WO2024012524A1 - 抗体药物偶联物及其制备方法和用途 - Google Patents

抗体药物偶联物及其制备方法和用途 Download PDF

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Publication number
WO2024012524A1
WO2024012524A1 PCT/CN2023/107220 CN2023107220W WO2024012524A1 WO 2024012524 A1 WO2024012524 A1 WO 2024012524A1 CN 2023107220 W CN2023107220 W CN 2023107220W WO 2024012524 A1 WO2024012524 A1 WO 2024012524A1
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seq
sequence
antibody
drug conjugate
pharmaceutically acceptable
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PCT/CN2023/107220
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English (en)
French (fr)
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肖亮
蔡家强
薛彤彤
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苏州宜联生物医药有限公司
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Publication of WO2024012524A1 publication Critical patent/WO2024012524A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the present disclosure belongs to the field of medical technology and relates to a variety of antibodies, antibody-drug conjugates and preparation methods thereof, as well as their use in preventing and/or treating diseases related to abnormal cell activity, including but not limited to preventing and/or treating tumor diseases. uses in.
  • ADC is a conjugate of antibodies and small molecule drugs. It combines the targeting effect of antibodies and the activity of bioactive molecules to become a biological missile with very promising efficacy and safety advantages.
  • the antibody guides the ADC to bind to the target cell and is subsequently internalized by the cell.
  • the small molecule drug is released in the cell through enzymatic decomposition under the action of specific enzymes to treat the disease.
  • ADC drugs have developed very rapidly in recent years, and there are 14 types of ADCs on the market.
  • target ADCs such as antibodies/ADC drugs targeting NaPi2b are in the clinical research stage.
  • ADC drugs can provide tumor patients with broader and better drug options, and also has broad market prospects.
  • the present disclosure provides an antibody drug conjugate represented by formula (XV) or a pharmaceutically acceptable salt or solvent thereof
  • the compound contains a biologically active molecule (drug molecule), a linker and a targeting moiety, and the targeting moiety is connected to the linker through an active group (such as a sulfhydryl group) to form an antibody drug conjugate.
  • the present disclosure also develops anti-Napi2b variable light (VL) and/or variable heavy (VH) domain-containing antibodies derived from Human antibodies.
  • the present disclosure provides an antibody drug conjugate represented by Formula XV,
  • Tb is an anti-NaPi2b antibody or its antigen-binding fragment
  • q is the drug-antibody coupling ratio
  • D is a biologically active molecule fragment
  • L 1 is the extension unit
  • L 2 does not exist or is a connected unit
  • L 3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues
  • L 4 is absent or present, and when L 4 is present, L 4 is selected from Bit 1 is connected to L 3 and bit 2 is connected to D.
  • the 1 position of L 1 is connected to Tb through an S atom
  • the 1 position of L 1 is connected to the open disulfide bond (for example, through the reducing agent TCEP Reducing the disulfide bond can open the disulfide bond and connect the sulfhydryl groups contained in Tb (such as an antibody) after generating a thiol group -SH).
  • the -S- between L 1 and Tb is not an additional external sulfur atom.
  • -S- is not another external sulfur atom, but the thiol group and L 1 contained in Tb itself after opening the disulfide bond.
  • 1 bits are connected to form -S-.
  • the extension unit is a component of the antibody drug conjugate or drug linker conjugate or linker, and its function is to connect the remaining parts of the antibody drug conjugate or the remaining parts of the linker that bind to the target site.
  • the extension unit can connect the Tb unit to L 2 (if present) or L 3. Specific examples include but are not limited to (where 1 position is connected to the target-binding antibody portion and 2 position is connected to L 2 or L 3 ):
  • L is selected from:
  • Each Z is independently selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond, C6-10 aryl group, 5-10 membered heteroaryl group, amide group, sulfonamide group, imine group and CF 2 ;
  • Rx and Ry are independently selected from H and C1-4 alkyl
  • Each m is independently selected from 0, 1, 2, 3, 4, 5 and 6;
  • y1, y2, y3 and y4 are independently selected from any integer between 0 and 20;
  • Position 1 is connected to Tb through the S atom, and position 2 is connected to L 2 or L 3 .
  • the linking unit is a component of an antibody-drug conjugate or a drug-linker conjugate or a linker, and its function is to combine the extension unit with an amino acid residue or a fragment or short peptide composed of 1-10 amino acid residues.
  • the presence of the connecting unit can connect L 1 to L 3 . Specific examples include but are not limited to (where 1 bit is connected to the extension unit and 2 bits are connected to L 3 ):
  • L is absent or present, and when L is present, L is selected from:
  • y1, y2, y3, and y4 are independently selected from any integer between 0 and 20, with bit 1 connected to L 1 and bit 2 connected to L 3 .
  • L1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • L is selected from Each Z is independently selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond, C6-10 aryl group, 5-10 membered heteroaryl and amide group (preferably selected from direct bond, carbon-carbon triple bond, carbon-carbon double bond bond); Rx and Ry are independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1 is selected from any integer between 1-6 (such as 4, 5, 6); each y2 is independently selected from any integer between 0-15 (such as 6-15); each y3 is independently selected from 1, 2 and 3; each y4 is independently selected from 0 and 1; 1
  • the bit is connected to Tb through the S atom, and the 2 bit is connected to L 2 or L 3 .
  • L is selected from m is selected from 2, 3, 4, y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 Independently selected from 1 or 2, position 1 is connected to Tb through an S atom and position 2 is connected to L 2 or L 3 .
  • L is selected from
  • L is selected from Position 1 is connected to Tb through the S atom, and position 2 is connected to L 2 or L 3 .
  • L 2 is absent or present, and when L 2 is present, L 2 is selected from y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, with bit 1 connected to L 1 and bit 2 connected to L 3 .
  • L 2 is absent or present, and when L 2 is present, L 2 is selected from Bit 1 is connected to L 1 and bit 2 is connected to L 3 .
  • L2 is absent.
  • L2 is selected from
  • L3 is selected from an amino acid residue or a short peptide consisting of 2-10 amino acid residues; the amino acid residue is selected from a natural amino acid residue, a non-natural amino acid residue, or selected from AA The amino acid residue shown in 1 or its stereoisomer.
  • L 3 is selected from the amino acid residue Val, D-Val, Cit, Phe, Lys, Lys(Ac), Leu, Gly, Ala, Asn, Asp, Arg, AA 1 or consists of 2-10 A short peptide composed of amino acid residues selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Asp, and AA 1 .
  • L3 is selected from Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, AA1 , Val-Cit, Cit-Val, Cit-Ala, Val-Ala, Lys -Val, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), Ala-Ala, Val-AA 1 , Ala-AA 1 , Gly-AA 1 , AA 1 -Gly, Ala-Ala-Ala , Ala-Ala-Asn, Ala-Ala-Asp, Val-AA 1 -Gly, Ala-AA 1 -Gly, Gly-AA 1 -Gly, Lys-Ala-Ala-Asn, Lys-Ala-Ala-Asp, Gly-Phe-Gly, Gly-Gly-Phe-Gly, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-A
  • L 3 is selected from AA 1 , AA 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn, and Gly-Gly-Phe-Gly.
  • L3 is selected from AA1 and Val- AA1 -Gly.
  • L3 is selected from Val- AA1 -Gly.
  • L3 is selected from X - is selected from halide ions, carboxylate ions, sulfate ions, hydrogen sulfate ions and OH - , the 1-position is connected to L 1 or L 2 , and the 2-position is connected to L 4 or D.
  • L3 is selected from Bit 1 is connected to L 1 or L 2 , bit 2 is connected to L 4 or D.
  • L3 is selected from Bit 1 is connected to L 1 or L 2 , bit 2 is connected to L 4 or D.
  • the structure of the amino acid residue represented by AA 1 is as follows,
  • R a and R b are each independently selected from H, And R a and R b are not H at the same time;
  • R a and R b and the carbon atoms to which they are commonly connected form a 4-10 membered heterocycle, which is optionally substituted by one or more R 0 ;
  • r and r 1 are each independently selected from any integer from 0 to 20;
  • R m1 and R n1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl and -COOR x1 ;
  • R x1 is selected from C1-6 alkyl
  • R m1 and R n1 together with the nitrogen atom to which they are commonly connected form a 4-10 membered heterocycle, which is optionally substituted by one or more R 0' ;
  • Rz is selected from C1-6 alkyl
  • R 0 and R 0' are each independently selected from C1-6 alkyl, C3-6 cycloalkyl, -NR m2 R n2 and 4-10 membered heterocyclyl optionally substituted by C1-6 alkyl;
  • R m2 and R n2 are each independently selected from H and C1-6 alkyl.
  • any one of R a and R b is H, and the other is selected from
  • any one of R a and R b is H, and the other is selected from
  • Ra and Rb together with the carbon atom to which they are commonly attached, form a 5-6 membered heterocycle substituted with R0 .
  • R a and R b together with the carbon atom to which they are commonly attached form a piperidine ring or piperazine ring substituted by R 0 .
  • Ra and Rb together with the carbon atom to which they are commonly attached, form a piperidine ring substituted with R0 .
  • r, r 1 are each independently selected from 0, 1, 2, 3, 4, and 5.
  • r, r 1 are each independently selected from 0 and 4.
  • any one of r and r 1 is 0 and the other is 4.
  • R m1 , R n1 are each independently selected from H, methyl, ethyl, n-propyl, n-butyl, -COOCH3, -COOCH2CH3, -COOCH2CH2CH3, -COOCH(CH3)2, -COOC (CH3)3 and -COOCH2CH2CH2CH3.
  • R m1 , R n1 are each independently selected from H, C1-6 alkyl, C3-6 cycloalkyl, and tert-butoxycarbonyl.
  • R m1 , R n1 are each independently selected from H and C1-6 alkyl.
  • R m1 , R n1 are each independently selected from H, methyl, ethyl, and n-propyl.
  • R m1 and R n1 are each independently selected from H, C1-6 alkyl (such as H, methyl); r is 0 , when r 1 is 4, R m1 and R n1 are each independently selected from C1-6 alkyl (such as methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (such as ethyl, n-propyl) base).
  • R m1 and R n1 together with the nitrogen atom to which they are commonly attached form a 5-6 membered heterocycle optionally substituted with R 0' .
  • R m1 and R n1 together with the nitrogen atom to which they are commonly attached form a piperidine or piperazine ring optionally substituted by R 0' .
  • R m1 and R n1 together with the nitrogen atom to which they are commonly attached form Nitrogen atom No. 1 is a nitrogen atom commonly connected to R m1 and R n1 .
  • Rz is methyl
  • R 0 , R 0′ are each independently selected from C1-6 alkyl, -NR m2 R n2 and 5-6 membered heterocyclyl optionally substituted by C1-6 alkyl.
  • R0 is selected from C1-6 alkyl and 5-6 membered heterocyclyl substituted by C1-6 alkyl, the 5-6 membered heterocyclyl is selected from piperidinyl and piperazinyl .
  • R0 is selected from methyl, ethyl, and 5-6 membered heterocyclyl substituted by methyl, the 5-6 membered heterocyclyl being piperidinyl.
  • R0 is selected from methyl and a 5-6 membered heterocyclyl substituted by a methyl group, the 5-6 membered heterocyclyl being piperidinyl.
  • R0 is selected from methyl, ethyl, and
  • R0 is selected from methyl and
  • R 0' is selected from C1-6 alkyl and -NR m2 R n2 .
  • R0 ' is selected from methyl and -NRm2Rn2 .
  • Rm2 , Rn2 is methyl
  • amino acid residue represented by AA 1 is selected from
  • amino acid residue represented by AA 1 is selected from
  • amino acid residue represented by AA 1 is selected from
  • L 4 is absent or present, and when L 4 is present, L 4 is Bit 1 is connected to L 3 and bit 2 is connected to D.
  • L 4 is absent.
  • L4 is selected from Bit 1 is connected to L 3 and bit 2 is connected to D.
  • L4 is selected from Bit 1 is connected to L 3 and bit 2 is connected to D.
  • the structure of is selected from the following structural fragments:
  • q is selected from any value between 0.1 and 16.0; in preferred embodiments, q is selected from any integer between 0.1 and 16.0.
  • q is selected from any value between 0.1 and 8.0, and in preferred embodiments, q is selected from any integer between 0.1 and 8.0.
  • q is selected from any value between 2-8.
  • q is selected from any value between 3-8.
  • q is selected from any value between 4-8.
  • q is selected from any value between 6-8.
  • q is selected from any integer between 2-8.
  • q is selected from any integer between 3-8.
  • q is selected from any integer between 4-8.
  • q is selected from any integer between 6-8.
  • q is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.
  • q is selected from 2, 4, 6, and 8.
  • the biologically active molecule fragment refers to the antibody-drug conjugate (or antibody-drug conjugate (ADC)) known in the art, which is present in tumor tissues or tumor cells.
  • ADC antibody-drug conjugate
  • biologically active drugs such as small molecule cytotoxic drugs, which include groups after losing one atom or atomic group
  • derivatives thereof such as their precursors
  • D is a molecule fragment having anti-tumor biological activity.
  • D is a molecule fragment with anti-tumor biological activity, wherein the biologically active molecule is selected from the group consisting of cytotoxic agents or derivatives thereof, such as DNA topoisomerase inhibitors (such as camptothecin-based biologically active molecules). , such as camptothecin, DXD, camptothecin with modified substituents or DXD with modified substituents) or tubulin inhibitors (such as MMAF tubulin inhibitors, MMAE tubulin inhibitors).
  • cytotoxic agents or derivatives thereof such as DNA topoisomerase inhibitors (such as camptothecin-based biologically active molecules).
  • DNA topoisomerase inhibitors such as camptothecin-based biologically active molecules.
  • tubulin inhibitors such as MMAF tubulin inhibitors, MMAE tubulin inhibitors.
  • the antibody drug conjugate has the structure of Formula I:
  • R 1 and R 2 are each independently selected from H, halogen, -OH, optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or,
  • R 1 and R 2 together with the carbon atom to which they are connected form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring containing 1 or more O, S, N, carbonyl, sulfoxide or sulfone base or any combination thereof;
  • R 3 is selected from H, halogen, -OH, -NH 2 , optionally substituted C1-6 alkyl and optionally substituted C1-6 alkoxy, or,
  • R 3 and R 2 and the carbon atoms to which they are connected together form a 5-7 membered carbocyclic ring or a 5-7 membered heterocyclic ring, which contains 1 or more O, S, N, carbonyl, sulfoxide or sulfone group or any combination thereof;
  • W is absent or present.
  • W is selected from -O-, -S-, -NR 4 -, Bit 1 is connected to X, bit 2 is connected to L 4 or L 3 ;
  • X is selected from direct bonds, optionally substituted -O-(CH 2 ) n3 -, -N(R 4 )-(CH 2 ) n3 -, -S-(CH 2 ) n3 -, carbonyl-(CH 2 ) n3 , -SO 2 -(CH 2 ) n3 -, -(CH 2 ) n1 -, C3-6 cycloalkyl, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, the 1-position is connected to the parent ring, and the 2-position is connected to W or L 4 connected; the substituent is selected from one or more C1-4 alkyl groups, C3-6 cycloalkyl groups, or multiple C1-4 alkyl groups and the carbon atoms connected to them simultaneously form a C3-6 cycloalkyl group. ;
  • Each M is independently selected from the group consisting of direct bonds and -CR 5a R 5b -;
  • R 4 , R 5 , R 5a , R 5b , R 6 and R 7 are each independently selected from H, optionally substituted C1-4 alkyl, optionally substituted C1-4 alkoxy and optionally substituted C3 -6 cycloalkyl;
  • n, n’, n1, n2, n3 are each independently selected from any integer between 0 and 6;
  • L 4 is absent or present, and when L 4 is present, L 4 is selected from Bit 1 is connected to L 3 , bit 2 is connected to W or X.
  • Tb, L1 , L2 , L3, and q have the meanings provided above and in any embodiment specifically described herein.
  • R 1 , R 2 are each independently selected from H, halogen, and C1-4 alkyl.
  • R 1 and R 2 together with the carbon atom to which they are attached form a 5-6 membered heterocycle containing 1, 2 or 3 O, S or N or any combination thereof.
  • R1 is selected from H and halogen
  • R2 is selected from H and C1-4 alkyl.
  • R 1 and R 2 and the carbon atom to which they are attached form The dashed line indicates the position where the heterocycle is fused to the benzene ring.
  • R 1 is H or F and R 2 is H or methyl.
  • R 1 is F
  • R 2 is methyl or R 1 and R 2 together with the carbon atom to which they are attached form
  • R1 is F and R2 is methyl.
  • R 1 and R 2 together with the carbon atom to which they are attached form
  • R3 is selected from H and C1-4 alkyl.
  • R3 and X together with the carbon atom to which they are attached form a 5-6 membered carbocyclic ring.
  • R3 is H or R3 and X together with the carbon atom to which it is attached form
  • the dashed lines indicate where the carbocyclic rings are fused with the benzene and pyridine rings.
  • R3 is H.
  • W is absent or present, and when W is present, W is selected from -O-, -S-, -NR 4 -, Bit 1 is connected to X, bit 2 is connected to L 4 or L 3 .
  • W is absent or present, and when W is present, W is selected from -O-, -S-, -NR 4 -, Bit 1 is connected to X, bit 2 is connected to L 4 or L 3 .
  • W is selected from -O-, -NR 4 -, and Bit 1 is connected to X, bit 2 is connected to L 4 or L 3 .
  • W is selected from -O- and -NR 4 -, with position 1 attached to X and position 2 attached to L 4 or L 3 .
  • X is selected from optionally substituted -(CH 2 ) n1 -, C6-10 aryl, 5-10 membered heteroaryl and 4-10 membered heterocyclyl, 1-position is connected to the parent ring, 2-position is connected to W or L 4 ; the substituent is selected from 1 or 2 C1 -4 alkyl, or two C1-4 alkyl groups and the carbon atoms connected to them at the same time together form a C3-6 cycloalkyl group.
