Ligands of integrin [alpha] v [beta] 6 protein receptor for delivery, conjugates thereof, and uses thereof

Abstract

The purpose of the present invention is to provide a ligand of an integrin [alpha] v [beta] 6 protein receptor capable of binding to the integrin [alpha] v [beta] 6 protein receptor, a conjugate thereof with a molecule to be transported (e.g., an oligonucleotide conjugate, e.g., a ligand-siRNA conjugate), and methods of making and using the same. The present disclosure also relates to compositions containing such ligand conjugates of integrin [alpha] v [beta] 6 protein receptors, methods of making and uses thereof.

Description

Technical Field

[0001] This disclosure relates to ligands of the integrin αvβ6 protein receptor capable of binding to the integrin αvβ6 protein receptor, conjugates thereto with the target molecule (e.g., oligonucleotide conjugates, such as ligand-siRNA conjugates), methods for their preparation, and applications. This disclosure also relates to compositions containing such ligand conjugates of the integrin αvβ6 protein receptor, methods for their preparation, and uses. Background Technology

[0002] The integrin αvβ6 protein receptor (also known as integrin receptor αvβ6) is a molecular target and epithelial-specific cell surface receptor expressed only in epithelial cells. It is primarily regulated by the integrin β6 (ITGB6) gene, increasing integrin ligand expression, macrophage infiltration, pro-inflammatory cytokine secretion, signal transduction, and activation of the STAT1 (activator of transcription 1) signaling pathway. Its expression is upregulated in damaged tissues, including fibrotic lung. Currently, several small molecule integrin inhibitors in clinical and preclinical stages are used to treat lung-related diseases, including but not limited to primary sclerosing cholangitis, idiopathic pulmonary fibrosis, and acute respiratory distress syndrome. Furthermore, molecules targeted by integrin αvβ6 are widely used in integrin-related cancer imaging and diagnosis.

[0003] Over the past two decades, research on RNA interference (RNAi) has translated this biological breakthrough into a powerful new drug platform for regulating mRNA expression. Of the six currently approved siRNA drugs, except for the first siRNA drug, Patisiran, which uses LNP delivery, the subsequent five approved siRNA drugs all use GalNac as a ligand for effective liver-targeted delivery. Furthermore, oligonucleotide modification technologies have been continuously upgraded, improving the stability of siRNA drugs to nucleases, RNA binding affinity, and pharmacokinetic properties. However, the delivery of siRNA drugs to extrahepatic cells, tissues, and organs (such as the lungs) still faces significant challenges. Currently, there are many types of lung diseases urgently requiring effective drugs, such as severe asthma and other mucosal obstructive lung diseases (such as cystic fibrosis). Currently, siRNA drugs can target disease-related sites (e.g., MUC5AC, MUC5B, and RAGE), knocking down the mRNA of relevant target genes to achieve therapeutic effects; however, drug delivery systems are currently lacking.

[0004] The ligands (especially ligand-siRNA conjugates) of the integrin αvβ6 protein receptor disclosed herein will provide a feasible approach for the construction of drug delivery systems for the lungs and will also provide an effective solution for the treatment of related lung diseases. Summary of the Invention

[0005] This invention provides an oligonucleotide conjugate with high delivery efficiency, which can effectively deliver oligonucleotides to target organs or tissues, such as the lungs, and exhibits high gene silencing activity and other pharmaceutical activities.

[0006] In one aspect, this disclosure provides a ligand for the integrin αvβ6 protein receptor, comprising a targeting group and a linking group, and having the structure shown in Formula I:

[0007]

[0008] Among them, R 1 Selected from C6-C 14 Aryl or 5-10 heteroaryl, the C6-C 14 Aryl and 5-10 heteroaryl groups are selectively R 1a replace;

[0009] R 0 Selected from hydrogen or -R 0a -L;

[0010] -R 0a -L is selected from -R 3 NR 17 -L、-R 3 NR 17 R 18 -L、-R 3 C(O)NR 17 R 18 -L、-R 3 OC(O)R 17 NR 18 -L、-R 3 NR 17 C(O)R 18 -L、-R 3 OC(O)R 17 -L、-R 3 C(O)OR 17 -L、-R 3 NR 17 C(O)OR 18 -L or -R 3 OC(O)(NR 17 )R 18 -L;

[0011] R 2 For hydrogen; deuterium; optionally R 2a Substituted C1-C6 alkyl; -OH; optionally R 2b Substituted C3-C6 cycloalkyl groups; or -S(O)2R 2c ;

[0012] Or, R 2 It is a single key, preferably used to connect with L;

[0013] Where L is a linking group that links the transported molecule.

[0014] Where L is selected from

[0015] L1 and L2 are each independently selected from: -(R 20 ) m -、-(R 20 ) m R 21 R 22 -、-(R 20 ) m R 21 -、-(R 20 O) m -、-(R 20 O) m R 21 -、-(R 20 O) m R 21 R 22 -、-R 20 C(O)R 21 -、-R 20 SSR 21 -、-R 20 C(O)R 21 R 22 -、-C(O)R 21 -、-C(O)R 21 R 22 -、-R 20 OC(O)R 21 -、-R 20 OC(O)R 21 R 22 -、-R 20 C(O)OR 21 -or-R 20 C(O)OR 21 R 22 - where m is an integer from 0 to 30;

[0016] Each R 1a Independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C8 cycloalkyl, deuterium, halogen, -CN, -OR 3 -NR 4 R 5 -C(O)R 3-OC(O)R 3 -C(O)OR 3 -C(O)NR 4 R 5 , where each R 1a Independently and optionally by deuterium, halogen, oxy group, or -OR, where possible. 6 -NR 6 R 7 -C(O)R 6 C1-C6 alkyl substitutions with -CN or halogen substitutions;

[0017] Each R 2a R 2b R 2e and R 2f Independent of an oxygen group or R 1a ;

[0018] R 2c For optional use by R 2e Substituted C1-C6 alkyl groups or optionally R 2f Substituted C3-C5 cycloalkyl groups;

[0019] R 3 R 17 R 18 and R 19 Each of these elements independently represents a bond, hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, or C6-C 14 aryl, 5- to 10-membered heteroaryl, or 3- to 12-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl and 3- to 12-membered heterocyclic groups are independently and optionally converted by halogens, deuterium, oxo groups, -CN, -OR 8 -NR 8 R 9 -P(O)(OR) 8 (OR) 9 (or optionally substituted with C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo groups;)

[0020] R 20 R 21 and R 22 Each is independently hydrogen, deuterium, C1-C 12 Alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl group, C1-C 12 Heteroalkyl, C2-C 12Heterene, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, halogen, -CN, -OR 3 -SR 3 -NR 4 R 5 -C = NH (OR 3 -C(O)R 3 -OC(O)R 3 -C(O)OR 3 -C(O)NR 4 R 5 -NR 3 C(O)R 4 -NR 3 C(O)OR 4 -NR 3 C(O)NR 4 R 5 -S(O)R 3 -S(O)2R 3 -NR 3 S(O)R 4 -NR 3 S(O)2R 4 -S(O)NR 4 R 5 -S(O)2NR 4 R 5 or -OP(=O)(OR) 4 (OR) 5 ), where R 20 R 21 and R 22 Where possible, it can be independently and optionally replaced by halogen, deuterium, oxo group, -CN, -OR 8 -NR 8 R 9 -OP(=S)(OR) 8 (OR) 9 -OP(=O)(OR) 8 (OR) 9 -OP(=S)(SR) 8 (OR) 9 (or optionally, C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo groups);

[0021] R 4 and R 5 Each of the following is independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 4 and R 5 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 6-membered heteroaryl, and 3- to 6-membered heterocyclic groups are independently and optionally replaced by deuterium, halogen, oxo group, -CN, or -OR. 8 -NR 8 R 9 Alternatively, it may be replaced by C1-C6 alkyl groups that are substituted with deuterium, halogen, -OH, or oxo groups;

[0022] R 6 and R 7 Each is independently hydrogen, deuterium, C1-C6 alkyl group optionally substituted with deuterium, halogen or oxo group, C2-C6 alkenyl group optionally substituted with deuterium, halogen or oxo group, or C2-C6 alkynyl group optionally substituted with deuterium, halogen or oxo group.

[0023] R 8 and R 9 Each is independently hydrogen, deuterium, C1-C6 alkyl group optionally substituted with deuterium, halogen or oxo group, C2-C6 alkenyl group optionally substituted with deuterium, halogen or oxo group, or C2-C6 alkynyl group optionally substituted with deuterium, halogen or oxo group.

[0024] Each R 10 R 11 R 12 and R 13 Independently hydrogen or deuterium;

[0025] R 14 It is deuterium;

[0026] q can be 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0027] Each R 15 Independently selected from hydrogen, deuterium, or halogen;

[0028] Each R 16 Independently selected from hydrogen, deuterium, or halogen; and

[0029] p is 3, 4, 5, 6, 7, 8, or 9.

[0030] Among them, R 2 and R 0 At least one of the molecules is connected to one or more transported molecules via L.

[0031] In one aspect, this disclosure also provides oligonucleotide conjugates comprising the above-described ligands, particularly siRNA.

[0032] In another aspect, this disclosure also provides a pharmaceutical composition comprising the oligonucleotide conjugate of this disclosure and a pharmaceutically acceptable carrier thereof.

[0033] In another aspect, this disclosure also provides the use of the oligonucleotide conjugates and / or pharmaceutical compositions of this disclosure in the preparation of medicaments for inhibiting the expression of target mRNAs for the purpose of inhibiting the expression of target genes in cells.

[0034] In another aspect, this disclosure also provides a method for inhibiting the expression of lung target genes in an animal model, the method comprising administering an effective amount of the oligonucleotide conjugate of this disclosure and / or the pharmaceutical composition of this disclosure to the lungs of said animal. Detailed Implementation

[0035] This disclosure provides ligands for the integrin αvβ6 protein receptor that have an affinity for the integrin αvβ6 protein receptor and specifically bind to it, thereby enabling the efficient delivery of conjugated molecules (particularly oligonucleotide molecules, such as RNAi agents) to desired cells or tissues expressing the integrin αvβ6 protein receptor, such as epithelial cells. This disclosure also provides methods for delivering the transported molecule to tissues and / or cells expressing the integrin αvβ6 protein receptor, for example, including delivery of an active pharmaceutical ingredient to a target organ or tissue (e.g., the lungs). Therefore, this disclosure also provides methods for treating a disease, symptom, or disorder in a subject, wherein delivery of a therapeutically transported molecule (e.g., an active pharmaceutical ingredient) to cells expressing the integrin αvβ6 protein receptor can treat the subject, wherein the method includes administering one or more ligands of the integrin αvβ6 protein receptor disclosed herein, which are conjugated to one or more therapeutically transported molecules, to the subject.

[0036] ligands of integrin αvβ6 protein receptor

[0037] In one aspect, this disclosure provides a ligand for the integrin αvβ6 protein receptor, comprising a targeting group and a linking group, and having the structure shown in Formula I:

[0038]

[0039] Among them, R 1 Selected from C6-C 14 Aryl or 5-10 heteroaryl, the C6-C 14 Aryl and 5-10 heteroaryl groups are selectively R 1a replace;

[0040] R 0 Selected from hydrogen or -R 0a -L;

[0041] -R 0a -L is selected from -R 3 NR 17 -L、-R 3 NR 17 R 18 -L、-R 3 C(O)NR 17 R 18 -L、-R 3 OC(O)R 17 NR 18 -L、-R 3 NR 17 C(O)R 18 -L、-R 3 OC(O)R 17 -L、-R 3 C(O)OR 17 -L、-R 3 NR 17 C(O)OR 18 -L or -R 3 OC(O)(NR 17 )R 18 -L;

[0042] R 2 For hydrogen; deuterium; optionally R 2a Substituted C1-C6 alkyl; -OH; optionally R 2b Substituted C3-C6 cycloalkyl groups; or -S(O)2R 2c ;

[0043] Or, R 2 It is a single key, preferably used to connect with L;

[0044] Where L is a linking group that links the transported molecule.

[0045] Where L is selected from

[0046] L1 and L2 are each independently selected from: -(R 20 ) m -、-(R 20 ) m R 21 R 22 -、-(R 20 ) m R 21 -、-(R 20 O) m -、-(R 20 O) m R21 -、-(R 20 O) m R 21 R 22 -、-R 20 C(O)R 21 -、-R 20 SSR 21 -、-R 20 C(O)R 21 R 22 -、-C(O)R 21 -、-C(O)R 21 R 22 -、-R 20 OC(O)R 21 -、-R 20 OC(O)R 21 R 22 -、-R 20 C(O)OR 21 -or-R 20 C(O)OR 21 R 22 - where m is an integer from 0 to 30;

[0047] Each R 1a Independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C8 cycloalkyl, deuterium, halogen, -CN, -OR 3 -NR 4 R 5 -C(O)R 3 -OC(O)R 3 -C(O)OR 3 -C(O)NR 4 R 5 , where each R 1a Independently and optionally by deuterium, halogen, oxy group, or -OR, where possible. 6 -NR 6 R 7 -C(O)R 6 C1-C6 alkyl substitutions with -CN or halogen substitutions;

[0048] Each R 2a R 2b R 2e and R 2f Independent of an oxygen group or R 1a ;

[0049] R 2c For optional use by R 2e Substituted C1-C6 alkyl groups or optionally R 2fSubstituted C3-C5 cycloalkyl groups;

[0050] R 3 R 17 R 18 and R 19 Each of these elements independently represents a bond, hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, or C6-C 14 aryl, 5- to 10-membered heteroaryl, or 3- to 12-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl and 3- to 12-membered heterocyclic groups are independently and optionally converted by halogens, deuterium, oxo groups, -CN, -OR 8 -NR 8 R 9 -P(O)(OR) 8 (OR) 9 (or optionally substituted with C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo groups;)

[0051] R 20 R 21 and R 22 Each is independently hydrogen, deuterium, C1-C 12 Alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl group, C1-C 12 Heteroalkyl, C2-C 12 Heterene, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, halogen, -CN, -OR 3 -SR 3 -NR 4 R 5 -C = NH (OR 3 -C(O)R 3 -OC(O)R 3 -C(O)OR 3 -C(O)NR 4 R 5 -NR 3 C(O)R 4 -NR 3 C(O)OR 4 -NR 3 C(O)NR 4 R 5 -S(O)R 3 -S(O)2R 3 -NR3 S(O)R 4 -NR 3 S(O)2R 4 -S(O)NR 4 R 5 -S(O)2NR 4 R 5 or -OP(=O)(OR) 4 (OR) 5 ), where R 20 R 21 and R 22 Where possible, it can be independently and optionally replaced by halogen, deuterium, oxo group, -CN, -OR 8 -NR 8 R 9 -OP(=S)(OR) 8 (OR) 9 -OP(=O)(OR) 8 (OR) 9 -OP(=S)(SR) 8 (OR) 9 (or optionally, C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo groups);

[0052] R 4 and R 5 Each of the following is independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14 aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 4 and R 5 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 6-membered heteroaryl, and 3- to 6-membered heterocyclic groups are independently and optionally replaced by deuterium, halogen, oxo group, -CN, or -OR. 8 -NR 8 R 9 Alternatively, it may be replaced by C1-C6 alkyl groups that are substituted with deuterium, halogen, -OH, or oxo groups;

[0053] R 6 and R 7 Each is independently hydrogen, deuterium, C1-C6 alkyl group optionally substituted with deuterium, halogen or oxo group, C2-C6 alkenyl group optionally substituted with deuterium, halogen or oxo group, or C2-C6 alkynyl group optionally substituted with deuterium, halogen or oxo group.

[0054] R 8 and R 9Each is independently hydrogen, deuterium, C1-C6 alkyl group optionally substituted with deuterium, halogen or oxo group, C2-C6 alkenyl group optionally substituted with deuterium, halogen or oxo group, or C2-C6 alkynyl group optionally substituted with deuterium, halogen or oxo group.

[0055] Each R 10 R 11 R 12 and R 13 Independently hydrogen or deuterium;

[0056] R 14 It is deuterium;

[0057] q can be 0, 1, 2, 3, 4, 5, 6, 7, or 8;

[0058] Each R 15 Independently selected from hydrogen, deuterium, or halogen;

[0059] Each R 16 Independently selected from hydrogen, deuterium, or halogen; and

[0060] p is 3, 4, 5, 6, 7, 8, or 9.

[0061] Among them, R 2 and R 0 At least one of the molecules is connected to one or more transported molecules via L.

[0062] The ligands of the integrin αvβ6 protein receptor include pharmaceutically acceptable salts, in the form of isoforms (e.g., stereoisomers).

[0063] In some embodiments, the ligand of the integrin αvβ6 protein receptor may have a structure selected from the following formula:

[0064]

[0065] or

[0066]

[0067] Among them, R 0 R 0a R 1 R 2 R 10 R 11 R 12 R 13 R 14 R 15 R 16 L, p, and q are defined as above.

[0068] In some implementations, R 1It may be selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thienopyrimidinyl, pyridyl, pyrrolopyrimidinyl, quinoxalinyl, indazoleyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl; and optionally substituted with deuterium, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 perhaloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 halocycloalkyl, C3-C8 cycloalkoxy, cyano, amino, alkylamino, or dialkylamino.

[0069] In a specific implementation, R 1 Selectable from pyrimidin-2-yl, pyrimidin-4-yl, quinazolin-4-yl, 1H-pyrazolo[3,4-d]pyrimidin-4-yl, 1H-pyrazolo[4,3-d]pyrimidin-7-yl, pyrazin-2-yl, quinolin-4-yl, pyrido[2,3-d]pyrimidin-4-yl, pyrido[3,2-d]pyrimidin-4-yl, pyrido[3,4-d]pyrimidin-4-yl, thieno[2,3-d]pyrimidin-4-yl, thieno[3,2-d]pyrimidin-4-yl, thieno[2,3-d]pyrimidin-4-yl, thieno[3,2-d]pyrimidin-4-yl, thienopyrimidin-4-yl, Pyridin-2-yl, pyridin-3-yl, 7H-pyrrolo[2,3-d]pyrimidin-4-yl, quinoxalin-2-yl, 1H-indazole-3-yl, benzo[d]thiazolyl-2-yl, naphthyl-1-yl, 9H-purine-6-yl or isoquinoline-1-yl; and optionally substituted with one or more of the following groups: deuterium; methyl; cyclopropyl; fluorine; chlorine; bromine; difluoromethyl; trifluoromethyl; methyl and fluorine; methyl and trifluoromethyl; methoxy; cyano; dimethylamino; phenyl; pyridin-3-yl; or pyridin-4-yl.

[0070] In a specific implementation, R 1 For optional use by R 1a Substituted quinazolin-4-yl.

[0071] In a specific implementation, R 1 It is a quinazoline-4-yl group optionally substituted with a halogen, or a quinazoline-4-yl group optionally substituted with a C1-C6 alkyl or C1-C6 alkoxy group.

[0072] In a specific implementation, R 1 It is a quinazolin-4-yl group optionally substituted with fluorine, chlorine, methyl, trifluoromethyl, or methoxy. In a preferred embodiment, R 1 It is an unsubstituted quinazolin-4-yl group.

[0073] In some implementations, R 0 and R 2 One of them has a linking group L. For example, when R 0 When it is hydrogen, R 2For a single key and connected to L; or, when R 0 Selected from -R 0a When -L, R 2 Selected from hydrogen, deuterium, and optionally R 2a Substituted C1-C6 alkyl groups, -OH groups, optionally R 2b Substituted C3-C6 cycloalkyl groups, or -S(O)2R 2c .

[0074] In some implementations, R 0a Optional from -C 1-6 Alkyl-NH-, optionally substituted with halogen, deuterium, oxo group, or C1-C3 alkyl.

[0075] In some preferred embodiments, the ligand of the integrin αvβ6 protein receptor may have a structure selected from the following formula:

[0076]

[0077] or,

[0078]

[0079] Among them, R 0 R 0a R 2 R 10 R 11 R 12 R 13 R 14 R 15 R 16 L, p, and q are defined above.

[0080] And among them, structural units Can be optionally R 1a replace.

[0081] In a specific implementation, each R 1a Independently C1-C6 alkyl, deuterium, halogen, -CN, -OH, -NH2, or -NO2, wherein each R 1a It can be independently and optionally replaced by deuterium or halogens, where possible.

[0082] In a specific implementation, R 2 Optional and can be R 2a Substituted C1-C6 alkyl; -OH; optionally R 2b Substituted C3-C6 cycloalkyl groups; or -S(O)2R 2c .

[0083] In a specific implementation, R 2Optional and can be R 2a Substituted C1-C6 alkyl groups, -OH groups, and optionally R groups 2b Substituted C3-C6 cycloalkyl groups. Preferably, R 2 Optional and can be R 2a Substituted C1-C6 alkyl groups and optionally R 2b Substituted C3-C6 cycloalkyl groups. Most preferably, R 2 Optional and can be R 2a Substituted C1-C6 alkyl groups.

[0084] In some implementations, R 2a The derivatives can be selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, deuterium, oxoyl, halogen, -CN, -OH, -NH2, -C(O)-C1-C6 alkyl, -OC(O)-C1-C6 alkyl, -C(O)O-C1-C6 alkyl, -C(O)NH-C1-C6 alkyl, -C(O)N(C1-C6 alkyl)2, wherein each R 2a Where possible, it may be independently and optionally substituted with C1-C6 alkyl groups substituted with deuterium, halogen, oxo group, -OH, -NH2, -C(O)-C1-C6 alkyl, -CN or halogen.

[0085] In some implementations, R 2a It can be selected from C1-C6 alkyl, -O-C1-C6 alkyl, deuterium, oxo group, -CN, -OH, or -NH2.

[0086] In a preferred embodiment, R 2 The group can be selected from methyl, ethyl, propyl, cyclopropyl, or cyclobutyl; each of which is optionally substituted by one or more of the following groups: hydroxyl, methoxy, ethoxy, acetamyl, fluorine, fluoroalkyl, or dimethylamido. In a more specific embodiment, R 2 It can be a methyl, ethyl, or propyl group substituted with hydroxyl, methoxy, or ethoxy groups. In the most preferred embodiment, R 2 It can be methyl, ethyl, or propyl substituted with methoxy or ethoxy, such as ethyl substituted with methoxy.

[0087] In a specific implementation, R 3 Independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14 aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14Aryl, 5- to 6-membered heteroaryl, and 3- to 6-membered heterocyclic groups are independently and optionally converted by halogens, deuterium, oxo groups, -CN, or -OR. 8 -NR 8 R 9 -P(O)(OR) 8 (OR) 9 (or optionally, C1-C6 alkyl groups substituted with deuterium, halogen, -OH, or oxo groups.)

[0088] In a specific implementation, R 3 Independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14 aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 The aryl, 5- to 6-membered heteroaryl and 3- to 6-membered heterocyclic groups are independently and optionally substituted with halogen, deuterium, oxo, -CN, -OH, -NH2, -P(O)(OH)(OH) or optionally with C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo.

[0089] In a specific implementation, m is selected from an integer from 1 to 12, such as an integer from 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, or 10).

[0090] In a specific implementation, R 10 R 11 R 12 and R 13 It is hydrogen independently.

[0091] In a specific implementation, q is 0.

[0092] In a specific implementation, R 15 and R 16 It is hydrogen, and p is 3 or 4, preferably 3.

[0093] In some preferred embodiments, the ligand of the integrin αvβ6 protein receptor may have the following structure:

[0094]

[0095] or,

[0096] Among them, R 0a R 2 And L is as defined above,

[0097] And among them, structural units Can be optionally R 1a replace.

[0098] In some implementations, R 0a Optional from -C 1-6 Alkyl group -NH-, optionally substituted with halogen, deuterium, oxo group, or C1-C3 alkyl group. Preferably, R 0a It can be -CH2-NH- or -CH2-CH2-NH-.

[0099] In some embodiments, the ligand of the integrin αvβ6 protein receptor may have a structure of formula I-A2, wherein R 0a R 2 And L as defined in this paper, and wherein, structural unit Can be optionally R 1a replace.

[0100] In some embodiments, the ligand of the integrin αvβ6 protein receptor may have a structure of formula I-A2, wherein R 0a Selected from -CH2-NH- or -CH2-CH2-NH-, and L as defined herein.

[0101] In specific embodiments, the ligands of the integrin αvβ6 protein receptor of this disclosure can be linked by phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, amino carbonate bonds, and carbonate bonds, thereby linking multiple ligands of this disclosure together to deliver multiple transported molecules simultaneously. The transported molecules may be the same or different, or multiple ligands of this disclosure may be linked to a single molecule to be transported, so as to more efficiently transport the molecules to tissues and / or cells expressing the integrin αvβ6 protein receptor.

[0102] Alternatively, the link between the ligand of this disclosure and the transported molecule can be determined according to the structure of the transported molecule, and can typically be linked via phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds. For example, when the transported molecule is an oligonucleotide (e.g., an RNAi agent), the ligand of this disclosure can be linked to the ribose group, base, or phosphate backbone of the nucleotide via phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds.

[0103] Linking group

[0104] The linker group L can be selected from:

[0105]

[0106] Among them, L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C1-C 12 Heteroalkyl-,-C(O)-C1-C 12 Alkyl-, -C(O)-C1-C 12 Heteroalkyl-, -C1-C 12 Alkyl-SS-C1-C 12 Alkyl-, -C1-C 12 Alkyl-SS-C1-C 12 Heteroalkyl-, -C1-C 12 Heteroalkyl-SS-C1-C 12 Heteroalkyl-, -(CH2CH2O) m -C1-C 12 Alkyl-, -C1-C 12 Alkyl-5 to 6-membered heterocyclic group - or -C1-C 12 Heteroalkyl-5 to 6-membered heterocyclic-, optionally substituted with a halogen, deuterium, oxo group, -NH2, -NO2, -CN, -OH, or optionally substituted with a halogen or -OH C1-C3 alkyl group, optionally substituted with a halogen C1-C3 alkoxy group, wherein m is selected from an integer from 1 to 10.

[0107] R 19 Each is independently hydrogen, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl or 3- to 12-membered heterocyclic group.

[0108] In some implementations, L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C1-C 12 Heteroalkyl-,-C(O)-C1-C 12 Alkyl-, -C(O)-C1-C 12 Heteroalkyl-, -C(O)-C1-C2alkyl-(CH2CH2O) m -、-C(O)-(CH2CH2O) m -C1-C2 alkyl-, -(CH2CH2O) m -C1-C 12 Alkyl-, -C1-C 12 Alkyl-5 to 6-membered heterocyclic group - or -C1-C 12The heteroalkyl-5 to 6-membered heterocyclic group is optionally substituted with a halogen, deuterium, oxo group, -NH2, -NO2, -CN, -OH, or a C1-C3 alkyl group optionally substituted with a halogen or -OH, or a C1-C3 alkoxy group optionally substituted with a halogen, wherein m is selected from integers from 1 to 5.

[0109] In some implementations, L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C(O)-C1-C 12 Alkyl group -, -C(O)-CH2-(CH2CH2O) m -、-C(O)-(CH2CH2O) m -CH2CH2-, -(CH2CH2O) m -C1-C 12 Alkyl-, -C1-C 12 Alkyl-5 to 6-membered heterocyclic group - or -C1-C 12 Heteroalkyl-5 to 6-membered heterocyclic-, wherein m is selected from an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5).

[0110] In some implementations, L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C(O)-C1-C 12 Alkyl group -, -C(O)-CH2-(CH2CH2O) m -、-C(O)-(CH2CH2O) m -CH2CH2-, -(CH2CH2O) m -C1-C 12 Alkyl-, -C1-C 12 Alkyl-5 to 6-membered heterocyclic group - or -CH2-(CH2CH2O) m -5 to 6-membered heterocyclic base-, where m is selected from integers from 1 to 5.

