can be a quaternized nitrogen atom. The heterocyclyl radical is partially or fully saturated. The heterocyclyl radical can be attached to the remainder of the molecule through any available carbon atom. Examples of such heterocyclyl radicals include, but are not limited to: dioxanyl, thienyl[l,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocyclylenyl is a subset of heterocyclyl, referring to the same radicals, but with two points of attachment.

[0037] As used in the present disclosure, "alkoxy" refers to an alkyl group of the indicated number of carbon atoms linked via an oxygen bridge, e.g., methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, t-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, or 3-methylpentoxy. Alkoxy groups typically have from 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms linked via an oxygen bridge. "Alkyleneoxy" is a subset of alkoxy, referring to the same radicals, but with two points of attachment.

[0038] Various protecting groups, such as hydroxyl protecting groups or amino protecting groups, can be used in the present disclosure. Generally, protecting groups render a chemical functional group, such as a hydroxyl or an amino group, insensitive to specific reaction conditions, and can be added to and removed from the functional group in a molecule without substantially damaging the rest of the molecule. Representative protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, New York, 1991, each of which is incorporated by reference in its entirety into the present disclosure. In some embodiments, the protecting groups are stable under basic conditions, but can be removed under acidic conditions. In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present disclosure include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthyl (Pixyl), and 9-(p-methoxyphenyl)xanthyl (Mox). In some embodiments, nonexclusive examples of hydroxyl protecting groups that can be used in the present disclosure include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).

[0039] In the present disclosure, A, U, C, G and T refer to adenine nucleotide, uracil nucleotide, cytosine nucleotide, guanine nucleotide and thymine nucleotide, respectively, unless otherwise specified. 5-methylcytosine nucleotide refers to a nucleotide in which the hydrogen at the 5' position of the cytosine nucleotide base is replaced by a methyl group. The structure of these nucleotides is well known to those skilled in the art.

[0040] In the context of the present disclosure, unless otherwise specified, "conjugation reaction" refers to a reaction between two or more chemical moieties each having a specific function, in which the moieties are connected to each other by covalent linkage, for example, the conjugation reaction can include, but is not limited to, a coupling reaction, a condensation reaction, an addition reaction, a substitution reaction and a polymerization reaction; accordingly, "conjugate" refers to a compound formed by covalent linkage between the chemical moieties. Further, "conjugate" means a compound in which one or more chemical moieties having a specific function are covalently linked to a functional group. Conjugate should be understood in the context of the present disclosure as a general term for a plurality of conjugates or a conjugate represented by a certain chemical formula. In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to a functional group by a reaction to ultimately form a conjugate of the present disclosure. "Active functional group" refers to a functional group that can undergo a conjugation reaction to form a covalent linkage to obtain a conjugate of the present disclosure.

[0041] The term "subject," as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, rabbits, sheep, rats, and any species of poultry. In some embodiments, a "subject" refers to a mammal, such as a rodent or a primate. In some embodiments, a "subject" refers to a mouse, a rat, or a non-human primate. In some embodiments, a "subject" refers to a human subject.

[0042] As used herein, "treatment" refers to an approach for obtaining beneficial or desired results, including but not limited to therapeutic benefit. A "therapeutic benefit" means ablation or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the improvement of one or more established physiological signs or symptoms of the underlying disorder being treated, even though the subject can still be afflicted with the disorder.

[0043] As used herein, "prevention" refers to an approach for obtaining beneficial or desired results, including but not limited to prophylactic benefit. To achieve a "prophylactic benefit," an active ingredient containing a functional group can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though the subject can not yet be diagnosed with the disease. In some embodiments, "prevention" includes reducing or eliminating the risk of a particular disease by intervening in the mRNA or protein level by administering a conjugate, a pharmaceutically acceptable salt, or a pharmaceutical composition of the present disclosure to a subject at risk of developing the particular disease before the risk progresses to an overt disease process.

[0044] Compounds of the present disclosure

[0045] In one aspect, the present disclosure provides a compound having a structure represented by Formula (1):

[0046] wherein:

[0047] A1, A2, A3, and A4 are each CR 11 or N, and at least one of A1and A3is CR 11 , at least one of A2and A4is CR 11 each R 11 is independently selected from H, OH, SH, halogen, and one of a straight-chain or branched hydrocarbon group having 1 to 6 carbon atoms; 10

[0048] R 21 , R 22 , and R 23 ​Whether the two are the same or different, each is independently selected from one of O, S and NH;

[0049] R 31 Each R 32 and R 33 Whether the groups are the same or different, each group is independently selected from one of H, C3-C6 cycloalkyl, C3-C6 substituted cycloalkyl, C1-C6 straight-chain or branched hydrocarbon groups and C1-C6 straight-chain or branched substituted hydrocarbon groups;

[0050] R 41 R 42 R 51 R 52 and each R 53 Whether the elements are the same or different, they are each independently selected from H, OH, SH, NH2, halogens, and C3-C. 10 cycloalkyl, C3-C 10 Substituted cycloalkyl, C1-C 10 Straight-chain or branched hydrocarbon groups and C1-C 10 One of the straight-chain or branched substituted hydrocarbon groups;

[0051] R 10 R 61 R 62 R 63 R 64 R 65 and R 66 Whether they are the same or different, each is independently R. p Or R q ;R p Selected from hydrogen, hydroxyl, halogen, C4-C 20 Polyoxyethyl, C4-C 20 Substituted polyoxyethyl, C3-C 10 cycloalkyl, C3-C 10 Substituted cycloalkyl, C1-C 20 Straight-chain or branched hydrocarbon groups and C1-C 20 One of the straight-chain or branched substituted hydrocarbon groups, R q Selected from groups containing active functional groups or protected active functional groups;

[0052] Y is selected from OR k 、N(R k )2 and SR k One of them, wherein each R k Independently, it can be H or a protecting group;

[0053] m is an integer selected from 1 to 10, and n is an integer selected from 0 to 2.

[0054] The present disclosure provides compounds having the structure of formula (1) as described above, which can specifically bind to integrin ανβ6 and have good binding activity, and further can deliver various functional groups to cells expressing integrin ανβ6, thereby producing therapeutic, diagnostic and / or prophylactic effects on related diseases, symptoms and / or disorders.

[0055] According to some embodiments of the compounds of the present disclosure, m can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is selected from an integer from 1 or 3-10.

[0056] In some embodiments, m is selected from an integer from 1-5. For example, in different embodiments of the present disclosure, m is 1, 2, 3, 4, or 5. In some embodiments, m is selected from an integer from 1-3. In some embodiments, m is 1 or 3. In some embodiments, m is 1.

[0057] In some embodiments, n is 0 or 1.

[0058] In some embodiments, each R 11 is independently selected from one of H, OH, SH, halogen, and C1-C3 linear or branched alkyl. In some embodiments, each R 11 is H.

[0059] In some embodiments, R 31 , each R 32 , and R 33 are the same or different, and are each independently selected from one of H, C3-C5 cycloalkyl, C3-C5 substituted cycloalkyl, C2-C5 alkyl, and C2-C5 substituted alkyl. In some embodiments, each R 32 and R 33 is H; R 31 is H, C2-C5 alkyl, or C3-C6 cycloalkyl. In some embodiments, R 31 is H, cyclopropyl, t-butyl, n-propyl, or i-propyl.

[0060] In some embodiments, R 41 , R 42 , R 51 , R 52 , and each R 53 are the same or different, and are each independently selected from one of H, OH, SH, NH2, halogen, C3-C6 cycloalkyl, C3-C6 substituted cycloalkyl, C1-C6 alkyl, and C1-C6 substituted alkyl. In some embodiments, R 41 , R 42 , R 51 , R 52 , and each R53 All are H.

[0061] The conjugates disclosed herein may or may not have a group R containing an active functional group. q In some implementations, R 10 R 61 R 62 R 63 R 64 R 65 and R 66 At least one of them is R q In some implementations, R 10 R 61 R 62 R 63 R 64 R 65 and R 66 One of them is R q In some implementations, R 10 For R q ;R 61 R 62 R 63 R 64 R 65 and R 66 For R p In some implementations, each R p Independently selected from H, OH, C4-C 20 Polyoxyethyl, C4-C 20 Substituted polyoxyethyl, C1-C 20 Straight-chain or branched hydrocarbon groups and C1-C 20 One of the straight-chain or branched substituted hydrocarbon groups. In some embodiments, C4-C 20 The polyoxyethyl group has the following structure: -(CH₂CH₂O) nj - where nj is an integer between 2 and 10; in some embodiments, nj is 3-7; in some embodiments, nj is 5. In this disclosure, substituted polyoxyethyl means that one or more hydrogen atoms in the polyoxyethyl group are substituted. In some embodiments, Rq, in addition to the aforementioned active functional group or protected active functional group, further comprises C6-C linked to said active functional group or protected active functional group. 14 Polyoxyethylidene, C6-C 14 Substituted polyoxyethylidene, C8-C 14 alkeneoxy groups and C8-C 14one of substituted alkyleneoxy groups. These linking groups can enable the compounds of the present disclosure to maintain a suitable spatial configuration when forming conjugates, thereby effectively delivering the functional groups in the conjugates to the integrin ανβ6-expressing cells and / or tissues. On the other hand, these linking groups can be able to affect the biocompatibility, stability, and / or metabolic speed of the conjugates, thereby obtaining favorable bioavailability and pharmacokinetic characteristics. In some embodiments, R q is 8-15 atoms in length, the length referring to the longest chain of atoms from the active functional group in R q to the point of attachment of the naphthalene ring in the structure shown in Formula (1).

[0062] In some embodiments, R q has a structure shown in any one of Formulas (31) to (36):

[0063] wherein, represents the point of covalent attachment of the group; and Z is an active functional group.

[0064] In some embodiments, the active functional group Z is selected from one of azido, alkynyl, dibenzocyclooctyne, thiol, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, dithio, ortho-pyridyl dithio, maleimide, tosylate, tetrazine, trans-cyclooctene, hydrazide, and phosphoramidite. By including the active functional group, the compounds of the present disclosure are able to form conjugate linkages with the functional groups by undergoing conjugation reactions, thereby obtaining the conjugates provided by the present disclosure.

[0065] In some embodiments, R 10 is R q In some embodiments, R 61 , R 62 , R 63 , R 64 , R 65 , and R 66 are independently H or methyl. In some embodiments, R 61 , R 62 , R 63 , R 64 , R 65 , and R 66 are all H.

[0066] In some embodiments, the compound has a structure shown in Formula (2):

[0067] In some embodiments, R 21 , R 22 , and R 23each of A1, A2, A3, and A4 is CH or N. In some embodiments, each of A1, A2, A3, and A4 is CH or N, and at least one of A1and A3is CH, and at least one of A2and A4is CH. In some embodiments, at least one of A3and / or A4of A1, A2, A3, and A4is N, and the others are CH. In some embodiments, Y is hydroxyl or hydroxyl protected by a hydroxyl protecting group.

[0068] In some embodiments, the compound has a structure represented by any one of Formulas (21) to (30):

[0069] In different embodiments of the present disclosure, the different structures represented by Formulas (21) to (30) above are enantiomers, and the left-handed or right-handed structures of each structure can be obtained by chiral resolution according to the actual needs.

[0070] For example, in different embodiments of the present disclosure, the following compounds having structures represented by Formulas (21-1) to (30-1) can be further used:

[0071] In some embodiments, Z is azido.

[0072] In some embodiments, the hydroxyl protecting group is selected from at least one of benzoyl, benzyl, tert-butyloxycarbonyl, trityl, substituted silyl, and alkylacyl.

[0073] Preparation method of the compound of the present disclosure

[0074] In another aspect, the present disclosure also provides a preparation method of the compound represented by Formula (1) above. In different embodiments of the present disclosure, any reasonable synthetic route can be used to prepare the compound represented by Formula (1).

[0075] In some embodiments, the preparation method of the compound of the present disclosure is carried out according to the following preparation process and will be further described in the example part, and the preparation method provided in the present disclosure is only an example of the preparation process of the compound of the present disclosure, and is not used to limit the compound of the present disclosure.

[0076] First preparation method

[0077] In some embodiments, the compound represented by Formula (1) can be prepared by the first preparation method represented by the following formula, which comprises:

[0078] contacting a compound having the structure of Formula (101) with a compound having the structure of Formula (102) in an organic solvent under coupling reaction conditions and in the presence of a coupling catalyst to isolate a compound having the structure of Formula (1):

[0079] wherein A1-A4, R 10 , R 21 , R 23 , R 31 , R 33 , R 41 , R 42 , R 51 , R 53 , R 61 , R 66 , Y, m, and n are each as defined and optionally varied above.

[0080] The X1group in Formula (101) and the X2group in Formula (102) can be selected from any group that can undergo a coupling reaction such that the compound having the structure of Formula (101) and the compound having the structure of Formula (102) react to form the compound having the structure of Formula (1) via the coupling reaction. In some embodiments, the coupling reaction in this step is a Suzuki coupling reaction, and X1may be a halogen, and X2may be selected from one of a boronic acid, a boronic acid salt, and a boronic acid ester.

[0081] In some embodiments, the molar ratio of the compound having the structure of Formula (101) to the compound having the structure of Formula (102) is 0.3-3:1. In some embodiments, the molar ratio is 0.3-1:1.

[0082] In the present disclosure, the type and amount of the organic solvent is not limited as long as it can achieve sufficient dissolution of the reactants and does not cause undesirable side reactions with the reactants. In some embodiments, the organic solvent can be selected from at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol, and tetrahydrofuran. In some embodiments, the organic solvent is tetrahydrofuran. In some embodiments, the volume ratio of the organic solvent to the compound having the structure of Formula (102) is 2-6:1, for example, it can be 2.5-4.5:1.

[0083] In some embodiments, the reaction conditions of the above coupling reaction include that the reaction temperature is 0-100°C, for example, it can be 80-100°C; and the reaction time is 1-20 hours, for example, it can be 15-20 hours.

[0084] In some embodiments, the coupling catalyst can be a palladium catalyst, such as palladium acetate or tetrakis(triphenylphosphine)palladium. In some embodiments, the molar ratio of the palladium catalyst to the compound of structure (101) is 0.02-0.08:1, for example, it can be 0.04-0.06:1. In some embodiments, palladium acetate is used as the catalyst in the coupling reaction.

[0085] In some embodiments, the process for separating the compound of structure (101) and the compound of structure (102) after the coupling reaction includes: extracting the product of the coupling reaction to separate the organic phase, washing and drying the organic phase to remove impurities, filtering to remove the drying agent, concentrating the filtrate, and column chromatography separation and purification treatment to obtain the compound of structure (1).

[0086] In some embodiments, the extractant used in the extraction process can be selected from one of ethyl acetate, dichloromethane, chloroform, diethyl ether, and methyl tert-butyl ether. In different embodiments of the present disclosure, the amount of the extractant used in the extraction process and the number of times of extraction are sufficient to extract and separate the product. In some embodiments, the molar volume ratio of the amount of the organic solvent used in each extraction process to the amount of the reaction product is 1-20:1 L / mol, and the number of times of extraction can be 2-6 times.

[0087] Any conventional washing agent can be used to wash the organic phase. For example, the washing agent is selected from saturated brine or dilute hydrochloric acid, and the volume ratio of the washing agent to the organic phase is 1:2-4.

[0088] Any drying agent that can achieve the purpose of drying the organic phase can be used, for example, anhydrous sodium sulfate can be used as the drying agent.

[0089] The filtration and concentration processes can be achieved by using the filtration and concentration processes well known in the art.

[0090] The elution reagent used in the column chromatography separation process can be methanol and / or dichloromethane elution reagent, ethyl acetate and / or petroleum ether elution reagent. In some embodiments, methanol and / or dichloromethane elution reagent is used, wherein the volume of methanol is 0-6%:1 relative to the volume of dichloromethane. In some embodiments, the column chromatography separation process can use gradient elution, for example, a mixture of ethyl acetate and petroleum ether is used as the eluent, wherein the volume ratio of ethyl acetate to petroleum ether is 0:100-50:50.

[0091] The compound of structure (102) can be obtained by any reasonable means by those skilled in the art. In some embodiments, X2is B(OH)2, R 61 - R 66 are H, R 10For benzyloxy, 1-benzyloxynaphthalene-4-boronic acid represented by formula (113) can be obtained via condensation reaction of benzyloxy-4-bromonaphthalene represented by formula (112) and isopropyl borate. Both isopropyl borate and benzyloxy-4-bromonaphthalene represented by formula (112) are readily prepared by those skilled in the art, or are commercially available.

[0092] In some embodiments, the condensing reagent can be selected from n-butyllithium.

[0093] In some embodiments, the reaction conditions for the condensation reaction include: the reaction temperature is 0-100°C, for example, it can be 40-100°C; the reaction time is 1-20 hours, for example, it can be 6-20 hours.

[0094] In some embodiments, the method further comprises reduction of the benzyloxy protecting group in the presence of Pd / C to obtain a hydroxyl group, which is readily achieved by those skilled in the art. In some embodiments, the method further comprises further linking other chemical moieties from the hydroxyl group to obtain the ligand compound of the present application.

[0095] The compound represented by formula (101) can be obtained by any reasonable means by those skilled in the art. In some embodiments, the compound represented by formula (101) is commercially available or custom-made. In some embodiments, for the compound represented by formula (101) above, the compound represented by formula (101) can be obtained by contacting the compound represented by formula (103) and the compound represented by formula (202) in an organic solvent under condensation reaction conditions and in the presence of a condensing reagent, and then isolating the compound represented by formula (101):

[0096] wherein, in formula (103) and formula (202), A1-A4, R 21 -R 23 , R 31 -R 33 , R 41 -R 42 , R 51 -R 53 , Y, m, n and X1are each as defined and optionally substituted as described above. The X3group in formula (103) and the X4group in formula (202) are selected from any groups that can undergo condensation reaction, and by the condensation reaction, the compound represented by formula (103) and the compound represented by formula (202) are contacted to obtain the compound represented by formula (101).

[0097] In some embodiments, the X3group can be selected from one of amino, imino, halogenated amino, and N-tert-butoxycarbonyl amino, and the X4group can be selected from one of hydroxyl and hydroxyl protected by a protecting group.

[0098] In some embodiments, the molar ratio of the compound having the structure shown in formula (103) to the compound having the structure shown in formula (202) in the condensation reaction step is 0.5-3:1, for example, can be 0.8-1.5:1.

[0099] In the present disclosure, the kind and amount of the organic solvent used are not limited as long as the solubility of the reactants is sufficient and no undesired side reactions occur. In some embodiments, the organic solvent can be selected from at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol, and tetrahydrofuran. In some embodiments, the volume ratio of the organic solvent to the compound having the structure shown in formula (202) is 2-6:1, for example, can be 2.5-4.5:1.

[0100] In some embodiments, the reaction conditions of the condensation reaction described above include that the reaction temperature is 0-100°C, for example, can be 20-30°C; and the reaction time is 1-20 hours, for example, can be 2-4 hours.

[0101] In some embodiments, the condensation reagent can be a polypeptide condensation reagent. The polypeptide condensation reagent is a condensation reagent for forming an acyl bond. In some embodiments, the condensation reagent can be 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or O-benzotriazol-tetramethyluronium hexafluorophosphate (HBTU).

[0102] In some embodiments, the molar ratio of the condensation reagent to the compound having the structure shown in formula (202) is 0.5-5:1, for example, can be 0.8-1.2:1.

[0103] In some embodiments, the operation process of separating the product of the condensation reaction in the preparation process is the same as the operation process of separating the reaction product obtained after the reaction of the compound having the structure shown in formula (101) and the compound having the structure shown in formula (102).