  • X is selected from optionally substituted Position 1 is connected to the parent ring, position 2 is connected to W or L4 ; the substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl ) and the carbon atoms to which they are attached together form a C3-6 cycloalkyl group (such as cyclopropyl).
  • X is selected from Position 1 is connected to the mother ring, position 2 is connected to W or L 4 .
  • X is selected from Bit 1 is connected to the mother ring, bit 2 is connected to W.
  • X when W is absent, X is selected from Position 1 is connected to the mother ring, position 2 is connected to L 4 ; when W exists, X is selected from Bit 1 is connected to the mother ring, bit 2 is connected to W.
  • W is selected from -O-, -NR 4 -, and 1 bit is connected to X, 2 bits are connected to L 4 or L 3 ; X is selected from Bit 1 is connected to the mother ring, bit 2 is connected to W.
  • R 4 and R 5 are each independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl.
  • each R 4 is independently selected from H, C1-4 alkyl, and C3-6 cycloalkyl, and R 5 is H.
  • each R 4 is independently selected from H, methyl, ethyl, n-propyl, isopropyl, tert-butyl, and cyclopropyl, and R 5 is H.
  • R 5a , R 5b are each independently selected from H and C1-4 alkyl.
  • R 5a , R 5b are each independently selected from H and methyl.
  • each R is independently selected from H and C1-4 alkyl.
  • R 7 is H.
  • n is selected from 1, 2, and 3.
  • n 1
  • n1 is selected from 1, 2, 3, and 4.
  • n2 is 1.
  • n3 is 0.
  • L3 is selected from X - is selected from halide ions, carboxylate ions, sulfate ions, hydrogen sulfate ions and OH - , the 1-position is connected to L 1 or L 2 , and the 2-position is connected to L 4 or W.
  • L3 is selected from Bit 1 is connected to L 1 or L 2 , bit 2 is connected to L 4 or W.
  • L3 is selected from Bit 1 is connected to L 1 or L 2 , bit 2 is connected to L 4 or W.
  • L 4 is absent or present, and when L 4 is present, L 4 is Bit 1 is connected to L 3 , bit 2 is connected to W or X.
  • L 4 is absent.
  • L4 is selected from Bit 1 is connected to L 3 , bit 2 is connected to W or X.
  • L4 is selected from Bit 1 is connected to L 3 , bit 2 is connected to W or X.
  • the structure of is selected from the following structural fragments:
  • 1 bit is connected to Tb and 2 bits are connected to W.
  • D is Structural fragment shown; position 1 is connected to L 3 or L 4 ; e.g.
  • the structure of is selected from the following structural fragments:
  • 1 bit is connected to L 4 ; when L 4 does not exist, 1 bit is connected to L 3 .
  • W is absent or present.
  • W is selected from -O-, -S-, -NR 4 -,
  • -O-, -NR 4 - or R 4 and R 5 are each independently selected from H and C1-4 alkyl;
  • n is independently selected from 0, 1, 2, 3 and 4;
  • X is selected from
  • R 1 is selected from H, halogen
  • R 2 is selected from H, C1-4 alkyl, or, R 1 and R 2 and the carbon atoms connected to them form The dotted line indicates the position where the heterocyclic ring is fused with the benzene ring;
  • R 3 is selected from H and C1-4 alkyl, or R 3 and X and the carbon atom to which they are connected together form a 5-6 membered carbocyclic ring;
  • W is absent or present, and when W is present, W is selected from -O-, -NR 4 - (for example -NH-, -N(CH 3 )-, -N(C 2 H 5 )-), R 4 is independently selected from H, methyl, ethyl, isopropyl, n-propyl, tert-butyl and cyclopropyl;
  • AA 1 In, r is selected from 0, 1, 2, 3, 4 and 5;
  • R a and R b either one is H, and the other one is selected from Alternatively, R a and R b together with the carbon atoms to which they are commonly attached, Forming a 5-6 membered heterocycle substituted by R 0 ;
  • R 1 is selected from H, halogen
  • R 2 is selected from H, C1-4 alkyl, or, R 1 and R 2 and the carbon atoms connected to them form
  • R 3 is selected from H and C1-4 alkyl, or R 3 and X and the carbon atom to which it is connected together form R 3 and X and the carbon atom to which it is connected together to form
  • W is selected from -O- and -NR 4 -, the 1-position is connected to X, and the 2-position is connected to L 4 or L 3 ;
  • X is selected from Position 1 is connected to the mother ring, position 2 is connected to W;
  • R 1 is F
  • R 2 is methyl or R 1 and R 2 together with the carbon atom to which they are attached form
  • R3 is H.
  • the antibody drug conjugate has the structure of Formula I-1:
  • Tb Tb, L 1 , L 2 , L 3 , L 4 , The meaning of offering.
  • the antibody drug conjugate has the structure of Formula I-1A or I-1B:
  • Tb, L2 , L3 , L4 , X, Ri , R2 , R3 , R4, and q have the meanings provided above and in any embodiment specifically described herein.
  • the antibody drug conjugate has the structure of Formula I-2:
  • Tb, L1 , L2 , L3 , L4 , X, R1 , R2 , R3, and q have the meanings provided above and in any embodiment specifically described herein.
  • the antibody drug conjugate has the structure of Formula I-2A or I-2B:
  • Tb, L2 , L3 , L4 , X, Ri , R2 , R3, and q have the meanings provided above and in any embodiment specifically described herein.
  • the antibody drug conjugate has the structure of Formula I-3:
  • Tb, L1 , L2 , L3 , L4 , X, R1 , R2 , R3 , R4 , R5 , n and q have the values provided above and in any embodiment specifically described herein meaning.
  • the antibody drug conjugate has the structure of Formula I-3A or I-3B:
  • Tb, L2 , L3 , L4 , X, Ri , R2 , R3 , R4, and q have the meanings provided above and in any embodiment specifically described herein.
  • the antibody drug conjugate has the structure of Formula IA:
  • the antibody drug conjugate has the structure of Formula IB:
  • the antibody drug conjugate is selected from the following:
  • Tb is an anti-NaPi2b antibody or antigen-binding fragment thereof.
  • the antibodies or antigen-binding fragments thereof and monoclonal antibodies or antigen-binding fragments thereof include: Fab, Fab', F(ab')2, Fd, Fv (e.g., scFv), dAb, complementarity determining Region fragment, non-human antibody, humanized antibody, chimeric antibody, fully human antibody, probody, monoclonal antibody, bispecific antibody or multispecific antibody.
  • Tb is an anti-Napi2b antibody or antigen-binding fragment thereof that has endocytosis, no endocytosis activity, or weak endocytosis.
  • Tb is an anti-Napi2b antibody or antigen-binding fragment thereof with endocytic activity.
  • Tb is an anti-Napi2b antibody or antigen-binding fragment thereof that has no endocytic activity, or has weak endocytic activity.
  • Tb is an anti-NaPi2b antibody or antigen-binding fragment thereof.
  • the anti-Napi2b antibody is a non-human antibody, a humanized antibody, a chimeric antibody, a fully human antibody.
  • the anti-Napi2b antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.
  • the anti-Napi2b antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof.
  • the anti-NaPi2b antibody or antigen-binding fragment thereof is selected from: lifastuzumab or antigen-binding fragment thereof, upifitamab or antigen-binding fragment thereof, antibody C1.
  • the anti-NaPi2b antibody is selected from: 16G5B3, 60A12B3, 22H9C4, 66C12D12 and 34F2A10 murine antibodies, or humanized antibodies thereof.
  • the anti-NaPi2b antibody is selected from the group consisting of: 60A12B3-hz1 antibody, 22H9C4-hz1 antibody, 66C12D12-hz1 antibody and 34F2A10-hz1 antibody.
  • the Tb antibody or its antigen-binding fragment can be prepared by various methods known in the art, for example, obtained through genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present disclosure are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells.
  • the transfected host cells are then cultured under specific conditions and express the antibodies of the present disclosure.
  • Tb in the antibody-drug conjugate, is the antibody Napi2b antibody or antigen-binding fragment thereof described in the second aspect.
  • the present disclosure provides an anti-Napi2b antibody or antigen-binding fragment thereof.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):
  • LCDR1 or a variant of its sequence LCDR2 or a variant of its sequence contained in the light chain variable region (VL) represented by SEQ ID NO: 2, 18, 20, 35, 37, 53, 55, 72, 75 or 51 variants, and variants of LCDR3 or its sequence.
  • VL light chain variable region
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence contained in the VH shown in SEQ ID NO: 1 or 17, and HCDR3 or Variants of its sequence; and/or variants of LCDR1 or its sequence, LCDR2 or its sequence, and LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 2 or 18.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises a VH contained in SEQ ID NO: 19 or 34.
  • HCDR1 or variants of its sequence HCDR2 or variants of its sequence, and HCDR3 or variants of its sequence; and/or variants of LCDR1 or its sequence contained in the VL shown in SEQ ID NO: 20 or 35 , LCDR2 or a variant of its sequence and LCDR3 or a variant of its sequence.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence contained in the VH shown in SEQ ID NO: 36 or 52, and HCDR3 or Variants of its sequence; and/or LCDR1 or variants of its sequence, LCDR2 or variants of its sequence, and LCDR3 or variants of its sequence contained in the VL shown in SEQ ID NO: 37 or 53.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence contained in the VH set forth in SEQ ID NO: 54, 71 or 73, and HCDR3 or a variant of its sequence; and/or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO: 55 or 72.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence contained in the VH shown in SEQ ID NO: 74 or 33, and HCDR3 or Variants of its sequence; and/or LCDR1 or variants of its sequence, LCDR2 or variants of its sequence, and LCDR3 or variants of its sequence contained in the VL shown in SEQ ID NO: 75 or 51.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or its sequence contained in the VH shown in SEQ ID NO: 17
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or its sequence contained in the VH shown in SEQ ID NO:34
  • Variants of LCDR1 or its sequence, LCDR2 or its sequence, and LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 35
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or its sequence contained in the VH shown in SEQ ID NO:52
  • Variants of LCDR1 or its sequence, LCDR2 or its sequence, and LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 53 are also included in the anti-Napi2b antibody or antigen-binding fragment thereof.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence contained in the VH set forth in SEQ ID NO: 71 or 73, and HCDR3 or Variants of its sequence; and/or LCDR1 or variants of its sequence, LCDR2 or variants of its sequence, and LCDR3 or variants of its sequence contained in the VL shown in SEQ ID NO:72.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises HCDR1 or a variant of its sequence, HCDR2 or a variant of its sequence, and HCDR3 or its sequence contained in the VH shown in SEQ ID NO:33 Variants of LCDR1 or its sequence, LCDR2 or its sequence, and LCDR3 or its sequence contained in the VL shown in SEQ ID NO: 51.
  • the variant of the sequence is one or several amino acid substitutions, deletions, or additions (eg, 1, 2, or 3 amino acid substitutions, deletions, or additions) compared to its source CDR. CDR.
  • the substitutions are conservative substitutions.
  • the CDRs are defined according to the AbM, Chothia, Kabat, or IMGT numbering systems.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) and/or a light chain variable region (VL).
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
  • HCDR1 of a sequence with acid substitutions, deletions or additions e.g.
  • the sequence is SEQ ID NO: 4 or has one or several amino acid substitutions compared thereto, HCDR2 of a sequence with deletions or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids), the sequence is SEQ ID NO: 6 or compared thereto with one or several amino acid substitutions, deletions, or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO:7 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 9 or having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:10 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 3 or has one or more amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 5 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 6 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 9 or having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:10 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 21 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 22 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 25 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 26 or having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:27 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 21 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 23 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 24 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • LCDR1 of a sequence with substitutions, deletions or additions e.g. substitutions, deletions or additions of 1, 2 or 3 amino acids
  • the sequence is SEQ ID NO: 26 or compared thereto with one or several amino acid substitutions, deletions or additions
  • LCDR2 of a sequence with additions e.g., substitution, deletion or addition of 1, 2 or 3 amino acids
  • the sequence is SEQ ID NO: 27 or compared thereto with substitution, deletion or addition of one or several amino acids (e.g. 1 LCDR3 of a sequence with 2 or 3 amino acid substitutions, deletions or additions);
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 38 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO:39 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 41 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 42 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 44 or having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:45 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 38 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 40 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 41 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 42 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 44 or having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:45 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 56 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO: 57 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 60 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 61 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of the sequence SEQ ID NO: 63 or with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2 of the sequence SEQ ID NO:64 or a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • HCDR1 of a sequence with acid substitutions, deletions or additions e.g.
  • the sequence is SEQ ID NO: 58 or has one or several amino acid substitutions compared thereto, HCDR2 of a sequence with deletions or additions (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 60 or compared thereto with one or several amino acid substitutions, deletions or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence of HCDR3; and/or,
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 56 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO:59 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 60 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • LCDR1 of the sequence SEQ ID NO: 63 or with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions)
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined according to the Kabat numbering system:
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:3, HCDR2 whose sequence is SEQ ID NO:4, and HCDR3 whose sequence is SEQ ID NO:6; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:7, LCDR2 whose sequence is SEQ ID NO:9, and LCDR3 whose sequence is SEQ ID NO:10;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:3, HCDR2 whose sequence is SEQ ID NO:5, and HCDR3 whose sequence is SEQ ID NO:6; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:8, LCDR2 whose sequence is SEQ ID NO:9, and LCDR3 whose sequence is SEQ ID NO:10;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:21, HCDR2 whose sequence is SEQ ID NO:22, and HCDR3 whose sequence is SEQ ID NO:24; and/or,
  • the VL includes: LCDR1 with a sequence of SEQ ID NO:25, LCDR2 with a sequence of SEQ ID NO:26, and LCDR3 with a sequence of SEQ ID NO:27;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:21, HCDR2 whose sequence is SEQ ID NO:23, and HCDR3 whose sequence is SEQ ID NO:24; and/or,
  • the VL includes: LCDR1 with a sequence of SEQ ID NO:25, LCDR2 with a sequence of SEQ ID NO:26, and LCDR3 with a sequence of SEQ ID NO:27;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:38, HCDR2 whose sequence is SEQ ID NO:39, and HCDR3 whose sequence is SEQ ID NO:41; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:42, LCDR2 with the sequence SEQ ID NO:44, and LCDR3 with the sequence SEQ ID NO:45;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:38, HCDR2 whose sequence is SEQ ID NO:40, and HCDR3 whose sequence is SEQ ID NO:41; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:43, LCDR2 with the sequence SEQ ID NO:44, and LCDR3 with the sequence SEQ ID NO:45;
  • the VH includes: HCDR1 with the sequence SEQ ID NO:56, HCDR2 with the sequence SEQ ID NO:57, and HCDR3 with the sequence SEQ ID NO:60; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:61, LCDR2 whose sequence is SEQ ID NO:63, and LCDR3 whose sequence is SEQ ID NO:64;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:56, HCDR2 whose sequence is SEQ ID NO:58, and HCDR3 whose sequence is SEQ ID NO:60; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:62, LCDR2 with the sequence SEQ ID NO:63, LCDR3 with the sequence SEQ ID NO:64; or
  • the VH includes: HCDR1 with the sequence SEQ ID NO:56, HCDR2 with the sequence SEQ ID NO:59, and HCDR3 with the sequence SEQ ID NO:60; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:62, LCDR2 with the sequence SEQ ID NO:63, and LCDR3 with the sequence SEQ ID NO:64.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 11 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 12 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 14 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is WAS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 10 or an LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • VH heavy chain variable region containing the following 3 CDRs: the sequence is SEQ ID NO: 11 or has one or more amino groups compared with it HCDR1 of a sequence with acid substitutions, deletions or additions (e.g. substitutions, deletions or additions of 1, 2 or 3 amino acids), the sequence is SEQ ID NO: 13 or has one or several amino acid substitutions compared thereto, HCDR2 of a sequence with deletions or additions (e.g.
  • substitution, deletion or addition of 1, 2 or 3 amino acids the sequence is SEQ ID NO: 14 or compared thereto with one or several amino acid substitutions, deletions or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is WAS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 10 or an LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 28 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 29 or compared thereto with one or more substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO:31 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 32 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is YTS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 27 or an LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 28 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO:30 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO:31 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 32 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is YTS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 27 or an LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 46 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO: 47 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 49 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 46 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a sequence of one or more amino acids (substitution, deletion or addition of amino acids), the sequence is SEQ ID NO: 48 or compared thereto with one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 49 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 50 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is SAS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 45 or an LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 65 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO: 66 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 68 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 69 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is WAS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 64 or LCDR3 having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto;
  • a heavy chain variable region (VH) containing the following 3 CDRs the sequence is SEQ ID NO: 65 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3) HCDR1 with a substitution, deletion or addition of one amino acid), the sequence is SEQ ID NO: 67 or has one or several substitutions, deletions or additions of amino acids (e.g. substitution of 1, 2 or 3 amino acids) , deletion or addition), the sequence is SEQ ID NO: 68 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) ) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO: 69 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is WAS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence of which is SEQ ID NO: 64 or an LCDR3 having a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; or
  • substitutions, deletions or additions of 1, 2 or 3 amino acids the sequence is SEQ ID NO:77 or has one or several amino acid substitutions compared thereto, HCDR2 of a sequence with deletions or additions (e.g., substitution, deletion, or addition of 1, 2, or 3 amino acids), the sequence of which is SEQ ID NO: 78 or compared thereto with one or several amino acid substitutions, deletions, or additions ( For example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence of HCDR3; and/or,
  • a light chain variable region (VL) containing the following 3 CDRs the sequence is SEQ ID NO:79 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is HTS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2 or 3 amino acid substitutions, deletions or additions) LCDR2, the sequence is SEQ ID NO: 16 or an LCDR3 having a sequence with one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto.
