WO2023186072A1 - 配体-药物偶联物及其用途 - Google Patents

配体-药物偶联物及其用途 Download PDF

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WO2023186072A1
WO2023186072A1 PCT/CN2023/085394 CN2023085394W WO2023186072A1 WO 2023186072 A1 WO2023186072 A1 WO 2023186072A1 CN 2023085394 W CN2023085394 W CN 2023085394W WO 2023186072 A1 WO2023186072 A1 WO 2023186072A1
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seq
antibody
alkyl
ligand
pharmaceutically acceptable
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PCT/CN2023/085394
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English (en)
French (fr)
Inventor
付雅媛
柴晓鹃
刘力锋
陈常艳
严玉玺
唐锋
曹卓晓
唐任宏
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江苏先声药业有限公司
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Publication of WO2023186072A1 publication Critical patent/WO2023186072A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/44—Glucocorticosteroids; Drugs increasing or potentiating the activity of glucocorticosteroids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J71/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton is condensed with a heterocyclic ring

Definitions

  • the present disclosure belongs to the field of biomedicine and relates to a class of novel structural ligand-drug conjugates, their preparation methods, pharmaceutical compositions containing the conjugates, and their use in the treatment of autoimmune diseases.
  • Glucocorticoid is an extremely important type of regulatory molecule in the body. It plays an important role in regulating the body's development, growth, metabolism and immune function. It is the most important regulatory hormone for the body's stress response and is also The most widely used and effective anti-inflammatory and immunosuppressant in clinical practice. Glucocorticoids are used to treat many autoimmune and inflammatory diseases, including rheumatoid arthritis (RA), psoriasis, and inflammatory bowel disease. However, long-term use of glucocorticoids can easily cause side effects such as osteoporosis, muscle atrophy, hypertension, suppression of the body's immunity, and aggravation of infection. The use of hormones in the treatment of autoimmune diseases is therefore limited.
  • TNF ⁇ anti-tumor necrosis factor ⁇
  • IL-4R IL-4R
  • Anti-TNF ⁇ biologics eg, adalimumab, etanercept, golimumab, and infliximab
  • anti-IL-4R biologics eg, dupilumab
  • ADA anti-drug antibodies
  • the present disclosure combines the treatment of biological agents and hormone drugs to provide a new type of glucocorticoid receptor agonist, which is combined with a ligand to form a ligand-hormone drug conjugate for the treatment of autoimmune diseases.
  • the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ;
  • Pc is the ligand unit
  • L is the connecting subunit
  • D is a drug unit represented by the following formula (DI):
  • R 1 and R 2 are each independently selected from H, CH 3 or halogen
  • Ring A is selected from phenyl, 5-10 membered heteroaryl or C 3 -C 10 cycloalkyl, which phenyl, 5-10 membered heteroaryl or C 3 -C 10 cycloalkyl is optionally replaced by one or Multiple R 1a substitutions;
  • X is selected from O, S, C 1 -C 3 alkylene-O, C 1 -C 3 alkylene -S, NR 6 or C(R 7 )(R 8 );
  • R 6 is selected from H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl, said C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or The 4-7 membered heterocyclyl group is optionally substituted by one or more R b ;
  • R 7 and R 8 are each independently selected from H, halogen, CN, OH, NH 2 , C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl, or R 7 , R 8 and the atoms to which they are connected together form a C 3 -C 6 cycloalkyl group or a 4-7 membered heterocyclyl group, and the OH, NH 2 , C 1 -C 6 alkyl group, C 3 -C 6 cycloalkyl group or The 4-7 membered heterocyclyl group is optionally substituted by one or more R b ;
  • R 3 is selected from H, OH or NHR 9 , R 4 , R 5 and the atoms to which they are connected together form a C 5 -C 6 cycloalkenyl group, a 5-6 membered heterocyclyl group or a 5-6 membered heteroaryl group, said C 5 -C 6 cycloalkenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted by one or more R 4a ; alternatively, R 5 is selected from H, OH or NHR 9 , R 3 , R 4 and the atoms to which they are connected together form a C 5 -C 6 cycloalkenyl group, a 5-6 membered heterocyclyl group or a 5-6 membered heteroaryl group.
  • the C 5 -C 6 cycloalkenyl group, 5-6 membered heteroaryl group Heterocyclyl or 5-6 membered heteroaryl is optionally substituted by one or more R 4a
  • R 9 is selected from H, C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl, said C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or The 4-7 membered heterocyclyl group is optionally substituted by one or more R d ;
  • R 13 and R 14 are each independently selected from H, halogen, CN, OH, NH 2 , O(C 1 -C 3 alkyl) or C 1 -C 6 alkyl;
  • C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by one or more R d ;
  • R 11 and R 12 are each independently selected from H or C 1 -C 6 alkyl
  • C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by one or more R b ;
  • C 1 -C 6 alkyl, C 3 -C 6 cycloalkyl or 4-7 membered heterocyclyl is optionally substituted by one or more R c ;
  • n 1 ⁇ 16
  • linker unit L is covalently connected to any one of the -OH, -NH 2 or -NH- groups present in R 3 , R 4 , R 5 , R 13 , and R 14 of the compound of formula (DI).
  • each of R 1 and R 2 is independently selected from H or F.
  • Ring A is selected from phenyl or 5-6 membered heteroaryl, which is optionally substituted with one or more R 1a .
  • Ring A is selected from phenyl, optionally substituted with one or more R 1a .
  • R 1a is selected from NH 2 .
  • Ring A is selected from
  • Ring A is selected from
  • X is selected from O, S, C 1 -C 3 alkylene-O, C 1 -C 3 alkylene-S, or C(R 7 )(R 8 ).
  • R 7 and R 8 are each independently selected from H, halogen, CN, OH, NH 2 or C 1 -C 3 alkyl, or R 7 , R 8 and the atom to which they are connected together form C 3 -C 6 cycloalkyl, the OH, NH 2 , C 1 -C 3 alkyl or C 3 -C 6 cycloalkyl is optionally substituted by one or more R b .
  • R 7 and R 8 are each independently selected from H, halogen, or C 1 -C 3 alkyl, or R 7 , R 8 and the atoms to which they are attached together form a C 3 -C 6 cycloalkyl group.
  • R 7 , R 8 are each independently selected from H, F, or methyl, or R 7 , R 8 and the atom to which they are attached together form a cyclopropyl group.
  • X is selected from O, S, CH2O , CH2S , CH2 , CF2 , CHCH3 , or
  • X is selected from CH2 .
  • R 11 , R 12 are each independently selected from H, methyl, or ethyl.
  • R 10 is selected from OH or
  • R 9 is selected from H or C 1 -C 6 alkyl.
  • R 9 is selected from H.
  • R 3 is selected from H, OH or NH 2
  • R 4 , R 5 and the atoms to which they are connected together form a C 5 -C 6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heterocyclyl group.
  • Heteroaryl, the C 5 -C 6 cycloalkenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted by one or more R 4a .
  • R 5 is selected from H, OH or NH 2 , and R 3 and R 4 together with the atoms to which they are connected form a C 5 -C 6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heterocyclyl group.
  • Heteroaryl, the C 5 -C 6 cycloalkenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted by one or more R 4a .
  • R 3 is selected from H, R 4 , R 5 and the atom to which they are connected together form a 5-6 membered heterocyclyl group or a 5-6 membered heteroaryl group, the 5-6 membered heterocyclyl group or The 5-6 membered heteroaryl group is optionally substituted with one or more R 4a .
  • R 5 is selected from H or NH 2 , and R 3 , R 4 and the atom to which they are connected together form a C 5 -C 6 cycloalkenyl, a 5-6 membered heterocyclyl or a 5-6 membered heteroaryl. group, the C 5 -C 6 cycloalkenyl, 5-6 membered heterocyclyl or 5-6 membered heteroaryl is optionally substituted by one or more R 4a .
  • R 3 is selected from H, OH, or NH 2 , and R 4 , R 5 and the atoms to which they are attached together form described Optionally substituted by one or more R 4a .
  • R 3 is selected from H, and R 4 , R 5 and the atoms to which they are attached together form described Optionally substituted by one or more R 4a .
  • R 5 is selected from H, OH, or NH 2 , and R 3 and R 4 together with the atoms to which they are attached form described Optionally substituted by one or more R 4a .
  • R 5 is selected from H or NH 2 , and R 3 and R 4 together with the atoms to which they are attached form described Optionally substituted by one or more R 4a .
  • R 3 is selected from H, and R 4 , R 5 and the atoms to which they are attached together form
  • R 5 is selected from H or NH 2 , and R 3 and R 4 together with the atoms to which they are connected form
  • each of R 13 and R 14 is independently selected from H or NH 2 .
  • R 13 is selected from H.
  • R14 is selected from H.
  • the compound represented by formula (DI) or a pharmaceutically acceptable salt thereof is selected from the following compounds represented by formula (D-II) or a pharmaceutically acceptable salt thereof:
  • R 1 and R 2 are each independently selected from H or F;
  • R 10 is selected from OH or
  • R 3 , R 4 , R 5 , R 13 and R 14 are as defined above.
  • the compound represented by formula (DI) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n :
  • Pc, L, n are as defined above;
  • D is selected from the following compounds:
  • L 1 is a dipeptide residue, and one of the amino acids constituting the dipeptide residue is glycine.
  • the L1 is a dipeptide residue selected from Gly-Lys or Gly-Glu.
  • linker unit L is selected from The a end is covalently connected to the ligand unit Pc, the b end is covalently connected to the drug unit D, and the L 1 is selected from the dipeptide residue of Gly-Lys or Gly-Glu.
  • linker unit L is selected from Its a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D.
  • linker unit L is selected from Its a-end is covalently connected to the ligand unit Pc, and its b-end is covalently connected to the drug unit D.
  • the ligand-drug conjugate of the present disclosure having the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the Pc is an antibody or antigen-binding fragment that specifically binds TNF ⁇ or IL-4R; the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) or/and a light chain variable region (VH). VL);
  • the heavy chain variable region includes the VH shown in SEQ ID NO. 4-9, 12-16, 18, 20, 22, 57, 68, 70, 72, 74 or 76 Contained HCDR1, HCDR2 and HCDR3; or/and the light chain variable region includes the VL shown in SEQ ID NO.
  • the heavy chain variable region or/and the light chain variable region are included in the HCDR1-3 or/and LCDR1-3 described in group (1) Compared with each CDR, a sequence with at least 80% identity, or a sequence with at most 3 insertion, deletion or substitution mutations; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the HCDR1-3 and the LCDR1-3 are determined according to the Kabat numbering system, Chothia numbering system or IMGT numbering system.
  • the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, or/and the light chain variable region includes LCDR1, LCDR2, and LCDR3, wherein, the HCDR1-3 or/and the LCDR1 -3 selected from the following;
  • the HCDR1-3 is SEQ ID NO.24-26; or/and the LCDR1-3 is SEQ ID NO.27-29;
  • the HCDR1-3 is SEQ ID NO.30-32; or/and the LCDR1-3 is SEQ ID NO.33-35;
  • the HCDR1-3 is SEQ ID NO.36-38; or/and the LCDR1-3 is SEQ ID NO.39-41;
  • the HCDR1-3 is SEQ ID NO.42-44; or/and the LCDR1-3 is SEQ ID NO.45-47;
  • the HCDR1-3 is SEQ ID NO.48-50; or/and the LCDR1-3 is SEQ ID NO.51-53;
  • the HCDR1-3 is SEQ ID NO.60-62; or/and the LCDR1-3 is SEQ ID NO.63-65;
  • the HCDR1-3 is SEQ ID NO.78-80; or/and the LCDR1-3 is SEQ ID NO.81-83;
  • the HCDR1-3 is SEQ ID NO.84-86; or/and the LCDR1-3 is SEQ ID NO.87-89;
  • the HCDR1-3 is SEQ ID NO.90-92; or/and the LCDR1-3 is SEQ ID NO.93-95;
  • the HCDR1-3 is SEQ ID NO.96-98; or/and the LCDR1-3 is SEQ ID NO.99-101;
  • the HCDR1-3 is SEQ ID NO.102-104; or/and the LCDR1-3 is SEQ ID NO.105-107; or,
  • Said HCDR1-3 or/and said LCDR1-3 has at least 80% identity with each CDR of said HCDR1-3 and LCDR1-3 in any of groups (1)-(11) sequence, or a sequence with at most 3 insertion, deletion or substitution mutations; preferably, the HCDR1-3 or/and the LCDR1-3 have the same characteristics as any one of the groups (1)-(11)
  • Each CDR in HCDR1-3 and LCDR1-3 has at least 80% identity; further, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment includes: the heavy chain variable region includes SEQ ID NO. 4-9, 12-16, 18, 20, 22, 57, 68, 70, 72, 74 Or the sequence shown in 76, or/and the light chain variable region includes the sequence shown in SEQ ID NO. 1-3, 17, 19, 21, 23, 58, 69, 71, 73, 75 or 77; or the The heavy chain variable region or/and the light chain variable region include sequences with at least 80% identity compared to the heavy chain variable region and the light chain variable region in any of the above groups; further , the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the antibody or antigen-binding fragment includes a heavy chain constant region sequence and/or a light chain constant region sequence selected from a complete constant region sequence or a sequence thereof.
  • the constant region fragment includes CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region Selected from the human or mouse kappa constant region or lambda constant region; optionally, the antibody or antigen-binding fragment includes a complete heavy chain and a light chain, the heavy chain is composed of the VH and the heavy chain constant region, the The heavy chain constant region has the sequence shown in SEQ ID NO: 10 or 59, the light chain is composed of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO: 11.
  • the antibody is selected from the group consisting of Nerelimomab, Certolizumab, Infliximab, Golimumab, Adalimumab, Dupixent, 002Mab, 003Mab, 004Mab, 005Mab, or 006Mab.
  • the antibody is selected from the group consisting of Nerelimomab, Certolizumab, Infliximab, Golimumab, Adalimumab, or Dupixent.
  • the antibody or antigen-binding fragment is monospecific, bispecific, trispecific, or tetraspecific.
  • the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof wherein n is selected from 1 to 16, for example, n is selected from 1 to 10, for example n is selected from 1 to 8, for example, n is selected from 2 to 8, for example, n is selected from 2 to 6, for example, n is selected from 6 to 8.
  • n is selected from 2 to 6, for example, n is 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.
  • n is selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.
  • the present disclosure also provides a drug-linker compound or a pharmaceutically acceptable salt thereof, the general structural formula of which is XLD, wherein:
  • Drug unit D is as defined above;
  • connection subunit L is selected from The a end is covalently connected to X, the b end is covalently connected to the drug unit D, and L 1 is as defined in any one of the above;
  • X is selected from halogen, OS(O) 2 CH 3 or OS(O) 2 CF 3 .
  • L is selected from The a-terminal is covalently connected to X, the b-terminal is covalently connected to the drug unit D, and the L 1 is selected from the dipeptide residue of Gly-Lys or Gly-Glu.
  • L is selected from The a end is covalently connected to X, and the b end is covalently connected to drug unit D.
  • L is selected from The a end is covalently connected to X, and the b end is covalently connected to drug unit D.
  • X is selected from Br or I.
  • X is selected from Br.
  • Drug unit D is selected from the following compounds:
  • L is selected from Its a end is covalently connected to X, and its b end is covalently connected to drug unit D;
  • X is selected from Br or I.
  • the drug-linker compound of the present disclosure having the general formula XLD or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 10 , R 13 and R 14 are as defined above.
  • the compound represented by formula (DH) or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:
  • the present disclosure provides a pharmaceutical composition, which includes the ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients. .
  • the present disclosure provides a method for treating inflammatory diseases or autoimmune diseases in mammals, which includes administering a therapeutically effective amount of a formula of the general formula Pc-(LD) n to a mammal in need of the treatment, preferably a human. body-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.
  • the present disclosure provides the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the preparation and treatment of inflammatory diseases or autoimmune diseases. uses in medicines.
  • the present disclosure provides the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for use in the treatment of inflammatory diseases or autoimmune diseases. the use of.
  • the present disclosure provides the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating inflammatory diseases or autoimmune diseases.
  • the present disclosure provides a pharmaceutical composition, which includes the compound represented by the aforementioned formula (D-H) of the present disclosure or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable excipients.
  • the present disclosure provides a method for treating inflammatory diseases or autoimmune diseases in mammals, which includes administering a therapeutically effective amount of a compound represented by the aforementioned formula (D-H) or a pharmaceutical thereof to a mammal in need of such treatment, preferably a human. acceptable salts, or pharmaceutical compositions thereof.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the preparation of a drug for treating inflammatory diseases or autoimmune diseases.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in the treatment of inflammatory diseases or autoimmune diseases.
  • the present disclosure provides a compound represented by the aforementioned formula (D-H) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating inflammatory diseases or autoimmune diseases.
  • the inflammatory or autoimmune disease may be selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, atopic dermatitis, urticaria, myocarditis, encephalitis, uveitis, chronic obstructive pulmonary disease, vasculitis, scleroderma, psoriasis, plaque Lumpy psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis, collagenous colitis, polypoidal colitis, necrheuma
  • the present disclosure provides a method for preparing a ligand-drug conjugate with the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, including using the aforementioned drug-linker with the general formula XLD The step of coupling the compound to the aforementioned ligand.
  • the ligand is an antibody or an antigen-binding fragment.
  • the present disclosure provides a method for preparing a ligand-drug conjugate with the general formula Pc-(LD) n or a pharmaceutically acceptable salt thereof, which includes combining the aforementioned drug unit D of the present disclosure with the aforementioned ligand unit Pc
  • the step of connecting; optionally, connecting through the aforementioned linker unit L; optionally, the ligand is an antibody or an antigen-binding fragment.
  • the present disclosure provides the use of the compound represented by the aforementioned formula (DH) or a pharmaceutically acceptable salt thereof in the preparation of the aforementioned ligand-drug conjugate of the general formula Pc-(LD) n or its pharmaceutically acceptable salt.
  • the ligand-drug conjugate provided by the present disclosure has significant anti-inflammatory activity, excellent plasma stability and reduced toxic and side effects.
  • the present disclosure provides an antibody or antigen-binding fragment that specifically binds human TNF ⁇ , which includes a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising SEQ.
  • VH heavy chain variable region
  • VL light chain variable region
  • the sequence shown in any one of ID NO. 5-9 and 12-15, and the light chain variable region includes the sequence shown in SEQ ID NO. 2 or 3.
  • an antibody or antigen-binding fragment that specifically binds human TNF ⁇ includes a heavy chain variable region (VH) and a light chain variable region (VL) as shown below:
  • VH includes SEQ ID NO.5
  • VL includes SEQ ID NO.2
  • VH includes SEQ ID NO.6, and the VL includes SEQ ID NO.2;
  • VH includes SEQ ID NO.7
  • VL includes SEQ ID NO.2
  • VH includes SEQ ID NO.8, and the VL includes SEQ ID NO.2;
  • the VH includes SEQ ID NO.9, and the VL includes SEQ ID NO.2;
  • VH includes SEQ ID NO.5
  • VL includes SEQ ID NO.3
  • VH includes SEQ ID NO.6, and the VL includes SEQ ID NO.3;
  • the VH includes SEQ ID NO.7, and the VL includes SEQ ID NO.3;
  • VH includes SEQ ID NO.8, and the VL includes SEQ ID NO.3;
  • VH includes SEQ ID NO.9
  • VL includes SEQ ID NO.3
  • the VH includes SEQ ID NO.12, and the VL includes SEQ ID NO.2;
  • the VH includes SEQ ID NO.13, and the VL includes SEQ ID NO.2;
  • the VH includes SEQ ID NO.14, and the VL includes SEQ ID NO.2; or,
  • the VH includes SEQ ID NO. 15, and the VL includes SEQ ID NO. 2.
  • the antibody or antigen-binding fragment that specifically binds human TNF ⁇ includes a heavy chain constant region sequence and/or a light chain constant region sequence selected from an intact constant region.
  • region sequence or fragment thereof, the constant region fragment includes CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, the The light chain constant region is selected from the group consisting of human or mouse kappa constant region or lambda constant region; optionally, the antibody or antigen-binding fragment includes a complete heavy chain and a light chain, and the heavy chain is composed of the VH and heavy chain constant regions.
  • the heavy chain constant region has the sequence shown in SEQ ID NO:10, the light chain is composed of the VL and the light chain constant region, the light chain constant region has the sequence shown in SEQ ID NO:11 .
  • the antibody or antigen-binding fragment that specifically binds human TNF ⁇ is monospecific, bispecific, trispecific, or tetraspecific.
  • the present disclosure provides a pharmaceutical composition, which includes the aforementioned antibody or antigen-binding fragment of the present disclosure that specifically binds to human TNF ⁇ and pharmaceutically acceptable excipients.
  • the present disclosure provides a method for treating inflammatory diseases or autoimmune diseases in mammals, comprising administering a therapeutically effective amount of the aforementioned antibody or antigen-binding fragment that specifically binds human TNF ⁇ to a mammal in need of the treatment, preferably a human. , or pharmaceutical compositions thereof.
  • the present disclosure provides the use of the aforementioned antibody or antigen-binding fragment that specifically binds to human TNF ⁇ , or a pharmaceutical composition thereof in the preparation of medicaments for treating inflammatory diseases or autoimmune diseases.
  • the present disclosure provides the use of the aforementioned antibody or antigen-binding fragment that specifically binds to human TNF ⁇ , or a pharmaceutical composition thereof in the treatment of inflammatory diseases or autoimmune diseases.
  • the present disclosure provides the aforementioned antibody or antigen-binding fragment that specifically binds human TNF ⁇ , or a pharmaceutical composition thereof for treating inflammatory diseases or autoimmune diseases.
  • the inflammatory or autoimmune disease may be selected from the group consisting of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, atopic dermatitis, urticaria, myocarditis, encephalitis, uveitis, chronic obstructive pulmonary disease, vasculitis, scleroderma, psoriasis, plaque Lumpy psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, microscopic colitis, collagenous colitis, polypoidal colitis, bad
  • ligand refers to a macromolecular compound that recognizes and binds to an antigen or receptor associated with a target cell.
  • the function of the ligand is to present the drug to the target cell population bound to the ligand.
  • ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or other molecules that can bind to cells.
  • the ligand or ligand unit is represented as Pc, and the ligand can form a connection bond with the linker unit through a heteroatom on the ligand.
  • the ligand is selected from an antibody or antigen-binding fragment selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody; in some embodiments of the disclosure,
  • the antibodies are monoclonal antibodies.
  • linker or “linker unit” refers to a chemical structural segment or chemical bond that is connected to a ligand at one end and a drug at the other end.
  • drug refers to a small molecule compound that is biologically active in an organism.
  • the drug is a glucocorticoid receptor agonist or its corresponding phosphate ester molecule with anti-inflammatory function.
  • ligand-drug conjugate refers to a ligand connected to a biologically active drug through a stable linker unit.
  • the "ligand-drug conjugate” is an antibody-drug conjugate (ADC).
  • ADC refers to the combination of a monoclonal antibody or an antibody fragment with a stable linker unit. Biologically active drugs are linked.
  • DAR drug-to-antibody ratio
  • Pc-(LD) n the DAR is defined by the variable "n", which can be either an integer or a decimal.
  • antibody is used in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable domain of an immunoglobulin heavy chain and/or sufficient sequence from the variable domain of an immunoglobulin light chain to be capable of specifically binding to an antigen or Peptide combination.
  • Antibody herein encompasses various forms and various structures so long as they exhibit the desired antigen-binding activity.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers.
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.
  • Antibody herein includes a typical “quadruple chain antibody”, which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which It consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from the N end to the C end; and, When the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant in the N-terminal to C-terminal direction.
  • VH heavy chain variable region
  • CH1 domain a heavy chain constant region
  • HR hinge region
  • CH2 domain heavy chain constant region
  • CH3 domain heavy chain constant region in the direction from the N end to the C end
  • the full-length antibody is of the IgE isotype, it
  • Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have a kappa chain or a lambda chain.
  • Antibodies herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos, etc., and immunoglobulin neoantigen receptors (Ig new antigen receptor, IgNAR) discovered in sharks and other cartilaginous fishes.
  • HCAbs Heavy-chain antibodies
  • Ig new antigen receptor, IgNAR immunoglobulin neoantigen receptors
  • the "antibodies” herein can be derived from any animal, including but not limited to humans and non-human animals.
  • the non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • Antibodies herein include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (e.g., containing naturally occurring mutations or arising during the production of the preparation), such variants are usually present in small amounts. ), each antibody comprising the population is identical and/or binds the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.
  • the modifier "monoclonal" herein should not be construed as requiring production of the antibody or antigen-binding molecule by any particular method.
  • monoclonal antibodies can be produced by a variety of techniques, including (but not limited to) hybridoma technology, recombinant DNA methods, phage library display technology, and the use of transgenic animals containing all or part of the human immunoglobulin locus. methods and other methods known in the art.
  • natural antibody refers to antibodies made and paired by the immune system of a multicellular organism.
  • engineered antibody herein refers to non-natural antibodies obtained through genetic engineering, antibody engineering and other technologies.
  • engineered antibodies include humanized antibodies, small molecule antibodies (such as scFv, etc.), bis(antibodies), etc. Specific antibodies, etc.
  • the term "monospecific" is intended to mean having one or more binding sites, where each binding site binds the same epitope of the same antigen.
  • multispecific antibody refers to an antibody having at least two antigen-binding sites, each of the at least two antigen-binding sites binding to a different epitope of the same antigen or to a different epitope of a different antigen. combine.
  • terms such as “bispecific,” “trispecific,” “tetraspecific,” etc. refer to the number of different epitopes to which the antibody/antigen-binding molecule can bind.
  • valency indicates the presence of a specified number of binding sites in the antibody/antigen binding molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites respectively in the antibody/antigen binding molecule. existence of points.
  • Fully-length antibody “intact antibody” and “intact antibody” are used interchangeably herein and refer to having a structure that is substantially similar to the structure of a native antibody.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities.
  • Antigen-binding fragment or “antibody fragment” herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2 and scFv.
  • Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab” fragments, each containing the heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ).
  • Fab fragment herein refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain.
  • Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region.
  • Fab’-SH is a Fab’ fragment in which the cysteine residues of the constant domain carry free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen binding sites (two Fab fragments) and part of the Fc region.
  • scFv single-chain variable fragment
  • linker see, e.g., Bird et al., Science 242:423- 426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Volume 113, Roseburg and Moore, eds., Springer-Verlag, New York , pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, you can use A linker with the amino acid sequence (GGGGS) 4 was used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present disclosure are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al.
  • a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
  • diabody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, forcing the domain to be separated from another
  • the complementary domains of one chain pair and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123(1994)).
  • chimeric antibody refers to an antibody in which part of the light chain or/and heavy chain is derived from an antibody (which can be derived from a specific species or belong to a specific antibody class or subclass). ), and another part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the target Antigen binding activity (U.S.P. 4,816,567 of Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).
  • chimeric antibody may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody) and the heavy and light chain variable regions of the antibody are The light chain constant region is derived from a secondary antibody (eg, a human antibody).
  • a first antibody e.g., a murine antibody
  • a secondary antibody eg, a human antibody
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance immune response, etc.
  • the term "fully human antibody” refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region refers to the region of an antibody heavy or light chain involved in allowing the antibody to bind to an antigen.
  • "Heavy chain variable region” is used interchangeably with “VH” and “HCVR”, and “light chain variable region” Used interchangeably with “VL” and “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • complementarity determining region and “CDR” are used interchangeably in this article, and usually refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is due to its spatial structure. It can form precise complementarity with the antigenic epitope, so it is also called complementarity determining region.
  • the heavy chain variable region CDR can be abbreviated as HCDR
  • the light chain variable region CDR can be abbreviated as LCDR.
  • framework region or "FR region” is used interchangeably and refers to those amino acid residues other than CDRs in the heavy or light chain variable region of an antibody.
  • a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results).
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • heavy chain constant region herein refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, that are relative to the antibody's Variable domains have more conserved amino acid sequences.
  • "Heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variations or fragments thereof.
  • "Heavy chain constant region” includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter includes only "full-length heavy chain constant region"part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain When an antibody is used, it does not include the CH1 domain.
  • a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxyl-terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen.
  • the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.
  • Fc refers to the carboxyl-terminal portion of the antibody resulting from papain hydrolysis of the intact antibody, which typically contains the CH3 and CH2 domains of the antibody.
  • Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions.
  • the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain.
  • the Fc region can include Or excluding Lys447.
  • identity herein may be calculated by aligning two amino acid sequences or two nucleic acid sequences for the purpose of optimal comparison (e.g., may be the optimal).
  • the alignment introduces gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • the percent identity between two sequences varies as a function of the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program of the GCG software package (available at www.gcg.com), use the Blossum 62 matrix or The PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 determine the percent identity between two amino acid sequences.
  • the GAP program in the GCG software package uses the NWSgapdna.CMP matrix with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • tautomer refers to a functional group isomer resulting from the rapid movement of an atom in a molecule between two positions.
  • Compounds of the present disclosure may exhibit tautomerism.
  • Tautomeric compounds can exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to isolate a single tautomer usually yield a mixture whose physical and chemical properties are consistent with the mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form is dominant; in phenols, the enol form is dominant. This disclosure encompasses all tautomeric forms of the compounds.
  • stereoisomer refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.
  • the compounds of the present disclosure may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms, or asymmetric double bonds, and therefore the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms.
  • Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S) )-enantiomer, non- Enantiomers, (D)-isomers, (L)-isomers, and racemic or other mixtures thereof, such as enantiomeric or diastereomerically enriched mixtures, all of the above
  • isomers and mixtures thereof are within the definition of compounds of the present disclosure.
  • the compounds of the present disclosure containing asymmetric atoms can be isolated in an optically active pure form or in a racemic form.
  • the optically active pure form can be resolved from a racemic mixture or synthesized by using chiral starting materials or chiral reagents. .
  • substituted means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, as long as the valence state of the specific atom is normal and the substituted compound is stable.
  • the ethyl group is "optionally" substituted by halogen, which means that the ethyl group can be unsubstituted (CH 2 CH 3 ), monosubstituted (CH 2 CH 2 F, CH 2 CH 2 Cl, etc.), or polysubstituted. (CHFCH 2 F, CH 2 CHF 2 , CHFCH 2 Cl, CH 2 CHCl 2, etc.) or completely substituted (CF 2 CF 3 , CF 2 CCl 3 , CCl 2 CCl 3, etc.). It will be understood by those skilled in the art that any substitution or substitution pattern that is sterically impossible and/or cannot be synthesized will not be introduced for any group containing one or more substituents.
  • any variable eg, R a , R b
  • its definition in each instance is independent. For example, if a group is replaced by 2 R b , there are independent options for each R b .
  • linking group When the number of a linking group is 0, such as -(CH 2 ) 0 -, it means that the linking group is a bond.
  • the direction of connection is arbitrary.
  • the structural unit When X in is selected from “C 1 -C 3 alkylene-O", at this time O-ring B", you can also connect ring A and ring B from right to left to form “ring AOC 1 -C 3 alkylene-ring B”.
  • substituents When a substituent's bond is cross-linked to two atoms on a ring, the substituent can be bonded to any atom on the ring.
  • structural unit Indicates that R 5 can be substituted at any position on the benzene ring.
  • Cm - Cn refers to having an integer number of carbon atoms in the range of mn.
  • C 1 -C 10 means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms or 10 carbon atoms.
  • alkyl refers to a hydrocarbon group of the general formula C n H 2n+1 , which alkyl group may be straight or branched.
  • C 1 -C 6 alkyl is understood to mean a straight-chain or branched saturated hydrocarbon radical having 1, 2, 3, 4, 5 or 6 carbon atoms.
  • alkyl group examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl base, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, etc.
  • C 1 -C 3 alkyl is understood to mean a straight-chain or branched saturated alkyl group having 1 to 3 carbon atoms.
  • the "C 1 -C 6 alkyl group” may further include "C 1 -C 3 alkyl group”.
  • alkoxy refers to a group produced by losing a hydrogen atom on a hydroxyl group of a straight-chain or branched alcohol, and can be understood as “alkyloxy” or “alkyl-O-”.
  • C 1 -C 6 alkoxy is understood to mean “C 1 -C 6 alkyloxy” or “C 1 -C 6 alkyl-O-”.
  • the "C 1 -C 6 alkoxy group” may further include "C 1 -C 3 alkoxy group”.
  • cycloalkyl refers to a carbocyclic group that is fully saturated and exists in the form of a single ring, a branched ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is generally 3 to 10 membered.
  • C 3 -C 10 cycloalkyl can be understood to mean saturated monocyclic, double-ring, Spiro ring or bridged ring, which has 3 to 10 carbon atoms.
  • cycloalkyl group examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2 .1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, etc.
  • C 3 -C 10 cycloalkyl may include “C 3 -C 6 cycloalkyl”, and the term “C 3 -C 6 cycloalkyl” is understood to mean a saturated monocyclic or bicyclic hydrocarbon ring having 3 to 6 carbon atoms, specific examples include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, etc.
  • cycloalkyloxy is understood to mean “cycloalkyl-O-”.
  • cycloalkenyl refers to a non-aromatic carbocyclic group that is not completely saturated and exists in the form of a single ring, a condensed ring, a bridged ring or a spiro ring. Unless otherwise indicated, the carbocyclic ring is typically 5 to 8 membered.
  • C 5 -C 6 cycloalkenyl refers to a cycloalkenyl group with 5 or 6 ring carbon atoms. Specific examples include but are not limited to cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl Alkenyl etc.
  • heterocyclyl refers to a fully saturated or partially saturated (not aromatic heteroaromatic as a whole) monocyclic, paracyclic, spirocyclic or bridged cyclic group containing 1 to 5 ring atoms.
  • Heteroatom or heteroatom group that is, an atomic group containing heteroatoms
  • 4-14 membered heterocyclyl refers to a heterocyclyl with a number of ring atoms of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and its ring atoms contain 1-5 are independently selected from the heteroatoms or heteroatom groups described above.
  • 4--14-membered heterocyclyl includes “4-10-membered heterocyclyl” or “4-7-membered heterocyclyl", etc., wherein specific examples of 4-membered heterocyclyl include but are not limited to azetidine base or oxetanyl; specific examples of 5-membered heterocyclyl include but are not limited to tetrahydrofuryl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4 ,5-dihydrooxazolyl or 2,5-dihydro-1H-pyrrolyl; specific examples of 6-membered heterocyclic groups include but are not limited to tetrahydropyranyl, piperidyl, morpholinyl, dithiane base, thiomorpholinyl, piperazinyl, trithialkyl, tetrahydropyridinyl or 4H-[1,3,4]thiadia
  • the heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include but are not limited to hexahydrocyclopenta[c]pyrrole-2(1H)-yl; 5,6-membered bicyclic groups. Specific examples include, but are not limited to, hexahydropyrro[1,2-a]pyrazin-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4 ,3-a]pyrazinyl or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl.
  • the heterocyclic group may be a benzo-fused cyclic group of the above-mentioned 4-7 membered heterocyclic group, and specific examples include but are not limited to dihydroisoquinolyl and the like.
  • “4-10-membered heterocyclyl” may include “5-10-membered heterocyclyl", “4-7-membered heterocyclyl”, “5-6-membered heterocyclyl”, “6-8-membered heterocyclyl” , "4-10 membered heterocycloalkyl”, “5-10 membered heterocycloalkyl”, “4-7 membered heterocycloalkyl”, “5-6 membered heterocycloalkyl”, “6-8 membered "Heterocycloalkyl” and other scopes, "4-7 membered heterocyclyl” may further include "4-6 membered heterocyclyl", “5-6 membered heterocyclyl", “4-7 membered heterocyclyl” , "4-6 membered heterocycloalky
  • aryl refers to an all-carbon monocyclic or fused polycyclic aromatic ring group having a conjugated ⁇ electron system.
  • C 6 -C 10 aryl is understood to mean an aryl group having 6 to 10 carbon atoms.
  • a ring with 6 carbon atoms (“C 6 aryl”), for example phenyl; or a ring with 9 carbon atoms (“C 9 aryl”), for example indanyl or indenyl; or a ring with 10
  • a ring of 10 carbon atoms such as tetrahydronaphthyl, dihydronaphthyl or naphthyl.
  • aryloxy is understood to mean “aryl-O-”.
  • heteroaryl refers to an aromatic monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and S, and the remaining ring atoms are C.
  • heteroaryl refers to an aromatic monocyclic or fused polycyclic ring system containing at least one ring atom selected from N, O, and S, and the remaining ring atoms are C.
  • 5-10 membered heteroaryl is understood to include monocyclic or bicyclic aromatic ring systems having 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and it contains 1-5, preferably 1-3 heteroatoms independently selected from N, O and S.
  • heteroaryl group is selected from the group consisting of thienyl, furyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiazolyl Diazolyl, etc.
  • benzo derivatives such as benzofuryl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazole base, indazolyl, indolyl or isoindolyl, etc.; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, etc. and their benzo derivatives, such as quinolyl, quinazole Phyllinyl or isoquinolinyl, etc.; or azocinyl, indolizinyl, purinyl, etc.
  • 5-6 membered heteroaryl refers to an aromatic ring system having 5 or 6 ring atoms and containing 1-3, preferably 1-2 heteroatoms independently selected from N, O and S.
  • heteroaryloxy is understood to mean “heteroaryl-O-”.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • hydroxy refers to the -OH group.
  • cyano refers to the -CN group.
  • mercapto refers to the -SH group.
  • amino refers to the -NH group .
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent or slow down (reduce) the progression of undesirable physiological changes or pathologies in the subject, such as cancer, autoimmune diseases, and viral infections.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • an effective amount refers to an amount of a therapeutic agent that is effective when administered alone or in combination with another therapeutic agent to a cell, tissue or subject to prevent or alleviate the symptoms of a disease or the progression of that disease.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • subject refers to an organism undergoing treatment for a particular disease or condition as described in this disclosure.
  • subjects and patients include mammals such as humans, primates (eg, monkeys), or non-primate mammals undergoing treatment for a disease or condition.
  • the amount of a compound of the present disclosure that constitutes a "therapeutically effective amount” will vary depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art. based on its own knowledge and the contents of this disclosure.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without multiple toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salts refers to salts of pharmaceutically acceptable acids or bases, including salts of compounds with inorganic or organic acids, and salts of compounds with inorganic or organic bases.
  • composition refers to a mixture of one or more compounds of the present disclosure or salts thereof and pharmaceutically acceptable excipients.
  • the purpose of pharmaceutical compositions is to facilitate administration of the compounds of the present disclosure to an organism.
  • pharmaceutically acceptable excipients refers to those excipients that have no obvious irritating effect on the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.
  • the phosphate ester compounds described in this application (such as R 10 is selected from The compound) is an ester prodrug, which releases biologically active compounds after hydrolysis by phosphatase in the organism.
  • phosphate compounds Glucocorticoid receptor stimulator is released after hydrolysis by phosphatase in the body.
  • Motive agent Therefore, those skilled in the art can understand or expect that if compound 009 has certain biological activity (such as glucocorticoid receptor agonistic activity), the corresponding ester compound 009-p will also have the same or similar activity in vivo. biological activity.
  • the present disclosure also includes isotopically labeled compounds that are the same as those described herein, but in which one or more atoms are replaced by an atom having an atomic weight or mass number different from that typically found in nature.
  • isotopes that may be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H, 3 H, 11 C, 13 C, 14 C, 13 respectively N, 15 N, 15 O, 17 O, 18 O, 31 P, 32 P, 35 S, 18 F, 123 I, 125 I and 36 Cl, etc.
  • isotopically labeled compounds of the present disclosure can be used in compound and/or substrate tissue distribution analyses. Tritiated (ie 3 H) and carbon-14 (ie 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron-emitting isotopes such as 15 O, 13 N, 11 C, and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the present disclosure can generally be prepared by substituting an isotopically labeled reagent for a non-isotopically labeled reagent by following procedures similar to those disclosed in the Schemes and/or Examples below.
  • compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms.
  • pharmaceutical compositions of the present disclosure may be in the form of sterile injectable aqueous solutions for intramuscular or subcutaneous administration.
  • the pharmaceutical compositions of the present disclosure may accept other vehicles or solvents when used, such as water, Ringer's solution or isotonic sodium chloride solution.
  • the daily dose is 0.001 mg/kg to 600 mg/kg body weight, preferably 0.05 mg/kg to 200 mg/kg body weight, more preferably 0.1 mg/kg to 100 mg/kg body weight, In single or divided dose form.
  • the compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by their combination with other chemical synthesis methods, and methods well known to those skilled in the art. Equivalent alternatives, preferred embodiments include, but are not limited to, the embodiments of the present disclosure.
  • Figure 1 is a COZY diagram of intermediate 1-5 in Example 1 of the present disclosure.
  • Figure 2 is a diagram showing the efficacy test results of the compound of formula (D-H) in Test Example 6 of the present disclosure on the CHS mouse model.
  • Figure 3 is a diagram showing the activity test results of the anti-human TNF ⁇ antibody-drug conjugate in the GRE reporter gene in Test Example 9 of the present disclosure.
  • Figure 4 is a diagram showing the activity test results of the anti-mouse TNF ⁇ antibody-drug conjugate in the GRE reporter gene in Test Example 9 of the present disclosure.
  • Figure 5 is a graph showing the inhibitory activity test results of anti-mouse TNF ⁇ -ADC in the LPS-stimulated mouse PBMC cytokine release assay in Test Example 11 of the present disclosure.
  • Figure 6 is a graph showing the efficacy test results of anti-mouse TNF ⁇ -ADC in the mouse CHS model in Test Example 12 of the present disclosure.
  • Figure 7 is a graph showing the efficacy test results of anti-mouse TNF ⁇ -ADC in the DBA/1 mouse CAIA model in Test Example 13 of the present disclosure.
  • Figure 8 shows the efficacy test of the anti-human TNF ⁇ -ADC in the TNF ⁇ humanized mouse CAIA model in Test Example 14 of the present disclosure. Test result graph.
  • Figure 9 is a graph showing the test results of the inhibitory effect of the anti-IL-4R antibody-drug conjugate on the proliferation of human primary CD4 + T cells in Test Example 20 of the present disclosure.
  • Figure 10 shows the detection results of middle ear thickness of the anti-IL-4R antibody-drug conjugate in the atopic dermatitis mouse model in Test Example 21 of the present disclosure.
  • Figure 11 shows the serum IgE detection results of the anti-IL-4R antibody-drug conjugate in the atopic dermatitis mouse model in Test Example 21 of the present disclosure.
  • Figures 12-13 show that the administration of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure significantly improved the pathological damage and inflammation of the ear patch and skin tissue at the modeling site of the atopic dermatitis mouse model.
  • Figures 14-15 show that the administration of the anti-IL-4R antibody-drug conjugate in Test Example 21 of the present disclosure significantly inhibited the infiltration of inflammatory CD4 + T cells in the ear patch and skin tissue of the modeling site of the atopic dermatitis mouse model.
  • the ratios expressed for mixed solvents are volumetric mixing ratios.
  • % refers to wt%.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the units of NMR shifts are 10 -6 (ppm).
  • the solvents measured by NMR are deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS);
  • TMS tetramethylsilane
  • IC 50 refers to the half inhibitory concentration, which refers to the concentration when half of the maximum inhibitory effect is achieved.
  • Concentration, "EC 50” refers to the concentration that causes half of the maximum effect concentration and 50% of the maximum effect.
  • Example 1 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl (base)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro Synthesis of -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (compound 001)
  • Step 3 (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl )Synthesis of tert-butyl carbamate 1-4
  • Step 4 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-7-hydroxy-8b-(2-hydroxy Acetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5] Synthesis of indeno[1,2-d][1,3]dioxalan-4(2H)-one 1-5
  • reaction solution was quenched with sodium bicarbonate aqueous solution (20mL), extracted with ethyl acetate (100mL x 2), the organic phase was washed with saturated sodium chloride aqueous solution (50mL), the organic phase was dried, filtered and concentrated under reduced pressure.
  • Step 5 tert-butyl (4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxalan-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)carbamic acid 1-6 synthesis of esters
