Drug conjugates of glucocorticoid receptor agonists and their medical use

Abstract

Drug conjugates of glucocorticoid receptor agonists and their medical use. In particular, an antibody-drug conjugate of formula (I): Ab-(L-D) k (I), wherein Ab is an antibody or antigen binding fragment thereof, L is a linker covalently linking Ab to D, k is 1 to 20, and D is of formula (II-A) or (II-B) as follows: wherein the various groups are as defined in the specification. The antibody-drug conjugates are effective in treating immune disorders.

Description

Technical Field

[0001] This disclosure pertains to the pharmaceutical field, specifically relating to a drug conjugate of a glucocorticoid receptor agonist and its pharmaceutical applications. Background Technology

[0002] Rheumatoid arthritis (RA) is a common type of arthritis, an autoimmune disease with an incidence rate of 0.3-1% in the population. If left untreated, it can lead to bone destruction and joint damage. Multiple pro-inflammatory cytokines are involved in the pathogenesis of RA, such as tumor necrosis factor-α (TNFα) and interleukins like IL-1, IL-6, and IL-8. Therefore, inhibiting the production of pro-inflammatory cytokines or blocking their physiological effects is currently a hot topic in RA research. In recent years, many newly developed biological agents have been developed to control disease progression by blocking or downregulating the activity of pro-inflammatory cytokines, such as TNFα inhibitors and anti-IL-6R antibodies. Currently, TNFα is considered one of the most important pro-inflammatory cytokines among the many cytokines involved in the inflammatory response of RA, playing a crucial role in the development of RA, local inflammatory responses, and tissue damage. Currently, TNFα inhibitors approved by the US FDA include: the soluble receptor antagonist etanercept, the human-mouse chimeric antibody infliximab, and the fully human monoclonal antibody adalimumab. TNFα inhibitors include the fully human monoclonal antibody golimumab and the pegylated humanized Fab' fragment cetolizumab pegol. Despite their clinical success, TNFα inhibitors remain limited by their maximum achievable efficacy in patients, necessitating the identification and development of more potent and effective agents. Patients treated with TNFα inhibitors may also develop immunogenic responses to the agents, further limiting their effectiveness.

[0003] Glucocorticoid receptor agonists are also relatively effective drugs for treating rheumatoid arthritis. Representative glucocorticoid receptor agonists include those synthesized in vivo from cortisol and corticosterone, as well as synthetic glucocorticoid receptor agonists such as dexamethasone, prednisone, and prednisolone. Because these glucocorticoid receptor agonists have a steroid structure, they are collectively referred to as steroids and are used in the treatment of various diseases. However, due to their use, these steroids sometimes exhibit side effects such as steroid-induced peptic ulcers, steroid-induced purpura, steroid-induced pancreatitis, steroid-induced diabetes, steroid-induced cataracts, and steroid-induced glaucoma.

[0004] Antibody-drug conjugates (ADCs) are monoclonal antibodies or antibody fragments linked to a biologically active drug via a stable chemical linker compound. Most ADCs in preclinical and clinical development are for oncology indications, where cytotoxic payloads target cancer cells expressing antigens. However, modulating pathogenic cell activity through ADC-mediated delivery of bioactive small molecules is also attractive for non-oncology indications, leading to the widespread adoption of this technology.

[0005] Existing technologies have disclosed some drug conjugates of glucocorticoid receptor agonists, such as WO2017210471 and WO2019106609. Summary of the Invention

[0006] This disclosure provides, in one aspect, an antibody-drug conjugate (ADC) of formula (I),

[0007] Ab-(LD) k

[0008] (I)

[0009] Wherein, Ab represents an antibody or its antigen-binding fragment.

[0010] L is a connector that covalently links Ab to D, and k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any value between any two values).

[0011] D is shown in equation (II-A) or (II-B) below:

[0012]

[0013] in,

[0014] Indicates a single bond or a double bond;

[0015] R 1a Each is independently selected from hydrogen, alkyl, and alkoxy, wherein each alkyl and alkoxy group is optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, and hydroxyalkyl.

[0016] Ring A is an aryl or heteroaryl group optionally substituted by one or more substituents Q1;

[0017] Ring B is an aryl or heteroaryl group that may be optionally substituted by one or more substituents Q1;

[0018] X1 is -(CR)5a R 5b )m- and aryl or heteroaryl groups optionally substituted by one or more substituents Q1;

[0019] R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, and optionally substituted by one or more substituents Q1: alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k alkoxy, alkylthio, alkenyl, and alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds;

[0020] Ring C and ring D are each independently selected from aryl and heteroaryl groups optionally substituted by one or more substituents Q1, and at least one of ring C and ring D is selected from fused-ring aryl or fused-heteroaryl groups optionally substituted by one or more substituents Q1;

[0021] X2 is selected from -(CR) 6a R 6b )n-, aryl or heteroaryl groups optionally substituted by one or more substituents Q1, -O-, -S-, -S(O)-, -S(O)(O)-, -NR 6c -, -CH2S-, -CH2O-, -NHCR 6d R 6e -、-CR 6f =CR 6g - and -C≡C-, or X2 does not exist;

[0022] R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, or optionally substituted by one or more substituents Q1, namely: alkyl, -NR. i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)Rk alkoxy, alkylthio, alkenyl, and alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds;

[0023] R 6c R 6d R 6e R 6f and R 6g Each is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy;

[0024] R1 is independently selected from hydrogen, alkyl and alkoxy, wherein each of the alkyl and alkoxy groups is optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl.

[0025] R2 is independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0026]

[0027] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0028] R 2b Each is independently a C1-C6 alkyl or C1-C6 alkoxy;

[0029] R 2c Each is independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0030] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0031] R3 can be hydrogen or halogen independently;

[0032] R4 is independently selected from hydrogen, halogen, and hydroxyl;

[0033] m and n are each independent integers from 1 to 6;

[0034] The substituents Q1 are each independently selected from C1-C6 alkyl, halogen, deuterium, hydroxyl, mercapto, -NR. i Rj Oxygenation, thioation, -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k Nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused-ring aryl and 5- to 12-membered fused-heteroaryl;

[0035] R i and R j Each is independently selected from hydrogen atoms, hydroxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0036] R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j The alkyl, alkoxy, and haloalkyl groups thereon are each optionally selected independently from C1-C6 alkyl, halogen, hydroxyl, mercapto, -NR i R j The substituted group is substituted by one or more substituents selected from oxo, thio, carboxyl, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic, 6- to 10-membered aryl, and 5- to 10-membered heteroaryl; and

[0037] The condition is when R 5a When R is hydrogen or alkyl, 5b It is not hydrogen or alkyl.

[0038] In some implementations, R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy, wherein each alkyl and alkoxy group is optionally substituted by one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, deuterium, amino, cyano, and hydroxyl.

[0039] In some implementations, R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy.

[0040] In some embodiments, ring A is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0041] In some embodiments, ring A is optionally substituted with one or more substituents Q1.

[0042] In some embodiments, ring B is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0043] In some embodiments, ring B is optionally substituted with one or more substituents Q1.

[0044] In some implementations, X1 is -(CR 5a R 5b )m- or optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0045] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0046] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k-S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0047] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)(O)R k C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0048] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0049] In some embodiments, ring C and ring D are each independently selected from 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused-ring aryl, or 5- to 12-membered fused-heteroaryl, optionally substituted by one or more substituents Q1, wherein the heteroaryl or fused-heteroaryl contains at least one nitrogen atom.

[0050] In some embodiments, ring C and ring D are each independently selected from the following groups optionally substituted by one or more substituents Q1:

[0051]

[0052] In some embodiments, ring C is selected from those optionally substituted with one or more substituents Q1:

[0053]

[0054] In some embodiments, ring D is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0055] In some embodiments, ring D is optionally substituted with one or more substituents Q1.

[0056] In some implementations, X2 is selected from -(CR 6a R 6b -n-, -O-, -S-, -NR 6c -, -CH2S-, -CH2O-, -NHCR 6d R 6e - and optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, which is optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0057] In some implementations, R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0058] In some implementations, R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k-S(O)(O)R k C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0059] In some implementations, R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0060] In some embodiments, R1 is independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy, preferably hydrogen.

[0061] In some implementations, R4 is hydrogen on its own.

[0062] In some embodiments, the substituents Q1 are each independently selected from halogens, hydroxyl groups, mercapto groups, deuterium groups, oxo groups, thio groups, cyano groups, amino groups, carboxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups.

[0063] In some implementations, R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j .

[0064] In some implementations, D is shown as in equation (II-A') or (II-B'):

[0065]

[0066] in,

[0067] R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy;

[0068] Ring A is The ring A may be optionally replaced by one or more substituents Q1;

[0069] Ring B is The ring B may be optionally replaced by one or more substituents Q1;

[0070] X1 is -(CR) 5a R 5b )m- or optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom;

[0071] R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 5a and R 5b Together they form oxidized or thiolated compounds;

[0072] Ring C is selected from The ring C is optionally replaced by one or more substituents Q1;

[0073] Ring D is The ring D may be optionally replaced by one or more substituents Q1;

[0074] X2 is selected from -(CR) 6a R 6b -n-, -O-, -S-, -NR 6c -, -CH2S-, -CH2O-, -NHCR 6d R 6e - and optionally 6 to 10 aryl or 5 to 10 heteroaryl groups substituted with one or more substituents Q1;

[0075] R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds;

[0076] R 6c R 6d and R 6eEach is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy;

[0077] R2 is independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0078]

[0079] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0080] R 2b Each is independently a C1-C6 alkyl or C1-C6 alkoxy;

[0081] R 2c Each is independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0082] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0083] R3 can be hydrogen or halogen independently;

[0084] m and n are each independent integers from 1 to 6;

[0085] The substituents Q1 are each independently selected from halogens, hydroxyl groups, mercapto groups, deuterium groups, oxo groups, thio groups, cyano groups, amino groups, carboxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0086] R i and R j Each is independently selected from hydrogen atoms, hydroxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0087] R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j ;and

[0088] The condition is when R 5a When R is hydrogen or alkyl, 5b It is not hydrogen or alkyl.

[0089] In some implementations, R 1a It is hydrogen.

[0090] In some implementations, X1 is selected from -(CR5a R 5b m-, optionally substituted by one or more substituents Q1:

[0091] In some implementations, R 5a and R 5b Both are fluorine.

[0092] In some implementations, R 5a and R 5b Together they form oxo or thio, with oxo being preferred.

[0093] In some implementations, X2 is selected from -(CR 6a R 6b n-, optionally substituted by one or more substituents Q1:

[0094] In some implementations, R 6a and R 6b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, C1-C6 alkyl, -NR i R j -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0095] In some implementations, R2 is independently selected from -CH2OH, -CH2SH, -OH, and

[0096] In some implementations, R3 is hydrogen.

[0097] In some implementations, R3 is fluorine.

[0098] In some implementations, k is any value between 1 and 10, preferably any value between 2 and 5. k can be an integer or a decimal.

[0099] In some embodiments, the linker is stable outside the cell, allowing the ADC to remain intact in its extracellular environment but cleave upon internalization within the cell. In some embodiments, when the ADC enters a cell expressing an antigen specific to the antibody portion of the ADC, the glucocorticoid receptor agonist drug portion cleaves from the antibody portion, releasing the unmodified form of the glucocorticoid receptor agonist.

[0100] In some embodiments, the cleavable portion of the linker is a cleavable peptide portion. In some embodiments, ADCs containing cleavable peptide portions exhibit lower aggregation levels and improved antibody-to-drug ratios compared to ADCs containing other cleavable portions. In some embodiments, adding a cleavable portion increases cytotoxicity and / or potency compared to non-cleavable linkers. In some embodiments, the cleavable peptide portion is enzymatically cleavable, and the linker is an enzyme-cleavable linker. In some embodiments, the enzyme is a cathepsin, and the linker is a cathepsin-cleavable linker. In some embodiments, enzyme-cleavable linkers (e.g., cathepsin-cleavable linkers) exhibit one or more of the aforementioned improved properties compared to other cleavage mechanisms.

[0101] In some embodiments, the linker comprises an amino acid unit L1, said amino acid unit L1 preferably comprising 2 to 7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, aspartic acid, homolysine, n-methylvaline, ... Peptide residues composed of amino acids (q being an integer from 1 to 6), exemplary amino acid units including but not limited to valine-citrulline (Val-Cit), alanine-phenylalanine (Ala-Phe); phenylalanine-lysine (Phe-Lys), phenylalanine-homolysine (Phe-Homolys), n-methyl-valine-citrulline (Me-Val-Cit), alanine-alanine (Ala-Ala), glycine-glutamic acid (Gly-Glu), glutamic acid-alanine-alanine (Glu-Ala-Ala), glycine-lysine (Gly-Lys), glycine-valine-citrulline (Glv-Val-Cit), and glycine-glycine-glycine (Gly-Gly-Gly).

