Ligand-drug conjugates and uses thereof

Abstract

The present disclosure relates to a class of ligand-drug conjugates having a novel structure, or pharmaceutically acceptable salts thereof, and in particular, to a ligand-drug conjugate having a novel structure, the invention provides a ligand-drug conjugate with a structural general formula of Pc-(L-D) n or a pharmaceutically acceptable salt thereof, a preparation method thereof, a pharmaceutical composition containing the conjugate, and an application of the conjugate in treatment of autoimmune diseases or inflammatory diseases.

Description

Ligand-drug conjugates and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to Chinese patent application No. 202210339497.4 filed on April 1, 2022, Chinese patent application No. 202210395481.5 filed on April 15, 2022, and Chinese patent application No. 202211392212.X filed on November 8, 2022, all of which are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present disclosure belongs to the field of biomedicine and relates to a class of ligand-drug conjugates with novel structures, a preparation method thereof, a pharmaceutical composition containing the conjugate, and the use thereof in treating autoimmune diseases. Background Art

[0004] Glucocorticoids (GC) are an extremely important class of regulatory molecules in the body. They play an important regulatory role in the body's development, growth, metabolism, and immune function. They are the most important regulatory hormone for the body's stress response and are also the most widely used and effective anti-inflammatory and immunosuppressant in clinical practice. Glucocorticoids can be used to treat many autoimmune and inflammatory diseases, including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. However, long-term use of glucocorticoids can easily cause side effects such as osteoporosis, muscle atrophy, hypertension, and suppression of the body's immunity, leading to aggravated infections. Therefore, the use of hormones in the treatment of autoimmune diseases is limited.

[0005] In addition to hormonal agents, a variety of biologics are used to treat autoimmune and inflammatory diseases, such as those targeting tumor necrosis factor α (TNFα) and IL-4R. TNFα is a cytokine involved in systemic inflammation and plays a critical role in many human diseases. Anti-TNFα biologics (such as adalimumab, etanercept, golimumab, and infliximab) and anti-IL-4R biologics (such as dupilumab) have been clinically proven effective in treating autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and atopic dermatitis and are widely used. However, long-term use of these drugs can lead to the development of anti-drug antibodies (ADAs) in some patients, resulting in drug resistance and reduced efficacy. Furthermore, long-term use of these drugs may also increase the risk of infection and malignancy.

[0006] Therefore, there is still a huge unmet clinical need in the treatment of autoimmune and inflammatory diseases, and more effective and safer treatments need to be developed.

[0007] Summary of the Invention

[0008] The present disclosure combines the treatment of biological agents and hormone drugs to provide a novel glucocorticoid receptor agonist, which is combined with a ligand to form a ligand-hormone drug conjugate for the treatment of autoimmune diseases.

[0009] The present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ;

[0010] in,

[0011] Pc is the ligand unit;

[0012] L is a linker unit;

[0013] D is a drug unit represented by the following formula (DI):

[0014] in,

[0015] R 1 、R 2 are each independently selected from H, CH3 or halogen;

[0016] Ring A is selected from phenyl, 5-10 membered heteroaryl or C3-C 10 Cycloalkyl, the phenyl, 5-10 membered heteroaryl or C3-C 10 The cycloalkyl group is optionally substituted with one or more R 1a replace;

[0017] X is selected from O, S, C1-C3 alkylene-O, C1-C3 alkylene-S, NR 6 or C(R 7 )(R 8 );

[0018] R 6 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally substituted by one or more R b replace;

[0019] R 7 、R 8 Each is independently selected from H, halogen, CN, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, or R 7 、R 8Together with the atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by one or more R b replace;

[0020] R 3 Selected from H, OH or NHR 9 , R 4 、R 5 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a Replace; or, R 5 Selected from H, OH or NHR 9 , R 3 、R 4 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace;

[0021] R 9 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally substituted by one or more R d replace;

[0022] R 13 、R 14 Each is independently selected from H, halogen, CN, OH, NH2, O(C1-C3 alkyl) or C1-C6 alkyl;

[0023] Every R 4a independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R d replace;

[0024] Every R d Independently selected from halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2;

[0025] R 10 Selected from OH, SH, O (C1-C6 alkyl), S (C1-C6 alkyl), OC (=O) - (C1-C6 alkyl) or The O(C1-C6 alkyl), S(C1-C6 alkyl), OC(=O)-(C1-C6 alkyl) are optionally substituted with one or more halogens or CN;

[0026] R 11 、R 12 Each independently selected from H or C1-C6 alkyl;

[0027] Every R 1a independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R b replace;

[0028] Every R b independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R c replace;

[0029] Every R c is independently selected from halogen, CN, ═O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; and,

[0030] n is 1 to 16;

[0031] Wherein, the linker unit L and the compound R of formula (DI) 3 、R 4 、R 5 、R 13 、R 14 Any one of the -OH, -NH2 or -NH- groups present in the is covalently linked.

[0032] In some embodiments, R 1 、R 2 are each independently selected from H or F.

[0033] In some embodiments, ring A is selected from phenyl or 5-6 membered heteroaryl, wherein the phenyl or 5-6 membered heteroaryl is optionally substituted by one or more R 1a replace.

[0034] In some embodiments, Ring A is selected from phenyl, which is optionally substituted with one or more R 1a replace.

[0035] In some embodiments, R 1aSelected from halogen, CN, =O, OH, NH2 or C1-C6 alkyl.

[0036] In some embodiments, R 1a Selected from NH2.

[0037] In some embodiments, Ring A is selected from

[0038] In some embodiments, Ring A is selected from

[0039] In some embodiments, X is selected from O, S, C1-C3 alkylene-O, C1-C3 alkylene-S, or C(R 7 )(R 8 ).

[0040] In some embodiments, R 7 、R 8 are each independently selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl, or R 7 、R 8 Together with the atoms to which they are attached, they form a C3-C6 cycloalkyl group, wherein the OH, NH2, C1-C3 alkyl group or C3-C6 cycloalkyl group is optionally replaced by one or more R b replace.

[0041] In some embodiments, R 7 、R 8 are each independently selected from H, halogen or C1-C3 alkyl, or R 7 、R 8 Together with the atoms to which they are attached, they form a C3-C6 cycloalkyl group.

[0042] In some embodiments, R 7 、R 8 are each independently selected from H, F or methyl, or R 7 、R 8 Together with the atoms to which they are attached, they form a cyclopropyl group.

[0043] In some embodiments, X is selected from O, S, CHO, CHS, CH, CF, CHCH, or

[0044] In some embodiments, X is selected from CH2.

[0045] In some embodiments, R 10 Selected from OH, SH, O(C1-C3 alkyl), OC(=O)-(C1-C3 alkyl) or The O(C1-C3 alkyl) or OC(=O)-(C1-C3 alkyl) is optionally substituted with one or more halogens.

[0046] In some embodiments, R 11 、R 12 Each is independently selected from H, methyl or ethyl.

[0047] In some embodiments, R 10 Selected from OH, SH, OCH2F, O(C=O)CH3,

[0048] In some embodiments, R 10 Selected from OH or

[0049] In some embodiments, R 9 Selected from H or C1-C6 alkyl.

[0050] In some embodiments, R 9 Selected from H.

[0051] In some embodiments, R 3 is selected from H, OH or NH2, R 4 、R 5 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace.

[0052] In some embodiments, R 5 is selected from H, OH or NH2, R 3 、R 4 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace.

[0053] In some embodiments, R 3 Selected from H, R 4 、R 5 Together with the atoms to which they are attached, they form a 5-6 membered heterocyclic group or a 5-6 membered heteroaryl group, wherein the 5-6 membered heterocyclic group or the 5-6 membered heteroaryl group is optionally substituted by one or more R 4a replace.

[0054] In some embodiments, R 5 Selected from H or NH2, R 3 、R 4Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace.

[0055] In some embodiments, R 3 is selected from H, OH or NH2, R 4 、R 5 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

[0056] In some embodiments, R 3 Selected from H, R 4 、R 5 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

[0057] In some embodiments, R 5 is selected from H, OH or NH2, R 3 、R 4 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

[0058] In some embodiments, R 5 Selected from H or NH2, R 3 、R 4 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

[0059] In some embodiments, R 4a Selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl or C3-C6 cycloalkyl.

[0060] In some embodiments, R 4a is selected from halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

[0061] In some embodiments, R 4aSelected from =O or NH2.

[0062] In some embodiments, R 3 Selected from H, R 4 、R 5 Together with the atoms they are connected to form

[0063] In some embodiments, R 5 Select H or NH2, R 3 、R 4 Together with the atoms they are connected to form

[0064] In some embodiments, R 13 、R 14 are each independently selected from H or NH2.

[0065] In some embodiments, R 13 Selected from H.

[0066] In some embodiments, R 14 Selected from H.

[0067] In some embodiments, the compound represented by formula (DI) or a pharmaceutically acceptable salt thereof is selected from the compound represented by formula (D-II) or a pharmaceutically acceptable salt thereof:

[0068] in,

[0069] R 1 、R 2 are each independently selected from H or F;

[0070] R 10 Selected from OH or

[0071] R 3 、R 4 、R 5 、R 13 、R 14 As defined above.

[0072] In some embodiments, the structural unit Selected from

[0073] In some embodiments, the structural unit Selected from

[0074] In some embodiments, the compound represented by formula (DI) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0075] In some embodiments, the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD) n :

[0076] in,

[0077] Pc, L, and n are as defined above;

[0078] D is selected from the following compounds:

[0079] In some embodiments, the linker unit L is selected from Its a-end is covalently linked to the ligand unit Pc, and its b-end is covalently linked to the drug unit D, wherein L 1 It is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl.

[0080] In some embodiments, the L 1 It is a peptide residue consisting of 2, 3 or 4 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl.

[0081] In some embodiments, the L 1 It is a dipeptide residue, and one of the amino acids constituting the dipeptide residue is glycine.

[0082] In some embodiments, the L 1 A dipeptide residue selected from Gly-Lys or Gly-Glu.

[0083] In some embodiments, the linker unit L is selected from Its a end is covalently linked to the ligand unit Pc, and its b end is covalently linked to the drug unit D. 1 A dipeptide residue selected from Gly-Lys or Gly-Glu.

[0084] In some embodiments, the linker unit L is selected from Its a-terminal is covalently linked to the ligand unit Pc, and its b-terminal is covalently linked to the drug unit D.

[0085] In some embodiments, the linker unit L is selected from Its a-terminal is covalently linked to the ligand unit Pc, and its b-terminal is covalently linked to the drug unit D.

[0086] In some embodiments, the disclosed formula is Pc-(LD) n The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0087] where Pc and n are as defined above.

[0088] In some embodiments, the aforementioned general formula is Pc-(LD) n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand unit Pc can be selected from a polypeptide, an antibody or an antigen-binding fragment;

[0089] In some embodiments, the Pc is an antibody or antigen-binding fragment that specifically binds to TNFα or IL-4R; the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL);

[0090] In some embodiments, (1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO. 4-9, 12-16, 18, 20, 22, 57, 68, 70, 72, 74 or 76; or / and the light chain variable region comprises SEQ ID NO. NO.1-3, 17, 19, 21, 23, 58, 69, 71, 73, 75 or 77; (2) the heavy chain variable region or / and the light chain variable region comprise a sequence having at least 80% identity, or a sequence having at most 3 insertion, deletion or substitution mutations, compared to each CDR in the HCDR1-3 or / and LCDR1-3 in group (1); further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. Optionally, the HCDR1-3 and the LCDR1-3 are determined according to the Kabat numbering system, the Chothia numbering system or the IMGT numbering system.

[0091] In some embodiments, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 and / or the LCDR1-3 are selected from the following:

[0092] (1) the HCDR1-3 are SEQ ID NOs. 24-26; or / and the LCDR1-3 are SEQ ID NOs. 27-29;

[0093] (2) the HCDR1-3 are SEQ ID NOs. 30-32; or / and the LCDR1-3 are SEQ ID NOs. 33-35;

[0094] (3) the HCDR1-3 are SEQ ID NOs. 36-38; or / and the LCDR1-3 are SEQ ID NOs. 39-41;

[0095] (4) the HCDR1-3 are SEQ ID NOs. 42-44; or / and the LCDR1-3 are SEQ ID NOs. 45-47;

[0096] (5) the HCDR1-3 are SEQ ID NOs. 48-50; or / and the LCDR1-3 are SEQ ID NOs. 51-53;

[0097] (6) the HCDR1-3 are SEQ ID NOs. 60-62; or / and the LCDR1-3 are SEQ ID NOs. 63-65;

[0098] (7) the HCDR1-3 are SEQ ID NOs. 78-80; or / and the LCDR1-3 are SEQ ID NOs. 81-83;

[0099] (8) the HCDR1-3 are SEQ ID NOs. 84-86; or / and the LCDR1-3 are SEQ ID NOs. 87-89;

[0100] (9) the HCDR1-3 are SEQ ID NOs. 90-92; or / and the LCDR1-3 are SEQ ID NOs. 93-95;

[0101] (10) the HCDR1-3 are SEQ ID NOs. 96-98; or / and the LCDR1-3 are SEQ ID NOs. 99-101;

[0102] (11) the HCDR1-3 are SEQ ID NOs. 102-104; or / and the LCDR1-3 are SEQ ID NOs. 105-107; or,

[0103] The HCDR1-3 and / or the LCDR1-3 have a sequence that is at least 80% identical to each CDR in any one of the HCDR1-3 and LCDR1-3 in groups (1) to (11), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the HCDR1-3 and / or the LCDR1-3 have a sequence that is at least 80% identical to each CDR in any one of the HCDR1-3 and LCDR1-3 in groups (1) to (11); further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0104] In some embodiments, the antibody or antigen-binding fragment comprises: the heavy chain variable region comprises the sequence shown in SEQ ID NO.4-9, 12-16, 18, 20, 22, 57, 68, 70, 72, 74 or 76, or / and the light chain variable region comprises the sequence shown in SEQ ID NO.1-3, 17, 19, 21, 23, 58, 69, 71, 73, 75 or 77; or the heavy chain variable region and / or the light chain variable region comprise a sequence having at least 80% identity compared to the heavy chain variable region and light chain variable region in any of the above groups; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0105] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence, and the heavy chain constant region and / or light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO: 10 or 59, and the light chain is composed of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO: 11.

[0106] In some embodiments, the antibody is selected from Nerelimomab, Certolizumab, Infliximab, Golimumab, Adalimumab, Dupixent, 002Mab, 003Mab, 004Mab, 005Mab or 006Mab.

[0107] In some embodiments, the antibody is selected from Nerelimomab, Certolizumab, Infliximab, Golimumab, Adalimumab, or Dupixent.

[0108] In some embodiments, the antibody or antigen-binding fragment is monospecific, bispecific, trispecific, or tetraspecific.

[0109] In some embodiments, the aforementioned general formula is Pc-(LD) n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is selected from 1 to 16, for example, n is selected from 1 to 10, for example, n is selected from 1 to 8, for example, n is selected from 2 to 8, for example, n is selected from 2 to 6, for example, n is selected from 6 to 8.

[0110] In some embodiments, n is selected from 2 to 6, for example, n is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.

[0111] In some embodiments, n is selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0112] The present disclosure also provides a drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structural formula XLD, wherein:

[0113] The drug unit D is as defined above;

[0114] The linker unit L is selected from Its a end is covalently linked to X, its b end is covalently linked to the drug unit D, and L 1 Any of the definitions above;

[0115] X is selected from halogen, OS(O)2CH3 or OS(O)2CF3.

[0116] In some embodiments, L is selected from Its a end is covalently linked to X, and its b end is covalently linked to the drug unit D. 1 A dipeptide residue selected from Gly-Lys or Gly-Glu.

[0117] In some embodiments, L is selected from Its a-terminal is covalently linked to X, and its b-terminal is covalently linked to the drug unit D.

[0118] In some embodiments, L is selected from Its a-terminal is covalently linked to X, and its b-terminal is covalently linked to the drug unit D.

[0119] In some embodiments, X is selected from Br or I.

[0120] In some embodiments, X is selected from Br.

[0121] In some embodiments, the present disclosure provides a drug-linker compound of the aforementioned formula XLD or a pharmaceutically acceptable salt thereof, wherein:

[0122] The drug unit D is selected from the following compounds:

[0123] L is selected from Its a-terminal is covalently linked to X, and its b-terminal is covalently linked to the drug unit D;

[0124] X is selected from Br or I.

[0125] In some embodiments, the drug-linker compound of the general formula XLD of the present disclosure or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0126] The present disclosure also provides a compound represented by the following formula (DH) or a pharmaceutically acceptable salt thereof:

[0127] in,

[0128] R 1 、R 2 、R 3 、R 4 、R 5 、R 10 、R 13 、R 14As defined above.

[0129] In some embodiments, the compound represented by formula (DH) or a pharmaceutically acceptable salt thereof is selected from the following compounds or pharmaceutically acceptable salts thereof:

[0130] On the other hand, the present disclosure provides a pharmaceutical composition comprising the aforementioned general formula Pc-(LD) n ligand-drug conjugate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0131] In another aspect, the present disclosure provides a method for treating an inflammatory disease or an autoimmune disease in a mammal, comprising administering to a mammal, preferably a human, in need of such treatment a therapeutically effective amount of the aforementioned compound of the formula Pc-(LD) n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0132] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in preparing a drug for treating inflammatory diseases or autoimmune diseases.

[0133] On the other hand, the present disclosure provides the aforementioned general formula Pc-(LD) n Use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating inflammatory diseases or autoimmune diseases.

[0134] On the other hand, the present disclosure provides a compound of the aforementioned formula Pc-(LD) for treating inflammatory diseases or autoimmune diseases. n A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0135] On the other hand, the present disclosure provides a pharmaceutical composition comprising the compound represented by the aforementioned formula (DH) of the present disclosure or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0136] On the other hand, the present disclosure provides a method for treating inflammatory diseases or autoimmune diseases in mammals, comprising administering a therapeutically effective amount of a compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

[0137] In another aspect, the present disclosure provides use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating inflammatory diseases or autoimmune diseases.

[0138] In another aspect, the present disclosure provides use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating inflammatory diseases or autoimmune diseases.

[0139] In another aspect, the present disclosure provides a compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating inflammatory diseases or autoimmune diseases.

[0140] In some embodiments, the inflammatory disease or autoimmune disease can be selected from rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, atopic dermatitis, urticaria, myocarditis, encephalitis, uveitis, chronic obstructive pulmonary disease, vasculitis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis, transmural colitis, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma.

[0141] In another aspect, the present disclosure provides a compound of the formula Pc-(LD) n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof comprises the step of coupling the drug-linker compound of the general formula XLD disclosed above with the aforementioned ligand, optionally, the ligand is an antibody or antigen-binding fragment.

[0142] In another aspect, the present disclosure provides a compound of the formula Pc-(LD) n A method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, comprising the step of connecting the aforementioned drug unit D of the present disclosure to the aforementioned ligand unit Pc; optionally, the connection is through the aforementioned linker unit L; optionally, the ligand is an antibody or antigen-binding fragment.

[0143] On the other hand, the present disclosure provides a compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof for preparing the aforementioned general formula Pc-(LD) n The invention also relates to the use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, and / or the use of a compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof in the preparation of a drug-linker compound of the aforementioned general formula XLD or a pharmaceutically acceptable salt thereof.

[0144] The ligand-drug conjugates provided by the present disclosure have significant anti-inflammatory activity, excellent plasma stability and reduced toxic side effects.

[0145] On the other hand, the present disclosure provides an antibody or antigen-binding fragment that specifically binds to human TNFα, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs. 5-9 and 12-15, and the light chain variable region comprises the sequence shown in SEQ ID NO. 2 or 3.

[0146] In some embodiments, the antibody or antigen-binding fragment that specifically binds to human TNFα comprises the following heavy chain variable region (VH) and light chain variable region (VL):

[0147] (1) the VH comprises SEQ ID NO. 5, and the VL comprises SEQ ID NO. 2;

[0148] (2) the VH comprises SEQ ID NO. 6, and the VL comprises SEQ ID NO. 2;

[0149] (3) the VH comprises SEQ ID NO. 7, and the VL comprises SEQ ID NO. 2;

[0150] (4) the VH comprises SEQ ID NO. 8, and the VL comprises SEQ ID NO. 2;

[0151] (5) the VH comprises SEQ ID NO. 9, and the VL comprises SEQ ID NO. 2;

[0152] (6) the VH comprises SEQ ID NO. 5, and the VL comprises SEQ ID NO. 3;

[0153] (7) the VH comprises SEQ ID NO. 6, and the VL comprises SEQ ID NO. 3;

[0154] (8) the VH comprises SEQ ID NO. 7, and the VL comprises SEQ ID NO. 3;

[0155] (9) the VH comprises SEQ ID NO. 8, and the VL comprises SEQ ID NO. 3;

[0156] (10) the VH comprises SEQ ID NO. 9, and the VL comprises SEQ ID NO. 3;

[0157] (11) the VH comprises SEQ ID NO. 12, and the VL comprises SEQ ID NO. 2;

[0158] (12) the VH comprises SEQ ID NO. 13, and the VL comprises SEQ ID NO. 2;

[0159] (13) the VH comprises SEQ ID NO. 14, and the VL comprises SEQ ID NO. 2; or,

[0160] (14) The VH comprises SEQ ID NO.15, and the VL comprises SEQ ID NO.2.

[0161] In some embodiments, the antibody or antigen-binding fragment that specifically binds to human TNFα includes a heavy chain constant region sequence and / or a light chain constant region sequence, wherein the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, wherein the constant region fragment includes CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment includes a complete heavy chain and a light chain, wherein the heavy chain is composed of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO: 10, and the light chain is composed of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO: 11.

[0162] In some embodiments, the antibody or antigen-binding fragment that specifically binds human TNFα is monospecific, bispecific, trispecific, or tetraspecific.

[0163] In another aspect, the present disclosure provides a pharmaceutical composition comprising the aforementioned antibody or antigen-binding fragment that specifically binds to human TNFα and a pharmaceutically acceptable excipient.

[0164] In another aspect, the present disclosure provides a method for treating inflammatory diseases or autoimmune diseases in mammals, comprising administering a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment that specifically binds to human TNFα, or a pharmaceutical composition thereof, to a mammal, preferably a human, in need of such treatment.

[0165] In another aspect, the present disclosure provides use of the aforementioned antibody or antigen-binding fragment that specifically binds to human TNFα, or a pharmaceutical composition thereof, in the preparation of a medicament for treating inflammatory diseases or autoimmune diseases.

[0166] In another aspect, the present disclosure provides use of the aforementioned antibody or antigen-binding fragment that specifically binds to human TNFα, or a pharmaceutical composition thereof, in treating inflammatory diseases or autoimmune diseases.

[0167] In another aspect, the present disclosure provides the aforementioned antibody or antigen-binding fragment that specifically binds to human TNFα, or a pharmaceutical composition thereof, for treating inflammatory diseases or autoimmune diseases.

[0168] In some embodiments, the inflammatory disease or autoimmune disease can be selected from rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjögren's syndrome, polymyositis, dermatomyositis, atopic dermatitis, urticaria, myocarditis, encephalitis, uveitis, chronic obstructive pulmonary disease, vasculitis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis, transmural colitis, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma.

[0169] Definitions and Explanations of Terms

[0170] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0171] The term "ligand" refers to a macromolecular compound that can recognize and bind to an antigen or receptor associated with a target cell. The function of the ligand is to present the drug to the target cell population bound to the ligand. These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells. In the embodiments of the present disclosure, the ligand or ligand unit is represented by Pc, and the ligand can form a connection bond with the linker unit through a heteroatom on the ligand. In some embodiments of the present disclosure, the ligand is selected from an antibody or an antigen-binding fragment, and the antibody is selected from a chimeric antibody, a humanized antibody, a fully human antibody or a murine antibody; in some embodiments of the present disclosure, the antibody is a monoclonal antibody.

[0172] The term "linker" or "linker unit" refers to a chemical structure fragment or chemical bond that is connected to the ligand at one end and to the drug at the other end.

[0173] The term "drug" refers to a small molecule compound that has biological activity in an organism. In some embodiments of the present disclosure, the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule with anti-inflammatory function.