  • Step 6 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl)methyl (base)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro Synthesis of -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one 001
  • Example 2 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((1H-benzo[d]imidazol-6-yl)methyl)phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho Synthesis of [2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 002)
  • Step 2 Synthesis of 6-(4-formylbenzyl)-1H-benzo[d]imidazole-1-carboxylic acid tert-butyl ester 2-3
  • Step 3 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-benzo[d]imidazol-6-yl)methyl)phenyl) -7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho Synthesis of [2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one
  • Dissolve 16alpha-hydroxyprednisolone F1 230 mg, 0.61 mmol
  • anhydrous magnesium sulfate (279 mg, 2.32 mmol)
  • Dissolve 2-3 (308 mg, 0.92 mmol) in anhydrous acetonitrile (1.5 mL) and add it to the reaction system, cool the temperature to 0°C, add trifluoromethanesulfonic acid (431 mg, 2.87 mmol) dropwise, and stir at 25°C for 2 hours.
  • LCMS detects that the reaction is complete.
  • Example 3 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((1H-indol-6-yl)methyl)phenyl)-7-hydroxy -8b-(2-Hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2', Synthesis of 1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 003)
  • Example 4 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-10-(4-(indoline-6 -methyl)phenyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1' :Synthesis of 4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 004)
  • Step 1 Synthesis of 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline 4-2
  • Step 2 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indoline-1-carboxylic acid tert-butyl ester 4-3 synthesis
  • Step 3 Synthesis of 6-(4-formylbenzyl)indoline-1-carboxylic acid tert-butyl ester 4-4
  • Step 4 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-10-(4-(indoline-6 -methyl)phenyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1' :Synthesis of 4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one 004
  • Example 5 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4] Oxazin-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl1,2,6a,6b,7,8,8a,8b ,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H) -Synthesis of ketone (compound 005)
  • Step 2 Synthesis of 4-((3,4-dihydro-2H-benzo[b][1,4]oxazin-7-yl)methyl)benzene(formal)aldehyde 5-3
  • Azin-7-yl)methyl)benzaldehyde 5-3 (397 mg).
  • Step 3 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1,4] Synthesis of oxazin-7-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl
  • LCMS detects that the reaction is complete. Add saturated aqueous sodium bicarbonate solution (15 mL) to quench the reaction, extract with ethyl acetate (50 mL x 3), wash the organic phase with saturated aqueous sodium chloride solution (20 mL), dry the organic phase, filter and concentrate under reduced pressure, and the crude product is prepared -HPLC [ column : ,21.1%).
  • the first step (6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl base)-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene -Synthesis of 3-keto 6-2
  • LCMS detects that the reaction is complete. Add 40 mL of water and stir for 5 minutes, then wash the filter residue with water (10 mL -6,9-Difluoro-11,16,17-trihydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-6,7,8,9,10,11,12,13 ,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-3-one 6-2 (0.7g).
  • Step 2 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-dihydro-2H-benzo[b][1, 4]oxazin-7-yl)methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7 ,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxapenta Synthesis of cyclo-4(2H)-one 006
  • LCMS detects that the reaction is complete. Add saturated aqueous sodium bicarbonate solution (10mL) to quench the reaction, use ethyl acetate (50mL x 2) for extraction, wash the organic phase with saturated aqueous sodium chloride solution (20mL), dry the organic phase, filter and concentrate under reduced pressure, and the crude product is prepared by -HPLC [Column: Phenomenex Luna C18 100*30mm*3 ⁇ m; mobile phase: A: water (0.225% FA); B: MeCN, B%: 40%-70%, 8min] separation and purification, obtaining compound 006 (82.7 mg ,34.6%).
  • Example 7 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((4-aminobenzo[d]oxazol-7-yl)methyl)benzene base)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H- Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 007)
  • Step 2 7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzo[d]oxazole-4-amine 7-3 synthesis
  • Step 3 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-aminobenzo[d]oxazol-7-yl)methyl)benzene base)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H- Synthesis of naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 007)
  • Example 8 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolane-4 -(yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a, 12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (compound 008) synthesis
  • Step 2 Synthesis of tert-butyl (7-bromobenzo[d][1,3]dioxolane-4-yl) carbamate 8-3
  • Step 4 tert-butyl (7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-hydroxy-8b-(2-hydroxyacetyl)-6a, 8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1' :4,5]indeno[1,2-d][1,3]dioxalan-10-yl)benzo[d][1,3]dioxalan-4-yl)benzo[d][1,3]dioxalan-4-yl )Synthesis of carbamate 8-5
  • Step 5 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-aminobenzo[d][1,3]dioxolane-4 -(yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a, 12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (compound 008) synthesis
  • Example 9 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl (base)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro Synthesis of -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (compound 009)
  • Step 1 Synthesis of tert-butyl(5-bromo-2,3-dihydrobenzofuran-7-yl)carbamate 9-2
  • reaction solution was filtered and concentrated, and the residue was separated and purified by column chromatography (0-10% tetrahydrofuran/petroleum ether) to obtain the intermediate tert-butyl (5-(4,4,5,5-tetramethyl-1,3 , 2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-yl)carbamate 9-3 (990 mg, 71.8%).
  • Step 3 tert-butyl (5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS))-7-hydroxy-8b-(2-hydroxyacetyl)-6a ,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1 ':4,5]indeno[1,2-d][1,3]dioxalan-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)aminomethyl Synthesis of acid ester 9-4
  • Step 4 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5-yl)methyl (base)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro Synthesis of -1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one (compound 009)
  • LCMS detects that the reaction is complete.
  • the reaction solution was blown dry with nitrogen, and the obtained residue was prepared by high-efficiency liquid phase preparation [column: YMC-Pack CN150*30mm*5 ⁇ m; mobile phase: A, 0.1% formic acid aqueous solution; B: acetonitrile, B%, 40%-60 %, 12min] was separated and purified to obtain compound (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran- 5-yl)methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a ,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]
  • Step 1 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(bromomethyl)phenyl)-2,6b-difluoro-7-hydroxy -8b-(2-Hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-decahydro-1H-naphtho[2', Synthesis of 1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one 010-3
  • Step 2 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2,3-dihydrobenzofuran-7-amine010 Synthesis of -2 4-Bromo-2,3-dihydrobenzofuran-7-amine 1-3 (7.30g, 34.1mmol) was dissolved in 1,4-dioxane (150mL), and diazona was added Alcohol borate (20.0g, 78.8mmol), 1,1-bis(diphenylphosphorus)ferrocene palladium chloride (2.50g, 3.4mmol) and potassium acetate (10.0g, 102.3mmol), under nitrogen atmosphere Stir at 80°C for 16 hours. LCMS showed that the reaction was completed.
  • reaction solution was filtered and concentrated.
  • Step 3 (2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-4-yl) )Methyl)phenyl)-2,6b-difluoro-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a ,12,12a,12b-decahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-4(2H)-one Synthesis of 010
  • Example 11 (6aR, 6bS, 7S, 8aS, 8bS, 10R, 11aR, 12aS, 12bS)-10-(4-((7-amino-2,3-dihydro-1H-inden-4-yl) Methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-ten Synthesis of Hydrogen-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one (compound 011)
  • reaction solution was filtered and concentrated, and the obtained residue was separated and purified by column chromatography (0-60% ethyl acetate/petroleum ether) to obtain the intermediate 7-(4,4,5,5-tetramethyl-1,3 , 2-dioxaborolan-2-yl)-2,3-dihydro-1H-indene-4-amine 11-3 (77.0 mg, 63.0%).
  • Step 3 (6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydro-1H-indene-4-yl) Methyl)phenyl)-7-hydroxy-8b-(2-hydroxyacetyl)-6a,8a-dimethyl-6a,6b,7,8,8a,8b,11a,12,12a,12b-ten Synthesis of Hydrogen-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxalan-4(2H)-one 011
  • Example 12 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b -Dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-2-oxoethyl Synthesis of dihydrogen phosphate (compound 009-p)
  • Step 2 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-amino-2,3-dihydrobenzofuran-5- methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b -Dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-8b-yl)-2-oxoethyl Synthesis of dihydrogen phosphate 009-p
  • Step 1 Synthesis of: (S)-1-benzyl 5-tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)acetamido)pentadiene ester
  • Step 3 (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-(tert-butoxy)-5-oxo Synthesis of valeric acid INT-1
  • Example 13 (S)-4-(2-(2-bromoacetamido)acetamido)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS ,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10- Synthesis of (yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-001)
  • LCMS detects that the reaction is complete.
  • the reaction solution was diluted with ethyl acetate (250mL), washed with saturated aqueous ammonium chloride solution (50mL*2) and saturated aqueous sodium chloride solution (50mL), dried, filtered, concentrated, and the residue was subjected to column chromatography (Spherical C18 40-60 ⁇ m, Compound L1-001-1 (2.80 g) was obtained using a mixture of decreasing polarity of water and acetonitrile as eluent; acetonitrile gradient ratio 80%).
  • reaction solution was diluted with ethyl acetate (100 mL), washed with dilute hydrobromic acid (10 mL) and saturated aqueous sodium bicarbonate solution (30 mL*3), dried, filtered, concentrated, and the residue was subjected to reversed-phase column chromatography (Spherical C18 40 -60 ⁇ m, A mixture of water and acetonitrile with decreasing polarity was used as eluent; acetonitrile gradient ratio 80%) to obtain (S)-tert-butyl 4-(2-(2-bromoacetamido)acetamido)-5-((4 -(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxy)oxo)acetyl)- 7-hydroxy-6a,8a-dimethyl-4-oxo-2,4,6
  • Step 4 (S)-4-(2-(2-bromoacetamido)acetamido)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS ,12bS)-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxy)acetyl)-2,4,6a,6b,7,8,8a, 8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1,3]dioxolane-10- Synthesis of (yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-001)
  • Example 14 2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-((S))-6-amino-2-(2- (2-bromoacetamido)acetamido)caproylamino)-2,3-dihydrobenzofuran-4-yl)methyl)phenyl)-7-hydroxy-6a,8a-dimethyl-4- Oxo-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-8bH-naphtho[2',1':4,5]indeno[1 ,2-d][1,3]dioxalan-8b-yl)-2-oxoethyl phosphate (L2-001)
  • Step 1 Synthesis of N 2 -(((benzyloxy)carbonyl)glycine)-N 6 -(tert-butoxycarbonyl)-L-lysine benzyl ester (L2-001-2)
  • Second step L2-001-3 synthesis of (S)-2-(2-aminoacetamido)-6-((tert-butoxycarbonyl)amino)hexanoic acid
  • Step 3 (S)-2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-6-((tert-butoxycarbonyl)amino)caproic acid Synthesis of L2-001-4
  • LCMS detects that the reaction is complete.
  • the reaction solution was diluted with anhydrous dichloromethane (200mL), the organic phase was washed with water (50mL Ether) was separated and purified, and the crude product obtained was subjected to preparative high-performance liquid chromatography (column: C18-1 150*30mm*5 ⁇ m; a mixture of water (containing 0.05% TFA) and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient ratio 42%-82%, elution time 9 minutes) was separated and purified to obtain L2-001-5 (424 mg, 46.4%).
  • reaction solution was diluted with ethyl acetate (100mL), and the organic phase was washed with dilute hydrobromic acid (10mL), saturated aqueous sodium bicarbonate solution (20mL x 2) and saturated brine (20mL), dried, filtered, concentrated, and the residue Separate and purify through silica gel column chromatography (0-65% tetrahydrofuran/petroleum ether) to obtain compound L2-001-8 (65.0 mg).
  • reaction solution was blown dry with nitrogen, and the resulting residue was subjected to preparative high-performance liquid chromatography (column: Boston Green ODS 150*30mm*5 ⁇ m; a mixture of water (containing 0.05% TFA) and acetonitrile with decreasing polarity was used as the eluent; Acetonitrile gradient ratio 22%-37%, elution time 10 minutes) was separated and purified to obtain compound L2-001 (5.32 mg, 12.9%).
  • LCMS detects that the reaction is complete.
  • the reaction solution was diluted with ethyl acetate (250mL), washed with saturated aqueous ammonium chloride solution (50mL*2) and saturated aqueous sodium chloride solution (50mL), dried, filtered, concentrated, and the residue was subjected to column chromatography (Spherical C18 40 -60 ⁇ m, A mixture of water and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient ratio 80%) was separated and purified to obtain compound L1-009-1 (2.60 g, 98.5%).
  • Step 2 (S)-tert-butyl 4-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)-5-((5-(4- ((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((di-tert-butoxyphosphoryl)oxo)acetyl)-7-hydroxy-6a,8a -Dimethyl-4-oxyylidene-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1': 4,5]indeno[1,2-d][1,3]dioxalan-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5 -Synthesis of oxyylid
  • Aqueous sodium sulfite solution (20 mL) was added at 0°C to quench the reaction, extracted with ethyl acetate (200 mL), and the organic phase was washed with saturated brine (50 mL), dried, filtered, and concentrated to obtain a crude product (2.80 g).
  • the obtained product was dissolved in acetonitrile (30 mL), piperidine (938 mg, 11.0 mmol) was added at 0°C, and stirred at 25°C for 30 minutes.
  • reaction solution was concentrated, and the residue was subjected to column chromatography (Spherical C18 40-60 ⁇ m, A mixture of water and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient ratio 40%) was separated and purified to obtain compound L1-009-02 (1.40 g, 60.0%).
  • reaction solution was diluted with ethyl acetate (100mL), washed with dilute hydrobromic acid (10mL) and saturated aqueous sodium bicarbonate solution (30mL*3), dried, filtered and concentrated, and the residue was subjected to column chromatography (Spherical C18 40- 60 ⁇ m, A mixture of water and acetonitrile with decreasing polarity was used as the eluent; acetonitrile gradient ratio 80%) was separated and purified to obtain compound L1-009-03 (1.00 g, 64.0%).
  • Example 16 (S)-4-(2-(2-bromoacetamido)acetamido)-5-((4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR) ,12aS,12bS)-2,6b-difluoro-7-hydroxy-6a,8a-dimethyl-4-oxo-8b-(2-(phosphonooxo)acetyl)-2,4, 6a,6b,7,8,8a,8b,11a,12,12a,12b-dodecahydro-1H-naphtho[2',1':4,5]indeno[1,2-d][1 Synthesis of ,3]dioxalan-10-yl)benzyl)-2,3-dihydrobenzofuran-7-yl)amino)-5-oxopentanoic acid (L1-010)
  • reaction solution was blown dry under nitrogen flow to remove the solvent, and the residue was subjected to high performance liquid chromatography [column: Boston Prime C18 150*30mm*5 ⁇ m; a mixture of water (containing 0.05% NH 4 HCO 3 ) and acetonitrile with decreasing polarity was used as a wash Deliquidation; acetonitrile gradient ratio 13%-33%, elution time 11 minutes] separation and purification to obtain compound L1-010 (3.25 mg).
  • Example 17-1 Humanized design of Infliximab antibody
  • IMGT http://imgt.cines.fr
  • human antibody heavy chain and light chain variable region germline gene database we selected heavy chain and light chain variable region germlines with high homology to mouse antibodies.
  • the gene is used as a template, and the CDRs of the mouse antibody are transplanted into the corresponding human template respectively to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • humanized monoclonal antibodies are obtained by performing reverse mutations on embedded residues, residues that directly interact with the CDR region, and residues in the framework region that have an important impact on the conformation of VL and VH. .
  • the humanized light chain template of the human-mouse chimeric antibody Infliximab is IGKV6-21*01, and the humanized heavy chain template is IGHV3-73*01.
  • the CDRs of Infliximab are transplanted into their human templates to obtain the corresponding Humanized version.
  • the key amino acids in the FR region sequence of Infiliximab's humanized antibody are back-mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • the specific reverse mutation design is shown in Table 1.
  • variable regions of Infliximab and its humanized antibodies are as follows:
  • variable region combinations of each antibody are as follows:
  • the antibody constant region sequence is as follows:
  • heavy chain (HC) and light chain (LC) plasmids were constructed (the nucleic acid sequences encoding antibody VH and VL were recombined into expression vectors with signal peptide and heavy chain constant region/light chain constant region sequences to obtain Recombinant plasmid expressing VH-CH1-Fc/VL-CL).
  • Add the heavy chain and light chain plasmids corresponding to the antibody and the transfection reagent PEI Polysciences, Catalog No.: 24765-1) into OPTI-MEM (Gibco, Catalog No.: 11058021), mix well, let it stand for 15 minutes, and add Expi293 cells (Thermofisher, Catalog No.
  • Human TNF ⁇ protein (Acro Biosystems, product number: TNA-H5228) was coated overnight at 4°C, rinsed three times with 0.05% Tween20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5 h. Rinse three times with 0.05% Tween 20-PBS solution, add the diluted sample, and incubate at 37°C for 1 hour. Rinse three times with 0.05% Tween 20-PBS solution, add secondary antibody HRP goat anti-human IgG Fc (Merck, Cat. No.: AP113), and incubate at 37°C for 1 hour.
  • HRP goat anti-human IgG Fc Merck, Cat. No.: AP113
  • Table 4 ELISA binding experiment of Infliximab humanized antibody and human TNF ⁇ protein
  • the BIAcore 8K instrument is used to determine the affinity of the antibody and the antigen using a multi-cycle kinetic method. In each cycle, the antibody to be tested is first captured using a Protein A chip (GE, Cat. No.: 29-1275-56), and then the antigen human TNF ⁇ is injected. Protein (Acro Biosystems, Cat.
  • TNA-H5228 record the binding and dissociation process of antibody and antigen protein, and finally use Glycine pH1.5 to complete chip regeneration, in which the mobile phase is HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20), the flow rate is 30 ⁇ L/min, the binding time is 240s, the dissociation time is 900s, the regeneration time is 30s, the detection temperature is 25°C, and the antigen concentration is 10-0.15625nM.
  • HBS-EP+ 10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20
  • the flow rate is 30 ⁇ L/min
  • the binding time is 240s
  • the dissociation time is 900s
  • the regeneration time is 30s
  • the detection temperature is 25°C
  • the antigen concentration is 10-0.15625nM.
  • Infliximab-Hu01 Based on the binding affinity results, Infliximab-Hu01 with the smallest number of back mutations was selected. Because the heavy chain variable region Infliximab.VH1 contains amino acid DD, there is a risk of isomerization during the antibody expression process and needs to be repaired. Design the following 4 variants of Infliximab.VH1, the specific sequences are as follows:
  • variable region sequence Use the aforementioned method to construct the corresponding heavy chain plasmid, and co-transfect Expi293 cells with the light chain plasmid containing Infliximab.VL1 as the variable region sequence.
  • the supernatant is collected and purified to obtain four hotspot repaired humanized antibodies.
  • Each antibody can The amino acid sequences of the variable regions correspond to the following:
  • the amino acid sequences of the variable region and constant region of the antibody used to prepare the anti-human TNF ⁇ antibody-drug conjugate are shown in Table 8.
  • the CDR amino acid sequence of the antibody is shown in Table 9.
  • Infliximab-h is the humanized antibody Infliximab-Hu01c. .
  • heavy chain (HC) and light chain (LC) plasmids for the production of anti-human TNF ⁇ antibodies were constructed (the nucleic acid sequences encoding the antibodies VH and VL were recombined to contain the signal peptide and heavy chain constant region/light chain constant region On the expression vector of region sequence, a recombinant plasmid expressing VH-CH1-Fc/VL-CL was obtained).
  • Anti-human TNF ⁇ antibodies Nerelimomab, Certolizumab, Infliximab-h, Golimumab and Adalimumab were prepared according to the antibody expression and purification method in Example 17-1.
  • amino acid sequences of the antibody variable region and constant region used to prepare anti-IL-4R antibody-drug conjugates are as follows, and their sequences are referred to patent PCT/US2007/021210.
  • Dupilumab heavy chain (HC, SEQ ID NO.55): ( The underlined and bold part corresponds to VH-SEQ ID NO.57; The shaded area corresponds to CH-SEQ ID NO.59)
  • Dupilumab HCDR1 (SEQ ID NO.60): DYAMT
  • LC Dupilumab light chain (LC, SEQ ID NO.56): ( The underlined and bold part corresponds to VL-SEQ ID NO.58; the shaded part corresponds to CL-SEQ ID NO.11)
  • LCDR2 (SEQ ID NO.64): LGSNRAS
  • the heavy chain (HC) and light chain (LC) plasmids for the production of anti-human IL-4R antibodies were constructed (the nucleic acid sequences encoding the antibodies VH and VL were recombined into a plasmid with a signal peptide and a heavy chain constant region/light chain constant region).
  • a recombinant plasmid expressing VH-CH1-Fc/VL-CL was obtained).
  • Dupilumab antibody was prepared according to the antibody expression and purification method in Example 17-1.
  • Table 9-1 Amino acid sequence of VH, VL and CDR of anti-human IL4R antibody (Kabat)
  • the anti-mouse TNF ⁇ antibody (hereinafter referred to as "001Mab") used to prepare the anti-mouse TNF ⁇ antibody-drug conjugate was purchased from BioXcell with the catalog number BE0058.
  • Anti-Hel-hIgG4 used to prepare the isotype control of anti-human IL-4R antibody-drug conjugate was purchased from Baiying Biotechnology, the catalog number is B669701.
  • the anti-human TNF ⁇ antibody used to prepare the anti-human TNF ⁇ antibody-drug conjugate and the anti-human IL-4R antibody used to prepare the anti-human IL-4R antibody-drug conjugate were prepared through Example 17-2.
  • the drug-linker compound dissolved in DMSO was added to the reaction system and coupled at 25°C for 16 hours.
  • the reaction product was concentrated by ultrafiltration and replaced with phosphate (PBS) buffer to remove unreacted free small molecules.
  • PBS phosphate
  • ADC SEC Purity Analysis Apply SEC-HPLC method to analyze the protein sample to be tested, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein.
  • the ADC molecules to be tested are treated with PNGase F to remove the N-sugar modification, and then treated with Dithiothreitol (DTT), incubated at 37°C for 1 hour, and reduced into light and heavy chains, and then used Thermo Vanquish UHPLC -Q Exactive Plus mass spectrometry system for analysis, and 2 ⁇ g of protein was injected into Waters ACQUITY Protein BEH size exclusion chromatography column, the mobile phase is an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile, the flow rate is 0.2mL/min, the analysis time is 30 minutes, the mass spectrometer is Thermo Q Exactive Plus, and the main parameters of the mass spectrometer are: The spray voltage is
  • the mass spectrometry data analysis software Biopharma Finder 4.1 is used, and the light and heavy chain mass spectra are calculated through the Respect algorithm deconvolution process.
  • the molecular weight information of the peak and the mass spectrum response signal of each component are used to calculate the DAR value of the ADC sample to be tested.
  • the antibody-drug conjugates or pharmaceutically acceptable salts thereof as shown in Table 10 below were prepared.
  • Example 19 Biological activity and related property testing
  • Test example 1 K562-GRE reporter gene detects the activity of compound of formula (DH)
  • K562 cells were seeded at 5 ⁇ 10 5 cells/well into a 6-well culture dish (Brand Costar, No. 3516), incubate at 37°C, 5% CO2 for 24 hours. The next day, dilute 3 ⁇ g of pNL2.2 [NLucP/MMTV/Hygro-NANO] (Promega) and 3 ⁇ l of PLUS reagent (Brand Invitrogen, Cat. No. 11514-015) into 150 ⁇ L Opti-MEM (Brand Gibco, Cat. No. 11058021) and incubate at room temperature for 5 minutes.
  • the pNL2.2 [NLucP/MMTV/Hygro-NANO] vector contains the MMTV LTR (murine mammary tumor virus long terminal repeat), which responds to and drives activation of several nuclear receptors, such as the glucocorticoid receptor and the androgen receptor Transcription of the luciferase reporter gene NanoLuc.
  • the diluted DNA solution was mixed and pre-incubated with Lipofectamine LTX solution (Brand Invitrogen, Cat. No. 15338-100) (6 ⁇ l Lipofectamine LTX + 144 ⁇ l Opti-MEM) in a 1:1 ratio, and incubated at room temperature for 15 minutes to form DNA-Lipofectamine LTX complex.
  • K562 cells were transfected for 24 h at 37°C, 5% CO2 . After transfection, cells were washed with 3 mL of PBS and grown selectively for two weeks with complete growth medium containing 125 ⁇ g/mL hygromycin B (Brand Invitrogen, Cat. No. 10687010). "K562-GRE(pNL2.2[NLucP/MMTV/Hygro-NANO])" cells were generated.
  • K562-GRE (pNL2.2 [NLucP/MMTV/Hygro-NANO) cells were cultured in 50 ⁇ l of assay medium (RPMI1640 medium, 1% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acids and 1% penicillin- Streptomycin) was seeded into a 96-well tissue culture-treated white plate (Brand Costar, Cat. No. 3917) with 50,000 cells per well, and 50 ⁇ L (2 ⁇ ) of a compound diluted 5-fold in the assay medium was added to treat the cells, each There are a total of 8 concentrations of the compound, and the final concentration of the compound in the system is 0.0000128 ⁇ M-1 ⁇ M.
  • assay medium RPMI1640 medium, 1% FBS, 1% sodium pyruvate, 1% MEM non-essential amino acids and 1% penicillin- Streptomycin
  • Test Example 2 Determination of binding activity of compound of formula (D-H) to glucocorticoid receptor GR
  • Polarscreen Glucocorticoid Receptor Assay Kit Red (Brand Thermo, Cat. No. A15898) kit was used to determine the binding activity of the compound to GR.
  • the compound to be tested was diluted 10 times in a 96-well V-bottom plate (Brand Nunc, Cat. No. 249944) with DMSO, with the highest concentration being 100 ⁇ M, for a total of 8 concentrations. Then use the detection buffer Complete GR Screening buffer provided in the kit to further dilute the compound 50 times, and transfer 10 ⁇ l of the diluted compound to a 384 microwell plate (brand Corning, Cat. No.
  • Test Example 3 Determination of binding activity of compound of formula (D-H) to estrogen receptor ER
  • TR-FRET ER Alpha Coactivator Assay kit (Brand Thermo, Cat. No. A15885) kit measures the binding activity of compounds to ER.
  • the ER receptor agonist Estradiol (Brand Sigma, Cat. No. E8875-25) was diluted 10 times with DMSO in a 96-well V-bottom plate, with the highest concentration being 100 ⁇ M, for a total of 8 concentrations.
  • the compound to be tested was diluted 10-fold with DMSO, with the highest concentration being 3000 ⁇ M, for a total of 8 concentrations.
  • Test Example 4 Determination of binding activity of compound of formula (DH) to androgen receptor AR
  • the TR-FRET Androgen Receptor Coactivator Assay kit (Brand Thermo, Cat. No. A15878) determines the binding activity of compounds to AR.
  • the AR receptor agonist dihydrotestosterone (DHT) (Brand Sigma, Cat. No. D-073) was diluted 10 times with DMSO in a 96-well V-bottom plate, with the highest concentration being 100 ⁇ M, for a total of 8 concentrations.
  • the compound to be tested was diluted 10-fold with DMSO, with the highest concentration being 3000 ⁇ M, for a total of 8 concentrations.
  • Test Example 5 Determination of binding activity of compound of formula (D-H) to progesterone receptor PR
  • TR-FRET Progesterone Receptor Coactivator Assay kit (Brand Thermo, Cat. No. A15903) kit determines the binding activity of compounds to PR.
  • the PR receptor agonist Progesterone (Brand Sigma, Cat. No. P0130) was diluted 10 times with DMSO in a 96-well V-bottom plate, with the highest concentration being 100 ⁇ M, for a total of 8 concentrations.
  • the compound to be tested was diluted ten-fold with DMSO, with the highest concentration being 3000 ⁇ M, for a total of 8 concentrations. Then the compound was further diluted 50 times with the detection buffer Nuclear Receptor Buffer F (containing 5mM DTT) provided in the kit, and 10 ⁇ l of the dilution was transferred.
  • test results show that the test compound has strong binding activity to GR and weak binding activity to PR. It does not bind to ER and AR, and is expected to reduce the side effects caused by acting on other hormone receptors.
  • CHS mouse model 6-8 week old C57BL/6N female mice (Beijing Vitong Lever Experimental Animal Technology Co., Ltd.) were selected, and the hair on the abdomen of each mouse was removed with small animal electric clippers. Use a micropipette to absorb 400 ⁇ l of sensitizer and apply it evenly on the mouse abdomen for epidermal sensitization. Hold the mouse for 3-5 seconds after application and allow the solvent to evaporate from the skin as much as possible.
  • the specific preparation method of the sensitizer is: weigh 0.5g of FITC (fluorescein isothiocyanate; sigma-Aldrich) powder, use 50mL of acetone (acetone; Sinopharm Reagent) and 50mL of DBP (dibutyl phthalate; sigma -Aldrich) The solvent mixed in equal proportions is fully dissolved to obtain a sensitizer with a FITC content of 0.5%. On the 6th day after sensitization, first use a dial thickness gauge to measure the thickness of the mouse's right ear as the baseline value, and then use a micropipette to absorb the freshly prepared sensitizer and apply it evenly on the inner and outer sides of the mouse's right ear.
  • FITC fluorescein isothiocyanate
  • acetone acetone
  • DBP dibutyl phthalate
  • Treatment plan for CHS model mice Group the mice into blank group, negative control group, positive control group and experimental group.
  • the dosage of Compound 001 and Compound 009 in the experimental group was 3 ⁇ g/animal
  • the positive control group was Compound A (synthesized according to the method reported in patent WO2017210471 Compound 41) at 3 ⁇ g/animal
  • the negative control group was blank vehicle (0.5% DMSO/PBS ).
  • Example 17-2 The anti-human TNF ⁇ antibody prepared in Example 17-2 was subjected to ELISA and Biacore detection, and the method was the same as in Example 17-1.
  • the ELISA detection EC 50 value and Biacore detection results are shown in Table 12.
  • Mouse TNF ⁇ protein (Novoprotein, Cat. No.: CF09) was coated overnight at 4°C, rinsed three times with 0.05% Tween 20-PBS solution, added 2% BSA blocking solution, and blocked at 37°C for 1.5 hours. Rinse three times with 0.05% Tween 20-PBS solution, add the diluted sample, and incubate at 37°C for 1 hour. Rinse three times with 0.05% Tween 20-PBS solution, add secondary antibody HRP goat anti-Rat IgG (Jackson Immuno, Cat. No.: 112-035-003), and incubate at 37°C for 1 hour.
  • HRP goat anti-Rat IgG Jackson Immuno, Cat. No.: 112-035-003
  • Test Example 8 Test of binding activity of antibody-drug conjugates to TNF ⁇ -expressing cells
  • the nucleotide sequence encoding the TACE-resistant human transmembrane TNF ⁇ amino acid sequence was cloned into the pcDNA5/FRT vector (Thermofisher) to obtain pcDNA5/FRT-human TNF ⁇ FL. (77-88del) plasmid.
  • This plasmid was co-transfected with pOG44 plasmid into Flp-In-CHO host cells (Thermofisher).
  • Flp-In-CHO-human TNF ⁇ FL (77-88del) cells were digested and counted, and then plated into a 96-well plate (Corning, 3795) at 1.5 ⁇ 10 5 cells/well. Add gradient dilutions of the sample to be tested and incubate at 4°C for 1 hour. After washing twice with PBS, Alexa Fluor-647Goat anti-human Fc secondary antibody (JacksonImmuno, 109-605-098) was added and incubated at 4°C for 1 hour. After washing twice with PBS, the cells were resuspended and the mean fluorescence intensity (MFI) was analyzed using a flow cytometer (BD FACSCanto TM II).
  • MFI mean fluorescence intensity
  • the nucleotide sequence encoding the TACE-resistant human transmembrane TNF ⁇ amino acid sequence was cloned into the pLVX-IRES-Puro lentiviral vector, and the virus was prepared in HEK293T cells Particles.
  • the K562-human TNF ⁇ -GRE-Luc reporter cell line the K562-GRE cells constructed in Test Example 1 were seeded into a 6-well culture dish containing 1.5 mL of complete growth medium (Costar, 3516), culture was continued for 24 hours at 37°C and 5% CO2 .
  • K562-GRE cells were infected for 24 h at 37°C, 5% CO2 . After incubation, cells were washed with 3 mL of PBS and selected for 2 weeks with complete growth medium containing 125 ⁇ g/mL hygromycin B (Invitrogen, 10687-010) and 1 ⁇ g/mL puromycin (Gibco, A1113802) to generate K562-human TNF ⁇ -GRE-Luc reporter cells.
  • K562-human TNF ⁇ -GRE-Luc cells were resuspended in assay medium (RPMI1640, 10% carbon-adsorbed FBS, 1% Penicillin-Streptomycin), and seeded at 5 ⁇ 10 4 cells/well (50 ⁇ l) in a 96-well plate (Corning , 3610). Dilute the anti-TNF ⁇ antibody-drug conjugate with the above assay medium, add 50 ⁇ l per well to a 96-well plate, and incubate for 24 hours at 37°C, 5% CO2 .
  • assay medium RPMI1640, 10% carbon-adsorbed FBS, 1% Penicillin-Streptomycin
  • Test Example 10 Activity inhibition test of antibody-drug conjugates in LPS-stimulated human monocyte cytokine release model
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • the cells were plated in cell assay plates (Corning, 7007) in RPMI1640 medium (Gibco, 72400-047) containing 15140-122) and 1 ⁇ 10 5 cells per well.
  • the anti-human TNF ⁇ antibody-drug conjugate can significantly inhibit the release of pro-inflammatory cytokines IL-6 and IL-1 ⁇ from monocytes stimulated and activated by LPS.
  • Test Example 11 Activity inhibition test of antibody-drug conjugates in LPS-stimulated mouse PBMC cytokine release model
  • mice The anticoagulated blood of 6-8 week old female C57BL/6 mice (Shanghai Lingchang Biotechnology Co., Ltd.) was taken, the mouse PBMC cells were isolated using FICOLL-PAQUE (Cytiva, 17544602), and resuspended in 10% FBS (Gibco, 10091-148) and 1% penicillin-streptomycin (Gibco, 15140-122) in RPMI1640 medium (Gibco, 72400-047) and plated in cell assay plates (Corning, 7007), each Well 4 x 10 5 cells. Cells were incubated with different concentrations of anti-mouse TNF ⁇ antibody-drug conjugates at 37°C, 5% CO2 incubator for 4 h.
  • lipopolysaccharide LPS (Sigma, L4516) with a final concentration of 1000ng/mL was added to stimulate the cells. After incubation for 40 hours in a 37°C, 5% CO2 incubator, the plate was centrifuged at 350g for 5 minutes, and 100 ⁇ L of the supernatant was carefully transferred to Another clean 96-well plate was used to detect IL-6 concentration (Dakewi, 1210603).
  • Use GraphPad Prism software to perform a four-parameter nonlinear regression fitting curve on the dose response data, and calculate the IC 50 value. The results are shown in Table 17 and Figure 5.
  • the anti-mouse TNF ⁇ antibody-drug conjugate can significantly inhibit the release of pro-inflammatory cytokine IL-6 from mouse PBMC stimulated and activated by LPS.
  • Test Example 12 Antibody-drug conjugate CHS mouse model efficacy test
  • Treatment plan for CHS model mice Group the mice into normal control group, negative control group and drug administration group.
  • the negative control group was blank vehicle (0.5% DMSO/PBS), and the administration group was anti-mouse TNF ⁇ antibody 001Mab (BioXcell, BE0058), ADC-L1-001-5 and ADC-L1-009-5, and the dosage was 10 mg. /kg.
  • Test Example 13 DBA/1 mouse collagen antibody-induced arthritis (CAIA) drug efficacy model test
  • DBA/1 male mice aged 6 to 8 weeks (Beijing Viton River Experimental Animal Technology Co., Ltd.) were taken.
  • day 0 five monoclonal antibodies against type II collagen
  • the mixture of 5-Clone Cocktail Kit (Chrondex) was injected intraperitoneally into DBA/1 mice, and 150 ⁇ l was injected into each mouse.
  • the concentration of the antibody mixture is 10 mg/ml, that is, the total amount of antibody mixture injected into each mouse is 1.5 mg; on the third day after type II collagen antibody induction, the mice were intraperitoneally injected with 100 ⁇ l of lipopolysaccharide at a concentration of 0.5 mg/ml. (LPS), that is, the total amount of LPS injected into each mouse is 50 ⁇ g.
  • LPS lipopolysaccharide
  • mice developed disease symptoms around day 5, with the disease reaching its peak on days 7-8. The animals were scored based on their clinical symptoms. 0: No erythema and swelling; 1: Erythema or mild redness and swelling near the tarsus or ankle joint or metatarsal bone, and one toe is red and swollen; 2: Slight erythema and swelling of the ankle joint and metatarsal bone, or redness and swelling of more than two toes; 3: Moderate erythema and swelling of the ankle, wrist joint, and metatarsal bones; 4: Severe redness and swelling of the ankle, wrist joint, metatarsal bones, and toes.
  • mice were randomly divided into normal control group, negative control group and drug administration group according to body weight.
  • the administration groups were anti-mouse TNF ⁇ antibodies 001Mab, ADC-L1-001-5 and ADC-L1-009-5, and the administration dose was 10 mg/kg; the negative control group was blank vehicle (PBS).
  • the mice were intraperitoneally injected on the 3rd, 6th, 10th and 13th days after collagen antibody induction.
  • the negative control group was intraperitoneally injected with an equal volume of vehicle at the same time points. The clinical symptom scores and body weight of the mice were recorded every day.
  • Test Example 14 TNF ⁇ humanized mouse collagen antibody-induced arthritis (CAIA) efficacy model test
  • TNF ⁇ humanized C57BL/6 male mice (B-hTNF ⁇ , Biocytogen) were selected.
  • day 0 five monoclonal antibodies of type II collagen (Arthrogen- CIA 5-Clone Cocktail Kit; Chrondex) mixture was intraperitoneally injected into B-hTNF ⁇ -C57BL/6 mice, 500 ⁇ l was injected into each mouse.
  • the concentration of the antibody mixture is 10 mg/ml, that is, the total amount of antibody mixture injected into each mouse is 5 mg; on the third day after type II collagen antibody induction, the mice were intraperitoneally injected with 100 ⁇ l of lipopolysaccharide with a concentration of 0.5 mg/ml ( LPS), that is, the total amount of LPS injected into each mouse was 50 ⁇ g.
  • LPS lipopolysaccharide
  • mice developed disease symptoms around day 5, with the disease reaching its peak on days 7-9. The animals were scored based on their clinical symptoms. 0: No erythema and swelling; 1: Erythema or mild redness and swelling near the tarsus or ankle joint or metatarsal bone, and one toe is red and swollen; 2: Slight erythema and swelling of the ankle joint and metatarsal bone, or redness and swelling of more than two toes; 3: Moderate erythema and swelling of the ankle, wrist joint, and metatarsal bones; 4: Severe redness and swelling of the ankle, wrist joint, metatarsal bones, and toes.
  • mice were randomly divided into normal control group, negative control group and drug administration group according to their body weight.
  • the administration group is Adalimumab and ADC-L1-009-6, and the administration dose is 10 mg/kg; the negative control group is blank vehicle (PBS).
  • the mice were intraperitoneally injected on the 3rd, 6th, 10th and 13th days after collagen antibody induction.
  • the negative control group was intraperitoneally injected with an equal volume of vehicle at the same time points. The clinical symptom scores and body weight of the mice were recorded every day.
  • Test Example 15 Plasma stability test of anti-TNF ⁇ antibody-drug conjugate
  • ADC was diluted in human plasma (Aoneng Bio, PB021F-C), mouse plasma and monkey plasma (final concentration was 100 ⁇ g/ml), and incubated in a 37°C incubator. The day of incubation was marked as day 0, and samples were taken out on days 7 and 14 for detection of free small molecules.
  • Test Example 16 Enzyme-linked immunoreaction (ELISA) to detect the binding level of anti-IL-4R antibody-drug conjugate to IL-4R protein
  • the anti-human IL-4R antibody-drug conjugate prepared in Example 18 was subjected to ELISA detection.
  • the human IL-4R protein (Acro Biosystems, Cat. No.: ILR-H5221) was mixed with PBS (purchased from Hyclone, Cat. No. SH30256.01). Dilute to a final concentration of 2 ⁇ g/mL, add 50 ⁇ L per well to a 96-well ELISA plate (purchased from Corning, Cat. No. 9018), coat at 37°C for 1 hour, discard the supernatant, and add blocking solution containing 5% (w/w ) Skimmed milk powder (purchased from Sangon, Cat. No.
  • PBST PBS
  • Tween 20 purchased from Sangon, product number A100777-0500.
  • PBST PBS
  • Tween 20 purchased from Sangon, product number A100777-0500.
  • PBST PBS
  • Tween 20 purchased from Sangon, product number A100777-0500.
  • the dilution solution contains 1% (w/w) BSA (purchased from Sangon, Cat. No. A500023- 0100) in PBS, incubate at 37°C for 1 hour, and then wash the plate three times with PBST.
  • HRP horseradish peroxidase
  • Test Example 17 Flow cytometry experiment (FACS) to detect the binding level of anti-IL-4R antibody-drug conjugate to Ramos cells
  • Ramos cells were expanded and cultured in a T-75 cell culture flask (purchased from Corning, Cat. No. 430720), centrifuged at 1000 rpm for 5 minutes at room temperature to collect the cells, and washed twice with PBS (purchased from Hyclone, Cat. No. SH30256.01) buffer. Cells were resuspended in PBS and counted. According to 1 ⁇ 10 5 cells per well, 50 ⁇ L was added to the U-shaped bottom 96-well FACS plate. The anti-human IL-4R antibody-drug conjugate was serially diluted in PBS containing 1% (w/w) BSA (purchased from Sangon, Cat. No.
  • the ability of the molecules to be tested to bind to IL-4R on the surface of Ramos cells is equivalent to the monoclonal antibodies from which they are derived.
  • the connecting small molecules have no effect on the binding of the monoclonal antibodies to the cells.
  • the data MFI in Table 20 is the maximum average fluorescence intensity value of the measured cell population.
  • Test Example 18 Detection of endocytosis level of anti-IL-4R antibody-drug conjugate on Ramos cells
  • Ramos cells were expanded and cultured in T-75 cell culture flasks, centrifuged at 1000 rpm for 5 minutes at room temperature to collect the cells, and washed twice with PBS buffer. According to 4 ⁇ 10 5 cells per well, 100 ⁇ L was added to the U-shaped bottom 96-well FACS reaction plate and kept at 4°C for later use. Prepare 2% FBS (w/w) PBS to dilute the anti-IL-4R antibody-drug conjugate to be tested, add 100 ⁇ L to each well and mix the cells to a final concentration of 100 nM, and incubate at 4°C for 1 hour.
  • Endocytosis rate (%) (MFI 4°C-MFI 37°C)/MFI 4°C ⁇ 100%. The results are shown in Table 21.
  • the endocytosis rate of the molecules to be tested on Ramos cells is comparable to the monoclonal antibodies from which they were derived, and the connecting small molecules have no significant effect on the endocytosis rate.
  • Test Example 19 K562-GRE reporter gene detection activity determination of anti-IL-4R antibody-drug conjugate
  • the nucleotide sequence encoding the TACE-resistant human transmembrane IL-4R amino acid sequence was cloned into the pLVX-IRES-Puro lentiviral vector, and viral particles were prepared in HEK293T cells.
  • the K562-human IL-4R-GRE-Luc reporter cell line the K562-GRE cells constructed in Test Example 1 were seeded into a 6-well culture dish containing 1.5 mL of complete growth medium at 5 ⁇ 10 5 cells/well ( Costar, 3516), incubate at 37°C, 5% CO2 for 24 hours.
  • K562 -GRE cells were infected for 24 hours at 37°C, 5% CO2 . After incubation, cells were washed with 3 mL of PBS and treated with 125 ⁇ g/mL hygromycin B (Invitrogen, 10687-010) and 1 ⁇ g/mL puro( Gibco, A1113802) complete growth medium for two weeks to generate K562-human IL-4R-GRE-Luc reporter cells.
  • K562-human IL4R-GRE-Luc cells were collected and resuspended in assay medium (RPM1640+1% carbon-adsorbed fetal bovine serum+1% sodium pyruvate+1% non-essential amino acids+ at a density of 1 ⁇ 10 7 /ml 1% penicillin/streptomycin). Cells were seeded in a 96-well plate (Costar, 3917) at 5 ⁇ 10 4 cells/well (50 ⁇ l).
  • the conjugate and the isotype control conjugate ADC-L1-009-8 started from 40 nM and were diluted 6 points at a 5-fold gradient, and the diluent was the above-mentioned assay medium.
  • the PE Envision instrument-US Lumi module was used for detection. Fluorescence signal value in the well plate. Draw the induction curve of the drug with the drug concentration as the abscissa and the fluorescence signal value RLU as the ordinate. Four-parameter fitting (GraphPad Prism9) was used to calculate the EC 50 and maximum luminescence intensity of the induction curve. The results are shown in Table 22.
  • Test Example 20 Inhibitory effect of anti-IL-4R antibody-drug conjugate on the proliferation of human primary CD4+ T cells
  • T cells take out an appropriate number of T cells, add 2 ml of PBS to wash the cells twice, and centrifuge at 350g for 5 minutes. After centrifugation and discarding the supernatant, add the pre-prepared CellTrace Violet (Invitrogen, C34557A) working solution to a final concentration of 5 ⁇ M. Flow and mix the T cell suspension added with CellTrace Violet dye, place it in a 37°C incubator and incubate for 10 minutes, shaking gently once to mix. After CTV labeling, add 5 times the volume of complete culture medium to the labeled cell suspension to terminate the reaction. After leaving it at room temperature for 5 minutes, centrifuge at 350g for 5 minutes.
  • CellTrace Violet Invitrogen, C34557A
  • the drug groups are set as follows: (1) Anti-Hel-hIgG4 (isotype control), (2) Dupilumab, (3) anti-IL-4R antibody-drug conjugate single drug group to be tested.
  • the above (1)-(3) groups were all given anti-CD3/CD28 immunocult stimulation.
  • the platemap add CTV-labeled T cells/100 ⁇ L/well (1 ⁇ 10 5 cells/well) and drug working solution 50 ⁇ L/well (4 ⁇ final concentration) in a 96-U well plate (Corning, Cat. No.: 3799). ), anti-CD3/CD28 immunocult 50 ⁇ L/well, the final volume of the system is 200 ⁇ L/well.
  • both the Dupilumab monoclonal antibody group and the test molecule group can inhibit the proliferation of human CD4 + T cells. And compared with the Dupilumab group, the molecule group to be tested has a stronger inhibitory effect on the proliferation of human CD4 + T cells, and there is a statistical difference.
  • Test Example 21 Pharmacological and pharmacodynamic effects of anti-IL-4R antibody-drug conjugate on atopic dermatitis mouse model
  • mice in the normal control group were evenly applied with 25 ⁇ L of acetone and olive oil mixture (4:1) for stimulation; the backs of the mice in the model group were stimulated. and right ear, apply 25 ⁇ L of 0.4% OXA (the same solvent as before) solution evenly on each ear for stimulation.
  • Drug intervention design Drug administration will begin on the 6th day after the initial OXA sensitization. Normal control mice will be divided into separate groups; the mice in the modeling group will be divided into 3 groups according to ear thickness, with 8 mice in each group, and they will be the vehicle control group. (PBS), Dupilumab-10mpk group, anti-IL-4R antibody-drug conjugate to be tested-10mpk group. Mice in each group were intraperitoneally administered twice a week.
  • mice in vivo drug efficacy experiment was completed on the 26th day, and serum, right ear skin tissue, and back skin tissue were collected for subsequent testing. During the entire in vivo experiment, the ear thickness of mice in each group was measured before each OXA induction and at the end of the experiment. As shown in Figure 10, compared with mice in the vehicle control group, both Dupilumab and anti-IL-4R antibody-drug conjugates can significantly inhibit the ear thickening of mice, and the same dose of anti-IL-4R antibody-drug conjugates The inhibitory effect is stronger; at the same time, as shown in Figure 11, both drugs can also inhibit the production of IgE in mice.
  • Histopathological evaluation Take the ear and back skin soaked in fixative, fix it, embed it in paraffin, section it at a thickness of 5 ⁇ m, dewax it and perform gradient hydration before H&E staining. After staining, the pathological sections were scanned by a scanning machine and blinded by an independent second person for pathological scoring. H&E staining pathology scoring standards, epidermis: stromal cell proliferation (0.5-2 points), erosion (0.5-2 points), appearance of spinous processes (0.5-2 points), hyperkeratosis with parakeratosis (0.5-2 points); Dermis and subcutaneous: mixed inflammatory cell infiltration (0.5-2 points), peripheral eosinophil infiltration (0.5-2 points), total 12 points.
  • Test Example 22 Plasma stability test of anti-IL-4R antibody-drug conjugate
  • ADC was diluted in human plasma (Aoneng Biotechnology, PB021F-C) (final concentration was 100 ⁇ g/ml) and placed in a 37°C incubator for incubation. The day of incubation was marked as day 0, and samples were taken out on days 7, 14, and 21 for detection of free small molecules.
  • Test Example 23 Anti-TNF ⁇ antibody-drug conjugate cell incubation experiment
  • the anti-TNF ⁇ antibody-drug conjugate binds to TNF ⁇ on the cell surface, it is endocytosed into the cell. Enzymes in cell lysosomes cleave ADC and release small hormone molecules, thereby exerting the anti-inflammatory effect of small hormone molecules. In order to confirm the form of small molecules released after ADC endocytosis into cells and the enzymatic hydrolysis efficiency, cell incubation experiments were performed.
  • the K562-human TNF ⁇ -GRE-Luc cells (1 ⁇ 10 4 /well) constructed in Test Example 9 were seeded in a 96-well plate (Corning, 3599). After adding 15 ⁇ g/ml ADC, the cells were cultured at 37°C. Box incubation. Combine cells with ADC The samples at the time of mixing are marked as 0 h, and then the samples were taken out at 24 h and 48 h respectively, and acetonitrile (100 ⁇ L/well) was added and mixed by pipetting.
  • Test Example 24 Inhibitory effect of anti-IL-4R antibody-drug conjugate on CD23 expression on B cells in human primary T and B cell co-culture system
  • PBMC cryopreserved cells (Stemexpress, Cat: PBMNC050C) from the liquid nitrogen tank, place them in a 37°C water bath and shake gently to dissolve the cells. Transfer the cell suspension to a centrifuge tube containing 8 ml of preheated 1640 medium (Gibco, Cat: 72400047) and centrifuge for 8 minutes (centrifugation conditions are 300g, 25°C), and discard the supernatant.
  • human primary T cells 50,000 cells/50 ⁇ L/well
  • human primary B cells 25,000 cells/50 ⁇ L/well
  • anti-IL- 4R antibody-drug conjugate 50 ⁇ L/well
  • the above-mentioned cell pellets co-cultured with primary human T and B cells were resuspended in 200 ⁇ L/well PBS, centrifuged for 5 min (centrifugation conditions were 350 g, 25°C), and the supernatant was discarded.
  • Use the fixable near-infrared dead and live cell staining kit dye (Thermofisher, Cat: L10119) to incubate at room temperature for 10 minutes, add 100 ⁇ L/well of staining buffer (2% FBS+PBS), and centrifuge for 5 minutes (centrifugation conditions are 350g, 25°C) Then discard the supernatant.

