Antibody-glucocorticoid conjugates and their uses

Abstract

This invention provides an antibody-glucocorticoid conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, and its uses. The structural formula of the ADC or its pharmaceutically acceptable salt or solvate is shown in the text. The ADC provided by this invention can significantly affect the activation level of glucocorticoid-mediated signaling pathways, thereby producing significant anti-inflammatory activity; it has good plasma stability, which can avoid premature release of glucocorticoid molecules in systemic circulation; it has good endocytic capacity and human lysosomal cleavage capacity, which can achieve targeted absorption of the ADC and rapid release of small glucocorticoid molecules, thereby improving efficacy. In mouse contact hypersensitivity models and rat rheumatoid arthritis models, it showed excellent anti-inflammatory activity, significantly superior to adalimumab and the control group.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antibody-glucocorticoid conjugate and its uses, including but not limited to rheumatoid arthritis, inflammatory bowel disease, psoriasis, ankylosing spondylitis, bullous pemphigus, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, and hidradenitis suppurativa. Background Technology

[0002] Tumor necrosis factor-alpha (TNF-α) is a cellular signaling protein (cytokine) involved in systemic inflammation, primarily regulating the function of immune cells. Anti-TNF-α biologics (e.g., adalimumab, etanercept, and infliximab) have demonstrated clinically proven efficacy in treating autoimmune disorders and inflammatory disorders such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease. Despite their clinical success, anti-TNF-α biologics remain limited by their maximum achievable efficacy in patients. Patients treated with anti-TNF-α biologics may also develop immunogenic responses to the agents, further limiting their effectiveness. Therefore, the identification and development of more potent and effective treatments are necessary.

[0003] Glucocorticoids have anti-inflammatory properties and are widely used to treat inflammatory disorders or diseases, such as respiratory diseases, rheumatic immune diseases, kidney diseases, and skin diseases. They can control disease symptoms, slow disease progression, and have significant clinical effects. However, they can also cause many adverse reactions, such as obesity, moon face, buffalo hump, edema, spontaneous fractures, osteoporosis and femoral head necrosis, peptic ulcer disease, acute gastrointestinal bleeding, insomnia, hirsutism, and decreased immunity. Therefore, the clinical application of glucocorticoid drugs is limited.

[0004] Currently, although antibody-corticosteroid conjugates are used to treat diseases or symptoms such as inflammation, they still suffer from low anti-inflammatory efficacy and short duration of action. Therefore, there is a need to develop therapeutic agents with enhanced efficacy and longer duration of action. Summary of the Invention

[0005] The main objective of this invention is to provide an antibody-glucocorticoid conjugate and its use, in order to at least partially solve at least one of the above-mentioned technical problems.

[0006] As a first aspect of the present invention, the present invention provides an antibody-glucocorticoid conjugate having the formula (I) shown herein, wherein the structural formula (I) is as follows:

[0007]

[0008] Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment;

[0009] L1 is selected from:

[0010]

[0011] m is an integer from 1 to 8, p is an integer from 1 to 8, and s is an integer from 1 to 6;

[0012] L2 does not exist, or is selected from

[0013] r is an integer from 1 to 6, and q is an integer from 1 to 8;

[0014] D is a glucocorticoid derivative with the formula (II):

[0015]

[0016] Glucocorticoid derivative D uses the oxygen atom of the 21-position hydroxyl group as the linking site, in formula (II),

[0017] R1 is selected from H, F, or Cl;

[0018] R2 is selected from H, methyl, or F;

[0019] R3 and R4 are each independently selected from H, OH, alkyl groups containing 1-6 carbons, OR6, OCOR7, or R3 and R4 together. That is, C16 and C17 are also connected by an oxygen bridge;

[0020] R6 is selected from H or alkyl groups containing 1-6 carbons or halogen-substituted alkyl groups;

[0021] R7 is selected from alkyl groups containing 1-6 carbons, alkenyl groups containing 2-6 carbons, alkynyl groups containing 2-6 carbons, aryl groups containing 6-10 carbons, or heteroaryl or heterocyclic groups containing 4-10 carbons.

[0022] R8 and R9 are each independently selected from H, alkyl groups containing 1-6 carbons, cycloalkyl groups containing 3-10 carbons, alkenyl groups containing 2-6 carbons, alkynyl groups containing 2-6 carbons, aryl groups containing 6-10 carbons, heteroaryl or heterocyclic groups containing 4-10 carbons.

[0023] Halogens are selected from F or Cl;

[0024] R5 is selected from H, F, or Cl;

[0025] D cannot be selected.

[0026]

[0027] n is an integer or decimal between 1 and 8.

[0028] Furthermore, L1-L2 are selected from:

[0029]

[0030] Where m is an integer from 1 to 8, p is an integer from 1 to 8, s is an integer from 1 to 6, q is an integer from 1 to 8, and r is an integer from 1 to 6.

[0031] Furthermore, L1 is selected from:

[0032]

[0033]

[0034] Furthermore, L2 does not exist, or is selected from:

[0035]

[0036] Furthermore, L1-L2 are selected from:

[0037]

[0038]

[0039] Furthermore, in the glucocorticoid derivative D,

[0040] R1 is selected from H or F;

[0041] R2 is selected from H, methyl, or F;

[0042] R3 and R4 are independently selected from H, OH, alkyl groups containing 1-6 carbons, OCOR7, or R3 and R4 together. That is, C16 and C17 are also connected by an oxygen bridge;

[0043] R7 is selected from heterocyclic groups with 4-10 carbon atoms;

[0044] R8 and R9 are each independently selected from H and alkyl groups containing 1-6 carbons;

[0045] R5 is selected from H, F, or Cl;

[0046] Preferably, the glucocorticoid derivative D is selected from:

[0047]

[0048] Extensive exploration and research have revealed that when antibody-glucocorticoid conjugates (ADCs) are constructed using glucocorticoid fragments such as beclomethasone 17-monopropionate or desisobutylcyclosporine, they cannot achieve the same stability, endocytosis, or anti-inflammatory activity as the ADCs provided in this invention. The ADCs provided in this invention achieve their effects through the combined action of glucocorticoids, linkers, antibodies or their antigen-binding fragments, and the conjugation ratio. Even minor changes in any of these components can directly affect the performance of the ADC, leading to significant variations in activity and efficacy.

[0049] Furthermore, the L1-L2-D is selected from:

[0050]

[0051]

[0052]

[0053]

[0054] Furthermore, the compound represented by formula (I) is selected from:

[0055]

[0056]

[0057]

[0058]

[0059] Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment;

[0060] n is an integer or decimal from 1 to 8, preferably an integer or decimal from 3 to 6. Further, the compound represented by formula (I) is selected from:

[0061]

[0062]

[0063] Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment;

[0064] n is an integer or decimal of 3-6, preferably an integer or decimal of 4-6. Further, the compound represented by formula (I) is selected from:

[0065]

[0066]

[0067]

[0068]

[0069] Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment;

[0070] Furthermore, the compound represented by formula (I) is selected from:

[0071]

[0072]

[0073] Ab represents an anti-TNF-α antibody or its antigen-binding fragment.

[0074] The present invention also includes variants and equivalents that are substantially homologous to anti-TNF-α antibodies. These may contain, for example, conserved substitution mutations, where one or more amino acids are substituted with similar amino acids. For example, a conserved substitution refers to the substitution of one amino acid with another amino acid from the same class, such as the substitution of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. The intention of conserved amino acid substitution is well known in the art.

[0075] Furthermore, the antibody Ab is selected from anti-TNF-α antibodies.

[0076] Furthermore, the antibody Ab is selected from adalimumab, infliximab, cetocilizumab, or golimumab. It also contains sequences of adalimumab, infliximab, cetocilizumab, or golimumab, such as complementarity-determining regions, heavy chain domains, and / or light chain domains.

[0077] Furthermore, the antibody Ab is selected from adalimumab. The anti-TNF-α antibody or its antigen-binding fragment competitively inhibits the binding of adalimumab to TNF-α, or binds to the same TNF-α epitope as adalimumab; or it is adalimumab or its antigen-binding fragment.

[0078] As a second aspect of the invention, the invention provides a pharmaceutical composition comprising the above-described antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt or solvate thereof and optionally one or more inert carriers and / or diluents.

[0079] As a third aspect of the invention, the invention provides the use of the above-described antibody-glucocorticoid conjugate or its pharmaceutically acceptable salt or solvate, or the above-described pharmaceutical composition, in the preparation of a medicament for treating glucocorticoid receptor-mediated diseases.

[0080] Furthermore, the disease is selected from inflammatory diseases or autoimmune diseases.

[0081] Furthermore, the diseases are selected from rheumatoid arthritis, inflammatory bowel disease, psoriasis, ankylosing spondylitis, bullous pemphigus, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, and hidradenitis suppurativa.

[0082] Compared with the prior art, the present invention has the following beneficial effects:

[0083] The antibody-glucocorticoid conjugate (ADC) provided by this invention can significantly affect the activation level of glucocorticoid-mediated signaling pathways, thereby producing significant anti-inflammatory activity. It exhibits good plasma stability, preventing premature release of glucocorticoid molecules in systemic circulation. It also possesses good endocytosis and lysosomal cleavage capabilities, enabling targeted absorption of the ADC and rapid release of small glucocorticoid molecules, thus improving therapeutic efficacy. In mouse contact hypersensitivity models and rat rheumatoid arthritis models, it demonstrated excellent anti-inflammatory activity, significantly superior to adalimumab and the control group. Attached Figure Description

[0084] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0085] Figure 1 The HIC-HPLC spectrum of ADC-II-1;

[0086] Figure 2 The HIC-HPLC spectrum of ADC-II-2;

[0087] Figure 3 The results are the average fluorescence intensity measurements obtained by flow cytometry.