  • the sequence is SEQ ID NO:79 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acids) LCDR1 of a sequence with a substitution, deletion or addition), a sequence that is HTS or a sequence with one or several amino acid substitutions, deletions or additions (e.g. 1, 2
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein the CDRs are defined by the IMGT numbering system:
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:11, HCDR2 whose sequence is SEQ ID NO:12, and HCDR3 whose sequence is SEQ ID NO:14; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:15, LCDR2 with the sequence WAS, and LCDR3 with the sequence SEQ ID NO:10;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:11, HCDR2 whose sequence is SEQ ID NO:13, and HCDR3 whose sequence is SEQ ID NO:14; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:15, LCDR2 with the sequence WAS, and LCDR3 with the sequence SEQ ID NO:10;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:28, HCDR2 whose sequence is SEQ ID NO:29, and HCDR3 whose sequence is SEQ ID NO:31; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:32, LCDR2 whose sequence is YTS, and LCDR3 whose sequence is SEQ ID NO:27;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:28, HCDR2 whose sequence is SEQ ID NO:30, and HCDR3 whose sequence is SEQ ID NO:31; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:32, LCDR2 whose sequence is YTS, and LCDR3 whose sequence is SEQ ID NO:27;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:46, HCDR2 whose sequence is SEQ ID NO:47, and HCDR3 whose sequence is SEQ ID NO:49; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:50, LCDR2 whose sequence is SAS, and LCDR3 whose sequence is SEQ ID NO:45;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:46, HCDR2 whose sequence is SEQ ID NO:48, and HCDR3 whose sequence is SEQ ID NO:49; and/or,
  • the VL includes: LCDR1 whose sequence is SEQ ID NO:50, LCDR2 whose sequence is SAS, and LCDR3 whose sequence is SEQ ID NO:45;
  • the VH includes: HCDR1 with the sequence SEQ ID NO:65, HCDR2 with the sequence SEQ ID NO:66, and HCDR3 with the sequence SEQ ID NO:68; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:69, LCDR2 with the sequence WAS, and LCDR3 with the sequence SEQ ID NO:64;
  • the VH includes: HCDR1 whose sequence is SEQ ID NO:65, HCDR2 whose sequence is SEQ ID NO:67, and HCDR3 whose sequence is SEQ ID NO:68; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:69, LCDR2 with the sequence WAS, and LCDR3 with the sequence SEQ ID NO:64;
  • the VH includes: HCDR1 with the sequence SEQ ID NO:76, HCDR2 with the sequence SEQ ID NO:77, and HCDR3 with the sequence SEQ ID NO:78; and/or,
  • the VL includes: LCDR1 with the sequence SEQ ID NO:79, LCDR2 with the sequence HTS, and LCDR3 with the sequence SEQ ID NO:16.
  • anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the following heavy chain variable region (VH) and/or light chain variable region (VL), wherein, consistent with the aforementioned Kabat numbering system or IMGT Compared with the CDRs defined by the numbering system, at least one CDR in the heavy chain variable region (VH) and/or light chain variable region (VL) contains a mutation, and the mutation is a substitution, deletion or deletion of one or several amino acids. Addition or any combination thereof (eg substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof) and still have Napi2b binding activity.
  • VH heavy chain variable region
  • VL light chain variable region
  • substitutions described in this disclosure are conservative substitutions.
  • the anti-Napi2b antibody or antigen-binding fragment thereof binds human Napi2b, monkey Napi2b, and/or rat Napi2b.
  • the VH of an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and/or the antibody or The VL of its antigen-binding fragment contains the framework region (FR) derived from the light chain variable region (VL) of human immunoglobulin.
  • FR framework region
  • the antibodies of the present disclosure, or antigen-binding fragments thereof are fully human.
  • the antibodies of the present disclosure, or antigen-binding fragments thereof are humanized.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the disclosure is humanized to a degree of at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • VH Heavy chain variable region
  • VL light chain variable region
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a VH set forth in SEQ ID NO: 1 or 17, and/or, a VL set forth in SEQ ID NO: 2 or 18.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a VH set forth in SEQ ID NO: 19 or 34, and/or, a VL set forth in SEQ ID NO: 20 or 35.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a VH set forth in SEQ ID NO: 36 or 52, and/or, a VL set forth in SEQ ID NO: 37 or 53.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a VH set forth in SEQ ID NO: 54, 71, or 73, and/or, a VL set forth in SEQ ID NO: 55 or 72.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a VH set forth in SEQ ID NO: 74 or 33, and/or, a VL set forth in SEQ ID NO: 75 or 51.
  • the anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure includes the above-mentioned 5 regimen groups, and the VH ratio of any group is at least 70%, at least 80%, at least 85%, at least 90%, A VH that has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or, with the set of VL Compared to having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or a VL with at least 99% sequence identity; or
  • the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure comprise the VH of any of the above-mentioned 5 scheme groups having one or several amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, A VH with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; and/or a VL with one or several substitutions, deletions or additions of amino acids or any combination thereof, e.g. VL with substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof; in certain preferred embodiments , the substitution is a conservative substitution.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • Heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are independently associated with (a) to (k) ) VH and VL described in any group have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% respectively , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or
  • VH and VL Heavy chain variable region (VH) and light chain variable region (VL), wherein said heavy chain variable region (VH) and light chain variable region (VL) are consistent with any of (a) to (k)
  • the VH and VL described in a group independently have one or several amino acid substitutions, deletions or additions or any combination thereof (for example, 1, 2, 3, 4, 5, 6 , substitution, deletion or addition of 7, 8, 9 or 10 amino acids or any combination thereof). In certain preferred embodiments, the substitutions are conservative substitutions.
  • the anti-Napi2b antibodies of the present disclosure are chimeric, humanized, or fully human antibodies.
  • the antibodies of the present disclosure or antigen-binding fragments thereof are selected from the group consisting of Fab, Fab', (Fab') 2 , Fv fragments such as scFv or disulfide-linked Fv (dsFv), diabodies, and Multispecific antibodies.
  • anti-Napi2b antibodies of the disclosure are scFv.
  • the heavy chain of an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises the heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof that is identical to the wild type from which it is derived.
  • CH heavy chain constant region
  • Sequence comparisons with conservative substitutions of up to 50 amino acids e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids
  • Conservative substitutions for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).
  • the light chain of an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof that is identical to the wild-type CL from which it is derived.
  • CL light chain constant region
  • Sequence comparisons with conservative substitutions of up to 50 amino acids e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids
  • Conservative substitutions for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).
  • the constant region is altered, e.g., mutated, to modify properties of the anti-Napi2b antibody molecule (e.g., alters one or more of the following: Fc receptor binding, antibody glycosylation, cysteine residues number of bases, effector cell function, or complement function).
  • Functional changes can be produced by replacing at least one amino acid residue in the constant region of an antibody with a different residue, e.g., altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function (e.g., decreasing ).
  • the Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis (ADCP), CDC, etc.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure has a heavy chain constant region (CH) selected from the group consisting of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE.
  • CH heavy chain constant region
  • Chain constant region in particular a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, more particularly selected from the group consisting of IgG1 (eg human IgG1).
  • the human IgG1 heavy chain constant region is set forth in SEQ ID NO:70.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region) .
  • the light chain constant region has the sequence set forth in SEQ ID NO:80.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises the CH set forth in SEQ ID NO:70 or a variant thereof that has up to 20 amino acids conserved compared to SEQ ID NO:70 Substitutions (e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or a conservative substitution of 10 amino acids), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% compared to SEQ ID NO:70 , at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • substitutions e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or a conservative substitution of 10 amino acids
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof.
  • the light chain constant region comprises a kappa light chain constant region.
  • the light chain constant region comprises the light chain constant region (CL) set forth in SEQ ID NO:80 or a variant thereof having at most 20 amino acids compared to SEQ ID NO:80 Conservative substitutions of at most 20, at most 15, at most 10 or at most 5 amino acids; for example, 1, 2, 3, 4, 5, 6, 7, 8, Conservative substitution of 9 or 10 amino acids), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least compared to SEQ ID NO:80 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • the anti-Napi2b antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) set forth in SEQ ID NO:70 and the light chain constant region (CL) set forth in SEQ ID NO:80.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • a heavy chain comprising or consisting of an amino acid sequence selected from:
  • (ii) Compared with the sequence shown in (i), it has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof); or
  • a light chain comprising an amino acid sequence selected from:
  • substitution, deletion or addition of one or several amino acids or any combination thereof for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • a heavy chain comprising or consisting of an amino acid sequence selected from:
  • (ii) Compared with the sequence shown in (i), it has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof); or
  • a light chain comprising or consisting of an amino acid sequence selected from:
  • substitution, deletion or addition of one or several amino acids or any combination thereof for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises:
  • a heavy chain comprising or consisting of an amino acid sequence selected from:
  • (ii) Compared with the sequence shown in (i), it has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof); or
  • a light chain comprising or consisting of an amino acid sequence selected from:
  • substitution, deletion or addition of one or several amino acids or any combination thereof for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; or
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain
  • the heavy chain includes or consists of the following sequence:
  • (ii) Compared with the sequence shown in (i), it has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof); or
  • the light chain includes or consists of the following sequence:
  • substitution, deletion or addition of one or several amino acids or any combination thereof for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; or
  • sequence shown in (vi) and (iv) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% sequence identity;
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure comprises a heavy chain and a light chain
  • the heavy chain includes or consists of the following sequence:
  • (ii) Compared with the sequence shown in (i), it has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof); or
  • the light chain includes or consists of the following sequence:
  • substitution, deletion or addition of one or several amino acids or any combination thereof for example, up to 50, up to 45, up to 40, up to 35, up to 30 , substitution, deletion or addition of up to 25, up to 20, up to 15, up to 10 or up to 5 amino acids or any combination thereof; for example 1, 2, 3, 4, 5, 6 , a sequence with substitutions, deletions or additions of 7, 8, 9 or 10 amino acids or any combination thereof; or
  • sequence shown in (vi) and (iv) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, at least 98% or at least 99% sequence identity;
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the anti-Napi2b antibody or antigen-binding fragment thereof wherein the antibody comprises a heavy chain and a light chain selected from the group consisting of:
  • Light chain preferably includes the heavy chain shown in SEQ ID NO:81 and the light chain shown in SEQ ID NO:82;
  • the antibody provided by the present disclosure is a multispecific antibody that binds Napi2b as well as one or more other antigens.
  • the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • the multispecific antibodies of the present disclosure comprise an anti-Napi2b antibody or antigen-binding fragment thereof as described above, and an additional antibody or fragment thereof or antibody analog.
  • the anti-Napi2b antibodies of the disclosure, or antigen-binding fragments thereof can be derivatized, such as linked to another molecule (eg, another polypeptide or protein).
  • another molecule eg, another polypeptide or protein
  • derivatization e.g., labeling
  • the anti-Napi2b antibodies or antigen-binding fragments thereof of the present disclosure are also intended to include such derivatized forms.
  • an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure can be linked (by chemical coupling, genetic fusion, non-covalent linkage, or other means) to one or more other molecular moieties, such as another antibody (e.g., forming Bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or an antigen-binding fragment thereof to another molecule (e.g., avidin or a polyhistidine tag).
  • another antibody e.g., forming Bispecific antibodies
  • detection reagents e.g., pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or an antigen-binding fragment thereof to another molecule (e.g., avidin or a polyhistidine tag).
  • bispecific antibody is produced by cross-linking 2 or more antibodies (either of the same type or of different types).
  • Methods for obtaining bispecific antibodies are well known in the art, and examples thereof include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods) or genetic engineering methods.
  • Another type of derivatized antibody is a labeled antibody.
  • the antibodies of the present disclosure, or antigen-binding fragments thereof can be linked to a detectable label.
  • a detectable label described in this disclosure can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • Such labels include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.
  • radionuclides fluorescein e.g., 3H, 125I, 35S, 14C
  • radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a light detector to detect the emitted light.
  • Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and thermometric markers are detected by simply visualizing a colored marker.
  • such labels can be adapted for immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • detectable labels as described above can be linked to the antibodies or antigen-binding fragments thereof of the present disclosure via linkers of varying lengths to reduce potential steric hindrance.
  • the antibodies of the present disclosure or antigen-binding fragments thereof can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.
  • chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of the antibody, such as increasing serum half-life.
  • the antibodies of the present disclosure can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy chain and light chain genes of the antibodies of the present disclosure are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions and express the antibodies of the present disclosure.
  • Antigen-binding fragments of the present disclosure can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the disclosure provides an isolated nucleic acid molecule comprising an antibody encoding the second aspect of the disclosure or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof, or one or Nucleotide sequences of multiple CDRs; in certain embodiments, the nucleotide sequences are replaceable based on codon degeneracy. In certain embodiments, the nucleotide sequence is codon optimized.
  • an isolated nucleic acid molecule of the present disclosure comprises: (i) a first sequence encoding the heavy chain variable region and the light chain variable region, respectively, of the antibody of the second aspect of the disclosure, or an antigen-binding fragment thereof; nucleic acid and a second nucleic acid, or (ii) each encoding a second aspect of the disclosure a first nucleic acid of a heavy chain variable region and a heavy chain constant region of an antibody or an antigen-binding fragment thereof, and a second nucleic acid of a light chain variable region and a light chain constant region, respectively, or (iii) encoding a second nucleic acid of the present disclosure, respectively.
  • first and second nucleic acids comprise degenerate sequences or nucleic acids that are substantially identical to any of the first and second nucleic acids in (i)-(iii) above.
  • the degenerate sequence or substantially identical sequence refers to having at least about 85%, 90%, 95%, 99% or more of the nucleic acid molecule compared to the nucleic acid molecule described in (i)-(iii). Sequences with sequence identity or sequences with one or more nucleotide substitutions, or sequences that differ by no more than 3, 6, 15, 30, or 45 nucleotides.
  • a vector comprising the isolated nucleic acid molecule of the third aspect of the present disclosure.
  • vectors of the present disclosure are cloning vectors or expression vectors.
  • vectors of the present disclosure are, for example, plasmids, cosmids, phages, lentiviruses, and the like.
  • the vector is capable of expressing an antibody or antigen-binding fragment thereof of the present disclosure in a subject (eg, a mammal, eg, a human).
  • a host cell comprising the isolated nucleic acid molecule of the third aspect of the disclosure or the vector of the fourth aspect of the disclosure.
  • the host cell may be a eukaryotic cell (eg, mammalian cell, insect cell, yeast cell) or prokaryotic cell (eg, E. coli).
  • Suitable eukaryotic cells include, but are not limited to, NSO cells, SP2/0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • host cells of the present disclosure are mammalian cells, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).
  • the sixth aspect provides a method for preparing the antibody of the second aspect of the present disclosure or the antigen-binding fragment thereof, or the multispecific antibody of the present disclosure, which includes, allowing the antibody or the antigen-binding fragment thereof, or the multispecific
  • the host cells of the present disclosure are cultured under conditions for expression of specific antibodies, and the antibodies or antigen-binding fragments thereof are recovered from the cultured host cells.
  • the seventh aspect provides an anti-Napi2b antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, wherein the antibody is the above-mentioned Napi2b-conjugated of antibodies or antigen-binding fragments thereof.
  • the antibody drug conjugate is selected from:
  • Tb 1 is an anti-Napi2b antibody or its antigen-binding fragment, such as: lifastuzumab, upifitamab, 60A12B3-hz1, 22H9C4-hz1, 66C12D12-hz1, 34F2A10-hz1, C1, 16G5B3-hz2 and 16G5B3-hz1 antibodies.
  • q is selected from any value between 0.1 and 16.0, preferably between 2 and 8. In some embodiments, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some preferred embodiments, q is 2, 4, 6 or 8.
  • the present disclosure provides a method for preparing the aforementioned antibody drug conjugate, which includes:
  • Tb has the meaning provided above and in any embodiment specifically described herein;
  • R 1 , R 2 , R 3 , X, W, L 1 , L 2 , L 3 , L 4 have the meanings provided above and in any embodiment specifically described herein.
  • Lg is a leaving group, and Lg is selected from halogen, sulfone group, tertiary amine base (Me 3 N + , Et 3 N + ), diazonium base, -OMs, MeSO 2 -, CF 3 SO 3 -.
  • Lg is selected from F, Cl, MeSO2-.
  • Lg is selected from F, MeSO2-.
  • the method includes conjugating Tb with a drug linker represented by Formula III
  • the steps of coupling reaction to form CS bonds are carried out under suitable solvents and conditions.
  • the ratio of the amount of substances of the Tb to the drug linker conjugate is 1: (1-20), such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:14, 1:16, 1:18, 1:(10-20), 1:(12-20 ), 1:(14-20), 1:(16-20) or 1:(18-20).
  • the coupling reaction is performed in water and/or organic solvents.
  • the organic solvent is selected from N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, nitriles (such as acetonitrile), alcohols (such as methanol, ethanol) or any combination thereof.
  • the method further includes the step of purifying the coupling product.
  • the coupling product is purified by chromatography.
  • the chromatography method includes one or more of ion exchange chromatography, hydrophobic chromatography, reversed phase chromatography, or affinity chromatography.
  • the present disclosure provides a group of antibody drug conjugates, which includes the aforementioned antibody drug conjugates of the present disclosure, their stereoisomers, their prodrugs, and their pharmaceutically acceptable salts or pharmaceutically acceptable solvates thereof, wherein the antibody-drug conjugate has one, two or more q values.
  • the q value and the DAR value are equal.
  • the antibody drug conjugates in the population have two or more q values
  • the antibody drug conjugates with a specific q value account for 10% of all antibody drug conjugates in the population.
  • the proportion is greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%.
