Glucocorticoid receptor agonist and ligand-drug conjugate thereof

Abstract

Provided in the present invention is a glucocorticoid receptor agonist, which is a compound as represented by the following formula I. The glucocorticoid receptor agonist of the present invention has the advantages of a high efficiency and low toxic side effects.

Description

Glucocorticoid receptor agonists and their ligand drug conjugates Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to glucocorticoid receptor agonists and their ligand drug conjugates. Background Technology

[0002] Glucocorticoids (GC), also known as adrenocortical hormones, are a class of steroid hormones secreted by the adrenal cortex, possessing a wide range of physiological and pharmacological effects. Medicinal glucocorticoids such as hydrocortisone, dexamethasone, methylprednisolone, and budesonide have strong anti-inflammatory effects and are widely used to treat autoimmune and inflammatory diseases.

[0003] Glucocorticoids exert their effects primarily by binding to intracellular glucocorticoid receptors (GR). After binding to the receptor, glucocorticoids form the GC-GR complex, which interacts with hormone-response elements and other transcription factors in the cell nucleus, regulating the transcription and expression of related genes, thereby achieving anti-inflammatory and immunosuppressive effects. However, high-dose or long-term use of glucocorticoids can easily lead to various toxic side effects or complications, limiting the application of this class of drugs. Therefore, there is an urgent need to develop novel glucocorticoid drugs with higher efficacy and fewer side effects to meet clinical needs.

[0004] Antibody-drug conjugates (ADCs) consist of three parts: an antibody, a bioactive molecule (drug molecule), and a linker. They are designed to deliver the bioactive molecule into cells in a target-dependent manner, exerting effective therapeutic activity against abnormal cells expressing the target. To further improve the efficacy of glucocorticoid drugs and reduce side effects, there is also an urgent need to develop glucocorticoid ADCs to meet clinical needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, one objective of this invention is to provide a glucocorticoid receptor agonist, which is a compound represented by Formula I.

[0006] The glucocorticoid receptor agonist of the present invention has one or more effects selected from the group consisting of: (1) the ability to continuously activate glucocorticoid receptors; (2) the ability to affect the transcription of IFN signaling pathway response genes; (3) good plasma stability; (4) the ability to affect immune cell activity; (5) the conjugate thereof has a targeting effect; (6) biocompatibility; (7) the ability to affect the release of cytokines from immune cells; (8) the ability to affect the degree of skin fibrosis; (9) the ability to affect the number and / or proportion of dendritic cells; (10) the ability to affect GRE expression levels; (11) the ability to affect the release of cytokines from monocytes; (12) the ability to affect contact hypersensitivity reactions; (13) the ability to affect arthritis symptoms; (14) lower toxicity; and (15) the antibody conjugate thereof has a stronger ability to affect the release of cytokines from immune cells.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A compound of Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof.

[0009] in,

[0010] R1 and R2 are each independently selected from H, halogens, and C1-C6 alkyl groups;

[0011] R is Where X is selected from C and chemical bonds, Y is selected from C, N, O and S, and Z is selected from C and chemical bonds;

[0012] R3 and R4 are each independently selected from H, halogen, amino, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and R5R6NC1-C6 alkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy and amino are each independently optionally substituted by one or more selected from halogen, hydroxy, cyano and amino.

[0013] or,

[0014] When Y is selected from N, O and S, R3 and R4 together with the attached C atom form a 6-8 membered aromatic ring, a 5-8 membered heteroaromatic ring and a 3-6 membered heterocycle, wherein each of the 6-8 membered aromatic ring, the 5-8 membered heteroaromatic ring and the 3-6 membered heterocycle is independently and optionally substituted by one or more selected from C1-C6 alkyl, halogen, hydroxy, cyano and amino groups.

[0015] R5 and R6 are each independently selected from H, C1-C6 alkyl, halogen and C1-C6 alkoxy;

[0016] in,

[0017] R3 and R4 are not both H;

[0018] When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and

[0019] When R is When R3 is not an ethyl group, R3 is not an ethyl group.

[0020] According to the present invention, the compounds represented by Formula I, their stereoisomers or pharmaceutically acceptable salts thereof, wherein R1 and R2 are each independently selected from H, F, Cl, Br and methyl; in some embodiments, R1 and R2 are each independently selected from H and F.

[0021] According to the present invention, the stereoisomer of the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from H, halogen, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy, and di(C1-C6 alkyl)aminomethyl; in some embodiments, R3 is selected from H, F, trifluoromethyl, aminosulfonyl, methyl, methoxy, and dimethylaminomethyl.

[0022] According to the present invention, the compound represented by Formula I, its stereoisomer or pharmaceutically acceptable salt thereof, wherein R4 is selected from H, amino, C1-C6 alkyl and C1-C6 alkoxy, and in some embodiments, R4 is selected from H, amino, methyl and methoxy.

[0023] According to the present invention, the compound represented by Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein R is... R3 and R4 are defined as above.

[0024] According to the present invention, the stereoisomer of the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from H, F, trifluoromethyl, methyl, methoxy, and dimethylaminomethyl, and / or R4 is selected from H, amino, methyl, and methoxy.

[0025] According to the present invention, the stereoisomer of the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, has the structure shown in Formula II:

[0026] in,

[0027] R1, R2, R3, and R4 are defined as above;

[0028] in,

[0029] R3 and R4 are not both H;

[0030] When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and

[0031] R3 is not an ethyl group.

[0032] In some embodiments, compounds with structures as shown in Formula II, their stereoisomers, or pharmaceutically acceptable salts thereof, wherein:

[0033] R1 and R2 are each independently selected from H, halogens, and C1-C6 alkyl groups;

[0034] R3 is selected from H, halogen, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and di(C1-C6)alkylaminomethyl;

[0035] R4 is selected from H, amino, C1-C6 alkyl, and C1-C6 alkoxy.

[0036] In some embodiments, compounds with structures as shown in Formula II, their stereoisomers, or pharmaceutically acceptable salts thereof, wherein:

[0037] R1 and R2 are each independently selected from H and F;

[0038] R3 is selected from H, F, trifluoromethyl, aminosulfonyl, methyl, methoxy, and dimethylaminomethyl;

[0039] R4 is selected from H, amino, methyl, and methoxy.

[0040] According to the present invention, the stereoisomer of the compound represented by Formula I, or its pharmaceutically acceptable salt thereof, wherein when Y is selected from N, O, and S, R3 and R4 together with the attached C atom form a 6-8 membered aromatic ring or a 5-8 membered nitrogen-containing heteroaromatic ring.

[0041] According to the present invention, the compound represented by Formula I, its stereoisomer or its pharmaceutically acceptable salt, wherein one of X and Z is a chemical bond and the other is C, and R3 and R4 together with the C atom to which they are attached form a benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, pyridazine ring or pyrazole ring.

[0042] According to the present invention, the stereoisomer of the compound represented by Formula I, or a pharmaceutically acceptable salt thereof, wherein R is selected from one of the following:

[0043] According to the present invention, the stereoisomer of the compound represented by Formula I or a pharmaceutically acceptable salt thereof, wherein the compound represented by Formula I is selected from the following compounds:

[0044] Another object of the present invention is to provide a method for preparing the compound represented by Formula I, comprising the following steps:

[0045] Compound A reacts with compound B to produce compound C; compound C is prepared and separated to obtain compound I (target compound) and compound IS, wherein R1, R2 and R are as described above.

[0046] According to the method for preparing the compound of formula I according to the present invention, in the step of preparing the compound of formula C from the compound of formula A, the reaction solvent is one or more of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and N,N-dimethylacetamide.

[0047] According to the method for preparing the compound of formula I according to the present invention, in the step of preparing the compound of formula C from the compound of formula A, the reaction is carried out under the catalysis of a Lewis acid, wherein the Lewis acid is selected from trifluoromethanesulfonic acid and / or perchloric acid.

[0048] According to the method for preparing the compound of formula I according to the present invention, in the step of preparing the compound of formula C from the compound of formula A, the reaction is stirred in an ice bath.

[0049] According to the method for preparing the compound represented by Formula I of the present invention, in the step of preparing the compound of Formula C from the compound of Formula A, the molar ratio of the compound of Formula A to the compound of Formula B is 1:1.

[0050] According to the method for preparing the compound represented by Formula I of the present invention, in the step of preparing the compound of Formula C from the compound of Formula A, the molar volume ratio of the compound of Formula A to the solvent is 1:2 to 1:18, preferably 1:3 to 1:15, more preferably 1:3 to 1:10.

[0051] According to the method for preparing the compound of formula I according to the present invention, in the step of preparing the compound of formula C from the compound of formula A, the molar ratio of the compound of formula A to the Lewis acid is 1:2 to 1:6, preferably 1:3 to 1:5.

[0052] According to the method for preparing the compound of formula I according to the present invention, in the step of preparing the compound of formula C from the compound of formula A, after the reaction is complete, the reaction solution is extracted, washed, and concentrated to obtain the compound of formula C.

[0053] According to the method for preparing the compound represented by Formula I of the present invention, the compound of Formula C is purified and separated by C18 preparative chromatography to obtain the compound of Formula I and the compound of Formula I.

[0054] Another object of the present invention is to provide a ligand-drug conjugate or a pharmaceutically acceptable salt thereof having the structure shown in Formula III:

[0055] ligand-(LD)n

[0056] Formula III

[0057] Wherein, the ligand is selected from polypeptides, proteins and antibodies, L is a linker, D is a drug, the drug is a compound represented by Formula I as described above, its stereoisomer or a pharmaceutically acceptable salt thereof, and n is an integer or decimal between 1 and 10.

[0058] In some implementations, the ligand is an antibody;

[0059] In some embodiments, the antibody specifically binds to antigens selected from the group consisting of: BDCA2, IL-4R, TROP2, AXL, BAFFR, BCMA, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, differentiation antigen cluster 40 (C D40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.2. CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, C XCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA type II (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR1), IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL6R, IL5R, IL7R, IL9R, integrins, LAG3, LIFR, sialic acid-binding immunoglobulin-like lectin-4 (MAG / Siglec-4), MMR, MSR1, NCR3LG1, NKG2 D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, sialic acid-binding immunoglobulin-like lectin-1 (Siglec-1), Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPα, SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, tumor necrosis factor α (TNFα), TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-γ, and VTCN1.