[0088] Figure 4 Changes in the thickness of middle ear swelling in a contact hypersensitivity model;

[0089] Figure 5 Changes in middle ear weight in a contact hypersensitivity model;

[0090] Figure 6 This describes changes in paw swelling in a rat collagen antibody-induced arthritis model. Detailed Implementation

[0091] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions were applied in the examples. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0092] Terminology section

[0093] Unless otherwise specified, all technical and scientific terms used in this invention are consistent with the common understanding of one of ordinary skill in the art to which this invention pertains. While this invention may be tested using any methods and materials similar to or equivalent to those used herein, preferred methods and materials are described. In describing and claiming this invention, the following terms are used in accordance with the definitions below.

[0094] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two heavy chains and two light chains linked by interchain disulfide bonds. Based on the amino acid composition and sequence of the constant region of the heavy chain, immunoglobulins can be classified into five classes, or isotypes: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ or λ chains based on their constant region. Each of the five classes of Ig can have either a κ or λ chain.

[0095] The term "anti-TNF-α protein" refers to a protein capable of (1) binding to TNF-α and (2) inhibiting the binding of soluble TNF-α to TNF-α receptors on the cell surface and / or lysing cells expressing surface TNF-α or TNF-α receptors in vitro in the presence of complement. In some embodiments, anti-TNF antibodies may bind to and be internalized on the cell surface TNF-α receptor. Anti-TNF-α proteins include anti-TNF-α antibodies (e.g., adalimumab, infliximab, cetocilizumab, and golimumab). Anti-TNF-α antibodies are actively internalized upon binding to transmembrane TNF receptors on monocyte-derived dendritic cells (DCs) and rapidly enter and degrade in lysosomes.

[0096] The term "anti-TNF-α antibody" or "antibody that binds to TNF-α" refers to an antibody that can bind to TNF-α with sufficient affinity, for example, so that the antibody can be used as a therapeutic agent targeting TNF-α.

[0097] The terms “conjugate,” “antibody-drug conjugate,” or “ADC” refer to compounds or derivatives thereof that are conjugated to proteins such as cell-binding agents (e.g., anti-TNF-α antibodies).

[0098] The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies.

[0099] In this invention, the term "mouse antibody" refers to an antibody prepared using mice based on knowledge and skills in the art. Preparation involves injecting a test subject with a specific antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics.

[0100] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody. It can reduce the immune response induced by murine antibodies. To create a chimeric antibody, a hybridoma that secretes murine-specific monoclonal antibodies must first be established. Then, the variable region gene is cloned from the murine hybridoma cells. Next, the constant region gene of the human antibody is cloned as needed. The murine variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

[0101] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. This overcomes the heterologous response induced by chimeric antibodies carrying a large amount of mouse protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or from publicly available references. To avoid a decrease in activity along with a decrease in immunogenicity, the human antibody variable region framework sequence can be subjected to minimal reverse or reversion mutations to maintain activity. The humanized antibody of this invention also includes a humanized antibody further maturated with affinity for the CDR by phage display.

[0102] The term "fully human antibody," also known as a "fully human monoclonal antibody," refers to an antibody whose variable and constant regions are both human-derived, eliminating immunogenicity and toxicity. Related technologies for the preparation of fully human antibodies mainly include: human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody preparation technology, and single B cell antibody preparation technology.

[0103] The term "linker" refers to a chemical moiety that can link an anti-TNF-α protein (such as an antibody) to a glucocorticoid. Linker groups may be prone to cleavage, thereby promoting the release of glucocorticoids. For example, such cleavable linker groups may be susceptible to peptidase-induced cleavage under conditions where glucocorticoids and / or antibodies remain active.

[0104] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0105] The term "expression vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the expression vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the expression vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The expression vectors of the present invention can replicate autonomously in host cells that have been introduced therein or can integrate into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome.

[0106] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.

[0107] The engineered antibodies or antigen-binding fragments of this invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. In a prior art, mammalian expression systems lead to antibody glycosylation, particularly at the N-terminal site of the Fc region. Positive clones are scaled up in a bioreactor medium to produce antibodies. The culture medium secreting antibodies can be purified using conventional techniques, such as using an A or G Sepharose FF column. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient, and antibody fragments are detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, e.g., at -70°C, or lyophilized.

[0108] The term "peptide" refers to a compound fragment that lies between amino acids and proteins. It is composed of two or more amino acid molecules linked together by peptide bonds and is a structural and functional segment of a protein.

[0109] The term "glucocorticoid" refers to a naturally occurring or synthetic steroid hormone that interacts with glucocorticoid receptors, and specific glucocorticoids are disclosed in detail in this invention. A "glucocorticoid group" is obtained by removing one or more hydrogen atoms from the parent glucocorticoid. The removal of hydrogen atoms facilitates the attachment of the parent glucocorticoid to the linking group.

[0110] The term "heterofunctional group" refers to the chemical moiety that connects a linker group to an anti-TNF-α protein (such as an antibody). Heterofunctional groups are characterized by having different reactive groups at either end of the chemical moiety.

[0111] The terms “drug / antibody ratio,” “conjugate ratio,” or “DAR” refer to the number of drug molecules (e.g., glucocorticoids or their derivatives) linked to an antibody (e.g., an antibody). Therefore, in conjugates having the general formula (DL-)n-Ab, DAR is defined by the amount of drug loaded per antibody-drug conjugate, e.g., “n.”

[0112] When referring to compounds having the formula (DL-)n-Ab representing individual conjugates, the term "compound DAR" refers to the number of Ds linked to the individual Ab (e.g., drug loading or n being an integer from 1 to 10).

[0113] When referring to compounds of the formula (DL-)n-Ab representing a group of conjugates, the term “group DAR” refers to the average number of Ds linked to Ab (e.g., drug loading or n being an integer or decimal of 1 to 10 ± 0.5, ± 0.4, ± 0.3, ± 0.2, ± 0.1).

[0114] The term "subject" refers to humans, non-human primates, etc., who will become recipients of a specific treatment. Generally, the terms "subject" and "patient" are used interchangeably in this invention to refer to human subjects.

[0115] The “effective amount” of a coupling agent is the amount sufficient to achieve the explicitly described purpose. The “effective amount” can be determined relative to the stated purpose.

[0116] The term "therapeutic effective amount" refers to the amount of a conjugate that effectively "treats" a disease or symptom in a subject or mammal. "Prophylactic effective amount" refers to the amount that effectively achieves the desired preventative outcome.

[0117] Terms such as “treating,” “treatment,” or “to treat” or “alleviating,” refer to therapeutic measures that cure, reduce, alleviate, or slow or stop the progression of a diagnosed pathological condition or symptom (“therapeutic treatment”). Therefore, subjects requiring therapeutic treatment include those who have been diagnosed with or are suspected of having the condition. Preventive or preventative measures refer to measures that prevent the development of a target pathological condition or symptom (“preventive treatment”). Therefore, subjects requiring preventive treatment include those susceptible to the condition and those seeking to prevent the condition.

[0118] Adalimumab is described in U.S. Patent No. 6,258,562, which is incorporated herein by reference in its entirety. Infliximab is described in U.S. Patent No. 5,656,272, which is incorporated herein by reference in its entirety. Citopizumab is discussed in WO01 / 94585, which is incorporated herein by reference in its entirety. Golimumab is discussed in WO2013 / 087912, and its sequences are provided in GenBank: DI496971.1 and GenBank: DI496970.1, each of which is incorporated herein by reference in its entirety.

[0119] The sequences of exemplary anti-TNF-α antibodies or their antigen-binding fragments are provided below.

[0120] Variable heavy chain CDR amino acid sequence:

[0121]

[0122] Variable light chain CDR amino acid sequence:

[0123]

[0124]

[0125] Heavy chain amino acid sequence:

[0126]

[0127] Light chain amino acid sequence:

[0128]

[0129]

[0130] To achieve the synthetic objective of this invention, the following synthetic technique is employed:

[0131] Example I

[0132] Example 1.1 Synthesis of N-((10S)-10-phenyl-1-((6aR,7S,8aS,8bS,11aR)-7-hydroxy-6a,8a-dimethyl-4-acyl-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-6-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)hexamamide (L-01)

[0133]

[0134] first step

[0135] N-fluorenemethoxycarbonyl-glycyl-glycine (4.3 g, 12.13 mmol), 150 mL of tetrahydrofuran, and 40 mL of toluene were added to a reaction flask. Pyridine (1.6 mL, 19.8 mmol) and lead tetraacetate (6.53 g, 14.7 mmol) were added, and the mixture was heated to 70 °C and reacted for 10 hours. After the reaction was complete, the mixture was cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate. The organic phase was concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1 / 1) to give 3.4 g of (2-(9H-fluorenemethoxycarbonylamino)acetamido)methyl acetate, yield: 76.1%. MS m / z (ESI): 391.2 [M+Na] +

[0136] Step 2

[0137] (2-(9H-fluorenylmethoxycarbonylamino)acetamido)methyl acetate (0.12 g, 0.33 mmol), budesonide (0.22 g, 0.5 mmol), and 20 mL of dichloromethane were added to a reaction flask, followed by pyridine p-toluenesulfonate (42 mg, 0.17 mmol). The mixture was heated to 40 °C and reacted for 24 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 1a 0.1 g, yield: 40%. MS m / z (ESI): 739.6 [M+H] +

[0138] Step 3

[0139] Compound 1a (90 mg, 0.12 mmol) and 4 mL of N,N-dimethylformamide were added to a reaction flask, followed by 22 μL of 1,8-diazabicycloundec-7-ene (0.15 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 1b 53 mg, yield: 85%. MS m / z (ESI): 517.4 [M+H] +