  • the drug to antibody ratio (DAR) in the antibody drug conjugates in the population is selected from an integer or decimal number from 1 to 10.
  • the drug to antibody ratio (DAR) of the population is selected from the group consisting of 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5;
  • the drug to antibody ratio (DAR) of the population is selected from the group consisting of about 2.0, 4.0, 6.0, and 8.0;
  • the drug to antibody ratio (DAR) in the antibody drug conjugates in the population is selected from the group consisting of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2 , 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.
  • the population contains ADCs with a distribution of DARs from 1 to 8, for example, 1.5, 2, 4, 6, and 8 (i.e., a drug load of 1.5, 2, 4, 6, and 8 type).
  • degradation products can be generated such that DARs 1, 3, 5 and 7 can also be included in the population.
  • the group may also have a DAR greater than 8.
  • the antibody-drug conjugate is produced by reduction and subsequent coupling of interchain disulfides.
  • the antibody drug conjugate includes both: the antibody drug conjugate with a DAR of 4 or less (i.e., a drug loading species of 4 or less) and a DAR of 6 or higher (i.e., the drug loading category is 6 or higher).
  • the present disclosure provides a pharmaceutical composition, which includes the above-mentioned first or seventh aspect antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable A salt or a pharmaceutically acceptable solvate thereof, an anti-Napi2b antibody or an antigen-binding fragment thereof of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the ninth aspect A group of antibody drug conjugates, and optionally one or more pharmaceutical excipients.
  • the pharmaceutical composition includes the above-mentioned first or seventh aspect antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable salt.
  • the accepted solvate, the anti-Napi2b antibody or antigen-binding fragment thereof of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, or the antibody-drug conjugate of the ninth aspect The doses of the group are all therapeutically effective.
  • the pharmaceutical composition includes the above-mentioned first or seventh aspect antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable salt. Acceptable solvates, and pharmaceutically acceptable carriers and/or excipients.
  • the pharmaceutical composition of the present disclosure includes the antibody-drug conjugate group of the ninth aspect, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition of the present disclosure includes the above-mentioned antibody drug conjugate group, or includes an antibody drug conjugate group and a buffer.
  • pharmaceutical compositions of the present disclosure further include excipients and/or surfactants.
  • compositions of the present disclosure comprise an anti-Napi2b antibody or antigen-binding fragment thereof of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient.
  • pharmaceutical compositions of the present disclosure comprise an anti-Napi2b antibody, or antigen-binding fragment thereof, and a buffer.
  • pharmaceutical compositions of the present disclosure further include excipients and/or surfactants.
  • the buffer is a histidine buffer. In certain preferred embodiments, the buffer is a 20 mM histidine buffer with a pH of 6.0.
  • compositions of the disclosure comprise a host cell of the disclosure, and a pharmaceutically acceptable carrier and/or excipient, wherein the host cell comprises an isolated nucleic acid molecule as described above or carrier.
  • compositions of the disclosure comprise a multispecific antibody of the disclosure, and a pharmaceutically acceptable carrier and/or excipient.
  • the drug to antibody ratio (DAR) in the pharmaceutical composition is selected from an integer or decimal number from 1 to 10.
  • the drug to antibody ratio (DAR) in the pharmaceutical composition is selected from the group consisting of 1.5-2.5, 3.5-4.5, 5.5-6.5, and 7.5-8.5;
  • the drug to antibody ratio (DAR) in the pharmaceutical composition is selected from the group consisting of about 2.0, 4.0, 6.0, and 8.0;
  • the drug to antibody ratio (DAR) in the antibody drug conjugate in the pharmaceutical composition is selected from the group consisting of 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 , 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9 and 9.
  • the disclosure provides a diagnostic or therapeutic kit comprising one or more of the following: an antibody or antigen-binding fragment thereof of the disclosure, a nucleic acid, a vector, a host cell, a multispecific antibody, Antibody drug conjugates, antibody drug conjugate groups or pharmaceutical compositions.
  • the diagnostic or therapeutic kit further includes instructions for use.
  • the present disclosure provides the aforementioned antibody drug conjugate composition or the aforementioned pharmaceutical composition in the preparation of medicaments for treating and/or preventing diseases related to abnormal cell activity (such as cancer diseases). the use of.
  • the antibody-drug conjugate composition or the aforementioned pharmaceutical composition may be in a therapeutically effective amount.
  • an anti-Napi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, or multispecific antibody of the present disclosure is provided in the preparation of a medicament for modulating (inhibiting or blocking) Activity of Napi2b.
  • anti-Napi2b antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, or multispecific antibodies of the present disclosure in the preparation of medicaments for treating or preventing activity with Napi2b is provided. related diseases.
  • an anti-Napi2b antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, antibody drug conjugate, or multispecific antibody of the present disclosure is provided in the preparation of a medicament for treating or prevent tumors associated with Napi2b activity.
  • the above-mentioned first or seventh aspect antibody-drug conjugates of the present disclosure are provided, their stereoisomers, their prodrugs, A pharmaceutically acceptable salt thereof or a pharmaceutically acceptable solvate thereof, an anti-Napi2b antibody or an antigen-binding fragment thereof in the second aspect, the nucleic acid described in the third aspect, the vector described in the fourth aspect, and the host described in the fifth aspect
  • the above-mentioned first or seventh aspect of the present disclosure provides an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate.
  • the above-mentioned first or seventh aspect of the present disclosure provides an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate.
  • the above-mentioned first or seventh aspect of the present disclosure provides an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable salt.
  • Solvate, the anti-Napi2b antibody or antigen-binding fragment thereof of the second aspect, the nucleic acid of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the antibody-drug conjugate group of the ninth aspect Use of the pharmaceutical composition of the ten aspects in the preparation of a medicament for treating or preventing Napi2b-related tumors.
  • the present disclosure provides an antibody-drug conjugate of the first aspect or the seventh aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutical
  • the group of compounds, or the pharmaceutical composition of the tenth aspect are methods for treating and/or preventing diseases (eg, tumors) associated with abnormal cell activity.
  • the tumor is selected from the group consisting of esophageal cancer (such as esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer or lung cancer).
  • esophageal cancer such as esophageal adenocarcinoma and esophageal squamous cell carcinoma
  • brain tumor such as esophageal adenocarcinoma and esophageal squamous cell carcinoma
  • lung cancer such as small cell lung cancer, non-small cell lung cancer or lung cancer.
  • adenocarcinoma squamous cell carcinoma
  • bladder cancer gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colon cancer (such as human colon adenocarcinoma), rectum cancer, colorectal cancer, liver cancer, renal cancer, urothelial cancer, epidermal cancer, non-Hodgkin lymphoma, central nervous system tumors (such as glioma, glioblastoma multiforme, glioma, or sarcoma), prostate cancer, or thyroid cancer.
  • the tumor is a NaPi2b-related tumor.
  • the tumor is a Napi2b-associated tumor.
  • examples of the term "pharmaceutically acceptable salts” are organic acid adduct salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formates, acetates, propionates Salt, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, alkyl sulfonate Or aryl sulfonate; preferably, the alkyl sulfonate is methanesulfonate or ethyl sulfonate; the aryl sulfonate is benzene sulfonate or p-toluene sulfonate.
  • Suitable inorganic salts may also be formed including, but not limited to, hydrochlorides, hydrobromides, hydroiodates, nitrates, bicarbonates and carbonates, sulfates or phosphates, and the like
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, reagents to maintain osmotic pressure, Agents that delay absorption, preservatives.
  • compositions can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid that provides a pharmaceutically acceptable anion.
  • the pharmaceutical excipients refer to the excipients and additives used in the production of drugs and preparation of prescriptions. They refer to excipients and additives that have been reasonably evaluated in terms of safety in addition to active ingredients and are included in pharmaceutical preparations. substance in.
  • pharmaceutical excipients also have important functions such as solubilization, dissolution, and sustained and controlled release. They are important ingredients that may affect the quality, safety, and effectiveness of drugs. According to their origin, they can be divided into natural products, semi-synthetic products and fully synthetic products.
  • solvents can be divided into: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, Glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surface active agents Agents, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, release retardants, etc.; can be divided according to their route of administration It is available for oral administration, injection, mucosal administration, transdermal or topical administration, nasal or oral inhalation administration and ocular administration.
  • the same pharmaceutical excipients can be used in pharmaceutical preparations with different
  • the pharmaceutical composition can be prepared into various suitable dosage forms according to the route of administration.
  • suitable dosage forms for example, tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants agents, aerosols, powder mist, sprays, etc.
  • the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the compound of the present disclosure or its pharmaceutically acceptable salt or conjugate, suitably 0.1 mg to 800 mg, preferably 0.5-500 mg, preferably 0.5-500 mg. Containing 0.5 to 350 mg, particularly preferably 1 to 250 mg.
  • the pharmaceutical composition can be administered in the form of injection, including injection liquid, sterile powder for injection and concentrated solution for injection.
  • carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may be used as solvents or suspending media, such as mono- or diglycerides.
  • treatment generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing the disease or its symptoms; and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or side effects due to the disease.
  • treatment encompasses any treatment of a disease in a patient, including: (a) preventing the disease or symptoms in a patient who is susceptible to the disease or symptoms but has not yet been diagnosed with the disease; (b) suppressing the symptoms of the disease, i.e., arresting its progression; or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptoms.
  • the term "individual” includes humans or non-human animals.
  • Exemplary human subjects include human subjects (referred to as patients) suffering from a disease, such as those described herein, or normal subjects.
  • non-human animals in this disclosure includes all vertebrate animals, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).
  • the term "effective dose” refers to an amount of a compound that, when administered, alleviates to a certain extent one or more symptoms of the condition being treated.
  • biologically active refers to inhibiting or preventing the function of a cell and/or Substances that cause cell death or destruction.
  • biologically active substance, biologically active molecule or drug molecule in the conjugate is a molecule with anti-tumor biological activity.
  • radioactive isotopes such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and Lu radioactive isotopes; metal complexes, such as metal platinum complexes , metal gold complexes, oxaliplatin, etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxyl Treeline, 9-aminocamptothecin, SN-38, irinotecan, topotecan, bellotecan, rubotecan, topoisomerase II inhibitor, actinomycin D, doxorubicin, poly Ruubicin, docarmicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs
  • linker refers to a fragment that connects a biologically active molecule (drug molecule) to a targeting moiety.
  • targeting moiety refers to the portion of the conjugate that is capable of specifically binding to a target (or portion of a target) on the cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.
  • antibodies or antigen-binding fragments thereof include derivatized antibodies or antigen-binding fragments thereof, such as antibodies or antigen-binding fragments having a sulfhydryl group, wherein the derivatization causes the antibody to have a group that reacts with a drug linker conjugate. group or ability.
  • the sulfhydryl group-SH can be derivatized by opening the disulfide bond (eg, reduction by the reducing agent TCEP).
  • cancer and “tumor” are used herein with the same meaning.
  • gene as used herein includes not only DNA but also its mRNA, its cDNA and its cRNA.
  • polynucleotide is used herein in the same sense as nucleic acid and also includes DNA, RNA, probes, oligonucleotides and primers.
  • polypeptide and “protein” are used herein without distinction.
  • cell as used herein also includes cells within an individual animal and cells in culture.
  • Napi2b involved in the anti-Napi2b antibodies described in the present disclosure can be Napi2b conventional in the art, and of course also represents Napi2b variants.
  • NaPi2b NaPi2b is encoded by the SLC34A2 gene, consists of 690 amino acids, spans the membrane 8 times in structure, and has 4 extracellular loops.
  • Sodium ion-dependent phosphate transporter 2B NaPi2b maintains Pi homeostasis in the body by selectively transporting divalent Pi (HPO 4 2- ).
  • Human NaPi2b has 85.3% homology with rat and 95.8% homology with cynomolgus monkey.
  • NaPi2b has high amino acid sequence similarity with NaPi2a/2c of this family (51.29% homology with NaPi2a and 44.64% homology with NaPi2c), especially the homology in the transmembrane region is >80%.
  • KD refers to the ratio obtained from Kd (the off-rate of a specific binding molecule-target protein interaction) to Ka (the on-rate of a specific binding molecule-target protein interaction) (or Kd/Ka, expressed in molar concentration (M)) dissociation constant.
  • KD values can be determined using methods well established in the art. The preferred method of determining the KD of a bound molecule is by using surface plasmon resonance, such as a biosensor system such as the BiacoreTM (GE Healthcare Life Sciences) system.
  • percent homology between two amino acid sequences is equal to percent identity between the two sequences.
  • Sequence comparisons and determination of percent identity between sequences can be performed using methods generally known in the art, and mathematical algorithms can be used to accomplish such sequence comparisons and determination of percent identity. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci.
  • ALIGN program version 2.0
  • the GAP program in the GCG software package available online from Accelrys can be used to determine percent identity between amino acid sequences or between nucleotide sequences.
  • the two sequences are of equal length.
  • epitope refers to a portion of an antigenic polypeptide or protein that has antigenic or immunogenic activity in an animal body, preferably a mammal.
  • the epitopes of the antibodies of the present disclosure or antigen-binding fragments thereof can be determined by existing techniques, such as synthetic peptide methods, immunoinformatics prediction, determination of polypeptide activity, epitope peptide scanning methods, phage display technology, X-ray diffraction and nuclear magnetic resonance Analysis, antibody homology modeling and protein docking prediction method.
  • the phrase "antibodies that bind the same epitope” as used herein means different antibodies that bind a common epitope. If the second antibody binds to part of the peptide or part of the tertiary structure bound to the first antibody, then it can be determined that the first antibody and the second antibody bind to the same epitope.
  • antibody is taken in its broadest interpretation and includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, as long as they have the required required biological activity.
  • antibody and “immunoglobulin” are used interchangeably.
  • Antibody molecule or “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen-binding site that immunospecifically binds an antigen.
  • antibody broadly encompasses not only intact antibody molecules, but also fragments of said antibodies and variants (including derivatives) of said antibodies and antibody fragments.
  • antibody molecule refers to an intact antibody molecule or full-length antibody.
  • antibody molecule used in this specification include, but are not limited to, single chain Fv (scFv), Fab fragment, Fab' fragment, F(ab')2, disulfide-linked Fv (sdFv), Fv, and intact antibodies. or full-length antibodies.
  • single chain Fv or “scFv” refers to a polypeptide comprising the VL domain of an antibody linked to the VH domain of the antibody.
  • antibodies that immunospecifically bind Napi2b can cross-react with other antigens.
  • the antibody that immunospecifically binds Napi2b does not cross-react with other antigens. Immunospecific binding can be identified, for example, by immunoassays or other methods known to those skilled in the art.
  • An "intact" antibody or “full-length” antibody refers to a protein containing two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds, the protein containing: ( 1) in the case of a heavy chain, a variable region (herein abbreviated as "VH”) and a heavy chain constant region containing three domains CH1, CH2, CH3; and (2) in the case of a light chain, a light chain
  • VH variable region
  • VL variable region
  • the light chain constant region contain one domain CL.
  • Antibodies of the disclosure include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, anti-idiotypic (anti-Id) antibodies (including, for example, antibodies of the disclosure anti-Id antibodies), and epitope-binding fragments of any of the above antibodies.
  • the immunoglobulin molecules of the present disclosure may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin.
  • the antibody of the present disclosure includes or consists of a VH domain, a VH CDR (mostly represented by HCDR in this article), a VL domain, or a VL domain having any amino acid sequence described in the sequence and its specific information table, or a fragment or variant thereof.
  • CDR (LCDR is mostly used in this article) represents) composition.
  • the term "monoclonal antibody” refers to an antibody derived from a population of antibodies that is substantially homogeneous, that is, the individual antibodies that make up the population are identical except for the possible presence of a small number of natural mutations.
  • Monoclonal antibodies have high specificity for one determinant (epitope) of an antigen, whereas polyclonal antibodies contain different antibodies directed against different determinants (epitope).
  • epitopope epitope
  • the advantage of monoclonal antibodies is that they can be synthesized without contamination from other antibodies.
  • the modifier "monoclonal” here indicates that the antibody is characterized by being derived from a substantially homogeneous population of antibodies and should not be construed as requiring that it be prepared by a specific method.
  • monoclonal antibodies also specifically include chimeric antibodies, that is, a part of the heavy chain and/or light chain is identical or homologous to a certain type, type or subtype of antibody, and the remaining part is identical to another type of antibody.
  • Antibodies of one, another, or subclass are identical or homologous as long as they possess the desired biological activity (see, e.g., US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855).
  • Chimeric antibodies useful in the present disclosure include primatized antibodies comprising variable region antigen-binding sequences and human constant region sequences from non-human primates (eg, monkeys, orangutans, etc.).
  • antigen-binding fragment refers to a portion of an antibody, preferably the antigen-binding region or variable region.
  • antibody fragments include Fab, Fab', F(ab') 2 , Fd, Fv, dAb and complementarity determining region fragments, diabodies, linear antibodies and single chain antibody molecules.
  • antigen-binding fragment refers to a partial fragment of an antibody with antigen-binding activity, wherein the fragment has all or part of the functions of the antibody, including but not limited to single-chain Fv (scFv), Fab, Fab', F( ab')2, disulfide-linked Fv (sdFv), Fv, di-scFv, etc.
  • the term also includes Fab', which is a monovalent fragment of the variable region of an antibody resulting from treatment of F(ab')2 under reducing conditions.
  • Fab' is a monovalent fragment of the variable region of an antibody resulting from treatment of F(ab')2 under reducing conditions.
  • the term is not limited to these molecules as long as the fragment has binding affinity for the antigen.
  • these functional fragments include not only fragments obtained by treating the full-length molecule of the antibody protein with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes.
  • Fab' refers to a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above.
  • Fab' of the present disclosure also includes Fab' produced using genetically modified antibody genes.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker or directly (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.
  • a linker having the amino acid sequence (GGGGS) 4 can be 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 VH and VL of scFv.