[0060] According to the present invention, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is represented by formula IV as follows:

[0061] Where Ab is the antibody, L is the linker, n is an integer or decimal between 1 and 10, and R, R1 and R2 are as defined above.

[0062] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula IV, wherein,

[0063] R1 and R2 are each independently selected from H, halogens, and C1-C6 alkyl groups;

[0064] R is Where X is selected from C and chemical bonds, Y is selected from C, N, O and S, and Z is selected from C and chemical bonds;

[0065] R3 and R4 are each independently selected from H, halogen, amino, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and R5R6NC1-C6 alkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy and amino are each independently optionally substituted by one or more selected from halogen, hydroxy, cyano and amino.

[0066] or,

[0067] When Y is selected from N, O and S, R3 and R4 together with the attached C atom form a 6-8 membered aromatic ring, a 5-8 membered heteroaromatic ring and a 3-6 membered heterocycle, wherein each of the 6-8 membered aromatic ring, the 5-8 membered heteroaromatic ring and the 3-6 membered heterocycle is independently and optionally substituted by one or more selected from C1-C6 alkyl, halogen, hydroxy, cyano and amino groups.

[0068] R5 and R6 are each independently selected from H, C1-C6 alkyl, halogen and C1-C6 alkoxy;

[0069] in,

[0070] R3 and R4 are not both H;

[0071] When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and

[0072] When R is When R3 is not an ethyl group, R3 is not an ethyl group.

[0073] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula IV, wherein,

[0074] R1 and R2 are each independently selected from H, halogens, and C1-C6 alkyl groups;

[0075] R is Where X is selected from C and chemical bonds, Y is selected from C, N, O and S, and Z is selected from C and chemical bonds;

[0076] R3 and R4 are each independently selected from H, halogen, amino, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and R5R6NC1-C6 alkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy and amino are each independently optionally substituted by one or more selected from halogen, hydroxy, cyano and amino.

[0077] R5 and R6 are each independently selected from H, C1-C6 alkyl, halogen and C1-C6 alkoxy;

[0078] in,

[0079] R3 and R4 are not both H;

[0080] When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and

[0081] When R is When R3 is not an ethyl group, R3 is not an ethyl group.

[0082] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula IV, wherein,

[0083] R1 and R2 are each independently selected from H, F, Cl, Br and methyl groups;

[0084] R3 is selected from H, halogen, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and di(C1-C6)alkylaminomethyl;

[0085] R4 is selected from H, amino, C1-C6 alkyl, and C1-C6 alkoxy.

[0086] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula IV, wherein,

[0087] R1 and R2 are each independently selected from H and F;

[0088] R3 is selected from H, F, trifluoromethyl, aminosulfonyl, methyl, methoxy, and dimethylaminomethyl;

[0089] R4 is selected from H, amino, methyl, and methoxy.

[0090] According to the present invention, a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate is represented by the following formula V:

[0091] Where Ab is the antibody, L is the linker, n is an integer or decimal between 1 and 10, and R1, R2, R3 and R4 are as defined above.

[0092] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula V, wherein...

[0093] R1 and R2 are each independently selected from H, F, Cl, Br and methyl groups;

[0094] R3 is selected from H, halogen, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and di(C1-C6 alkyl)aminomethyl;

[0095] R4 is selected from H, amino, C1-C6 alkyl, and C1-C6 alkoxy.

[0096] In some embodiments, the ligand-drug conjugate or its pharmaceutically acceptable salt, as shown in Formula V, wherein...

[0097] R1 and R2 are each independently selected from H and F;

[0098] R3 is selected from H, F, trifluoromethyl, aminosulfonyl, methyl, methoxy, and dimethylaminomethyl;

[0099] R4 is selected from H, amino, methyl, and methoxy.

[0100] According to the ligand-drug conjugate or its pharmaceutically acceptable salt as described in the present invention, wherein the L has -L a -L b- L c -L d - structure, where L a Linked to ligands, L d Related to drugs, including:

[0101] L a For coupling unit; L b It either does not exist or is a connected segment; L c It is a polypeptide fragment; L d It can be either non-existent or any unit.

[0102] According to the ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the present invention, wherein:

[0103] L a Selected from The wavy line represents the connection point with the antibody, and * represents the connection point with L. b The connection point;

[0104] L bThe components are selected from -C(=O)-, -(CH2)mC(=O)-, -C(=O)-(CH2)mC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, -(CH2)mOC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, -NH-(CH2)m-(CH2CH2O)pC(=O)-, -(CH2)m-(CH2CH2O)pC(=O)- and -(CH2)mC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, wherein: m is an integer from 1 to 10, preferably m is 2, 3 or 5; p is an integer from 4 to 32, preferably p is an integer from 10 to 14;

[0105] L c It is a polypeptide fragment composed of 1 to 7 amino acids, wherein the amino acids are selected from valine (V), alanine (A), citrulline (Cit), serine (S), isoleucine (I), lysine (K), glycine (G), phenylalanine (F), glutamic acid (E) and aspartic acid (D).

[0106] L d Selected from the following structures:

[0107] Among them, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

[0108] According to the ligand-drug conjugate or its pharmaceutically acceptable salt as described in the present invention, wherein...

[0109] L a Selected from The wavy line represents the connection point with the antibody, and * represents the connection point with L. b The connection point;

[0110] L b Selected from -C(=O)- and -(CH2)mC(=O)-, where m is 2, 3 or 5;

[0111] L cThe following are examples of amino acids: valine-alanine (VA), valine-glycine (VG), valine-citrulline (VC), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine-glycine (AAAG), glycine-glycine-glycine-glycine (GGGG), valine-alanine-glycine (VAG), valine-citrulline-glycine (VCG), alanine-alanine-glycine (AAG), alanine-alanine-alanine (AAA), and alanine-alanine. (AA), glutamic acid-alanine-glycine-glycine (EAGG), glycine-glutamic acid-alanine-glycine (GEAG), glycine-glutamic acid-glycine-glycine (GEGG), glutamic acid-glycine-glycine (EGG), glutamic acid-alanine-glycine (EAG), valine-lysine-glycine (VKG), glycine-glutamic acid-glycine (GEG), glutamic acid-alanine (EA), glutamic acid-glycine (EG) or glycine-glutamic acid (GE) polypeptide fragments;

[0112] L d Selected from the following structures: Among them, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

[0113] According to the present invention, the ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the ligand-drug conjugate has the structure shown in Formula VI:

[0114] According to the ligand-drug conjugate or a pharmaceutically acceptable salt thereof of the present invention, wherein the antibody is an antibody that specifically binds to BDCA2, IL-4R or TNFα; in some embodiments, the antibody is an antibody that specifically binds to BDCA2; in some embodiments, the antibody comprises the heavy chain shown in SEQ ID NO:1 and the light chain shown in SEQ ID NO:2; or comprises the heavy chain shown in SEQ ID NO:3 and the light chain shown in SEQ ID NO:4.

[0115] In some embodiments, the antibody that specifically binds to BDCA2 comprises the CDR region of the heavy chain shown in SEQ ID NO:7 and the CDR region of the light chain shown in SEQ ID NO:8.

[0116] According to the ligand-drug conjugate or its pharmaceutically acceptable salt, the antibody is an antibody that specifically binds to BDCA2, and the antibody comprises the heavy chain variable region shown in SEQ ID NO:5 and the light chain variable region shown in SEQ ID NO:6.

[0117] In some embodiments, the antibody that specifically binds to BDCA2 comprises the heavy chain shown in SEQ ID NO:7 and the light chain shown in SEQ ID NO:8.

[0118] In some embodiments, the antibody is antibody 24F4, which specifically binds to BDCA2, and is prepared according to the sequence disclosed in patent WO2014093396. The sequences of its light chain and heavy chain are shown below:

[0119] 24F4 heavy chain (SEQ ID NO:1):

[0120] 24F4 light chain (SEQ ID NO:2):

[0121] In some embodiments, the antibody is antibody 3E5, which specifically binds to BDCA2, and is prepared according to the sequence disclosed in patent WO2021023793. The sequences of its light chain and heavy chain are shown below:

[0122] 3E5 heavy chain (SEQ ID NO:3):

[0123] 3E5 light chain (SEQ ID NO:4):

[0124] In some embodiments, the antibody is 10C9-VH6VL5(LS), an antibody that specifically binds to BDCA2, and its preparation process is as follows:

[0125] Balb / c mice (6-8 weeks old) were immunized with 100 μg / mouse of hBDCA2 ECD-Fc Tag protein, followed by immunization with 50 μg / mouse on days 14, 28, and 42. One week after the fourth immunization, the antibody titer in serum collected from the tail vein of each mouse was measured by ELISA. Mice with sufficient titers were boosted with 50 μg of protein, and 3 days later, the mice were sacrificed and spleen cells were collected. These cells were then fused with myeloma cells SP2 / 0 (purchased from Shanghai Cell Bank) using PEG (Sigma; catalog number: P7306) according to standard methods. After 10 days of fusion culture, the binding activity of human BDCA2 protein in the hybridoma cell supernatant was detected by ELISA, and positive hybridoma cells were selected for cryopreservation.

[0126] RNA was extracted from the obtained positive hybridoma cells using standard methods and reverse transcribed into cDNA. Sequencing yielded the heavy chain variable region and light chain variable region sequences of the positive clones. Further analysis of the amino acid sequences was performed, and the CDR amino acid sequences were determined based on databases such as Kabat (Kabat, EA et al. 1991).

[0127] Humanization of murine antibodies was achieved through CDR transplantation. The nucleic acid sequences of the heavy and light chain variable regions of the murine antibody were compared with the human IgG gene sequence database to identify the best-matching human IgG gene sequence. Then, the CDR regions of the heavy and light chains of the chimeric antibody were transplanted into the framework sequences of the heavy and light chain variable regions of the matching human IgG gene, respectively. Individual amino acid reversion mutations and PTM (post-translational modification) removal were then performed to obtain the humanized antibody.