[0140] Step 4

[0141] N-fluorenemethoxycarbonyl-glycyl-L-phenylalanine (73 mg, 0.15 mmol), N-hydroxysuccinimide (17 mg, 0.15 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (37 mg, 0.19 mmol), and 5 mL of N,N-dimethylformamide were added to a reaction flask and reacted at room temperature for 1 hour. Compound 1b (50 mg, 0.1 mmol) and N,N-diisopropylethylamine (32 μL, 0.19 mmol) were added, and the reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 1 c 53 mg of compound, yield: 55%. MS m / z (ESI): 1000.7 [M+H) +

[0142] Step 5

[0143] Compound 1c (50 mg, 0.05 mmol) and 5 mL of N,N-dimethylformamide were added to a reaction flask, followed by 9 μL of 1,8-diazabicycloundec-7-ene (0.06 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 1d 33 mg, yield: 85%. MS m / z (ESI): 778.5 [M+H] +

[0144] Step 6

[0145] Add 12 mg (0.058 mmol) of 6-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid and 2 mL of N,N-dimethylformamide to a reaction flask, then add compound 1d (30 mg, 0.039 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (15 mg, 0.077 mmol), N-hydroxysuccinimide (6.6 mg, 0.058 mmol), and N,N-diisopropylethylamine (12 μL, 0.077 mmol). React at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-01 24mg, yield: 65%. MS m / z (ESI): 969.6 [MH] -

[0146] Example 1.2 Synthesis of N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17R)-2-chloro-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-6-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)hexamamide (L-02)

[0147]

[0148] first step

[0149] (2-(9H-fluorenylmethoxycarbonylamino)acetamido)methyl acetate (0.12 g, 0.33 mmol), halometasone (0.22 g, 0.5 mmol), and 20 mL of dichloromethane were added to a reaction flask, followed by pyridine p-toluenesulfonate (42 mg, 0.17 mmol). The mixture was heated to 40 °C and reacted for 24 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 2a 0.12 g, yield: 48%. MS m / z (ESI): 775.2 [M+Na]+

[0150] Step 2

[0151] Compound 2a (0.1 g, 0.13 mmol) and 4 mL of N,N-dimethylformamide were added to a reaction flask, followed by 24 μL of 1,8-diazabicycloundec-7-ene (0.16 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give 60 mg of compound 2b, yield: 85%. MS m / z (ESI): 531.2 [M+H] +

[0152] Step 3

[0153] N-fluorenylmethoxycarbonyl-glycyl-L-phenylalanine (71 mg, 0.14 mmol), N-hydroxysuccinimide (16 mg, 0.14 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (37 mg, 0.19 mmol), and 5 mL of N,N-dimethylformamide were added to a reaction flask and reacted at room temperature for 1 hour. Compound 2b (50 mg, 0.094 mmol) and N,N-diisopropylethylamine (32 μL, 0.19 mmol) were added, and the reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 2c40mg of compound, yield: 42%. MS m / z (ESI): 1036.3 [M+Na] +

[0154] Step 4

[0155] Compound 2c (50 mg, 0.05 mmol) and 5 mL of N,N-dimethylformamide were added to a reaction flask, followed by 9 μL of 1,8-diazabicycloundec-7-ene (0.06 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 2d 33 mg, yield: 85%. MS m / z (ESI): 814.5 [M + Na] +

[0156] Step 5

[0157] Add 6-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid (13 mg, 0.062 mmol) and 2 mL of N,N-dimethylformamide to a reaction flask, then add compound 2d (33 mg, 0.04 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (16 mg, 0.083 mmol), N-hydroxysuccinimide (7.2 mg, 0.062 mmol), and N,N-diisopropylethylamine (14 μL, 0.083 mmol). React at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-02 18mg, yield: 45%. MS m / z (ESI): 983.5 [MH] -

[0158] Example 1.3N-((10S)-10-phenyl-1-((2S,6aS,6bR,7S,8aS,8bS,11aR)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1 Synthesis of ':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-03)

[0159]

[0160]

[0161] first step

[0162] (2-(9H-fluorenylmethoxycarbonylamino)acetamido)methyl acetate (0.1 g, 0.27 mmol), fluocinolone acetonide (0.14 g, 0.31 mmol), and 5 mL of dichloromethane were added to a reaction flask, followed by pyridine p-toluenesulfonate (35 mg, 0.14 mmol). The mixture was heated to 40 °C and reacted for 24 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 3a 100 mg, yield: 48.7%. MS m / z (ESI): 783.7 [M+Na] +

[0163] Step 2

[0164] Compound 3a (0.1 g, 0.13 mmol) and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by 26 μL of 1,8-diazabicycloundec-7-ene (0.17 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give 65 mg of compound 3b, yield: 92.8%. MS m / z (ESI): 561.1 [M+Na] +

[0165] Step 3

[0166] N-fluorenemethoxycarbonyl-glycyl-glycyl-L-phenylalanine (0.12 g, 0.24 mmol), N-hydroxysuccinimide (21 mg, 0.18 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (35 mg, 0.18 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask and reacted at room temperature for 1 hour. Compound 3b (65 mg, 0.12 mmol) and N,N-diisopropylethylamine (60 μL, 0.36 mmol) were added, and the reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound 3c43mg, yield: 35%. MS m / z (ESI): 1044.3 [M+Na] +

[0167] Step 4

[0168] Compound 3c (43 mg, 0.042 mmol) and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by 10 μL of 1,8-diazabicycloundec-7-ene (0.066 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 3d 30 mg, yield: 89.3%. MS m / z (ESI): 800.3 [M+H] +

[0169] Step 5

[0170] Compound 3d (30 mg, 0.038 mmol), 2,5-diacypyrrolyl 1-(2,5-diacy-2,5-dihydro-1H-pyrrolo-1-yl)-3-acyl-7,10,13,16-tetraoxa-4-azanonadecanate (35 mg, 0.068 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by N,N-diisopropylethylamine (20 μL, 0.12 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-03 18.2mg, yield: 40%. MS m / z (ESI): 1196.4 [MH] -

[0171] Example 1.4 Synthesis of N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl-1-(6-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3,6,9,12-tetraoxapentadecanylamide (L-04)

[0172]

[0173] first step

[0174] Add 0.79 g (3.73 mmol) of 6-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)hexanoic acid, 1 g (3.11 mmol) of tert-butyl-1-amino-3,6,9,12-tetraoxapentadecanyl ester, 1.8 g (4.7 mmol) of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1 mL (6.22 mmol) of N,N-diisopropylethylamine, and 30 mL of dichloromethane to a reaction flask. Cool to 0 °C and react for 2 hours, then allow to rise to room temperature and react overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 1.0 g of tert-butyl 22-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadocosanoate, yield: 65%). MS m / z (ESI): 515.4 [M+H] +

[0175] Step 2

[0176] Add 0.8 g (1.55 mmol) of tert-butyl 22-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadodocarboxylate and 10 mL of dichloromethane to a reaction flask, cool to 0 °C, slowly add 5 mL of trifluoroacetic acid, raise to room temperature, and react overnight. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 0.52g of 22-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadocarboxylic acid, yield: 73%). MS m / z (ESI): 459.4 [M+H] +

[0177] Step 3

[0178] 22-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadoacenoic acid (0.5 g, 1 mmol), N-hydroxysuccinimide (0.14 g, 1.2 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.29 g, 1.5 mmol), 4-dimethylaminopyridine (12 mg, 0.1 mmol), and 20 mL of dichloromethane were added to a reaction flask and reacted at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 0.34 g of 2,5-diacylopyrrole-1-yl 22-(2,5-diacylopyrrole-2,5-dihydro-1H-pyrrole-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadocosanoate, yield: 62%. MS m / z (ESI): 556.4 [M+H] +

[0179] Step 4

[0180] (2-(9H-fluorenylmethoxycarbonylamino)acetamido)methyl acetate (0.37 g, 1 mmol), diflucolone (0.59 g, 1.5 mmol), and 20 mL of dichloromethane were added to a reaction flask, followed by pyridine p-toluenesulfonate (0.12 g, 0.5 mmol). The mixture was heated to 40 °C and reacted for 24 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 4a 0.24 g, yield: 35%. MS m / z (ESI): 725.4 [M + Na] +

[0181] Step 5

[0182] Compound 4a (0.24 g, 0.34 mmol) and 4 mL of N,N-dimethylformamide were added to a reaction flask, followed by 62 μL of 1,8-diazabicycloundec-7-ene (0.41 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 4b (0.14 g), yield: 88%. MS m / z (ESI): 481.3 [M+H] +

[0183] Step 6

[0184] N-fluorenylmethoxycarbonyl-glycyl-L-phenylalanine (0.22 g, 0.44 mmol), N-hydroxysuccinimide (50 mg, 0.44 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.11 g, 0.58 mmol), and 5 mL of N,N-dimethylformamide were added to a reaction flask and reacted at room temperature for 1 hour. Compound 4b (0.14 g, 0.29 mmol) and N,N-diisopropylethylamine (96 μL, 0.58 mmol) were added, and the reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound 4c 0.15g, yield: 55%. MS m / z (ESI): 986.4 [M+Na] +

[0185] Step 7

[0186] Compound 4c (0.15 g, 0.15 mmol) and 5 mL of N,N-dimethylformamide were added to a reaction flask, followed by 28 μL of 1,8-diazabicycloundec-7-ene (0.19 mmol). The mixture was cooled to 0 °C and reacted for 1 hour. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound 4d 0.1 g, yield: 88%. MS m / z (ESI): 764.3 [M + Na]+

[0187] Step 8

[0188] Compound 4d (0.1 g, 0.13 mmol), 2,5-diacypyrrole-1-yl 22-(2,5-diacy-2,5-dihydro-1H-pyrrole-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadodocarboxylate (0.11 g, 0.2 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by N,N-diisopropylethylamine (67 μL, 0.4 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-04 51mg, yield: 32%. MS m / z (ESI): 1180.6 [MH] -

[0189] Example 1.5N-((10S)-10-phenyl-1-((2S,6aS,6bR,7S,8aS,8bS,11aR)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1 Synthesis of ':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(6-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)hexamido)-3,6,9,12-tetraoxapentadecanylamide (L-05)

[0190]

[0191] first step

[0192] Compound 3a was prepared according to the preparation method in the first step of Example 1.3.

