WO2022166779A1 - 糖皮质激素受体激动剂的药物偶联物及其在医药上的应用 - Google Patents

糖皮质激素受体激动剂的药物偶联物及其在医药上的应用 Download PDF

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WO2022166779A1
WO2022166779A1 PCT/CN2022/074455 CN2022074455W WO2022166779A1 WO 2022166779 A1 WO2022166779 A1 WO 2022166779A1 CN 2022074455 W CN2022074455 W CN 2022074455W WO 2022166779 A1 WO2022166779 A1 WO 2022166779A1
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alkyl
hydrogen
substituents
alkoxy
group
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PCT/CN2022/074455
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English (en)
French (fr)
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祝令建
洪敏�
唐满平
粟璐
邓梦蝶
张敬杨
任文明
林侃
黄建
廖成
张连山
Original Assignee
上海森辉医药有限公司
上海盛迪医药有限公司
江苏恒瑞医药股份有限公司
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Priority to US18/264,072 priority Critical patent/US20240189438A1/en
Priority to JP2023547354A priority patent/JP2024506012A/ja
Priority to CN202280009295.1A priority patent/CN116761820A/zh
Priority to CA3207416A priority patent/CA3207416A1/en
Priority to EP22749058.8A priority patent/EP4289857A1/en
Publication of WO2022166779A1 publication Critical patent/WO2022166779A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6845—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a cytokine, e.g. growth factors, VEGF, TNF, a lymphokine or an interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring
    • C07J71/0005—Oxygen-containing hetero ring
    • C07J71/0026—Oxygen-containing hetero ring cyclic ketals
    • C07J71/0031—Oxygen-containing hetero ring cyclic ketals at positions 16, 17
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present disclosure belongs to the field of medicine, and in particular relates to a drug conjugate of a glucocorticoid receptor agonist and its application in medicine.
  • RA Rheumatoid arthritis
  • TNF ⁇ tumor necrosis factor- ⁇
  • IL-1 interleukins
  • IL-6 interleukins
  • IL-8 interleukins
  • TNF ⁇ inhibitors that have been approved by the US FDA include: soluble receptor antagonist-etanercept (Etanercept), human-mouse chimeric antibody-infliximab (Infliximab), fully human monoclonal antibody adalimumab (Adalimumab, ), fully human monoclonal antibody Golimumab (Golimumab) and polyethylene glycol humanized Fab' fragment Certolizumab pegol.
  • Etanercept soluble receptor antagonist-etanercept
  • Infliximab human-mouse chimeric antibody-infliximab
  • Adalimumab fully human monoclonal antibody adalimumab
  • Golimumab fully human monoclonal antibody Golimumab
  • polyethylene glycol humanized Fab' fragment Certolizumab pegol polyethylene glycol humanized Fab' fragment Certolizumab pegol.
  • TNF ⁇ inhibitors are still limited by the maximum efficacy they can achieve in patients, requiring the identification and development of more potent and effective therapeutics. Patients treated with TNF ⁇ inhibitors may also develop immunogenic responses to the therapeutic agent, limiting its effectiveness.
  • Glucocorticoid receptor agonists are also more effective drugs in the treatment of rheumatoid arthritis.
  • glucocorticoid receptor agonists produced in vivo such as cortisol and corticosterone
  • synthetic glucocorticoids such as dexamethasone, prednisone, and prednisolone are known Hormone receptor agonists.
  • These glucocorticoid receptor agonists are collectively referred to as steroids because of their steroid structure, and are used in the treatment of various diseases. However, these steroids sometimes exhibit side effects such as steroid peptic ulcer, steroid purpura, steroid pancreatitis, steroid diabetes, steroid cataract, and steroid glaucoma due to their use.
  • ADC Antibody drug conjugate
  • ADC refers to a monoclonal antibody or antibody fragment linked to a biologically active drug through a stable chemical linker compound.
  • Most ADCs in preclinical and clinical development are used in oncology indications where cytotoxic payloads target antigen-expressing cancer cells.
  • modulation of pathogenic cell activity through ADC-mediated delivery of bioactive small molecules is also attractive for non-oncological indications, leading to widespread use of this technology.
  • glucocorticoid receptor agonists such as WO2017210471, WO2019106609 and so on.
  • ADC antibody-drug conjugate
  • Ab is an antibody or an antigen-binding fragment thereof
  • L is a linker covalently linking Ab to D
  • 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 number in between)
  • R 1a are each independently selected from hydrogen, alkyl and alkoxy, each independently optionally selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro substituted with one or more substituents in hydroxy, hydroxy and hydroxyalkyl;
  • Ring A is an aryl or heteroaryl group optionally substituted with one or more substituents Q 1 ;
  • Ring B is an aryl or heteroaryl optionally substituted with one or more substituents Q;
  • X 1 is -(CR 5a R 5b )m- and an aryl or heteroaryl group optionally substituted with one or more substituents Q 1 ;
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano and the following groups optionally substituted with one or more substituents Q : 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) ORk , -C(S) Rk , alkoxy, alkylthio, alkenyl and alkynyl, or R 5a and R 5b together form oxo or thio;
  • Ring C and Ring D are each independently selected from aryl and heteroaryl groups optionally substituted by one or more substituents Q 1 , and at least one of Ring C and Ring D is selected from optionally substituted by one or more The fused ring aryl or fused heteroaryl substituted by the substituent Q 1 ;
  • R 6a and R 6b are each independently selected from hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano or the following groups optionally substituted by one or more substituents Q : 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 6a , R 6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkane group, or R 6a and R 6b together form oxo or thio;
  • R 6c , R 6d , R 6e , R 6f and R 6g are each independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and C 1 -C 6 alkoxy;
  • R 1 are each independently selected from hydrogen, alkyl and alkoxy, wherein said alkyl and alkoxy are each independently optionally selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, substituted with one or more substituents of nitro, hydroxy and hydroxyalkyl;
  • R2 is each independently selected from -CH2OH , -CH2SH , -CH2Cl , -SCH2Cl , -SCH2F , -SCH2CF3 , -OH , -OCH2CN , -OCH2Cl , -OCH 2 F, -OCH 3 , -OCH 2 CH 3 , -SCH 2 CN,
  • R 2a is each independently hydrogen or C 1 -C 6 alkyl
  • R 2b is each independently C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • R 2c are each independently selected from hydrogen, C 1 -C 6 alkyl, -CH 2 OH and C 1 -C 6 alkoxy;
  • R 2d and R 2e are each independently hydrogen or C 1 -C 6 alkyl
  • R3 is each independently hydrogen or halogen
  • R 4 are each independently selected from hydrogen, halogen and hydroxyl
  • n are each independently an integer from 1 to 6;
  • the substituent groups Q 1 are each independently selected from C 1 -C 6 alkyl, halogen, deuterium, hydroxyl, mercapto, -NR i R j , oxo, thio, -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, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused ring aryl, and 5- to
  • R i and R j are each independently selected from a hydrogen atom, a hydroxyl group, a C 1 -C 6 alkyl group, and a C 1 -C 6 alkoxy group;
  • R k is independently selected from hydrogen atoms, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, hydroxyl and -NR i R j , wherein said alkyl, alkane Oxy and haloalkyl are each independently optionally selected from C 1 -C 6 alkyl, halogen, hydroxy, mercapto, -NR i R j , oxo, thio, carboxyl, nitro, cyano, C 1 - C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, 3 to 10 membered cycloalkyl, 3 to 10 membered heterocyclyl, 6 to 10 membered substituted with one or more substituents in aryl and 5- to 10-membered heteroaryl; and
  • the proviso is that when R5a is hydrogen or alkyl, R5b is not hydrogen or alkyl.
  • each R 1a is independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy, each independently optionally selected from Substituted with one or more substituents of C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halogen, deuterium, amino, cyano and hydroxy.
  • each R 1a is independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy.
  • Ring A is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring A is optionally substituted with one or more substituents Q 1
  • Ring B is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring B is optionally substituted with one or more substituents Q
  • X 1 is -(CR 5a R 5b )m- or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more substituents Q , whereby The heteroaryl group contains at least one nitrogen atom.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, deuterium, nitro, cyano and optionally substituted with one or more substituents Q 1 Group: C 1 -C 6 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 , C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 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 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl , or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k , -S(O)R k , -S(O)(O)R k , C 1 -C 6 alkoxy, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 - C6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 5a and R 5b together form oxo or sulfur generation.
  • Ring C and Ring D are each independently selected from 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered optionally substituted with one or more substituents Q A fused ring aryl group or a 5- to 12-membered fused heteroaryl group containing at least one nitrogen atom.
  • Ring C and Ring D are each independently selected from the following groups optionally substituted with one or more substituents Q:
  • Ring C is selected from optionally substituted with one or more substituents Q:
  • Ring D is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring D is optionally substituted with one or more substituents Q
  • X 2 is selected from -(CR 6a R 6b )n-, -O-, -S-, -NR 6c -, -CH 2 S-, -CH 2 O-, -NHCR 6d R 6e - and a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 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 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl , or R 6a and R 6b together with the carbon atoms to which they are attached form a 3- to 10-membered cycloalkyl, or R 6a and R 6b together form oxo or
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k , -S(O)R k , -S(O)(O)R k , C 1 -C6alkoxy, C2 - C6alkenyl and C2 - C6alkynyl , or R6a , R6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl, or R6a and R 6b together form oxo or thio.
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 - C6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 6a and R 6b together form oxo or sulfur generation.
  • each R 1 is independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 alkoxy, preferably hydrogen.
  • each R4 is independently hydrogen.
  • the substituent groups Q 1 are each independently selected from halogen, hydroxyl, sulfhydryl, deuterium, oxo, thio, cyano, amino, carboxyl, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy.
  • R k is independently selected from hydrogen atoms, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, hydroxy, and -NR i R j .
  • D is represented by the following formula (II-A') or (II-B'):
  • R 1a is each independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 alkoxy;
  • Ring A is The ring A is optionally substituted with one or more substituents Q 1 ;
  • Ring B is The ring B is optionally substituted with one or more substituents Q 1 ;
  • X 1 is -(CR 5a R 5b )m- or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group contains at least one Nitrogen atom;
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 5a and R 5b together form oxo or thio;
  • Ring C is selected from The ring C is optionally substituted by one or more substituents Q;
  • Ring D is The ring D is optionally substituted with one or more substituents Q 1 ;
  • X 2 is selected from -(CR 6a R 6b )n-, -O-, -S-, -NR 6c -, -CH 2 S-, -CH 2 O-, -NHCR 6d R 6e - and optionally by a or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group substituted by multiple substituents Q 1 ;
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6alkoxy, or R 6a and R 6b together form oxo or thio;
  • R 6c , R 6d and R 6e are each independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and C 1 -C 6 alkoxy;
  • R2 is each independently selected from -CH2OH , -CH2SH , -CH2Cl , -SCH2Cl , -SCH2F , -SCH2CF3 , -OH , -OCH2CN , -OCH2Cl , -OCH 2 F, -OCH 3 , -OCH 2 CH 3 , -SCH 2 CN,
  • R 2a is each independently hydrogen or C 1 -C 6 alkyl
  • R 2b is each independently C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • R 2c are each independently selected from hydrogen, C 1 -C 6 alkyl, -CH 2 OH and C 1 -C 6 alkoxy;
  • R 2d and R 2e are each independently hydrogen or C 1 -C 6 alkyl
  • R3 is each independently hydrogen or halogen
  • n are each independently an integer from 1 to 6;
  • the substituent groups Q 1 are each independently selected from halogen, hydroxyl, mercapto, deuterium, oxo, thio, cyano, amino, carboxyl, C 1 -C 6 alkyl and C 1 -C 6 alkoxy;
  • R i and R j are each independently selected from a hydrogen atom, a hydroxyl group, a C 1 -C 6 alkyl group, and a C 1 -C 6 alkoxy group;
  • R k is independently selected from a hydrogen atom, a C 1 -C 6 alkyl group, a C 1 -C 6 haloalkyl group, a C 1 -C 6 alkoxy group, a hydroxyl group, and -NR i R j ;
  • the proviso is that when R5a is hydrogen or alkyl, R5b is not hydrogen or alkyl.
  • R 1a is hydrogen
  • X 1 is selected from -(CR 5a R 5b )m-, optionally substituted with one or more substituents Q 1 :
  • both R 5a and R 5b are fluoro.
  • R 5a and R 5b together form oxo or thio, preferably oxo.
  • X 2 is selected from -(CR 6a R 6b )n-, optionally substituted with one or more substituents Q 1 :
  • R 6a and R 6b are each independently selected from hydrogen, halogen, hydroxy, mercapto, deuterium, cyano, C 1 -C 6 alkyl, -NR i R j , -C(O)OR k and C 1 -C 6 alkoxy, or R 6a and R 6b together form oxo or thio.
  • each R 2 is independently selected from -CH 2 OH, -CH 2 SH, -OH and
  • R3 is hydrogen
  • R3 is fluoro
  • k is any value between 1-10, preferably any value between 2-5. k can be an integer or a decimal.
  • the linker is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but is capable of cleavage when internalized in the cell.
  • the glucocorticoid receptor agonist drug moiety is cleaved from the antibody portion, and the glucocorticoid receptor agonist-releasing glucocorticoid receptor agonist is cleaved. Unmodified form.
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • ADCs comprising cleavable peptide moieties exhibit lower levels of aggregation, improved antibody to drug ratios relative to ADCs comprising other cleavable moieties.
  • the addition of a cleavable moiety increases cytotoxicity and/or potency relative to a non-cleavable linker.
  • the cleavable peptide moiety is cleavable by an enzyme
  • the linker is a linker that is cleavable by the enzyme.
  • the enzyme is a cathepsin and the linker is a linker that is cleaved by the cathepsin.
  • an enzyme-cleavable linker eg, a cathepsin-cleavable linker
  • the linker comprises an amino acid unit L 1 preferably comprising from 2 to 7 selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine , glutamic acid, aspartic acid, homolysine, n-methyl-valine, (q is an integer from 1 to 6) peptide residues consisting of amino acids, exemplary amino acid units include, but are 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), gly
  • the linker comprises a stretch unit, which is a chemical structural fragment covalently linked to the antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptide chemical moiety at the other end.
  • stretch units include, but are not limited to
  • the stretching unit is selected from
  • p is each independently 1, 2, 3, 4, 5, or 6.
  • the linker is selected from
  • the linker is selected from
  • the antibody-drug conjugate is selected from:
  • the antibody-drug conjugate is selected from
  • k is selected from 1 to 10 and can be an integer or a decimal.
  • the antibody or antigen-binding fragment thereof in the antibody-drug conjugate (ADC) of the present disclosure is selected from murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies or antigen-binding fragments thereof.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of anti-TNF ⁇ 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 antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof binds to human and/or mouse TNF ⁇ .
  • Antibodies and antigen-binding fragments that bind TNF ⁇ are known in the art.
  • the anti-TNF ⁇ antibody or antigen-binding fragment does not bind to TNF- ⁇ .
  • Anti-TNF ⁇ antibodies and antigen-binding fragments thereof include, for example, adalimumab, infliximab, certolizumab pegol, afelimumab, nerelimomab, ozolacizumab Antigen (ozoralizumab), placulumab (placulumab) and golimumab (golimumab) or antigen-binding fragments thereof. Additional anti-TNFa antibodies and antigen-binding fragments are provided, for example, in WO 2013/087912, WO 2014/152247 and WO 2015/073884, each of which is incorporated herein by reference in its entirety.
  • Anti-TNF ⁇ antibodies and antigen-binding fragments thereof also include competitive inhibition of adalimumab, infliximab, certolizumab, afelimumab, neremumab, ozolacizumab, pram Antibodies and antigen-binding fragments thereof that bind kulumab or golimumab to TNF ⁇ .
  • Anti-TNF ⁇ antibodies and antigen-binding fragments thereof also include combinations with adalimumab, infliximab, certolizumab, afelimumab, neremumab, ozolacizumab, praccuron Antibodies and antigen-binding fragments that bind mAb or golimumab to the same TNF ⁇ epitope.
  • the anti-TNF ⁇ antibody or antigen-binding fragment thereof competitively inhibits the binding of adalimumab to TNF ⁇ . In certain embodiments, the anti-TNF ⁇ antibody or antigen-binding fragment thereof binds the same TNF ⁇ epitope as adalimumab. In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof is adalimumab or an antigen-binding fragment thereof. In certain embodiments, the anti-TNFa antibody or antigen-binding fragment thereof is adalimumab.
  • the anti-TNF ⁇ antibody or antigen-binding fragment thereof comprises adalimumab, infliximab, certolizumab, afelimumab, neremumab, ozolacizumab , praculumab or golimumab sequences, eg, complementarity determining regions (CDRs), variable heavy chain domains (VH) and/or variable light chain domains (VL).
  • CDRs complementarity determining regions
  • VH variable heavy chain domains
  • VL variable light chain domains
  • the present disclosure also provides a compound represented by formula (III-A) or (III-B) or a pharmaceutically acceptable salt thereof,
  • Ring A is an aryl or heteroaryl group optionally substituted with one or more substituents Q 1 ;
  • Ring B is an aryl or heteroaryl optionally substituted with one or more substituents Q;
  • X 1 is -(CR 5a R 5b )m- or an aryl or heteroaryl group optionally substituted by one or more substituents Q 1 ;
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano and the following groups optionally substituted with one or more substituents Q : 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) ORk , -C(S) Rk , alkoxy, alkylthio, alkenyl and alkynyl, or R 5a and R 5b together form oxo or thio;
  • Ring C and Ring D are each independently selected from aryl and heteroaryl or fused heteroaryl optionally substituted by one or more substituents Q, and at least one of Ring C and Ring D is selected from optionally Condensed aryl or condensed heteroaryl substituted by one or more substituents Q 1 ;
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, nitro, cyano and the following groups optionally substituted with one or more substituents Q : 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) ORk , -C(S) Rk , alkoxy, alkylthio, alkenyl and alkynyl, or R6a , R6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkane group, or R 6a and R 6b together form oxo or thio;
  • R 6c , R 6d , R 6e , R 6f and R 6g are each independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and C 1 -C 6 alkoxy;
  • R 1 are each independently selected from hydrogen, alkyl and alkoxy, wherein said alkyl and alkoxy are each independently optionally selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, substituted with one or more substituents of nitro, hydroxy and hydroxyalkyl;
  • R2 is each independently selected from -CH2OH , -CH2SH , -CH2Cl , -SCH2Cl , -SCH2F , -SCH2CF3 , -OH , -OCH2CN , -OCH2Cl , -OCH 2 F, -OCH 3 , -OCH 2 CH 3 , -SCH 2 CN,
  • R 2a is each independently hydrogen or C 1 -C 6 alkyl
  • R 2b is each independently C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • R 2c are each independently selected from hydrogen, C 1 -C 6 alkyl, -CH 2 OH and C 1 -C 6 alkoxy;
  • R 2d and R 2e are each independently hydrogen or C 1 -C 6 alkyl
  • R3 is each independently hydrogen or halogen
  • R 4 are each independently selected from hydrogen, halogen and hydroxyl
  • n are each independently selected from integers from 1 to 6;
  • the substituent groups Q 1 are each independently selected from C 1 -C 6 alkyl, halogen, deuterium, hydroxyl, mercapto, -NR i R j , oxo, thio, -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, C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, 3- to 10-membered cycloalkyl, 3- to 10-membered heterocyclyl, 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered fused ring aryl, and 5- to
  • R i and R j are each independently selected from a hydrogen atom, a hydroxyl group, a C 1 -C 6 alkyl group, and a C 1 -C 6 alkoxy group;
  • R k is independently selected from hydrogen atoms, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, hydroxyl and -NR i R j , wherein said alkyl, alkane Oxy and haloalkyl are each independently optionally selected from C 1 -C 6 alkyl, halogen, hydroxy, mercapto, -NR i R j , oxo, thio, carboxyl, nitro, cyano, C 1 - C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, 3 to 10 membered cycloalkyl, 3 to 10 membered heterocyclyl, 6 to 10 membered substituted with one or more substituents in aryl and 5- to 10-membered heteroaryl;
  • R 1a are each independently selected from hydrogen, alkyl and alkoxy, each independently optionally selected from alkyl, alkoxy, halogen, deuterium, amino, cyano, nitro substituted with one or more substituents in hydroxy, hydroxy and hydroxyalkyl;
  • R 1b is each independently selected from hydrogen, PG-, HL 1 -, PG-L 1 -, or
  • R 1a and R 1b together with their attached nitrogen atoms form: or R 1a and R 1b together with the nitrogen atom to which they are attached form a nitro group;
  • p is each independently 1, 2, 3, 4, 5 or 6;
  • L1 is an amino acid unit, preferably -glycine-glutamic acid- or
  • X is halogen
  • PG is an amino protecting group
  • the proviso is that when R5a is hydrogen or alkyl, R5b is not hydrogen or alkyl.