[0128] Heavy chain variable region of 10C9-VH6VL5(LS) (SEQ ID NO:5):

[0129] The light chain variable region of 10C9-VH6VL5(LS) (SEQ ID NO:6):

[0130] Heavy chain sequence of 10C9-VH6VL5(LS) (SEQ ID NO:7):

[0131] The light chain sequence of 10C9-VH6VL5(LS) (SEQ ID NO:8):

[0132] Another object of the present invention is to provide a method for preparing the ligand-drug conjugate shown in Formula III above, comprising the following steps:

[0133] (1) Provide ligands, treat the ligands with a reducing agent to reduce all cysteine ​​disulfide residues;

[0134] (2) The reduced ligand from step (1) is coupled with a drug linker (LD) to obtain the ligand-drug conjugate.

[0135] According to the method for preparing the ligand-drug conjugate of Formula III of the present invention, in step (1),

[0136] The reducing agent is selected from tris(2-carboxyethyl)phosphonic acid hydrochloride (TECP);

[0137] The molar ratio of reducing agent to ligand is 8:1 to 12:1, for example, 8:1, 9:1, 10:1, 11:1, 12:1.

[0138] According to the method for preparing the ligand-drug conjugate of Formula III of the present invention, in step (2),

[0139] The molar ratio of drug linker to ligand is 1:1 to 20:1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1 or 20:1.

[0140] Another object of the present invention is to provide a pharmaceutical composition comprising a compound of formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of formula III, IV, V or VI or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0141] According to the pharmaceutical composition of the present invention, the content of the compound represented by Formula I, or the ligand drug conjugate represented by Formula III, Formula IV, Formula V or Formula VI, is a safe and effective amount, such as 0.001-99 wt%, preferably 0.01-90 wt%, and more preferably 0.1-80 wt%.

[0142] According to the pharmaceutical composition of the present invention, the pharmaceutically acceptable carrier includes one or more of the following: diluent, preservative, filler, flow conditioner, disintegrant, wetting agent, emulsifier, suspending agent, sweetener, flavoring agent, aromatizer, lubricant, and dispersant.

[0143] Another object of the present invention is to provide the use of a compound of Formula I, its stereoisomers or pharmaceutically acceptable salts thereof, or a ligand-drug conjugate of Formula III, IV, V or VI or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for the prevention or treatment of a disease. In some embodiments, the disease is an autoimmune disease or an inflammatory disease; in some embodiments, the disease is selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjögren's syndrome, ankylosing spondylitis, Wegener's granulomatosis and systemic sclerosis, autoimmune hemolytic anemia, pernicious anemia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenic purpura and vasculitis, multiple sclerosis, myasthenia gravis and Guillain-Barré syndrome, ulcerative colitis. Enteritis, Crohn's disease, autoimmune diseases and atrophic gastritis, IgA nephropathy, primary nephrotic syndrome, autoimmune glomerulonephritis, pulmonary renal hemorrhage syndrome and lupus nephritis, type I diabetes, toxic diffuse goiter, Hashimoto's thyroiditis, primary adrenal atrophy and chronic thyroiditis, psoriasis, pemphigus vulgaris, cutaneous lupus erythematosus, dermatomyositis and polymyalgia rheumatica and asthma; in some embodiments, the diseases described are those mediated by dysregulation of the glucocorticoid receptor signaling pathway.

[0144] Another object of the present invention is to provide a compound of Formula I, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of Formula III, IV, V or VI or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for use as a medicine.

[0145] Another object of the present invention is to provide a compound of Formula I, its stereoisomers or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of Formula III, IV, V or VI or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, for the prevention or treatment of a disease; in some embodiments, the disease is an autoimmune disease or an inflammatory disease; in some embodiments, the disease is selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjögren's syndrome, ankylosing spondylitis, Wegener's granulomatosis and systemic sclerosis, autoimmune hemolytic anemia, pernicious anemia, idiopathic hemolytic anemia. Thrombocytopenic purpura, idiopathic thrombocytopenic purpura and vasculitis, multiple sclerosis, myasthenia gravis and Guillain-Barré syndrome, ulcerative colitis, Crohn's disease, autoimmune diseases and atrophic gastritis, IgA nephropathy, primary nephrotic syndrome, autoimmune glomerulonephritis, pulmonary renal hemorrhage syndrome and lupus nephritis, type I diabetes, toxic diffuse goiter, Hashimoto's thyroiditis, primary adrenal atrophy and chronic thyroiditis, psoriasis, pemphigus vulgaris, cutaneous lupus erythematosus, dermatomyositis and polymyalgia rheumatica and asthma; in some embodiments, the diseases described are those mediated by dysregulation of the glucocorticoid receptor signaling pathway.

[0146] Another object of the present invention is to provide a method for preventing or treating a disease, comprising administering to a subject a therapeutically or preventively effective amount of a compound of formula I, its stereoisomers, or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate of formula III, IV, V, or VI, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof; in some embodiments, the disease is an autoimmune disease or an inflammatory disease; in some embodiments, the disease is selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjögren's syndrome, ankylosing spondylitis, Wegener's granulomatosis and systemic sclerosis, autoimmune hemolytic anemia, etc. Pernicious anemia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenic purpura and vasculitis, multiple sclerosis, myasthenia gravis and Guillain-Barré syndrome, ulcerative colitis, Crohn's disease, autoimmune diseases and atrophic gastritis, IgA nephropathy, primary nephrotic syndrome, autoimmune glomerulonephritis, pulmonary renal hemorrhage syndrome and lupus nephritis, type I diabetes, toxic diffuse goiter, Hashimoto's thyroiditis, primary adrenal atrophy and chronic thyroiditis, psoriasis, pemphigus vulgaris, cutaneous lupus erythematosus, dermatomyositis and polymyalgia rheumatica and asthma; in some embodiments, the diseases described are those mediated by dysregulation of the glucocorticoid receptor signaling pathway.

[0147] definition

[0148] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0149] "Halogen" refers to F, Cl, Br or I.

[0150] "Alkyl" is a straight-chain or branched saturated aliphatic hydrocarbon group with 1 to 20 carbon atoms, preferably an alkyl group with 1 to 15 carbon atoms, more preferably an alkyl group with 1 to 8 carbon atoms, and even more preferably an alkyl group with 1, 2, 3, 4, 5, or 6 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and their various branched isomers. The alkyl group may optionally be further substituted with one or more substituents.

[0151] "Alkoxy" refers to a group formed by the bonding of an alkyl group with an oxygen atom. The definition of alkyl is the same as that of "alkyl" as described above. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, n-hexoxy, cyclopropoxy, cyclobutoxy, etc. The alkoxy group may optionally be further substituted by one or more substituents.

[0152] "Heterocyclic" or "heterocyclic group" refers to a substituted or unsubstituted saturated non-aromatic cyclic group, which can be a 3- to 8-membered (e.g., 3, 4, 5, 6, 7, 8-membered) monocyclic, a 6- to 12-membered (e.g., 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1, 2, or 3 heteroatoms selected from N, O, or S, preferably a 3- to 8-membered heterocyclic group. The "heterocyclic group" can be attached to a heteroatom or a carbon atom; the "heterocyclic group" can be a bridged ring or a spirocyclic ring. Non-limiting examples of "heterocyclic groups" include epoxyethyl, aziridinepropyl, oxacyclobutyl, aziridinebutyl, 1,3-dioxopentyl, 1,4-dioxopentyl, 1,3-dioxahexacycloyl, aziridineheptyl, piperidinyl, piperinyl, morpholinyl, thiomorpholinyl, 1,3-dithiaalkyl, tetrahydrofuranyl, tetrahydropyrroleyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, aziridine[3.2.1]octyl, aziridine[5.2.0]nonyl, oxacyclo[5.3.1.1]dodecyl, aziridine, and oxaspiro[3.3]heptyl, etc. The heterocyclic group may optionally be further substituted with one or more substituents.

[0153] "Aromatic ring" or "aryl" refers to a substituted or unsubstituted aromatic ring, which can be a 6- to 8-membered monocyclic ring (e.g., 6, 7, 8-membered), a 6- to 12-membered (e.g., 6, 7, 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system. It can be a bridged ring or a spirocyclic ring. Non-limiting examples include phenyl, naphthyl, etc. The aromatic ring may optionally be further substituted by one or more substituents.

[0154] "Heteroaromatic ring" or "heteroaryl" refers to an aromatic ring having a conjugated planar ring system and containing heteroatoms. It can be a 5- to 8-membered (e.g., 5, 6, 7, 8-membered) monocyclic ring, an 8- to 12-membered (e.g., 8, 9, 10, 11, 12-membered) bicyclic ring, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, 15-membered) tricyclic system, and contains 1 to 6 (e.g., 1, 2, 3, 4, 5, 6) heteroatoms selected from N, O, or S. Examples include 5-8-membered nitrogen-containing heteroaromatic rings, 5-8-membered oxygen-containing heteroaromatic rings, and 5-8-membered sulfur-containing heteroaromatic rings. Non-limiting examples of heteroaryl groups include oxazolyl, triazolyl, pyridyl, furanyl, thiophenyl, pyranyl, pyrroloyl, pyrazolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, benzimidazolyl, benzopyridyl, pyrrolopyridyl, etc. The heterocyclic ring may optionally be further substituted by one or more substituents.

[0155] The carbon, hydrogen, oxygen, sulfur, nitrogen, F, Cl, Br, or I involved in the groups and compounds described in this invention include their isotopes, and the carbon, hydrogen, oxygen, sulfur, or nitrogen involved in the groups and compounds described in this invention may optionally be further replaced by one or more of their corresponding isotopes, wherein the isotopes of carbon include 12C, 13C, and 14C, the isotopes of hydrogen include protium (H), deuterium (D, also called heavy hydrogen), and tritium (T, also called superheavy hydrogen), the isotopes of oxygen include 16O, 17O, and 18O, the isotopes of sulfur include 32S, 33S, 34S, and 36S, the isotopes of nitrogen include 14N and 15N, the isotopes of fluorine include 17F and 19F, the isotopes of chlorine include 35Cl and 37Cl, and the isotopes of bromine include 79Br and 81Br.

[0156] "Pharmaceutical composition" refers to a mixture of one or more compounds described in this invention, their pharmaceutically acceptable salts or prodrugs, and other chemical components, wherein "other chemical components" refers to pharmaceutically acceptable carriers, excipients, and / or one or more other therapeutic agents.