[0111] In some embodiments, the linking group L is selected from... In a preferred embodiment, the linking group L is selected from...

[0112] In this invention, L1 and L2 are merely descriptive terms and do not imply that L1 as defined in the context of this document cannot be located in the position of L2. For example, The L1 and L2 in the text are only for the purpose of making the description clearer, and do not mean that the L1 option described cannot be in the L2 position.

[0113] Furthermore, there is no particular limitation on the direction of L; that is, the left-to-right order described in this article does not imply that the ligand's target group and the transported molecule are linked in that order, and they can be linked in either the left-to-right or reverse direction. For example, In this process, a target group can be attached to the L1 end and the transported molecule can be attached to the L2 end, or vice versa.

[0114] In some preferred embodiments, multiple linking groups L may be present; for example, the groups L may be linked together to form a branched structure, thereby linking multiple target groups. In some embodiments, the multiple L are linked by phosphate ester bonds or thiophosphate ester bonds.

[0115] In specific embodiments, the link between L1 and / or L2 in the ligands of this disclosure and the transported molecule can be established via phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds. For example, when the transported molecule is an oligonucleotide (e.g., an RNAi agent), L1 and / or L2 in the ligands of this disclosure can be linked to the ribose group, base, or phosphate backbone of the nucleotide, or the 5' and / or 3' hydroxyl or phosphate group or phosphate-derived group at the 5' and / or 3' ends via phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds.

[0116] In a specific implementation, L1 and / or L2 are linked to nucleotides via phosphate ester bonds or thiophosphate ester bonds.

[0117] In another embodiment, L1 and / or L2 of the ligands disclosed herein can be linked by phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds, thereby linking multiple ligands of the present disclosure together to simultaneously deliver multiple transported molecules, wherein the transported molecules may be the same or different, or multiple ligands of the present disclosure may be linked to a single molecule to be transported, to more efficiently transport the molecules to tissues and / or cells expressing the integrin αvβ6 protein receptor. In a specific embodiment, one end of L1 and / or L2 can be linked to a target group by a single bond, an N-substituted or unsubstituted amide bond, an aminocarbonate bond, or a carbonate bond.

[0118] In some embodiments, the 5- to 6-membered heterocyclic groups in L1 and L2 may be selected from 5- to 6-membered heterocyclic alkyl groups containing 1, 2, or 3 heteroatoms selected from N, O, or S as ring atoms, such as tetrahydropyrrole, dioxanecycloyl, morpholinyl, tetrahydropyranyl, piperidinyl, or tetrahydrofuranyl, preferably tetrahydropyrrole or morpholinyl.

[0119] In some implementations, L1 and / or L2 may each be independently selected from: C1-C 12 Alkylene;

[0120]

[0121] In some implementations, L1 and / or L2 may each be independently selected from: C1-C 12 Alkylene;

[0122]

[0123] In this invention, one end of L1 and / or L2 may be connected to a target group or the transported molecule. For example, L1 and / or L2 may be linked to the transported molecule via a phosphodiester bond, a phosphotriester bond, a thiophosphate diester bond, a thiophosphate triester bond, an N-substituted or unsubstituted amide bond, an aminocarbonate bond, or a carbonate bond. Alternatively, in a specific embodiment, one end of L1 and / or L2 may be connected to the target group via a single bond, an N-substituted or unsubstituted amide bond, an aminocarbonate bond, or a carbonate bond.

[0124] In a further preferred embodiment, L1 and / or L2 may also be linked to additional ligands (e.g., the same or different ligands, such as those disclosed herein) or additional transported molecules (e.g., the same or different active drug molecules), for example, via phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, amino carbonate bonds, or carbonate bonds. For example, with For example, when a portion of the five-membered ring is linked to the transported molecule, another transported molecule or another ligand can be linked through the hydroxyl group on the five-membered ring.

[0125] In some implementations, L1 and / or L2 may each be independently selected from: C1-C 12 Alkylene;

[0126] For clarity, some or all of the connection structures are shown in the above formula.

[0127] In some implementations, L1 and / or L2 may each be independently selected from: C1-C 12 Alkylene;

[0128]

[0129] or

[0130] L1 and / or L2 can be independently selected from: C1-C12 Alkylene;

[0131]

[0132]

[0133] in, Connect to the target group, Linking the transported molecule (e.g., RNAi agents); for clarity, and / or Partial or complete depiction of the linking structure is shown; wherein the unpointed end is directly connected to the central ring of the linking group L. For example, and / or This indicates that the terminator is linked to the target group or the transported molecule by a single bond at the indicated end; and / or This indicates that the terminator is linked to the target group or the transported molecule via a phosphodiester bond at the indicated end. This indicates that the terminator is linked to the target group or the transported molecule via a phosphodiester bond at the indicated end. or This indicates that the terminator is linked to the target group or the transported molecule via an ester or amide bond at the indicated end.

[0134] In some implementations, L1 and / or L2 may each be independently selected from: C4-C 10 Alkylene, preferably C4-C7 alkylene, such as C4-C5 alkylene or C6-C7 alkylene; or For example or or For example, or For example, or For example or For example or or For example, or or For example or or For example, or or For example, or For example or For example, or

[0135] in, Connect to the target group, Linking the transported molecule (e.g., RNAi agents).

[0136] In some embodiments, the linking group L may be selected from:

[0137]

[0138] in, Connect to the target group, Connect the transported molecules;

[0139] in, Connect to the target group, Connecting the transported molecules; for example,

[0140]

[0141] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0142] in, Connect to the target group, Connecting the transported molecules, for example,

[0143]

[0144] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0145] in, Connect to the target group, Connecting the transported molecules, for example,

[0146]

[0147] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0148] in, Connect to the target group, Connecting the transported molecules, for example,

[0149] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0150] in, Connect to the target group, Connecting the transported molecules, for example,

[0151] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0152] in, Connect to the target group, Connecting the transported molecules, for example,

[0153] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0154] in, Connect to the target group, Connecting the transported molecules, for example,

[0155] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0156] in, Connect to the target group, Connecting the transported molecules, for example,

[0157] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O; or

[0158] in, Connect to the target group, Connecting the transported molecules, for example,

[0159] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O.

[0160] When multiple ligands of this disclosure are linked together by phosphodiester bonds, phosphotriester bonds, thiophosphate diester bonds, thiophosphate triester bonds, N-substituted or unsubstituted amide bonds, aminocarbonate bonds, or carbonate bonds, the linking group may be two or more linking groups L linked together. In some embodiments, the linking group may be two or three linking groups L linked together.

[0161] In some preferred embodiments, the linking group L may be selected from the following:

[0162]

[0163] in, Connect to the target group, Connect the transported molecules;

[0164]

[0165] in, Connect to the target group, Connect the transported molecules;

[0166]

[0167] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0168]

[0169] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0170]

[0171] in, Connect to the target group, Connect the transported molecules;

[0172]

[0173] in, Connect to the target group, Connect the transported molecule, where X is selected from S or O;

[0174]

[0175] in, Connect to the target group, Connect the transported molecules;

[0176]

[0177] in, Connect to the target group, Connect the transported molecules;

[0178]

[0179] in, Connect to the target group, Connect the transported molecules;

[0180]

[0181] in, Connect to the target group, Connect the transported molecules;

[0182]

[0183] in, Connect to the target group, Connect the transported molecules.

[0184] Targeting group

[0185] In a preferred embodiment, the targeting group may be selected from the following:

[0186]

[0187] In a preferred embodiment, the targeting group may be selected from the following:

[0188]

[0189] In particular, this disclosure provides a ligand for the integrin αvβ6 protein receptor, comprising a targeting group and a linker group, and having the structure shown in Formula I:

[0190]

[0191] Among them, R 1 Selected from C6-C 14 Aryl or 5-10 heteroaryl, the C6-C 14 Aryl and 5-10 heteroaryl groups are selectively R 1a Replace; R 1The derivatives can be selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thiophenopyrimidinyl, pyridyl, pyrrolopyrimidinyl, quinoxalinyl, indazoleyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl, optionally with R 1a replace;

[0192] R 0 Selected from hydrogen or -R 0a -L;

[0193] -R 0a -L is selected from -C 1-6 Alkyl-NH-L, wherein the structural moiety is -C 1-6 The alkyl group -NH- is optionally replaced by a halogen, deuterium, oxo group, or C1-C3 alkyl group;

[0194] R 2 For hydrogen; deuterium; optionally R 2a Substituted C1-C6 alkyl; -OH; optionally R 2b Substituted C3-C6 cycloalkyl groups; or -S(O)2R 2c ;

[0195] Or, R 2 It is a single key, preferably used to connect with L;

[0196] Where L is a linking group that links the transported molecule.

[0197] Where L is selected from

[0198]

[0199] L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C1-C 12 Heteroalkyl-,-C(O)-C1-C 12 Alkyl-, -C(O)-C1-C 12 Heteroalkyl-, -C1-C 12 Alkyl-SS-C1-C 12 Alkyl-, -C1-C 12 Alkyl-SS-C1-C 12 Heteroalkyl-, -C1-C 12 Heteroalkyl-SS-C1-C 12 Heteroalkyl-, -(CH2CH2O) m -C1-C 12 Alkyl- or -C1-C 12Alkyl-5 to 6-membered heterocyclic group, optionally substituted with halogen, deuterium, oxo group, -NH2, -NO2, -CN, -OH, or C1-C3 alkyl group optionally substituted with halogen or -OH, or C1-C3 alkoxy group optionally substituted with halogen, wherein m is selected from an integer from 1 to 10.

[0200] Each R 1a Independently, it is deuterium, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 perhaloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 halocycloalkyl, C3-C8 cycloalkoxy, cyano, amino, alkylamino or dialkylamino;

[0201] Each R 2a R 2b R 2e and R 2f Independent of an oxygen group or R 1a ;

[0202] R 2c For optional use by R 2e Substituted C1-C6 alkyl groups or optionally R 2f Substituted C3-C5 cycloalkyl groups;

[0203] R 19 Each is independently hydrogen, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl or 3- to 12-membered heterocyclic group;

[0204] Each R 10 R 11 R 12 and R 13 It can be independently hydrogen or deuterium, preferably independently hydrogen;

[0205] R 14 It is deuterium;

[0206] q can be 0, 1, 2, 3, 4, 5, 6, 7 or 8, preferably q can be 0, 1 or 2, and more preferably q can be 0;

[0207] Each R 15 It is independently selected from hydrogen, deuterium or halogen, preferably independently hydrogen;

[0208] Each R 16 It is independently selected from hydrogen, deuterium, or halogen, preferably independently hydrogen; and

[0209] p is 3, 4, 5, 6, 7, 8 or 9, preferably 3, 4, 5 or 6;

[0210] Among them, R 2 and R 0At least one of the molecules is connected to one or more transported molecules via L.

[0211] This disclosure provides a ligand for the integrin αvβ6 protein receptor, comprising a targeting group and a linking group, and having the structure shown in Formula I:

[0212]

[0213] Among them, R 1 Selected from C6-C 14 Aryl or 5-10 heteroaryl, the C6-C 14 Aryl and 5-10 heteroaryl groups are selectively R 1a Replace; R 1 The derivatives can be selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thiophenopyrimidinyl, pyridyl, pyrrolopyrimidinyl, quinoxalinyl, indazoleyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl, optionally with R 1a replace;

[0214] R 0 Selected from -R 0a -L;

[0215] -R 0a -L is selected from -C 1-6 Alkyl-NH-L, wherein the structural moiety is -C 1-6 The alkyl group -NH- is optionally replaced by a halogen, deuterium, oxo group, or C1-C3 alkyl group;

[0216] R 2 For hydrogen; deuterium; optionally R 2a Substituted C1-C6 alkyl; -OH; optionally R 2b Substituted C3-C6 cycloalkyl groups; or -S(O)2R 2c ;

[0217] Where L is a linking group that links the transported molecule.

[0218] Where L is selected from

[0219] L1 and L2 are each independently selected from: -C1-C 12 Alkyl-, -C1-C 12 Heteroalkyl-,-C(O)-C1-C 12 Alkyl-, -C(O)-C1-C 12 Heteroalkyl-, -C1-C 12 Alkyl-SS-C1-C 12 Alkyl-, -C1-C 12 Alkyl-SS-C1-C12 Heteroalkyl-, -C1-C 12 Heteroalkyl-SS-C1-C 12 Heteroalkyl-, -(CH2CH2O) m -C1-C 12 Alkyl- or -C1-C 12 Alkyl-5 to 6-membered heterocyclic group, optionally substituted with halogen, deuterium, oxo group, -NH2, -NO2, -CN, -OH, or C1-C3 alkyl group optionally substituted with halogen or -OH, or C1-C3 alkoxy group optionally substituted with halogen, wherein m is selected from an integer from 1 to 10.

[0220] Each R 1a Independently, it is deuterium, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 perhaloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 halocycloalkyl, C3-C8 cycloalkoxy, cyano, amino, alkylamino or dialkylamino;

[0221] Each R 2a R 2b R 2e and R 2f Independent of an oxygen group or R 1a ;

[0222] R 2c For optional use by R 2e Substituted C1-C6 alkyl groups or optionally R 2f Substituted C3-C5 cycloalkyl groups;

[0223] Each R 10 R 11 R 12 and R 13 It can be independently hydrogen or deuterium, preferably independently hydrogen;

[0224] R 14 It is deuterium;

[0225] q can be 0, 1, or 2, preferably 0;

[0226] Each R 15 It is hydrogen;

[0227] Each R 16 It is hydrogen; and

[0228] p is 3, 4, 5 or 6;

[0229] Among them, R 0 One or more transported molecules are connected by an L-link.

[0230] In this disclosure, the transported molecule may be, but is not limited to, small molecules, antibodies, antibody fragments, immunoglobulins, monoclonal antibodies, labels or markers, lipids, natural or modified oligonucleotide-based compounds (e.g., antisense oligonucleotides or RNAi agents), natural or modified nucleic acids, peptides, nucleic acid aptamers, polymers, polyamines, proteins, toxins, vitamins, polyethylene glycol, haptens, digoxigenin, biotin, radioactive atoms or molecules, or fluorophores. In some embodiments, the transported molecule includes a pharmaceutically active ingredient or a prodrug. In some embodiments, the transported molecule includes an oligonucleotide-based compound as a pharmaceutically active ingredient. In some embodiments, the transported molecule includes an RNAi agent as a pharmaceutically active ingredient.

[0231] The double-stranded oligonucleotide comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to the target gene; and the sense strand is complementary to the antisense strand.

[0232] In some implementations, the antisense strand comprises a sequence whose inverse complementary sequence to at least 15 consecutive nucleotides in the target sequence differs by no more than 3 nucleotides. Specifically, a start nucleotide is selected in the target sequence along the 5' end to the 3' end, and at least 15 nucleotides extending in the 3' direction, including the start nucleotide, serve as the binding region of the siRNA. The antisense strand comprises the inverse complementary sequence of the nucleotide sequence corresponding to the binding region. It should be noted that the start nucleotide can be a nucleotide at any position in the target sequence, as long as extending in the 3' direction from the start nucleotide yields at least 15 consecutive nucleotides (including the nucleotide at the start position).

[0233] In this disclosure, the nucleotide sequence of the antisense strand can be completely complementary or substantially complementary to the target sequence. When the nucleotide sequence of the antisense strand is substantially complementary to the target sequence, the nucleotide sequence of the antisense strand contains no more than three mismatched bases with the target sequence. For example, the number of mismatched bases is one, two, or three. When the nucleotide sequence of the antisense strand is completely complementary to the target sequence, the nucleotide sequence of the antisense strand does not contain any mismatched bases with the target sequence.

[0234] Furthermore, the antisense strand consists of at least 15 nucleotides. In some embodiments, the antisense strand consists of 15-28 nucleotides. For example, the length of the antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the antisense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0235] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the antisense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the antisense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the antisense strand.

[0236] In some embodiments, the sense strand comprises a sequence differing from at least 15 consecutive nucleotides in the target sequence by no more than 3 nucleotides. The sense strand includes regions complementary to the antisense strand, and the nucleotide sequence of the sense strand is identical or substantially identical to the sequence of the antisense strand binding region on the target sequence. Therefore, the nucleotide sequence of the sense strand is at least 15 consecutive nucleotides in the target sequence that bind the antisense strand; or, the nucleotide sequence of the sense strand differs from at least 15 consecutive nucleotides in the target sequence that bind the antisense strand by 1, 2, or 3 bases.

[0237] Furthermore, the positive sense strand consists of at least 15 nucleotides. In some embodiments, the positive sense strand consists of 15-28 nucleotides. For example, the length of the positive sense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides. Preferably, the positive sense strand consists of 18-25 nucleotides, more preferably 18-23 nucleotides, and most preferably 19, 21, or 23 nucleotides.

[0238] In some specific embodiments, the nucleotide that differs from the target sequence is preferably located at the end of the sense strand. For example, in some specific embodiments, the differing nucleotide is located at the 3' end of the sense strand. In other specific embodiments, the differing nucleotide is located at the 5' end of the sense strand.

[0239] In this disclosure, the length of the justice chain and the length of the antisense chain may be the same or different.

[0240] In some implementations, the lengths of the justice chain and the antisense chain are the same. Specifically, the length ratio of the justice chain to the antisense chain is 15 / 15, 16 / 16, 17 / 17, 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, 25 / 25, 26 / 26, 27 / 27, or 28 / 28. Preferably, the length ratio of the justice chain to the antisense chain is 18 / 18, 19 / 19, 20 / 20, 21 / 21, 22 / 22, 23 / 23, 24 / 24, or 25 / 25; more preferably, it is 19 / 19, 20 / 20, 21 / 21, 22 / 22, or 23 / 23; and most preferably, it is 19 / 19, 21 / 21, or 23 / 23.

[0241] In some implementations, the lengths of the justice chain and the antisense chain are different. For example, the justice chain / antisense chain length ratio is 18 / 19, 18 / 20, 18 / 21, 18 / 22, 18 / 23, 18 / 24, 18 / 25, 18 / 26, 19 / 18, 19 / 20, 19 / 21, 19 / 22, 19 / 23, 19 / 24, 19 / 25, 19 / 26, 20 / 19, 20 / 21, 20 / 22, 20 / 23, 20 / 24, 20 / 25. Examples of possible lengths include 20 / 26, 21 / 18, 21 / 19, 21 / 20, 21 / 22, 21 / 23, 21 / 24, 21 / 25, 21 / 26, 22 / 18, 22 / 19, 22 / 20, 22 / 21, 22 / 23, 22 / 24, 22 / 25, 22 / 26, 23 / 19, 23 / 20, 23 / 21, 23 / 22, 23 / 24, 23 / 25, or 23 / 26. In some preferred embodiments, the length ratio of the justice chain to the antisense chain is 19 / 21 or 21 / 23.

[0242] In this disclosure, the sense strand and the antisense strand can be fully complementary or substantially complementary. When they are substantially complementary, there are no more than 3 mismatched bases in the double-stranded region formed by the sense strand and the antisense strand.

[0243] In some embodiments, after the sense and antisense strands are at least partially complementary to form a double-stranded region, the sense, antisense, or combination thereof has protruding nucleotides extending out of the double-stranded region. The number of protruding nucleotides can be one or more, for example, one or two. Furthermore, the one or two protruding nucleotides can be located at the 5' end, 3' end, or both ends of any antisense or sense strand, and each protruding nucleotide can be of any type. For example, in the case where the sense strand consists of a 21-bit sequence A+D and the antisense strand consists of a 21-bit sequence B+E, the nucleotides at positions 1-19 of sequence A are completely anticomplementary to those at positions 1-19 of sequence B in the direction from the 5' end to the 3' end, thereby forming protruding nucleotides at the 3' end of sequences D and E, respectively. For example, when the sense strand consists of a 19-position sequence A and the antisense strand consists of a 21-position sequence B+E, the nucleotides 1-19 of sequence A are completely anticomplementary to those 1-19 of sequence B in the direction from the 5' end to the 3' end, thus forming a protruding nucleotide at the 3' end of sequence E on the antisense strand.

[0244] In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the sense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the antisense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and the 3' end of the antisense strand has 1-2 protruding nucleotides extending out of the double-stranded region, while the 3' end of the sense strand is blunt. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have 1-2 protruding nucleotides extending out of the double-stranded region at their 3' ends. In some embodiments, the sense strand and the antisense strand are at least partially complementary to form the double-stranded region, and both the sense strand and the antisense strand have blunt 3' ends.

[0245] In this document, the double-stranded oligonucleotides are modified in the sense strand and / or antisense strand, and such modified double-stranded oligonucleotides are referred to herein as "double-stranded oligonucleotide modifiers".

[0246] In some embodiments, each nucleotide of the sense strand is independently a modified or unmodified nucleotide. In some embodiments, each nucleotide of the antisense strand is independently a modified or unmodified nucleotide.

[0247] In some embodiments, any two linked nucleotides in the sense strand are linked by a phosphodiester bond or a phosphothiodiester bond. In some embodiments, any two linked nucleotides in the antisense strand are linked by a phosphodiester bond or a phosphothiodiester bond.

[0248] In some embodiments, the ribosome group of the 5' terminal nucleotide of the positive strand is a 5' hydroxyl group.

[0249] In some embodiments, the ribosome of the 5' terminal nucleotide of the sense strand may have a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand has a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group. The type of group at the 5' position of the ribosome depends on the preparation method used, which is known to those skilled in the art based on the corresponding preparation method.

[0250] For example, the structure of the 5' phosphate group is as follows: The structure of the 5' phosphate derivative group includes, but is not limited to: wait.

[0251] Furthermore, the double-stranded oligonucleotide is modified with siRNA.

[0252] In some embodiments, the siRNA modifier comprises a modification of at least one nucleotide. The nucleotide modification is selected from at least one of ribose group modification and base modification. In some embodiments, "nucleotide modification" refers to a nucleotide or nucleotide derivative formed by replacing the 2' hydroxyl group of the ribose group with another group, or a nucleotide whose base is a modified base. The nucleotide modification does not result in a significant weakening or loss of the siRNA's ability to suppress gene expression. For example, modified nucleotides disclosed in JK Watts, G.F. Deleavey, and MJ Damha, Chemically Modified siRNA: Tools and Applications. Drug Discov Today, 2008, 13(19-20):842-55 can be selected. Nucleotide modification can improve the stability of siRNA and maintain its high inhibitory efficiency against target genes.

[0253] For example, the modified nucleosides have the following structures:

[0254] Wherein, Base represents a base, such as A, U, G, C, or T. The hydroxyl group at the 2' position of the ribosome is replaced by R. These hydroxyl groups at the 2' position of the ribosome can be replaced by various groups known to those skilled in the art; for example, R can be selected from halogens, alkyl groups, alkoxy groups, substituted alkyl groups, and substituted alkoxy groups. For example, in some specific embodiments, the modified nucleotide includes, but is not limited to, 2'-fluoro(2'-F) modified nucleotides, 2'-alkoxy modified nucleotides, 2'-substituted alkoxy modified nucleotides, 2'-alkyl modified nucleotides, 2'-substituted alkyl modified nucleotides, and 2'-deoxyribonucleotides.

[0255] In some embodiments, the 2'-alkoxy modified nucleotide is a nucleotide modified with 2'-methoxy (2'-OMe, 2'-O-CH3), etc.

[0256] In some embodiments, the 2'-substituted alkoxy-modified nucleotide is a nucleotide modified with 2'-methoxyethoxy (2'-O-CH2-CH2-O-CH3), a nucleotide modified with 2'-O-CH2-CH=CH2, etc.

[0257] In some embodiments, the 2'-substituted alkyl-modified nucleotide is a 2'-CH2-CH2-CH=CH2 modified nucleotide, etc.

[0258] In some embodiments, the modification of the nucleotide is a modification of its bases. Base modifications can be of various types known to those skilled in the art. Exemplary examples include, but are not limited to, m... 6 A、Ψ、m 1 A、m 5 A, ms 2 i 6 A、i 6 A、m 3 C, m 5 C、ac 4 C, m 7 G, m 2,2 G, m 2 G, m 1 G, Q, m 5 U、mcm 5 U、ncm 5 U、ncm 5 Um, D, mcm 5 s 2 U, Inosine(I), hm 5 C, s 4 U、s 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives.

[0259] In some embodiments, a nucleotide derivative refers to a compound that can replace a nucleotide in a nucleic acid but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide derivative can be an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. A BNA refers to a restricted or inaccessible nucleotide. A BNA can contain a bridging structure with a "fixed" C3'-endoglucan condensation, such as a five-membered, six-membered, or seven-membered ring. This bridge is typically incorporated into the 2',4' position of the ribose to provide a 2',4'-BNA nucleotide, such as LNA, ENA, cET, etc.

[0260] The nucleosides of LNA are shown in formula (1), the nucleosides of ENA are shown in formula (2), and the nucleosides of cET are shown in formula (3):

[0261]

[0262] Here, "Base" refers to a base, such as A, U, G, C, or T.

[0263] Acyclic nucleotides are a class of nucleotides formed by opening the sugar ring of a nucleotide, such as unopened nucleic acids (UNA) or glycerol nucleic acids (GNA). UNA is shown in formula (4), and GNA is shown in formula (5).

[0264]

[0265]

[0266] In formulas (4) and (5) above, Base refers to a base (e.g., A, U, G, C or T), and R is selected from H, OH or alkoxy (-O-alkyl).

[0267] In some embodiments, nucleotide derivative modification refers to the replacement of nucleotides in nucleic acids with nucleotide derivatives. Exemplary examples include isonucleotides, LNA, ENA, cET, UNA, or GNA.

[0268] In some embodiments, the nucleotides in the nucleic acid are replaced with isonucleotides, also referred to as isonucleotide modification in the context of this disclosure. In some embodiments, isonucleotide modification includes incorporating an isonucleotide at one or more sites on the sense and / or antisense strands of the siRNA to be modified, in place of the native nucleoside for coupling at the corresponding positions.

[0269] In some embodiments, the isonucleoside modification is D-isonucleoside modification. In other embodiments, the isonucleoside modification is L-isonucleoside modification. In still other embodiments, the isonucleoside modification is a combination of D-isonucleoside and L-isonucleoside modification.

[0270] In some embodiments, the 5' and / or 3' ends of the antisense strand may include a debase residue (Ab), which may also be referred to as a "debase site" or "debase nucleotide". A debase residue (Ab) is a nucleotide or nucleoside lacking a nucleobase at the 1' position of the sugar moiety. In some embodiments, the debase residue may be located inside the nucleotide sequence. In some embodiments, Ab or AbAb may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may include one or more additional debase residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab is added to the 3' end of the sense strand. In some embodiments, a debase (deoxyribose) residue may be replaced with a ribitol (debase ribose) residue.

[0271] In some embodiments, one or more inverse debased deoxyribose residues (invAb) are added to the 3' end of the positive strand. In some embodiments, one or more inverse debased deoxyribose residues (invAb) are added to both the 5' end and the 3' end of the positive strand. The inverse debased deoxyribose residues may be linked via phosphate ester bonds, thiophosphate ester bonds, or other nucleoside internucleotide bonds. When describing modification sites in the modification method, (invAb) is not counted as the first site of the sequence. (invAb) may have the following structure:

[0272] When located at the 3' end, it is When it is at the 5' end, it is

[0273] In some embodiments, one or more inverted adenosine nucleotides may be added to the 5' or 3' end of the positive strand. In some embodiments, one or more inverted adenosine nucleotides may be inserted between the nucleobase sequences of the positive strand. In some embodiments, including one or more inverted adenosine nucleotides at or near the end of the positive strand may allow for enhanced activity of the RNAi agent or other desired properties.