  • each R 1a is independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy, each independently optionally selected from Substituted with one or more substituents of C 1 -C 6 alkyl, C 1 -C 6 alkoxy, halogen, deuterium, amino, cyano and hydroxy.
  • each R 1a is independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy.
  • L is selected from -glycine - glutamic acid- or
  • each R 1b is independently selected from hydrogen, PG-, HL 1 -, PG-L 1 -,
  • PG is Boc or Cbz.
  • Ring A is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring A is optionally substituted with one or more substituents Q 1
  • Ring B is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring B is optionally substituted with one or more substituents Q
  • X 1 is -(CR 5a R 5b )m- or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more substituents Q , whereby the heteroaryl group contains at least one nitrogen atom;
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxy, mercapto, deuterium, nitro, cyano and optionally substituted with one or more substituents Q 1 Group: C 1 -C 6 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 , C 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 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 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl , or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k , -S(O)R k , -S(O)(O)R k , C 1 -C 6 alkoxy, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl, or R 5a and R 5b together form oxo or thio.
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 - C6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 5a and R 5b together form oxo or sulfur generation.
  • Ring C and Ring D are each independently selected from 6- to 10-membered aryl, 5- to 10-membered heteroaryl, 8- to 12-membered optionally substituted with one or more substituents Q Fused ring aryl groups and 5- to 12-membered fused heteroaryl groups containing at least one nitrogen atom.
  • Ring C is selected from the following groups optionally substituted with one or more substituents Q:
  • Ring C is selected from optionally substituted with one or more substituents Q:
  • Ring D is a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group containing at least one nitrogen atom.
  • Ring D is optionally substituted with one or more substituents Q
  • X 2 is -(CR 6a R 6b )n- or a 6- to 10-membered aryl or 5- to 10-membered heteroaryl optionally substituted with one or more substituents Q , whereby The heteroaryl group contains at least one nitrogen atom.
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 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 1 -C 6 alkoxy, C 1 -C 6 alkylthio, C 2 -C 6 alkenyl and C 2 -C 6 alkynyl , or R 6a and R 6b together with the carbon atoms to which they are attached form a 3- to 10-membered cycloalkyl, or R 6a and R 6b together form oxo or
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k , -S(O)R k , -S(O)(O)R k , C 1 -C6alkoxy, C2 - C6alkenyl and C2 - C6alkynyl , or R6a , R6b together with the carbon atom to which they are attached form a 3- to 10-membered cycloalkyl, or R6a and R 6b together form oxo or thio.
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, sulfhydryl, deuterium, cyano, and the following groups optionally substituted with one or more substituents Q : C 1 - C6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 6a and R 6b together form oxo or sulfur generation.
  • each R 1 is independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 alkoxy, preferably hydrogen.
  • each R4 is independently hydrogen.
  • the substituent groups Q 1 are each independently selected from halogen, hydroxyl, mercapto, deuterium, cyano, amino, carboxyl, C 1 -C 6 alkyl, and C 1 -C 6 alkoxy.
  • R k is independently selected from hydrogen atoms, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, hydroxy, and -NR i R j .
  • the compound of formula (III-A) or (III-B) is a compound of formula (III-A') or (III-B') below
  • Ring A is The ring A is optionally substituted with one or more substituents Q 1 ;
  • Ring B is The ring B is optionally substituted with one or more substituents Q 1 ;
  • X 1 is selected from -(CR 5a R 5b )m- or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group optionally substituted with one or more substituents Q 1 , the heteroaryl group comprising at least a nitrogen atom;
  • R 5a and R 5b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6 alkoxy, or R 5a and R 5b together form oxo or thio;
  • Ring C is selected from The ring C is optionally substituted by one or more substituents Q;
  • Ring D is The ring D is optionally substituted with one or more substituents Q 1 ;
  • X 2 is selected from -(CR 6a R 6b )n-, -O-, -S-, -NR 6c -, -CH 2 S-, -CH 2 O-, -NHCR 6d R 6e - and optionally by a or a 6- to 10-membered aryl group or a 5- to 10-membered heteroaryl group substituted by multiple substituents Q 1 ;
  • R 6a and R 6b are each independently selected from the group consisting of hydrogen, halogen, hydroxyl, mercapto, deuterium, cyano and the following groups optionally substituted with one or more substituents Q : C 1 -C 6 alkyl, -NR i R j , -C(O)R k , -C(O)OR k and C 1 -C 6alkoxy, or R 6a and R 6b together form oxo or thio;
  • R 6c , R 6d and R 6e are each independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl and C 1 -C 6 alkoxy;
  • R2 is each independently selected from -CH2OH , -CH2SH , -CH2Cl , -SCH2Cl , -SCH2F , -SCH2CF3 , -OH , -OCH2CN , -OCH2Cl , -OCH 2 F, -OCH 3 , -OCH 2 CH 3 , -SCH 2 CN,
  • R 2a is each independently hydrogen or C 1 -C 6 alkyl
  • R 2b is each independently C 1 -C 6 alkyl or C 1 -C 6 alkoxy
  • R 2c are each independently selected from hydrogen, C 1 -C 6 alkyl, -CH 2 OH and C 1 -C 6 alkoxy;
  • R 2d and R 2e are each independently hydrogen or C 1 -C 6 alkyl
  • R3 is each independently hydrogen or halogen
  • n are each independently an integer from 1 to 6;
  • the substituent groups Q 1 are each independently selected from halogen, hydroxyl, mercapto, deuterium, oxo, thio, cyano, amino, carboxyl, C 1 -C 6 alkyl and C 1 -C 6 alkoxy;
  • R i and R j are each independently selected from a hydrogen atom, a hydroxyl group, a C 1 -C 6 alkyl group, and a C 1 -C 6 alkoxy group;
  • R k is independently selected from hydrogen atoms, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, hydroxyl and -NR i R j ;
  • R 1a are each independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 alkoxy;
  • R 1b is each independently selected from hydrogen, PG-, HL 1 -, PG-L 1 -,
  • p is each independently 1, 2, 3, 4, 5 or 6;
  • L1 is an amino acid unit selected from -glycine-glutamic acid- or
  • X is halogen
  • PG is an amino protecting group
  • the proviso is that when R5a is hydrogen or alkyl, R5b is not hydrogen or alkyl.
  • R 1a is hydrogen
  • each R 1b is independently selected from hydrogen, PG-, HL 1 -, PG-L 1 -, preferably hydrogen or
  • both R 1a and R 1b are hydrogen.
  • X 1 is selected from -(CR 5a R 5b )m-, optionally substituted with one or more substituents Q 1 :
  • both R 5a and R 5b are fluoro.
  • R 5a and R 5b together form oxo or thio, preferably oxo.
  • X 2 is selected from -(CR 6a R 6b )n-, optionally substituted with one or more substituents Q 1 :
  • R 6a and R 6b are each independently selected from hydrogen, halogen, hydroxy, mercapto, deuterium, cyano, C 1 -C 6 alkyl, -NR i R j , -C(O)OR k and C 1 -C 6 alkoxy, or R 6a and R 6b together form oxo or thio.
  • each R 2 is independently selected from -CH 2 OH, -CH 2 SH, -OH and
  • R3 is hydrogen
  • R3 is fluoro
  • each p is independently 1 or 2, preferably 1.
  • the compound of formula (III-A) or (III-B) is selected from
  • the compound of formula (III-A) or (III-B) is selected from
  • X is halogen, preferably chlorine or bromine, more preferably bromine.
  • R 5a and R 5b are selected from oxo or thio, the other is not present.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the aforementioned antibody-drug conjugates, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the unit dose of the pharmaceutical composition is 0.001 mg-1000 mg.
  • the pharmaceutical composition contains from 0.01% to 99.99% of the aforementioned antibody-drug conjugate, based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of the aforementioned antibody-drug conjugates. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of the aforementioned antibody-drug conjugates. In certain embodiments, the pharmaceutical composition contains 1%-99% of the aforementioned antibody-drug conjugates. In certain embodiments, the pharmaceutical composition contains 2%-98% of the aforementioned antibody-drug conjugates.
  • the pharmaceutical composition contains 0.01%-99.99% of a pharmaceutically acceptable carrier, diluent or excipient based on the total weight of the composition. In certain embodiments, the pharmaceutical composition contains 0.1%-99.9% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 0.5%-99.5% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 1%-99% of a pharmaceutically acceptable carrier, diluent or excipient. In certain embodiments, the pharmaceutical composition contains 2%-98% of a pharmaceutically acceptable carrier, diluent or excipient.
  • the antibody-drug conjugates and/or pharmaceutical compositions comprising the antibody-drug conjugates described in the present disclosure can be used to lyse surface TNF ⁇ -expressing cells (in vitro or in vivo) and/or for the treatment of increased TNF ⁇ (eg, , a disease or condition characterized by increased TNF ⁇ in synovial fluid.
  • 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.
  • the antibody-drug conjugates and/or compositions are used to treat Crohn's disease.
  • the antibody-drug conjugates and/or compositions are used to treat ulcerative colitis.
  • the antibody-drug conjugates and/or compositions are used to treat rheumatoid arthritis (RA). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat juvenile idiopathic arthritis (JA). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat psoriatic arthritis (PsA). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat spondyloarthritis, such as ankylosing spondylitis (AS) or axial spondyloarthritis (axSpA). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat Crohn's disease (CD) in adults.
  • RA rheumatoid arthritis
  • JA juvenile idiopathic arthritis
  • PsA psoriatic arthritis
  • the antibody-drug conjugates and/or compositions are used to treat spondyloarthritis, such as anky
  • the antibody-drug conjugates and/or compositions are used to treat pediatric Crohn's disease. In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat ulcerative colitis (UC). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat plaque psoriasis (Ps). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat hidradenitis suppurativa (HS). In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat uveitis. In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat Behce's disease. In certain embodiments, the antibody-drug conjugates and/or compositions are used to treat psoriasis, including plaque psoriasis.
  • the present disclosure also provides a method of delivering a glucocorticoid receptor agonist to a TNF ⁇ -expressing cell, comprising the step of contacting the TNF ⁇ -expressing cell with an antibody-drug conjugate described in this disclosure.
  • the present disclosure also provides a method of determining the anti-inflammatory activity of an antibody-drug conjugate.
  • Such methods can include the step of contacting a TNF ⁇ -expressing cell with an antibody-drug conjugate as described herein.
  • Some embodiments comprise contacting TNF ⁇ -expressing cells with an antibody-drug conjugate as described herein, and determining the reduced release of proinflammatory cytokines from the cells as compared to control cells.
  • Some embodiments comprise in vitro methods for determining the anti-inflammatory activity of antibody-drug conjugates.
  • Some embodiments include screening methods (eg, in vitro methods) comprising contacting cells (eg, cells expressing TNF ⁇ ) with an antibody-drug conjugate, directly or indirectly, and determining whether the antibody-drug conjugate modulates the cell's Activity or function, as reflected, for example, by changes in cell morphology or viability, expression of markers, 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
  • an indirect interaction includes, for example, the action of a composition on an intermediate molecule, which in turn acts on a reference entity (eg, a cell or cell culture).
  • the present disclosure also provides a pharmaceutical composition
  • 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 agent.
  • the compounds or their pharmaceutically acceptable salts and pharmaceutical compositions thereof can be used for the treatment of immune diseases.
  • the present disclosure further provides a kit comprising the antibody-drug conjugate described in the present disclosure, or a pharmaceutical composition.
  • linker refers to a chemical structural fragment or bond that is linked to a ligand at one end and a drug at the other end, and may also be linked to other linkers. Reconnect with the drug.
  • a linker may comprise one or more linker members.
  • Exemplary linker members include 6-maleimidohexanoyl (MC), maleimidopropionyl (MP), valine-citrulline (Val-Cit or vc), alanine-benzene Alanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), and those derived from coupling with linker reagents: N-succinimidyl 4-(2-pyridylthio)valerate ( SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, also referred to herein as MCC) and N-succinimidyl ( 4-iodo-acetyl)aminobenzoate (SIAB).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • Vcitrulline Val-Cit or
  • Linkers can include stretch units, spacer units, amino acid units, and stretch units. It can be synthesized by methods known in the art, such as described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers eg, hydrazones
  • protease-sensitive linkers eg, peptidase-sensitive linkers
  • photolabile linkers eg, dimethyl linkers, or disulfide-containing linkers
  • disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • stretch unit refers to a chemical structural fragment that is covalently linked to an antibody through a carbon atom at one end and to an amino acid unit, disulfide moiety, sulfonamide moiety, or non-peptide chemical moiety at the other end.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple amino acid units and glucocorticoids to form antibody-drug conjugates, which can selectively link glucocorticoids to amino acid units superior.
  • amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
  • the general formula is H 2 NCHRCOOH, R is H, substituted or unsubstituted alkyl, and the like. According to the position where the amino group is attached to the carbon atom in the carboxylic acid, it can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • amino acids that constitute natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids, including glycine, alanine, and valine.
  • ⁇ -amino acids including glycine, alanine, and valine.
  • Glutamine Leucine
  • Isoleucine Isoleucine
  • Phenylalanine Phenylalanine
  • Tryptophan Tryptophan
  • Tyrosine Tyrosine
  • Aspartic acid Aspartic acid
  • Histidine Asparagine, Glutamic acid, Lysine, Glutamine, Methionine, Arginine , Serine (Serine), Threonine (Threonine), Cysteine (Cysteine), Proline (Proline) and the like.
  • Unnatural amino acids such as citrulline. As is well known to those skilled in the art, unnatural amino acids do not constitute natural proteins and thus do not participate in the synthesis of antibodies in the present disclosure.
  • the three-letter and one-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).
  • antibody-drug conjugate refers to a ligand linked to a biologically active drug through a stable linking unit.
  • antibody-drug conjugate (antibody drug conjugate, ADC) refers to linking a monoclonal antibody or antibody fragment with a biologically active glucocorticoid through a stable linking unit. Wherein the antibody or antibody fragment can be combined with the glucocorticoid molecule containing the linker through specific groups therein (eg, interchain disulfide bonds).
  • drug loading refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the drug loading may range from 1-20, preferably 1-10 glucocorticoids (D) linked per antibody (Ab).
  • the drug load is expressed as k, and exemplary can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the mean of the values in between.
  • the average amount of drug per ADC molecule after the conjugation reaction can be characterized by conventional methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC.
  • the molecular size variant assay (CE-SDS) of the monoclonal antibody of the present disclosure can use the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia” 0542), quantitative determination of the purity of recombinant monoclonal antibody products.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • the glucocorticoid is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through the linking unit.
  • the glucocorticoid can be coupled to the antibody in the coupling reaction.
  • the number of drug molecules will be less than the theoretical maximum.
  • Antibody-drug conjugate loading can be controlled by the following non-limiting methods, including:
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions.
  • the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies, preferably humanized antibodies and fully human antibodies.
  • murine antibody in the present disclosure refers to the use of murine antibodies in accordance with the knowledge and skill in the art. In preparation, test subjects are injected with a specific antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • To build a chimeric antibody first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the mouse variable region gene from the mouse hybridoma cell. After connecting with human constant region gene into chimeric gene, it is inserted into expression vector, and finally chimeric antibody molecule is expressed in eukaryotic system or prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody antibodies produced in framework sequences.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database, and in Kabat, E.A. et al., 1991 Sequences of Proteins of Immunological Interest, 5th ed.
  • the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that are further subjected to affinity maturation of the CDRs by phage display. Documents further describing methods involved in humanization using mouse antibodies include, for example, Queen et al., Proc., Natl. Acad. Sci. USA, 88, 2869, 1991 and the method of Winter and colleagues [Jones et al., Nature, 321, 522 (1986), Riechmann, et al, Nature, 332, 323-327 (1988), Verhoeyen, et al, Science, 239, 1534 (1988)].
  • the development of monoclonal antibodies has gone through four stages, namely: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • the present disclosure is a fully human monoclonal antibody.
  • the technologies related to the preparation of fully human antibodies mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies. 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, comprising A bivalent fragment of two Fab fragments linked by a disulfide bridge; (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv fragment composed of the one-armed VH and VL domains of an antibody; (v ) a single domain or dAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VH domain; and (vi) isolated complementarity determining regions (CDRs) or (vii) optionally A combination of two or more isolated CDRs
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included within the term "antigen-binding fragment" of an antibody.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating IgG antibody molecules with the protease papain (which cleaves the amino acid residue at the 224th position of the H chain), wherein the N-terminal side of the H chain is an antibody fragment. About half and the entire L chain is held together by disulfide bonds.
  • F(ab')2 is an antibody with a molecular weight of about 100,000 obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG with the enzyme pepsin and having antigen-binding activity and comprising two Fab regions linked at the hinge position Fragment.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of the above-mentioned F(ab')2.
  • the Fab' fragment of the antibody can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic or eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.
  • single chain antibody is intended to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker molecule.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof, eg using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain, and to the heavy chain variable domain CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).
  • CDR1, HCDR2, HCDR3 there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acid residue numbers in VH are approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3)
  • CDR amino acid residue numbers in VL are approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation (see, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996)).