[0157] The term "pharmaceutically acceptable salt" refers to derivatives obtained from the compounds of this invention prepared with relatively non-toxic acids or bases. These salts can be prepared during the synthesis, isolation, and purification of the compounds, or by reacting the purified free form of the compounds with suitable acids or bases. When the compounds contain relatively acidic functional groups, they react with alkali metal, alkaline earth metal hydroxides, or organic amines to yield base addition salts, including cations based on alkali metals and alkaline earth metals. When the compounds contain relatively basic functional groups, they react with organic or inorganic acids to yield acid addition salts.

[0158] In the chemical structure of the compound described in this invention, the bond... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration representing the center of a solid.

[0159] The term "stereoisomer" refers to an isomer formed by at least one asymmetric center. In compounds having one or more asymmetric centers, racemic mixtures, single enantiomers, mixtures of diastereomers, and individual diastereomers can be produced. Specific individual molecules may also exist as geometric isomers (cis / trans). Unless otherwise stated, when the stereochemistry of a disclosed compound is not explicitly described in its name or structure and it has one or more asymmetric centers, it should be understood that all possible stereoisomers of the compound are represented.

[0160] The stereoisomers of the compounds of this invention can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. For example, an enantiomer of a compound of this invention can be prepared by asymmetric catalysis or chiral derivative derivatization. Alternatively, a single stereoisomer can be obtained from a mixture using chiral resolution techniques. Alternatively, it can be prepared directly from chiral starting materials. The separation of optically pure compounds in this invention is typically accomplished using preparative chromatography, employing chiral chromatographic columns to achieve the separation of chiral compounds.

[0161] The term "pharmaceutically acceptable carrier" refers to a medium generally acceptable in the art for delivering a bioactive pharmaceutical agent to animals, particularly mammals. Depending on the route of administration and dosage form, this includes adjuvants, excipients, or excipients such as diluents, preservatives, fillers, flow modifiers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatizers, lubricants, and dispersants. Pharmaceutically acceptable carriers are formulated based on a multitude of factors, including but not limited to: the type and nature of the active pharmaceutical agent, the target population to which the composition containing the pharmaceutical agent will be administered, the intended route of administration of the composition, and the target therapeutic indication.

[0162] The term "glucocorticoid" generally refers to naturally occurring or synthetic steroid hormones that interact with glucocorticoid receptors.

[0163] Ligands are any large molecules (peptides, proteins, peptides, typically antibodies) commonly used in drug conjugation techniques, or small molecules (such as folic acid or aptamers) that can be covalently conjugated to linkers or drug-linkers using bioconjugation techniques (Greg T. Hermanson, Bioconjugate Techniques, 3rd Edition, 2013, Academic Press). Ligands are traditionally selected based on their targeting ability. A non-exhaustive list of ligands includes: proteins, peptides, peptides, antibodies, full-length antibodies and their antigen-binding fragments, interferons, lymphokines, hormones, growth factors, vitamins, transferrin, or any other cell-binding molecule or substance. The primary class of ligands used to prepare conjugates is antibodies.

[0164] The term "antibody-drug conjugate" refers to an antibody (Ab) linked to a biologically active drug via a stable linker. The chemical structural fragment or bond linked to the Ab is called a "linker".

[0165] The term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. For example, a natural IgG antibody is a heterotetraglycosyl protein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to the C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N to the C-terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL). The light chains are either κ or λ. The heavy chain is divided into γ, μ, α, δ, or ε, and antibody isotypes are defined as IgG, IgM, IgA, IgD, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are linked by a “J” region of approximately 12 or more amino acids, and the heavy chain also includes a “D” region of approximately 10 or more amino acids. (Generally, see *Basic Immunology*, Paul W., ed., 2nd ed. Raven Press, NY, 1989, Ch. 7; all are incorporated herein by reference for various purposes.)

[0166] The term "antibody fragment" refers to a molecule that is distinct from the complete antibody but contains a portion of the complete antibody that retains the antigen-binding ability of the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibodies, single-chain Fab (scFab), biantibodies, linear antibodies, and single-chain antibody molecules (e.g., scFv). The fragments are: (i) Fab fragments having VL, CL, VH, and CH1 domains and a disulfide bond between the heavy and light chains; (ii) Fab' fragments having one or more cysteine ​​residues at the C-terminus of the CH1 domain; (iii) Fd fragments having VH and CH1 domains; (iv) Fd' fragments having VH and CH1 domains and one or more cysteine ​​residues at the C-terminus of the CH1 domain; (v) Fv fragments having VL and VH domains in one arm of the antibody; (vi) dAb fragments consisting of a VH domain; (vii) hingeless antibodies containing at least VL, VH, CL, and CH1 domains and lacking a hinge region; (vii) ii) F(ab)2 fragment, which is a bivalent fragment containing two Fab' fragments connected by a disulfide bridge in the hinge region; (ix) single-chain antibody molecule (e.g., single-chain Fv; scFv); (x) "diabodies" having two antigen-binding sites, including a heavy chain variable domain (VH) and a light chain variable domain (VL) connected to each other in the same polypeptide chain; (xi) "linear antibody" containing a pair of tandem Fd fragments (VH-CH1-VH-CH1), which together with the complementary light chain polypeptide form a pair of antigen-binding regions; (xii) dsFv refers to a fragment formed by replacing one amino acid residue in each of VH and VL with a cysteine ​​residue and then linking the polypeptides together via an SS bond between the cysteine ​​residues.

[0167] The term "linker" refers to a chemical structural fragment that links an active drug substance and an antibody (Ab). In this application, the term "linker" generally refers to any chemical portion capable of linking a protein (e.g., an antibody, an antibody fragment (e.g., an antigen-binding fragment), or a functional equivalent) to a glucocorticoid. Linkers may be cleavable ("cleavable linkers"), thereby facilitating the release of glucocorticoids. For example, under conditions where the glucocorticoid and / or antibody remain active, such cleavable linkers may be sensitive to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Alternatively, linkers may be substantially cleavable ("uncleavable linkers").

[0168] The term "drug-antibody ratio" or "DAR" refers to the number of groups derived from a small molecule glucocorticoid receptor agonist (e.g., glucocorticoid) linked to an Ab (i.e., a protein, such as an antibody or its antigen-binding fragment, an anti-TNF protein, an anti-TNF-α antibody or a fragment thereof, a soluble receptor, or a soluble TNF receptor). It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably selected from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplarily, the drug loading can be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and can be a decimal or an integer. In this application, DAR is defined by the variable "n," and the drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC.

[0169] The drug loading of ligand-drug conjugates can be controlled using the following non-limiting methods, including:

[0170] (1) Control the molar ratio of drug linker fragments to monoclonal antibodies,

[0171] (2) Control the reaction time and temperature.

[0172] (3) Choose different reaction reagents.

[0173] The term "variable region" or "variable domain" generally refers to the structural domain of the antibody heavy or light chain involved in antibody-antigen binding. In this application, the term "variable" generally refers to the fact that certain portions of the sequence of the antibody's variable domain vary significantly, resulting in various specific antibody binding and specificity to their respective antigens. This variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the light and heavy chain variable regions, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, connected by three CDR structural loop regions. The CDRs in each chain are closely packed together through the FR region and together with the CDRs from the other chain, they form the antigen-binding site of the antibody.

[0174] In this field, various methods can be used to encode the variable region of an antibody or to delineate the antibody's CDR, such as the Kabat numbering scheme and definition rules based on sequence variability (see Kabat et al., Protein Sequence in Immunology, 5th Edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia numbering scheme and definition rules based on the location of structural loop regions (see Al-Lazikani et al., JMol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V gene by franc et al., as well as Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc., see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018.

[0175] The terms "treatment" and "treating" generally refer to a method for achieving a beneficial or desired outcome, including but not limited to therapeutic benefits. Therapeutic benefits include, but are not limited to, eradicating, suppressing, reducing, or improving the underlying disorder being treated. Additionally, therapeutic benefits are achieved by eradicating, suppressing, reducing, or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the patient, although the patient may still have the underlying disorder.

[0176] The terms “prevention” and “preventing” generally refer to methods for achieving a beneficial or desired outcome, including but not limited to preventive benefits. For the purpose of preventing benefits, a drug may be administered to a patient at risk of developing a particular disease or to a patient who reports having one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0177] The term "therapeutic or preventative effective dose" refers to a compound of the present invention, its stereoisomers, or pharmaceutically acceptable salts thereof, in an amount sufficient to provide a reasonable benefit / risk ratio for any medical treatment and / or prevention of the disorder. However, it should be understood that the total daily dosage of the compounds of the present invention, their stereoisomers, or pharmaceutically acceptable salts thereof must be determined by the attending physician within the bounds of reliable medical judgment. For any given patient, the specific therapeutically effective dose level must be determined based on a number of factors, including the disorder being treated and its severity; the activity of the specific compound used; the patient's age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with or concurrently with the specific compound used; and similar factors known in the medical field. For example, it is practiced in the art to start with a dose of the compound below the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0178] In this application, the terms “subject” or “patient” generally refer to humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quails, and / or turkeys.

[0179] In this application, the term "comprising" and its variations, including "containing," "including," and other forms, generally refers to including other components, elements, values, steps, etc. Attached Figure Description

[0180] Figure 1A shows the inhibition of TLR9-induced IFN-α secretion by antibody-drug conjugates.

[0181] Figure 1B shows the inhibition of TLR9-induced TNF-α secretion by antibody-drug conjugates.

[0182] Figure 1C shows the inhibition of TLR9-induced IL-6 secretion by the antibody-drug conjugate.

[0183] Figure 1D shows the inhibition of TLR9-induced IL-8 secretion by the antibody-drug conjugate. Detailed Implementation

[0184] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). NMR shifts (δ) were expressed in 10⁻¹⁰ ohms. -6The unit (ppm) is given. NMR measurements were performed using an AVANCE NEO 300MHz, AVANCE NEO 400MHz, AVANCE III 300MHz, or AVANCE III HD 400MHz NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD). The internal standard was tetramethylsilane (TMS).

[0185] MS measurements were performed using an Agilent G6125B mass spectrometer.

[0186] The CombiFlash rapid preparation instrument uses a Yamazen medium-pressure preparative chromatograph.

[0187] The known starting materials of this invention can be synthesized using or according to methods known in the art, or can be purchased from companies such as McLean, Aladdin, ACMEC, Shaanxi Didu Co., Ltd., Jiangsu Aikon, Leyan, and Alpha Chemical.