[0174] The term "ligand-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker unit. In some embodiments of the present disclosure, the "ligand-drug conjugate" is an antibody-drug conjugate (ADC), which is a monoclonal antibody or antibody fragment linked to a biologically active drug via a stable linker unit.

[0175] The term "DAR" or "drug-antibody ratio" refers to the average number of small molecule glucocorticoid receptor agonist drugs attached per antibody molecule. n In the antibody-drug conjugates, the DAR is defined by the variable "n", which can be either an integer or a decimal.

[0176] The term "antibody" is used in the broadest sense to refer to a polypeptide or combination of polypeptides that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and / or sufficient sequence from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen. "Antibodies" herein encompass various forms and structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003); Roque et al., Biotechnol. Prog. 20: 639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art that can be used to graft CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0177] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0178] The term "antibody" herein also includes antibodies that do not contain light chains, for example, heavy-chain antibodies (HCAbs) produced by dromedary camels (Camelus dromedarius), Bactrian camels (Camelus bactrianus), llamas (Lama glama), guanacos (Lama guanicoe) and alpacas (Vicugna pacos), as well as immunoglobulin new antigen receptors (Ig new antigen receptor, IgNAR) found in cartilaginous fish such as sharks.

[0179] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, cassowaries, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0180] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, whole antibodies, fragments of whole antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0181] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically existing in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.

[0182] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and the like.

[0183] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0184] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0185] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0186]

[0014] "Full-length antibody," "intact antibody," and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0187] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, and scFv.

[0188] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0189] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond can also exist between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0190] The term "diabody" refers to a antibody whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0191] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen (Cabilly et al. USP 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0192] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0193] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0194] The term "variable region" refers to the region of an antibody heavy or light chain that is involved in binding the antibody to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). These regions are also called complementarity determining regions because they form precise spatial complementarity with antigenic epitopes. The heavy chain variable region CDRs can be abbreviated as HCDRs, and the light chain variable region CDRs can be abbreviated as LCDRs. The terms "framework region" or "FR region" are used interchangeably and refer to the amino acid residues in the heavy chain variable region or light chain variable region of an antibody, excluding the CDRs. A typical antibody variable region is composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0195] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0196] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CHI domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter comprises only "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CHI domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CHI domain. For example, a typical "heavy chain constant region fragment" can be selected from the CHI, Fc, or CH3 domains.

[0197] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0198] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can be, for example, removed during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and therefore, the Fc region may or may not include Lys447.

[0199] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0200] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0201] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been integrated into the GAP program in the GCG software package (available at www.gcg.com), is used with a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used with a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0202] In this article Indicates the junction site.

[0203] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0204] The term "tautomer" refers to functional group isomers resulting from the rapid shift of an atom in a molecule between two positions. The compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist as two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates, while in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

[0205] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0206] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0207] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0208] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0209] When any variable (such as R a 、R b ) appears more than once in the composition or structure of a compound, its definition is independent in each case. For example, if a group is represented by two R b is replaced, then each Rb There are independent options.

[0210] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a bond.

[0211] When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in ALZ represents a bond, it means that the structure is actually AZ.

[0212] When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. When X is selected from "C1-C3 alkylene-O", X can connect ring A and ring B from left to right to form "ring A-C1-C3 alkylene-O-ring B", or can connect ring A and ring B from right to left to form "ring A0-C1-C3 alkylene-ring B".

[0213] When a substituent's bond crosses two atoms in a ring, the substituent may be bonded to any atom in the ring. Represents R 5 Substitution can occur at any position on the benzene ring.

[0214] In this article, C m -C n It means having an integer number of carbon atoms in the range mn. For example, "C1-C 10 ” means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, or 10 carbon atoms.

[0215] The term "alkyl" refers to a group of the formula C n H 2n+1 The term "C1-C6 alkyl" may be understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc. The term "C1-C3 alkyl" may be understood to mean a straight-chain or branched saturated alkyl group having 1 to 3 carbon atoms. The "C1-C6 alkyl" may further include "C1-C3 alkyl".

[0216] The term "alkoxy" refers to a group derived from a straight-chain or branched alcohol by the loss of a hydrogen atom from a hydroxyl group, and can be understood as "alkyloxy" or "alkyl-O-." The term "C1-C6 alkoxy" can be understood as "C1-C6 alkyloxy" or "C1-C6 alkyl-O-." The "C1-C6 alkoxy" may further include "C1-C3 alkoxy."

[0217] The term "cycloalkyl" refers to a fully saturated carbocyclic group that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 3- to 10-membered ring. The term "C3-C 10 The term "cycloalkyl" is understood to mean a saturated monocyclic, bicyclic, spirocyclic or bridged ring having 3 to 10 carbon atoms. Specific examples of the cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, and the like. The term "C3-C 10 "Cycloalkyl" may include "C3-C6 cycloalkyl". The term "C3-C6 cycloalkyl" may be understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms. Specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[0218] The term "cycloalkyloxy" may be understood as "cycloalkyl-O-".

[0219] The term "cycloalkenyl" refers to a non-aromatic carbocyclic group that is not fully saturated and exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is typically a 5- to 8-membered ring. The term "C5-C6 cycloalkenyl" refers to a cycloalkenyl group having 5 or 6 ring carbon atoms, and specific examples include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, and cyclohexadienyl.

[0220] The term "heterocyclyl" refers to a fully saturated or partially saturated (heteroaromatic group that is not aromatic as a whole) monocyclic, fused, spiro or bridged ring group, which contains 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatom-containing groups) in its ring atoms, wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-. The term "4-14 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, and containing 1 to 5 heteroatoms or heteroatomic groups independently selected from the above-mentioned heteroatoms in its ring atoms. “4-14 membered heterocyclyl” includes “4-10 membered heterocyclyl” or “4-7 membered heterocyclyl”, etc., wherein specific examples of 4 membered heterocyclyl include but are not limited to azetidinyl or oxetanyl; specific examples of 5 membered heterocyclyl include but are not limited to tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl or 2,5-dihydro-1H-pyrrolyl; specific examples of 6 membered heterocyclyl include but are not limited to tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7 membered heterocyclyl include but are not limited to diazepanyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include but are not limited to hexahydrocyclopenta[c]pyrrol-2(1H)-yl; specific examples of 5,6-membered bicyclic groups include but are not limited to hexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclic group may be a benzo-fused ring group of the above-mentioned 4-7-membered heterocyclic groups, specific examples of which include but are not limited to dihydroisoquinolinyl and the like. "4-10 membered heterocyclyl" may include "5-10 membered heterocyclyl", "4-7 membered heterocyclyl", "5-6 membered heterocyclyl", "6-8 membered heterocyclyl", "4-10 membered heterocycloalkyl", "5-10 membered heterocycloalkyl", "4-7 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", "6-8 membered heterocycloalkyl", etc., and "4-7 membered heterocyclyl" may further include "4-6 membered heterocyclyl", "5-6 membered heterocyclyl", "4-7 membered heterocycloalkyl", "4-6 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", etc. Although some bicyclic heterocyclyl groups in the present disclosure partially contain a benzene ring or a heteroaromatic ring, the heterocyclyl group as a whole is still non-aromatic.

[0221] The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated π electron system. 10 "Aryl" is understood to be an aromatic radical having 6 to 10 carbon atoms. In particular, a ring having 6 carbon atoms ("C6 aryl"), such as phenyl; or a ring having 9 carbon atoms ("C9 aryl"), such as indanyl or indenyl; or a ring having 10 carbon atoms ("C 10 "aryl"), for example tetrahydronaphthyl, dihydronaphthyl or naphthyl.

[0222] The term "aryloxy" may be understood as "aryl-O-".

[0223] The term "heteroaryl" refers to a monocyclic or fused polycyclic ring system with aromatic character, which contains at least one ring atom selected from N, O, S, and the remaining ring atoms are C. The term "5-10 membered heteroaryl" is understood to include monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and containing 1 to 5, preferably 1 to 3 heteroatoms independently selected from N, O and S. In particular, the heteroaryl group is selected from thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl or isoquinolyl, and the like; or acinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl or phenoxazinyl, and the like. The term "5-6 membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms and containing 1-3, preferably 1-2, heteroatoms independently selected from N, O and S.

[0224] The term "heteroaryloxy" may be understood as "heteroaryl-O-".

[0225] The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

[0226] The term "hydroxy" refers to an -OH group.

[0227] The term "cyano" refers to a -CN group.

[0228] The term "mercapto" refers to a -SH group.

[0229] The term "amino" refers to a -NH2 group.

[0230] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0231] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0232] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0233] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0234] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0235] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0236] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0237] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0238] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0239] The phosphate compounds described in this application (such as R 10 Selected from Compounds) are ester prodrugs, which are hydrolyzed by phosphatase in the body to release biologically active compounds. For example, phosphate compounds Glucocorticoid receptor agonist is released after being hydrolyzed by phosphatase in the body Therefore, those skilled in the art can understand or anticipate that if compound 009 has a certain biological activity (such as glucocorticoid receptor agonist activity), the corresponding ester compound 009-p also has the same or similar biological activity in vivo.

[0240] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure 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 et al.

[0241] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 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. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0242] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0243] In all administration methods of the compounds described herein, the daily dosage is 0.001 mg / kg to 600 mg / kg body weight, preferably 0.05 mg / kg to 200 mg / kg body weight, more preferably 0.1 mg / kg to 100 mg / kg body weight, in single or divided doses.

[0244] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0245] The chemical reactions of the embodiments of the present disclosure are carried out in a suitable solvent that is compatible with the chemical transformations of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0246] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0247] FIG1 is a COSY diagram of intermediate 1-5 of Example 1 of the present disclosure.

[0248] FIG2 is a graph showing the results of the efficacy test of the compound of formula (DH) in Test Example 6 of the present disclosure on a CHS mouse model.

[0249] FIG3 is a graph showing the activity test results of the anti-human TNFα antibody-drug conjugate in the GRE reporter gene in Test Example 9 of the present disclosure.

[0250] FIG4 is a graph showing the activity test results of the anti-mouse TNFα antibody-drug conjugate in the GRE reporter gene in Test Example 9 of the present disclosure.

[0251] FIG5 is a graph showing the inhibitory activity test results of the anti-mouse TNFα-ADC in Test Example 11 of the present disclosure in the LPS-stimulated mouse PBMC cytokine release assay.

[0252] FIG6 is a graph showing the efficacy test results of the anti-mouse TNFα-ADC in Test Example 12 of the present disclosure in the mouse CHS model.

[0253] FIG7 is a graph showing the efficacy test results of the anti-mouse TNFα-ADC in Test Example 13 of the present disclosure in the DBA / 1 mouse CAIA model.

[0254] FIG8 is a graph showing the efficacy test results of the anti-human TNFα-ADC in Test Example 14 of the present disclosure in the TNFα humanized mouse CAIA model.

[0255] FIG9 shows the anti-IL-4R antibody-drug conjugate in Test Example 20 of the present disclosure for human primary CD4 + Figure 2 shows the results of a T cell proliferation inhibition test.

[0256] FIG10 shows the ear thickness test results of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure in an atopic dermatitis mouse model.

[0257] FIG11 shows the serum IgE detection results of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure in an atopic dermatitis mouse model.

[0258] Figures 12-13 show that the administration of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure significantly improved the pathological damage and inflammation of the ear and skin tissues at the modeling site of the atopic dermatitis mouse model.

[0259] Figures 14-15 show that the administration of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure significantly inhibited the expression of inflammatory CD4 in the ear and skin tissue of the atopic dermatitis mouse model. + T cell infiltration. DETAILED DESCRIPTION

[0260] The invention is described in detail below by way of examples, but is not intended to limit the present disclosure in any way. While the present disclosure has been described in detail herein, including specific embodiments thereof, it will be apparent to those skilled in the art that various modifications can be made to the specific embodiments without departing from the spirit and scope of the present disclosure. All reagents used in the present disclosure were commercially available and used without further purification.

[0261] Unless otherwise specified, the ratios expressed for mixed solvents are volume ratios.

[0262] Unless otherwise stated, % refers to wt%.

[0263] Compounds are manually or Software naming, commercially available compounds use supplier catalog names.

[0264] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). The units of NMR shifts are 10 -6 (ppm). The solvents for NMR determination are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC 50 " refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximum inhibitory effect is achieved. 50 "half maximal effect concentration" refers to the concentration that causes 50% of the maximum effect.

[0265] Example 1: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 001)

[0266] Synthesis route and specific synthesis steps:

[0267] Step 1: Synthesis of 4-bromo-2,3-dihydrobenzofuran-7-amine 1-2

[0268] Following the method reported in the literature (WO2016169504A1), 2,3-dihydrobenzofuran-7-amine (1.0 g, 7.40 mmol) was dissolved in dimethylformamide (20 mL), and N-bromosuccinimide (1.48 g, 8.33 mmol) was added. The mixture was stirred at 25°C for 3 hours. LCMS indicated completion of the reaction. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (100 mL x 2). The organic phase was washed with saturated sodium chloride solution (30 mL x 2), dried, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 4) to obtain 4-bromo-2,3-dihydrobenzofuran-7-amine 1-2 (1.0 g).

[0269] LC-MS:Rt:0.444min;MS m / z(ESI):213.9[M+H] + ;

[0270] Step 2: Synthesis of tert-butyl (4-bromo-2,3-dihydrobenzofuran-7-yl)carbamate

[0271] 4-Bromo-2,3-dihydrobenzofuran-7-amine 1-2 (1.0 g, 4.67 mmol) was dissolved in dichloromethane (20 mL), and di-tert-butyl dicarbonate (1.41 g, 6.46 mmol) and 4-dimethylaminopyridine (571 mg, 4.67 mmol) were added. The mixture was stirred at 25°C for 16 hours. LCMS indicated the reaction was complete, and the mixture was diluted with water (20 mL). The mixture was extracted with ethyl acetate (100 mL x 2), and the organic phase was washed with saturated aqueous sodium chloride (30 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 5) to give tert-butyl (4-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 1-3 (600 mg, 38.8%).

[0272] LC-MS:Rt:1.756min; MS m / z(ESI):258.1[M-56+H] + ;

[0273] 1H NMR (400MHz, CHLOROFORM-d) δ = 7.71-7.54 (m, 1H), 6.87 (d, J = 8.6Hz, 1H), 6.44 (s, 1H), 4.55 (t, J = 8.8Hz, 2H), 3.15 (t, J = 8.8Hz, 2H), 1.43 (s, 9H).

[0274] Step 3: Synthesis of tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 1-4

[0275] Tert-butyl (4-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 1-3 (450 mg, 1.43 mmol) was dissolved in dioxane (10 mL). Bispinacol boronate (909 mg, 3.58 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (105 mg, 0.14 mmol), and potassium acetate (422 mg, 4.30 mmol) were added. The mixture was stirred at 80°C under a nitrogen atmosphere for 16 hours. LCMS indicated the reaction was complete. The reaction solution was filtered and concentrated, and the residue was purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 6) to obtain compound 1-4 (410 mg, 79.2%).

[0276] LC-MS:Rt:1.937min;MS m / z(ESI):306.2[M-56+H] + .

[0277] Step 4: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 1-5

[0278] The reactants 16α-hydroxyprednisolone F1 (1 g, 2.66 mmol) and anhydrous magnesium sulfate (1.22 g, 10.1 mmol) were dissolved in acetonitrile (20 mL) and stirred at 25 ° C for 1 hour. 4-(Bromomethyl)benzaldehyde (529 mg, 2.66 mmol) was dissolved in acetonitrile (2 mL) and added to the reaction solution. The solution was cooled to 0 ° C and trifluoromethanesulfonic acid (1.88 g, 12.5 mmol) was added dropwise. The mixture was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete, and the reaction solution was quenched with aqueous sodium bicarbonate solution (20 mL), extracted with ethyl acetate (100 mL x 2), and the organic phase was washed with saturated aqueous sodium chloride solution (50 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 2). The crude product was separated and purified by SFC [column: DAICEL CHIRALCEL OD (250 mm*50 mm, 10 μm); mobile phase: A: 0.1% ammonia methanol solution; B: CO2, 50%] to give intermediate 1-5 (1.2 g, 80.9%).

[0279] LC-MS:Rt:1.551min; MS m / z(ESI):557.1[M+H] + ;

[0280] 1 H NMR (400MHz, DMSO-d6) δ = 7.45 (s, 3H), 7.31 (d, J = 10.1Hz, 1H), 6.16 (dd, J = 1.8, 10.1Hz, 1H), 5.93 (s, 1H) ),5.75(s,1H),5.45(s,1H),5.16-5.03(m,1H),4.94(d,J=4.9Hz,1H),4.79(d,J=3.0Hz,1H),4.68(s,2 H),4.53(d,J=19.5Hz,1H),4.30(s,1H),4.19(d,J=19.4Hz,1H),2.61-2.53(m,1H),2.31-2.25(m,1H), 2.17-2.07(m,1H),2.06-1.98(m,1H),1.81-1.61(m,5H),1.39(s,3H),1.10-0.99(m,2H),0.87(s,3H).

[0281] The absolute configuration of intermediate 1-5 was confirmed by 2D NMR (see Figure 1).

[0282] Step 5: Synthesis of tert-butyl (4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamate 1-6

[0283] Compound 1-5 (300 mg, 0.54 mmol) was dissolved in tetrahydrofuran (6 mL) and water (0.6 mL). Tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate (194 mg, 0.54 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (78.8 mg, 0.11 mmol), and potassium carbonate (223 mg, 1.61 mmol) were added. The mixture was stirred at 80°C under a nitrogen atmosphere for 16 hours. LCMS indicated the reaction was complete. The reaction solution was filtered and concentrated, and the residue was isolated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 1) to obtain compound 1-6 (300 mg, 54.8%).

[0284] LC-MS:Rt:0.584min;MS m / z(ESI):712.4[M+H] + ;

[0285] Step 6: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 001

[0286] 1-6 (280 mg, 0.39 mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at 25°C for 1 hour. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative HPLC [columns: Phenomenex luna 30*30 mm*10 μm; YMC AQ 100*30*10 μm; mobile phase: A: water (HCl); B: ACN, B%, 30%-60%, 15 min] to afford compound 001 (10 mg, 19.0%).

[0287] LC-MS:Rt:1.511min; MS m / z(ESI):612.5[M+H] + ;

[0288] 1 H NMR (400MHz, DMSO-d6) δ=9.52-8.19(m,2H),7.38(d,J=8.1Hz,2H),7.32(d,J=10.1Hz,1H),7.20(d,J=8.0Hz,2H),6.89(d,J =8.0Hz,1H),6.65(d,J=8.0Hz,1H),6.17(dd,J=1.8,10.1Hz,1H),5.93(s,1H),5.41(s,1H),4.92(d,J=5.0Hz,1H),4.81(br s,1H),4.60(t,J=8.8Hz,2H),4.50(d,J=19.4Hz,1H),4.29(br s,1H),4.18(d,J=19.4Hz,1H),3.86(s,2H),3.16-3.08(m,2H),2.62-2.54(m,1H),2.30(m,1H),2.1 9-2.08(m,1H),2.07-1.97(m,1H),1.81-1.59(m,5H),1.40(s,3H),1.12-0.96(m,2H),0.87(s,3H).

[0289] Example 2: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-benzo[d]imidazol-6-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 002)

[0290] Synthesis route and specific synthesis steps:

[0291] Step 1: Synthesis of tert-butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole-1-carboxylate 2-2

[0292] 6-(4,4,5-Trimethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazole (1.00 g, 4.10 mmol), N,N-diisopropylethylamine (1.06 g, 8.19 mmol), 4-dimethylaminopyridine (50 mg, 0.41 mmol), and di-tert-butyl dicarbonate (894 mg, 4.10 mmol) were dissolved in anhydrous dichloromethane (20 mL) and reacted at 25°C for 6 hours. LCMS confirmed the reaction was complete. The reaction was quenched by the addition of water (20 mL) and extracted with dichloromethane (50 mL x 2). The organic phase was washed with saturated sodium chloride (30 mL), dried, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 5) to afford 2-2 (1.0 g, 69.5%).

[0293] LC-MS:Rt:1.793min; MS m / z(ESI):345.3[M+H] + ;

[0294] 1 H NMR (400MHz, DMSO-d6) δ = 8.50–8.24 (m, 2H), 8.00-7.75 (m, 2H), 1.70 (d, J = 3.5Hz, 9H), 1.36 (d, J = 5.1Hz, 12H).

[0295] Step 2: Synthesis of tert-butyl 6-(4-formylbenzyl)-1H-benzo[d]imidazole-1-carboxylate 2-3

[0296] 2-2 (500 mg, 1.45 mmol), 4-(bromomethyl)benzaldehyde (376 mg, 1.89 mmol), potassium carbonate (602 mg, 4.36 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (106 mg, 0.14 mmol) were dissolved in anhydrous tetrahydrofuran (10 mL) and reacted at 80°C under a nitrogen atmosphere for 16 hours. LCMS confirmed the reaction was complete. The reaction solution was filtered and concentrated, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 5) to afford 2-3 (380 mg, crude).

[0297] LC-MS: Rt: 1.563 min and 1.593; MS m / z (ESI): 337.3 [M+H]+ ;

[0298] Step 3: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-benzo[d]imidazol-6-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one

[0299] Dissolve 16alpha-hydroxyprednisolone F1 (230 mg, 0.61 mmol) in anhydrous acetonitrile (1.5 mL), add anhydrous magnesium sulfate (279 mg, 2.32 mmol), and stir at 25°C for 1 hour. Dissolve 2-3 (308 mg, 0.92 mmol) in anhydrous acetonitrile (1.5 mL) and add the reaction system. Cool to 0°C, add trifluoromethanesulfonic acid (431 mg, 2.87 mmol) dropwise, and stir at 25°C for 2 hours. LCMS indicates the reaction is complete. The reaction was quenched by the addition of saturated sodium bicarbonate solution (5 mL), and the mixture was extracted with ethyl acetate (30 mL x 2). The organic phase was washed with saturated aqueous sodium chloride solution (20 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was purified by Prep-HPLC [column: Phenomenex Luna 30*30mm*10μm; YMC AQ 100*30*10μm; mobile phase: A: water (containing 0.225% formic acid); B: MeCN, B%, 15%-45%, 20 mins] to give compound 002 (35.8 mg, 9.75%).

[0300] LC-MS: Rt: 1.390 min; MS m / z (ESI): 595.5 [M+H]; Chiral-HPLC: Rt: 2.211 min, chiral purity = 97.56%;

[0301] 1H NMR(400MHz,DMSO-d6)δ=8.39(br s,1H),7.52(d,J=8.3Hz,1H),7.44(s,1H),7.40-7.35(m,2H),7.33-7.26(m,3H),7. 12(d,J=8.4Hz,1H),6.16(dd,J=1.6,10.1Hz,1H),5.93(s,1H),5.40(s,1H),5.08(br s,1H),4.92(d,J=4.9Hz,1H),4.78(d,J=3.0Hz,1H),4.50(br d,J= 16.0Hz,1H),4.28(s,1H),4.17(d,J=19.5Hz,1H),4.05(s,2H),2.30(s,1H),2.1 5-1.98(m,2H),1.81-1.60(m,5H),1.39(s,3H),1.10-0.97(m,2H),0.86(s,3H).

[0302] Example 3: Synthesis of (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((1H-indol-6-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-decahydro-1H-naphtho[2', 1': 4, 5] indeno[1, 2-d][1, 3] dioxolane-4(2H)-one (Compound 003)

[0303] Synthesis route and specific synthesis steps:

[0304] Compound 1-7 (500 mg, 0.89 mmol) was dissolved in tetrahydrofuran (5 mL) and water (0.5 mL), and 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole 3-1 (262 mg, 1.08 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (66 mg, 0.09 mmol) and potassium carbonate (372 mg, 2.69 mmol) were added, and the mixture was stirred at 80 ° C for 16 hours under a nitrogen atmosphere. LCMS showed the reaction was complete, and the reaction mixture was diluted with ethyl acetate (100 mL). The organic phase was washed with saturated aqueous sodium chloride (30 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 1). The crude product was separated and purified by Prep-HPLC [column: Phenomenex luna 30*30mm*10μm+YMC AQ 100*30*10μm; mobile phase: A: water (0.225% FA); B: MeCN, B%, 50%-80%, 20 min] to give Compound 003 (60 mg, 11.2%).