[0104] The compound having the structure shown in formula (103) can be obtained by any reasonable means by those skilled in the art. In some embodiments, the compound having the structure shown in formula (103) is commercially available or commercially custom-made. In different embodiments of the present disclosure, for the compound having the structure shown in formula (103), when A1, A2, A3, and A4 are all -CH-, i.e., having the structure shown in formula (104):

[0105] In some embodiments, a compound having a structure of Formula (105) is contacted with a compound having a structure of Formula (106) in an organic solvent under condensation reaction conditions and in the presence of a condensation reagent to isolate a compound having a structure of Formula (104):

[0106] wherein X1, X3, Y, R 21 , R 23 , R 31 are defined and have the value ranges as described previously.

[0107] In some embodiments, the organic solvent used and its amount, the reaction conditions and the condensation reagent used in the condensation reaction of a compound having a structure of Formula (105) with a compound having a structure of Formula (106) are the same as those described above for the condensation reaction of a compound having a structure of Formula (103) with a compound having a structure of Formula (202). The difference is that the molar ratio of the compound having a structure of Formula (105) to the compound having a structure of Formula (106) is 0.5-3: 1, for example, it can be 0.5-1.5: 1, further it can be 0.8-1.2: 1; the molar ratio of the condensation reagent to the compound having a structure of Formula (105) is 0.5-5: 1, for example, it can be 0.5-1.5: 1, further it can be 0.8-1.2: 1.

[0108] A compound having a structure of Formula (105) can be obtained by any reasonable means by one skilled in the art. In some embodiments, X1is Br, R 23 is O, Y is OH, and the compound having a structure of Formula (105) is 3-amino-3-(4-bromophenyl)-propionic acid, which is readily commercially available.

[0109] A compound having a structure of Formula (106) can be obtained by any reasonable means by one skilled in the art. In some embodiments, the compound having a structure of Formula (106) is commercially available or custom ordered. In different embodiments, the compound having a structure of Formula (106) can be selected from a readily commercially available protected amino acid, for example, Boc-glycine, or a Boc-glycine with different substituents. For example, in some embodiments, Boc-L-cyclopropylglycine can be used to introduce a cyclopropyl group into the compound.

[0110] In different embodiments of the present disclosure, for a compound having a structure of Formula (103), when one of A1, A2, A3and A4is -N-, for example, it has a structure of Formula (107) as follows:

[0111] The compound of formula (107) can be obtained by any reasonable means by one skilled in the art. In some embodiments, the compound of formula (107) is commercially available or custom ordered. In some embodiments, the compound of formula (107) can be prepared using the same preparation method as the compound of formula (104), except that the compound of formula (105) is replaced by the compound of formula (109) in the reaction:

[0112] wherein X1, Y and R 23 are as defined and selected above, and the molar ratio of the compound of formula (109) to the compound of formula (106) is 0.3-3:1, for example, 1.5-2.5:1. In some embodiments of the present disclosure, the molar ratio of the compound of formula (109) to the condensing reagent is 0.3-3:1, for example, 1.5-2.5:1.

[0113] In some embodiments, R 23 is O, and Y is OH, in which case, the compound of formula (109) can be obtained by contacting the compound of formula (108) with malonic acid or a malonic acid salt containing different substituent groups in the presence of an organic solvent and ammonium acetate.

[0114] wherein X1is as defined and selected above.

[0115] In some embodiments, the amount of malonic acid or a malonic acid salt containing different substituent groups is 0.5-4:1, for example, 1-2.5:1, further 1.5-2:1, relative to the amount of the compound of formula (108).

[0116] In some embodiments, the amount of ammonium acetate is 0.5-4:1, for example, 1-2.5:1, further 1.5-2:1, relative to the amount of the compound of formula (108).

[0117] The compound of formula (108) can be obtained by any reasonable means by one skilled in the art. In some embodiments, X1is Br, and the compound of formula (108) is 2-bromopyridine-5-carboxaldehyde, which is readily commercially available.

[0118] The kind and amount of the organic solvent used in the reaction process are not limited, as long as the reactants can be fully dissolved and no undesired side reactions occur. In some embodiments, the organic solvent can be selected from at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol and tetrahydrofuran. In some embodiments, the volume ratio of the organic solvent to the compound of formula (108) is 2-6: 1, for example, it can be 2.5-4.5: 1.

[0119] In some embodiments, the reaction conditions for preparing the compound of formula (109) include that the reaction temperature is 0-100°C, for example, it can be 20-30°C; and the reaction time is 1-20 hours, for example, it can be 10-15 hours.

[0120] The compound of formula (202) can be obtained by any reasonable means by those skilled in the art. In some embodiments, the compound of formula (202) is commercially available or commercially custom-made. In some embodiments, after the compound of formula (110) and the compound of formula (111) are contacted and separated in the presence of an organic solvent and an acidic catalyst, the compound of formula (202) is further obtained by a hydrogenation reduction reaction.

[0121] wherein, in formula (110) and formula (111), R 22 , R 32 , R 33 , R 41 -R 42 , R 51 -R 53 , X4, m and n are defined and optionally selected as described above.

[0122] In some embodiments, the molar ratio of the compound of formula (110) to the compound of formula (111) is 0.3-3: 1, for example, it can be 0.8-1.2: 1.

[0123] In some embodiments, the acidic catalyst can be selected from naturally occurring L-proline, and the molar ratio of L-proline to the compound of formula (110) is 0.3-2: 1, for example, it can be 0.5-1.5: 1.

[0124] The kind and amount of the organic solvent in this step are not limited, as long as the reactants can be sufficiently dissolved and no undesired side reactions occur. In some embodiments, the organic solvent can be selected from at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol and tetrahydrofuran. In some embodiments, the volume ratio of the organic solvent to the compound of formula (110) is 2-6: 1, for example, it can be 2.5-4.5: 1.

[0125] The compound of formula (110) can be obtained by any reasonable means by those skilled in the art. In some embodiments, R 51 - R 53 are H, R 42 is H, n is 1, and the compound of formula (110) is commercially available 2-amino-3-pyridinecarboxaldehyde.

[0126] The compound of formula (111) can be obtained by any reasonable means by those skilled in the art. In some embodiments, R 41 is H, R 33 is H, R 32 is H, m = 1, R 22 is O, X4is O-CH2-CH3, and the compound of formula (111) is commercially available methyl levulinate.

[0127] In some embodiments, the above hydrogenation reduction reaction is carried out in the presence of a hydrogenation catalyst. In some embodiments, the hydrogenation catalyst is platinum oxide. In some embodiments, the weight of the hydrogenation catalyst is 5%-20%, for example, it can be 10%, based on the reaction product generated by contacting the compound of formula (110) with the compound of formula (111).

[0128] In different embodiments of the present disclosure, for each of the above reactions, the operation process for separating the reaction product is the same as that for separating the reaction product obtained after the reaction of the compound of formula (101) with the compound of formula (102).

[0129] Second preparation method

[0130] In some embodiments, the compound of formula (1) can be prepared by the following second preparation method, which comprises:

[0131] In an organic solvent, under condensation reaction conditions and in the presence of a condensation reagent, contacting a compound of formula (201) with a compound of formula (202) to separate a compound of formula (1):

[0132] wherein, in formula (201) and formula (202), A1to A4, R 10 , R 21 , R 23 , R 31 , R 33 , R 41 , R 42 , R 51 , R 53 , R 61 , R 66 , Y, X3, X4, m and n are each as defined and optionally substituted as described above.

[0133] In some embodiments, the organic solvent, reaction conditions and condensing reagent used in the condensation reaction are the same as those used in the condensation reaction of the compound of formula (103) and the compound of formula (202) in the first preparation method described above. The difference is that the molar ratio of the compound of formula (201) to the compound of formula (202) is 0.3-3:1, for example, it can be 0.5-1:1; the molar ratio of the condensing reagent to the compound of formula (202) is 0.5-5:1, for example, it can be 0.8-1.2:1.

[0134] In some embodiments, the process of separating the product of the condensation reaction in the preparation process is the same as the process of separating the compound of formula (1) described above.

[0135] The compound of formula (201) can be obtained by any reasonable means by those skilled in the art. In some embodiments, the compound of formula (201) is commercially available or custom ordered. In some embodiments, the compound of formula (201) is obtained by contacting a compound of formula (203) and a compound of formula (106) in an organic solvent under condensation reaction conditions and in the presence of a condensing reagent, and then separating.

[0136] In formula (203), A1to A4, R 10 , R 21 , R 23 , R 31 , R 61 , R 66 , X3, Y are each as defined and optionally substituted as described above.

[0137] In some embodiments, the selection of the organic solvent, the reaction conditions, and the condensation reagent used in the condensation reaction of the compound having the structure of formula (203) and the compound having the structure of formula (106) is the same as that described above for the condensation reaction of the compound having the structure of formula (103) and the compound having the structure of formula (202). The difference is that the molar ratio of the compound having the structure of formula (203) to the compound having the structure of formula (106) is 0.3-3: 1, for example, it can be 0.5-1.5: 1; the molar ratio of the compound having the structure of formula (203) to the condensation reagent is 0.5-5: 1, for example, it can be 0.8-1.5: 1.

[0138] The compound having the structure of formula (203) can be obtained by any reasonable means by those skilled in the art. In some embodiments, the compound having the structure of formula (203) is commercially available or custom ordered. In some embodiments, the compound having the structure of formula (203) can be obtained by contacting the compound having the structure of formula (204) with malonic acid or a malonic acid salt containing different substituents in the presence of an organic solvent and ammonium acetate.

[0139] In some embodiments, the amount of malonic acid or a malonic acid salt containing different substituents is 0.5-4: 1, for example, it can be 1-2.5: 1, and further can be 1.5-2: 1, relative to the amount of the compound having the structure of formula (204).

[0140] In some embodiments, the amount of ammonium acetate is 0.5-4: 1, for example, it can be 1-2.5: 1, and further can be 1.5-2: 1, relative to the amount of the compound having the structure of formula (204).

[0141] The type and amount of the organic solvent used in the reaction is not limited, as long as it can achieve sufficient dissolution of the reactants and does not cause undesirable side reactions with the reactants. In some embodiments, the organic solvent can be selected from at least one of anhydrous dichloromethane, anhydrous methanol, anhydrous ethanol, anhydrous tetrahydrofuran, N,N-dimethylformamide, methanol, and tetrahydrofuran. In some embodiments, the volume ratio of the organic solvent to the compound having the structure of formula (204) is 2-6: 1, for example, it can be 2.5-4.5: 1.

[0142] In some embodiments, the reaction conditions for preparing the compound having the structure of formula (203) described above include that the reaction temperature is 0-100°C, for example, it can be 70-100°C; and the reaction time is 1-20 hours, for example, it can be 12-15 hours.

[0143] The compound of formula (204) can be obtained by any reasonable means by one skilled in the art. In some embodiments, the compound of formula (204) is readily commercially available or custom ordered. In some embodiments, the compound of formula (102) and the compound of formula (205) are contacted under coupling reaction conditions and in the presence of a coupling catalyst to isolate the compound of formula (204).

[0144] wherein each of A1-A4, R 10 , R 61 -R 66 and X2are as defined and optionally substituted as described above. In formula (205), X5is selected from one of an oxygen atom, a sulfur atom, or a nitrogen atom.

[0145] In some embodiments, in formula (205), A3and A4are both N.

[0146] The compound of formula (205) can be obtained by any reasonable means by one skilled in the art. In some embodiments, A1and A2are both CH, A3and A4are both N, and X5is S, the compound of formula (204) is readily commercially available as 2-methylthiopyrimidine-5-carboxaldehyde.

[0147] In some embodiments, the selection of the organic solvent, the reaction conditions, and the coupling reagent used in the coupling reaction of the compound of formula (205) and the compound of formula (102) are the same as those described above for the coupling reaction of the compound of formula (101) and the compound of formula (102). The difference is that the molar ratio of the compound of formula (205) to the compound of formula (102) is 1.5-4: 1, for example, it can be 1.8-2.2: 1; and the molar ratio of the compound of formula (205) to the coupling catalyst is 10-30: 1, for example, it can be 15-25: 1. In some embodiments, the coupling catalyst can be tetrakis(triphenylphosphine)palladium.

[0148] In various embodiments of the present disclosure, for each of the above reactions, the process for isolating the reaction product is the same as that described above for isolating the reaction product obtained after the reaction of the compound of formula (101) and the compound of formula (102).

[0149] Conjugates

[0150] In another aspect, the present disclosure provides a conjugate comprising at least one ligand group and at least one functional group, the ligand group being formed by removing one hydrogen atom and / or one functional group from a compound of the present disclosure, the ligand group and the functional group being connected via a covalent bond or through a linking group. In some embodiments, the ligand group is formed by removing one hydrogen atom and / or one functional group from a compound of formula (1) 10 .

[0151] The conjugates provided in the present disclosure containing ligand groups based on the compounds have a higher inhibitory rate on the level of target mRNA in cells expressing integrin αvβ6 for a longer period of time.

[0152] In some embodiments, the conjugate provided in the present disclosure has a structure shown in formula (3):

[0153] wherein each R L is the same or different, independently represents one of the ligand groups; R j represents a covalent bond or a linking group; each A0group is the same or different, independently represents one of the functional groups;

[0154] m0is an integer from 1 to 6; n0is an integer from 1 to 6.

[0155] In some embodiments, m0is an integer from 1 to 6, i.e., the conjugate shown in formula (3) contains 1 to 6 functional groups A0. From the perspective of delivery efficiency and cost, in some embodiments, m0is an integer from 1 to 4, i.e., the conjugate shown in formula (3) contains 1 to 4 functional groups A0. In some embodiments, m0is 1, i.e., the conjugate shown in formula (3) contains 1 functional group A0.

[0156] In some embodiments, n0is an integer from 1 to 6, i.e., the conjugate shown in formula (3) contains 1 to 6 R L groups. From the perspective of delivery efficiency and cost, in some embodiments, n0is an integer from 1 to 3, i.e., the conjugate shown in formula (3) contains 1 to 3 R L groups. In some embodiments, n0is 1, i.e., the conjugate shown in formula (3) contains 1 R L group.

[0157] R j The role of the R L group is to connect the ligand group R LThe specific binding to integrin αvβ6 delivers the functional group A0 specifically to cells and / or tissues expressing integrin αvβ6. Therefore, any mechanism capable of achieving this linkage without affecting the ligand group R... L The specific binding of R to integrin αvβ6 and the effect of the functional group A0 j All functional groups can achieve the purpose of this invention and solve the technical problems to be solved by this invention. In some embodiments, after the conjugate shown in formula (3) reaches cells and / or tissues expressing integrin αvβ6, the R j Cleavage occurs, releasing the individual functional group A0 corresponding to the active pharmaceutical molecule. In some embodiments, the R... j It does not cleave in vivo, and at this point, R in the conjugate... j Groups and R L The presence of the functional group A0 does not affect its diagnostic and / or therapeutic effects.

[0158] In some embodiments, the conjugate has the structure shown in formula (4):

[0159] Where n0 is 2 or 3. In this embodiment, m0 is 1, in which case the conjugate shown in formula (4) contains one functional group A0 and two or three ligand groups R. L Each ligand group R L They are covalently linked to the functional group A0.

[0160] In some implementations, R j The linking group is used. In some embodiments, both m0 and n0 are 1, in which case the conjugate shown in formula (3) contains one functional group A0 and one ligand group R. L ligand group R L via R j Connected to the functional group A0. In some embodiments, m0 is 1 and n0 is an integer greater than 2, in which case multiple ligand groups R L via R j Linked to the functional group A0. In some embodiments, each R L The group is attached to the same atom of the functional group A0. In some embodiments, each R L The group is attached to different atoms of the functional group A0.

[0161] In some implementations, R j As a linking group, the linking group R j It includes the main chain, the side chain, and the connecting part.

[0162] The main chain portion is connected to a conjugation linker and a side chain portion, respectively. In some embodiments, the main chain portion is a straight chain alkylene of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene is replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 aldosidyl, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclylene, and C5-C 10 heteroarylene; and wherein the straight chain alkylene can have any one or more substituents selected from the group consisting of C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C10 alkyl, -C(O)C1-C 10 alkyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl).

[0163] chain portion is covalently linked to the backbone portion and R L In some embodiments, each side chain portion is independently a covalent bond, or a straight chain alkylene of 1-70 carbon atoms, or one or more carbon atoms of the straight chain alkylene is replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 aldosidyl, C2-C 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclyl, and C5-C 10 heteroarylene; and the straight chain alkylene can have any one or more substituents selected from the group consisting of C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkyl, -OC1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkyl, -SC1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -NH(C1-C

[0164] The conjugation linkers are connected to the backbone moiety and the functional group A0. In some embodiments, each conjugation linker is independently a covalent bond or a combination of one or more of the following linking structures: C1-C 10 linear alkylene, phosphonate bond, thiophosphonate bond, amide bond, ester bond, ether bond, disulfide bond, 1,2,3-triazole, polyethylene glycol, pyrrolidine, 2-oxopyrrolidine, phenylene, cyclohexylene, 2-succinimide, 2- thiosuccinimide, amino acid, nucleotide.

[0165] In some embodiments, the number of conjugation linkers is m0, and the number of side chain moieties is n0, each of the conjugation linkers is connected to the backbone moiety and one of the functional groups A0, and each of the side chain moieties is connected to the backbone moiety and one of the R L groups. Thus, each functional group A0and R L group is independently connected to a linker group R j .

[0166] In some embodiments, all side chain moieties are attached to the same atom in the backbone moiety; or, each side chain moiety is attached to a different atom in the backbone moiety.

[0167] In some embodiments, m0 is 1, the linking group R j comprises a structure as shown in formula (301):

[0168] wherein k = n0; L C is the backbone moiety, L A is the side chain moiety, L B is the conjugation linker, denotes the site of covalent attachment of a group.

[0169] the backbone moiety L C is a covalent bond or a 2-7 valent, straight-chained or branched C1-C 25 saturated hydrocarbon group, or one or more carbon atoms in the saturated hydrocarbon group are replaced by one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 alkylenoside, C2-C5 alkenylen, C2-C5 alkynylen, C6-C 10 arylen, C3-C8 heterocyclylen, and C5-C 10 heteroarylen; wherein the saturated hydrocarbon group can have any one or more substituents selected from the group consisting of C1-C5 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -C(O)O(C1-C5 alkyl), -CON(C1-C5 alkyl)(C1-C5 alkyl), -CONH(C1-C5 alkyl), -CONH2, -NHC(O)(C1-C5 alkyl), -NHC(O)(phenyl), -N(C1-C5 alkyl)C(O)(C1-C5 alkyl), -N(C1-C5 alkyl)C(O)(phenyl), -C(O)C1-C5 alkyl, -C(O)C1-C5 alkylphenyl, -OC(O)C1-C5 alkyl, -SO2(C1-C5 alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5 alkyl), -SO2NH(phenyl), -NHSO2(C1-C5 alkyl), and -NHSO2(phenyl).

[0170] In some embodiments, L C is a 2-7 valent C5-C 20saturated hydrocarbon group, or one or more carbon atoms in said saturated hydrocarbon group is / are replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 alkylenedioxy, C2-C5 alkenylen, C2-C5 alkynylen, C6-C 10 arylen, C3-C8 heteroarylen, and C5-C 10 heteroarylen; wherein said saturated hydrocarbon group can have one or more substituents selected from the group consisting of C1-C5 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, -O-C1-C5 alkyl, -OC1-C5 alkylphenyl, -C1-C5 alkyl-OH, -SC1-C5 alkyl, nitro, -CONH2.

[0171] In some embodiments, L C is 5-30 atoms in length, wherein said L C is 5-30 atoms in length. C In some embodiments, L A is 5-30 atoms in length. B In some embodiments, L C is 8-25 atoms in length.

[0172] each said side chain moiety L A is independently a covalent bond, or a straight chain alkylene of 1-70 carbon atoms in length, or one or more carbon atoms in said straight chain alkylene is / are replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 alkylenedioxy, C2-C 10 alkenylen, C2-C 10 alkynylen, C6-C 10 arylen, C3-C 18 heteroarylen, and C5-C 10 heteroarylen; and said straight chain alkylene can have one or more substituents selected from the group consisting of C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C10 alkyl, -SC1-C 10 alkyl, -SC1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halo substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl).