[0188] Unless otherwise specified in the examples, the reaction can be carried out in an argon atmosphere or a nitrogen atmosphere, which means that the reaction flask is connected to an argon balloon or a nitrogen balloon with a volume of about 1L.

[0189] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0190] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0191] In the examples, TLC was used to monitor the reaction progress. The developing solvents used in the reaction were: A: dichloromethane / methanol system, B: petroleum ether / ethyl acetate. The volume ratio of the solvents was adjusted according to the polarity of the compounds. Small amounts of basic or acidic reagents such as ammonia and acetic acid could also be added for adjustment.

[0192] HPLC can also be used to monitor the reaction process. The HPLC method is as follows: column: Aglient, Eclipse XDB-C18, 5um, 4.6mm×150mm column, wavelength 220~254nm, flow rate 1mL / min, phases A and B are eluted according to a certain gradient program A: 0.1% TFA aqueous solution, B acetonitrile.

[0193] C18 preparation and chromatographic purification uses acetonitrile and water as solvents and elution according to a certain gradient program. The gradient can be adjusted according to the HPLC elution time of the compound.

[0194] Preferred embodiments of the present invention will now be described in more detail. While preferred embodiments of the present invention are described below, it should be understood that one should not be limited to the embodiments set forth herein. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0195] Example 1. Preparation of the compound

[0196] Example 1-1. Synthesis of BD0166

[0197] Compound 1A (16-α-hydroxy-prednisolone (purchased from ACMEC, the same below) 0.564 g (1.5 mmol) was added to a single-necked flask and dissolved in 15 mL of acetonitrile. Compound 1B (16-α-hydroxy-prednisolone (purchased from Jiangsu Aikon, the same below)) 0.210 g (1.5 mmol) was added, along with 2-amino-4-fluoro-benzaldehyde (purchased from Jiangsu Aikon, the same below). The flask was placed in an ice bath, and 1.126 g (7.5 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, the mixture was diluted with ethyl acetate, washed with sodium bicarbonate solution, and the organic phase was concentrated to obtain crude BD0166-A with a mixed configuration. C18 preparative chromatography was used to purify the crude product to give 0.250 g of compound BD0166.

[0198] MS (ESI) m / z of compound BD0166: 498.2 [M+H] + .

[0199] Examples 1-2. Synthesis of BD0172

[0200] 0.623 g (1.5 mmol) of compound 2A (dihydroxyfluocinolone acetonide, purchased from Shaanxi Didu) and 0.21 g (1.5 mmol) of compound 1B were dissolved in 5 mL of acetonitrile. 1.126 g (7.5 mmol) of trifluoromethanesulfonic acid was added in an ice bath, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed three times with sodium bicarbonate. After drying and concentration, crude BD0172-A with a mixed configuration was obtained. C18 preparative chromatography was used to purify the crude product to give 0.337 g of compound BD0172.

[0201] MS-ESI of compound BD0172: m / z 534.2 [M+H] + .

[0202] Examples 1-3. Synthesis of BD0173

[0203] Compound 3A (triamcinolone, purchased from Shaanxi Didu) 0.8 g (2.03 mmol) and compound 1B 0.28 g (2.03 mmol) were added to a single-necked flask, 10 mL of acetonitrile was added, and the mixture was stirred to dissolve. The flask was placed in an ice bath, and trifluoromethanesulfonic acid (10.15 mmol) was added. The mixture was stirred in an ice bath for 40 min. After the reaction was complete, ethyl acetate was added to dissolve the mixture, and the solution was washed with saturated sodium bicarbonate solution until neutral. The organic phase was collected and concentrated to obtain crude BD0173-A with a mixed configuration. The crude product was purified by C18 preparative chromatography to obtain 415 mg of compound BD0173.

[0204] MS-ESI of compound BD0173: m / z 516.2 [M+H] + .

[0205] Examples 1-4. Synthesis of BD0184

[0206] 350 mg (0.93 mmol) of compound 1A was added to a single-necked flask and dissolved in 15 mL of acetonitrile. 113 mg (0.93 mmol, 2-amino-benzaldehyde) of compound 4A was added, and the flask was placed in an ice bath. 558 mg (3.72 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0184-A with a mixed configuration. C18 preparative chromatography was used for purification to obtain 294 mg of compound BD0184.

[0207] MS-ESI of compound BD0184: m / z 480.2 [M+H] + .

[0208] Examples 1-5. Synthesis of BD0230

[0209] 0.565 g (1.5 mmol) of compound 1A was dissolved in 12 mL of acetonitrile, and 261 mg (1.5 mmol) of compound 5A (4-(trifluoromethyl)benzaldehyde, purchased from Leyan) was added. The mixture was placed in an ice bath, and 675 mg (4.5 mmol) of trifluoromethanesulfonic acid was added. The mixture was stirred in an ice bath for 1 h. After the reaction was completed, the mixture was diluted with ethyl acetate, washed three times with sodium bicarbonate, dried and concentrated to obtain crude compound BD0230-A with mixed configuration. C18 preparative chromatography was used to purify the crude compound BD0230 to obtain 472 mg of compound BD0230.

[0210] MS-ESI of compound BD0230: m / z 619.3 [M+H] + .

[0211] Examples 1-6. Synthesis of BD0231

[0212] Compound 1A 500 mg (1.32 mmol) and compound 6A (4-formylbenzenesulfonamide, purchased from Alpha Chemicals) 245 mg (1.32 mmol) were added to a single-necked flask and dissolved in 20 mL of acetonitrile. Trifluoromethanesulfonic acid 597 mg (3.98 mmol) was added dropwise under ice bath conditions. The mixture was stirred under ice bath conditions for about 1 hour. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with sodium bicarbonate, and the organic phase was concentrated to obtain crude compound BD0231-A with mixed configuration. C18 preparative chromatography was used to purify the crude compound BD0231 to obtain 449 mg of compound BD0231.

[0213] MS-ESI of compound BD0231: m / z 544.2 [M+H] + .

[0214] Examples 1-7. Synthesis of BD0260

[0215] 200 mg (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 66 mg (0.531 mmol) of compound 7A (p-fluorobenzaldehyde) was added, and the flask was placed in an ice bath. 0.239 g (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0260-A with a mixed configuration. C18 preparative chromatography was used for purification to obtain 0.083 g of compound BD0260.

[0216] MS-ESI of compound BD0260: m / z 483.2 [M+H] + .

[0217] 1H NMR (400MHz, DMSO) δ7.57–7.48(m,2H),7.32(d,J=10.1Hz,1H),7.26–7.17(m,2H),6.17(dd,J=10.1,1.9Hz,1H) ,5.94(s,1H),5.47(s,1H),5.09(t,J=5.9Hz,1H),4.94(d,J=4.9Hz,1H),4.80(d,J=3.2Hz,1H),4.53(dd,J=19.5 ,6.4Hz,1H),4.34–4.27(m,1H),4.19(dd,J=19.5,5.5Hz,1H),2.55(dd,J=13.7,8.7Hz,1H),2.32(dd,J=13.3,2. 6Hz,1H),2.19–2.08(m,1H),2.06–1.98(m,1H),1.84–1.58(m,5H),1.40(s,3H),1.14–0.97(m,2H),0.87(s,3H).

[0218] Examples 1-8. Synthesis of BD0261

[0219] 0.200 g (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 64 mg (0.531 mmol) of compound 8A (p-tolualdehyde, purchased from Maclean's) was added, and the flask was placed in an ice bath. 0.239 g (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0261-A with mixed configuration. C18 preparative chromatography was used for purification to give 0.134 g of compound BD0261.

[0220] MS-ESI of compound BD0261: m / z 479.2 [M+H] + .

[0221] Examples 1-9. Synthesis of BD0262

[0222] 200 mg (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 71 mg (0.531 mmol) of compound 9A (2,4-xyleneformaldehyde, purchased from Aladdin) was added, and the flask was placed in an ice bath. 0.239 g (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0262-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 0.109 g of compound BD0262.

[0223] MS–ESI of compound BD0262: m / z 493.2 [M+H] + .

[0224] Examples 1-10. Synthesis of BD0263

[0225] 200 mg (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 64 mg (0.531 mmol) of compound 10A (3,4-xyleneformaldehyde, purchased from Aladdin) was added, and the flask was placed in an ice bath. 239 mg (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound 1BD0263-A with a mixed configuration. C18 preparative chromatography was used for purification to give 0.143 g of compound BD0263.

[0226] MS-ESI of compound BD0263: m / z 493.2 [M+H] + .

[0227] Examples 1-11. Synthesis of BD0264

[0228] 500 mg (1.33 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 178 mg (1.33 mmol) of compound 11A (3,5-xyleneformaldehyde, purchased from Aladdin) was added, and the flask was placed in an ice bath. 600 mg (4 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0264-A with a mixed configuration. C18 preparative chromatography was used for purification to give 0.291 g of compound BD0264.

[0229] MS-ESI of compound BD0264: m / z 493.2 [M+H] + .

[0230] Examples 1-12. Synthesis of BD0265

[0231] 500 mg (1.32 mmol) of compound 1A was added to a single-necked flask and dissolved in 15 mL of acetonitrile. 159 mg (1.32 mmol) of compound 12A (m-tolualdehyde, purchased from Maclean's) was added, and the flask was placed in an ice bath. 0.600 mg (4 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0265-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 0.340 g of compound BD0265.

[0232] MS-ESI of compound BD0265: m / z 479.2 [M+H] + .

[0233] Examples 1-13. Synthesis of BD0266

[0234] 200 mg (0.531 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 64 mg (0.531 mmol) of compound 13A, o-tolualdehyde (purchased from Aladdin), was added. The flask was placed in an ice bath, and 0.239 g (1.593 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0266-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 0.131 g of compound BD0266.

[0235] MS-ESI-m / z 479.2 for compound BD0266 [M+H] + .

[0236] Examples 1-14. Synthesis of BD0267

[0237] 226 mg (0.602 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 100 mg (0.602 mmol) of compound 14A (3,4-dimethoxytolualdehyde, purchased from Maclean's) was added, and the flask was placed in an ice bath. 271 mg (1.806 mmol) of trifluoromethanesulfonic acid was added dropwise, and the mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0267-A with mixed configuration. C18 preparative chromatography was used for purification to give 0.147 g of compound BD0267.