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Abstract

本公开涉及一类结构新颖的配体-药物偶联物或其药学上可接受的盐，具体而言，本公开提供了结构通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、其制备方法、含有该偶联物的药物组合物、以及其在治疗自身免疫性疾病或炎性疾病中的用途。

Description

配体-药物偶联物及其用途
相关申请的交叉引用
本申请要求于2022年4月1日提交的申请号为202210339497.4的中国专利申请、2022年4月15日提交的申请号为202210395481.5的中国专利申请以及2022年11月8日提交的申请号为202211392212.X的中国专利申请的优先权权益，这些专利申请文件通过引用整体并入本文，用于所有目的。
技术领域
本公开属于生物医药领域，涉及一类结构新颖的配体-药物偶联物、其制备方法、含有该偶联物的药物组合物、以及其在治疗自身免疫性疾病中的用途。
背景技术
糖皮质激素(Glucocorticoid，GC)是机体内极为重要的一类调节分子，它对机体的发育、生长、代谢以及免疫功能等起着重要调节作用，是机体应激反应最重要的调节激素，也是临床上使用最为广泛而有效的抗炎和免疫抑制剂。糖皮质激素可用于治疗许多自身免疫性疾病和炎性疾病，包括类风湿关节炎(RA)、银屑病和炎性肠病等。但长期使用糖皮质激素，易造成骨质疏松、肌肉萎缩、高血压、抑制机体免疫力引起加重感染等副作用。因此激素在自身免疫性疾病治疗中的使用是受限的。
除激素药物外，多种生物制剂被用于治疗自身免疫性疾病和炎性疾病，如靶向抗肿瘤坏死因子α(TNFα)和靶向IL-4R的生物制剂。TNFα是一种涉及到系统性炎症的细胞因子，在许多人类疾病中具有十分关键的作用。抗TNFα生物制剂(如阿达木单抗、依那西普、戈利木单抗和英夫利昔单抗)及抗IL-4R生物制剂(如度普利尤单抗)治疗类风湿性关节炎、炎性肠病、特应性皮炎等自身免疫性疾病的疗效已在临床得到验证，并被广泛使用。然而，一些患者在长期使用这类药物后产生了抗药抗体(anti-drug antibodies,ADA)，从而产生耐药性，降低药物疗效。此外，这类药物的长期使用，也可能增加感染和恶性肿瘤的风险。
因此，在自身免疫性疾病和炎性疾病治疗领域，仍有巨大的未满足的临床需求，需要开发更有效更安全的治疗方法。
发明内容
本公开将生物制剂和激素药物的治疗结合起来，提供一种新型的糖皮质激素受体激动剂，并与配体组合成配体-激素药物偶联物，用于治疗自身免疫性疾病。
本公开提供一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n；
其中，
Pc为配体单元；
L为连接子单元；
D为以下式(D-I)所示的药物单元：
其中，
R1、R2各自独立地选自H、CH3或卤素；
环A选自苯基、5-10元杂芳基或C3-C10环烷基，所述苯基、5-10元杂芳基或C3-C10环烷基任选被一个或多个R1a取代；
X选自O、S、C1-C3亚烷基-O、C1-C3亚烷基-S、NR6或C(R7)(R8)；
R6选自H、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
R7、R8各自独立地选自H、卤素、CN、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，或者R7、R8与它们连接的原子共同形成C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
R3选自H、OH或NHR9，R4、R5与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代；或者，R5选自H、OH或NHR9，R3、R4与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代；
R9选自H、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rd取代；
R13、R14各自独立地选自H、卤素、CN、OH、NH2、O(C1-C3烷基)或C1-C6烷基；
每一个R4a独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rd取代；
每一个Rd独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；
R10选自OH、SH、O(C1-C6烷基)、S(C1-C6烷基)、O-C(＝O)-(C1-C6烷基)或所述O(C1-C6烷基)、S(C1-C6烷基)、O-C(＝O)-(C1-C6烷基)任选被一个或多个卤素或CN取代；
R11、R12各自独立地选自H或C1-C6烷基；
每一个R1a独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
每一个Rb独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rc取代；
每一个Rc独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；并且，
n为1～16；
其中，连接子单元L与式(D-I)化合物R3、R4、R5、R13、R14中存在的-OH、-NH2或-NH-基团中的任意一个共价连接。
在一些实施方案中，R1、R2各自独立地选自H或F。
在一些实施方案中，环A选自苯基或5-6元杂芳基，所述苯基或5-6元杂芳基任选被一个或多个R1a取代。
在一些实施方案中，环A选自苯基，所述苯基任选被一个或多个R1a取代。
在一些实施方案中，R1a选自卤素、CN、＝O、OH、NH2或C1-C6烷基。
在一些实施方案中，R1a选自NH2。
在一些实施方案中，环A选自
在一些实施方案中，环A选自
在一些实施方案中，X选自O、S、C1-C3亚烷基-O、C1-C3亚烷基-S或C(R7)(R8)。
在一些实施方案中，R7、R8各自独立地选自H、卤素、CN、OH、NH2或C1-C3烷基，或者R7、R8与它们连接的原子共同形成C3-C6环烷基，所述OH、NH2、C1-C3烷基或C3-C6环烷基任选被一个或多个Rb取代。
在一些实施方案中，R7、R8各自独立地选自H、卤素或C1-C3烷基，或者R7、R8与它们连接的原子共同形成C3-C6环烷基。
在一些实施方案中，R7、R8各自独立地选自H、F或甲基，或者R7、R8与它们连接的原子共同形成环丙基。
在一些实施方案中，X选自O、S、CH2O、CH2S、CH2、CF2、CHCH3或
在一些实施方案中，X选自CH2。
在一些实施方案中，R10选自OH、SH、O(C1-C3烷基)、O-C(＝O)-(C1-C3烷基)或所述O(C1-C3烷基)或O-C(＝O)-(C1-C3烷基)任选被一个或多个卤素取代。
在一些实施方案中，R11、R12各自独立地选自H、甲基或乙基。
在一些实施方案中，R10选自OH、SH、OCH2F、O(C＝O)CH3、
在一些实施方案中，R10选自OH或
在一些实施方案中，R9选自H或C1-C6烷基。
在一些实施方案中，R9选自H。
在一些实施方案中，R3选自H、OH或NH2，R4、R5与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
在一些实施方案中，R5选自H、OH或NH2，R3、R4与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
在一些实施方案中，R3选自H，R4、R5与它们连接的原子共同形成5-6元杂环基或5-6元杂芳基，所述5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
在一些实施方案中，R5选自H或NH2，R3、R4与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
在一些实施方案中，R3选自H、OH或NH2，R4、R5与它们连接的原子共同形成 所述任选被一个或多个R4a取代。
在一些实施方案中，R3选自H，R4、R5与它们连接的原子共同形成 所述任选被一个或多个R4a取代。
在一些实施方案中，R5选自H、OH或NH2，R3、R4与它们连接的原子共同形成 所述 任选被一个或多个R4a取代。
在一些实施方案中，R5选自H或NH2，R3、R4与它们连接的原子共同形成 所述 任选被一个或多个R4a取代。
在一些实施方案中，R4a选自卤素、CN、＝O、OH、NH2、C1-C6烷基或C3-C6环烷基。
在一些实施方案中，R4a选自卤素、CN、＝O、OH、NH2或C1-C3烷基。
在一些实施方案中，R4a选自＝O或NH2。
在一些实施方案中，R3选自H，R4、R5与它们连接的原子共同形成
在一些实施方案中，R5选H或NH2，R3、R4与它们连接的原子共同形成
在一些实施方案中，R13、R14各自独立地选自H或NH2。
在一些实施方案中，R13选自H。
在一些实施方案中，R14选自H。
在一些实施方案中，式(D-I)所示化合物或其药学上可接受的盐选自以下式(D-II)所示化合物或其药学上可接受的盐：
其中，
R1、R2各自独立地选自H或F；
R10选自OH或
R3、R4、R5、R13、R14如前文所述的任一定义。
在一些实施方案中，结构单元选自
在一些实施方案中，结构单元选自
在一些实施方案中，式(D-I)所示化合物或其药学上可接受的盐选自以下所示化合物或其药学上可接受的盐：



在一些实施方案中，本公开提供一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n：
其中，
Pc、L、n如前文定义；
D选自以下化合物：

在一些实施方案中，所述连接子单元L选自其a端与配体单元Pc共价连接，b端与药物单元D共价连接，其中L1为由1至8个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
在一些实施方案中，所述L1为由2、3或4个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代。
在一些实施方案中，所述L1为二肽残基，构成二肽残基中的一个氨基酸为甘氨酸。
在一些实施方案中，所述L1选自Gly-Lys或Gly-Glu的二肽残基。
在一些实施方案中，连接子单元L选自其a端与配体单元Pc共价连接，b端与药物单元D共价连接，所述L1选自Gly-Lys或Gly-Glu的二肽残基。
在一些实施方案中，连接子单元L选自 其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
在一些实施方案中，连接子单元L选自 其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
在一些实施方案中，本公开通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：

其中Pc和n如前文定义。
在一些实施方案中，前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐，其中所述配体单元Pc可选自多肽、抗体或抗原结合片段；
在一些实施方案中，所述Pc为特异性结合TNFα或IL-4R的抗体或抗原结合片段；所述抗体或抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)；
在一些实施方案中，(1)所述重链可变区包括SEQ ID NO.4-9、12-16、18、20、22、57、68、70、72、74或76所示VH中所含的HCDR1、HCDR2和HCDR3；或/和所述轻链可变区包括SEQ ID NO.1-3、17、19、21、23、58、69、71、73、75或77所示VL中所含的LCDR1、LCDR2和LCDR3；(2)所述重链可变区或/和所述轻链可变区包括与第(1)组中所述HCDR1-3或/和LCDR1-3中的每个CDR相比，具有至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列；进一步的，所述至少80％同一性为85％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的同一性。可选的，所述HCDR1-3和所述LCDR1-3根据Kabat编号系统、Chothia编号系统或IMGT编号系统确定。
在一些实施方案中，所述重链可变区包括HCDR1、HCDR2和HCDR3，或/和所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3或/和所述LCDR1-3选自以下；
(1)所述HCDR1-3为SEQ ID NO.24-26；或/和所述LCDR1-3为SEQ ID NO.27-29；
(2)所述HCDR1-3为SEQ ID NO.30-32；或/和所述LCDR1-3为SEQ ID NO.33-35；
(3)所述HCDR1-3为SEQ ID NO.36-38；或/和所述LCDR1-3为SEQ ID NO.39-41；
(4)所述HCDR1-3为SEQ ID NO.42-44；或/和所述LCDR1-3为SEQ ID NO.45-47；
(5)所述HCDR1-3为SEQ ID NO.48-50；或/和所述LCDR1-3为SEQ ID NO.51-53；
(6)所述HCDR1-3为SEQ ID NO.60-62；或/和所述LCDR1-3为SEQ ID NO.63-65；
(7)所述HCDR1-3为SEQ ID NO.78-80；或/和所述LCDR1-3为SEQ ID NO.81-83；
(8)所述HCDR1-3为SEQ ID NO.84-86；或/和所述LCDR1-3为SEQ ID NO.87-89；
(9)所述HCDR1-3为SEQ ID NO.90-92；或/和所述LCDR1-3为SEQ ID NO.93-95；
(10)所述HCDR1-3为SEQ ID NO.96-98；或/和所述LCDR1-3为SEQ ID NO.99-101；
(11)所述HCDR1-3为SEQ ID NO.102-104；或/和所述LCDR1-3为SEQ ID NO.105-107；或，
所述HCDR1-3或/和所述LCDR1-3具有与第(1)-(11)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列；优选地，所述HCDR1-3或/和所述LCDR1-3具有与第(1)-(11)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性；进一步的，所述至少80％同一性为85％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的同一性。
在一些实施方案中，所述抗体或抗原结合片段包括：所述重链可变区包括SEQ ID NO.4-9、12-16、18、20、22、57、68、70、72、74或76所示序列，或/和所述轻链可变区包括SEQ ID NO.1-3、17、19、21、23、58、69、71、73、75或77所示序列；或所述重链可变区或/和所述轻链可变区包括与上述任一组中所述重链可变区和轻链可变区相比，具有至少80％同一性的序列；进一步的，所述至少80％同一性为85％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％的同一性。
在一些实施方案中，所述抗体或抗原结合片段包括重链恒定区序列和/或轻链恒定区序列，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；可选的，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选的，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:10或59所示序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:11所示序列。
在一些实施方案中，所述抗体选自Nerelimomab，Certolizumab，Infliximab，Golimumab，Adalimumab，Dupixent，002Mab，003Mab，004Mab，005Mab或006Mab。
在一些实施方案中，所述抗体选自Nerelimomab，Certolizumab，Infliximab，Golimumab，Adalimumab或Dupixent。
在一些实施方案中，所述抗体或抗原结合片段为单特异性、双特异性、三特异性、或四特异性。
在一些实施方案中，前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐，其中n选自1～16，例如n选自1～10，例如n选自1～8，例如n选自2～8，例如n选自2～6，例如n选自6～8。
在一些实施方案中，n选自2～6，例如n为2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9或6.0。
在一些实施方案中，n选自6～8，例如n为6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8.0。
本公开还提供了一种药物-连接子化合物或其药学上可接受的盐，其结构通式为X-L-D，其中：
药物单元D如前文所述任一定义；
连接子单元L选自其a端与X共价连接，b端与药物单元D共价连接，L1如前文所述任一定义；
X选自卤素、OS(O)2CH3或OS(O)2CF3。
在一些实施方案中，L选自其a端与X共价连接，b端与药物单元D共价连接，所述L1选自Gly-Lys或Gly-Glu的二肽残基。
在一些实施方案中，L选自 其a端与X共价连接，b端与药物单元D共价连接。
在一些实施方案中，L选自 其a端与X共价连接，b端与药物单元D共价连接。
在一些实施方案中，X选自Br或I。
在一些实施方案中，X选自Br。
在一些实施方案中，本公开前述通式为X-L-D的药物-连接子化合物或其药学上可接受的盐，其中：
药物单元D选自以下化合物：