[0173] each said conjugate linker L B is independently one or more of a covalent bond or the following linking combinations: C1-C 10a straight-chain alkylene, a phosphonate bond, a phosphorothioate bond, an amide bond, an ester bond, an ether bond, a disulfide bond, a 1,2,3-triazoleylene, a polyethylene glycolylene, a pyrrolidineylene, a 2-oxopyrrolidineylene, a phenylene, a cyclohexylene, a 2-succinimideylene, a 2-thiosuccinimideylene, an amino acidylene, a nucleotideylene.

[0174] In some embodiments, k is an integer from 1 to 3; L C contains any one of the groups as shown in formulae (L1)-(L3) via an ether bond in the group as shown in formulae (L1)-(L3) to L A are directly connected.

[0175] denotes the site at which the group is attached to the remainder of the molecule;

[0176] In some embodiments, k = 1, L C contains the group as shown in formula (L1), the O atom in the group (L1) and L A are directly connected. In some embodiments, k = 2, L C contains the group as shown in formula (L2), each of the 2 O atoms in the group (L1) and 1 L A are directly connected. In some embodiments, k = 4, L C contains the group as shown in formula (L3), each of the 3 O atoms in the group (L3) and 1 L A are directly connected.

[0177] L B is a phosphonate bond or a disulfide bond;

[0178] each L A is a covalent bond, or each L A is selected from the group consisting of the groups (L4)-(L23) and combinations of their connections:

[0179] wherein each j1 is an integer from 1 to 10;

[0180] each R' is a C1-C 10 alkyl group;

[0181] each Ra is a hydrogen atom, a C1-C 10 alkyl group, or is selected from the group consisting of the groups (L24)-(L37):

[0182] In some embodiments, L A has a length of 3-35 atoms, wherein the L A length refers to the L A group.C directly connected atoms to L A directly connected atoms to R L the number of chain atoms in the longest chain of atoms formed by the directly connected atoms. In some embodiments, each L A is a linking combination of at least 2 of groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each L A is a linking combination of at least 2 of groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).

[0183] In some embodiments, L A has a structure comprising an amide bond as shown in formula (302), L B has a structure comprising an N-acyl pyrrolidine as shown in formula (303), containing a carbonyl and an oxygen atom, L C is a linking group based on hydroxymethyl aminomethane, dimethylol aminomethane, or trimethylol aminomethane:

[0184] wherein n 302 , q 302 and p 302 are each independently an integer from 2 to 6, optionally n 302 , q 302 and p 302 are each independently 2 or 3; n 303 is an integer from 4 to 16, optionally n 303 is an integer from 8 to 12, denotes the site of covalent attachment of a group.

[0185] In some embodiments, each of said side chain moieties L A is attached to one R L group via a phosphodiester linkage, an ether linkage, or an ester linkage, and is attached to said backbone moiety L C via the oxygen atom of a hydroxyl group in said backbone moiety L C forms an ether linkage with said backbone moiety L B forms an amide bond with the nitrogen atom of an amino group in said backbone moiety L C forms a phosphodiester linkage, an ether linkage, or an ester linkage with said functional group A0 via the oxygen atom in formula (303). In some embodiments, the backbone moiety L C is a linking group based on hydroxymethyl aminomethane, dimethylol aminomethane, or trimethylol aminomethane, said backbone moiety L C is attached to each of said side chain moieties L Aare connected via ether bonds and via the nitrogen atom of the amino group to the conjugation linker L B are connected via amide bonds. Thus, the linker group R j 1-3 side chains are attached to the same carbon atom of the aminomethyl group and are connected via the conjugation linker L B to R L the ligand group.

[0186] In some embodiments, the conjugate has a structure according to Formula (305):

[0187] In some embodiments, the linker group R j comprises a structure according to Formula (306):

[0188] wherein n 306 = n0-1, each p 306 is independently an integer from 1 to 6, denotes the point of covalent attachment of the group; the combination of linkages formed by all pyrrolidinylene groups and any possible phosphodiester groups constitutes the backbone portion, each side chain portion is constituted by the atomic chain between the carbonyl group attached to the nitrogen atom of the pyrrolidinylene group and the oxygen atom marked with * and is connected to the ligand group R L by a phosphoester bond, ether bond or ester bond; at least one of the oxygen atoms marked with # is a conjugation linker and is connected to the functional group A0by an ether bond, ester bond or phosphoester bond, the remaining oxygen atoms marked with # are connected to a hydrogen atom to form a hydroxyl group or to a C1-C3alkyl group to form a C1-C3alkoxy group. Thus, the linker group R j 1-3 side chain portions are attached to different carbon atoms of the backbone portion and are connected to the ligand group R L by an oxygen atom.

[0189] In some embodiments, the linker group R j comprises a structure according to Formula (306-1):

[0190] In some embodiments, the conjugate provided by the present disclosure has a structure according to Formula (307a), (307b) or (307c):

[0191] In some embodiments, the conjugate provided by the present disclosure has a structure according to Formula (307a-1), (307b-1) or (307c-1):

[0192] In some embodiments, the conjugate provided by the present disclosure has a structure as shown in formula (308):

[0193] wherein n 308 = m0+ n0- 1. In some embodiments, n 308 is an integer from 1 to 6, considering the easiness of synthesis, the cost of structure / process, and the specificity of tumor cells, etc. In some embodiments, n 308 is an integer from 2 to 6. Further, in some embodiments, n 308 is 3 or 4.

[0194] each R3is independently a functional group A0, or is an R L group. In some embodiments, at least one R3is a functional group A0, and at least one R3is an R L group. In some embodiments, one R3is a functional group A0, and the rest of R3is an R L group. Further, in some embodiments, 2-4 R3is an R L group, and the rest of R3is a functional group A0.

[0195] In some embodiments, when each m 308 is independently selected from an integer from 2 to 10, it is considered that the spatial position between the plurality of ligand groups R L in the conjugate is more suitable for binding to integrin αvβ6. In order to make the compound represented by formula (308) simpler, easier to synthesize and / or reduce the cost, according to some embodiments of the present disclosure, each m 308 is independently an integer from 2 to 5, and in some embodiments, each m 308 is the same.

[0196] It can be understood by those skilled in the art that when each R 308 is independently selected from H, C1-C 10 alkyl, C1-C 10 haloalkyl and C1-C 10 alkoxy, the properties of the conjugate represented by formula (308) are not changed, and the purpose of the present disclosure can be achieved. In some embodiments, each R 308 is independently selected from H, methyl or ethyl. In some embodiments, each R 308 is H.

[0197] each L1connected to the functional group A0represents the conjugate connection, and each L1connected to the R L represents the side chain moiety. In some embodiments, one R3is the functional group A0, and the rest of R3is the RL group. In some embodiments, one or more L1s serve as the side chain moiety, linking the R L group to the N atom on the nitrogen-containing backbone; and another one or more L1s serve as the conjugation linker, linking the functional group A0 to the N atom on the nitrogen-containing backbone. The nitrogen-containing backbone collectively constitutes the backbone portion of the linking group R j In the context of the present disclosure, "nitrogen-containing backbone" refers to the chain structure in the structure of Formula (308) in which the carbon atom of R 308 is linked to the N atom.

[0198] In some embodiments, each L1 is independently 3-25 atoms in length, from the perspective of delivery efficiency and synthesis cost. In some embodiments, each L1 is independently 4-17 atoms in length. In some embodiments, each L1 is independently 4-15 atoms in length. Those skilled in the art will appreciate that, although L1 is defined as a linear alkylene for convenience, it can not be a linear group or an amine or alkenyl group resulting from the above-mentioned substitutions and / or replacements, for example, due to the different name. For the purpose of the present disclosure, the length of L1 is the number of atoms in the chain connecting the two connection points. For this purpose, a ring (such as a heterocyclylene or heteroarylene) resulting from the replacement of a carbon atom of the linear alkylene is counted as the length of the corresponding portion of the ring in the chain in terms of the minimum number of atoms between the connection points on the ring.

[0199] In some embodiments, L1 is selected from the group consisting of the groups of Formulae (L4)-(L23) above and any connecting combination thereof. In some embodiments, each L1 is independently selected from the group consisting of connecting combinations of at least two of groups (L4)-(L9), (L13), (L14), (L18). In some embodiments, each L1 is independently a connecting combination of at least two of groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).

[0200] In some embodiments, in the conjugate of Formula (308), each L1 contains both a connection site to the N atom on the nitrogen-containing backbone and a connection site to the functional group A0 or the R L group, and the site connected to the N atom on the nitrogen-containing backbone forms an amide bond with the N atom. In some embodiments, the one or more L1s connected to the functional group A0 are selected from B5, B6, B5', or B6':

[0201] wherein, the site of covalent attachment of the represented group, and q2 is an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.

[0202] In some embodiments, the compound of Formula (308) contains multiple functional groups. In some embodiments, each functional group in the compound of Formula (308) is the same functional group. In some embodiments, each functional group in the compound of Formula (308) is a functional group for the same purpose and function. In some embodiments, the compound of Formula (308) contains different kinds of functional groups for different purposes and functions.

[0203] In some embodiments, the compound of Formula (308) has a structure of Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423), (424), (425), (426), or (427):

[0204] In some embodiments, the linking group R j In some embodiments, the linking group R j is selected from one or more of N-succinimidyl 4-(2- dithiopyridyl)butanoate (SPDB), N-succinimidyl 4-(2- thiopyridyl)pentanoate (SPP), (S)-2-((S)-2-amino-3- methylbutyramido)-5-ureidopentanoate (Val-Cit), an activatable enzyme linker, a sulfatase-cleavable linker, a galactoside-cleavable linker, a lysosomal protease- sensitive linker, a peptidyl linker, a glucuronide linker, an acid-sensitive cleavable linker, a glutathione-sensitive disulfide linker, or a noncleavable linker. Such linking groups R jDescribed in the literature“Mckertish CM, Kayser V. Advances and Limitations of Antibody Drug Conjugates for Cancer. Biomedicines. 2021 Jul 23;9(8):872”, the entire contents of which are incorporated herein by reference.

[0205] One or more functional groups can be included in the conjugates of the present disclosure. In the context of the present disclosure, a functional group refers to a radical containing a free radical formed by removal of one atom, functional group, or moiety from an active agent molecule having corresponding functionality (e.g., a delivery aid compound, a diagnostic agent compound, a therapeutic agent compound, and / or a functional oligonucleotide compound that modulates expression levels of a relevant gene), such as removal of one hydrogen atom, and the functional group forms a covalent linkage with the rest of the molecule of the conjugates of the present disclosure through the free radical. In some embodiments, each of the functional groups is independently selected from at least one of a diagnostic agent group, a therapeutic agent group, an oligonucleotide group, and a delivery aid group.

[0206] In some embodiments, at least one of the functional groups is a delivery aid group selected from one or more of a C8-C 30 alkyl or alkenyl group, a cholesterol group, a lipid group, a palmitic acid group, and a cholic acid group. The conjugates of the present disclosure with a delivery aid group are better compatible with the intracellular environment of the integrin ανβ6-expressing cells, have better bioavailability, and / or enable more efficient delivery of the conjugates of the present disclosure into the relevant targeted cells.

[0207] In some embodiments, at least one of the functional groups is a diagnostic agent group, each of which is independently selected from at least one of a contrast agent group, an isotope tracer, and a fluorescent tracer. By including a diagnostic agent group, the conjugates of the present disclosure are able to deliver the diagnostic agent group to the relevant targeted cells and / or tissues, thereby specifically, efficiently, and accurately diagnosing the progression status and / or symptom information of the relevant disease.

[0208] In some embodiments, at least one of the functional groups is a therapeutic agent group, each of which is selected from at least one of a cytotoxic group, an antibiotic group, an angiogenesis inhibitor, an antibody drug group, and a group comprising a radioisotope. By including the disease therapeutic agent group, the conjugate of the present disclosure is able to specifically deliver the therapeutic agent group to the targeted cells and / or tissues, thereby treating and / or alleviating the relevant disease process or symptoms through the action of the therapeutic agent group. For example, by specifically delivering a cytotoxic group to the relevant cells and / or tissues via the conjugate of the present disclosure, the targeted cells are specifically eliminated, thereby significantly reducing the number of targeted cells while reducing the side effects of low targeting of the cytotoxic itself, thereby treating the relevant disease, symptoms, and / or disorders.

[0209] In some embodiments, at least one of the functional groups is an oligonucleotide group. The oligonucleotide group is able to modulate the level of a target mRNA in a cell that expresses integrin αvβ6. By including the oligonucleotide group, the conjugate of the present disclosure is able to specifically deliver the oligonucleotide group to the cell that expresses integrin αvβ6, thereby modulating the level of the relevant target mRNA in the cell, such as inhibiting the expression of an oncogene, through the action of the oligonucleotide group, such as an RNA interference effect, thereby treating and / or alleviating the relevant disease process or symptoms. In some embodiments, the functional oligonucleotide group is an siRNA group. The siRNA group refers to a chemical moiety formed by the loss of one or more atoms or functional groups from an siRNA molecule.

[0210] In some embodiments, the ligand group is linked to the siRNA group via a linker group in the conjugate. In some embodiments, the linker group is linked to the sense strand or the antisense strand of the siRNA. In some embodiments, the linker group is linked to the sense strand of the siRNA, thereby minimizing the impact on the activity of the siRNA. In some embodiments, the linker group is linked to the end of the sense strand or the antisense strand of the siRNA, which refers to the first 4 nucleotides from the end of the sense strand or the antisense strand. In some embodiments, the linker group is linked to the terminal nucleotide of the sense strand or the antisense strand, which refers to the first nucleotide from the end of the sense strand or the antisense strand. In some embodiments, the linker group is linked to the interior of the sense strand or the antisense strand of the siRNA, which refers to any nucleotide in the sense strand or the antisense strand other than the terminal nucleotide.

[0211] The siRNA group can be linked to the nucleotide at any position. In some embodiments, the linking group is linked to the ribose ring 2', 3' or 5' position of the nucleotide by replacing one hydrogen atom. In some embodiments, the linking group is linked to the base or phosphate group of the nucleotide.

[0212] The siRNA group included in the conjugate of the present disclosure can be any siRNA group capable of modulating the level of target mRNA in integrin ανβ6-expressing cells. In some embodiments, the siRNA includes, but is not limited to, one of the siRNAs targeting ACE2, ENaC a, SOD1, SARS-CoV-2 or RAGE mRNA. In some embodiments, the siRNA is an siRNA targeting SOD1 mRNA. In some embodiments, the siRNA is an siRNA having the following SEQ ID NO: 5 and SEQ ID NO: 6 for the sense and antisense strands, respectively. In some embodiments, the siRNA is an siRNA targeting ENaC a or RAGE mRNA.

[0213] In some embodiments, the integrin ανβ6-expressing cell described above is an epithelial cell or a muscle cell. In some embodiments, the epithelial cell is selected from one or more of alveolar epithelial 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 tumor cells. In some embodiments, the integrin ανβ6-expressing cell described above is an alveolar epithelial cell. In some embodiments, the muscle cell is, for example, a skeletal muscle cell.

[0214] The functional group can be included in the conjugate of the present disclosure by any suitable means. For example, the functional group A0may be linked to the backbone moiety by the conjugation linker described above.

[0215] One of skill in the art can employ any reasonable synthetic route to prepare the conjugate provided by the present disclosure.

[0216] In some embodiments, the method for synthesizing the conjugate provided by the present disclosure comprises contacting a protected conjugate with a deprotection reagent in a solvent under deprotection reaction conditions, and isolating to obtain the conjugate provided by the present disclosure. The protected conjugate is a compound in which any reactive functional group in the conjugate provided by the present disclosure is protected by a protecting group. In some embodiments, the reactive functional group includes, but is not limited to, a hydroxyl group, an amino group, and / or a phosphoric acid group, and the protecting group is a hydroxyl protecting group, an amino protecting group, and / or a phosphoric acid protecting group (e.g., a cyanoethyl protecting group), respectively. The solvent, deprotection reaction conditions, and deprotection reagent are selected and determined according to the protecting group. In some embodiments, the method comprises adding the protected conjugate into a mixed solution of an aqueous methylamine solution and aqueous ammonia, and the deprotection reaction conditions include reacting at room temperature and atmospheric pressure for 1-5 h. In some embodiments, the mixed solution is obtained by mixing the aqueous methylamine solution and saturated concentrated aqueous ammonia in equal volume, and the amount of the solution relative to the protected conjugate is 0.1-10 mL / μmol. In some embodiments, the isolation comprises purification by column chromatography separation, and the product eluate is collected and the solvent is removed. The purification conditions can be, for example, using a preparative ion chromatography purification column, and eluting with a gradient eluent of an aqueous sodium chloride solution and an aqueous sodium phosphate solution. In some embodiments, eluent A: 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); eluent B: 1.5 M sodium chloride, 20 mM sodium phosphate (pH 8.1), solvent: water / acetonitrile = 9:1 (volume ratio); elution gradient: eluent A:eluent B = 100:0-50:50 gradient elution.

[0217] In some embodiments, the conjugate provided by the present disclosure has a structure shown in formula (3), and the method for synthesizing the protected conjugate comprises: contacting a compound comprising an active group R x1 and a ligand group with a compound comprising an active group R x2 and a functional group in an organic solvent under coupling reaction conditions, and reacting to obtain the protected conjugate. Wherein the ligand group is formed by removing one hydrogen atom or one functional group from a compound provided by formula (1) below, and each of the functional groups is independently one of a diagnostic agent group, a therapeutic agent group having a therapeutic effect on a pulmonary disease, and a functional oligonucleotide group having a therapeutic effect on a pulmonary disease, wherein any active group in the ligand group and the functional group is protected by a protecting group, and the active group R x1 and the active group R x2 are groups capable of forming a covalent bond or a linking group R j through reaction. In some embodiments, the active group R x1 may be an azido group, and the active group Rx2 may be an alkyne group; or, the active group R x1 may be an amino group, the active group R x2 may be a hydroxyl group. In some embodiments, the active group R x1 is an active functional group in the compound of the present disclosure. In some embodiments, the molar ratio of the ligand group with the active group R x1 and the functional group with the active group R x2 is 1.5m0-3m0: n0, in some embodiments, the molar ratio of the ligand group with the active group R x1 and the functional group with the active group R x2 is 1.5-3: 1, or 3: 1.

[0218] In some embodiments, the coupling reaction conditions are condensation reaction conditions or conditions of a thiol-disulfide exchange reaction.

[0219] In some embodiments, the coupling reaction conditions are condensation reaction conditions, the condensation reaction conditions are acylation condensation reaction conditions, dehydration condensation reaction conditions, or conditions of a click chemistry reaction, the active group R x1 and the active group R x2 are groups capable of undergoing the aforementioned condensation reaction. In some embodiments, the condensation reaction conditions are conditions of an acylation condensation reaction, the active group R x1 and R x2 are groups capable of undergoing an acylation condensation reaction to form R j . In some embodiments, the condensation reaction conditions are conditions of a dehydration condensation reaction, one of the active groups R x1 and R x2 is a group comprising an acyl halide group or a carboxyl group, and the other is a group comprising an amino group or a hydroxyl group. In some embodiments, the condensation reaction conditions are conditions of a click chemistry reaction, one of the active groups R x1 and R x2 is a group comprising an alkyne group, and the other is a group comprising an azido group. In some embodiments, the condensation reaction conditions are conditions of a Michael addition reaction, one of the active groups R x1 and R x2 is a group comprising a thiol group, and the other is a group comprising a succinimidyl group. In some embodiments, the condensation reaction conditions are conditions of an N-hydroxysuccinimidyl-carbodiimide (NHS-EDC) coupled reaction, one of the active groups R x1 and R x2 is a group comprising an N-hydroxysuccinimidyl (NHS) group, and the other is a group comprising a carbodiimidyl (EDC) group.