[0238] MS-ESI of compound BD0267: m / z 525.2 [M+H] + .

[0239] Examples 1-15. Synthesis of BD0268

[0240] 450 mg (1.2 mmol) of compound 1A was added to a single-necked flask and dissolved in 10 mL of acetonitrile. 175 mg (1.2 mmol) of compound 15A (1H-pyrrolo[2,3-b]pyridine-2-carboxaldehyde, purchased from ACMEC) was added. The flask was placed in an ice bath, and 540 mg (3.6 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0268-A with mixed configuration. C18 preparative chromatography was used for purification to give 0.229 g of compound BD0268.

[0241] MS-ESI of compound BD0268: m / z 505.2 [M+H] + .

[0242] Examples 1-16. Synthesis of BD0269

[0243] 450 mg (1.2 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 0.196 g (1.2 mmol) of compound 16A (thieno[2,3-b]pyridine-2-carboxaldehyde, purchased from ACMEC) was added. The flask was placed in an ice bath, and 540 mg (3.6 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0269-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 0.109 g of compound BD0269.

[0244] MS-ESI of compound BD0269: m / z 522.2 [M+H] + .

[0245] Examples 1-17. Synthesis of BD0270

[0246] 400 mg (1.06 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 172 mg (1.06 mmol) of compound 17A (1-benzothiaphen-2-carboxaldehyde, purchased from ACMEC) was added. The flask was placed in an ice bath, and 0.477 g (3.18 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0270-A with mixed configuration. C18 preparative chromatography was used for purification to give 0.116 g of compound BD0270.

[0247] MS-ESI of compound BD0270: m / z 521.2 [M+H] + .

[0248] Examples 1-18. Synthesis of BD0272

[0249] 500 mg (1.32 mmol) of compound 1A was added to a single-necked flask and dissolved in 6 mL of acetonitrile. 192 mg (1.32 mmol) of compound 18A (1H-pyrrolo[2,3-B]pyridine-3-carboxaldehyde, purchased from ACMEC) was added. The flask was placed in an ice bath, and 600 mg (4 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0272-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 242 mg of compound BD0272.

[0250] MS-ESI of compound BD0272: m / z 505.3 [M+H] + .

[0251] Examples 1-19. Synthesis of BD0273

[0252] 500 mg (1.32 mmol) of compound 1A was added to a single-necked flask and dissolved in 10 mL of acetonitrile. 192 mg (1.32 mmol) of compound 19A (1H-pyrrolo[2,3-c]pyridine-3-carboxaldehyde, purchased from ACMEC) was added. The flask was placed in an ice bath, and 600 mg (4.0 mmol) of trifluoromethanesulfonic acid was added dropwise. The mixture was stirred in an ice bath for about 1 hour. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate solution. The organic phase was concentrated to obtain crude compound BD0273-A with mixed configuration. C18 preparative chromatography was used for purification to obtain 252 mg of compound BD0273.

[0253] MS-ESI of compound BD0273: m / z 505.1 [M+H]+ .

[0254] Synthesis of BD0275 (Examples 1-20)

[0255] (1) Synthesis of compound 20B

[0256] 0.5 g of methyl 4-(bromomethyl)-2-methylbenzoate (purchased from Jiangsu Aikon) and 0.44 g of sodium carbonate were mixed and dissolved in 20 mL of THF (tetrahydrofuran). The mixture was cooled to 0 °C and stirred. 2 mL of 2.0 M dimethylamine THF solution (purchased from Aladdin) was added, and the mixture was heated to 45 °C and stirred for 1 h. The mixture was filtered, and the filtrate was concentrated to dryness. Diethyl ether was added and the mixture was stirred and filtered. The filtrate was the product, and the product was concentrated to obtain 0.395 g of compound 20B.

[0257] MS-ESI of compound 20B: m / z 208.1 [M+H] + .

[0258] (2) Synthesis of compound 20C

[0259] 0.192 g of compound 20B was dissolved in 2 mL of dichloromethane. The solution was allowed to dissolve completely, and the solution was cooled to -78 °C under nitrogen. A mixed solution of 1.05 mL of 1.5 M diisobutylaluminum hydride (DIBAL-H) ​​and 8 mL of dichloromethane was added. The reaction mixture was stirred at -78 °C for another 2 hours, and then 33 mL of dry methanol was added dropwise. The reaction mixture was heated to approximately -20 °C, and then 60 mL of hydrochloric acid solution was added. The mixture was stirred vigorously for 2 hours, and the product was in the aqueous phase. 0.062 g of compound 20C was obtained by C18 preparative chromatography.

[0260] MS-ESI of compound 20C: m / z 178.1 [M+H] + .

[0261] (3) Synthesis of BD0275

[0262] 0.65 g of compound 1A was dissolved in 5 mL of acetonitrile, and 0.306 g of compound 20C was added. The mixture was placed in an ice bath, and 0.9 mL of perchloric acid was added. The mixture was stirred in an ice bath for 1 h. After the reaction was complete, ethyl acetate was added for dilution, and the mixture was washed with sodium bicarbonate and concentrated to obtain the mixed configuration compound BD0275-D. C18 was prepared and purified to obtain 96 mg of compound BD0275.

[0263] MS-ESI of compound BD0275: m / z 536.3 [M+H] + .

[0264] Example 2. Synthesis of Drug Linker (LD)

[0265] Example 2-1. Synthesis of LD-1

[0266] 2.0 g of compound 1N-[fluorenylmethoxycarbonyl]-L-valine-L-alanine (purchased from Jier Biochemical) was added to a reaction flask, dissolved in 50 mL of dichloromethane and 10 mL of LDMF. Then, 1.27 g of glycine tert-butyl ester (purchased from Maclean), 4.0 g of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), and 3.2 mL of 2,4,6-trimethylpyridine were added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, 100 mL of dichloromethane was added for dilution, and the mixture was washed five times with water. After separation, the dichloromethane phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give 4.57 g of compound 2. MS (ESI) m / z: 524.3 [M+H] + .

[0267] 2.50 g of compound 2 was dissolved in 20 mL of dichloromethane, and 20 mL of trifluoroacetic acid was added. After the reaction was completed as monitored by HPLC, ethyl acetate was added and the mixture was stirred for 2 h. The mixture was filtered and dried under vacuum to obtain 2.41 g of compound 3. MS (ESI) m / z: 468.2 [M+H] + .

[0268] 2.41 g of compound 3, 5 mg of copper acetate, and 0.7 mL of acetic acid were added to a reaction flask. 2.64 g of lead tetraacetate (purchased from Aladdin) was dissolved in 50 mL of LDM and added dropwise to the reaction flask. The reaction system was purged with nitrogen, heated to 70 °C, and reacted for 4 hours. After the reaction was complete, ethyl acetate was added, the mixture was filtered, the filtrate was washed with water, separated, and the organic phase was concentrated to give 1.14 g of compound 4. MS (ESI) m / z: 482.2 [M+H] + .

[0269] 0.939 g of compound 5 (C-1) and 1.07 g of compound 4 were added to a reaction flask and dissolved in 50 mL of tetrahydrofuran. 0.436 g of potassium tert-butoxide was dissolved in 20 mL of tetrahydrofuran. The reaction system was purged with nitrogen. The tetrahydrofuran solution of potassium tert-butoxide was added dropwise to the reaction flask under ice bath conditions. After the addition was complete, the reaction was continued under ice bath conditions for 2 hours. After the reaction was complete, water was added to quench the reaction mixture. The mixture was extracted with ethyl acetate, and the organic phase was concentrated to give 1.234 g of compound 6. MS (ESI) m / z: 886.4 [M+H] + .

[0270] 0.825 g of compound 6 was added to a reaction flask, dissolved in 30 mL of dichloromethane, and then 1.1 mL of piperidine was added. The reaction was carried out at room temperature for 4 hours. After the reaction was complete, 30 mL of diethyl ether was added and the mixture was stirred. After filtration and vacuum drying, 0.801 g of compound 7 was obtained. MS (ESI) m / z: 664.3 [M+H] + .

[0271] 0.72 g of compound 7, 0.40 g of compound 8, and 0.596 g of 2-bromo-1-ethylpyridine boron tetrafluoride salt were added to a reaction flask and dissolved in 3 mL of LDMF. Then, 0.38 mL of diisopropylethylamine was added, and the mixture was allowed to react overnight at room temperature. After the reaction was complete, the solution was purified by C18 preparative chromatography to obtain LD-1, 0.534 g, MS (ESI) m / z: 1000.2 [M+H]. + .

[0272] Example 2-2. Preparation of LD-2

[0273] 0.678 g of compound 9 (BD0260) and 0.680 g of compound 4 were added to a reaction flask and dissolved in 50 mL of tetrahydrofuran. 0.318 g of potassium tert-butoxide was dissolved in 15 mL of tetrahydrofuran. The reaction system was purged with nitrogen. The tetrahydrofuran solution of potassium tert-butoxide was added dropwise to the reaction flask under ice bath conditions. After the addition was complete, the reaction was continued under ice bath conditions for 2 hours. After the reaction was complete, water was added to quench the reaction mixture. The mixture was extracted with 80 mL of ethyl acetate, and the organic phase was concentrated to obtain 0.653 g of compound 10. MS (ESI) m / z: 904.4 [M+H] + .

[0274] 0.630 g of compound 10 was added to a reaction flask, dissolved in 10 mL of dichloromethane, followed by 2 mL of piperidine. The reaction was carried out at room temperature for 4 hours. After the reaction was complete, 100 mL of diethyl ether was added, and the mixture was stirred and slurried. The mixture was filtered and dried under vacuum to obtain 0.487 g of compound 11. MS (ESI) m / z: 681.3 [M+H] + .

[0275] 0.470 g of compound 11, 0.251 g of compound 8, and 0.379 g of 2-bromo-1-ethylpyridine boron tetrafluoride salt were added to a reaction flask, dissolved in 15 mL of LDMF, and then 0.24 mL of diisopropylethylamine was added. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete, the product was purified by C18 preparative chromatography to obtain 0.380 g of LD-2. MS (ESI) m / z: 1017.2 [M+H] + .