[0102] In some embodiments, the linker includes a stretching unit, which is a chemical structural fragment with one end covalently linked to an antibody via a carbon atom and the other end linked to an amino acid unit, a disulfide moiety, a sulfonamide moiety, or a non-peptide chemical moiety. Exemplary stretching units include, but are not limited to, those shown below.

[0103]

[0104] In some embodiments, the stretching element is selected from... Each of p can be 1, 2, 3, 4, 5, or 6 independently.

[0105] In some implementations, the connector is selected from

[0106]

[0107]

[0108]

[0109] In some implementations, the connector is selected from

[0110]

[0111] In some embodiments, the antibody-drug conjugate is selected from:

[0112]

[0113]

[0114]

[0115] In this context, Ab, D, and k are defined as previously, and p is independently 1, 2, 3, 4, 5, or 6.

[0116] In some embodiments, the antibody-drug conjugate is selected from...

[0117]

[0118]

[0119]

[0120]

[0121] k is selected from 1 to 10, and can be an integer or a decimal.

[0122] On the other hand, the antibody or its antigen-binding fragment described in the antibody-drug conjugate (ADC) of this disclosure is selected from murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies or their antigen-binding fragments.

[0123] In some embodiments, the antibody or its antigen-binding fragment is selected from anti-TNFα antibody, anti-IL-4R antibody, anti-IL-6 / IL-6R antibody, anti-IL-13R antibody, anti-IL-17 / IL-17R antibody, anti-IL-23 / IL23R antibody, anti-IL-36R antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD28 antibody, anti-CD40 antibody, anti-TSLP antibody, or its antigen-binding fragment.

[0124] In some embodiments, the antibody or its antigen-binding fragment binds to human and / or mouse TNFα. Antibodies and antigen-binding fragments that bind TNFα are known in the art.

[0125] In some implementations, the anti-TNFα antibody or antigen-binding fragment does not bind to TNF-β.

[0126] Anti-TNFα antibodies and their antigen-binding fragments include, for example, adalimumab, infliximab, certolizumab pegol, afimomab, nerelimomab, ozoralizumab, placulumab, and golimumab, or their antigen-binding fragments. Other anti-TNFα antibodies and antigen-binding fragments are provided in, for example, WO 2013 / 087912, WO 2014 / 152247, and WO 2015 / 073884, each of which is incorporated herein by reference in its entirety.

[0127] Anti-TNFα antibodies and their antigen-binding fragments also include antibodies and their antigen-binding fragments that competitively inhibit the binding of adalimumab, infliximab, sertuzumab, afenomumab, neremomab, ozolazumab, prakurumumab, or golimumab to TNFα. Anti-TNFα antibodies and their antigen-binding fragments also include antibodies and antigen-binding fragments that bind to the same TNFα epitope as adalimumab, infliximab, sertuzumab, afenomumab, neremomab, ozolazumab, prakurumumab, or golimumab.

[0128] In some embodiments, the anti-TNFα antibody or its antigen-binding fragment competitively inhibits the binding of adalimumab to TNFα. In some embodiments, the anti-TNFα antibody or its antigen-binding fragment binds to the same TNFα epitope as adalimumab. In some embodiments, the anti-TNFα antibody or its antigen-binding fragment is adalimumab or its antigen-binding fragment. In some embodiments, the anti-TNFα antibody or its antigen-binding fragment is adalimumab.

[0129] In some embodiments, the anti-TNFα antibody or its antigen-binding fragment contains sequences of adalimumab, infliximab, cetozumab, afimomab, neremomab, ozolazumab, pralkurumab, or golimumab, such as complementarity-determining regions (CDRs), variable heavy chain domains (VHs), and / or variable light chain domains (VLs).

[0130] This disclosure also provides a compound of formula (III-A) or (III-B) or a pharmaceutically acceptable salt thereof.

[0131]

[0132] in,

[0133] Indicates a single bond or a double bond;

[0134] Ring A is an aryl or heteroaryl group optionally substituted by one or more substituents Q1;

[0135] Ring B is an aryl or heteroaryl group that may be optionally substituted by one or more substituents Q1;

[0136] X1 is -(CR) 5a R 5b )m- or optionally aryl or heteroaryl groups substituted with one or more substituents Q1;

[0137] R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, and optionally substituted by one or more substituents Q1: alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k alkoxy, alkylthio, alkenyl, and alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds;

[0138] Ring C and ring D are each independently selected from aryl and heteroaryl or fused heteroaryl groups optionally substituted by one or more substituents Q1, and at least one of ring C and ring D is selected from fused aryl or fused heteroaryl groups optionally substituted by one or more substituents Q1.

[0139] X2 is selected from -(CR) 6a R 6b )n-, aryl or heteroaryl groups optionally substituted by one or more substituents Q1, -O-, -S-, -S(O)-, -S(O)(O)-, -NR 6c -, -CH2S-, -CH2O-, -NHCR 6d R 6e -、-CR 6f =CR 6g - and -C≡C-, or X2 does not exist;

[0140] R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, and optionally substituted by one or more substituents Q1: alkyl, -NR i Rj -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k alkoxy, alkylthio, alkenyl, and alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds;

[0141] R 6c R 6d R 6e R 6f and R 6g Each is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy;

[0142] R1 is independently selected from hydrogen, alkyl and alkoxy, wherein each of the alkyl and alkoxy groups is optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy and hydroxyalkyl.

[0143] R2 is independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0144]

[0145] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0146] R 2b Each is independently a C1-C6 alkyl or C1-C6 alkoxy;

[0147] R 2c Each is independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0148] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0149] R3 can be hydrogen or halogen independently;

[0150] R4 is independently selected from hydrogen, halogen, and hydroxyl;

[0151] m and n are each independently selected from integers from 1 to 6;

[0152] The substituents Q1 are each independently selected from C1-C6 alkyl, halogen, deuterium, hydroxyl, mercapto, -NR. i R j Oxygenation, thioation, -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k Nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused-ring aryl and 5- to 12-membered fused-heteroaryl;

[0153] R i and R j Each is independently selected from hydrogen atoms, hydroxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0154] R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j The alkyl, alkoxy, and haloalkyl groups thereon are each optionally selected independently from C1-C6 alkyl, halogen, hydroxyl, mercapto, -NR i R j It is substituted by one or more substituents selected from oxo, thio, carboxyl, nitro, cyano, C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, C2-C6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclic, 6- to 10-membered aryl and 5- to 10-membered heteroaryl.

[0155] R 1a Each is independently selected from hydrogen, alkyl, and alkoxy, wherein each alkyl and alkoxy group is optionally substituted by one or more substituents selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro, hydroxy, and hydroxyalkyl.

[0156] R 1b Each is independently selected from hydrogen, PG-, H-L1-, PG-L1-, or

[0157] R 1a and R 1b Together with the nitrogen atom it is attached to, it forms: Or R 1a and R 1b Together with the nitrogen atom attached to it, it forms a nitro group;

[0158] p can be 1, 2, 3, 4, 5 or 6 independently;

[0159] L1 is an amino acid unit, preferably -glycine, -glutamic acid, or -

[0160] X is a halogen;

[0161] PG is an amino protecting group; and

[0162] The condition is when R 5a When R is hydrogen or alkyl, 5b It is not hydrogen or alkyl.

[0163] In some implementations, R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy, wherein each alkyl and alkoxy group is optionally substituted by one or more substituents selected from C1-C6 alkyl, C1-C6 alkoxy, halogen, deuterium, amino, cyano, and hydroxyl.

[0164] In some implementations, R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy.

[0165] In some embodiments, L1 is selected from glycine-glutamic acid- or

[0166] In some implementations, R 1b Each is independently selected from hydrogen, PG-, H-L1-, PG-L1-,

[0167]

[0168] In some implementations, PG is Boc or Cbz.

[0169] In some embodiments, ring A is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0170] In some embodiments, ring A is optionally substituted with one or more substituents Q1.

[0171] In some embodiments, ring B is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0172] In some embodiments, ring B is optionally substituted with one or more substituents Q1.

[0173] In some implementations, X1 is -(CR 5a R 5b )m- or optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom;

[0174] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k -C(S)R k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0175] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0176] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)(O)R k C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0177] In some implementations, R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 5a and R 5b Together they form oxidized or thiolated compounds.

[0178] In some embodiments, ring C and ring D are each independently selected from 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused-ring aryl, and 5- to 12-membered fused-heteroaryl, optionally substituted by one or more substituents Q1, wherein the heteroaryl or fused-heteroaryl contains at least one nitrogen atom.

[0179] In some embodiments, ring C is selected from the following groups optionally substituted by one or more substituents Q1:

[0180]

[0181] In some embodiments, ring C is selected from those optionally substituted with one or more substituents Q1:

[0182]

[0183] In some embodiments, ring D is a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl, optionally substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0184] In some embodiments, ring D is optionally substituted with one or more substituents Q1.

[0185] In some implementations, X2 is -(CR 6a R 6b )n- or optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom.

[0186] In some implementations, R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)OR k -S(O)(O)R k -S(O)(O)OR k C1-C6 alkoxy, C1-C6 alkylthio, C2-C6 alkenyl, and C2-C6 alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0187] In some implementations, R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k -S(O)R k -S(O)(O)R k C1-C6 alkoxy, C2-C6 alkenyl and C2-C6 alkynyl, or R 6a R 6b Together with the carbon atom attached to it, it forms a 3- to 10-membered cycloalkyl group, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0188] In some implementations, R 6a and R 6bEach group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0189] In some embodiments, R1 is independently selected from hydrogen, C1-C6 alkyl and C1-C6 alkoxy, preferably hydrogen.

[0190] In some implementations, R4 is hydrogen on its own.

[0191] In some embodiments, the substituents Q1 are each independently selected from halogens, hydroxyl groups, mercapto groups, deuterium groups, cyano groups, amino groups, carboxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups.

[0192] In some implementations, R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j .

[0193] In some embodiments, the compound of formula (III-A) or (III-B) is a compound of formula (III-A') or (III-B').

[0194]

[0195] in,

[0196] Ring A is The ring A may be optionally replaced by one or more substituents Q1;

[0197] Ring B is The ring B may be optionally replaced by one or more substituents Q1;

[0198] X1 is selected from -(CR) 5a R 5b )m- or optionally a 6- to 10-membered aryl or a 5- to 10-membered heteroaryl substituted with one or more substituents Q1, wherein the heteroaryl contains at least one nitrogen atom;

[0199] R 5a and R 5b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR iR j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 5a and R 5b Together they form oxidized or thiolated compounds;

[0200] Ring C is selected from The ring C is optionally replaced by one or more substituents Q1;

[0201] Ring D is The ring D may be optionally replaced by one or more substituents Q1;

[0202] X2 is selected from -(CR) 6a R 6b -n-, -O-, -S-, -NR 6c -, -CH2S-, -CH2O-, -NHCR 6d R 6e - and optionally 6 to 10 aryl or 5 to 10 heteroaryl groups substituted with one or more substituents Q1;

[0203] R 6a and R 6b Each group is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, and optionally substituted by one or more substituents Q1: C1-C6 alkyl, -NR i R j -C(O)R k -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds;

[0204] R 6c R 6d and R 6e Each is independently selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl, and C1-C6 alkoxy;

[0205] R2 is independently selected from -CH2OH, -CH2SH, -CH2Cl, -SCH2Cl, -SCH2F, -SCH2CF3, -OH, -OCH2CN, -OCH2Cl, -OCH2F, -OCH3, -OCH2CH3, -SCH2CN,

[0206]

[0207] R 2a Each is independently hydrogen or C1-C6 alkyl;

[0208] R 2b Each is independently a C1-C6 alkyl or C1-C6 alkoxy;

[0209] R 2c Each is independently selected from hydrogen, C1-C6 alkyl, -CH2OH and C1-C6 alkoxy;

[0210] R 2d and R 2e Each is independently hydrogen or C1-C6 alkyl;

[0211] R3 can be hydrogen or halogen independently;

[0212] m and n are each independent integers from 1 to 6;

[0213] The substituents Q1 are each independently selected from halogens, hydroxyl groups, mercapto groups, deuterium groups, oxo groups, thio groups, cyano groups, amino groups, carboxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0214] R i and R j Each is independently selected from hydrogen atoms, hydroxyl groups, C1-C6 alkyl groups, and C1-C6 alkoxy groups;

[0215] R k Independently selected from hydrogen atom, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, hydroxyl and -NR i R j ;

[0216] R 1a Each is independently selected from hydrogen, C1-C6 alkyl, and C1-C6 alkoxy;

[0217] R 1b Each is independently selected from hydrogen, PG-, H-L1-, PG-L1-,

[0218] p can be 1, 2, 3, 4, 5 or 6 independently;

[0219] L1 is an amino acid unit selected from -glycine, -glutamic acid, or...

[0220] X is a halogen;

[0221] PG is an amino protecting group; and

[0222] The condition is when R 5a When R is hydrogen or alkyl, 5b It is not hydrogen or alkyl.

[0223] In some implementations, R 1aIt is hydrogen.