[0274] In some embodiments, the 5' and / or 3' ends of the sense and / or antisense strands may have a cyclopropylphosphonate-containing nucleotide (cPrpN), or may be located within the nucleotide sequence. In some embodiments, cPrpN may be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand may include one or more additional cPrpNs. In some embodiments, cPrpN is added to the 3' end of the sense strand. In some embodiments, cPrpN is added to the 5' and / or 3' ends of the sense strand via a phosphothioester bond. In some embodiments, cPrpN is added to the 5' and / or 3' ends of the antisense strand via a phosphothioester bond.

[0275] In some embodiments, the double-stranded ribonucleic acid (siRNA) modifier includes modification of a phosphodiester bond at at least one position. In some embodiments, the modification of the phosphodiester bond refers to the substitution of at least one oxygen atom in the phosphodiester bond by a sulfur atom to form a phosphothiodiester bond. The phosphothiodiester bond can stabilize the double-stranded structure of siRNA and maintain the specificity of base pairing. An exemplary phosphothiodiester bond structure is shown below:

[0276]

[0277] In some embodiments, the siRNA modifier comprises at least one of the following chemical modifications:

[0278] (1) Modification of at least one nucleotide in the positive strand,

[0279] (2) Modification of the phosphodiester bond at at least one position in the positive chain.

[0280] (3) Modification of at least one nucleotide in the antisense strand,

[0281] (4) Modification of phosphodiester bonds at at least one position in the antisense chain.

[0282] Furthermore, the double-stranded RNA modifier is an siRNA modifier containing at least one of the chemical modifications in (1)-(4).

[0283] In some embodiments, the positive strand of the siRNA modifier includes the following modifications along the 5' end toward the 3' end: at least three ribonucleotides at positions 7, 9, 10, and 11 of the positive strand are 2'-fluoromodified ribonucleotides; and at most 3, 2, or 1 ribonucleotides at other positions of the positive strand are not 2'-methoxymodified.

[0284] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end toward the 3' end: at least three ribonucleotides at positions 7, 9, 10, and 11 of the sense strand are 2'-fluoromodified ribonucleotides; and at most two or one ribonucleotide at other positions of the sense strand are not 2'-methoxymodified ribonucleotides.

[0285] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end toward the 3' end: at least three ribonucleotides at positions 9, 10, and 11 of the sense strand are 2'-fluoromodified ribonucleotides; and at most two or one ribonucleotide at other positions of the sense strand are not 2'-methoxymodified ribonucleotides.

[0286] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 9, 10, and 11 of the sense strand are 2'-fluoro-modified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxy-modified ribonucleotides.

[0287] In some embodiments, the sense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 7, 9, and 11 of the sense strand are 2'-fluoro-modified ribonucleotides; and the ribonucleotides at other positions of the sense strand are 2'-methoxy-modified ribonucleotides.

[0288] In this paper, the 5' nucleotide of the positive strand has a 5' hydroxyl group.

[0289] In this paper, the nucleotide structure of the ribosome with a 5' hydroxyl group at the 5' end of the positive strand is shown in Formula X:

[0290]

[0291] Wherein, Base represents a base, such as A, U, G, C or T; R is a hydroxyl group or is substituted by various groups known to those skilled in the art, for example, R can be 2'-fluoro(2'-F), 2'-alkoxy, 2'-substituted alkoxy, 2'-alkyl, 2'-substituted alkyl, 2'-amino, 2'-substituted amino, 2'-deoxynucleotide.

[0292] In some embodiments, the positive strand of the siRNA modifier, along the 5'-to-3' direction, includes a phosphothioester bond at one or more of the following positions: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the third and fourth nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and / or between the second and third nucleotides starting at the 3' end. In some embodiments, the positive strand of the siRNA modifier, along the 5'-to-3' direction, includes a phosphothioester bond at one or more of the following positions: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and between the second and third nucleotides starting at the 3' end.

[0293] In some embodiments, the positive strand of the siRNA modifier includes a phosphothioester bond at the following positions along the 5' end toward the 3' end: between the first and second nucleotides starting at the 5' end, and / or between the second and third nucleotides starting at the 5' end.

[0294] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end direction: the ribonucleotides at any even-numbered positions in the antisense strand are 2'-fluoromodified ribonucleotides, and at most 3, 2, or 1 ribonucleotides at any odd-numbered positions in the antisense strand are not 2'-methoxymodified.

[0295] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at any odd-numbered positions in the antisense strand are 2'-methoxy modified ribonucleotides, and the ribonucleotides at any even-numbered positions in the antisense strand are 2'-fluoro modified ribonucleotides.

[0296] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0297] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 8, 9, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0298] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 5, 7, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0299] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 12, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0300] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 6 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0301] In some embodiments, the antisense strand of the siRNA modifier includes the following modifications along the 5' end to the 3' end: the ribonucleotides at positions 2, 6, 14, and 16 of the antisense strand are 2'-F modified ribonucleotides, the ribonucleotide at position 7 of the antisense strand is a ribonucleotide modified with the nucleotide derivative GNA, and the ribonucleotides at the remaining positions of the antisense strand are 2'-methoxy modified ribonucleotides.

[0302] In some embodiments, the ribosome of the 5' terminal nucleotide of the antisense strand may have a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group, such as EVP.

[0303] In some embodiments, the first nucleotide of the 5' segment of the antisense strand is a nucleotide containing cyclopropylphosphonate (cPrpN). For example, when the first nucleotide is U, the nucleotide at the first position of the antisense strand has the following structure:

[0304] In some embodiments, the antisense strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the third and fourth nucleotides starting at the 5' end, between the first and second nucleotides starting at the 3' end, and / or between the second and third nucleotides starting at the 3' end.

[0305] In some embodiments, the antisense strand of the siRNA modifier, along the 5'-to-3' direction, includes phosphothioester bonds at the following positions: between the first and second nucleotides starting at the 5' end, between the second and third nucleotides starting at the 5' end, between the third and fourth nucleotides starting at the 5' end, and between the first and second nucleotides starting at the 3' end.

[0306] In some embodiments, the antisense strand of the siRNA modifier includes phosphothioester bonds at the following positions along the 5' end to the 3' end: between the first nucleotide cPrpN and the second nucleotide starting at the 5' end, between the second nucleotide and the third nucleotide starting at the 5' end, between the third nucleotide and the fourth nucleotide starting at the 5' end, and between the first nucleotide and the second nucleotide starting at the 3' end.

[0307] In some embodiments, when neither the 5' nor the 3' end of the positive chain is connected (invAb), the positive chain contains a phosphothioester bond located at the following positions:

[0308] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0309] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0310] Between the first nucleotide and the second nucleotide starting at the 3' end of the positive strand; and / or

[0311] Between the second and third nucleotides starting at the 3' end of the positive strand;

[0312] or,

[0313] The positive chain contains phosphothiophosphate diester bonds located at the following positions:

[0314] Between the first nucleotide and the second nucleotide starting at the 5' end of the positive strand; and / or

[0315] Between the second and third nucleotides starting at the 5' end of the positive strand.

[0316] In some embodiments, when only one (invAb) is attached to the 5' end of the positive chain, the positive chain contains a phosphothiophosphate diester bond located at the following positions:

[0317] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0318] Between the first and second nucleotides starting at the 5' end of the positive strand.

[0319] In some embodiments, when only one (invAb) is attached to the 3' end of the positive chain, the positive chain contains a phosphothiophosphate diester bond located at the following positions:

[0320] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0321] Between the second and third nucleotides starting at the 5' end of the positive strand;

[0322] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0323] In some embodiments, when the 5' end and 3' end of the positive chain are each connected to an (invAb), the positive chain contains a phosphothiophosphate diester bond located at the following positions:

[0324] Between (invAb) at the 5' end of the positive strand and the first nucleotide starting at the 5' end;

[0325] Between the first and second nucleotides starting at the 5' end of the positive strand;

[0326] Between (invAb) at the 3' end of the positive strand and the first nucleotide starting at the 3' end.

[0327] In this paper, the 5' nucleotide of the positive strand has a 5' hydroxyl group.

[0328] In an embodiment, the ribosome of the 5' terminal nucleotide of the antisense strand may have: a 5' hydroxyl group, a 5' phosphate group, or a 5' phosphate-derived group.

[0329] In some embodiments, the ligands of this disclosure are conjugated to the 5' end of the sense strand or the 5' end of the antisense strand of the oligonucleotide.

[0330] In some embodiments, the ligands of this disclosure are conjugated to the 3' end of the sense strand or the 3' end of the antisense strand of the oligonucleotide.

[0331] In some embodiments, the ligands of this disclosure are attached to both ends of the justice chain.

[0332] In some embodiments, the ligands of this disclosure are fused to both ends of the antisense chain.

[0333] In some embodiments, the ligands of this disclosure are conjugated to internal positions on the sense or antisense strand. In some embodiments, one or more ligands of this disclosure are conjugated to ribose, nucleotides, and / or internucleotide bonds. In some embodiments, one or more ligands of this disclosure are conjugated to ribose at the 2', 3', 4', and / or 5' positions. In some embodiments, one or more ligands of this disclosure are conjugated to native nucleotides (such as A, T, G, C, or U) or modified nucleotides as defined herein. In some embodiments, one or more ligands of this disclosure are conjugated to phosphate groups or modified phosphate groups as defined herein.

[0334] In some embodiments, the ligands of this disclosure are conjugated to the 5' or 3' end of the sense strand and to the 5' or 3' end of the antisense strand.

[0335] In some embodiments, at least one ligand of this disclosure is located at one or more terminal positions of the sense or antisense strand. In one embodiment, at least one ligand of this disclosure is located at the 3' or 5' end of the sense strand. In one embodiment, at least one ligand of this disclosure is located at the 3' or 5' end of the antisense strand.

[0336] In some embodiments, the oligonucleotide conjugates of this disclosure contain ligands described herein that are conjugated to one or more internal positions on at least one chain. An internal position on the chain refers to a nucleotide at any position on the chain, excluding the 3' and 5' end positions (e.g., excluding two positions: position 1 counting from the 3' end and position 1 counting from the 5' end).

[0337] In one embodiment, at least one ligand of the present disclosure is located at one or more internal locations on at least one chain, including all locations except the two terminal locations at each end of the chain (e.g., excluding four locations: locations 1 and 2 counted from the 3' end and locations 1 and 2 counted from the 5' end). In another embodiment, the ligand of the present disclosure is located at one or more internal locations on at least one chain, including all locations except the three terminal locations at each end of the chain (e.g., excluding six locations: locations 1, 2, and 3 counted from the 3' end and locations 1, 2, and 3 counted from the 5' end).

[0338] In one embodiment, at least one ligand of the present disclosure is located at one or more positions at at least one end of the double-stranded region, including all positions within the double-stranded region but excluding overhanging regions or vectors that replace the terminal nucleotide at the 3' end of the sense strand.

[0339] In one embodiment, at least one ligand of the present disclosure is located on the sense strand within the first five, four, three, two, or one base pairs of the antisense strand 5' end of the double-stranded region.

[0340] In one embodiment, one or more ligands of this disclosure are located in one or more of the following internal positions: counting from the 5' end of each chain, positions 4-8 and 13-18 on the sense chain, and positions 6-10 and 15-18 on the antisense chain.

[0341] In one embodiment, one or more ligands of this disclosure are located at one or more of the following internal positions: counting from the 5' end of each chain, positions 5, 6, 7, 15 and 17 on the sense chain, and positions 15 and 17 on the antisense chain.

[0342] All sites in a given compound do not require uniform modification. Instead, more than one modification can be incorporated into a single RNAi agent or even into a single nucleotide. The sense and antisense strands of the RNAi agents of this invention can be synthesized and / or modified by methods known in the art. A modification on one nucleotide is independent of a modification on another nucleotide.

[0343] In a specific embodiment, the oligonucleotide conjugate is selected from:

[0344]

[0345]

[0346]

[0347]

[0348] Preparation methods of ligands and oligonucleotide conjugates

[0349] This disclosure also provides a method for preparing the ligands described herein, comprising:

[0350] 1) Prepare precursor compound I comprising a targeting group and an L1 moiety, wherein the precursor compound comprises an azide group or a tetrazine group;

[0351] 2) Prepare precursor compound II containing the L2 moiety, wherein the precursor compound II contains an alkynyl group or a ring-strained alkynyl group (such as DBCO, BCN, etc.);

[0352] 3) The precursor compound I and precursor compound II are reacted to obtain the ligand described herein through one or more of the following reactions:

[0353] a. CuAAc click chemistry reaction of azide and alkynyl groups;

[0354] b. SPAAC click chemistry of azide groups and cyclostynyl groups (Huisgen cyclization addition);

[0355] c. Diels-Alder Cycloaddition (SPIEDAC) of alkynyl (strained alkynyl) and tetrazine.

[0356] In another aspect, this disclosure also provides a method for preparing oligonucleotide conjugates, comprising conjugating the oligonucleotide with a ligand of this disclosure. Alternatively, in a preferred embodiment, the oligonucleotide may first be conjugated with the aforementioned precursor compound II, and then reacted with precursor compound I.

[0357] In a specific embodiment, the precursor compound I may be selected from one or more of the following:

[0358]

[0359]

[0360] In a specific embodiment, the precursor compound II may be selected from one or more of the following:

[0361]

[0362] Pharmaceutical compositions, uses and treatment methods

[0363] On the other hand, the present invention provides a pharmaceutical composition comprising the oligonucleotide conjugates described above. In some embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0364] In some embodiments, the pharmaceutically acceptable excipient may be, for example, a carrier, transporter, diluent, and / or delivery polymer.

[0365] On the other hand, this disclosure provides the use of the above-described oligonucleotide conjugate or the above-described pharmaceutical composition in the preparation of a medicament.

[0366] In some embodiments, the drug is used to prevent and / or treat diseases associated with lung disorders and diseases associated with muscle tissue (including skeletal muscle and cardiac muscle). In some embodiments, the drug is used to prevent and / or treat lung-related diseases.

[0367] In some embodiments, the drug is used to prevent and / or treat tumor-related diseases.

[0368] In this disclosure, the ligand of the integrin αvβ6 protein receptor serves as a transport and delivery tool to deliver various oligonucleotide agents (e.g., RNAi reagents) to target sites.

[0369] As an example, the oligonucleotide conjugates of this disclosure may be MUC5AC RNAi reagents, for example, reagents may contain a ligand of the integrin αvβ6 protein receptor of this disclosure and siRNA modifiers targeting MUC5AC, which are designed to target a specific location on the MUC5AC gene (e.g., NM_001304359.2).

[0370] MUC5AC is a transcriptionally regulated secretory mucin expressed in the pulmonary airway epithelium and in other mucosal tissues (e.g., gastrointestinal tract, genitourinary system, eye, and ear) (Lillehoj et al., Int Rev CellMolBiol, 2013). In the airways, MUC5AC and MUC5B are the major gel-forming mucins. MUC5B is constitutively expressed and is essential for mucociliary clearance (Roy et al., Nature 2014). In normal subjects, MUC5B expression is relatively higher than MUC5AC expression in the trachea and proximal airways, with this ratio further increasing in the distal airways, and MUC5AC expression is almost undetectable in the distal and terminal bronchioles (Okuda et al., AJRCCM 2019). MUC5AC is typically expressed at low levels in the airways. MUC5AC expression can be robustly induced by external stressors such as pro-inflammatory mediators (including, for example, type II cytokines: IL-4, IL-9, IL-17, IL-23, and IL-13), harmful inhaled substances (e.g., cigarette smoke, acrolein, toxic gases), viral infections, and allergens. The resulting excessive mucus secretion and over-concentration are understood as a common pathogenic mechanism associated with airway obstruction in severe asthma and other mucooblocclusion lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (NCFB), and primary ciliary dyskinetic disorder (PCD) (Boucher, NEJM 2019). In patients with asthma, COPD, and NCFB, overexpression and secretion of MUC5AC lead to airway luminal narrowing, airway obstruction, and exacerbation (Dunican et al., JCI 2017; Bonser et al., JCI). 2016; Kesimer et al., NEJM 2017; Ramsey et al., AJRCCM 2019). A genome-wide association study (GWAS) identified a novel MUC5AC allele that was associated with increased MUC5AC expression and moderate to severe asthma (Shrine et al., Lancet RespirMed 2019). Experimental evidence from MUC5AC-deficient mice confirms that MUC5AC-mediated airway obstruction is a major cause of airway hyperresponsiveness to allergens, independent of inflammation and bronchoconstriction (Evans et al., Nat Commun, 2015).

[0371] Increased MUC5AC expression has been observed in malignant tumors such as lung adenocarcinoma, pancreatic cancer, salivary gland cancer, breast cancer, cholangiocarcinoma, ovarian cancer, and other tumors (Krishn et al., Carcinogenesis 2018), where it has been associated with tumor cell migration and invasiveness. Loss-of-function mutations in MUC5AC and other mucin genes are significantly deficient in tumor cells, suggesting that mucin overexpression may prevent tumors from being recognized by immune cells (Gorlov et al., Cancer Genetics 2019). Tumor MUC5AC overexpression is associated with progression and poor survival in patients with lung adenocarcinoma (Bauer et al., JCI Insight 2018). MUC5AC overexpression is also associated with many other conditions, including allergic rhinitis, chronic rhinitis and sinusitis, otitis media, Barrett's esophagus, pancreatitis, and inflammatory bowel disease (Krishn et al., Carcinogenesis 2018).

[0372] The use of MUC5AC RNAi reagents provides a method for the therapeutic (including preventative) treatment of diseases or disorders, where reduced MUC5AC gene expression and / or reduced MUC5AC protein levels can provide therapeutic benefits for said diseases or disorders. The MUC5AC RNAi reagents disclosed herein can be used to treat a variety of diseases, including mucoobulent lung diseases (such as asthma, CF, COPD, NCFB, PCD), allergic bronchopulmonary aspergillosis, interstitial lung disease, cancers (such as lung adenocarcinoma, pancreatic cancer, salivary gland cancer, breast cancer, cholangiocarcinoma, ovarian cancer, and other tumors), respiratory infections (such as respiratory syncytial virus, influenza, rhinovirus), otitis media, inflammatory bowel disease, cholelithiasis, allergic rhinitis, chronic rhinitis and sinusitis, and nasal polyposis. In some embodiments, the MUC5AC RNAi reagents disclosed herein can be used to treat mucoobulent lung diseases, such as severe asthma or COPD. The MUC5AC RNAi reagents can be further used to treat, for example, various cancers. Such treatments involve administering a MUC5AC RNAi agent to humans or animals with elevated or enhanced MUC5AC gene expression and / or MUC5AC protein levels above the desired level.

[0373] In some embodiments, the MUC5AC RNAi reagent D-ER-FY004-mN010 disclosed herein exhibits an inhibition rate of over 75% against the MUC5AC target gene in C57BL / 6j mice; while the MUC5AC RNAi reagent D-ER-FY004-m005 disclosed herein, under the same experimental conditions, exhibits an inhibition rate of less than 56% against the MUC5AC target gene in C57BL / 6j mice.

[0374] In some embodiments, the oligonucleotide conjugates disclosed herein relate to RAGE RNAi reagents and MMP7 RNAi reagents.

[0375] The receptor for advanced glycation end products (RAGE) is a transmembrane receptor containing an extracellular ligand-binding domain and an amino acid cytoplasmic tail, used for intracellular signaling activation. RAGE is a well-known multi-ligand receptor that can interact with various ligands, such as advanced glycation end products (AGEs), S-100 protein, and HMGB1, leading to the production of reactive oxygen species (ROS), inflammatory pathogenesis, cell proliferation, and autophagy. Therefore, in lung epithelial cells, reduced RAGE expression may lead to a reduction in RAGE-dependent inflammatory pathways, thereby slowing the frequency and progression of asthma attacks in patients.

[0376] Matrix metalloproteinase 7 (“MMP7” or “matrix lysozyme”) is the smallest member (28 kDa) of the metalloproteinase (MMP) family. Composed of 24 associated zinc-dependent secretory endopeptidases with diverse substrates and functions, it is capable of degrading components of the extracellular matrix (e.g., elastin, proteoglycans, type IV collagen, fibronectin, and core proteins (proteoglycans)) and cleaving and regulating the activity of non-extracellular matrix substrates, such as cytokines (Fujishima et al., Arch Pathol Lab Med (2010); Craig et al., Am J Respir Cell Mol Biol (2015)). These functional roles in extracellular matrix remodeling and regulation of cytokine signaling link MMP family members to the pathogenesis of cancer, chronic inflammation, and fibrosis. MMP7 is constitutively expressed and secreted by epithelial cells throughout the body (including glandular epithelium of the skin, lungs, liver, intestine, pancreas, salivary glands, and reproductive tract) and plays a role in epithelial cell repair (Pilcher et al., Ann NY Acad). Sci (1999)). Increased MMP7 expression is associated with pathogenic pulmonary fibrosis (Rosas et al., plos (2008)). Idiopathic pulmonary fibrosis (IPF) is a specific type of fibrosis, a chronic lung disease that is often fatal, with a relatively unpredictable clinical course and rate of disease progression (Zuo et al., Proc NatlAcad). SciUSA (2002)). Increased expression of MMP7 in peripheral blood, bronchoalveolar lavage fluid, and lung tissue is known in patients with IPF. Serum MMP7 expression is a potent serum biomarker for IPF, associated with its severity and progression. Consistent with its known mechanisms, MMP7 regulates multiple pathways leading to abnormal function of epithelial cells, fibroblasts, and immune cells in IPF. Notably, MMP7 knockout mice exhibit protective effects against bleomycin-mediated lung injury (a standard rodent model of IPF), demonstrating reduced pulmonary inflammation, fibrosis, and mortality, suggesting a pathogenic role for MMP7 in IPF (Craig et al., Journal of Respiratory Cell & Biology (2015)). Genome-wide association studies (GWAS) have shown that gain-of-function of the MMP7 gene variant rs1 1568818AA is associated with IPF risk. Furthermore, in such GWAS studies, MMP7 gene variants have been associated with a variety of cancers and sclerosis (Moreno-Ortiz et al., Genet MolRes (2014)).

[0377] Effective delivery of RNAi agents to cells in vivo requires specific targeting and substantial protection of the extracellular environment, especially serum proteins. RNA interference-based therapies have provided promising clinical data for the treatment of liver-related diseases. However, RNAi delivery to extrahepatic tissues remains a barrier, limiting the application of RNAi-based therapies. The integrin αvβ6 protein receptor ligand-RNAi drug conjugate disclosed herein can not only effectively deliver RNAi drugs to the lungs to treat various lung diseases, but also achieve effective extrahepatic delivery to other sites, such as muscle tissue, including skeletal and cardiac muscle tissue.

[0378] Integrin αvβ6 protein receptor ligand-RNAi drug conjugates can be used for the treatment and / or prevention of diseases such as myosin-associated muscle hypertrophy, congenital myasthenia gravis, myotonic dystrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular atrophy (SMA), Pompe disease, PLN cardiomyopathy, muscle spasms, obstructive hypertrophic cardiomyopathy (HOCM), familial hypertrophic cardiomyopathy (FHC), heart failure with preserved ejection fraction (HFPEF), atrial fibrillation (AFIB), ventricular fibrillation (VFIB), angina pectoris, myocardial infarction (MI), heart failure or heart failure with reduced ejection fraction (HFREF), supraventricular tachycardia (SVT), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), or arrhythmias or congestive heart failure (CHF).

[0379] Target genes involved may include, but are not limited to: adrenaline receptor β1 (ADRB1), calcium voltage-gated channel subunit αC (CACNAIC), calcium voltage-gated channel subunit G (CACNAIG) (T-type calcium channel), angiotensin receptor type 1 (AGTR1), sodium voltage-gated channel α subunit 2 (SCN2A), hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (HCN1), hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4), hyperpolarization-activated cyclic nucleotide-gated potassium channel 3 (HCN3), potassium voltage-gated channel subfamily A member 5 (KCNA5), potassium intrarectifier channel subfamily J member 3 (KCNJ3), potassium intrarectifier channel subfamily J member 4 (KCNJ4), and phosphatidylcholine receptors. The following proteins are listed: white protein (PLN), calcium / calmodulin-dependent protein kinase IIδ (CAMK2D), phosphodiesterase 1 (PDE1), myostatin (MSTN), cholinergic receptor nicotinic α1 subunit, cholinergic receptor nicotinic β1 subunit (CHRNB1), cholinergic receptor nicotinic δ subunit, cholinergic receptor nicotinic Epsilon subunit, cholinergic receptor nicotinyl γ subunit (CHRNE), type XIII collagen α1 chain (COL13A1), docking protein 7 (DOK7), LDL receptor-associated protein 4 (LRP4), muscle-associated receptor tyrosine kinase (MUSK), synaptic receptor-associated protein (RAPSN), sodium voltage-gated channel α subunit 4 (SCN4A), dual Homeobox 4 (DUX4), myotonic kinase (DMPK), glycogen synthase 1 (GYS1), motor neuron 1 (SMN1), α-glucosidase (GAA), etc.

[0380] In another aspect, the present invention further provides a method for delivering the disclosed conjugates to a specific target in a subject via subcutaneous or intravenous administration. The present invention further provides a method for using the compounds of the present invention to deliver the active pharmaceutical molecule in the conjugates to a specific target in a subject via subcutaneous or intravenous administration.

[0381] Another aspect of the present invention relates to a method for reducing the expression of a target gene in a cell, the method comprising contacting the cell with a conjugate of the present disclosure, the conjugate comprising an antisense strand complementary to the target gene; a sense strand at least partially complementary to the antisense strand; and one or more ligands as described in the present disclosure.

[0382] In aspects of this disclosure relating to conjugates, all the foregoing examples relating to ligands of this disclosure, conjugates containing said ligands, and conjugates of oligonucleotides to ligands of this disclosure are suitable for this aspect of the disclosure in relation to methods for reducing the expression of target genes in cells.

[0383] In one implementation, the cells are extrahepatic cells.

[0384] In one implementation, the cell is not a liver cell.

[0385] Another aspect of the present invention relates to a method for reducing the expression of a target gene in a subject, the method comprising administering a conjugate of the present disclosure to the subject, including contacting the cells with the conjugate, the conjugate comprising an antisense strand complementary to the target gene; a sense strand at least partially complementary to the antisense strand; and one or more ligands as described in the present disclosure.

[0386] definition

[0387] Unless otherwise stated, the following terms as used in this application shall have the following meanings. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient.

[0388] As used in this article, the term "oligonucleotide" refers to a polymer of linked nucleosides, each of which may be independently modified or unmodified.

[0389] As used herein, “RNAi reagent” (also referred to as “RNAi trigger”) means a composition containing RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecules capable of degrading or inhibiting (e.g., under appropriate conditions, degrading or inhibiting) the translation of a targeted messenger RNA (mRNA) transcript in a sequence-specific manner. RNAi reagents as used herein can function via RNA interference mechanisms (i.e., by inducing RNA interference through interaction with RNA interference pathways in mammalian cells, such as RNA-induced silencing complexes or RISC), or via alternative mechanisms or pathways. Although it is believed, as the terminology is used herein, that RNAi reagents function primarily through RNA interference mechanisms, the disclosed RNAi reagents are not bound by or limited to any particular pathway or mechanism of action. The RNAi reagents disclosed herein contain a sense strand and an antisense strand, and include, but are not limited to, small (or short) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrates. The antisense strand of the RNAi reagent described herein is at least partially complementary to the targeted mRNA (e.g., MUC5AC mRNA). The RNAi reagent may include one or more modified nucleotides and / or one or more non-phosphodiester bonds, as well as the ligands disclosed herein.