[0193] Step 2

[0194] Compound 3b was prepared according to the preparation method in step 2 of Example 1.3.

[0195] Step 3

[0196] Compound 3c was prepared according to the preparation method in step 3 of Example 1.3.

[0197] Step 4

[0198] Compound 3d was prepared according to the preparation method in step 4 of Example 1.3.

[0199] Step 5

[0200] Compound 3d (30 mg, 0.038 mmol), 2,5-diacypyrrole-1-yl 22-(2,5-diacy-2,5-dihydro-1H-pyrrole-1-yl)-17-acyl-4,7,10,13-tetraoxa-16-azadodocarboxylate (31 mg, 0.056 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by N,N-diisopropylethylamine (19 μL, 0.11 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-05 21mg, yield: 45%. MS m / z (ESI): 1238.7 [MH] -

[0201] Example 1. Synthesis of 6N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17R)-2-chloro-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12-tetraoxapentadecanylamide (L-06)

[0202]

[0203] first step

[0204] Compound 2a was prepared according to the preparation method in the first step of Example 1.2.

[0205] Step 2

[0206] Compound 2b was prepared according to the preparation method in step 2 of Example 1.2.

[0207] Step 3

[0208] Compound 2c was prepared according to the preparation method in step 3 of Example 1.2.

[0209] Step 4

[0210] Compound 2d was prepared according to the preparation method in step 4 of Example 1.2.

[0211] Step 5

[0212] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (21 mg, 0.061 mmol) and 2 mL of N,N-dimethylformamide were added to a reaction flask, followed by compound 2d (24 mg, 0.03 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (18 mg, 0.094 mmol), N-hydroxysuccinimide (7 mg, 0.061 mmol), and N,N-diisopropylethylamine (15 μL, 0.093 mmol). The reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm, mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-06 21.4mg, yield: 63.7%. MS m / z (ESI): 1117.3 [MH] -

[0213] Example 1.7 Synthesis of N-((10S)-10-phenyl-1-((6aR,7S,8aS,8bS,11aR)-7-hydroxy-6a,8a-dimethyl-4-acyl-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12-tetraoxapentadecanylamide (L-07)

[0214]

[0215] first step

[0216] Compound 1a was prepared according to the preparation method in step 2 of Example 1.1.

[0217] Step 2

[0218] Compound 1b was prepared according to the preparation method in step 3 of Example 1.1.

[0219] Step 3

[0220] Compound 1c was prepared according to the preparation method in step 4 of Example 1.1.

[0221] Step 4

[0222] Compound 1d was prepared according to the preparation method in step 5 of Example 1.1.

[0223] Step 5

[0224] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (0.14 g, 0.4 mmol) and 6 mL of N,N-dimethylformamide were added to a reaction flask, followed by compound 1d (0.15 g, 0.19 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.12 g, 0.6 mmol), N-hydroxysuccinimide (46 mg, 0.4 mmol), and N,N-diisopropylethylamine (0.1 mL, 0.6 mmol). The reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-07 46mg, yield: 10.4%. MS m / z (ESI): 1105.5 [M+H] +

[0225] Example 1. Synthesis of 8N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17S)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(2,5-diacyl-2,5-dihydro-1H-pyrrole-1-yl)-3,6,9,12-tetraoxapentadecanylamide (L-08)

[0226]

[0227] first step

[0228] Compound 4a was prepared according to the preparation method in step 4 of Example 1.4.

[0229] Step 2

[0230] Compound 4b was prepared according to the preparation method in step 5 of Example 1.4.

[0231] Step 3

[0232] Compound 4c was prepared according to the preparation method in step 6 of Example 1.4.

[0233] Step 4

[0234] Compound 4d was prepared according to the preparation method in step 7 of Example 1.4.

[0235] Step 5

[0236] 1-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)-3,6,9,12-tetraoxapentadecan-15-acid (90 mg, 0.26 mmol) and 4 mL of N,N-dimethylformamide were added to a reaction flask, followed by compound 4d (0.1 g, 0.13 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (77 mg, 0.4 mmol), N-hydroxysuccinimide (30 mg, 0.26 mmol), and N,N-diisopropylethylamine (40 μL, 0.4 mmol). The reaction was carried out at room temperature for 5 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give compound L-08 50mg, yield: 36%. MS m / z (ESI): 1067.6 [MH] -

[0237] Example 1.9 Synthesis of (2S)-2-(2-(2-(2-bromoacetamide)acetamide)acetamide)-N-(2-(((2-((2S,6aS,6bR,7S,8aS,8bS,11aR)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-2-acylethoxy)methyl)amino)-2-acylethyl)-3-phenylpropionamide (L-09)

[0238]

[0239] first step

[0240] Compound 3c was prepared according to the preparation method in step 3 of Example 1.3.

[0241] Step 2

[0242] Compound 3d was prepared according to the preparation method in step 4 of Example 1.3.

[0243] Step 3

[0244] Compound 3d (25 mg, 0.031 mmol), perfluorophenyl 2-bromoethyl ester (14 mg, 0.047 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by N,N-diisopropylethylamine (15 μL, 0.09 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound L-09 8.9 mg, yield: 31%. MS m / z (ESI): 920.3 [M+H] +

[0245] Example 1. Synthesis of 10(2S)-2-(2-(2-(2-(2-bromoacetamide)acetamide)acetamide)acetamide)-N-(2-(((2-((6S,9R,10S,11S,13S,16R,17R)-2-chloro-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentyl[a]phenanthrene-17-yl)-2-acylethoxy)methyl)amino)-2-acylethyl)-3-phenylpropionamide (L-10)

[0246]

[0247] first step

[0248] Glycine (63 mg, 0.85 mmol), 2,5-diacylopyrrolidone-1-yl bromoacetic acid (0.2 g, 0.85 mmol), and 20 mL of dichloromethane were added to a reaction flask and reacted at room temperature for one hour. Then, N,N-diisopropylcarbodiimide (0.2 mL, 1.3 mmol) was added, and the reaction was continued at room temperature for three hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to obtain 0.16 g of 2,5-diacylopyrrolidone-1-yl (2-bromoacetyl)glycine ester, yield: 65%. MS m / z (ESI): 293.1 [M+H] +

[0249] Step 2

[0250] Compound 2c was prepared according to the preparation method in step 3 of Example 1.2.

[0251] Step 3

[0252] Compound 2d was prepared according to the preparation method in step 4 of Example 1.2.

[0253] Step 4

[0254] Compound 2d (35 mg, 0.04 mmol), 2,5-diacylopyrrolidone-1-yl(2-bromoacetyl)glycine ester (19 mg, 0.066 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask. N,N-diisopropylethylamine (15 μL, 0.09 mmol) was added, and the reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound L-10 14 mg, yield: 32%. MS m / z (ESI): 967.2 [MH] -

[0255] Example 1.1 Synthesis of 11N-((10S)-10-phenyl-1-((2S,6aS,6bR,7S,8aS,8bS,11aR)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecylhydro-8bH-naphtho[2',1':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-6-(2-bromoacetamide)hexamethylenetetramine (L-11)

[0256]

[0257] first step

[0258] Compound 3c was prepared according to the preparation method in step 3 of Example 1.3.

[0259] Step 2

[0260] Compound 3d was prepared according to the preparation method in step 4 of Example 1.3.

[0261] Step 3

[0262] Compound 3d (25 mg, 0.031 mmol), perfluorophenyl 6-(2-bromoacetamide)hexanoate (20 mg, 0.047 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask, followed by N,N-diisopropylethylamine (15 μL, 0.09 mmol). The reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound L-11 11 mg, yield: 35%. MS m / z (ESI): 1033.4 [M+H] +

[0263] Example 1. Synthesis of 12N-((10S)-10-phenyl-1-((2S,6aS,6bR,7S,8aS,8bS,11aR)-2,6b-difluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(2-bromoacetamide)-3,6,9,12-tetraoxapentadecanylamide (L-12)

[0264]

[0265]

[0266] first step

[0267] 1-Amino-3,6,9,12-tetraoxapentadecanoic acid (0.22 g, 0.85 mmol), 2,5-diacylpyrrolidine-1-yl bromoacetic acid (0.2 g, 0.85 mmol), and 20 mL of dichloromethane were added to a reaction flask and reacted at room temperature for one hour. Then, N,N-diisopropylcarbodiimide (0.2 mL, 1.3 mmol) was added, and the reaction was continued at room temperature for three hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5μm 21.2×250mm; mobile phase: A-water (10mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding fractions were collected and concentrated under reduced pressure to give 0.22g of 2,5-diacylopyrrolidone-1-yl-1-bromo-2-acyl-6,9,12,15-tetraoxa-3-azaoctadecanoate, yield: 54%. MS m / z (ESI): 483.1 [M+H] +

[0268] Step 2

[0269] Compound 3c was prepared according to the preparation method in step 3 of Example 1.3.

[0270] Step 3

[0271] Compound 3d was prepared according to the preparation method in step 4 of Example 1.3.