[0220] In some embodiments, the active group R is included x1 Compounds with ligand groups are formed by coupling reactions in which the active group R is attached. 10 The compound provided in this disclosure is prepared by contacting it with a crosslinking agent, wherein the crosslinking agent contains an active group R. x1 and acylated groups. The active group R 10 The active group R is covalently linked to the acylated group through a coupling reaction, thereby forming a covalent bond. x1 Linked to compounds disclosed herein. In some embodiments, an active group R is included. x2 The compound containing functional groups is prepared by contacting a compound having condensation-reactive groups and functional groups with a crosslinking agent under coupling reaction conditions, wherein the crosslinking agent contains the active group R. x2 And acylated groups, wherein the condensation reactive group can react with the acylated group to form a covalent bond.

[0221] In some embodiments, the active group R x1 Or R x2 It is an active group containing 1-3 click-active chemical groups at its terminal, wherein the click-active chemical groups include a terminal alkynyl group. In some embodiments, the acylation group is a carboxyl group, acyl chloride group, or active ester group, such as one of NHS ester group, imine ester group, and pentafluorophenyl ester group. Those skilled in the art can obtain the crosslinking agent by various methods. For example, when the acylation group is a pentafluorophenyl ester group and the click-active chemical group includes a terminal alkynyl group, the crosslinking agent can be prepared as follows: The 5'-GalNAc conjugated antisense oligonucleotides were prepared by the method described in Scheme 1a(A) of Michael E., et al., "Efficient synthesis and biological evaluation of 5'-GalNAc conjugated antisense oligonucleotides." Bioconjugate Chemistry 26.8(2015):1451-1455, the entire contents of which are incorporated herein by reference. In some embodiments, the active group R... 10 It is an amino group. In some embodiments, the coupling condition is a basic condition. In some embodiments, the basic condition is the presence of a weakly alkaline aqueous solution, such as an aqueous solution of sodium bicarbonate.

[0222] In some embodiments, the coupling reaction conditions are one of the mercapto-disulfide bond exchange reactions, wherein the active group Rx1 and R x2 one is a group comprising a thiol group and the other a leaving group linked via a disulfide bond.

[0223] The compound comprising an active group R x2 and a functional group can be obtained by the skilled person in various ways. In some embodiments, the coupling reaction conditions are thiol-disulfide exchange reaction conditions, the active group R x2 comprises a thiol group, and the compound comprising an active group R x2 and a functional group can be obtained by the skilled person in various known ways, for example by preparation using a phosphoramidite monomer comprising a thiol group by phosphoramidite solid phase synthesis methods, or by being commercially available. In some embodiments, the functional group is a functional oligonucleotide group, the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions, the active group R x1 is a hydroxyl group, and the active group R x2 is a phosphoramidite group, the method of preparation comprising sequentially linking nucleoside monomers to a solid support linked to a ligand group and an active group R x1 in accordance with the nucleic acid sequence of the functional oligonucleotide in accordance with the phosphoramidite solid phase synthesis reaction. In some embodiments, the functional group is a diagnostic agent group or a therapeutic agent group, the coupling reaction conditions are phosphoramidite solid phase synthesis reaction conditions, the active group R x2 is a phosphoramidite group, the compound comprising an active group R x2 and a functional group can be, for example, a compound comprising a phosphoramidite group and a fluorescent group or a therapeutic agent group, which is readily commercially available. In some embodiments, the coupling reaction conditions are Michael addition reaction conditions, the active group R x2 is an N-succinimidyl group, and the compound comprising an active group R x2 and a functional group can be, for example, a compound comprising an N-succinimidyl group and a therapeutic agent group, which is readily commercially available. In some embodiments, the functional group is a functional oligonucleotide group, the active group R x1 is an N-succinimidyl group, and the compound comprising an active group R x2nucleotide sequence I and a nucleotide sequence II, each comprising 5-25 modified or unmodified nucleotides, the nucleotide sequence I and the nucleotide sequence II being at least partially reverse complementary, the ligand group being attached to the nucleotide sequence I, the functional group being attached to the nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II not eliciting an immune response or a toxic response in a subject, the coupling reaction condition being a reaction condition for annealing to form a double-stranded nucleic acid. In some embodiments, the functional oligonucleotide group is an siRNA group, the 5' end of the nucleotide sequence I is attached to the ligand group via a phosphodiester bond, and the 3' end of the nucleotide sequence II is attached to the 5' end of the siRNA via a phosphodiester bond. In some embodiments, the nucleotide sequence I and the nucleotide sequence II each consist of 17 nucleotides and are fully reverse complementary. In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively:

[0224] 5'-GUACAUUCUAGAUAGCC-3' (SEQ ID NO: 1)

[0225] 5'-GGCUAUCUAGAAUGUAC-3' (SEQ ID NO: 2).

[0226] In some embodiments, the nucleotide sequence I and the nucleotide sequence II have the sequences set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively:

[0227] 5'-GmUfAmCfAmUfUfCfUfAmGmAmUfAmGmCfCf-3' (SEQ ID NO: 3)

[0228] 5'-GmGmCfUfAmUfCfUfAmGmAmAmUfGmUfAmCf-3' (SEQ ID NO: 4).

[0229] wherein capital letters C, G, U, A represent the base composition of the nucleotides; lower case letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; and lower case letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluoro modified nucleotide.

[0230] In some embodiments, when the functional group in the conjugate is an siRNA group, the synthesis process for siRNA can be carried out according to the description in Example 1 section of WO2019010274A1, with the only difference that the sense strand and antisense strand sequences of siRNA involved in the conjugate according to the present disclosure are synthesized respectively for the sense strand and antisense strand of siRNA.

[0231] In some embodiments, the conjugate can also be used in the present disclosure in the form of its pharmaceutically acceptable salt or precursor compound. In the context of the present disclosure, "pharmaceutically acceptable salt" refers to the formation of a corresponding salt of a drug in order to increase the stability, solubility and / or bioavailability of the drug, which does not produce additional side effects on the human body in pharmacy, such as potassium salt, sodium salt or carboxylic acid salt. In some embodiments, the conjugate of the present disclosure contains free carboxyl or amino groups. In some embodiments, the free carboxyl or amino groups are protected by a protecting group. These carboxyl or amino groups can form a salt with a corresponding base or acid. In some embodiments, in the conjugate described in the present disclosure, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate bond between each adjacent nucleotide carries a negative charge, which can exist in the form of hydroxyl or mercapto, and the hydrogen ion in the hydroxyl or mercapto can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, an ammonium ion NH4 +The compound is an organic ammonium cation. For the purpose of improving solubility and / or bioavailability, in some embodiments, the pharmaceutically acceptable salt is a partial or complete water-soluble salt of the conjugate. In some embodiments, the water-soluble salt may be an amine salt, an alkali metal salt, or an alkaline earth metal salt. In some embodiments, the amine salt is selected from one or more of ammonium salts, methylamine salts, tertiary amine salts, and quaternary ammonium salts; the alkali metal salt is selected from potassium salts or sodium salts; and the alkaline earth metal salt is selected from calcium salts or magnesium salts. In some embodiments, the tertiary amine salt is triethylamine salt, triisopropylamine salt, or N,N-diisopropylethylamine salt. In some embodiments, the pharmaceutically acceptable salt of the conjugate is a sodium salt or a partial sodium salt of the conjugate. A "precursor compound" refers to a compound that, although not structurally or functionally identical to the conjugate, can react upon entering the body or in a body fluid environment to form the conjugate comprising this disclosure, thereby exerting its effects and achieving the purpose of this disclosure. In some cases, these precursor compounds can increase drug stability, prolong sustained-release time, and increase bioavailability. In some embodiments, the precursor compound includes a precursor group that can react in vivo to form all functional groups A0 in the conjugate. In some embodiments, the precursor compound includes a compound formed by replacing all active hydroxyl groups in the conjugate with acetoxy groups. In some embodiments, the precursor compound includes a pharmaceutical precursor group, which is a residue formed from a delivery adjuvant, diagnostic agent, therapeutic agent, and / or oligonucleotide precursor compound corresponding to the functional group in the conjugate. In some embodiments, the pharmaceutical precursor group can be, for example, a group formed by replacing the active hydrogen in the hydroxyl or amino functional group of the functional group with an acyl, alkyl, or phosphoryl group. Those skilled in the art will understand that the use of these pharmaceutically acceptable salts and precursor compounds is also within the scope of this disclosure.

[0232] Pharmaceutical Composition

[0233] In another aspect, this disclosure also provides a pharmaceutical composition comprising the conjugates of this disclosure and a pharmaceutically acceptable carrier.

[0234] The pharmaceutically acceptable carrier can be a carrier conventionally used in the art, for example, but not limited to, water, physiological saline, magnetic nanoparticles (e.g., nanoparticles based on Fe3O4or Fe2O3), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine) (PLL), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly(D&L-lactic / glycolic acid) copolymer (PLGA), poly(2-aminoethyl ethylene phosphate) (PPEEA), and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA), and derivatives thereof.

[0235] In some embodiments, the pharmaceutically acceptable carrier contains a physiologically acceptable compound that functions, for example, to stabilize the pharmaceutical composition or to increase or decrease the absorption of the conjugate and / or pharmaceutical composition. The physiologically acceptable compound is selected from one or more of the following: carbohydrates, such as glucose, sucrose, and / or dextrans; antioxidants, such as ascorbic acid and / or glutathione; chelating agents; low molecular weight proteins; compositions that decrease the clearance or hydrolysis of any co-administered substances; excipients; stabilizers and buffers. A detergent can also be used to stabilize the composition or to increase or decrease the absorption of the pharmaceutical composition. The physiologically acceptable compound can also include one or more of a humectant, an emulsifier, a dispersant, or a preservative specifically to prevent the growth or action of microorganisms. The physiologically acceptable compound is known to one of skill in the art, and the present disclosure does not further elaborate. It is readily understood by one of skill in the art that the selection of the pharmaceutically acceptable carrier and the physiologically acceptable compound depends on, for example, the route of administration and the specific physiochemical properties of any co-administered substances.

[0236] In some embodiments, the pharmaceutically acceptable carrier is sterile and generally free of undesirable matter. The pharmaceutical compositions of the present disclosure can further comprise, as desired, pharmaceutically-acceptable auxiliary substances to approximate a physiologic condition, including but not limited to one or more of a pH adjusting agent, a buffer, and a toxicity adjusting agent, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, or sodium lactate, for example, and the concentration of the conjugate of the present disclosure in the pharmaceutical composition can vary within wide limits and is generally selected according to the special mode of administration, depending on the fluid volume, the viscosity, the body weight, and the like.

[0237] In some embodiments, the content of the conjugate and the pharmaceutically acceptable carrier in the pharmaceutical composition is not particularly limited, and in some embodiments, the weight ratio of the conjugate to the pharmaceutically acceptable carrier can be 1 : (1-500), and in some embodiments, the above weight ratio is 1 : (1-50).

[0238] In some embodiments, the pharmaceutical composition can further comprise a pharmaceutically acceptable other auxiliary material, which is one or more of various ingredients conventionally used in the art, such as one or more of a diluent, a binder, a disintegrant, a lubricant, a capsule, a protective agent, an osmotic pressure adjusting agent, or a solvent.

[0239] For example, when the pharmaceutical composition is an injection solution, the pharmaceutically acceptable auxiliary material includes a solvent such as deionized water, water for injection, ethanol, or a pH buffer.

[0240] The pH buffer can be a tris-hydroxymethyl aminomethane hydrochloride buffer having a pH of 7.5-8.5 and / or a phosphate buffer having a pH of 5.5-8.5, and for example, can be a phosphate buffer having a pH of 5.5-8.5. The amount of the solvent is adjusted according to the desired concentration of the solution, and the concentration of the siRNA or siRNA group in the injection solution is 0.01 mg / mL-5 mg / mL siRNA, 0.1 mg / mL-5 mg / mL, or 0.5 mg / mL-3 mg / mL of the solution, based on the siRNA or siRNA group.

[0241] The protective agent protects the siRNA or siRNA conjugate by weakening crystallization or reducing physical damage. The protective agent can be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose, and glucose. The content of the protective agent can be 0.01-30% by weight, based on the total weight of the pharmaceutical composition.

[0242] The osmotic pressure adjusting agent can be sodium chloride and / or potassium chloride. The content of the osmotic pressure adjusting agent is such that the osmotic pressure of the pharmaceutical composition is 200-700 milliosmoles per kilogram (mOsm / kg). The content of the osmotic pressure adjusting agent can be readily determined by one skilled in the art according to the desired osmotic pressure. In some embodiments, the dosage of the pharmaceutical composition during administration is adjusted according to the mode of administration.

[0243] In some embodiments, the pharmaceutical composition can be a liquid formulation, such as an injection solution, or a lyophilized powder formulation that is mixed with a liquid excipient to prepare a liquid formulation for administration. The liquid formulation can be, but is not limited to, used for subcutaneous, intramuscular, or intravenous injection administration, and can be, but is not limited to, used for delivery of the pharmaceutical composition through puncture injection, or through oropharyngeal inhalation or nasal administration. In some embodiments, the pharmaceutical composition is used for subcutaneous, intramuscular, or intravenous injection administration.

[0244] In some embodiments, the pharmaceutical composition can be in the form of a liposome formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (hereinafter can also be referred to as an organic amine), a helper lipid, and / or a pegylated lipid. Among them, the organic amine, the helper lipid, and the pegylated lipid can be selected from one or more of the amine-containing transfection compounds or pharmaceutically acceptable salts or derivatives thereof, the helper lipids, and the pegylated lipids described in Chinese Patent Application CN103380113A (which is incorporated by reference in its entirety into the present disclosure).

[0245] In some embodiments, the organic amine can be a compound as shown in formula (201) or a pharmaceutically acceptable salt thereof described in Chinese Patent Application CN103380113A:

[0246] wherein:

[0247] X 101 and X 102 each independently is O, S, N-A, or C-A, wherein A is hydrogen or a C1-C 20 hydrocarbon chain;

[0248] Y 101 and Z 101 each independently is C=O, C=S, S=O, CH-OH, or SO2;

[0249] R 101 , R 102 , R 103 , R 104 , R 105 , R 106 and R107 each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group, a substituted or unsubstituted, branched or unbranched acyl group, a substituted or unsubstituted, branched or unbranched aryl group, a substituted or unsubstituted, branched or unbranched heteroaryl group;

[0250] x is an integer from 1 to 10;

[0251] n is an integer from 1 to 3, m is an integer from 0 to 20, and p is 0 or 1; wherein, if m = p = 0, then R 102 is hydrogen;

[0252] and, if at least one of n or m is 2, then R 103 and the nitrogen in formula (401) forms a structure as shown in formula (402) or formula (403):

[0253] wherein g, e, and f are each independently an integer from 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (401).

[0254] In some embodiments, R 103 is a polyamine. In other embodiments, R 103 is a ketal. In some embodiments, R 101 and R 102 each independently is any substituted or unsubstituted, branched or unbranched alkyl or alkenyl group having from 3 to about 20 carbon atoms, such as from 8 to about 18 carbon atoms, and from 0 to 4 double bonds, such as from 0 to 2 double bonds.

[0255] In some embodiments, if each of n and m independently has a value of 1 or 3, then R 103 may be any one of the following formulae (404)-(413):

[0256] wherein in formulae (404)-(413), g, e, and f are each independently an integer from 1 to 6, each "HCC" represents a hydrocarbon chain, and each * shows a possible point of attachment to the nitrogen atom in formula (401), wherein each H at any * location can be replaced to effect attachment to the nitrogen atom in formula (401). 103

[0257] ​The compound of formula (401) can be obtained by any reasonable method by one skilled in the art. In some embodiments, the compound of formula (401) can be prepared according to the description in Chinese patent application CN103380113A.

[0258] In some embodiments, the organic amine is an organic amine of formula (414) and / or an organic amine of formula (415):

[0259] The helper lipid is cholesterol, an analog of cholesterol, and / or a derivative of cholesterol;

[0260] The PEGylated lipid is l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)]-2000.

[0261] In some embodiments, the molar ratio between the organic amine, the helper lipid, and the PEGylated lipid in the pharmaceutical composition is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).

[0262] In some embodiments, the pharmaceutical composition particles formed by the conjugate of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150, or 160 nm.

[0263] In some embodiments, in the pharmaceutical composition formed by the conjugate of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight / weight ratio) of the conjugate to all lipids (e.g., organic amine, helper lipid, and / or PEGylated lipid) is in the range of from about 1: 1 to about 1:50, from about 1: 1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the conjugate of the present disclosure to all lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, or 1:18.

[0264] In some embodiments, the pharmaceutical composition can exist in separate components at the time of sale, and can exist in the form of a liquid preparation at the time of use. In some embodiments, the pharmaceutical composition of the conjugate provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, only by replacing the existing conjugate with the conjugate provided by the present disclosure; in some embodiments, it can be prepared according to the following method:

[0265] The organic amine, auxiliary lipid and polyethylene glycol lipid are suspended in alcohol according to the above-mentioned molar ratio and mixed to obtain a lipid solution; the amount of alcohol is such that the total mass concentration of the obtained lipid solution is 2-25 mg / mL, for example, it can be 8-18 mg / mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid near room temperature, for example, one or more of ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, for example, it can be ethanol.

[0266] The conjugate provided by the present disclosure is dissolved in a buffered salt solution to obtain a conjugate aqueous solution. The concentration of the buffered salt solution is 0.05-0.5 M, for example, it can be 0.1-0.2 M, the pH of the buffered salt solution is adjusted to 4.0-5.5, for example, it can be 5.0-5.2, the amount of buffered salt solution is such that the concentration of the conjugate does not exceed 0.6 mg / mL, for example, it can be 0.2-0.4 mg / mL. The buffered salt is selected from one or more of soluble acetate and soluble citrate, for example, it can be sodium acetate and / or potassium acetate.

[0267] The lipid solution and the conjugate aqueous solution are mixed, and the product obtained after mixing is incubated at 40-60°C for at least 2 minutes, for example, it can be 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the conjugate aqueous solution is 1:(2-5), for example, it can be 1:4.

[0268] The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilized to obtain the pharmaceutical composition provided by the present disclosure, and the physicochemical parameters are as follows: the pH value is 6.5-8, the encapsulation efficiency is not less than 80%, the particle size is 40-200 nm, the polydispersity index is not higher than 0.30, and the osmotic pressure is 250-400 mOsm / kg; for example, the physicochemical parameters can be as follows: the pH value is 7.2-7.6, the encapsulation efficiency is not less than 90%, the particle size is 60-100 nm, the polydispersity index is not higher than 0.20, and the osmotic pressure is 300-400 mOsm / kg.

[0269] The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The method of removing impurities can employ various methods available, for example, a flow fractionation system, a hollow fiber column, ultrafiltration under 100 KDa conditions, and the exchange solution for ultrafiltration is phosphate buffer (PBS) at pH 7.4. The method of sterilization can employ various methods available, for example, sterilization by filtration on a 0.22 μιη filter.

[0270] Use of the conjugate and / or the pharmaceutical composition of the present disclosure

[0271] In yet another aspect, the present disclosure also provides use of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for diagnosing a disease, a symptom and / or a disorder, wherein at least one of the functional groups is a diagnostic agent group that can be used to diagnose the disease, the symptom and / or the disorder upon delivery to a cell expressing ανβ6. In some embodiments, each of the diagnostic agent groups is independently selected from one of a contrast agent group, an isotope tracer, and a fluorescent tracer.

[0272] In yet another aspect, the present disclosure also provides use of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, a symptom and / or a disorder, wherein at least one of the functional groups is a therapeutic agent group that can be used to treat the disease, the symptom and / or the disorder upon delivery to a cell expressing ανβ6. In some embodiments, each of the therapeutic agent groups is selected from one of a cytotoxic agent group, an antibiotic agent group, an angiogenesis inhibitor, an antibody drug group, and a group comprising a radioisotope.