  • antibodies bind with an affinity (KD) of less than about 10-7 M, eg, about less than 10-8 M, 10-9 M, or 10-10 M or less.
  • KD affinity
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequences can be obtained from the Journal of Immunoglobulins, 2001 ISBN012441351 by aligning with the IMGT human antibody variable region germline gene database and MOE software.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques. For example, use an A or G Sepharose FF column with adjusted buffer.
  • Non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Amino acid sequence identity refers to the alignment of amino acid sequences and, where necessary, the introduction of gaps to achieve a maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity, in the first sequence to the second sequence.
  • the percentage of amino acid residues that are identical For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • anti-TNF ⁇ antibody or "antibody that binds TNF ⁇ ” refers to an antibody capable of binding TNF ⁇ , eg, with sufficient affinity, such that the antibody is useful as a therapeutic agent targeting TNF ⁇ .
  • the extent of binding of an anti-TNF ⁇ antibody to an unrelated non-TNF ⁇ protein may be less than about 10% of the binding of the antibody to TNF ⁇ as measured, for example, by radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the antibody that binds to TNF ⁇ has a dissociation constant (Kd) ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
  • peptide refers to a fragment of a compound between an amino acid and a protein, consisting of two or more amino acid molecules connected to each other by peptide bonds, and is a structural and functional fragment of a protein, such as hormones, enzymes, etc. All are peptides.
  • sucrose refers to a biological macromolecule composed of three elements, C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.
  • fluorescent probe refers to characteristic fluorescence in the ultraviolet-visible-near-infrared region, and its fluorescence properties (excitation and emission wavelength, intensity, lifetime and polarization, etc.) can vary with the properties of the environment, such as polarity, refractive index A class of fluorescent molecules that are sensitively changed by changes in viscosity, etc., which non-covalently interact with nucleic acids (DNA or RNA), proteins or other macromolecular structures to change one or several fluorescent properties, which can be used for research The properties and behavior of macromolecular substances.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain 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-methylp
  • alkyl groups containing 1 to 6 carbon atoms 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-dimethyl Butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl , 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane, which are A straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene ( -CH2- ), 1,1-ethylene (-CH( CH3 )-), 1,2-ethylene ( -CH2) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • Alkylene groups can be substituted or unsubstituted, and when substituted, the substituents can be substituted at any available point of attachment.
  • alkenylene refers to a linear alkene comprising 2 to 8 carbon atoms, preferably 2 to 6 carbon atoms, more preferably 2 to 4 carbon atoms and at least one double bond at any position radicals, including, for example, vinylene, allylene, propenylene, butenylene, prenylene, butadienylene, pentenylene, pentylene Alkenyl, hexenylene, hexadienylene, etc.
  • alkynylene includes linear alkynes 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 radicals, including, for example, ethynylene, propynylene, butynylene, pentynylene, hexynylene, and the like.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 6 carbon atoms carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups. "Carbocycle” refers to the ring system in a cycloalkyl group.
  • spirocycloalkyl refers to a 5- to 20-membered monocyclic polycyclic group sharing one carbon atom (called a spiro atom), which may contain one or more double bonds, but none of the rings are fully conjugated The pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of spiro atoms shared between the rings, spirocycloalkyl groups are classified into mono-spirocycloalkyl groups, double-spirocycloalkyl groups or poly-spirocycloalkyl groups, preferably mono-spirocycloalkyl groups and double-spirocycloalkyl groups.
  • Spirocarbocycle refers to the ring system in a spirocycloalkyl.
  • Non-limiting examples of spirocycloalkyl include:
  • fused cycloalkyl refers to an all-carbon polycyclic group of 5 to 20 members in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or more rings. Multiple double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused cycloalkyl, preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl. "Fused carbocycle” refers to the ring system in a fused cycloalkyl. Non-limiting examples of fused cycloalkyl groups include:
  • bridged cycloalkyl refers to an all-carbon polycyclic group of 5 to 20 members, any two rings sharing two non-directly connected carbon atoms, which may contain one or more double bonds, but none of the rings have complete Conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan. According to the number of constituent rings, it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
  • bridged cycloalkyl include:
  • the cycloalkyl ring can be fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein the ring linked to the parent structure is a cycloalkyl, non-limiting examples include indanyl, tetrahydronaphthalene base, benzocycloheptyl, etc.
  • Cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • ring atoms excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably 3 to 6 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidine group, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc., preferably piperidinyl and pyrrolidinyl.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. "Heterocycle" refers to a ring system in a heterocyclyl group.
  • spiroheterocyclyl refers to a 5- to 20-membered monocyclic polycyclic heterocyclic group sharing one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O ) m (where m is an integer from 0 to 2) heteroatoms and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl and bis-spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl group.
  • Spiroheterocycle refers to the ring system in a spiroheterocyclyl group.
  • Non-limiting examples of spiroheterocyclyl include:
  • fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more Double bonds, but none of the rings have a fully conjugated pi-electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), the remaining rings Atom is carbon. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic groups.
  • fused heterocycle refers to a ring system in a fused heterocyclyl.
  • fused heterocyclyl groups include:
  • bridged heterocyclyl refers to a 5- to 14-membered, polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings have a complete common A pi-electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon.
  • m is an integer from 0 to 2
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • bridged heterocyclyl groups include:
  • heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:
  • Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo, carboxyl or carboxylate.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, preferably 6 to 10 membered, such as benzene base and naphthyl.
  • the aryl ring can be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring.
  • “Aromatic ring” refers to the ring system in an aryl group.
  • Non-limiting examples of aryl groups include:
  • Aryl may be substituted or unsubstituted, and when substituted, the substituents are 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, heterocycle
  • fused ring aryl may be an unsaturated aromatic fused ring structure containing 8-14 ring atoms, which is formed by two or more ring structures sharing two adjacent atoms.
  • the atoms are preferably 8-12.
  • it includes all unsaturated fused-ring aryl groups, such as naphthalene, phenanthrene, etc., and also includes partially saturated fused-ring aryl groups, such as benzo 3-8 membered saturated monocyclic cycloalkyl, benzo 3-8 membered partially saturated monocyclic ring alkyl.
  • fused aromatic ring refers to the ring system in a fused ring aryl group.
  • fused-ring aryl group examples include 2,3-dihydro-1H-indenyl, 1H-indenyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl and the like.
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 12 membered, eg imidazolyl, furanyl, thienyl, thiazolyl, pyrazolyl, oxazolyl, pyrrolyl, tetrazolyl, pyridyl, pyrimidinyl, thiadiazole, pyrazine base, etc., preferably imidazolyl, pyrazolyl, pyrimidinyl or thiazolyl; more preferably pyrazolyl or thiazolyl.
  • heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring.
  • Heteroaromatic ring refers to the ring system in a heteroaryl group.
  • Non-limiting examples of heteroaryl groups include:
  • Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.
  • fused heteroaryl may be an unsaturated heteroaryl group containing 5-14 ring atoms (including at least one heteroatom) formed by two or more cyclic structures that share two adjacent atoms connected to each other.
  • Aromatic condensed ring structure, including carbon atom, nitrogen atom and sulfur atom can be oxo, preferably "5-12-membered condensed heteroaryl", “7-12-membered condensed heteroaryl”, “9-12-membered heteroaryl” fused heteroaryl” etc., such as benzofuranyl, benzoisofuranyl, benzothienyl, indolyl, isoindole, benzoxazolyl, benzimidazolyl, indazolyl, benzotri azolyl, quinolinyl, 2-quinolinone, 4-quinolinone, 1-isoquinolinone, isoquinolinyl, acridine, phenanthridine, benzopyridaziny
  • the fused heteroaryl group can be optionally substituted or unsubstituted, and when substituted, the substituents are 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, cycloalkanethio group, heterocycloalkylthio group, carboxyl group or carboxylate group.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, carboxyl or carboxylate.
  • alkylthio refers to -S-(alkyl) and -S-(unsubstituted cycloalkyl), wherein alkyl is as defined above.
  • alkylthio include: methylthio, ethylthio, propylthio, butylthio, cyclopropylthio, cyclobutylthio, cyclopentylthio, cyclohexylthio.
  • Alkylthio can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , substituted with one or more substituents in the heterocycloalkylthio group.
  • hydroxyalkyl refers to an alkyl group substituted with hydroxy, wherein alkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with halogen, wherein alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with a deuterium atom, wherein alkyl is as defined above.
  • hydroxyl refers to the -OH group.
  • a carbon atom is linked to an oxygen atom by a double bond, in which a ketone or aldehyde group is formed.
  • a carbon atom is linked to a sulfur atom by a double bond to form a thiocarbonyl-C(S)-.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • cyano refers to -CN.
  • nitro refers to -NO2 .
  • aldehyde group refers to -CHO.
  • carboxylate refers to -C(O)O(alkyl) or -C(O)O(cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
  • acyl halide refers to a compound containing a group -C(O)-halogen.
  • sulfonyl refers to -S(O)(O)-.
  • amino protecting groups are suitable groups known in the art for amino protection, see the literature ("Protective Groups in Organic Synthesis", 5 Th . Ed. TW Greene & P. GMWuts) for amino protecting groups, preferably , the amino protecting group can be (C 1-10 alkyl or aryl) acyl, such as: formyl, acetyl, benzoyl, etc.; can be (C 1-6 alkyl or C 6-10 aryl) can also be (C 1-6 alkoxy or C 6-10 aryloxy) carbonyl, such as: Boc or Cbz; can also be substituted or unsubstituted alkyl, such as: trityl group (Tr), 2,4-dimethoxybenzyl (DMB), p-methoxybenzyl (PMB) or benzyl (Bn).
  • Tr trityl group
  • DMB 2,4-dimethoxybenzyl
  • PMB p-methoxybenzyl
  • Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.
  • a heterocycloalkyl group optionally substituted with an alkyl group means that an alkyl group may, but need not, be present, and the specification includes the case where the heterocycloalkyl group is substituted with an alkyl group and the case where the heterocycloalkyl group is not substituted with an alkyl group replaced situation.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • drug carrier is used for the drugs of the present disclosure, and refers to a system capable of changing the way the drug enters the human body and its distribution in the body, controlling the release rate of the drug, and delivering the drug to the targeted organs.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient refers to an addition other than the main drug in a pharmaceutical preparation, and may also be called an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in ointments and creams of semi-solid preparations; preservatives, antioxidants, flavorings, fragrances in liquid preparations, Solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • the term "diluent” is also known as filler, the main purpose of which is to increase the weight and volume of the tablet.
  • the addition of the diluent not only ensures a certain volume, but also reduces the dosage deviation of the main components and improves the compression moldability of the drug.
  • the medicine in the tablet contains oily components, it is necessary to add an absorbent to absorb the oily substances, so as to keep the "dry” state, so as to facilitate the tableting.
  • Compounds of the present disclosure may contain one or more asymmetric centers, and thus may give rise to enantiomers, diastereomers, and may be defined as (R)- or (S)- according to absolute stereochemistry or as (D)- or (L)- other stereoisomeric forms of amino acids.
  • the present 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 conventional methods can be used For example chromatography and fractional crystallization preparation.
  • the bond Indicates an unspecified configuration, i.e. if a chiral isomer exists in the chemical structure, the bond can be or both Two configurations.
  • the bond The configuration is not specified, i.e. it can be either the Z configuration or the E configuration, or both.
  • Stereoisomers refer to compounds composed of the same atoms bonded by the same bonds but with different three-dimensional structures, which are not interchangeable.
  • Various stereoisomers and mixtures thereof are contemplated in this disclosure, and includes “enantiomers,” which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.
  • Tautomer refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. Tautomers of any of the compounds are included in this disclosure.
  • Atoms capable of being isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be replaced by isotopic isotopes 2 H(D), 3 H, 11 C, 13 C, 14 C, 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125 I, respectively.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have an abundance of at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) of deuterium (ie, at least 45 % of deuterium incorporated).
  • Figure 1 Anti-inflammatory activity of small-molecule steroids in lipopolysaccharide-stimulated human PBMC to secrete cytokines
  • FIG. 1 Activity of anti-TNF-ADCs in membrane-bound TNF ⁇ -mediated GRE reporter gene system
  • Figure 3 Activity of anti-TNF-ADC in lipopolysaccharide-stimulated human monocytes to secrete cytokines
  • FIG. 6 Activity of anti-TNF-ADC in mouse collagen antibody-induced arthritis (CAIA) model
  • FIG. 7 Activity of anti-TNF-ADC at different time points in mouse collagen antibody-induced arthritis (CAIA) model
  • NMR shifts ([delta]) are given in units of 10<" 6 > (ppm). NMR was measured by Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was four Methylsilane (TMS).
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methylsilane
  • MS was measured with a Shimadzu 2010 Mass Spectrometer or an Agilent 6110A MSD mass spectrometer.
  • HPLC uses Shimadzu LC-20A systems, Shimadzu LC-2010HT series or Agilent Agilent 1200 LC high pressure liquid chromatograph (Ultimate XB-C18 3.0*150mm chromatographic column or Xtimate C18 2.1*30mm chromatographic column).
  • the chiral preparative column uses DAICEL CHIRALPAK IC (250mm*30mm, 10um) or Phenomenex-Amylose-1 (250mm*30mm, 5um).
  • the CombiFlash rapid preparation instrument uses Combiflash Rf150 (TELEDYNE ISCO).
  • the average inhibition rate and IC 50 value of kinases were measured with NovoStar microplate reader (BMG, Germany).
  • the known starting materials of the present disclosure can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui chemical companies.
  • Argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.
  • Hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenation apparatus and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenation apparatus.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction used a CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, which is 20°C to 30°C.
  • the monitoring of the reaction progress in the embodiment adopts thin layer chromatography (TLC), the developing solvent used in the reaction, the eluent system of the column chromatography used for purifying the compound and the developing solvent system of the thin layer chromatography method include: A: Dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system, D: petroleum ether/ethyl acetate/methanol, the volume ratio of the solvent depends on the polarity of the compound For adjustment, a small amount of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.
  • TLC thin layer chromatography
  • DAST diethylaminosulfur trifluoride
  • THF tetrahydrofuran
  • NMP N-methylpyrrolidone
  • DCM dichloromethane
  • m-CPBA m-chloroperoxybenzoic acid
  • DIEA N,N-diisopropyl ethylamine
  • TEA triethylamine
  • Boc tert-butoxycarbonyl, MeOH: methanol
  • Et2O diethyl ether.
  • compound 3-A (90.0 mg, 0.145 mmol, 1.0 eq) was added to 2 mL of toluene, and then BOC acid anhydride (63.3 mg, 0.290 mmol, 2.0 eq) was added, the system was heated to 100 ° C to react to complete, directly reduce It was concentrated to dryness under pressure and subjected to column chromatography to obtain 48.0 mg of compound 3-A-1.
  • 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 was added.
  • - Diethylphosphoramidite di-tert-butyl ester (187.0 mg, 0.756 mmol), reacted at room temperature for 2 hours. Cool to 0°C, slowly add H 2 O 2 (50.0 mg, 0.82 mmol), stir at room temperature after the addition is complete, add 2 mL of water, filter, and dry the filter cake to obtain about 40.0 mg of compound 3-A-2 , directly into the next reaction.
  • compound 3-A-2 (40.0 mg, 0.043 mmol) was dissolved in dichloromethane (1.0 mL), cooled to 0°C, added with trifluoroacetic acid (0.3 mL), and reacted at room temperature for 3 hours. After concentration, it was separated by preparative HPLC ( CH3CN / H2O , + 0.1% trifluoroacetic acid) to give about 12.0 mg of compound 3-B.
  • compound 4-A (45.0 mg, 0.074 mmol, 1.0 eq) was added to 2 mL of toluene, then BOC acid anhydride (32.0 mg, 0.158 mmol, 2.0 eq) was added, and the system was heated to 100 °C to react until the reaction was complete, and then directly It was concentrated under reduced pressure and subjected to column chromatography to obtain 45.2 mg of compound 4-A-1.
  • compound 4-A-2 (40.0 mg, 0.042 mmol) was dissolved in dichloromethane (1.0 mL), cooled to 0°C, added trifluoroacetic acid (0.3 mL), and reacted at room temperature for 3 hours. After concentration, it was separated using preparative HPLC ( CH3CN / H2O , + 0.1% trifluoroacetic acid) to give about 15.0 mg of compound 4-B.
  • compound 3-8 (830 mg, 0.650 mmol) was dissolved in acetonitrile (20 mL), piperidine (302 ⁇ L, 3.250 mmol) was added, and the reaction was carried out at room temperature. After concentration, beating with 15 mL of petroleum ether was repeated three times to obtain a total of 645 mg of compound 3-9, which was directly used in the next reaction.
  • compound 3-10 (830 mg, 0.706 mmol) was dissolved in dichloromethane (8 mL), cooled to 0° C., trifluoroacetic acid (4 mL) was added, and the reaction was carried out at room temperature. After concentration, preparative HPLC was used for separation (CH 3 CN/H 2 O, +0.1% trifluoroacetic acid) to obtain 220 mg of compound 3-B00 in total, three-step yield: 33.6%.
  • the aqueous solution (2.5 mM, 356.8 ⁇ L, 891.24 nmol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the aqueous solution (2.5 mM, 405.4 ⁇ L, 1012.77 nmol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • Test Example 1 In Vitro Activity of Small Molecular Steroids
  • Small molecule steroids were tested for binding activity to the glucocorticoid receptor (GR) using a human glucocorticoid NHR binding (radiolabeled agonist) assay (#232020, eurofins).
  • the experimental principle was to incubate various concentrations of small molecule steroids and 5 nM [3H]dexamethasone with human recombinant GR at 4°C for 24 hours.
  • Small-molecule steroids bind human GR competitively with [3H]dexamethasone.
  • the binding activity of small-molecule steroids to GR can be calculated by counting the [3H]dexamethasone specifically bound to the receptor. The results are shown in Table 1.
  • NHR nuclear translocation assays test small molecule steroids for mineralocorticoid receptor (MR) agonist activity.
  • PathHunter NHR CHO-K1 cells were plated in 384-well white-walled microplates, and small molecule steroids to be tested were added to co-incubate at 37°C, 5% CO 2 to induce the reaction.
  • Assay signals were generated with the PathHunter detection reagent mix and chemiluminescent signals were detected after one hour incubation at room temperature. Data were analyzed using a four-parameter curve fit to generate EC50 values. The results are shown in Table 1.
  • A549 cells were plated (40,000 cells/96-well plate), pGL4.36 (MMTV-Luc, 100ng/well) was transfected with lipo3000, incubated overnight with different concentrations of small molecule steroids, and then incubated with Bright-Glo after 24 hours. (promega) assay reagents to detect the expression of luciferase in the reporter gene system.
  • Test Example 2 Anti-inflammatory activity of small molecular steroids in lipopolysaccharide (LPS)-stimulated human PBMC to secrete cytokines
  • PBMCs Primary human peripheral blood mononuclear cells
  • RPMI 2% FBS, 1% penicillin-streptomycin
  • IL-6 was detected by alpha LISA (Cisbio).