L选自其a端与X共价连接，b端与药物单元D共价连接；
X选自Br或I。
在一些实施方案中，本公开通式为X-L-D的药物-连接子化合物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：

本公开还提供了以下式(D-H)所示的化合物或其药学上可接受的盐：
其中，
R1、R2、R3、R4、R5、R10、R13、R14如前文所述任一定义。
在一些实施方案中，式(D-H)所示的化合物或其药学上可接受的盐选自以下所示化合物或其药学上可接受的盐：


另一方面，本公开提供一种药物组合物，其包含本公开前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐和药学上可接受的辅料。
另一方面，本公开提供治疗哺乳动物炎性疾病或自身免疫性疾病的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物在制备治疗炎性疾病或自身免疫性疾病的药物中的用途。
另一方面，本公开提供前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物在治疗炎性疾病或自身免疫性疾病中的用途。
另一方面，本公开提供治疗炎性疾病或自身免疫性疾病的前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供一种药物组合物，其包含本公开前述式(D-H)所示的化合物或其药学上可接受的盐和药学上可接受的辅料。
另一方面，本公开提供治疗哺乳动物炎性疾病或自身免疫性疾病的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物。
另一方面，本公开提供前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物在制备治疗炎性疾病或自身免疫性疾病的药物中的用途。
另一方面，本公开提供前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物在治疗炎性疾病或自身免疫性疾病中的用途。
另一方面，本公开提供治疗炎性疾病或自身免疫性疾病的前述式(D-H)所示的化合物或其药学上可接受的盐、或其药物组合物。
在一些实施方案中，所述炎性疾病或自身免疫性疾病疾病可选自类风湿关节炎、强直性脊柱炎、骨关节炎、脊椎炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、特应性皮炎、荨麻疹、心肌炎、脑炎、葡萄膜炎、慢性阻塞性肺疾病、血管炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、微观结肠炎、胶原性结肠炎、息肉状结肠炎、坏死性小肠结肠炎、透壁性结肠炎、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘。
另一方面，本公开提供通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐的制备方法，包括将本公开前述通式为X-L-D的药物-连接子化合物与前述配体偶联的步骤，可选的，所述配体为抗体或抗原结合片段。
另一方面，本公开提供通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐的制备方法，包括将本公开前述药物单元D与前述配体单元Pc连接的步骤；可选的，通过前述连接子单元L连接；可选的，配体为抗体或抗原结合片段。
另一方面，本公开提供前述式(D-H)所示的化合物或其药学上可接受的盐在制备前述通式为Pc-(L-D)n的配体-药物偶联物或其药学上可接受的盐中的用途，和/或前述式(D-H)所示的化合物或其药学上可接受的盐在制备前述通式为X-L-D的药物-连接子化合物或其药学上可接受的盐中的用途。
本公开提供的配体-药物偶联物具有显著的抗炎活性、优异的血浆稳定性和降低的毒副作用。
另一方面，本公开提供一种特异性结合人TNFα的抗体或抗原结合片段，其包括重链可变区(VH)和轻链可变区(VL)，所述重链可变区包括SEQ ID NO.5-9和12-15中任一个所示的序列，并且所述轻链可变区包括SEQ ID NO.2或3所示的序列。
在一些实施方案中，特异性结合人TNFα的抗体或抗原结合片段，包括以下所示的重链可变区(VH)和轻链可变区(VL)：
(1)所述VH包括SEQ ID NO.5，并且所述VL包括SEQ ID NO.2；
(2)所述VH包括SEQ ID NO.6，并且所述VL包括SEQ ID NO.2；
(3)所述VH包括SEQ ID NO.7，并且所述VL包括SEQ ID NO.2；
(4)所述VH包括SEQ ID NO.8，并且所述VL包括SEQ ID NO.2；
(5)所述VH包括SEQ ID NO.9，并且所述VL包括SEQ ID NO.2；
(6)所述VH包括SEQ ID NO.5，并且所述VL包括SEQ ID NO.3；
(7)所述VH包括SEQ ID NO.6，并且所述VL包括SEQ ID NO.3；
(8)所述VH包括SEQ ID NO.7，并且所述VL包括SEQ ID NO.3；
(9)所述VH包括SEQ ID NO.8，并且所述VL包括SEQ ID NO.3；
(10)所述VH包括SEQ ID NO.9，并且所述VL包括SEQ ID NO.3；
(11)所述VH包括SEQ ID NO.12，并且所述VL包括SEQ ID NO.2；
(12)所述VH包括SEQ ID NO.13，并且所述VL包括SEQ ID NO.2；
(13)所述VH包括SEQ ID NO.14，并且所述VL包括SEQ ID NO.2；或，
(14)所述VH包括SEQ ID NO.15，并且所述VL包括SEQ ID NO.2。
在一些实施方案中，特异性结合人TNFα的抗体或抗原结合片段包括重链恒定区序列和/或轻链恒定区序列，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区片段包括CH1，铰链区，CH2，CH3或Fc；可选的，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选的，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由所述VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:10所示序列，所述轻链由所述VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:11所示序列。
在一些实施方案中，特异性结合人TNFα的抗体或抗原结合片段为单特异性、双特异性、三特异性、或四特异性。
另一方面，本公开提供一种药物组合物，其包含本公开前述特异性结合人TNFα的抗体或抗原结合片段和药学上可接受的辅料。
另一方面，本公开提供治疗哺乳动物炎性疾病或自身免疫性疾病的方法，包括对需要该治疗的哺乳动物，优选人类，给予治疗有效量的前述特异性结合人TNFα的抗体或抗原结合片段、或其药物组合物。
另一方面，本公开提供前述特异性结合人TNFα的抗体或抗原结合片段、或其药物组合物在制备治疗炎性疾病或自身免疫性疾病的药物中的用途。
另一方面，本公开提供前述特异性结合人TNFα的抗体或抗原结合片段、或其药物组合物在治疗炎性疾病或自身免疫性疾病中的用途。
另一方面，本公开提供治疗炎性疾病或自身免疫性疾病的前述特异性结合人TNFα的抗体或抗原结合片段、或其药物组合物。
在一些实施方案中，所述炎性疾病或自身免疫性疾病疾病可选自类风湿关节炎、强直性脊柱炎、骨关节炎、脊椎炎、系统性红斑狼疮、皮肤红斑狼疮、狼疮性肾炎、IgA肾病、干燥综合征、多肌炎、皮肌炎、特应性皮炎、荨麻疹、心肌炎、脑炎、葡萄膜炎、慢性阻塞性肺疾病、血管炎、硬皮病、银屑病、斑块状银屑病、斑秃、多发性硬化症、肌萎缩侧索硬化症、炎性肠病、溃疡性结肠炎、克罗恩病、微观结肠炎、胶原性结肠炎、息肉状结肠炎、坏 死性小肠结肠炎、透壁性结肠炎、移植物抗宿主病、器官移植排斥、自身免疫肝炎、I型糖尿病、自身免疫性血管炎、湿疹或哮喘。
术语定义和说明
除非另有说明，本公开中所用的术语具有下列含义，本公开中记载的基团和术语定义，包括其作为实例的定义、示例性的定义、优选的定义、表格中记载的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“配体”是指能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞结合的分子。在本公开实施方案中，配体或配体单元表示为Pc，配体可通过配体上的杂原子与连接子单元形成连接键。在本公开的一些实施方案中，配体选自抗体或抗原结合片段，所述抗体选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；在本公开的一些实施方案中，抗体为单克隆抗体。
术语“连接子”或“连接子单元”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化学键。
术语“药物”指在生物体内具有生物活性的小分子化合物。在本公开的一些实施方案中，药物为具有抗炎功能的糖皮质激素受体激动剂或其相应的磷酸酯分子。
术语“配体-药物偶联物”，指配体通过稳定的连接子单元与具有生物活性的药物相连。在本公开的一些实施方案中，“配体-药物偶联物”为抗体-药物偶联物(antibody drug conjugate，ADC)，ADC指将单克隆抗体或者抗体片段通过稳定的连接子单元与具有生物活性的药物相连。
术语“DAR”或“药物抗体比率”是指每个抗体分子连接的小分子糖皮质激素受体激动剂药物的平均数目。在通式为Pc-(L-D)n的抗体-药物偶联物中，DAR由变量“n”定义，n既可以是整数，也可以是小数。
术语“抗体”按最广义使用，是指包含来自免疫球蛋白重链可变区的足够序列和/或来自免疫球蛋白轻链可变区的足够序列，从而能够特异性结合至抗原的多肽或多肽组合。本文“抗体”涵盖各种形式和各种结构，只要它们展现出期望的抗原结合活性。本文“抗体”包括具有移植的互补决定区(CDR)或CDR衍生物的替代蛋白质支架或人工支架。此类支架包括抗体衍生的支架(其包含引入以例如稳定化抗体三维结构的突变)以及包含例如生物相容性聚合物的全合成支架。参见，例如Korndorfer等人,2003,Proteins:Structure,Function,and Bioinformatics,53(1):121-129(2003)；Roque等人,Biotechnol.Prog.20:639-654(2004)。此类支架还可以包括非抗体衍生的支架，例如本领域已知可用于移植CDR的支架蛋白，包括但不限于肌腱蛋白、纤连蛋白、肽适体等。
本文“抗体”包括一种典型的“四链抗体”，其属于由两条重链(HC)和两条轻链(LC)组成的免疫球蛋白；重链是指这样的多肽链，其在N端到C端的方向上由重链可变区(VH)、重链恒定区CH1结构域、铰链区(HR)、重链恒定区CH2结构域、重链恒定区CH3结构域组成；并且，当所述全长抗体为IgE同种型时，任选地还包括重链恒定区CH4结构域；轻链是在N端到C端方向上由轻链可变区(VL)和轻链恒定区(CL)组成的多肽链；重链与重链之间、重链与轻链之间通过二硫键连接，形成“Y”字型结构。由于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，可将本文“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，IgA可分为IgA1和IgA2。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。
本文“抗体”还包括不包含轻链的抗体，例如，由单峰驼(Camelus dromedarius)、双峰驼(Camelus bactrianus)、大羊驼(Lama glama)、原驼(Lama guanicoe)和羊驼(Vicugna pacos)等产生的重链抗体(heavy-chain antibodies,HCAbs)以及在鲨等软骨鱼纲中发现的免疫球蛋白新抗原受体(Ig new antigen receptor,IgNAR)。
本文“抗体”可以来源于任何动物，包括但不限于人和非人动物，所述非人动物可选自灵长类动物、哺乳动物、啮齿动物和脊椎动物，例如骆驼科动物、大羊驼、原鸵、羊驼、羊、兔、小鼠、大鼠或软骨鱼纲(例如鲨)。
本文“抗体”包括但不限于单克隆抗体、多克隆抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、完整抗体、完整抗体的片段、裸抗体、缀合抗体、嵌合抗体、人源化抗体或全人抗体。
术语“单克隆抗体”是指从基本上同质的抗体群体获得的抗体，即，除了可能的变异体(例如含有天然存在的突变或在制剂的生产过程中产生，此类变体通常以少量存在)之外，包含所述群体的各个抗体是相同的和/或结合相同的表位。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制剂相反，单克隆抗体制剂中的每种单克隆抗体针对抗原上的单一决定簇。本文修饰语“单克隆”不应解释为需要通过任何特定方法产生所述抗体或抗原结合分子。举例来说，单克隆抗体可通过多种技术制得，包括(但不限于)杂交瘤技术、重组DNA方法、噬菌体库展示技术和利用含有全部或部分人免疫球蛋白基因座的转殖基因动物的方法和其它本领域已知的方法。
术语“天然抗体”是指通过多细胞生物体的免疫系统制造和配对的抗体。本文术语“工程化抗体”的抗体是指通过基因工程、抗体工程等技术获得的非天然抗体，示例性地，“工程化抗体”包括人源化抗体、小分子抗体(例如scFv等)、双特异性抗体等等。
术语“单特异性”是指表示具有一个或多个结合位点，其中每个结合位点结合相同抗原的相同表位。
术语“多特异性抗体”是指具有至少两个抗原结合位点，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。因此，诸如“双特异性”、“三特异性”、“四特异性”等术语是指抗体/抗原结合分子可以结合的不同表位的数目。
术语“价”表示抗体/抗原结合分子中规定数目的结合位点的存在。因此,术语“单价”、“二价”、“四价”和“六价”分别表示抗体/抗原结合分子中一个结合位点、两个结合位点、四个结合位点和六个结合位点的存在。
本文“全长抗体”、“完好抗体”和“完整抗体”在本文中可互换使用，是指具有基本上与天然抗体结构相似的结构。
本文“抗原结合片段”和“抗体片段”在本文中可互换使用，其不具备完整抗体的全部结构，仅包含完整抗体的局部或局部的变体，所述局部或局部的变体具备结合抗原的能力。本文“抗原结合片段”或“抗体片段”包括但不限于Fab、Fab’、Fab’-SH、F(ab’)2和scFv。
完整抗体的木瓜蛋白酶消化生成两个同一的抗原结合片段，称作“Fab”片段，每个含有重和轻链可变域，还有轻链的恒定域和重链的第一恒定域(CH1)。如此,本文术语“Fab片段”指包含轻链的VL域和恒定域(CL)的轻链片段,和重链的VH域和第一恒定域(CH1)的抗体片段。Fab’片段因在重链CH1域的羧基末端增加少数残基而与Fab片段不同,包括来自抗体铰链区的一个或多个半胱氨酸。Fab’-SH是其中恒定域的半胱氨酸残基携带游离硫醇基团的Fab’片段。胃蛋白酶处理产生具有两个抗原结合位点(两个Fab片段)和Fc区的一部分的F(ab’)2片段。
术语“scFv”(single-chain variable fragment)是指包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun,The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-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，可使 用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。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。在一些情况下，scFv的VH与VL之间还可以存在二硫键，形成二硫键连接的Fv(dsFv)。
术语“双抗体(diabody)”，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(Cabilly等人的U.S.P 4,816,567；Morrison等人,Proc.Natl.Acad.Sci.USA,81:6851 6855(1984))。例如，术语“嵌合抗体”可包括这样的抗体(例如人鼠嵌合抗体)，其中抗体的重链和轻链可变区来自第一抗体(例如鼠源抗体)，而抗体的重链和轻链恒定区来自第二抗体(例如人抗体)。
术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留或部分保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性、提高免疫细胞活性的能力、增强免疫应答的能力等。
术语“全人抗体”是指具有其中FR和CDR二者都源自人种系免疫球蛋白序列的可变区的抗体。此外，如果抗体包含恒定区，则恒定区也源自人种系免疫球蛋白序列。本文全人抗体可以包括不由人种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机或位点特异性诱变或通过体内体细胞突变引入的突变)。然而，本文“全人抗体”不包括其中来源于另一个哺乳动物物种(例如小鼠)的种系的CDR序列已被移植到人框架序列上的抗体。
术语“可变区”是指抗体重链或轻链中牵涉使抗体结合抗原的区域，“重链可变区”与“VH”、“HCVR”可互换使用，“轻链可变区”与“VL”、“LCVR”可互换使用。天然抗体的重链和轻链的可变域(分别是VH和VL)一般具有相似的结构,每个域包含四个保守的框架区(FR)和三个高变区(HVR)。参见例如Kindt等人,Kuby Immunology,第6版,W.H.Freeman and Co.,p.91(2007)。单个VH或VL域可足以赋予抗原结合特异性。本文术语“互补决定区”与“CDR”可互换使用，通常指重链可变区(VH)或轻链可变区(VL)的高变区(HVR)，该部位因在空间结构上可与抗原表位形成精密的互补，故又称为互补决定区，其中，重链可变区CDR可缩写为HCDR，轻链可变区CDR可缩写为LCDR。本术语“构架区”或“FR区”可互换，是指抗体重链可变区或轻链可变区中除CDR以外的那些氨基酸残基。通常典型的抗体可变区由4个FR区和3个CDR区按以下顺序组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
本文“CDR”可由本领域公知的方式加以标注和定义，包括但不限于Kabat编号系统、Chothia编号系统或IMGT编号系统，使用的工具网站包括但不限于AbRSA网站(http://cao.labshare.cn/AbRSA/cdrs.php)、abYsis网站(www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi)和IMGT网站(http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign.cgi#results)。本文CDR包括不同定义方式的氨基酸残基的重叠(overlap)和子集。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域， 或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
本文术语“Fc”是指完整抗体经木瓜蛋白酶水解而成的抗体羧基端部分，典型地，其包含抗体的CH3和CH2结构域。Fc区包括例如天然序列Fc区、重组Fc区和变体Fc区。尽管免疫球蛋白重链的Fc区的边界可以略微变化，但是人IgG重链的Fc区通常被定义为从Cys226位置的氨基酸残基或从Pro230延伸至其羧基末端。Fc区的C末端赖氨酸(根据Kabat编号系统的残基447)可以例如在抗体的产生或纯化过程中，或通过对编码抗体重链的核酸重组工程化而除去，因此，Fc区可包括或不包括Lys447。
本文术语“同一性”可通过以下方式计算获得：为确定两个氨基酸序列或两个核酸序列的“同一性”百分数，将所述序列出于最佳比较目的比对(例如，可以为最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
考虑到为最佳比对这两个序列而需要引入的空位的数目和每个空位的长度，两个序列之间的同一性百分数随所述序列共有的相同位置变化而变化。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。例如，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。又例如，使用GCG软件包中的GAP程序(在www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum62评分矩阵。
本文中表示连接位点。
本文中消旋体或者对映体纯的化合物的图示法来自Maehr，J.Chem.Ed.1985，62:114-120。除非另有说明，用楔形键和虚楔键表示一个立体中心的绝对构型，用黑实键和虚键表示一个立体中心的相对构型(如脂环化合物的顺反构型)。
术语“互变异构体”是指因分子中某一原子在两个位置迅速移动而产生的官能团异构体。本公开化合物可表现出互变异构现象。互变异构的化合物可以存在两种或多种可相互转化的种类。互变异构体一般以平衡形式存在，尝试分离单一互变异构体时通常产生一种混合物，其理化性质与化合物的混合物是一致的。平衡的位置取决于分子内的化学特性。例如，在很多脂族醛和酮如乙醛中，酮型占优势；而在酚中，烯醇型占优势。本公开包含化合物的所有互变异构形式。
术语“立体异构体”是指由分子中原子在空间上排列方式不同所产生的异构体，包括顺反异构体、对映异构体和非对映异构体。
本公开的化合物可以具有不对称原子如碳原子、硫原子、氮原子、磷原子或不对称双键，因此本公开的化合物可以存在特定的几何或立体异构体形式。特定的几何或立体异构体形式可以是顺式和反式异构体、E型和Z型几何异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非 对映异构体、(D)-异构体、(L)-异构体，以及其外消旋混合物或其它混合物，例如对映异构体或非对映体富集的混合物，以上所有这些异构体以及它们的混合物都属于本公开化合物的定义范围之内。烷基等取代基中可存在另外的不对称碳原子、不对称硫原子、不对称氮原子或不对称磷原子，所有取代基中涉及到的这些异构体以及它们的混合物，也均包括在本公开化合物的定义范围之内。本公开的含有不对称原子的化合物可以以光学活性纯的形式或外消旋形式被分离出来，光学活性纯的形式可以从外消旋混合物拆分，或通过使用手性原料或手性试剂合成。
术语“被取代”是指特定原子上的任意一个或多个氢原子被取代基取代，只要特定原子的价态是正常的并且取代后的化合物是稳定的。当取代基为氧代(即＝O)时，意味着两个氢原子被取代，氧代不会发生在芳香基上。
术语“任选”或“任选地”是指随后描述的事件或情况可以发生或不发生，该描述包括发生所述事件或情况和不发生所述事件或情况。例如，乙基“任选”被卤素取代，是指乙基可以是未被取代的(CH2CH3)、单取代的(CH2CH2F、CH2CH2Cl等)、多取代的(CHFCH2F、CH2CHF2、CHFCH2Cl、CH2CHCl2等)或完全被取代的(CF2CF3、CF2CCl3、CCl2CCl3等)。本领域技术人员可理解，对于包含一个或多个取代基的任何基团，不会引入任何在空间上不可能存在和/或不能合成的取代或取代模式。
当任何变量(例如Ra、Rb)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。例如，如果一个基团被2个Rb所取代，则每个Rb都有独立的选项。
当一个连接基团的数量为0时，比如-(CH2)0-，表示该连接基团为键。
当其中一个变量选自化学键或不存在时，表示其连接的两个基团直接相连，比如A-L-Z中L代表键时表示该结构实际上是A-Z。
当本文中涉及到的连接基团若没有指明其连接方向，则其连接方向是任意的。例如当结构单元中的X选自“C1-C3亚烷基-O”时，此时X既可以按照与从左到右的方向连接环A和环B构成“环A-C1-C3亚烷基-O-环B”，也可以按照从右到左的方向连接环A和环B构成“环A-O-C1-C3亚烷基-环B”。
当一个取代基的键交叉连接到一个环上的两个原子时，这种取代基可以与这个环上的任意原子相键合。例如，结构单元表示R5可在苯环上的任意一个位置发生取代。
本文中的Cm-Cn是指具有m-n范围中的整数个碳原子。例如“C1-C10”是指该基团可具有1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子、6个碳原子、7个碳原子、8个碳原子、9个碳原子或10个碳原子。
术语“烷基”是指通式为CnH2n+1的烃基，该烷基可以是直链或支链的。术语“C1-C6烷基”可理解为表示具有1、2、3、4、5或6个碳原子的直链或支链饱和烃基。所述烷基的具体实例包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、1-甲基丁基、2-甲基丁基、3-甲基丁基、新戊基、己基、2-甲基戊基等。术语“C1-C3烷基”可理解为表示具有1至3个碳原子的直链或支链饱和烷基。所述“C1-C6烷基”可以进一步包含“C1-C3烷基”。
术语“烷氧基”是指直链或支链醇类失去羟基上的氢原子产生的基团，可理解为“烷基氧基”或“烷基-O-”。术语“C1-C6烷氧基”可理解为“C1-C6烷基氧基”或“C1-C6烷基-O-”。所述“C1-C6烷氧基”可以进一步包含“C1-C3烷氧基”。
术语“环烷基”是指完全饱和的且以单环、并环、桥环或螺环等形式存在的碳环基。除非另有指示，该碳环通常为3至10元环。术语“C3-C10环烷基”可理解为表示饱和的单环、并环、 螺环或桥环，其具有3～10个碳原子。所述环烷基的具体实例包括但不限于环丙基、环丁基、环戊基、环己基、环庚基、环辛基、环壬基、环癸基，降冰片基(双环[2.2.1]庚基)、双环[2.2.2]辛基、金刚烷基、螺[4.5]癸烷基等。术语“C3-C10环烷基”可以包含“C3-C6环烷基”，术语“C3-C6环烷基”可理解为表示饱和的单环或双环烃环，其具有3～6个碳原子，具体实例包括但不限于环丙基、环丁基、环戊基或环己基等。
术语“环烷基氧基”可理解为“环烷基-O-”。
术语“环烯基”是指不完全饱和的且以单环、稠环、桥环或螺环等形式存在的非芳香族碳环基。除非另有指示，该碳环通常为5至8元环。术语“C5-C6环烯基”是指环碳原子数为5或6的环烯基，具体实例包括但不限于环戊烯基、环戊二烯基、环己烯基、环己二烯基等。
术语“杂环基”是指完全饱和的或部分饱和的(整体上不是具有芳香性的杂芳族)单环、并环、螺环或桥环基团，其环原子中含有1-5个杂原子或杂原子团(即含有杂原子的原子团)，所述“杂原子或杂原子团”包括但不限于氮原子(N)、氧原子(O)、硫原子(S)、磷原子(P)、硼原子(B)、-S(＝O)2-、-S(＝O)-、-P(＝O)2-、-P(＝O)-、-NH-、-S(＝O)(＝NH)-、-C(＝O)NH-或-NHC(＝O)NH-等。术语“4-14元杂环基”是指环原子数目为4、5、6、7、8、9、10、11、12、13或14的杂环基，且其环原子中含有1-5个独立选自上文所述的杂原子或杂原子团。“4-14元杂环基”包括“4-10元杂环基”或“4-7元杂环基”等，其中，4元杂环基的具体实例包括但不限于氮杂环丁烷基或氧杂环丁烷基；5元杂环基的具体实例包括但不限于四氢呋喃基、二氧杂环戊烯基、吡咯烷基、咪唑烷基、吡唑烷基、吡咯啉基、4,5-二氢噁唑基或2,5-二氢-1H-吡咯基；6元杂环基的具体实例包括但不限于四氢吡喃基、哌啶基、吗啉基、二噻烷基、硫代吗啉基、哌嗪基、三噻烷基、四氢吡啶基或4H-[1,3,4]噻二嗪基；7元杂环基的具体实例包括但不限于二氮杂环庚烷基。所述杂环基还可以是双环基，其中，5,5元双环基的具体实例包括但不限于六氢环戊并[c]吡咯-2(1H)-基；5,6元双环基的具体实例包括但不限于六氢吡咯并[1,2-a]吡嗪-2(1H)-基、5,6,7,8-四氢-[1,2,4]三唑并[4,3-a]吡嗪基或5,6,7,8-四氢咪唑并[1,5-a]吡嗪基。任选地，所述杂环基可以是上述4-7元杂环基的苯并稠合环基，具体实例包括但不限于二氢异喹啉基等。“4-10元杂环基”可以包含“5-10元杂环基”、“4-7元杂环基”、“5-6元杂环基”、“6-8元杂环基”、“4-10元杂环烷基”、“5-10元杂环烷基”、“4-7元杂环烷基”、“5-6元杂环烷基”、“6-8元杂环烷基”等范围，“4-7元杂环基”进一步可以包含“4-6元杂环基”、“5-6元杂环基”、“4-7元杂环烷基”、“4-6元杂环烷基”、“5-6元杂环烷基”等范围。本公开中尽管有些双环类杂环基部分地含有一个苯环或一个杂芳环，但所述杂环基整体上仍是无芳香性的。
术语“芳基”是指具有共轭的π电子体系的全碳单环或稠合多环的芳香环基团。术语“C6-C10芳基”可理解为具有6～10个碳原子的芳基。特别是具有6个碳原子的环(“C6芳基”)，例如苯基；或者具有9个碳原子的环(“C9芳基”)，例如茚满基或茚基；或者具有10个碳原子的环(“C10芳基”)，例如四氢化萘基、二氢萘基或萘基。
术语“芳基氧基”可理解为“芳基-O-”。
术语“杂芳基”是指具有芳香性的单环或稠合多环体系，其中含有至少一个选自N、O、S的环原子，其余环原子为C的芳香环基。术语“5-10元杂芳基”可理解为包括这样的单环或双环芳族环系：其具有5、6、7、8、9或10个环原子，特别是5或6或9或10个环原子，且其包含1-5个，优选1-3个独立选自N、O和S的杂原子。特别地，杂芳基选自噻吩基、呋喃基、吡咯基、噁唑基、噻唑基、咪唑基、吡唑基、异噁唑基、异噻唑基、噁二唑基、三唑基或噻二唑基等以及它们的苯并衍生物，例如苯并呋喃基、苯并噻吩基、苯并噻唑基、苯并噁唑基、苯并异噁唑基、苯并咪唑基、苯并三唑基、吲唑基、吲哚基或异吲哚基等；或吡啶基、哒嗪基、嘧啶基、吡嗪基或三嗪基等以及它们的苯并衍生物，例如喹啉基、喹唑啉基或异喹啉基等；或吖辛因基、吲嗪基、嘌呤基等以及它们的苯并衍生物；或噌啉基、酞嗪基、喹唑啉基、喹喔啉基、萘啶基、蝶啶基、咔唑基、吖啶基、吩嗪基、吩噻嗪基或吩噁嗪基等。术语“5-6元杂芳基”指具有5或6个环原子的芳族环系，且其包含1-3个，优选1-2个独立选自N、O和S的杂原子。
术语“杂芳基氧基”可理解为“杂芳基-O-”。
术语“卤”或“卤素”是指氟、氯、溴或碘。
术语“羟基”是指-OH基团。
术语“氰基”是指-CN基团。
术语“巯基”是指-SH基团。
术语“氨基”是指-NH2基团。
术语“治疗”是指外科手术或药物处理(surgical or therapeutic treatment)，其目的是预防、减缓(减少)治疗对象中不希望的生理变化或病变，如癌症、自身免疫性疾病和病毒感染的进展。有益的或所希望的临床结果包括但不限于症状的减轻、疾病程度减弱、疾病状态稳定(即，未恶化)、疾病进展的延迟或减慢、疾病状态的改善或缓和、以及缓解(无论是部分缓解或完全缓解)，无论是可检测的或不可检测的。需要治疗的对象包括已患有病症或疾病的对象以及易于患上病症或疾病的对象或打算预防病症或疾病的对象。当提到减缓、减轻、减弱、缓和、缓解等术语时，其含义也包括消除、消失、不发生等情况。
术语“有效量”指单独给予或与另一治疗剂组合给予细胞、组织或对象时能有效防止或缓解疾病病症或该疾病进展的治疗剂用量。“有效量”还指足以缓解症状，例如治疗、治愈、防止或缓解相关医学病症，或治疗、治愈、防止或缓解这些病症的速度增加的化合物用量。当将活性成分单独给予个体时，治疗有效剂量单指该成分。当应用某一组合时，治疗有效剂量指产生治疗作用的活性成分的组合用量，而无论是组合、连续或同时给予。
术语“受试者”是指接受对如本公开所述的特定疾病或病症的治疗的生物体。对象和患者的实例包括接受疾病或病症治疗的哺乳动物，如人、灵长类动物(例如，猴)或非灵长类哺乳动物。
构成“治疗有效量”的本公开化合物的量取决于该化合物、疾病状态及其严重性、给药方式以及待被治疗的哺乳动物的年龄而改变，但可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指药学上可接受的酸或碱的盐，包括化合物与无机酸或有机酸形成的盐，以及化合物与无机碱或有机碱形成的盐。
术语“药物组合物”是指一种或多种本公开的化合物或其盐与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对有机体给予本公开的化合物。
术语“药学上可接受的辅料”是指对有机体无明显刺激作用，而且不会损害该活性化合物的生物活性及性能的那些辅料。合适的辅料是本领域技术人员熟知的，例如碳水化合物、蜡、水溶性和/或水可膨胀的聚合物、亲水性或疏水性材料、明胶、油、溶剂、水等。
词语“包括(comprise)”或“包含(comprise)”及其英文变体例如comprises或comprising可理解为开放的、非排他性的意义，即“包括但不限于”。
本申请中记载的磷酸酯类化合物(如R10选自的化合物)为酯类前药，其经生物体内的磷酸酯酶水解后释放出具有生物活性的化合物。例如，磷酸酯类化合物经生物体内的磷酸酯酶水解后释放得到糖皮质激素受体激 动剂因此，本领域技术人员可以理解或可以预期，若化合物009具备某种生物学活性(如糖皮质激素受体激动活性)，则对应的酯类化合物009-p在生物体内也具备相同或相似的生物学活性。
本公开还包括与本文中记载的那些相同的，但一个或多个原子被原子量或质量数不同于自然中通常发现的原子量或质量数的原子置换的同位素标记的本公开化合物。可结合到本公开化合物的同位素的实例包括氢、碳、氮、氧、磷、硫、氟、碘和氯的同位素，诸如分别为2H、3H、11C、13C、14C、13N、15N、15O、17O、18O、31P、32P、35S、18F、123I、125I和36Cl等。
某些同位素标记的本公开化合物(例如用3H及14C标记)可用于化合物和/或底物组织分布分析中。氚化(即3H)和碳-14(即14C)同位素对于由于它们易于制备和可检测性是尤其优选的。正电子发射同位素，诸如15O、13N、11C和18F可用于正电子发射断层扫描(PET)研究以测定底物占有率。通常可以通过与公开于下文的方案和/或实施例中的那些类似的下列程序，通过同位素标记试剂取代未经同位素标记的试剂来制备同位素标记的本公开化合物。
本公开的药物组合物可适用于肠胃外给药，如合适的单位剂型的无菌溶液剂、混悬剂或冻干产品。例如，本公开的药物组合物可以是用于肌内或皮下给药的无菌注射水溶液形式。本公开的药物组合物在使用时可接受其它溶媒或溶剂，如水、林格氏液或等渗氯化钠溶液。
本文所述化合物的所有施用方法中，每天给药的剂量为0.001mg/kg到600mg/kg体重，优选为0.05mg/kg到200mg/kg体重，更优选0.1mg/kg到100mg/kg体重，以单独或分开剂量的形式。
本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其它化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。
本公开具体实施方式的化学反应是在合适的溶剂中完成的，所述的溶剂须适合于本公开的化学变化及其所需的试剂和物料。为了获得本公开的化合物，有时需要本领域技术人员在已有实施方式的基础上对合成步骤或者反应流程进行修改或选择。
本领域合成路线规划中的一个重要考量因素是为反应性官能团(如本公开中的氨基、羧基)选择合适的保护基，例如，可参考Greene's Protective Groups in Organic Synthesis(4th Ed).Hoboken,New Jersey:John Wiley & Sons,Inc.本公开引用的所有参考文献整体上并入本公开。
附图说明
图1为本公开实施例1中间体1-5的COSY图。
图2为本公开测试例6中式(D-H)化合物在CHS小鼠模型上的药效试验结果图。
图3为本公开测试例9中抗人TNFα抗体-药物偶联物在GRE报告基因中的活性测试结果图。
图4为本公开测试例9中抗鼠TNFα抗体-药物偶联物在GRE报告基因中的活性测试结果图。
图5为本公开测试例11中抗鼠TNFα-ADC在LPS刺激的小鼠PBMC细胞因子释放测定中的抑制活性测试结果图。
图6为本公开测试例12中抗鼠TNFα-ADC在小鼠CHS模型中的药效测试结果图。
图7为本公开测试例13中抗鼠TNFα-ADC在DBA/1小鼠CAIA模型中的药效试验结果图。
图8为本公开测试例14中抗人TNFα-ADC在TNFα人源化小鼠CAIA模型中的药效试 验结果图。
图9为本公开测试例20中抗IL-4R抗体-药物偶联物对于人原代CD4+T细胞增殖的抑制作用试验结果图。
图10为本公开测试例21中抗IL-4R抗体-药物偶联物在特应性皮炎小鼠模型中耳厚检测结果。
图11为本公开测试例21中抗IL-4R抗体-药物偶联物在特应性皮炎小鼠模型中血清IgE检测结果。
图12-13为本公开测试例21中抗IL-4R抗体-药物偶联物给药显著改善特应性皮炎小鼠模型造模部位耳片和皮肤组织病理学损伤及炎症程度。
图14-15为本公开测试例21中抗IL-4R抗体-药物偶联物给药显著抑制特应性皮炎小鼠模型造模部位耳片和皮肤组织中致炎性CD4+T细胞浸润。
具体实施方式
下面通过实施例对发明进行详细描述，但并不意味着对本公开有任何的不利限制。本文已经详细地描述了本公开，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本公开精神和范围的情况下针对本公开具体实施方式进行各种改变将是显而易见的。本公开所使用的所有试剂是市售的，无需进一步纯化即可使用。
除非另作说明，混合溶剂表示的比例是体积混合比例。
除非另作说明，否则，％是指wt％。
化合物经手工或软件命名，市售化合物采用供应商目录名称。
化合物的结构是通过核磁共振(NMR)和/或质谱(MS)来确定的。NMR位移的单位为10-6(ppm)。NMR测定的溶剂为氘代二甲基亚砜、氘代氯仿、氘代甲醇等，内标为四甲基硅烷(TMS)；“IC50”指半数抑制浓度，指达到最大抑制效果一半时的浓度,“EC50”指能半数最大效应浓度，引起50％最大效应的浓度。
实施例1：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物001)的合成
合成路线和具体合成步骤：
第一步：4-溴-2,3-二氢苯并呋喃-7-胺1-2的合成
按照文献报道的方法(WO2016169504A1)，将2,3-二氢苯并呋喃-7-胺(1.0g,7.40mmol)溶于二甲基甲酰胺(20mL)，加入N-溴代丁二酰亚胺(1.48g,8.33mmol)，25℃搅拌3小时。LCMS显示反应完毕，加水(20mL)稀释，使用乙酸乙酯(100mL x 2)进行萃取，有机相用饱和氯化钠水(30mL x2)溶液洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(四氢呋喃/石油醚＝1/4)分离纯化，得到4-溴-2,3-二氢苯并呋喃-7-胺1-2(1.0g)。
LC-MS:Rt:0.444min；MS m/z(ESI):213.9[M+H]+；
第二步：(4-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸叔丁酯的合成
将4-溴-2,3-二氢苯并呋喃-7-胺1-2(1.0g,4.67mmol)溶于二氯甲烷(20mL)，加入二碳酸二叔丁酯(1.41g,6.46mmol)和4-二甲氨基吡啶(571mg,4.67mmol)，25℃搅拌16小时。LCMS显示反应完毕，加水(20mL)稀释，使用乙酸乙酯(100mL x 2)进行萃取，有机相用饱和氯化钠水溶液(30mL)洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(四氢呋喃/石油醚＝1/5)分离纯化，得到(4-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸叔丁酯1-3(600mg,38.8％)。
LC-MS:Rt:1.756min；MS m/z(ESI):258.1[M-56+H]+；
1H NMR(400MHz,CHLOROFORM-d)δ＝7.71-7.54(m,1H),6.87(d,J＝8.6Hz,1H),6.44(s,1H),4.55(t,J＝8.8Hz,2H),3.15(t,J＝8.8Hz,2H),1.43(s,9H)。
第三步：(4-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-基)氨基甲酸叔丁酯1-4的合成
将(4-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸叔丁酯1-3(450mg,1.43mmol)溶于二氧六环(10mL)，加入双联频哪醇硼酸酯(909mg,3.58mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(105mg,0.14mmol)和乙酸钾(422mg,4.30mmol)，氮气氛围下80℃搅拌16小时。LCMS显示反应完毕，反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/6)分离纯化，得到化合物1-4(410mg,79.2％)。
LC-MS:Rt:1.937min；MS m/z(ESI):306.2[M-56+H]+。
第四步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-7-羟基-8b-(2-羟基 乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮1-5的合成
将反应物16α-羟基泼尼松龙F1(1g,2.66mmol)和无水硫酸镁(1.22g,10.1mmol)溶于乙腈(20mL)中，25℃搅拌1小时，再将4-(溴甲基)苯甲醛(529mg,2.66mmol)溶于乙腈(2mL)并加入到反应液中，冷却至0℃滴加三氟甲磺酸(1.88g,12.5mmol)，25℃搅拌2小时。LCMS显示反应完毕，反应液用碳酸氢钠水溶液(20mL)淬灭，使用乙酸乙酯(100mL x 2)进行萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(四氢呋喃/石油醚＝1/2)分离纯化，得到的粗产品经SFC[柱子:DAICEL CHIRALCEL OD(250mm*50mm,10μm)；流动相:A:0.1％氨甲醇溶液；B:CO2,50％]分离纯化，得到中间体1-5(1.2g,80.9％)。
LC-MS:Rt:1.551min；MS m/z(ESI):557.1[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.45(s,3H),7.31(d,J＝10.1Hz,1H),6.16(dd,J＝1.8,10.1Hz,1H),5.93(s,1H),5.75(s,1H),5.45(s,1H),5.16-5.03(m,1H),4.94(d,J＝4.9Hz,1H),4.79(d,J＝3.0Hz,1H),4.68(s,2H),4.53(d,J＝19.5Hz,1H),4.30(s,1H),4.19(d,J＝19.4Hz,1H),2.61-2.53(m,1H),2.31-2.25(m,1H),2.17-2.07(m,1H),2.06-1.98(m,1H),1.81-1.61(m,5H),1.39(s,3H),1.10-0.99(m,2H),0.87(s,3H)。
中间体1-5的绝对构型经二维核磁验证(见图1)。
第五步：叔丁基(4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯的1-6合成
将化合物1-5(300mg,0.54mmol)溶于四氢呋喃(6mL)和水(0.6mL)，加入叔丁基(4-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯(194mg,0.54mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(78.8mg,0.11mmol)和碳酸钾(223mg,1.61mmol)，氮气氛围下80℃搅拌16小时。LCMS显示反应完毕，反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/1)分离纯化纯化，得到化合物1-6(300mg,54.8％)。
LC-MS:Rt:0.584min；MS m/z(ESI):712.4[M+H]+；
第六步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮001的合成
将1-6(280mg,0.39mmol)溶于二氯甲烷(6mL)，加入三氟乙酸(2mL)，25℃搅拌1小时。LCMS显示反应完毕，反应液浓缩，粗产品经制备-HPLC[柱子:Phenomenex luna 30*30mm*10μm；YMC AQ 100*30*10μm；流动相:A:水(HCl)；B:ACN,B％，30％-60％,15min]分离纯化，得到化合物001(10mg,19.0％)。
LC-MS:Rt:1.511min；MS m/z(ESI):612.5[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝9.52-8.19(m,2H),7.38(d,J＝8.1Hz,2H),7.32(d,J＝10.1Hz,1H),7.20(d,J＝8.0Hz,2H),6.89(d,J＝8.0Hz,1H),6.65(d,J＝8.0Hz,1H),6.17(dd,J＝1.8,10.1Hz,1H),5.93(s,1H),5.41(s,1H),4.92(d,J＝5.0Hz,1H),4.81(br s,1H),4.60(t,J＝8.8Hz,2H),4.50(d,J＝19.4Hz,1H),4.29(br s,1H),4.18(d,J＝19.4Hz,1H),3.86(s,2H),3.16-3.08(m,2H),2.62-2.54(m,1H),2.30(m,1H),2.19-2.08(m,1H),2.07-1.97(m,1H),1.81-1.59(m,5H),1.40(s,3H),1.12-0.96(m,2H),0.87(s,3H)。
实施例2：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-苯并[d]咪唑-6-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物002)的合成
合成路线和具体合成步骤：
第一步：6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-1H-苯并[d]咪唑-1-甲酸叔丁酯2-2的合成
将6-(4,4,5-三甲基-1,3,2-二噁硼戊环-2-基)-1H-苯并[d]咪唑(1.00g,4.10mmol)，N,N-二异丙基乙胺(1.06g,8.19mmol)，4-二甲氨基吡啶(50mg,0.41mmol)和二碳酸二叔丁酯(894mg,4.10mmol)溶于无水二氯甲烷(20mL)中，25℃反应6小时。LCMS检测反应完毕。加入水(20mL)淬灭反应，使用二氯甲烷(50mL x 2)萃取，有机相用饱和氯化钠水溶液(30mL)洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(乙酸乙酯/石油醚＝1/5)分离纯化，得到2-2(1.0g,69.5％)。
LC-MS:Rt:1.793min；MS m/z(ESI):345.3[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝8.50–8.24(m,2H),8.00-7.75(m,2H),1.70(d,J＝3.5Hz,9H),1.36(d,J＝5.1Hz,12H)。
第二步：6-(4-甲酰基苯甲基)-1H-苯并[d]咪唑-1-甲酸叔丁酯2-3的合成
将2-2(500mg,1.45mmol)，4-(溴甲基)苯甲醛(376mg,1.89mmol)，碳酸钾(602mg,4.36mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(106mg,0.14mmol)溶于无水四氢呋喃(10mL)中，氮气氛围下80℃反应16小时。LCMS检测反应完毕。将反应液过滤浓缩，残留物经柱层析(乙酸乙酯/石油醚＝1/5)分离纯化，得到2-3(380mg,crude)。
LC-MS:Rt:1.563min和1.593；MS m/z(ESI):337.3[M+H]+；
第三步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-苯并[d]咪唑-6-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮的合成
将16alpha-羟基泼尼松龙F1(230mg,0.61mmol)溶于无水乙腈(1.5mL)，加入无水硫酸镁(279mg,2.32mmol)，25℃下搅拌1小时。将2-3(308mg,0.92mmol)溶于无水乙腈(1.5mL)中加入反应体系，降温至0℃滴加三氟甲磺酸(431mg,2.87mmol)，25℃搅拌2小时。LCMS检测反应完毕。加入饱和碳酸氢钠溶液(5mL)淬灭反应，用乙酸乙酯(30mL x 2)萃取，有机相用饱和氯化钠水溶液(20mL)洗涤，有机相干燥过滤减压浓缩，粗产物经Prep-HPLC[柱子:Phenomenex Luna 30*30mm*10μm；YMC AQ 100*30*10μm；流动相:A:水(含0.225％的甲酸)；B:MeCN,B％,15％-45％,20mins]，得到化合物002(35.8mg,9.75％)。
LC-MS:Rt:1.390min；MS m/z(ESI):595.5[M+H]；Chiral-HPLC:Rt:2.211min，手性纯度＝97.56％；
1H NMR(400MHz,DMSO-d6)δ＝8.39(br s,1H),7.52(d,J＝8.3Hz,1H),7.44(s,1H),7.40-7.35(m,2H),7.33-7.26(m,3H),7.12(d,J＝8.4Hz,1H),6.16(dd,J＝1.6,10.1Hz,1H),5.93(s,1H),5.40(s,1H),5.08(br s,1H),4.92(d,J＝4.9Hz,1H),4.78(d,J＝3.0Hz,1H),4.50(br d,J＝ 16.0Hz,1H),4.28(s,1H),4.17(d,J＝19.5Hz,1H),4.05(s,2H),2.30(s,1H),2.15-1.98(m,2H),1.81-1.60(m,5H),1.39(s,3H),1.10-0.97(m,2H),0.86(s,3H)。
实施例3:(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((1H-吲哚-6-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物003)的合成
合成路线和具体合成步骤：
将化合物1-7(500mg,0.89mmol)溶于四氢呋喃(5mL)和水(0.5mL)，加入6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-1H-吲哚3-1(262mg,1.08mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(66mg,0.09mmol)和碳酸钾(372mg,2.69mmol)，氮气氛围下80℃下搅拌16小时。LCMS显示反应完毕，用乙酸乙酯(100mL)稀释，有机相用饱和氯化钠水溶液(30mL)洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(四氢呋喃/石油醚＝1/1)分离纯化，得到的粗产品经Prep-HPLC[柱子:Phenomenex luna 30*30mm*10μm+YMC AQ 100*30*10μm；流动相:A:水(0.225％FA)；B:MeCN,B％,50％-80％,20min]分离纯化，得到化合物003(60mg 11.2％)。
LC-MS:Rt:2.098min；MS m/z(ESI):594.3[M+H]+；Chiral-HPLC:Rt:1.204min,手性纯度＝100％；
1H NMR(400MHz,DMSO-d6)δ＝10.92(s,1H),7.41(d,J＝8.1Hz,1H),7.37(d,J＝8.0Hz,2H),7.30(d,J＝10.1Hz,1H),7.28-7.23(m,3H),7.18(s,1H),6.85(dd,J＝1.3,8.1Hz,1H),6.34(s,1H),6.16(dd,J＝1.8,10.1Hz,1H),5.93(s,1H),5.40(s,1H),5.08(t,J＝5.9Hz,1H),4.92(d,J＝5.0Hz,1H),4.78(d,J＝3.1Hz,1H),4.50(dd,J＝6.3,19.4Hz,1H),4.29(s,1H),4.17(dd,J＝5.4,19.4Hz,1H),4.00(s,2H),2.59-2.53(m,1H),2.36-2.26(m,1H),2.18-2.06(m,1H),2.05-1.96(m,1H),1.80-1.54(m,5H),1.39(s,3H),1.11-0.97(m,2H),0.86(s,3H)。
实施例4：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-10-(4-(二氢吲哚-6-基甲基)苯基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物004)的合成
合成路线和具体合成步骤：
第一步：6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)二氢吲哚4-2的合成
将6-溴二氢吲哚(1.0g,5.05mmol)溶于二氧六环(20mL)中，加入双联频哪醇硼酸酯(2.56g,10.1mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(369mg,504μmol)和乙酸钾(1.49g,15.2mmol)，氮气氛围下80℃反应12小时。LCMS检测反应完毕。反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/3)分离纯化，得到6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)二氢吲哚4-2(1.2g,96.9％)。
LC-MS:Rt:1.509min；MS m/z(ESI):246.3[M+H]+；
第二步：6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)二氢吲哚-1-甲酸叔丁酯4-3的合成
将4-2(500mg,2.04mmol)溶于二氯甲烷(8mL)中，加入二碳酸二叔丁酯(891mg,4.08mmol)，二异丙基乙胺(528mg,4.08mmol)和4-二甲氨基吡啶(49.8mg,408μmol)，25℃搅拌6小时。LCMS显示反应完毕。加入二氯甲烷稀释(50mL)，有机相用饱和氯化钠水溶液(15mL)洗涤，有机相干燥过滤减压浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/5)分离纯化，得到4-3(260mg,36.9％)。
LC-MS:Rt:2.340min；MS m/z(ESI):290.3[M-56+H]；
第三步：6-(4-甲酰基苯甲基)二氢吲哚-1-甲酸叔丁酯4-4的合成
将6-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)二氢吲哚-1-甲酸叔丁酯(260mg,753μmol)溶于四氢呋喃(5mL)中，加入4-(溴甲基)苯甲醛(299mg,1.51mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(165mg,226μmol)和碳酸钾(520mg,3.77mmol)，氮气氛围下80℃搅拌12小时。LCMS显示反应完毕。反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚为1/5)分离纯化，得到4-4(190mg,74.8％)。
LC-MS:Rt:1.810min；MS m/z(ESI):282.2[M-56+H]+；
第四步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-10-(4-(二氢吲哚-6-基甲基)苯基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮004的合成
将16alpha-羟基泼尼松龙F1(165mg,438μmol)溶于乙腈(3mL)中，加入无水硫酸镁 (258mg,2.15mmol)，25℃搅拌1小时，将6-(4-甲酰基苯甲基)二氢吲哚-1-甲酸叔丁酯(178mg,526μmol)溶于乙腈(1mL)后加入反应液，降温至0℃后滴加三氟甲磺酸(322mg,2.15mmol)，25℃搅拌2小时。LCMS显示反应完毕。加入饱和碳酸氢钠水溶液(10mL)淬灭反应，使用乙酸乙酯(30mL x 3)进行萃取，有机相用饱和氯化钠水溶液(20mL)洗涤，有机相干燥过滤减压浓缩，粗产物经Prep-HPLC[柱子:Phenomenex luna 30*30mm*10μm+YMC AQ 100*30*10μm；流动相:A:水(0.225％FA)；B:MeCN,B％,18％-48％,20min]分离纯化，得到化合物004(42.9mg,6.45％)。
LC-MS:Rt:1.532min；MS m/z(ESI):596.3[M+H]+；Chiral-HPLC:Rt:2.502min,手性纯度＝100％；
1H NMR(400MHz,DMSO-d6)δ＝7.37-7.33(m,2H),7.30(d,J＝10.1Hz,1H),7.20(d,J＝8.0Hz,2H),6.88(d,J＝7.3Hz,1H),6.37(d,J＝7.4Hz,1H),6.28(s,1H),6.18-6.13(m,1H),5.95-5.91(m,1H),5.39(s,2H),5.12-5.06(m,1H),4.91(d,J＝5.0Hz,1H),4.80-4.75(m,1H),4.54-4.45(m,1H),4.32-4.25(m,1H),4.21-4.12(m,1H),3.75(s,2H),3.38-3.34(m,2H),2.84-2.77(m,2H),2.55-2.47(m 1H),2.34-2.27(m,1H),2.16-2.07(m,1H),2.06-1.97(m,1H),1.79-1.59(m,5H),1.39(s,3H),1.12-0.97(m,2H),0.89-0.82(m,3H)。
实施例5：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基1,2,6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物005)的合成
合成路线和具体合成步骤：
第一步：7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-3,4-二氢-2H-苯并[b][1,4]噁嗪5-2的合成
将7-溴-3,4-二氢-2H-苯并[b][1,4]噁嗪(1.00g,4.67mmol)5-1溶于四氢呋喃(20mL)中，加入双联频哪醇硼酸酯(1.30g,5.14mmol)，乙酸钾(1.38g,14.0mmol)和[1,1'-双(二苯基膦基)二茂铁]二氯化钯(341mg,467μmol)，氮气氛围下80℃反应16小时。LCMS检测反应完毕。反应液过滤浓缩，残留物经柱层析(乙酸乙酯/石油醚＝1/5)纯化，得到7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-3,4-二氢-2H-苯并[b][1,4]噁嗪5-2(950mg,77.9％)。
LC-MS:Rt:1.454min；MS m/z(ESI):262.3[M+H]+；
第二步：4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯(甲)醛5-3的合成
将5-2(900mg,3.45mmol)溶于四氢呋喃(20mL)中，加入4-(溴甲基)苯甲醛(1.03g,5.17mmol)，碳酸钾(1.43g,10.3mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(252mg,345μmol)，氮气氛围下80℃反应16小时。LCMS检测反应完毕。反应液过滤浓缩，残留物经柱层析(乙酸乙酯/石油醚＝1/3)纯化，得到4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯甲醛5-3(397mg)。
LC-MS:Rt:1.202min；MS m/z(ESI):254.1[M+H]+；
第三步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基的合成
将16alpha-羟基泼尼松龙F1(350mg,929μmol)溶于乙腈(15mL)中，加入硫酸镁(425mg,3.53mmol)，25℃搅拌1小时后，将4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯甲醛(235mg,929μmol)5-3溶于乙腈(5mL)后加入至反应体系，降温至0℃滴加三氟甲磺酸(655mg,4.37mmol)，25℃搅拌2小时。LCMS检测反应完毕。加入饱和碳酸氢钠水溶液(15mL)淬灭反应，使用乙酸乙酯(50mL x 3)进行萃取，有机相用饱和氯化钠水溶液(20mL)洗涤，有机相干燥过滤减压浓缩，粗产物经prep-HPLC[柱子:Xtimate C18 150*40mm*10μm；流动相:A:水(10mM NH4HCO3)；B:MeCN,B％,40％-70％,10min]分离纯化，得到化合物005(20mg,21.1％)。
LC-MS:Rt:1.874min；MS m/z(ESI):612.3[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.41-7.27(m,3H),7.19(br d,J＝7.9Hz,2H),6.56-6.38(m,3H),6.16(dd,J＝1.1,10.0Hz,1H),5.93(s,1H),5.52(br s,1H),5.39(s,1H),5.05(t,J＝5.9Hz,1H),4.92(br d,J＝5.0Hz,1H),4.76(br d,J＝2.6Hz,1H),4.49(br dd,J＝6.3,19.4Hz,1H),4.29(br s,1H),4.17(br dd,J＝5.6,19.4Hz,1H),4.10-4.01(m,2H),3.70(s,2H),3.29-3.25(m,1H),3.21(br s,2H),2.37-2.25(m,1H),2.18-1.96(m,2H),1.84-1.56(m,5H),1.39(s,3H),1.14-0.97(m,2H),0.86(s,3H)。
实施例6：(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物006)的合成
合成路线和具体合成步骤：
第一步：(6S,8S,9R,10S,11S,13S,14S,16R,17S)-6,9-二氟-11,16,17-三羟基-17-(2-羟基乙酰 基)-10,13-二甲基-6,7,8,9,10,11,12,13,14,15,16,17-十二氢-3H-环戊二烯并[a]菲-3-酮6-2的合成
将化合物(2S,6aS,6bR,7S,8aS,8bS,11aR,12aS,12bS)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a,10,10-四甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮6-1(1.0g,2.21mmol)溶于30％氟硼酸水溶液(20mL)中，室温搅拌48小时。LCMS检测反应完毕。加入40mL水搅拌5分钟，过滤得到的用水(10mL x 3)和乙醇(10mL x 3)依次洗涤滤渣，得到粗产品(6S,8S,9R,10S,11S,13S,14S,16R,17S))-6,9-二氟-11,16,17-三羟基-17-(2-羟基乙酰基)-10,13-二甲基-6,7,8,9,10,11,12,13,14,15,16,17-十二氢-3H-环戊二烯并[a]菲-3-酮6-2(0.7g)。
LC-MS:Rt:0.426min；MS m/z(ESI):413.1[M+H]+；
第二步：(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮006的合成
将6-2(150mg,0.36mmol)溶于无水乙腈(1.5mL)中，加入硫酸镁(166mg,1.38mmol)，25℃下搅拌1小时后，将4-((3,4-二氢-2H-苯并[b][1,4]噁嗪-7-基)甲基)苯甲醛(138mg,0.55mmol)溶于无水乙腈(1.5mL)后加入至反应体系中，降温至0℃滴加三氟甲磺酸(257mg,1.71mmol)，25℃反应2小时。LCMS检测反应完毕。加入饱和碳酸氢钠水溶液(10mL)淬灭反应，使用乙酸乙酯(50mL x 2)进行萃取，有机相用饱和氯化钠水溶液(20mL)洗涤，有机相干燥过滤减压浓缩，粗产物经Prep-HPLC[柱子:Phenomenex Luna C18 100*30mm*3μm；流动相:A:水(0.225％FA)；B:MeCN,B％:40％-70％,8min]分离纯化，得到化合物006(82.7mg,34.6％)。
LC-MS:Rt:1.553min；MS m/z(ESI):648.2[M+H]+；Chiral-HPLC:Rt:3.241min,手性纯度＝100％；
1H NMR(400MHz,DMSO-d6)δ＝7.32(d,J＝8.1Hz,2H),7.26(d,J＝10.1Hz,1H),7.20(d,J＝8.1Hz,2H),6.53-6.48(m,1H),6.47-6.43(m,2H),6.33-6.27(m,1H),6.13(s,1H),5.72-5.57(m,1H),5.50(d,J＝3.0Hz,1H),5.43(s,1H),4.94(d,J＝4.9Hz,1H),4.51(d,J＝19.5Hz,1H),4.19-4.04(m,2H),4.07-4.03(m,2H),3.69(s,2H),3.22-3.19(m,2H),2.65-2.56(m,1H),2.28-2.07(m,2H),2.05-1.98(m,1H),1.75-1.65(m,3H),1.51-1.49(m,1H),1.50(s,3H),0.86(s,3H)。
实施例7：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-氨基苯并[d]噁唑-7-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物007)的合成
合成路线和具体合成步骤：
第一步：7-溴苯并[d]噁唑-4-胺7-2的合成
按照文献报道的方法(CN110903258)，将苯并[d]噁唑-4-胺(1.0g,7.5mmol)溶于二甲基甲酰胺(20mL)中，加入NBS(1.3g,7.1mmol)，25℃搅拌1小时。LCMS检测反应完毕。反应液加入水(20mL)稀释，使用乙酸乙酯(100mL x 2)萃取，有机相用饱和氯化钠水溶液(30mL x2)洗涤，有机相干燥过滤减压浓缩，粗产物经柱层析(四氢呋喃/石油醚＝1/2)分离纯化，得到7-溴苯并[d]噁唑-4-胺7-2(1.3g,64.5％)。
LC-MS:Rt:1.181min；MS m/z(ESI):214.9[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝8.59(s,1H),7.24(d,J＝8.5Hz,1H),6.52(d,J＝8.6Hz,1H),5.86(s,2H)。
第二步：7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯并[d]噁唑-4-胺7-3的合成
将7-溴苯并[d]噁唑-4-胺7-2(1.3g,6.1mmol)和双联频哪醇硼酸酯(3.1g,12.2mmol)溶于无水二氧六环(26mL)中，加入乙酸钾(1.8g,18.3mmol)，[1,1'-双(二苯基膦基)二茂铁]二氯化钯(447mg,0.6mmol)，80℃氮气保护下搅拌16小时。LCMS检测反应完毕。反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/2)纯化，得到7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯并[d]噁唑-4-胺7-3(1.4g,64.3％)。