[0276] Example 3. Coupling of ADC

[0277] DAR value detection:

[0278] The DAR value of the ADC of this invention was analyzed using hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). The ADC was separated in the column using a MabPac HIC-Butyl analytical column (4.6 x 100 mm, 5 μm, catalog number 088558, ThermoFisher, USA).

[0279] Chromatographic conditions: Mobile phase A was 25 mM sodium phosphate buffer (pH 6.8) containing 1.5 M ammonium sulfate, and mobile phase B was 25 mM sodium phosphate buffer (pH 6.8) containing 25% acetonitrile. Gradient elution: 0–2 min 15% buffer B, 2–16 min 15%–80% buffer B, 16–18 min 80%–100% buffer B, 18–20 min 100%–15% buffer B, 20–21 min 15% buffer B. Flow rate and temperature were set to 0.5 mL / min and 25 °C, respectively. ADC drug distribution was detected at 248 nm and 280 nm for DAR value calculation.

[0280] ADC fabrication

[0281] Antibody-adjuvant (ADC) drugs are prepared by reacting antibodies with drug linkers (LDs). In simple terms, the antibody is treated with the reducing agent tris(2-carboxyethyl)phosphonic acid hydrochloride (TECP) to reduce all cysteine ​​disulfide residues, forming a highly nucleophilic cysteine ​​thiol group (-CH2SH). This fully reduced antibody then reacts with the electrophilic functional group (bromoacetyl) of the drug linker to ultimately prepare the ADC.

[0282] Antibodies dissolved in PBS (pH 7.2), such as 24F4, were mixed with 2 mM EDTA and reduced with TCEP (TCEP:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, drug linkers LD-1 or LD-2 were added to the reduced antibody (drug linker:antibody molar ratio 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-1 or ADC-2, which were then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-1 or ADC-2 was between 3.7 and 4.3. The structures of ADC-1 and ADC-2 are as follows:

[0283] Antibody 10C9-VH6VL5(LS) dissolved in PBS at pH 7.2 was supplemented with 2 mM EDTA and reduced with TCEP (TCEP:antibody molar ratio 10:1). After incubation at 37°C for approximately 120 minutes, drug linker LD-1 or LD-2 was added to the reduced antibody (drug linker:antibody molar ratio 5:1). The mixture was purified and desalted by elution with G25 resin at room temperature for 1 hour, and filtered aseptically through a 0.2 μm filter to obtain ADC-3 or ADC-4, which were then frozen and stored. Analysis using the above hydrophobic interaction chromatography-high performance liquid chromatography (HPLC) determined that the average DAR value of ADC-3 or ADC-4 was between 3.7 and 4.3. The structures of ADC-3 and ADC-4 are as follows:

[0284] The comparative compounds used in this invention are as follows:

[0285] Comparative Example 1: Compound C-1

[0286] The compound was synthesized according to the synthesis method of Compound 41 in Example 41 of Patent WO2005028495A1.

[0287] Comparative Example 2: Compound C-5

[0288] The compound was synthesized according to the synthesis method of step 5 of Example 2A in patent CN109476699A.

[0289] Bioactivity test

[0290] Test Example 1. Assay of Glucocorticoid Receptor Reporter Gene (GRE) Activity

[0291] 1.1 Cell line construction

[0292] After digesting 293T cells with trypsin, at a dose of 2×10⁻⁶... 5Inoculate one cell per well into a 6-well culture dish and incubate overnight in an incubator. 2.5 μg of pGL4.35 [luc2P / 9XGAL4UAS / Hygro] (Promega; catalog number: E1370), 2.5 μg of pBIND-GR (Promega; catalog number: E1581), and 18 μL of Lipofectamine 2000 (Invitrogen; catalog number: 11668-019) were added to Opti-MEM (Gibco; catalog number: 11058-021) and incubated at room temperature for 30 min. The mixture was then added to 6-well cell culture dishes. After 24 hours, 0.5 mg / mL hygromycin B (Gibco; catalog number: 10687-010) and 1 mg / mL LG418 (Gibco; catalog number: 11811-031) were added and the cells were selected under pressure for 2 weeks to obtain the 293T-GR-GAL4 cell line.

[0293] 1.2 Glucocorticoid receptor GRE reporter gene assay

[0294] 293T-GR-GAL4 cells were digested with trypsin to adjust the cell density to 3.3 × 10⁻⁶ cells / year. 5 Samples were inoculated at a concentration of 90 μL / well into 96-well white microplates and incubated overnight. The initial concentration of the test samples was 1200 nM, and the initial concentration of prednisolone (manufacturer: MCE; catalog number: HY-17463) was 20 μM. After 4-fold dilution for 3 concentration points, and then 3-fold dilution for 4 concentration points, 10 μL / well was added to the plate. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours. Bright-GLo luciferase detection solution (manufacturer: Promega; catalog number: E2620) was added at 100 μL / well for detection. The results are shown in Table 1.

[0295] The results showed that, compared with prednisolone, the compounds of this invention can effectively activate the GRE reporter gene.

[0296] Table 1. Glucocorticoid receptor GRE reporter gene assay Note: N / A indicates no activity.

[0297] Test Example 2. Detection of the ability of compounds to inhibit TLR9-induced cytokines

[0298] The inhibitory effect of the compound on TLR9 agonist (ODN2216)-induced IFN-α was detected using human peripheral blood mononuclear cells (PBMCs). PBMCs were resuscitated and resuspended in complete culture medium (RPMI 1640 + 10% FBS + 1×NEAA + 1×Sodium Pyruvate + 1×GlutaMax), at a concentration of 0.5–1×10⁻⁶ cells per well. 6 Cells were seeded into 96-well cell culture plates; the test samples were diluted, with an initial concentration of 1067 nM, followed by 4-fold serial dilutions before being added to the plates, and incubated at 37°C for 7 hours; then 0.5 μM of ODN2216 (manufacturer: Invivogen; catalog number: tlrl-2216-1) was added to the plates, and the plates were incubated overnight (18 hours) at 37°C. The supernatant was collected, and the IFN-α concentration in the supernatant was detected using an IFN-α ELISA kit (manufacturer: Dayou; catalog number: 1110013). The results are shown in Table 2.

[0299] The results showed that the test sample had a significant inhibitory effect on TLR9-induced IFN-α secretion.

[0300] Table 2. Inhibitory effect of glucocorticoids on TLR9-induced IFN-α

[0301] Test Example 3: Glucocorticoid Receptor GRE Reporter Gene Elution Assay

[0302] 293T-GR-GAL4 cells were digested with trypsin to adjust the cell density to 1.1 × 10⁻⁶. 5 The sample was inoculated at a rate of 90 μL / well into a 96-well white microplate and incubated overnight. The test sample, C-5, and dexamethasone (US Pharmacopeia) were diluted to 80 μM, then serially diluted 6-fold, and added to the plate at 10 μL / well. After incubation at 37°C and 5% CO2 for 30 min, 1 h, 2 h, and 4 h, respectively, the supernatant was slowly removed by pipette, the plate was slowly washed with culture medium, and then 100 μL / well of culture medium was added. The plate was incubated at 37°C and 5% CO2 for 24 h. Bright-GLo luciferase detection solution (Promega; catalog number: E2620) was added at 100 μL / well for detection. The results are shown in Table 3.

[0303] The results showed that, compared with C-5 and dexamethasone, the compounds of the present invention bind more tightly to glucocorticoid receptors, are less likely to be eluted, and have a longer-lasting ability to activate glucocorticoid receptors.

[0304] Table 3 Results of GRE reporter gene elution assay

[0305] Test Example 4: Detection of the ability of antibody-drug conjugates to inhibit TLR9-induced secretion of IFN-α, TNF-α, IL-6 and IL-8

[0306] The inhibitory effect of antibody-drug conjugates on TLR9 agonist-induced cytokines was detected using human peripheral blood mononuclear cells (PBMCs). PBMCs were resuscitated and resuspended in complete culture medium (RPMI 1640 + 10% FBS + 1×NEAA + 1×Sodium Pyruvate + 1×GlutaMax), and cultured at a density of 0.5–1×10⁻⁶ cells per well. 6 Cells were seeded into 96-well cell culture plates. The test samples were diluted to target concentrations of 534 nM and 267 nM, and then added to the plates, incubated at 37°C for 7 hours. Next, 0.5 μM of ODN2216 (Invivogen; catalog number: tlrl-2216-1) was added to the plates, and the plates were incubated at 37°C for 40 hours. The supernatant was collected, and the concentrations of each cytokine in the supernatant were detected using the IFN-α ELISA kit (manufacturer: Dayou; catalog number: 1110013), TNF-α kit (manufacturer: R&D; catalog number: VAL105G), IL-6 kit (manufacturer: R&D; catalog number: VAL102C), and IL-8 kit (manufacturer: R&D; catalog number: VAL103). The results are shown in Figures 1A-1D.

[0307] The results showed that the antibody-drug conjugate ADC-4 conjugated with BD0260 of the present invention had a significant inhibitory effect on the secretion of TLR9-induced IFN-α, TNF-α, IL-6 and IL-8, and the inhibitory activity of the antibody-drug conjugate ADC-4 conjugated with BD0260 of the present invention on cytokine secretion was significantly better than that of the antibody-drug conjugate ADC-3 conjugated with the comparative compound C-1.

Claims

1. A compound of Formula I, its stereoisomer or a pharmaceutically acceptable salt thereof, in, R1 and R2 are each independently selected from H, halogens, and C1-C6 alkyl groups; R is Where X is selected from C and chemical bonds, Y is selected from C, N, O and S, and Z is selected from C and chemical bonds; R3 and R4 are each independently selected from H, halogen, amino, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and R5R6NC1-C6 alkyl, wherein the C1-C6 alkyl, C1-C6 alkoxy and amino are each independently optionally substituted by one or more selected from halogen, hydroxy, cyano and amino. or, When Y is selected from N, O and S, R3 and R4 together with the attached C atom form a 6-8 membered aromatic ring, a 5-8 membered heteroaromatic ring and a 3-6 membered heterocycle, each of which is independently and optionally substituted by one or more selected from C1-C6 alkyl, halogen, hydroxyl, cyano and amino groups. R5 and R6 are each independently selected from H, C1-C6 alkyl, halogen and C1-C6 alkoxy; in, R3 and R4 are not both H; When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and When R is When R3 is not an ethyl group, R3 is not an ethyl group.