[0273] In yet another aspect, the present disclosure also provides use of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure in the manufacture of a medicament for treating a disease, a symptom and / or a disorder, wherein at least one of the functional groups is an oligonucleotide group that treats the disease, the symptom and / or the disorder by adjusting the level of the target mRNA in the cell expressing ανβ6. In some embodiments, the oligonucleotide group is an siRNA group.

[0274] In yet another aspect, the present disclosure also provides a method of diagnosing a disease, a symptom or a disorder, comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure, wherein the functional group is a diagnostic agent group that can be used to diagnose the disease, the symptom and / or the disorder upon delivery to a cell expressing ανβ6.

[0275] In yet another aspect, the present disclosure also provides a method of treating a disease, a symptom, or a disorder, comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure, wherein the functional group is a therapeutic group, the therapeutic group is useful for treating the disease, the symptom, and / or the disorder upon delivery to the avb6-expressing cell.

[0276] In yet another aspect, the present disclosure also provides a method of treating a disease, a symptom, or a disorder, comprising administering to a subject in need thereof an effective amount of the conjugate of the present disclosure and / or the pharmaceutical composition of the present disclosure, wherein the functional group is an oligonucleotide group, the oligonucleotide group is useful for treating the disease, the symptom, and / or the disorder by adjusting the level of the target mRNA in the avb6-expressing cell.

[0277] In some embodiments, the avb6-expressing cell is an epithelial cell or a muscle cell. In some embodiments, the epithelial cell is one or more of an alveolar epithelial cell, a secretory epithelial cell, a ciliated epithelial cell, a corneal and conjunctival epithelial cell, a dermal epithelial cell, a bile duct epithelial cell, an intestinal epithelial cell, a ductal epithelial cell, a glandular epithelial cell, or an epithelial tumor cell. In some embodiments, the muscle cell is a skeletal muscle cell.

[0278] In some embodiments, the disease, the symptom, and / or the disorder comprises at least one of pulmonary fibrosis, asthma, chronic pulmonary obstruction, pneumonia, a tumor, and a muscle-related disease.

[0279] In some embodiments, the functional group in the conjugate of the present disclosure is an oligonucleotide group, the oligonucleotide group is capable of adjusting the level of a target mRNA in the epithelial cell or the muscle cell, the target mRNA comprising but not limited to SOD1 mRNA, ACE-2 mRNA, ENaC mRNA, and SARS-CoV-2 mRNA.

[0280] In some embodiments, the disease, the symptom, and / or the disorder comprises at least one of pulmonary fibrosis, asthma, chronic pulmonary obstruction, pneumonia, a tumor, and a muscle-related disease.

[0281] In some embodiments, the methods of the present disclosure are effective to treat, diagnose, and / or prevent diseases, conditions, and / or disorders associated with epithelial cells or muscle cells by administering a conjugate and / or a pharmaceutical composition comprising the same. In some embodiments, the conjugate in the methods comprises an oligonucleotide group as a functional group. The conjugate of the present disclosure can reduce the distribution of the diagnostic agent and / or the therapeutic agent at other undesired organs / tissues of the body, and reduce potential side effects, due to its high specificity in targeting integrin ανβ6. This is particularly important and valuable for radiotherapy and / or chemotherapy drugs that are commonly used in the field of related disease treatment and have significant known side effects.

[0282] The term "administering" as used herein refers to the placement of a conjugate and / or a pharmaceutical composition into a subject's body by a method or route that results, at least in part, in localization of the conjugate and / or the pharmaceutical composition to a desired site to produce a desired effect. Suitable routes of administration for the methods of the present disclosure include local and systemic administration. In general, local administration results in delivery of more of the conjugate and / or the pharmaceutical composition to a particular site as compared to the entire body of the subject, while systemic administration results in delivery of the conjugate and / or the pharmaceutical composition to substantially the entire body of the subject.

[0283] The subject can be administered by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including buccal and sublingual administration). The frequency of administration can be one or more times per day, per week, per two weeks, per three weeks, per month, or per year.

[0284] The dosage of the conjugate and / or the pharmaceutical composition of the present disclosure can be a conventional dosage in the art, which can be determined based on various parameters, especially the age, weight, and gender of the subject. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage range of the human use can be derived from the data obtained from the cell culture assays and animal studies.

[0285] The amount of the conjugate used can be 0.001 to 100 mg / kg of body weight, in some embodiments 0.01 to 50 mg / kg of body weight, in further embodiments 0.05 to 20 mg / kg of body weight, in still further embodiments 0.1 to 15 mg / kg of body weight, in yet further embodiments 0.1 to 10 mg / kg of body weight, in terms of the amount of the conjugate in the conjugate and / or pharmaceutical composition, when the conjugate and / or pharmaceutical composition of the present disclosure is administered, for example, to a male or female, 6-12 week old, 18-25 g body weight C57BL / 6J or C3H / HeNCrlVr mouse. The above-mentioned amount can be preferred when the conjugate and / or pharmaceutical composition of the present disclosure is administered.

[0286] Kit

[0287] The present disclosure also provides a kit containing the conjugate and / or pharmaceutical composition of the present disclosure.

[0288] In some embodiments, the kit of the present disclosure can provide the conjugate and / or pharmaceutical composition in one container. In some embodiments, the kit of the present disclosure can include a container providing a pharmaceutically acceptable excipient. In some embodiments, the kit can further include other ingredients, such as a stabilizer or a preservative. In some embodiments, the kit of the present disclosure can include at least one other therapeutic agent in a container different from the container providing the conjugate and / or pharmaceutical composition of the present disclosure. In some embodiments, the kit can include an instruction for mixing the conjugate and / or pharmaceutical composition with a pharmaceutically acceptable carrier and / or an excipient or other ingredients, if any.

[0289] In the kit of the present disclosure, the conjugate and the pharmaceutically acceptable carrier and / or excipient and the pharmaceutical composition, and / or the pharmaceutically acceptable excipient can be provided in any form, for example, in a liquid form, a dry form or a lyophilized form. In some embodiments, the conjugate and the pharmaceutically acceptable carrier and / or excipient and the pharmaceutical composition and the optional pharmaceutically acceptable excipient are substantially pure and / or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.

[0290] The present disclosure will be further illustrated by the following examples, but the present disclosure is not in any way limited thereto.

[0291] Example

[0292] Unless otherwise specified, the reagents, media used in the following examples are commercially available, and the nucleic acid electrophoresis, real-time PCR and other operations are performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).

[0293] The reagents and sources of reagents used in the embodiments of the present disclosure are as follows:

[0294] (S)-3-amino-3-(4-bromophenyl)-propionic acid, purchased from Beijing Coupling Technology Co., Ltd.;

[0295] Boc-L-cyclopropylglycine (N-tert-butoxycarbonyl-L-cyclohexylglycine), purchased from Beijing Coupling Technology Co., Ltd.;

[0296] Boc-glycine (N-(tert-butoxycarbonyl) amino acetic acid), purchased from Beijing Coupling Technology Co., Ltd.;

[0297] 2-methylthiopyrimidine-5-carboxaldehyde, purchased from Beijing Coupling Technology Co., Ltd.;

[0298] 2-bromopyridine-5-carboxaldehyde, purchased from Beijing Coupling Technology Co., Ltd.;

[0299] 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (abbreviation XPhos), purchased from Beijing Coupling Technology Co., Ltd.;

[0300] Tetrakis triphenylphosphine palladium (abbreviation Pd(Ph3P)4), purchased from Beijing Coupling Technology Co., Ltd.;

[0301] 2-(7-azobenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (abbreviation HATU), purchased from Beijing Coupling Technology Co., Ltd.;

[0302] N,N,-diisopropyl ethylamine (abbreviation DIPEA), purchased from Beijing Coupling Technology Co., Ltd.;

[0303] Cuprous (I) thiophene-2-carboxylate (abbreviation Cu u T), purchased from Beijing Coupling Technology Co., Ltd.;

[0304] Azido-pentaethylene glycol-p-toluenesulfonyl ester (abbreviation N3-PEG5-Tos), purchased from Beijing Coupling Technology Co., Ltd.;

[0305] Azido-pentaethylene glycol-p-toluenesulfonyl ester (abbreviation N3-PEG5-Tos), purchased from Beijing Coupling Technology Co., Ltd.;

[0306] Thionyl chloride (abbreviation: SOCl2), purchased from Beijing Coupling Technology Co., Ltd.

[0307] Preparation Example 1-6 Synthesis process of the ligand compound provided by the present disclosure

[0308] Preparation Example 1 Synthesis of ligand compound 1

[0309] In this preparation example, the synthesis process of ligand compound 1 is as follows:

[0310] Compound 4 (0.5 g, 1.44 mmol), compound 5 (0.356 g, 1.73 mmol) and HATU (0.657 g, 1.73 mmol) were added to 10 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (0.558 g, 4.32 mmol) was slowly added dropwise, and the resulting reaction solution was stirred at room temperature for 2 hours. Then 20 mL of water was added to the reaction solution, and extracted with ethyl acetate three times (3 x 10 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered, concentrated, and then column chromatography was performed, and the elution solvent was methanol in dichloromethane at a volume ratio of 0-6%, to obtain 0.5 g of white solid powder, which was compound 6, with a yield of 70%. ESI-MS (m / z): 503.1 [M+H] + .

[0311] In this example, compound 4 can be synthesized as follows:

[0312] Compound 1 (2.42 g, 10 mmol) was added to 30 mL of methanol, and the resulting reaction solution was stirred at 0°C, and thionyl chloride (3.57 g, 30 mmol) was slowly added dropwise to the reaction solution. After the reaction solution returned to room temperature, it was stirred overnight. Then, the reaction solution was concentrated under reduced pressure to remove the solvent, to obtain 2.4 g of white solid powder, which was compound 2, with a yield of 95%. It was directly used in the next step without purification. ESI-MS (m / z): 258.1 [M+H] + .

[0313] Compound 2 (2 g, 7.8 mmol), Boc-glycine (1.6 g, 9.36 mmol) and HATU (3.54 g, 9.36 mmol) were added into 50 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (3 g, 23.4 mmol) was slowly added dropwise into the mixture, and the resulting reaction solution was stirred at room temperature for 2 hours. Then, 30 mL of water was added into the reaction solution, and the reaction solution was extracted with ethyl acetate for three times (3 x 30 mL), and the organic phase was separated. The organic phase was washed with 20 mL of saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and then subjected to column chromatography separation, and eluted with a gradient of ethyl acetate to petroleum ether (0:100-50:50) to obtain 2.57 g of white solid powder, which was compound 3, with a yield of 80%. ESI-MS (m / z): 415.1 [M+H] + .

[0314] Compound 3 (2 g, 4.8 mmol) was dissolved in 4 M, 10 mL of hydrogen chloride in dioxane, and the resulting reaction solution was stirred at room temperature for 2 hours, and then concentrated to obtain 1.6 g of light yellow foamy solid compound 4, with a yield of 100%, which was directly used in the next step without purification.

[0315] In this embodiment, compound 5 can be synthesized by the following route:

[0316] 2-Amino-3-pyridine carboxaldehyde (5 g, 41 mmol), methyl acetoacetate (6 g, 41 mmol) and L-proline (2.35 g, 20.5 mmol) were dissolved in 120 mL of anhydrous ethanol, respectively, and the resulting reaction solution was heated to reflux and stirred overnight. After the reaction solution was returned to room temperature, it was concentrated, 100 mL of ethyl acetate and 50 mL of brine were added, and the resulting organic phase was washed with 50 mL of saturated brine, dried with anhydrous sodium sulfate, filtered, concentrated, and then subjected to column chromatography separation, and eluted with a gradient of ethyl acetate to petroleum ether (0-50%) to obtain 4.5 g of white solid, which was compound 5-1, with a yield of 50%. ESI-MS (m / z): 231.1 [M+H] + .

[0317] Compound 5-1 (4 g) was dissolved in 20 mL of methanol, and 20 mL of 1 N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 3.5 hours. After the reaction was completed, most of the methanol was evaporated, and the pH of the reaction solution was adjusted to 6-7 with 1 N hydrochloric acid solution under ice bath, and a large amount of white solid was precipitated. The white solid was filtered to obtain 3 g of white solid, which was compound 5-2, with a yield of 83%. ESI-MS (m / z): 203.1 [M+H] + .

[0318] Compound 5-2 (3 g) was dissolved in methanol, 10% platinum oxide was added, and the reaction solution was stirred at room temperature overnight after three times of hydrogen exchange. After the reaction was completed, the reaction solution was filtered and concentrated to obtain 3 g of light yellow solid, which was compound 5, with a yield of 95%. ESI-MS (m / z): 213.1 [M+H] + . 1 H-NMR (400MHz, DMSO-d6) δ 7.03 (d, J = 7.3 Hz, 1H), 6.31 (s, 1H), 6.28 (d, J = 7.3 Hz, 1H), 3.24 (d, J = 5.7 Hz, 2H), 2.67 (t, J = 7.7 Hz, 2H), 2.61 (t, J = 6.3 Hz, 2H), 2.47 (t, J = 7.6 Hz, 2H), 1.76 (p, J = 6.0 Hz, 2H).

[0319] Compound 6 (500 mg, 1 mmol), compound 7 (554 mg, 2 mmol), XPhos (24 mg, 0.05 mmol), palladium acetate (11 mg, 0.05 mmol) and potassium phosphate (633 mg, 3 mmol) were added to 15 mL of a mixed solvent of tetrahydrofuran and water (volume ratio of tetrahydrofuran: water = 3:1) to obtain a reaction solution. The reaction solution was heated to 90°C under argon protection and stirred for 16 hours until the reaction was complete. The reaction solution was concentrated after being cooled to room temperature. 20 mL of water was added to the concentrated reaction solution, which was extracted with ethyl acetate three times (3 x 10 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried with anhydrous sodium sulfate, and filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 490 mg of light yellow solid powder, which was compound 8, with a yield of 75%. ESI-MS (m / z): 657.3 [M+H] + .

[0320] Compound 8 (490 mg, 0.75 mmol) was dissolved in 10 mL of anhydrous methanol, and palladium-carbon was added, with the weight ratio of palladium-carbon to compound 8 being 10%, and then three times of hydrogen exchange was performed, and the reaction solution was stirred at room temperature overnight. After filtration and concentration, 423 mg of off-white solid powder was obtained, which was compound 9, with a yield of 100%. ESI-MS (m / z): 567.3 [M+H] + .

[0321] Compound 9 (400 mg, 0.71 mmol), compound 10 (383 mg, 0.92 mmol) and potassium carbonate (294 mg, 2.13 mmol) were dissolved in 10 mL of DMF to obtain a reaction solution. The reaction solution was heated to 100°C and stirred for 6 hours, and then after the reaction solution was cooled to room temperature, 10 mL of water was added and extracted with 200 mL of ethyl acetate to separate the organic phase. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 450 mg of a light yellow foamy solid, which was compound 11, with a yield of 78%. ESI-MS (m / z): 812.4 [M+H] + .

[0322] Compound 11 (400 mg, 0.49 mmol) was dissolved in 3 mL of methanol, and 3 mL of 1N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 1 hour, and then the pH of the reaction solution was adjusted to 6-7 with 1N hydrochloric acid solution in an ice bath. 10 mL of dichloromethane was added to the reaction solution to extract the organic phase. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered, concentrated to obtain 380 mg of a white solid, which was ligand compound 1, with a yield of 95%.

[0323] ESI-MS (m / z): 798.4 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.74-8.62 (m, 2H), 8.27 (m, 2H), 8.16-8.07 (m, 2H), 7.86 (dd, J = 8.2, 2.4 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.56-7.47 (m, 3H), 7.08 (dd, J = 7.9, 2.1 Hz, 2H), 6.87 (s, 1H), 6.30 (d, J = 7.3 Hz, 1H), 5.27 (q, J = 7.2 Hz, 1H), 4.35 (t, J = 4.6 Hz, 2H), 3.93 (t, J = 4.5 Hz, 2H), 3.81-3.65 (m, 5H), 3.61-3.54 (m, 6H), 3.52 (d, J = 3.7 Hz, 6H), 3.36 (t, J = 4.9 Hz, 3H), 3.22 (d, J = 6.1 Hz, 3H), 2.82 (dd, J = 6.9, 3.5 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 2.46 (dd, J = 7.9, 3.0 Hz, 2H), 1.73 (p, J = 6.1 Hz, 2H).

[0324] Synthesis of ligand compound 2

[0325] In this preparation example, the synthesis process of ligand compound 2 is as follows:

[0326] Compound 2 (0.5 g, 2 mmol), Boc-L-cyclopropylglycine (0.5 g, 2.4 mmol) and HATU (0.9 g, 2.4 mmol) were added to 40 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (0.75 g, 6 mmol) was slowly added dropwise. The resulting reaction solution was stirred at room temperature for 2 hours. Then, 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate three times (3 x 10 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed, and the eluent solvent was ethyl acetate in petroleum ether at a volume ratio of 0-50%, to obtain 0.7 g of white solid powder, which was compound 12, with a yield of 70%. ESI-MS (m / z): 455.1 [M+H] + .

[0327] Compound 12 (0.6 g, 1.3 mmol) was dissolved in 4 M, 5 mL of hydrogen chloride in dioxane, and the resulting reaction solution was stirred at room temperature for 2 hours and concentrated to obtain a light yellow foamy solid, which was compound 13, which was directly used in the next step without purification. Compound 13 (0.468 g, 1.2 mmol), compound 5 (0.3 g, 1.44 mmol) and HATU (0.65 g, 1.44 mmol) were added to 20 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (0.62 g, 4.8 mmol) was slowly added dropwise. The resulting reaction solution was stirred at room temperature for 2 hours. 10 mL of water was added to the reaction solution, which was extracted with ethyl acetate three times (3 x 10 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed, and the eluent solvent was methanol in dichloromethane at a volume ratio of 0-6%, to obtain 0.488 g of white solid powder, which was compound 14, with a yield of 75%. ESI-MS (m / z): 543.15 [M+H] + .

[0328] Compound 14 (0.4 g, 0.74 mmol), compound 7 (0.41 g, 1.48 mmol), XPhos (18 mg, 0.037 mmol), palladium acetate (10 mg, 0.037 mmol) and potassium phosphate (0.47 g, 2.22 mmol) were added into 15 mL of a mixed solvent of tetrahydrofuran and water (volume ratio of tetrahydrofuran: water = 3: 1) respectively to obtain a reaction solution. The reaction solution was heated to 90°C under argon protection and stirred for 16 hours until the reaction was completed. The reaction solution was cooled to room temperature and concentrated, 10 mL of water was added to the concentrated reaction solution, extracted with ethyl acetate three times (3 x 10 mL), the organic phase was combined, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, the volume ratio of methanol to dichloromethane was 0-6%, to obtain 0.36 g of light yellow solid powder, which was compound 15, with a yield of 70%. ESI-MS (m / z): 697.3 [M+H] + .

[0329] Compound 15 (0.3 g, 0.43 mmol) was dissolved in 6 mL of anhydrous methanol, and palladium-carbon was added, wherein the weight ratio of palladium-carbon to compound 15 was 10%, and then hydrogen was introduced three times, the obtained reaction solution was stirred at room temperature overnight, and then filtered and concentrated to obtain 0.26 g of off-white solid powder, which was compound 16, with a yield of 100%. ESI-MS (m / z): 607.3 [M+H] + .

[0330] Compound 16 (200 mg, 0.33 mmol), compound 10 (179 mg, 0.43 mmol) and potassium carbonate (137 mg, 0.99 mmol) were respectively dissolved in 10 mL of DMF, the obtained reaction solution was heated to 100°C and stirred for 6 hours, after cooling to room temperature, 10 mL of water was added and extracted with 20 mL of ethyl acetate, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, the volume ratio of methanol to dichloromethane was 0-6%, to obtain 196 mg of light yellow foamy solid, which was compound 17, with a yield of 70%. ESI-MS (m / z): 852.4 [M+H] + .