[0305] LC-MS:Rt:2.098min; MS m / z(ESI):594.3[M+H] + ;Chiral-HPLC: Rt: 1.204 min, chiral purity = 100%;

[0306] 1 H NMR (400MHz, DMSO-d6) δ = 10.92 (s, 1H), 7.41 (d, J = 8.1Hz, 1H), 7.37 (d, J = 8.0Hz, 2H), 7.30 (d, J = 10.1Hz, 1H), 7.28-7.23 (m, 3H), 7.18 (s,1H),6.85(dd,J=1.3,8.1Hz,1H),6.34(s,1H),6.16(dd,J=1.8,10.1Hz,1H),5.93(s,1H),5.40(s,1H),5.08(t,J=5.9Hz,1H),4.92 (d,J=5.0Hz,1H),4.78(d,J=3.1Hz,1H),4.50(dd,J=6.3,19.4Hz,1H),4.29(s,1H),4.17(dd,J=5.4,19.4Hz,1H),4.00(s,2H),2.59-2 .53(m,1H),2.36-2.26(m,1H),2.18-2.06(m,1H),2.05-1.96(m,1H),1.80-1.54(m,5H),1.39(s,3H),1.11-0.97(m,2H),0.86(s,3H).

[0307] Example 4: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-10-(4-(dihydroindole-6-ylmethyl)phenyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 004)

[0308] Synthesis route and specific synthesis steps:

[0309] Step 1: Synthesis of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline 4-2

[0310] 6-Bromoindoline (1.0 g, 5.05 mmol) was dissolved in dioxane (20 mL), and bis-pinacol boronate (2.56 g, 10.1 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (369 mg, 504 μmol), and potassium acetate (1.49 g, 15.2 mmol) were added. The mixture was reacted at 80°C under a nitrogen atmosphere for 12 hours. LCMS confirmed the completion of the reaction. The reaction solution was filtered and concentrated, and the residue was purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 3) to obtain 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline 4-2 (1.2 g, 96.9%).

[0311] LC-MS:Rt:1.509min; MS m / z(ESI):246.3[M+H] + ;

[0312] Step 2: Synthesis of tert-butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-carboxylate 4-3

[0313] 4-2 (500 mg, 2.04 mmol) was dissolved in dichloromethane (8 mL), and di-tert-butyl dicarbonate (891 mg, 4.08 mmol), diisopropylethylamine (528 mg, 4.08 mmol), and 4-dimethylaminopyridine (49.8 mg, 408 μmol) were added. The mixture was stirred at 25°C for 6 hours. LCMS indicated the reaction was complete. Dichloromethane (50 mL) was added to dilute the mixture, and the organic phase was washed with saturated aqueous sodium chloride (15 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 5) to afford 4-3 (260 mg, 36.9%).

[0314] LC-MS: Rt: 2.340min; MS m / z (ESI): 290.3[M-56+H];

[0315] Step 3: Synthesis of tert-butyl 6-(4-formylbenzyl)indoline-1-carboxylate 4-4

[0316] tert-Butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-carboxylate (260 mg, 753 μmol) was dissolved in tetrahydrofuran (5 mL). 4-(Bromomethyl)benzaldehyde (299 mg, 1.51 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (165 mg, 226 μmol), and potassium carbonate (520 mg, 3.77 mmol) were added. The mixture was stirred at 80°C under a nitrogen atmosphere for 12 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (tetrahydrofuran / petroleum ether, 1 / 5) to afford 4-4 (190 mg, 74.8%).

[0317] LC-MS:Rt:1.810min; MS m / z(ESI):282.2[M-56+H] + ;

[0318] Step 4: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-10-(4-(dihydroindole-6-ylmethyl)phenyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 004

[0319] 16alpha-Hydroxyprednisolone F1 (165 mg, 438 μmol) was dissolved in acetonitrile (3 mL), and anhydrous magnesium sulfate (258 mg, 2.15 mmol) was added. The mixture was stirred at 25°C for 1 hour. Tert-butyl 6-(4-formylbenzyl)indoline-1-carboxylate (178 mg, 526 μmol) was dissolved in acetonitrile (1 mL) and added to the reaction mixture. The temperature was cooled to 0°C, and trifluoromethanesulfonic acid (322 mg, 2.15 mmol) was added dropwise. The mixture was stirred at 25°C for 2 hours. LCMS indicated the reaction was complete. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution (10 mL), extracted with ethyl acetate (30 mL x 3), and the organic phase was washed with saturated aqueous sodium chloride solution (20 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was separated and purified by Prep-HPLC [column: Phenomenex luna 30*30mm*10μm+YMC AQ 100*30*10μm; mobile phase: A: water (0.225% FA); B: MeCN, B%, 18%-48%, 20 min] to give compound 004 (42.9 mg, 6.45%).

[0320] LC-MS:Rt:1.532min; MS m / z(ESI):596.3[M+H] + ;Chiral-HPLC: Rt: 2.502 min, chiral purity = 100%;

[0321] 1 H NMR (400MHz, DMSO-d6) δ = 7.37-7.33 (m, 2H), 7.30 (d, J = 10.1Hz, 1H), 7.20 (d, J = 8.0Hz, 2H), 6.88 (d,J=7.3Hz,1H),6.37(d,J=7.4Hz,1H),6.28(s,1H),6.18-6.13(m,1H),5.95-5.91(m,1H),5.3 9(s,2H),5.12-5.06(m,1H),4.91(d,J=5.0Hz,1H),4.80-4.75(m,1H),4.54-4.45(m,1H),4.32- 4.25(m,1H),4.21-4.12(m,1H),3.75(s,2H),3.38-3.34(m,2H),2.84-2.77(m,2H),2.55-2.47(m 1H),2.34-2.27(m,1H),2.16-2.07(m,1H),2.06-1.97(m,1H),1.79-1.59(m,5H),1.39(s,3H),1.12-0.97(m,2H),0.89-0.82(m,3H).

[0322] Example 5: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 005)

[0323] Synthesis route and specific synthesis steps:

[0324] Step 1: Synthesis of 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine 5-2

[0325] 7-Bromo-3,4-dihydro-2H-benzo[b][1,4]oxazine 5-1 (1.00 g, 4.67 mmol) was dissolved in tetrahydrofuran (20 mL). Bis-pinacol boronate (1.30 g, 5.14 mmol), potassium acetate (1.38 g, 14.0 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (341 mg, 467 μmol) were added. The mixture was reacted at 80°C under a nitrogen atmosphere for 16 hours. LCMS confirmed the completion of the reaction. The reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 5) to afford 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazine 5-2 (950 mg, 77.9%).

[0326] LC-MS:Rt:1.454min; MS m / z(ESI):262.3[M+H] + ;

[0327] Step 2: Synthesis of 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde 5-3

[0328] 5-2 (900 mg, 3.45 mmol) was dissolved in tetrahydrofuran (20 mL), and 4-(bromomethyl)benzaldehyde (1.03 g, 5.17 mmol), potassium carbonate (1.43 g, 10.3 mmol), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (252 mg, 345 μmol) were added. The mixture was reacted at 80°C under a nitrogen atmosphere for 16 hours. LCMS confirmed the completion of the reaction. The reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (ethyl acetate / petroleum ether = 1 / 3) to obtain 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde 5-3 (397 mg).

[0329] LC-MS:Rt:1.202min; MS m / z(ESI):254.1[M+H] + ;

[0330] Step 3: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl

[0331] 16alpha-Hydroxyprednisolone F1 (350 mg, 929 μmol) was dissolved in acetonitrile (15 mL), and magnesium sulfate (425 mg, 3.53 mmol) was added. After stirring at 25°C for 1 hour, 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde (235 mg, 929 μmol) 5-3 was dissolved in acetonitrile (5 mL) and added to the reaction system. The temperature was cooled to 0°C, and trifluoromethanesulfonic acid (655 mg, 4.37 mmol) was added dropwise. The mixture was stirred at 25°C for 2 hours. The reaction was complete by LCMS. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (15 mL), extracted with ethyl acetate (50 mL x 3), and the organic phase was washed with saturated aqueous sodium chloride solution (20 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was separated and purified by prep-HPLC [column: Xtimate C18 150*40mm*10μm; mobile phase: A: water (10mM NH4HCO3); B: MeCN, B%, 40%-70%, 10 min] to give compound 005 (20 mg, 21.1%).

[0332] LC-MS:Rt:1.874min;MS m / z(ESI):612.3[M+H] + ;

[0333] 1H NMR (400MHz, DMSO-d6) δ=7.41-7.27(m,3H),7.19(br d,J=7.9Hz,2H),6.56-6.38(m,3H),6.16(dd,J=1.1,10.0Hz,1H),5.93(s,1H),5.52(br s,1H),5.39(s,1H),5.05(t,J=5.9Hz,1H),4.92(br d,J=5.0Hz,1H),4.76(br d,J=2.6Hz,1H),4.49(br dd,J=6.3,19.4Hz,1H),4.29(br s,1H),4.17(br dd,J=5.6,19.4Hz,1H),4.10-4.01(m,2H),3.70(s,2H),3.29-3.25(m,1H),3.21(br s,2H),2.37-2.25(m,1H),2.18-1.96(m,2H),1.84-1.56(m,5H),1.39(s,3H),1.14-0.97(m,2H),0.86(s,3H).

[0334] Example 6: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 006)

[0335] Synthesis route and specific synthesis steps:

[0336] Step 1: Synthesis of (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-one 6-2

[0337] Compound (2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a,10,10-tetramethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 6-1 (1.0 g, 2.21 mmol) was dissolved in 30% aqueous fluoroboric acid (20 mL) and stirred at room temperature for 48 hours. The reaction was complete by LCMS. 40 mL of water was added and stirred for 5 minutes. The filtered residue was washed with water (10 mL x 3) and ethanol (10 mL x 3) to give a crude product (6S,8S,9R,10S,11S,13S,14S,16R,17S))-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-one 6-2 (0.7 g).

[0338] LC-MS:Rt:0.426min;MS m / z(ESI):413.1[M+H] + ;

[0339] Step 2: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 006

[0340] 6-2 (150 mg, 0.36 mmol) was dissolved in anhydrous acetonitrile (1.5 mL), and magnesium sulfate (166 mg, 1.38 mmol) was added. After stirring at 25°C for 1 hour, 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzaldehyde (138 mg, 0.55 mmol) was dissolved in anhydrous acetonitrile (1.5 mL) and added to the reaction system. The temperature was cooled to 0°C, and trifluoromethanesulfonic acid (257 mg, 1.71 mmol) was added dropwise. The mixture was reacted at 25°C for 2 hours. The reaction was complete by LCMS. The reaction was quenched by adding saturated aqueous sodium bicarbonate solution (10 mL), extracted with ethyl acetate (50 mL x 2), and the organic phase was washed with saturated aqueous sodium chloride solution (20 mL). The organic phase was dried, filtered, and concentrated under reduced pressure. The crude product was separated and purified by Prep-HPLC [column: Phenomenex Luna C18 100*30mm*3μm; mobile phase: A: water (0.225% FA); B: MeCN, B%: 40%-70%, 8 min] to give compound 006 (82.7 mg, 34.6%).

[0341] LC-MS:Rt:1.553min; MS m / z(ESI):648.2[M+H] + ;Chiral-HPLC: Rt: 3.241 min, chiral purity = 100%;

[0342] 1 H NMR (400MHz, DMSO-d6) δ = 7.32 (d, J = 8.1Hz, 2H), 7.26 (d, J = 10.1Hz, 1H), 7.20 (d, J = 8.1Hz, 2H), 6.53-6.48 (m, 1H), 6 .47-6.43(m,2H),6.33-6.27(m,1H),6.13(s,1H),5.72-5.57(m,1H),5.50(d,J=3.0Hz,1H),5.43(s,1H),4.94(d,J =4.9Hz,1H),4.51(d,J=19.5Hz,1H),4.19-4.04(m,2H),4.07-4.03(m,2H),3.69(s,2H),3.22-3.19(m,2H),2.65-2 .56(m,1H),2.28-2.07(m,2H),2.05-1.98(m,1H),1.75-1.65(m,3H),1.51-1.49(m,1H),1.50(s,3H),0.86(s,3H).

[0343] Example 7: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminobenzo[d]oxazol-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 007)

[0344] Synthesis route and specific synthesis steps:

[0345] Step 1: Synthesis of 7-bromobenzo[d]oxazol-4-amine 7-2

[0346] Following the method reported in the literature (CN110903258), benzo[d]oxazol-4-amine (1.0 g, 7.5 mmol) was dissolved in dimethylformamide (20 mL), and NBS (1.3 g, 7.1 mmol) was added. The mixture was stirred at 25°C for 1 hour. LCMS confirmed the reaction was complete. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (100 mL x 2). The organic phase was washed with saturated sodium chloride solution (30 mL x 2), dried, filtered, and concentrated under reduced pressure. The crude product was isolated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 2) to afford 7-bromobenzo[d]oxazol-4-amine 7-2 (1.3 g, 64.5%).

[0347] LC-MS:Rt:1.181min; MS m / z(ESI):214.9[M+H] + ;

[0348] 1 H NMR (400MHz, DMSO-d6) δ = 8.59 (s, 1H), 7.24 (d, J = 8.5Hz, 1H), 6.52 (d, J = 8.6Hz, 1H), 5.86 (s, 2H).

[0349] Step 2: Synthesis of 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-4-amine 7-3

[0350] 7-Bromobenzo[d]oxazol-4-amine 7-2 (1.3 g, 6.1 mmol) and bis-pinacol boronate (3.1 g, 12.2 mmol) were dissolved in anhydrous dioxane (26 mL). Potassium acetate (1.8 g, 18.3 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (447 mg, 0.6 mmol) were added. The mixture was stirred at 80°C under nitrogen for 16 hours. LCMS confirmed the completion of the reaction. The reaction mixture was filtered and concentrated, and the residue was purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 2) to afford 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazol-4-amine 7-3 (1.4 g, 64.3%).

[0351] LC-MS: Rt: 1.368min; MS m / z (ESI): 261.2[M+H];

[0352] Step 3: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminobenzo[d]oxazol-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 007)

[0353] Compound 7-3 (112 mg, 0.4 mmol) and compound 1-5 (200 mg, 0.4 mmol) were dissolved in tetrahydrofuran (4 mL) and water (0.4 mL). Potassium carbonate (149 mg, 1.1 mmol) and tetrakis(triphenylphosphine)palladium (124 mg, 0.1 mmol) were added, and the mixture was stirred at 80°C under a nitrogen atmosphere for 16 hours. LCMS confirmed the completion of the reaction. The reaction solution was filtered, and the residue was purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 2). The crude product was further purified by preparative HPLC [column: Phenomenex C18 75*30 mm*3 μm; mobile phase: A: water (NH4HCO3); B: acetonitrile, B%, 34%-58%, 10 min] to afford compound 007 (37.7 mg, 20.52%).

[0354] LC-MS:Rt:1.832min; MS m / z(ESI):611.5[M+H] + ;

[0355] 1H NMR (400MHz, DMSO-d6) δ = 8.45 (s, 1H), 7.36 (d, J = 8.1Hz, 2H), 7.31 (d, J = 10.1Hz, 1H), 7.25 (d,J=8.1Hz,2H),6.93(d,J=8.0Hz,1H),6.48(d,J=8.0Hz,1H),6.17(dd,J=1.8,10.1Hz,1H ),5.94(s,1H),5.48(s,2H),5.39(s,1H),5.08(s,1H),4.92(d,J=5.0Hz,1H),4.78(d,J=2 .9Hz,1H),4.50(dd,J=3.0,19.3Hz,1H),4.29(s,1H),4.17(d,J=18.4Hz,1H),4.03(s,2H), 2.61-2.53(m,1H),2.37-2.29(m,1H),2.16-1.97(m,2H),1.82-1.59(m,5H),1.40(s,3H),1.13-1.00(m,2H),0.86(s,3H).

[0356] Example 8: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolane-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 008)

[0357] Synthesis route and specific synthesis steps:

[0358] Step 1: Synthesis of 7-bromobenzo[d][1,3]dioxolane-4-amine 8-2

[0359] The starting material, benzo[d][1,3]dioxolane-4-amine 8-1 (2.00 g, 14.6 mmol), was dissolved in N,N-dimethylformamide (50 mL). N-bromosuccinimide (2.50 g, 13.9 mmol) was added at 0°C and stirred for 1 hour. LCMS confirmed the reaction was complete. The reaction solution was diluted with ethyl acetate (200 mL), washed with water (100 mL x 2) and saturated brine (50 mL). The organic phase was dried, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether / tetrahydrofuran = 2 / 1) to afford 7-bromobenzo[d][1,3]dioxolane-4-amine 8-2 (1.30 g, 94.8%).

[0360] LC-MS:Rt:1.311min; MS m / z(ESI):218.1[M+H] + ;

[0361] Step 2: Synthesis of tert-butyl (7-bromobenzo[d][1,3]dioxolane-4-yl)carbamate 8-3

[0362] 7-Bromobenzo[d][1,3]dioxolane-4-amine 8-2 (1.00 g, 4.6 mmol) and di-tert-butyl dicarbonate (3.0 g, 13.9 mmol) were dissolved in 1,4-dioxane (20 mL) and stirred at 100°C for 16 hours. LCMS confirmed the reaction was complete. The reaction solution was concentrated, and the residue was purified by column chromatography (petroleum ether / ethyl acetate = 6 / 1) to afford tert-butyl (7-bromobenzo[d][1,3]dioxolane-4-yl)carbamate 8-3 (820 mg, 43.5%).

[0363] LC-MS:Rt:1.707min;MS m / z(ESI):262.1[M+H-56] + ;

[0364] 1 H NMR (400MHz, DMSO-d6) δ = 8.95 (s, 1H), 6.98-6.89 (m, 2H), 6.10 (s, 2H), 1.44 (s, 9H).

[0365] Step 3: Synthesis of tert-butyl (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxolane-4-yl)carbamate 8-4

[0366] Dissolve tert-butyl (7-bromobenzo[d][1,3]dioxolane-4-yl)carbamate 8-3 (820 mg, 2.60 mmol) and bis(1,4-dioxane) boronate (1.30 g, 5.20 mmol) in 1,4-dioxane (25 mL). Add potassium acetate (764 mg, 7.80 mmol) and 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (190 mg, 0.30 mmol). Stir at 80°C under a nitrogen atmosphere for 16 hours. The reaction is complete by LCMS. The reaction solution was filtered and concentrated, and the residue was purified by column chromatography (petroleum ether / tetrahydrofuran = 6 / 1) to give tert-butyl (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-4 (1.00 g, 83.8%).

[0367] LC-MS:Rt:1.782min;MS m / z(ESI):308.3[M+H-56] + ;

[0368] Step 4: Synthesis of tert-butyl (7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)benzo[d][1,3]dioxolane-4-yl)carbamate 8-5

[0369] Tert-butyl (7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-4 (489 mg, 1.35 mmol) and (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7 ,8,8a,8b,11a,12,12a,12b-Decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 1-5 (500 mg, 0.9 mmol) was dissolved in a mixed solvent of tetrahydrofuran (20 mL) and water (2 mL), potassium carbonate (372 mg, 2.70 mmol) and tetrakistriphenylphosphine palladium (311 mg, 0.3 mmol) were added, and stirred at 80 ° C for 16 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was diluted with water and extracted with ethyl acetate (100 mL). The organic phase was washed with saturated brine (30 mL), dried, filtered, and concentrated. The residue was purified by column chromatography (petroleum ether / tetrahydrofuran = 1 / 1) and then purified by high-performance preparative liquid chromatography [column: Gemini NX C18 The reaction mixture was stirred for 2 h at 4 ℃ for 10 min. The reaction mixture was stirred for 2 h at 4 ℃ for 30 min. The reaction mixture was stirred for 10 min for 30 min. The reaction mixture was stirred for 2 h. The reaction mixture was stirred for 30 min. The reaction mixture was stirred for 30 min. The reaction mixture was stirred for 30 min. The reaction mixture was stirred for 40 min. The reaction mixture was stirred for 30 min. The reaction mixture was stirred for 30 min. The reaction mixture was stirred for 30 min.

[0370] LC-MS:Rt:1.742min; MS m / z(ESI):714.4[M+H] + ;

[0371] Step 5: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolane-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 008)

[0372] Tert-butyl (7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)benzo[d][1,3]dioxolan-4-yl)carbamate 8-5 (20 mg, 0.03 mmol) was dissolved in dichloromethane (4 mL). Trifluoroacetic acid (1 mL) was added and the mixture was stirred at 25°C for 0.5 h. The reaction was complete by LCMS. The reaction solution was concentrated and the residue was purified by high performance preparative liquid chromatography [column: Xtimate C18 The product was separated and purified by HPLC-MS / MS: 100*30mm*10μm; mobile phase: A: 0.1% ammonia solution; B: acetonitrile, B%, 20%-70%, 35min] to obtain (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolane-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 008) (3.1 mg, 17.3%).

[0373] LC-MS:Rt:1.681min; MS m / z(ESI):614.5[M+H] + ;

[0374] 1H NMR (400MHz, DMSO-d6) δ = 7.36 (d, J = 8.0Hz, 2H), 7.32 (d, J = 10.1Hz, 1H), 7.20 (d, J = 7.9Hz, 2H), 6.42 (d, J = 8.3Hz ,1H),6.22-6.15(m,2H),5.94(s,1H),5.88(s,2H),5.40(s,1H),5.11(t,J=5.9Hz,1H),4.92(d,J=5.0Hz,1H),4 .83-4.75(m,3H),4.51(dd,J=6.3,19.4Hz,1H),4.30(s,1H),4.18(dd,J=5.6,19.4Hz,1H),3.73(s,2H),2.31(d ,J=2.3Hz,1H),2.15-2.00(m,2H),1.81-1.61(m,5H),1.40(s,3H),1.25(s,1H),1.13-1.01(m,2H),0.87(s,3H).

[0375] Example 9: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 009)

[0376] Synthesis route and specific synthesis steps:

[0377] Step 1: Synthesis of tert-butyl (5-bromo-2,3-dihydrobenzofuran-7-yl) carbamate 9-2

[0378] 5-Bromo-2,3-dihydrobenzofuran-7-carboxylic acid 9-1 (1.00 g, 4.11 mmol) was dissolved in toluene (10 mL) and tert-butanol (10 mL), and then added at room temperature. Molecular sieves (200 mg) and triethylamine (1.25 g, 12.3 mmol) were stirred at 110°C for half an hour, then cooled to room temperature. Diphenylphosphine azide (1.70 g, 6.17 mmol) was added dropwise, and the temperature was raised to 110°C and stirred for 10 hours. LCMS confirmed the reaction was complete. The reaction solution was filtered and diluted with ethyl acetate (150 mL). The organic phase was washed with saturated brine (30 mL), dried, filtered, and concentrated. The residue was purified by column chromatography (0-40% ethyl acetate / petroleum ether) to obtain the intermediate tert-butyl (5-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 9-2 (1.03 g, 79.7%).

[0379] LC-MS:Rt:1.065min; MS m / z(ESI):259.7[M+H-56] + ;

[0380] 1 H NMR (400MHz, DMSO-d6) δ = 8.59 (s, 1H), 7.50 (br s, 1H), 7.13-7.11 (m, 1H), 4.61-4.50 (m, 2H), 3.24-3.17 (m, 2H), 1.48-1.41 (m, 9H).

[0381] Step 2: Synthesis of tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3

[0382] Dissolve tert-butyl (5-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 9-2 (600 mg, 1.91 mmol), bis(naphthalene) boronate (533 mg, 2.10 mmol), potassium acetate (562 mg, 5.73 mmol), and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (139 mg, 190 μmol) in anhydrous 1,4-dioxane (10 mL) and react at 80°C under a nitrogen atmosphere for 16 hours. LCMS analysis confirmed the reaction was complete. The reaction solution was filtered and concentrated, and the residue was separated and purified by column chromatography (0-10% tetrahydrofuran / petroleum ether) to give the intermediate tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3 (990 mg, 71.8%).