[0272] Step 4

[0273] Compound 3d (50 mg, 0.06 mmol), 2,5-diacylopyrrolidone-1-yl-1-bromo-2-acyl-6,9,12,15-tetraoxa-3-azaoctadecanoate (45 mg, 0.09 mmol), and 3 mL of N,N-dimethylformamide were added to a reaction flask. N,N-diisopropylethylamine (31 μL, 0.19 mmol) was added, and the reaction was carried out at room temperature for 3 hours. After the reaction was complete, the reaction solution was concentrated under reduced pressure. The crude product was purified by high-performance liquid chromatography (HPLC) (column: Phenomenex C18 5 μm 21.2 × 250 mm; mobile phase: A-water (10 mmol NH4OAc); B-acetonitrile, gradient elution, flow rate: 18 mL / min). The fractions were collected and concentrated under reduced pressure to give compound L-1217 mg, yield: 24%. MS m / z (ESI): 1165.4 [MH] -

[0274] Example 1. Synthesis of 13N-((10S)-10-phenyl-1-((6aR,7S,8aS,8bR)-7-hydroxy-6a,8a-dimethyl-4-acyl-10-propyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5]indeno[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-13)

[0275]

[0276] Following the preparation method in Example 1.3, budesonide was replaced with fluocinolone acetonide to prepare compound L-13 with an MSm / z (ESI) of 1174.4 [MH]. - .

[0277] Example 1.14N-((10S)-10-phenyl-1-((6aS,6bR,7S,8aS,8bR)-6b-fluoro-7-hydroxy-6a,8a,10,10-tetramethyl-4-acyl-1,2,4,6a,6b,7,8,8a,11a,12,12a,12b-dodecano-8bH-naphtho[2',1':4,5] Synthesis of indo[1,2-d]][1,3]dioxolane-8b-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-14)

[0278]

[0279] Following the preparation method in Example 1.3, triamcinolone was substituted for fluocinolone acetonide to prepare compound L-14 with an MSm / z (ESI) of 1178.4 [MH]. - .

[0280] Example 1. Synthesis of 15N-((10S)-10-phenyl-1-((9R,10S,11S,13S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-15)

[0281]

[0282] Following the preparation method in Example 1.3, dexamethasone was replaced with fluocinolone acetonide to prepare compound L-15 with an MSm / z (ESI) of 1136.4 [MH]. - .

[0283] Example 1. Synthesis of 16N-((10S)-10-phenyl-1-((9R,10S,11S,13S,16S,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl))-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-16)

[0284]

[0285] Following the preparation method in Example 1.3, by replacing fluocinolone acetonide with betamethasone, compound L-16 was prepared with an MSm / z (ESI) of 1136.4 [MH]. - .

[0286] Example 1. Synthesis of 17N-((10S)-10-phenyl-1-((6S,10R,11S,13S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl))-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-17)

[0287]

[0288] Following the preparation method in Example 1.3, methylprednisolone was replaced with fluocinolone to prepare compound L-17 with MSm / z (ESI): 1118.4 [MH]. - .

[0289] Example 1. Synthesis of 18N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17R)-2-chloro-6,9-difluoro-11,17-dihydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propionamidyl)-3,6,9,12-tetraoxapentadecanylamide (L-18)

[0290]

[0291] Following the preparation method in Example 1.3, halometasone was substituted for fluocinolone acetonide to prepare compound L-18 with an MSm / z (ESI) of 1188.3 [MH]. - .

[0292] Example 1. Synthesis of 19N-((10S)-10-phenyl-1-((6S,9R,10S,11S,13S,16R,17R)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecano-3H-cyclopentyl[a]phenanthrene-17-yl)-1,6,9,12,15-pentaacyl-3-oxa-5,8,11,14-tetraazahexadecyl)-1-(3-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-3,6,9,12-tetraoxapentadecanylamide (L-19)

[0293]

[0294] Following the preparation method in Example 1.3, by replacing fluocinolone with diflubenzuron, compound L-19 was prepared with an MSm / z (ESI) of 1138.4 [MH]. - .

[0295] Example 1. Synthesis of 20(6S,9R,10S,11S,13S,16R,17R)-17-((S)-10-phenyl-36-(2,5-diacyl-2,5-dihydro-1H-pyrrolo-1-yl)-6,9,12,15,18,34-hexaoxa-3,21,24,27,30-pentaoxa-5,8,11,14,17,33-hexaazahexadecyl)-6,9-difluoro-11-hydroxy-10,13,16-trimethyl-3-acyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecylhydro-3H-cyclopentyl[a]phenanthrene-17-ylfuran-2-carboxylic acid ester (L-20)

[0296]

[0297] Following the preparation method in Example 1.3, fluocinolone acetonide was replaced with 17-furfuryl ester difluorometholone to prepare compound L-20 (MS m / z (ESI): 1248.4 [MH)). - .

[0298] Example II: ADC Coupling Method

[0299] General Method 1: Preparation of ADC by Maleimide Coupling

[0300] Add 2–2.5 molar equivalents of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP·HCl) solution (10 mM) to adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL), mix thoroughly, and react at 37 °C for 120 min. The reduction product directly enters the coupling reaction without TCEP removal. Add 8 molar equivalents of LD solution containing maleimide coupling group (DMSO, 10 mg / mL, where D is the glucocorticoid drug and L is the linker) to the reaction solution, react at 25 °C for 120 min, and then terminate the reaction by adding 12 molar equivalents of L-cysteine ​​solution (10 mM). Use a HITRAP CM FF cation exchange column to remove unreacted small molecules and potential aggregates, and replace the buffer system with 20 mM phosphate buffer (pH 6.5) using a 30 kDa ultrafiltration tube. Finally, the ultrafiltration collection solution was adjusted to 20mM phosphate buffer, pH 6.5, and stored at -80℃ for later use.

[0301] General Method 2: Preparation of ADC by Hydrolysis of Thiosuccinimide

[0302] 0.7 M arginine (pH 9.0) solution was slowly added to the ADC PBS buffer prepared by maleimide coupling to achieve a total arginine concentration of 50 mM. The mixture was then incubated at 25 °C for 72 hours, and hydrolysis was quenched by adding 0.1 M acetic acid solution. Potential aggregates were removed using a HITRAP CM FF cation exchange column, and the buffer system was replaced with 20 mM phosphate buffer (pH 6.5) using a 30 kDa ultrafiltration tube. Finally, the ultrafiltration collection solution was adjusted to 20 mM phosphate buffer, pH 6.5, and stored at -80 °C for later use.

[0303] General Method 3: Preparation of ADC via Halogenated Alkyl Coupling

[0304] Add 2 molar equivalents of TCEP·HCl solution (10 mM) to adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL), mix thoroughly, and react at 37 °C for 120 min. The reduction product directly enters the coupling reaction without TCEP removal. Add 8 molar equivalents of DL solution containing a haloalkane coupling group (DMSO, 10 mg / mL, where D is the glucocorticoid drug and L is the linker) to the reaction solution, react at 25 °C for 120 min, and then terminate the reaction by adding 12 molar equivalents of L-cysteine ​​solution (10 mM). Use a HITRAP CM FF cation exchange column to remove unreacted small molecules and potential aggregates, and replace the buffer system with 20 mM phosphate buffer (pH 6.5) using a 30 kDa ultrafiltration tube. Finally, adjust the ultrafiltration collection system to 20 mM phosphate buffer, pH 6.5, and store at -80 °C for later use.

[0305] Example II-1:

[0306]

[0307] Following the method described in General Method 1, 90 μL of TCEP·HCl solution (10 mM, 900 nmol) was added to 6.0 mL of adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL, 405 nmol), and the mixture was thoroughly mixed and reacted at 37 °C for 120 min. Then, 314 μL of DMSO solution (10 mg / mL, 3.24 μM) of compound L-01 from Example 1.1 was added to the reaction solution, and the mixture was reacted at 25 °C for 120 min. Finally, 486 μL of L-cysteine ​​solution (10 mM, 4.86 μM) was added to terminate the reaction.

[0308] Unreacted small molecules and potential aggregates were removed using a HITRAP CM FF cation exchange column. The chromatographic purification conditions were as follows:

[0309] Chromatographic column: HITRAP CM FF column (brand: GE);

[0310] Buffer A: 20mM pH 6.5 phosphate buffer;

[0311] Buffer B: 20mM pH 6.5 phosphate buffer + 500mM NaCl solution;

[0312] Elution method: First, rinse the column with Buffer A, then dilute the sample to 3 times its volume with 40 mM L-histidine hydrochloride (pH 5.5) and inject it. Elute with a linear gradient of 0-100% Buffer B solution, collect the eluent with UV absorbance greater than 50 mAU, and finally adjust the ultrafiltration collection system to 20 mM phosphate buffer to prepare Example ADC-II-1 (2 mg / ml, 21 mL), which is stored at -80℃ for later use.

[0313] The conjugation ratio (DAR) of the antibody-glucocorticoid conjugate was determined using hydrophobic interaction chromatography (HIC-HPLC). The HIC-HPLC chromatogram of ADC-II-1 is shown below. Figure 1 The heterogeneous mixture contains antibodies with zero DL molecules ("E0" peak), two DL molecules ("E2" peak), four DL molecules ("E4" peak), six DL molecules ("E6" peak), and eight DL molecules ("E8" peak). Based on the peak areas of this spectrum, the average antibody-to-drug ratio (n) was calculated to be 4.3. The calculation formula is: n is determined by multiplying the sum of the percentage areas of each peak by its corresponding drug load, summing the results, and dividing by 100, i.e., n = (0 * A E0 +2*A E2 +4*A E4 +6*A E6 +8*A E8 ) / 100, and other embodiments refer to the same calculation method, which will not be repeated here.

[0314] Example II-2:

[0315]

[0316] Following the method described in General Method 1, 180 μL of TCEP·HCl solution (10 mM, 1800 nmol) was added to 12.0 mL of adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL, 810 nmol), and the mixture was thoroughly mixed and reacted at 37 °C for 120 min. Then, 778 μL of DMSO solution of compound L-03 (10 mg / mL, 7.78 μM) was added to the reaction solution, and the reaction was continued at 25 °C for 120 min. Finally, 972 μL of L-cysteine ​​solution (10 mM, 9.72 μM) was added to terminate the reaction. Unreacted small molecules and potential aggregates were removed using a HITRAP CM FF cation exchange column under the purification conditions described in Example II-1. The ultrafiltration collection solution was adjusted to 20 mM phosphate buffer to prepare Example ADC-II-2 (2 mg / mL, 48 mL), which was stored at -80 °C for later use.