[0224] In some implementations, R 1b Each is independently selected from hydrogen, PG-, H-L1-, PG-L1-, Preferred hydrogen or

[0225] In some implementations, R 1a and R 1b Both are hydrogen.

[0226] In some implementations, X1 is selected from -(CR 5a R 5b m-, optionally substituted by one or more substituents Q1:

[0227] In some implementations, R 5a and R 5b Both are fluorine.

[0228] In some implementations, R 5a and R 5b Together they form oxo or thio, with oxo being preferred.

[0229] In some implementations, X2 is selected from -(CR 6a R 6b n-, optionally substituted by one or more substituents Q1:

[0230] In some implementations, R 6a and R 6b Each is independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano, C1-C6 alkyl, -NR i R j -C(O)OR k and C1-C6 alkoxy groups, or R 6a and R 6b Together they form oxidized or thiolated compounds.

[0231] In some implementations, R2 is independently selected from -CH2OH, -CH2SH, -OH, and

[0232] In some implementations, R3 is hydrogen.

[0233] In some implementations, R3 is fluorine.

[0234] In some implementations, p is 1 or 2 independently, preferably 1.

[0235] In some embodiments, the compound of formula (III-A) or (III-B) is selected from...

[0236]

[0237] Or its medicinal salt.

[0238] In some embodiments, the compound of formula (III-A) or (III-B) is selected from...

[0239]

[0240]

[0241]

[0242] Or a pharmaceutically usable salt thereof, wherein X is a halogen, preferably chlorine or bromine, more preferably bromine.

[0243] In the structure described in this disclosure Indicates a single bond or a double bond.

[0244] As will be understood by those skilled in the art, for example when R 5a and R 5b If one of them is selected from oxo or thio, then the other does not exist.

[0245] This disclosure also provides a pharmaceutical composition comprising at least one of the aforementioned antibody-drug conjugates, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0246] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0247] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% of the aforementioned antibody-drug conjugate based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% of the aforementioned antibody-drug conjugate. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% of the aforementioned antibody-drug conjugate. In some embodiments, the pharmaceutical composition contains 1% to 99% of the aforementioned antibody-drug conjugate. In some embodiments, the pharmaceutical composition contains 2% to 98% of the aforementioned antibody-drug conjugate.

[0248] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable carriers, diluents, or excipients based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable carriers, diluents, or excipients.

[0249] The antibody-drug conjugates and / or pharmaceutical compositions comprising antibody-drug conjugates described herein can be used to lyse cells expressing surface TNFα (in vitro or in vivo) and / or to treat diseases or conditions characterized by increased TNFα (e.g., increased TNFα in synovial fluid). In some embodiments, the antibody-drug conjugates and / or compositions can be used to inhibit cytokine release (in vitro or in vivo) and / or to treat autoimmune or inflammatory diseases. In some embodiments, the antibody-drug conjugates and / or compositions are used to treat Crohn's disease. In some embodiments, the antibody-drug conjugates and / or compositions are used to treat ulcerative colitis. In some embodiments, the antibody-drug conjugates and / or compositions are used to treat rheumatoid arthritis (RA). In some embodiments, the antibody-drug conjugates and / or compositions are used to treat juvenile idiopathic arthritis (JA). In some embodiments, the antibody-drug conjugates and / or compositions are used to treat psoriatic arthritis (PsA). In some embodiments, the antibody-drug conjugate and / or composition is used to treat spondyloarthritis, such as ankylosing spondylitis (AS) or axial spondyloarthritis (axSpA). In some embodiments, the antibody-drug conjugate and / or composition is used to treat adult Crohn's disease (CD). In some embodiments, the antibody-drug conjugate and / or composition is used to treat pediatric Crohn's disease. In some embodiments, the antibody-drug conjugate and / or composition is used to treat ulcerative colitis (UC). In some embodiments, the antibody-drug conjugate and / or composition is used to treat plaque psoriasis (Ps). In some embodiments, the antibody-drug conjugate and / or composition is used to treat hidradenitis suppurativa (HS). In some embodiments, the antibody-drug conjugate and / or composition is used to treat uveitis. In some embodiments, the antibody-drug conjugate and / or composition is used to treat Behcet's disease. In some embodiments, the antibody-drug conjugate and / or composition is used to treat psoriasis, including plaque psoriasis.

[0250] This disclosure also provides a method for delivering a glucocorticoid receptor agonist to cells expressing TNFα, comprising the step of contacting the cells expressing TNFα with the antibody-drug conjugate described in this disclosure.

[0251] This disclosure also provides a method for determining the anti-inflammatory activity of an antibody-drug conjugate. Such a method may include the step of contacting cells expressing TNFα with an antibody-drug conjugate as described herein. Some embodiments include contacting cells expressing TNFα with an antibody-drug conjugate as described herein and determining a reduced release of pro-inflammatory cytokines from the cells compared to control cells. Some embodiments include in vitro methods for determining the anti-inflammatory activity of an antibody-drug conjugate.

[0252] Some embodiments include screening methods (e.g., in vitro methods) that involve directly or indirectly contacting cells (e.g., cells expressing TNFα) with an antibody-drug conjugate and determining whether the antibody-drug conjugate modulates cell activity or function, as reflected, for example, by changes in cell morphology or viability, marker expression, differentiation or dedifferentiation, cellular respiration, mitochondrial activity, membrane integrity, maturation, proliferation, viability, apoptosis, or cell death. An example of a direct interaction is a physical interaction, while indirect interactions include, for example, the action of the composition on an intermediate molecule that in turn acts on a reference entity (e.g., cells or cell cultures).

[0253] This disclosure also provides a pharmaceutical composition comprising at least one of the aforementioned compounds of formula (III-A) or (III-B) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent, or excipient. The compound or its pharmaceutically acceptable salt, and the pharmaceutical composition thereof, may be used to treat immune diseases.

[0254] This disclosure further provides a kit comprising the antibody-drug conjugate or pharmaceutical composition described herein.

[0255] Terminology Definition

[0256] Unless otherwise specified, all technical and scientific terms used in this disclosure are consistent with the common understanding of one of ordinary skill in the art to which this disclosure pertains. While this disclosure may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred methods and materials are described herein. In describing and claiming protection for this disclosure, the following terms are used in accordance with the definitions below.

[0257] When a trade name is used in this disclosure, the applicant intends to include formulations of products under that trade name, generic drugs of products under that trade name, and active pharmaceutical ingredients.

[0258] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0259] The terms “linker,” “linker unit,” “connector unit,” “connector,” or “linker fragment” refer to a chemical structural segment or bond that is connected to a ligand at one end and to a drug at the other end. It can also be connected to other linkers before being connected to a drug.

[0260] The linker may comprise one or more linker components. Exemplary linker components include 6-maleiminohexanoyl (MC), maleiminopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with a linker reagent: N-succinimino-4-(2-pyridylthio)valerate (SPP), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (SMCC, also referred to herein as MCC), and N-succinimino-4-iodo-acetyl)aminobenzoate (SIAB). The linker may comprise stretching units, spacer units, amino acid units, and extension units. It can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile adapters (e.g., hydrazone), protease-sensitive (e.g., peptidase-sensitive) adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0261] The term "stretching unit" refers to a chemical structural segment that is covalently linked to an antibody at one end by a carbon atom and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptide chemical moiety at the other end.

[0262] The term "spacer unit" is a bifunctional compound structural fragment that can be used to couple amino acid units and glucocorticoids to ultimately form antibody-drug conjugates. This coupling method allows glucocorticoids to be selectively linked to amino acid units.

[0263] The term "amino acid" refers to an organic compound whose molecular structure contains both an amino group and a carboxyl group, with both groups directly attached to the -CH- structure. The general formula is H₂NCHRCOOH, where R is H, a substituted or unsubstituted alkyl group, etc. Based on the position of the amino group attached to the carbon atom in the carboxylic acid, amino acids can be classified into α, β, γ, δ, ε…-amino acids. In the biological world, the amino acids that constitute natural proteins have specific structural characteristics, namely, their amino groups are directly attached to the α-carbon atom, i.e., α-amino acids, including glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, aspartic acid, histidine, asparagine, glutamic acid, lysine, glutamine, methionine, arginine, serine, threonine, cysteine, and proline. Non-natural amino acids, such as citrulline, are also present. As is known to those skilled in the art, non-natural amino acids do not constitute natural proteins and therefore do not participate in the synthesis of the antibodies disclosed herein. The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0264]

[0265]

[0266] The term "antibody-drug conjugate" refers to a ligand linked to a biologically active drug via a stable linker. In this disclosure, "antibody-drug conjugate" (ADC) refers to a monoclonal antibody or antibody fragment linked to a biologically active glucocorticoid via a stable linker. The antibody or antibody fragment may bind to a glucocorticoid molecule containing a linker via specific groups therein (e.g., interchain disulfide bonds).

[0267] The term "drug loading" refers to the average amount of drug loaded in each antibody-drug conjugate molecule within a population of antibody-drug conjugates, and can also be expressed as the ratio of drug amount to antibody amount. The drug loading ranges from 1 to 20, preferably 1 to 10, glucocorticoids (D) linked to each antibody (Ab). In embodiments of this disclosure, the drug loading is represented by k, which can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the average of any two values. Preferably, it is 1-10, more preferably 1-8, or 2-8, or 2-7, or 3-8, or 3-7, or 3-6, or 4-7, or 4-6, or the average of 4-5. The average amount of drug per ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, CE-SDS (monoclonal antibody size variant assay), and HPLC characterization.

[0268] The present invention discloses a method for determining the molecular size variant of monoclonal antibodies (CE-SDS), which uses sodium dodecyl sulfate capillary electrophoresis (CE-SDS) with ultraviolet detection. Under reducing and non-reducing conditions, the purity of recombinant monoclonal antibody products is quantitatively determined according to molecular weight using capillary electrophoresis (2015 edition of the Chinese Pharmacopoeia 0542).

[0269] In one embodiment of this disclosure, the glucocorticoid is coupled to the ε-amino group of the N-terminal amino group and / or lysine residue of the ligand via a linker unit. Generally, the number of drug molecules that can be coupled to the antibody in the coupling reaction will be less than the theoretical maximum value.

[0270] The loading of antibody-drug conjugates can be controlled using the following non-restrictive methods, including:

[0271] (1) Control the molar ratio of the ligation reagent and the monoclonal antibody.

[0272] (2) Control the reaction time and temperature.

[0273] (3) Choose different reaction reagents.

[0274] The term "antibody" refers to immunoglobulin, a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being 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 divide them into 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 differences in the constant region. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0275] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved backbone regions (FRs). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDRs of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0276] The antibodies disclosed herein include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, with humanized antibodies and fully human antibodies being preferred.

[0277] The term "mouse antibody" in this disclosure refers to antibodies prepared using mice in accordance with the 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.

[0278] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody generated by grafting a mouse CDR sequence into a human antibody variable region framework, i.e., a human germline antibody framework sequence of different types. 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 or publicly available references that include germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database, as well as in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. 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 antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Further literature describing methods that can be used to participate in humanization includes, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and Winter et al. [Jones et al., Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

[0279] 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. The development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies, and fully human monoclonal antibodies. This disclosure pertains to fully human monoclonal antibodies. 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.

[0280] The term “antigen-binding fragment” refers to one or more fragments of an antibody that maintain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in “antigen-binding fragments” include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VH and VL domains of a single arm of the antibody; (v) single-domain or dAb fragments (Ward et al., (1989) Nature 341: 544-546) consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs) or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be linked by synthetic linkers using recombinant methods, thereby enabling the production of a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0281] Fab is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at position 224 of the H chain). Approximately half of the N-terminal side of the H chain and the entire L chain are linked together by disulfide bonds.

[0282] F(ab')2 is an antibody fragment with a molecular weight of approximately 100,000, possessing antigen-binding activity, and containing two Fab regions connected at the hinge position, obtained by digesting the portion below the two disulfide bonds in the hinge region of IgG with the enzyme pepsin.

[0283] Fab' is an antibody fragment with a molecular weight of approximately 50,000 and antigen-binding activity obtained by cleaving the disulfide bonds in the hinge region of the aforementioned F(ab')2.

[0284] In addition, the Fab' can be produced by inserting DNA encoding the Fab' fragment of an antibody into a prokaryotic or eukaryotic expression vector and then introducing the vector into a prokaryote or eukaryote to express the Fab'.

[0285] The terms “single-chain antibody,” “single-chain Fv,” or “scFv” refer to molecules containing a variable domain (or region; VH) of the antibody heavy chain and a variable domain (or region; VL) of the antibody light chain linked by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example, using variants with 1–4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444–6448). Other connectors that may be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

[0286] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR applies only to CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3) of the heavy chain variable domain. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable domain and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable domain. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the “Kabat” numbering rule (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the “Chothia” numbering rule (see Al-Lazikani et al., (1997) JMB 273: 927-948), and the ImMunoGenTics (IMGT) numbering rule (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)), etc. For example, in the classic format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).Combining the CDR definitions from Kabat and Chothia, the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). Following IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0287] The term "antibody framework" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0288] The term “epitope” or “antigenic determinant” refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996)).