  • test results are shown in FIG. 1 , the small molecule steroid compounds described in the present disclosure can significantly inhibit LPS-induced IL-6 release.
  • Test Example 3 Activity of anti-TNF-ADC in membrane-bound TNF ⁇ -mediated GRE reporter gene system
  • Humira-3-B00, Humira-4-B00, Humira-A-B00 prepared according to Example 7 of WO2019106609).
  • a reporter cell line stably transfected with MMLV-Luc was established with lentivirus in Hela cells.
  • the stably transfected Hela-MMTV-Luc cell line was plated in a 96-well plate (30,000 cells/well), and lipo3000 was used in each well to transfect the empty vector plasmid or human TNF ⁇ mutant plasmid (TNF ⁇ 12), and the restriction site of TACE was removed. , 50ng/well). After overnight incubation with different concentrations of anti-TNF-ADC, Bright-Glo (promega) detection reagent was used to detect the expression of luciferase in the reporter gene system after 24 hours.
  • Test Example 4 Activity of anti-TNF-ADC in lipopolysaccharide (LPS) stimulation of human monocytes to secrete cytokines
  • Mononuclear cells were sorted and enriched from cryopreserved primary human peripheral blood PBMC using EsaySep TM Human CD14 Sorting Kit and plated in 96-well plates. After co-incubating monocytes with different concentrations of anti-TNF-ADC for 4 hours at 37°C, 5% CO2 , they were stimulated with 0.01 ng/mL LPS overnight. The medium supernatant was collected the next day and the concentration of IL-6 was detected by alpha LISA (cisbio).
  • the ADCs of the present disclosure potently inhibit LPS-induced IL-6 secretion at high concentrations (4-100 nM) (as shown in Figure 3).
  • the anti-inflammatory activity of Humira-3-B00 and Humira-4-B00 at 4 nM was significantly stronger than that of the control ADC molecule Humira-A-B00 at this concentration (as shown in Figure 4).
  • Test Example 5 Mouse collagen antibody-induced arthritis (CAIA) model
  • mice Male balb/c mice, 6w, were purchased from the Laboratory Animal Management Department of Shanghai Institute of Family Planning. Breeding environment: SPF; production license: SCXK (Shanghai) 2018-0006; Balb/c mouse certificate number: 20180006023393.
  • mice in each group except the control group were injected intraperitoneally with 1.5 mg/mice of type II collagen antibody mixture (purchased from Chondrex Company).
  • mice in each group were intraperitoneally injected with LPS 50 ⁇ g/mice (100 ⁇ L) to enhance the immune response.
  • the mice in each group were treated with drug administration from day 5, and the arthritis scores of the limbs were performed every 1-3 days.
  • the specific experimental process is shown in Figure 5. Mice were administered by the following dosing regimen, and semi-quantitative scoring of arthritis severity was performed on each group of mice every 1-3 days to evaluate the anti-inflammatory activity of anti-TNF-ADC.
  • Humira had weak anti-inflammatory activity in the CAIA model, and Humira-4-B00 had a rapid onset of action (starting on day 5) and durable reduction in arthritis inflammation.
  • the control ADC molecule, Humira-A-B00 showed a relieving effect on arthritis only at the later stage of the arthritis progression (from day 11 onwards) (as shown in Figure 6). From the 8th day to the 14th day after the arthritis modeling, Humira-4-B00 significantly reduced the mouse arthritis score compared with the model group (*, p ⁇ 0.05), while the inhibitory effect of the control ADC molecule Humira-A-B00 There was no significant difference in inflammatory activity relative to the model group (as shown in Figure 7).
  • mice Male ICR mice, 6w, were purchased from the Laboratory Animal Management Department of Shanghai Institute of Family Planning. Breeding environment: SPF; production license: SCXK (Shanghai) 2018-0006, certificate number: 20180006023622.
  • Humira-3-B00, Humira-4-B00, Humira-A-B00, Humira-A-A00 prepared according to WO2017210471 example 2 and WO2019106609 example 7).
  • mice were housed adaptively for 7 days after arrival and were randomized into groups. On the 0th day, 50 ⁇ L of 1% DNFB (2,4-dinitrofluorobenzene) solution was applied to the abdomen of the mice for immunization. 20 ⁇ L) were challenged, the mice were sacrificed on day 6 (24 h later), and the bilateral 8 mm diameter ear pieces were removed using a punch and weighed. Mice in each group were treated with administration on day 0 and day 4, respectively. The specific experimental process is shown in Figure 8. Mice were administered by the following dosing regimen, and the ear pieces on the control side and the model side of the mice were weighed (indicating the degree of swelling) to evaluate the anti-inflammatory activity of anti-TNF-ADC.
  • DNFB 2,4-dinitrofluorobenzene
  • Test Example 7 Stability of ADC Samples in Plasma
  • test molecules were prepared as a 1 mg/mL solution in PBS and sterilized by filtration through a 0.22 ⁇ m filter. Add 15 ⁇ L of 1 mg/mL sample to 135 ⁇ L of reaction matrix to make a final concentration of 100 ⁇ g/mL. Incubate at 37°C in the dark for 0 days, 7 days, 14 days, and 21 days, and the samples are tested for free toxins.
  • Humira-3-B00 and Humira-4-B00 showed excellent stability in human, cynomolgus monkey, mouse, rat plasma and 1% BSA, and the free toxins were all below the detection limit ;
  • Humira-A-A00 has a little free toxin released in rat plasma;
  • Humira-A-B00 has a little free toxin in rat and mouse plasma.

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Abstract

糖皮质激素受体激动剂的药物偶联物及其在医药上的应用。具体而言，涉及一种式(I)所示的抗体-药物偶联物：Ab-(L-D)k(I)，其中，Ab为抗体或其抗原结合片段，L为将Ab共价连接于D的连接子，k为1至20，且D如式(II-A)或(II-B)所示。其中式中各基团如说明书中所定义。所述抗体-药物偶联物可有效治疗免疫性疾病。

Description

糖皮质激素受体激动剂的药物偶联物及其在医药上的应用 技术领域
本公开属于医药领域，具体涉及一种糖皮质激素受体激动剂的药物偶联物及其在医药上的应用。
背景技术
类风湿关节炎(RA)是一种常见的关节炎，属于自身免疫疾病，在人群中的发病率为0.3-1％，如不及时治疗，可导致骨破坏和关节损伤。在RA发病中有多种促炎性细胞因子参与，如肿瘤坏死因子-α(TNFα)、白细胞介素如IL-1、IL-6、IL-8等。因此，抑制促炎性细胞因子的产生或阻断其生理作用，是目前RA研究领域的热点。近年来许多新研发的生物制剂可通过阻断或下调促炎性细胞因子的活性而控制病情的进展，如TNFα抑制剂、抗IL-6R抗体等目前认为，在诸多RA炎症反应的细胞因子中，TNFα是最重要的促炎性细胞因子之一，其在RA的病情发展、局部炎症反应和组织损伤中均起着重要作用。目前已被美国FDA批准的TNFα抑制剂包括：可溶性受体拮抗剂-依那西普(Etanercept)，人鼠嵌合抗体-英夫利昔单抗(Infliximab)，全人源单抗阿达木单抗(Adalimumab，
Figure PCTCN2022074455-appb-000001
)，全人源单抗戈利木单抗(Golimumab)和聚乙二醇人源化Fab’片段赛妥珠单抗(Certolizumab pegol)。尽管它们在临床上取得了成功，但TNFα抑制剂仍然受限于它们在患者中可以达到的最大功效，需要鉴定和开发更强力有效的治疗剂。用TNFα抑制剂治疗的患者也可能对治疗剂产生免疫原性应答，从而限制其有效性。
糖皮质激素受体激动剂也是治疗类风湿关节炎较为有效的药物。作为代表性的糖皮质激素受体激动剂，已知皮质醇、皮质酮等在生物体内制成的糖皮质激素受体激动剂以及地塞米松、泼尼松、泼尼松龙等合成糖皮质激素受体激动剂。这些糖皮质激素受体激动剂由于具有类固醇结构，因此总称为类固醇类，应用在各种疾病的治疗中。但是，这些类固醇类由于其使用，有时会表现出类固醇消化性溃疡、类固醇紫斑、类固醇胰炎、类固醇糖尿病、类固醇白内障、类固醇青光眼等副作用。
抗体药物偶联物(antibody drug conjugate，ADC)，是指单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的药物相连。临床前和临床开发中的大多数ADC都用于肿瘤适应症，其中细胞毒性有效载荷靶向表达抗原的癌细胞。但是，通过ADC介导的生物活性小分子的传递来调节病原性细胞活性对于非肿瘤学适应症也是有吸引力的，从而导致了该技术的广泛应用。
现有技术已经公开了一些糖皮质激素受体激动剂的药物偶联物，例如WO2017210471，WO2019106609等。
发明内容
本公开一方面提供了一种式(I)所示的抗体-药物偶联物(ADC)，
Ab-(L-D) k
(I)
其中，Ab为抗体或其抗原结合片段，
L为将Ab共价连接于D的连接子，且k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
D如下式(II-A)或(II-B)所示：
Figure PCTCN2022074455-appb-000002
其中，
Figure PCTCN2022074455-appb-000003
表示单键或双键；
R 1a各自独立地选自氢、烷基和烷氧基，所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
环A为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
环B为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
X 1为-(CR 5aR 5b)m-和任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 5a和R 5b一起形成氧代或硫代；
环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的芳基和杂芳基，且环C和环D中至少有一个选自任选被一个或多个取代基Q 1所取代的稠环芳基或稠杂芳基；；
X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的芳基或杂芳基、-O-、-S-、-S(O)-、-S(O)(O)-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-、-CR 6f＝CR 6g-和-C≡C-，或者X 2不存在；
R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基或任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基， 或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代；
R 6c、R 6d、R 6e、R 6f和R 6g各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
R 1各自独立地选自氢、烷基和烷氧基，其中所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
Figure PCTCN2022074455-appb-000004
R 2a各自独立地为氢或C 1-C 6烷基；
R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
R 3各自独立地为氢或卤素；
R 4各自独立地选自氢、卤素和羟基；
m和n各自独立地为1至6的整数；
取代基团Q 1各自独立地选自C 1-C 6烷基、卤素、氘、羟基、巯基、-NR iR j、氧代、硫代、-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、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基、5至10元杂芳基、8至12元稠环芳基和5至12元稠杂芳基；
R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j，其中所述的烷基、烷氧基和卤代烷基各自独立地任选被选自C 1-C 6烷基、卤素、羟基、巯基、-NR iR j、氧代、硫代、羧基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基和5至10元杂芳基中的一个或多个取代基所取代；且
条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
在某些实施方案中，R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，所述的烷基和烷氧基各自独立地任选被选自C 1-C 6烷基、C 1-C 6烷氧基、卤素、氘、氨基、氰基和羟基中的一个或多个取代基所取代。
在某些实施方案中，R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基。
在某些实施方案中，环A为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环A为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000005
Figure PCTCN2022074455-appb-000006
在某些实施方案中，环B为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环B为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000007
Figure PCTCN2022074455-appb-000008
在某些实施方案中，X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，环C和环D各自独立地选自任选被一个或多个取代基Q 1 所取代的6至10元芳基、5至10元杂芳基、8至12元稠环芳基或5至12元稠杂芳基，所述杂芳基或稠杂芳基包含至少一个氮原子。
在某些实施方案中，环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的下述基团：
Figure PCTCN2022074455-appb-000009
在某些实施方案中，环C选自任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000010
在某些实施方案中，环D为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环D为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000011
Figure PCTCN2022074455-appb-000012
在某些实施方案中，X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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 1-C 6烷氧基、C 1-C 6 烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 1各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，优选氢。
在某些实施方案中，R 4各自独立地为氢。
在某些实施方案中，取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基。
在某些实施方案中，R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j。
在某些实施方案中，D如下式(II-A’)或(II-B’)所示：
Figure PCTCN2022074455-appb-000013
其中，
R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基；
环A为
Figure PCTCN2022074455-appb-000014
所述环A任选被一个或多个取代基Q 1所取代；
环B为
Figure PCTCN2022074455-appb-000015
所述环B任选被一个或多个取代基Q 1所取代；
X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子；
R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6 烷氧基，或者R 5a和R 5b一起形成氧代或硫代；
环C选自
Figure PCTCN2022074455-appb-000016
Figure PCTCN2022074455-appb-000017
所述环C任选被一个或多个取代基Q 1所取代；
环D为
Figure PCTCN2022074455-appb-000018
所述环D任选被一个或多个取代基Q 1所取代；
X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基；
R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代；
R 6c、R 6d和R 6e各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
Figure PCTCN2022074455-appb-000019
R 2a各自独立地为氢或C 1-C 6烷基；
R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
R 3各自独立地为氢或卤素；
m和n各自独立地为1至6的整数；
取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基；
R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j； 且
条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
在某些实施方案中，R 1a为氢。
在某些实施方案中，X 1选自-(CR 5aR 5b)m-、任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000020
在某些实施方案中，R 5a和R 5b均为氟。
在某些实施方案中，R 5a和R 5b一起形成氧代或硫代，优选氧代。
在某些实施方案中，X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000021
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基、C 1-C 6烷基、-NR iR j、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 2各自独立地选自-CH 2OH、-CH 2SH、-OH和
Figure PCTCN2022074455-appb-000022
在某些实施方案中，R 3为氢。
在某些实施方案中，R 3为氟。
在某些实施方案中，k为1-10之间的任意数值，优选2-5之间的任意数值。k可以为整数，也可以为小数。