[0317] The HIC-HPLC chromatogram of ADC-II-2 is shown below. Figure 2 Based on the peak area of ​​the spectrum, the average antibody-to-conjugate ratio n was calculated to be 5.5.

[0318] Example II-3:

[0319]

[0320] Following the method described in General Method 1, 180 μL of TCEP·HCl solution (10 mM, 1800 nmol) was added to 12.0 mL of adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL, 810 nmol), and the mixture was thoroughly mixed and reacted at 37 °C for 120 min. Then, 692 μL of DMSO solution (10 mg / mL, 6.48 μM) of compound L-08 from Example 1.8 was added to the reaction solution, and the mixture was reacted at 25 °C for 120 min. Finally, 972 μL of L-cysteine ​​solution (10 mM, 9.72 μM) was added to terminate the reaction. Unreacted small molecules and potential aggregates were removed using a HITRAP CM FF cation exchange column under the purification conditions described in Example II-1. Finally, the ultrafiltration collection solution system was adjusted to 20mM phosphate buffer to prepare Example ADC-II-3 (2mg / ml, 44mL), which was stored at -80℃ for later use.

[0321] The average antibody-to-conjugate ratio (n) was calculated to be 4.3 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-3.

[0322] Example II-4:

[0323]

[0324] Following the method described in General Method 2, 1.4 mL of 0.7 M arginine solution was slowly added to 20 mL (2 mg / mL) of ADC-II-2 in Example 1 to achieve a total arginine concentration of 50 mM. The mixture was then incubated at 25 °C for 72 hours, and the hydrolysis reaction was quenched by adding 1.0 mL of 0.1 M acetic acid solution. Potential aggregates were removed using a HITRAP CM FF cation exchange column under the purification conditions described in Example II-1. Finally, the ultrafiltration collection was adjusted to 20 mM phosphate buffer to prepare ADC-II-4 (2 mg / mL, 16.5 mL), which was stored at -80 °C for later use.

[0325] The average antibody-to-conjugate ratio (n) was calculated to be 5.3 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-4.

[0326] Example II-5:

[0327]

[0328] Following the method described in General Method 3, 90 μL of TCEP·HCl solution (10 mM, 900 nmol) was added to 6.0 mL of adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL, 405 nmol), and the mixture was thoroughly mixed and reacted at 37 °C for 120 min. Then, 378 μL of DMSO solution (10 mg / mL, 3.24 μM) of compound L-12 from Example 1.12 was added to the reaction solution, and the mixture was reacted at 25 °C for 120 min. Finally, 486 μL of L-cysteine ​​solution (10 mM, 4.86 μM) was added to terminate the reaction. Unreacted small molecules and potential aggregates were removed using a HITRAP CM FF cation exchange column under the purification conditions described in Example II-1. The ultrafiltration collection solution was adjusted to 20 mM phosphate buffer to prepare ADC-II-5 (2 mg / mL, 25.5 mL), which was stored at -80 °C for later use.

[0329] The average antibody-to-conjugate ratio (n) was calculated to be 4.9 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-5.

[0330] Example II-6:

[0331]

[0332] Following the method described in General Method 3, 120 μL of TCEP·HCl solution (10 mM, 1200 nmol) was added to 8.0 mL of adalimumab (40 mM phosphate, 2 mM EDTA buffer, pH 7.0 ± 0.1, 10 mg / mL, 538 nmol), and the mixture was thoroughly mixed and reacted at 37 °C for 120 min. Then, 444 μL of DMSO solution (10 mg / mL, 4.31 μM) of compound L-11 from Example 1.11 was added to the reaction solution, and the mixture was reacted at 25 °C for 120 min. Finally, 646 μL of L-cysteine ​​solution (10 mM, 6.46 μM) was added to terminate the reaction. Unreacted small molecules and potential aggregates were removed using a HITRAP CM FF cation exchange column under the purification conditions described in Example II-1. Finally, the ultrafiltration collection solution system was adjusted to 20 mM phosphate buffer to prepare ADC-II-6 (2 mg / ml, 32.0 mL), which was stored at -80℃ for later use.

[0333] The average antibody-to-conjugate ratio (n) was calculated to be 4.0 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-6.

[0334] Example II-7:

[0335]

[0336] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-13 to prepare compound ADC-II-7 (2 mg / mL, 42.5 mL).

[0337] The average antibody-to-conjugate ratio (n) was calculated to be 5.1 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-7.

[0338] Example II-8:

[0339]

[0340] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-14 to prepare compound ADC-II-8 (2 mg / mL, 45.0 mL).

[0341] The average antibody-to-conjugate ratio (n) was calculated to be 5.5 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-8.

[0342] Example II-9:

[0343]

[0344] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-15 to prepare compound ADC-II-9 (2 mg / mL, 41.3 mL).

[0345] The average antibody-to-conjugate ratio (n) was calculated to be 4.9 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-9.

[0346] Example II-10:

[0347]

[0348] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-16 to prepare compound ADC-II-10 (2 mg / mL, 39.6 mL).

[0349] The average antibody-to-conjugate ratio (n) was calculated to be 5.0 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-10.

[0350] Example II-11:

[0351]

[0352] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-17 to prepare compound ADC-II-11 (2 mg / mL, 42.0 mL).

[0353] The average antibody-to-conjugate ratio (n) was calculated to be 4.7 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-11.

[0354] Example II-12:

[0355]

[0356] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-18 to prepare compound ADC-II-12 (2 mg / mL, 46.2 mL).

[0357] The average antibody-to-conjugate ratio (n) was calculated to be 4.9 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-12.

[0358] Example II-13:

[0359]

[0360] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-19 to prepare compound ADC-II-13 (2 mg / mL, 42.0 mL).

[0361] The average antibody-to-conjugate ratio (n) was calculated to be 5.3 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-13.

[0362] Example II-14:

[0363]

[0364] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-20 to prepare compound ADC-II-14 (2 mg / mL, 36.50 mL).

[0365] The average antibody-to-conjugate ratio (n) was calculated to be 4.5 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-14.

[0366] Example II-15:

[0367]

[0368] Following the preparation method of Example II-1, compound L-01 was replaced with compound L-02 to prepare compound ADC-II-15 (2 mg / mL, 40.3 mL).

[0369] The average antibody-to-conjugate ratio (n) was calculated to be 4.3 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-15.

[0370] Example II-16:

[0371]

[0372] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-04 to prepare compound ADC-II-16 (2 mg / mL, 40.3 mL).

[0373] The average antibody-to-conjugate ratio (n) was calculated to be 4.6 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-16.

[0374] Example II-17:

[0375]

[0376] Following the preparation method of Example II-2, compound L-03 was replaced with compound L-05 to prepare compound ADC-II-17 (2 mg / mL, 38.7 mL).

[0377] The average antibody-to-conjugate ratio (n) was calculated to be 5.1 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-17.

[0378] Example II-18:

[0379]

[0380] Following the preparation method of Example II-3, compound L-08 was replaced with compound L-06 to prepare compound ADC-II-18 (2 mg / mL, 44.0 mL).

[0381] The average antibody-to-conjugate ratio (n) was calculated to be 4.7 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-18.

[0382] Example II-19:

[0383]

[0384] Following the preparation method of Example II-3, compound L-08 was replaced with compound L-07 to prepare compound ADC-II-19 (2 mg / mL, 44.3 mL).

[0385] The average antibody-to-conjugate ratio (n) was calculated to be 3.9 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-19.

[0386] Example II-20:

[0387]

[0388] Following the preparation method of Example II-4, ADC-II-2 was replaced with ADC-II-3 to prepare compound ADC-II-20 (2 mg / mL, 20.6 mL).

[0389] The average antibody-to-conjugate ratio (n) was calculated to be 4.0 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-20.

[0390] Example II-21:

[0391]

[0392] Following the preparation method of Example II-6, compound L-11 was replaced with compound L-09 to prepare compound ADC-II-21 (2 mg / mL, 45.8 mL).

[0393] The average antibody-to-conjugate ratio (n) was calculated to be 4.5 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-21.

[0394] Example II-22:

[0395]

[0396] Following the preparation method of Example II-6, compound L-11 was replaced with compound L-10 to prepare compound ADC-II-22 (2 mg / mL, 37.3 mL).

[0397] The average antibody-to-conjugate ratio (n) was calculated to be 4.4 based on the peak area of ​​the HIC-HPLC chromatogram of ADC-II-22.

[0398] Comparative Example:

[0399] Comparative Example 1.1

[0400]

[0401] The control ADC4 was prepared according to the preparation method of Example 7 of WO2019106609.

[0402] The average antibody-to-conjugate ratio (n) was calculated to be 4.2 based on the peak area of ​​the HIC-HPLC chromatogram of ADC4.

[0403] Example III: Biological Evaluation

[0404] ADC-II-1 to ADC-II-22 Phosphate Buffer: 20 mM phosphate buffer solution, 2 mg / ml, store at -80℃ for later use.

[0405] Reference ADC4: 20mM phosphate buffer solution, 2mg / ml, stored at -80℃ for later use.

[0406] Adalimumab injection: Humira, 40mg / 0.8ml, AbbVie, batch number 15107XH23.

[0407] Example 3-1: Activity of antibody-glucocorticoid conjugates (ADCs) in membrane-bound TNF-α-mediated GRE reporter gene systems

[0408] 3.1.1 Test Sample

[0409] Phosphate buffer for ADC-II-1 to ADC-II-14 groups and phosphate buffer for control ADC4.

[0410] 3.1.2 Grouping and Dosage Setting

[0411] ADC-II-1 to ADC-II-14 groups and a control ADC4 group were set up, and the drug dosage for each ADC group was set to 1000.0, 200.0, 40.0, 8.0, 1.6, 0.32, 0.064, and 0.013 nM.