[0383] LC-MS:Rt:4.417min; MS m / z(ESI):306.2[M+H-56] + ;

[0384] 1H NMR (400MHz, CHLOROFORM-d)δ=8.17-8.27(m,1H)7.38(s,1H)7.25-7.31(m,1H)4.59-4.66(m,2H)3.20-3.29(m,2H)1.33(s,12H)1.31(s,9H)

[0385] Step 3: Synthesis of tert-butyl (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-4

[0386] (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 1-5(300 A mixture of tert-butyl (5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3 (300 mg, 830 μmol), tetrakistriphenylphosphine palladium (62.2 mg, 53.8 μmol), and potassium carbonate (148 mg, 1.08 mmol) was dissolved in anhydrous tetrahydrofuran (5 mL) and water (1 mL) and stirred at 80°C for 2 hours under a nitrogen atmosphere. The reaction was complete by LCMS. The reaction solution was extracted with ethyl acetate (50 mL), and the organic phase was washed with saturated brine (10 mL), dried, filtered, and concentrated. The residue was separated and purified by column chromatography (0-50% tetrahydrofuran / petroleum ether) to obtain the intermediate (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl) -6a,8a-dimethyl-4-oxoylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-4 (300 mg, 78.3%).

[0387] LC-MS:Rt:2.156min; MS m / z(ESI):712.3[M+H] + ;

[0388] Step 4: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 009)

[0389] Intermediate tert-butyl (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-4-oxylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolan-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-4 (220 mg, 309 μmol) was dissolved in anhydrous dichloromethane (4 mL). Trifluoroacetic acid (1 mL) was added and the mixture was stirred at 25°C for 40 minutes. The reaction was complete by LCMS. The reaction solution was blown dry with nitrogen, and the obtained residue was purified by high-efficiency preparative liquid chromatography [column: YMC-Pack CN150*30mm*5μm; mobile phase: A, 0.1% formic acid aqueous solution; B: acetonitrile, B%, 40%-60%, 12min] separation and purification were carried out to obtain compound (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 009 (2.00 mg, 1.06%).

[0390] LC-MS:Rt:2.576min; MS m / z(ESI):612.3[M+H] + ;

[0391] 1H NMR (400MHz, DMSO-d6) δ = 7.33-7.38 (m, 2H) 7.31 (d, J = 10.13Hz, 1H) 7.19 (d, J = 8.13Hz, 2H) 6.29 (d, J = 17.26Hz, 2H)6.17(dd,J=10.13,1.88Hz,1H)5.94(s,1H)5.39(s,1H)5.08(s,1H)4.90-4.94(m,1H)4.77-4.81(m,1H)4.4 5-4.73(m,3H)4.42(t,J=8.63Hz,2H)4.29(d,J=2.88Hz,1H)4.13-4.23(m,1H)3.71(s,2H)3.04(t,J=8.69Hz,2 H)2.30(s,1H)2.09-2.17(m,1H)1.98-2.06(m,1H)1.56-1.84(m,6H)1.40(s,3H)0.97-1.11(m,2H)0.86(s,3H).

[0392] Example 10: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (Compound 010)

[0393] Synthesis route and specific synthesis steps:

[0394] Step 1: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 010-3

[0395] The raw material fluocinolone SM-1 (1.00 g, 2.42 mmol) was dissolved in anhydrous acetonitrile (10 mL), and anhydrous magnesium sulfate (1.46 g, 12.1 mmol) was added. After stirring at 25°C for 1 hour, a solution of 4-bromomethylbenzaldehyde (579 mg, 2.91 mmol) in acetonitrile (2 mL) was added at 0°C, and then a solution of trifluoromethanesulfonic acid (1.82 g, 12.12 mmol) in acetonitrile (2 mL) was slowly added dropwise. The mixture was stirred at 0°C for five minutes. LCMS detected that the reaction was complete. The reaction was quenched with saturated sodium bicarbonate aqueous solution (20 mL), extracted with ethyl acetate (50 mL*2), and the organic phase was washed with saturated sodium chloride aqueous solution (20 mL), dried, filtered, and concentrated. The residue was purified by Prep-HPLC [column: YMC-Pack CN The reaction mixture was separated and purified by a 1% 4-[(4-bromomethyl)phenyl]-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 010-3 (640 mg, 42%).

[0396] LC-MS:Rt:2.845min; MS m / z(ESI):595.1[M+H] + ;

[0397] 1 H NMR (400MHz, DMSO-d6) δ = 7.62-7.39 (m, 4H), 7.27 (d, J = 10.3Hz, 1H), 6.30 (dd, J = 1.8, 10.0Hz,1H),6.13(s,1H),5.77-5.45(m,3H),5.01-4.95(m,1H),4.68(s,2H),4.55(br d,J=19.3Hz,1H),4.22(br d,J=19.3Hz,2H),2.74-2.56(m,1H),2.37-2.12(m,3H),2.09-1.95(m,1H),1.77-1.64(m,3H),1.56-1.42(m,4H),0.87(s,3H);

[0398] 19 F NMR (376MHz, DMSO-d6) δ = -164.99 (s, 1F), -186.39 (s, 1F).

[0399] Step 2: Synthesis of 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine 010-2 4-Bromo-2,3-dihydrobenzofuran-7-amine 1-3 (7.30 g, 34.1 mmol) was dissolved in 1,4-dioxane (150 mL), and bis(1,1-bis(diphenylphosphino)ferrocenepalladium chloride) (20.0 g, 78.8 mmol) was added, and the mixture was stirred at 80 ° C for 16 hours under a nitrogen atmosphere. LCMS showed that the reaction was complete. The reaction solution was filtered and concentrated. The residue was separated and purified by column chromatography (tetrahydrofuran / petroleum ether = 1 / 6) to give the intermediate 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine 010-2 (9.50 g, 96.0%).

[0400] LC-MS: Rt: 1.470min; MS m / z(ESI): 262.0[M+H];

[0401] Step 3: Synthesis of (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 010

[0402] (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4-( 2H)-ketone 010-3 (150 mg, 253 μmol) was dissolved in tetrahydrofuran (2 mL) and water (0.5 mL). 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine (79 mg, 303 μmol), potassium carbonate (79 mg, 303 μmol), and tetrakistriphenylphosphine palladium (29 mg, 25.3 μmol) were added sequentially. The mixture was reacted at 80°C under a nitrogen atmosphere for two hours. LCMS analysis indicated that the reaction was complete. The reaction solution was extracted with ethyl acetate (10 mL x 3). The organic phase was washed with saturated sodium chloride solution (5 mL), dried, filtered, and concentrated. The residue was purified by preparative chromatography [column: Phenomenex C18 The product was separated and purified by HPLC-MS / MS [75*30mm*3μm; mobile phase: A, 0.1% ammonia solution; B: acetonitrile, B%, 30%-70%, 9min] to obtain compound (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 010 (20.0 mg).

[0403] LC-MS:Rt:2.092min; MS m / z(ESI):648.3[M+H] + ;

[0404] 1H NMR (400MHz, DMSO-d6) δ = 7.33 (d, J = 8.0Hz, 2H), 7.27 (d, J = 9.8Hz, 1H), 7.18 (d, J = 8. 0Hz,2H),6.42-6.37(m,2H),6.30(dd,J=1.8,10.3Hz,1H),6.13(s,1H),5.75-5.56(m ,1H),5.52(d,J=2.8Hz,1H),5.45(s,1H),5.11(t,J=6.0Hz,1H),4.95(d,J=4.8Hz,1H ),4.56-4.38(m,5H),4.25-4.15(m,2H),3.73(s,2H),2.97(t,J=8.7Hz,2H),2.44(br s,1H),2.32-2.19(m,2H),2.10-2.00(m,1H),1.78-1.64(m,3H),1.50(s,4H),0.86(s,3H);

[0405] 19 F NMR (376MHz, DMSO-d6) δ = -164.96 (s, 1F), -186.39 (s, 1F).

[0406] Example 11: Synthesis of (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-amino-2,3-dihydro-1H-inden-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-6a, 6b, 7, 8, 8a, 8b, 11a, 12, 12a, 12b-decahydro-1H-naphtho[2', 1': 4, 5] indeno[1, 2-d][1, 3] dioxolane-4(2H)-one (Compound 011)

[0407] Synthesis route and specific synthesis steps:

[0408] Step 1: Synthesis of 7-bromo-2,3-dihydro-1H-inden-4-amine 11-2

[0409] Dissolve 2,3-dihydro-1H-inden-4-amine (2.00 g, 15.0 mmol) in N,N-dimethylformamide (20 mL). Add N-bromosuccinimide (NBS) (2.67 g, 15.0 mmol) at 0°C and stir at 0°C for 1 hour. LCMS confirms the reaction is complete. Quench with saturated sodium bicarbonate solution (50 mL) at 0°C and extract with ethyl acetate (100 mL x 2). The organic phase is washed with water (50 mL x 3) and saturated brine (50 mL), dried, filtered, and concentrated. The resulting residue is purified by column chromatography (0-15% ethyl acetate / petroleum ether) to afford 7-bromo-2,3-dihydro-1H-inden-4-amine 11-2 (1.70 g, 53.4%).

[0410] LC-MS:Rt:0.800min; MS m / z(ESI):213.8[M+H] + ;

[0411] Step 2: Synthesis of 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-4-amine 11-3

[0412] 7-Bromo-2,3-dihydro-1H-inden-4-amine 11-2 (100 mg, 471 μmol) and bis-indole boronate (239 mg, 943 μmol) were dissolved in 1,4-dioxane (5 mL). 1,1-Bis(diphenylphosphino)ferrocenepalladium chloride (34.5 mg, 47.2 μmol) and potassium acetate (92.5 mg, 943 μmol) were added. The mixture was stirred at 80°C under a nitrogen atmosphere for 12 hours. LCMS confirmed the completion of the reaction. The reaction solution was filtered and concentrated, and the resulting residue was purified by column chromatography (0-60% ethyl acetate / petroleum ether) to afford the intermediate 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-4-amine 11-3 (77.0 mg, 63.0%).

[0413] LC-MS:Rt:0.914min;MS m / z(ESI):260.3[M+H] + ;

[0414] Step 3: Synthesis of (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydro-1H-inden-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 011

[0415] 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydro-1H-inden-4-amine 11-3 (77 mg, 295.98 μmol) and (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,1 1a,12,12a,12b-Decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one 1-5 (110 mg, 197 μmol) was dissolved in tetrahydrofuran (5 mL) and water (1 mL). 1,1-Bis(diphenylphosphino)ferrocenepalladium chloride (14.4 mg, 19.7 μmol) and potassium carbonate (54.5 mg, 394 μmol) were added. The mixture was stirred at 80°C under a nitrogen atmosphere for 2 hours. LCMS confirmed the reaction was complete. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 2). The organic phase was washed with saturated brine (20 mL), dried, filtered, and concentrated. The resulting residue was purified by high performance preparative liquid chromatography (HPLC) using a Boston Prime C18 column. The product was separated and purified by HPLC-MS / MS (150*30mm*5μm; mobile phase: A, 0.1% formic acid aqueous solution; B, ACN, 29%-49%, 12 min) to give (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydro-1H-inden-4-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (55.0 mg, 45.7%).

[0416] LC-MS:Rt:0.856min;MS m / z(ESI):610.4[M+H] + ;

[0417] 1 H NMR (400MHz, DMSO-d6) δ=7.37-7.28(m,3H),7.16-7.11(m,1H),7.14(d,J=7.8Hz,1H),6.66(d,J=7.8Hz,1H),6.33(d,J=8.0Hz,1H) ,6.16(dd,J=1.6,10.2Hz,1H),5.93(s,1H),5.39(s,1H),5.08(s,1H),4.91(d,J=5.3Hz,1H),4.80-4.75(m,1H),4.75-4.55(m,2H), 4.50(d,J=17.8Hz,1H),4.29(s,1H),4.17(d,J=18.1Hz,1H),3.73(s,2H),2.66(t,J=7.5Hz,2H),2.63-2.57(m,2H),2.57-2.53(m,1 H),2.30(s,1H),2.16-1.98(m,2H),1.91(quin,J=7.4Hz,2H),1.81-1.60(m,5H),1.40(s,3H),1.11-0.97(m,2H),0.90-0.81(m,3H)

[0418] Example 12: Synthesis of 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-2-oxoethyl dihydrogen phosphate (Compound 009-p)

[0419] Synthesis route and specific synthesis steps:

[0420] Step 1: Synthesis of tert-butyl (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphinoyl)oxy)acetyl)-7-hydroxy-6a,8a-dimethyl-4-oxyde-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamate (009-p-1)

[0421] Intermediate 9-4 (80.0 mg, 112 μmol) was dissolved in N,N-dimethylformamide (4 mL), and di-tert-butyl N,N-diethylphosphoramidite (336 mg, 1.35 mmol) and tetrazole (78.7 mg, 1.12 mmol) were added. The mixture was stirred at room temperature for 30 minutes. TLC confirmed the reaction was complete. The mixture was cooled to 0°C, and hydrogen peroxide (580 mg, 5.12 mmol) was added. The mixture was returned to room temperature and stirred for 1 hour. LCMS confirmed the reaction was complete. The reaction was quenched by the addition of aqueous sodium sulfite (10 mL) at 0°C and extracted with ethyl acetate (30 mL). The organic phase was washed with saturated brine (10 mL), dried, filtered, and concentrated. The resulting residue was purified by column chromatography (0-60% ethyl acetate / petroleum ether) to afford Intermediate 009-p-1 (50.0 mg, 49.2%).

[0422] LC-MS:Rt:1.216min; MS m / z(ESI):904.6[M+H] + ;

[0423] Step 2: Synthesis of 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-2-oxoethyl dihydrogen phosphate 009-p

[0424] Intermediate 009-p-1 (50.0 mg, 55.3 μmol) was dissolved in dichloromethane (3 mL), and trifluoroacetic acid (630 mg, 5.53 mmol) was added. The mixture was stirred at room temperature for 30 minutes. LCMS confirmed the reaction was complete. The reaction solution was dried with nitrogen, and the resulting residue was purified by preparative HPLC (column: Boston Prime C18 150*30 mm*5 μm; mobile phase: A, 0.1% formic acid in water; B, acetonitrile, B%, 38%-58%, 12 min) to yield compound 009-p (10.0 mg, 26.1%).

[0425] LC-MS: Rt: 2.752min; MS m / z (ESI): 692.3[M+H];

[0426] 1 H NMR (400MHz, DMSO-d6) δ = 7.37-7.24 (m, 3H), 7.14 (d, J = 6.8Hz, 2H), 6.27 (s, 2H), 6.16 (dd,J=1.6,10.0Hz,1H),5.93(s,1H),5.47(s,1H),4.94-4.84(m,2H),4.66-4.56(m, 1H),4.40(t,J=8.6Hz,2H),4.28(s,1H),3.68(s,2H),3.00(t,J=8.7Hz,2H),2.29(d, J=2.9Hz,1H),2.16-1.94(m,3H),1.83-1.60(m,5H),1.39(s,3H),1.04-0.83(m,5H).

[0427] Synthesis of the intermediate (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butyloxy)-5-oxopentanoic acid INT-1

[0428] Synthesis route and specific synthesis steps:

[0429] Step 1: Synthesis of (S)-1-benzyl 5-tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)pentanediol

[0430] Dissolve (S)-1-benzyl-5-tert-butyl-2-aminopentanediol (5.00 g, 15.2 mmol) in dichloromethane (50 mL). Add 50% n-propyl cyclic phosphoric anhydride in ethyl acetate (10.1 g, 15.9 mmol), N,N-diisopropylethylamine (5.88 g, 45.5 mmol) and 2-(((benzyloxy)carbonyl)amino)acetic acid (3.49 g, 16.7 mmol) and stir at 25°C for 16 hours. The mixture was diluted with dichloromethane (300 mL), and the organic phase was washed with saturated aqueous sodium bicarbonate (100 mL) and saturated brine (50 mL), filtered, dried, and concentrated. The residue was separated and purified by column chromatography (0-30% tetrahydrofuran / petroleum ether) to give (S)-1-benzyl-5-tert-butyl-2-(2-(((benzyloxy)carbonyl)amino)acetamido)pentanediol (7.13 g).

[0431] LC-MS:Rt:0.991min; MS m / z(ESI):429.1[M-56+H] + ;

[0432] 1 H NMR (400MHz, DMSO-d6) δ = 8.32 (d, J = 7.5Hz, 1H), 7.43-7.28 (m, 10H), 5.75 (s, 1H), 5.13 (s, 2H), 5.0 5(s,2H),4.45-4.30(m,1H),3.72-3.59(m,2H),2.32-2.19(m,2H),2.04-1.79(m,2H),1.38(s,9H).

[0433] Step 2: Synthesis of (S)-2-(2-aminoacetylamino)-5-(tert-butoxy)-5-oxopentanoic acid

[0434] (S)-1-Benzyl-5-tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)pentanediol (7.13 g, 14.7 mmol) was dissolved in tetrahydrofuran (80 mL), 10% palladium on carbon (1.57 g) was added, and the mixture was stirred at 25°C under a hydrogen atmosphere (15 psi) for 16 hours. TLC confirmed the completion of the reaction. The reaction solution was diluted with water (200 mL), filtered, concentrated, and lyophilized to yield (S)-2-(2-aminoacetamido)-5-(tert-butyloxy)-5-oxopentanoic acid (3.83 g).

[0435] 1H NMR (400MHz, DMSO-d6) δ = 8.08 (s, 1H), 4.00 (s, 1H), 3.35 (s, 1H), 2.24-2.08 (m, 2H), 1.99-1.88 (m, 1H), 1.81-1.71 (m, 1H), 1.39 (s, 9H).

[0436] Step 3: Synthesis of (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butyloxy)-5-oxopentanoic acid INT-1

[0437] Dissolve (S)-2-(2-aminoacetamido)-5-(tert-butoxy)-5-oxopentanoic acid (3.83 g, 14.7 mmol) in a mixture of tetrahydrofuran (40 mL) and water (40 mL). Add 9-fluorenylmethylsuccinimidyl carbonate (Fmoc-Osu, 5.96 g, 17.7 mmol) and sodium bicarbonate (3.71 g, 44.1 mmol). Stir at 25°C for 16 hours. The reaction is complete by TLC. The reaction solution was adjusted to pH 3 with 1N dilute hydrochloric acid and extracted with ethyl acetate (150 mL x 2). The organic phase was washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered, and concentrated. The residue was separated and purified by column chromatography (0-54% tetrahydrofuran / petroleum ether) to give the intermediate (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-(tert-butoxy)-5-oxopentanoic acid (6.00 g).

[0438] LC-MS:Rt:0.947min; MS m / z(ESI):427.0[M-56+H] + ;

[0439] 1 H NMR (400MHz, DMSO-d6) δ = 12.70 (s, 1H), 8.11 (d, J = 7.8Hz, 1H), 7.90 (d, J = 7.5Hz, 2H), 7.72 (d, J = 7.5Hz, 2H), 7.54 (t, J = 6.0Hz, 1H), 7.45-7. 40(m,2H),7.36-7.31(m,2H),4.30-4.20(m,4H),3.73-3.63(m,2H),2 .29-2.22(m,2H),2.01-1.91(m,1H),1.38(s,9H),1.32-1.28(m,1H).

[0440] Example 13: Synthesis of (S)-4-(2-(2-bromoacetylamino)acetylamino)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-001)

[0441] Synthesis route and specific synthesis steps:

[0442] Step 1: Synthesis of compound L1-001-1

[0443] Compound 001 (2.00 g, 3.27 mmol) and (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid (Intermediate INT-1, 1.74 g, 3.60 mmol) were dissolved in dichloromethane (10 mL) and N,N-dimethylformamide (20 mL). 2,6-Lutidine (1.05 g, 9.81 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (HATU, 1.49 g, 3.92 mmol) were added sequentially, and the mixture was reacted at 25°C for 12 hours. The reaction was complete by LCMS. The reaction solution was diluted with ethyl acetate (250 mL), washed with saturated aqueous ammonium chloride solution (50 mL*2) and saturated aqueous sodium chloride solution (50 mL), dried, filtered, concentrated, and the residue was purified by column chromatography (Spherical C18 40-60 μm, Using decreasingly polar mixtures of water and acetonitrile as eluent; acetonitrile gradient ratio 80%) gave compound L1-001-1 (2.80 g).

[0444] LC-MS: Rt:5.510min; MS m / z(ESI):1077.2[M+H] + ;

[0445] SFC:Rt:3.241min.

[0446] Step 2: Synthesis of compound L1-001-2

[0447] Intermediate L1-001-1 (2.80 g, 2.60 mmol) was dissolved in N,N-dimethylformamide (30 mL). 1H-tetrazole (1.82 g, 26.0 mmol) and di-tert-butyl N,N-diethylphosphoramidite (7.78 g, 31.2 mmol) were added at 0°C and stirred at 25°C for 30 minutes. TLC confirmed the reaction was complete. Hydrogen peroxide (2.21 g, 30% w / w) was added at 0°C and stirred at 25°C for 1 hour. LCMS confirmed the reaction was complete. Aqueous sodium sulfite (20 mL) was added at 0°C to quench the reaction. The mixture was extracted with ethyl acetate (200 mL). The organic phase was washed with saturated brine (50 mL), dried, filtered, and concentrated to yield a crude product (3.3 g). The crude product was dissolved in acetonitrile (30 mL), piperidine (1.11 g, 13.0 mmol) was added at 0°C, and stirred at 25°C for 30 minutes. LCMS confirmed the reaction was complete. The reaction solution was concentrated under reduced pressure, and the residue was purified by column chromatography (Spherical C18 40-60 μm, Using decreasingly polar mixtures of water and acetonitrile as eluent; acetonitrile gradient ratio 40%) to give (S)-tert-butyl-4-(2-aminoacetamido)-5-((4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a -dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoate (L1-001-2) (1.80 g).

[0448] LC-MS:Rt:0.979min; MS m / z(ESI):1048.0[M+H] + ;

[0449] Step 3: Synthesis of compound L1-001-3

[0450] 2-Bromoacetic acid (478 mg, 3.44 mmol) was dissolved in N,N-dimethylformamide (20 mL), and ethyl 2-ethoxyquinoline-1(2H)-carboxylate (EEDQ, 851 mg, 3.44 mmol) was added. The mixture was stirred at 25°C for 1 hour, and a solution of intermediate L1-001-2 (1.80 g, 1.72 mmol) in N,N-dimethylformamide (10 mL) was added dropwise. The mixture was stirred at 25°C for 2 hours. LCMS confirmed the completion of the reaction. The reaction solution was diluted with ethyl acetate (100 mL), washed with diluted hydrobromic acid (10 mL) and saturated sodium bicarbonate solution (30 mL*3), dried, filtered, and concentrated. The residue was purified by reverse phase column chromatography (Spherical C18 40-60 μm, Using decreasingly polar mixtures of water and acetonitrile as eluent; acetonitrile gradient ratio 80%) to give (S)-tert-butyl 4-(2-(2-bromoacetylamino)acetylamino)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)oxy)acetyl)-7-hydroxy- 6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoate (L1-001-3) (1.40 g).

[0451] LC-MS:Rt:2.413min; MS m / z(ESI):1112.5[M-56+H] + .

[0452] Step 4: Synthesis of (S)-4-(2-(2-bromoacetylamino)acetylamino)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-001)

[0453] Intermediate L1-001-3 (0.65 g, 0.56 mmol) was dissolved in anhydrous dichloromethane (6 mL). Trifluoroacetic acid (2 mL) was added at 25°C and stirred for 30 minutes. LCMS confirmed the reaction was complete. The reaction solvent was dried with nitrogen, and the residue was dissolved in dichloromethane (5 mL) and dried with nitrogen three times. The residue was purified by preparative HPLC using a Waters Xbridge BEH C18 column (100 x 30 mm x 10 μm); eluents were a decreasingly polar mixture of water (containing 0.05% NH₄HCO₃) and acetonitrile; the acetonitrile gradient was 8% to 38% over 11 minutes. Compound L1-001 (0.31 g) was obtained.

[0454] LC-MS:Rt:2.937min; MS m / z(ESI):998.2 / 1000.2[M+H] + ;

[0455] 1 H NMR (400MHz, D2O) δ = 7.35 (d, J = 10.0Hz, 1H), 7.21 (d, J = 7.7Hz, 1H), 7.03 (d, J = 7.8Hz, 2H), 6.59 (d, J = 7.9Hz, 2H), 6.4 4(d,J=8.3Hz,1H),6.12-6.01(m,1H),5.69(s,1H),5.52(s,1H),5.00(s,1H),4.92-4.82(m,1H),4.78-4.74(m,1H),4 .43(dd,J=5.4,8.4Hz,1H),4.18(s,2H),4.10-3.99(m,1H),3.90(s,2H),3.86(s,2H),3.58-3.30(m,2H),2.49(s,1H) ,2.32(t,J=7.6Hz,4H),2.10-1.86(m,5H),1.85-1.54(m,4H),1.34-1.11(m,4H),0.81(s,3H),0.25(d,J=9.7Hz,2H).