[0289] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example: approximately less than 10 -8 M, 10 - 9 M or 10 -10 M or lower affinity (KD) binding.

[0290] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0291] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the 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 vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0292] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this invention utilize genetic engineering methods to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained by comparing the IMGT Human Antibody Variable Region Germline Gene Database and using MOE software, from the Journal of Immunoglobulins, 2001 ISBN012441351.

[0293] 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; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO and NSO cells.

[0294] The engineered antibody or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or GSepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody 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 should be immediately frozen, e.g., at -70°C, or lyophilized.

[0295] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence, after aligning the amino acid sequences and, where necessary, introducing gaps to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0296] The term "anti-TNFα antibody" or "TNFα-binding antibody" refers to an antibody capable of binding to TNFα with sufficient affinity, for example, such that the antibody can be used as a therapeutic agent targeting TNFα. The degree to which an anti-TNFα antibody binds to unrelated non-TNFα proteins can be less than, for example, about 10% of the antibody binding to TNFα as measured by radioimmunoassay (RIA). In some embodiments, the TNFα-binding antibody has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.

[0297] 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. It is a structural and functional fragment of proteins, such as hormones and enzymes, which are essentially peptides.

[0298] The term "sugar" refers to a biological macromolecule composed of three elements: C, H, and O. It can be classified into monosaccharides, disaccharides, and polysaccharides.

[0299] The term "fluorescent probe" refers to a class of fluorescent molecules that exhibit characteristic fluorescence in the ultraviolet-visible-near-infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, and polarization, etc.) can be sensitively altered by changes in the properties of their environment, such as polarity, refractive index, and viscosity. These fluorescent probes interact non-covalently with nucleic acids (DNA or RNA), proteins, or other macromolecular structures, causing changes in one or more fluorescence properties. They can be used to study the properties and behavior of macromolecules.

[0300] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-Dimethylpentyl, 2,2-Dimethylpentyl, 3,3-Dimethylpentyl, 2-Ethylpentyl, 3-Ethylpentyl, n-Octyl, 2,3-Dimethylhexyl, 2,4-Dimethylhexyl, 2,5-Dimethylhexyl, 2,2-Dimethylhexyl, 3,3-Dimethylhexyl, 4,4-Dimethylhexyl, 2-Ethylhexyl, 3-Ethylhexyl, 4-Ethylhexyl, 2-Methyl-2-Ethylpentyl, 2-Methyl-3-Ethylpentyl, n-Nonyl, 2-Methyl-2-Ethylhexyl, 2-Methyl-3-Ethylhexyl, 2,2-Diethylpentyl, n-Decyl, 3,3-Diethylhexyl, 2,2-Diethylhexyl, and their various branched isomers, etc. More preferably, alkyl groups containing 1 to 6 carbon atoms are used. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, etc. The alkyl group can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0301] The term "alkylene" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane. It is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), etc. Alkylenes can be substituted or unsubstituted; when substituted, the substituent can be replaced at any usable linking point.

[0302] The term "alkenyl" refers to a linear alkenyl group having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms, and having at least one double bond at any position, including, for example, vinylene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentenylene, hexenylene, hexadienylene, etc.

[0303] The term "subchain ynyl" includes linear subchain ynyl groups having 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms and having at least one triple bond at any position, including, for example, ethynylene, propynylene, butynylene, pentylyne, hexynylene, etc.

[0304] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups. "Carbocyclic" refers to the ring system within the cycloalkyl group.

[0305] The term "spirocycloalkyl" refers to a polycyclic group consisting of 5 to 20 quintile rings sharing a single carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6 to 14 quintiles, more preferably 7 to 10 quintiles. Spirocycloalkyl groups are classified into monospirocycloalkyl, bispirocycloalkyl, or polyspirocycloalkyl groups based on the number of shared spiro atoms between the rings, with monospirocycloalkyl and bispirocycloalkyl groups being preferred. More preferably, it is a 4-quintile, 4-quintile, 4-quintile, 5-quintile, or 5-quintile / 6-quintile monospirocycloalkyl group. "Spirocarbon ring" refers to the ring system within the spirocycloalkyl group. Non-limiting examples of spirocycloalkyl groups include:

[0306]

[0307] The term "fused-ring alkyl" refers to a 5- to 20-membered polycyclic carbon group in which each ring shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more rings may contain one or more double bonds, but no ring has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of constituent rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused-ring alkyl, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic alkyl. "Fused-carbon ring" refers to the ring system within a fused-ring alkyl group. Non-limiting examples of fused-ring alkyl groups include:

[0308]

[0309] The term "bridged cycloalkyl" refers to a 5- to 20-membered polycyclic carbon group in which any two rings share two non-directly bonded carbon atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, it is 6- to 14-membered, more preferably 7- to 10-membered. Depending on the number of rings, it can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged cycloalkyl groups include:

[0310]

[0311] The cycloalkyl ring may be fused to an aryl, heteroaryl, or heterocycloalkyl ring, wherein the ring connected to the parent structure is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. The cycloalkyl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0312] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m The heteroatom (where m is an integer from 0 to 2) excluding the ring portion of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl or pyrrolidinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups. "Heterocyclic" refers to the ring system within the heterocyclic group.

[0313] The term "spiroheterocyclic group" refers to a polycyclic heterocyclic group consisting of 5 to 20 member monocyclic rings sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O). m The rings consist of heteroatoms (where m is an integer from 0 to 2), with the remaining ring atoms being carbon. They may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. Preferably, they are 6 to 14 fused, more preferably 7 to 10 fused. Spiroheterocyclic groups are classified into monospirocyclic, bispirocyclic, or multispirocyclic groups based on the number of shared spiroatoms between rings, with monospirocyclic and bispirocyclic groups being preferred. More preferably, they are 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospirocyclic groups. "Spiroheterocyclic" refers to the ring system within the spirocyclic group. Non-limiting examples of spirocyclic groups include:

[0314]

[0315] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic group, preferably bicyclic or tricyclic, more preferably 5-membered / 5-membered or 5-membered / 6-membered bicyclic fused heterocyclic group. "Fused heterocyclic" refers to the ring system in the fused heterocyclic group. Non-limiting examples of fused heterocyclic groups include:

[0316]

[0317] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two non-directly bonded atoms. It may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, it consists of 6 to 14 rings, more preferably 7 to 10 rings. Depending on the number of rings, it can be classified as a bicyclic, tricyclic, tetracyclic, or polycyclic bridged heterocyclic group, preferably bicyclic, tricyclic, or tetracyclic, and more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0318]

[0319] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0320] wait.

[0321] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0322] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. "Aromatic ring" refers to the ring system within the aryl group. Non-limiting examples of aryl groups include:

[0323]

[0324] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group, preferably phenyl.

[0325] The term "fused-ring aryl" can refer to an unsaturated aromatic fused-ring structure containing 8-14 ring atoms, formed by two or more ring structures sharing two adjacent atoms. Preferably, it contains 8-12 ring atoms. Examples include fully unsaturated fused-ring aryl groups such as naphthalene and phenanthrene, as well as partially saturated fused-ring aryl groups such as benzo[3-8] saturated monocyclic cycloalkyl groups and benzo[3-8] partially saturated monocyclic cycloalkyl groups. "Fused aromatic ring" refers to the ring system within the fused-ring aryl group. Specific examples of fused-ring aryl groups include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, and 1,4-dihydronaphthyl.

[0326] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, such as imidazolyl, furanyl, thiophenel, thiazolyl, pyrazolyl, oxazolyl, pyrrololyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl or thiazolyl. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. "Heteroaryl ring" refers to the ring system within the heteroaryl group. Non-limiting examples of heteroaryl groups include:

[0327]

[0328] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0329] The term "fused aryl" can refer to an unsaturated aromatic fused ring structure containing 5-14 ring atoms (including at least one heteroatom) formed by two or more ring structures sharing two adjacent atoms. The carbon, nitrogen, and sulfur atoms can be substituted with oxygen. Preferably, it includes "5-12 fused aryl", "7-12 fused aryl", "9-12 fused aryl", etc., such as benzofuranyl, benzoisofuranyl, benzothiopheneyl, indole, isoindole, benzoxazolyl, benzoimidazolyl, indazole, benzotriazolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridinel, phenanthridinel, benzopyridinyl, phthalazinyl, quinazolinyl, quinoxalinyl, quinoxalinyl, phenoxalinyl, phenazinyl, pteridinel, purinel, naphthidyl, phenazine, phenothiazine, etc. "Dense aromatic rings" refers to the ring system in dense aromatic groups.

[0330] The fused heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0331] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0332] The term "alkathio" refers to -S- (alkyl) and -S- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkathio groups include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, and cyclohexylthio. Alkathio groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkathio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkathio, and heterocycloalkathio.

[0333] The term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group, wherein the alkyl group is as defined above.

[0334] The term "halogenated alkyl" refers to an alkyl group that has been substituted with a halogen, wherein the alkyl group is as defined above.

[0335] The term “deuterated alkyl” refers to an alkyl group that has been replaced by a deuterium atom, wherein the alkyl group is as defined above.

[0336] The term "hydroxyl group" refers to the -OH group.

[0337] The term "oxo" refers to an =O group. For example, a carbon atom is connected to an oxygen atom by a double bond, forming a ketone or aldehyde group.

[0338] The term "thio" refers to the =S group. For example, a carbon atom and a sulfur atom are linked by a double bond to form a thiocarbonyl group -C(S)-.

[0339] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0340] The term "amino" refers to -NH2.

[0341] The term "cyano" refers to -CN.

[0342] The term "nitro" refers to -NO2.

[0343] The term "carboxyl group" refers to -C(O)OH.

[0344] The term "aldehyde group" refers to -CHO.

[0345] The term "carboxylic acid ester group" refers to -C(O)O (alkyl) or -C(O)O (cycloalkyl), where alkyl and cycloalkyl are as defined above.

[0346] The term "acyl halide" refers to a compound containing a -C(O)-halogen group.

[0347] The term "sulfonyl" refers to -S(O)(O)-.

[0348] The term "sulfinyl" refers to -S(O)-.

[0349] "Amino protecting group" is a suitable group known in the art for amino protection, see reference ("Protective Groups in Organic Synthesis", 5). Th The amino protecting group in Ed.TW Greene & P. ​​GMWuts, preferably, is a (C 1-10 Alkyl or aromatic acyl group, such as formyl, acetyl, benzoyl, etc.; can be (C 1-6 Alkyl or C 6-10 aryl)sulfonyl; or (C 1-6 Alkoxy or C 6-10 Aryloxy)carbonyl, such as Boc or Cbz; can also be substituted or unsubstituted alkyl, such as triphenylmethyl (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).

[0350] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the cases in which the event or environment occurs or does not occur. For example, "optionally alkyl-substituted heterocyclic alkyl group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic alkyl group is substituted with an alkyl group and cases where the heterocyclic alkyl group is not substituted with an alkyl group.

[0351] The term "pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0352] The term "drug carrier" is used in the context of the drugs disclosed herein, referring to a system that can alter the way a drug enters the human body and its distribution within the body, control the rate of drug release, and deliver the drug to a target organ. Drug carrier release and targeting systems can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, high-molecular-weight surfactants, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates, preferably such as micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules while also exhibiting good membrane permeability, making them excellent drug carriers.

[0353] The term "excipient" refers to any additive in a pharmaceutical preparation other than the active pharmaceutical ingredient (API). Examples of excipients include binders, fillers, disintegrants, and lubricants in tablets; the base portion in semi-solid preparations such as ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solvents, osmotic pressure regulators, and colorants in liquid preparations.

[0354] The term "diluent," also known as a filler, is primarily used to increase the weight and volume of tablets. The addition of diluents not only ensures a specific volume but also reduces dosage deviations of the main components and improves the compressibility of the drug. When the tablet contains oily components, absorbents are added to absorb the oil and maintain a "dry" state, facilitating tablet formation.

[0355] The compounds disclosed herein may contain one or more asymmetric centers, thus producing enantiomers, diastereomers, and other stereoisomers as defined by absolute stereochemistry (R)- or (S)- or (D)- or (L)- for amino acids. This disclosure includes all possible isomers as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)- isomers can be prepared using chiral synthons or chiral reagents, or using conventional methods such as chromatography and fractional crystallization. Conventional methods for preparing / separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution using racemates (or racemates of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain an alkene double bond or other geometrically asymmetric centers, unless otherwise stated, it means that the compounds include E and Z geometric isomers. Furthermore, all tautomeric forms are also included.

[0356] In the chemical structure of the compounds described in this disclosure, the bonds... This indicates that the configuration is not specified; that is, if chiral isomers exist in the chemical structure, the bond... It can be Or simultaneously include Two configurations. In the chemical structure of the compounds described in this disclosure, the bonds... No configuration is specified, meaning it can be Z configuration, E configuration, or both configurations.