  • di-scFv refers to an antibody fragment formed by the joining of two scFvs.
  • the antibody or antigen-binding fragment thereof contains variants, amino acid substitutions, deletions, or additions that still have the activity to bind the antigen.
  • bispecific antibody is also called “bifunctional antibody conjugate” and refers to a antibody composed of a first antibody (fragment) and a second antibody (fragment).
  • the conjugate formed by the coupling arm retains the activity of the respective antibodies, so it has bifunctionality and bispecificity.
  • multispecific antibody includes, for example, trispecific antibodies, which are antibodies with three different antigen-binding specificities, and tetraspecific antibodies, which are antibodies with four different antigen-binding specificities.
  • intact antibody or “full-length antibody” refers to an antibody that contains an antigen-binding variable region and a light chain constant region (CL), heavy chain constant region (CH1, CH2, and CH3).
  • the constant region may be a native sequence (eg, a human native constant region sequence) or an amino acid sequence variant thereof.
  • Intact antibodies are preferably intact antibodies with one or more effector functions.
  • antibody is a modified antibody, including an antibody or an antibody fragment that can specifically bind to its target and can be coupled with a masking group, where the masking group refers to the antibody or antibody fragment with which it binds
  • the cleavage constant of the binding ability of the target site is at least 100 times, 1000 times, or 10000 times greater than the cleavage constant of the binding ability of the antibody or antibody fragment without the coupling masking group to its target site.
  • a "humanized" form of a non-human (eg, murine) antibody refers to a chimeric antibody that contains a minimal amount of non-human immunoglobulin sequence.
  • Most humanized antibodies are human recipient immunoglobulins whose hypervariable region residues have been replaced with non-human (e.g., mouse, rat, rabbit, or non-human primate) species that have the desired specificity, affinity, and function. Hypervariable region residues (donor antibody).
  • donor antibody In some embodiments, framework region (FR) residues of human immunoglobulins are also replaced with non-human residues.
  • humanized antibodies may also contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are intended to further optimize the antibody's performance.
  • Humanized antibodies generally contain at least one, and usually two, variable regions, in which all or nearly all hypervanable loops correspond to those of non-human immunoglobulins, while the FRs are entirely or almost entirely human immunoglobulins. Protein sequence.
  • the humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc, typically a human immunoglobulin Fc).
  • Fc immunoglobulin constant region
  • Intact antibodies can be divided into different "classes” based on the amino acid sequence of the heavy chain constant region.
  • the five main classes are IgA, IgD, IgE, IgG and IgM, several of which can also be divided into different "subclasses” (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
  • the heavy chain constant regions of different classes of antibodies are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art.
  • the CDRs contained in the antibodies of the present disclosure or antigen-binding fragments thereof can be determined according to various numbering systems known in the art.
  • the antibodies of the disclosure, or antigen-binding fragments thereof contain CDRs preferably determined by the Kabat, Chothia, or AbM or IMGT numbering systems.
  • framework residue region or "FR residue” refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • germline antibody gene is a gene encoding a non-lymphocyte-expressed immunoglobulin that has not undergone the maturation process leading to genetic rearrangement and maturation that results in the expression of a specific immunoglobulin.
  • amino acid substitutions conservative amino acid substitutions are preferred in this specification.
  • Conservative amino acid substitutions represent substitutions that occur within a set of amino acids relative to amino acid side chains.
  • Preferred amino acid sets are as follows: acidic set (aspartic acid and glutamic acid); basic set (lysine, arginine and histidine); non-polar set (alanine, valine, leucine Amino acid, isoleucine, proline, phenylalanine acid, methionine, and tryptophan); and the uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine).
  • More preferred sets of amino acids are as follows: aliphatic hydroxyl groups (serine and threonine); amide-containing sets (asparagine and glutamine); aliphatic sets (alanine, valine, leucine and isoleucine). amino acids); and aromatic sets (phenylalanine, tryptophan, and tyrosine). Such amino acid substitutions are preferably made within a set that does not impair the properties of the material having the original amino acid sequence.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art.
  • alanine can be represented by A or Ala
  • arginine can be represented by R or Arg
  • glycine can be represented by G or Gly
  • glutamine can be represented by Q or Gln.
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom (eg, tumors and infectious diseases) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no further worsening) of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the symptoms of the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a non-human primate mammal, or a human.
  • the subject eg, human
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent disease refers to an amount that is sufficient to prevent, prevent, or delay the occurrence of a disease (for example, tumors and infectious diseases);
  • an effective amount to treat a disease refers to an amount that is sufficient to cure or at least partially cure Amount to prevent disease and its complications in patients who already have the disease. Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • effector function refers to those biological activities attributable to the Fc region of an antibody (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with the antibody. Vary according to the same type.
  • pharmaceutically acceptable means that the molecule itself, molecule fragments or compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • substances that can serve as pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.
  • antibody-drug conjugate group refers to a group or group of antibody-drug conjugates of the present invention, their stereoisomers, their prodrugs, their pharmaceutically acceptable salts, their tautomers, or a mixture of pharmaceutically acceptable solvates thereof, wherein the antibody drug conjugate
  • the q's can be the same or different. In addition, it may also be called an "antibody-drug conjugate mixture.”
  • the in vivo therapeutic effect of antibodies and antibody-drug conjugates on cancer can be determined using experimental animals, for example, by administering antibodies to nude mice implanted with a Napi2b-expressing tumor cell line and measuring any changes in cancer cells.
  • the amino acid substitution in the antibody is by L-amino acid, it is not limited thereto.
  • one or more D-amino acids may be included in the antibody peptide chain. Peptides containing D-amino acids are more stable and less susceptible to degradation in the oral cavity, intestine, or plasma than peptides containing only L-amino acids.
  • Monoclonal antibodies used in the present disclosure can be produced by a number of methods.
  • monoclonal antibodies useful in the present disclosure can be obtained by hybridoma methods using cells from a number of species, including mouse, hamster, rat, and human (see, e.g., Kohler et al., 1975, Nature, 256:495). Either prepared by recombinant DNA technology (see, e.g., US 4,816,567), or isolated from a phage antibody library (see, e.g., Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology , 222:581-597).
  • direct bond means that the groups on both sides are directly connected, for example, the compound represented by formula II , if X is a direct bond, its structural formula is The remaining direct keys can be understood by referring to the above content.
  • absent means that the group is not present, for example, in the compound of formula II , if W does not exist, its structural formula is
  • the drug linker conjugate represented by formula III in, L 4 is When , the labels 1 and 2 indicate the connecting positions of L 4 and other groups. Specifically, position 1 is connected to L 3 , and position 2 is connected to the drug molecule, that is, When connected, they form The remaining numbers 1 and 2 can be understood with reference to the foregoing content.
  • X is selected from optionally substituted
  • the substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl)"
  • X can be, for example
  • Other similar definitions of X can be understood with reference to the foregoing content.
  • X is selected from optionally substituted
  • the substituent is selected from two C1-4 alkyl groups (such as methyl) and the carbon atoms connected to them simultaneously form a C3-6 cycloalkyl group (such as cyclopropyl)", then X can be, for example Other similar definitions of X can be understood with reference to the foregoing content.
  • the structure of the amino acid residue shown in AA 1 if R a and R b together with the carbon atoms to which they are commonly attached form a 4-10 membered heterocycle, the 4-10 membered heterocycle is optionally substituted by one or more R 0 , where the term "The 4-10 membered heterocycle is optionally substituted by one or more R 0 " means that the 4-10 membered heterocycle may not be substituted or may be substituted by one or more R 0 , and among the plurality of R 0 , the definition of each R 0 may be the same or different. Other similar definitions can be understood with reference to the foregoing content.
  • L 3 is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala-Asn
  • the distal amino group of lysine (Lys) is optionally replaced by 1, 2 or 3 substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O
  • substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl)
  • substituted is that the distal amino group of Lys in each option of L 3 is optionally replaced by 1, 2 or 3 selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O substituted by substituents.
  • “Lys far "Amino terminal” refers to the lysine residue exposed amino group -NH 2 .
  • the distal amino group of lysine (Lys) is optionally substituted by 1, 2 or 3 substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O” It means that the distal amino group of lysine (Lys) may not be substituted, or may be substituted by 1, 2 or 3 selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O group, for example, it can be substituted by 1 tert-butoxycarbonyl group, which becomes Alternatively, it can be substituted by 2 methyl groups, which becomes Or be replaced by 2 methyl groups and 1 O at the same time, which becomes It should be noted that "the distal amino group of lysine (Lys) is substituted by O” means that the distal amino group of lysine (Lys) is oxo-substit
  • substituents of the compounds of the present disclosure are disclosed by group type or range.
  • this disclosure includes each and every individual subcombination of individual members of these radical classes and ranges.
  • C1-6 alkyl refers specifically to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.
  • C1-6 alkyl refers to a linear or branched alkyl group containing 1 to 6 carbon atoms, including, for example, “C1-3 alkyl” or “C1-4 alkyl", methyl , ethyl, etc. Specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl.
  • C 1-4 alkyl refers to a straight-chain or branched alkyl group containing 1-4 carbon atoms, including, for example, "C 1-3 alkyl", methyl, ethyl, etc., specifically Examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
  • C 2-6 alkenyl refers to a linear, branched or cyclic alkenyl group containing at least one double bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 alkenyl "wait.
  • Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl , 2-hexenyl, 3-hexenyl, 1,4-hexenyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexenyl Key et al.
  • C 2-6 alkynyl refers to a linear or branched chain alkynyl group containing at least one triple bond and having 2 to 6 carbon atoms, including, for example, "C 2-4 alkynyl” and the like. Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 -Hexynyl, 5-methyl-2-hexynyl, etc.
  • halogen includes fluorine, chlorine, bromine, and iodine.
  • 3-6 membered cycloalkyl or "C 3-6 cycloalkyl” refers to a saturated cyclic alkyl group containing 3 to 6 carbon atoms, including cyclopropyl (i.e., cyclopropyl) , cyclobutyl (ie cyclobutyl), cyclopentyl (ie cyclopentyl), cyclohexyl.
  • 3-7 membered carbocycloalkyl or "C 3-7 cycloalkyl” refers to a saturated cyclic alkyl group containing 3-7 carbon atoms, including cyclopropyl, cyclobutanyl , cyclopentyl, cyclohexyl, cycloheptyl.
  • C1-6 alkoxy refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentoxy base, hexyloxy group, etc.
  • C 1-4 alkoxy refers to an alkyl group as defined above attached to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.
  • the term "4-10 membered heterocyclyl” refers to a cyclic group containing 4-10 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom).
  • the term "4-6 membered heterocyclyl” refers to a cyclic group containing 4-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom).
  • ring atoms (eg carbon atoms, nitrogen atoms or sulfur atoms) in the cyclic structure can be oxo-substituted.
  • “4-8 membered heterocyclic group” includes, for example, “4-8 membered nitrogen-containing heterocyclic group”, “4-8 membered oxygen-containing heterocyclic group”, “4-7 membered heterocyclic group”, “4-7 membered heterocyclic group” "Oxygen-containing heterocyclyl", “4-7-membered heterocyclyl”, “4-6-membered heterocyclyl”, “5-7-membered heterocyclyl”, “5-6-membered heterocyclyl”, “5- “6-membered nitrogen-containing heterocyclic group” includes but is not limited to oxocyclobutanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.
  • the term "4-10 membered heterocycle” refers to a ring containing 4-10 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom).
  • the term “5-6 membered heterocycle” refers to a ring containing 5-6 ring atoms (at least one of which is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, Piperidine, piperazine, tetrahydropyran and other rings.
  • aryl refers to an aromatic monocyclic or polycyclic hydrocarbon group, such as a 6-10-membered aryl group, a 5-8-membered aryl group, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, etc.
  • the "6-10 membered aryl group” refers to an aryl group containing 6-10 ring atoms.
  • C6-10 aryl group refers to an aryl group containing 6-10 carbon atoms.
  • heteroaryl refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom.
  • ring atoms eg carbon atoms, nitrogen atoms or sulfur atoms
  • cyclic structure can be oxo-substituted.
  • bonds in the structural formulas represented by wavy lines " ⁇ " herein are intended to indicate that the structure represents cis or trans isomers, or a mixture of cis and trans isomers in any proportion.
  • DAR drug to antibody ratio
  • the term "drug to antibody ratio” or “DAR” refers to the ratio of drug and antibody in a population (or mixture) or composition or ADC molecule, e.g., a small molecule toxin attached to the antibody of the ADC.
  • the DAR of an ADC can range from 1 to 16, but depending on the number of attachment sites on the antibody, higher loadings (e.g. 20) are also possible.
  • the term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. It should be understood that the latter is often referred to as average DAR.
  • the antibody has been reduced to separated heavy and light chains.
  • DAR1 represents a conjugate containing a light chain or heavy chain coupled to one toxin molecule
  • DAR2 represents a conjugate containing a light chain or heavy chain coupled.
  • DAR3 represents conjugates containing light or heavy chains coupled to 3 toxin molecules.
  • the conjugate obtained according to the above method has better solubility and excellent chemical stability. If the reversible Michael addition reaction caused by the maleimide connection method in traditional ADC does not occur, a high drug-antibody ratio can be obtained. In some embodiments, the DAR value of the conjugate can reach 6-8;
  • the coupling efficiency can reach or exceed 90%
  • linker has high plasma stability but can also be cleaved in the tumor microenvironment (both within tumor cells and outside tumor cells). Therefore, it can be used in tumors with low antigen expression or no antigen expression. produce good anti-tumor effects;
  • the conjugate (ADC) obtained according to the above method improves the exposure of the entire ADC molecule in a relatively acidic tumor environment by adjusting the physical and chemical properties of the linker and the overall ADC molecule, so the ADC has better tumor tissue targeting.
  • property that is, the ability to enrich in the tumor microenvironment, increases the concentration ratio of bioactive molecules in the tumor to blood, and reduces the mechanism-related toxicity of ADC molecules (produced after ADC binds to cell surface antigens in non-tumor tissues and is endocytosed) toxicity, or "on-target toxicity", therefore has a higher therapeutic index;
  • the conjugate obtained according to the above method has high stability in the body circulation, reduces the shedding of drug molecules in non-target tissues, and reduces the "off-target” toxicity caused by the shedding of toxins in non-target tissues;
  • the bioactive molecules of the conjugate have higher anti-tumor cell activity and therefore have excellent by-stander effect.
  • the ADC can more effectively kill tumor cells with high antigen expression and low antigen expression in tumor tissues. Tumor cells that express or do not express the antigen;
  • the toxin-linker disclosed in the present disclosure can utilize the extracellular cleavage ability of its linker in the tumor microenvironment to form an antibody-conjugated drug with an antibody without cell endocytosis ability.
  • This type of antibody-conjugated drug still has high anti-tumor activity.
  • the toxin-linker of the present disclosure by utilizing the extracellular cleavage ability of the linker in the tumor microenvironment and its enrichment ability in the tumor microenvironment, can bind to antibodies without cell endocytosis ability and tumor extracellular antigens.
  • the anti-Napi2b antibodies provided by the present disclosure have an extremely high degree of humanization or fully human antibodies, and thus can be safely administered to human subjects without triggering immunogenic reactions.
  • the ADC of the present invention has significant clinical value.
  • FIG. 3A Affinity detection of 66C12D12-hz1 antibody in serum
  • the measuring instrument of nuclear magnetic resonance uses a Bruker 400MHz nuclear magnetic resonance instrument; the measuring solvent is deuterated methanol (CD 3 OD), deuterated chloroform (CDCl 3 ) or hexadeuterated dimethyl sulfoxide (DMSO-d 6 ); the internal standard substance is tetramethylsilane (TMS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • ESI Agilent 6120B
  • Step 1 4-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (4.6g) is dissolved in anhydrous tetrahydrofuran (50ml). The above solution is treated with dry ice-acetone under nitrogen protection. Cool to -78°C, then add n-butyllithium (12ml, 2M) dropwise, stir the reaction solution at -78°C for 20 minutes, then add 2-amino-4-fluoro-5-methylbenzoic acid methyl ester (1.83g ), after the addition is completed, the reaction naturally rises to room temperature, and the reaction is continued with stirring for 5 hours.
  • Step 2 (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indeno-3,6,10(4H)-trione (2.63g), (2-amino-4-fluoro-5-methylphenyl)(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-4-yl)methanone( 3.03g), p-toluenesulfonic acid (1.74g) was dissolved in dichloromethane (50ml), then the solvent was removed, and the mixture was heated to 120°C under nitrogen protection for 4 hours.
  • Step 1 (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxola[4, Synthesis of 5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione
  • reaction solution was stirred at 0°C for 5 minutes, then raised to room temperature, and stirred for 3 hours.
  • the reaction solution was diluted with water (50 mL), extracted with ethyl acetate (80 mL Aqueous solution: 5%-60%) was further purified to obtain the target compound (yellow solid, 400 mg, yield: 67%).
  • Step 2 (S)-14-(3-azidopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxola[ Synthesis of 4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione
  • Step 3 (S)-14-(3-aminopropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxola[4, Synthesis of 5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-8,11(7H)-dione
  • Example A1.6 (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxetane Penta[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)-2-hydroxyethyl Synthesis of Amide (A1.6)
  • Step 1 (S)-2-(ethyl(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3 ]dioxol[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)amino)-2 -Preparation of oxyethyl acetate (A1.6-B):
  • Step 2 (S)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1 ,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl Synthesis of 2-hydroxyacetamide (A1.6)
  • Step 1 Preparation of the compound methylbenzyl (3-(6-nitrobenzo[d][1,3]dioxetane-5-yl)-3-oxopropyl) carbamate
  • the reaction solution was extracted with ethyl acetate (30 mL ⁇ 95%) to obtain the target compound (A1.7-B, 180 mg, yield 23%).