LC-MS:Rt:1.368min；MS m/z(ESI):261.2[M+H]；
第三步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((4-氨基苯并[d]噁唑-7-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物007)的合成
将化合物7-3(112mg,0.4mmol)和化合物1-5(200mg,0.4mmol)溶于四氢呋喃(4mL)和水(0.4mL)中，加入碳酸钾(149mg,1.1mmol)和四(三苯基膦)钯(124mg,0.1mmol)，氮气氛围下80℃搅拌16小时。LCMS检测反应完毕。反应液过滤，残留物经柱层析(四氢呋喃/石油醚＝1/2)分离纯化，得到的粗产物再经制备HPLC[柱子:Phenomenex C18 75*30mm*3μm；流动相:A:水(NH4HCO3)；B：乙腈，B％,34％-58％,10min]分离纯化，得到化合物007(37.7mg,20.52％)。
LC-MS:Rt:1.832min；MS m/z(ESI):611.5[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝8.45(s,1H),7.36(d,J＝8.1Hz,2H),7.31(d,J＝10.1Hz,1H),7.25(d,J＝8.1Hz,2H),6.93(d,J＝8.0Hz,1H),6.48(d,J＝8.0Hz,1H),6.17(dd,J＝1.8,10.1Hz,1H),5.94(s,1H),5.48(s,2H),5.39(s,1H),5.08(s,1H),4.92(d,J＝5.0Hz,1H),4.78(d,J＝2.9Hz,1H),4.50(dd,J＝3.0,19.3Hz,1H),4.29(s,1H),4.17(d,J＝18.4Hz,1H),4.03(s,2H), 2.61-2.53(m,1H),2.37-2.29(m,1H),2.16-1.97(m,2H),1.82-1.59(m,5H),1.40(s,3H),1.13-1.00(m,2H),0.86(s,3H)。
实施例8：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基苯并[d][1,3]二噁戊环-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物008)的合成
合成路线和具体合成步骤：
第一步：7-溴苯并[d][1,3]二噁戊环-4-胺8-2的合成
将起始物料苯并[d][1,3]二噁戊环-4-胺8-1(2.00g,14.6mmol)溶于N,N-二甲基甲酰胺(50mL)中，0℃加入N-溴代丁二酰亚胺(2.50g,13.9mmol)，0℃搅拌1小时。LCMS检测反应完毕。反应液加乙酸乙酯(200mL)稀释，用水(100mL*2)和饱和食盐水(50mL)洗涤，有机相干燥过滤浓缩，残留物经柱层析(石油醚/四氢呋喃＝2/1)纯化，得到7-溴苯并[d][1,3]二噁戊环-4-胺8-2(1.30g,94.8％)。
LC-MS:Rt:1.311min；MS m/z(ESI):218.1[M+H]+；
第二步：叔丁基(7-溴苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-3的合成
将7-溴苯并[d][1,3]二噁戊环-4-胺8-2(1.00g,4.6mmol)和二碳酸二叔丁酯(3.0g,13.9mmol)溶于1,4-二氧六环(20mL)中，100℃搅拌16小时。LCMS检测反应完毕。反应液浓缩，残留物经柱层析(石油醚/乙酸乙酯＝6/1)纯化，得到叔丁基(7-溴苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-3(820mg,43.5％)。
LC-MS:Rt:1.707min；MS m/z(ESI):262.1[M+H-56]+；
1H NMR(400MHz,DMSO-d6)δ＝8.95(s,1H),6.98-6.89(m,2H),6.10(s,2H),1.44(s,9H)。
第三步：叔丁基(7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-4的合成
将叔丁基(7-溴苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-3(820mg,2.60mmol)和双联嚬哪醇硼酸酯(1.30g,5.20mmol)溶于1,4-二氧六环(25mL)中，加入乙酸钾(764mg,7.80mmol)，1,1’-双(二苯基膦基)二茂铁]二氯化钯(190mg,0.30mmol)，在氮气氛围下80℃搅拌16小时。LCMS检测反应完毕。反应液过滤浓缩，残留物经柱层析(石油醚/四氢呋喃＝6/1)纯 化，得到叔丁基(7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-4(1.00g,83.8％)。
LC-MS:Rt:1.782min；MS m/z(ESI):308.3[M+H-56]+；
第四步：叔丁基(7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-5的合成
将叔丁基(7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-4(489mg,1.35mmol)和(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮1-5(500mg,0.9mmol)溶于四氢呋喃(20mL)和水(2mL)混合溶剂中，加入碳酸钾(372mg,2.70mmol)和四三苯基膦钯(311mg,0.3mmol)，氮气氛围下80℃搅拌16小时。LCMS检测反应完毕。反应液加入水稀释，使用乙酸乙酯(100mL)萃取，有机相用饱和食盐水(30mL)洗涤，干燥过滤浓缩，残留物经柱层析(石油醚/四氢呋喃＝1/1)纯化，再经高效制备液相[柱子：Gemini NX C18 5μm*10*150mm；流动相:A:0.1％甲酸水溶液；B:乙腈,49％-79％,10min]分离纯化，得到中间体叔丁基(7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-5(20mg,2.8％)。
LC-MS:Rt:1.742min；MS m/z(ESI):714.4[M+H]+；
第五步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基苯并[d][1,3]二噁戊环-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物008)的合成
将叔丁基(7-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)苯并[d][1,3]二噁戊环-4-基)氨基甲酸酯8-5(20mg,0.03mmol)溶于二氯甲烷(4mL)中，加入三氟乙酸(1mL)，25℃搅拌0.5小时。LCMS检测反应完毕。反应液浓缩，残留物经高效制备液相[柱子：Xtimate C18 100*30mm*10μm；流动相:A:0.1％氨水溶液；B:乙腈,B％,20％-70％,35min]分离纯化，得到(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基苯并[d][1,3]二噁戊环-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物008)(3.1mg,17.3％)。
LC-MS:Rt:1.681min；MS m/z(ESI):614.5[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.36(d,J＝8.0Hz,2H),7.32(d,J＝10.1Hz,1H),7.20(d,J＝7.9Hz,2H),6.42(d,J＝8.3Hz,1H),6.22-6.15(m,2H),5.94(s,1H),5.88(s,2H),5.40(s,1H),5.11(t,J＝5.9Hz,1H),4.92(d,J＝5.0Hz,1H),4.83-4.75(m,3H),4.51(dd,J＝6.3,19.4Hz,1H),4.30(s,1H),4.18(dd,J＝5.6,19.4Hz,1H),3.73(s,2H),2.31(d,J＝2.3Hz,1H),2.15-2.00(m,2H),1.81-1.61(m,5H),1.40(s,3H),1.25(s,1H),1.13-1.01(m,2H),0.87(s,3H)。
实施例9：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-5-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物009)的合成
合成路线和具体合成步骤：
第一步：叔-丁基(5-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-2的合成
将5-溴-2,3-二氢苯并呋喃-7-羧酸9-1(1.00g,4.11mmol)溶于甲苯(10mL)和叔丁醇(10mL)的中，室温加入分子筛(200mg)和三乙胺(1.25g,12.3mmol)，110℃搅拌半小时后，冷却至室温，滴加磷叠氮化酯二苯基(1.70g,6.17mmol)，升温至110℃搅拌10小时。LCMS检测反应完毕。将反应液过滤，用乙酸乙酯(150mL)稀释，有机相用饱和食盐水(30mL)洗涤，干燥过滤浓缩，残留物经柱层析(0～40％乙酸乙酯/石油醚)分离纯化，得到中间体叔丁基(5-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-2(1.03g,79.7％)。
LC-MS:Rt:1.065min；MS m/z(ESI):259.7[M+H-56]+；
1H NMR(400MHz,DMSO-d6)δ＝8.59(s,1H),7.50(br s,1H),7.13-7.11(m,1H),4.61-4.50(m,2H),3.24-3.17(m,2H),1.48-1.41(m,9H)。
第二步：叔-丁基(5-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-3的合成
将叔-丁基(5-溴-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-2(600mg,1.91mmol)，双联嚬哪醇硼酸酯(533mg,2.10mmol)，醋酸钾(562mg,5.73mmol)，1,1-双(二苯基磷)二茂铁氯化钯(139mg,190μmol)溶于无水1,4-二氧六环(10mL)中，氮气氛围下80℃反应16小时。LCMS检测反应完毕。将反应液过滤浓缩，残留物经柱层析(0～10％四氢呋喃/石油醚)分离纯化，得到中间体叔丁基(5-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-3(990mg,71.8％)。
LC-MS:Rt:4.417min；MS m/z(ESI):306.2[M+H-56]+；
1H NMR(400MHz,CHLOROFORM-d)δ＝8.17-8.27(m,1H)7.38(s,1H)7.25-7.31(m,1H)4.59-4.66(m,2H)3.20-3.29(m,2H)1.33(s,12H)1.31(s,9H)
第三步：叔-丁基(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-4的合成
将(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮1-5(300mg,538μmol)，叔-丁基(5-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-3(300mg,830μmol)，四三苯基磷钯(62.2mg,53.8μmol)和碳酸钾(148mg,1.08mmol)溶于无水四氢呋喃(5mL)和水(1mL)中，氮气氛围下80℃搅拌2小时。LCMS检测反应完毕。反应液用乙酸乙酯(50mL)萃取，有机相用饱和食盐水(10mL)洗涤，干燥过滤浓缩，残留物经柱层析(0～50％四氢呋喃/石油醚)分离纯化，得到中间体(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧 亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-4(300mg,78.3％)。
LC-MS:Rt:2.156min；MS m/z(ESI):712.3[M+H]+；
第四步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-5-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物009)的合成
将中间体叔丁基(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯9-4(220mg,309μmol)溶于无水二氯甲烷(4mL)中，加入三氟乙酸(1mL)，25℃搅拌40分钟。LCMS检测反应完毕。将反应液用氮气吹干，得到的残留物经高效制备液相[柱子:YMC-Pack CN150*30mm*5μm；流动相:A,0.1％甲酸水溶液；B:乙腈,B％,40％-60％,12min]分离纯化，得到化合物(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-5-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮009(2.00mg,1.06％)。
LC-MS:Rt:2.576min；MS m/z(ESI):612.3[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.33-7.38(m,2H)7.31(d,J＝10.13Hz,1H)7.19(d,J＝8.13Hz,2H)6.29(d,J＝17.26Hz,2H)6.17(dd,J＝10.13,1.88Hz,1H)5.94(s,1H)5.39(s,1H)5.08(s,1H)4.90-4.94(m,1H)4.77-4.81(m,1H)4.45-4.73(m,3H)4.42(t,J＝8.63Hz,2H)4.29(d,J＝2.88Hz,1H)4.13-4.23(m,1H)3.71(s,2H)3.04(t,J＝8.69Hz,2H)2.30(s,1H)2.09-2.17(m,1H)1.98-2.06(m,1H)1.56-1.84(m,6H)1.40(s,3H)0.97-1.11(m,2H)0.86(s,3H)。
实施例10：(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-4-基)甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物010)的合成
合成路线和具体合成步骤：
第一步：(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮010-3的合成
将原料双羟氟轻松SM-1(1.00g,2.42mmol)溶于无水乙腈(10mL)中，加入无水硫酸镁(1.46g,12.1mmol)，25℃搅拌1小时后，0℃加入4-溴甲基苯甲醛(579mg,2.91mmol)的乙腈(2mL)溶液，再缓慢滴加三氟甲磺酸(1.82g,12.12mmol)的乙腈(2mL)溶液，0℃搅拌五分钟。LCMS检测反应完毕。用饱和碳酸氢钠水溶液(20mL)淬灭反应，用乙酸乙酯(50mL*2)萃取，有机相用饱和氯化钠水溶液(20mL)洗涤，干燥过滤浓缩，残留物经Prep-HPLC[柱子:YMC-Pack CN 150*30mm*5μm；流动相:A,0.1％甲酸水溶液；B,乙腈,B％，53％-63％,12min]分离纯化，得到中间体(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮010-3(640mg,42％)。
LC-MS:Rt:2.845min；MS m/z(ESI):595.1[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.62-7.39(m,4H),7.27(d,J＝10.3Hz,1H),6.30(dd,J＝1.8,10.0Hz,1H),6.13(s,1H),5.77-5.45(m,3H),5.01-4.95(m,1H),4.68(s,2H),4.55(br d,J＝19.3Hz,1H),4.22(br d,J＝19.3Hz,2H),2.74-2.56(m,1H),2.37-2.12(m,3H),2.09-1.95(m,1H),1.77-1.64(m,3H),1.56-1.42(m,4H),0.87(s,3H)；
19F NMR(376MHz,DMSO-d6)δ＝-164.99(s,1F),-186.39(s,1F)。
第二步：4-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-胺010-2的合成4-溴-2,3-二氢苯并呋喃-7-胺1-3(7.30g,34.1mmol)溶于1,4-二氧六环(150mL)，加入双联嚬哪醇硼酸酯(20.0g,78.8mmol)，1,1-双(二苯基磷)二茂铁氯化钯(2.50g,3.4mmol)和乙酸钾(10.0g,102.3mmol)，氮气氛围下80℃搅拌16小时。LCMS显示反应完毕，反应液过滤浓缩，残留物经柱层析(四氢呋喃/石油醚＝1/6)分离纯化，得到中间体4-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-胺010-2(9.50g,96.0％)。
LC-MS:Rt:1.470min；MS m/z(ESI):262.0[M+H]；
第三步：(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-4-基)甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮010的合成
将(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮010-3(150mg,253μmol)溶于四氢呋喃(2mL)和水(0.5mL)中，依次加入4-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢苯并呋喃-7-胺(79mg,303μmol)，碳酸钾(79mg,303μmol)，四三苯基膦钯(29mg,25.3μmol)，氮气氛围下80℃反应两个小时。LCMS检测反应完全。反应液用乙酸乙酯(10mL*3)萃取，有机相用饱和氯化钠水溶液(5mL)洗涤，干燥过滤浓缩，残留物经药效制备液相[柱子:Phenomenex C18 75*30mm*3μm；流动相:A,0.1％氨水溶液；B:乙腈,B％,30％-70％,9min]分离纯化，得到化合物(2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-4-基)甲基)苯基)-2,6b-二氟-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮010(20.0mg)。
LC-MS:Rt:2.092min；MS m/z(ESI):648.3[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.33(d,J＝8.0Hz,2H),7.27(d,J＝9.8Hz,1H),7.18(d,J＝8.0Hz,2H),6.42-6.37(m,2H),6.30(dd,J＝1.8,10.3Hz,1H),6.13(s,1H),5.75-5.56(m,1H),5.52(d,J＝2.8Hz,1H),5.45(s,1H),5.11(t,J＝6.0Hz,1H),4.95(d,J＝4.8Hz,1H),4.56-4.38(m,5H),4.25-4.15(m,2H),3.73(s,2H),2.97(t,J＝8.7Hz,2H),2.44(br s,1H),2.32-2.19(m,2H),2.10-2.00(m,1H),1.78-1.64(m,3H),1.50(s,4H),0.86(s,3H)；
19F NMR(376MHz,DMSO-d6)δ＝-164.96(s,1F),-186.39(s,1F)。
实施例11：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢-1H-茚-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(化合物011)的合成
合成路线和具体合成步骤：
第一步：7-溴-2,3-二氢-1H-茚-4-胺11-2的合成
将2,3-二氢-1H-茚-4-胺(2.00g,15.0mmol)溶于N,N-二甲基甲酰胺(20mL)中，0℃下加入N-溴代丁二酰亚胺(NBS)(2.67g,15.0mmol)，0℃搅拌1小时。LCMS检测反应完毕。0℃加入饱和碳酸氢钠溶液(50mL)进行淬火，用乙酸乙酯(100mL*2)萃取，有机相用水(50mL*3)和饱和食盐水(50mL)洗涤，干燥过滤浓缩，得到的残留物经柱层析(0～15％乙酸乙酯/石油醚)分离纯化，得到7-溴-2,3-二氢-1H-茚-4-胺11-2(1.70g,53.4％)。
LC-MS:Rt:0.800min；MS m/z(ESI):213.8[M+H]+；
第二步：7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢-1H-茚-4-胺11-3的合成
将7-溴-2,3-二氢-1H-茚-4-胺11-2(100mg,471μmol)和双联嚬哪醇硼酸酯(239mg,943μmol)溶于1,4-二氧六环(5mL)中，加入1,1-双(二苯基磷)二茂铁氯化钯(34.5mg,47.2μmol)和醋酸钾(92.5mg,943μmol)，氮气氛围下80℃搅拌12小时。LCMS检测反应完毕。将反应液过滤浓缩，得到的残留物经柱层析(0～60％乙酸乙酯/石油醚)分离纯化，得到中间体7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢-1H-茚-4-胺11-3(77.0mg,63.0％)。
LC-MS:Rt:0.914min；MS m/z(ESI):260.3[M+H]+；
第三步：(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢-1H-茚-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮011的合成
将7-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)-2,3-二氢-1H-茚-4-胺11-3(77mg,295.98μmol)和(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-(溴甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮1-5(110mg,197μmol)溶于四氢呋喃(5mL)和水(1mL)中，加入1,1-双(二苯基磷)二茂铁氯化钯(14.4mg,19.7μmol)和碳酸钾(54.5mg,394μmol)，氮气氛围下80℃搅拌反应2小时。LCMS检测反应完毕。加水(20mL)稀释，然后用乙酸乙酯(50mL*2)萃取，有机相用饱和食盐水(20mL)洗涤，干燥过滤浓缩，得到的残留物经高效制备液相[柱子：Boston Prime C18 150*30mm*5μm；流动相:A,0.1％甲酸水溶液；B,ACN,29％-49％,12min)分离纯化，得到(6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢-1H-茚-4-基)甲基)苯基)-7-羟基-8b-(2-羟基乙酰基)-6a,8a-二甲基-6a,6b,7,8,8a,8b,11a,12,12a,12b-十氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-4(2H)-酮(55.0mg,45.7％)。
LC-MS:Rt:0.856min；MS m/z(ESI):610.4[M+H]+；
1H NMR(400MHz,DMSO-d6)δ＝7.37-7.28(m,3H),7.16-7.11(m,1H),7.14(d,J＝7.8Hz,1H),6.66(d,J＝7.8Hz,1H),6.33(d,J＝8.0Hz,1H),6.16(dd,J＝1.6,10.2Hz,1H),5.93(s,1H),5.39(s,1H),5.08(s,1H),4.91(d,J＝5.3Hz,1H),4.80-4.75(m,1H),4.75-4.55(m,2H),4.50(d,J＝17.8Hz,1H),4.29(s,1H),4.17(d,J＝18.1Hz,1H),3.73(s,2H),2.66(t,J＝7.5Hz,2H),2.63-2.57(m,2H),2.57-2.53(m,1H),2.30(s,1H),2.16-1.98(m,2H),1.91(quin,J＝7.4Hz,2H),1.81-1.60(m,5H),1.40(s,3H),1.11-0.97(m,2H),0.90-0.81(m,3H)
实施例12：2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-5-基)甲基)苯基)-7-羟基-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-8b-基)-2-氧代乙基磷酸二氢酯(化合物009-p)的合成
合成路线和具体合成步骤：
第一步：叔丁基(5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((二-叔丁氧基膦酰爪基)氧代)乙酰基)-7-羟基-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基甲酸酯(009-p-1)的合成
将中间体9-4(80.0mg,112μmol)溶于N,N-二甲基甲酰胺(4mL)中，加入N,N-二乙基亚磷酰胺二叔丁酯(336mg,1.35mmol)和四氮唑(78.7mg,1.12mmol)，室温下搅拌30分钟。TLC监测反应完全，冷却至0℃，向反应液加入过氧化氢(580mg,5.12mmol)，恢复至室温下搅拌1小时。LCMS检测反应完毕。在0℃加入亚硫酸钠水溶液(10mL)淬灭反应，用乙酸乙酯(30mL)萃取，有机相用饱和食盐水(10mL)洗涤，干燥过滤浓缩，得到的残留物经柱层析(0～60％乙酸乙酯/石油醚)分离纯化，得到中间体009-p-1(50.0mg,49.2％)。
LC-MS:Rt:1.216min；MS m/z(ESI):904.6[M+H]+；
第二步：2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-氨基-2,3-二氢苯并呋喃-5-基)甲基)苯基)-7-羟基-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-8b-基)-2-氧代乙基磷酸二氢酯009-p的合成
将中间体009-p-1(50.0mg,55.3μmol)溶于二氯甲烷(3mL)中，加入三氟乙酸(630mg,5.53mmol)，室温下搅拌反应30分钟。LCMS检测反应完毕。将反应液用氮气吹干，得到的残留物经制备高效液相[柱子:Boston Prime C18 150*30mm*5μm；流动相:A,0.1％甲酸水溶液；B,乙腈,B％,38％-58％,12min)分离纯化，得到化合物009-p(10.0mg,26.1％)。
LC-MS:Rt:2.752min；MS m/z(ESI):692.3[M+H]；
1H NMR(400MHz,DMSO-d6)δ＝7.37-7.24(m,3H),7.14(d,J＝6.8Hz,2H),6.27(s,2H),6.16(dd,J＝1.6,10.0Hz,1H),5.93(s,1H),5.47(s,1H),4.94-4.84(m,2H),4.66-4.56(m,1H),4.40(t,J＝8.6Hz,2H),4.28(s,1H),3.68(s,2H),3.00(t,J＝8.7Hz,2H),2.29(d,J＝2.9Hz,1H),2.16-1.94(m,3H),1.83-1.60(m,5H),1.39(s,3H),1.04-0.83(m,5H)。
中间体(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸INT-1的合成
合成路线和具体合成步骤：
第一步：(S)-1-苯甲基5-叔-丁基2-(2-(((苄氧基)羰基)氨基)乙酰氨基)戊二酯的合成
将(S)-1-苯甲基-5-叔丁基-2-氨基戊二酯(5.00g,15.2mmol)溶于二氯甲烷(50mL)中，加入质量分数为50％正丙基环磷酸酐的乙酸乙酯溶液(10.1g,15.9mmol)，N,N-二异丙基乙胺(5.88g,45.5mmol)和2-(((苄氧基)羰基)氨基)乙酸(3.49g,16.7mmol)，25℃搅拌16小时。用二氯甲烷(300mL)稀释，有机相用饱和碳酸氢钠水溶液(100mL)和饱和食盐水(50mL)洗涤，过滤，干燥，浓缩，残留物经柱层析(0～30％四氢呋喃/石油醚)分离纯化，得到(S)-1-苯甲基-5-叔-丁基-2-(2-(((苄氧基)羰基)氨基)乙酰氨基)戊二酯(7.13g)。
LC-MS:Rt:0.991min；MS m/z(ESI):429.1[M-56+H]+；
1H NMR(400MHz,DMSO-d6)δ＝8.32(d,J＝7.5Hz,1H),7.43-7.28(m,10H),5.75(s,1H),5.13(s,2H),5.05(s,2H),4.45-4.30(m,1H),3.72-3.59(m,2H),2.32-2.19(m,2H),2.04-1.79(m,2H),1.38(s,9H)。
第二步：(S)-2-(2-氨基乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸的合成
将(S)-1-苯甲基-5-叔-丁基2-(2-(((苄氧基)羰基)氨基)乙酰氨基)戊二酯(7.13g,14.7mmol)溶于四氢呋喃(80mL)中，加入10％钯/碳(1.57g)，在氢气氛围(15psi)下25℃搅拌16小时。TLC检测反应完毕。反应液用水(200mL)稀释，过滤，浓缩，冻干，得到(S)-2-(2-氨基乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸(3.83g)。
1H NMR(400MHz,DMSO-d6)δ＝8.08(s,1H),4.00(s,1H),3.35(s,1H),2.24-2.08(m,2H),1.99-1.88(m,1H),1.81-1.71(m,1H),1.39(s,9H)。
第三步：(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸INT-1的合成
将(S)-2-(2-氨基乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸(3.83g,14.7mmol)溶于四氢呋喃(40mL)和水(40mL)混合溶剂中，加入9-芴甲基琥珀酰亚氨基碳酸酯(Fmoc-Osu,5.96g,17.7mmol)和碳酸氢钠(3.71g,44.1mmol)，25℃搅拌16小时。TLC检测反应完毕。反应液用1N稀盐酸调节pH＝3，用乙酸乙酯(150mL x 2)萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，干燥，过滤，浓缩，残留物经柱层析(0～54％四氢呋喃/石油醚)分离纯化，得到中间体(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸(6.00g)。
LC-MS:Rt:0.947min；MS m/z(ESI):427.0[M-56+H]+；
1H NMR(400MHz,DMSO-d6)δ＝12.70(s,1H),8.11(d,J＝7.8Hz,1H),7.90(d,J＝7.5Hz,2H),7.72(d,J＝7.5Hz,2H),7.54(t,J＝6.0Hz,1H),7.45-7.40(m,2H),7.36-7.31(m,2H),4.30-4.20(m,4H),3.73-3.63(m,2H),2.29-2.22(m,2H),2.01-1.91(m,1H),1.38(s,9H),1.32-1.28(m,1H)。
实施例13：(S)-4-(2-(2-溴乙酰氨基)乙酰氨基)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-8b-(2-(膦酰氧基)乙酰基)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸(L1-001)的合成
合成路线和具体合成步骤：
第一步：化合物L1-001-1的合成
将化合物001(2.00g,3.27mmol)和(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸(中间体INT-1,1.74g,3.60mmol)溶于二氯甲烷(10mL)和N,N-二甲基甲酰胺(20mL)中，依次加入2,6-二甲基吡啶(1.05g,9.81mmol)和O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(HATU,1.49g,3.92mmol)，25℃反应12小时。LCMS检测反应完毕。反应液用乙酸乙酯(250mL)稀释，用饱和氯化铵水溶液(50mL*2)和饱和氯化钠水溶液(50mL)洗涤，干燥，过滤，浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例80％)得到化合物L1-001-1(2.80g)。
LC-MS:Rt:5.510min；MS m/z(ESI):1077.2[M+H]+；
SFC:Rt:3.241min。
第二步：化合物L1-001-2的合成
将中间体L1-001-1(2.80g,2.60mmol)溶于N,N-二甲基甲酰胺(30mL)，0℃下加入1H-四氮唑(1.82g,26.0mmol)和N,N-二乙基亚磷酰胺二叔丁酯(7.78g,31.2mmol)，25℃搅拌30分钟。TLC检测反应完全。在0℃加入双氧水(2.21g,30％w/w)，25℃搅拌1小时。LCMS检测反应完毕。在0℃加入亚硫酸钠水溶液(20mL)淬灭反应，用乙酸乙酯(200mL)萃取，有机相用饱和食盐水(50mL)洗涤，干燥，过滤，浓缩，得到粗产品(3.3g)。将粗产品溶于乙腈(30mL)中，在0℃下加入哌啶(1.11g,13.0mmol)，25℃搅拌30分钟。LCMS检测反应完毕。 将反应液减压浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例40％)得到(S)-叔丁基-4-(2-氨基乙酰氨)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((二叔丁氧基磷酰基)氧代)乙酰基)-7-羟基-6a,8a-二甲基-4-氧代-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸酯(L1-001-2)(1.80g)。
LC-MS:Rt:0.979min；MS m/z(ESI):1048.0[M+H]+；
第三步：化合物L1-001-3的合成
将2-溴乙酸(478mg,3.44mmol)溶于N,N-二甲基甲酰胺(20mL)中，加入2-乙氧基喹啉-1(2H)-甲酸乙酯(EEDQ,851mg,3.44mmol)，25℃搅拌1小时，再滴加溶有中间体L1-001-2(1.80g,1.72mmol)的N,N-二甲基甲酰胺(10mL)溶液，25℃搅拌2小时。LCMS检测反应完毕。反应液用乙酸乙酯(100mL)稀释，用稀释的氢溴酸(10mL)饱和碳酸氢钠水溶液(30mL*3)洗涤，干燥，过滤，浓缩，残留物经反相柱层析(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例80％)得到(S)-叔-丁基4-(2-(2-溴乙酰氨基)乙酰氨基)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((二叔丁氧基磷酰基)氧基)氧代)乙酰基)-7-羟基-6a,8a-二甲基-4-氧代-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸酯(L1-001-3)(1.40g)。
LC-MS:Rt:2.413min；MS m/z(ESI):1112.5[M-56+H]+。
第四步：(S)-4-(2-(2-溴乙酰氨基)乙酰氨基)-5-((4-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-8b-(2-(膦酰氧基)乙酰基)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸(L1-001)的合成
将中间体L1-001-3(0.65g,0.56mmol)溶于无水二氯甲烷(6mL)中，25℃下加入三氟乙酸(2mL)，25℃搅拌30分钟。LCMS检测反应完毕。用氮气将反应溶剂吹干，残留物用二氯甲烷(5mL)溶解再用氮气吹干，重复3次。残留物经制备高效液相色谱[柱子:Waters Xbridge BEH C18 100*30mm*10μm；用水(含有0.05％NH4HCO3)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例8％-38％,洗脱时间11分钟]分离纯化，得到化合物L1-001(0.31g)。
LC-MS:Rt:2.937min；MS m/z(ESI):998.2/1000.2[M+H]+；
1H NMR(400MHz,D2O)δ＝7.35(d,J＝10.0Hz,1H),7.21(d,J＝7.7Hz,1H),7.03(d,J＝7.8Hz,2H),6.59(d,J＝7.9Hz,2H),6.44(d,J＝8.3Hz,1H),6.12-6.01(m,1H),5.69(s,1H),5.52(s,1H),5.00(s,1H),4.92-4.82(m,1H),4.78-4.74(m,1H),4.43(dd,J＝5.4,8.4Hz,1H),4.18(s,2H),4.10-3.99(m,1H),3.90(s,2H),3.86(s,2H),3.58-3.30(m,2H),2.49(s,1H),2.32(t,J＝7.6Hz,4H),2.10-1.86(m,5H),1.85-1.54(m,4H),1.34-1.11(m,4H),0.81(s,3H),0.25(d,J＝9.7Hz,2H)。
实施例14：2-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-10-(4-((7-((S)-6-氨基-2-(2-(2-溴乙酰氨基)乙酰氨基)己酰氨基)-2,3-二氢苯并呋喃-4-基)甲基)苯基)-7-羟基-6a,8a-二甲基-4-氧代-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-8bH-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-8b-基)-2-氧代乙基磷酸酯(L2-001)
合成路线和具体合成步骤：
第一步：N2-(((苄氧基)羰基)甘氨酸)-N6-(叔丁氧基羰基)-L-赖氨酸苄基酯(L2-001-2)的合成
将N6-(叔丁氧羰基)-L-赖氨酸苄酯单盐酸盐(5.00g,13.4mmol)溶于无水二氯甲烷(50mL)中，加入1-丙基磷酸酐(T3P,8.96g,质量分数50％的乙酸乙酯溶液)，N,N-二异丙基乙胺(5.20g,40.2mmol)和苄氧羰基甘氨酸(3.09g,14.8mmol)，25℃搅拌2小时。LCMS检测反应完毕。在0℃加入碳酸氢钠水溶液(20mL)淬灭反应，用二氯甲烷(150mL*3)萃取，有机相用饱和氯化钠水溶液(60mL)洗涤，干燥，过滤浓，缩，残留物经硅胶柱层析(0～32％四氢呋喃/石油醚)分离纯化，得到L2-001-2(6.39g)。
LC-MS:Rt:0.957min；MS m/z(ESI):428.1[M-100+H]+；
1H NMR(400MHz,DMSO-d6)δ＝8.27(d,J＝7.5Hz,1H),7.46-7.40(m,1H),7.39-7.27(m,10H),6.76(t,J＝5.2Hz,1H),5.12(s,2H),5.01(br s,2H),4.33-4.22(m,1H),3.74-3.63(m,2H),2.87(q,J＝6.3Hz,2H),1.73-1.55(m,2H),1.37(s,9H),1.35-1.19(m,4H)。
第二步：(S)-2-(2-氨基乙酰氨基)-6-((叔-丁氧羰基)氨基)己酸的L2-001-3合成
将中间体L2-001-2(7.95g,15.1mmol)溶于四氢呋喃(90mL)中，加入10％湿钯碳(1.60g)，用氢气置换3次，在氢气氛围下(15PSI)25℃反应16小时。TLC显示原料消失。反应液用水(100mL)和甲醇(50mL)稀释，过滤，浓缩，得到(S)-2-(2-氨基乙酰氨基)-6-((叔-丁氧羰 基)氨基)己酸L2-001-3(3.82g)。
1H NMR(400MHz,DMSO-d6)δ＝8.08(br s,1H),6.76(br s,1H),4.02(br s,1H),3.33-3.19(m,2H),2.86(q,J＝6.5Hz,2H),1.70-1.51(m,2H),1.37-1.29(m,11H),1.22(d,J＝6.3Hz,2H)。
第三步：(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-6-((叔丁氧羰基)氨基)己酸L2-001-4的合成
将(S)-2-(2-氨基乙酰氨基)-6-((叔-丁氧羰基)氨基)己酸L2-001-3(3.82g,12.6mmol)溶于四氢呋喃(40mL)和水(40mL)混合溶剂中，加入碳酸氢钠(3.17g,37.8mmol)和9-芴甲基-N-琥珀酰亚胺基碳酸酯(5.10g,15.1mmol)，25℃搅拌16小时。LCMS检测反应完毕。在0℃下滴加1M稀盐酸调节pH＝7，用乙酸乙酯(150mL*3)萃取，有机相用饱和氯化钠水溶液(50mL)洗涤，干燥，过滤，浓缩，残留物经硅胶柱层析(0～36％四氢呋喃/石油醚)分离纯化，得到中间体(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-6-((叔-丁氧羰基)氨基)己酸L2-001-4(6.07g)。
LC-MS:Rt:0.960min；MS m/z(ESI):426.1[M-100+H]+；
1H NMR(400MHz,DMSO-d6)δ＝12.60(br s,1H),8.07(d,J＝7.8Hz,1H),7.89(d,J＝7.5Hz,2H),7.72(d,J＝7.5Hz,2H),7.52(t,J＝6.1Hz,1H),7.44-7.40(m,2H),7.35-7.31(m,2H),6.76(t,J＝5.2Hz,1H),4.30-4.17(m,4H),3.73-3.63(m,2H),2.88(q,J＝6.5Hz,2H),1.73-1.55(m,2H),1.36(s,11H),1.31-1.24(m,2H)。
第四步：化合物L2-001-5的合成
将化合物001和中间体(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-6-((叔丁氧羰基)氨基)己酸L2-001-4(644mg，1.23mmol)溶于无水二氯甲烷(30mL)和无水N,N-二甲基甲酰胺(5mL)中，0℃加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N’,N’-四甲基脲六氟磷酸酯(435mg,1.14mmol)和2,6-二甲基吡啶(263mg,2.45mmol)，25℃搅拌16小时。LCMS检测反应完毕。反应液用无水二氯甲烷(200mL)稀释，有机相用水(50mL x 2)和饱和氯化钠水溶液(30mL)洗涤，干燥过滤浓缩，残留物经柱层析(0～50％四氢呋喃/石油醚)分离纯化，得到的粗产品经制备高效液相色谱(柱子:C18-1 150*30mm*5μm；用水(含有0.05％TFA)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例42％-82％,洗脱时间9分钟)分离纯化，得到L2-001-5(424mg,46.4％)。
LC-MS:Rt:1.081min；MS m/z(ESI):1019.4[M+H]；
1H NMR(400MHz,DMSO-d6)δ＝9.34(s,1H),8.03(br d,J＝8.0Hz,1H),7.89(d,J＝7.4Hz,2H),7.71(d,J＝7.5Hz,2H),7.54(t,J＝6.2Hz,1H),7.47-7.26(m,8H),7.19(d,J＝8.0Hz,2H),6.73(s,1H),6.59(d,J＝8.3Hz,1H),6.16(dd,J＝1.6,10.1Hz,1H),5.92(s,1H),5.40(s,1H),5.08(t,J＝5.9Hz,1H),4.92(d,J＝5.0Hz,1H),4.78(d,J＝3.0Hz,1H),4.56-4.45(m,4H),4.33-4.12(m,5H),3.84(s,2H),3.67(d,J＝5.8Hz,2H),3.06(t,J＝8.8Hz,2H),2.87(d,J＝5.9Hz,2H),2.73-2.55(m,1H),2.35-2.24(m,2H),2.17-1.94(m,3H),1.81-1.49(m,7H),1.42-1.31(m,14H),1.09-0.96(m,2H),0.86(s,3H)。
第五步：化合物L2-001-6的合成
将中间体L2-001-5(200mg,179μmol)和1H-四氮唑(125mg,1.79mmol)溶于无水N,N-二甲基甲酰胺(2mL)中，0℃加入用无水N,N-二甲基甲酰胺(5mL)稀释的N,N-二乙基亚磷酰胺二叔丁酯(535mg,2.14mmol)，25℃搅拌1小时。0℃加入用无水N,N-二甲基甲酰胺(0.5mL)稀释的过氧化氢溶液(111mg,30％w/w)，25℃下搅拌1小时。LCMS检测反应完毕。0℃加入亚硫酸钠水溶液(2mL)淬灭反应，使用乙酸乙酯(150mL)萃取，有机相用水(30mL x 3)和饱和氯化钠水溶液(30mL)洗涤，干燥，过滤，浓缩，残留物经柱层析(0～55％四氢呋喃/石油醚)分离纯化，得到化合物L2-001-6(196mg,83.6％)。
LC-MS:Rt:1.153min；MS m/z(ESI):1212.6[M-100+H]+。
第六步：化合物L2-001-7的合成
将中间体L2-001-6(150mg,114μmol)溶于乙腈(2mL)中，加入哌啶(20.0mg,234μmol)，0℃搅拌10分钟。LCMS检测反应完毕。反应液浓缩，得到化合物L2-001-7(124mg)。LC-MS:Rt:0.989min；MS m/z(ESI):1091.0[M+H]；
第七步：化合物L2-001-8的合成
将中间体L2-001-7(120mg,110μmol)溶于无水N,N-二甲基甲酰胺(2mL)中，加2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ，33.0mg,132μmol)和溴乙酸(23.0mg,165μmol)，25℃搅拌2小时。LCMS检测反应完毕。反应液用乙酸乙酯(100mL)稀释，有机相用稀释的氢溴酸(10mL)、饱和碳酸氢钠水溶液(20mL x 2)和饱和食盐水(20mL)洗涤，干燥，过滤，浓缩，残留物经硅胶柱层析(0～65％四氢呋喃/石油醚)分离纯化，得到化合物L2-001-8(65.0mg)。
LC-MS:Rt:1.110min；MS m/z(ESI):1111.3[M-100+H]+；
第八步：化合物L2-001的合成
将中间体L2-001-8(50.0mg,41.3μmol)溶于无水二氯甲烷(0.5mL)中，加入三氟乙酸(0.2mL)，25℃搅拌40分钟。LCMS检测反应完毕。将反应液用氮气吹干，得到的残留物经制备高效液相色谱(柱子:Boston Green ODS 150*30mm*5μm；用水(含有0.05％TFA)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例22％-37％,洗脱时间10分钟)分离纯化，得到化合物L2-001(5.32mg，12.9％)。
LC-MS:Rt:0.822min；MS m/z(ESI):996.9/998.9[M+H]；
1H NMR(400MHz,DMSO-d6)δ＝9.36(s,1H),8.61-8.39(m,1H),8.19(d,J＝7.9Hz,1H),7.67(s,3H),7.54(d,J＝8.1Hz,1H),7.26-7.15(m,3H),6.91(s,2H),6.56(d,J＝8.3Hz,1H),6.10(d,J＝10.4Hz,1H),5.83(s,1H),5.43(s,1H),4.91(d,J＝4.9Hz,1H),4.82-4.73(m,2H),4.59-4.50(m,1H),4.46-4.38(m,2H),4.17(s,1H),3.94(s,2H),3.88-3.63(m,7H),2.90-2.72(m,5H),2.17(d,J＝10.5Hz,1H),2.03(s,2H),1.89(s,1H),1.78-1.65(m,3H),1.55(d,J＝12.9Hz,5H),1.35(s,5H),0.85(s,3H),0.73-0.56(m,1H)。
实施例15：(S)-4-(2-(2-溴乙酰氨基)乙酰氨基)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-7-羟基-6a,8a-二甲基-4-氧代-8b-(2-(膦酸基氧代)乙酰基)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸(L1-009)
合成路线和具体合成步骤：
第一步：化合物L1-009-1的合成
将化合物009(1.50g,2.45mmol)和中间体(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸INT-1(1.30g,2.70mmol)溶于二氯甲烷(12mL)和N,N-二甲基甲酰胺(24mL)中，在0℃下加入O-(7-氮杂苯并三氮唑-1-基)-N,N,N,N-四甲基脲六氟膦盐(1.31g,3.43mmol)和2,6-二甲基吡啶(788mg,7.36mmol)，反应液在25℃下搅拌16小时。LCMS检测反应完毕。反应液乙酸乙酯(250mL)稀释，用饱和氯化铵水溶液(50mL*2)和饱和氯化钠水溶液(50mL)洗涤，干燥，过滤，浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例80％)分离纯化，得到化合物L1-009-1(2.60g,98.5％)。
LC-MS:Rt:5.134min；MS m/z(ESI):1076.5[M+H]+；
第二步：(S)-叔-丁基4-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-((5-(4-((6aR,6bS,7S,8aS,8bS,10R,11aR,12aS,12bS)-8b-(2-((二叔丁氧基磷酰基)氧代)乙酰基)-7-羟基-6a,8a-二甲基-4-氧亚基-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧亚基戊酯L1-009-02的合成
将化合物L1-009-1(2.40g,2.23mmol)和(1H-四氮唑)(1.56g,22.3mmol)溶于N,N-二甲基甲酰胺(10mL)中，在0℃下加入N,N-二乙基亚磷酰胺二叔丁酯(6.67g,26.8mmol)，25℃搅拌30分钟。TLC监测反应完全。在0℃加入过氧化氢(3.12g,30％w/w)，25℃搅拌1小时。LCMS检测反应完毕。在0℃加入亚硫酸钠水溶液(20mL)淬灭反应，用乙酸乙酯(200mL)萃取，有机相用饱和食盐水(50mL)洗涤，干燥，过滤，浓缩，得到粗产品(2.80g)。将所得产品溶于乙腈(30mL)中，在0℃下加入哌啶(938mg,11.0mmol)，25℃搅拌30分钟。将反应液浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例40％)分离纯化分离纯化，得到化合物L1-009-02(1.40g,60.0％)。
LC-MS:Rt:0.972min；MS m/z(ESI):1046.3[M+H]+；
第三步：化合物L1-009-03的合成
将中间体L1-009-02(1.40g,1.34mmol)溶于N,N-二甲基甲酰胺(5mL)中，加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ,530mg,2.14mmol)和溴乙酸(372mg,2.68mmol)，25℃搅拌2小时。LCMS检测反应完毕。反应液用乙酸乙酯(100mL)稀释，用稀释的氢溴酸(10mL)和饱和碳酸氢钠水溶液(30mL*3)洗涤，干燥过滤浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例80％)分离纯化，得到化合物L1-009-03(1.00g,64.0％)。
LC-MS:Rt:5.260min；MS m/z(ESI):1168.4[M+H]；
1H NMR(400MHz,DMSO-d6)δ＝9.36(s,1H),8.49(t,J＝5.6Hz,1H),8.19(d,J＝8.1Hz,1H),7.44(s,1H),7.39-7.31(m,3H),7.23(d,J＝8.0Hz,2H),6.85(s,1H),6.17(dd,J＝1.8,10.0 Hz,1H),5.98-5.85(m,1H),5.93(s,1H),5.49(s,1H),5.00-4.88(m,3H),4.62(dd,J＝9.1,17.9Hz,1H),4.56-4.48(m,3H),4.31(s,1H),3.94(s,2H),3.83-3.77(m,3H),3.15(t,J＝8.6Hz,2H),2.31-2.18(m,3H),2.16-1.86(m,5H),1.78-1.66(m,5H),1.47-1.38(m,30H),1.11-1.00(m,2H),0.89(s,3H)。
第四步：化合物LI-009的合成
将中间体L1-009-03(700mg,0.6mmol)溶于二氯甲烷(6mL)中，加入三氟乙酸(2mL)，25℃搅拌30分钟。LCMS检测反应完毕。将反应液用氮气吹干，用氮气将反应溶剂吹干，残留物用二氯甲烷(5mL)溶解再用氮气吹干，重复3次。残留物经制备高效液相色谱[柱子:Phenomenex C18 150*40mm*5μm；用水(含有0.05％NH4HCO3)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例0％-30％,洗脱时间10分钟]分离纯化，得到化合物LI-009(314mg)。
LC-MS:Rt:3.018min；MS m/z(ESI):998.1/1000.2[M+H]；
1H NMR(400MHz,DEUTERIUM OXIDE)δ＝7.35(d,J＝9.8Hz,1H),7.17(s,1H),6.97(d,J＝7.8Hz,2H),6.59(d,J＝7.8Hz,2H),6.21(s,1H),6.12(d,J＝10.8Hz,1H),5.77(s,1H),5.48(s,1H),4.95(d,J＝4.5Hz,1H),4.90-4.81(m,1H),4.69-4.64(m,2H),4.48-4.42(m,1H),4.39-4.28(m,2H),4.14(s,1H),3.88-3.82(m,3H),3.56-3.40(m,2H),2.64-2.42(m,2H),2.40-2.23(m,3H),2.03-1.79(m,5H),1.77-1.45(m,4H),1.28-1.04(m,4H),0.77(s,3H),0.16-0.05(m,2H)。
实施例16：(S)-4-(2-(2-溴乙酰氨基)乙酰氨基)-5-((4-(4-((2S,6aS,6bR,7S,8aS,8bS,10R,11aR,12aS,12bS)-2,6b-二氟-7-羟基-6a,8a-二甲基-4-氧代-8b-(2-(膦酸基氧代)乙酰基)-2,4,6a,6b,7,8,8a,8b,11a,12,12a,12b-十二氢-1H-萘并[2',1':4,5]茚并[1,2-d][1,3]二噁戊环-10-基)苯甲基)-2,3-二氢苯并呋喃-7-基)氨基)-5-氧代戊酸(L1-010)的合成
合成路线和具体合成步骤：
第一步：化合物L1-010-01的合成
将化合物010(650mg,1.00mmol)和(S)-2-(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)-5-(叔-丁氧基)-5-氧代戊酸(533mg,1.10mmol)溶于二氯甲烷(2.5mL)和无水N,N-二甲基甲酰胺(5mL)的混合溶剂中，0℃下加入2,6-二甲基吡啶(323mg,3.01mmol)和2-(7-偶氮苯并三氮唑)-N,N,N,N-四甲基脲六氟磷酸酯(HATU,458mg,1.20mmol)，25℃搅拌12个小时。LCMS检测反应完毕。反应液加入水(10mL)淬灭，用乙酸乙酯(50mL*2)萃取，有机相用饱和食盐水(30mL)洗涤，干燥过滤浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例30％-70％)分离纯化，得化合物L1-010-01(500mg)。
LC-MS:Rt:2.351min；MS m/z(ESI):1112.7[M+H]。
第二步：化合物L1-010-02的合成
将化合物L1-010-01(300mg,0.27mmol)溶于无水N,N-二甲基甲酰胺(6mL)中，0℃下加入1H-四氮唑(189mg,2.70mmol)和N,N-二乙基亚磷酰胺二叔丁酯(807mg,3.24mmol)，25℃下搅拌1小时。TLC检测反应完毕。0℃滴加双氧水(210mg,30％w/w)的N,N-二甲基甲酰胺(1mL)溶液，25℃搅拌2小时。LCMS检测反应完毕。0℃加入饱和亚硫酸钠水溶液(3mL)淬灭反应，用乙酸乙酯(50mL)萃取，有机相用水(20mL*3)和饱和食盐水(30mL)洗涤，干燥过滤浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例30％-70％)分离纯化，得化合物L1-010-02(170mg)。
LC-MS:Rt:6.129min；MS m/z(ESI):1304.8[M+H]；
第三步：化合物L1-010-03的合成
将化合物L1-010-02(170mg,130μmol)溶于乙腈中(6mL)，加入哌啶(55.5mg,652μmol)，25℃搅拌1小时。LCMS检测反应完毕。将反应液浓缩，得到化合物L1-010-03(140mg)。
LC-MS:Rt:1.785min；MS m/z(ESI):1046.5[M+H]。
第四步：化合物L1-010-04的合成
将2-溴乙酸(36.0mg,259μmol)溶于无水N,N-二甲基甲酰胺(1mL)中，0℃加入2- 乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ,64.0mg,259μmol)，25℃搅拌1小时后，加入中间体L1-010-03(140mg,129μmol)的无水N,N-二甲基甲酰胺(0.5mL)溶液，25℃搅拌2小时。LCMS检测反应完毕。0℃滴加氢溴酸水溶液(1M,1mL)淬灭反应，用乙酸乙酯(50mL)萃取，有机相用水(10mL*2)和饱和碳酸氢钠水溶液(10mL)洗涤，干燥，过滤，浓缩，残留物经柱层析色谱法(Spherical C18 40-60μm,用水和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例30％-70％)分离纯化，得化合物L1-010-04(24.4mg,15.9％)。
LC-MS:Rt:2.003min；MS m/z(ESI):1204.4[M+H]。
第五步：化合物L1-010合成
将中间体L1-010-04(24.4mg,20.3μmol)溶于无水二氯甲烷(1mL)中，25℃下加入三氟乙酸(308mg,2.70mmol)，25℃搅拌1小时。LCMS检测反应完毕。将反应液用氮气流下吹干除去溶剂，残留物经高效液相色谱[柱子:Boston Prime C18 150*30mm*5μm；用水(含有0.05％NH4HCO3)和乙腈的极性递减的混合物作为洗脱液；乙腈梯度比例13％-33％，洗脱时间11分钟]分离纯化，得到化合物L1-010(3.25mg)。
LC-MS:Rt:2.671min；MS m/z(ESI):1034.2/1036.3[M+H]；
1H NMR(400MHz,DMSO-d6)δ＝9.56-9.32(m,1H),8.54-8.38(m,1H),8.21(d,J＝7.9Hz,1H),7.44(d,J＝8.2Hz,1H),7.36(d,J＝7.9Hz,2H),7.29-6.96(m,5H),6.65-6.60(m,1H),6.28(d,J＝11.6Hz,1H),6.12(s,1H),5.77-5.55(m,1H),5.46(s,1H),4.96(d,J＝4.6Hz,1H),4.66(s,2H),4.59-4.49(m,3H),4.18(d,J＝9.8Hz,1H),3.94(s,1H),3.87-3.78(m,4H),3.77-3.64(m,2H),3.07(t,J＝8.7Hz,2H),2.30-2.21(m,4H),2.08-1.89(m,4H),1.84-1.65(m,3H),1.52(s,4H),0.93(s,3H)。
实施例17-1：Infliximab抗体人源化设计
通过比对IMGT(http://imgt.cines.fr)人类抗体重链轻链可变区种系基因数据库，分别挑选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。基于抗体的三维结构，对包埋残基、与CDR区有直接相互作用的残基以及对VL和VH的构象有重要影响的构架区的残基进行回复突变，即得到人源化单克隆抗体。人鼠嵌合抗体Infliximab的人源化轻链模板为IGKV6-21*01，人源化重链模板为IGHV3-73*01，将Infliximab的CDR分别移植到其人源模板中，即获得对应的人源化版本。根据需要，将Infiliximab的人源化抗体的FR区序列中关键氨基酸进行回复突变为鼠源抗体对应的氨基酸，以保证原有的亲和力。具体回复突变设计见表1。
表1：Infliximab人源化抗体回复突变设计
Infliximab及其人源化抗体可变区具体序列如下：
Infliximab.VL氨基酸序列如SEQ ID NO.1所示：
Infliximab.VL1氨基酸序列如SEQ ID NO.2所示：
Infliximab.VL2氨基酸序列如SEQ ID NO.3所示：
Infliximab.VH氨基酸序列如SEQ ID NO.4所示：
Infliximab.VH1氨基酸序列如SEQ ID NO.5所示：
Infliximab.VH2氨基酸序列如SEQ ID NO.6所示：
Infliximab.VH3氨基酸序列如SEQ ID NO.7所示：
Infliximab.VH4氨基酸序列如SEQ ID NO.8所示：
Infliximab.VH5氨基酸序列如SEQ ID NO.9所示：
将上述不同的轻链和重链序列进行交叉组合，获得多种Infliximab人源化抗体，各抗体的可变区组合方式如下：
表2：Infliximab人源化抗体可变区对应氨基酸序列
抗体恒定区序列如下：
表3：重链恒定区和轻链恒定区序列
人源化抗体的表达纯化
根据上述序列，构建重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。将抗体相应的重链、轻链质粒和转染试剂PEI(Polysciences，货号：24765-1)加入OPTI-MEM(Gibco，货号：11058021)中混匀后静置15min，加入Expi293细胞(Thermofisher，货号：A14527)中，放入5％CO2，120rpm，37℃摇床培养。转染第二天， 加入OPM-293 ProFeed(上海奥浦迈，货号：F081918-001)和葡萄糖(Sigma，货号:G7528)。转染第六天，收集细胞上清，用Protein A(GE，货号：28985254)纯化，洗脱后的样品透析至PBS缓冲液，PH7.4，得到Infliximab人源化抗体。
人源化抗体与人TNFα蛋白ELISA结合实验
人TNFα蛋白(Acro Biosystems，货号：TNA-H5228)于4℃包被过夜，用0.05％Tween20-PBS溶液漂洗3次，加入2％BSA封闭液，37℃封闭1.5h。用0.05％Tween 20-PBS溶液漂洗3次后加入倍比稀释的样品，37℃孵育1h。用0.05％Tween 20-PBS溶液漂洗3次后加入二抗HRP goat anti-human IgG Fc(Merck，货号：AP113)，37℃孵育1h。用0.05％Tween20-PBS溶液漂洗3次后加入TMB溶液(Seracare，货号：5120-0077)，室温反应10min后加入1M盐酸终止反应，酶标仪450nm波长读板。人源化分子与人TNFα蛋白的结合能力与Infliximab相当，结合EC50值见表4。
表4：Infliximab人源化抗体与人TNFα蛋白ELISA结合实验
人源化抗体与人TNFα蛋白的Biacore结合实验
应用BIAcore 8K仪器，采用多循环动力学法测定抗体与抗原的亲和力，在每一个循环中，首先用Protein A芯片(GE，货号：29-1275-56)捕获待测抗体，然后注入抗原人TNFα蛋白(Acro Biosystems，货号：TNA-H5228)，记录抗体和抗原蛋白的结合和解离过程，最后用Glycine pH1.5完成芯片再生，其中流动相为HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA,0.05％surfactant P20)，流速30μL/min，结合时间为240s，解离时间为900s，再生时间为30s，检测温度为25℃，抗原浓度为10-0.15625nM。应用Biacore 8K分析软件(版本号2.0)根据1:1binding模型，对数据进行分析，拟合抗体抗原结合动力学参数，包括结合速率常数ka、解离速率常数kd、平衡解离常数KD。结果见表5，人源化抗体的亲和力与Infliximab相近。
表5：Infliximab人源化抗体与人TNFα蛋白的结合亲和力
人源化抗体Hotspot修复
根据结合亲和力的结果，选择回复突变数最少的Infliximab-Hu01。因其重链可变区Infliximab.VH1存在氨基酸DD，在抗体表达过程中有异构化(isomerization)的风险，需进行修复。设计以下4个Infliximab.VH1的变体，具体序列如下：
Infliximab.VH1a氨基酸序列如SEQ ID NO.12所示：
Infliximab.VH1b氨基酸序列如SEQ ID NO.13所示：
Infliximab.VH1c氨基酸序列如SEQ ID NO.14所示：
Infliximab.VH1d氨基酸序列如SEQ ID NO.15所示：
采用前述方法，构建相应的重链质粒，与Infliximab.VL1为可变区序列的轻链质粒共同转染Expi293细胞，收集上清后纯化得到4个hotspot修复后的人源化抗体，各抗体可变区氨基酸序列对应如下：
表6：Hotspot修复的Infliximab人源化抗体可变区对应氨基酸序列
对这四个抗体进行ELISA和Biacore检测，具体方案如上所述。ELISA检测结果EC50值和Biacore检测结果见表7。除Infliximab-Hu01d外，其余三个抗体与Infliximab-Hu01的亲和力相当。
表7：Hotspot修复的Infliximab人源化抗体亲和力检测
实施例17-2：抗体制备
17-2-1抗人TNFα抗体制备
用于制备抗人TNFα抗体-药物偶联物的抗体可变区和恒定区氨基酸序列见表8，抗体的CDR氨基酸序列见表9，其中Infliximab-h为Infliximab人源化后的抗体Infliximab-Hu01c。
根据表8的序列，构建生产抗人TNFα抗体的重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。根据实施例17-1中抗体表达纯化方法制备得到抗人TNFα抗体Nerelimomab、Certolizumab、Infliximab-h、Golimumab和Adalimumab。
表8：抗人TNFα抗体的氨基酸序列