2. The compound of formula I according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R1 and R2 are each independently selected from H, F, Cl, Br and methyl; preferably, R1 and R2 are each independently selected from H and F.

3. The compound of formula I according to claim 1 or 2, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R3 is selected from H, halogen, halogenated C1-C6 alkyl, aminosulfonyl, C1-C6 alkyl, C1-C6 alkoxy and di(C1-C6)alkylaminomethyl; preferably, R3 is selected from H, F, trifluoromethyl, aminosulfonyl, methyl, methoxy and dimethylaminomethyl.

4. The compound of formula I according to any one of claims 1-3, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R4 is selected from H, amino, C1-C6 alkyl and C1-C6 alkoxy, preferably, R4 is selected from H, amino, methyl and methoxy.

5. The compound of formula I according to any one of claims 1-4, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R is 6. The compound of formula I according to claim 5, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R3 is selected from H, F, trifluoromethyl, methyl, methoxy, and dimethylaminomethyl, and / or R4 is selected from H, amino, methyl, and methoxy.

7. The compound of Formula I according to claim 1, its stereoisomer or pharmaceutically acceptable salt thereof, having the structure shown in Formula II: in, R1, R2, R3 and R4 as defined in claim 1; in, R3 and R4 are not both H; When R1, R2, and R3 are all halogens, R4 is not hydrogen (H); and R3 is not an ethyl group.

8. The compound of formula I according to claim 1 or 2, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, When Y is selected from N, O, and S, R3 and R4 together with the C atoms they are attached to form a 6-8 membered aromatic ring or a 5-8 membered nitrogen-containing heteroaromatic ring.

9. The compound of formula I according to claim 8, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, One of X and Z is a chemical bond, and the other is C. R3 and R4 together with the C atoms they are attached to form a benzene ring, pyridine ring, pyrimidine ring, pyrazine ring, pyridazine ring, or pyrazole ring.

10. The compound of formula I according to any one of claims 1-5, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, R is selected from one of the following:

11. The compound of formula I according to any one of claims 1-10, its stereoisomer or a pharmaceutically acceptable salt thereof, wherein, The compound represented by Formula I is selected from the following compounds:

12. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, having the structure shown in Formula III: ligand-(LD)n Formula III in, The ligand is selected from polypeptides, proteins and antibodies, L is a linker, D is a drug, the drug is a compound of Formula I as described in any one of claims 1-11, its stereoisomer or a pharmaceutically acceptable salt thereof, and n is an integer or decimal between 1 and 10. Preferably, the ligand is an antibody; more preferably, the antibody specifically binds to an antigen selected from the group consisting of: BDCA2, IL-4R, TROP2, AXL, BAFFR, BCMA, BDCA4, BTLA, BTNL2, BTNL3, BTNL8, BTNL9, C10, CCR1, CCR3, CCR4, CCR5, CCR6, CCR7, CCR9, CCR10, CD11c, CD137, CD138, CD14, CD163, CD168, CD 177, CD19, CD20, CD209, CD209L, CD22, CD226, CD248, CD25, CD27, CD274, CD276, CD28, CD30, CD300A, CD33, CD37, CD38, CD4, differentiation antigen cluster 40 (C D40), CD44, CD45, CD46, CD47, CD48, CD5, CD52, CD55, CD56, CD59, CD62E, CD68, CD69, CD70, CD74, CD79a, CD79b, CD8, CD80, CD86, CD90.

2. CD96, CLEC12A, CLEC12B, CLEC7A, CLEC9A, CR1, CR3, CRTAM, CSF1R, CTLA4, CXCR1 / 2, CXCR4, C XCR5, DDR1, DDR2, DEC-205, DLL4, DR6, FAP, FCamR, FCMR, FcR's, Fire, GITR, HHLA2, HLA type II (HLA class II), HVEM, ICOSLG, IFNAR, type I interferon receptor subunit (IFNAR1), IFNLR1, IL10R1, IL10R2, IL12R, IL13RA1, IL13RA2, IL15R, IL17RA, IL17RB, IL17RC, IL17RE, IL20R1, IL20R2, IL21R, IL22R1, IL22RA, IL23R, IL27R, IL29R, IL2Rg, IL31R, IL36R, IL3RA, IL6R, IL5R, IL7R, IL9R, integrins, LAG3, LIFR, sialic acid-binding immunoglobulin-like lectin-4 (MAG / Siglec-4), MMR, MSR1, NCR3LG1, NKG 2D, NKp30, NKp46, OX40 (CD134), PDCD1, PROKR1, PVR, PVRIG, PVRL2, PVRL3, RELT, SIGIRR, sialic acid-binding immunoglobulin-like lectin-1 (Siglec-1), Siglec-10, Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, SIRPα, SLAMF7, TACI, PTCRA, TCRb, CD3z, CD3, TEK, TGFBR1, TGFBR2, TGFBR3, TIGIT, TLR2, TLR4, tumor necrosis factor α (TNFα), TROY, TSLPR, TYRO, VLDLR, VSIG4, IL2R-γ, and VTCN1.

13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12, wherein, The ligand-drug conjugate is shown in Formula IV below: Wherein, Ab is an antibody, L is a linker, n is an integer or decimal between 1 and 10, and R, R1 and R2 are as defined in claim 1.

14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 13, wherein, The ligand-drug conjugate is shown in formula V below: Wherein, Ab is an antibody, L is a linker, n is an integer or decimal between 1 and 10, and R1, R2, R3 and R4 are as defined in claim 1.

15. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12-14, wherein, The L has -L a -L b- L c -L d - structure, where L a Linked to ligands, L d Related to drugs, including: L a For coupling unit; L b It either does not exist or is a connected segment; L c It is a polypeptide fragment; L d It can be either non-existent or any unit.

16. The ligand-drug conjugate of claim 15 or a pharmaceutically acceptable salt thereof, wherein: L a Selected from The wavy line represents the connection point with the antibody, and * represents the connection point with L. b The connection point; L b The components are selected from -C(=O)-, -(CH2)mC(=O)-, -C(=O)-(CH2)mC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, -(CH2)mOC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, -NH-(CH2)m-(CH2CH2O)pC(=O)-, -(CH2)m-(CH2CH2O)pC(=O)- and -(CH2)mC(=O)-NH-(CH2)m-(CH2CH2O)pC(=O)-, where m is an integer from 1 to 10, preferably m is 2, 3 or 5; p is an integer from 4 to 32, preferably p is an integer from 10 to 14; L c It is a polypeptide fragment composed of 1 to 7 amino acids, wherein the amino acids are selected from valine (V), alanine (A), citrulline (Cit), serine (S), isoleucine (I), lysine (K), glycine (G), phenylalanine (F), glutamic acid (E) and aspartic acid (D). L d Selected from the following structures: Among them, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 15 or 16, wherein, L a Selected from The wavy line represents the connection point with the antibody, and * represents the connection point with L. b The connection point; L b Selected from -C(=O)- and -(CH2)mC(=O)-, where m is 2, 3 or 5; L c The following are examples of amino acids: valine-alanine (VA), valine-glycine (VG), valine-citrulline (VC), glycine-glycine-phenylalanine-glycine (GGFG), alanine-alanine-alanine-glycine (AAAG), glycine-glycine-glycine-glycine (GGGG), valine-alanine-glycine (VAG), valine-citrulline-glycine (VCG), alanine-alanine-glycine (AAG), alanine-alanine-alanine (AAA), and alanine-alanine. (AA), glutamic acid-alanine-glycine-glycine (EAGG), glycine-glutamic acid-alanine-glycine (GEAG), glycine-glutamic acid-glycine-glycine (GEGG), glutamic acid-glycine-glycine (EGG), glutamic acid-alanine-glycine (EAG), valine-lysine-glycine (VKG), glycine-glutamic acid-glycine (GEG), glutamic acid-alanine (EA), glutamic acid-glycine (EG) or glycine-glutamic acid (GE) polypeptide fragments; L d Selected from the following structures: Among them, a * Indicates with L c The connection point, b * Indicates the connection point with the drug.

18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12-17, wherein, The ligand-drug conjugate has the structure shown in Formula VI:

19. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 12-18, wherein, The antibody is an antibody that specifically binds to BDCA2, IL-4R, or TNFα; preferably, the antibody is an antibody that specifically binds to BDCA2; more preferably, the antibody comprises the heavy chain shown in SEQ ID NO:1 and the light chain shown in SEQ ID NO:2; or comprises the heavy chain shown in SEQ ID NO:3 and the light chain shown in SEQ ID NO:4; or comprises the heavy chain variable region shown in SEQ ID NO:5 and the light chain variable region shown in SEQ ID NO:6; optionally, the antibody comprises the heavy chain shown in SEQ ID NO:7 and the light chain shown in SEQ ID NO:

8.

20. A pharmaceutical composition comprising a compound of Formula I as claimed in any one of claims 1-11, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a ligand-drug conjugate as claimed in any one of claims 12-19 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

21. The use of the compound of Formula I as claimed in any one of claims 1-11, its stereoisomer or a pharmaceutically acceptable salt thereof, or the ligand-drug conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 12-19, or the pharmaceutical composition of claim 20 in the preparation of a medicament for the prevention or treatment of a disease; Preferably, the disease is an autoimmune disease or an inflammatory disease; More preferably, the disease is selected from one or more of the following: rheumatoid arthritis, systemic lupus erythematosus, scleroderma, Sjögren's syndrome, ankylosing spondylitis, Wegener's granulomatosis and systemic sclerosis, autoimmune hemolytic anemia, pernicious anemia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenic purpura and vasculitis, multiple sclerosis, myasthenia gravis and Guillain-Barré syndrome, ulcerative colitis, Crohn's disease, autoimmune diseases and atrophic gastritis, IgA nephropathy, primary nephrotic syndrome, autoimmune glomerulonephritis, pulmonary renal hemorrhage syndrome and lupus nephritis, type I diabetes, toxic diffuse goiter, Hashimoto's thyroiditis, primary adrenal atrophy and chronic thyroiditis, psoriasis, pemphigus vulgaris, cutaneous lupus erythematosus, dermatomyositis and polymyalgia rheumatica and asthma; Most preferably, the disease is a disease mediated by dysregulation of the glucocorticoid receptor signaling pathway.

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