[0412] 3.1.3 Test Methods

[0413] HeLa cells stably transfected with the MMTV-Luc reporter gene using lentivirus were purchased from ECACC (Catalog No.: HeLa / MMTV-Luc). To generate transmembrane TNF-α reporter cells, stably transfected HeLa-MMTV-Luc cells were seeded in 96-well plates at 30,000 cells per well. Human TNF-α mutant plasmids (TNF-αΔ1–12, with TACE restriction sites removed) were transfected using Lipo3000 according to the kit instructions. After 12 hours, the cells were incubated with different concentrations of ADC. After 24 hours, the luminescence signal values ​​were read using a multi-mode microplate reader according to the luciferase assay kit instructions. The data were processed and fitted to obtain ECACC results. 50 value.

[0414] 3.1.4 Test Results

[0415] Table 1. Activity in the membrane-bound TNF-α-mediated GRE reporter gene system.

[0416]

[0417]

[0418] The results showed that ADC-II-1 to ADC-II-14 groups all had the ability to affect the activation level of cellular GRE and could affect the activation level of glucocorticoid-mediated signaling pathways.

[0419] Other ADC compounds of this invention (ADC-II-15 to ADC-II-22) all exhibited the ability to affect the level of GRE activation in cells (ECG) under the same experimental conditions. 50 (between 0.1-3 nM), which will not be elaborated further here.

[0420] Example 3-2: Activity of antibody-glucocorticoid conjugate (ADC) in lipopolysaccharide (LPS)-induced cytokine release

[0421] 3.2.1 Test Sample

[0422] ADC-II-1 to ADC-II-14 phosphate buffer, control ADC4 phosphate buffer, adalimumab injection (Humira, 40 mg / 0.8 ml, AbbVie, batch number 15107XH23).

[0423] 3.2.2 Grouping and Dosage Setting

[0424] The following groups were set up: culture medium blank group, PBMC cell control group, ADC-II-1 to ADC-II-14 groups, control ADC4 group and adalimumab group. The drug dosage of each ADC group and adalimumab group was set to 1000.0, 333.3, 111.1, 37.0, 12.4, 4.1 and 1.4 ng / mL.

[0425] 3.2.3 Test Methods

[0426] Primary human peripheral blood mononuclear cells (PBMCs) (purchased from Lonza) were washed in 50 ml of phosphate-buffered saline (PBS), resuspended in 5% DMSO-contaminated fetal bovine serum (FBS), aliquoted, and cryopreserved in liquid nitrogen. The PBMCs were thawed, resuspended in cell culture medium (e.g., RPMI cell culture medium) containing 2% FBS and 1% penicillin and streptomycin, and seeded in 96-well plates. The cells were then incubated at 37°C and 5% CO2 with 50 μL of different concentrations of ADC and adalimumab for 4 hours. After adding 100 ng / mL lipopolysaccharide (LPS) and culturing for 24 hours, the supernatant was collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was directly transferred to another 96-well plate, and the IL-1β content in the culture supernatant was determined using an enzyme-linked immunosorbent assay (ELISA) kit (Immunoway) according to the manufacturer's instructions. The data were processed, and the IC50 was obtained. 50 Values. The inhibitory activity of LPS-stimulated PBMC cytokine release is shown in Table 2.

[0427] 3.2.4 Test Results

[0428] Table 2. Activity of inhibiting LPS-stimulated PBMC cytokine release.

[0429]

[0430]

[0431] The results showed that groups ADC-II-1 to ADC-II-14 all had the ability to affect the release of cytokines, such as IL-1β, from PBMC cells.

[0432] Other ADC compounds of this invention (ADC-II-15 to ADC-II-22) all exhibited the ability to influence the release of cytokines from PBMC cells (IC50) under the same experimental conditions. 50 (IL-1β) levels were between 10 and 300 ng / ml.

[0433] Example 3-3: Assay of the endocytic capacity of antibody-glucocorticoid conjugates (ADCs) in membrane-bound TNF-α-overexpressing cells

[0434] 3.3.1 Test Sample

[0435] ADC-II-1 to ADC-II-6 phosphate buffer, control ADC4 phosphate buffer, adalimumab injection (Humira, 40 mg / 0.8 ml, AbbVie, batch number 15107XH23).

[0436] 3.3.2 Grouping and Dosage Setting

[0437] The following groups were set up: blank group (K562 cell line without hTNF gene transfection), ADC-II-1 to ADC-II-6 groups, control ADC4 group and adalimumab group. The dosage of each ADC group and adalimumab group was set to 3 μg / mL.

[0438] 3.3.3 Test Methods

[0439] This experiment used a stable K562 cell line overexpressing the hTNF gene (purchased from Cyagen). Each ADC group was diluted to a concentration of 12 μg / mL using RPMI Medium 1640 medium as the antibody working solution. Zenon... TM (Invitrogen, lot number 2365674) diluted 25 times as Zenon TM Working solution; take 25 μL each of antibody working solution and Zenon. TM The working solution was mixed and incubated at room temperature for 5 minutes to form a labeled complex. The number of K562 cells was adjusted to 2 × 10⁻⁶. 6 Cells / mL were seeded into 96-well plates, 50 μL per well, along with 50 μL of the labeled complex. The 96-well plates were incubated at 37°C for 6–48 h, and flow cytometry was performed at 6 h, 16 h, 24 h, 30 h, and 48 h. The mean fluorescence intensity (MFI) was analyzed using FlowJo software.

[0440] 3.3.4 Test Results

[0441] Flow cytometry mean fluorescence intensity measurement results as follows Figure 3 As shown.

[0442] The results show that: Figure 3It was found that the mean fluorescence intensity (MFI) of the ADC-II-1 to ADC-II-6 groups, the control ADC4 group, and the adalimumab group were all higher than those of the blank group at 6h, 16h, 24h, and 30h, indicating that they have endocytosis activity in K562 cells. Among them, the mean fluorescence intensity of the ADC-II-2 to ADC-II-6 groups (1.9 to 2.2 times that of the blank group at 24h) was significantly stronger than that of the ADC-II-1 group and the control ADC4 group (1.3 to 1.5 times that of the blank group at 24h).

[0443] Other ADC compounds of the present invention (ADC-II-7 to ADC-II-22) showed an average fluorescence intensity of 1.9 to 2.2 times that of the blank group and 1.3 to 1.5 times that of the control ADC4 group and ADC-II-1 group after incubation in K562 cells for 24 h under the same experimental conditions.

[0444] Examples 3-4: Stability of antibody-glucocorticoid conjugates (ADCs) in plasma

[0445] 3.4.1 Test Sample

[0446] Take 2 mg / mL of ADC-II-1 to ADC-II-6 phosphate buffer as working solutions and place them on ice before use.

[0447] 3.4.2 Test Methods

[0448] 980 μL of human and rat (heparin sodium anticoagulant) samples (n=2) were pre-incubated at 37℃ for 5 min, and 25 μL of each test sample was added. The mixture was vortexed to obtain a final compound concentration of 50 μg / mL and incubated at 37℃. At days 1, 3, 7, 14, 21, and 28, 50 μL of the incubated sample was collected and placed in an EP tube containing 100 μL of ice-cold methanol. The sample was vortexed to inactivate the compounds. The collected samples were stored at -60 to -90℃, and the release of glucocorticoids was detected using LC-MS / MS.

[0449] 3.4.3 Test Results

[0450] Table 3 Release in different plasmas

[0451]

[0452] The results showed that at a concentration of 50 μg / mL, the ADC-II-2 to ADC-II-6 groups exhibited good stability after 28 days of incubation in human and rat plasma, with free glucocorticoid levels all below the detection limit. The ADC-II-1 group showed significant release of free glucocorticoids after 14 days of incubation in human and rat plasma.

[0453] Other ADC compounds of the present invention (ADC-II-7 to ADC-II-22) showed good stability after incubation in human and rat plasma for 28 days under the same experimental conditions, and the free glucocorticoids were all below the detection limit.

[0454] Examples 3-5: Release of glucocorticoid molecules from antibody-glucocorticoid conjugates (ADCs) in human lysosomes

[0455] 3.5.1 Test Sample

[0456] Take 2 mg / mL of ADC-II-1 to ADC-II-6 phosphate buffer and control ADC4 phosphate buffer as working solutions and place them on ice for use.

[0457] 50mM sodium acetate buffer: Weigh 409mg sodium acetate and dissolve it in 100mL of ultrapure water. Adjust the pH to 5.18 with concentrated hydrochloric acid, mix well, and store at 2-8℃.

[0458] 2mM TCEP 50mM Sodium Acetate Buffer: Weigh 5.93mg TCEP·HCl (purchased from TCI, batch number: 5TJLE-QP) and dissolve it in 10.37mL 50mM sodium acetate buffer. Prepare fresh before use.

[0459] 3.5.2 Test Methods

[0460] Human lysosomes (purchased from XENOTECH, batch number: 2010245) were added to 2 mM TCEP 50 mM sodium acetate buffer to prepare a 0.125 mg / mL human lysosome solution. After pre-incubation at 37°C for 5 min, 50 μg / mL of the test sample was added, and incubation continued. At 0, 1, 2, 4, 8, and 24 h, 50 μL of the reaction solution was taken from the reaction system and added to an EP tube pre-filled with 100 μL of ice-cold methanol. The reaction was immediately terminated by vortexing. The release of glucocorticoids was detected by LC-MS / MS.

[0461] The negative control group consisted of an ADC incubation system without human lysosomes, replaced with an equal volume of buffer solution, and samples were taken for analysis at 0, 1, 2, 4, 8, and 24 hours.