[0331] Compound 17 (150 mg, 0.18 mmol) was dissolved in 3 mL of methanol, and 3 mL of 1 N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to 6-7 with 1 N hydrochloric acid solution in an ice bath, and 10 mL of dichloromethane was added for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain 140 mg of a white solid, which was ligand compound 2, with a yield of 95%. ESI-MS (m / z): 838.4 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.77 (d, J = 8.0 Hz, 1H), 8.67 (d, J = 2.3 Hz, 1H), 8.38-8.24 (m, 2H), 8.11 (dd, J = 7.2, 2.4 Hz, 1H), 7.86 (dd, J = 8.2, 2.4 Hz, 1H), 7.61-7.48 (m, 5H), 7.07 (dd, J = 9.5, 7.7 Hz, 2H), 6.64 (s, 1H), 6.30 (d, J = 7.3 Hz, 1H), 5.26 (q, J = 7.3 Hz, 1H), 4.34 (t, J = 4.5 Hz, 2H), 3.93 (dd, J = 5.7, 3.4 Hz, 2H), 3.83 (t, J = 8.2 Hz, 1H), 3.69 (dd, J = 5.9, 3.7 Hz, 2H), 3.63-3.48 (m, 12H), 3.36 (m, 4H), 3.23 (d, J = 5.8 Hz, 2H), 2.81 (d, J = 7.1 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 1.74 (t, J = 5.9 Hz, 2H), 1.04-0.95 (m, 1H), 0.45 (td, J = 10.7, 9.9, 5.4 Hz, 2H), 0.39 (dd, J = 8.3, 4.9 Hz, 2H).

[0332] Synthesis of ligand compound 3 of Preparation Example 3

[0333] In this preparation example, the synthesis of ligand compound 3 was as follows:

[0334] Compound 18 (18.5 g, 100 mmol), malonic acid (20.8 g, 200 mmol) and ammonium acetate (15.4 g, 200 mmol) were dissolved in 150 mL of anhydrous ethanol, respectively, and the resulting reaction solution was heated to reflux and stirred overnight to generate a large amount of white solid. After returning to room temperature, the reaction solution was concentrated, slurried with 50 mL of ethyl acetate, and filtered to obtain 25 g of white solid, which was compound 19, with a yield of 100%. ESI-MS (m / z): 244.98 [M+H] + .

[0335] Compound 19 (25 g, 100 mmol) was added to 100 mL of methanol, and the resulting reaction solution was stirred at 0°C. Sulfurous acid chloride (35.7 g, 300 mmol) was slowly added dropwise to the reaction solution, which was returned to room temperature and stirred overnight. The solvent was removed by reduced pressure concentration to obtain a white solid powder, which was compound 20, with a yield of 95%, which was directly used in the next step without purification. ESI-MS (m / z): 259.1 [M+H] + .

[0336] Compound 20 (10 g, 39 mmol), Boc-L-glycine (8.2 g, 46.8 mmol) and HATU (17.78 g, 46.8 mmol) were added to 70 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (15 g, 117 mmol) was slowly added dropwise thereto. The resulting reaction solution was stirred at room temperature for 2 hours. 30 mL of water was added to the reaction solution, which was extracted with ethyl acetate three times (3 x 60 mL), and the organic phase was separated. The organic phase was washed with 30 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed with an eluent of ethyl acetate to petroleum ether in a volume ratio of 0-50% to obtain 12 g of white solid powder with a yield of 75%. Chiral resolution (column type: CHIRALPAK AD, column length: 3 cm x 25 cm, 5 μm, mobile phase A: carbon dioxide, mobile phase B: isopropyl alcohol, flow rate: 200 mL / min, wavelength: UV 220 nm, temperature: 25°C, and Prep-SFC-350 type preparative chromatography) was performed to obtain 5 g of compound 22 with a yield of 80%. ESI-MS (m / z): 416.1 [M+H] + .

[0337] Compound 22 (500 mg, 1.2 mmol) was dissolved in 4 M hydrogen chloride in dioxane (5 mL) and the resulting reaction solution was stirred at room temperature for 2 hours. After the solvent was concentrated, a light yellow foamy solid was obtained, which was compound 23, and was directly used in the next step without purification. Compound 23 (450 mg, 1.2 mmol), compound 5 (316 mg, 1.44 mmol) and HATU (550 mg, 1.44 mmol) were added to 20 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (621 mg, 4.8 mmol) was slowly added dropwise. The resulting reaction solution was stirred at room temperature for 2 hours. 20 mL of water was added to the reaction solution, which was extracted with ethyl acetate three times (3 x 15 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0-6%, to obtain 424 mg of white solid powder, which was compound 24, with a yield of 70%. ESI-MS (m / z): 504.1 [M+H] + .

[0338] Compound 24 (400 mg, 0.8 mmol), compound 7 (445 mg, 1.6 mmol), XPhos (20 mg, 0.04 mmol), palladium acetate (10 mg, 0.04 mmol) and potassium phosphate (509 mg, 2.4 mmol) were added to 10 mL of a mixed solvent of tetrahydrofuran and water (volume ratio of tetrahydrofuran: water = 3:1) to obtain a reaction solution. The reaction solution was heated to 90°C under argon protection and stirred for 16 hours until the reaction was complete. After the reaction solution was cooled to room temperature, it was concentrated, 20 mL of water was added, and the organic phase was extracted with ethyl acetate three times (3 x 30 mL), combined, washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 360 mg of light yellow solid powder, which was compound 25, with a yield of 70%. ESI-MS (m / z): 658.3 [M+H] + .

[0339] Compound 25 (300 mg, 0.46 mmol) was dissolved in 10 mL of anhydrous methanol and palladium-carbon was added, with the weight ratio of palladium-carbon to compound 25 being 10%, and then hydrogen was introduced three times. The resulting reaction solution was stirred at room temperature overnight, and then filtered and concentrated to obtain 250 mg of off-white solid powder, which was compound 26, with a yield of 95%. ESI-MS (m / z): 568.2 [M+H] + .

[0340] Compound 26 (200 mg, 0.35 mmol), compound 10 (194 mg, 0.7 mmol) and potassium carbonate (145 mg, 1.05 mmol) were dissolved in 10 mL of DMF, respectively, and the resulting reaction solution was heated to 100°C and stirred for 6 hours. The reaction solution was cooled to room temperature, 10 mL of water and 20 mL of ethyl acetate were added for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed for separation, and the elution solvent was methanol in dichloromethane at a volume ratio of 0-7%. 198 mg of light yellow foamy solid was obtained, which was compound 27, and the yield was 70%. ESI-MS (m / z): 813.4 [M+H] + .

[0341] Compound 27 (150 mg, 0.18 mmol) was dissolved in 3 mL of methanol, 3 mL of 1N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to 6-7 with 1N hydrochloric acid solution in an ice bath, 15 mL of dichloromethane was added for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 140 mg of white solid, which was ligand compound 3, and the yield was 95%. ESI-MS (m / z): 799.4 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 8.74-8.62 (m, 2H), 8.27 (dd, J = 6.6, 3.0 Hz, 2H), 8.16-8.07 (m, 1H), 7.86 (dd, J = 8.2, 2.4 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.56-7.47 (m, 3H), 7.08 (dd, J = 7.9, 2.1 Hz, 2H), 6.87 (s, 1H), 6.30 (d, J = 7.3 Hz, 1H), 5.27 (q, J = 7.2 Hz, 1H), 4.35 (t, J = 4.6 Hz, 2H), 3.93 (t, J = 4.5 Hz, 2H), 3.81-3.65 (m, 5H), 3.61-3.54 (m, 6H), 3.52 (d, J = 3.7 Hz, 6H), 3.36 (t, J = 4.9 Hz, 3H), 3.22 (m, 2H), 2.82 (dd, J = 6.9, 3.5 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 1.73 (p, J = 6.1 Hz, 2H).

[0342] Synthesis of ligand compound 4

[0343] In the preparation example, the synthesis process of the ligand compound 4 is as follows:

[0344] 2-methyl mercapto pyrimidine-5-formaldehyde (15.4 g, 100 mmol), compound 7 (55.6 g, 200 mmol), CuCl2(28.7 g, 150 mmol) and (Ph3P)4Pd (5.7 g, 5 mmol) were added into 300 mL of anhydrous tetrahydrofuran respectively, heated to 70°C under argon protection, stirred for 16 hours until the reaction was complete to obtain a reaction solution. After the reaction solution was cooled to room temperature, 100 mL of water and 150 mL of ethyl acetate were added for extraction, filtration, separation of the organic phase in the filtrate. The organic phase was washed with 50 mL of saturated brine, dried with anhydrous sodium sulfate, and filtered, concentrated, and then column chromatography separation was performed with elution solvent, the volume ratio of ethyl acetate to petroleum ether was 0-50%, to obtain 20 g of light yellow solid powder, which was compound 28, with a yield of 60%. ESI-MS (m / z): 341.1 [M+H] + .

[0345] Compound 28 (20 g, 59 mmol), malonic acid (12.2 g, 118 mmol) and ammonium acetate (9.1 g, 118 mmol) were dissolved in 120 mL of anhydrous ethanol respectively to obtain a reaction solution which was heated to reflux and stirred overnight to generate a large amount of white solid. After being restored to room temperature, the reaction solution was concentrated, slurried with 50 mL of ethyl acetate, and filtered to obtain 9 g of white solid, which was compound 29, with a yield of 40%, which was directly used in the next step without purification. ESI-MS (m / z): 400.1 [M+H] + .

[0346] Compound 29 (9 g, 22 mmol) was added to 50 mL of methanol, and thionyl chloride (8 g, 66 mmol) was slowly added dropwise under stirring at 0°C to obtain a reaction solution which was stirred overnight after being restored to room temperature. Then the solvent was removed by reduced pressure concentration to obtain white solid powder, which was compound 30, with a yield of 95%, which was directly used in the next step without purification. ESI-MS (m / z): 414.2 [M+H] + .

[0347] Compound 30 (9 g, 22 mmol), Boc-glycine (4.23 g, 26 mmol) and HATU (10 g, 26 mmol) were added into 150 mL of anhydrous dichloromethane, and N,N-diisopropyl ethylamine (8.5 g, 66 mmol) was slowly added dropwise. The resulting reaction solution was stirred at room temperature for 2 hours and concentrated. 20 mL of water was added to the concentrated reaction solution, and extracted with ethyl acetate three times (3 x 30 mL), and the organic phase was separated. The organic phase was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed, and the eluent was methanol in dichloromethane at a volume ratio of 0:100-50:50, to obtain 8.7 g of white solid powder, which was compound 31, with a yield of 70%. Compound 31 was subjected to chiral resolution to obtain 4 g of compound 32, with a yield of 90%. ESI-MS (m / z): 416.1 [M+H] + .

[0348] In the reaction process, the chiral resolution method includes: the column type is CHIRALPAK AD, the column length is 3 cm x 25 cm, 5 μm, the mobile phase A is carbon dioxide, the mobile phase B is isopropyl alcohol, the flow rate is 200 mL / min, the wavelength is UV 220 nm, and the temperature is 25 °C. The preparation chromatography model is Prep-SFC-350.

[0349] Compound 32 (500 mg, 0.88 mmol) was dissolved in 4 M, 5 mL of hydrogen chloride in dioxane, and the resulting reaction solution was stirred at room temperature for 2 hours and concentrated to remove the solvent to obtain a light yellow foamy solid, which was compound 33, which was directly used in the next step without purification. Compound 33 (446 mg, 0.88 mmol), compound 5 (217 mg, 1 mmol) and HATU (401 mg, 1 mmol) were added into 30 mL of anhydrous dichloromethane, and N,N-diisopropyl ethylamine (454 mg, 3.52 mmol) was slowly added dropwise. The resulting reaction solution was stirred at room temperature for 2 hours, concentrated to remove the solvent, and then 20 mL of water was added, extracted with ethyl acetate three times (3 x 20 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed, and the eluent was methanol in dichloromethane at a volume ratio of 0-6%, to obtain 405 mg of white solid powder, which was compound 34, with a yield of 70%. ESI-MS (m / z): 659.3 [M+H] + .

[0350] Compound 34 (400 mg, 0.61 mmol) was dissolved in 10 mL of anhydrous methanol, and palladium-carbon was added, wherein the weight ratio of the added palladium-carbon to compound 34 was 10%, and then hydrogen was introduced for three times, and the obtained reaction solution was stirred at room temperature overnight. Then after filtration and concentration, 340 mg of off-white solid powder was obtained, which was compound 35, and the yield was 95%. ESI-MS (m / z): 569.2 [M+H] + .

[0351] Compound 35 (300 mg, 0.53 mmol), compound 10 (287 mg, 0.69 mmol) and potassium carbonate (219 mg, 1.59 mmol) were respectively dissolved in 10 mL of DMF, and the obtained reaction solution was heated to 100°C and stirred for 6 hours. After the reaction solution was cooled to room temperature, 15 mL of water and 30 mL of ethyl acetate were added for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried with anhydrous sodium sulfate, and then filtered, concentrated, and then subjected to column chromatography separation, and the elution solvent was methanol with respect to dichloromethane at a volume ratio of 0-6%, to obtain 280 mg of light yellow foamy solid, which was compound 36, and the yield was 65%. ESI-MS (m / z): 814.4 [M+H] + .

[0352] Compound 36 (200 mg) was dissolved in 3 mL of methanol, and 3 mL of 1N NaOH solution was added, and the obtained reaction solution was stirred at room temperature for 1 hour, and then the pH of the reaction solution was adjusted to 6-7 with 1N hydrochloric acid solution in an ice bath. 20 mL of dichloromethane was added to the reaction solution for extraction, and the organic phase was separated. The organic phase was washed with 15 mL of saturated brine, dried with anhydrous sodium sulfate, and then filtered and concentrated, to obtain 186 mg of white solid, which was ligand compound 4, and the yield was 95%. ESI-MS (m / z): 800.36 [M+H] + . 1H NMR (500 MHz, DMSO-d6) δ 8.91 (s, 2H), 8.82 (d, J = 8.2 Hz, 2H), 8.37-8.25 (m, 2H), 8.10 (d, J = 8.2 Hz, 1H), 7.57 (p, J = 6.8 Hz, 2H), 7.13 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 7.00 (s, 1H), 6.31 (d, J = 7.3 Hz, 1H), 5.23 (q, J = 6.9 Hz, 1H), 4.37 (t, J = 4.5 Hz, 2H), 3.94 (t, J = 4.6 Hz, 2H), 3.71 (dt, J = 20.9, 5.6 Hz, 4H), 3.63-3.46 (m, 12H), 3.36 (t, J = 4.9 Hz, 3H), 3.22 (d, J = 5.8 Hz, 3H), 2.85 (dd, J = 10.6, 6.6 Hz, 2H), 2.72 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 6.3 Hz, 2H), 1.81-1.64 (m, 2H).

[0353] Synthesis of Ligand Compound 5

[0354] In this preparation example, the synthesis process of ligand compound 5 is as follows:

[0355] Compound 26 (100 mg, 0.176 mmol), compound 37 (75 mg, 0.23 mmol) and potassium carbonate (73 mg, 0.53 mmol) were dissolved in 10 mL of DMF respectively, and the resulting reaction solution was heated to 100°C and stirred for 6 hours. After the reaction solution was cooled to room temperature, 10 mL of water and 20 mL of ethyl acetate were added for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed for separation, and the elution solvent was methanol in dichloromethane at a volume ratio of 0-6%. 90 mg of light yellow foamy solid was obtained, which was compound 38, and the yield was 70%. ESI-MS (m / z): 725.3 [M+H] + .

[0356] Compound 38 (80 mg, 0.11 mmol) was dissolved in 3 mL of methanol, and 3 mL of 1N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to 6-7 with 1N hydrochloric acid solution in an ice bath. 10 mL of dichloromethane was added to the reaction solution for extraction, and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and 70 mg of white solid was obtained, which was ligand compound 5, and the yield was 95%. ESI-MS (m / z): 711.3 [M+H]+ . 1 H NMR (500 MHz, DMSO-d6) δ 8.74-8.62 (m, 2H), 8.27 (dd, J = 6.6, 3.0 Hz, 2H), 8.16-8.07 (m, 1H), 7.86 (dd, J = 8.2, 2.4 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.56-7.47 (m, 3H), 7.08 (dd, J = 7.9, 2.1 Hz, 2H), 6.87 (s, 1H), 6.30 (d, J = 7.3 Hz, 1H), 5.27 (q, J = 7.2 Hz, 1H), 4.35 (t, J = 4.6 Hz, 2H), 3.93 (t, J = 4.5 Hz, 2H), 3.81-3.65 (m, 5H), 3.61-3.54 (m, 4H), 3.36 (t, J = 4.9 Hz, 3H), 3.22 (d, J = 6.1 Hz, 3H), 2.82 (dd, J = 6.9, 3.5 Hz, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.60 (t, J = 6.3 Hz, 2H), 2.46 (dd, J = 7.9, 3.0 Hz, 2H), 1.73 (p, J = 6.1 Hz, 2H).

[0357] Synthesis of ligand compound 6 in Preparation Example 6

[0358] In this preparation example, the synthesis process of ligand compound 6 is as follows:

[0359] The preparation process of this preparation example is carried out according to the method for preparing ligand compound 3 in Preparation Example 3, except that the step of chiral resolution is not carried out to obtain ligand compound 6 with racemic configuration, and the yield of ligand compound 6 obtained in the preparation process is 95%.

[0360] wherein compound 45 is obtained after acidification reaction of compound 21 in Preparation Example 3, and the reaction process comprises: dissolving compound 21 (500 mg, 1.2 mmol) in 4 M, 5 mL hydrogen chloride solution in dioxane, stirring the obtained reaction solution at room temperature for 2 hours, and obtaining light yellow foamy solid after concentrating the solvent, which is compound 45, and directly used in subsequent reaction without purification.

[0361] Synthesis of control ligand compound 1 in Comparative Preparation Example 1

[0362] In this preparation example, the synthesis process of control ligand compound 1 is as follows:

[0363] To compound 1 (2.42 g, 10 mmol) was added 30 mL of methanol, the resulting reaction solution was stirred at 0 °C, and sulfurous anhydride (3.57 g, 30 mmol) was slowly added dropwise to the reaction solution. After the reaction solution returned to room temperature, it was stirred overnight. Then, the reaction solution was concentrated under reduced pressure, and the solvent was removed to obtain 2.4 g of white solid powder, which was compound 2, with a yield of 95%. It was directly used in the next step without purification. ESI-MS (m / z): 258.1 [M+H] + .

[0364] Compound 2 (2 g, 7.8 mmol), Boc-glycine (1.6 g, 9.36 mmol), and HATU (3.54 g, 9.36 mmol) were added to 50 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (3 g, 23.4 mmol) was slowly added dropwise to the mixture. The resulting reaction solution was stirred at room temperature for 2 hours. Then, 30 mL of water was added to the reaction solution, and the reaction solution was extracted with ethyl acetate three times (3 x 30 mL), and the organic phase was separated. The organic phase was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of ethyl acetate to petroleum ether being 0:100-50:50 gradient elution to obtain 2.57 g of white solid powder, which was compound 3, with a yield of 80%. ESI-MS (m / z): 415.1 [M+H] + .

[0365] Compound 3 (2 g, 4.8 mmol) was dissolved in 4 M, 10 mL of hydrogen chloride in dioxane, and the resulting reaction solution was stirred at room temperature for 2 hours. After concentration, 1.6 g of light yellow foamy solid compound 4 was obtained, with a yield of 100%, which was directly used in the next step without purification.