  • Step 2 Compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(methyl)carbamate (A1.7 Preparation of -C)
  • Step 3 Benzyl (S)-(2-(7-ethyl-7-hydroxy-8,11-dioxy-7,8,11,13-tetrahydro-10H-[1,3]dioxy Heterocycle[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(methyl)aminomethyl
  • acid ester A1.7-D
  • Step 4 (S)7-ethyl-7-hydroxy-14-(2-(methylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxetane[4, Preparation of 5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-8,11(7H)-dione (A1.7-E)
  • Step 5 Compound (S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3] Dioxyol [4,5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-14-yl)ethyl)(methyl)amino)
  • Step 6 Compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]di Oxyheterocycle[4,5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N -Preparation of methylacetamide (A1.7)
  • Example A1.8 (S)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro- 10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14 Synthesis of -ethyl)-2-hydroxyacetamide (A1.8)
  • Step 1 Compound isopropylbenzyl (3-(6-nitrobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)carbamate (A1.8 - Preparation of B)
  • Step 2 Compound benzyl (3-(6-aminobenzo[d][1,3]dioxin-5-yl)-3-oxypropyl)(isopropyl)carbamate (A1. Preparation of 8-C)
  • Step 3 (S)7-ethyl-7-hydroxy-14-(2-(isopropylamino)ethyl)-10,13-dihydro-11H-[1,3]dioxetane[4, Preparation of 5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinoline-8,11(7H)-dione (A1.8-D)
  • the reaction was carried out at 120 The reaction was carried out under vacuum for 2 hours.
  • the reaction solution was brought to room temperature, water (50 mL) was added, and extracted with dichloromethane (30 mL
  • Step 4 Compound ((S)-2-((2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3 ]dioxa[4,5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-14-yl)ethyl)(isopropyl)
  • Step 5 Compound (S)-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]di Oxo[4,5-g]pyrano[3',4':6,7]indolazine[1,2-b]quinolin-14-yl)ethyl)-2-hydroxy-N- Preparation of isopropylacetamide (A1.8)
  • the combined organic phases were dried and filtered over anhydrous sodium sulfate in sequence, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • Dissolve compound A1.9 (200mg, 0.444mmol) in dimethyl sulfoxide (2mL), add IBX (311mg, 1.11mmol), stir at room temperature for 2 hours, then add IBX (186mg, 0.666mmol) to the reaction solution Then, tetrahydropyrrole (6.3 mg, 0.089 mmol) and acetonitrile (3 mL) were added to the reaction solution, and the reaction solution was stirred at room temperature overnight. LCMS detects that the reaction is complete.
  • reaction solution was brought to room temperature, water (50 mL) was added, and extracted with dichloromethane (30 mL ⁇ 3). The organic phases were combined and dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain compound A1.15-E (390 mg , yield 76%) as a brown solid.
  • the compound was further purified by high-performance liquid phase (acetonitrile/water containing 0.05% formic acid) to obtain the target compound (S)-4-ethyl Base-8-fluoro-4-hydroxy-11-(3-isopropylaminopropyl)-9-methyl-1,12-dihydro-14H-pyrano[3',4':6,7 ] Indolozino[1,2-b]quinoline-3,14(4H)-dione (A1.16, 2.03 mg).
  • Raney Nickel (609 mg, 10.5 mmol) was added to a mixed solution of compound A1.18C (1.0 g, 2.1 mmol) in methanol (25 mL) and tetrahydrofuran (50 mL), and the reaction solution was stirred under a hydrogen atmosphere at room temperature for 5 hours.
  • Example B1.1 (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4 ,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)butoxy)methyl)ethyl Synthesis of Amide (B1.1)
  • Example B1.2 (S)-2-((2-aminoacetamido)methoxy)-N-ethyl-N-(2-(7-ethyl-7-hydroxy-8,11- Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxola[4,5-g]pyrano[3',4':6,7] Synthesis of indolozino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.2)
  • Example B1.3 (S)-2-amino-N-((2-((((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3, 4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)amino)-2 Synthesis of -Oxoethoxy)methyl)acetamide (B1.3)
  • Example B1.4 (S)-2-amino-N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14 -Synthesis of tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)methyl)acetamide (B1.4)
  • DIPEA (200ul) and HBTU (420mg) were added to the DMF (5ml) solution of compound (B1.4-A) (175mg) and compound (B1.3-A) (409mg).
  • the compound was reacted with gentle stirring at room temperature for 20 hours.
  • the mixture was added with ethyl acetate (100ml), washed with water (100ml x 3), the organic solvent was removed under reduced pressure, and then 1:1 DCM/TFA (10ml) was added to the residue and left at room temperature for 20 minutes.
  • Example B1.6 (S)-2-amino-N-((4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14- Synthesis of (1H-pyrazol-1-yl)methyl)acetamide (B1.6)
  • Example B1.7 (S)-2-amino-N-((2-((((7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro -10H-[1,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline- Synthesis of 14-yl)methyl)amino)-2-oxoethoxy)methyl)acetamide (B1.7)
  • Example B1.8 (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl )Synthesis of phenyl)acetamide (B1.8)
  • Example B1.9 (S)-2-amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl )Synthesis of phenyl)acetamide (B1.9)
  • the crude product is purified by preparative chromatography (0.01% trifluoroacetic acid aqueous solution, acetonitrile) to obtain the target product (S)-2- Amino-N-(4-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]-dioxolane And[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-14-yl)phenyl)acetamide (B1.9) (26.3 mg, yield 38%) was a yellow solid.
  • Example B1.10 (S)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]-dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14- Synthesis of acetyl)propyl)acetamide (B1.10)
  • Example B1.11 (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11 -Dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxola[4,5-g]pyrano[3',4':6,7 Synthesis of indolozino[1,2-b]quinolin-14-yl)ethyl)acetamide (B1.11)
  • Step 2 (S)-2-((2-aminoacetamido)methoxy)-N-isopropyl-N-(2-(7-ethyl-7-hydroxy-8,11-dioxo) Generation-7,8,11,13-tetrahydro-10H-[1,3]dioxola[4,5-g]pyrano[3',4':6,7]indole Preparation of Azino[1,2-b]quinolin-14-yl)ethyl)acetamide
  • Example B1.12 (S)-2-amino-N-((4-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4 ,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)propoxy)methyl)ethyl Synthesis of Amide (B1.12)
  • Step 2 Dissolve B1.12-A (240 mg) in DMF (5 ml), add piperidine (1 ml), stir the compound for 20 minutes, dissolve it under reduced pressure and remove low boiling point components, and the residue is directly used in the next step of synthesis.
  • a small amount of the crude product was purified by reverse phase chromatography (acetonitrile/0.05% FA in water: 5% to 50%) to obtain the target compound.
  • Example B1.14 (S)-2-amino-N-((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H -[1,3]dioxola[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14- Synthesis of methyl)propoxy)methyl)acetamide (B1.14)
  • PPTS pyridinium p-toluenesulfonate
  • Step 2 Dissolve B1.14-A (240 mg) in DMF (5 ml), add piperidine (1 ml), stir the compound for 20 minutes, dissolve it under reduced pressure and remove the low boiling point components, and the residue is directly used in the next step of synthesis. A small amount of the crude product was purified by reverse phase chromatography (acetonitrile/0.05% FA in water: 5% to 50%) to obtain the target compound.
  • Example B1.15 (S,E)-2-amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro- 10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-14 -yl)allyl)acetamide
  • Example B1.16 (S,E)-2-amino-N-(((3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydrofuran) Hydrogen-10H-[1,3]dioxolo[4,5-g]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -14-yl)allyl)oxy)methyl)acetamide
  • raw material A1.13 (150 mg, 0.335 mmol) was dissolved in toluene (3 mL), and (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido was added ) methyl acetate (B1.1-A, 308 mg, 0.84 mmol), then zinc acetate (123 mg, 0.67 mmol) was added to the reaction solution, and the reaction solution was stirred at 100°C overnight. LCMS detects that the reaction is complete.
  • the reaction solution was directly purified by reversed-phase chromatography (acetonitrile/0.05% formic acid aqueous solution: 5% to 50%) to obtain the target compound (9H-fluoren-9-yl)methyl (S, E)-(2-(( (3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxeterocycle[4,5-g] Pyran[3',4':6,7]indolazine[1,2-b]quinolin-14-yl)allyl)oxy)methyl)amino)-2-oxoethyl) Carbamate (B1.16-A, 80 mg, yield 31%) is a white solid.
  • Example B1.17 (S,E)-2-amino-N-(((3-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-oxo-3 ,4,12,14-Tetrahydro- 1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-11-yl)allyl)oxy)methyl)acetamide
  • Example C1.1 N 6 -(tert-butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo- 5,8,11,14,17,20,23,26,29-Naoxa-2-aza-31-alkanoyl)-L-valine)-L-lysine (compound C1.1 )
  • Step 1 29-azido-3,6,9,12,15,18,21,24,27-nonoxa-29-alkanoic acid tert-butyl ester
  • Step 2 29-amino-3,6,9,12,15,18,21,24,27-nonoxa-29-alkanoic acid tert-butyl ester
  • Step 3 1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonoxa-2-nitrogen Hetero-31-alkanoic acid
  • Step 4 N 6 -(tert-butoxycarbonyl)-N 2 -((1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17 ,20,23,26,29-nona-2-aza-31-alkanoyl)-L-valine)-L-lysine
  • Example C1.2 N 6 -(tert-butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl))-1H-1,2,3- Triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonoxa-29-alkanoyl)-L-valine)-L-lysine (compound C1.2)
  • Step 1 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18 ,21,24,27-Naoxa-29-alkanoate tert-butyl ester
  • reaction solution was stirred at room temperature for 2 hours, then added ethyl acetate (100ml), washed with water (100ml x 3), dried and removed the organic solvent under reduced pressure, and the crude product was separated by silica gel column chromatography to obtain the target product 29-(4-(2 -(Methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonoxa -29-tert-butylalkanoate.
  • Step 2 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18 ,21,24,27-nonoxa-29-alkanoic acid
  • the mixture was allowed to stand at room temperature for 1 hour, and then the low-boiling components were removed under reduced pressure to obtain the target product 29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazole-1 -base)-3,6,9,12,15,18,21,24,27-nonoxa-29-alkanoic acid.
  • Step 3 N 6 -(tert-butoxycarbonyl)-N 2 -((29-(4-(2-(methylthio)pyrimidin-5-yl))-1H-1,2,3-triazole- 1-yl)-3,6,9,12,15,18,21,24,27-nonoxa-29-alkanoyl)-L-valine)-L-lysine
  • Example C1.3 (1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8,11,14,17,20,23,26,29-nonaoxa -2-Aza-31-alkanoyl)-glycine-glycine-L-phenylalanine (compound C1.3)
  • the target product (1-(2-(methylthio)pyrimidin-5-yl)-1-oxo-5,8, 11,14,17,20,23,26,29-Naoxa-2-aza-31-alkanoyl)-glycine-glycine-L-phenylalanine (compound C1.3).
  • Example C1.4 (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nonaoxa -30-Aza-36-(35-yne)-acyl)glycineglycinephenylalanine (compound C1.4)
  • the target product (36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3 is obtained ,6,9,12,15,18,21,24,27-nona-30-aza-36-(35-yne)-acyl)glycineglycinephenylalanine (compound C1.4) .
  • Example C1.5 N 6 -(tert-butoxycarbonyl)-N 2 -((36-(2-(methylthio)pyrimidin-5-yl)-31-oxo-3,6,9, 12,15,18,21,24,27-nona-30-aza-36-(35-yne)-acyl)-L-valine)-L-lysine (C1.5 )
  • the target product N 6 -(tert-butoxycarbonyl)-N 2 -((36-(2-( Methylthio)pyrimidin-5-yl)-31-oxo-3,6,9,12,15,18,21,24,27-nona-30-aza-36-(35- Alkyne)-acyl)-L-valine)-L-lysine (compound C1.5).
  • Example C1.6 N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)-hex-5-ynyl)-L-valer Acid)-L-lysine (compound C1.6)
  • Example C1.7 N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)-hex-5-ynyl)-L-valer Acid)-L-lysine (compound C1.7)
  • Potassium peroxymonosulfonate (1.414g) was added to a mixed solution of compound C1.6 (260 mg) in tetrahydrofuran (3 mL) and water (3 mL), and the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered, and the filtrate was purified using a C18 column (acetonitrile/0.05% formic acid aqueous solution: 5%-60%) to obtain the target compound N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-( Methanesulfonyl)pyrimidin-5-yl)-hex-5-ynyl)-L-valine)-L-lysine (compound C1.7) (120 mg).
  • Example C1.8 N 6 -(tert-butoxycarbonyl)-N 2 -((6-(2-(methylthio)pyrimidine-5-carboxamido)hexanoyl)-L-valine) -L-Lai Acid (compound C1.8)
  • Example C1.9 N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3- Triazol-1-yl)hexanoyl)-L-valine)-L-lysine (compound C1.9)
  • Step 1 6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)hexanoic acid
  • Step 2 N 6 -(tert-butoxycarbonyl)-N 2 -((6-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazole- 1-yl)hexanoyl)-L-valine)-L-lysine
  • Example C1.10 (6-(2-(methylthio)pyrimidin-5-yl)hex-(5-yne)-acyl)glycineglycine-L-phenylalanine (compound C1.10)
  • Example C1.6 Using the same method and reaction conditions as in Example C1.6 and using different reaction raw materials, the target product in the table below was obtained.
  • Example C1.13 (29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)-3,6,9,12 ,15,18,21,24,27-Naoxa29alkanoyl)glycineglycine-L-phenylalanine (compound C1.13)
  • the target product (29-(4-(2-(methylthio)pyrimidin-5-yl)-1H-1,2, 3-Triazol-1-yl)-3,6,9,12,15,18,21,24,27-nonazoyl)glycineglycine-L-phenylalanine (compound C1.13) .
  • Example C1.14 N 6 -(tert-butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9 ,12,15,18,21,24,27,30-nonoxa-6,33-diazatrinonadecan-38-ynyl)-L-valine)-L-lysine ( C1.14)
  • the solvent was concentrated to remove the crude product, which was separated and purified using a C18 column reverse phase (acetonitrile/0.01% FA aqueous solution: 5%-50%) to obtain the target compound (C1.14B, 290 mg) as a colorless oil.
  • the crude product was separated and purified by reverse-phase C18 column (acetonitrile/0.01% FA aqueous solution: 5%-65%)
  • the target compound N 6 -(tert-butoxycarbonyl)-N 2 -((39-(2-(methylthio)pyrimidin-5-yl)-5,34-dioxo-3,9,12, 15,18,21,24,27,30-nonaza-6,33-diazatrinonadecan-38-ynyl)-L-valine)-L-lysine (C1.14 )(170mg).