[0390] In this art, “G,” “C,” “A,” “T,” and “U” typically represent the bases of guanine, cytosine, adenine, thymine, and uracil, respectively. However, it is also generally known in the art that each of “G,” “C,” “A,” “T,” and “U” typically also represents a nucleotide containing guanine, cytosine, adenine, thymine, and uracil as a base, respectively. This is a common practice in representing deoxyribonucleic acid (DNA) sequences and / or ribonucleic acid (RNA) sequences. Therefore, in the context of this disclosure, the meanings of “G,” “C,” “A,” “T,” and “U” include all of the above-mentioned possible cases. However, it should be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide (as further detailed below) or one with an alternative substitution. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted with other parts without substantially altering the base-pairing properties of an oligonucleotide (including a nucleotide with such a substitution). For example, without limitation, a nucleotide including inosine as its base can be base-paired with a nucleotide including adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced in the nucleotide sequence of the dsRNA characterized in this disclosure by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU swing base pairing with the target mRNA. Sequences containing such replacement portions are suitable for the compositions and methods characterized in this disclosure. The terms “double-stranded oligonucleotide,” “double-stranded RNA (dsRNA) molecule,” and “dsRNA” as used in the context of this disclosure are used interchangeably. The term “dsRNA” refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, described as having “sense” and “antisense” orientation relative to the target gene. In some embodiments, double-stranded ribonucleic acid (dsRNA) triggers the degradation of target RNA, such as mRNA, through a post-transcriptional gene silencing mechanism (referred to herein as RNA interference or RNAi).

[0391] Typically, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, “double-stranded ribonucleic acid” can include chemically modified ribonucleotides, phosphate backbones, etc. These modifications can include all types of modifications disclosed herein or known in the art.

[0392] In some embodiments, the double-stranded ribonucleic acid (dsRNA) of this disclosure is siRNA, which interacts with the mRNA sequence transcribed from the target gene to guide the cleavage of the target RNA. Not wishing to be bound by theory, long double-stranded RNA introduced into the cell is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short 19-23 base pairs of interfering RNA with a characteristic dibase 3' overhang (Bernstein et al., (2001) Nature, 409:363). These siRNAs are then incorporated into the RNA-inducible silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling complementary antisense strands to guide target recognition (Nykanen et al., (2001) Cell 107:309). Once bound to a suitable target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev. 15:188).

[0393] As used in the context of this disclosure, the term "protruding nucleotide" refers to one or more unpaired nucleotides that protrude from the double-stranded structure of a dsRNA when one 3' end of one strand extends beyond the 5' end of the other strand, or vice versa. "Flat-ended" or "blunt-terminated" means that there are no unpaired nucleotides at the said end of the double-stranded ribonucleic acid, i.e., no nucleotide protrusions. A "flat-terminated" double-stranded ribonucleic acid is a dsRNA that is double-stranded throughout its entire length, i.e., without nucleotide protrusions at either end of the molecule.

[0394] The term "antisense strand" refers to a strand of double-stranded RNA that has a region that is substantially complementary to the target sequence. Mismatches at the terminal regions are the most tolerable when the complementary region is not perfectly complementary to the target sequence, and if mismatches do occur, they are typically within one or more terminal regions, such as within 5, 4, 3, 2, or 1 nucleotides at the 5' and / or 3' ends.

[0395] The term "sense chain" refers to a double-stranded RNA strand containing a region that is substantially complementary to the antisense chain region.

[0396] When referring to the expression of a given gene, the terms “silence,” “reduction,” “inhibition,” or “downregulation” as used herein mean that, when a cell, cell assembly, tissue, organ, or subject is treated with the RNAi reagent described herein, the expression of said gene is reduced compared to a second cell, cell assembly, tissue, organ, or subject that has not been so treated, measured by the level of RNA transcribed from said gene or the level of polypeptide, protein, or protein subunit translated from mRNA in the cell, cell assembly, tissue, organ, or subject in which said gene is transcribed.

[0397] As used in this article, the terms "sequence" and "nucleotide sequence" refer to a series or order of nucleobases or nucleotides described by consecutive letters using standard nomenclature. Unless otherwise indicated, nucleotide sequences are written from left to right in a 5' to 3' orientation.

[0398] As used herein and unless otherwise stated, the term "complementary" when used to describe a first nucleobase or nucleotide sequence (e.g., the positive strand of an RNAi reagent or the targeted mRNA) relative to a second nucleobase or nucleotide sequence (e.g., an antisense strand of an RNAi reagent or a single-stranded antisense oligonucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide comprising the second nucleotide sequence (forming base-pair hydrogen bonds under mammalian physiological conditions (or other suitable in vivo or in vitro conditions)) and to form a double-stranded or double-helix structure under certain standard conditions. Those skilled in the art will be able to select the set of conditions most suitable for the hybridization assay. A complementary sequence contains either a Watson-Crick base pair or a non-Watson-Crick base pair and contains, at least to the extent that the hybridization requirements above are satisfied, a native or modified nucleotide or nucleotide mimic. Sequence identity or complementarity is independent of modification. For example, for the purpose of determining identity or complementarity, a and Af, as defined herein, are complementary to U (or T) and identical to A.

[0399] As used in this article, "perfect complementarity" means that in a hybridization pair of nucleobase or nucleotide sequences, all (100%) bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0400] As used in this article, "partial complementarity" means that in a hybridization pair of nucleobase or nucleotide sequences, at least 70% (but not all) of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0401] As used herein, "substantially complementary" means that in a hybridization pair of nucleobase or nucleotide sequences, at least 85% (but not all) of the bases in the sequential sequence of the first oligonucleotide will hybridize with the same number of bases in the sequential sequence of the second oligonucleotide. The sequential sequence may contain all or part of the first or second nucleotide sequence.

[0402] As used herein, the terms “complementary,” “fully complementary,” “partially complementary,” and “substantially complementary” are used to describe nucleobase or nucleotide matching between the sense and antisense strands of the RNAi reagent or between the antisense strand of the RNAi reagent and the sequence of the targeted mRNA (e.g., MUC5AC mRNA).

[0403] When referring to RNAi reagents, the phrase “delivered to / introduced into cells” as used herein means the functional delivery of RNAi reagents into cells. The phrase “functional delivery” means the delivery of RNAi reagents to cells in a manner that enables them to have the intended biological activity (e.g., sequence-specific inhibition of gene expression).

[0404] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0405] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, the ethyl group “optionally” being halogenated means that the ethyl group can be unsubstituted (-CH2CH3), monosubstituted (e.g., -CH2CH2F), polysubstituted (e.g., -CHFCH2F, -CH2CHF2, etc.), or fully substituted (-CF2CF3). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0406] In this article, () m -n indicates that the part has an integer number of repeating units within a given range. For example, "-(CH2CH2O)". 1-12 "-" indicates that the group can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 repeats of the units in parentheses.

[0407] When any variable (e.g., R) 14 When a group appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is surrounded by two R...14 Replaced, then each R 14 Each has its own independent options.

[0408] As used herein, unless otherwise stated, “alkyl” means and comprises having a specified number of carbon atoms (i.e., C1-C1). 12 A saturated straight-chain (i.e., unbranched) or branched monovalent hydrocarbon chain, or a combination thereof, having one to twelve carbon atoms. A specific alkyl group is an alkyl group having 1 to 20 carbon atoms (“C1-C2”). 20 Alkyl groups ("C1-C1") having 1 to 10 carbon atoms 10 Alkyl groups (“C6-C10”) having 6 to 10 carbon atoms 10 Alkyl groups include, but are not limited to, alkyl groups having 1 to 6 carbon atoms (“C1-C6 alkyl”), alkyl groups having 2 to 6 carbon atoms (“C2-C6 alkyl”), alkyl groups having 1 to 4 carbon atoms (“C1-C4 alkyl”), or alkyl groups having 1 to 3 carbon atoms (“C1-C3 alkyl”). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0409] As used herein, "alkylene" refers to the same residue as an alkyl group but with a divalent valence. Specific alkylene groups are those having 1 to 20 carbon atoms ("C1-C2"). 20 Alkylenes (“C1-C1”) and alkylenes having 1 to 10 carbon atoms. 10 Alkylenes (“C6-C10”) and alkylenes having 6 to 10 carbon atoms. 10 Alkylenes (“C1-C6 alkylenes”), alkylenes (“C1-C5 alkylenes”), alkylenes (“C1-C4 alkylenes”), or alkylenes (“C1-C3 alkylenes”) having 1 to 5 carbon atoms. Examples of alkylenes include, but are not limited to, groups such as methylene (-CH2-), ethylene (-CH2CH2-), propylene (-CH2CH2CH2-), isopropylene (-CH2CH(CH3)-), butylene (-CH2(CH2)2CH2-), isobutylene (-CH2CH(CH3)CH2-), pentylene (-CH2(CH2)3CH2-), hexylene (-CH2(CH2)4CH2-), heptylene (-CH2(CH2)5CH2-), and octylene (-CH2(CH2)6CH2-).

[0410] As used herein, unless otherwise stated, "alkenyl" means and includes having at least one alkene unsaturated site (i.e., having at least one C=C portion) and having a specified number of carbon atoms (i.e., C2-C). 10 Alkenes are unsaturated straight-chain (i.e., unbranched) or branched monovalent hydrocarbon chains, or combinations thereof, having two to ten carbon atoms. Alkenes can have a "cis" or "trans" configuration, or alternatively, an "E" or "Z" configuration. Specific alkenes are alkenes having 2 to 20 carbon atoms ("C2-C..."). 20 alkenyl groups ("C6-C"), alkenyl groups having 6 to 10 carbon atoms ("C6-C") 10 Alkenes are groups having 2 to 8 carbon atoms (“C2-C8 alkenes”), 2 to 6 carbon atoms (“C2-C6 alkenes”), or 2 to 4 carbon atoms (“C2-C4 alkenes”). Examples of alkenes include, but are not limited to, vinyl (or vinyl), propenyl, propenyl (or allyl), 2-methylpropenyl, butenyl, butenyl, butenyl, butenyl, 1,3-dienyl, 2-methylbutenyl, pentenyl, pentenyl, hexenyl, hexenyl, hexenyl, hexenyl, and hexenyl-3-alkenyl groups.

[0411] As used herein, unless otherwise stated, "alkynyl" means and includes having at least one alkynyl unsaturated site (i.e., having at least one part of the formula C≡C) and having a specified number of carbon atoms (i.e., C2-C). 10 A straight-chain (i.e., unbranched) or branched monovalent hydrocarbon chain (two to ten carbon atoms) or a combination thereof. A specific alkynyl group is an alkynyl group having 2 to 20 carbon atoms (“C2-C”). 20 Alkyne group ("C6-C"), and alkynyl groups with 6 to 10 carbon atoms ("C6-C") 10 The alkynyl group includes, but is not limited to, groups with 2 to 8 carbon atoms ("C2-C8 alkynyl"), 2 to 6 carbon atoms ("C2-C6 alkynyl"), or 2 to 4 carbon atoms ("C2-C4 alkynyl"). Examples of alkynyl groups include, but are not limited to, ethynyl (or ethynyl), propynyl-1-alkynyl, propynyl-2-alkynyl (or propynyl), butynyl-1-alkynyl, butynyl-2-alkynyl, butynyl-3-alkynyl, etc.

[0412] As used herein, unless otherwise stated, "cycloalkyl" means and comprises a saturated cyclic monovalent hydrocarbon structure having a specified number of carbon atoms (i.e., C3-C10 represents three to ten carbon atoms). A cycloalkyl group may consist of one ring (such as cyclohexyl) or multiple rings (such as adamantyl). Cycloalkyl groups comprising more than one ring may be fused, spirocyclic, or bridged, or combinations thereof. Specific cycloalkyl groups are those having 3 to 12 cyclic carbon atoms. Preferred cycloalkyl groups are cyclic hydrocarbons having 3 to 8 cyclic carbon atoms ("C3-C8 cycloalkyl"), cyclic hydrocarbons having 3 to 6 cyclic carbon atoms ("C3-C6 cycloalkyl"), or cyclic hydrocarbons having 3 to 4 cyclic carbon atoms ("C3-C4 cycloalkyl"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, etc.

[0413] As used herein, “aryl” refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple fused rings (e.g., naphthyl or anthracene), which may or may not be aromatic. A specific aryl group is an aryl group having 6 to 14 cyclic carbon atoms (“C6-C”). 14 Aryl groups ("aryl") can be attached to the parent structure at an aromatic ring position or at a non-aromatic ring position. In one variation, an aryl group with more than one ring (at least one non-aromatic ring) is attached to the parent structure at an aromatic ring position.

[0414] As used herein, "heteroaryl" refers to an unsaturated aromatic cyclic group having 1 to 14 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14) cyclic carbon atoms and at least one cyclic heteroatom, said cyclic heteroatom including, but not limited to, heteroatoms such as nitrogen, oxygen, and sulfur. Heteroaryl groups may have a single ring (e.g., pyridyl, furanyl) or multiple fused rings (e.g., indazinyl, benzothiopheneyl), said fused rings may be aromatic or may not be aromatic. The specific heteroaryl group is a 5- to 14-membered ring having 1 to 12 cyclic carbon atoms and 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 5- to 10-membered ring having 1 to 8 cyclic carbon atoms and 1 to 4 (e.g., 1, 2, 3, or 4) cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 5-, 6-, or 7-membered ring having 1 to 5 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In one variation, the specific heteroaryl group is a monocyclic aromatic 5-, 6-, or 7-membered ring having 1 to 6 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In another variation, the specific heteroaryl group is a polycyclic aromatic ring having 1 to 12 cyclic carbon atoms and 1 to 6 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. A heteroaryl group having more than one ring (at least one of which is non-aromatic) can be attached to the parent structure at an aromatic ring position or at a non-aromatic ring position. In one variation, the heteroaryl group having more than one ring (at least one of which is non-aromatic) is attached to the parent structure at an aromatic ring position. The heteroaryl group can be attached to the parent structure at a ring carbon atom or at a ring heteroatom.

[0415] As used herein, unless otherwise stated, "cycloalkyl" means and comprises a saturated cyclic monovalent hydrocarbon structure having a specified number of carbon atoms (i.e., C3-C8 represents three to eight carbon atoms). A cycloalkyl group may consist of one ring (such as cyclohexyl) or multiple rings. Cycloalkyl groups comprising more than one ring may be fused, spirocyclic, or bridged, or combinations thereof. Specific cycloalkyl groups are cyclic hydrocarbons having 3 to 6 cyclic carbon atoms ("C3-C6 cycloalkyl") or cyclic hydrocarbons having 3 to 4 cyclic carbon atoms ("C3-C4 cycloalkyl"). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0416] The term "heterocyclic alkyl" refers to a fully saturated cyclic group that may exist as a monocyclic, bridged, or spirocyclic ring. Unless otherwise indicated, the heterocycle is typically a 3- to 7-membered ring containing 1 to 3 (preferably 1 or 2) heteroatoms independently selected from sulfur, oxygen, and / or nitrogen. Examples of 3-membered heterocyclic alkyl groups include, but are not limited to, ethylene oxide, cyclothioethylene, and cycloazoethylene; non-limiting examples of 4-membered heterocyclic alkyl groups include, but are not limited to, acridine, oxadiazolyl, and thiobutyl; examples of 5-membered heterocyclic alkyl groups include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, imidazolyl, and tetrahydropyrazolyl; examples of 6-membered heterocyclic alkyl groups include, but are not limited to, piperidinyl, tetrahydropyranyl, tetrahydrothiaranyl, morpholinyl, piperazine, 1,4-thiaoxane, 1,4-dioxane, thiomorpholinyl, 1,3-dithiaalkyl, and 1,4-dithiaalkyl; and examples of 7-membered heterocyclic alkyl groups include, but are not limited to, azirheptanyl, oxeheptanyl, and thioheptanyl. Monocyclic heterocyclic alkyl groups having 5 or 6 ring atoms are preferred.

[0417] As used herein, "heterocyclic" or "heterocyclic group" refers to a saturated or unsaturated non-aromatic cyclic group having a single ring or multiple fused rings and having 1 to 14 cyclic carbon atoms and 1 to 6 cyclic heteroatoms (such as nitrogen, sulfur, or oxygen). Heterocyclic groups comprising more than one ring can be fused, bridged, or spirocyclic, or any combination thereof, but do not include heteroaryl groups. Heterocyclic groups may optionally be independently substituted by one or more substituents described herein. The specific heterocyclic group is a 3- to 14-membered ring having 1 to 13 cyclic carbon atoms and 1 to 6 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 3- to 12-membered ring having 1 to 11 cyclic carbon atoms and 1 to 6 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 3- to 10-membered ring having 1 to 9 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; a 3- to 8-membered ring having 1 to 7 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur; or a 3- to 6-membered ring having 1 to 5 cyclic carbon atoms and 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur. In one variation, the heterocyclic group comprises a monocyclic 3-, 4-, 5-, 6-, or 7-membered ring having 1 to 2, 1 to 3, 1 to 4, 1 to 5, or 1 to 6 cyclic carbon atoms and 1 to 2, 1 to 3, or 1 to 4 cyclic heteroatoms independently selected from nitrogen, oxygen, and sulfur.

[0418] As used herein, unless otherwise specified, "heteroalkyl" or its subordinate concepts (such as heteroalkyl, heteroalkenyl, heteroynyl, heteroaryl, etc.) either on their own or in combination with another term represent a stable linear, branched or cyclic hydrocarbon group or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom.

[0419] As used herein, unless otherwise specified, "heteroalkyl" on its own or in combination with another term refers to a stable, straight-chain, branched hydrocarbon group or combination thereof, consisting of a number of carbon atoms and at least one heteroatom. In a typical example, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heteroatom or heterogroup can be located at any internal position of the heteroalkyl group, including where the hydrocarbon group is attached to the rest of the molecule, but the terms "alkoxy," "alkamino," and "alkithioyl" (or thioalkoxy) are conventional expressions referring to those alkyl groups that are attached to the rest of the molecule, respectively, by an oxygen atom, amino atom, or sulfur atom. Examples of "heteroalkyl" include, but are not limited to, -OCH3, -OCH2CH3, -OCH2CH2CH3, -OCH(CH3)2, -N(CH3)2, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-SS-CH2-CH3, -CH2-CH2-S(O)-CH3, -S(O)2-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. At most two heteroatoms can be consecutive, for example, -CH2-NH-OCH3.

[0420] "Halogen" refers to fluorine, chlorine, bromine, and iodine.

[0421] "Hydroxy" refers to the -OH group.

[0422] "O" refers to the =O group.

[0423] Cyclic strained alkynes refer to cyclic alkyne types such as DBCO and BCN, which can achieve cycloaddition reactions of alkyne-azides under conditions without copper ion catalysis.

[0424] CuAAC reaction is an abbreviation for copper(I)-alkyne / azide cycloaddition, which refers to the copper-catalyzed cycloaddition reaction of alkynyl-azide.

[0425] SPAAC reaction is an abbreviation for Strain-promoted alkyne-azide cycloaddition, which refers to strain-promoted cycloaddition of alkynyl-azide groups.

[0426] Tetraazine is a six-membered heterocyclic compound containing four nitrogen atoms, usually referring to 1,2,4,5-tetraazine with or without substituted 3,6 positions.

[0427] The Diels-Alder reaction (DA reaction), also known as the Diels-Alder reaction or diene addition reaction, is a type of cycloaddition reaction.

[0428] In this article, the term "room temperature" generally refers to 25°C, and is usually a temperature condition in the range of 15-25°C.

[0429] When one of the variables is selected as a covalent bond, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a covalent bond, it means that the structure is actually AZ.

[0430] When a substituent is cross-bonded to two atoms on a ring, it can bond to any atom on that ring. For example, structural units. This indicates that it can be substituted at any position on the cyclohexyl group or cyclohexadiene.

[0431] When referring to a connection between two compounds or molecules, the terms “connection” or “combination” as used herein mean that the two compounds or molecules are linked by a covalent bond. Unless otherwise stated, the terms “connection” and “combination” as used herein may refer to a connection between a first compound and a second compound, with or without any inserted atoms or groups.

[0432] The term "treatment" means administering the compound or preparation described in this application to prevent, improve, or eliminate a disease or one or more symptoms related to said disease, and includes:

[0433] (i) To prevent the occurrence of disease or disease state in mammals, especially when such mammals are susceptible to the disease state but have not yet been diagnosed with the disease state;

[0434] (ii) To suppress the disease or disease state, that is, to curb its development;

[0435] (iii) Alleviate the disease or disease state, even if the disease or disease state subsides.

[0436] The term "therapeutic effective amount" means the amount of the compound of this application used to treat or prevent a particular disease, condition, or disorder; (ii) to reduce, improve, or eliminate one or more symptoms of a particular disease, condition, or disorder; or (iii) to prevent or delay the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of the compound of this application constituting a "therapeutic effective amount" varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the present disclosure.

[0437] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0438] Pharmaceutically acceptable salts may include, for example, metal salts, ammonium salts, salts formed with organic bases, salts formed with inorganic acids, salts formed with basic or acidic amino acids, etc. In this application, oligonucleotides may also be in the form of pharmaceutically acceptable salts. For example, pharmaceutically acceptable salts mentioned in this application include, but are not limited to, sodium salts, triethylamine salts, and hexylamine salts.

[0439] The term "pharmaceutical composition" refers to a mixture of one or more compounds of this application or their salts with pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compounds of this application to an organism.

[0440] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0441] The word “comprise” or “include” and its English variants such as comprises or comprising should be understood in an open, non-exclusive sense, meaning “including but not limited to”.

[0442] The compounds and intermediates of this application may also exist in different tautomer forms, and all such forms are included within the scope of this application. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton transfer tautomers) include interconversions via proton migration, such as keto-enol and imine-enamine isomerization. A specific example of a proton tautomer is the imidazole moiety, where a proton can migrate between two ring nitrogens. Valence tautomers include interconversions via the recombination of some bonding electrons.

[0443] This application also includes compounds of this application that are identical to those described herein, but with one or more atoms replaced by isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this application include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as... 2 H,3 H, 11 C 13 C 14 C 13 N、 15 N、 15 O、 17 O、 18 O、 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.

[0444] Certain isotope-labeled compounds of this application (e.g., using...) 3 H and 14 Those labeled with C can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e. 3 H) and carbon-14 (i.e. 14 C) Isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes, such as... 15 O、 13 N、 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. The isotopically labeled compounds of this application can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0445] In addition, heavier isotopes (such as deuterium) are used. 2 H)) substitution can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., increased in vivo half-life or reduced dose requirement), and may therefore be preferred in certain situations, where deuterium substitution can be partial or complete, with partial deuterium substitution referring to at least one hydrogen being replaced by at least one deuterium.

[0446] The compounds of this application may be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers include, such as enantiomers and diastereomers. The compounds containing asymmetric carbon atoms of this application can be isolated in optically active pure form or in racemic form. The optically active pure form can be resolved from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

[0447] The pharmaceutical compositions of this application can be prepared by combining the compounds of this application with suitable pharmaceutically acceptable excipients, for example, in solid, semi-solid, liquid or gaseous formulations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalers, gels, microspheres and aerosols.

[0448] Typical routes of administration of the compound of this application, its isomers, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, vaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, and intravenous administration.

[0449] The pharmaceutical composition of this application can be manufactured using methods well known in the art, such as conventional mixing, dissolving, granulation, sugar-coated pill making, grinding, emulsification, freeze drying, etc.

[0450] In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of this application to be formulated into tablets, pills, lozenges, sugar-coated tablets, capsules, liquids, gels, pastes, suspensions, etc., for oral administration to patients.

[0451] Solid oral compositions can be prepared using conventional mixing, filling, or tableting methods. For example, they can be obtained by mixing the active compound with solid excipients, optionally milling the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into granules to obtain the core of a tablet or sugar-coated formulation. Suitable excipients include, but are not limited to, binders, diluents, disintegrants, lubricants, glidants, sweeteners, or flavoring agents.

[0452] The pharmaceutical composition may also be suitable for parenteral administration, such as in suitable unit dosage forms of sterile solutions, suspensions or lyophilized products.

[0453] For purposes of description and disclosure, all patents, patent applications and other identified publications are expressly incorporated herein by reference. Any reference to these publications herein does not constitute an endorsement that such publication is part of the general knowledge in the art.

[0454] The compounds of this application can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of this application.

[0455] The chemical reactions in the specific embodiments of this application are carried out in a suitable solvent, which must be suitable for the chemical changes and the reagents and materials required in this application. In order to obtain the compounds of this application, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction process based on existing embodiments.

[0456] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino groups in this application). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this application are incorporated herein by reference in their entirety.

[0457] Other objects, features, aspects and advantages of the invention will become apparent from the following detailed description and from the claims.

[0458] Abbreviations: EA: Ethyl acetate; PE: Petroleum ether; DIPEA: N,N-diisopropylethylamine; DCM: Dichloromethane; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; MeOH: Methanol; THF: Tetrahydrofuran; NBS: N-bromosuccinimide; Boc: tert-butyloxycarbonyl; TEA: Triethylamine; DMF: N,N-dimethylformamide; DIBAL-H: Diisobutylaluminum hydride; ACN: Acetonitrile; TBDMSCl: tert-butyldimethylchlorosilane; TBDMSO: tert-butyldimethyloxysilyl; ODMTr: 4, 4'-Dimethoxytriphenylmethyloxy; TBAF: Tetrabutylammonium fluoride; DMTrCl: 4,4'-Dimethoxytriphenylmethyl chloride; LAH: Lithium aluminum hydride; BnO: Benzyloxy; TsNHBoc: Tert-Butoxycarbonyl-p-Toluenesulfonic acid amine; TEBA: Benzyltriethylammonium chloride; MsCl: Methylsulfonyl chloride; NMM: N-Methylmorpholine; TCEP: Tris(2-carbonylethyl)phosphine; TEAA: Triethylamine / acetic acid; Py-SS-Py: 2,2'-Disulfide dipyridine; DMSO: Dimethyl sulfoxide; TBTA: Tris[(1-Benzyl-1H-1,2,3-Triazol-4-yl)methyl]amine.

[0459] Example

[0460] The present invention will now be described in further detail through embodiments, but the present invention is not limited to these embodiments.

[0461] All reagents used in this application are commercially available and can be used without further purification.

[0462] Preparation Example 1: Synthesis of compound D-ER-FY004002-P1

[0463]

[0464] (1-1) Synthesis of D-ER-FY004001-A1

[0465]

[0466] At room temperature, 100.0 g (0.63 mol) of 4-acetylbutyl ethyl ester was dissolved in 1000 mL of ethanol, followed by the addition of 77.2 g (0.63 mol) of 2-amino-3-pyridinecarboxaldehyde and 36.2 g (0.315 mol) of proline. The mixture was refluxed for 10 h. After the reaction was complete, the ethanol was removed by vacuum concentration, diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (EA / PE = 1 / 10-1 / 5) to give compound D-ER-FY004001-A1 (120.0 g, yield: 77.7%). MS ESI [M+H] + 245.12.

[0467] (1-2) Synthesis of D-ER-FY004001-A2

[0468]

[0469] Compound D-ER-FY004001-A1 (120.0 g, 0.49 mol) was dissolved in 500 mL of ethanol at room temperature, followed by the addition of 10% (w / w) 15% palladium on carbon. The reaction was carried out under a hydrogen atmosphere for 24 h. After the reaction was complete, the palladium on carbon was removed by diatomaceous earth filtration, and the ethanol was removed by concentration under reduced pressure to obtain compound D-ER-FY004001-A2 (118.0 g, yield: 96.7%). MS ESI [M+H] + 249.15.