表9：抗人TNFα抗体的CDR氨基酸序列(Kabat)
17-2-2抗人IL-4R抗体制备
用于制备抗IL-4R抗体-药物偶联物的抗体可变区和恒定区氨基酸序列如下，其序列参考专利PCT/US2007/021210。
Dupilumab heavy chain(HC,SEQ ID NO.55)：(下划线加粗部分对应VH-SEQ ID NO.57； 阴影部分对应CH-SEQ ID NO.59)
Dupilumab HCDR1(SEQ ID NO.60)：DYAMT
Dupilumab HCDR2(SEQ ID NO.61)：SISGSGGNTYYADSVKG
Dupilumab HCDR3(SEQ ID NO.62)：DRLSITIRPRYYGLDV
Dupilumab light chain(LC,SEQ ID NO.56)：(下划线加粗部分对应VL-SEQ ID NO.58；阴影部分对应CL-SEQ ID NO.11)
Dupilumab LCDR1(SEQ ID NO.63)：RSSQSLLYSIGYNYLD
Dupilumab LCDR2(SEQ ID NO.64)：LGSNRAS
Dupilumab LCDR3(SEQ ID NO.65)：MQALQTPYT
根据上述序列，构建生产抗人IL-4R抗体的重链(HC)和轻链(LC)质粒(将编码抗体VH和VL的核酸序列重组至带有信号肽和重链恒定区/轻链恒定区序列的表达载体上，得到表达VH-CH1-Fc/VL-CL的重组质粒)。根据实施例17-1中抗体表达纯化方法制备得到Dupilumab抗体。
利用上述制备Dupilumab抗体的方法制备另外的抗人IL-4R抗体，其具体的可变区序列、CDR序列以及抗体来源请见下表。根据实施例17-1中抗体表达纯化方法制备得到002Mab抗体、003Mab抗体、004Mab抗体、005Mab抗体和006Mab抗体。
表9-1：抗人IL4R抗体的VH、VL和CDR的氨基酸序列(Kabat)