[0357] "Stereoisomers" refer to compounds composed of identical atoms bonded by the same bonds but having different three-dimensional structures, and are not interchangeable. Various stereoisomers and mixtures thereof are contemplated in this disclosure, and include "enantiomers," which refer to two stereoisomers whose molecules are non-overlapping mirror images of each other.

[0358] "Tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. This disclosure includes tautomers of any of the said compounds.

[0359] The compounds described in this disclosure, or any isotopically labeled derivatives thereof, or isomers thereof, are covered by this disclosure. Atoms capable of being isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine. They can be labeled with isotopes. 2 H(D), 3 H, 11 C 13 C 14 C 15 N、 18 F, 31 P, 32 P, 35S, 36 Cl and 125 I, etc. are used instead. Unless otherwise stated, when a position is specifically designated as deuterium (D), the position shall be understood as having a deuterium abundance of at least 3,000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 45% deuterium incorporation). Attached Figure Description

[0360] Figure 1 Anti-inflammatory activity of small molecule steroids in experiments stimulating lipopolysaccharide-induced cytokine secretion by human PBMCs

[0361] Figure 2 Activity of anti-TNF-ADCs in the membrane-bound TNFα-mediated GRE reporter gene system

[0362] Figure 3 Activity of anti-TNF-ADC in lipopolysaccharide-stimulated human monocytes secreting cytokines

[0363] Figure 4 Activity of anti-TNF-ADC (4 nM) in LPS-induced human monocyte cytokine release assay

[0364] Figure 5 Experimental procedure for test example 5

[0365] Figure 6 Activity of anti-TNF-ADC in a mouse model of collagen antibody-induced arthritis (CAIA)

[0366] Figure 7 Activity of anti-TNF-ADC at different time points in a mouse model of collagen antibody-induced arthritis (CAIA)

[0367] Figure 8 Experimental procedure for test example 6

[0368] Figure 9 Activity of anti-TNF-ADCs in a delayed-type hypersensitivity (DTH) model Detailed Implementation

[0369] The following examples further describe the preparation of the compounds and pharmaceutically acceptable salts described in this disclosure, but these examples are not intended to limit the scope of this disclosure.

[0370] Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents whose specific source is not specified are commercially available conventional reagents.

[0371] NMR shift (δ) with 10 -6The unit (ppm) is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO) as the solvent. 6 ), deuterated chloroform (CDCl3), deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0372] MS measurements were performed using a Shimadzu 2010 Mass Spectrometer or an Agilent 6110A MSD mass spectrometer.

[0373] HPLC determinations were performed using a Shimadzu LC-20A system, Shimadzu LC-2010HT series, or Agilent 1200 LC high-performance liquid chromatograph (Ultimate XB-C18 3.0*150mm column or Ultimate C18 2.1*30mm column).

[0374] Chiral HPLC analysis was performed using Chiralpak IC-3 100×4.6mm ID, 3um, Chiralpak AD-3 150×4.6mm ID, 3um, Chiralpak AD-3 50×4.6mm ID, 3um, Chiralpak AS-3 150×4.6mm ID, 3um, Chiralpak AS-3 100×4.6mm ID, 3μm, ChiralCel OD-3 150×4.6mmI.D.,3um, Chiralcel OD-3 100×4.6mm ID,3μm, Chiralcel OJ-H 150×4.6mm ID,5um, Chiralcel OJ-3 150×4.6mm ID, 3um chromatographic column; Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates are used for thin layer chromatography. The silica gel plates used for thin layer chromatography (TLC) are 0.15mm to 0.2mm in diameter, and the diameter of the silica gel plates used for thin layer chromatography separation and purification is 0.4mm to 0.5mm.

[0375] Column chromatography typically uses Yantai Huanghai silica gel of 100-200 mesh, 200-300 mesh, or 300-400 mesh as the carrier.

[0376] Chiral preparation columns used were DAICL CHIRALPAK IC (250 mm * 30 mm, 10 μm) or Phenomenex-Amylose-1 (250 mm * 30 mm, 5 μm).

[0377] The CombiFlash rapid preparation system uses a CombiFlash Rf150 (TELEDYNE ISCO).

[0378] Mean inhibition rate of kinases and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG GmbH, Germany).

[0379] The known starting materials disclosed herein can be synthesized using or in accordance with methods known in the art, or can be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, AccelaChemBio Inc, and Darui Chemicals.

[0380] Unless otherwise specified in the examples, the reactions can be carried out under an argon or nitrogen atmosphere.

[0381] Argon or nitrogen atmosphere refers to a reaction flask connected to an argon or nitrogen gas balloon with a volume of approximately 1L.

[0382] A hydrogen atmosphere refers to a reaction flask connected to a hydrogen balloon with a volume of approximately 1L.

[0383] The pressurized hydrogenation reaction was performed using a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0384] The hydrogenation reaction is usually carried out under vacuum, filled with hydrogen gas, and repeated 3 times.

[0385] The microwave reaction was performed using a CEM Discover-S 908860 microwave reactor.

[0386] Unless otherwise specified in the examples, "solution" refers to an aqueous solution.

[0387] Unless otherwise specified in the examples, the reaction temperature is room temperature, which is 20℃~30℃.

[0388] The reaction process in the examples was monitored using thin-layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compounds, and the developing solvent system for TLC included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate system, C: petroleum ether / ethyl acetate system, and D: petroleum ether / ethyl acetate / methanol. The volume ratio of the solvent was adjusted according to the polarity of the compounds, and small amounts of basic or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0389] Preparation of 1.0M Tris buffer at pH 8.30 ± 0.1:

[0390] Weigh 6.0g of tris into a 50mL volumetric flask, add 40mL of purified water, shake to dissolve, add 1.2mL of concentrated hydrochloric acid dropwise to adjust the pH to 8.30, and then make up to volume with purified water.

[0391] Preparation of buffer A:

[0392] In a 2.0L container, add KH2PO4 (8.50g), K2HPO4 (8.56g), NaCl (5.86g), and EDTA (1.50g), then add 1.6L of water for injection. Stir for half an hour until completely dissolved, then bring the volume up to 2.0L with water for injection. The pH value is measured to be 6.30±0.1.

[0393] The abbreviations used in the following experiments have the following meanings:

[0394] DAST: Diethylaminosulfur trifluoride; THF: Tetrahydrofuran; NMP: N-Methylpyrrolidone; DCM: Dichloromethane; m-CPBA: m-chloroperoxybenzoic acid; DIEA: N,N-Diisopropylethylamine; TEA: Triethylamine; Boc: Tert-Butoxycarbonyl; MeOH: Methanol; Et2O: Diethyl ether.

[0395] Example 1

[0396]

[0397] first step

[0398] Compound 1-1 (500 mg, 1.9 mmol, 1.0 eq), compound 1-2 (730 mg, 2.3 mmol, 1.2 eq), Pd(PPh3)4 (660 mg, 0.57 mmol, 0.3 eq), K2CO3 (960 mg, 6.9 mmol, 3.6 eq), and DMF (35 mL, 70 V) were added to a 100 mL single-necked flask. The mixture was purged three times under nitrogen protection and stirred at 80 °C until the reaction was complete. EA (50 mL) and H2O (130 mL) were added to the reaction mixture. The mixture was separated, and the aqueous phase was extracted with EA (30 mL x 2). The combined organic phases were washed successively with H2O (50 mL x 3), saturated LiCl (50 mL), and saturated NaCl solution (30 mL). The mixture was dried over anhydrous Na2SO4, filtered, and the solvent was removed by rotary evaporation. The crude product was obtained by column chromatography, and then pulped with PE:EA = 2:1. After filtration, compounds 1-3 (220 mg, yield 31%) were obtained.

[0399] Ms(ESI): m / z 318[M-55] + .

[0400] Step 2

[0401] Compounds 1-3 (100 mg, 0.268 mmol, 1.1 eq), compounds 1-4 (92 mg, 0.243 mmol, 1.0 eq), anhydrous MgSO4 (146 mg, 1.215 mmol, 5.0 eq), and anhydrous CH3CN (5 mL) were added to a 50 mL three-necked flask and stirred at room temperature for 0.7 h under nitrogen protection. The mixture was then cooled to 0 °C in an ice bath, and CF3SO3H (182 mg, 1.215 mmol, 5.0 eq) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm naturally to room temperature. Once the reaction was complete, the mixture was quenched in an ice bath with EA and saturated sodium bicarbonate solution, and the pH was adjusted to greater than 8. The aqueous phase was then extracted with EA, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The crude product was then subjected to prep-HPLC to obtain compound 1-A (64.5 mg, yield 42%).

[0402] Ms(ESI): m / z 632.3[M+H] + .

[0403] 1 H NMR (400MHz, DMSO) δ7.73(dd,J=8.3,2.5Hz,4H),7.64(d,J=8.3Hz,2H),7.57(d,J=8.2Hz,2H),7.33(d,J=10.1Hz,1H),7.11(t,J=7.8 Hz,1H),6.89(s,1H),6.83(d,J=7.8Hz,1H),6.57(d,J=7.9Hz,1H),6.17(d,J=10.1Hz,1H),5.95(s,1H),5.52(s,1H),5.17(s,2H),5. 12(t,J=5.9Hz,1H),4.97(d,J=5.1Hz,1H),4.82(d,J=2.8Hz,1H),4.56(dd,J=19.6,6.4Hz,1H),4.32(s,1H),4.22(dd,J=19.6,5.5Hz ,1H),2.61-2.53(m,1H),2.34(d,J=10.4Hz,1H),2.20-2.01(m,2H),1.90–1.60(m,5H),1.41(s,3H),1.10-1.01(m,2H),0.89(s,3H).

[0404] Example 2

[0405]

[0406] Under a nitrogen atmosphere, compounds 1-4 (176 mg, 0.47 mmol, 1.0 eq) and anhydrous magnesium sulfate (282 mg, 2.34 mmol, 5.0 eq) were added to a 25 mL reaction flask, followed by anhydrous acetonitrile (5 mL). The mixture was stirred at room temperature for 90 minutes. Then, compound 2-1 (160 mg, 0.49 mmol, 1.05 eq) was added to the mixture, and the mixture was cooled to 0–5 °C in an ice bath. Trifluoromethanesulfonic acid (351 mg, 2.34 mmol, 5.0 eq) was added via syringe, and the reaction was continued with stirring in an ice bath until completion. The reaction solution was filtered through diatomaceous earth, washed with ethyl acetate, and the filtrate was further treated with ethyl acetate and water. The mixture was separated, and the organic phase was washed once more with saturated brine. After drying, the solution was concentrated under reduced pressure to obtain a crude product, which was purified by preparative-HPLC to give compound 2-A (88 mg, yield 32.2%).

[0407] Ms(ESI): m / z 584.42[M+1] + .

[0408] Example 3

[0409]

[0410] first step

[0411] Under nitrogen protection, compound 3-1 (10.0 g, 45.03 mmol), (Bpin)2 (pinacol diboronate, 18.3 g, 72.05 mmol), X-Phos (1.36 g, 2.70 mmol), and KOAc (8.84 g, 90.06 mmol) were dissolved in 1,4-dioxane (150 mL). Pd2(dba)3 (1.65 g, 1.80 mmol) was added, and the reaction was heated to 90 °C. After cooling to room temperature, the filtrate was filtered, concentrated, and separated by column chromatography (PE / EA) to give 13 g of compound 3-2.

[0412] MS-ESI: m / z 270.2 [M+H] + .

[0413] Step 2

[0414] Compound 3-2 (13 g, 45.03 mmol) was dissolved in toluene (100 mL), and Boc2O (13.4 mL, 58.54 mmol) was added. The reaction was heated to 100 °C. After cooling to room temperature, the solution was concentrated and separated by column chromatography (PE / EA) to give 16 g of compound 3-3. The two-step yield was 96.2%.

[0415] MS-ESI: m / z 396.1 [M+Na] + .

[0416] Step 3

[0417] Under nitrogen protection, compounds 3-3 (2.0 g, 5.42 mmol), 3-4 (2.16 g, 10.84 mmol), and K₂CO₃ (3.75 g, 27.10 mmol) were dissolved in tetrahydrofuran (50 mL). Pd(dppf)Cl₂·DCM (441 mg, 0.54 mmol) was added, and the reaction was heated to 80 °C. After cooling to room temperature, the reaction was quenched with water, extracted with ethyl acetate, concentrated, and separated by column chromatography (PE / EA) to give 1.33 g of compounds 3-5, yield: 67.9%.

[0418] MS-ESI: m / z 384.1 [M+Na] + .

[0419] Step 4

[0420] Under nitrogen protection, compounds 1-4 (123 mg, 0.327 mmol) and MgSO4 (197 mg, 1.635 mmol) were dissolved in acetonitrile (10 mL), and the mixture was reacted at room temperature for 1 hour. A solution of compound 3-5 (130 mg, 0.360 mmol) in acetonitrile (10 mL) was added, and the mixture was cooled to 0 °C. Trifluoromethanesulfonic acid (145 μL, 1.635 mmol) was slowly added dropwise. After the addition was complete, the mixture was allowed to return to room temperature naturally. The filtrate was concentrated and separated using preparative HPLC (CH3CN / H2O) to give 90 mg of compound 3-A, with a yield of 44.4%.