[0470] (1-3) Synthesis of D-ER-FY004001-A3

[0471]

[0472] Compound D-ER-FY004001-A2 (118.0 g, 0.47 mol) was dissolved in 1000 mL of 6N HCl at room temperature and reacted at 50 °C for 12 h. After the reaction was complete, the solvent was removed by concentration under reduced pressure to obtain compound D-ER-FY004001-A3 (98.2 g, yield: 93.6%). MS ESI [M+H] + 221.12.

[0473] (1-4) Synthesis of D-ER-FY004002-P1-A1

[0474]

[0475] At room temperature, compound D-ER-FY004001-A3 (98.2 g, 0.44 mol) and DIPEA (287.3 g, 2.23 mol) were dissolved in 1000 mL of DCM. HATU (253.9 g, 0.67 mol) was then added and the mixture was stirred for 30 minutes. 2-Methoxyethane-1-amine (50.1 g, 0.67 mol) was then added and the reaction proceeded for 2 hours. After the reaction was complete, the mixture was diluted with water, extracted with DCM, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 80) to give compound D-ER-FY004002-P1-A1 (106.4 g, yield: 86.0%). MS ESI [M+H] + 278.18.

[0476] (1-5) Synthesis of D-ER-FY004002-P1-A2

[0477]

[0478] At room temperature, compound D-ER-FY004002-P1-A1 (100.0 g, 0.36 mol) was dissolved in 1000 mL of THF. The solution was cooled to 0 °C, and a turbid solution of LiAlH4 (20.6 g, 0.54 mol) in 500 mL of THF was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 14 h. After the reaction was complete, the solution was quenched with 20 mL of water at low temperature. Then, 20 mL of 15 wt% NaOH aqueous solution and 60 mL of water were slowly added dropwise with stirring. Solid magnesium sulfate was then added and stirred for 10 minutes to absorb the residual water. The mixture was filtered, and the filtrate was extracted with DCM. The organic phase was concentrated, and the solution was purified by column chromatography (MeOH / DCM = 1 / 100 - 1 / 20) to give compound D-ER-FY004002-P1-A2 (36.3 g, yield: 38.2%). MS ESI [M+H] + 264.20.(1-6) Synthesis of D-ER-FY004002-P1-B1

[0479]

[0480] At room temperature, compound D-ER-FY004002-P1-A2 (36.3 g, 0.14 mol) was dissolved in 200 mL of acetic acid. The solution was cooled to 0 °C, and Br2 (24.0 g, 0.15 mol) was slowly added dropwise. After the addition was complete, the reaction was allowed to proceed at room temperature for 1 h. After the reaction was complete, the solution was quenched with sodium thiosulfate at low temperature, and the pH was adjusted to 8 with saturated sodium bicarbonate. The solution was extracted with DCM, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM = 1 / 100 - 1 / 20) to give compound D-ER-FY004002-P1-B1 (38.8 g, yield: 81.0%). MS ESI [M+H] + 342.11.

[0481] (1-7) Synthesis of D-ER-FY004002-P1-B2

[0482]

[0483] At room temperature, compound D-ER-FY004002-P1-B1 (30.0 g, 87.97 mmol), zinc cyanide (30.9 g, 0.26 mol), tetraphenylphosphine palladium (30.5 g, 26.4 mmol), and 300 mL of DMF were added sequentially. The mixture was substituted with N2 three times, and then the temperature was raised to 100 °C for 5 h. After the reaction was complete, insoluble matter was removed by filtration, the filtrate was diluted with water, extracted with DCM, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 40) to give compound D-ER-FY004002-P1-B2 (15.0 g, yield: 59.2%). MSESI[M+H] + 289.20.

[0484] (1-8) Synthesis of D-ER-FY004002-P1-B3

[0485]

[0486] At room temperature, compound D-ER-FY004002-P1-B2 (15.0 g, 52.1 mmol) and TEA (10.5 g, 104.2 mmol) were dissolved in 100 mL of ethanol, and Boc anhydride (13.6 g, 62.5 mmol) was added dropwise, and the reaction was carried out for 1 h. After the reaction was complete, the ethanol was removed by vacuum concentration, followed by dilution with water, extraction with DCM, concentration of the organic phase, and purification by column chromatography (MeOH / DCM = 1 / 100) to give compound D-ER-FY004002-P1-B3 (16.5 g, yield: 81.6%). MS ESI [M+H] + 389.25.

[0487] Synthesis of (1-9)D-ER-FY004002-P1-B4

[0488]

[0489] At room temperature, compound D-ER-FY004002-P1-B3 (16.5 g, 42.5 mmol) was dissolved in 200 mL of diethyl ether. The solution was cooled to 0 °C, and a turbid solution of LiAlH4 (8.1 g, 212.6 mmol) in 80 mL of diethyl ether was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After the reaction was complete, the solution was quenched with water at low temperature. Then, 30 mL of water and 10 mL of 15 wt% NaOH aqueous solution were slowly added dropwise with stirring. Sodium sulfate solid was then added, stirred, and filtered. The filtrate was extracted with DCM, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 10) to give compound D-ER-FY004002-P1-B4 (6.4 g, yield: 38.4%). MS ESI [M+H] + 393.28.

[0490] (1-10) Synthesis of D-ER-FY004002-P1-D1

[0491]

[0492] At room temperature, compound D-ER-FY004002-P1-B4 (2.0 g, 5.1 mmol) and DIPEA (1.97 g, 15.3 mmol) were dissolved in 20 mL of DCM. The solution was cooled to 0 °C, and a DCM solution of 6-azidohexanoate succinimide ester (1.94 g, 7.65 mmol) in 10 mL was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After the reaction was complete, the solution was directly concentrated and purified by column chromatography (MeOH / DCM = 1 / 100) to give compound D-ER-FY004002-P1-D1 (2.1 g, yield: 77.5%). MS ESI [M+H] + 532.35.

[0493] (1-11) Synthesis of D-ER-FY004002-P1-D2

[0494]

[0495] At room temperature, compound D-ER-FY004002-P1-D1 (2.1 g, 3.95 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and a solution of hydrochloric acid (1.4 g, 39.5 mmol) in 10 mL of EA was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P1-D2 (1.63 g, yield: 95.6%). MS ESI [M+H] + 432.30.

[0496] (1-12) Synthesis of D-ER-FY004001-B1

[0497]

[0498] Weigh out 10.0 g (45.66 mmol) of D-ER-FY004001-B0 and dissolve it in 50 mL of DMF. Cool to 0 °C, add potassium carbonate (12.6 g, 90.32 mmol), and then add methyl iodide (32.4 g, 228.3 mmol). Restore the reaction to room temperature and react for 14 h. TLC monitoring showed no residue of the starting material. Extract with ethyl acetate (3 × 300 mL). Wash the combined organic extracts with water (5 × 200 mL), dry with anhydrous MgSO4, filter, concentrate under reduced pressure, and purify by column chromatography (PE / EA = 10 / 1-3 / 1) to obtain D-ER-FY004001-B1 (9.2 g, yield: 86.5%). MS ESI [M+Na]+ 234.13.

[0499] (1-13) Synthesis of D-ER-FY004001-D2

[0500]

[0501] Weigh D-ER-FY004001-B1 (9.2 g, 39.5 mmol) and dissolve it in 50 mL of pyridine. Cool the solution to 0 °C. Then, add p-toluenesulfonyl chloride (37.5 g, 197 mmol) (dissolved in 20 mL of anhydrous acetonitrile) dropwise to the above reaction system. After the addition is complete, heat the solution to room temperature and react for 8 h. HPLC monitoring showed that no raw material remained. Add 300 mL of diethyl ether and stir for 1 h. Wash the organic extract with water (3 × 200 mL), saturated sodium bisulfate (3 × 200 mL), saturated sodium bicarbonate (3 × 200 mL), and saturated brine (3 × 200 mL). Dry the extract in anhydrous MgSO4, filter, concentrate under reduced pressure, and purify by column chromatography (PE / EA = 5 / 1) to obtain D-ER-FY004001-D2 (6.4 g, yield: 41.9%). Synthesis of MS ESI[M+Na]+388.14.(1-14)D-ER-FY004002-P1-D3

[0502]

[0503] Compounds D-ER-FY004002-P1-D2 (1.63 g, 3.78 mmol), D-ER-FY004001-D2 (2.93 g, 7.56 mmol), potassium carbonate (1.21 g, 8.8 mmol), and DMF (10 mL) were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 40) to give compound D-ER-FY004002-P1-D3 (1.71 g, yield: 76.4%). MS ESI [M+H] + 647.42.

[0504] (1-15) Synthesis of D-ER-FY004002-P1-D4

[0505]

[0506] At room temperature, compound D-ER-FY004002-P1-D3 (1.71 g, 2.89 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and a 10 mL solution of hydrochloric acid (1.03 g, 28.9 mmol) in EA was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P1-D4 (1.40 g, yield: 96.9%). MS ESI [M+H] + 547.36.

[0507] (1-16) Synthesis of D-ER-FY004002-P1-D5

[0508]

[0509] Compound D-ER-FY004002-P1-D4 (1.40 g, 2.56 mmol), TEA (0.77 g, 7.69 mmol), 4-chloroquinazoline (0.63 g, 3.85 mmol), and 5 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 40) to give compound D-ER-FY004002-P1-D5 (1.35 g, yield: 78.1%). MS ESI [M+H] + 675.40.

[0510] (1-17) Synthesis of D-ER-FY004002-P1

[0511]

[0512] At room temperature, compound D-ER-FY004002-P1-D5 (135 mg, 0.20 mmol) was dissolved in a mixed solvent of THF (1 mL) and water (1 mL), followed by the addition of lithium hydroxide (96 mg, 4.00 mmol). The mixture was then heated to 40 °C and reacted for 2 h. After the reaction was complete, compound D-ER-FY004002-P1 (40 mg, yield: 30.2%) was prepared by HPLC. MS ESI [M+H] + 661.39.

[0513] 1 H NMR(400MHz,Chloroform-d)δ9.47(s,1H),8.61(s,1H),8.25–8.12(m,1H),8.10–8.00(m,2H),7.85–7.71(m,2H),7.55 –7.45(m,2H),4.68–4.60(m,1H),4.18(d,J=5.6Hz,2H),3.73(t,J=4.8Hz,2H),3.60(s,1H),3.43(t,J=5.6Hz,2H),3.37 –3.28(m,5H),3.27–3.18(m,2H),3.17–3.07(m,2H),2.89(t,J=7.8Hz,2H),2.78(t,J=8.2Hz,2H),2.69(t,J=6.2Hz,2H) ,2.45–2.27(m,2H),2.23(t,J=7.5Hz,2H),1.98–1.82(m,4H),1.80–1.67(m,2H),1.60–1.53(m,2H),1.42–1.30(m,4H).

[0514] Synthesis of Preparation Example 2D-ER-FY004004-P1

[0515]

[0516] (2-1) Synthesis of D-ER-FY004001-E1

[0517]

[0518] Compound D-ER-FY004001-A2 (1.40 g, 5.65 mmol) was dissolved in 10 mL of THF, then cooled to -78 °C. A hexane solution (6.2 mL) of DIBAL-H (0.88 g, 6.21 mmol) was slowly added dropwise. After the addition was complete, the reaction was maintained at this temperature for 2 h. After the reaction was complete, 4 mL of water and 1 mL of 15 wt% NaOH aqueous solution were slowly added dropwise under controlled temperature and stirring. Sodium sulfate was then added and dried. The mixture was stirred and filtered. The filtrate was extracted with DCM, the organic phase was concentrated, and purified by column chromatography (EA / PE = 1 / 3-1 / 1) to give compound D-ER-FY004001-E1 (0.73 g, yield: 63.4%). MS ESI [M+H]+205.13.

[0519] (2-2) Synthesis of D-ER-FY004001-E2

[0520]

[0521] At room temperature, compound D-ER-FY004001-E1 (0.73 g, 3.58 mmol) was dissolved in 10 mL of DCM, followed by the addition of 17-azido-3,6,9,12,15-pentaheptadecane-1-amine (1.64 g, 5.37 mmol) and TEA (0.72 g, 7.16 mmol). After stirring for 10 minutes, the mixture was cooled to 0 °C, and sodium triacetoxyborohydride (2.28 g, 10.73 mmol) was slowly added. The mixture was then allowed to react at room temperature for 2 h. After the reaction was complete, the mixture was quenched with water at low temperature, extracted with DCM, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100 - 1 / 20) to give compound D-ER-FY004001-E2 (0.33 g, yield: 18.7%). MS ESI [M+H] +495.32

[0522] (2-3) Synthesis of D-ER-FY004001-E3

[0523]

[0524] Compounds D-ER-FY004001-E2 (0.33 g, 0.67 mmol), D-ER-FY004001-D2 (0.52 g, 1.34 mmol), potassium carbonate (0.28 g, 2.00 mmol), and 7 mL of DMF were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 80) to give compound D-ER-FY004001-E3 (0.35 g, yield: 73.9%). MS ESI [M+H]+710.44.

[0525] (2-4) Synthesis of D-ER-FY004001-E4

[0526]

[0527] At room temperature, compound D-ER-FY004001-E3 (0.35 g, 0.49 mmol) was dissolved in EA, cooled to 0 °C, and a solution of EA (4 mL) containing hydrochloric acid (0.18 g, 4.9 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004001-E4 (0.27 g, yield: 89.8%). MS ESI [M+H]+610.38.

[0528] (2-5) Synthesis of D-ER-FY004001-E5

[0529]

[0530] Compound D-ER-FY004001-E4 (0.27 g, 0.44 mmol), TEA (0.13 g, 1.33 mmol), 4-chloroquinazoline (0.11 g, 0.66 mmol), and 3 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 60) to give compound D-ER-FY004001-E5 (0.24 g, yield: 73.4%). MS ESI [M+H] +738.42.

[0531] (2-6) Synthesis of D-ER-FY004004-P1

[0532]

[0533] At room temperature, compound D-ER-FY004001-E5 (100 mg, 0.13 mmol) was dissolved in a mixed solvent of THF (1 mL) and water (1 mL), followed by the addition of lithium hydroxide (64 mg, 2.6 mmol). The mixture was then heated to 40 °C and reacted for 2 h. After the reaction was complete, compound D-ER-FY004004-P1 (24 mg, yield: 24.5%) was prepared by liquid chromatography. MS ESI [M+H] +724.41.

[0534] 1H NMR(400MHz,Chloroform-d)δ9.89(s,1H),9.82(s,1H),8.73(s,1H),8.58(d,J=8.7Hz,1H),7.9 3(d,J=8.6Hz,1H),7.96-7.87(m,1H),7.86-7.77(m,1H),7.30(d,J=7.1Hz,1H),6.34(d,J=7.2Hz ,1H),4.93(s,1H),3.87-3.80(m,2H),3.67-3.55(m,21H),3.50-3.40(m,4H),3.39-3.32(m,4H) ,2.75-2.63(m,4H),2.60-2.47(m,2H),1.95-1.87(m,2H),1.87-1.77(m,2H),1.76-1.67(m,2H).

[0535] Synthesis of Preparation Example 3D-ER-FY004004-P2

[0536]

[0537] (3-1) Synthesis of D-ER-FY004001-G1

[0538]

[0539] At room temperature, compound D-ER-FY004001-E1 (1.77 g, 8.68 mmol) was dissolved in DCM, followed by the addition of amino-pentaethylene glycol-azide (3.4 g, 13.0 mmol) and TEA (1.75 g, 17.35 mmol). After stirring for 10 minutes, the mixture was cooled to 0°C, and sodium triacetoxyborohydride (5.5 g, 26.0 mmol) was slowly added. Once the addition was complete, the mixture was allowed to react at room temperature for 2 hours. After the reaction was complete, the mixture was quenched with water at low temperature, extracted with DCM, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100 - 1 / 20) to give compound D-ER-FY004001-G1 (0.7 g, yield: 17.9%). MS ESI [M+H] +451.30.

[0540] (3-2) Synthesis of D-ER-FY004001-G2

[0541]

[0542] Compounds D-ER-FY004001-G1 (0.7 g, 1.56 mmol), D-ER-FY004001-D2 (1.2 g, 3.11 mmol), potassium carbonate (0.64 g, 4.67 mmol), and DMF (7 mL) were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 60) to give compound D-ER-FY004001-G2 (0.65 g, yield: 62.8%). MS ESI [M+H] +666.41.

[0543] (3-3) Synthesis of D-ER-FY004001-G3

[0544]

[0545] At room temperature, compound D-ER-FY004001-G2 (0.65 g, 0.98 mmol) was dissolved in EA (7 mL), cooled to 0 °C, and a solution of hydrochloric acid (0.35 g, 9.8 mmol) in EA (3 mL) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, compound D-ER-FY004001-G3 (0.45 g, yield: 81.5%) was directly concentrated. MS ESI [M+H] +566.36.

[0546] (3-4) Synthesis of D-ER-FY004001-G4

[0547]

[0548] Compound D-ER-FY004001-G3 (0.35 g, 0.62 mmol), TEA (0.19 g, 1.86 mmol), 4-chloroquinazoline (0.15 g, 0.93 mmol), and 3 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM = 1 / 100-1 / 60) to give compound D-ER-FY004001-G4 (0.31 g, yield: 72.2%). MS ESI [M+H] +694.40.

[0549] (3-5) Synthesis of D-ER-FY004004-P2

[0550]

[0551] At room temperature, compound D-ER-FY004001-G4 (100 mg, 0.14 mmol) was dissolved in a mixed solvent of THF (1 mL) and water (1 mL), followed by the addition of lithium hydroxide (67 mg, 2.8 mmol). The mixture was then heated to 40 °C and reacted for 2 h. After the reaction was complete, compound D-ER-FY004004-P2 (29 mg, yield: 29.6%) was prepared by liquid chromatography. MS ESI [M+H] +68 0.38.

[0552] Synthesis of Preparation Example 4D-ER-FY004008-P1

[0553]

[0554] (4-1) Synthesis of D-ER-FY003101-A2

[0555]

[0556] Weigh (3R,5S)-5-{[bis(4-methoxyphenyl)(phenyl)methoxy]methyl}pyrrolidine-3-ol (D-ER-FY003101-A1) (5.0 g, 11.93 mmol) and imidazole (4.06 g, 59.66 mmol) and dissolve them in 50 mL of DCM. Then, add TBDMSCl (2.70 g, 17.90 mmol) dropwise to the above reaction system. After the addition is complete, continue the reaction at room temperature for 5 h. TLC monitoring shows that there is no starting material remaining. Add 10 mL of water and wash the reaction system three times with DCM (100 mL * 3). Combine the organic phases, then concentrate under reduced pressure and purify by column chromatography (EA / PE = 1 / 2 - 1 / 1) to obtain product D-ER-FY003101-A2 (5.5 g, yield: 86.5%). MS ESI [M+H + 534.3.

[0557] (4-2) Synthesis of D-ER-FY004008-A1

[0558]

[0559] Weigh D-ER-FY003101-A2 (3.5 g, 6.57 mmol) and dissolve it in 10 mL of anhydrous acetonitrile. Cool the solution to 0 °C. Slowly add potassium carbonate (1.81 g, 13.13 mmol) to the reaction system. Stir at 0 °C for 30 minutes. Then add 8-bromo-1-octyne (2.48 g, 13.13 mmol) dropwise to the reaction system. After the addition is complete, heat to room temperature and react for 10 h. TLC monitoring showed no residue of the starting material. Concentrate under reduced pressure to remove acetonitrile. Add 10 mL of water. Wash the reaction system three times with ethyl acetate (100 mL * 3). Combine the organic phases and wash with saturated brine (100 mL). Concentrate under reduced pressure to obtain crude product D-ER-FY004008-A1 (4.5 g, unpurified). MS ESI [M+H] + 642.39.

[0560] (4-3) Synthesis of D-ER-FY004008-A2

[0561]

[0562] D-ER-FY004008-A1 (crude product, 4.5 g, 1.0 equiv.) was weighed and dissolved in 10 mL of tetrahydrofuran. Then, TBAF (9.16 g, 35.1 mmol) was added dropwise to the reaction system. The mixture was reacted at room temperature for 18 h. TLC monitoring showed no residue of the starting material. The tetrahydrofuran was removed by concentration under reduced pressure, and the product was purified by column chromatography (EA / PE = 1 / 2-2 / 1) to obtain product D-ER-FY004008-A2 (2.8 g, two-step yield: 81.8%). MS ESI [M+H] + 528.30.

[0563] (4-4) Synthesis of D-ER-FY004008-P1

[0564]

[0565] Weigh out bis(diisopropylamino)(2-cyanoethoxy)phosphine (514 mg, 1.71 mmol) and tetrazolium (40 mg, 0.57 mmol) and dissolve them in 2 mL of DCM. Replace the nitrogen gas three times. Then add a 2 mL solution of D-ER-FY004008-A2 (300 mg, 0.57 mmol) in DCM to the above reaction system and react at room temperature for 2 h. TLC monitoring showed that there was no residue of the starting material. The reaction system was purified by rapid column chromatography (PE / EA = 1 / 0-10 / 1) to obtain product D-ER-FY004008-P1 (196 mg, yield: 47.4%).

[0566] 1H NMR(400MHz,Chloroform-d)δ7.52–7.44(m,2H),7.40–7.19(m,7H),6.89–6.81(m ,4H),3.90–3.84(m,2H),3.83(s,6H),3.70–3.50(m,3H),3.05–2.90(m,2H),2.82 –2.75(m,1H),2.67(t,J=6.6Hz,2H),2.43–2.27(m,2H),2.19(td,J=7.1,2.6Hz,2 H),2.13–2.01(m,1H),1.99–1.85(m,2H),1.60–1.25(m,10H),1.24–1.19(m,12H).

[0567] Synthesis of Preparation Example 5D-ER-FY004006-P1

[0568]

[0569] (5-1) Synthesis of D-ER-FY004006-B1

[0570]

[0571] N4-acetylcytidine (5.0 g, 19.38 mmol) was dissolved in 35 mL of pyridine and cooled to 0 °C. DMTrCl (9.85 g, 29.07 mmol) was then slowly added to the reaction system. After the addition was complete, the reaction was continued at room temperature for 12 h. TLC monitoring showed no residual starting material. 10 mL of water was added, and the reaction system was extracted with DCM (100 mL * 3). The organic phases were combined, washed with saturated brine (10 mL), and then concentrated under reduced pressure and purified by column chromatography (EA / Pe = 1 / 3-1 / 1) to obtain product D-ER-FY004006-B1 (7.8 g, yield: 75.7%). MS ESI [M+H] +588.23.

[0572] (5-2) Synthesis of D-ER-FY004006-B2

[0573]

[0574] Weigh out 7.1 g (12.09 mmol) of D-ER-FY004006-B1, dissolve it in 70 mL of anhydrous THF, and cool to 0 °C. Then, slowly add 0.97 g (24.19 mmol) of sodium hydride to the reaction system. After addition, stir at 0 °C for 30 minutes. Next, add 3.43 g (18.14 mmol) dropwise to the reaction system. After the addition is complete, heat to 40 °C and react for 20 h. Cool to room temperature, add 30 mL of water, and extract the reaction system using DCM (100 mL * 3). Combine the organic phases, wash with saturated brine (100 mL), and then concentrate under reduced pressure and purify by column chromatography (EA / Pe = 1 / 5-1 / 3) to obtain product D-ER-FY004006-B2 (1.3 g, yield: 15.5%). MS ESI [M+H] + 696.32.

[0575] (5-3) Synthesis of D-ER-FY004006-P1

[0576]

[0577] 173 mg (0.58 mmol) of bis(diisopropylamino)(2-cyanoethoxy)phosphine and 20 mg (0.29 mmol) of tetrazolium were weighed and dissolved in 2 mL of DCM. Nitrogen gas was purged three times. Then, 200 mg (0.29 mmol) of D-ER-FY004006-B2 was added dropwise to the reaction system. After addition, the reaction was carried out at room temperature for 2 h. TLC monitoring showed no residual starting material. The reaction system was purified by rapid column chromatography (PE / EA = 10 / 1-5 / 1) to obtain product D-ER-FY004006-P1 (150 mg, yield: 58.2%). MS ESI [M-83+H]+813.2.

[0578] Synthesis of Preparation Example 6N-ER-FY017002-P1

[0579]

[0580] (6-1) Synthesis of N-ER-FY017002-P1-A1

[0581] 1.0 g (8.58 mmol) of 6-chlorohexyne was dissolved in 5 mL of DMF. Potassium thioacetate (1.47 g, 12.86 mmol) was then added to the system. The mixture was purged with nitrogen three times, and the temperature was raised to 50 °C for 3.5 h. HPLC monitoring showed no residue of the starting material. The mixture was cooled to room temperature and extracted with diethyl ether (3 × 30 mL). The combined organic extracts were washed with water (5 × 20 mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to obtain N-ER-FY017002-P1-A1 (1.1 g, yield: 81.8%). MS ESI [M+Na] +179.03.

[0582] (6-2) Synthesis of N-ER-FY017002-P1

[0583] Weigh out 0.27 g (7.05 mmol) of lithium aluminum hydride, dissolve it in 7 mL of anhydrous THF, and cool to 0 °C. Then, add 0.55 g (3.53 mmol) of N-ER-FY017002-P1-A1 (dissolved in 2 mL of anhydrous acetonitrile) dropwise to the above reaction system. After the addition is complete, heat to room temperature and react for 20 minutes. HPLC monitoring shows no residual starting material. Then, add H2O until no more gas is released. Adjust the pH to 6 with citric acid, extract with diethyl ether (3 × 30 mL), wash the combined organic extracts with water (2 × 20 mL), dry with anhydrous MgSO4, filter, and concentrate under reduced pressure to obtain N-ER-FY017002-P1 (0.34 g, yield: 84.6%). MS ESI [M+Na] +137.05.

[0584] Synthesis of Preparation Example 7D-ER-FY004009-P1

[0585]

[0586] (7-1) Synthesis of D-ER-FY003117-P1-A1

[0587]

[0588] Compound (S)-benzyloxymethyl ethylene oxide (38.0 g, 231.0 mmol, 10 eq), TsNHBoc (75.4 g, 278.0 mmol, 12 eq), K₂CO₃ (6.40 g, 46.3 mmol, 2.0 eq), and TEBA (5.27 g, 23.1 mmol, 1.0 eq) were added to a three-necked flask at room temperature. The reaction was carried out at 95 °C for 2 hours. After cooling to room temperature, the mixture was diluted with water (500 mL), extracted with dichloromethane (200 mL x 3), dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent PE:EA = 3:1, v / v) to give compound D-ER-FY003117-P1-A1 (40.0 g, yield: 39.7%). MS: m / z [M+1] + 436.2.

[0589] (7-2) Synthesis of D-ER-FY003117-P1-A2

[0590]

[0591] Compound D-ER-FY003117-P1-A1 (79.0 g, 181 mmol, 1.20 eq), (R)-benzyloxymethyl ethylene oxide (24.7 g, 150.8 mmol, 1.0 eq), potassium carbonate (4.21 g, 30.5 mmol, 0.20 eq), and TEBA (3.47 g, 15.2 mmol, 0.10 eq) were added to a single-necked flask and reacted at 95 °C for 3 hours. After cooling to room temperature, the reaction mixture was extracted with water (500 mL) and dichloromethane (200 mL * 3). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (eluent PE:EA = 3:1, v / v) to give compound D-ER-FY003117-P1-A2 (40.0 g, yield 43.8%). MS: m / z [M+1] + 600.2.

[0592] (7-3) Synthesis of D-ER-FY003117-P1-A3

[0593]

[0594] Compound D-ER-FY003117-P1-A2 (77.0 g, 128 mmol, 1.0 eq) and triethylamine (28.6 mL, 205 mmol, 1.60 eq) were added sequentially to dichloromethane (800 mL) under ice-water bath conditions, followed by the slow addition of methanesulfonyl chloride (24.2 g, 212 mmol, 1.65 eq). The reaction was carried out at 25 °C for 2 hours, and LC-MS showed that the reaction was complete. The reaction solution was washed with water (800 mL), and the organic phase was concentrated under reduced pressure to give compound D-ER-FY003117-P1-A3 (90.0 g). MS: m / z [M+1] + 678.2.