[0456] Example 14: 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-((S)-6-amino-2-(2-(2-bromoacetamido)acetamido)hexanoylamino)-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-2-oxoethyl phosphate (L2-001)

[0457] Synthesis route and specific synthesis steps:

[0458] Step 1: N 2 -(((Benzyloxy)carbonyl)glycine)-N 6 Synthesis of -(tert-Butoxycarbonyl)-L-lysine benzyl ester (L2-001-2)

[0459] N 6 Benzyl-(tert-Butyloxycarbonyl)-L-lysine monohydrochloride (5.00 g, 13.4 mmol) was dissolved in anhydrous dichloromethane (50 mL). 1-Propylphosphonic anhydride (T3P, 8.96 g, 50% by weight in ethyl acetate), N,N-diisopropylethylamine (5.20 g, 40.2 mmol), and benzyloxycarbonylglycine (3.09 g, 14.8 mmol) were added and stirred at 25°C for 2 hours. LCMS confirmed the reaction was complete. The reaction was quenched by the addition of aqueous sodium bicarbonate (20 mL) at 0°C and extracted with dichloromethane (150 mL x 3). The organic phase was washed with saturated aqueous sodium chloride (60 mL), dried, filtered, concentrated, and the residue was purified by silica gel column chromatography (0-32% tetrahydrofuran / petroleum ether) to afford L2-001-2 (6.39 g).

[0460] LC-MS:Rt:0.957min;MS m / z(ESI):428.1[M-100+H] + ;

[0461] 1H NMR (400MHz, DMSO-d6) δ = 8.27 (d, J = 7.5Hz, 1H), 7.46-7.40 (m, 1H), 7.39-7.27 (m, 10H), 6.76 (t, J = 5.2Hz, 1H), 5.12 (s, 2H), 5.01 (br s,2H),4.33-4.22(m,1H),3.74-3.63(m,2H),2.87(q,J=6.3Hz,2H),1.73-1.55(m,2H),1.37(s,9H),1.35-1.19(m,4H).

[0462] Step 2: Synthesis of L2-001-3 of (S)-2-(2-aminoacetamido)-6-((tert-butoxycarbonyl)amino)hexanoic acid

[0463] Intermediate L2-001-2 (7.95 g, 15.1 mmol) was dissolved in tetrahydrofuran (90 mL), and 10% wet palladium on carbon (1.60 g) was added. The atmosphere was replaced with hydrogen three times, and the mixture was allowed to react at 25°C under a hydrogen atmosphere (15 PSI) for 16 hours. TLC indicated the disappearance of the starting material. The reaction mixture was diluted with water (100 mL) and methanol (50 mL), filtered, and concentrated to yield (S)-2-(2-aminoacetylamino)-6-((tert-butoxycarbonyl)amino)hexanoic acid L2-001-3 (3.82 g).

[0464] 1 H NMR (400MHz, DMSO-d6) δ = 8.08 (br s, 1H), 6.76 (br s, 1H), 4.02 (br s,1H),3.33-3.19(m,2H),2.86(q,J=6.5Hz,2H),1.70-1.51(m,2H),1.37-1.29(m,11H),1.22(d,J=6.3Hz,2H).

[0465] Step 3: Synthesis of (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-6-((tert-butoxycarbonyl)amino)hexanoic acid L2-001-4

[0466] (S)-2-(2-Aminoacetylamino)-6-((tert-butoxycarbonyl)amino)hexanoic acid L2-001-3 (3.82 g, 12.6 mmol) was dissolved in a mixture of tetrahydrofuran (40 mL) and water (40 mL). Sodium bicarbonate (3.17 g, 37.8 mmol) and 9-fluorenylmethyl-N-succinimidyl carbonate (5.10 g, 15.1 mmol) were added and stirred at 25°C for 16 hours. The reaction was complete by LCMS. 1 M dilute hydrochloric acid was added dropwise at 0°C to adjust the pH to 7, and the mixture was extracted with ethyl acetate (150 mL*3). The organic phase was washed with saturated aqueous sodium chloride solution (50 mL), dried, filtered, and concentrated. The residue was separated and purified by silica gel column chromatography (0-36% tetrahydrofuran / petroleum ether) to give the intermediate (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-6-((tert-butoxycarbonyl)amino)hexanoic acid L2-001-4 (6.07 g).

[0467] LC-MS:Rt:0.960min;MS m / z(ESI):426.1[M-100+H] + ;

[0468] 1 H NMR(400MHz,DMSO-d6)δ=12.60(br s,1H),8.07(d,J=7.8Hz,1H),7.89(d,J=7.5Hz,2H),7.72(d,J=7.5Hz,2H),7.52(t,J=6.1Hz,1H),7.44-7.40(m,2H),7.35-7.31(m,2H), 6.76(t,J=5.2Hz,1H),4.30-4.17(m,4H),3.73-3.63(m,2H),2.88(q,J=6.5Hz,2H),1.73-1.55(m,2H),1.36(s,11H),1.31-1.24(m,2H).

[0469] Step 4: Synthesis of compound L2-001-5

[0470] Compound 001 and intermediate (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-6-((tert-butoxycarbonyl)amino)hexanoic acid L2-001-4 (644 mg, 1.23 mmol) were dissolved in anhydrous dichloromethane (30 mL) and anhydrous N,N-dimethylformamide (5 mL). O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (435 mg, 1.14 mmol) and 2,6-lutidine (263 mg, 2.45 mmol) were added at 0°C, and the mixture was stirred at 25°C for 16 hours. The reaction was complete by LCMS. The reaction solution was diluted with anhydrous dichloromethane (200 mL), the organic phase was washed with water (50 mL x 2) and saturated aqueous sodium chloride solution (30 mL), dried, filtered, and concentrated. The residue was separated and purified by column chromatography (0-50% tetrahydrofuran / petroleum ether). The crude product was separated and purified by preparative high performance liquid chromatography (column: C18-1 150*30mm*5μm; a decreasingly polar mixture of water (containing 0.05% TFA) and acetonitrile as the eluent; an acetonitrile gradient ratio of 42%-82%, elution time 9 minutes) to give L2-001-5 (424 mg, 46.4%).

[0471] LC-MS: Rt: 1.081min; MS m / z (ESI): 1019.4[M+H];

[0472] 1H NMR (400MHz, DMSO-d6) δ = 9.34 (s, 1H), 8.03 (br d,J=8.0Hz,1H),7.89(d,J=7.4Hz,2H),7.71(d,J=7.5Hz,2H),7.54(t,J=6.2Hz,1H),7.47-7.26(m,8H),7.19(d,J=8.0Hz,2H),6.73(s ,1H),6.59(d,J=8.3Hz,1H),6.16(dd,J=1.6,10.1Hz,1H),5.92(s,1H),5.40(s,1H),5.08(t,J=5.9Hz,1H),4.92(d,J=5.0Hz,1H),4.7 8(d,J=3.0Hz,1H),4.56-4.45(m,4H),4.33-4.12(m,5H),3.84(s,2H),3.67(d,J=5.8Hz,2H),3.06(t,J=8.8Hz,2H),2.87(d,J=5.9Hz, 2H),2.73-2.55(m,1H),2.35-2.24(m,2H),2.17-1.94(m,3H),1.81-1.49(m,7H),1.42-1.31(m,14H),1.09-0.96(m,2H),0.86(s,3H).

[0473] Step 5: Synthesis of compound L2-001-6

[0474] Intermediate L2-001-5 (200 mg, 179 μmol) and 1H-tetrazole (125 mg, 1.79 mmol) were dissolved in anhydrous N,N-dimethylformamide (2 mL). Di-tert-butyl N,N-diethylphosphoramidite (535 mg, 2.14 mmol) diluted in anhydrous N,N-dimethylformamide (5 mL) was added at 0°C, and the mixture was stirred at 25°C for 1 hour. Hydrogen peroxide solution (111 mg, 30% w / w) diluted in anhydrous N,N-dimethylformamide (0.5 mL) was added at 0°C, and the mixture was stirred at 25°C for 1 hour. The reaction was complete by LCMS. Aqueous sodium sulfite solution (2 mL) was added at 0°C to quench the reaction, and the mixture was extracted with ethyl acetate (150 mL). The organic phase was washed with water (30 mL x 3) and saturated aqueous sodium chloride solution (30 mL), dried, filtered, and concentrated. The residue was separated and purified by column chromatography (0-55% tetrahydrofuran / petroleum ether) to give compound L2-001-6 (196 mg, 83.6%).

[0475] LC-MS:Rt:1.153min;MS m / z(ESI):1212.6[M-100+H] + .

[0476] Step 6: Synthesis of compound L2-001-7

[0477] Intermediate L2-001-6 (150 mg, 114 μmol) was dissolved in acetonitrile (2 mL), and piperidine (20.0 mg, 234 μmol) was added. The mixture was stirred at 0°C for 10 minutes. LCMS confirmed the reaction was complete. The reaction solution was concentrated to afford compound L2-001-7 (124 mg). LC-MS: Rt: 0.989 min; MS m / z (ESI): 1091.0 [M+H];

[0478] Step 7: Synthesis of compound L2-001-8

[0479] Intermediate L2-001-7 (120 mg, 110 μmol) was dissolved in anhydrous N,N-dimethylformamide (2 mL), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 33.0 mg, 132 μmol) and bromoacetic acid (23.0 mg, 165 μmol) were added. The mixture was stirred at 25°C for 2 hours. LCMS confirmed the reaction was complete. The reaction solution was diluted with ethyl acetate (100 mL), and the organic phase was washed with diluted hydrobromic acid (10 mL), saturated aqueous sodium bicarbonate (20 mL x 2), and saturated brine (20 mL), dried, filtered, and concentrated. The residue was isolated and purified by silica gel column chromatography (0-65% tetrahydrofuran / petroleum ether) to afford compound L2-001-8 (65.0 mg).

[0480] LC-MS:Rt:1.110min; MS m / z(ESI):1111.3[M-100+H] + ;

[0481] Step 8: Synthesis of compound L2-001

[0482] Intermediate L2-001-8 (50.0 mg, 41.3 μmol) was dissolved in anhydrous dichloromethane (0.5 mL), trifluoroacetic acid (0.2 mL) was added, and the mixture was stirred at 25°C for 40 minutes. LCMS confirmed the reaction was complete. The reaction solution was dried with nitrogen, and the resulting residue was purified by preparative HPLC (column: Boston Green ODS 150*30 mm*5 μm; eluent: decreasingly polar mixtures of water (containing 0.05% TFA) and acetonitrile; acetonitrile gradient ratio 22%-37%, elution time 10 minutes) to afford compound L2-001 (5.32 mg, 12.9%).

[0483] LC-MS: Rt:0.822min; MS m / z(ESI):996.9 / 998.9[M+H];

[0484] 1 H NMR (400MHz, DMSO-d6) δ = 9.36 (s, 1H), 8.61-8.39 (m, 1H), 8.19 (d, J = 7.9Hz, 1H), 7.67 (s, 3H), 7.54 (d, J = 8.1Hz, 1H), 7.26-7.1 5(m,3H),6.91(s,2H),6.56(d,J=8.3Hz,1H),6.10(d,J=10.4Hz,1H),5.83(s,1H),5.43(s,1H),4.91(d,J=4.9Hz,1H),4.82-4. 73(m,2H),4.59-4.50(m,1H),4.46-4.38(m,2H),4.17(s,1H),3.94(s,2H),3.88-3.63(m,7H),2.90-2.72(m,5H),2.17(d,J=1 0.5Hz,1H),2.03(s,2H),1.89(s,1H),1.78-1.65(m,3H),1.55(d,J=12.9Hz,5H),1.35(s,5H),0.85(s,3H),0.73-0.56(m,1H).

[0485] Example 15: (S)-4-(2-(2-bromoacetamido)acetamido)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-009)

[0486] Synthesis route and specific synthesis steps:

[0487] Step 1: Synthesis of compound L1-009-1

[0488] Compound 009 (1.50 g, 2.45 mmol) and intermediate (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid INT-1 (1.30 g, 2.70 mmol) were dissolved in dichloromethane (12 mL) and N,N-dimethylformamide (24 mL). O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (1.31 g, 3.43 mmol) and 2,6-lutidine (788 mg, 7.36 mmol) were added at 0°C, and the reaction solution was stirred at 25°C for 16 hours. LCMS detection indicated that the reaction was complete. The reaction solution was diluted with ethyl acetate (250 mL), washed with saturated aqueous ammonium chloride solution (50 mL*2) and saturated aqueous sodium chloride solution (50 mL), dried, filtered, concentrated, and the residue was purified by column chromatography (Spherical C18 40-60 μm, The mixture of water and acetonitrile with decreasing polarity was used as the eluent; the gradient ratio of acetonitrile was 80%) to separate and purify the product to give compound L1-009-1 (2.60 g, 98.5%).

[0489] LC-MS:Rt:5.134min; MS m / z(ESI):1076.5[M+H] + ;

[0490] Step 2: (S)-tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)acetyl)-7-hydroxy-6a,8a- Synthesis of dimethyl-4-oxydecahydro-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxydecahydropentyl ester L1-009-02

[0491] Compound L1-009-1 (2.40 g, 2.23 mmol) and (1H-tetrazole) (1.56 g, 22.3 mmol) were dissolved in N,N-dimethylformamide (10 mL). Di-tert-butyl N,N-diethylphosphoramidite (6.67 g, 26.8 mmol) was added at 0°C and stirred at 25°C for 30 minutes. TLC confirmed the reaction was complete. Hydrogen peroxide (3.12 g, 30% w / w) was added at 0°C and stirred at 25°C for 1 hour. LCMS confirmed the reaction was complete. Aqueous sodium sulfite (20 mL) was added at 0°C to quench the reaction. The mixture was extracted with ethyl acetate (200 mL). The organic phase was washed with saturated brine (50 mL), dried, filtered, and concentrated to yield the crude product (2.80 g). The resulting product was dissolved in acetonitrile (30 mL), piperidine (938 mg, 11.0 mmol) was added at 0°C, and stirred at 25°C for 30 minutes. The reaction solution was concentrated and the residue was purified by column chromatography (Spherical C18 40-60 μm, The resulting product was separated and purified using decreasingly polar mixtures of water and acetonitrile as eluents; the acetonitrile gradient ratio was 40%) to afford compound L1-009-02 (1.40 g, 60.0%).

[0492] LC-MS:Rt:0.972min; MS m / z(ESI):1046.3[M+H] + ;

[0493] Step 3: Synthesis of compound L1-009-03

[0494] The intermediate L1-009-02 (1.40 g, 1.34 mmol) was dissolved in N,N-dimethylformamide (5 mL), and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 530 mg, 2.14 mmol) and bromoacetic acid (372 mg, 2.68 mmol) were added and stirred at 25°C for 2 hours. LCMS detection showed that the reaction was complete. The reaction solution was diluted with ethyl acetate (100 mL), washed with diluted hydrobromic acid (10 mL) and saturated sodium bicarbonate aqueous solution (30 mL*3), dried, filtered, and concentrated. The residue was purified by column chromatography (Spherical C18 40-60 μm, The mixture of water and acetonitrile with decreasing polarity was used as the eluent; the acetonitrile gradient ratio was 80%) to separate and purify the product to give compound L1-009-03 (1.00 g, 64.0%).

[0495] LC-MS: Rt: 5.260min; MS m / z (ESI): 1168.4[M+H];

[0496] 1H NMR (400MHz, DMSO-d6) δ = 9.36 (s, 1H), 8.49 (t, J = 5.6Hz, 1H), 8.19 (d, J = 8.1Hz, 1H), 7. 44(s,1H),7.39-7.31(m,3H),7.23(d,J=8.0Hz,2H),6.85(s,1H),6.17(dd,J=1.8,10.0 Hz,1H),5.98-5.85(m,1H),5.93(s,1H),5.49(s,1H),5.00-4.88(m,3H),4 .62(dd,J=9.1,17.9Hz,1H),4.56-4.48(m,3H),4.31(s,1H),3.94(s,2H),3 .83-3.77(m,3H),3.15(t,J=8.6Hz,2H),2.31-2.18(m,3H),2.16-1.86(m,5 H),1.78-1.66(m,5H),1.47-1.38(m,30H),1.11-1.00(m,2H),0.89(s,3H).

[0497] Step 4: Synthesis of compound LI-009

[0498] Intermediate L1-009-03 (700 mg, 0.6 mmol) was dissolved in dichloromethane (6 mL), trifluoroacetic acid (2 mL) was added, and the mixture was stirred at 25°C for 30 minutes. LCMS confirmed the reaction was complete. The reaction solution was dried with nitrogen, and the reaction solvent was blown dry with nitrogen. The residue was dissolved in dichloromethane (5 mL) and then blown dry with nitrogen three times. The residue was purified by preparative HPLC (Phenomenex C18 column, 150 x 40 mm x 5 μm; eluent: a decreasingly polar mixture of water (containing 0.05% NH4HCO3) and acetonitrile; acetonitrile gradient from 0% to 30% over 10 minutes) to yield compound LI-009 (314 mg).

[0499] LC-MS: Rt: 3.018min; MS m / z (ESI): 998.1 / 1000.2[M+H];

[0500] 1H NMR(400MHz,DEUTERIUM OXIDE)δ=7.35(d,J=9.8Hz,1H),7.17(s,1H),6.97(d,J=7.8Hz,2H),6.59(d,J=7.8Hz,2H),6.21(s,1H),6.1 2(d,J=10.8Hz,1H),5.77(s,1H),5.48(s,1H),4.95(d,J=4.5Hz,1H),4.90-4.81(m,1H),4.69-4.64(m,2H),4 .48-4.42(m,1H),4.39-4.28(m,2H),4.14(s,1H),3.88-3.82(m,3H),3.56-3.40(m,2H),2.64-2.42(m,2H), 2.40-2.23(m,3H),2.03-1.79(m,5H),1.77-1.45(m,4H),1.28-1.04(m,4H),0.77(s,3H),0.16-0.05(m,2H).

[0501] Example 16: Synthesis of (S)-4-(2-(2-bromoacetamido)acetamido)-5-((4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-010)

[0502] Synthesis route and specific synthesis steps:

[0503] Step 1: Synthesis of compound L1-010-01

[0504] Compound 010 (650 mg, 1.00 mmol) and (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxopentanoic acid (533 mg, 1.10 mmol) were dissolved in a mixed solvent of dichloromethane (2.5 mL) and anhydrous N,N-dimethylformamide (5 mL). 2,6-Lutidine (323 mg, 3.01 mmol) and 2-(7-azobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate (HATU, 458 mg, 1.20 mmol) were added at 0°C, and the mixture was stirred at 25°C for 12 hours. The reaction was complete by LCMS. The reaction solution was quenched by adding water (10 mL) and extracted with ethyl acetate (50 mL*2). The organic phase was washed with saturated brine (30 mL), dried, filtered, and concentrated. The residue was purified by column chromatography (Spherical C18 40-60 μm, The mixture of water and acetonitrile with decreasing polarity was used as the eluent; the gradient ratio of acetonitrile was 30%-70%) to separate and purify the compound L1-010-01 (500 mg).

[0505] LC-MS: Rt: 2.351min; MS m / z (ESI): 1112.7[M+H].

[0506] Step 2: Synthesis of compound L1-010-02

[0507] Compound L1-010-01 (300 mg, 0.27 mmol) was dissolved in anhydrous N,N-dimethylformamide (6 mL). 1H-tetrazole (189 mg, 2.70 mmol) and di-tert-butyl N,N-diethylphosphoramidite (807 mg, 3.24 mmol) were added at 0°C and stirred at 25°C for 1 hour. TLC confirmed the completion of the reaction. A solution of hydrogen peroxide (210 mg, 30% w / w) in N,N-dimethylformamide (1 mL) was added dropwise at 0°C and stirred at 25°C for 2 hours. LCMS confirmed the completion of the reaction. Saturated aqueous sodium sulfite solution (3 mL) was added at 0°C to quench the reaction. The mixture was extracted with ethyl acetate (50 mL). The organic phase was washed with water (20 mL*3) and saturated brine (30 mL), dried, filtered, and concentrated. The residue was purified by column chromatography (Spherical C18 40-60 μm, The mixture of water and acetonitrile with decreasing polarity was used as the eluent; the gradient ratio of acetonitrile was 30%-70%) to separate and purify the mixture to obtain compound L1-010-02 (170 mg).

[0508] LC-MS: Rt: 6.129min; MS m / z (ESI): 1304.8[M+H];

[0509] Step 3: Synthesis of compound L1-010-03

[0510] Compound L1-010-02 (170 mg, 130 μmol) was dissolved in acetonitrile (6 mL), and piperidine (55.5 mg, 652 μmol) was added. The mixture was stirred at 25°C for 1 hour. LCMS confirmed the reaction was complete. The reaction solution was concentrated to afford compound L1-010-03 (140 mg).

[0511] LC-MS: Rt: 1.785min; MS m / z (ESI): 1046.5[M+H].

[0512] Step 4: Synthesis of compound L1-010-04

[0513] 2-Bromoacetic acid (36.0 mg, 259 μmol) was dissolved in anhydrous N,N-dimethylformamide (1 mL). 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ, 64.0 mg, 259 μmol) was added at 0°C. After stirring at 25°C for 1 hour, a solution of intermediate L1-010-03 (140 mg, 129 μmol) in anhydrous N,N-dimethylformamide (0.5 mL) was added and stirred at 25°C for 2 hours. LCMS confirmed the completion of the reaction. Aqueous hydrobromic acid (1 M, 1 mL) was added dropwise at 0°C to quench the reaction. The mixture was extracted with ethyl acetate (50 mL). The organic phase was washed with water (10 mL x 2) and saturated aqueous sodium bicarbonate (10 mL), dried, filtered, and concentrated. The residue was purified by column chromatography (Spherical C18 40-60 μm, The mixture of water and acetonitrile with decreasing polarity was used as the eluent; the gradient ratio of acetonitrile was 30%-70%) to separate and purify the product to obtain compound L1-010-04 (24.4 mg, 15.9%).

[0514] LC-MS: Rt: 2.003min; MS m / z (ESI): 1204.4[M+H].

[0515] Step 5: Synthesis of compound L1-010

[0516] Intermediate L1-010-04 (24.4 mg, 20.3 μmol) was dissolved in anhydrous dichloromethane (1 mL). Trifluoroacetic acid (308 mg, 2.70 mmol) was added at 25°C and stirred at 25°C for 1 hour. LCMS confirmed the reaction was complete. The solvent was removed by evaporation under a stream of nitrogen. The residue was purified by HPLC (column: Boston Prime C18 150*30 mm*5 μm; eluent: decreasingly polar mixtures of water (containing 0.05% NH4HCO3) and acetonitrile; acetonitrile gradient: 13% to 33% over 11 minutes) to yield compound L1-010 (3.25 mg).

[0517] LC-MS: Rt: 2.671min; MS m / z (ESI): 1034.2 / 1036.3[M+H];

[0518] 1 H NMR (400MHz, DMSO-d6) δ=9.56-9.32(m,1H),8.54-8.38(m,1H),8.21(d,J=7.9Hz,1H),7.44(d,J=8.2Hz,1H),7.36(d,J =7.9Hz,2H),7.29-6.96(m,5H),6.65-6.60(m,1H),6.28(d,J=11.6Hz,1H),6.12(s,1H),5.77-5.55(m,1H),5.46(s,1H ),4.96(d,J=4.6Hz,1H),4.66(s,2H),4.59-4.49(m,3H),4.18(d,J=9.8Hz,1H),3.94(s,1H),3.87-3.78(m,4H),3.77- 3.64(m,2H),3.07(t,J=8.7Hz,2H),2.30-2.21(m,4H),2.08-1.89(m,4H),1.84-1.65(m,3H),1.52(s,4H),0.93(s,3H).