[0421] MS-ESI: m / z 620.3 [M+H] + .

[0422] 1H NMR (400MHz, DMSO) δ7.96(dd,J=7.0,2.2Hz,1H),7.38–7.26(m,5H),7.20(d,J=8.2Hz,3H),7.17–7.09(m,1H),6.67(d,J =7.2Hz,1H),6.14(d,J=10.1Hz,1H),5.91(s,1H),5.36(s,1H),4.90(d,J=4.9Hz,1H),4.75(brs,1H),4.47(d,J=19.4Hz, 1H),4.32(s,2H),4.27(brs,1H),4.15(d,J=19.4Hz,1H),2.58–2.50(m,3H),2.34–2.23(m,1H),2.08(dd,J=16.2,6.0Hz ,1H),2.03–1.93(m,1H),1.81–1.53(m,5H),1.38(s,3H),1.23(s,1H),1.03(ddd,J=27.9,11.3,2.6Hz,2H),0.84(s,3H).

[0423] Example 4

[0424]

[0425] first step

[0426] In a 1000 mL single-necked flask, add compound 4-1 (48 g, 176.4 mmol, 1.0 eq), pinacol diboronate (71.7 g, 282.2 mmol, 1.6 eq), potassium acetate (34.6 g, 352.8 mmol, 2.0 eq), PdCl2 (dppf) (6.4 g, 8.82 mmol, 0.05 eq), and dioxane (500 mL). Under nitrogen protection, heat to 95 °C and stir until the reaction is complete. Stop the reaction, cool the reaction solution, and then add compound 4-2 (80.8 g, 352.8 mmol, 2.0 eq), potassium carbonate (48.8 g, 352.8 mmol, 2.0 eq), PdCl2 (dppf) (6.4 g, 8.82 mmol, 0.05 eq), and water (100 mL). Stir and heat to 80 °C under nitrogen protection until the reaction is complete. After the reaction solution was cooled, ethyl acetate and water were added and stirred. The mixture was separated, dried over anhydrous sodium sulfate, concentrated, and the crude product was passed through a column to give approximately 54 g of compound 4-3 (yield 90%).

[0427] Ms(ESI): m / z 342.1[M+1] + .

[0428] Step 2

[0429] Compound 4-3 (7.0 g, 20.5 mmol, 1.0 eq), potassium permanganate (9.7 g, 61.6 mmol, 3.0 eq), and tetra-tert-butylammonium bromide (20.0 g, 61.6 mmol, 3.0 eq) were added to a 500 mL three-necked flask, followed by 140 mL of dichloroethane. The mixture was stirred at room temperature under nitrogen protection until the reaction was complete. The reaction solution was then cooled with ice water, and 10% sodium bisulfite and acetic acid were added and stirred. The mixture was separated into liquid and liquid layers. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and the crude product was passed through a column chromatography to give approximately 6.2 g of compound 4-4, with a yield of 85%.

[0430] Ms(ESI): m / z 378.1 [M+23] + .

[0431] Step 3:

[0432] Compound 4-4 (20.0 g, 56.0 mmol, 1.0 eq), propylene dithiol (12.1 g, 112.0 mmol, 2.0 eq), and boron trifluoride diethyl ether (23.8 g, 168.0 mmol, 3.0 eq) were added to a 500 mL three-necked flask, followed by chloroform (100 mL). The mixture was heated under nitrogen protection and stirred until the reaction was complete. The reaction solution was then cooled with ice water, and water was added and stirred until the solid was completely dissolved. The mixture was separated into layers, and the organic layer was dried over anhydrous sodium sulfate. The mixture was then filtered and concentrated. The crude product was added to petroleum ether and stirred. After filtration, approximately 22 g of compound 4-5 was obtained and directly added to the next step.

[0433] Ms(ESI): m / z 346.0 [M+1] + .

[0434] Step 4:

[0435] Compound 4-5 (22.0 g, 1.0 eq, based on 56.0 mmol) and di-tert-butyl dicarbonate (24.4 g, 112.0 mmol, 2.0 eq) were added to a 100 mL three-necked flask, followed by ethanol (60 mL). The mixture was heated to 50 °C under nitrogen protection and stirred until the reaction was complete. The reaction was then stopped, the reaction solution was concentrated, and the solution was filtered through a column to obtain approximately 18.5 g of compound 4-6, with a yield of 71%.

[0436] Ms(ESI): m / z 468.1 [M+23] + .

[0437] Step 5:

[0438] Compound 4-6 (4.7 g, 13.6 mmol, 1.0 eq) and DAST (6.6 g, 40.9 mmol, 3.0 eq) were added to a 100 mL three-necked flask, followed by 50 mL of dichloromethane. The mixture was heated to 50 °C under nitrogen protection and stirred until the reaction was complete. The reaction was then stopped, and the mixture was quenched with water under ice-water cooling. The mixture was separated, and the organic layer was dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and column filtered to give approximately 3.9 g of compound 4-7, with a yield of 76%.

[0439] Ms(ESI): m / z 400.1 [M+23] + .

[0440] Step 6:

[0441] Compound 4-7 (2.0 g, 5.3 mmol, 1.0 eq) was added to a 100 mL three-necked flask and dissolved in tetrahydrofuran (25 mL). Under nitrogen protection, the mixture was cooled to about 0 °C, and 1.0 M lithium aluminum hydride tetrahydrofuran solution (8.0 mL, 8.0 mmol, 1.5 eq) was slowly added dropwise. The mixture was stirred at about 0 °C until the reaction was complete. The reaction was stopped, and the mixture was quenched with water (0.8 mL) under ice water cooling. Then, 3.0 M potassium hydroxide aqueous solution (0.8 mL) was added, followed by water (1.6 mL). The mixture was stirred for 15 min, filtered, and the filtrate was dried with anhydrous sodium sulfate. The filtrate was then filtered again and concentrated to obtain about 2.2 g of compound 4-8, which was directly added to the next step.

[0442] Ms(ESI): m / z 372.1[M+23] + .

[0443] Step 7:

[0444] Compound 4-8 (2.2 g, 5.3 mmol, 1.0 eq) was added to a 100 mL three-necked flask and dissolved in ethyl acetate (25 mL). Under nitrogen protection, the mixture was cooled to about 5 °C, and Dess-Martin oxidant (6.7 g, 15.9 mmol, 3.0 eq) was added. The mixture was stirred at about 10 °C until the reaction was complete. The reaction was stopped, the mixture was filtered, the filtrate was concentrated, and the crude product was purified by column chromatography to obtain about 1.5 g of compound 4-9, with a yield of 82%.

[0445] Ms(ESI): m / z 370.1 [M+23] + .

[0446] Step 8:

[0447] Compounds 1-4 (1.2 g, 3.0 mmol, 1.0 eq) and 4-9 (1.1 g, 3.17 mmol, 1.05 eq) were added to a 100 mL three-necked flask, followed by anhydrous magnesium sulfate (1.8 g, 15.0 mmol, 5.0 eq), and then acetonitrile (25 mL). The mixture was stirred and cooled to below 0 °C under nitrogen protection. Trifluoromethanesulfonic acid (2.3 g, 15.0 mmol, 5.0 eq) was added, and the mixture was stirred at approximately 0 °C until the reaction was complete. The reaction was then stopped, and the mixture was filtered. The filtrate was used directly to prepare approximately 1.5 g of compound 4-A, with a yield of 70%.

[0448] Ms(ESI): m / z 606.3[M+1] + .

[0449] 1 H-NMR(400MHz,MeOD)δ7.53(m,4H),7.44(m,2H),7.28(m,3H),6.23(dd,1H),5.99(t,1H),5.52(s,1H),5.08(d,1H),4.63(d,1H),4. 37(m,2H),2.65(td,1H),2.36(d,1H),2.25(m,1H),2.13(m,1H),1.95(dd,1H),1.80(m,4H),1.49(s,3H),1.11(dt,1H),1.10(m,4H).

[0450] Example 5

[0451]

[0452] Compound 5-1 (85.3 mg, 0.262 mmol, 1.1 eq), compound 5-2 (94 mg, 0.238 mmol, 1.0 eq, Maclean / C10492138 / P>98%), anhydrous MgSO4 (143 mg, 1.19 mmol, 5.0 eq), and anhydrous CH3CN (4 mL) were added to a 25 mL Schiller flask and stirred at room temperature for 0.7 h under nitrogen protection. The mixture was then cooled to 0 °C in an ice bath, and CF3SO3H (179 mg, 1.19 mmol, 5.0 eq) was slowly added dropwise. After the addition was complete, the mixture was allowed to warm naturally until the reaction was complete. The reaction was quenched in an ice bath with EA and saturated sodium bicarbonate solution, and then the aqueous phase was extracted with EA. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. Prep-HPLC yielded compound 5-A (67.1 mg, yield: 42.6%).

[0453] Ms(ESI): m / z 602.3[M+H] + .

[0454] 1 H NMR (400MHz, DMSO) δ7.71(d,J=8.1Hz,2H),7.59(d,J=8.2Hz,2H),7.29(d,J=10.2Hz,1H),7.16(t,J=7.7Hz,1H),6.92(s, 1H),6.87–6.76(m,2H),6.22(d,J=10.2Hz,1H),6.02(s,1H),5.59(s,1H),5.47(d,J=2.9Hz,1H),5.39(s,2H),5.13(t,J=5 .9Hz,1H),5.00(d,J=4.3Hz,1H),4.57(dd,J=19.6,6.5Hz,1H),4.30-4.15(m,2H),2.71-2.56(m,2H),2.38-2.31(m,1H), 2.20-2.12(m,1H),2.09-2.01(m,1H),1.90-1.80(m,1H),1.78-1.64(m,3H),1.50(s,3H),1.45-1.34(m,1H),0.89(s,3H).

[0455] Example 6

[0456]

[0457] first step

[0458] Under nitrogen protection, 2 mL of toluene was added to compound 3-A (90.0 mg, 0.145 mmol, 1.0 eq), followed by the addition of BOC anhydride (63.3 mg, 0.290 mmol, 2.0 eq). The system was heated to 100 °C and reacted until complete. The mixture was then concentrated to dryness under reduced pressure and subjected to column chromatography to obtain 48.0 mg of compound 3-A-1.

[0459] MS-ESI: m / z 742.3 [M+Na] + .

[0460] Step 2

[0461] Under nitrogen protection, compound 3-A-1 (45.0 mg, 0.063 mmol) and tetrazolium (66.0 mg, 0.945 mmol) were dissolved in N,N-dimethylacetamide (1.5 mL), and N,N-diethylphosphamide di-tert-butyl ester (187.0 mg, 0.756 mmol) was added. The reaction was carried out at room temperature for 2 hours. After cooling to 0 °C, H₂O₂ (50.0 mg, 0.82 mmol) was slowly added. After the addition was complete, the mixture was stirred at room temperature until the reaction was complete. 2 mL of water was added, the mixture was filtered, and the filter cake was dried to obtain approximately 40.0 mg of compound 3-A-2, which was directly added to the next reaction step.

[0462] MS-ESI: m / z 943.3 [M+Na] + .

[0463] Step 3

[0464] Under nitrogen protection, compound 3-A-2 (40.0 mg, 0.043 mmol) was dissolved in dichloromethane (1.0 mL), cooled to 0 °C, and trifluoroacetic acid (0.3 mL) was added. The reaction was carried out at room temperature for 3 hours. After concentration, the mixture was separated by preparative HPLC (CH3CN / H2O, +0.1% trifluoroacetic acid) to give approximately 12.0 mg of compound 3-B.

[0465] ESI: m / z 700.3 [M+H] + .

[0466] Example 7

[0467]

[0468] first step

[0469] Under nitrogen protection, compound 4-A (45.0 mg, 0.074 mmol, 1.0 eq) was added to 2 mL of toluene, followed by BOC anhydride (32.0 mg, 0.158 mmol, 2.0 eq). The system was heated to 100 °C and reacted until complete. The mixture was then concentrated under reduced pressure and subjected to column chromatography to obtain 45.2 mg of compound 4-A-1.

[0470] MS-ESI: m / z 728.2 [M+Na] + .

[0471] Step 2

[0472] Under nitrogen protection, compound 4-A-1 (45.0 mg, 0.063 mmol) and tetrazolium (66.0 mg, 0.945 mmol) were dissolved in N,N-dimethylacetamide (1.0 mL), and N,N-diethylphosphite di-tert-butyl ester (249.0 mg, 0.756 mmol) was added. The reaction was carried out at room temperature for 2 hours. After cooling to 0 °C, H₂O₂ (50.0 mg, 0.82 mmol) was slowly added. After the addition was complete, the mixture was stirred at room temperature until the reaction was complete. 2 mL of water was added, the mixture was filtered, and the filter cake was dried to obtain approximately 43.0 mg of compound 4-A-2, which was directly added to the next reaction step.

[0473] MS-ESI: m / z 920.3 [M+Na] + .