[0595] (7-4) Synthesis of D-ER-FY003117-P1-A4

[0596]

[0597] Compound D-ER-FY003117-P1-A3 (90.0 g, 133 mmol, 1.0 eq) and potassium carbonate (91.8 g, 664 mmol, 5.0 eq) were added to methanol (900 mL) at room temperature. The reaction was carried out at 66 °C for 2 hours. The organic phase was concentrated under reduced pressure, and the reaction solution was diluted with water (200 mL). Extraction was performed using dichloromethane (200 mL x 3), and the organic phase was concentrated under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5 / 1-2 / 1, v / v) to give compound D-ER-FY003117-P1-A4 (64.0 g, yield 99.9%). MS m / z [M+1] + 482.2.

[0598] (7-5) Synthesis of D-ER-FY003117-P1-A5

[0599]

[0600] Compound D-ER-FY003117-P1-A4 (69.0 g, 143 mmol, 1.0 eq) and magnesium filings (54.7 g, 228 mol, 1.60 eq) were added to methanol (400 mL) and reacted at 66 °C for 1 hour. The reaction solution was diluted with water (3000 mL) and saturated ammonium chloride aqueous solution (3000 mL), extracted with dichloromethane (1000 mL * 3), and the organic phase was washed with saturated sodium bicarbonate (300 mL * 3). The organic phase was concentrated under reduced pressure to obtain D-ER-FY003117-P1-A5 (32.0 g, yield: 68.2%). MS: m / z [M+1] + 328.2.

[0601] (7-6) Synthesis of D-ER-FY004009-P1-B1

[0602]

[0603] Weigh 2.6 g (7.95 mmol, 1 eq) of D-ER-FY003117-P1-A5 into a reaction flask, add concentrated hydrochloric acid (40 mL, 11.9 mol / L), and react at 50 °C for 6 h. The reaction was completed by TLC and mass spectrometry. The reaction system was concentrated, 30 mL of water was added, and the mixture was lyophilized to obtain D-ER-FY004009-P1-B1 (3.0 g), MS: m / z 148.2 [M+1]+.

[0604] (7-7) Synthesis of D-ER-FY004009-P1-A3

[0605]

[0606] Weigh D-ER-FY004009-P1-B1 (2.0 g, 13.6 mmol, 1.0 eq) into a reaction flask, dissolve in acetonitrile (30 mL), add potassium carbonate (5.6 g, 40.5 mmol, 3 eq) and 8-bromo-1-octyne (3.8 g, 20.2 mmol, 1.5 eq), stir overnight at 40 °C, and concentrate the reaction solution under reduced pressure. Purify by column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound D-ER-FY004009-P1-A3 (780 mg). MS: m / z 256.2 [M+H]+.

[0607] (7-8) Synthesis of D-ER-FY004009-P1-A4

[0608]

[0609] At room temperature, compounds D-ER-FY004009-P1-A3 (580 mg, 2.27 mmol, 1.0 eq) and NMM (1.15 g, 11.35 mmol, 5.0 eq) were weighed and added to a reaction flask, dissolved in DCM (15 mL), and DMTrCl (1.51 g, 4.45 mmol, 2.0 eq) was added with stirring at room temperature. The reaction was allowed to proceed overnight. The reaction mixture was poured into water (20 mL), extracted three times with DCM (10 mL), and the organic phases were combined and concentrated to obtain the crude product. The crude product was purified by column chromatography (petroleum ether / ethyl acetate = 2 / 1) to give compound D-ER-FY004009-P1-A4 (500 mg, yield: 40.00%). MS: m / z 558.4 [M+H]+.

[0610] (7-9) Synthesis of D-ER-FY004009-P1

[0611]

[0612] Weigh D-ER-FY004009-P1-A4 (280 mg, 0.50 mmol, 1.0 eq) into a reaction flask, add tetrazolium (212.2 mg, 3.01 mmol, 6 eq), dissolve in anhydrous DCM (8 mL), and protect the system by fully purging with N2. Add bis(diisopropylamino)(2-cyanoethoxy)phosphine (907.8 mg, 3.01 mmol, 6 eq) with stirring at room temperature. React at room temperature for 2.5 h. After the reaction is complete as monitored by TLC, concentrate the crude product to dryness under N2 protection and purify by column chromatography (petroleum ether / ethyl acetate = 3:1) to obtain compound D-ER-FY004009-P1 (250.0 mg, yield: 65.8%). MS ESI [M-83+H] + m / z: 675.4

[0613] Synthesis of Preparation Example 8D-ER-FY004008-P2

[0614]

[0615] (8-1) Synthesis of D-ER-FY004008-P2-A1

[0616]

[0617] Weigh D-ER-FY003101-A2 (1.0 g, 1.88 mmol) and dissolve it in 5 mL of anhydrous acetonitrile. Cool the solution to 0 °C. Slowly add potassium carbonate (0.52 g, 3.76 mmol) to the reaction system. Stir at 0 °C for 30 minutes. Then, add propargyl-PEG2-bromo (0.78 g, 3.76 mmol) (CAS No.: 1287660-82-5) dropwise to the reaction system. After the addition is complete, heat to room temperature and react for 12 h. TLC monitoring showed no residue of the starting material. Concentrate under reduced pressure to remove acetonitrile. Add 50 mL of water. Wash the reaction system three times with ethyl acetate (50 mL * 3). Combine the organic phases and wash with saturated brine (50 mL). Concentrate under reduced pressure to obtain crude product D-ER-FY004008-P2-A1 (1.3 g), which was used directly in the next step without purification. MS ESI [M+H] + m / z: 660.4.

[0618] (8-2) Synthesis of D-ER-FY004008-P2-A2

[0619]

[0620] Weigh out 1.3 g (1.0 equiv.) of D-ER-FY004008-P2-A1 (crude product) and dissolve it in 10 mL of tetrahydrofuran. Then, add 3.1 g (12 mmol) dropwise to the above reaction system and react at room temperature for 18 h. TLC monitoring showed no residue of the starting material. The tetrahydrofuran was removed by concentration under reduced pressure, and the product was purified by column chromatography (EA / PE (v / v) = 1 / 2-2 / 1) to obtain product D-ER-FY004008-P2-A2 (0.7 g, two-step yield: 68.2%). MS ESI [M+H] + m / z:546.4.

[0621] (8-3) Synthesis of D-ER-FY004008-P2

[0622]

[0623] 1.03 g (3.42 mmol) of bis(diisopropylamino)(2-cyanoethoxy)phosphine and 80 mg (1.14 mmol) of tetrazolium were weighed and dissolved in 5 mL of DCM. Nitrogen gas was purged three times. Then, a 5 mL solution of D-ER-FY004008-P2-A2 (622 mg, 1.14 mmol) in DCM was added dropwise to the above reaction system. The reaction was carried out at room temperature for 2 h. TLC monitoring showed no residual starting material. The reaction system was purified by rapid column chromatography (PE / EA(v / v) = 1 / 0 - 10 / 1) to obtain product D-ER-FY004008-P2 (1.03 g, yield: 40.2%). MS ESI [M-83+H] + m / z: 663.4.

[0624] Synthesis of Preparation Example 9D-ER-FY004002-P2

[0625]

[0626] (9-1) Synthesis of D-ER-FY004002-P2-A1

[0627]

[0628] At room temperature, compound D-ER-FY004002-P1-B4 (2.0 g, 5.1 mmol) and DIPEA (1.97 g, 15.3 mmol) were dissolved in 20 mL of DCM. The solution was cooled to 0 °C, and a 10 mL solution of azido-diethylene glycol-acetic acid-succinimide ester (CAS No.: 945550-23-2) (2.19 g, 7.65 mmol) in DCM was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After the reaction was complete, the solution was directly concentrated and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100) to give compound D-ER-FY004002-P2-A1 (2.0 g, yield: 69.5%). MS ESI [M+H] + 564.4.

[0629] (9-2) Synthesis of D-ER-FY004002-P2-A2

[0630]

[0631] At room temperature, compound D-ER-FY004002-P2-A1 (2.0 g, 3.55 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and an EA solution of 1.3 g (35.5 mmol) in 8 mL was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P2-A2 (1.49 g, yield: 90.1%). MS ESI [M+H] + 464.4.

[0632] (9-3) Synthesis of D-ER-FY004002-P2-A3

[0633]

[0634] Compounds D-ER-FY004002-P2-A2 (1.49 g, 3.20 mmol), D-ER-FY004001-D2 (2.48 g, 6.40 mmol), potassium carbonate (1.03 g, 7.46 mmol), and acetonitrile (10 mL) were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P2-A3 (1.58 g, yield: 72.5%). MS ESI [M+H] + 679.6.

[0635] (9-4) Synthesis of D-ER-FY004002-P2-A4

[0636]

[0637] At room temperature, compound D-ER-FY004002-P2-A3 (1.58 g, 2.32 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and a 10 mL solution of EA containing hydrochloric acid (0.85 g, 23.2 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P2-A4 (1.24 g, yield: 92.3%). MS ESI [M+H] + 579.4.

[0638] Synthesis of (9-5)D-ER-FY004002-P2-A5

[0639]

[0640] Compound D-ER-FY004002-P2-A4 (1.24 g, 2.14 mmol), TEA (0.64 g, 6.43 mmol), 4-chloroquinazoline (0.53 g, 3.22 mmol), and 4 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P2-A5 (1.07 g, yield: 71.2%). MS ESI [M+H + 707.2.

[0641] (9-6) Synthesis of D-ER-FY004002-P2

[0642]

[0643] At room temperature, compound D-ER-FY004002-P2-A5 (1.07 g, 1.52 mmol) was dissolved in a mixed solvent of THF (3 mL) and water (3 mL), followed by the addition of lithium hydroxide (730 mg, 30.4 mmol), and the mixture was heated to 40 °C for 2 h. After the reaction was complete, compound D-ER-FY004002-P2 (370 mg, yield: 35.2%) was prepared by HPLC. MS ESI [M+H] + 693.2.

[0644] Synthesis of Preparation Example 10D-ER-FY004002-P3

[0645] Synthesis of (10-1)D-ER-FY004002-P3-A1

[0646]

[0647] At room temperature, compound D-ER-FY004002-P1-B4 (1.0 g, 2.5 mmol) and DIPEA (0.99 g, 7.6 mmol) were dissolved in 10 mL of DCM. The solution was cooled to 0 °C, and a 10 mL solution of azido-tetraethylene glycol-vinyl succinimide (CAS No.: 1807534-82-2) (1.43 g, 3.82 mmol) in DCM was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After the reaction was complete, the solution was directly concentrated and purified by column chromatography (MeOH / DCM = 1 / 100) to give compound D-ER-FY004002-P3-A1 (1.54 g, yield: 61.5%). MS ESI [M+H] + 652.6.

[0648] (10-2) Synthesis of D-ER-FY004002-P3-A2

[0649]

[0650] At room temperature, compound D-ER-FY004002-P3-A1 (1.54 g, 2.36 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and a 5 mL solution of EA containing hydrochloric acid (0.86 g, 23.6 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P3-A2 (1.20 g, yield: 92.4%). MS ESI [M+H] + 552.4.

[0651] Synthesis of (10-3)D-ER-FY004002-P3-A3

[0652]

[0653] Compounds D-ER-FY004002-P3-A2 (1.20 g, 2.18 mmol), D-ER-FY004001-D2 (1.69 g, 4.36 mmol), potassium carbonate (0.70 g, 5.08 mmol), and acetonitrile (10 mL) were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P3-A3 (1.26 g, yield: 75.2%). MS ESI [M+H + 767.6.

[0654] Synthesis of (10-4)D-ER-FY004002-P3-A4

[0655]

[0656] At room temperature, compound D-ER-FY004002-P3-A3 (1.26 g, 1.64 mmol) was dissolved in 10 mL of EA. The solution was cooled to 0 °C, and an EA solution of 0.60 g (16.4 mmol) in 8 mL was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P3-A4 (1.02 g, yield: 93.5%). MS ESI [M+H] + 667.6.

[0657] Synthesis of (10-5)D-ER-FY004002-P3-A5

[0658]

[0659] Compound D-ER-FY004002-P3-A4 (1.02 g, 1.53 mmol), TEA (0.46 g, 4.59 mmol), 4-chloroquinazoline (0.38 g, 2.30 mmol), and 5 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P3-A5 (0.84 g, yield: 68.5%). MS ESI [M+H + 795.4.

[0660] Synthesis of (10-6)D-ER-FY004002-P3

[0661]

[0662] At room temperature, compound D-ER-FY004002-P3-A5 (0.84 g, 1.05 mmol) was dissolved in a mixed solvent of THF (3 mL) and water (3 mL), followed by the addition of lithium hydroxide (504 mg, 21 mmol). The mixture was then heated to 40 °C and reacted for 2 h. After the reaction was complete, compound D-ER-FY004002-P3 (234 mg, yield: 28.6%) was obtained by HPLC. MS ESI [M+H] + 781.4.

[0663] Synthesis of Preparation Example 11D-ER-FY004002-P4

[0664]

[0665] (11-1) Synthesis of D-ER-FY004002-P4-A1

[0666]

[0667] At room temperature, compounds D-ER-FY004002-P1-B4 (2.0 g, 5.0 mmol) and DIPEA (2.0 g, 15.2 mmol) were dissolved in 10 mL of DCM. The solution was cooled to 0 °C, and a 20 mL solution of Azido-PEG4-PFP ester (CAS No.: 1353012-00-6) (3.49 g, 7.64 mmol) in DCM was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 1 h. After the reaction was complete, the solution was directly concentrated and purified by column chromatography (MeOH / DCM (v / v) = 1 / 100) to give compound D-ER-FY004002-P4-A1 (2.18 g, yield: 65.5%). MS ESI [M+H] + 666.4.

[0668] (11-2) Synthesis of D-ER-FY004002-P4-A2

[0669]

[0670] At room temperature, compound D-ER-FY004002-P4-A1 (2.18 g, 3.28 mmol) was dissolved in 20 mL of EA. The solution was cooled to 0 °C, and a 10 mL solution of EA containing hydrochloric acid (1.20 g, 32.8 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P4-A2 (1.75 g, yield: 94.1%). MS ESI [M+H] + 566.4.

[0671] (11-3) Synthesis of D-ER-FY004002-P4-A3

[0672]

[0673] Compounds D-ER-FY004002-P4-A2 (1.75 g, 3.09 mmol), D-ER-FY004001-D2 (2.39 g, 6.18 mmol), potassium carbonate (0.99 g, 7.20 mmol), and acetonitrile (20 mL) were added sequentially at room temperature, and the mixture was then heated to 80 °C and reacted for 10 h. After the reaction was complete, the mixture was diluted with water, extracted with EA, concentrated the organic phase, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P4-A3 (1.58 g, yield: 65.4%). MS ESI [M+H] + 781.4.

[0674] (11-4) Synthesis of D-ER-FY004002-P4-A4

[0675]

[0676] At room temperature, compound D-ER-FY004002-P4-A3 (1.58 g, 2.02 mmol) was dissolved in 20 mL of EA. The solution was cooled to 0 °C, and a 10 mL solution of EA containing hydrochloric acid (0.74 g, 20.2 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to react at room temperature for 2 h. After the reaction was complete, the solution was directly concentrated to give compound D-ER-FY004002-P4-A4 (1.30 g, yield: 94.2%). MS ESI [M+H] + 681.4.

[0677] (11-5) Synthesis of D-ER-FY004002-P4-A5

[0678]

[0679] Compound D-ER-FY004002-P4-A4 (1.30 g, 1.90 mmol), TEA (0.57 g, 5.70 mmol), 4-chloroquinazoline (0.47 g, 2.86 mmol), and 8 mL of isopropanol were added sequentially at room temperature, and the mixture was then heated to 85 °C and reacted for 1 h. After the reaction was complete, the isopropanol was removed by concentration under reduced pressure, diluted with water, extracted with EA, the organic phase was concentrated, and purified by column chromatography (MeOH / DCM(v / v) = 1 / 100-1 / 40) to give compound D-ER-FY004002-P4-A5 (1.0 g, yield: 65.3%). MS ESI [M+H + 809.4.

[0680] Synthesis of (11-6)D-ER-FY004002-P4

[0681]

[0682] At room temperature, compound D-ER-FY004002-P4-A5 (1.0 g, 1.24 mmol) was dissolved in a mixed solvent of THF (5 mL) and water (5 mL), followed by the addition of lithium hydroxide (595 mg, 24.8 mmol), and the mixture was heated to 40 °C for 2 h. After the reaction was complete, compound D-ER-FY004002-P4 (252 mg, yield: 25.6%) was obtained by HPLC. MS ESI [M+H] + 795.4

[0683] 1 H NMR (400MHz, DMSO-d6) δ8.41(s,1H),8.36(s,1H),8.23(d,J=8.3Hz,1H),8.11(s,1H),8.03(t,J=5.6Hz,1H),7.79–7.70(m,1H ),7.69–7.64(m,1H),7.50(t,J=7.6Hz,1H),6.98(s,1H),6.68(s,1H),4.02(d,J=5.5Hz,2H),3.58–3.53(m,4H),3.52–3.40(m ,16H),3.34(t,J=4.9Hz,2H),3.19(t,J=5.5Hz,2H),2.98–2.88(m,2H),2.87–2.75(m,3H),2.71–2.63(m,1H),2.55(t,J=6.2H z,2H),2.48–2.44(m,4H),2.29(t,J=6.4Hz,2H),2.19–2.09(m,1H),2.08–2.00(m,1H),1.75–1.64(m,2H),1.55–1.43(m,4H).

[0684] Table 1. Compounds prepared in Preparation Examples 1-11

[0685]

[0686]

[0687]

[0688] Example 1: Synthesis of siRNA conjugate D-ER-FY004-mN010

[0689] (1-1) Preparation of siRNA

[0690] 1.1 Synthesis of the Justice Chain (SS Chain)

[0691] The oligonucleotide was synthesized using a solid-phase phosphoramide method, employing a blank CPG solid-phase support as the starting cycle. Nucleoside monomers were sequentially linked from the 3'-5' direction according to the nucleotide arrangement of the positive strand. Each linkage of a nucleoside monomer involved four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesis scale was 1 μmol of oligonucleotides. The synthetic conditions are as follows:

[0692] The nucleoside monomers were provided in 1.0 g / 10 mL acetonitrile solution. The reaction conditions were the same for each step, i.e., the temperature was 25 °C. Deprotection was performed three times using a 3% trichloroacetic acid-dichloromethane solution.

[0693] The activator used in the coupling reaction was an acetonitrile solution of 0.25 mol / L 5-ethylthiotetrazole, and the coupling reaction was carried out for 8 min.

[0694] Oxidation was performed using 0.05 mol / L iodine in tetrahydrofuran / pyridine / water (70 / 20 / 10, v / v / v) for 0.5 min; or thiolation was performed using 0.05 mol / L N,N-dimethyl-N'-(3-thio-3H-1,2,4-dithiazo-5-yl)formamidin in acetonitrile / pyridine (2 / 3, v / v) for 8 min.

[0695] Use a 20% acetic anhydride-acetonitrile solution and 15% N-methylimidazole / acetonitrile to cap the product, and cap it twice.

[0696] 1.2 Synthesis of the antisense chain (AS chain)

[0697] The solid-phase phosphoramide synthesis method utilizes a blank CPG solid-phase support as the starting cycle, and nucleoside monomers are sequentially linked from the 3'-5' direction according to the nucleotide arrangement sequence of the antisense strand. Each linkage of a nucleoside monomer involves four steps: deprotection, coupling, oxidation or thiolation, and capping. The synthesis conditions for 1 μmol of oligonucleotides in the antisense strand are the same as those for the sense strand.

[0698] 1.3 Purification and Annealing of Oligonucleotides

[0699] 1.3.1 Ammonolysis

[0700] The synthesized solid-phase support (sense or antisense chain) was added to a 1.5 mL centrifuge tube, and 1.0 mL of 50% methylamine / ammonia water (v / v) was added. The mixture was reacted in a metal bath shaker at 25 °C for 10 hours. The supernatant was collected, and the solid-phase support was washed twice with enzyme-free water, 200 μL each time. The supernatants were combined, centrifuged and concentrated, and the crude product was purified.

[0701] 1.3.2 Purification

[0702] Purification was performed using a reverse chromatography system with a Shim-pack GIS C18 column and gradient elution with a hexylamine acetate-acetonitrile system. The prepared liquid was collected and lyophilized to obtain the single-chain product.

[0703] 1.3.3 Annealing

[0704] The sense chain (SS chain) and the antisense chain (AS chain) were mixed in a molar ratio (SS chain / AS chain = 1 / 1), reacted in a metal bath shaker at 37°C for 4 hours, naturally cooled to room temperature, and the system was freeze-dried to obtain the product.

[0705] Detection: Purity was determined by HPLC; molecular weight was analyzed by LC-MS and compared with theoretical values.

[0706] Thus, the sense and antisense strands of the synthesized siRNA were confirmed.

[0707] (1-2) Synthesis of D-ER-FY004-mSS010

[0708] The positive siRNA strand of the siRNA conjugate D-ER-FY004-mN010 in Table 2 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, the synthesized phosphoramide monomer D-ER-FY004008-P1 was linked to the 5' end of the nucleic acid sequence in a continuous three-linked manner. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS010 as shown in the figure below.

[0709]

[0710] (1-3) Synthesis of D-ER-FY004-mNSS010-A1

[0711]

[0712] Dissolve 1400 nmol of D-ER-FY004-mSS010 prepared in steps (1-2) of Example 1 in 3.0 mL of purified water. Add 300 μL of acetonitrile, 600 μL of 20 mM copper sulfate solution, and 1200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 11.1 mg of D-ER-FY004002-P1 molecules in 0.5 mL of DMF and add this DMF solution to the reaction system. Finally, add 700 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions used are: Phase A: 50 mM TEAA (pH 1-2). 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks and concentrate to obtain the nucleotide product D-ER-FY004-mNSS010-A1 (720 nmol, yield 51.4%) shown in Table 2 SEQ ID NO: 1 (calculated molecular weight: 9975.7; measured molecular weight: 9979.0).

[0713] (1-4) Synthesis of D-ER-FY004-mN010

[0714]

[0715] Following the method described in (1-1) of Example 1, the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-mN010 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was sequentially linked with nucleotide monomers according to the order of SEQ ID NO: 8 nucleotides shown in Table 2. The sense strand D-ER-FY004-mNSS010-A1 obtained in step (1-3) and the antisense strand prepared by solid-phase synthesis in step (1-4) were dissolved in an equimolar amount using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY004-mN010 in Table 2.

[0716] Example 2 Synthesis of siRNA conjugate D-ER-FY004-m011

[0717] (2-1) Synthesis of D-ER-FY004-mSS011

[0718] Following the method described in Example 1 (1-1), the siRNA positive strand of the siRNA conjugate D-ER-FY004-m011 in Table 2 was synthesized by solid-phase synthesis. During solid-phase synthesis, the synthesized phosphoramide monomer D-ER-FY004009-P1 was linked to the 5' end of the positive strand nucleic acid sequence in a continuous three-linked manner. After solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS011 as shown in the following formula.

[0719]

[0720] (2-2) Synthesis of Justice Chain D-ER-FY004-mSS011-A1

[0721]

[0722] Dissolve 230 nmol of D-ER-FY004-mSS011 prepared in step (2-1) in 1.0 mL of purified water. Add 60 μL of acetonitrile, 100 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 11 mg of D-ER-FY004002-P1 molecules in 1 mL of DMF. Add 165.5 μL of this small molecule DMF solution to the reaction system. Finally, add 100 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-mSS011-A1 (85 nmol, yield 37.0%) shown in Table 2 (calculated molecular weight: 10065.7; measured molecular weight: 10069.0).

[0723] (2-3) Synthesis of D-ER-FY004-m011

[0724]

[0725] Following the method described in (1-1) of Example 1, the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-m011 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was sequentially linked with nucleotide monomers according to the order of SEQ ID NO: 8 nucleotides shown in Table 2. The sense strand D-ER-FY004-mSS011-A1 obtained in step (2-2) and the antisense strand prepared by solid-phase synthesis in step (2-3) were dissolved in an equimolar amount using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY004-m011 in Table 2.

[0726] Example 3 Synthesis of siRNA conjugate D-ER-FY004-m005

[0727] (3-1) Synthesis of D-ER-FY004-mSS009

[0728] Following the method described in Example 1 (1-1), the positive siRNA strand of the siRNA conjugate D-ER-FY004-m005 in Table 2 was synthesized by solid-phase synthesis. During solid-phase synthesis, the phosphoramidite monomer D-ER-FY004006-P1 was solid-phase synthesized at the position of the 6th base from the 5' end. After solid-phase synthesis, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS009 as shown in the following formula.

[0729]

[0730] (3-2) Synthesis of Justice Chain D-ER-FY004-mSS009-A1

[0731]

[0732] Dissolve 400 nmol of D-ER-FY004-mSS009 prepared in step (3-1) in 2.0 mL of purified water. Add 150 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 400 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 6.5 mg of D-ER-FY004002-P1 molecules in 1 mL of DMF. Add 162 μL of this DMF solution to the reaction system. Finally, add 400 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-mSS009-A1 (100 nmol, yield 25.0%) shown in Table 2 as SEQ ID NO: 3 (calculated molecular weight: 7888.3, ​​measured molecular weight: 7890.0).

[0733] (3-3) Synthesis of siRNA conjugate D-ER-FY004-m005

[0734]

[0735] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-m005 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 8 shown in Table 2, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-mSS009-A1 obtained in step (3-2) and the antisense strand prepared by solid-phase synthesis in step (3-3) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-m005 in Table 2.

[0736] Example 4: Synthesis of siRNA conjugate D-ER-FY004-mN019

[0737] (4-1) Synthesis of D-ER-FY004-mSS019

[0738] Following the method described in Example 1 (1-1), the positive siRNA strand of the siRNA conjugate D-ER-FY004-mN019 in Table 2 was synthesized by solid-phase synthesis. During solid-phase synthesis, the phosphoramidite monomer D-ER-FY004009-P1 was synthesized before the first nucleotide from the 3' end and after the first nucleotide from the 5' end. After solid-phase synthesis, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS019 as shown in the following formula.

[0739]

[0740] (4-2) Synthesis of D-ER-FY004-mNSS019-A1

[0741]

[0742] Dissolve 350 nmol of D-ER-FY004-mSS019 prepared in step (4-1) in 2.0 mL of purified water. Add 120 μL of acetonitrile, 100 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 11 mg of D-ER-FY004002-P1 molecules in 1 mL of DMF. Add 168 μL of this DMF solution to the reaction system. Finally, add 100 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-mNSS019-A1 (200 nmol, yield 57.1%) shown in Table 2 as SEQ ID NO: 4 (calculated molecular weight: 8893.9, measured molecular weight: 8893.2).

[0743] (4-3) Synthesis of siRNA conjugate D-ER-FY004-mN019

[0744]

[0745] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-mN019 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 8 shown in Table 2, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-mNSS019-A1 obtained in step (4-2) and the antisense strand prepared by solid-phase synthesis in step (4-3) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the siRNA conjugate D-ER-FY004-mN019 in Table 2.