在某些实施方案中，连接子在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在细胞中内化时能够裂解。在某些实施方案中，当ADC进入表达对ADC的抗体部分具有特异性的抗原的细胞时，糖皮质激素受体激动剂药物部分从抗体部分裂解，且裂解释放糖皮质激素受体激动剂的未修饰形式。
在某些实施方案中，连接子中的可裂解部分为可裂解肽部分。在某些实施方案中，相对于包含其他可裂解部分的ADC，包含可裂解肽部分的ADC显示较低的聚集水平，改善的抗体与药物比率。在某些实施方案中，相对于不可裂解的连接子，添加可裂解部分增加细胞毒性和/或效力。在某些实施方案中，可裂解肽部分能够由酶裂解，且连接子为酶能够裂解的连接子。在某些实施方案中，酶为组织蛋白酶，且连接子为组织蛋白酶能够裂解的连接子。在某些实施方案中，与其它分裂机制相比，酶能够裂解的连接子(例如组织蛋白酶能够裂解的连接子)显示上述改善特性中的一种或多种。
在某些实施方案中，连接子包含氨基酸单元L 1，所述氨基酸单元L 1优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸、高赖氨酸、n-甲基-缬氨酸、
Figure PCTCN2022074455-appb-000023
(q为1-6的整数)的氨基酸构成的肽残基，示例性氨基酸单元包括但不限于缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-苯丙氨酸(Ala-Phe)；苯丙氨酸-赖氨酸(Phe-Lys)、苯丙氨酸-高赖氨酸(Phe-Homolys)、n-甲基-缬氨酸-瓜氨酸(Me-Val-Cit)、丙氨酸-丙氨酸(Ala-Ala)、甘氨酸-谷氨酸(Gly-Glu)、谷氨酸-丙氨酸-丙氨酸(Glu-Ala-Ala)、甘氨酸-赖氨酸(Gly-Lys)、甘氨酸-缬氨酸-瓜氨酸(Glv-Val-Cit)和甘氨酸-甘氨酸-甘氨酸(Gly-Gly-Gly)和
Figure PCTCN2022074455-appb-000024
在某些实施方案中，连接子包含拉伸单元，为一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分连接的化学结构片段。示例性拉伸单元包括但不限于
Figure PCTCN2022074455-appb-000025
在某些实施方案中，所述拉伸单元选自
Figure PCTCN2022074455-appb-000026
其中p各自独立地为1、2、3、4、5或6。
在某些实施方案中，连接子选自
Figure PCTCN2022074455-appb-000027
Figure PCTCN2022074455-appb-000028
Figure PCTCN2022074455-appb-000029
在某些实施方案中，连接子选自
Figure PCTCN2022074455-appb-000030
在某些实施方案中，所述的抗体-药物偶联物选自：
Figure PCTCN2022074455-appb-000031
Figure PCTCN2022074455-appb-000032
Figure PCTCN2022074455-appb-000033
其中，Ab、D、k如前所定义，p各自独立地为1、2、3、4、5或6。
在某些实施方案中，所述的抗体-药物偶联物选自
Figure PCTCN2022074455-appb-000034
Figure PCTCN2022074455-appb-000035
Figure PCTCN2022074455-appb-000036
Figure PCTCN2022074455-appb-000037
k选自1至10，可以为整数，也可以为小数。
另一方面，本公开抗体-药物偶联物(ADC)中所述抗体或其抗原结合片段选自鼠源抗体、嵌合抗体、人源化抗体和全人源抗体或其抗原结合片段。
在某些实施方案中，抗体或其抗原结合片段选自抗TNFα抗体、抗IL-4R抗体、抗IL-6/IL-6R抗体、抗IL-13R抗体、抗IL-17/IL-17R抗体、抗IL-23/IL23R抗体、抗IL-36R抗体、抗CD20抗体、抗CD22抗体、抗CD28抗体、抗CD40抗体、抗TSLP抗体或其抗原结合片段。
在某些实施方案中，抗体或其抗原结合片段与人和/或小鼠TNFα结合。结合TNFα的抗体和抗原结合片段是本领域已知的。
在某些实施方案中，抗TNFα抗体或抗原结合片段不与TNF-β结合。
抗TNFα抗体及其抗原结合片段包括例如阿达木单抗、英利昔单抗、赛妥珠单抗(certolizumab pegol)、阿非莫单抗、奈瑞莫单抗(nerelimomab)、奥左拉珠单抗(ozoralizumab)、普拉库鲁单抗(placulumab)和戈利木单抗(golimumab)或其抗原结合片段。另外的抗TNFα抗体和抗原结合片段提供在例如WO 2013/087912、WO 2014/152247和WO 2015/073884中，将其各自通过引用以其整体并入本文。
抗TNFα抗体及其抗原结合片段还包括竞争性抑制阿达木单抗、英利昔单抗、赛妥珠单抗、阿非莫单抗、奈瑞莫单抗、奥左拉珠单抗、普拉库鲁单抗或戈利木单抗与TNFα的结合的抗体及其抗原结合片段。抗TNFα抗体及其抗原结合片段还包括与阿达木单抗、英利昔单抗、赛妥珠单抗、阿非莫单抗、奈瑞莫单抗、奥左拉珠单抗、普拉库鲁单抗或戈利木单抗结合相同TNFα表位的抗体和抗原结合片段。
在某些实施方案中，抗TNFα抗体或其抗原结合片段竞争性抑制阿达木单抗与TNFα的结合。在某些实施方案中，抗TNFα抗体或其抗原结合片段与阿达木单抗结合相同的TNFα表位。在某些实施方案中，抗TNFα抗体或其抗原结合片段是阿达木单抗或其抗原结合片段。在某些实施方案中，抗TNFα抗体或其抗原结合片段是阿达木单抗。
在某些实施方案中，抗TNFα抗体或其抗原结合片段包含阿达木单抗、英利昔单抗、赛妥珠单抗、阿非莫单抗、奈瑞莫单抗、奥左拉珠单抗、普拉库鲁单抗或戈利木单抗的序列，例如互补决定区(CDR)、可变重链结构域(VH)和/或可变轻链结构域(VL)。
本公开还提供了一种式(III-A)或(III-B)所示化合物或其可药用盐，
Figure PCTCN2022074455-appb-000038
其中，
Figure PCTCN2022074455-appb-000039
表示单键或双键；
环A为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
环B为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 5a和R 5b一起形成氧代或硫代；
环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的芳基和杂芳基或稠杂芳基，且环C和环D中至少有一个选自任选被一个或多个取代基Q 1所取代的稠环芳基或稠杂芳基；
X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的芳基或杂芳基、-O-、-S-、-S(O)-、-S(O)(O)-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-、-CR 6f＝CR 6g-和-C≡C-，或者X 2不存在；
R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代；
R 6c、R 6d、R 6e、R 6f和R 6g各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
R 1各自独立地选自氢、烷基和烷氧基，其中所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
Figure PCTCN2022074455-appb-000040
R 2a各自独立地为氢或C 1-C 6烷基；
R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
R 3各自独立地为氢或卤素；
R 4各自独立地选自氢、卤素和羟基；
m和n各自独立地选自1至6的整数；
取代基团Q 1各自独立地选自C 1-C 6烷基、卤素、氘、羟基、巯基、-NR iR j、氧代、硫代、-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、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基、5至10元杂芳基、8至12元稠环芳基和5至12元稠杂芳基；
R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j，其中所述的烷基、烷氧基和卤代烷基各自独立地任选被选自C 1-C 6烷基、卤素、羟基、巯基、-NR iR j、氧代、硫代、羧基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基和5至10元杂芳基中的一个或多个取代基所取代；
R 1a各自独立地选自氢、烷基和烷氧基，所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
Figure PCTCN2022074455-appb-000041
Figure PCTCN2022074455-appb-000042
或者
R 1a和R 1b与其相连的氮原子一起形成：
Figure PCTCN2022074455-appb-000043
或者R 1a和R 1b与其相连的氮原子一起形成硝基基团；
p各自独立地为1、2、3、4、5或6；
L 1是氨基酸单元，优选-甘氨酸-谷氨酸-或
Figure PCTCN2022074455-appb-000044
X为卤素；
PG为氨基保护基；且
条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
在某些实施方案中，R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，所述的烷基和烷氧基各自独立地任选被选自C 1-C 6烷基、C 1-C 6烷氧基、卤素、氘、氨基、氰基和羟基中的一个或多个取代基所取代。
在某些实施方案中，R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基。
在某些实施方案中，L 1选自-甘氨酸-谷氨酸-或
Figure PCTCN2022074455-appb-000045
在某些实施方案中，R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
Figure PCTCN2022074455-appb-000046
在某些实施方案中，PG为Boc或Cbz。
在某些实施方案中，环A为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环A为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000047
Figure PCTCN2022074455-appb-000048
在某些实施方案中，环B为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环B为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000049
Figure PCTCN2022074455-appb-000050
在某些实施方案中，X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子；
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代。
在某些实施方案中，环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的6至10元芳基、5至10元杂芳基、8至12元稠环芳基和5至12元稠杂芳基，所述杂芳基或稠杂芳基包含至少一个氮原子。
在某些实施方案中，环C选自任选被一个或多个取代基Q 1所取代的下述基团：
Figure PCTCN2022074455-appb-000051
在某些实施方案中，环C选自任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000052
在某些实施方案中，环D为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，环D为任选被一个或多个取代基Q 1所取代的
Figure PCTCN2022074455-appb-000053
Figure PCTCN2022074455-appb-000054
在某些实施方案中，X 2为-(CR 6aR 6b)n-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，或者 R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 1各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，优选氢。
在某些实施方案中，R 4各自独立地为氢。
在某些实施方案中，取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基。
在某些实施方案中，R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j。
在某些实施方案中，所述式(III-A)或(III-B)化合物为下式(III-A’)或(III-B’)化合物
Figure PCTCN2022074455-appb-000055
其中，
环A为
Figure PCTCN2022074455-appb-000056
所述环A任选被一个或多个取代基Q 1所取代；
环B为
Figure PCTCN2022074455-appb-000057
所述环B任选被一个或多个取代基Q 1所取代；
X 1选自-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子；
R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代；
环C选自
Figure PCTCN2022074455-appb-000058
Figure PCTCN2022074455-appb-000059
所述环C任选被一个或多个取代基Q 1所取代；
环D为
Figure PCTCN2022074455-appb-000060
所述环D任选被一个或多个取代基Q 1所取代；
X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基；
R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代；
R 6c、R 6d和R 6e各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
Figure PCTCN2022074455-appb-000061
R 2a各自独立地为氢或C 1-C 6烷基；
R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
R 3各自独立地为氢或卤素；
m和n各自独立地为1至6的整数；
取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基；
R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j；
R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基；
R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
Figure PCTCN2022074455-appb-000062
Figure PCTCN2022074455-appb-000063
p各自独立地为1、2、3、4、5或6；
L 1是氨基酸单元，选自-甘氨酸-谷氨酸-或
Figure PCTCN2022074455-appb-000064
X为卤素；
PG为氨基保护基；且
条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
在某些实施方案中，R 1a为氢。
在某些实施方案中，R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
Figure PCTCN2022074455-appb-000065
优选氢或
Figure PCTCN2022074455-appb-000066
在某些实施方案中，R 1a和R 1b均为氢。
在某些实施方案中，X 1选自-(CR 5aR 5b)m-、任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000067
在某些实施方案中，R 5a和R 5b均为氟。
在某些实施方案中，R 5a和R 5b一起形成氧代或硫代，优选氧代。
在某些实施方案中，X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的：
Figure PCTCN2022074455-appb-000068
在某些实施方案中，R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基、C 1-C 6烷基、-NR iR j、-C(O)OR k和C 1-C 6烷氧基，或者R 6a和R 6b一起形成氧代或硫代。
在某些实施方案中，R 2各自独立地选自-CH 2OH、-CH 2SH、-OH和
Figure PCTCN2022074455-appb-000069
在某些实施方案中，R 3为氢。
在某些实施方案中，R 3为氟。
在某些实施方案中，p各自独立地为1或2，优选1。
在某些实施方案中，所述式(III-A)或(III-B)化合物选自
Figure PCTCN2022074455-appb-000070
或其可药用盐。
在某些实施方案中，所述式(III-A)或(III-B)化合物选自
Figure PCTCN2022074455-appb-000071
Figure PCTCN2022074455-appb-000072
Figure PCTCN2022074455-appb-000073
或其可药用盐，其中X为卤素，优选氯或溴，更优选溴。
本公开所述的结构中，
Figure PCTCN2022074455-appb-000074
表示单键或双键。
本领域技术人员可以理解的，例如当R 5a和R 5b的其中一个选自氧代或硫代时，则另一个不存在。
本公开还提供了一种药物组合物，包括至少一种前述抗体-药物偶联物，以及药学上可接受的载体、稀释剂或赋形剂。
在某些实施方案中，所述的药物组合物的单位剂量为0.001mg-1000mg。
在某些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01％-99.99％的前述抗体-药物偶联物。在某些实施方案中，所述的药物组合物含有0.1％-99.9％的前述抗体-药物偶联物。在某些实施方案中，所述的药物组合物含有0.5％-99.5％的前述抗体-药物偶联物。在某些实施方案中，所述的药物组合物含有1％-99％的前述抗体-药物偶联物。在某些实施方案中，所述的药物组合物含有2％-98％的前述抗体-药物偶联物。
在某些实施方案中，基于组合物的总重量，所述的药物组合物含有0.01％-99.99％的药学上可接受的载体、稀释剂或赋形剂。在某些实施方案中，所述的药物组合物含有0.1％-99.9％的药学上可接受的载体、稀释剂或赋形剂。在某些实施方案中，所述的药物组合物含有0.5％-99.5％的药学上可接受的载体、稀释剂或赋形剂。在某些实施方案中，所述的药物组合物含有1％-99％的药学上可接受的载体、稀释剂或赋形剂。在某些实施方案中，所述的药物组合物含有2％-98％的药学上可接受的载体、稀释剂或赋形剂。
本公开所述的抗体-药物偶联物和/或包含抗体-药物偶联物的药物组合物可用于裂解表达表面TNFα的细胞(体外或体内)和/或用于治疗以增加的TNFα(例如，滑液中增加的TNFα)为特征的疾病或病症。在某些实施方案中，抗体-药物偶联物和/或组合物可用于抑制细胞因子释放(体外或体内)和/或用于治疗自身免疫性或炎性疾病。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗克罗恩病。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗溃疡性结肠炎。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗类风湿性关节炎(RA)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗幼年特发性关节炎(JA)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗银屑病关节炎(PsA)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗脊椎关节病，例如强直性脊柱炎(AS)或轴型脊椎关节炎(axSpA)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗成人克罗恩病(CD)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗小儿克罗恩病。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗溃疡性结肠炎(UC)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗斑块型银屑病(Ps)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗化脓性汗腺炎(HS)。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗葡萄膜炎。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗白塞斯病。在某些实施方案中，抗体-药物偶联物和/或组合物用于治疗银屑病，包括斑块型银屑病。
本公开还提供了一种向表达TNFα的细胞递送糖皮质激素受体激动剂的方法，包括使表达TNFα的细胞与本公开所述的抗体-药物偶联物接触的步骤。
本公开还提供了一种确定抗体-药物偶联物的抗炎活性的方法。此类方法可以包括使表达TNFα的细胞与如本文所描述的抗体-药物偶联物接触的步骤。一些实施例包含使表达TNFα的细胞与如本文所描述的抗体-药物偶联物接触，并确定与 对照细胞相比，促炎细胞因子从细胞中的减少的释放。一些实施例包含确定抗体-药物偶联物的抗炎活性的体外方法。
一些实施例包含筛选方法(例如体外方法)，其包括使细胞(例如，表达TNFα的细胞)直接地或间接地与抗体-药物偶联物接触，并且确定抗体-药物偶联物是否调节细胞的活性或功能，如例如通过细胞形态或生存力、标记物的表达、分化或去分化、细胞呼吸、线粒体活性、膜完整性、成熟、增殖、生存力、凋亡或细胞死亡的变化所反映的。直接相互作用的一个示例是物理相互作用，而间接相互作用包括例如组合物对中间分子的作用，所述中间分子反过来作用于参考实体(例如，细胞或细胞培养物)。
本公开还提供了一种药物组合物，包括至少一种前述的式(III-A)或(III-B)化合物或其可药用盐，以及药学上可接受的载体、稀释剂或赋形剂。所述化合物或其可药用盐及其药物组合物可用于治疗免疫性疾病。
本公开进一步提供了一种试剂盒，其包含本公开所述的抗体-药物偶联物，或药物组合物。
术语定义
除非另有限定，本公开所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本公开所述相似或等同的任何方法和材料实施或测试本公开，但本公开描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“连接子”、“连接单元”、“接头单元”、“接头”或“连接片段”是指一端与配体连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。例示性的接头构件包括6-马来酰亚氨基己酰基(MC)、马来酰亚氨基丙酰基(MP)、缬氨酸-瓜氨酸(Val-Cit或vc)、丙氨酸-苯丙氨酸(ala-phe)、对氨基苄氧羰基(PAB)，及那些源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯(SMCC，在本文中也称作MCC)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(SIAB)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元。可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利 No.5,208,020)。
术语“拉伸单元”指一端通过碳原子与抗体共价连接而另一端与氨基酸单元、二硫化物部分、磺酰胺部分或非肽化学部分连接的化学结构片段。
术语“间隔单元”是一种双功能化合结构片段，可用于偶联氨基酸单元和糖皮质激素最终形成抗体-药物偶联物，这种偶联方式可以将糖皮质激素选择性的连接到氨基酸单元上。
术语“氨基酸”是指分子结构中含有氨基和羧基，并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H 2NCHRCOOH，R为H、取代或未取代烷基等。根据氨基连结在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸，包括甘氨酸(Glycine)、丙氨酸(Alanine)、缬氨酸(Valine)、亮氨酸(Leucine)、异亮氨酸(Isoleucine)、苯丙氨酸(Phenylalanine)、色氨酸(Tryptophan)、酪氨酸(Tyrosine)、天冬氨酸(Aspartic acid)、组氨酸(Histidine)、天冬酰胺(Asparagine)、谷氨酸(Glutamic acid)、赖氨酸(Lysine)、谷氨酰胺(Glutamine)、甲硫氨酸(Methionine)、精氨酸(Arginine)、丝氨酸(Serine)、苏氨酸(Threonine)、半胱氨酸(Cysteine)、脯氨酸(Proline)等。非天然氨基酸如瓜氨酸。如本领域技术人员所公知的，非天然氨基酸并不构成天然蛋白质，因此也不参与本公开中抗体的合成。本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