[0366] Compound 4 (1.67 g, 4.8 mmol), compound 39 (1.6 g, 5.76 mmol), and HATU (2.2 g, 5.76 mmol) were added to 30 mL of anhydrous dichloromethane, respectively, and N,N-diisopropyl ethylamine (2.2 g, 16.8 mmol) was slowly added dropwise to the mixture. The resulting reaction solution was stirred at room temperature for 2 hours. Then, 20 mL of water was added to the reaction solution, and the reaction solution was extracted with ethyl acetate three times (3 x 30 mL), and the organic phase was separated. The organic phase was washed with 20 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 2.16 g of white solid powder, which was compound 40, with a yield of 78%. ESI-MS (m / z): 577.2 [M+H] + .

[0367] Compound 40 (1 g, 1.74 mmol), compound 7 (0.96 g, 3.84 mmol), XPhos (41 mg, 0.087 mmol), palladium acetate (20 mg, 0.087 mmol) and potassium phosphate (1.1 g, 5.22 mmol) were added into 50 mL of a mixed solvent of tetrahydrofuran and water (volume ratio of tetrahydrofuran: water = 3:1) to obtain a reaction solution. The reaction solution was heated to 70°C under argon protection and stirred for 16 hours until the reaction was complete. After the reaction solution was cooled to room temperature, it was concentrated. 20 mL of water was added to the concentrated reaction solution, which was extracted with ethyl acetate three times (3 x 30 mL), and the organic phase was separated. The organic phase was washed with 10 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 968 mg of a light yellow solid powder, which was compound 41, with a yield of 78%. ESI-MS (m / z): 745.4 [M+H] + .

[0368] Compound 41 (0.968 g, 1.3 mmol) was dissolved in 10 mL of anhydrous methanol, and palladium-carbon was added, with the weight ratio of palladium-carbon to compound 41 being 10%, and then hydrogen was introduced three times. The obtained reaction solution was stirred at room temperature overnight, and after filtration and concentration, 870 mg of an off-white solid powder was obtained, which was compound 42, with a yield of 100%. ESI-MS (m / z): 655.3 [M+H] + .

[0369] Compound 42 (0.87 g, 1.3 mmol), compound 10 (0.72 g, 1.7 mmol) and potassium carbonate (0.55 g, 3.9 mmol) were respectively dissolved in 15 mL of DMF, and the obtained reaction solution was heated to 100°C and stirred for 6 hours. After cooling to room temperature, 20 mL of water was added and extracted with 40 mL of ethyl acetate, and the organic phase was separated. The organic phase was washed with 15 mL of saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and then column chromatography was performed to separate and elute the solvent, with the volume ratio of methanol to dichloromethane being 0:100-50:50, to obtain 0.96 g of a light yellow foamy solid, which was compound 43, with a yield of 80%. ESI-MS (m / z): 900.4 [M+H] + .

[0370] Compound 43 (0.96 g, 1 mmol) was dissolved in 5 mL of methanol, and 5 mL of 1 N NaOH solution was added, and the resulting reaction solution was stirred at room temperature for 1 hour. The pH of the reaction solution was adjusted to 6-7 with 1 N hydrochloric acid solution in an ice bath. 20 mL of dichloromethane was added to the reaction solution for extraction, and the organic phase was separated. The organic phase was washed with 15 mL of saturated brine, dried over anhydrous sodium sulfate, and filtered and concentrated to obtain 0.9 g of a white solid, which was compound 44, with a yield of 95%. ESI-MS (m / z): 886.4 [M+H] + .

[0371] Compound 44 (0.9 g, 1 mmol) was dissolved in 4 M, 5 mL of hydrogen chloride in dioxane, and the resulting reaction solution was stirred at room temperature for 2 hours and concentrated to obtain 0.78 g of a light yellow foamy solid, which was the control ligand compound 1, with a yield of 100%. ESI-MS (m / z): 786.4 [M+H] + . 1 H NMR (500 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.56 (d, J = 8.3 Hz, 1H), 8.27 (dd, J = 7.8, 1.8 Hz, 1H), 8.20 (t, J = 5.9 Hz, 1H), 7.81-7.73 (m, 2H), 7.53 (dqd, J = 8.2, 6.8, 1.5 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.9 Hz, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.80 (s, 1H), 6.67 (d, J = 6.4 Hz, 1H), 5.30 (q, J = 7.5 Hz, 1H), 4.33 (t, J = 4.6 Hz, 2H), 3.99-3.87 (m, 2H), 3.77 (d, J = 5.8 Hz, 2H), 3.69 (dd, J = 5.9, 3.7 Hz, 2H), 3.62-3.49 (m, 10H), 3.49-3.34 (m, 4H), 3.30 (m, 2H), 2.79 (d, J = 7.3 Hz, 2H), 2.29 (s, 3H), 1.81 (p, J = 7.2 Hz, 2H), 1.36-1.13 (m, 2H).

[0372] Synthesis of the conjugate provided by the present disclosure

[0373] The siRNA contained in the conjugate provided by the present disclosure is an siSOD1 sequence, and the sense strand and the antisense strand of the siSOD1 sequence have the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively:

[0374] Sense strand: 5'-UmsUmsUmUmAmAmUfCfCfUmCmAmCmUmCmUmAmAmAm-3' (SEQ ID NO: 5)

[0375] Antisense strand: 5'-UmsUfsUmAmGmAfGmUmGmAmGmGmAmUfUmAfAmAmAmsUmsGm-3' (SEQ ID NO: 6)

[0376] wherein, in the siSOD1 sequence, capital letters C, G, U, A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a 2'-methoxy modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a 2'-fluorine modified nucleotide; and lowercase letter s represents that the linkage between the two nucleotides adjacent to the left and right of the letter s is a phosphorothioate linkage.

[0377] wherein, in the conjugate provided by the present disclosure, the crosslinking agent for connecting the siRNA and the ligand group is as shown in the following formula (N-1):

[0378] The crosslinking agent contains 3 click chemistry groups containing terminal alkynyl groups and 1 acylation group containing a carboxyl group.

[0379] In the present preparation example, the siRNA, the crosslinking agent and the ligand group are sequentially connected to form a conjugate, and the preparation of the conjugate comprises the following processes:

[0380] (7-1) The connection process of siRNA and crosslinking agent (N-1) is as follows:

[0381] The crosslinking agent (N-1) is synthesized according to the preparation process of Example 14 in CN111698995A, and further connected with a phosphoramidite monomer containing an NH2C6- group. Then, according to the nucleotide types and sequences of the above-mentioned siSOD1 sequence sense strand and antisense strand, the nucleoside monomers are sequentially connected in the direction of 3' to 5' by the phosphoramidite solid-phase synthesis method, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, oxidation or sulfuration, and capping. After the connection of the nucleoside monomer at the 5' terminal of the sense strand is completed, the above-mentioned phosphoramidite monomer is further connected. The siSOD1 sense strand and antisense strand are separated and annealed to obtain siSOD1-(N-1) group. Wherein, the crosslinking agent group formed by the crosslinking agent (N-1) is connected to the 5' terminal of the siSOD1 sense strand. And the 5' terminal of the siSOD1 sense strand and the crosslinking agent group are conjugated and connected by the amide formed by the NH2C6- group at the terminal of the NH2C6- group, and the amine group at the terminal of the NH2C6- group can promote the conjugation of the required targeting ligand.

[0382] For the above specific synthesis procedure, it can be carried out according to the method of Example 1 in WO2019010274A1, the entire content of which is incorporated herein by reference, which will not be repeated here.

[0383] (7-2) Conjugate connection of siSOD1-(N-1) group and ligand group

[0384] Take the conjugate connection of the control ligand compound 1 and the siSOD1-(N-1) group to obtain the reference conjugate; take the conjugate connection of the ligand compound 1 and the siSOD1-(N-1) group to obtain the conjugate 1; take the conjugate connection of the ligand compound 2 and the siSOD1-(N-1) group to obtain the conjugate 2; take the conjugate connection of the ligand compound 3 and the siSOD1-(N-1) group to obtain the conjugate 3; take the conjugate connection of the ligand compound 4 and the siSOD1-(N-1) group to obtain the conjugate 4; take the conjugate connection of the ligand compound 5 and the siSOD1-(N-1) group to obtain the conjugate 5.

[0385] Wherein, the conjugate connection process of each ligand compound and siSOD1-(N-1) group is as follows:

[0386] (7-2-1) Take 150 μL of H2O, 70 μL of 0.2 M carbonate buffer solution (pH = 9.2), and 70 μL of N,N-dimethylformamide (DMF) to dissolve the siSOD1-(N-1) group to obtain a siSOD1-(N-1) solution, and the amount is taken as 1.0 eq.

[0387] (7-2-2) Take 6.0 eq of ligand compound dissolved in 70 μL of DMF, and then dissolve the ligand compound solution in the siSOD1-(N-1) solution obtained in step (1) to obtain a reactant mixture.

[0388] (7-2-3) Take 10.0 eq of tris(3-hydroxypropyl triazolylmethyl)amine (THPTA) and 3.0 eq of CuSO4·5H2O according to the volume ratio THPTA:CuSO4·5H2O = 10:3, shake for 5 min at 40°C, take 37 μL and add to the reactant mixture obtained in the above step (2), and perform vortex shaking to obtain an intermediate product mixture, and the pH of the intermediate product mixture is measured to be 8. Then, take 25.0 eq of sodium ascorbate and quickly add to the intermediate product mixture, and perform vortex shaking treatment, and react for 1 h at 40°C to obtain a product mixture.

[0389] 3 μL of the product mixture was diluted with a mixed solution of DMF and H2O (volume ratio of DMF to H2O was 1:5), and then separated and purified by HPLC treatment. In the HPLC treatment, a C18 column was used as the chromatographic column, and an ammonium bicarbonate buffer solution was used as the mobile phase.

[0390] The product after the HPLC treatment was subjected to freeze-drying treatment to obtain the conjugate product.

[0391] The conjugate 1, the conjugate 2, the conjugate 3, the conjugate 4, the conjugate 5 and the reference conjugate were prepared according to the above procedures, respectively, except that the ligand compound used in the above step (2) was replaced by the different ligand compound contained in each conjugate.

[0392] After each conjugate was diluted to a concentration of 0.2 mg / mL (calculated as siRNA) using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25 °C)), the molecular weight was detected by liquid chromatography-mass spectrometry (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters Company, model: LCT Premier). The measured value was consistent with the theoretical value, indicating that the synthesized conjugates 1-5 and the reference conjugate were the target designed double-stranded nucleic acid sequences, and these conjugates all had the structure shown in formula (305), wherein each ligand group R L connected to the ribose 5' position of the 5' terminal nucleotide of the siRNA sense strand through the linker group.

[0393] In vitro activity of the ligand compound provided by the present disclosure

[0394] In the present disclosure, the binding activity of the ligand compound provided by the present disclosure and integrin αvβ6 was determined by fluorescence polarization method. The determination process is as follows:

[0395] (A-1) The ligand compound 1 and the ligand compounds 3-6 obtained in the above preparation example were dissolved in PBS solution to obtain ligand compound solutions with a concentration of 100 μM, respectively. Each of the ligand compound solutions was prepared into 10 different concentrations of ligand compound working solutions with concentrations of 25 μM, 6.25 μM, 1.5625 μM, 0.3906 μM, 0.09765 μM, 0.02441 μM, 0.006104 μM, 0.001526 μM, 0.0003815 μM, 0.00009538 μM (calculated as the amount of ligand compound) by using dimethyl sulfoxide (DMSO).

[0396] (A-2) Take 25 mM pH = 7.4 HEPES, 150 mM NaCl, 1 mM CHAPS and 400 mM MgCl2to prepare a buffer solution, prepare 4x ligand compound working solution with 1x buffer solution, and take 3.5 μL of ligand compound working solution into different experimental hole plates respectively.

[0397] (A-3) Prepare 4x integrin αvβ6 working solution with 1x buffer solution, and take 3.5 μL of integrin αvβ6 working solution into the experimental hole plates containing different ligand compound working solutions respectively, to obtain mixed working solution respectively.

[0398] (A-4) Centrifuge the mixed working solution obtained in step (A-3) at 1000 rpm for 1 minute. After centrifugation, incubate the experimental hole plate in a 25℃ incubator for 15 minutes.

[0399] (A-5) Prepare 2x fluorescence RGD-containing peptide substrate working solution with 1x buffer solution, and take 7 μL of substrate working solution into the above centrifuged mixed working solution respectively to obtain substrate mixed working solution.

[0400] (A-6) Centrifuge the substrate mixed working solution obtained in step (A-5) at 1000 rpm for 1 minute. After centrifugation, incubate the experimental hole plate in a 25℃ incubator for 1 hour. And use Envision plate reader (Perkin Elmer) to detect fluorescence polarization with 531 nm excitation and 590 nm emission measurement, read the FP fluorescence signal value. And use Graphpad prism5.0 statistical analysis software to analyze the data, and then obtain the IC 50 .

[0401] In this embodiment, the IC 50 values of the binding activity of ligand compound 1 and ligand compounds 3-6 to integrin αvβ6 can be reflected, which are shown in Table 1.

[0402] Table 1 IC 50 values of ligand compounds binding to integrin αvβ6 (nM)

[0403] As can be seen from Table 1, the IC 50 values of the ligand compounds provided by the present disclosure are low when binding to integrin αvβ6, and in particular, the IC 50The range is between 0.8-0.92 nM, indicating that the ligand compound of the present disclosure can form stable binding with integrin αvβ6 at a lower concentration, showing high integrin αvβ6 binding activity. Thus, the ligand compound of the present disclosure can deliver different functional groups into target cells expressing integrin αvβ6 for the treatment or diagnosis of related diseases or symptoms.

[0404] Inhibition of SOD1 mRNA in vivo by conjugate of experimental example 2

[0405] The conjugate 2, conjugate 3 and reference conjugate were respectively dissolved in PBS to a solution with a concentration of 0.2 mg / mL (calculated as the conjugate), and stored at -20°C.

[0406] C57BL / 6j mice (all female, weighing about 20 g, 6-8 weeks old, purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.) were randomly divided into groups, with 5 mice in each group, and numbered respectively. Each mouse was administered with the above conjugate solution by pulmonary spray administration, wherein the concentration of the conjugate solution was 0.2 mg / mL (calculated as siRNA), the administration volume was 50 μL, the single administration dose was 0.5 mg / kg (calculated as siRNA), and the administration was performed once at the first administration (referred to as day 1) and day 4, and the group of mice was recorded as the test group. The mice administered with the reference conjugate solution in the same manner were recorded as the positive control group. In addition, another group of mice was administered with 50 μL of PBS per mouse each time by pulmonary spray administration, and the administration was performed once at day 1 and day 4, and the group of mice was recorded as the blank control group.

[0407] The first administration time point was taken as day 1 (i.e. D1), and the mice were observed for abnormalities 10-15 minutes after administration. The mice were sacrificed 7 days after the last administration (i.e. D11), and the lung tissues of each mouse were collected and cut into several blocks with a volume of 2 mm 3 left and right, and stored in RNA later (Sigma Aldrich).

[0408] Add 1 mL Trizol (Sigma) to each lung tissue, crush 3 times for 30 s in a Tissuelyser II automatic tissue homogenizer, obtain lung tissue homogenate, add 0.2 mL chloroform to the lung tissue homogenate, and stand for 3 min. Centrifuge at 12000 rpm for 10 min at 4°C, and take 0.4 mL supernatant. Add 0.5 mL isopropanol to the supernatant, and stand for 10 min at room temperature. Centrifuge at 12000 rpm for 10 min at 4°C, and discard the supernatant. Wash the precipitate with 1 mL ethanol, centrifuge at 12000 rpm for 5 min at 4°C, and discard the supernatant. Add 70 μL DEPC-treated water to the precipitate to obtain the extracted total RNA solution.

[0409] For each lung tissue total RNA of the mouse, take 10.5 μL total RNA aqueous solution containing 1 μg total RNA, and use a reverse transcription kit Reverse Transcription System (purchased from Promega, product number A3500) to prepare a reverse transcription reaction system 20 μL according to the reverse transcription operation steps in the kit instructions, and perform reverse transcription on the total RNA. The reverse transcription conditions are as follows: for each reverse transcription reaction system, incubate the reverse transcription reaction system at 42°C for 30 min, then at 95°C for 5 min, and finally at 4°C for 5 min. After the reaction is completed, add 80 μL DEPC water to the reverse transcription reaction system to obtain a cDNA-containing solution.

[0410] For each reverse transcription reaction system, take 5 μL of the above cDNA-containing solution as a template, and use the reagents provided in a SYBR select Master Mix kit (Applied biosystem) to prepare a qPCR reaction system 20 μL. The PCR primer sequences for amplifying the target gene mSOD1 and the internal reference gene mGAPDH are shown in Table 2 below, and the final concentration of each primer is 0.25 μM. Place each qPCR reaction system on an ABI StepOnePlus Real-Time PCR instrument, and use a three-step method for amplification. The amplification program is as follows: pre-denaturation at 95°C for 10 min, then denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. Repeat the above denaturation, annealing, and extension processes for a total of 40 times, and then obtain product W containing the amplified target gene mSOD1 and internal reference gene mGAPDH. Incubate product W at 95°C for 1 min, at 55°C for 30 s, and at 95°C for 30 s in turn, and collect the melting curves of the target gene mSOD1 and the internal reference gene mGAPDH in product W using a real-time fluorescence quantitative PCR instrument to obtain the Ct values of the target gene mSOD1 and the internal reference gene mGAPDH.

[0411] Table 2 primer sequence information

[0412] The expression levels of the target gene SOD1 mRNA in each test group were relatively quantitatively calculated by using comparative Ct (ΔΔCt) method, and the calculation method was as follows:

[0413] ΔCt (test group) = Ct (target gene in test group) - Ct (internal reference gene in test group)

[0414] ΔCt (control group) = Ct (target gene in control group) - Ct (internal reference gene in control group)

[0415] ΔΔCt (test group) = ΔCt (test group) - ΔCt (average of control group)

[0416] ΔΔCt (control group) = ΔCt (control group) - ΔCt (average of control group)

[0417] Wherein, ΔCt (average of control group) is the arithmetic mean of the ΔCt (control group) of each of the 5 mice in the control group. Thus, each mouse in the test group and the control group corresponds to a ΔΔCt value.

[0418] The expression level of SOD1 mRNA (mSOD1) in the test group was normalized based on the control group, and the expression level of SOD1 mRNA in the blank control group was defined as 100%,

[0419] Relative expression level of SOD1 mRNA in test group = 2-ΔΔCt (test group) x 100%

[0420] Inhibition rate of SOD1 mRNA in test group = (1 - relative expression level of SOD1 mRNA in test group) x 100%.

[0421] Figure 1 is a scatter plot of the relative expression level of SOD1 mRNA in the whole lung of C57BL / 6j mice after being given 0.5 mg / kg (calculated by siRNA) conjugate 2, conjugate 3 and reference conjugate, and PBS, respectively.

[0422] As can be seen from FIG. 1, in the present experimental example, the inhibition rate of the reference conjugate on the SOD1 mRNA in the lung of the test group of mice was 38% on day 7 after the last administration (D11), the inhibition rate of conjugate 2 on the SOD1 mRNA in the lung of the test group of mice was 41%, and the inhibition rate of conjugate 3 on the SOD1 mRNA in the lung of the test group of mice was 44%. The present example shows that the inhibitory activities of conjugate 2 and conjugate 3 provided by the present disclosure on the SOD1 mRNA in the lung of mice are all comparable to or even better than the inhibitory activity of the positive control group provided by the reference conjugate, and the conjugates can effectively inhibit the expression level of the target gene in the lung of mice, and thus can further achieve the effect of treating diseases. In summary, the conjugates provided by the present disclosure can effectively deliver functional groups to integrin ανβ6-expressing cells and / or tissues, and achieve the purpose of treating diseases.

[0423] The above describes some embodiments of the present disclosure in detail, but the present disclosure is not limited to the specific details in the above-described embodiments. Within the technical concept range of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection range of the present disclosure.