[0519] Example 17-1: Humanized Design of Infliximab Antibody

[0520] By comparing the IMGT (http: / / imgt.cines.fr) database of human antibody heavy and light chain variable region germline genes, we selected heavy and light chain variable region germline genes with high homology to mouse antibodies as templates. The mouse antibody CDRs were then transplanted into the corresponding humanized templates, resulting in a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Based on the antibody's three-dimensional structure, we backmutated buried residues, residues that directly interact with the CDRs, and residues in the framework regions that significantly influence VL and VH conformations, thereby generating humanized monoclonal antibodies. The humanized light chain template for the chimeric antibody Infliximab is IGKV6-21*01, and the humanized heavy chain template is IGHV3-73*01. The CDRs of Infliximab were transplanted into the corresponding humanized templates to generate the corresponding humanized versions. As needed, key amino acids in the FR region of the humanized infiliximab antibody were backmutated to their mouse counterparts to maintain the original affinity. Detailed backmutation design is shown in Table 1.

[0521] Table 1: Back mutation design of Infliximab humanized antibody

[0522] The specific sequences of the variable regions of Infliximab and its humanized antibody are as follows:

[0523] The amino acid sequence of Infliximab.VL is shown in SEQ ID NO.1:

[0524] The amino acid sequence of Infliximab.VL1 is shown in SEQ ID NO.2:

[0525] The amino acid sequence of Infliximab.VL2 is shown in SEQ ID NO.3:

[0526] The amino acid sequence of Infliximab.VH is shown in SEQ ID NO.4:

[0527] The amino acid sequence of Infliximab.VH1 is shown in SEQ ID NO.5:

[0528] The amino acid sequence of Infliximab.VH2 is shown in SEQ ID NO.6:

[0529] The amino acid sequence of Infliximab.VH3 is shown in SEQ ID NO.7:

[0530] The amino acid sequence of Infliximab.VH4 is shown in SEQ ID NO.8:

[0531] The amino acid sequence of Infliximab.VH5 is shown in SEQ ID NO.9:

[0532] The above-mentioned different light chain and heavy chain sequences were cross-combined to obtain a variety of infliximab humanized antibodies. The variable region combinations of each antibody are as follows:

[0533] Table 2: Amino acid sequences corresponding to the variable regions of the humanized Infliximab antibody

[0534] The antibody constant region sequences are as follows:

[0535] Table 3: Heavy chain constant region and light chain constant region sequences

[0536] Expression and purification of humanized antibodies

[0537] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector containing a signal peptide and heavy chain constant region / light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). The corresponding heavy chain and light chain plasmids and the transfection reagent PEI (Polysciences, Catalog No.: 24765-1) were added to OPTI-MEM (Gibco, Catalog No.: 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293 cells (Thermofisher, Catalog No.: A14527) and cultured in a shaking incubator at 37°C with 5% CO2 and 120 rpm. The day after transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No.: F081918-001) and glucose (Sigma, Catalog No.: G7528) were added. On the sixth day of transfection, the cell supernatant was collected and purified with Protein A (GE, Catalog No.: 28985254). The eluted sample was dialyzed into PBS buffer, pH 7.4, to obtain the Infliximab humanized antibody.

[0538] ELISA binding experiment of humanized antibody and human TNFα protein

[0539] Human TNFα protein (Acro Biosystems, Catalog No. TNA-H5228) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS, and blocked with 2% BSA blocking buffer at 37°C for 1.5 hours. After rinsing three times with 0.05% Tween 20-PBS, serially diluted samples were added and incubated at 37°C for 1 hour. After rinsing three times with 0.05% Tween 20-PBS, secondary antibody HRP goat anti-human IgG Fc (Merck, Catalog No. AP113) was added and incubated at 37°C for 1 hour. After rinsing three times with 0.05% Tween 20-PBS, TMB solution (Seracare, Catalog No. 5120-0077) was added. The reaction was incubated at room temperature for 10 minutes, and then terminated with 1M hydrochloric acid. The plate was read at 450 nm using a microplate reader. The humanized molecule has a comparable binding capacity to human TNFα protein as Infliximab and binds to EC 50 See Table 4 for values.

[0540] Table 4: ELISA binding assay of humanized Infliximab antibody and human TNFα protein

[0541] Biacore binding assay of humanized antibodies and human TNFα protein

[0542] A multi-cycle kinetic method was used to determine the affinity of antibodies to antigens using a BIAcore 8K instrument. In each cycle, the antibody to be tested was first captured using a Protein A chip (GE, Catalog No.: 29-1275-56). Then, the antigen, human TNFα protein (Acro Biosystems, Catalog No.: TNA-H5228), was injected, and the binding and dissociation processes of the antibody and antigen protein were recorded. Finally, the chip was regenerated with Glycine pH 1.5. The mobile phase was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20), the flow rate was 30 μL / min, the association time was 240 s, the dissociation time was 900 s, the regeneration time was 30 s, the detection temperature was 25°C, and the antigen concentration ranged from 10 to 0.15625 nM. The data were analyzed using Biacore 8K analysis software (version 2.0) based on a 1:1 binding model, and the kinetic parameters of antibody-antigen binding, including the association rate constant ka, the dissociation rate constant kd, and the equilibrium dissociation constant KD, were fitted. The results are shown in Table 5. The affinity of the humanized antibody was similar to that of infliximab.

[0543] Table 5: Binding affinity of humanized infliximab antibody to human TNFα protein

[0544] Humanized Antibody Hotspot Repair

[0545] Based on the binding affinity results, Infliximab-Hu01, which has the fewest back mutations, was selected. Because the heavy chain variable region of Infliximab.VH1 contains amino acid DD, there is a risk of isomerization during antibody expression and this requires repair. The following four Infliximab.VH1 variants were designed, with the following sequences:

[0546] The amino acid sequence of Infliximab.VH1a is shown in SEQ ID NO.12:

[0547] The amino acid sequence of Infliximab.VH1b is shown in SEQ ID NO.13:

[0548] The amino acid sequence of Infliximab.VH1c is shown in SEQ ID NO.14:

[0549] The amino acid sequence of Infliximab.VH1d is shown in SEQ ID NO.15:

[0550] The corresponding heavy chain plasmids were constructed using the aforementioned method and co-transfected into Expi293 cells with a light chain plasmid containing the variable region sequence of Infliximab.VL1. The supernatant was collected and purified to obtain four hotspot-repaired humanized antibodies. The amino acid sequences of the variable regions of each antibody are as follows:

[0551] Table 6: Hotspot repaired amino acid sequences of the variable regions of the humanized Infliximab antibody

[0552] These four antibodies were tested by ELISA and Biacore, and the specific protocols were as described above. 50 The values ​​and Biacore test results are shown in Table 7. Except for Infliximab-Hu01d, the affinities of the other three antibodies were comparable to those of Infliximab-Hu01.

[0553] Table 7: Affinity detection of hotspot repaired Infliximab humanized antibody

[0554] Example 17-2: Antibody Preparation

[0555] Preparation of 17-2-1 anti-human TNFα antibody

[0556] The amino acid sequences of the variable and constant regions of the antibodies used to prepare anti-human TNFα antibody-drug conjugates are shown in Table 8, and the amino acid sequences of the CDRs of the antibodies are shown in Table 9, where Infliximab-h is the humanized antibody Infliximab-Hu01c.

[0557] Based on the sequences in Table 8, heavy chain (HC) and light chain (LC) plasmids for producing anti-human TNFα antibodies were constructed (nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector containing a signal peptide and heavy chain constant region / light chain constant region sequences to obtain a recombinant plasmid expressing VH-CH1-Fc / VL-CL). The anti-human TNFα antibodies Nerelimomab, Certolizumab, Infliximab-h, Golimumab, and Adalimumab were prepared according to the antibody expression and purification method of Example 17-1.

[0558] Table 8: Amino acid sequences of anti-human TNFα antibodies

[0559] Table 9: CDR amino acid sequences of anti-human TNFα antibodies (Kabat)

[0560] Preparation of 17-2-2 anti-human IL-4R antibody

[0561] The amino acid sequences of the antibody variable and constant regions used to prepare anti-IL-4R antibody-drug conjugates are as follows, and their sequences are referenced in patent PCT / US2007 / 021210.

[0562] Dupilumab heavy chain (HC, SEQ ID NO. 55): (The underlined bold portion corresponds to VH-SEQ ID NO. 57; the shaded portion corresponds to CH-SEQ ID NO. 59)

[0563] Dupilumab HCDR1 (SEQ ID NO.60): DYAMT

[0564] Dupilumab HCDR2(SEQ ID NO.61):SISGSGGNTYYADSVKG

[0565] Dupilumab HCDR3 (SEQ ID NO.62): DRLSITIRPRYYGLDV

[0566] Dupilumab light chain (LC, SEQ ID NO. 56): (The underlined bold portion corresponds to VL-SEQ ID NO. 58; the shaded portion corresponds to CL-SEQ ID NO. 11)

[0567] Dupilumab LCDR1 (SEQ ID NO.63): RSSQSLLYSIGYNYLD

[0568] Dupilumab LCDR2 (SEQ ID NO.64): LGSNRAS

[0569] Dupilumab LCDR3 (SEQ ID NO.65): MQALQTPYT

[0570] Based on the above sequences, heavy chain (HC) and light chain (LC) plasmids for producing anti-human IL-4R antibodies were constructed (nucleic acid sequences encoding the antibody VH and VL were recombined into an expression vector carrying a signal peptide and heavy chain constant region / light chain constant region sequences to produce a recombinant plasmid expressing VH-CH1-Fc / VL-CL). Dupilumab antibody was prepared according to the antibody expression and purification method described in Example 17-1.

[0571] Additional anti-human IL-4R antibodies were prepared using the above-described method for preparing dupilumab. The specific variable region sequences, CDR sequences, and antibody sources are shown in the table below. Antibodies 002Mab, 003Mab, 004Mab, 005Mab, and 006Mab were prepared using the antibody expression and purification method described in Example 17-1.

[0572] Table 9-1: Amino acid sequences of VH, VL and CDRs of anti-human IL4R antibodies (Kabat)

[0573] Example 18: Preparation of Antibody-Drug Conjugates

[0574] The anti-mouse TNFα antibody used to prepare the anti-mouse TNFα antibody-drug conjugate (hereinafter referred to as "001Mab") was purchased from BioXcell with the product number BE0058.

[0575] Anti-Hel-hIgG4, used to prepare isotype control for anti-human IL-4R antibody-drug conjugates, was purchased from Bio-Innovation with the catalog number B669701.

[0576] The anti-human TNFα antibody used to prepare the anti-human TNFα antibody-drug conjugate and the anti-human IL-4R antibody used to prepare the anti-human IL-4R antibody-drug conjugate were prepared according to Example 17-2.

[0577] Conjugation: The above-mentioned antibody was added to an Amicon-Ultra-30kD ultrafiltration tube and concentrated to 20 mM phosphate buffer, pH 7.0. 10 mM diphenylphosphinoacetic acid was added, and the mixture was placed on a thermostatic metal shaker at 37°C to reduce the antibody for 3 hours. The drug-linker compound dissolved in DMSO was added to the reaction system and conjugated at 25°C for 16 hours. The reaction product was concentrated by ultrafiltration to phosphate buffer (PBS) to remove unreacted free small molecules. The ADC product was analyzed for purity and DAR value using SEC and LC-MS.

[0578] ADC SEC Purity Analysis: SEC-HPLC was used to analyze the protein samples to characterize the size uniformity of the recombinant protein and determine its purity. The HPLC used in this method was an Agilent 1260, with a TSKgel G3000SWXL column (from Tosoh Bioscience). The mobile phase consisted of a 200 mM pH 7.0 phosphate buffer / isopropanol (v / v 9:1) mixture. The detection temperature was 25°C, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 minutes.

[0579] Measurement of ADC DAR value by mass spectrometry: Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) was used to determine the DAR value of ADC molecules. First, the ADC molecule to be tested was treated with PNGase F to remove the N-glycan modification, and then treated with dithiothreitol (DTT) and incubated at 37°C for 1 hour to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected into a Waters ACQUITY Protein BEH molecular exclusion chromatography column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min and the analysis time was 30 minutes. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, and parent ion scan range 800-3000. Finally, the mass spectrometry data analysis software Biopharma Finder was used. 4.1. Deconvolution processing is performed using the Respect algorithm to calculate the molecular weight information of the light and heavy chain mass spectrum peaks and the mass spectrum response signals of each component, respectively, and thus calculate the DAR value of the ADC sample to be tested.

[0580] According to the above general preparation method, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof shown in Table 10 below was prepared.

[0581] Table 10: Antibody-drug conjugates, their SEC purity and DAR values

[0582] Example 19: Biological activity and related properties testing

[0583] The compounds in the following test examples were all prepared according to the methods of the above embodiments of the present disclosure.

[0584] Test Example 1. Activity of Compound (DH) Using K562-GRE Reporter Gene Assay

[0585] To generate the parental K562-GRE cell line, K562 cells were plated at 5 × 10 5Cells were seeded per well in a 6-well dish (Costar, Cat. No. 3516) containing 2 mL of complete culture medium (RPM1640, 10% FBS, penicillin-streptomycin) and incubated at 37°C, 5% CO₂ for 24 hours. The next day, 3 μg of pNL2.2[NLucP / MMTV / Hygro-NANO] (Promega) and 3 μl of PLUS reagent (Invitrogen, Cat. No. 11514-015) were diluted into 150 μL of Opti-MEM (Gibco, Cat. No. 11058021) and incubated at room temperature for 5 minutes. The pNL2.2[NLucP / MMTV / Hygro-NANO] vector contains the MMTV LTR (murine mammary tumor virus long terminal repeat), which responds to activation of several nuclear receptors (such as the glucocorticoid receptor and the androgen receptor) and drives transcription of the luciferase reporter gene NanoLuc. After incubation, the diluted DNA solution was mixed with Lipofectamine LTX solution (Invitrogen, Cat. No. 15338-100) (6 μl Lipofectamine LTX + 144 μl Opti-MEM) in a 1: 1 ratio and pre-incubated, and incubated at room temperature for 15 minutes to form a DNA-Lipofectamine LTX complex. After incubation, 300 μl of DNA-Lipofectamine complex was added directly to the cell wells. K562 cells were transfected at 37° C., 5% CO 2 for 24 hours. After transfection, the cells were washed with 3 mL of PBS and selected for growth for two weeks with complete growth medium containing 125 μg / mL hygromycin B (Invitrogen, Cat. No. 10687010). "K562-GRE (pNL2.2 [NLucP / MMTV / Hygro-NANO])" cells were generated.

[0586] K562-GRE (pNL2.2 [NLucP / MMTV / Hygro-NANO]) cells were seeded at 50,000 cells per well in 96-well tissue culture-treated white plates (Costar, Cat. No. 3917) in 50 μL of assay medium (RPMI 1640 medium, 1% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acids, and 1% penicillin-streptomycin). Cells were treated with 50 μL (2×) of a compound serially diluted 5-fold in assay medium. Each compound was treated at a total of eight concentrations, with a final concentration of 0.0000128 μM to 1 μM. After mixing, the cells were incubated at 37°C for 24 hours. After 24 hours of incubation, an equal volume of 100 μL of the Nano-Glo Luciferase Assay System (Promega, Cat. No. N1120) was added to the cells, and the reaction was shaken at 500 g for 10 minutes. Chemiluminescence was measured using a PHERAstar instrument. The antibody binding curve was drawn with the logarithm of the antibody concentration as the abscissa and the corresponding chemiluminescence reading as the ordinate, and the EC was calculated by four-parameter fitting (GraphPad Prism9). 50 The results are shown in Table 11.

[0587] Test Example 2. Determination of the binding activity of the compound of formula (DH) to the glucocorticoid receptor GR

[0588] The binding activity of compounds to the GR was determined using the Polarscreen Glucocorticoid Receptor Assay Kit, Red (Thermo Fisher Scientific, Catalog No. A15898). Test compounds were serially diluted 10-fold in DMSO in a 96-well V-bottom plate (Nunc, Catalog No. 249944), with a maximum concentration of 100 μM for a total of eight concentrations. The compounds were then further diluted 50-fold in the Complete GR Screening buffer provided in the kit. 10 μl of these diluted compounds were transferred to a 384-well microplate (Corning, Catalog No. 4514). 5 μl of Fluormone GS Red (4× concentration) was added to the test compounds, followed by 5 μl of the GR Full Length mixture (4× concentration). The assay was repeated in duplicate. The 384-well plate was incubated in the dark at room temperature for 2 hours. Fluorescence polarization (mP) was measured using an EnVision multi-function microplate reader (PerkinElmer). The log value of the final concentration of the compound was plotted on the X-axis and the mP value on the Y-axis. The data were entered into the processing software Graphpad Prism 9 for four-parameter fitting and calculation of EC 50 , the results are shown in Table 11.

[0589] Test Example 3. Determination of the binding activity of the compound of formula (DH) to estrogen receptor ER

[0590] use The TR-FRET ER Alpha Coactivator Assay kit (Thermo, Cat. No. A15885) was used to measure the ER binding activity of compounds. The ER receptor agonist, Estradiol (Sigma, Cat. No. E8875-25), was serially diluted 10-fold with DMSO in a 96-well V-bottom plate, reaching a maximum concentration of 100 μM for a total of eight concentrations. The test compound was serially diluted 10-fold with DMSO, reaching a maximum concentration of 3000 μM for a total of eight concentrations. The compound was then further diluted 50-fold with the provided Nuclear Receptor Buffer E (containing 5 mM DTT). 10 μl of this diluted compound was transferred to a 96-well half-area microplate (Corning, Cat. No. 3694). 5 μl of ER-LBD protein (4× concentration) was added to the test compound. Then, 5 μl of a mixture of fluorescein coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added. The assay was performed in duplicate. The plate was incubated at room temperature for 2 hours. Fluorescence values ​​were read using a PHERAstar instrument (Excitation 337, Emission 520 / 495 nm) and the 520:495 ratio was calculated. The logarithm of the final compound concentration was plotted on the X-axis and the 520 / 495 ratio on the Y-axis. The data were entered into Graphpad Prism 9 software for four-parameter fitting and calculation of the EC. 50 The results are shown in Table 11. The tested compounds have weak binding activity to estrogen receptors.

[0591] Test Example 4. Determination of the binding activity of the compound of formula (DH) to androgen receptor AR

[0592] use The TR-FRET Androgen Receptor Coactivator Assay kit (Thermo, Catalog No. A15878) was used to determine the AR binding activity of compounds. The AR receptor agonist dihydrotestosterone (DHT) (Sigma, Catalog No. D-073) was serially diluted 10-fold in DMSO to a maximum concentration of 100 μM for a total of eight concentrations in a 96-well V-bottom plate. The test compound was serially diluted 10-fold in DMSO to a maximum concentration of 3000 μM for a total of eight concentrations. The compound was further diluted 50-fold in the provided Nuclear Receptor Buffer A (containing 5 mM DTT). 10 μl of the diluted compound was transferred to a 96-well half-area microplate. 5 μl of AR-LBD protein (4× concentration) was added to the test compound. Then, 5 μl of a mixture of fluorescein coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added. The assay was performed in duplicate. The plate was incubated at room temperature for 2 hours, and fluorescence values ​​were read using a PHERAstar instrument (Excitation 337, Emission 520 / 495 nm). The 520:495 ratio was calculated. The log value of the final compound concentration was plotted on the X-axis, and the 520 / 495 ratio was plotted on the Y-axis. The data were entered into Graphpad Prism 9 for four-parameter fitting and EC was calculated. 50 The results are shown in Table 11. The test compound has weak binding activity to androgen receptor.

[0593] Test Example 5. Determination of the binding activity of the compound of formula (DH) to progesterone receptor PR

[0594] use The TR-FRET Progesterone Receptor Coactivator Assay kit (Thermo, Cat. No. A15903) was used to measure compound binding to PR. Progesterone (Sigma, Cat. No. P0130), a PR receptor agonist, was serially diluted 10-fold in DMSO to a maximum concentration of 100 μM for a total of eight concentrations. Test compounds were serially diluted 10-fold in DMSO to a maximum concentration of 3000 μM for a total of eight concentrations. The compounds were then further diluted 50-fold in the provided Nuclear Receptor Buffer F (containing 5 mM DTT). 10 μl of these diluted compounds were transferred to a 96-well half-area microplate. 5 μl of PR-LBD protein (4× concentration) was added to the test compounds. Then, 5 μl of a mixture of fluorescein coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added. The assay was performed in duplicate. The plate was incubated at room temperature for 2 hours. Fluorescence values ​​were read using a PHERAstar instrument (Excitation 337, Emission 520 / 495 nm) and the 520:495 ratio was calculated. The log value of the final compound concentration was plotted on the X-axis and the 520 / 495 ratio was plotted on the Y-axis. The data were entered into Graphpad Prism 9 for four-parameter fitting and EC was calculated. 50 The results are shown in Table 11. The tested compound has a certain binding activity with the progesterone receptor.

[0595] Table 11 In vitro activity results of compounds of formula (DH) N / A means not tested.

[0596] The test results show that the test compound has strong binding activity to GR, weak binding activity to PR, and does not bind to ER and AR, which is expected to reduce the side effects caused by acting on other hormone receptors.

[0597] Test Example 6. Efficacy test of compound (DH) in CHS mouse model

[0598] CHS mouse model construction: 6-8 week old female C57BL / 6N mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected. The abdominal hair of each mouse was shaved cleanly with small animal clippers. 400 μl of sensitizer was aspirated using a micropipette and evenly applied to the mouse's abdomen for epidermal sensitization. After application, the mouse was restrained for 3-5 seconds to allow the solvent to evaporate from the skin as much as possible. The sensitizer was prepared as follows: 0.5 g of FITC (fluorescein isothiocyanate; Sigma-Aldrich) powder was weighed and thoroughly dissolved in a mixture of 50 mL of acetone (Sinopharm Reagent) and 50 mL of DBP (dibutyl phthalate; Sigma-Aldrich) in equal proportions to obtain a sensitizer with a FITC content of 0.5%. On day 6 after sensitization, the right ear thickness of the mice was measured using a dial thickness gauge as a baseline value. Then, freshly prepared sensitizer was evenly applied to the inner and outer sides of the right ear using a micropipette for epidermal stimulation, with 10 μL applied to each side. Twenty-four hours after stimulation, the right ear thickness was measured again using a dial thickness gauge. The change in right ear thickness was calculated as Δear thickness = (right ear thickness 24 hours after stimulation) - (baseline right ear thickness).

[0599] Treatment plan for CHS model mice: Mice were divided into a blank group, a negative control group, a positive control group, and an experimental group. The experimental group received 3 μg / mouse of Compound 001 and Compound 009, the positive control group received 3 μg / mouse of Compound a (synthesized according to the method reported in patent WO2017210471, Compound 41), and the negative control group received blank vehicle (0.5% DMSO / PBS). The drug was administered via a single intraperitoneal injection 1 hour prior to epidermal stimulation of the right ear.

[0600] The test results are shown in Figure 2. Compounds 001 and 009 significantly reduced the thickness of the right ear of CHS mice compared to the negative control group. Compounds 001 and 009 were more effective than the positive reference compound a at the same dose. (* indicates statistical significance of the difference between each experimental group and the negative control group, using one-way ANOVA. ** indicates P value < 0.01, and **** indicates P value < 0.0001.)

[0601] Test Example 7. Verification of Anti-TNFα Antibody Binding Activity

[0602] Anti-human TNFα antibody binding activity

[0603] The anti-human TNFα antibody prepared in Example 17-2 was subjected to ELISA and Biacore detection using the same method as in Example 17-1. 50 The values ​​and Biacore detection results are shown in Table 12.

[0604] Table 12 Anti-human TNFα antibody ELISA and Biacore binding activity

[0605] Binding activity of anti-mouse TNFα antibody (001Mab)

[0606] Mouse TNFα protein (Novoprotein, Catalog No.: CF09) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, and blocked with 2% BSA blocking solution at 37°C for 1.5 hours. After rinsing three times with 0.05% Tween 20-PBS solution, serially diluted samples were added and incubated at 37°C for 1 hour. After rinsing three times with 0.05% Tween 20-PBS solution, secondary antibody HRP goat anti-Rat IgG (Jackson Immuno, Catalog No.: 112-035-003) was added and incubated at 37°C for 1 hour. After rinsing three times with 0.05% Tween 20-PBS solution, TMB solution (Seracare, Catalog No.: 5120-0077) was added. After reacting at room temperature for 10 minutes, the reaction was terminated by adding 1M hydrochloric acid and the plate was read at a wavelength of 450nm on a microplate reader. 001Mab binds to mouse TNFα protein EC 50 The value is 1.391nM.