[0462] 3.5.3 Test Results

[0463] Table 4 Release in human lysosomes

[0464]

[0465] The results showed that at a concentration of 50 μg / mL, the ADC-II-2 to ADC-II-6 groups could release 50-95% of free glucocorticoids after incubation in human lysosomes for 24 h, while the ADC-II-1 group and the control ADC4 group could only release 10-30% of free glucocorticoids under the same incubation conditions.

[0466] Other ADC compounds of the present invention (ADC-II-7 to ADC-II-22) can release 50-95% of free glucocorticoids under the same experimental conditions.

[0467] Examples 3-6: Bioactivity assay of antibody-glucocorticoid conjugates (ADCs) in a contact hypersensitivity model

[0468] 3.6.1 Laboratory Animals

[0469] Male balb / c mice, 6 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in an SPF environment.

[0470] 3.6.2 Test Sample

[0471] ADC-II-1 to ADC-II-5 phosphate buffer, control ADC4 phosphate buffer, adalimumab injection (Humira, 40 mg / 0.8 ml, AbbVie, batch number 15107XH23).

[0472] 3.6.3 Test Methods

[0473] After arrival, the experimental animals were acclimatized for 7 days and then randomly divided into groups of 10. One day before the experiment, the fur on the abdomen of the mice was removed, covering an area of ​​approximately 3×3 cm. 2 Mice were sensitized on days 0 and 1 by applying 50 μL of 0.5% DNFB (2,4-dinitrofluorobenzene, prepared with acetone and olive oil in a 4:1 ratio) solution to the shaved areas. Mice in each group were administered the drug intraperitoneally on days 0 and 4 at a dose of 10 mg / kg. On day 5, 20 μL of 0.2% DNFB solution was applied to the inner and outer sides of the right ear to challenge the mice. On day 6, mice were euthanized by cervical dislocation, and bilateral 8 mm diameter ear flaps were removed from the same location using a punch. The thickness was measured with calipers, and the weight was determined by an electronic balance. The difference in thickness and weight between the left and right ear flaps was used to assess the anti-inflammatory activity of the antibody-glucocorticoid conjugate.

[0474] Statistical analysis was performed using SPSS software, and the measurement data were expressed as follows: One-way ANOVA was used for comparisons between groups. The LSD test was used for groups with homogeneous variances, and the Dunnett-t test was used for groups with unequal variances. A p-value < 0.05 was considered statistically significant.

[0475] 3.6.4 Test Results

[0476] The changes in ear swelling thickness and ear weight in the contact hypersensitivity model are shown respectively in Figure 4 and Figure 5 .

[0477] The results showed that: from Figure 4 and Figure 5 , it can be seen that compared with the model group, the ADC-II-1 to ADC-II-5 groups could significantly reduce the change values of ear swelling and ear weight in mice (P < 0.001); among them, the inhibitory effects of ADC-II-2 to ADC-II-5 were better than those of the ADC-II-1 group, the adalimumab group, and the control ADC4 group.

[0478] Under the same test conditions, compared with the model group, other ADC compounds (ADC-II-6 to ADC-II-22) of the present invention could significantly reduce the change values of ear swelling and ear weight in mice, with significant differences (P < 0.01).

[0479] Example 3 - 7: Bioactivity determination of antibody - glucocorticoid conjugate (ADC) in a rat collagen antibody - induced arthritis model

[0480] 3.7.1 Experimental animals

[0481] SD rats, male, 80 - 100 g, animal certificate number: SCXK(Beijing)2019 - 0010, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., feeding environment: SPF.

[0482] 3.7.2 Test samples

[0483] Phosphate buffer solutions of ADC-II-1 to ADC-II-5, phosphate buffer solution of control ADC4, adalimumab injection (Humira, 40 mg / 0.8 ml, AbbVie, batch number 15107XH23).

[0484] 3.7.3 Test methods

[0485] Preparation of collagen - adjuvant emulsion: Mix chicken type II collagen and Freund's complete adjuvant in equal volumes, and pipette in an ice - water bath to thoroughly emulsify the solution into a stable emulsion. The final collagen concentration in the emulsion is 1.0 mg / kg.

[0486] One hundred rats were randomly divided into a normal control group and a model group. The normal control group consisted of 10 rats. Rats in the model group received an intradermal injection of approximately 0.2 mL of collagen-adjuvant emulsion in their left hind paw. Twenty-one days after modeling, paw edema was measured. When the paw edema volume exceeded 2.50 mL, the rats were randomly divided into eight groups of 10 rats each: the model group, the adalimumab group, the control ADC4 group, and the ADC-II-1 to ADC-II-5 groups. Each group received a single intraperitoneal injection of 10 mg / kg. Following intraperitoneal administration, paw edema was measured every 3 days using a paw edema meter according to the water displacement method, with continuous observation for 28 days.

[0487] The formula for calculating changes in foot swelling is as follows:

[0488] Foot swelling change value (ΔmL) = Average foot swelling value of the model group - Average foot swelling value of each treatment group.

[0489] Statistical analysis was performed using SPSS software, and the measurement data were expressed as follows: One-way ANOVA was used for comparisons between groups. The LSD test was used for groups with homogeneous variances, and the Dunnett-t test was used for groups with unequal variances. A p-value < 0.05 was considered statistically significant.

[0490] 3.7.4 Test Results

[0491] Changes in paw swelling in a rat collagen antibody-induced arthritis model are shown in the figure. Figure 6 .

[0492] The results show that: Figure 6 It was found that, compared with the model group, the ADC-II-2 to ADC-II-5 groups could significantly reduce rat paw edema, and the effect could last for 28 days (P<0.001); the adalimumab group, ADC-II-1 group and the control ADC4 group could reduce rat paw edema within 0-12 days, and the inhibitory activity gradually weakened after 12 days; the paw edema inhibitory activity of the ADC-II-2 to ADC-II-5 groups was significantly better than that of the adalimumab group, ADC-II-1 group and the control ADC4 group.

[0493] Other ADC compounds of the present invention (ADC-II-6 to ADC-II-22) significantly reduced rat paw swelling compared with the model group under the same experimental conditions, and the efficacy lasted for 28 days, with statistical differences (P < 0.05).

[0494] The ADC compound of this invention was well tolerated by animals throughout the study, with controllable safety, and showed normal weight gain.

[0495] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody-glucocorticoid conjugate having the formula (I) or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of equation (I) is as follows: Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment; The L1-L2 are selected from: D is a glucocorticoid derivative with the formula (II): In glucocorticoid derivative D, R1 is selected from H or F; R2 is selected from H, methyl, or F; R3 and R4 are independently selected from H, OH, alkyl groups containing 1-6 carbons, OCOR7, or R3 and R4 together. That is, C16 and C17 are also connected by an oxygen bridge; R7 is selected from heteroaryl groups with 4-10 carbon atoms; R8 and R9 are each independently selected from H and alkyl groups containing 1-6 carbons; R5 is selected from H, F, or Cl; n is an integer or decimal between 1 and 8.

2. The antibody-glucocorticoid conjugate of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The glucocorticoid derivative D is selected from:

3. The antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof as described in claim 1 or 2, characterized in that, The L1-L2-D is selected from:

4. The antibody-glucocorticoid conjugate of claim 3 or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula (I) is selected from: Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment; n is an integer or decimal between 1 and 8.

5. The antibody-glucocorticoid conjugate of claim 4 or a pharmaceutically acceptable salt thereof, characterized in that, n is an integer or decimal between 3 and 6.

6. The antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof as described in claim 4 or 5, characterized in that, The compound represented by formula (I) is selected from: Wherein, Ab is an anti-TNF-α antibody or its antigen-binding fragment; n is an integer or decimal between 3 and 6.

7. The antibody-glucocorticoid conjugate of claim 6 or a pharmaceutically acceptable salt thereof, characterized in that, n is an integer or decimal between 4 and 6.

8. The antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof as described in claim 4 or 5, characterized in that, The compound represented by formula (I) is selected from: Ab represents an anti-TNF-α antibody or its antigen-binding fragment.

9. The antibody-glucocorticoid conjugate of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, The compound represented by formula (I) is selected from: Ab represents an anti-TNF-α antibody or its antigen-binding fragment.

10. The antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1, 2, 4, 5, 7, or 9, characterized in that, The antibody Ab is selected from adalimumab, infliximab, sertozumab, golimumab, or their antigen-binding fragments.

11. The antibody-glucocorticoid conjugate of claim 3 or a pharmaceutically acceptable salt thereof, characterized in that, The antibody Ab is selected from adalimumab, infliximab, sertozumab, golimumab, or their antigen-binding fragments.

12. The antibody-glucocorticoid conjugate of claim 6 or a pharmaceutically acceptable salt thereof, characterized in that, The antibody Ab is selected from adalimumab, infliximab, sertozumab, golimumab, or their antigen-binding fragments.

13. The antibody-glucocorticoid conjugate of claim 8 or a pharmaceutically acceptable salt thereof, characterized in that, The antibody Ab is selected from adalimumab, infliximab, sertozumab, golimumab, or their antigen-binding fragments.

14. The antibody-glucocorticoid conjugate of claim 10 or a pharmaceutically acceptable salt thereof, characterized in that, The antibody Ab is selected from adalimumab or its antigen-binding fragment.

15. The antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 11-13, characterized in that, The antibody Ab is selected from adalimumab or its antigen-binding fragment.

16. A pharmaceutical composition, characterized in that, It comprises the antibody-glucocorticoid conjugate as described in any one of claims 1-15 or a pharmaceutically acceptable salt thereof and optionally one or more inert carriers and / or diluents.

17. Use of the antibody-glucocorticoid conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-15 or the pharmaceutical composition of claim 16 in the preparation of a medicament for treating glucocorticoid receptor-mediated diseases; said diseases being selected from rheumatoid arthritis, inflammatory bowel disease, psoriasis, ankylosing spondylitis, bullous pemphigus, multiple sclerosis, systemic lupus erythematosus, Sjögren's syndrome, and hidradenitis suppurativa.

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