实施例18：抗体-药物偶联物制备
用于制备抗鼠TNFα抗体-药物偶联物的抗鼠TNFα抗体(下文称“001Mab”)购自BioXcell，货号为BE0058。
用于制备抗人IL-4R抗体-药物偶联物同型对照的Anti-Hel-hIgG4购自百英生物，货号为B669701。
用于制备抗人TNFα抗体-药物偶联物的抗人TNFα抗体、以及制备抗人IL-4R抗体-药物偶联物的抗人IL-4R抗体通过实施例17-2制备获得。
偶联：将上述抗体加入超滤管Amicon-Ultra-30kD并浓缩换液至20mM的pH＝7.0磷酸缓冲溶液，加入10mM二苯膦乙酸，将混合溶液放置恒温金属摇床37℃还原抗体3h。将DMSO溶解的药物-连接子化合物加入反应体系，25℃偶联16h。对反应产物超滤浓缩换液至磷酸盐(PBS)缓冲液，去除未反应的游离小分子。采用SEC和LC-MS方法对ADC产品进行纯度和DAR值分析。
ADC SEC纯度分析:应用SEC-HPLC法分析待测蛋白样品，表征重组蛋白的分子大小均一性，测定重组蛋白的纯度。本法所用的HPLC为Agilent 1260，色谱柱为TSKgel G3000SWXL(来自Tosoh Bioscience)，流动相为200mM的pH＝7.0磷酸缓冲液/异丙醇(v/v9:1)混合液，检测温度为25℃，流速为0.5mL/min，检测波长为280nm，目标蛋白上样量为50μg，分析时间为40分钟。
用质谱测量ADC DAR值：应用超高效液相色谱-质谱(UHPLC-MS)联用法测定ADC分子的DAR值。首先将待测ADC分子经过PNGase F处理，脱掉N糖修饰，再用二硫苏糖醇(Dithiothreitol，DTT)处理，37℃孵育1小时，还原成轻链和重链，然后用Thermo Vanquish UHPLC-Q Exactive Plus质谱系统进行分析，取2μg蛋白注射到Waters ACQUITY Protein  BEH分子排阻色谱柱，流动相为含0.1％甲酸、0.05％TFA、25％乙腈的水溶液，流速为0.2mL/min，分析时间30分钟，质谱仪为Thermo Q Exactive Plus，质谱主要参数分别为喷雾电压3.8kV，毛细管加热温度300℃，鞘气流速35arb，母离子扫描范围800-3000等，最后，应用质谱数据分析软件Biopharma Finder 4.1，通过Respect算法去卷积处理，分别计算出轻重链质谱峰的分子量信息和各组分的质谱响应信号，以此计算出待测ADC样品的DAR值。
根据以上通用制备方法，制备得到如下表10所示的抗体-药物偶联物或其药学上可接受的盐。
表10：抗体-药物偶联物、其SEC纯度和DAR值


实施例19：生物学活性及相关性质测试
以下测试例中的化合物均根据本公开上述实施例的方法制备获得。
测试例1.K562-GRE报告基因检测式(D-H)化合物的活性
为了产生亲本K562-GRE细胞系，将K562细胞以5×105个细胞/孔接种到含2mL完全培养基(RPM1640、10％FBS、青霉素-链霉素)的6孔培养皿(品牌Costar，货号3516)，在37℃、5％CO2下培养24小时。第二天，将3μg的pNL2.2[NLucP/MMTV/Hygro-NANO](Promega)和3μl的PLUS试剂(品牌Invitrogen，货号11514-015)稀释到150μL Opti-MEM(品牌Gibco，货号11058021)中并在室温下孵育5分钟。pNL2.2[NLucP/MMTV/Hygro-NANO]载体含有MMTV LTR(鼠乳腺肿瘤病毒长末端重复)，其响应于若干种核受体(如糖皮质激素受体和雄激素受体)的激活并驱动荧光素酶报告基因NanoLuc的转录。孵育之后，将稀释的DNA溶液与Lipofectamine LTX溶液(品牌Invitrogen，货号15338-100)(6μl Lipofectamine LTX+144μl Opti-MEM)以1:1比例混合并预孵育，并且在室温下孵育15分钟以形成DNA-Lipofectamine LTX复合物。孵育之后，将300μl的DNA-Lipofectamine复合物直接添加到细胞孔中。将K562细胞在37℃、5％CO2下转染24小时。转染之后，将细胞用3mL的PBS洗涤，并且用含有125μg/mL潮霉素B(品牌Invitrogen，货号10687010)的完全生长培养基选择生长两周。产生“K562-GRE(pNL2.2[NLucP/MMTV/Hygro-NANO])”细胞。
将K562-GRE(pNL2.2[NLucP/MMTV/Hygro-NANO)细胞在50μl的测定培养基(RPMI1640培养基,1％FBS,1％丙酮酸钠,1％MEM非必需氨基酸和1％青霉素-链霉素)中以每孔50000个细胞接种于96孔组织培养处理的白板(品牌Costar，货号3917)中，加入50μL(2×)用测定培养基5倍系列稀释的化合物处理细胞，每个化合物共8个浓度，化合物在体系中的终浓度为0.0000128μM-1μM。混匀后，放于37℃培养箱中培养24小时。在孵育24小时后，向细胞中加入等体积100μL的Nano-Glo荧光素酶测定系统(品牌Promega，货号N1120)，放于摇床500g反应10分钟，并用PHERAstar仪器进行读取化学发光。以抗体浓度的对数值为横坐标，对应的化学发光读值为纵坐标绘制抗体的结合曲线，四参数拟合(GraphPad Prism9)，计算EC50值，结果见表11。
测试例2.式(D-H)化合物与糖皮质激素受体GR的结合活性测定
使用Polarscreen Glucocorticoid Receptor Assay Kit,Red(品牌Thermo,货号A15898)试剂盒测定化合物与GR的结合活性。将待测化合物在96孔V底板(品牌Nunc，货号249944)中用DMSO进行10倍梯度稀释，最高浓度为100μM，共8个浓度。然后用试剂盒中提供的检测缓冲液Complete GR Screening buffer进一步50倍稀释化合物，并转移10μl稀释好的化合物至384微孔板(品牌Corning，货号4514)，将5μl Fluormone GS Red(4×浓度)添加到待测化合物中，然后加入5μl GR Full length的混合物(4×浓度)，实验做复孔。将384孔板室温避光孵育2个小时，用EnVision多功能酶标仪(厂家：Perkinelmer)检测荧光偏振(mP)。以化合物终浓度的log值为X轴，mP值为Y轴，将数据录入处理软件Graphpad Prism 9进行四参数拟合并计算EC50，结果见表11。
测试例3.式(D-H)化合物与雌激素受体ER的结合活性测定
使用TR-FRET ER Alpha Coactivator Assay kit(品牌Thermo,货号A15885)试剂盒测定化合物与ER的结合活性。将ER受体激动剂Estradiol(品牌Sigma，货号E8875-25)在96孔V底板中用DMSO进行10倍梯度稀释，最高浓度为100μM，共8个浓度。待测化合物用DMSO进行10倍梯度稀释，最高浓度为3000μM，共8个浓度。然后用试剂盒中提供的检测缓冲液Nuclear Receptor Buffer E(含5mM DTT)进一步50倍稀释化合物，并转移10μl稀释好的化合物加至96孔半区微孔板(品牌Corning，货号3694)，将5μl ER-LBD蛋白(4×浓度)添加到待测化合物中，然后加入5μl荧光素辅激活物肽和Tb标记的anti-GST抗体的混合物(4×浓度)，实验做复孔。将板室温孵育2个小时，用PHERAstar仪器读取荧光值(Excitation 337,Emission 520/495nm)，计算520:495比值。以化合物终浓度的log值为X轴，520/495比值为Y轴，将数据录入软件Graphpad Prism 9进行四参数拟合并计算EC50。结果见表11，待测化合物对雌激素受体结合活性较弱。
测试例4.式(D-H)化合物与雄激素受体AR的结合活性测定
使用TR-FRET Androgen Receptor Coactivator Assay kit(品牌Thermo,货号A15878)试剂盒测定化合物与AR的结合活性。将AR受体激动剂dihydrotestosterone(DHT)(品牌Sigma，货号D-073)在96孔V底板中用DMSO进行10倍梯度稀释，最高浓度为100μM，共8个浓度。待测化合物用DMSO进行10倍梯度稀释，最高浓度为3000μM，共8个浓度。用试剂盒中提供的检测缓冲液Nuclear Receptor Buffer A(含5mM DTT)进一步50倍稀释化合物，并转移10μl稀释好的化合物加至96孔半区微孔板，将5μl AR-LBD蛋白(4×浓度)添加到待测化合物中，然后加入5μl荧光素辅激活物肽和Tb标记的anti-GST抗体的混合物(4×浓度)，实验做复孔。将板室温孵育2个小时，用PHERAstar仪器读取荧光值(Excitation337,Emission 520/495nm)，计算520:495比值。以化合物终浓度的log值为X轴，520/495比值为Y轴，将数据录入处理软件Graphpad Prism 9进行四参数拟合并计算EC50。结果见表11，待测化合物对雄激素受体结合活性较弱。
测试例5.式(D-H)化合物与孕激素受体PR的结合活性测定
使用TR-FRET Progesterone Receptor Coactivator Assay kit(品牌Thermo,货号A15903)试剂盒测定化合物与PR的结合活性。将PR受体激动剂Progesterone(品牌Sigma，货号P0130)在96孔V底板中用DMSO进行10倍梯度稀释，最高浓度为100μM，共8个浓度。待测化合物用DMSO进行十倍梯度稀释，最高浓度为3000μM，共8个浓度，然后用试剂盒中提供的检测缓冲液Nuclear Receptor Buffer F(含5mM DTT)进一步50倍稀释化合物，并转移10μl稀释好的化合物加至96孔半区微孔板，将5μl PR-LBD蛋白(4×浓度)添加到检测化合物中，然后加入5μl荧光素辅激活物肽和Tb标记的anti-GST抗体的混合物(4×浓度)，实验做复孔。将板室温孵育2个小时，用PHERAstar仪器读取荧光值(Excitation 337,Emission 520/495nm)，计算520:495比值。以化合物终浓度的log值为X轴，520/495比值为Y轴，将数据录入处理软件Graphpad Prism 9进行四参数拟合并计算EC50。结果见表11，待测化合物与孕激素受体有一定的结合活性。
表11式(D-H)化合物体外活性结果

N/A表示未测试。
测试结果表明，测试化合物对于GR结合活性较强，对于PR结合活性弱，不结合ER和AR，有望减少作用于其它激素受体带来的副作用。
测试例6.式(D-H)化合物CHS小鼠模型药效试验
CHS小鼠模型构建：选择6-8周龄C57BL/6N雌性小鼠(北京维通利华实验动物技术有限公司)，将每只小鼠腹部的毛发用小动物电推剪剔除干净。用微量移液器吸取400μl致敏剂，将其均匀涂抹在小鼠腹部进行表皮致敏。涂抹后控制住小鼠3-5秒，并让溶剂从皮肤上尽量蒸发干净。致敏剂的具体配制方法为：称取FITC(异硫氰酸荧光素；sigma-Aldrich)粉末0.5g，用50mL丙酮(acetone；国药试剂)与50mL DBP(邻苯二甲酸二丁酯；sigma-Aldrich) 等比例混合后的溶剂充分溶解，得到FITC含量为0.5％的致敏剂。致敏后第6天，先用刻度盘式测厚仪测量小鼠右耳厚度作为基线值，然后用微量移液器吸取新鲜配制的致敏剂分别均匀涂抹在小鼠右耳的内外两侧进行表皮刺激，每侧涂抹10μL。刺激后24h，用刻度盘式测厚仪再次测量小鼠右耳厚度，得到小鼠右耳厚度变化值Δ耳朵厚度＝(刺激后24h右耳厚度)-(右耳厚度基线值)。
CHS模型小鼠治疗方案：将小鼠分组为空白组、阴性对照组、阳性对照组及实验组。其中实验组化合物001和化合物009给药剂量为3μg/只，阳性对照组为3μg/只的化合物a(根据专利WO2017210471化合物41报道的方法合成)，阴性对照组为空白溶媒(0.5％DMSO/PBS)。在右耳表皮刺激前1h，通过腹腔注射一次性给药。
试验结果见图2。经化合物001和009给药后，与阴性对照组相比，CHS小鼠的右耳厚度有显著降低；化合物001和009的药效均优于同等剂量的阳性参照化合物a。(*表示各实验组与阴性对照组相比统计学差异的显著性，统计方法为one-way ANOVA，**表示P值<0.01，****表示P值<0.0001)。
测试例7.抗TNFα抗体结合活性验证
抗人TNFα抗体结合活性
将实施例17-2制备得到的抗人TNFα抗体进行ELISA和Biacore检测，方法同实施例17-1。ELISA检测EC50值和Biacore检测结果见表12。
表12抗人TNFα抗体ELISA和Biacore结合活性
抗鼠TNFα抗体(001Mab)结合活性
鼠TNFα蛋白(Novoprotein，货号：CF09)4℃包被过夜，用0.05％Tween 20-PBS溶液漂洗3次，加入2％BSA封闭液，37℃封闭1.5h。用0.05％Tween 20-PBS溶液漂洗3次后加入倍比稀释的样品，37℃孵育1h。用0.05％Tween 20-PBS溶液漂洗3次后加入二抗HRP goat anti-Rat IgG(Jackson Immuno，货号：112-035-003)，37℃孵育1h。用0.05％Tween 20-PBS溶液漂洗3次后加入TMB溶液(Seracare，货号：5120-0077)，室温反应10min后加入1M盐酸终止反应，酶标仪450nm波长读板。001Mab与鼠TNFα蛋白结合EC50值为1.391nM。
测试例8.抗体-药物偶联物与表达TNFα细胞的结合活性测试
将编码TACE抗性的人跨膜TNFα氨基酸序列(在SEQ ID NO.54序列基础上缺少氨基酸77-88)的核苷酸序列克隆到pcDNA5/FRT载体(Thermofisher)，得到pcDNA5/FRT-human TNFαFL(77-88del)质粒。将该质粒与pOG44质粒共转染Flp-In-CHO宿主细胞(Thermofisher)。转染24小时后，洗涤细胞并用含有600μg/mL潮霉素B(Invitrogen，10687010)的完全生长培养基选择两周，产生Flp-In-CHO-human TNFαFL(77-88del)细胞。
将Flp-In-CHO-human TNFαFL(77-88del)细胞消化后计数，以1.5×105个/孔铺于加入96孔板(Corning，3795)。加入梯度稀释的待测样品，于4℃孵育1小时。用PBS洗两遍后，加入Alexa Fluor-647Goat anti-human Fc二抗(JacksonImmuno，109-605-098)，于4℃孵育1小时。用PBS洗两遍后，重悬细胞，用流式细胞仪(BD FACSCantoTM II)分析平均荧光强度(MFI)。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值。结果见表13，不同的抗TNFα抗体-药物偶联物与表达人跨膜TNFα的细胞均有较强的结合活性。
表13抗TNFα抗体-药物偶联物与Flp-In-CHO-human TNFαFL(77-88del)细胞的结合活性

SEQ ID NO.54
测试例9.K562-GRE报告基因检测抗TNFα抗体-药物偶联物的活性
将编码TACE抗性的人跨膜TNFα氨基酸序列(在SEQ ID NO.54基础上缺少氨基酸77-88)的核苷酸序列克隆到pLVX-IRES-Puro慢病毒载体，并在HEK293T细胞中制备病毒颗粒。为了构建K562-human TNFα-GRE-Luc报告基因细胞系，将测试例1构建的K562-GRE细胞以5×105个/孔接种到含1.5mL完全生长培养基的6孔培养皿(Costar，3516)中，在37℃、5％CO2下持续培养24小时。第二天，将提前制备好的人TNFα慢病毒颗粒溶液1.5ml加入细胞皿中，并补以终浓度8μg/ml polybrene(Santa Cruz，sc-134220)进行慢病毒感染。将K562-GRE细胞在37℃、5％CO2下感染24小时。孵育之后，将细胞用3mL的PBS洗涤，并且用含有125μg/mL潮霉素B(Invitrogen，10687-010)和1μg/mL嘌呤霉素(Gibco，A1113802)的完全生长培养基选择两周，产生K562-human TNFα-GRE-Luc报告基因细胞。
将K562-human TNFα-GRE-Luc细胞重悬于测定培养基(RPMI1640，10％碳吸附FBS，1％Penicillin-Streptomycin)，以5×104个/孔(50μl)接种于96孔板(Corning，3610)。用上述测定培养基稀释抗TNFα抗体-药物偶联物，加入96孔板中，每孔50μl，在37℃，5％CO2下孵育24小时。在孵育24小时后，用萤光素酶报告基因检测系统(Promega，N1120)处理10分钟，并用Envision仪器(PerkinElmer)检测发光强度。使用Graphpad Prism软件对数据进行四参数曲线拟合分析，得到EC50值。结果见表14和图3。
表14抗人TNFα抗体-药物偶联物在GRE报告基因测定中的活性
将上述编码TACE抗性的人跨膜TNFα氨基酸序列的核苷酸序列替换为编码小鼠TNFα氨基酸序列(SEQ ID NO.67)的核苷酸序列，并按照测试例9上述相同方法构建产生K562-mouse TNFα-GRE-Luc报告基因细胞。采用与上述相同的报告基因细胞培养和检测方法，检测抗鼠TNFα抗体-药物偶联物在GRE报告基因测定中的活性，结果见表15和图4。
表15抗鼠TNFα抗体-药物偶联物在GRE报告基因测定中的活性
SEQ ID NO.67
测试例10.抗体-药物偶联物在LPS刺激的人单核细胞细胞因子释放模型中的活性抑制测试
将冻存的原代人外周血单核细胞(PBMC，Sailybio，XFB-HP025B)解冻复苏。使用单核细胞分选试剂盒(Stemcell，19058)从PBMC细胞中分选单核细胞并将单核细胞重悬于补充有2％人AB血清(GEMINI，100-512)和1％青霉素-链霉素(Gibco，15140-122)的RPMI1640培养基(Gibco，72400-047)中，铺板在细胞测定板(Corning，7007)中，每孔1×105细胞。将细胞与不同浓度的抗人TNFα抗体-药物偶联物在37℃，5％CO2培养箱预孵育2小时。然后加入终浓度为100ng/mL的脂多糖LPS(Sigma，L4516)刺激细胞，在37℃，5％CO2培养箱孵育24小时。第二天将板子以350g，离心5min，小心吸取上清50μL转移到另外一块干净的96孔板中，检测IL-6(达科为，1110603)和IL-1β(达科为，1110123)浓度。使用GraphPad Prism软件对剂量响应数据进行四参数非线性回归拟合曲线，并计算IC50值。如表16显示，抗人TNFα抗体-药物偶联物可明显抑制LPS刺激活化的单核细胞促炎细胞因子IL-6和IL-1β的释放。
表16抗人TNFα抗体-药物偶联物在LPS刺激的人单核细胞细胞因子释放测定中的抑制活性
测试例11.抗体-药物偶联物在LPS刺激的小鼠PBMC细胞因子释放模型中的活性抑制测试
取6-8周龄雌性C57BL/6小鼠(上海灵畅生物科技有限公司)的抗凝血，用FICOLL-PAQUE(Cytiva，17544602)分离出小鼠的PBMC细胞，重悬于补充有10％FBS(Gibco，10091-148)和1％青霉素-链霉素(Gibco，15140-122)的RPMI1640培养基(Gibco，72400-047)中，并且铺板在细胞测定板(Corning，7007)中，每孔4×105个细胞。将细胞与不同浓度的抗鼠TNFα抗体-药物偶联物在37℃，5％CO2培养箱孵育4小时。然后加入终浓度为1000ng/mL的脂多糖LPS(Sigma，L4516)刺激细胞，在37℃、5％CO2培养箱孵育40小时后，将板子以350g，离心5min，小心吸取上清100μL转移到另外一块干净的96孔板中，用于检测IL-6浓度(达科为，1210603)。使用GraphPad Prism软件对剂量响应数据进行四参数非线性回归拟合曲线，并计算IC50值。结果如表17和图5显示，抗鼠TNFα抗体-药物偶联物可明显抑制LPS刺激活化的小鼠PBMC促炎细胞因子IL-6的释放。
表17抗鼠TNFα-药物偶联物在LPS刺激的小鼠PBMC细胞因子释放测定中的抑制活性
测试例12：抗体-药物偶联物CHS小鼠模型药效试验
CHS小鼠模型的构建方法参见测试例6。
CHS模型小鼠治疗方案：将小鼠分组为正常对照组、阴性对照组和给药组。阴性对照组为空白溶媒(0.5％DMSO/PBS)，给药组为抗鼠TNFα抗体001Mab(BioXcell，BE0058)、ADC-L1-001-5和ADC-L1-009-5，给药剂量为10mg/kg。在右耳表皮刺激前1h，通过腹腔注射一次性给药。
实验结果如图6所示，给药后CHS小鼠的右耳厚度有显著降低，与阴性对照组相比均有统计学差异(P值<0.0001，统计方法为one-way ANOVA)。抗鼠TNFα-药物偶联物(ADC-L1-001-5和ADC-L1-009-5)的药效明显优于抗鼠TNFα抗体001Mab组(*表示ADC组与001Mab组相比统计学差异的显著性，统计方法为one-way ANOVA，*表示P值<0.05，**表示P值<0.01)。
测试例13：DBA/1小鼠胶原抗体诱导关节炎(CAIA)药效模型试验
取6-8周龄DBA/1雄性小鼠(北京维通利华实验动物技术有限公司)，在实验开始之日(第0天)，将II型胶原的5种单克隆抗体(Arthrogen-CIA 5-Clone Cocktail Kit；Chrondex)的混合液经腹腔注射到DBA/1小鼠体内，每只小鼠注射150μl。抗体混合液的浓度为10mg/ml，即每只小鼠体内注射抗体混合液总量为1.5mg；II型胶原抗体诱导后第3天，小鼠腹腔注射100μl浓度为0.5mg/ml的脂多糖(LPS)，即每只小鼠体内注射LPS的总量为50μg。
小鼠在第5天左右出现疾病症状，在第7-8天时疾病达到高峰期。根据动物临床症状进行评分。0：无红斑和红肿；1：近跗骨附近或踝关节或跖骨出现红斑或轻度红肿，1个脚趾红肿；2：踝关节和跖骨轻微红斑和肿胀，或超过两个脚趾红肿；3：踝、腕关节和跖骨中度红斑和肿胀；4：踝、腕关节，跖骨和脚趾全部严重红肿。
CAIA小鼠治疗方案：根据体重将小鼠随机分组为正常对照组、阴性对照组及给药组。给药组为抗鼠TNFα抗体001Mab、ADC-L1-001-5和ADC-L1-009-5，给药剂量为10mg/kg；阴性对照组为空白溶媒(PBS)。分别于胶原抗体诱导后的第3天、第6天、第10天、第13天腹腔注射给药，阴性对照组在相同时间点腹腔注射等体积的溶媒。每天记录小鼠临床症状评分及体重。
实验结果如图7所示，给药组CAIA小鼠的关节炎临床症状均有显著改善，临床评分与阴性对照组相比有统计学差异(*表示各实验组与阴性对照组相比统计学差异的显著性，统计方法为two-way ANOVA，**表示P值<0.01，****表示P值<0.0001)。抗鼠TNFα-ADC的药效明显优于抗鼠TNFα抗体组。
测试例14：TNFα人源化小鼠胶原抗体诱导关节炎(CAIA)药效模型试验
选择6-8周龄TNFα人源化C57BL/6雄性小鼠(B-hTNFα，百奥赛图)，在实验开始之日(第0天)，将II型胶原的5种单克隆抗体(Arthrogen-CIA 5-Clone Cocktail Kit；Chrondex)的混合液经腹腔注射到B-hTNFα-C57BL/6小鼠体内，每只小鼠注射500μl。抗体混合液的浓度为10mg/ml，即每只小鼠体内注射抗体混合液总量为5mg；II型胶原抗体诱导后第3天，小鼠腹腔注射100μl浓度为0.5mg/ml的脂多糖(LPS)，即每只小鼠体内注射LPS的总量为50μg。
小鼠在第5天左右出现疾病症状，在第7-9天时疾病达到高峰期。根据动物临床症状进行评分。0：无红斑和红肿；1：近跗骨附近或踝关节或跖骨出现红斑或轻度红肿，1个脚趾红肿；2：踝关节和跖骨轻微红斑和肿胀，或超过两个脚趾红肿；3：踝、腕关节和跖骨中度红斑和肿胀；4：踝、腕关节，跖骨和脚趾全部严重红肿。
CAIA小鼠治疗方案：
根据体重将小鼠随机分组为正常对照组、阴性对照组及给药组。给药组为Adalimumab和ADC-L1-009-6，给药剂量为10mg/kg；阴性对照组为空白溶媒(PBS)。分别于胶原抗体诱导后的第3天、第6天、第10天、第13天腹腔注射给药，阴性对照组在相同时间点腹腔注射等体积的溶媒。每天记录小鼠临床症状评分及体重。
试验结果如图8所示，给药组CAIA小鼠的关节炎临床症状均有显著改善，临床评分与阴性对照组相比有统计学差异；ADC-L1-009-6的药效优于Adalimumab单抗。(*表示各实验组与阴性对照组相比统计学差异的显著性，统计方法为two-way ANOVA，****表示P值<0.0001)
测试例15：抗TNFα抗体-药物偶联物的血浆稳定性测试
将ADC在人血浆(澳能生物，PB021F-C)、小鼠血浆和猴血浆中稀释(终浓度为100μg/ml)，置于37℃培养箱中孵育。将孵育当天标记为第0天，随后分别在第7天和14天取出样品，进行游离小分子的检测。
取20μL样品，加入300μL内标工作液(乙腈配置)，涡旋混匀5分钟，离心5分钟(14000rpm)，4μL上清液LC-MS/MS(API 6500+)进样分析，结果见表18。表18结果表明ADC在人、小鼠血浆和猴血浆中均较为稳定，游离小分子的释放率不超过0.5％。
表18抗TNFα抗体-药物偶联物的血浆稳定性