[0746] Example 5 Synthesis of siRNA conjugate D-ER-FY004-mN021

[0747] (5-1) Synthesis of D-ER-FY004-mSS021

[0748] Following the method described in Example 1 (1-1), the positive siRNA strand of D-ER-FY004-mN021 in Table 2 was synthesized by solid-phase synthesis. During solid-phase synthesis, before linking the phosphoramidite monomer (purchased from Shanghai Zhaowei Company) containing the HO(CH2)6-SS-(CH2)6- group to the first nucleotide monomer at the 3' end and after linking the last nucleotide monomer at the 5' end, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS021 as shown in the following formula.

[0749]

[0750] (5-2) Synthesis of D-ER-FY004-mSS021-A1

[0751]

[0752] Dissolve 100 nmol of D-ER-FY004-mSS021 prepared in step (5-1) in 1.0 mL of purified water. Add TCEP aqueous solution to the resulting solution at a mass ratio of TCEP (tris(2-carbonylethyl)phosphine) to D-ER-FY004-mSS021 of 1.5:1. After mixing, react at 25 °C for 2 hours. Dilute the reaction solution with 1.0 mL of enzyme-free water to obtain approximately 2.0 mL of reaction solution. Transfer the reaction solution to several 0.5 mL, 3K ultrafiltration tubes and centrifuge at 10,000 rpm for 10 minutes. Collect the liquid in the inner tubes. The solution was diluted again with enzyme-free water, and the ultrafiltration and centrifugation steps were repeated until most of the impurities except D-ER-FY004-mSS021-A1 were removed. Finally, the product inside the ultrafiltration tube was collected to obtain product D-ER-FY004-mSS021-A1 (95 nmol, yield 95%).

[0753] (5-3) Synthesis of D-ER-FY004-mSS021-A2

[0754]

[0755] The D-ER-FY004-mSS021-A1 (95 nmol) prepared in step (5-2) was dissolved in 50 μL of purified water in a 1.5 mL centrifuge tube. After complete dissolution, 50 μL of DMSO solution of Py-SS-Py (1.2 μmol, 0.26 mg) was added to the centrifuge tube. The mixture was vortexed and reacted at 25 °C for 4–6 hours, with the reaction progress monitored using a Waters mass spectrometer. After the reaction was complete, the reaction solution was diluted with a 20% (v / v) ethanol aqueous solution and purified using an Agilent 1260 HPLC system. The mobile phase conditions used were: Phase A: 50 mM TEAA (pH 7.0–7.4); Phase B: acetonitrile; gradient conditions: 0–30 min, Phase B: 5%–75%. The product peak was collected and concentrated to obtain product D-ER-FY004-mSS021-A2 (70 nmol, yield 74%).

[0756] (5-4) Synthesis of D-ER-FY004-mSS021-A3

[0757]

[0758] The D-ER-FY004-mSS021-A2 (70 nmol) obtained in step (5-3) was dissolved in 200 μL of 0.1 M ammonium acetate solution. After dissolution, 200 μL of LDMSO solution of N-ER-FY017002-P1 (350 nmol, 0.4 mg) was added to the reaction system, and the mixture was vortexed until homogeneous. The reaction was carried out at 25 °C for 4-6 hours, and the reaction progress was monitored using a Waters mass spectrometer. After the reaction was complete, the product was purified by Agilent 1260 HPLC. The mobile phase conditions used were: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions were: 0-30 min, Phase B: 5%-75%. The product peak was collected and concentrated to obtain product D-ER-FY004-mSS021-A3 (42 nmol, yield 60%).

[0759] (5-5) Synthesis of D-ER-FY004-mNSS021-A4

[0760]

[0761] Dissolve 400 nmol of D-ER-FY004-mSS021-A3 prepared in step (5-4) in 2.4 mL of purified water. Premix 200 μL of 20 mM copper sulfate solution, 400 μL of 200 mM sodium bicarbonate solution, and 200 μL of 20 mM TCEP aqueous solution, and add this mixture to the 2.4 mL D-ER-FY004-mSS021-A3 solution. Separately, dissolve 1.69 mg of D-ER-FY004002-P1 in 160 μL of DMF and add this solution to the reaction system. Vortex mix and react at 25 °C. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions used are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-mNSS021-A4 (150 nmol, yield 37.5%) shown in Table 2 as SEQ ID NO: 5 (calculated molecular weight: 8892.1, measured molecular weight: 8892.0).

[0762] (5-6) Synthesis of siRNA conjugate D-ER-FY004-mN021

[0763]

[0764] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-mN021 in Table 2 was synthesized by solid-phase synthesis. The nucleotide monomers were sequentially linked according to the nucleotide sequence of SEQ ID NO: 8 shown in Table 2. The sense strand D-ER-FY004-mNSS021-A4 obtained in step (5-5) and the antisense strand prepared by solid-phase synthesis in step (5-6) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-mN021 in Table 2.

[0765] Example 6 Synthesis of siRNA conjugate D-ER-FY004-m022

[0766] (6-1) Synthesis of D-ER-FY004-mSS011-B1

[0767]

[0768] Dissolve 230 nmol of D-ER-FY004-mSS011 prepared in step (2-1) of Example 2 in 1.0 mL of purified water. Add 60 μL of acetonitrile, 100 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 12 mg of D-ER-FY004004-P1 molecules in 1 mL of DMF. Add 165.5 μL of this small molecule DMF solution to the reaction system. Finally, add 100 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions used are: Phase A: 50 mM TEAA (pH 10.5). 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks and concentrate to obtain the nucleotide product D-ER-FY004-mSS011-B1 (98 nmol, yield 42.6%) shown in Table 2 as SEQ ID NO: 6 (calculated molecular weight: 10257.1; measured molecular weight: 10258.2).

[0769] (6-2) Synthesis of siRNA conjugate D-ER-FY004-m022

[0770]

[0771] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-m022 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 8 shown in Table 2, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-mSS011-B1 obtained in step (6-1) and the antisense strand prepared by solid-phase synthesis in step (6-2) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the siRNA conjugate D-ER-FY004-m022 in Table 2.

[0772] Example 7 Synthesis of siRNA conjugate D-ER-FY004-m023

[0773] (7-1) Synthesis of D-ER-FY004-mSS019-B1

[0774]

[0775] Dissolve 100 nmol of D-ER-FY004-mSS019 prepared in step (4-1) of Example 4 in 1.0 mL of purified water. Add 60 μL of acetonitrile, 50 μL of 20 mM copper sulfate solution, and 100 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 16.4 mg of D-ER-FY004004-P1 molecules in 1 mL of DMF. Add 35.2 μL of this small molecule DMF solution to the reaction system. Finally, add 100 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions used are: Phase A: 50 mM TEAA (pH 10.5). 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min, Phase B: 5%-75%; collect product peaks and concentrate to obtain the nucleotide product D-ER-FY004-mSS019-B1 (45 nmol, yield 45.0%) shown in SEQ ID NO: 7 in Table 2 (calculated molecular weight: 9019.9, measured molecular weight: 9021.0).

[0776] (7-2) Synthesis of siRNA conjugate D-ER-FY004-m023

[0777]

[0778] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-mAS001 of the siRNA in D-ER-FY004-m023 in Table 2 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 8 shown in Table 2, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-mSS019-B1 obtained in step (7-1) and the antisense strand prepared by solid-phase synthesis in step (7-2) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the siRNA conjugate D-ER-FY004-m023 in Table 2.

[0779] Example 8 Synthesis of siRNA conjugate D-ER-FY004-r102

[0780] (8-1) Synthesis of D-ER-FY004-rSS102

[0781] The positive siRNA strand of the siRNA conjugate D-ER-FY004-r102 in Table 3 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, the synthesized phosphoramide monomer D-ER-FY004008-P1 was linked to the 5' end of the nucleic acid sequence in a continuous three-linked manner. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-rSS102 as shown in the figure below.

[0782]

[0783] (8-2) Synthesis of D-ER-FY004-rSS102-A1

[0784]

[0785] Dissolve 380 nmol of D-ER-FY004-rSS102 prepared in step (8-1) of Example 8 in 1.5 mL of purified water. Add 120 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 3 mg of D-ER-FY004002-P1 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS102-A1 (195 nmol, yield 51.3%) shown in Table 3 as SEQ ID NO: 12 (calculated molecular weight: 10104.2; measured molecular weight: 10103.5).

[0786] (8-3) Synthesis of D-ER-FY004-r102

[0787]

[0788] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r102 in Table 3 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 13 shown in Table 3, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-rSS102-A1 obtained in step (8-2) and the antisense strand prepared by solid-phase synthesis in step (8-3) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-r102 in Table 3.

[0789] Example 9 Synthesis of siRNA conjugate D-ER-FY004-r105

[0790] (9-1) Synthesis of D-ER-FY004-rSS105-A1

[0791]

[0792] Dissolve 400 nmol of D-ER-FY004-rSS102 prepared in step (8-1) of Example 8 in 1.5 mL of purified water. Add 120 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 3.2 mg of D-ER-FY004002-P2 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS105-A1 (230 nmol, yield 57.5%) shown in Table 3 as SEQ ID NO: 16 (calculated molecular weight: 10200.2; measured molecular weight: 10200.0).

[0793] (9-2) Synthesis of D-ER-FY004-r105

[0794]

[0795] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r105 in Table 3 was synthesized by solid-phase synthesis. The sequence of its synthesis was followed by linking nucleotide monomers sequentially according to the nucleotide sequence of SEQ ID NO: 13 shown in Table 3. The sense strand D-ER-FY004-rSS105-A1 obtained in step (9-1) and the antisense strand prepared by solid-phase synthesis in step (9-2) were dissolved in an equimolar amount using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY004-r105 in Table 3.

[0796] Example 10 Synthesis of siRNA conjugate D-ER-FY004-r106

[0797] Synthesis of (10-1)D-ER-FY004-rSS106-A1

[0798]

[0799] Dissolve 350 nmol of D-ER-FY004-rSS102 prepared in step (8-1) of Example 8 in 1.5 mL of purified water. Add 120 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Dissolve 3.1 mg of D-ER-FY004002-P3 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS106-A1 (190 nmol, yield 54.2%) shown in Table 3 as SEQ ID NO: 17 (calculated molecular weight: 10464.5; measured molecular weight: 10465.0).

[0800] (10-2) Synthesis of D-ER-FY004-r106

[0801]

[0802] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r106 in Table 3 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 13 shown in Table 3, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-rSS106-A1 obtained in step (10-1) and the antisense strand prepared by solid-phase synthesis in step (10-2) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-r106 in Table 3.

[0803] Example 11 Synthesis of siRNA conjugate D-ER-FY004-r111

[0804] (11-1) Synthesis of D-ER-FY004-rSS104

[0805] The positive siRNA strand of the siRNA conjugate D-ER-FY004-r111 in Table 3 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, the synthesized phosphoramide monomer D-ER-FY004008-P2 was linked to the 5' end of the nucleic acid sequence in a continuous triple-linked manner. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-rSS104 as shown in the figure below.

[0806]

[0807] (11-2) Synthesis of D-ER-FY004-rSS111-A1

[0808]

[0809] Dissolve 300 nmol of D-ER-FY004-rSS104 prepared in step (11-1) of Example 11 in 1.5 mL of purified water. Add 100 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 3.1 mg of D-ER-FY004002-P2 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS111-A1 (155 nmol, yield 51.7%) shown in Table 3 as SEQ ID NO: 18 (calculated molecular weight: 10254.2; measured molecular weight: 10254.0).

[0810] (11-3) Synthesis of D-ER-FY004-r111

[0811]

[0812] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r111 in Table 3 was synthesized by solid-phase synthesis. The nucleotide monomers were sequentially linked according to the nucleotide sequence of SEQ ID NO: 13 shown in Table 3. The sense strand D-ER-FY004-rSS111-A1 obtained in step (11-2) and the antisense strand prepared by solid-phase synthesis in step (11-3) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-r111 in Table 3.

[0813] Example 12 Synthesis of siRNA conjugate D-ER-FY004-r119

[0814] Synthesis of (12-1)D-ER-FY004-rSS119-A1

[0815]

[0816] Dissolve 300 nmol of D-ER-FY004-rSS102 prepared in step (8-1) of Example 8 in 1.5 mL of purified water. Add 100 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Dissolve 2.8 mg of D-ER-FY004002-P4 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS119-A1 (160 nmol, yield 53.3%) shown in Table 3 as SEQ ID NO: 19 (calculated molecular weight: 10506.7; measured molecular weight: 10508.0).

[0817] (12-2) Synthesis of D-ER-FY004-r119

[0818]

[0819] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r119 in Table 3 was synthesized by solid-phase synthesis. The sequence of its synthesis was sequentially linked with nucleotide monomers according to the nucleotide sequence of SEQ ID NO: 13 shown in Table 3. The sense strand D-ER-FY004-rSS119-A1 obtained in step (12-1) and the antisense strand prepared by solid-phase synthesis in step (12-2) were dissolved in an equimolar amount using enzyme-free water, and then the two were annealed to obtain the conjugate D-ER-FY004-r119 in Table 3.

[0820] Example 13 Synthesis of siRNA conjugate D-ER-FY004-r107

[0821] (13-1) Synthesis of D-ER-FY004-rSS107-A1

[0822]

[0823] Dissolve 280 nmol of D-ER-FY004-rSS102 prepared in step (8-1) of Example 8 in 1.5 mL of purified water. Add 80 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Dissolve 2.6 mg of D-ER-FY004004-P1 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-rSS107-A1 (145 nmol, yield 51.8%) shown in Table 3 as SEQ ID NO: 20 (calculated molecular weight: 10293.4; measured molecular weight: 10293.0).

[0824] (13-2) Synthesis of D-ER-FY004-r107

[0825]

[0826] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY005-rAS002 of the siRNA in D-ER-FY004-r107 in Table 3 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 13 shown in Table 3, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-rSS107-A1 obtained in step (13-1) and the antisense strand prepared by solid-phase synthesis in step (13-2) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-r107 in Table 3.

[0827] Example 14 Synthesis of siRNA conjugate D-ER-FY004-m219

[0828] (14-1) Synthesis of D-ER-FY004-mSS202

[0829] The positive siRNA strand of the siRNA conjugate D-ER-FY004-m219 in Table 4-1 was synthesized using the solid-phase synthesis method described in Example 1 (1-1). During the solid-phase synthesis, the synthesized phosphoramide monomer D-ER-FY004008-P1 was linked to the 5' end of the nucleic acid sequence in a continuous three-linked manner. After the solid-phase synthesis was completed, the oligonucleotide single strand was cleaved from the solid-phase support to obtain the oligonucleotide single strand D-ER-FY004-mSS202 as shown in the figure below.

[0830]

[0831] (14-2) Synthesis of D-ER-FY004-mSS219-A1

[0832]

[0833] Dissolve 300 nmol of D-ER-FY004-mSS202 prepared in step (14-1) of Example 14 in 1.5 mL of purified water. Add 100 μL of acetonitrile, 200 μL of 20 mM copper sulfate solution, and 200 μL of 200 mM sodium bicarbonate solution to the system. Separately, dissolve 3 mg of D-ER-FY004002-P4 molecules in 0.4 mL of DMF and add it to the reaction system. Finally, add 200 μL of 20 mM TCEP (tris(2-carbonylethyl)phosphorus) aqueous solution, vortex to mix, and react at 25 °C for 4-6 h. Monitor the reaction progress using a Waters mass spectrometer. After the reaction is complete, purify the solution using an Agilent 1260 HPLC system. The mobile phase conditions are: Phase A: 50 mM TEAA (pH 7.0-7.4); Phase B: acetonitrile; gradient conditions: 0-30 min. Phase B: 5%-75%; collect the product peak and concentrate to obtain the nucleotide product D-ER-FY004-mSS219-A1 (145 nmol, yield 48.3%) shown in Table 4-1 as SEQ ID NO: 21 (calculated molecular weight: 10279.6; measured molecular weight: 10281.0).

[0834] (14-3) Synthesis of D-ER-FY004-m219

[0835]

[0836] Following the method described in Example 1 (1-1), the antisense strand D-ER-FY004-mAS201 of the siRNA in D-ER-FY004-m219 in Table 4-1 was synthesized by solid-phase synthesis. The sequence of its synthesis was based on the nucleotide sequence of SEQ ID NO: 22 shown in Table 4-1, where nucleotide monomers were sequentially linked. The sense strand D-ER-FY004-mSS219-A1 obtained in step (14-2) and the antisense strand prepared by solid-phase synthesis in step (14-3) were dissolved in an equimolar amount using enzyme-free water. The two strands were then annealed to obtain the conjugate D-ER-FY004-m219 in Table 4-1.

[0837] Example 15 Synthesis of siRNA conjugate D-ER-FY004-h102

[0838] The synthesis process of the conjugate D-ER-FY004-h102 is the same as that of D-ER-FY004-r102 in Example 8, except that the sequence of D-ER-FY004-h102 is shown in Table 4-2, the sense strand is SEQ ID NO: 25, and the antisense strand sequence is SEQ ID NO: 26.

[0839] Example 16 Synthesis of siRNA conjugate D-ER-FY004-h105

[0840] The synthesis process of the conjugate D-ER-FY004-h105 is the same as that of D-ER-FY004-r105 in Example 9, except that the sequence of D-ER-FY004-h105 is shown in Table 4-2, the sense strand is SEQ ID NO: 27, and the antisense strand sequence is SEQ ID NO: 26.

[0841] Example 17 Synthesis of siRNA conjugate D-ER-FY004-h106

[0842] The synthesis process of the conjugate D-ER-FY004-h106 is the same as that of D-ER-FY004-r106 in Example 10, except that the D-ER-FY004-h106 sequence is shown in Table 4-2, the sense strand is SEQ ID NO: 28, and the antisense strand sequence is SEQ ID NO: 26.

[0843] Example 18 Synthesis of siRNA conjugate D-ER-FY004-h119

[0844] The synthesis process of the conjugate D-ER-FY004-h119 is the same as that of D-ER-FY004-r119 in Example 12, except that the D-ER-FY004-h119 sequence is shown in Table 4-2, the sense strand is SEQ ID NO: 29, and the antisense strand sequence is SEQ ID NO: 26.

[0845] Table 2. Sequences of siRNA conjugates (targeting the MUC5AC gene, the basic sequences targeted are SS: 5'-CCAUACAGCAGUACAGUUACA-3', SEQ ID NO: 10; AS: 5'-UGUAACUGUACUGCUGUAUGG-3', SEQ ID NO: 11)

[0846]

[0847] Table 3. Sequences of siRNA conjugates (targeting the RAGE gene (rat source), the basic sequences targeted are SS: 5'-CGGAAUGGAAACUGAACACAA-3', SEQ ID NO: 14; AS: 5'-UUGUGUUCAGUUUCCAUUCCG-3', SEQ ID NO: 15)

[0848]

[0849]

[0850] Table 4-1. Sequences of siRNA conjugates (targeting the SCNN1A gene, the basic sequences targeted are SS: 5'-ACUGUGCAACCAGAACAAAUA-3', SEQ ID NO: 23; AS: 5'-UAUUUGUUCUGGUUGCACAGUUG-3', SEQ ID NO: 24)

[0851] Table 4-2. Sequences of siRNA conjugates (targeting the RAGE gene (human), the basic sequences targeted are SS: 5'-GAGUAGGUGCUCAAAACAUCA-3', SEQ ID NO: 30; AS: 5'-UGAUGUUUUGAGCACCUACUC-3', SEQ ID NO: 31)

[0852]

[0853] In Tables 2, 3, 4-1, and 4-2, lowercase letters a, u, c, and g represent 2'-methoxy modified adenosine, uridine, cytidine, and guanosine; Af, Uf, Cf, and Gf represent 2'-F modified adenosine, uridine, cytidine, and guanosine; and lowercase letter s indicates that the two nucleotides to the left and right of the letter s are linked by a phosphothioester bond.

[0854] The structure of LA is shown as number 1 in Table 5;

[0855] The structure of L2 is shown as number 2 in Table 5;

[0856] The structure of cL3 is shown as number 3 in Table 5;

[0857] The structure of LB is shown as number 4 in Table 5;

[0858] The structure of LC is shown in Table 5, number 5.

[0859] The structure of L6 is shown as number 6 in Table 5;

[0860] The structure of L7 is shown as number 7 in Table 5;

[0861] The structure of s(invAb) is shown as number 8 in Table 5;

[0862] The structure of cPrpus is shown as number 9 in Table 5;

[0863] The structure of L8 is shown as number 10 in Table 5;

[0864] The structure of L9 is shown as number 11 in Table 5;

[0865] The structure of L10 is shown as number 12 in Table 5;

[0866] The structure of L11 is shown as number 13 in Table 5;

[0867] The structure of L12 is shown as number 14 in Table 5;

[0868] The letter combination invAb represents an inverted debased residue; the letter combination s(invAb) represents an inverted debased residue linked by a thiophosphate diester bond, the structure of which is shown as number 8 in Table 5.

[0869] The letter combination cPrpu represents a nucleotide U containing cyclopropylphosphonate; the letter combination cPrpus represents a nucleotide U containing cyclopropylphosphonate linked by a phosphothioester bond, wherein the structure is shown as number 9 in Table 5.

[0870] The letter combination EVPu represents a trans-vinylphosphonate-containing nucleotide U; the letter combination EVPus represents a trans-vinylphosphonate-containing nucleotide U linked by a phosphothioester bond, wherein the structure is shown as number 15 in Table 5.

[0871] Table 5. Structure of integrin αvβ6 ligand small molecules and linker arms

[0872]

[0873]

[0874]

[0875]

[0876]

[0877]

[0878]

[0879] Experimental Example 1: Target gene repressive activity of the disclosed oligonucleotide conjugates in mice.

[0880] This experiment investigated the mRNA inhibitory activity of D-ER-FY004-mN010 and D-ER-FY004-m011 against MUC5AC gene expression in mice (especially in the lungs).

[0881] (1-1) Purchase and raising of animals

[0882] C57BL / 6j mice (male, 9–10 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental phase, animals were housed in reinforced and ventilated cages. Cages were changed at least weekly. Animals were housed in groups of 5–6 at a normal 12-hour light cycle (lights off at 08:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0883] (1-2) Construction of animal models

[0884] Mice were weighed and divided into groups of 5. Mice were sensitized (25 μg house dust mite protein HDM) on days 0 and 7, and stimulated (25 μg house dust mite protein HDM) on day 14. All house dust mites were administered intratracheally (it). In the sham-operated group, the HDM was replaced with saline on days 0, 7, and 14.

[0885] (1-3) Group setup and administration of drugs in animal models

[0886] Table 6 Animal drug administration groups

[0887]

[0888] (1-4) Collection and testing of animal samples

[0889] After the experiment ended on day 17, the left and right lungs of the animals were collected. The left lung was soaked in 10% formalin solution for pathological analysis. The right lung was ground up under liquid nitrogen and used for qPCR analysis of the expression of the target gene Muc5AC mRNA.

[0890] Calculate the inhibition rate using the following formula:

[0891] Inhibition rate (%) = (Muc5AC mRNA expression level in group G2 - Muc5AC mRNA expression level in the treatment group) / Muc5AC mRNA expression level in group G2

[0892] The results are shown in Table 7.

[0893] Table 7. Inhibition rate of Muc5AC gene expression in the right lung of mice by the conjugate.

[0894] Group Dosage Inhibition rate G1 solvent - G2 solvent 0％ G3 D-ER-FY004-mN010 77.5％ G4 D-ER-FY004-m011 58.3％

[0895] As shown in Table 7, the conjugate D-ER-FY004-mN010 of the present invention has a high inhibition rate against the target gene.

[0896] Experimental Example 2: Target gene repressive activity of the disclosed oligonucleotide conjugates in rats.

[0897] This experimental example investigated the mRNA inhibitory activity of D-ER-FY004-r102 and other components disclosed herein on RAGE gene expression in rats (especially in the lungs).

[0898] (2-1) Purchasing and raising animals

[0899] Male SD rats (6-8 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental period, the animals were housed in reinforced and ventilated cages. The cages were changed at least weekly. Animals were housed in groups of three, under a normal 12-hour light cycle (lights off at 8:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0900] (2-2) Group setup and administration of drugs to animals

[0901] Rats were weighed and divided into groups of 8 rats each. On day 0, the rats were administered the drugs intratracheally according to the experimental groups in Table 7. Group G1 was given an equal volume of solvent (1×PBS) as a negative control.

[0902] Table 8 Animal drug administration groups

[0903]

[0904] (2-3) Collection and testing of animal samples

[0905] After the experiment ended on day 8, the whole lungs of the animals were collected, ground up under liquid nitrogen, and used for qPCR experiments to analyze the expression of the target gene RAGE mRNA.

[0906] The results are shown in Table 9.

[0907] Table 9. Inhibition rate of RAGE gene expression in the lungs of rats by the conjugate.

[0908]

[0909]

[0910] As shown in Table 9, the conjugate D-ER-FY004-r102 of the present invention has a high inhibition rate against the target gene.

[0911] Experimental Example 3: Target protein inhibitory activity of the disclosed oligonucleotide conjugates in rats.

[0912] This experiment investigated the inhibitory activity of D-ER-FY004-r102 and other components disclosed herein on sRAGE protein in plasma expressing the RAGE gene in rats.

[0913] (3-1) Purchasing and raising animals

[0914] Male SD rats (6-8 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental period, the animals were housed in reinforced and ventilated cages. The cages were changed at least weekly. Animals were housed in groups of three, under a normal 12-hour light cycle (lights off at 8:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0915] (3-2) Group setup and administration of drugs to animals

[0916] Rats were weighed and divided into groups of 6 rats each. On day 0, the rats were administered the drugs intratracheally according to the experimental groups in Table 10. Group G1 was given an equal volume of solvent (1×PBS) as a negative control.

[0917] Table 10 Animal drug administration groups

[0918]

[0919] The compound in question is Tri-SM6.1-ανβ6-AD07475, as described in Arrowhead patent CN 117440817A. Its structure is as follows: its sense strand sequence is 5'-csggaauggAfAfAfcugaacacaas(invAb)-3' (SEQ ID NO: 32); its antisense strand sequence is 5-'cPrpusUfsgsUfgUfuCfaGfuUfuCfcAfuUfcCfsg-3' (SEQ ID NO: 13). The delivery portion, as shown in the figure below, is attached to the 5' end of the sense strand.

[0920]

[0921] (3-3) Collection and testing of animal samples

[0922] Blood samples were collected from animals on days 42, 91, and 105. Serum samples were analyzed by ELISA to detect the knockdown of sRAGE protein by the drug in the animals. The inhibition rate was obtained by comparing the plasma sRAGE protein level before administration on day 0.

[0923] The results are shown in Table 11.

[0924] Table 11 Inhibition rate of conjugates on sRAGE protein expression in rats

[0925]

[0926] As shown in Table 11, the conjugate D-ER-FY004-r102 of the present invention has the expected knockdown effect on the target gene. Moreover, unexpectedly, the conjugate D-ER-FY004-r102 of this application produced a long-lasting inhibitory effect on the target gene, and even after 105 days, it still had an inhibition rate comparable to that on day 42.

[0927] Experimental Example 4: Target protein inhibitory activity of the disclosed oligonucleotide conjugates in rats.

[0928] This experiment investigated the inhibitory activity of D-ER-FY004-r102, D-ER-FY004-r105, D-ER-FY004-r106, D-ER-FY004-r107, D-ER-FY004-r111 and Yangshen in rats against sRAGE protein expressed by the RAGE gene in plasma.

[0929] (4-1) Purchasing and raising animals

[0930] Male SD rats (6-8 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental period, the animals were housed in reinforced and ventilated cages. The cages were changed at least weekly. Animals were housed in groups of three, under a normal 12-hour light cycle (lights off at 8:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0931] (4-2) Group setup and administration of drugs to animals

[0932] Rats were divided into groups of 6 rats each according to their plasma sRAGE protein expression levels on day -1. On day 0, rats were administered the drugs intratracheally according to the experimental groups listed in Table 12 (it). Group G1 received an equal volume of solvent (1×PBS) as a negative control.