[0474] Step 3

[0475] Under nitrogen protection, compound 4-A-2 (40.0 mg, 0.042 mmol) was dissolved in dichloromethane (1.0 mL), cooled to 0 °C, and trifluoroacetic acid (0.3 mL) was added. The reaction was carried out at room temperature for 3 hours. After concentration, the product was separated by preparative HPLC (CH3CN / H2O, +0.1% trifluoroacetic acid) to give approximately 15.0 mg of compound 4-B.

[0476] ESI: m / z 686.2 [M+H] + .

[0477] Example 8

[0478]

[0479] first step

[0480] Under nitrogen protection, compounds 3-A (2.60 g, 4.20 mmol) and 3-6 (2.03 g, 4.20 mmol) were dissolved in N,N-dimethylacetamide (30 mL), and triethylamine (1.27 g, 12.60 mmol) was added. The mixture was cooled to 0 °C, and T3P (5.35 g, 8.40 mmol, 50% in DMF) was slowly added. The reaction was allowed to proceed to completion at room temperature. The reaction solution was directly purified by preparative HPLC to give 965 mg of product 3-7, yield: 21.2%.

[0481] MS-ESI: m / z 1106.5 [M+Na] + .

[0482] Step 2

[0483] Under nitrogen protection, compounds 3-7 (805 mg, 0.778 mmol) and tetrazolium (818 mg, 11.670 mmol) were dissolved in N,N-dimethylacetamide (10 mL), and N,N-diethylphosphite di-tert-butyl ester (2.6 mL, 9.336 mmol) was added. The reaction was carried out at room temperature for 2 hours. After cooling to 0 °C, H₂O₂ (437 μL, 4.279 mmol, 30% in water) was slowly added, and the mixture was stirred at room temperature for 1 hour after the addition was complete. The mixture was concentrated and separated by column chromatography (CH₃CN / H₂O) to give 692 mg of compounds 3-8, with a yield of 73.1%.

[0484] Step 3

[0485] Under nitrogen protection, compounds 3-8 (830 mg, 0.650 mmol) were dissolved in acetonitrile (20 mL), and piperidine (302 μL, 3.250 mmol) was added. The reaction was carried out at room temperature. After concentration, the mixture was stirred with 15 mL of petroleum ether. This process was repeated three times to obtain a total of 645 mg of compounds 3-9, which was directly used in the next reaction step.

[0486] MS-ESI: m / z 1054.5 [M+H] + .

[0487] Step 4

[0488] Under nitrogen protection, 2-bromoacetic acid (170 mg, 1.224 mmol) and EEDQ (305 mg, 1.224 mmol) were dissolved in N,N-dimethylacetamide (5 mL), and the reaction was carried out at room temperature for 1 hour. A solution of compound 3-9 (645 mg, 0.612 mmol) in N,N-dimethylacetamide (5 mL) was added, and the reaction was carried out at room temperature. The reaction solution was diluted with dichloromethane (200 mL), washed successively with 1 M hydrobromic acid aqueous solution, saturated sodium bicarbonate solution, and saturated brine. After concentrating the organic phase, 830 mg of compound 3-10 was obtained and used directly in the next reaction step.

[0489] MS-ESI: m / z 1196.4 [M+Na] + .

[0490] Step 5

[0491] Under nitrogen protection, compound 3-10 (830 mg, 0.706 mmol) was dissolved in dichloromethane (8 mL), cooled to 0 °C, and trifluoroacetic acid (4 mL) was added. The reaction was carried out at room temperature. After concentration, the mixture was separated by preparative HPLC (CH3CN / H2O, +0.1% trifluoroacetic acid) to give 220 mg of compound 3-B00. The three-step yield was 33.6%.

[0492] MS-ESI: m / z 1028.2 [M+Na] + .

[0493] 1 H NMR (400MHz, DMSO) δ9.98 (s, 1H), 8.55 (t, J = 5.4Hz, 1H), 8.36 (d, J = 7.4Hz, 1H), 7.97–7.81 (m, 2H), 7.55–7.46 (m,2H),7.45–7.38(m,2H),7.36–7.26(m,3H),7.23–7.16(m,2H),6.14(dd,J=10.0,1.1Hz,1H),5.90(s,1H), 5.44(s,1H),4.97–4.79(m,3H),4.65–4.50(m,2H),4.42(s,2H),4.27(brs,1H),3.94(s,2H),3.86(d,J=5.5H z,2H),2.41–2.22(m,4H),2.17–1.88(m,6H),1.81–1.56(m,6H),1.37(s,3H),1.06–0.92(m,2H),0.85(s,3H).

[0494] Example 9

[0495]

[0496] first step

[0497] Compound 4-A (0.329 g, 0.544 mmol, 1.05 eq) was added to a 25 mL three-necked flask, followed by compound 4-10 (0.25 g, 0.52 mmol, 1.0 eq), triethylamine (0.25 g, 1.56 mmol, 3.0 eq), and DMF (2 mL). After the addition was complete, the mixture was cooled in an ice bath for 5–10 min until the internal temperature reached -5 °C. Then, T3P (50% DMF) (0.9 mL, 1.82 mmol, 3.5 eq) was slowly added. After the addition was complete, the mixture was stirred under natural heating until the reaction was complete. The reaction solution was directly purified by pre-HPLC to obtain compound 4-11 (223 mg, yield 40%).

[0498] Ms(ESI): m / z 1092.4 [M+Na] + .

[0499] Step 2

[0500] Compound 4-11 (0.22 g, 0.206 mmol, 1.0 eq) was added to a 50 mL three-necked flask, followed by the starting materials tetrazolium (0.20 g, 2.87 mmol, 14.0 eq), N,N-diethylphosphamide di-tert-butyl ester (0.616 g, 2.47 mmol, 12.0 eq), and DMF (2.6 mL). After the addition was complete, the mixture was reacted at room temperature for 2 hours. The temperature was then lowered to 0 °C in an ice bath, and H2O2 (30%) (0.13 g, 0.57 mmol, 5.5 eq) was slowly added. The mixture was stirred at room temperature until the reaction was complete. The reaction solution was directly purified by pre-HPLC to obtain compound 4-12 (184.1 mg, yield 70.8%).

[0501] Ms(ESI): m / z 1284.6[M+Na] + .

[0502] Step 3

[0503] Compound 4-12 (0.285 g, 0.233 mmol, 1.0 eq) was added to a 25 mL single-necked flask, followed by piperidine (0.17 g, 1.96 mmol, 8.5 eq) and acetonitrile (5 mL). After the addition was complete, the mixture was stirred at room temperature. The mixture was concentrated under reduced pressure, and 5 mL of petroleum ether was added to form a slurry. The mixture was stirred at room temperature and then filtered. The filter cake was washed twice with 2 mL of petroleum ether to give compound 4-13 (209 mg, 91% yield).

[0504] Ms(ESI): m / z 1004.4[M+H] + .

[0505] Step 4

[0506] Add 0.074 g, 0.523 mmol, 2.6 eq of 2-bromoacetic acid to a 25 mL single-necked flask, followed by 0.13 g, 0.523 mmol, 2.6 eq of EEDQ and 1 mL of DMF. After addition, stir at room temperature for 1 hour. Add a 0.5 mL solution of DMF containing 0.21 g, 0.201 mmol, 1.0 eq of compound 4-13. Stir at room temperature until the reaction is complete. Dilute with 40 mL of dichloromethane, wash with 1 M HBr (10 mL x 2), wash with 20 mL x 2 of saturated sodium bicarbonate, and finally wash with saturated brine. Dry the organic phase with anhydrous sodium sulfate, filter, and concentrate under reduced pressure using an oil pump to obtain 230 mg of crude compound 4-14.

[0507] Ms(ESI): m / z 1182.4[M+Na] + .

[0508] Step 5

[0509] Compound 4-14 (0.240 g, 0.201 mmol, 1.0 eq) and DCM (2 mL) were added to a 25 mL single-necked flask. After the addition was complete, the temperature was lowered to 0 °C in an ice bath, and then trifluoroacetic acid (1 mL) was slowly added. After the addition was complete, the mixture was stirred at room temperature until the reaction was complete. The reaction solution was concentrated under reduced pressure in an ice bath and then purified by pre-HPLC. The solution was lyophilized to obtain 78 mg of compound 4-B00, with a yield of 39.2%.

[0510] Ms(ESI): m / z 1014.2[M+Na] + .

[0511] 1 H NMR (400MHz, DMSO) δ10.15(s,1H),8.53(t,J=5.2Hz,1H),8.29(br,d,J=7.5Hz,1H),7.83(s,1H),7.73(d,J=7.8Hz,1H),7.68–7.58 (m,2H),7.57–7.47(m,2H),7.43(t,J=7.9Hz,1H),7.32(d,J=9.9Hz,1H),7.20(d,J=8.2Hz,1H),6.17(d,J=10.1Hz,1H),5.92(s,1H ),5.58(s,1H),4.95–4.80(m,3H),4.58(br,dd,J=18.4,8.2Hz,1H),4.40–4.37(m,1H),4.33–4.27(m,1H),3.93(s,2H),3.82–3.78 (m,2H),2.30–2.25(m,3H),2.16–2.07(m,1H),2.10–1.90(m,2H),1.88–1.59(m,6H),1.39(s,3H),1.18–1.05(m,2H),0.89(s,3H).

[0512] Example 10

[0513]

[0514] first step

[0515] Compound 4-A (0.260 g, 0.430 mmol, 1.05 eq), compound 4-C-1 (0.193 g, 0.551 mmol, 1.32 eq), triethylamine (0.125 g, 1.23 mmol, 3.0 eq), and DMF (2 mL) were added to a 25 mL three-necked flask. After the addition was complete, the flask was cooled to -5 °C in an ice bath. Then, T3P (50% DMF) (0.5 mL, 1.024 mmol, 2.5 eq) was slowly added. The mixture was stirred until the reaction was complete under natural heating conditions. The reaction solution was directly purified by pre-HPLC (TFA (0.05%) water-acetonitrile) to give compound 4-C-2 (140 mg, yield 35.1%).

[0516] Ms(ESI): m / z 949.3[M+Na] + .

[0517] Step 2

[0518] Compound 4-C-2 (0.34 g, 0.366 mmol, 1.0 eq) was added to a 50 mL three-necked flask, followed by the starting materials tetrazolium (0.360 g, 5.13 mmol, 14.0 eq), N,N-diethylphosphamide di-tert-butyl ester (1.099 g, 4.40 mmol, 12.0 eq), and DMF (5 mL). After the addition was complete, the mixture was reacted at room temperature for 2 hours. The temperature was then lowered to 0 °C in an ice bath, and H2O2 (30%) (0.234 g, 2.02 mmol, 5.5 eq) was slowly added. The mixture was stirred at room temperature until the reaction was complete. The reaction solution was then purified directly using a medium-pressure Boston reversed-phase column to give compound 4-C-3 (304 mg, yield 64.1%).

[0519] Ms(ESI): m / z 1141.3[M+Na] + .

[0520] Step 3

[0521] Compound 4-C-3 (0.304 g, 0.272 mmol, 1.0 eq) was added to a 25 mL single-necked flask, followed by piperidine (0.255 g, 1.63 mmol, 9.0 eq) and acetonitrile (8 mL). After the addition was complete, the mixture was stirred at room temperature until the reaction was complete. The mixture was concentrated under reduced pressure, and 4 mL of petroleum ether was added to form a slurry. The mixture was stirred at 35 °C for 2 hours, then filtered. The filter cake was washed twice with 2 mL of petroleum ether to give compound 4-C-4 (243 mg, 99% yield).

[0522] Ms(ESI): m / z 897.6 [M+H] + .

[0523] Step 4

[0524] Add 0.112 g, 0.813 mmol, 3.0 eq of 2-bromoacetic acid to a 25 mL single-necked flask, followed by 0.201 g, 0.813 mmol, 3.0 eq of EEDQ and 2 mL of DMF. After addition, stir at room temperature for 0.6 hours. Add a 1 mL solution of DMF containing 0.243 g, 0.271 mmol, 1.0 eq of compound 4-C-4. Stir at room temperature until the reaction is complete. Dilute with 50 mL of dichloromethane, wash with 1 M HBr (15 mL x 2), wash with 15 mL x 2 of saturated sodium bicarbonate, and finally wash with saturated brine. Dry the organic phase with anhydrous sodium sulfate, filter, evaporate to dryness under reduced pressure using an oil pump, and purge with nitrogen for 10 minutes to obtain 275 mg of crude compound 4-C-5.

[0525] Ms(ESI): m / z 1039.1 [M+Na] + and 1041.1[M+Na+2] + .

[0526] Step 5

[0527] In a 25 mL single-necked flask, 0.275 g (0.271 mmol, 1.0 eq) of 4-C-5 and 2.5 mL of DCM were added. After the addition was complete, the mixture was cooled to 0 °C in an ice bath. Then, 1 mL of trifluoroacetic acid was slowly added. After the addition was complete, the mixture was stirred at room temperature until the reaction was complete. The reaction solution was concentrated under reduced pressure in an ice bath and then purified by pre-HPLC. The solution was lyophilized to obtain 120 mg of compound 4-C00, with a yield of 48.3%.