Figure PCTCN2022074455-appb-000075
Figure PCTCN2022074455-appb-000076
术语“抗体-药物偶联物”，指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“抗体-药物偶联物”(antibody drug conjugate，ADC)，指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的糖皮质激素相连。其中抗体或抗体片段可通过其中的特定基团(例如链间二硫键)与包含接头的糖皮质激素分子相结合。
术语“载药量”是指抗体-药物偶联物群体中，每个抗体-药物偶联物分子载有的药物平均数量，也可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个糖皮质激素(D)。在本公开的实施方案中，载药量表示为k，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选 1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征鉴定偶联反应后每个ADC分子的药物平均数量。
本公开单抗分子大小变异体测定法(CE-SDS)可采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛电泳法(2015年版《中国药典》0542)，定量测定重组单克隆抗体产品的纯度。
本公开的一个实施方案中，糖皮质激素通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制抗体-药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人源抗体，优选人源化抗体和全人源抗体。
术语“鼠源抗体”在本公开中为根据本领域知识和技能用鼠制备抗体。制备时用特定抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因 连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。进一步描述参与人源化可使用小鼠抗体的方法的文献包括，例如Queen等，Proc.，Natl.Acad.Sci.USA，88，2869，1991和Winter及其同事的方法[Jones等，Nature，321，522(1986)，Riechmann，等，Nature，332，323-327(1988)，Verhoeyen，等，Science，239，1534(1988)]。
术语“全人源抗体”、“全人抗体”或“完全人源抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。本公开为全人源单克隆抗体。全人抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature 341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA 85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功 用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1、IgG2、IgG3或IgG4亚型)、IgA1、IgA2、IgD、IgE或IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸 残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸(参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996))。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如：大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“抗TNFα抗体”或“与TNFα结合的抗体”是指能够例如以足够的亲和力结合TNFα的抗体，使得该抗体可用作靶向TNFα的治疗剂。抗TNFα抗体与不相关的非TNFα蛋白的结合程度可以小于如，例如，通过放射免疫测定(RIA)所测量的抗体与TNFα的结合的约10％。在某些实施方案中，与TNFα结合的抗体具有≤1μM、≤100nM、≤10nM、≤1nM或≤0.1nM的解离常数(Kd)。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、 粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代。
术语“亚链烯基”指包含具有2至8个碳原子，优选地具有2至6个碳原子，更优选地具有2至4个碳原子并在任何位置具有至少一个双键的线性链烯基，包括例如亚乙烯基、亚烯丙基(allylene)、亚丙烯基、亚丁烯基、亚异戊二烯基 (prenylene)、亚丁二烯基(butadienylene)、亚戊烯基、亚戊二烯基、亚己烯基、亚己二烯基等。
术语“亚链炔基”包括具有2至8个碳原子，优选地具有2至6个碳原子，更优选地具有2至4个碳原子且在任何位置具有至少一个叁键的线性亚链炔基，包括例如亚乙炔基、亚丙炔基、亚丁炔基、亚戊炔基、亚己炔基等。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。“碳环”指的是环烷基中的环系。
术语“螺环烷基”指5至20元的单环之间共用一个碳原子(称螺原子)的多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基或多螺环烷基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺环烷基。“螺碳环”指的是螺环烷基中的环系。螺环烷基的非限制性实例包括：
Figure PCTCN2022074455-appb-000077
术语“稠环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。“稠碳环”指的是稠环烷基中的环系。稠环烷基的非限制性实例包括：
Figure PCTCN2022074455-appb-000078
术语“桥环烷基”指5至20元，任意两个环共用两个不直接连接的碳原子的全碳多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更有选为双环或三环。桥环烷基的非限制性实例包括：
Figure PCTCN2022074455-appb-000079
所述环烷基环可以稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性实例包括茚满基、四氢萘基、苯并环庚烷基等。环烷基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至6个环原子。单环杂环基的非限制性实例包括吡咯烷基、咪唑烷基、四氢呋喃基、四氢噻吩基、二氢咪唑基、二氢呋喃基、二氢吡唑基、二氢吡咯基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等，优选哌啶基、吡咯烷基。多环杂环基包括螺环、稠环和桥环的杂环基。“杂环”指的是杂环基中的环系。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。“螺杂环”指的是螺杂环基中的环系。螺杂环基的非限制性实例包括：
Figure PCTCN2022074455-appb-000080
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7 至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。“稠杂环”指的是稠杂环基中的环系。稠杂环基的非限制性实例包括：
Figure PCTCN2022074455-appb-000081
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更有选为双环或三环。桥杂环基的非限制性实例包括：
Figure PCTCN2022074455-appb-000082
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2022074455-appb-000083
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基、羧基或羧酸酯基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。“芳环”指的是芳基中的环系。芳基非限制性实例包括：
Figure PCTCN2022074455-appb-000084
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基，优选苯基。
术语“稠环芳基”可以是含有8-14个环原子由两个或两个以上环状结构彼此共用两个相邻的原子连接起来形成的不饱和的具有芳香性的稠环结构，环原子优选8-12个。例如包括全部不饱和稠环芳基，例如萘、菲等，还包括部分饱和稠环芳基，例如苯并3-8元饱和单环环烷基、苯并3-8元部分饱和单环环烷基。“稠芳环”指的是稠环芳基中的环系。稠环芳基具体实例如2,3-二氢-1H-茚基、IH-茚基、1,2,3,4-四氢萘基、1,4-二氢萘基等。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至12元，例如咪唑基、呋喃基、噻吩基、噻唑基、吡唑基、噁唑基、吡咯基、四唑基、吡啶基、嘧啶基、噻二唑、吡嗪基等，优选为咪唑基、吡唑基、嘧啶基或噻唑基；更优选为吡唑基或噻唑基。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。“杂芳环”指的是杂芳基中的环系。杂芳基非限制性实例包括：
Figure PCTCN2022074455-appb-000085
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“稠杂芳基”可以是含有5-14个环原子(其中至少含有一个杂原子)由两个 或两个以上环状结构彼此共用两个相邻的原子连接起来形成的不饱和的具有芳香性的稠环结构，同时包括碳原子、氮原子和硫原子可以被氧代，优选"5-12元稠杂芳基"、"7-12元稠杂芳基"、"9-12元稠杂芳基"等，例如苯并呋喃基、苯并异呋喃基、苯并噻吩基、吲哚基、异吲哚、苯并噁唑基、苯并咪唑基、吲唑基、苯并三唑基、喹啉基、2-喹啉酮、4-喹啉酮、1-异喹啉酮、异喹啉基、吖啶基、菲啶基、苯并哒嗪基、酞嗪基、喹唑啉基、喹喔啉基、酚嗪基、喋啶基、嘌呤基、萘啶基、吩嗪、吩噻嗪等。“稠杂芳环”指的是稠杂芳基中的环系。
稠杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、羧基或羧酸酯基。
术语“烷硫基”指-S-(烷基)和-S-(非取代的环烷基)，其中烷基的定义如上所述。烷硫基的非限制性实例包括：甲硫基、乙硫基、丙硫基、丁硫基、环丙硫基、环丁硫基、环戊硫基、环己硫基。烷硫基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基中的一个或多个取代基所取代。
术语“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
术语“卤代烷基”指被卤素取代的烷基，其中烷基如上所定义。
术语“氘代烷基”指被氘原子取代的烷基，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“氧代”指＝O基团。例如，碳原子与氧原子通过双键连接，其中形成酮或醛基。
术语“硫代”指＝S基团。例如，碳原子与硫原子通过双键连接，形成硫代羰基-C(S)-。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“氰基”指-CN。
术语“硝基”指-NO 2。
术语“羧基”指-C(O)OH。
术语“醛基”指-CHO。
术语“羧酸酯基”指-C(O)O(烷基)或-C(O)O(环烷基)，其中烷基、环烷基如上所定义。
术语“酰卤”指含有-C(O)-卤素的基团的化合物。
术语“磺酰基”指-S(O)(O)-。
术语“亚磺酰基”指-S(O)-。
“氨基保护基”是本领域已知的适当的用于氨基保护的基团，参见文献(“Protective Groups in Organic Synthesis”，5 Th.Ed.T.W.Greene&P.G.M.Wuts)中的氨基保护基团，优选地，所述的氨基保护基可以是(C 1-10烷基或芳香基)酰基，例如：甲酰基，乙酰基，苯甲酰基等；可以是(C 1-6烷基或C 6-10芳基)磺酰基；也可以是(C 1-6烷氧基或C 6-10芳基氧基)羰基，例如：Boc或Cbz；还可以是取代或非取代的烷基，例如：三苯甲基(Tr)、2,4-二甲氧基苄基(DMB)、对甲氧基苄基(PMB)或苄基(Bn)。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环烷基团”意味着烷基可以但不必须存在，该说明包括杂环烷基团被烷基取代的情形和杂环烷基团不被烷基取代的情形。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药物载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩 成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。
本公开中化合物可以含有一个或多个不对称中心，因此可以产生对映异构体、非对映异构体，并且其可以根据绝对立体化学定义为(R)-或(S)-或用于氨基酸的(D)-或(L)-的其它立体异构形式。本公开包括所有可能异构体以及其外消旋和光学纯的形式。光学活性的(+)和(-)、(R)-和(S)-或(D)-和(L)-异构体可以使用手性合成子或手性试剂制备，或者可以使用常规方法例如色谱法和分级结晶制备。用于制备/分离各个对映体的常规方法包括从合适的的光学纯前体手性合成或使用例如手性高效液相色谱法(HPLC)的外消旋物(或盐或衍生物的外消旋物)拆分。当本文描述的化合物含有烯双键或其它几何不对称性中心，除非另有说明，否则其意味着所述化合物包括E和Z几何异构体。而且，所有的互变异构形式也意味着包括在内。
本公开所述化合物的化学结构中，键
Figure PCTCN2022074455-appb-000086
表示未指定构型，即如果化学结构中存在手性异构体，键
Figure PCTCN2022074455-appb-000087
可以为
Figure PCTCN2022074455-appb-000088
或者同时包含
Figure PCTCN2022074455-appb-000089
两种构型。本公开所述化合物的化学结构中，键
Figure PCTCN2022074455-appb-000090
并未指定构型，即可以为Z构型或E构型，或者同时包含两种构型。
“立体异构体”指通过相同的键键合但具有不同的三维结构的相同原子组成的化合物，其不可互换。本公开中预期各种立体异构体及其混合物，并且包括“对映异构体”，其指其分子彼此为不能重叠的镜像的两种立体异构体。
“互变异构体”指质子从分子的一个原子转移到同一分子的另一个原子。本公开中包括任何所述化合物的互变异构体。
本公开所述化合物或其可药用盐、或其异构体的任何同位素标记的衍生物都被本公开所覆盖。能够被同位素标记的原子包括但不限于氢、碳、氮、氧、磷、氟、氯、碘等。它们可分别被同位素同位素 2H(D)、 3H、 11C、 13C、 14C、 15N、 18F、 31P、 32P、 35S、 36Cl和 125I等代替。除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少3000倍的丰度的氘(即，至少45％的氘掺入)。
附图说明
图1：小分子类固醇在脂多糖刺激人PBMC分泌细胞因子实验中的抑炎活性
图2：抗TNF-ADC在膜结合型TNFα介导的GRE报告基因系统中的活性
图3：抗TNF-ADC在脂多糖刺激人单核细胞分泌细胞因子实验中的活性
图4：抗TNF-ADC(4nM)在LPS诱导的人单核细胞细胞因子释放实验中的活性
图5：测试例5的实验流程
图6：抗TNF-ADC在小鼠胶原抗体诱导的关节炎(CAIA)模型中的活性
图7：抗TNF-ADC在小鼠胶原抗体诱导的关节炎(CAIA)模型中不同时间点的活性
图8：测试例6的实验流程
图9：抗TNF-ADC在迟发型超敏反应(DTH)模型中的活性
具体实施方式
以下结合实施例进一步描述本公开所述的化合物、可药用盐的制备，但这些实施例并非限制本公开中的范围。
本公开中实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
NMR位移(δ)以10 -6(ppm)的单位给出。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)，氘代氯仿(CDCl 3)，氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用Shimadzu 2010 Mass Spectrometer或Agilent 6110A MSD质谱仪。
HPLC的测定使用Shimadzu LC-20A systems、Shimadzu LC-2010HT series或安捷伦Agilent 1200 LC高压液相色谱仪(Ultimate XB-C18 3.0*150mm色谱柱或Xtimate C18 2.1*30mm色谱柱)。
手性HPLC分析测定使用Chiralpak IC-3 100×4.6mm I.D.,3um、Chiralpak AD-3 150×4.6mm I.D.,3um、Chiralpak AD-3 50×4.6mm I.D.,3um、Chiralpak AS-3 150×4.6mm I.D.,3um、Chiralpak AS-3 100×4.6mm I.D.,3μm、ChiralCel OD-3 150×4.6mm I.D.,3um、Chiralcel OD-3 100×4.6mm I.D.,3μm、ChiralCel OJ-H 150×4.6mm I.D.,5um、Chiralcel OJ-3 150×4.6mm I.D.,3um色谱柱；薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm。
柱层析一般使用烟台黄海硅胶100～200目、200～300目或300～400目硅胶为载体。
手性制备柱使用DAICEL CHIRALPAK IC(250mm*30mm,10um)或Phenomenex-Amylose-1(250mm*30mm,5um)。
CombiFlash快速制备仪使用Combiflash Rf150(TELEDYNE ISCO)。
激酶平均抑制率及IC 50值的测定用NovoStar酶标仪(德国BMG公司)。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃～30℃。
实施例中的反应进程的监测采用薄层色谱法(TLC)，反应所使用的展开剂，纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，C：石油醚/乙酸乙酯体系，D：石油醚/乙酸乙酯/甲醇，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和醋酸等碱性或酸性试剂进行调节。
1.0M Tris缓冲液pH＝8.30±0.1的配制：
50mL容量瓶中称取tris 6.0g，加入40mL纯化水，振荡溶解，滴加入浓盐酸1.2mL，调pH至8.30，再用纯化水定容。
A缓冲液的配制：
在2.0L的容器中，加入KH 2PO 4(8.50g)、K 2HPO 4(8.56g)、NaCl(5.86g)和EDTA(1.50g)，加入1.6L注射用水，搅拌半小时，完全溶解后再用注射用水定容至2.0L，测定pH为6.30±0.1。
下述实验中所用缩写代表的含义如下:
DAST:二乙胺基三氟化硫；THF：四氢呋喃；NMP:N-甲基吡咯烷酮；DCM:二氯甲烷；m-CPBA:间氯过氧苯甲酸；DIEA:N,N-二异丙基乙胺；TEA：三乙胺；Boc：叔丁氧羰基，MeOH:甲醇；Et 2O:乙醚。
实施例1
Figure PCTCN2022074455-appb-000091
Figure PCTCN2022074455-appb-000092
第一步
将化合物1-1(500mg，1.9mmol，1.0eq)，化合物1-2(730mg，2.3mmol，1.2eq)，Pd(PPh 3) 4(660mg，0.57mmol，0.3eq)，K 2CO 3(960mg，6.9mmol，3.6eq)及DMF(35mL，70V)加入到100mL单口瓶中，氮气保护下置换三次，80℃搅拌至反应结束。将EA(50mL)及H 2O(130mL)加入反应液中，分液，水相用EA(30mLx 2)萃取。合并有机相，依次用H 2O(50mL x 3)，饱和LiCl(50mL)及饱和NaCl溶液洗涤(30mL)，无水Na 2SO 4干燥，过滤后旋蒸除去溶剂。过柱得到粗品，用PE:EA＝2:1打浆，过滤得到化合物1-3(220mg，收率31％)。
Ms(ESI):m/z 318[M-55] +。
第二步
将化合物1-3(100mg，0.268mmol，1.1eq)，化合物1-4(92mg，0.243mmol，1.0eq)，无水MgSO 4(146mg，1.215mmol，5.0eq)，无水CH 3CN(5mL)加入50mL三口瓶中，氮气保护下于室温搅拌0.7h。冰浴降温至0℃，缓慢滴加CF 3SO 3H(182mg，1.215mmol，5.0eq)，滴完后，自然升温到室温反应，反应完全后，在冰浴下，加入EA和饱和碳酸氢钠溶液进行淬灭，并调到pH值大于8，然后再用EA萃取水相，合并有机相，再用饱和食盐水洗涤，用无水硫酸钠干燥，浓缩后粗品经过prep-HPLC，得到化合物1-A(64.5mg，收率42％)。
Ms(ESI):m/z 632.3[M+H] +。
1H 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＝7.8Hz,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)。
实施例2
Figure PCTCN2022074455-appb-000093
氮气氛下，在一个25mL反应瓶中加入化合物1-4(176mg，0.47mmol，1.0eq)和无水硫酸镁(282mg，2.34mmol，5.0eq)，加入无水乙腈(5mL)，室温下搅拌反应90分钟。再向上述混合物中加入化合物2-1(160mg，0.49mmol，1.05eq)，冰浴冷却至0～5℃，由注射器加入三氟甲磺酸(351mg，2.34mmol，5.0eq)，维持冰浴搅拌反应至结束)。将反应液过滤硅藻土，乙酸乙酯润洗，滤液中再加入乙酸乙酯和水，分液，有机相再用饱和食盐水洗涤一次，干燥，减压浓缩得粗品，通过制备-HPLC纯化得化合物2-A(88mg，收率32.2％)。
Ms(ESI):m/z 584.42[M+1] +。
实施例3
Figure PCTCN2022074455-appb-000094
Figure PCTCN2022074455-appb-000095
第一步
氮气保护下，将化合物3-1(10.0g，45.03mmol)，(Bpin) 2(联硼酸频那醇酯，18.3g，72.05mmol)，X-Phos(1.36g，2.70mmol)和KOAc(8.84g，90.06mmol)溶于1，4-二氧六环(150mL)中。加入Pd 2(dba) 3(1.65g，1.80mmol)，升至90℃反应。冷却至室温，过滤后将滤液浓缩，柱层析分离(PE/EA)，得到化合物3-2共13g。
MS-ESI:m/z 270.2[M+H] +。
第二步
将化合物3-2(13g，45.03mmol)溶于甲苯(100mL)中，加入Boc 2O(13.4mL，58.54mmol)，升至100℃反应。冷却至室温，浓缩后柱层析分离(PE/EA)，得到化合物3-3共16g，两步收率：96.2％。
MS-ESI:m/z 396.1[M+Na] +。
第三步
氮气保护下，将化合物3-3(2.0g，5.42mmol)，化合物3-4(2.16g，10.84mmol)和K 2CO 3(3.75g，27.10mmol)溶于四氢呋喃(50mL)中。加入Pd(dppf)Cl 2·DCM(441mg，0.54mmol)，升至80℃反应。冷却至室温，加水淬灭，用乙酸乙酯提取，浓缩后柱层析分离(PE/EA)，得到化合物3-5共1.33g，收率：67.9％。
MS-ESI:m/z 384.1[M+Na] +。
第四步
氮气保护下，将化合物1-4(123mg，0.327mmol)和MgSO 4(197mg，1.635mmol)溶于乙腈(10mL)中，室温反应1小时。加入化合物3-5(130mg，0.360mmol)的乙腈(10mL)溶液，冷却至0℃，缓慢滴加三氟甲磺酸(145μL，1.635mmol)，加料完毕后，自然升至室温反应。过滤后将滤液浓缩，使用制备HPLC分离(CH 3CN/H 2O)，得到化合物3-A共90mg，收率44.4％。
MS-ESI:m/z 620.3[M+H] +。
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＝10.1 Hz,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)。
实施例4
Figure PCTCN2022074455-appb-000096
第一步
在1000mL单口瓶中加入化合物4-1(48g，176.4mmol，1.0eq)，联硼酸频哪醇酯(71.7g，282.2mmol，1.6eq)，醋酸钾(34.6g，352.8mmol，2.0eq)，PdCl 2(dppf)(6.4g，8.82mmol，0.05eq)，二氧六环(500mL)，氮气保护下升温至95℃搅拌至反应完全，停止反应，反应液冷却，再加入化合物4-2(80.8g，352.8mmol，2.0eq)，碳酸钾(48.8g，352.8mmol，2.0eq)，PdCl 2(dppf)(6.4g，8.82mmol，0.05eq)，再加水(100mL)搅拌，氮气保护下加热到80度搅拌至反应完全。反应液冷却后加乙酸乙酯和水搅拌，分液，无水硫酸钠干燥，浓缩后粗品过柱，得到化合物4-3约54g收率90％。