[0933] Table 12 Animal drug administration groups

[0934]

[0935] (4-3) Collection and testing of animal samples

[0936] Blood was collected from animals on days 42 and 98. Serum was analyzed by ELISA to detect the knockdown of sRAGE protein by the drug in animals. The inhibition rate was obtained by comparing the sRAGE protein expression level in the solvent group with that in the solvent group.

[0937] The results are shown in Table 13.

[0938] Table 13 Inhibition rate of conjugates on sRAGE protein expression in rats

[0939]

[0940] The genic ginseng described is derived from Arrowhead patent CN 117440817A, corresponding to the number Tri-SM6.1-ανβ6-AD07475. Its structure is as follows: its sense strand sequence is 5'-csggaauggAfAfAfcugaacacaas(invAb)-3' (SEQ ID NO: 32); its antisense strand sequence is 5-'cPrpusUfsgsUfgUfuCfaGfuUfuCfcAfuUfcCfsg-3' (SEQ ID NO: 13). Furthermore, the delivery portion, as shown in the figure below, is connected to the 5' end of the sense strand.

[0941]

[0942] As shown in Table 13, the conjugates of the present invention have the expected knockdown effect on the target genes. Among them, the inhibitory effect of conjugates D-ER-FY004-r107 and D-ER-FY004-r111 on sRAGE was significantly weakened before day 98, so the experiment was terminated early.

[0943] Experimental Example 5: Target protein inhibitory activity of the disclosed oligonucleotide conjugates in rats.

[0944] This experiment investigated the inhibitory activity of D-ER-FY004-r119 and *Gynostemma pentaphyllum* in rats against sRAGE protein expressed by the RAGE gene in plasma.

[0945] (5-1) Purchasing and raising animals

[0946] Male SD rats (6-8 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental period, the animals were housed in reinforced and ventilated cages. The cages were changed at least weekly. Animals were housed in groups of three, under a normal 12-hour light cycle (lights off at 8:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0947] (5-2) Group setup and administration of drugs to animals

[0948] Rats were divided into groups of 6 rats each according to their plasma sRAGE protein expression levels on day -1. On day 0, rats were administered the drugs intratracheally according to the experimental groups listed in Table 14 (it). Group G1 received an equal volume of solvent (1×PBS) as a negative control.

[0949] Table 14 Animal drug administration groups

[0950]

[0951] (5-3) Collection and testing of animal samples

[0952] Blood was collected from animals on days 28 and 98. Serum was analyzed by ELISA to detect the knockdown of sRAGE protein by the drug in animals. The inhibition rate was obtained by comparing the sRAGE protein expression level in the solvent group with that in the solvent group.

[0953] The results are shown in Table 15.

[0954] Table 15 Inhibition rate of conjugates on sRAGE protein expression in rats

[0955]

[0956] The genic ginseng described is derived from Arrowhead patent CN 117440817A, corresponding to the number Tri-SM6.1-ανβ6-AD07475. Its structure is as follows: its sense strand sequence is 5'-csggaauggAfAfAfcugaacacaas(invAb)-3' (SEQ ID NO: 32); its antisense strand sequence is 5-'cPrpusUfsgsUfgUfuCfaGfuUfuCfcAfuUfcCfsg-3' (SEQ ID NO: 13). Furthermore, the delivery portion, as shown in the figure below, is connected to the 5' end of the sense strand.

[0957]

[0958] As shown in Table 15, the conjugate of the present invention has the expected knockdown effect on the target gene, and the sRAGE inhibition rate at 98 days is significantly higher than that of the Yangshen group, demonstrating better long-term efficacy.

[0959] Experimental Example 6: Target gene repressive activity of the disclosed oligonucleotide conjugates in mice.

[0960] This experimental example investigated the mRNA inhibitory activity of D-ER-FY004-m219 disclosed herein on SCNN1A gene expression in mice (especially in the lungs).

[0961] (6-1) Purchasing and raising animals

[0962] C57BL / 6j mice (male, 9–10 weeks old) were purchased from a certified animal supplier and acclimatized to their environment for one week prior to the experiment. Throughout the experimental phase, animals were housed in reinforced and ventilated cages. Cages were changed at least weekly. Animals were housed in groups of 5–6 at a normal 12-hour light cycle (lights off at 08:00 PM), at 22±2°C and 50±10% relative humidity, with adequate food and water provided. All in vivo experimental procedures were approved by the Institute of Laboratory Animal Use and Management (IACUC), Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences.

[0963] (6-2) Group setup and administration of drugs in animal models

[0964] Mice were weighed and grouped into groups of 6. Rats were administered the drugs intratracheally (it) according to the experimental groups in Table 16 on day 0. Group G1 was given an equal volume of solvent (1×PBS) as a negative control.

[0965] Table 16 Animal drug administration groups

[0966]

[0967] (6-3) Collection and testing of animal samples

[0968] After the experiment ended on day 8, the left and right lungs of the animals were collected. The right lung was ground up under liquid nitrogen and used for qPCR analysis of the expression of the target gene SCNN1A mRNA.

[0969] The conjugate D-ER-FY004-m219 of the present invention has the desired inhibitory effect on the target gene.

[0970] Experimental Example 7: Repeated-dose toxicity test of subcutaneous injection of conjugate in SD rats

[0971] In this experiment, each conjugate was divided into two groups. Each experimental group consisted of five male and five female SD rats (from Guoke Saifu Hebei Pharmaceutical Technology Co., Ltd.). The rats were subcutaneously injected with the solvent control (0.9% sodium chloride injection), and the conjugates D-ER-FY004-h102, D-ER-FY004-h105, D-ER-FY004-h106, and D-ER-FY004-h119, respectively, every two weeks for a total of three administrations. On day 30 (D30), ten male and ten female animals from each group underwent planned end-of-treatment dissection.

[0972] The toxicity indicators in this study included: death / near death, general observation, body weight, food intake, ophthalmological examination, clinicopathological indicators (hematological and coagulation indicators, serum biochemical indicators, immune function indicators, urine indicators, bone marrow smear), gross necropsy, organ weight, histopathological examination, and tissue distribution.

[0973] During the experiment, all animals survived to the planned dissection. No obvious abnormalities were found in general observation, body weight, food intake, ophthalmological examination, cytokines, clinicopathological indicators (hematology, coagulation, serum biochemistry, immune function indicators, urine indicators, bone marrow smear), or gross necropsy of any of the animals that were related to the test group.

[0974] The results of the repeated-dose toxicity tests above show that the conjugate of the present invention as a lung delivery system has good safety.

Claims

1. A ligand of integrin ανβ6 protein receptor comprising a targeting group and a linker group, and having the structure of Formula I: ###0001### Formula I wherein L is a linker group that links a molecule to be transported, q is 0, 1, 2, 3, 4, 5, 6, 7, or 8; p is 3, 4, 5, 6, 7, 8, or 9, and the ligand of integrin ανβ6 protein receptor has a structure selected from the group consisting of: ###0002### ###0003### ###0004### ###0005### ###0006### ###0007### ###0008### ###0009### ###0010### ###0011### ###0012### ###0013### ###0014### ###0015### ###0016### ###0017### ###0018### ###0019### ###0020### ###0021### ###0022### ###0023### ###0024### ###0025### ###0026### ###0027### ###0028### ###0029### ###0030### ###0031### ###0032### ###0033### ###0034### ###0035### ###0036### ###0037### ###0038### ###0039### ###0040### ###0041### ###0042### ###0043### ###0044### ###0045### ###0046### ###0047### ###0048### ###0049### ###0050### ###0051### ###0052### ###0053### ###0054### ###0055### ###0056### ###0057### ###0058### ###0059### ###0060### ###0061### ###0062### ###0063### ###0064### ###0065### ###0066### ###0067### ###0068### ###0069### ###0070### ###0071### ###0072### ###0073### ###0074### ###0075### ###0076### ###0077### ###0078### ###0079### ###0080### ###0081### ###0082### ###0083### ###0084### ###0085### ###0086### ###0087### ###0088### ###0089### ###0090### ###0091### ###0092### ###0093### ###0094### ###0095### ###0096### ###0097### ###0098### ###0099### ###0100### ###0101### ###0102### ###0103### ###0104### ###0105### ###0106### ###0107### ###0108### ###0109### ###0110### ###0111### ###0112### ###0113### ###0114### ###0115### ###0116### ###0117### ###0118### ###0119### ###0120### ###0121### ###0122### ###0123### ###0124### ###0125### ###0126### ###0127### ###0128### ###0129### ###0130### ###0131### ###0132### ###0133### ###0134### ###0135### ###0136### ###0137### ###0138### ###0139### ###0140### ###0141### ###0142### ###0143### ###0144### ###0145### ###0146### ###0147### ###0148### ###0149### ###0150### ###0151### ###0152### ###0153### ###0154### ###0155### ###0156### ###0157### ###0158### ###0159### ###0160### ###0161### ###0162### ###0163### ###0164### ###0165### ###0166### ###0167### ###0168### ###0169### ###0170### ###0171### ###0172### ###0173### ###0174### ###0175### ###0176### ###0177### ###0178### ###0179### ###0180### ###0181### ###0182### ###0183### ###0184### ###0185### ###0186### ###0187### ###0188### ###0189### ###0190### ###0191### ###0192### ###0193### ###0194### ###0195### ###0196### ###0197### ###0198### ###0199### ###0200### ###0201### ###0202### ###0203### ###0204### ###0205### ###0206### ###0207### ###0208### ###0209### ###0210### ###0211### ###0212### ###0213### ###0214### ###021 wherein R 1 selected from C6-C 14 aryl or 5- to 10-membered heteroaryl, said C6-C 14 aryl and 5- to 10-membered heteroaryl are optionally substituted with R 1a substituents; R 0 selected from hydrogen or -R 0a -L; -R 0a -L is selected from -R 3 NR 17 -L, -R 3 NR 17 R 18 -L, -R 3 C(O)NR 17 R 18 -L, -R 3 OC(O)R 17 NR 18 -L, -R 3 NR 17 C(O)R 18 -L, -R 3 OC(O)R 17 -L, -R 3 C(O)OR 17 -L, -R 3 NR 17 C(O)OR 18 -L or -R 3 OC(O)(NR 17 )R 18 -L; R 2 is hydrogen; deuterium; Ci-C6alkyl optionally substituted with R 2a ; -OH; C3-C6cycloalkyl optionally substituted with R 2b ; or -S(0)2R 2c ; or R 2 is a single bond, preferably said single bond is used for the connection to L; ​ wherein L is selected from L1and L2are each independently selected from the group consisting of: -(R 20 ) m -(R 20 ) m R 21 R 22 -(R 20 ) m R 21 -(R 20 O) m -(R 20 O) m R 21 -(R 20 O) m R 21 R 22 -R 20 C(O)R 21 -R 20 SSR 21 -R 20 C(O)R 21 R 22 -C(O)R 21 -C(O)R 21 R 22 -R 20 OC(O)R 21 -R 20 OC(O)R 21 R 22 -R 20 C(O)OR 21 -or 20 C(O)OR 21 R 22 wherein m is an integer from 0 to 30; each R 1a independently C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, deuterium, halogen, -CN, -OR 3 , -NR 4 R 5 , -C(O)R 3 , -OC(O)R 3 , -C(O)OR 3 , -C(O)NR 4 R 5 , -S(O)2R 1a , -NR 6 R 6 R 7 , -C(O)R 6 , -CN, or halogen; and n is 1, 2, 3, or 4. each R 2a , R 2b , R 2e , and R 2f is independently oxo or R 1a ; R 2c C1-C6alkyl optionally substituted with R 2e C3-C5cycloalkyl optionally substituted with R 2f C3-C5cycloalkyl optionally substituted with R R 3 , R 17 , R 18 , and R 19 are each independently a bond, hydrogen, deuterium, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C6-C10aryl, 5-10 membered heteroaryl, or 3-12 membered heterocyclyl, wherein the C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C6-C10aryl, 5-10 membered heteroaryl, and 3-12 membered heterocyclyl of R 14 are each independently optionally substituted with halogen, deuterium, oxo, -CN, -OR 3 , -NR 14 R 8 , -P(O)(OR 8 )(OR 9 ), or C1-C6alkyl optionally substituted with deuterium, halogen, -OH, or oxo; 8 9 ;​ R 20 R 21 and R 22 Each is independently hydrogen, deuterium, C1-C 12 Alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl group, C1-C 12 Heteroalkyl, C2-C 12 Heterene, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl, 3- to 12-membered heterocyclic, halogen, -CN, -OR 3 -SR 3 -NR 4 R 5 -C = NH (OR 3 -C(O)R 3 -OC(O)R 3 -C(O)OR 3 -C(O)NR 4 R 5 -NR 3 C(O)R 4 -NR 3 C(O)OR 4 -NR 3 C(O)NR 4 R 5 -S(O)R 3 -S(O)2R 3 -NR 3 S(O)R 4 -NR 3 S(O)2R 4 -S(O)NR 4 R 5 -S(O)2NR 4 R 5 or -OP(=O)(OR) 4 (OR) 5 ), where R 20 R 21 and R 22 Independently and optionally by halogen, deuterium, oxo group, -CN, -OR, where possible. 8 -NR 8 R 9 -OP(=S)(OR) 8 (OR) 9 -OP(=O)(OR) 8 (OR) 9 -OP(=S)(SR) 8 (OR) 9 C1-C6 alkyl (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, 2-methylbutyl, 3-methylbutyl, R 4 and R 5 are each independently hydrogen, deuterium, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C6-C10aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocycloalkyl, wherein R 14 and R 4 are each independently hydrogen, deuterium, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C3-C6cycloalkyl, C6-C 5 aryl, 5- to 6-membered heteroaryl, and 3- to 6-membered heterocycloalkyl are independently optionally substituted with deuterium, halogen, oxo, -CN, -OR 14 , -NR 8 , -C(=O)R 8 , -C(=O)NR 9 , or C1-C6alkyl optionally substituted with deuterium, halogen, -OH, or oxo; R 6 and R 7 each independently is hydrogen, deuterium, C1-C6alkyl optionally substituted with deuterium, halogen, or oxo, C2-C6alkenyl optionally substituted with deuterium, halogen, or oxo, or C2-C6alkynyl optionally substituted with deuterium, halogen, or oxo; R 8 and R 9 each independently is hydrogen, deuterium, C1-C6alkyl optionally substituted with deuterium, halogen, or oxo, C2-C6alkenyl optionally substituted with deuterium, halogen, or oxo, or C2-C6alkynyl optionally substituted with deuterium, halogen, or oxo; each R 10 , R 11 , R 12 , and R 13 is independently hydrogen or deuterium; R 14 is deuterium; ​ Each R 15 Independently selected from hydrogen, deuterium, or halogen; Each R 16 Independently selected from hydrogen, deuterium, or halogen; and ​ wherein R 2 and at least one of R 0 is connected to one or more transported molecules by L.

2. A ligand of the integrin αvβ6 protein receptor according to claim 1, wherein, ​ wherein R 0 , R 0a , R 1 , R 2 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , L, p and q are as defined in claim 1.

3. A ligand of the integrin αvβ6 protein receptor according to claim 1 or 2, wherein, R 1 is selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thienopyrimidinyl, pyridinyl, pyrrolopyrimidinyl, quinoxalinyl, indazolyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl; and is optionally substituted with deuterium, hydroxy, C1-C6alkyl, C1-C6haloalkyl, C1-C6perhaloalkyl, C1-C6alkoxy, C3-C8cycloalkyl, C3-C8halocycloalkyl, C3-C8cycloalkoxy, cyano, amino, alkylamino, or dialkylamino; ​ R 0 and one of R 2 has the linker L; preferably, when R 0 is hydrogen, R 2 is a single bond and connects L; or, when R 0 is selected from -R 0a -L, R 2 is selected from hydrogen, deuterium, C1-C6alkyl optionally substituted with R 2a , -OH, C3-C6cycloalkyl optionally substituted with R 2b , or -S(O)2R 2c ; Or, R 0a Selected from -C 1-6 Alkyl-NH-, optionally substituted with halogen, deuterium, oxo group, or C1-C3 alkyl.

4. The ligand of the integrin αvβ6 protein receptor of claim 1, wherein, ​ wherein R 0 , R 0a , R 2 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , L, p and q are as defined in claim 1, and wherein the structural unit may optionally be substituted by R 1a substituted.

5. A ligand of the integrin αvβ6 protein receptor according to claim 4, wherein, each R is independently C1-C6alkyl, deuterium, halogen, -CN, -OH, -NH2, or -NO2, wherein each R 1a is independently C1-C6alkyl, deuterium, halogen, -CN, -OH, -NH2, or -NO2, wherein each R 1a is independently C1-C6alkyl, deuterium, halogen, -CN, -OH, -NH2, or -NO2, wherein each R or R 2 is selected from the group consisting of Ci-C6-alkyl optionally substituted by R 2a -OH; C3-C6-cycloalkyl optionally substituted by R 2b -OH; and -S(0)2R 2c , preferably R 2 may be selected from the group consisting of Ci-C6-alkyl optionally substituted by R 2a -OH; and C3-C6-cycloalkyl optionally substituted by R 2b , more preferably R 2 is selected from the group consisting of Ci-C6-alkyl optionally substituted by R 2a -OH; and C3-C6-cycloalkyl optionally substituted by R 2b , most preferably R 2 is selected from the group consisting of Ci-C6-alkyl optionally substituted by R 2a -OH. Or, R 2a Selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, deuterium, oxoyl, halogen, -CN, -OH, -NH2, -C(O)-C1-C6 alkyl, -OC(O)-C1-C6 alkyl, -C(O)O-C1-C6 alkyl, -C(O)NH-C1-C6 alkyl, -C(O)N(C1-C6 alkyl)2, wherein each R 2a Where possible, it may be independently and optionally substituted with deuterium, halogen, oxo group, -OH, -NH2, -C(O)-C1-C6 alkyl, -CN or halogen-substituted C1-C6 alkyl; or R is selected from methyl, ethyl, propyl, cyclopropyl, or cyclobutyl; each of which is optionally substituted with one or more of hydroxyl, methoxy, ethoxy, acetamido, fluoro, fluoroalkyl, or dimethylamido; preferably R 2 is methyl, ethyl, or propyl substituted with hydroxyl, methoxy, or ethoxy; more preferably R 2 is methyl, ethyl, or propyl substituted with hydroxyl, methoxy, or ethoxy; more preferably R 2 is methyl, ethyl, or propyl substituted with hydroxyl, methoxy, or ethoxy; more preferably R Or, R 3 Independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14 aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 6-membered heteroaryl, and 3- to 6-membered heterocyclic groups are independently and optionally converted by halogens, deuterium, oxo groups, -CN, or -OR. 8 -NR 8 R 9 -P(O)(OR) 8 (OR) 9 ) or optionally substituted with a C1-C6 alkyl group by deuterium, halogen, -OH or oxo group; preferably, R 3 Independently hydrogen, deuterium, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 ynyl, C3-C6 cycloalkyl, C6-C 14 aryl, 5- to 6-membered heteroaryl, or 3- to 6-membered heterocyclic, wherein R 3 C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C6 cycloalkyl, C6-C 14 The aryl, 5- to 6-membered heteroaryl and 3- to 6-membered heterocyclic groups are independently and optionally substituted with halogen, deuterium, oxo, -CN, -OH, -NH2, -P(O)(OH)(OH) or optionally with C1-C6 alkyl groups substituted with deuterium, halogen, -OH or oxo. ​ or R 10 , R 11 , R 12 and R 13 are independently hydrogen; ​ or R 15 and R 16 is hydrogen and p is 3 or 4, preferably 3.

6. A ligand of the integrin αvβ6 protein receptor according to any one of claims 1 to 5, wherein, The linking group L is selected from: wherein each of L1and L2is independently selected from: -C1-C 12 alkyl-, -C1-C 12 heteroalkyl-, -C(O)-C1-C 12 alkyl-, -C(O)-C1-C 12 heteroalkyl-, -C1-C 12 alkyl-S-S-C1-C 12 alkyl-, -C1-C 12 alkyl-S-S-C1-C 12 heteroalkyl-, -C1-C 12 heteroalkyl-S-S-C1-C 12 heteroalkyl-, -(CH2CH2O) m -C1-C 12 alkyl-, -C1-C 12 alkyl-5- to 6-membered heterocyclyl-, or -C1-C 12 heteroalkyl-5- to 6-membered heterocyclyl-, optionally substituted with halogen, deuterium, oxo, -NH2, -NO2, -CN, -OH, or C1-C3alkyl optionally substituted with halogen or -C1-C3alkoxy optionally substituted with halogen, wherein m is selected from an integer between 1-10, R 19 Each is independently hydrogen, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl or 3- to 12-membered heterocyclic group; -C1-C 12 alkyl-, -C(O)-C1-C 12 alkyl-, -C(O)-CH2-(CH2CH2O) m -, -C(O)-(CH2CH2O) m -CH2CH2-, -(CH2CH2O) m -C1-C 12 alkyl-, -C1-C 12 alkyl-5- to 6-membered heterocyclyl- or -C1-C 12 heteroalkyl-5- to 6-membered heterocyclyl-, wherein m is an integer selected from 1-5.

7. A ligand of the integrin αvβ6 protein receptor according to any one of claims 1 to 6, wherein, ​ wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, linking the targeting group, linking the transported molecule; or wherein, linking the targeting group, linking the transported molecule; ​ wherein, connecting the targeting group, connecting the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, connecting the targeting group, connecting the transported molecule, X is selected from S or O; wherein, linking the targeting group, linking the transported molecule, X is selected from S or O; wherein, linking the targeting group, linking the transported molecule; wherein, connecting the targeting group, connecting the transported molecule, X is selected from S or O; wherein, connecting the targeting group, connecting the transported molecule; wherein, linking the targeting group, linking the transported molecule; wherein, connecting targeting groups, connecting transported molecules; wherein, linking the targeting group, linking the transported molecule; wherein, connecting the targeting group, connecting the transported molecule.

8. The ligand of Integrin αvβ6 protein receptor as claimed in claim 1 wherein, ​ wherein R 1 selected from C6-C 14 aryl or 5- to 10-membered heteroaryl, said C6-C 14 aryl and 5- to 10-membered heteroaryl are optionally substituted with R 1a substituted with R 1 may be selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thienopyrimidinyl, pyridinyl, pyrrolopyrimidinyl, quinoxalinyl, indazolyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl, optionally substituted with R 1a substituted with R R 0 selected from hydrogen or -R 0a -L; -R 0a -L is selected from -C 1-6 alkyl-NH-L, wherein the moiety -C 1-6 alkyl-NH- is optionally substituted with halo, deuterium, oxo, or C1-C3 alkyl; R 2 is hydrogen; deuterium; Ci-C6alkyl optionally substituted with R 2a ; -OH; C3-C6cycloalkyl optionally substituted with R 2b ; or -S(0)2R 2c ; or R 2 is a single bond, preferably said single bond is used for the connection to L; ​ wherein L is selected from L1and L2are each independently selected from the group consisting of: -C1-C 12 alkyl-, -C1-C 12 heteroalkyl-, -C(O)-C1-C 12 alkyl-, -C(O)-C1-C 12 heteroalkyl-, -C1-C 12 alkyl-S-S-C1-C 12 alkyl-, -C1-C 12 alkyl-S-S-C1-C 12 heteroalkyl-, -C1-C 12 heteroalkyl-S-S-C1-C 12 heteroalkyl-, -(CH2CH2O) m -C1-C 12 alkyl-, or -C1-C 12 alkyl-5- to 6-membered heterocyclyl- optionally substituted with halogen, deuterium, oxo, -NH2, -NO2, -CN, -OH, or C1-C3 alkyl optionally substituted with halogen or -OH, wherein m is an integer selected from 1-10; Each R 1a Independently, it is deuterium, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 perhaloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 halocycloalkyl, C3-C8 cycloalkoxy, cyano, amino, alkylamino or dialkylamino; each R 2a , R 2b , R 2e , and R 2f is independently oxo or R 1a ; R 2c C1-C6alkyl optionally substituted with R 2e C1-C6alkyl optionally substituted with R 2f C3-C5cycloalkyl optionally substituted with R R 19 Each is independently hydrogen, deuterium, C1-C6 alkyl, C3-C6 cycloalkyl, C6-C 14 Aryl, 5- to 10-membered heteroaryl or 3- to 12-membered heterocyclic group; each R 10 , R 11 , R 12 and R 13 are independently hydrogen or deuterium, preferably each independently hydrogen; R 14 is deuterium; ​ Each R 15 It is independently selected from hydrogen, deuterium or halogen, preferably independently hydrogen; each R is independently selected from hydrogen, deuterium, or halogen, preferably independently hydrogen; and 16 is independently selected from hydrogen, deuterium, or halogen, preferably independently hydrogen; and ​ wherein R 2 and at least one of R 0 is connected to one or more transported molecules via L; ​ ​ wherein R 1 selected from C6-C 14 aryl or 5- to 10-membered heteroaryl, said C6-C 14 aryl and 5- to 10-membered heteroaryl are optionally substituted with R 1a R 1 may be selected from pyrimidinyl, quinazolinyl, pyrazolopyrimidinyl, pyrazinyl, quinolinyl, pyridopyrimidinyl, thienopyrimidinyl, pyridinyl, pyrrolopyrimidinyl, quinoxalinyl, indazolyl, benzothiazolyl, naphthyl, purinyl, or isoquinolinyl, optionally substituted with R 1a R R 0 selected from -R 0a -L; -R 0a -L is selected from -C 1-6 alkyl-NH-L, wherein the moiety -C 1-6 alkyl-NH- is optionally substituted with halo, deuterium, oxo, or C1-C3 alkyl; R 2 is hydrogen; deuterium; Ci-C6alkyl optionally substituted with R 2a ; -OH; C3-C6cycloalkyl optionally substituted with R 2b ; or -S(0)2R 2c ; ​ wherein L is selected from L1and L2are each independently selected from the group consisting of: -C1-C 12 alkyl-, -C1-C 12 heteroalkyl-, -C(O)-C1-C 12 alkyl-, -C(O)-C1-C 12 heteroalkyl-, -C1-C 12 alkyl-S-S-C1-C 12 alkyl-, -C1-C 12 alkyl-S-S-C1-C 12 heteroalkyl-, -C1-C 12 heteroalkyl-S-S-C1-C 12 heteroalkyl-, -(CH2CH2O) m -C1-C 12 alkyl-, or -C1-C 12 alkyl-5- to 6-membered heterocyclyl- optionally substituted with halogen, deuterium, oxo, -NH2, -NO2, -CN, -OH, or C1-C3 alkyl optionally substituted with halogen or -OH, wherein m is an integer selected from 1-10; Each R 1a Independently, it is deuterium, hydroxyl, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 perhaloalkyl, C1-C6 alkoxy, C3-C8 cycloalkyl, C3-C8 halocycloalkyl, C3-C8 cycloalkoxy, cyano, amino, alkylamino or dialkylamino; each R 2a , R 2b , R 2e , and R 2f are independently oxo or R 1a ; R 2c C1-C6alkyl optionally substituted with R 2e C3-C5cycloalkyl optionally substituted with R 2f C3-C5cycloalkyl optionally substituted with R each R 10 , R 11 , R 12 , and R 13 is independently hydrogen or deuterium, preferably each independently hydrogen; R 14 is deuterium; ​ Each R 15 It is hydrogen; Each R 16 It is hydrogen; and ​ wherein R 0 one or more transported molecules are linked by L. ​ 10. The double stranded oligonucleotide conjugate of claim 9, wherein, ​

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