[0424] In addition, it should be noted that the various specific technical features described in the above-described embodiments can be combined in any appropriate manner without contradiction, and in order to avoid unnecessary repetition, the present disclosure will not further describe various possible combination manners.

[0425] In addition, various different embodiments of the present disclosure can also be combined in any appropriate manner, as long as they do not deviate from the idea of the present disclosure, and they should also be considered as disclosed by the present disclosure.

Claims

1. A compound having the structure shown in Formula (1): ###0001### Formula (1) ​ wherein: A1, A2, A3, and A4 are each CR 11 Or N, and at least one of A1 and A3 is CR 11 At least one of A2 and A4 is CR 11 Each R 11 Each element is independently selected from H, OH, SH, halogens, and C1-C. 10 One of the straight-chain or branched hydrocarbon groups; R 21 , R 22 , and R 23 are the same or different, each independently selected from one of O, S, and NH; R 31 , each R 32 and R 33 is the same or different, each independently selected from one of H, C3-C6cycloalkyl, C3-C6substituted cycloalkyl, C1-C6straight or branched alkyl, and C1-C6straight or branched substituted alkyl; R 41 , R 42 , R 51 , R 52 and each R 53 are the same or different, each independently one of H, OH, SH, NH2, halogen, C3-C 10 cycloalkyl, C3-C 10 substituted cycloalkyl, C1-C 10 straight-chain or branched hydrocarbyl and C1-C 10 straight-chain or branched substituted hydrocarbyl; R 10 , R 61 , R 62 , R 63 , R 64 , R 65 and R 66 are the same or different, each independently R p or R q ; R p is selected from one of hydrogen, hydroxyl, halogen, C4-C 20 polyoxyethyl, C4-C 20 substituted polyoxyethyl, C3-C 10 cycloalkyl, C3-C 10 substituted cycloalkyl, C1-C 20 straight-chain or branched-chain alkyl and C1-C 20 straight-chain or branched-chain substituted alkyl, R q is selected from one of the groups containing active functional groups or protected active functional groups; Y is selected from one of OR k , N(R k )2, and SR k , wherein each R k is independently H or a protecting group; m is an integer selected from 1-10, and n is an integer selected from 0-2.

2. The compound of claim 1, wherein, m is an integer selected from 1 or 3-10; or, m is an integer selected from 1-3; or, m is 1 or 3; or, m is 1; and / or n is 0 or 1.

3. The compound of claim 1 or 2, wherein, R 31 , each R 32 and R 33 is the same or different, each independently selected from one of H, C3-C5 cycloalkyl, C3-C5 substituted cycloalkyl, C2-C5 alkyl and C2-C5 substituted alkyl.

4. The compound of claim 3, wherein, each R 32 and R 33 are H; R 31 is H, cyclopropyl, t-butyl, n-propyl or i-propyl.

5. The compound of any one of claims 1-4, wherein, R 41 , R 42 , R 51 , R 52 and each R 53 is the same or different, each being independently selected from one of H, OH, SH, NH2, halogen, C3-C6 cycloalkyl, C3-C6 substituted cycloalkyl, C1-C6 alkyl and C1-C6 substituted alkyl.

6. The compound of claim 5, wherein, R 41 , R 42 , R 51 , R 52 and each R 53 is H.

7. The compound of claim 1, wherein, R 10 , R 61 , R 62 , R 63 , R 64 , R 65 , and R 66 is R q .

8. The compound of claim 7, wherein, R 10 R q ; R 61 ; R 62 ; R 63 ; R 64 ; R 65 ; R 66 ; R p .

9. The compound of any one of claims 1, 7, and 8, wherein, Each R p Independently selected from H, hydroxyl, C4-C 20 Polyoxyethyl, C4-C 20 Substituted polyoxyethyl, C1-C 20 Straight-chain or branched hydrocarbon groups and C1-C 20 One of the straight-chain or branched substituted hydrocarbon groups.

10. The compound of any one of claims 1, 7, and 8, wherein, R q Further comprising one of a C6-C 14 polyoxyethylidene, C6-C 14 substituted polyoxyethylidene, C8-C 14 alkyleneoxy, and C8-C 14 substituted alkyleneoxy.

11. The compound of claim 10, wherein, R q having the structure of any one of Formulas (31) to (36): wherein denotes the site of covalent attachment of a group; Z is the reactive functional group.

12. The compound of claim 11, wherein, The reactive functional group Z is selected from one of azido, alkynyl, dibenzocyclooctyne, thiol, amino, hydroxyl, carboxyl, acyl halide, aldehyde, carbonate, aminooxy, active ester, disulfide, ortho-pyridyl disulfide, maleimide, tosylate, tetrazine, trans-cyclooctene, hydrazide, and phosphoramidite.

13. The compound of any one of claims 1, 7, and 8, wherein, R 10 R q ; R 61 , R 62 , R 63 , R 64 , R 65 and R 66 are independently H or methyl.

14. The compound of any one of claims 1, 7, and 8, wherein, The compound has a structure shown in formula (2):

15. The compound of claim 14, wherein, R 21 , R 22 , and R 23 are each O; and / or, A1, A2, A3, and A4 are each CH or N, and at least one of A1and A3is CH and at least one of A2and A4is CH; Y is hydroxyl or hydroxyl protected with a hydroxyl protecting group.

16. The compound of claim 15, wherein, The compounds have a structure according to any one of Formulas (21) to (30):

17. The compound of any one of claims 1-16, wherein, The hydroxyl protecting group is selected from one of benzoyl, benzyl, tert-butyloxycarbonyl, trityl, substituted silyl, and alkylacyl.

18. A conjugate comprising at least one ligand group formed by removing one hydrogen atom and / or one functional group from a compound of any one of claims 1-15, and at least one functional group, said ligand group and said functional group being connected by a covalent bond or through a linker.

19. The conjugate of claim 18, wherein, The conjugate has a structure represented by Formula (3): In formula (3), each R L identically or differently, independently represents one of the ligand groups; R j represents a covalent bond or a linking group; each A0group is the same or different, and independently represents one of the functional groups; m0 is an integer from 1-6; n0 is an integer from 1-6.

20. The conjugate of claim 19, wherein, m0 is an integer from 1-4, and / or n0 is an integer from 1-3.

21. The conjugate of claim 19 or 20, wherein, The conjugate has a structure represented by Formula (4): wherein n0 is 2 or 3.

22. The conjugate of claim 19 or 20, wherein, R j is a linker group, said linker group R j comprises a backbone moiety, a side chain moiety and a conjugation linker, said backbone moiety being connected to said conjugation linker and to said side chain moiety, respectively, said side chain moiety being connected to said backbone moiety and to said R L group, said conjugation linker being connected to said backbone moiety and to said functional group A0, wherein, the main chain portion is a straight chain alkylene of 1-70 carbon atoms, or one or more carbon atoms in the straight chain alkylene is replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 10 sugarosyl, C2-C 10 alkenylene, C6-C 10 arylene, C3-C 18 heterocyclylene, and C5-C 10 heteroarylene; and wherein the straight chain alkylene can have any one or more of the following substituents selected from the group consisting of C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl); each said side chain moiety is independently a covalent bond, or a straight chain alkylene of 1-70 carbon atoms, or one or more carbon atoms in said straight chain alkylene are replaced with one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8 10 alkenylene, C2-C 10 alkynylene, C6-C 10 arylene, C3-C 18 heterocyclylene, and C5-C 10 heteroarylene; and, said straight chain alkylene can have one or more substituents selected from the group consisting of C1-C 10 alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, C1-C 10 haloalkyl, -OC1-C 10 alkyl, -OC1-C 10 alkylphenyl, -C1-C 10 alkyl-OH, -OC1-C 10 haloalkyl, -SC1-C 10 alkyl, -SC1-C 10 alkylphenyl, -C1-C 10 alkyl-SH, -SC1-C 10 haloalkyl, halogen substituent, -OH, -SH, -NH2, -C1-C 10 alkyl-NH2, -N(C1-C 10 alkyl)(C1-C 10 alkyl), -NH(C1-C 10 alkyl), -N(C1-C 10 alkyl)(C1-C 10 alkylphenyl), -NH(C1-C 10 alkylphenyl), cyano, nitro, -CO2H, -C(O)O(C1-C 10 alkyl), -CON(C1-C 10 alkyl)(C1-C 10 alkyl), -CONH(C1-C 10 alkyl), -CONH2, -NHC(O)(C1-C 10 alkyl), -NHC(O)(phenyl), -N(C1-C 10 alkyl)C(O)(C1-C 10 alkyl), -N(C1-C 10 alkyl)C(O)(phenyl), -C(O)C1-C 10 alkyl, -C(O)C1-C 10 alkyl, -C(O)C1-C 10 haloalkyl, -OC(O)C1-C 10 alkyl, -SO2(C1-C 10 alkyl), -SO2(phenyl), -SO2(C1-C 10 haloalkyl), -SO2NH2, -SO2NH(C1-C 10 alkyl), -SO2NH(phenyl), -NHSO2(C1-C 10 alkyl), -NHSO2(phenyl), and -NHSO2(C1-C 10 haloalkyl). each said conjugate linkage is independently a covalent bond or a linking combination of one or more of the following linking structures: C1-C 10 linear alkylene, phosphonate, phosphorothioate, amide, ester, ether, disulfide, 1,2,3-triazole, polyethylene glycol, pyrrolidine, 2-oxopyrrolidine, phenylene, cyclohexylene, 2-succinimide, 2-thiosuccinimide, amino acid, nucleotide.

23. The conjugate of claim 22, wherein, The number of conjugation linkers is m0, the number of side chain moieties is n0, each of the conjugation linkers is connected to the main chain moiety and one of the functional groups A0, respectively; each side chain moiety is connected to the main chain moiety and one of the R L groups, respectively.

24. The conjugate of claim 22 or 23, wherein, All of the side chain moieties are attached to the same atom in the backbone moiety; or each of the side chain moieties is attached to a different atom in the backbone moiety.

25. The conjugate of claim 24, wherein, m0 is 1, the linking group R j comprising a structure according to Formula (301): wherein k = n0; L C L is the main chain portion, A L is the side chain portion, B L is the conjugation linker, denotes the site of covalent attachment of a group; said backbone moiety L C is a covalent bond or a 2-7 valent, straight-chained or branched C1-C 25 saturated hydrocarbon group, or one or more carbon atoms in said saturated hydrocarbon group are replaced by one or more selected from the group consisting of C(O), NH, O, S, CH=N, S(O)2, OP(O)2, C5-C8sugaroidene, C2-C5alkenylene, C2-C5alkynylene, C6-C 10 arylene, C3-C8heterocyclylene, and C5-C 10 heteroarylene; wherein said saturated hydrocarbon group can have any one or more substituents selected from the group consisting of C1-C5alkyl, C6-C 10 aryl, C5-C 10 heteroaryl, -O-C1-C5alkyl, -OC1-C5alkylphenyl, -C1-C5alkyl-OH, -SC1-C5alkyl, nitro, -C(O)O(C1-C5alkyl), -CON(C1-C5alkyl)(C1-C5alkyl), -CONH(C1-C5alkyl), -CONH2, -NHC(O)(C1-C5alkyl), -NHC(O)(phenyl), -N(C1-C5alkyl)C(O)(C1-C5alkyl), -N(C1-C5alkyl)C(O)(phenyl), -C(O)C1-C5alkyl, -C(O)C1-C5alkylphenyl, -OC(O)C1-C5alkyl, -SO2(C1-C5alkyl), -SO2(phenyl), -SO2NH2, -SO2NH(C1-C5alkyl), -SO2NH(phenyl), -NHSO2(C1-C5alkyl), and -NHSO2(phenyl).

26. The conjugate of claim 25, wherein, The conjugate has a structure as shown in formula (305):

27. The conjugate of claim 24, wherein, said linking group R j having a structure represented by Formula (306): wherein n 306 = n0-1, each p 306 is independently an integer from 1 to 6, represents the site of covalent attachment of the group; said backbone portion is composed of a structure formed by pyrrolidinylene and phosphodiester groups, each of said side chain portions is composed of an atomic chain between a carbonyl group attached to a nitrogen atom of a pyrrolidine group and the oxygen atom marked with an asterisk, said side chain portions are connected via the oxygen atom marked with an asterisk to said R L marked with a # is the conjugation linker and is connected to said functional group A0 forming an ether, ester or phosphonate bond, the remaining oxygen atoms marked with a # are connected to a hydrogen atom forming a hydroxyl group or to a C1-C3 alkyl group forming a C1-C3 alkoxy group.

28. The conjugate of claim 27, wherein, The conjugate has a structure as shown in formula (307a), (307b), or (307c):

29. The conjugate of claim 24, wherein, The conjugate has a structure represented by Formula (308): wherein: n 308 = m0+ n0- 1; each m 308 is independently an integer selected from 2-10; Each R 308 Independent of H, C1-C 10 Alkyl, C1-C 10 Halogenated alkyl or C1-C 10 Alkoxy; each R3is independently said functional group A0, or is said R L group, and at least one R3is said functional group A0, and at least one R3is said R L group; or one R3is said functional group A0, and the remaining R3are said R L group.

30. The conjugate of claim 29, wherein, each L1 is independently selected from the group consisting of linking combinations of groups (L4)-(L23), and any combinations thereof.

31. The conjugate of claim 30, wherein, each L1 is independently selected from the group consisting of linking combinations of at least two of groups (L4)-(L9), (L13), (L14), (L18); or each L1 is independently a linking combination of at least two of groups (L4), (L5), (L7), (L9), (L13), (L14), (L18).

32. The conjugate of any one of claims 29-31, wherein, each L1 is independently 3-25 atoms in length; or each L1 is independently 4-15 atoms in length.

33. The conjugate of any one of claims 29-32, wherein, n 308 is an integer from 2 to 6; and / or, 2 to 4 R3are said R L groups, the remaining R3being said functional groups.

34. The conjugate of any one of claims 29-33, wherein, each m 308 each independently is an integer from 2 to 5, and / or each m 308 are the same.

35. The conjugate of any one of claims 29-34, wherein, each L1simultaneously contains a linking site to a N atom on the nitrogen- containing backbone and a linking site to the functional group A0or the R L group forms an amide bond to this N atom; or L1, attached to the functional group A0, is selected from B5, B6, B5' or B6': wherein denotes the site of covalent attachment of a group, and q2 is an integer from 1-10; or q2 is an integer from 1-5.

36. The conjugate of claim 29, having a structure according to formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), (422), (423), (424), (425), (426), or (427).

37. The conjugate of any one of claims 18-36, wherein, each of the functional groups is independently selected from at least one of a diagnostic agent group, a therapeutic agent group, an oligonucleotide group, and a delivery aid group.

38. The conjugate of claim 37, wherein, at least one of the functional groups is a delivery aid group selected from C8-C 30 at least one of an alkyl or alkenyl group, a cholesteryl group, a lipid group, a palmitic acid group, and a cholic acid group.

39. The conjugate of claim 37 or 38, wherein, at least one of the functional groups is a diagnostic agent group, each of the diagnostic agent groups is independently selected from one of a contrast agent group, an isotopic tracer, and a fluorescent tracer.

40. The conjugate of claim 37 or 38, wherein, At least one of the functional groups is a therapeutic agent group, each of the therapeutic agent groups is selected from one of a cytotoxic group, an antibiotic group, an angiogenesis inhibitor, an antibody drug group, and a group comprising a radioisotope.

41. The conjugate of claim 37 or 38, wherein, At least one of the functional groups is an oligonucleotide group, the oligonucleotide group is capable of modulating the level of a target mRNA in a cell that expresses integrin αvβ6.

42. The conjugate of claim 41, wherein, The cell is selected from one or more of an alveolar epithelial cell, a secretory epithelial cell, a ciliated epithelial cell, a corneal and conjunctival epithelial cell, a dermal epithelial cell, a bile duct epithelial cell, an intestinal epithelial cell, a ductal epithelial cell, a glandular epithelial cell, an epithelial tumor cell, and a muscle cell.

43. The conjugate of claim 41 or 42, wherein, The oligonucleotide group is an siRNA group, the siRNA group is formed by removing one or more atoms from an siRNA molecule.

44. A pharmaceutical composition comprising the conjugate of any one of claims 18-43 and a pharmaceutically acceptable carrier.

45. Use of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44 in the manufacture of a medicament for the diagnosis of a disease, condition, and / or disorder, wherein, At least one of the functional groups is a diagnostic agent group, the diagnostic agent group is useful for diagnosing the disease, condition, and / or disorder upon delivery to a cell that expresses αvβ6.

46. The use of claim 45, wherein, Each of the diagnostic agent groups is independently selected from one of a contrast agent group, an isotope tracer, and a fluorescent tracer.

47. Use of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44 in the manufacture of a medicament for the treatment of a disease, condition, and / or disorder, wherein, At least one of the functional groups is a therapeutic agent group, the therapeutic agent group is useful for treating the disease, condition, and / or disorder upon delivery to a cell that expresses αvβ6.

48. The use of claim 47, wherein, Each of the therapeutic agent groups is selected from one of a cytotoxic group, an antibiotic group, an angiogenesis inhibitor, an antibody drug group, and a group comprising a radioisotope.

49. Use of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44 in the manufacture of a medicament for the treatment of a disease, condition, and / or disorder, wherein, At least one of the functional groups is an oligonucleotide group, the oligonucleotide group is useful for treating the disease, condition, and / or disorder by modulating the level of the target mRNA in the cell that expresses αvβ6.

50. The use of claim 49, wherein, The oligonucleotide group is an siRNA group.

51. The use of any one of claims 45-50, wherein, The cell is selected from one or more of an alveolar epithelial cell, a secretory epithelial cell, a ciliated epithelial cell, a corneal and conjunctival epithelial cell, a dermal epithelial cell, a bile duct epithelial cell, an intestinal epithelial cell, a ductal epithelial cell, a glandular epithelial cell, an epithelial tumor cell, and a muscle cell.

52. The use of any one of claims 45-51, wherein, The disease, condition, and / or disorder comprises at least one of pulmonary fibrosis, asthma, chronic pulmonary obstruction, pulmonary inflammation, and a muscle-related disease.

53. A method of diagnosing a disease, condition, or disorder comprising administering to a subject in need thereof an effective amount of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44, wherein, At least one of the functional groups is a diagnostic agent group, the diagnostic agent group is useful for diagnosing the disease, condition, and / or disorder upon delivery to a cell that expresses αvβ6.

54. The method of claim 53, wherein, Each of the diagnostic agent groups is independently selected from one of a contrast agent group, an isotope tracer, and a fluorescent tracer.

55. A method of treating a disease, condition, or disorder comprising administering to a subject in need thereof an effective amount of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44, wherein, At least one of the functional groups is a therapeutic agent group, the therapeutic agent group is useful for treating the disease, condition, and / or disorder upon delivery to a cell that expresses αvβ6.

56. The method of claim 55, wherein, Each of the therapeutic agent groups is selected from one of a cytotoxic group, an antibiotic group, an angiogenesis inhibitor, an antibody drug group, and a group comprising a radioisotope.

57. A method of treating a disease, condition, or disorder comprising administering to a subject in need thereof an effective amount of the conjugate of any one of claims 18-38 and / or the pharmaceutical composition of claim 44, wherein, At least one of the functional groups is an oligonucleotide group that treats the disease, condition and / or disorder by modulating the level of the target mRNA in the avb6 expressing cell.

58. The method of claim 57, wherein, The oligonucleotide group is an siRNA group.

59. The method of any of claims 53-58, wherein, The disease, condition and / or disorder comprises at least one of pulmonary fibrosis, asthma, chronic pulmonary obstruction, pulmonary inflammation, tumors and muscle related diseases.

60. The method of any of claims 53-59, wherein, The cell is one or more of alveolar epithelial 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, epithelial tumor cells and muscle cells.

61. A kit comprising the conjugate of any one of claims 18-43 and / or the pharmaceutical composition of claim 44.

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