Ms(ESI):m/z 342.1[M+1] +。
第二步
在500mL三口瓶中加入化合物4-3(7.0g，20.5mmol，1.0eq)，高锰酸钾(9.7g，61.6mmol，3.0eq)，四叔丁基溴化铵(20.0g，61.6mmol，3.0eq)，再加二氯乙烷(140mL)，氮气保护下室温搅拌至反应完全，停止反应，反应液冰水冷却，加入10％亚硫酸氢钠和醋酸搅拌，分液，有机层加无水硫酸钠干燥，抽滤，浓缩，粗品过柱，得到化合物4-4约6.2g，收率85％。
Ms(ESI):m/z 378.1[M+23] +。
第三步：
在500mL三口瓶中加入化合物4-4(20.0g，56.0mmol，1.0eq)，丙二硫醇(12.1g，112.0mmol，2.0eq)，三氟化硼乙醚(23.8g，168.0mmol，3.0eq)，再加氯仿(100mL)，氮气保护下加热回流搅拌至反应完全，停止反应，反应液冰水冷却，加入水搅拌，固体全溶，分液，有机层加无水硫酸钠干燥，抽滤，浓缩，粗品加石油醚搅拌，抽滤得到化合物4-5约22g，直接投入下一步。
Ms(ESI):m/z 346.0[M+1] +。
第四步：
在100mL三口瓶中加入化合物4-5(22.0g，以56.0mmol计，1.0eq)，二碳酸二叔丁酯(24.4g，112.0mmol，2.0eq)，再加乙醇(60mL)，氮气保护下加热50度搅拌至反应完全，停止反应，反应液浓缩，过柱，得到化合物4-6约18.5g，收率71％。
Ms(ESI):m/z 468.1[M+23] +。
第五步：
在100mL三口瓶中加入化合物4-6(4.7g，13.6mmol，1.0eq)，DAST(6.6g，40.9mmol，3.0eq)，再加二氯甲烷(50mL)，氮气保护下加热50℃搅拌至反应完全，停止反应，冰水冷却下加水淬灭，分液，有机层无水硫酸钠干燥，抽滤，浓缩，过柱，得到化合物4-7约3.9g，收率76％。
Ms(ESI):m/z 400.1[M+23] +。
第六步：
在100mL三口瓶中加入化合物4-7(2.0g，5.3mmol，1.0eq)，再加四氢呋喃(25mL)溶解，氮气保护下，冷却到0℃左右，缓慢滴加1.0M的氢化铝锂四氢呋喃溶液(8.0mL，8.0mmol，1.5eq)，保持0℃左右搅拌至反应完全，停止反应，冰水冷却下加水(0.8mL)淬灭，再加3.0M氢氧化钾水溶液(0.8mL)，再加水(1.6mL)搅拌15min，抽滤，滤液加无水硫酸钠干燥，抽滤，浓缩，得到化合物4-8约2.2g，直接投入下一步。
Ms(ESI):m/z 372.1[M+23] +。
第七步：
在100mL三口瓶中加入化合物4-8(2.2g，5.3mmol，1.0eq)，再加乙酸乙酯(25mL)溶解，氮气保护下，冷却到5℃左右，加入Dess-Martin氧化剂(6.7g， 15.9mmol，3.0eq)，保持10℃左右搅拌至反应完全，停止反应，抽滤，滤液浓缩，粗品过柱纯化，得到化合物4-9约1.5g，收率82％。
Ms(ESI):m/z 370.1[M+23] +。
第八步：
在100mL三口瓶中加入化合物1-4(1.2g，3.0mmol，1.0eq)，化合物4-9(1.1g，3.17mmol，1.05eq)，再加无水硫酸镁(1.8g，15.0mmol，5.0eq)，再加乙腈(25mL)搅拌，氮气保护下，冷却到0℃以下，加入三氟甲磺酸(2.3g，15.0mmol，5.0eq)，保持0℃左右搅拌至反应完全，停止反应，抽滤，滤液直接制备，得到化合物4-A约1.5g，收率70％。
Ms(ESI):m/z 606.3[M+1] +。
1H-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)。
实施例5
Figure PCTCN2022074455-appb-000097
将化合物5-1(85.3mg，0.262mmol，1.1eq)，化合物5-2(94mg，0.238mmol，1.0eq，麦克林/C10492138/P>98％)，无水MgSO 4(143mg，1.19mmol，5.0eq)，无水CH 3CN(4mL)加入25mL希莱克反应瓶中，氮气保护下于室温搅拌0.7h。冰浴降温至0℃，缓慢滴加CF 3SO 3H(179mg，1.19mmol，5.0eq)，滴完后自然升温反应至完全。在冰浴下，加入EA和饱和碳酸氢钠溶液进行淬灭，然后再用EA萃取水相，合并有机相，再用饱和食盐水洗涤，用无水硫酸钠干燥，浓缩后得到粗品，经prep-HPLC，得到化合物5-A(67.1mg，收率：42.6％)，
Ms(ESI):m/z 602.3[M+H] +。
1H 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)。
实施例6
Figure PCTCN2022074455-appb-000098
第一步
氮气保护下，将化合物3-A(90.0mg，0.145mmol,1.0eq)，加入甲苯2mL，然后加入BOC酸酐(63.3mg，0.290mmol，2.0eq)，体系加热到100℃反应至完全，直接减压浓缩干，柱层析，得到48.0mg化合物3-A-1。
MS-ESI:m/z 742.3[M+Na] +。
第二步
氮气保护下，将化合物3-A-1(45.0mg，0.063mmol)和四氮唑(66.0mg，0.945mmol)溶于N，N-二甲基乙酰胺(1.5mL)中，加入N，N-二乙基亚磷酰胺二 叔丁酯(187.0mg，0.756mmol)，室温下反应2小时。冷却至0℃，缓慢加入H 2O 2(50.0mg，0.82mmol)，加完后在室温下搅拌至反应完全，加水2mL，过滤，滤饼烘干，得到约40.0mg化合物3-A-2，直接投入下步反应。
MS-ESI:m/z 943.3[M+Na] +。
第三步
氮气保护下，将化合物3-A-2(40.0mg，0.043mmol)溶于二氯甲烷(1.0mL)中，冷却至0℃，加入三氟乙酸(0.3mL)，室温下反应3小时。浓缩后使用制备HPLC分离(CH 3CN/H 2O，+0.1％三氟乙酸)，得到化合物3-B约12.0mg。
ESI:m/z 700.3[M+H] +。
实施例7
Figure PCTCN2022074455-appb-000099
第一步
氮气保护下，将化合物4-A(45.0mg，0.074mmol,1.0eq)，加入甲苯2mL，然后加入BOC酸酐(32.0mg，0.158mmol，2.0eq)，体系加热到100℃反应至反应完全，直接减压浓缩，柱层析，得到45.2mg化合物4-A-1。
MS-ESI:m/z 728.2[M+Na] +。
第二步
氮气保护下，将化合物4-A-1(45.0mg，0.063mmol)和四氮唑(66.0mg，0.945mmol)溶于N，N-二甲基乙酰胺(1.0mL)中，加入N，N-二乙基亚磷酰胺二叔丁酯(249.0mg，0.756mmol)，室温下反应2小时。冷却至0℃，缓慢加入H 2O 2(50.0mg，0.82mmol)，加完后在室温下搅拌至反应完全，加水2mL，过滤，滤饼烘干，得到化合物4-A-2约43.0mg，直接投入下步反应。
MS-ESI:m/z 920.3[M+Na] +。
第三步
氮气保护下，将化合物4-A-2(40.0mg，0.042mmol)溶于二氯甲烷(1.0mL)中，冷却至0℃，加入三氟乙酸(0.3mL)，室温下反应3小时。浓缩后使用制备HPLC分离(CH 3CN/H 2O，+0.1％三氟乙酸)，得到化合物4-B约15.0mg。
ESI:m/z 686.2[M+H] +。
实施例8
Figure PCTCN2022074455-appb-000100
第一步
氮气保护下，将化合物3-A(2.60g，4.20mmol)和3-6(2.03g，4.20mmol)溶于N，N-二甲基乙酰胺(30mL)中，加入三乙胺(1.27g，12.60mmol)，冷却 至0℃，缓慢加入T 3P(5.35g，8.40mmol，50％in DMF)，室温反应至完全。反应液直接用制备HPLC纯化，得产品3-7共965mg，收率：21.2％。
MS-ESI:m/z 1106.5[M+Na] +。
第二步
氮气保护下，将化合物3-7(805mg，0.778mmol)和四氮唑(818mg，11.670mmol)溶于N，N-二甲基乙酰胺(10mL)中，加入N，N-二乙基亚磷酰胺二叔丁酯(2.6mL，9.336mmol)，室温下反应2小时。冷却至0℃，缓慢加入H 2O 2(437μL，4.279mmol，30％in water)，加完后在室温下搅拌1小时。浓缩后柱层析分离(CH 3CN/H 2O)，得到化合物3-8共692mg，收率：73.1％。
第三步
氮气保护下，将化合物3-8(830mg，0.650mmol)溶于乙腈(20mL)中，加入哌啶(302μL，3.250mmol)，室温下反应。浓缩后用15mL石油醚打浆，重复三次，得到化合物3-9共645mg，直接用于下一步反应。
MS-ESI:m/z 1054.5[M+H] +。
第四步
氮气保护下，将2-溴乙酸(170mg，1.224mmol)和EEDQ(305mg，1.224mmol)溶于N，N-二甲基乙酰胺(5mL)中，室温下反应1小时。加入化合物3-9(645mg，0.612mmol)的N，N-二甲基乙酰胺溶液(5mL)，室温下反应。反应液用二氯甲烷(200mL)稀释，依次用1M氢溴酸水溶液，饱和碳酸氢钠溶液和饱和食盐水洗涤，有机相浓缩后得到化合物3-10共830mg，直接用于下一步反应。
MS-ESI:m/z 1196.4[M+Na] +。
第五步
氮气保护下，将化合物3-10(830mg，0.706mmol)溶于二氯甲烷(8mL)中，冷却至0℃，加入三氟乙酸(4mL)，室温下反应。浓缩后使用制备HPLC分离(CH 3CN/H 2O，+0.1％三氟乙酸)，得到化合物3-B00共220mg，三步收率：33.6％。
MS-ESI:m/z 1028.2[M+Na] +。
1H 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＝5.5Hz,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)。
实施例9
Figure PCTCN2022074455-appb-000101
第一步
在25mL三口瓶中加入化合物4-A(0.329g，0.544mmol，1.05eq)，加化合物4-10(0.25g，0.52mmol，1.0eq)，三乙胺(0.25g，1.56mmol，3.0eq)，DMF(2mL)，加完后在冰浴下降温5-10min，使内温达到-5℃，然后缓慢加入T3P(50％DMF)(0.9mL,1.82mmol,3.5eq),加完后在自然升温条件下搅拌至反应完全，反应液直接经pre-HPLC纯化，得化合物4-11(223mg,收率40％。
Ms(ESI):m/z 1092.4[M+Na] +。
第二步
在50mL三口瓶中加入化合物4-11(0.22g，0.206mmol，1.0eq)，加原料四氮唑(0.20g，2.87mmol，14.0eq)，N,N-二乙基亚磷酰胺二叔丁酯(0.616g，2.47mmol，12.0eq)，DMF(2.6mL)，加完后在室温下反应2小时，冰浴下降温到0℃，然后缓慢加入H 2O 2(30％)(0.13g,0.57mmol,5.5eq)，加完后在室温下搅拌至反应完全，反应液直接经pre-HPLC纯化，得化合物4-12(184.1mg,收率70.8％。
Ms(ESI):m/z 1284.6[M+Na] +。
第三步
在25mL单口瓶中加入化合物4-12(0.285g，0.233mmol，1.0eq)，加原料piperidine(0.17g，1.96mmol，8.5eq)，乙腈(5mL)，加完后在室温条件下搅拌。减压浓缩，加入5mL石油醚打浆，在室温下搅拌，然后过滤，滤饼再用2mL石油醚洗涤两次，得化合物4-13(209mg,收率91％。
Ms(ESI):m/z 1004.4[M+H] +。
第四步
在25mL单口瓶中加入原料2-溴乙酸(0.074g，0.523mmol，2.6eq)，加原料EEDQ(0.13g，0.523mmol，2.6eq)，DMF(1mL)，加完后在室温下搅拌1小时，加入化合物4-13(0.21g，0.201mmol，1.0eq)的DMF(0.5mL)溶液，加完后在室温下搅拌至反应完全，先用二氯甲烷(40mL)稀释，再用1M HBr(10mL x 2)洗涤，然后饱和碳酸氢钠(20mL x 2)洗涤，最后用饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤，油泵减压浓缩，得化合物4-14(230mg,粗品。
Ms(ESI):m/z 1182.4[M+Na] +。
第五步
在25mL单口瓶中加入化合物4-14(0.240g，0.201mmol，1.0eq)，DCM(2mL)，加完后在冰浴下降温到0℃，然后缓慢加入三氟乙酸(1mL)，加完后在室温下搅拌至反应完全，反应液在冰浴下减压浓缩后经pre-HPLC纯化，冻干得化合物4-B00共78mg,收率39.2％。
Ms(ESI):m/z 1014.2[M+Na] +。
1H 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)。
实施例10
Figure PCTCN2022074455-appb-000102
第一步
在25mL三口瓶中加入化合物4-A(0.260g，0.430mmol，1.05eq)，化合物4-C-1(0.193g，0.551mmol，1.32eq)，三乙胺(0.125g，1.23mmol，3.0eq)，DMF(2mL)，加完后在冰浴下降温到-5℃，然后缓慢加入T3P(50％DMF)(0.5mL,1.024mmol,2.5eq)，自然升温条件下搅拌至反应完全，反应液直接经pre-HPLC纯化(TFA(0.05％)水-乙腈)，得化合物4-C-2(140mg,收率35.1％。
Ms(ESI):m/z 949.3[M+Na] +。
第二步
在50mL三口瓶中加入化合物4-C-2(0.34g，0.366mmol，1.0eq)，加原料四氮唑(0.360g，5.13mmol，14.0eq)，N,N-二乙基亚磷酰胺二叔丁酯(1.099g， 4.40mmol，12.0eq)，DMF(5mL)，加完后在室温下反应2小时，冰浴下降温到0℃，然后缓慢加入H 2O 2(30％)(0.234g,2.02mmol,5.5eq),加完后在室温下搅拌至反应完全，反应液直接经中压boston反相柱纯化，得化合物4-C-3(304mg,收率64.1％。
Ms(ESI):m/z 1141.3[M+Na] +。
第三步
在25mL单口瓶中加入化合物4-C-3(0.304g，0.272mmol，1.0eq)，加原料piperidine(0.255g，1.63mmol，9.0eq)，乙腈(8mL)，加完后在室温条件下搅拌至反应完全。减压浓缩，加入4mL石油醚打浆，在35℃下搅拌2小时，然后过滤，滤饼再用2mL石油醚洗涤两次，得化合物4-C-4(243mg,收率99％。
Ms(ESI):m/z 897.6[M+H] +。
第四步
在25mL单口瓶中加入原料2-溴乙酸(0.112g，0.813mmol，3.0eq)，加原料EEDQ(0.201g，0.813mmol，3.0eq)，DMF(2mL)，加完后在室温下搅拌0.6小时，加入化合物4-C-4(0.243g，0.271mmol，1.0eq)的DMF(1mL)溶液，加完后在室温下搅拌至反应完全，先用二氯甲烷(50mL)稀释，再用1M HBr(15mL x 2)洗涤，然后饱和碳酸氢钠(15mL x 2)洗涤，最后用饱和食盐水洗涤，有机相用无水硫酸钠干燥，过滤，油泵减压旋干，再用氮气吹10分钟，得化合物4-C-5共275mg,粗品。
Ms(ESI):m/z 1039.1[M+Na] +和1041.1[M+Na+2] +。
第五步
在25mL单口瓶中加入原料4-C-5(0.275g，0.271mmol，1.0eq)，DCM(2.5mL)，加完后在冰浴下降温到0℃，然后缓慢加入三氟乙酸(1mL),加完后在室温下搅拌至反应完全，反应液在冰浴下减压浓缩后经pre-HPLC纯化，冻干得化合物4-C00共120mg,收率48.3％。
Ms(ESI):m/z 905.2[M+H] +。
实施例11
Figure PCTCN2022074455-appb-000103
于37℃，向阿达木单抗的A缓冲液(pH＝6.3的0.05M缓冲水溶液；10.0mg/mL，6.0mL，405.11nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(2.5mM，356.8μL，891.24nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将1.0M Tris缓冲液(840μL)加入至上述反应液中，再将化合物3-B00(4.08mg，4051.10nmol)溶解于300μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：A缓冲液)，并用超滤管浓缩得到抗体-药物偶联物Humira-3-B00的A缓冲液(2.55mg/mL，23.5mL)，于4℃冷冻储存。
实施例12
Figure PCTCN2022074455-appb-000104
于37℃，向阿达木单抗的A缓冲液(pH＝6.3的0.05M缓冲水溶液；10.0mg/mL，6.0mL，405.11nmol)加入配制好的三(2-羧乙基)膦(TCEP)的水溶液(2.5mM，405.4μL，1012.77nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将1.0M Tris缓冲液(840μL)加入至上述反应液中，再将将化合物4-B00(4.02mg，4051.10nmol)溶解于300μL DMSO中，加入到上述反应液中，置于水浴 振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：A缓冲液)，并用超滤管浓缩得到标题产物抗体-药物偶联物Humira-4-B00的A缓冲液(2.58mg/mL，23.25mL)，于4℃冷冻储存。
生物学评价
以下结合测试例进一步描述解释本公开中，但这些实施例并非意味着限制本公开中的范围。
测试例1：小分子类固醇的体外活性
1、待测样品
化合物1-A至5-A，化合物A-A(根据WO2017210471实例2制备)。
Figure PCTCN2022074455-appb-000105
2、糖皮质激素受体结合测定
使用人糖皮质激素NHR结合(放射性标记的激动剂)实验测试小分子类固醇与糖皮质激素受体(GR)的结合活性(#232020，eurofins)。实验原理为将不同浓度的小分子类固醇和5nM[3H]地塞米松与人重组GR在4℃下孵育24小时。小分子类固醇与[3H]地塞米松竞争性地结合人GR。通过计数与受体特异性结合的[3H]地塞米松可计算出小分子类固醇与GR的结合活性。结果详见表1。
3、盐皮质激素受体激动剂活性测定
使用
Figure PCTCN2022074455-appb-000106
NHR核易位实验测试小分子类固醇的盐皮质激素受体(MR)激动剂活性。将PathHunter NHR CHO-K1细胞在384孔白壁微孔板中铺板，加入待测小分子类固醇在37℃，5％CO 2条件下共孵育以诱导反应。用PathHunter检测试剂混合物生成测定信号，室温孵育一小时后检测化学发光信号。使用四参数曲线拟合分析数据以生成EC 50值。结果详见表1。
4、GRE报告基因实验
将A549细胞铺板(40000个细胞/96孔板)，用lipo3000转染pGL4.36(MMTV-Luc，100ng/孔)，过夜培养后与不同浓度的小分子类固醇进行孵育，24h后用Bright-Glo(promega)检测试剂检测报告基因系统中荧光酶的表达。
表1.小分子类固醇的体外活性
Figure PCTCN2022074455-appb-000107
Figure PCTCN2022074455-appb-000108
测试例2：小分子类固醇在脂多糖(LPS)刺激人PBMC分泌细胞因子实验中的抑炎活性
1、待测样品
化合物1-A至5-A，化合物A-A。
2、测试方法
用RPMI(2％FBS，1％青霉素-链霉素)重悬冻存原代人外周血单核细胞(PBMC)并铺板于96孔板。将PBMC与不同浓度的小分子类固醇在37℃，5％CO 2条件下共孵育4小时后，加入0.01ng/mL LPS刺激过夜。第二天收集培养基上清并用alpha LISA(Cisbio)检测IL-6的浓度。
3、测试结果
测试结果如图1所示，本公开所述的小分子类固醇化合物能够显著抑制LPS诱导的IL-6释放。
测试例3：抗TNF-ADC在膜结合型TNFα介导的GRE报告基因系统中的活性
1、待测样品
Humira-3-B00、Humira-4-B00、Humira-A-B00(根据WO2019106609实例7制备)。
Figure PCTCN2022074455-appb-000109
2、测试方法
在Hela细胞中用慢病毒建立稳定转染MMLV-Luc的报告基因细胞系。将稳转Hela-MMTV-Luc细胞系铺板于96孔板中(30000个细胞/孔)，每孔用lipo3000转染空载质粒或人TNFα突变质粒(TNFα△12，去除TACE的酶切位点，50ng/孔)。过夜后与不同浓度的抗TNF-ADC孵育，24h后用Bright-Glo(promega)检测试剂检测报告基因系统中荧光酶的表达。
3、测试结果
测试结果如表2、图2所示。
表2抗TNF-ADC在膜结合型TNFα介导的GRE报告基因系统中的活性
hTNF△12 Humira-A-B00 Humira-3-B00 Humira-4-B00
EC 50(nM) 0.84 7.4 0.55
测试例4：抗TNF-ADC在脂多糖(LPS)刺激人单核细胞分泌细胞因子实验中的活性
使用EsaySep TM人CD14分选试剂盒从冻存原代人外周血PBMC中分选并富集单核细胞，铺板于96孔板。将单核细胞与不同浓度的抗TNF-ADC在37℃，5％CO 2条件下共孵育4小时后，用0.01ng/mL LPS刺激过夜。第二天收集培养基上清并用alpha LISA(cisbio)检测IL-6的浓度。
与阿达木单抗(Humira)相比，本公开的ADC在高浓度(4-100nM)时有效抑制LPS诱导的IL-6分泌(如图3所示)。另外，4nM的Humira-3-B00与Humira-4-B00的抗炎活性显著强于该浓度下的对照ADC分子Humira-A-B00(如图4所示)。
测试例5：小鼠胶原抗体诱导的关节炎(CAIA)模型
试验动物：
雄性balb/c小鼠，6w，购自上海市计划生育科学研究所实验动物经营部。饲养环境：SPF；生产许可证:SCXK(沪)2018-0006；Balb/c小鼠合格证编号：20180006023393。
待测样品：
Humira-3-B00、Humira-4-B00、Humira-A-B00。
试验方法：
试验动物到达后适应性饲养7天，随机分组。第0天，各组小鼠除对照组外，均腹腔注射II型胶原抗体混合物(购自Chondrex公司)1.5mg/只造模。第3天，各组小鼠除对照组外，腹腔注射LPS 50μg/只(100μL)以增强免疫反应。从第5天开始对各组小鼠进行给药处理，同时每1-3天进行四肢关节炎评分。具体实验流程如图5所示。通过以下给药方案给药小鼠，对各组小鼠每1-3天进行关节炎 病情严重程度半定量计分，进而评估抗TNF-ADC的抗炎活性。
表3：给药方案
Figure PCTCN2022074455-appb-000110
试验结果：
Humira在CAIA模型中的抗炎活性较弱，Humira-4-B00的起效快(从第5天开始)，并能持久地降低关节炎炎症。对照ADC分子Humira-A-B00仅在关节炎进程后期(从第11天起)显示出对关节炎的缓解作用(如图6所示)。从关节炎造模后的第8天至第14天，Humira-4-B00相比模型组显著降低小鼠关节炎评分(*,p<0.05)，而对照ADC分子Humira-A-B00的抑炎活性相对模型组没有达到显著性差异(如图7所示)。
测试例6：迟发型超敏反应(DTH)模型
试验动物：
雄性ICR小鼠，6w，购自上海市计划生育科学研究所实验动物经营部。饲养环境：SPF；生产许可证:SCXK(沪)2018-0006，合格证编号：20180006023622。
待测样品：
Humira-3-B00、Humira-4-B00、Humira-A-B00、Humira-A-A00(根据WO2017210471实例2和WO2019106609实例7制备)。
Figure PCTCN2022074455-appb-000111
测试方法：
动物到达后适应性饲养7天，随机分组。第0天，小鼠腹部去毛后涂抹1％DNFB(2，4二硝基氟苯)溶液50μL进行免疫，第5天时，在小鼠右耳内外侧分别涂抹0.5％的DNFB溶液10μL(共20μL)进行激发，第6天(24h后)处死小鼠，使用打孔器取下双侧直径8mm耳片称重。各组小鼠分别于第0天和第4天进行给药处理。具体实验流程如图8所示。通过以下给药方案给药小鼠，称重小鼠对照侧及造模侧耳片重量(提示肿胀度)，用于评估抗TNF-ADC的抗炎活性。
表4：给药方案
Figure PCTCN2022074455-appb-000112
测试结果：
各组小鼠未经处理的左耳重量之间没有差异。Humira-4-B00与阳性对照Humira-A-B00在该模型中的抑炎活性相当，Humira-3-B00的抑炎活性稍弱，但仍然显著强于Humira单抗(如图9所示)。
测试例7：ADC样品在血浆中的稳定性
待测样品：
Humira-3-B00、Humira-4-B00、Humira-A-B00、Humira-A-A00。
试验血浆：
人、食蟹猴、大鼠、小鼠以及1％BSA
试验方法：
将受试分子以PBS配置成1mg/mL溶液，并0.22μm滤膜过滤除菌。将15μL 1mg/mL样品加入135μL反应基质中，使终浓度为100μg/mL。37℃避光孵育0天、7天、14天、21天，样品检测游离毒素。
数据见表5。
表5：血浆稳定性数据
Figure PCTCN2022074455-appb-000113
Figure PCTCN2022074455-appb-000114
在100μg/mL的浓度下，Humira-3-B00、Humira-4-B00在人、食蟹猴、小鼠、大鼠血浆以及1％BSA中显示出优秀的稳定性，游离毒素均小于检测下限；Humira-A-A00在大鼠血浆中有少许的游离毒素释放；Humira-A-B00在大鼠和小鼠血浆中有少许的游离毒素。

Claims (23)

  1. 式(I)所示的抗体-药物偶联物，
    Ab-(L-D) k
    (I)
    其中，Ab为抗体或其抗原结合片段，
    L为将Ab共价连接于D的连接子，且k为1至20，
    D如下式(II-A)或(II-B)所示：