  • Example C1.15 N 6 -(tert-butoxycarbonyl)-N 2 -((32-(4-(2-(methylthio)pyrimidin-5-yl))-1H-1,2,3- Triazol-1-yl)-5-oxo-3,9,12,15,18,21,24,27,30-nonaoxa-6-azatriaconoyl)-L-valine Acid)-L-lysine (C1.15)
  • Example C1.16 N 6 ,N 6 -dimethyl-N 2 -((6-(2-(methylthio)pyrimidin-5-yl)hex-5-ynyl)-L-valine )-L-lysine
  • Example C1.18 N 2 -(tert-butoxycarbonyl)-N 6 -((6-(2-(methanesulfonyl)pyrimidin-5-yl)hex-5-ynyl)-L-valine acid)-L-lysine

Abstract

本公开涉及生物活性物偶联物及其制备方法和用途，具体公开了式XV所示的抗体药物偶联物、其制备方法，以及其在预防和/或治疗与细胞活动异常相关的疾病，包括但不限于在预防和/或***疾病中的用途。

Description

抗体药物偶联物及其制备方法和用途 技术领域
本公开属于医药技术领域，涉及多种抗体，抗体药物偶联物及其制备方法，以及其在预防和/或治疗与细胞活动异常相关的疾病，包括但不限于在预防和/或***疾病中的用途。
背景技术
利用细胞毒的化疗一度是癌症的标准疗法，但高杀伤力的细胞毒素分子会杀伤正常细胞，引起严重的毒副作用。靶向抗肿瘤药物由于同时具有靶向性和抗肿瘤活性，已成为当今肿瘤研究领域的热点，但经常由于靶向药物的靶点选择性问题产生比较大的毒副作用，从而限制了靶向药物的治疗效果。生物大分子药物，如抗体或抗体片段，虽然靶向性强，但对实体瘤的治疗效果有限，或者根本不具有治疗效果。ADC是抗体和小分子药物的偶联体，融合了抗体的靶向作用和生物活性分子的活性，成为一种生物导弹，具有非常可期待的疗效和安全性优势。抗体引导ADC结合到靶细胞，随后被细胞内化，小分子药物在细胞内通过特定酶作用下发生的酶解释放，治疗疾病。
ADC药物近几年发展非常迅速，已经上市的ADC有14种。在很多靶点的ADC中，如靶向NaPi2b的抗体/ADC药物均处于临床研究阶段。对很多靶点而言，开发具有差异性的、更优质的、安全性更佳的单抗或ADC药物，可以为肿瘤患者提供更广更优的用药选择，也具有广阔的市场前景。
发明内容
为了改进抗体药物偶联物(ADC)的治疗效果，降低药物毒副作用，提高治疗窗口，本公开提供一种式(XV)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物，其含有生物活性分子(药物分子)、连接体和靶向部分，所述靶向部分通过活性基团(例如巯基)与所述连接体连接形成抗体药物偶联物。本公开还开发了包含抗Napi2b的可变轻链结构域(VL)和/或可变重链(VH)结构域，所述可变轻链结构域和/或可变重链结构域抗体来自人源抗体。
为此，在本公开的第一方面，本公开提供了式XV所示的抗体药物偶联物，
或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，
其中：
Tb为抗NaPi2b的抗体或其抗原结合片段；
q为药物抗体偶联比；
D为生物活性分子片段；
L1为延伸单元；
L2不存在或为连接单元；
L3选自氨基酸残基或由2-10个氨基酸残基组成的短肽；
L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连。
另外，还需要说明的是，对于“L1的1位通过S原子与Tb相连”，本领域技术人员可以理解的是，L1的1位是与打开二硫键(例如，通过还原剂TCEP还原二硫键可以打开二硫键，生成巯基-SH)后的Tb(如抗体)自身所含有巯基进行连接，也就是说，L1与Tb之间的-S-并非另外再外接的硫原子。例如，中，-S-并非另外外接的硫原子，而是打开双硫键后的Tb自身所含有巯基与L1例如的1位进行连接后形成的-S-。
延伸单元为抗体药物偶联物或药物连接体偶联物或连接子的组分，其作用是将和靶点结合的抗体药物偶联物的其余部分或连接子的其余部分相连。延伸单元能够将Tb单元连接于L2(如果存在)或L3，具体实例包括但不限于(其中1位与靶点结合的抗体部分相连、2位与L2或L3相连)：
在一些实施方案中，L1选自：



各Z独立地选自直接键、碳碳三键、碳碳双键、C6-10芳基、5-10元杂芳基、酰胺基、磺酰胺基、亚胺基和CF2；
Rx和Ry独立地选自H和C1-4烷基；
各m独立地选自0、1、2、3、4、5和6；
y1、y2、y3和y4独立地选自0-20之间的任意整数；
1位通过S原子与Tb相连，2位与L2或L3相连。
连接单元为抗体药物偶联物或药物连接体偶联物或连接子的组分，其作用是用来结合延伸单元与氨基酸残基或由1-10个氨基酸残基组成的片段或短肽。连接单元存在时能够将L1连接于L3。具体实例包括但不限于(其中1位与延伸单元相连、2位与L3相连)：
在一些实施方案中，L2不存在或存在，L2存在时，L2选自：

y1、y2、y3和y4独立地选自0-20之间的任意整数，1位与L1相连，2位与L3相连。
在一些实施方案中，L1为
在一些实施方案中，L1选自 各Z独立地选自直接键、碳碳三键、碳碳双键、C6-10芳基、5-10元杂芳基和酰胺基(优选选自直接键、碳碳三键、碳碳双键)；Rx、Ry独立地选自H和C1-4烷基；各m独立地选自0、1、2、3、4、5和6；y1选自1-6之间任意整数(如4、5、6)；各y2独立地选自0-15(如6-15)之间任意整数；各y3独立地选自1、2和3；各y4独立地选自0和1；1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L1选自 m选自2、3、4，y1选自1-6之间任意整数(如4、5、6)，各y2独立地选自0-10(如6-10)之间任意整数，各y3独立地选自1或2，1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L1选自
在一些实施方案中，L1选自1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L2不存在或存在，L2存在时，L2选自 y1选自1-6之间任意整数(如4、5、6)，各y2独立地选自0-10(如6-10)之间任意整数，各y3独立地选自1或2，各y4独立地选自0或1，1位与L1相连，2位与L3相连。
在一些实施方案中，L2不存在或存在，L2存在时，L2选自 1位与L1相连，2位与L3相连。
在一些实施方案中，L2不存在。
在一些实施方案中，L2选自
在一些实施方案中，L3选自氨基酸残基或由2-10个氨基酸残基组成的短肽；所述的氨基酸残基选自天然氨基酸残基、非天然氨基酸残基、或选自AA1所示氨基酸残基或其立体异构体。
在一些实施方案中，L3选自氨基酸残基Val、D-Val、Cit、Phe、Lys、Lys(Ac)、Leu、Gly、Ala、Asn、Asp、Arg、AA1或由2-10个选自Val、Cit、Phe、Lys、D-Val、Leu、Gly、Ala、Asn、Asp、AA1的氨基酸残基组成的短肽。
在一些实施方案中，L3选自Val、Cit、Phe、Lys、D-Val、Leu、Gly、Ala、Asn、AA1、Val-Cit、Cit-Val、Cit-Ala、Val-Ala、Lys-Val、Val-Lys(Ac)、Phe-Lys、Phe-Lys(Ac)、Ala-Ala、Val-AA1、Ala-AA1、Gly-AA1、AA1-Gly、Ala-Ala-Ala、Ala-Ala-Asn、Ala-Ala-Asp、Val-AA1-Gly、Ala-AA1-Gly、Gly-AA1-Gly、Lys-Ala-Ala-Asn、Lys-Ala-Ala-Asp、Gly-Phe-Gly、Gly-Gly-Phe-Gly、D-Val-Leu-Lys、Gly-Gly-Arg、Ala-Ala-Asn、Gly-Gly-Phe、Val-Lys-Gly、Val-Lys-Gly-Gly、Val-Lys和Lys-Ala-Asn。
在一些实施方案中，L3选自AA1、AA1-Gly、Val-Cit、Val-AA1-Gly、AA1-Ala-Asn和Gly-Gly-Phe-Gly。
在一些实施方案中，L3选自AA1和Val-AA1-Gly。
在一些实施方案中，L3选自Val-AA1-Gly。
在一些实施方案中，L3选自 X-选自卤素离子、羧酸根离子、硫酸根离子、硫酸氢根离子和OH-，1位与L1或L2相连，2位与L4或D相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或D相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或D相连。
在一些实施方案中，AA1所示氨基酸残基的结构如下所示，
其中：
Ra、Rb各自独立地选自H、且Ra、Rb不同时为H；
或者，Ra与Rb和与它们共同相连的碳原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0所取代；
r、r1各自独立地选自0到20的任意整数；
Rm1、Rn1各自独立地选自H、C1-6烷基、C3-6环烷基和-COORx1；
Rx1选自C1-6烷基；
或者，Rm1与Rn1和与它们共同相连的氮原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0’所取代；
Rz选自C1-6烷基；
R0、R0’各自独立地选自C1-6烷基、C3-6环烷基、-NRm2Rn2和任选被C1-6烷基取代的4-10元杂环基；
Rm2、Rn2各自独立地选自H和C1-6烷基。
在一些实施方案中，Ra、Rb中，任一个为H，另一个选自
在一些实施方案中，Ra、Rb中，任一个为H，另一个选自
在一些实施方案中，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的5-6元杂环。
在一些实施方案中，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的哌啶环或哌嗪环。
在一些实施方案中，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的哌啶环。
在一些实施方案中，Ra与Rb和与它们共同相连的碳原子一起，形成1号碳原子为与Ra和Rb共同相连的碳原子。
在一些实施方案中，Ra与Rb和与它们共同相连的碳原子一起，形成1号碳原子为与Ra和Rb共同相连的碳原子。
在一些实施方案中，r、r1各自独立地选自0、1、2、3、4和5。
在一些实施方案中，r、r1各自独立地选自0和4。
在一些实施方案中，r、r1中，任一个为0，另一个为4。
在一些实施方案中，Rm1、Rn1各自独立地选自H、甲基、乙基、正丙基、正丁基、-COOCH3、-COOCH2CH3、-COOCH2CH2CH3、-COOCH(CH3)2、-COOC(CH3)3和-COOCH2CH2CH2CH3。
在一些实施方案中，Rm1、Rn1各自独立地选自H、C1-6烷基、C3-6环烷基和叔丁氧羰基。
在一些实施方案中，Rm1、Rn1各自独立地选自H和C1-6烷基。
在一些实施方案中，Rm1、Rn1各自独立地选自H、甲基、乙基和正丙基。
在一些实施方案中，r、r1中，r为4，r1为0时，Rm1、Rn1各自独立地选自H、C1-6烷基(如H、甲基)；r为0，r1为4时，Rm1、Rn1各自独立地选自C1-6烷基(如甲基、乙基、正丙基)，优选选自C2-6烷基(如乙基、正丙基)。
在一些实施方案中，Rm1与Rn1和与它们共同相连的氮原子一起，形成任选被R0’取代的5-6元杂环。
在一些实施方案中，Rm1与Rn1和与它们共同相连的氮原子一起，形成任选被R0’取代的哌啶环或哌嗪环。
在一些实施方案中，Rm1与Rn1和与它们共同相连的氮原子一起，形成1号氮原子为与Rm1和Rn1共同相连的氮原子。
在一些实施方案中，Rz为甲基。
在一些实施方案中，R0、R0’各自独立地选自C1-6烷基、-NRm2Rn2和任选被C1-6烷基取代的5-6元杂环基。
在一些实施方案中，R0选自C1-6烷基和被C1-6烷基取代的5-6元杂环基，所述5-6元杂环基选自哌啶基和哌嗪基。
在一些实施方案中，R0选自甲基、乙基和被甲基取代的5-6元杂环基，所述5-6元杂环基为哌啶基。
在一些实施方案中，R0选自甲基和被甲基取代的5-6元杂环基，所述5-6元杂环基为哌啶基。
在一些实施方案中，R0选自甲基、乙基和
在一些实施方案中，R0选自甲基和
在一些实施方案中，R0’选自C1-6烷基和-NRm2Rn2。
在一些实施方案中，R0’选自甲基和-NRm2Rn2。
在一些实施方案中，Rm2、Rn2为甲基。
在一些实施方案中，AA1所示氨基酸残基选自
在一些实施方案中，AA1所示氨基酸残基选自
在一些实施方案中，AA1所示氨基酸残基选自
在一些实施方案中，L4不存在或存在，L4存在时，L4为1位与L3相连，2位与D相连。
在一些实施方案中，L4不存在。
在一些实施方案中，L4选自1位与L3相连，2位与D相连。
在一些实施方案中，L4选自1位与L3相连，2位与D相连。
在一些实施方案中，的结构选自以下结构片段：
在一些实施方案中，q选自0.1-16.0之间的任意数值；在优选的实施方案中，q选自0.1-16.0之间的任意整数。
在一些实施方案中，q选自0.1-8.0之间的任意数值，在优选的实施方案中，q选自0.1-8.0之间的任意整数。
在一些实施方案中，q选自2-8之间的任意数值。
在一些实施方案中，q选自3-8之间的任意数值。
在一些实施方案中，q选自4-8之间的任意数值。
在一些实施方案中，q选自6-8之间的任意数值。
在一些实施方案中，q选自2-8之间的任意整数。
在一些实施方案中，q选自3-8之间的任意整数。
在一些实施方案中，q选自4-8之间的任意整数。
在一些实施方案中，q选自6-8之间的任意整数。
在一些实施方案中，q选自1、2、3、4、5、6、7、8、9、10、11和12。
在一些实施方案中，q选自2、4、6和8。
本公开中，所述的生物活性分子片段是指本领域中均知的，抗体-药物偶联物(或称抗体偶联药物(antibody-drug conjugate,ADC))中，在肿瘤组织间或肿瘤细胞内连接子裂解/降解/酶切后，能够形成具有生物活性的药物(例如小分子细胞毒药物，所述药物包括其失去一个原子或原子团后的基团)或其衍生物(例如其前体)的部分(片段或基团)。为了避免歧义，“药物”并非仅指已获得医药监管部门审批的“药品”，还包括临床中，或者研发和学术研究中任何有潜在治疗生物活性的分子。
在一些实施方案中，D为具有抗肿瘤生物活性分子片段。
在一些实施方案中，D为具有抗肿瘤生物活性分子片段，其中所述的生物活性分子选自细胞毒性剂或其衍生物，例如DNA拓扑异构酶抑制剂(例如喜树碱类生物活性分子，例如喜树碱、DXD、取代基被修饰的喜树碱或取代基被修饰的DXD)或微管蛋白抑制剂(例如MMAF类微管蛋白抑制剂，MMAE类微管蛋白抑制剂)。
在一些实施方案中，抗体药物偶联物具有式I所式结构：
其中，
R1、R2各自独立地选自H、卤素、-OH、任选取代的C1-6烷基和任选取代的C1-6烷氧基，或者，
R1和R2和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N，羰基，亚砜基或砜基或其任意组合；
R3选自H、卤素、-OH、-NH2、任选取代的C1-6烷基和任选取代的C1-6烷氧基，或者，
R3和X和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N，羰基，亚砜基或砜基或其任意组合，或者，
R3和R2和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N， 羰基，亚砜基或砜基或其任意组合；
W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
X选自直接键，任选取代的-O-(CH2)n3-、-N(R4)-(CH2)n3-、-S-(CH2)n3-、羰基-(CH2)n3、-SO2-(CH2)n3-、-(CH2)n1-、C3-6环烷基、C6-10芳基、5-10元杂芳基和4-10元杂环基，1位和母环相连，2位和W或L4相连；所述取代基选自一个或多个C1-4烷基、C3-6环烷基，或者多个C1-4烷基和与它们同时相连的碳原子一起形成C3-6环烷基；
各M独立地选自直接键和-CR5aR5b-；
R4、R5、R5a、R5b、R6、R7各自独立地选自H、任选取代的C1-4烷基、任选取代的C1-4烷氧基和任选取代的C3-6环烷基；
n、n’、n1、n2、n3各自独立地选自0到6之间的任意整数；
L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与W或X相连。
Tb、L1、L2、L3和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，R1、R2各自独立地选自H、卤素和C1-4烷基。
在一些实施方案中，R1和R2和与其相连的碳原子一起形成5-6元杂环，所述杂环含有1个、2个或3个O，S或N或其任意组合。
在一些实施方案中，R1选自H和卤素，R2选自H和C1-4烷基。
在一些实施方案中，R1和R2和与其相连的碳原子形成虚线表示所述杂环与苯环稠合的位置。
在一些实施方案中，R1为H或F，R2为H或甲基。
在一些实施方案中，R1为F，R2为甲基或R1和R2和与其相连的碳原子一起形成
在一些实施方案中，R1为F，R2为甲基。
在一些实施方案中，R1和R2和与其相连的碳原子一起形成
在一些实施方案中，R3选自H和C1-4烷基。
在一些实施方案中，R3和X和与其相连的碳原子一起形成5-6元碳环。
在一些实施方案中，R3为H或R3和X和与其相连的碳原子一起形成虚线表示所述碳环与苯环和吡啶环稠合的位置。
在一些实施方案中，R3为H。
在一些实施方案中，W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连。
在一些实施方案中，W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连。
在一些实施方案中，W选自-O-、-NR4-和1位X相连，2位与L4或L3相连。
在一些实施方案中，W选自-O-和-NR4-，1位X相连，2位与L4或L3相连。
在一些实施方案中，X选自任选取代的-(CH2)n1-、C6-10芳基、5-10元杂芳基和4-10元杂环基，1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基，或者2个C1-4烷基和与它们同时相连的碳原子一起形成C3-6环烷基。
在一些实施方案中，X选自任选取代的 1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基(如甲基)，或者2个C1-4烷基(如甲基)和与它们同时相连的碳原子一起形成C3-6环烷基(如环丙基)。
在一些实施方案中，X选自 1位和母环相连，2位和W或L4相连。
在一些实施方案中，X选自1位和母环相连，2位和W相连。
在一些实施方案中，W不存在时，X选自1位和母环相连，2位和L4相连；W存在时，X选自 1位和母环相连，2位和W相连。
在一些实施方案中，W选自-O-、-NR4-和1位X相连，2位与L4或L3相连；X选自1位和母环相连，2位和W相连。
在一些实施方案中，R4、R5各自独立地选自H、C1-4烷基和C3-6环烷基。
在一些实施方案中，各R4独立地选自H、C1-4烷基和C3-6环烷基，R5为H。
在一些实施方案中，各R4独立地选自H、甲基、乙基、正丙基、异丙基、叔丁基和环丙基，R5为H。
在一些实施方案中，R5a、R5b各自独立地选自H和C1-4烷基。
在一些实施方案中，R5a、R5b各自独立地选自H和甲基。
在一些实施方案中，各R7独立地选自H和C1-4烷基。
在一些实施方案中，R7为H。
在一些实施方案中，n选自1、2和3。
在一些实施方案中，n为1。
在一些实施方案中，n1选自1、2、3和4。
在一些实施方案中，n2为1。
在一些实施方案中，n3为0。
在一些实施方案中，L3选自 X-选自卤素离子、羧酸根离子、硫酸根离子、硫酸氢根离子和OH-，1位与L1或L2相连，2位与L4或W相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或W相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或W相连。
在一些实施方案中，L4不存在或存在，L4存在时，L4为1位与L3相连，2位与W或X相连。
在一些实施方案中，L4不存在。
在一些实施方案中，L4选自1位与L3相连，2位与W或X相连。
在一些实施方案中，L4选自1位与L3相连，2位与W或X相连。
需要说明的是，如前所述，W不存在或存在，由此，W不存在时，L4的1位与L3相连，2位与X相连；W存在时，L4的1位与L3相连，2位与W相连。以下关于L4的连接关系可参照前述内容进行理解。
在一些实施方案中，的结构选自以下结构片段：



其中，1位与Tb相连，2位与W相连。
在一些实施方案中，D为所示的结构片段；1位与L3或L4连接；例如
在一些实施方案中，的结构选自以下结构片段：


其中，1位与L4相连；当L4不存在时，1位与L3相连。
在一些实施方案中，
W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、例如不存在、-O-、-NR4-或R4和R5各自独立地选自H和C1-4烷基；n独立地选自0、1、2、3和4；
X选自
R1选自H、卤素，R2选自H、C1-4烷基，或，R1和R2和与其相连的碳原子形成 虚线表示所述杂环与苯环稠合的位置；
R3选自H和C1-4烷基，或，R3和X和与其相连的碳原子一起形成5-6元碳环；
优选地，W不存在或存在，W存在时，W选自-O-、-NR4-(例如-NH-、-N(CH3)-、-N(C2H5)-)、R4独立地选自H、甲基、乙基、异丙基、正丙基、叔丁基和环丙基；
W不存在时，X选自1位和母环相连；W存在时，X选自
AA1 中，r选自0、1、2、3、4和5；
Ra、Rb中，任一个为H，另一个选自或者，Ra与Rb和与它们共同相连的碳原子一起， 形成被R0取代的5-6元杂环；
R1选自H、卤素，R2选自H、C1-4烷基，或，R1和R2和与其相连的碳原子形成
R3选自H和C1-4烷基，或，R3和X和与其相连的碳原子一起形成R3和X和与其相连的碳原子一起形成
更优选地，W选自-O-和-NR4-，1位X相连，2位与L4或L3相连；
X选自1位和母环相连，2位和W相连；
为例如
R1为F，
R2为甲基或R1和R2和与其相连的碳原子一起形成
R3为H。
在一些实施方案中，抗体药物偶联物具有式I-1所式结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提 供的含义。
在一些实施方案中，抗体药物偶联物具有式I-1A或I-1B所式结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-2所式结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-2A或I-2B所式结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-3所式结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4、R5、n和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-3A或I-3B所式结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-A所式结构：
其中，Tb、X、R1、R2、R3、Ra、Rb和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-B所式结构：
其中，Tb、X、R1、R2、R3、Ra、Rb和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，所述抗体药物偶联物选自以下：


















在一些实施方案中，Tb为抗NaPi2b抗体或其抗原结合片段。
在一些实施方案中，所述抗体或其抗原结合片段以及单克隆抗体或其抗原结合片段包括：Fab、Fab'、F(ab')2、Fd、Fv(例如，scFv)、dAb、互补决定区片段、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗(Probody)、单克隆抗体、双特异性抗体或多特异性抗体。
在一些实施方案中，Tb为具有内吞,没有内吞活性的或内吞较弱的抗Napi2b的抗体或其抗原结合片段。
在一些实施方案中，Tb为具有内吞活性的抗Napi2b抗体或其抗原结合片段。
在一些实施方案中，Tb为没有内吞活性的、或内吞活性弱的抗Napi2b抗体或其抗原结合片段。
在一些实施方案中，Tb为抗NaPi2b抗体或其抗原结合片段。
在一些实施方案中，抗Napi2b抗体为非人抗体、人源化抗体、嵌合抗体、全人抗体。
在一些实施方案中，抗Napi2b抗体为单克隆抗体，双特异性抗体或多特异性抗体。
在一些实施方案中，抗Napi2b抗体或其抗原结合片段为单克隆抗体或其抗原结合片段。
在一些实施方案中，所述抗NaPi2b抗体或其抗原结合片段选自：lifastuzumab或其抗原结合片段，upifitamab或其抗原结合片段，抗体C1。
在一些优选地实施方案中，所述抗NaPi2b抗体选自：16G5B3、60A12B3、22H9C4、66C12D12和34F2A10鼠源抗体,或它们的人源化的抗体。
在一些优选地实施方案中，其中所述抗NaPi2b抗体选自：60A12B3-hz1抗体、22H9C4-hz1抗体、66C12D12-hz1抗体和34F2A10-hz1抗体。
所述Tb的抗体或其抗原结合的片段可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本公开抗体的重链和轻链基因的DNA分子。将所得DNA分子***表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本公开的抗体。
在一些优选实施方案中，在所述的抗体-药物偶联物中，Tb为第二方面所述的抗体Napi2b抗体或其抗原结合片段。
第二方面，本公开提供一种抗Napi2b抗体或其抗原结合片段。
在一些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含如下的互补决定区(CDR)：
SEQ ID NO:1、17、19、34、36、52、54、71、73、74或33所示的重链可变区(VH)中含有的HCDR1或其序列的变体、HCDR2或其序列的变体、以及HCDR3或其序列的变体；和/或
SEQ ID NO:2、18、20、35、37、53、55、72、75或51所示的轻链可变区(VL)中含有的LCDR1或其序列的变体、LCDR2或其序列的变体、以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:1或17所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:2或18所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:19或34所示的VH中含 有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:20或35所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:36或52所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:37或53所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:54、71或73所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:55或72所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:74或33所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:75或51所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:17所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:18所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:34所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:35所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:52所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:53所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:71或73所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:72所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:33所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:51所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些优选实施方案中，所述序列的变体为与其来源CDR相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个或3个氨基酸的置换、缺失或添加)的CDR。
在某些优选实施方案中，所述的置换为保守置换。
在某些实施方案中，所述CDR根据AbM、Chothia、Kabat或IMGT编号***定义。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含：重链可变区(VH)和/或轻链可变区(VL)。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号***定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:3或与其相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:4或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:6或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:7或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:9或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:3或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:5或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:6或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:8或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:9或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:21或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:22或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:25或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:26或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:27或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:21或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:23或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:24或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:25或与其相比具有一个或几个氨基酸的 置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:26或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:27或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:38或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:39或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:41或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:42或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:44或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:45或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:38或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:40或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:41或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:42或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:44或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:45或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(g)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:56或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:57或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:60或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:61或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:63或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(h)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:56或与其相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:58或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:60或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:62或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:63或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；或