BQL：低于定量限(<1ng/ml)。
测试例16：酶联免疫反应(ELISA)检测抗IL-4R抗体-药物偶联物与IL-4R蛋白结合水平
将实施例18制备得到的抗人IL-4R抗体-药物偶联物进行ELISA检测，将人IL-4R蛋白(Acro Biosystems，货号：ILR-H5221)用PBS(购自Hyclone，货号SH30256.01)稀释到终浓度2μg/mL，以50μL每孔加到96孔ELISA板(购自Corning，货号9018)中，37℃包被1h，弃上清，加入封闭液，即含有5％(w/w)脱脂奶粉(购自Sangon，货号A600669-0250)的PBS缓冲液，37℃封闭1小时。弃封闭液，用含0.05％Tween 20(购自Sangon，货号A100777-0500)PBS(下称PBST)洗板3次。每孔加入稀释的抗人IL-4R抗体-药物偶联物进50μL，浓度为100nM起始，3倍梯度稀释，稀释液为含有1％(w/w)BSA(购自Sangon，货号A500023-0100)的PBS，37℃孵育1小时后，用PBST洗板3次。每孔加入稀释的辣根过氧化物酶(HRP)标记的二抗(购自Bethyl，货号A80-304P)50μL，稀释液为1％BSA-PBS，室温孵育1小时后，用PBST洗板5次。每孔加入50μL TMB(购自KPL，货号5120-0077)底物，室温孵育5分钟，每孔加入50μL终止液(1M HCl)。用酶标仪(Ensight-HH3400，购自PerkinElmer)读取OD450nm数值。结合EC50值见表19，待测抗体-药物偶联物与人IL-4R蛋白的结合能力与其各自所来源的抗体相当。
表19抗体-药物偶联物与人IL-4R蛋白ELISA结合实验
测试例17:流式细胞实验(FACS)检测抗IL-4R抗体-药物偶联物与Ramos细胞的结合水平
将Ramos细胞在T-75细胞培养瓶(购自Corning，货号430720)中扩大培养，室温条件下1000rpm离心5分钟收集细胞，用PBS(购自Hyclone，货号SH30256.01)缓冲液洗涤2次，将细胞用PBS重悬并计数。按每孔1×105个细胞，50μL加入到U型底96孔FACS板中。用含有1％(w/w)BSA(购自Sangon，货号A500023-0100)的PBS梯度稀释抗人IL-4R抗体-药物偶联物，浓度为100nM起始，10倍梯度稀释，按每孔50μL加入细胞中混匀，4℃ 孵育1小时。每孔加入200μL PBS缓冲液，1500rpm离心5分钟洗涤，重复两次。弃上清后，每孔加入100μL稀释的荧光标记二抗(购自Jackson Immuno Research，货号109-605-098)，4℃孵育1小时。PBS离心洗涤2次，100μL每孔的PBS悬浮细胞。用FACS仪检测和分析结果。结果如表20所示，待测分子可结合Ramos细胞表面的IL-4R的能力与其各自所来源的单抗相当，连接小分子对单抗在细胞上的结合没有影响。表20中数据MFI为所测细胞群的最大平均荧光强度值。
表20抗体-药物偶联物与Ramos细胞的结合水平
测试例18：检测抗IL-4R抗体-药物偶联物在Ramos细胞上的内吞水平
将Ramos细胞在T-75细胞培养瓶中扩大培养，室温条件下1000rpm离心5分钟收集细胞，用PBS缓冲液洗涤2次。按每孔4×105个细胞，100μL加入到U型底96孔FACS反应板中，4℃备用。配置2％FBS(w/w)PBS稀释待测抗IL-4R抗体-药物偶联物，按每孔100μL加入细胞混匀，终浓度为100nM，4℃孵育1小时。用预冷的PBS离心洗涤两次，200μL完全培养基重悬后，等体积分到两个U型底96孔FACS反应板中。分别放置在4℃，37℃孵育2小时。板子取出后，加100μL预冷的PBS终止内吞。1500rpm离心5分钟，洗涤两次。弃上清，每孔加入100μL稀释的荧光标记二抗(购自Jackson Immuno Research，货号109-605-098)，4℃孵育1小时。预冷的PBS洗涤两次，用100μL的PBS悬浮细胞，FACS检测和分析结果。
内吞率(％)＝(MFI 4℃-MFI 37℃)/MFI 4℃×100％。结果如表21所示，待测分子在Ramos细胞上的内吞率与其各自所来源的单抗相当，且连接小分子对内吞率没有显著影响。
表21抗体-药物偶联物在Ramos细胞上的内吞

测试例19：K562-GRE报告基因检测抗IL-4R抗体-药物偶联物的活性测定
将编码TACE抗性的人跨膜IL-4R氨基酸序列(SEQ ID NO.66)的核苷酸序列克隆到pLVX-IRES-Puro慢病毒载体，并在HEK293T细胞中制备病毒颗粒。为了构建K562-human IL-4R-GRE-Luc报告基因细胞系，将测试例1构建的K562-GRE细胞以5×105个/孔接种到含1.5mL完全生长培养基的6孔培养皿(Costar，3516)中，在37℃、5％CO2下持续培养24小时。第二天，将提前制备好的人IL-4R慢病毒颗粒溶液1.5ml加入细胞皿中，并补以终浓度8(g/ml polybrene(Santa Cruz，sc-134220)进行慢病毒感染。将K562-GRE细胞在37℃、5％CO2下感染24小时。孵育之后，将细胞用3mL的PBS洗涤，并且用含有125μg/mL潮霉素B(Invitrogen，10687-010)和1μg/mL puro(Gibco，A1113802)的完全生长培养基选择两周，产生K562-human IL-4R-GRE-Luc报告基因细胞。
IL-4R氨基酸序列(SEQ ID NO.66)：
收集K562-human IL4R-GRE-Luc细胞，以1×107/ml的密度重悬在测定培养基中(RPM1640+1％碳吸附胎牛血清+1％丙酮酸钠+1％非必需氨基酸+1％青霉素/链霉素)中。将细胞以5×104个/孔(50μl)接种于96孔板(Costar，3917)。将待测抗IL-4R抗体-药物偶 联物和同型对照偶联物ADC-L1-009-8分别从40nM开始，以5倍比梯度稀释6个点，稀释液为上述测定培养基。将稀释好的药物，以50μl每孔加入到96孔细胞板中，混匀后，放置37℃、5％(v/v)CO2培养箱中孵育24小时。孵育结束后，将96孔板置于室温平衡5min，并向孔板中加入100μl/孔的Nano-Glo luciferase检测试剂(Promega，N1110)，室温振荡10min后，使用PE Envision仪器-US Lumi模块检测孔板中荧光信号值。以药物浓度为横坐标，荧光信号值RLU为纵坐标绘制药物的诱导曲线，四参数拟合(GraphPad Prism9)，计算诱导曲线的EC50及最大发光强度，结果见表22。
表22抗IL-4R抗体-药物偶联物在GRE报告基因测定中的活性
测试例20：抗IL-4R抗体-药物偶联物对于人原代CD4+T细胞增殖的抑制作用
利用FACS检测IL-4R抗体-药物偶联物对人原代CD4+T细胞增殖的抑制活性，方法如下：实验前一天复苏人PBMC细胞(stemexpress)，300g离心10分钟后，将细胞重悬于完全培养基中(RPMI1640+10％FBS+1×β-巯基乙醇+1％Pen/Srep)静置过夜。次日，收集静置过夜的PBMC细胞，用细胞计数仪进行细胞计数，并用T细胞分离试剂盒(Stemcell，货号：17951)分离人原代T细胞，并计数。首先，取出适量数目的T细胞，加入2ml PBS洗涤细胞两遍，350g离心5min。离心弃上清后，加入预先配制好的CellTrace Violet(Invitrogen，C34557A)工作液，使其终浓度为5μM。将加入CellTrace Violet染液的T细胞悬液吹匀混合，置于37℃培养箱孵育10分钟，期间轻轻摇晃混匀一次。CTV标记结束后，向标记的细胞悬液中加入5倍体积的完全培养基终止反应，室温放置5分钟后，350g离心5分钟，弃上清，用完全培养基重悬细胞并计数。根据计数结果，用完全培养基调整细胞密度为1×106/mL，待用。接着，用完全培养基稀释药物至200nM的工作液(4×终浓度)，待用。其次，配制ImmunoCult Human CD3/CD28 T Cell Activator(Stemcell，货号：10971)工作液，按1μL/0.1M细胞/孔取出相应体积的anti-CD3/CD28 immunocult加入完全培养基中混匀，待用。
设置药物组别如下：(1)Anti-Hel-hIgG4(同型对照)，(2)Dupilumab，(3)待测抗IL-4R抗体-药物偶联物单药组。以上(1)-(3)组均给予anti-CD3/CD28 immunocult刺激。按platemap，于96-U孔板(Corning，货号：3799)中，依次加入CTV标记的T细胞/100μL/孔(1×105个/孔)，药物工作液50μL/孔(4×终浓度)，anti-CD3/CD28 immunocult 50μL/孔，体系终体积为200μL/孔。将孔板置于振荡器上300rpm振荡1min后，放置于37℃，5％CO2培养箱中，将药物和细胞共孵育96h。孵育结束后，将96-U孔板中的细胞轻吹混匀，转移至96-V孔板，350g离心5分钟，弃上清。向孔板加入200μL/孔PBS洗细胞两遍后，弃上清。用PBS按1:1000比例配制细胞死活染料Live/Dead(Invitrogen，货号：L10119)工作液，并按100μL/孔加入细胞离心沉淀中吹匀，室温孵育20分钟。Live/Dead染料孵育结束后，加入150μL/孔的染色缓冲液(PBS+2％FBS)洗细胞两遍，弃上清。离心期间，配制细胞表面染色工作液，即向染色缓冲液中加入1μL/0.1M/孔FITC mouse anti-human CD4抗体(BD Bioscience，货号：555346)。向细胞离心沉淀中加入100μL/孔FITC mouse anti-human CD4抗体染色工作液，混匀，4℃避光孵育30分钟。染色结束后，向孔板加入150μL/孔的染色缓冲液洗细胞两遍后，弃上清，再用150μL/孔的染色缓冲液重悬细胞，用流式细胞仪检测细胞样品的增殖比例。通过FACS读取活的CD4+T细胞的增殖比例(即CTV阴性比例)，来评价药物对人原代CD4+T细胞增殖的抑制作用。结果如图9所示，相较于同型对照Anti-Hel-hIgG4组，Dupilumab单抗组和待测分子组均可抑制人CD4+T细胞增殖。且相较于Dupilumab组，待测分子组对人CD4+T细胞增殖的抑制作用更强，并具有统计学差异。
测试例21：抗IL-4R抗体-药物偶联物对特应性皮炎小鼠模型药理药效学作用
疾病模型构建：6-7周龄C57BL/6-hIL-4/h-IL-4RA双人源化小鼠(百奥赛图提供)。第0 天，正常对照组小鼠的背部和右耳，各均匀涂抹25μL的丙酮和橄榄油混合物(4:1)；其余组小鼠的背部和右耳，各均匀涂抹25μL 0.8％的噁唑酮(OXA)致敏(溶媒为丙酮和橄榄油混合物4:1)。在第7天到第25天，每周激发3次，正常对照组小鼠的背部和右耳，各均匀涂抹25μL的丙酮和橄榄油混合物(4:1)激发；造模组小鼠的背部和右耳，各均匀涂抹25μL0.4％的OXA(溶媒同前)溶液激发。
药物干预设计：从初次OXA致敏后第6天开始给药，正常对照小鼠单独成组；将造模组小鼠按照耳厚，平均分成3组，每组8只，分别为溶媒对照组(PBS)，Dupilumab-10mpk组，待测抗IL-4R抗体-药物偶联物-10mpk组。各组小鼠腹腔给药，每周2次。
第26天结束体内药效实验，并取血清、右耳皮肤组织和背部皮肤组织用于后续的检测。在整个体内实验期间，每次OXA诱导前及实验终点，对各组小鼠进行耳厚测量。如图10所示，与溶媒对照组小鼠相比，Dupilumab和抗IL-4R抗体-药物偶联物均能够明显抑制小鼠耳朵增厚，且同等剂量抗IL-4R抗体-药物偶联物抑制效果更强；同时，如图11所示两种药物也都能抑制小鼠体内IgE的产生。
组织病理学评价：取浸泡于固定液中的耳朵及背部皮肤，固定后石蜡包埋，以5μm厚度切片，脱腊和梯度水合后进行H&E染色。染色后，扫片机扫描病理切片，并由独立第二人盲法病理评分。H&E染色病理评分标准，表皮：基质细胞增生(0.5-2分)，糜烂(0.5-2分)，棘突出现(0.5-2分)，角化过度伴角化不全(0.5-2分)；真皮及皮下：混合炎细胞浸润(0.5-2分)，周围嗜酸性粒细胞浸润(0.5-2分)，总计12分。如图12-13所示，Dupilumab-10mg/kg及抗IL-4R抗体-药物偶联物-10mg/kg给药显著改善特应性皮炎小鼠模型造模部位耳片和皮肤组织病理学损伤及炎症程度，且同等剂量抗IL-4R抗体-药物偶联物的改善效果优于Dupilumab。
使用全自动免疫组化仪对小鼠耳片和背部皮肤组织切片对CD4+T细胞进行免疫组织化学染色(IHC)，每个耳朵和皮肤组织IHC切片随机选择4个200×视野，统计每个视野下阳性CD4细胞数量，计算每个视野的平均数。如图14-15所示，抗IL-4R抗体-药物偶联物给药显著抑制耳片和皮肤组织中致炎性CD4+T细胞浸润，而同剂量Dupilumab作用微弱。
测试例22：抗IL-4R抗体-药物偶联物的血浆稳定性测试
将ADC在人血浆(澳能生物，PB021F-C)中稀释(终浓度为100μg/ml)，置于37℃培养箱中孵育。将孵育当天标记为第0天，随后分别在第7天，14天和21天取出样品，进行游离小分子的检测。
取20μL样品，加入300μL内标工作液(乙腈配置)，涡旋混匀5分钟，离心5分钟(14000rpm)，4μL上清液LC-MS/MS(API 6500+)进样分析，结果见表23，结果表明ADC在人血浆中均较为稳定，游离小分子的释放率不超过0.5％。
表23抗IL-4R抗体-药物偶联物的血浆稳定性

BQL：低于定量限(<1ng/ml)。
测试例23：抗TNFα抗体-药物偶联物细胞孵育实验
抗TNFα抗体-药物偶联物与细胞表面的TNFα结合后，被内吞进入细胞。细胞溶酶体中的酶将ADC裂解后释放出激素小分子，从而发挥激素小分子的抗炎作用。为确认ADC内吞进入细胞后释放的小分子形式及酶解效率，进行了细胞孵育实验。
将测试例9中构建得到的K562-human TNFα-GRE-Luc细胞(1×104/孔)接种于96孔板(Corning，3599)，加入15μg/ml的ADC后，置于37℃细胞培养箱孵育。将细胞与ADC 混合当时的样品标记为0h，随后分别在24h和48h取出样品，加入乙腈(100μL/孔)吹打混匀。取20μL上述样品，加入300μL内标工作液(乙腈配置)，涡旋混匀5分钟，离心5分钟(14000rpm)，4μL上清液LC-MS/MS(API 6500+)进样分析，结果见表24。检测结果表明，检测到的小分子形式为化合物009，未检测到带磷酸基团的小分子化合物。本公开的ADC-L1-009-6进入细胞后，分子中的磷酸酯基转化为羟基，发挥药理活性的小分子化合物为化合物009。
表24抗TNFα抗体-药物偶联物在细胞内的小分子化合物释放浓度

BQL：低于定量限(<1ng/ml)
测试例24:抗IL-4R抗体-药物偶联物对人原代T、B细胞共培养体系中B细胞上的CD23表达的抑制作用
从液氮罐中取出PBMC冻存细胞(Stemexpress，Cat:PBMNC050C)，放入37℃水浴锅中轻柔震荡，使细胞溶解。将细胞悬液转移到含8ml预热1640培养基(Gibco,Cat:72400047)的离心管中离心8min(离心条件为300g，25℃)，弃上清。加入8ml的EasysepTM buffer缓冲液(Stemcell，Cat：20144)重悬细胞，用细胞计数仪(Beckman Coulter，Vi-Cell)计数，离心8min(离心条件为300g，25℃)后弃上清。根据计数结果，用EasysepTM buffer缓冲液重悬细胞，调整密度至5×107个细胞/mL。分别使用人T细胞分选试剂盒(Stemcell，Cat：17951)和人B细胞分选试剂盒(Stemcell，Cat：17954)分选人原代T细胞、B细胞，细胞计数后，待用。
配制如下试剂：1)调整人原代T细胞为1×106个细胞/mL，人原代B细胞为0.5×106个细胞/mL；2)用1640完全培养基配制抗IL-4R抗体-药物偶联物工作液(4倍终浓度)，使药物终浓度为500nM,100nM和20nM；3)配制金黄色葡萄球菌肠毒素A+人重组白介素-4+抗人CD3/CD28磁珠-混合工作液(4倍终浓度)，在1640完全培养基中加入人T细胞活化剂抗CD3/CD28磁珠(6250个磁珠/50μL/孔，Gibco，Cat：11131D)，人重组白介素-4(终浓度为10ng/mL，Peprotech，Cat：200-04)和-金黄色葡萄球菌肠毒素A(终浓度为1ng/mL，Toxin technology，Cat：AT101)，混匀待用。在96孔-U型板(Corning，Cat：3799)中依次加入人原代T细胞(50000个细胞/50μL/孔)，人原代B细胞(25000个细胞/50μL/孔)及抗IL-4R抗体-药物偶联物(50μL/孔)，轻吹混匀后，室温孵育10min。再加入金黄色葡萄球菌肠毒素A+人重组白介素-4+抗人CD3/CD28磁珠混合工作液，于37℃，5％CO2培养3天。孵育时间结束后，取出孔板将细胞混匀，离心5min(离心条件为350g，25℃)后吸取上清，用ELISA方法检测IL-13的分泌水平；细胞沉淀用FACS方法检测活的B细胞上CD23的表达情况。
上述人原代T、B细胞共培养的细胞沉淀用200μL/孔PBS重悬，离心5min(离心条件为350g，25℃)后弃上清。使用可固定近红外死活细胞染色试剂盒染料(Thermofisher，Cat：L10119)室温孵育10min后，加入100μL/孔的染色缓冲液(2％FBS+PBS)，离心5min(离心条件为350g，25℃)后弃上清。加入人Fc受体阻断剂(Biolegend，Cat：422302)，4℃孵育10min后加入2μL/孔PE抗人CD19抗体(Biolegend，Cat:302208)和2μL/孔APC抗人CD23抗体(Biolegend，Cat:338514)，4℃避光孵育30min，进行细胞表面染色。染色结束后，向细胞中加入100μL/孔的染色缓冲液，离心5min(离心条件为350g，25℃)后弃上清，用120μL/孔的染色缓冲液重悬细胞，使用流式细胞仪(BD,Canto II)检测活的CD19阳性细胞中CD23的阳性比例，并用Flowjo V10和Graphpad Prism 9软件分析数据。CD23的阳性比例越高，代表B细胞上CD23表达水平越高，即抗IL-4R抗体-药物偶联物的抑制活 性越弱。
表25 T-B系统中CD23阳性B细胞的百分比

Claims (37)

  1. 一种配体-药物偶联物或其药学上可接受的盐，其结构通式为Pc-(L-D)n，
    其中：
    Pc为配体单元；
    L为连接子单元；
    D为以下式(D-I)所示的药物单元：
    其中，
    R1、R2各自独立地选自H、CH3或卤素；
    环A选自苯基、5-10元杂芳基或C3-C10环烷基，所述苯基、5-10元杂芳基或C3-C10环烷基任选被一个或多个R1a取代；
    X选自O、S、C1-C3亚烷基-O、C1-C3亚烷基-S、NR6或C(R7)(R8)；
    R6选自H、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
    R7、R8各自独立地选自H、卤素、CN、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，或者R7、R8与它们连接的原子共同形成C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
    R3选自H、OH或NHR9，R4、R5与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代；或者，R5选自H、OH或NHR9，R3、R4与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代；
    R9选自H、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rd取代；
    R13、R14各自独立地选自H、卤素、CN、OH、NH2、O(C1-C3烷基)或C1-C6烷基；
    每一个R4a独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rd取代；
    每一个Rd独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；
    R10选自OH、SH、O(C1-C6烷基)、S(C1-C6烷基)、O-C(＝O)-(C1-C6烷基)或所述O(C1-C6烷基)、S(C1-C6烷基)、O-C(＝O)-(C1-C6烷基)任选被一个或多个卤素或CN取代；
    R11、R12各自独立地选自H或C1-C6烷基；
    每一个R1a独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rb取代；
    每一个Rb独立地选自卤素、CN、＝O、OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基，所述OH、NH2、C1-C6烷基、C3-C6环烷基或4-7元杂环基任选被一个或多个Rc取代；
    每一个Rc独立地选自卤素、CN、＝O、C1-C3烷基、OH、O(C1-C3烷基)、NH2、NH(C1-C3烷基)或N(C1-C3烷基)2；并且，
    n为1～16；
    其中，连接子单元L与式(D-I)化合物R3、R4、R5、R13、R14中存在的-OH、-NH2或-NH-基团中的任意一个共价连接。
  2. 根据权利要求1所述的配体-药物偶联物或其药学上可接受的盐，其中R1、R2各自独立地选自H或F。
  3. 根据权利要求1或2所述的配体-药物偶联物或其药学上可接受的盐，其中环A选自苯基或5-6元杂芳基，所述苯基或5-6元杂芳基任选被R1a取代。
  4. 根据权利要求1-3任一项所述的配体-药物偶联物或其药学上可接受的盐，其中X选自O、S、CH2O、CH2S、CH2、CF2、CHCH3或
  5. 根据权利要求1-4任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R10选自OH、SH、OCH2F、O(C＝O)CH3、
  6. 根据权利要求1-5任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R3选自H、OH或NH2，R4、R5与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
  7. 根据权利要求1-5任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R5选自H、OH或NH2，R3、R4与它们连接的原子共同形成C5-C6环烯基、5-6元杂环基或5-6元杂芳基，所述C5-C6环烯基、5-6元杂环基或5-6元杂芳基任选被一个或多个R4a取代。
  8. 根据权利要求1-5任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R3选自H、OH或NH2，R4、R5与它们连接的原子共同形成 所述任选被一个或多个R4a取代。
  9. 根据权利要求1-5任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R5选自H、OH或NH2，R3、R4与它们连接的原子共同形成 所述 任选被一个或多个R4a取代。
  10. 根据权利要求1-9任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R4a选自卤素、CN、＝O、OH、NH2、C1-C6烷基或C3-C6环烷基。
  11. 根据权利要求1-10任一项所述的配体-药物偶联物或其药学上可接受的盐，其中R13、R14各自独立地选自H或NH2。
  12. 根据权利要求1-5任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，结构单元选自
  13. 根据权利要求1所述的配体-药物偶联物或其药学上可接受的盐，其中式(D-I)所示化合物选自以下式(D-II)所示化合物：
    其中，
    R1、R2各自独立地选自H或F；
    R10选自OH或
  14. 根据权利要求1所述的配体-药物偶联物或其药学上可接受的盐，其中式(D-I)所示化合物选自以下所示化合物：


  15. 根据权利要求1-14任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，连接子单元L选自其a端与配体单元Pc共价连接，b端与药物单元D共价连接，所述L1为由1至8个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代；优选地，所述L1为二肽残基，构成二肽残基中的一个氨基酸为甘氨酸；更优选地，所述L1选自Gly-Lys或Gly-Glu的二肽残基。
  16. 根据权利要求1-14任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，连接子单元L选自其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
  17. 根据权利要求1-14任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，连接子单元L选自其a端与配体单元Pc共价连接，b端与药物单元D共价连接。
  18. 根据权利要求1所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体-药物偶联物或其药学上可接受的盐选自以下配体-药物偶联物或其药学上可接受的盐：

  19. 根据权利要求1-18任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体单元Pc选自多肽、抗体或抗原结合片段。
  20. 根据权利要求1-18任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述配体单元Pc为特异性结合TNFα或IL-4R的抗体或抗原结合片段。
  21. 根据权利要求20所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，其中：(1)所述重链可变区包括SEQ ID NO.4-9，12-16，18，20，22、57、68、70、72、74或76所示VH中所含的HCDR1、HCDR2和HCDR3；或/和所述轻链可变区包括SEQ ID NO.1-3，17，19，21，23、58、69、71、73、75或77所示VL中所含的LCDR1、LCDR2和LCDR3；(2)所述重链可变区和所述轻链可变区包括与第(1)组中所述HCDR1-3或/和LCDR1-3中的每个CDR相比，具有至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列。
  22. 根据权利要求20所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或抗原结合片段包含重链可变区(VH)或/和轻链可变区(VL)，所述重链可变区包括HCDR1、HCDR2和HCDR3，或/和所述轻链可变区包括LCDR1、LCDR2和LCDR3，其中，所述HCDR1-3或/和所述LCDR1-3选自以下；
    (1)所述HCDR1-3为SEQ ID NO.24-26；或/和所述LCDR1-3为SEQ ID NO.27-29；
    (2)所述HCDR1-3为SEQ ID NO.30-32；或/和所述LCDR1-3为SEQ ID NO.33-35；
    (3)所述HCDR1-3为SEQ ID NO.36-38；或/和所述LCDR1-3为SEQ ID NO.39-41；
    (4)所述HCDR1-3为SEQ ID NO.42-44；或/和所述LCDR1-3为SEQ ID NO.45-47；
    (5)所述HCDR1-3为SEQ ID NO.48-50；或/和所述LCDR1-3为SEQ ID NO.51-53；
    (6)所述HCDR1-3为SEQ ID NO.60-62；或/和所述LCDR1-3为SEQ ID NO.63-65；
    (7)所述HCDR1-3为SEQ ID NO.78-80；或/和所述LCDR1-3为SEQ ID NO.81-83；
    (8)所述HCDR1-3为SEQ ID NO.84-86；或/和所述LCDR1-3为SEQ ID NO.87-89；
    (9)所述HCDR1-3为SEQ ID NO.90-92；或/和所述LCDR1-3为SEQ ID NO.93-95；
    (10)所述HCDR1-3为SEQ ID NO.96-98；或/和所述LCDR1-3为SEQ ID NO.99-101；
    (11)所述HCDR1-3为SEQ ID NO.102-104；或/和所述LCDR1-3为SEQ ID NO.105-107；或
    所述HCDR1-3或/和所述LCDR1-3与第(1)-(11)组中任一组所述HCDR1-3和LCDR1-3中的每个CDR相比，具有至少80％同一性的序列，或至多发生3个插入、缺失或替换突变的序列。
  23. 根据权利要求20-22任一项所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体或抗原结合片段包括重链恒定区序列和/或轻链恒定区序列；可选的，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区序列或片段包括CH1，铰链区，和CH2，CH3或Fc；可选的，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选的，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:10或59所示序列，所述轻链由VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:11所示序列。
  24. 根据权利要求20所述的配体-药物偶联物或其药学上可接受的盐，其中，所述抗体选自Nerelimomab，Certolizumab，Infliximab，Golimumab，Adalimumab，Dupixent，002Mab，003Mab，004Mab，005Mab或006Mab。
  25. 一种药物-连接子化合物或其药学上可接受的盐，其结构通式为X-L-D，其中：
    药物单元D如权利要求1定义；
    连接子单元L选自其a端与X共价连接，b端与药物单元D共价连接，所述L1为由1至8个氨基酸构成的肽残基，所述肽残基进一步任选被卤素、CN、＝O、C1-C6烷基、OH、O(C1-C6烷基)、NH2、NH(C1-C6烷基)、N(C1-C6烷基)2、C3-C6环烷基和4-7元杂环基中的一个或多个取代基取代；
    X选自卤素、OS(O)2CH3或OS(O)2CF3。
  26. 根据权利要求25所述的药物-连接子化合物或其药学上可接受的盐，其中，L选自其a端与X共价连接，b端与药物单元D共价连接，所述L1选自Gly-Lys或Gly-Glu的二肽残基。
  27. 根据权利要求25所述的药物-连接子化合物或其药学上可接受的盐，其中，L选自其a端与X共价连接，b端与 药物单元D共价连接。
  28. 根据权利要求25-27任一项所述的药物-连接子化合物或其药学上可接受的盐，其中，X选自Br或I。
  29. 权利要求25所述通式为X-L-D的药物-连接子化合物或其药学上可接受的盐选自以下化合物或其药学上可接受的盐：

  30. 一种药物组合物，所述组合物包含权利要求1至24任一项所述配体-药物偶联物或其药学上可接受的盐和药学上可接受的辅料。
  31. 权利要求1至24任一项所述配体-药物偶联物或其药学上可接受的盐、或权利要求30所述的药物组合物在制备预防或者治疗炎性疾病或自身免疫性疾病的药物中的用途。
  32. 权利要求1至24任一项所述配体-药物偶联物或其药学上可接受的盐的制备方法，包括将权利要求25至29任一项所述药物-连接子化合物与配体偶联的步骤，可选的，所述配体为抗体或抗原结合片段。
  33. 一种特异性结合人TNFα的抗体或抗原结合片段，其包括重链可变区(VH)和轻链可变区(VL)，所述重链可变区包括SEQ ID NO.5-9和12-15中任一个所示的序列，并且所述轻链可变区包括SEQ ID NO.2或3所示的序列。
  34. 根据权利要求33所述的抗体或抗原结合片段，其包括以下所示的重链可变区(VH)和轻链可变区(VL)：
    (1)所述VH包括SEQ ID NO.5，并且所述VL包括SEQ ID NO.2；
    (2)所述VH包括SEQ ID NO.6，并且所述VL包括SEQ ID NO.2；
    (3)所述VH包括SEQ ID NO.7，并且所述VL包括SEQ ID NO.2；
    (4)所述VH包括SEQ ID NO.8，并且所述VL包括SEQ ID NO.2；
    (5)所述VH包括SEQ ID NO.9，并且所述VL包括SEQ ID NO.2；
    (6)所述VH包括SEQ ID NO.5，并且所述VL包括SEQ ID NO.3；
    (7)所述VH包括SEQ ID NO.6，并且所述VL包括SEQ ID NO.3；
    (8)所述VH包括SEQ ID NO.7，并且所述VL包括SEQ ID NO.3；
    (9)所述VH包括SEQ ID NO.8，并且所述VL包括SEQ ID NO.3；
    (10)所述VH包括SEQ ID NO.9，并且所述VL包括SEQ ID NO.3；
    (11)所述VH包括SEQ ID NO.12，并且所述VL包括SEQ ID NO.2；
    (12)所述VH包括SEQ ID NO.13，并且所述VL包括SEQ ID NO.2；
    (13)所述VH包括SEQ ID NO.14，并且所述VL包括SEQ ID NO.2；或，
    (14)所述VH包括SEQ ID NO.15，并且所述VL包括SEQ ID NO.2。
  35. 根据权利要求33或34所述的抗体或抗原结合片段，其中所述抗体或抗原结合片段包括重链恒定区序列和/或轻链恒定区序列；可选的，所述重链恒定区和/或轻链恒定区选自完整的恒定区序列或其片段，所述恒定区序列或片段包括CH1，铰链区，和CH2，CH3或Fc；可选的，所述重链恒定区选自人或鼠IgG1、IgG2、IgG3或IgG4恒定区，所述轻链恒定区选自人或鼠kappa恒定区或lamda恒定区；可选的，所述抗体或抗原结合片段包括完整的重链和轻链，所述重链由VH和重链恒定区组成，所述重链恒定区具有如SEQ ID NO:10所示序列，所述轻链由VL和轻链恒定区组成，所述轻链恒定区具有如SEQ ID NO:11所示序列。
  36. 一种药物组合物，所述组合物包含权利要求33至35任一项所述特异性结合人TNFα的抗体或抗原结合片段和药学上可接受的辅料。
  37. 权利要求33至35任一项所述特异性结合人TNFα的抗体或抗原结合片段，或权利要求36所述药物组合物在制备治疗炎性疾病或自身免疫性疾病的药物中的用途。
PCT/CN2023/085394 2022-04-01 2023-03-31 配体-药物偶联物及其用途 WO2023186072A1 (zh)

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WO2010126953A1 (en) * 2009-04-29 2010-11-04 Gilead Sciences, Inc. Corticosteroid linked beta-agonist compounds for use in therapy
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