    Figure PCTCN2022074455-appb-100001
    其中，
    Figure PCTCN2022074455-appb-100002
    表示单键或双键；
    R 1a各自独立地选自氢、烷基和烷氧基，所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代，优选R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基；
    环A为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    环B为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 5a和R 5b一起形成氧代或硫代；
    环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的芳基和杂芳基，且环C和环D中至少有一个为任选被一个或多个取代基Q 1所取代的稠环芳基或稠杂芳基；
    X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的芳基或杂芳基、-O-、-S-、-S(O)-、-S(O)(O)-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-、-CR 6f＝CR 6g-和-C≡C-，或者X 2不存在；
    R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，或者R 6a和R 6b一 起形成氧代或硫代；
    R 6c、R 6d、R 6e、R 6f和R 6g各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
    R 1各自独立地选自氢、烷基和烷氧基，其中所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代，优选R 1各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，更优选氢；
    R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
    Figure PCTCN2022074455-appb-100003
    R 2a各自独立地为氢或C 1-C 6烷基；
    R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
    R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
    R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
    R 3各自独立地为氢或卤素；
    R 4各自独立地选自氢、卤素和羟基，优选R 4各自独立地为氢；
    m和n各自独立地为1至6的整数；
    取代基团Q 1各自独立地选自C 1-C 6烷基、卤素、氘、羟基、巯基、-NR iR j、氧代、硫代、-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、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基、5至10元杂芳基、8至12元稠环芳基和5至12元稠杂芳基，优选Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基；
    R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
    R k各自独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j，其中所述的烷基、烷氧基和卤代烷基各自独立地任选被选自C 1-C 6烷基、卤素、羟基、巯基、-NR iR j、氧代、硫代、羧基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基和5至10元杂芳基中的一个或多个取代基所取代，优选R k各自独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j；且
    条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
  2. 根据权利要求1所述的抗体-药物偶联物，其中环A为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子，优选环A为任选被一个或多个取代基Q 1所取代的
    Figure PCTCN2022074455-appb-100004
  3. 根据权利要求1或2所述的抗体-药物偶联物，其中环B为任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子，优选环B为任选被一个或多个取代基Q 1所取代的
    Figure PCTCN2022074455-appb-100005
  4. 根据权利要求1-3中任意一项所述的抗体-药物偶联物，其中X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子，优选R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR 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 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基和C 2-C 6炔基，更优选R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，最优选R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代。
  5. 根据权利要求1-4中任意一项所述的抗体-药物偶联物，其中环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的6至10元芳基、5至10元杂芳基、8至12元稠环芳基或5至12元稠杂芳基，所述杂芳基或稠杂芳基包含至少一个氮原子，优选环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的下述基团：
    Figure PCTCN2022074455-appb-100006
    更优选环C选自任选被一个或多个取代基Q 1所取代的：
    Figure PCTCN2022074455-appb-100007
    Figure PCTCN2022074455-appb-100008
    且环D为任选被一个或多个取代基Q 1所取代的
    Figure PCTCN2022074455-appb-100009
  6. 根据权利要求1-5中任意一项所述的抗体-药物偶联物，其中X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子，优选R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k、-S(O)R k、-S(O)(O)R k、C 1-C 6烷氧基、C 2-C 6烯基和C 2-C 6炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基，更优选R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基。
  7. 根据权利要求1所述的抗体-药物偶联物，其中D如下式(II-A’)或(II-B’)所示：
    Figure PCTCN2022074455-appb-100010
    其中，
    R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基，优选氢；
    环A为
    Figure PCTCN2022074455-appb-100011
    所述环A任选被一个或多个取代基Q 1所取代；
    环B为
    Figure PCTCN2022074455-appb-100012
    所述环B任选被一个或多个取代基Q 1所取代；
    X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子；
    R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代；
    环C选自
    Figure PCTCN2022074455-appb-100013
    Figure PCTCN2022074455-appb-100014
    所述环C任选被一个或多个取代基Q 1所取代；
    环D为
    Figure PCTCN2022074455-appb-100015
    所述环D任选被一个或多个取代基Q 1所取代；
    X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基；
    R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基；
    R 6c、R 6d和R 6e各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
    R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
    Figure PCTCN2022074455-appb-100016
    R 2a各自独立地为氢或C 1-C 6烷基；
    R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
    R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
    R 2d和R 2e各自独立地为氢或C 1-C 6烷基，
    优选R 2各自独立地选自-CH 2OH、-CH 2SH、-OH和
    Figure PCTCN2022074455-appb-100017
    R 3各自独立地为氢或卤素，优选R 3各自独立地为氢或氟；
    m和n各自独立地为1至6的整数；
    取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基；
    R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
    R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j；且
    条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
  8. 根据权利要求1-7中任意一项所述的抗体-药物偶联物，其中X 1为-(CR 5aR 5b)m-，R 5a和R 5b均为氟，m为1、2或3，优选1；或
    X 1为-(CR 5aR 5b)m-，R 5a和R 5b一起形成氧代或硫代，优选氧代，m为1、2或3，优选1；或
    X 1选自任选被一个或多个取代基Q 1所取代的
    Figure PCTCN2022074455-appb-100018
    Figure PCTCN2022074455-appb-100019
    优选
    Figure PCTCN2022074455-appb-100020
  9. 根据权利要求1-8中任意一项所述的抗体-药物偶联物，其中X 2选自 -(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的：
    Figure PCTCN2022074455-appb-100021
    Figure PCTCN2022074455-appb-100022
    优选R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基、C 1-C 6烷基、-NR iR j、-C(O)OR k和C 1-C 6烷氧基。
  10. 根据权利要求1-9中任意一项所述的抗体-药物偶联物，其中k为1-10，优选2-5。
  11. 根据权利要求1-10中任意一项所述的抗体-药物偶联物，其中所述连接子包含氨基酸单元L 1，所述氨基酸单元L 1优选包含由2至7个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸、高赖氨酸、n-甲基-缬氨酸、
    Figure PCTCN2022074455-appb-100023
    的氨基酸构成的肽残基，q为1-6的整数，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-苯丙氨酸(Ala-Phe)；苯丙氨酸-赖氨酸(Phe-Lys)、苯丙氨酸-高赖氨酸(Phe-Homolys)、n-甲基-缬氨酸-瓜氨酸(Me-Val-Cit)、丙氨酸-丙氨酸(Ala-Ala)、甘氨酸-谷氨酸(Gly-Glu)、谷氨酸-丙氨酸-丙氨酸(Glu-Ala-Ala)和甘氨酸-赖氨酸(Gly-Lys)、甘氨酸-缬氨酸-瓜氨酸(Glv-Val-Cit)、甘氨酸-甘氨酸-甘氨酸(Gly-Gly-Gly)和
    Figure PCTCN2022074455-appb-100024
    最优选甘氨酸-谷氨酸(Gly-Glu)、
    Figure PCTCN2022074455-appb-100025
  12. 根据权利要求1-11中任意一项所述的抗体-药物偶联物，其中所述连接子包含拉伸单元，优选所述拉伸单元选自
    Figure PCTCN2022074455-appb-100026
    其中p各自独立地为1、2、3、4、5或6。
  13. 根据权利要求1-12中任意一项所述的抗体-药物偶联物，其中所述连接子选自
    Figure PCTCN2022074455-appb-100027
    Figure PCTCN2022074455-appb-100028
  14. 根据权利要求1-13中任意一项所述的抗体-药物偶联物，其选自：
    Figure PCTCN2022074455-appb-100029
    Figure PCTCN2022074455-appb-100030
    其中，p各自独立地为1、2、3、4、5或6，Ab、D和k如权利要求1中所定义。
  15. 根据权利要求1所述的抗体-药物偶联物，其选自
    Figure PCTCN2022074455-appb-100031
    Figure PCTCN2022074455-appb-100032
    Figure PCTCN2022074455-appb-100033
    k为1至10，Ab如权利要求1中所定义。
  16. 根据权利要求1-15中任意一项所述的抗体-药物偶联物，其中所述抗体选自鼠源抗体、嵌合抗体、人源化抗体和全人源抗体。
  17. 根据权利要求1-16中任意一项所述的抗体-药物偶联物，其中抗体或其抗原结合片段选自抗TNFα抗体、抗IL-4R抗体、抗IL-6/IL-6R抗体、抗IL-13R抗体、抗IL-17/IL-17R抗体、抗IL-23/IL23R抗体、抗IL-36R抗体、抗CD20抗体、抗CD22抗体、抗CD28抗体、抗CD40抗体、抗TSLP抗体或其抗原结合片段，优选阿达木单抗、英利昔单抗、赛妥珠单抗、阿非莫单抗、奈瑞莫单抗、奥左拉珠单抗、普拉库鲁单抗、戈利木单抗或其抗原结合片段，更优选阿达木单抗。
  18. 一种式(III-A)或(III-B)所示化合物或其可药用盐，
    Figure PCTCN2022074455-appb-100034
    其中，
    Figure PCTCN2022074455-appb-100035
    表示单键或双键；
    环A为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    环B为任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的芳基或杂芳基；
    R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 5a和R 5b一起形成氧代或硫代；
    环C和环D各自独立地选自任选被一个或多个取代基Q 1所取代的芳基和杂芳基或稠杂芳基，且环C和环D中至少有一个为任选被一个或多个取代基Q 1所取代的稠环芳基或稠杂芳基；
    X 2选自-(CR 6aR 6b)n-、任选被一个或多个取代基Q 1所取代的芳基或杂芳基、-O-、-S-、-S(O)-、-S(O)(O)-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-、-CR 6f＝CR 6g-和-C≡C-，或者X 2不存在；
    R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、硝基、氰基和任选被一个或多个取代基Q 1所取代的下述基团：烷基、-NR iR 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、烷氧基、烷硫基、烯基和炔基，或者R 6a、R 6b与其相连的碳原子一起形成3元至10元环烷基；
    R 6c、R 6d、R 6e、R 6f和R 6g各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
    R 1各自独立地选自氢、烷基和烷氧基，其中所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
    R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
    Figure PCTCN2022074455-appb-100036
    R 2a各自独立地为氢或C 1-C 6烷基；
    R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
    R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
    R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
    R 3各自独立地为氢或卤素；
    R 4各自独立地选自氢、卤素和羟基；
    m和n各自独立地选自1至6的整数；
    取代基团Q 1各自独立地选自C 1-C 6烷基、卤素、氘、羟基、巯基、-NR iR j、氧代、硫代、-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、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基、5至10元杂芳基、8至12元稠环芳基和5至12元稠杂芳基；
    R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
    R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j，其中所述的烷基、烷氧基和卤代烷基各自独立地任选被选自C 1-C 6烷基、卤素、羟基、巯基、-NR iR j、氧代、硫代、羧基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷硫基、C 2-C 6烯基、C 2-C 6炔基、3至10元环烷基、3至10元杂环基、6至10元芳基和5至10元杂芳基中的一个或多个取代基所取代；
    R 1a各自独立地选自氢、烷基和烷氧基，所述的烷基和烷氧基各自独立地任选被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基和羟烷基中的一个或多个取代基所取代；
    R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
    Figure PCTCN2022074455-appb-100037
    Figure PCTCN2022074455-appb-100038
    或者
    R 1a和R 1b与其相连的氮原子一起形成：
    Figure PCTCN2022074455-appb-100039
    或者R 1a和R 1b与其相连的氮原子一起形成硝基基团；
    p各自独立地为1、2、3、4、5或6；
    L 1是氨基酸单元，优选-甘氨酸-谷氨酸-或
    Figure PCTCN2022074455-appb-100040
    X为卤素；
    PG为氨基保护基；且
    条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
  19. 根据权利要求18所述的化合物或其可药用盐，其为式(III-A’)或(III-B’)所示化合物或其可药用盐：
    Figure PCTCN2022074455-appb-100041
    其中，
    环A为
    Figure PCTCN2022074455-appb-100042
    所述环A任选被一个或多个取代基Q 1所取代；
    环B为
    Figure PCTCN2022074455-appb-100043
    所述环B任选被一个或多个取代基Q 1所取代；
    X 1为-(CR 5aR 5b)m-或任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基，所述杂芳基包含至少一个氮原子；
    R 5a和R 5b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基，或者R 5a和R 5b一起形成氧代或硫代；
    环C选自
    Figure PCTCN2022074455-appb-100044
    Figure PCTCN2022074455-appb-100045
    所述环C任选被一个或多个取代基Q 1所取代；
    环D为
    Figure PCTCN2022074455-appb-100046
    所述环D任选被一个或多个取代基Q 1所取代；
    X 2选自-(CR 6aR 6b)n-、-O-、-S-、-NR 6c-、-CH 2S-、-CH 2O-、-NHCR 6dR 6e-和任选被一个或多个取代基Q 1所取代的6至10元芳基或5至10元杂芳基；
    R 6a和R 6b各自独立地选自氢、卤素、羟基、巯基、氘、氰基和任选被一个或多个取代基Q 1所取代的下述基团：C 1-C 6烷基、-NR iR j、-C(O)R k、-C(O)OR k和C 1-C 6烷氧基；
    R 6c、R 6d和R 6e各自独立地选自氢、C 1-C 6烷基、C 1-C 6卤代烷基和C 1-C 6烷氧基；
    R 2各自独立地选自-CH 2OH、-CH 2SH、-CH 2Cl、-SCH 2Cl、-SCH 2F、-SCH 2CF 3、-OH、-OCH 2CN、-OCH 2Cl、-OCH 2F、-OCH 3、-OCH 2CH 3、-SCH 2CN、
    Figure PCTCN2022074455-appb-100047
    R 2a各自独立地为氢或C 1-C 6烷基；
    R 2b各自独立地为C 1-C 6烷基或C 1-C 6烷氧基；
    R 2c各自独立地选自氢、C 1-C 6烷基、-CH 2OH和C 1-C 6烷氧基；
    R 2d和R 2e各自独立地为氢或C 1-C 6烷基；
    R 3各自独立地为氢或卤素；
    m和n各自独立地为1至6的整数；
    取代基团Q 1各自独立地选自卤素、羟基、巯基、氘、氧代、硫代、氰基、氨基、羧基、C 1-C 6烷基和C 1-C 6烷氧基；
    R i和R j各自独立地选自氢原子、羟基、C 1-C 6烷基和C 1-C 6烷氧基；
    R k独立地选自氢原子、C 1-C 6烷基、C 1-C 6卤代烷基、C 1-C 6烷氧基、羟基和-NR iR j；
    R 1a各自独立地选自氢、C 1-C 6烷基和C 1-C 6烷氧基；
    R 1b各自独立地选自氢、PG-、H-L 1-、PG-L 1-、
    Figure PCTCN2022074455-appb-100048
    Figure PCTCN2022074455-appb-100049
    p各自独立地为1、2、3、4、5或6；
    L 1是氨基酸单元，优选-甘氨酸-谷氨酸-或
    Figure PCTCN2022074455-appb-100050
    X为卤素；
    PG为氨基保护基；且
    条件是当R 5a为氢或烷基时，R 5b不为氢或烷基。
  20. 根据权利要求18所述的化合物或其可药用盐，其选自
    Figure PCTCN2022074455-appb-100051
    Figure PCTCN2022074455-appb-100052
    或其可药用盐。
  21. 根据权利要求18所述的化合物或其可药用盐，其选自
    Figure PCTCN2022074455-appb-100053
    Figure PCTCN2022074455-appb-100054
    Figure PCTCN2022074455-appb-100055
    Figure PCTCN2022074455-appb-100056
    或其可药用盐，其中X为卤素，优选氯或溴，更优选溴。
  22. 一种药物组合物，其包含根据权利要求1-17中任意一项所述的抗体-药物偶联物或根据权利要求18-21中任意一项所述的化合物或其可药用盐以及药学上可接受的赋形剂。
  23. 根据权利要求1-17中任意一项所述的抗体-药物偶联物或根据权利要求22所述的药物组合物在制备用于治疗免疫性疾病的药物中的用途，所述免疫性疾病优选自类风湿性关节炎、幼年特发性关节炎、银屑病性关节炎、强直性脊柱炎、成人克罗恩病、小儿克罗恩病、溃疡性结肠炎、化脓性汗腺炎、葡萄膜炎、白塞病、脊柱关节病和银屑病。
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