[0607] Test Example 8. Binding activity test of antibody-drug conjugates to cells expressing TNFα

[0608] The nucleotide sequence encoding the TACE-resistant human transmembrane TNFα amino acid sequence (lacking amino acids 77-88 based on SEQ ID NO. 54) was cloned into the pcDNA5 / FRT vector (Thermofisher) to generate the pcDNA5 / FRT-human TNFαFL(77-88del) plasmid. This plasmid was co-transfected with the pOG44 plasmid into Flp-In-CHO host cells (Thermofisher). 24 hours after transfection, the cells were washed and selected for two weeks with complete growth medium containing 600 μg / mL hygromycin B (Invitrogen, 10687010) to generate Flp-In-CHO-human TNFαFL(77-88del) cells.

[0609] Flp-In-CHO-human TNFαFL (77-88del) cells were digested and counted, with a count of 1.5×10 5Cells were plated in 96-well plates (Corning, 3795) with 100 cells / well. Serial dilutions of the sample to be tested were added and incubated at 4°C for 1 hour. After washing twice with PBS, Alexa Fluor-647 Goat anti-human Fc secondary antibody (Jackson Immuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with PBS, the cells were resuspended and analyzed by flow cytometry (BD FACSCanto TM II) Analyze the mean fluorescence intensity (MFI). Graphpad Prism software was used to perform four-parameter curve fitting analysis on the data to obtain the EC 50 The results are shown in Table 13. Different anti-TNFα antibody-drug conjugates all have strong binding activity to cells expressing human transmembrane TNFα.

[0610] Table 13 Binding activity of anti-TNFα antibody-drug conjugates to Flp-In-CHO-human TNFαFL (77-88del) cells

[0611] SEQ ID NO.54

[0612] Test Example 9. K562-GRE reporter gene assay for the activity of anti-TNFα antibody-drug conjugates

[0613] The nucleotide sequence encoding the TACE-resistant human transmembrane TNFα amino acid sequence (lacking amino acids 77-88 based on SEQ ID NO. 54) was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells. In order to construct the K562-human TNFα-GRE-Luc reporter gene cell line, the K562-GRE cells constructed in Test Example 1 were cultured at a rate of 5×10 5Each well was inoculated into a 6-well culture dish (Costar, 3516) containing 1.5 mL of complete growth medium and cultured at 37 ° C, 5% CO2 for 24 hours. The next day, 1.5 ml of the human TNFα lentiviral particle solution prepared in advance was added to the cell dish and supplemented with a final concentration of 8 μg / ml polybrene (Santa Cruz, sc-134220) for lentiviral infection. K562-GRE cells were infected at 37 ° C, 5% CO2 for 24 hours. After incubation, the cells were washed with 3 mL of PBS and selected for two weeks with complete growth medium containing 125 μg / mL hygromycin B (Invitrogen, 10687-010) and 1 μg / mL puromycin (Gibco, A1113802) to produce K562-human TNFα-GRE-Luc reporter gene cells.

[0614] K562-human TNFα-GRE-Luc cells were resuspended in assay medium (RPMI1640, 10% carbon-adsorbed FBS, 1% Penicillin-Streptomycin) and cultured at 5×10 4 50 μl / well (50 μl) were inoculated into 96-well plates (Corning, 3610). Anti-TNFα antibody-drug conjugate was diluted with the above assay medium and added to 96-well plates, 50 μl per well, and incubated at 37°C, 5% CO2 for 24 hours. The luciferase reporter gene assay system (Promega, N1120) was used for 10 minutes, and the luminescence intensity was detected using an Envision instrument (PerkinElmer). The data were analyzed by four-parameter curve fitting using Graphpad Prism software to obtain the EC 50 The results are shown in Table 14 and Figure 3.

[0615] Table 14 Activity of anti-human TNFα antibody-drug conjugates in GRE reporter gene assay

[0616] The nucleotide sequence encoding the TACE-resistant human transmembrane TNFα amino acid sequence was replaced with a nucleotide sequence encoding the mouse TNFα amino acid sequence (SEQ ID NO. 67), and K562-mouse TNFα-GRE-Luc reporter cells were constructed and generated according to the same method as described in Test Example 9. The activity of the anti-mouse TNFα antibody-drug conjugate in the GRE reporter gene assay was tested using the same reporter cell culture and detection methods as described above. The results are shown in Table 15 and Figure 4.

[0617] Table 15 Activity of anti-mouse TNFα antibody-drug conjugates in GRE reporter gene assay

[0618] SEQ ID NO.67

[0619] Test Example 10. Activity inhibition test of antibody-drug conjugates in LPS-stimulated human monocyte cytokine release model

[0620] The frozen primary human peripheral blood mononuclear cells (PBMC, Sailybio, XFB-HP025B) were thawed and revived. Monocytes were sorted from PBMC cells using a monocyte sorting kit (Stemcell, 19058) and resuspended in RPMI1640 medium (Gibco, 72400-047) supplemented with 2% human AB serum (GEMINI, 100-512) and 1% penicillin-streptomycin (Gibco, 15140-122), and plated in a cell assay plate (Corning, 7007), with 1×10 cells per well. 5 Cells. The cells were pre-incubated with different concentrations of anti-human TNFα antibody-drug conjugates at 37°C, 5% CO2 incubator for 2 hours. Lipopolysaccharide LPS (Sigma, L4516) with a final concentration of 100 ng / mL was then added to stimulate the cells and incubated at 37°C, 5% CO2 incubator for 24 hours. The next day, the plate was centrifuged at 350g for 5 minutes, and 50 μL of the supernatant was carefully transferred to another clean 96-well plate to detect the concentrations of IL-6 (Dakoway, 1110603) and IL-1β (Dakoway, 1110123). GraphPad Prism software was used to perform four-parameter nonlinear regression fitting of the dose-response data and calculate the IC 50 As shown in Table 16, the anti-human TNFα antibody-drug conjugate can significantly inhibit the release of proinflammatory cytokines IL-6 and IL-1β from monocytes activated by LPS stimulation.

[0621] Table 16 Inhibitory activity of anti-human TNFα antibody-drug conjugates in LPS-stimulated human monocyte cytokine release assay

[0622] Test Example 11. Activity inhibition test of antibody-drug conjugate in LPS-stimulated mouse PBMC cytokine release model

[0623] Anticoagulated blood was collected from 6-8 week old female C57BL / 6 mice (Shanghai Lingchang Biotechnology Co., Ltd.). PBMCs were isolated using FICOLL-PAQUE (Cytiva, 17544602), resuspended in RPMI1640 medium (Gibco, 72400-047) supplemented with 10% FBS (Gibco, 10091-148) and 1% penicillin-streptomycin (Gibco, 15140-122), and plated in cell assay plates (Corning, 7007) at 4 × 10 cells per well. 5 cells. The cells were incubated with different concentrations of anti-mouse TNFα antibody-drug conjugates at 37°C, 5% CO2 incubator for 4 hours. Then, lipopolysaccharide LPS (Sigma, L4516) with a final concentration of 1000 ng / mL was added to stimulate the cells. After incubation at 37°C, 5% CO2 incubator for 40 hours, the plate was centrifuged at 350g for 5 minutes, and 100 μL of the supernatant was carefully transferred to another clean 96-well plate for detection of IL-6 concentration (Dakoway, 1210603). GraphPad Prism software was used to perform four-parameter nonlinear regression fitting of the dose-response data and calculate the IC 50 The results are shown in Table 17 and Figure 5 , showing that the anti-mouse TNFα antibody-drug conjugate can significantly inhibit the release of the proinflammatory cytokine IL-6 from mouse PBMCs activated by LPS stimulation.

[0624] Table 17 Inhibitory activity of anti-murine TNFα-drug conjugates in LPS-stimulated mouse PBMC cytokine release assay

[0625] Test Example 12: Antibody-drug conjugate efficacy test in CHS mouse model

[0626] The method for constructing the CHS mouse model is shown in Test Example 6.

[0627] Treatment of CHS model mice: Mice were divided into a normal control group, a negative control group, and a drug-treated group. The negative control group received blank vehicle (0.5% DMSO / PBS). The drug-treated groups received the anti-mouse TNFα antibody 001Mab (BioXcell, BE0058), ADC-L1-001-5, and ADC-L1-009-5 at a dose of 10 mg / kg. The drug was administered via a single intraperitoneal injection 1 hour prior to epidermal stimulation of the right ear.

[0628] The experimental results are shown in Figure 6. After drug administration, the thickness of the right ear of CHS mice was significantly reduced, with statistically significant differences compared to the negative control group (P value < 0.0001, statistical method: one-way ANOVA). The anti-mouse TNFα-drug conjugates (ADC-L1-001-5 and ADC-L1-009-5) showed significantly better efficacy than the anti-mouse TNFα antibody 001Mab group (* indicates statistical significance between the ADC group and the 001Mab group, statistical method: one-way ANOVA, * indicates P value < 0.05, ** indicates P value < 0.01).

[0629] Test Example 13: DBA / 1 Mouse Collagen Antibody-Induced Arthritis (CAIA) Efficacy Model Test

[0630] 6-8 week old male DBA / 1 mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were intraperitoneally injected with 150 μl of a mixture of five monoclonal antibodies against type II collagen (Arthrogen-CIA 5-Clone Cocktail Kit; Chrondex) on the first day of the experiment (day 0). The concentration of the antibody mixture was 10 mg / ml, resulting in a total injection of 1.5 mg per mouse. Three days after induction of type II collagen antibodies, mice were intraperitoneally injected with 100 μl of lipopolysaccharide (LPS) at a concentration of 0.5 mg / ml, resulting in a total injection of 50 μg of LPS per mouse.

[0631] Mice develop disease symptoms around day 5, with disease reaching its peak around day 7-8. Animals are scored based on their clinical symptoms. 0: No erythema or swelling; 1: Erythema or mild swelling near the tarsal bones, ankles, or metatarsals, with swelling in one toe; 2: Mild erythema and swelling in the ankles and metatarsals, or swelling in more than two toes; 3: Moderate erythema and swelling in the ankles, wrists, and metatarsals; 4: Severe swelling in the ankles, wrists, metatarsals, and toes.

[0632] Treatment of CAIA mice: Mice were randomly divided into a normal control group, a negative control group, and a drug-treated group based on body weight. The drug-treated groups received 10 mg / kg of the anti-mouse TNFα antibodies 001Mab, ADC-L1-001-5, and ADC-L1-009-5; the negative control group received blank vehicle (PBS). Drugs were intraperitoneally injected on days 3, 6, 10, and 13 after collagen antibody induction. The negative control group received an equal volume of vehicle intraperitoneally at the same time points. Clinical symptom scores and body weights of the mice were recorded daily.

[0633] The experimental results, shown in Figure 7, show significant improvement in the clinical symptoms of arthritis in CAIA mice treated with the drug, with statistically significant differences in clinical scores compared to the negative control group (* indicates statistical significance of the difference between each experimental group and the negative control group, using two-way ANOVA; ** indicates P values ​​< 0.01, and **** indicates P values ​​< 0.0001). The efficacy of the anti-mouse TNFα-ADC was significantly superior to that of the anti-mouse TNFα antibody group.

[0634] Test Example 14: TNFα Humanized Mouse Collagen Antibody-Induced Arthritis (CAIA) Pharmacological Efficacy Model Test

[0635] 6-8 week old humanized TNFα C57BL / 6 male mice (B-hTNFα, Biocytogen) were selected. On the first day of the experiment (day 0), a mixture of five monoclonal antibodies against type II collagen (Arthrogen-CIA 5-Clone Cocktail Kit; Chrondex) was injected intraperitoneally (500 μl per mouse). The concentration of the antibody mixture was 10 mg / ml, resulting in a total of 5 mg injected per mouse. Three days after induction of type II collagen antibodies, mice were intraperitoneally injected with 100 μl of 0.5 mg / ml lipopolysaccharide (LPS), resulting in a total of 50 μg LPS injected per mouse.

[0636] Mice develop disease symptoms around day 5, with disease reaching its peak between days 7 and 9. Animals are scored based on their clinical symptoms. 0: No erythema or swelling; 1: Erythema or mild swelling near the tarsal bones, ankles, or metatarsals, with swelling in one toe; 2: Mild erythema and swelling in the ankles and metatarsals, or swelling in more than two toes; 3: Moderate erythema and swelling in the ankles, wrists, and metatarsals; 4: Severe swelling in the ankles, wrists, metatarsals, and toes.

[0637] CAIA mouse treatment regimen:

[0638] Mice were randomly divided into a normal control group, a negative control group, and a drug-treated group based on body weight. The drug-treated groups received adalimumab and ADC-L1-009-6 at a dose of 10 mg / kg; the negative control group received blank vehicle (PBS). Drugs were administered intraperitoneally on days 3, 6, 10, and 13 after collagen antibody induction. The negative control group received an equal volume of vehicle intraperitoneally at the same time points. Clinical symptom scores and body weights of the mice were recorded daily.

[0639] The experimental results are shown in Figure 8. Clinical symptoms of arthritis in CAIA mice in the treatment group were significantly improved, with clinical scores statistically different from those in the negative control group. ADC-L1-009-6 demonstrated superior efficacy to Adalimumab. (* indicates statistical significance of the difference between each experimental group and the negative control group, using two-way ANOVA. **** indicates P value < 0.0001.)

[0640] Test Example 15: Plasma Stability Test of Anti-TNFα Antibody-Drug Conjugate

[0641] The ADC was diluted in human plasma (Aoneng Bio, PB021F-C), mouse plasma, and monkey plasma (final concentration: 100 μg / ml) and incubated in a 37°C incubator. The day of incubation was marked as day 0, and samples were subsequently removed on days 7 and 14 for free small molecule detection.

[0642] A 20 μL sample was added to 300 μL of internal standard working solution (prepared in acetonitrile), vortexed for 5 minutes, centrifuged for 5 minutes (14,000 rpm), and 4 μL of the supernatant was injected for LC-MS / MS analysis (API 6500+). The results are shown in Table 18. The results in Table 18 show that the ADC is relatively stable in human, mouse, and monkey plasma, with the release rate of free small molecules not exceeding 0.5%.

[0643] Table 18 Plasma stability of anti-TNFα antibody-drug conjugates BQL: below the limit of quantification (<1 ng / ml).

[0644] Test Example 16: Enzyme-Linked Immunosorbent Assay (ELISA) to Detect Binding Levels of Anti-IL-4R Antibody-Drug Conjugates to IL-4R Protein

[0645] The anti-human IL-4R antibody-drug conjugate prepared in Example 18 was assayed by ELISA. Human IL-4R protein (Acro Biosystems, Catalog No. ILR-H5221) was diluted with PBS (Hyclone, Catalog No. SH30256.01) to a final concentration of 2 μg / mL. 50 μL of the protein was added to each well of a 96-well ELISA plate (Corning, Catalog No. 9018) and coated at 37°C for 1 hour. The supernatant was discarded, and blocking solution (PBS buffer containing 5% (w / w) skim milk powder (Sangon, Catalog No. A600669-0250)) was added and blocked at 37°C for 1 hour. The blocking solution was discarded, and the plate was washed three times with PBS containing 0.05% Tween 20 (Sangon, Catalog No. A100777-0500) (hereinafter referred to as PBST). Add 50 μL of diluted anti-human IL-4R antibody-drug conjugate to each well, starting at a concentration of 100 nM, and then dilute it 3-fold in PBS containing 1% (w / w) BSA (purchased from Sangon, product number A500023-0100). Incubate at 37°C for 1 hour, and then wash the plate three times with PBST. Add 50 μL of diluted horseradish peroxidase (HRP)-labeled secondary antibody (purchased from Bethyl, product number A80-304P) to each well in 1% BSA-PBS, incubate at room temperature for 1 hour, and then wash the plate five times with PBST. Add 50 μL of TMB (purchased from KPL, product number 5120-0077) substrate to each well, incubate at room temperature for 5 minutes, and then add 50 μL of stop solution (1 M HCl) to each well. Read the OD450nm value using a microplate reader (Ensight-HH3400, purchased from PerkinElmer). Binding EC 50 The values ​​are shown in Table 19. The binding abilities of the tested antibody-drug conjugates to human IL-4R protein are comparable to those of the antibodies from which they were derived.

[0646] Table 19 ELISA binding experiment of antibody-drug conjugates and human IL-4R protein

[0647] Test Example 17: Flow cytometry (FACS) assay to detect the binding level of anti-IL-4R antibody-drug conjugate to Ramos cells

[0648] Ramos cells were cultured in T-75 cell culture flasks (purchased from Corning, Cat. No. 430720), and the cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature. The cells were washed twice with PBS (purchased from Hyclone, Cat. No. SH30256.01) buffer, resuspended in PBS, and counted. 1×10 cells were counted per well. 550 μL of the diluted anti-human IL-4R antibody-drug conjugate was added to a U-bottom 96-well FACS plate. Anti-human IL-4R antibody-drug conjugate was serially diluted in PBS containing 1% (w / w) BSA (Sangon, Catalog No. A500023-0100) starting at 100 nM and then diluted 10-fold. 50 μL of the diluted solution was added to each well, mixed, and incubated at 4°C for 1 hour. 200 μL of PBS buffer was added to each well, and the cells were washed by centrifugation at 1500 rpm for 5 minutes. This was repeated twice. The supernatant was discarded, and 100 μL of the diluted fluorescently labeled secondary antibody (Jackson Immuno Research, Catalog No. 109-605-098) was added to each well. The cells were incubated at 4°C for 1 hour. Wash twice by centrifugation with PBS, and the cells were resuspended in 100 μL of PBS per well. Results were detected and analyzed using a FACS instrument. The results, as shown in Table 20, show that the ability of the test molecules to bind to IL-4R on the surface of Ramos cells is comparable to that of the monoclonal antibodies from which they were derived. The conjugated small molecule had no effect on monoclonal antibody binding to the cells. The MFI data in Table 20 represent the maximum mean fluorescence intensity of the measured cell population.

[0649] Table 20 Binding level of antibody-drug conjugates to Ramos cells

[0650] Test Example 18: Detecting the endocytosis level of anti-IL-4R antibody-drug conjugates in Ramos cells

[0651] Ramos cells were expanded and cultured in T-75 cell culture flasks. The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature and washed twice with PBS buffer. 4×10 cells were plated per well. 5 For each cell, add 100 μL to a U-bottom 96-well FACS plate and incubate at 4°C. Dilute the anti-IL-4R antibody-drug conjugate to be tested in 2% FBS (w / w) PBS and add 100 μL per well to the cells, mixing thoroughly to a final concentration of 100 nM. Incubate at 4°C for 1 hour. Wash twice with ice-cold PBS by centrifugation. Resuspend in 200 μL of complete medium and aliquot into two U-bottom 96-well FACS plates. Incubate at 4°C and 37°C for 2 hours, respectively. Remove the plate and add 100 μL of ice-cold PBS to terminate endocytosis. Centrifuge at 1500 rpm for 5 minutes and wash twice. Discard the supernatant and add 100 μL of diluted fluorescently labeled secondary antibody (Jackson Immuno Research, Cat. No. 109-605-098) to each well. Incubate at 4°C for 1 hour. Wash twice with ice-cold PBS, resuspend the cells in 100 μL of PBS, and analyze the results by FACS.

[0652] Endocytosis rate (%) = (MFI 4°C - MFI 37°C) / MFI 4°C x 100%. As shown in Table 21, the endocytosis rates of the test molecules in Ramos cells were comparable to those of their respective monoclonal antibodies, and the conjugated small molecules had no significant effect on the endocytosis rate.

[0653] Table 21 Internalization of Antibody-Drug Conjugates in Ramos Cells

[0654] Test Example 19: Activity Determination of Anti-IL-4R Antibody-Drug Conjugate Using K562-GRE Reporter Gene Assay

[0655] The nucleotide sequence encoding the TACE-resistant human transmembrane IL-4R amino acid sequence (SEQ ID NO. 66) was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells. In order to construct the K562-human IL-4R-GRE-Luc reporter gene cell line, the K562-GRE cells constructed in Test Example 1 were cultured at 5×10 5 Cells were seeded into 6-well culture dishes (Costar, 3516) containing 1.5 mL of complete growth medium and cultured at 37°C, 5% CO2 for 24 hours. The next day, 1.5 mL of the human IL-4R lentiviral particle solution prepared in advance was added to the cell dish and supplemented with a final concentration of 8 μg / mL polybrene (Santa Cruz, sc-134220) for lentiviral infection. K562-GRE cells were infected at 37°C, 5% CO2 for 24 hours. After incubation, the cells were washed with 3 mL of PBS and selected for two weeks with complete growth medium containing 125 μg / mL hygromycin B (Invitrogen, 10687-010) and 1 μg / mL puro (Gibco, A1113802) to generate K562-human IL-4R-GRE-Luc reporter gene cells.

[0656] IL-4R amino acid sequence (SEQ ID NO. 66):

[0657] K562-human IL4R-GRE-Luc cells were collected and cultured at 1×10 7 The cells were resuspended in assay medium (RPM1640 + 1% carbon-adsorbed fetal bovine serum + 1% sodium pyruvate + 1% non-essential amino acids + 1% penicillin / streptomycin) at a density of 5 × 10 4Each well (50 μl) was inoculated into a 96-well plate (Costar, 3917). The anti-IL-4R antibody-drug conjugate to be tested and the isotype control conjugate ADC-L1-009-8 were diluted in 6 points in a 5-fold gradient starting from 40 nM, and the diluent was the above-mentioned assay medium. The diluted drug was added to the 96-well cell plate at 50 μl per well, mixed, and incubated in a 37°C, 5% (v / v) CO2 incubator for 24 hours. After the incubation, the 96-well plate was placed at room temperature for 5 minutes, and 100 μl / well of Nano-Glo luciferase detection reagent (Promega, N1110) was added to the well plate. After shaking at room temperature for 10 minutes, the fluorescence signal value in the well plate was detected using the PE Envision instrument-US Lumi module. The drug induction curve was drawn with the drug concentration as the horizontal axis and the fluorescence signal value RLU as the vertical axis, and the four-parameter fitting (GraphPad Prism9) was used to calculate the EC of the induction curve. 50 and maximum luminous intensity. The results are shown in Table 22.

[0658] Table 22 Activity of anti-IL-4R antibody-drug conjugates in GRE reporter gene assay

[0659] Test Example 20: Inhibitory Effect of Anti-IL-4R Antibody-Drug Conjugate on Proliferation of Primary Human CD4+ T Cells

[0660] Detection of IL-4R antibody-drug conjugates on primary human CD4 +The inhibitory activity of T cell proliferation was assessed as follows: Human PBMCs (stemexpress) were resuspended the day before the experiment and centrifuged at 300g for 10 minutes. The cells were then resuspended in complete culture medium (RPMI1640 + 10% FBS + 1× β-mercaptoethanol + 1% Pen / Srep) and allowed to stand overnight. The next day, the PBMCs were collected and counted using a cell counter. Primary human T cells were isolated and counted using a T cell isolation kit (Stemcell, Cat. No. 17951). First, an appropriate number of T cells were removed, washed twice with 2ml of PBS, and centrifuged at 350g for 5 minutes. After centrifugation and discarding the supernatant, the pre-prepared CellTrace Violet working solution (Invitrogen, C34557A) was added to a final concentration of 5μM. The T cell suspension containing the CellTrace Violet dye was mixed by air-blowing and incubated in a 37°C incubator for 10 minutes, gently shaking once during the incubation. After CTV labeling, add 5 times the volume of complete medium to the labeled cell suspension to terminate the reaction. After standing at room temperature for 5 minutes, centrifuge at 350g for 5 minutes, discard the supernatant, resuspend the cells in complete medium and count them. According to the counting results, adjust the cell density to 1×10 6 / mL, set aside. Next, dilute the drug to a 200nM working solution (4× final concentration) in complete culture medium and set aside. Next, prepare the ImmunoCult Human CD3 / CD28 T Cell Activator (Stemcell, Cat. No. 10971) working solution, remove the corresponding volume of anti-CD3 / CD28 immunocult at 1μL / 0.1M cells / well, add it to the complete culture medium, mix thoroughly, and set aside.