[0528] Ms(ESI): m / z 905.2[M+H] + .

[0529] Example 11

[0530]

[0531] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (2.5 mM, 356.8 μL, 891.24 nmol) was added to adalimumab A buffer (0.05 M buffer aqueous solution, pH = 6.3; 10.0 mg / mL, 6.0 mL, 405.11 nmol). The mixture was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0532] Add 840 μL of 1.0 M Tris buffer to the above reaction solution, then dissolve compound 3-B00 (4.08 mg, 4051.10 nmol) in 300 μL of DMSO and add it to the above reaction solution. Place the mixture in a water bath and shake at 25 °C for 3 hours, then stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: buffer A), and concentrate it using an ultrafiltration tube to obtain buffer A of the antibody-drug conjugate Humira-3-B00 (2.55 mg / mL, 23.5 mL), which is then stored frozen at 4 °C.

[0533] Example 12

[0534]

[0535] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (2.5 mM, 405.4 μL, 1012.77 nmol) was added to adalimumab A buffer (0.05 M buffer aqueous solution, pH = 6.3; 10.0 mg / mL, 6.0 mL, 405.11 nmol). The mixture was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0536] Add 1.0 M Tris buffer (840 μL) to the above reaction solution. Then dissolve compound 4-B00 (4.02 mg, 4051.10 nmol) in 300 μL DMSO and add it to the above reaction solution. Place the mixture in a water bath and shake at 25 °C for 3 hours. Stop the reaction. Desalt and purify the reaction solution using a Sephadex G25 gel column (elution phase: buffer A), and concentrate it using an ultrafiltration tube to obtain buffer A (2.58 mg / mL, 23.25 mL) of the title product antibody-drug conjugate Humira-4-B00. Store frozen at 4 °C.

[0537] Biological evaluation

[0538] The present disclosure is further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present disclosure.

[0539] Test Example 1: In vitro activity of small molecule steroids

[0540] 1. Sample to be tested

[0541] Compounds 1-A to 5-A, and compound AA (prepared according to Example 2 of WO2017210471).

[0542]

[0543] 2. Glucocorticoid receptor binding assay

[0544] The binding activity of small molecule steroids to glucocorticoid receptors (GR) was tested using a human glucocorticoid NHR binding (radiolabeled agonist) assay (#232020, Eurofins). The experiment involved incubating different concentrations of small molecule steroids and 5 nM [3H]dexamethasone with recombinant human GR at 4°C for 24 hours. The small molecule steroids competitively bound to [3H]dexamethasone in human GR. The binding activity of the small molecule steroids to GR was calculated by counting the [3H]dexamethasone that specifically bound to the receptor. Results are detailed in Table 1.

[0545] 3. Mineralocorticoid receptor agonist activity assay

[0546] use The NHR nuclear translocation assay was used to test the mineralocorticoid receptor (MR) agonist activity of small molecule steroids. PathHunter NHR CHO-K1 cells were plated in 384-well white-walled microplates, and the target small molecule steroids were added and co-incubated at 37°C and 5% CO2 to induce the reaction. A PathHunter assay reagent mixture was used to generate the assay signal, and the chemiluminescence signal was detected after one hour of incubation at room temperature. Four-parameter curve fitting was used to analyze the data to generate EC. 50 Values. See Table 1 for details.

[0547] 4. GRE reporter gene experiment

[0548] A549 cells were plated (40,000 cells / 96-well plate), and pGL4.36 (MMTV-Luc, 100 ng / well) were transfected with lipo3000. After overnight culture, the cells were incubated with different concentrations of small molecule steroids. After 24 hours, the expression of luciferase in the reporter gene system was detected using Bright-Glo (promega) assay reagent.

[0549] Table 1. In vitro activity of small molecule steroids

[0550]

[0551] Test Example 2: Anti-inflammatory activity of small molecule steroids in the experiment of lipopolysaccharide (LPS)-stimulated cytokine secretion by human PBMCs.

[0552] 1. Sample to be tested

[0553] Compounds 1-A to 5-A, compound AA.

[0554] 2. Testing Methods

[0555] Primary human peripheral blood mononuclear cells (PBMCs) were resuspended in RPMI (2% FBS, 1% penicillin-streptomycin) and plated in 96-well plates. PBMCs were co-incubated with different concentrations of small molecule steroids at 37°C and 5% CO2 for 4 hours, followed by stimulation overnight with 0.01 ng / mL LPS. The supernatant was collected the next day, and the concentration of IL-6 was detected using alpha LISA (Cisbio).

[0556] 3. Test Results

[0557] Test results are as follows Figure 1 As shown, the small molecule steroid compounds described in this disclosure can significantly inhibit LPS-induced IL-6 release.

[0558] Test Example 3: Activity of anti-TNF-ADC in the membrane-bound TNFα-mediated GRE reporter gene system

[0559] 1. Sample to be tested

[0560] Humira-3-B00, Humira-4-B00, Humira-A-B00 (prepared according to Example 7 of WO2019106609).

[0561]

[0562] 2. Testing Methods

[0563] A stable reporter gene cell line transfected with MMLV-Luc was established in HeLa cells using lentivirus. The stably transfected HeLa-MMLV-Luc cell line was plated in 96-well plates (30,000 cells / well), and each well was transfected with either an empty vector plasmid or a human TNFα mutant plasmid (TNFαΔ12, with the TACE restriction site removed, 50 ng / well) using lipo3000. After overnight incubation, the cells were incubated with different concentrations of anti-TNF-ADC. After 24 hours, the expression of the luciferase in the reporter gene system was detected using a Bright-Glo (promega) assay.

[0564] 3. Test Results

[0565] The test results are shown in Table 2. Figure 2 As shown.

[0566] Table 2. Activity of anti-TNF-ADCs in the membrane-bound TNFα-mediated GRE reporter gene system.

[0567]

[0568]

[0569] Test Example 4: Activity of Anti-TNF-ADC in the Experiment of Cytokine Secretion by Lipopolysaccharide (LPS)-stimulated Human Monocytes

[0570] Use EsaySep TM Use the human CD14 sorting kit to sort and enrich monocytes from cryopreserved primary human peripheral blood PBMCs and plate them in 96-well plates. After co-incubating the monocytes with different concentrations of anti-TNF-ADC at 37°C and 5% CO2 for 4 hours, stimulate them overnight with 0.01 ng / mL LPS. Collect the culture medium supernatant the next day and detect the concentration of IL-6 using alpha LISA (cisbio).

[0571] Compared with adalimumab (Humira), the ADC of the present disclosure effectively inhibits LPS-induced IL-6 secretion at high concentrations (4 - 100 nM) (as Figure 3 shown). Additionally, the anti-inflammatory activities of Humira-3-B00 and Humira-4-B00 at 4 nM are significantly stronger than those of the control ADC molecule Humira-A-B00 at this concentration (as Figure 4 shown).

[0572] Test Example 5: Mouse Collagen Antibody-induced Arthritis (CAIA) Model

[0573] Test animals:

[0574] Male balb / c mice, 6 weeks old, purchased from the Experimental Animal Business Department of Shanghai Institute of Planned Parenthood Research. Breeding environment: SPF; Production License: SCXK (Shanghai) 2018 - 0006; Certificate number of Balb / c mice: 20180006023393.

[0575] Test samples:

[0576] Humira-3-B00, Humira-4-B00, Humira-A-B00.

[0577] Test method:

[0578] After the test animals arrive, adaptively raise them for 7 days and randomly group them. On day 0, except for the control group, inject each mouse intraperitoneally with a type II collagen antibody mixture (purchased from Chondrex) at 1.5 mg / mouse to establish the model. On day 3, except for the control group, inject each mouse intraperitoneally with 50 μg / mouse (100 μL) of LPS to enhance the immune response. Starting from day 5, administer the test samples to each group of mice, and at the same time, perform limb arthritis scoring every 1 - 3 days. The specific experimental procedure is as Figure 5As shown below. Mice were administered through the following dosing regimens, and the severity of arthritis in each group of mice was semi-quantitatively scored every 1 - 3 days to evaluate the anti-inflammatory activity of anti-TNF-ADC.

[0579] Table 3: Dosing Regimens

[0580]

[0581]

[0582] Test Results:

[0583] Humira has weak anti-inflammatory activity in the CAIA model. Humira-4-B00 has a rapid onset (starting from day 5) and can persistently reduce arthritis inflammation. The control ADC molecule Humira-A-B00 only shows a remission effect on arthritis in the late stage of the arthritis process (starting from day 11) (as Figure 6 shown). From day 8 to day 14 after arthritis induction, Humira-4-B00 significantly reduced the arthritis score in mice compared to the model group (*, p < 0.05), while the anti-inflammatory activity of the control ADC molecule Humira-A-B00 did not reach a significant difference compared to the model group (as Figure 7 shown).

[0584] Test Example 6: Delayed-Type Hypersensitivity (DTH) Model

[0585] Test Animals:

[0586] Male ICR mice, 6 weeks old, purchased from the Experimental Animal Business Department of Shanghai Institute of Planned Parenthood Research. Breeding environment: SPF; Production License: SCXK (Shanghai) 2018 - 0006, Certificate Number: 20180006023622.

[0587] Test Samples:

[0588] Humira-3-B00, Humira-4-B00, Humira-A-B00, Humira-A-A00 (prepared according to Example 2 of WO2017210471 and Example 7 of WO2019106609).

[0589]

[0590] Test Methods:

[0591] Animals were acclimatized for 7 days after arrival and then randomly divided into groups. On day 0, mice were immunized by applying 50 μL of 1% DNFB (2,4-dinitrofluorobenzene) solution to the shaved abdomen. On day 5, mice were challenged by applying 10 μL of 0.5% DNFB solution (total 20 μL) to the inner and outer sides of the right ear. On day 6 (24 hours later), mice were sacrificed, and 8 mm diameter ear flaps were removed from both sides using a punch and weighed. Mice in each group were treated with the drug on days 0 and 4, respectively. The specific experimental procedure is as follows: Figure 8 As shown. Mice were administered the drug according to the following regimen, and the weight of the control and model ear flaps was measured (to indicate the degree of swelling) to evaluate the anti-inflammatory activity of the anti-TNF-ADC.

[0592] Table 4: Dosing Regimen

[0593]

[0594]

[0595] Test results:

[0596] There was no difference in the weight of the untreated left ear among the groups of mice. Humira-4-B00 and the positive control Humira-A-B00 showed comparable anti-inflammatory activity in this model, while Humira-3-B00 exhibited slightly weaker anti-inflammatory activity, but was still significantly stronger than Humira monoclonal antibodies (e.g., Humira-4-B00). Figure 9 (As shown).

[0597] Test Example 7: Stability of ADC Samples in Plasma

[0598] Sample to be tested:

[0599] Humira-3-B00, Humira-4-B00, Humira-A-B00, Humira-A-A00.

[0600] Test plasma:

[0601] Humans, cynomolgus monkeys, rats, mice, and 1% BSA

[0602] Test method:

[0603] The test molecule was prepared into a 1 mg / mL solution with PBS and sterilized by filtration through a 0.22 μm filter membrane. 15 μL of the 1 mg / mL sample was added to 135 μL of the reaction matrix to achieve a final concentration of 100 μg / mL. The samples were incubated at 37°C in the dark for 0, 7, 14, and 21 days, and free toxins were detected.

[0604] The data is shown in Table 5.

[0605] Table 5: Plasma stability data

[0606]

[0607]

[0608] At a concentration of 100 μg / mL, Humira-3-B00 and Humira-4-B00 showed excellent stability in human, cynomolgus monkey, mouse, and rat plasma as well as in 1% BSA, with free toxin levels below the detection limit. Humira-A-A00 released a small amount of free toxin in rat plasma, and Humira-A-B00 released a small amount of free toxin in both rat and mouse plasma.

Claims

1. An antibody-drug conjugate selected from the group consisting of k is 2 to 5, and Ab is an anti-TNFa antibody or an antigen-binding fragment thereof.

2. The antibody-drug conjugate of claim 1, wherein the anti-TNFa antibody or an antigen-binding fragment thereof is selected from the group consisting of adalimumab, infliximab, certolizumab pegol, afelimomab, natalizumab, olaratumab, placulumab, golimumab, or an antigen-binding fragment thereof.

3. The antibody-drug conjugate of claim 1, wherein the anti-TNFa antibody or an antigen-binding fragment thereof is selected from the group consisting of adalimumab.

4. A compound of the formula:

5. A compound of the formula: wherein X is halogen.

6. The compound of claim 5, or a pharmaceutically acceptable salt thereof, wherein X is chloro or bromo.

7. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-3 and a pharmaceutically acceptable excipient.

8. The antibody-drug conjugate of any one of claims 1-3 or the use of the pharmaceutical composition of claim 7 for the manufacture of a medicament for the treatment of an immune disease selected from the group consisting of rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis.

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