[0661] The drug groups were set as follows: (1) Anti-Hel-hIgG4 (isotype control), (2) Dupilumab, and (3) the anti-IL-4R antibody-drug conjugate monotherapy group. Groups (1) to (3) were all stimulated with anti-CD3 / CD28 immunoculturing. According to the plate map, CTV-labeled T cells (1×10 cells / 100 μL / well) were added to a 96-U well plate (Corning, Cat. No. 3799). 5 / well), drug working solution 50μL / well (4× final concentration), anti-CD3 / CD28 immunocult 50μL / well, the final volume of the system is 200μL / well. Place the well plate on a shaker at 300rpm for 1 minute, place it in a 37°C, 5% CO2 incubator, and incubate the drug and cells for 96 hours. After the incubation is completed, the cells in the 96-U well plate are gently blown to mix, transferred to a 96-V well plate, centrifuged at 350g for 5 minutes, and the supernatant is discarded. Add 200μL / well PBS to the well plate to wash the cells twice, and discard the supernatant. Prepare the cell death dye Live / Dead (Invitrogen, cat. no.: L10119) working solution in PBS at a ratio of 1:1000, and add 100μL / well to the cell centrifugation pellet and blow it evenly, and incubate at room temperature for 20 minutes. After the Live / Dead dye incubation is completed, add 150μL / well staining buffer (PBS+2% FBS) to wash the cells twice and discard the supernatant. During the centrifugation period, prepare the cell surface staining working solution by adding 1μL / 0.1M / well FITC mouse anti-human CD4 antibody (BD Bioscience, Cat. No.: 555346) to the staining buffer. Add 100μL / well FITC mouse anti-human CD4 antibody staining working solution to the cell centrifugation pellet, mix well, and incubate at 4°C in the dark for 30 minutes. After the staining is completed, add 150μL / well staining buffer to the well plate to wash the cells twice, discard the supernatant, and resuspend the cells with 150μL / well staining buffer. Use flow cytometry to detect the proliferation ratio of the cell sample. Read the live CD4 by FACS. + The proliferation ratio of T cells (i.e., CTV negative ratio) is used to evaluate the effect of drugs on human primary CD4 + The results are shown in Figure 9. Compared with the isotype control Anti-Hel-hIgG4 group, both the Dupilumab monoclonal antibody group and the test molecule group can inhibit the proliferation of human CD4 + T cell proliferation. Compared with the Dupilumab group, the tested molecule group had a negative effect on human CD4 + The inhibitory effect on T cell proliferation was stronger and statistically significant.

[0662] Test Example 21: Pharmacological and pharmacodynamic effects of anti-IL-4R antibody-drug conjugates on atopic dermatitis mouse model

[0663] Disease model establishment: 6-7 week-old C57BL / 6-hIL-4 / h-IL-4RA dual-humanized mice (provided by Biocytogen). On day 0, 25 μL of a 4:1 mixture of acetone and olive oil was evenly applied to the back and right ear of normal control mice; mice in the other groups were sensitized with 25 μL of 0.8% oxazolone (OXA) (the vehicle was a 4:1 mixture of acetone and olive oil) applied evenly to the back and right ear. From day 7 to day 25, the mice were challenged three times per week with 25 μL of a 4:1 mixture of acetone and olive oil applied evenly to the back and right ear of normal control mice; and 25 μL of a 0.4% OXA solution (the vehicle was the same as above) applied evenly to the back and right ear of model mice.

[0664] Drug intervention design: Drug administration began six days after initial OXA sensitization. Normal control mice were grouped separately. Model-induced mice were evenly divided into three groups of eight based on ear thickness: vehicle control (PBS), dupilumab-10 mpk, and the anti-IL-4R antibody-drug conjugate-10 mpk group. Each group received intraperitoneal administration twice weekly.

[0665] The in vivo efficacy experiment was completed on day 26, and serum, right ear skin tissue, and back skin tissue were collected for subsequent testing. Throughout the in vivo experiment, ear thickness was measured in each group of mice before each OXA induction and at the end of the experiment. As shown in Figure 10, compared with the vehicle control group, both dupilumab and the anti-IL-4R antibody-drug conjugate significantly inhibited ear thickening in mice, with the anti-IL-4R antibody-drug conjugate having a stronger inhibitory effect at the same dose. Furthermore, as shown in Figure 11, both drugs also inhibited IgE production in mice.

[0666] Histopathological evaluation: Ear and dorsal skin samples were fixed in fixative, embedded in paraffin, and sectioned at 5 μm thickness. After dewaxing and gradient hydration, H&E staining was performed. After staining, the sections were scanned and scored by a second, blinded, independent investigator. The H&E staining scoring criteria for epidermis included: stromal cell proliferation (0.5-2 points), erosions (0.5-2 points), acanthosis (0.5-2 points), and hyperkeratosis with parakeratosis (0.5-2 points); and dermis and subcutaneous tissue: mixed inflammatory cell infiltration (0.5-2 points) and peripheral eosinophilic infiltration (0.5-2 points), for a total of 12 points. As shown in Figures 12-13, administration of Dupilumab-10 mg / kg and anti-IL-4R antibody-drug conjugate-10 mg / kg significantly improved the pathological damage and inflammation of the ear and skin tissues at the modeling site of the atopic dermatitis mouse model, and the improvement effect of the same dose of anti-IL-4R antibody-drug conjugate was better than that of Dupilumab.

[0667] Mouse ear pieces and back skin tissue sections were analyzed for CD4 + T cells were immunohistochemically stained (IHC). Four 200× fields of view were randomly selected for each ear and skin tissue IHC section. The number of positive CD4 cells in each field of view was counted and the average number of positive CD4 cells in each field of view was calculated. As shown in Figures 14-15, the administration of anti-IL-4R antibody-drug conjugate significantly inhibited the expression of inflammatory CD4 in ear and skin tissue. + T cell infiltration, while the same dose of Dupilumab had a weak effect.

[0668] Test Example 22: Plasma Stability Test of Anti-IL-4R Antibody-Drug Conjugate

[0669] The ADC was diluted in human plasma (Aoneng Bio, PB021F-C) (final concentration: 100 μg / ml) and incubated in a 37°C incubator. The day of incubation was marked as day 0, and samples were subsequently taken on days 7, 14, and 21 for free small molecule detection.

[0670] A 20 μL sample was added to 300 μL of internal standard working solution (prepared in acetonitrile), vortexed for 5 minutes, centrifuged for 5 minutes (14,000 rpm), and 4 μL of the supernatant was injected for LC-MS / MS (API 6500+) analysis. The results are shown in Table 23. The results show that the ADC is relatively stable in human plasma, and the release rate of free small molecules does not exceed 0.5%.

[0671] Table 23 Plasma Stability of Anti-IL-4R Antibody-Drug Conjugates BQL: below the limit of quantification (<1 ng / ml).

[0672] Test Example 23: Anti-TNFα Antibody-Drug Conjugate Cell Incubation Experiment

[0673] After binding to TNFα on the cell surface, the anti-TNFα antibody-drug conjugate is internalized and taken up by the cell. Lysosomal enzymes cleave the ADC, releasing the hormone small molecule, which exerts its anti-inflammatory effects. To confirm the form of the small molecule released after the ADC is internalized and the efficiency of enzymatic cleavage, a cell incubation experiment was performed.

[0674] The K562-human TNFα-GRE-Luc cells (1×10 4 / well) were inoculated in a 96-well plate (Corning, 3599), and after adding 15 μg / ml of ADC, they were placed in a 37 ° C cell culture incubator for incubation. The sample at the time of mixing the cells with ADC was marked as 0h, and then the samples were taken out at 24h and 48h, respectively, and acetonitrile (100 μL / well) was added and pipetted to mix. Take 20 μL of the above sample, add 300 μL of internal standard working solution (acetonitrile configuration), vortex mix for 5 minutes, centrifuge for 5 minutes (14000 rpm), and 4 μL of supernatant LC-MS / MS (API 6500 +) was sampled and analyzed. The results are shown in Table 24. The test results showed that the small molecule form detected was compound 009, and no small molecule compound with a phosphate group was detected. After the ADC-L1-009-6 disclosed in the present invention enters the cell, the phosphate group in the molecule is converted to a hydroxyl group, and the small molecule compound that exerts pharmacological activity is compound 009.

[0675] Table 24: Concentration of small molecule compounds released by anti-TNFα antibody-drug conjugates in cells BQL: below the limit of quantification (<1ng / ml)

[0676] Test Example 24: Inhibitory Effect of Anti-IL-4R Antibody-Drug Conjugate on CD23 Expression on B Cells in Human Primary T and B Cell Co-culture System

[0677] Remove PBMC cryopreserved cells (Stemexpress, Cat: PBMNC050C) from the liquid nitrogen tank and gently shake in a 37°C water bath to dissolve the cells. Transfer the cell suspension to a centrifuge tube containing 8 ml of preheated 1640 culture medium (Gibco, Cat: 72400047) and centrifuge for 8 minutes (centrifugation conditions are 300g, 25°C), and discard the supernatant. Add 8 ml of EasySep TM Resuspend the cells in a 4% PBS buffer (Stemcell, Cat: 20144), count them using a cell counter (Beckman Coulter, Vi-Cell), centrifuge for 8 min (centrifugation conditions: 300g, 25°C), and discard the supernatant. TM Resuspend the cells in buffer and adjust the density to 5×10 7 Human primary T cells and B cells were sorted using a human T cell sorting kit (Stemcell, Cat: 17951) and a human B cell sorting kit (Stemcell, Cat: 17954), respectively. After counting the cells, they were set aside for use.

[0678] Prepare the following reagents: 1) Adjust the number of primary human T cells to 1×10 6 cells / mL, and 0.5×10 primary human B cells 6cells / mL; 2) Use 1640 complete medium to prepare anti-IL-4R antibody-drug conjugate working solution (4 times the final concentration) to make the final drug concentration of 500nM, 100nM and 20nM; 3) Prepare Staphylococcus aureus enterotoxin A + human recombinant interleukin-4 + anti-human CD3 / CD28 magnetic beads-mixed working solution (4 times the final concentration), add human T cell activator anti-CD3 / CD28 magnetic beads (6250 magnetic beads / 50μL / well, Gibco, Cat: 11131D), human recombinant interleukin-4 (final concentration of 10ng / mL, Peprotech, Cat: 200-04) and -Staphylococcus aureus enterotoxin A (final concentration of 1ng / mL, Toxin technology, Cat: AT101) to 1640 complete medium, mix well and set aside. Human primary T cells (50,000 cells / 50 μL / well), human primary B cells (25,000 cells / 50 μL / well), and anti-IL-4R antibody-drug conjugate (50 μL / well) were added sequentially to a 96-well U-shaped plate (Corning, Cat: 3799). After gentle pipetting to mix, the cells were incubated at room temperature for 10 minutes. A working mixture of Staphylococcus aureus enterotoxin A, human recombinant interleukin-4, and anti-human CD3 / CD28 magnetic beads was then added, and the cells were incubated at 37°C, 5% CO2 for 3 days. After the incubation period, the plate was removed, the cells were mixed, and centrifuged for 5 minutes (350g, 25°C). The supernatant was aspirated and IL-13 secretion was assayed by ELISA. The cell pellet was then analyzed for CD23 expression on live B cells by FACS.

[0679] The cell pellet from the above-mentioned primary human T and B cell co-culture was resuspended in 200 μL / well PBS and centrifuged for 5 minutes (350g, 25°C). The supernatant was discarded. After incubation with a fixable near-infrared live / dead cell staining kit dye (Thermofisher, Cat: L10119) at room temperature for 10 minutes, 100 μL / well of staining buffer (2% FBS + PBS) was added and the cells were centrifuged for 5 minutes (350g, 25°C). The supernatant was discarded. Human Fc receptor blocker (Biolegend, Cat: 422302) was added and incubated at 4°C for 10 minutes. After that, 2 μL / well of PE anti-human CD19 antibody (Biolegend, Cat: 302208) and 2 μL / well of APC anti-human CD23 antibody (Biolegend, Cat: 338514) were added and incubated at 4°C in the dark for 30 minutes for cell surface staining. After staining, 100 μL / well of staining buffer was added to the cells, and the cells were centrifuged for 5 minutes (350g, 25°C). The supernatant was discarded, and the cells were resuspended in 120 μL / well of staining buffer. The CD23 positive ratio in live CD19-positive cells was measured using a flow cytometer (BD, Canto II), and the data were analyzed using Flowjo V10 and Graphpad Prism 9 software. A higher CD23 positive ratio indicates a higher level of CD23 expression on B cells, indicating weaker inhibitory activity of the anti-IL-4R antibody-drug conjugate.

[0680] Table 25 Percentage of CD23-positive B cells in the TB system

Claims

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the general structural formula Pc-(LD) n , in: Pc is the ligand unit; L is a linker unit; D is a drug unit represented by the following formula (DI): in, R 1 、R 2 are each independently selected from H, CH3 or halogen; Ring A is selected from phenyl, 5-10 membered heteroaryl or C3-C 10 Cycloalkyl, the phenyl, 5-10 membered heteroaryl or C3-C 10 The cycloalkyl group is optionally substituted with one or more R 1a replace; X is selected from O, S, C1-C3 alkylene-O, C1-C3 alkylene-S, NR 6 or C(R 7 )(R 8 ); R 6 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally substituted by one or more R b replace; R 7 、R 8 Each is independently selected from H, halogen, CN, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, or R 7 、R 8 Together with the atoms to which they are attached, they form a C3-C6 cycloalkyl or a 4-7 membered heterocyclic group, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally replaced by one or more R b replace; R 3 Selected from H, OH or NHR 9 , R 4 、R 5 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a Replace; or, R 5 Selected from H, OH or NHR 9 , R 3 、R 4 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace; R 9 is selected from H, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group, wherein the C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclic group is optionally substituted by one or more R d replace; R 13 、R 14 Each is independently selected from H, halogen, CN, OH, NH2, O(C1-C3 alkyl) or C1-C6 alkyl; Every R 4a independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R d replace; Every R d Independently selected from halogen, CN, =O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; R 10 Selected from OH, SH, O (C1-C6 alkyl), S (C1-C6 alkyl), OC (=O) - (C1-C6 alkyl) or The O(C1-C6 alkyl), S(C1-C6 alkyl), OC(=O)-(C1-C6 alkyl) are optionally substituted with one or more halogens or CN; R 11 、R 12 Each independently selected from H or C1-C6 alkyl; Every R 1a independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R b replace; Every R b independently selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl, wherein the OH, NH2, C1-C6 alkyl, C3-C6 cycloalkyl or 4-7 membered heterocyclyl is optionally replaced by one or more R c replace; Every R c is independently selected from halogen, CN, ═O, C1-C3 alkyl, OH, O(C1-C3 alkyl), NH2, NH(C1-C3 alkyl) or N(C1-C3 alkyl)2; and, n is 1 to 16; Wherein, the linker unit L and the compound R of formula (DI) 3 、R 4 、R 5 、R 13 、R 14 Any one of the -OH, -NH2 or -NH- groups present in the is covalently linked.

2. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 、R 2 are each independently selected from H or F.

3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein ring A is selected from phenyl or 5-6 membered heteroaryl, and the phenyl or 5-6 membered heteroaryl is optionally replaced by R 1a replace.

4. The ligand-drug conjugate according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein X is selected from O, S, CHO, CHS, CH, CF, CHCH or 5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R 10 Selected from OH, SH, OCH2F, O(C=O)CH3, 6. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 3 is selected from H, OH or NH2, R 4 、R 5 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace.

7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 5 is selected from H, OH or NH2, R 3 、R 4 Together with the atoms to which they are attached, they form a C5-C6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl, wherein the C5-C6 cycloalkenyl, the 5-6 membered heterocyclyl or the 5-6 membered heteroaryl is optionally substituted by one or more R 4a replace.

8. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 3 is selected from H, OH or NH2, R 4 、R 5 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

9. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R 5 is selected from H, OH or NH2, R 3 、R 4 Together with the atoms they are connected to form described Optionally one or more R 4a replace.

10. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein R 4a Selected from halogen, CN, =O, OH, NH2, C1-C6 alkyl or C3-C6 cycloalkyl.

11. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein R 13 、R 14 are each independently selected from H or NH2.

12. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein: Structural unit Selected from 13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula (DI) is selected from the compounds represented by the following formula (D-II): in, R 1 、R 2 are each independently selected from H or F; R 10 Selected from OH or 14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound represented by formula (DI) is selected from the following compounds:

15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein: The linker unit L is selected from Its a end is covalently linked to the ligand unit Pc, and its b end is covalently linked to the drug unit D. 1 is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl; preferably, L 1 is a dipeptide residue, wherein one of the amino acids constituting the dipeptide residue is glycine; more preferably, the L 1 A dipeptide residue selected from Gly-Lys or Gly-Glu.

16. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein: The linker unit L is selected from Its a-terminal is covalently linked to the ligand unit Pc, and its b-terminal is covalently linked to the drug unit D.

17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein: The linker unit L is selected from Its a-terminal is covalently linked to the ligand unit Pc, and its b-terminal is covalently linked to the drug unit D.

18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The ligand-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following ligand-drug conjugates or a pharmaceutically acceptable salt thereof:

19. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein: The ligand unit Pc is selected from a polypeptide, an antibody or an antigen-binding fragment.

20. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein: The ligand unit Pc is an antibody or antigen-binding fragment that specifically binds to TNFα or IL-4R.

21. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein: (1) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 contained in VH shown in SEQ ID NO.4-9, 12-16, 18, 20, 22, 57, 68, 70, 72, 74 or 76; or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 contained in VL shown in SEQ ID NO.1-3, 17, 19, 21, 23, 58, 69, 71, 73, 75 or 77; (2) the heavy chain variable region and the light chain variable region comprise sequences having at least 80% identity with each CDR in HCDR1-3 and / or LCDR1-3 in group (1), or sequences having at most 3 insertion, deletion or substitution mutations.

22. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein the HCDR1-3 and / or the LCDR1-3 are selected from the following: (1) the HCDR1-3 are SEQ ID NOs. 24-26; or / and the LCDR1-3 are SEQ ID NOs. 27-29; (2) the HCDR1-3 are SEQ ID NOs. 30-32; or / and the LCDR1-3 are SEQ ID NOs. 33-35; (3) the HCDR1-3 are SEQ ID NOs. 36-38; or / and the LCDR1-3 are SEQ ID NOs. 39-41; (4) the HCDR1-3 are SEQ ID NOs. 42-44; or / and the LCDR1-3 are SEQ ID NOs. 45-47; (5) the HCDR1-3 are SEQ ID NOs. 48-50; or / and the LCDR1-3 are SEQ ID NOs. 51-53; (6) the HCDR1-3 are SEQ ID NOs. 60-62; or / and the LCDR1-3 are SEQ ID NOs. 63-65; (7) the HCDR1-3 are SEQ ID NOs. 78-80; or / and the LCDR1-3 are SEQ ID NOs. 81-83; (8) the HCDR1-3 are SEQ ID NOs. 84-86; or / and the LCDR1-3 are SEQ ID NOs. 87-89; (9) the HCDR1-3 are SEQ ID NOs. 90-92; or / and the LCDR1-3 are SEQ ID NOs. 93-95; (10) the HCDR1-3 are SEQ ID NOs. 96-98; or / and the LCDR1-3 are SEQ ID NOs. 99-101; (11) the HCDR1-3 are SEQ ID NOs. 102-104; or / and the LCDR1-3 are SEQ ID NOs. 105-107; or The HCDR1-3 and / or the LCDR1-3 have a sequence that is at least 80% identical to each CDR in the HCDR1-3 and LCDR1-3 in any group of groups (1) to (11), or a sequence in which at most three insertion, deletion or substitution mutations occur.

23. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 22, wherein: The antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region sequence or fragment comprises CH1, a hinge region, and CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of VH and a heavy chain constant region, and the heavy chain constant region has a sequence as shown in SEQ ID NO: 10 or 59, and the light chain consists of VL and a light chain constant region, and the light chain constant region has a sequence as shown in SEQ ID NO:

11.

24. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 20, wherein: The antibody is selected from Nerelimomab, Certolizumab, Infliximab, Golimumab, Adalimumab, Dupixent, 002Mab, 003Mab, 004Mab, 005Mab or 006Mab.

25. A drug-linker compound or a pharmaceutically acceptable salt thereof, having the general structural formula XLD, wherein: Drug unit D as defined in claim 1; The linker unit L is selected from Its a end is covalently linked to X, and its b end is covalently linked to the drug unit D. 1 is a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted by one or more substituents selected from halogen, CN, ═O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl; X is selected from halogen, OS(O)2CH3 or OS(O)2CF3.

26. The drug-linker compound according to claim 25 or a pharmaceutically acceptable salt thereof, wherein: L is selected from Its a end is covalently linked to X, and its b end is covalently linked to the drug unit D. 1 A dipeptide residue selected from Gly-Lys or Gly-Glu.

27. The drug-linker compound according to claim 25 or a pharmaceutically acceptable salt thereof, wherein: L is selected from Its a-terminal is covalently linked to X, and its b-terminal is covalently linked to the drug unit D.

28. The drug-linker compound or a pharmaceutically acceptable salt thereof according to any one of claims 25 to 27, wherein: X is selected from Br or I.

29. The drug-linker compound of the general formula XLD according to claim 25, or a pharmaceutically acceptable salt thereof, is selected from the following compounds or pharmaceutically acceptable salts thereof:

30. A pharmaceutical composition comprising the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24 and a pharmaceutically acceptable excipient.

31. Use of the ligand-drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, or the pharmaceutical composition according to claim 30, in the preparation of a medicament for preventing or treating inflammatory diseases or autoimmune diseases.

32. A method for preparing the ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, comprising the step of coupling the drug-linker compound according to any one of claims 25 to 29 to a ligand, wherein the ligand is an antibody or an antigen-binding fragment.

33. An antibody or antigen-binding fragment that specifically binds to human TNFα, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises the sequence shown in any one of SEQ ID NOs. 5-9 and 12-15, and the light chain variable region comprises the sequence shown in SEQ ID NO. 2 or 3.

34. The antibody or antigen-binding fragment of claim 33, comprising the heavy chain variable region (VH) and light chain variable region (VL) shown below: (1) the VH comprises SEQ ID NO. 5, and the VL comprises SEQ ID NO. 2; (2) the VH comprises SEQ ID NO. 6, and the VL comprises SEQ ID NO. 2; (3) the VH comprises SEQ ID NO. 7, and the VL comprises SEQ ID NO. 2; (4) the VH comprises SEQ ID NO. 8, and the VL comprises SEQ ID NO. 2; (5) the VH comprises SEQ ID NO. 9, and the VL comprises SEQ ID NO. 2; (6) the VH comprises SEQ ID NO. 5, and the VL comprises SEQ ID NO. 3; (7) the VH comprises SEQ ID NO. 6, and the VL comprises SEQ ID NO. 3; (8) the VH comprises SEQ ID NO. 7, and the VL comprises SEQ ID NO. 3; (9) the VH comprises SEQ ID NO. 8, and the VL comprises SEQ ID NO. 3; (10) the VH comprises SEQ ID NO. 9, and the VL comprises SEQ ID NO. 3; (11) the VH comprises SEQ ID NO. 12, and the VL comprises SEQ ID NO. 2; (12) the VH comprises SEQ ID NO. 13, and the VL comprises SEQ ID NO. 2; (13) the VH comprises SEQ ID NO. 14, and the VL comprises SEQ ID NO. 2; or, (14) The VH comprises SEQ ID NO.15, and the VL comprises SEQ ID NO.

2.

35. The antibody or antigen-binding fragment of claim 33 or 34, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, the constant region sequence or fragment comprising CH1, a hinge region, and CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from a human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from a human or mouse kappa constant region or a lambda constant region; optionally, the antibody or antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consisting of a VH and a heavy chain constant region, the heavy chain constant region having the sequence shown in SEQ ID NO: 10, and the light chain consisting of a VL and a light chain constant region, the light chain constant region having the sequence shown in SEQ ID NO:

11. 36 . A pharmaceutical composition comprising the antibody or antigen-binding fragment that specifically binds to human TNFα according to any one of claims 33 to 35 and a pharmaceutically acceptable excipient.

37. Use of the antibody or antigen-binding fragment that specifically binds to human TNFα according to any one of claims 33 to 35, or the pharmaceutical composition according to claim 36, in the preparation of a medicament for treating inflammatory diseases or autoimmune diseases.

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