(i)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:56或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:59或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:60或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:62或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:63或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号***定义：
(a)所述VH包含：序列为SEQ ID NO:3的HCDR1，序列为SEQ ID NO:4的HCDR2，序列为SEQ ID NO:6的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:7的LCDR1，序列为SEQ ID NO:9的LCDR2，序列为SEQ ID NO:10的LCDR3；
(b)所述VH包含：序列为SEQ ID NO:3的HCDR1，序列为SEQ ID NO:5的HCDR2，序列为SEQ ID NO:6的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:8的LCDR1，序列为SEQ ID NO:9的LCDR2，序列为SEQ ID NO:10的LCDR3；
(c)所述VH包含：序列为SEQ ID NO:21的HCDR1，序列为SEQ ID NO:22的HCDR2，序列为SEQ ID NO:24的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:25的LCDR1，序列为SEQ ID NO:26的LCDR2，序列为SEQ ID NO:27的LCDR3；
(d)所述VH包含：序列为SEQ ID NO:21的HCDR1，序列为SEQ ID NO:23的HCDR2，序列为SEQ ID NO:24的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:25的LCDR1，序列为SEQ ID NO:26的LCDR2，序列为SEQ ID NO:27的LCDR3；
(e)所述VH包含：序列为SEQ ID NO:38的HCDR1，序列为SEQ ID NO:39的HCDR2，序列为SEQ ID NO:41的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:42的LCDR1，序列为SEQ ID NO:44的LCDR2，序列为SEQ ID NO:45的LCDR3；
(f)所述VH包含：序列为SEQ ID NO:38的HCDR1，序列为SEQ ID NO:40的HCDR2，序列为SEQ ID NO:41的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:43的LCDR1，序列为SEQ ID NO:44的LCDR2，序列为SEQ ID NO:45的LCDR3；
(g)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:57的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:61的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3；
(h)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:58的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:62的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3；或
(i)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:59的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:62的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号***定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:12或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:14或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:15或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为WAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:11或与其相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:13或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:14或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:15或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为WAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(c)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:29或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:31或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:32或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为YTS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:27或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(d)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:28或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:30或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:31或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:32或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为YTS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:27或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(e)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:46或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:47或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:49或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:50或与其相比具有一个或几个氨基酸的 置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:45或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(f)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:46或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:48或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:49或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:50或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:45或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(g)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:65或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:66或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:68或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:69或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为WAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(h)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:65或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:67或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:68或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:69或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为WAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:64或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；或
(i)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:76或与其相比具有一个或几个氨基 酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:77或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:78或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:79或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为HTS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:16或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按IMGT编号***定义：
(a)所述VH包含：序列为SEQ ID NO:11的HCDR1，序列为SEQ ID NO:12的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:15的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:10的LCDR3；
(b)所述VH包含：序列为SEQ ID NO:11的HCDR1，序列为SEQ ID NO:13的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:15的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:10的LCDR3；
(c)所述VH包含：序列为SEQ ID NO:28的HCDR1，序列为SEQ ID NO:29的HCDR2，序列为SEQ ID NO:31的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:32的LCDR1，序列为YTS的LCDR2，序列为SEQ ID NO:27的LCDR3；
(d)所述VH包含：序列为SEQ ID NO:28的HCDR1，序列为SEQ ID NO:30的HCDR2，序列为SEQ ID NO:31的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:32的LCDR1，序列为YTS的LCDR2，序列为SEQ ID NO:27的LCDR3；
(e)所述VH包含：序列为SEQ ID NO:46的HCDR1，序列为SEQ ID NO:47的HCDR2，序列为SEQ ID NO:49的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:50的LCDR1，序列为SAS的LCDR2，序列为SEQ ID NO:45的LCDR3；
(f)所述VH包含：序列为SEQ ID NO:46的HCDR1，序列为SEQ ID NO:48的HCDR2，序列为SEQ ID NO:49的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:50的LCDR1，序列为SAS的LCDR2，序列为SEQ ID NO:45的LCDR3；
(g)所述VH包含：序列为SEQ ID NO:65的HCDR1，序列为SEQ ID NO:66的HCDR2，序列为SEQ ID NO:68的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:69的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:64的LCDR3；
(h)所述VH包含：序列为SEQ ID NO:65的HCDR1，序列为SEQ ID NO:67的HCDR2，序列为SEQ ID NO:68的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:69的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:64的LCDR3；
或
(i)所述VH包含：序列为SEQ ID NO:76的HCDR1，序列为SEQ ID NO:77的HCDR2，序列为SEQ ID NO:78的HCDR3；和/或，
所述VL包含：序列为SEQ ID NO:79的LCDR1，序列为HTS的LCDR2，序列为SEQ ID NO:16的LCDR3。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中，与前述Kabat编号***或IMGT编号***定义的CDR相比，所述重链可变区(VH)和/或轻链可变区(VL)中至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)，且仍具有结合Napi2b活性。
在某些优选地实施方案中，本公开所述的置换为保守置换。
在某些实施方案中，所述抗Napi2b抗体或其抗原结合片段结合人Napi2b、猴Napi2b和/或大鼠Napi2b。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段的VH包含来源于人免疫球蛋白的重链可变区(VH)的构架区(FR)，和/或所述抗体或其抗原结合片段的VL包含来源于人免疫球蛋白的轻链可变区(VL)的构架区(FR)。因此，在某些实施方案中，本公开的抗体或其抗原结合片段是全人源的。在某些实施方案中，本公开的抗体或其抗原结合片段是人源化的。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)人免疫球蛋白的重链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)；和/或
(b)人免疫球蛋白的轻链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段的人源化程度为至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列或由其组成：
(i)SEQ ID NO:1、17、19、34、36、52、54、71、73、74或33所示的序列；
(ii)与SEQ ID NO:1、17、19、34、36、52、54、71、73、74或33所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与SEQ ID 1、17、19、34、36、52、54、71、73、74或33所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列或由其组成：
(iv)SEQ ID NO:2、18、20、35、37、53、55、72、75或51所示的序列；
(v)与SEQ ID NO:2、18、20、35、37、53、55、72、75或51所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与SEQ ID NO:2、18、20、35、37、53、55、72、75或51所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:1或17所示的VH，和/或,SEQ ID NO:2或18所示的VL。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:19或34所示的VH，和/或,SEQ ID NO:20或35所示的VL。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:36或52所示的VH，和/或,SEQ ID NO:37或53所示的VL。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:54、71或73所示的VH，和/或,SEQ ID NO:55或72所示的VL。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:74或33所示的VH，和/或,SEQ ID NO:75或51所示的VL。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含上述5个方案组中，任一组的VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与该组VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；或者
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含上述5个方案组中，任一组的VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、 7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与该组VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合的VL；在某些优选地实施方案中，所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)SEQ ID NO:1所示序列的VH和SEQ ID NO:2所示序列的VL；
(b)SEQ ID NO:17所示序列的VH和SEQ ID NO:18所示序列的VL；
(c)SEQ ID NO:19所示序列的VH和SEQ ID NO:20所示序列的VL；
(d)SEQ ID NO:34所示序列的VH和SEQ ID NO:35所示序列的VL；
(e)SEQ ID NO:36所示序列的VH和SEQ ID NO:37所示序列的VL；
(f)SEQ ID NO:52所示序列的VH和SEQ ID NO:53所示序列的VL；
(g)SEQ ID NO:54所示序列的VH和SEQ ID NO:55所示序列的VL；
(h)SEQ ID NO:71所示序列的VH和SEQ ID NO:72所示序列的VL；
(i)SEQ ID NO:73所示序列的VH和SEQ ID NO:72所示序列的VL；
(j)SEQ ID NO:74所示序列的VH和SEQ ID NO:75所示序列的VL；
(k)SEQ ID NO:33所示序列的VH和SEQ ID NO:51所示序列的VL；
(l)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)独立地与(a)至(k)任一组中所述的VH和VL相比分别具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或
(m)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)与(a)至(k)任一组中所述的VH和VL分别相比，独立地具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)。在某些优选地实施方案中，所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体是嵌合抗体、人源化抗体或全人源抗体。在某些实施方案中，本公开的抗体或其抗原结合片段选自Fab、Fab’、(Fab’)2、Fv片段例如scFv或二硫键连接的Fv(dsFv)、双抗体(diabody)和多特异性抗体。在某些实施方案中，本公开的抗Napi2b抗体是scFv。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换(例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换(例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在一些实施方案中，恒定区被改变，例如被突变，以修饰抗Napi2b抗体分子的性质(例如改变下列中的一个或多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低)。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用(ADCP)、CDC等。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段具有重链恒定区(CH)，其选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；特别地选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自IgG1(例如是人IgG1)的重链恒定区。在一些实施方案中，人IgG1重链恒定区如SEQ ID NO:70所示。在一些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段具有轻链恒定区，其选自例如κ或λ轻链恒定区，优选κ轻链恒定区(例如人κ轻链恒定区)。在一些实施方案中，轻链恒定区具有如SEQ ID NO:80所示的序列。
在一些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:70所示的CH或其变体，所述变体与SEQ ID NO:70相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO:70相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含轻链恒定区或其变体。在一些实施方案中，所述轻链恒定区包含κ轻链恒定区。在一些实施方案中，所述轻链恒定区包含SEQ ID NO:80所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO:80相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO:80相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗Napi2b抗体或其抗原结合片段包含SEQ ID NO:70所示的重链恒定区(CH)和SEQ ID NO:80所示的轻链恒定区(CL)。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列或由其组成：
(i)包含SEQ ID NO:71所示的VH序列和SEQ ID NO:70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO:72所示的VL序列和SEQ ID NO:80所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列或由其组成：
(i)包含SEQ ID NO:73所示的VH序列和SEQ ID NO:70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列或由其组成：
(iv)包含SEQ ID NO:72所示的VL序列和SEQ ID NO:80所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列或由其组成：
(i)包含SEQ ID NO:17所示的VH序列和SEQ ID NO:70所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列或由其组成：
(iv)包含SEQ ID NO:18所示的VL序列和SEQ ID NO:80所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含重链和轻链，
所述重链包含或由下述序列组成：
(i)SEQ ID NO:81所示的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
所述轻链包含或由下述序列组成：
(iv)SEQ ID NO:82所示的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
在某些优选地实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗Napi2b抗体或其抗原结合片段包含重链和轻链，
所述重链包含或由下述序列组成：
(i)SEQ ID NO:83所示的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少 94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
所述轻链包含或由下述序列组成：
(iv)SEQ ID NO:84所示的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
在某些优选地实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述的抗Napi2b抗体或其抗原结合片段，其中，所述抗体包含选自以下组的重链和轻链：
(a)包括SEQ ID NO:71所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:72所示的VL和SEQ ID NO:80所示的CL的轻链；
(b)包括SEQ ID NO:73所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:72所示的VL和SEQ ID NO:80所示的CL的轻链；优选包括SEQ ID NO:81所示的重链和SEQ ID NO:82所示的轻链；
(c)包括SEQ ID NO:17所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:18所示的VL和SEQ ID NO:80所示的CL的轻链。优选包括SEQ ID NO:83所示的重链和SEQ ID NO:84所示的轻链；
在某些实施方案中，本公开提供的抗体是一种多特异性抗体，其结合Napi2b以及一种或多种其他抗原。在某些优选地实施方案中，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。在一些实施方案中，本公开的多特异性抗体包含上述所述的抗Napi2b抗体或其抗原结合片段，和另外的抗体或其片段或抗体类似物。
本公开抗体衍生物
本公开的抗Napi2b抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会对其与Napi2b的结合产生不利影响。因此，本公开的抗Napi2b抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本公开的抗Napi2b抗体或其抗原结合片段连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或其抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
一种类型的衍生化抗体(例如，双特异性抗体)是通过交叉连接2个或更多个抗体(属于同一类型或不同类型)而产生的。获得双特异性抗体的方法是本领域公知的，其实例包括但不限于，化学交联法、细胞工程法(杂交瘤法)或基因工程法。
另一种类型的衍生化抗体是标记的抗体。例如，可以将本公开的抗体或其抗原结合片段连接至可检测的标记。本公开所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测的标记连接至本公开的抗体或其抗原结合片段，以降低潜在的位阻。
此外，本公开的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
抗体的制备
本公开的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本公开抗体的重链和轻链基因的DNA分子。将所得DNA分子***表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本公开的抗体。
本公开的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
第三方面，本公开提供一种分离的核酸分子，其包含编码本公开的第二方面抗体或其抗原结合片段、或其重链可变区和/或轻链可变区、或其一个或多个CDR的核苷酸序列；在某些实施方案中，所述核苷酸序列是可以根据密码子简并性进行替换的。在某些实施方案中，所述核苷酸序列是密码子最优化的。
在某些实施方案中，本公开所述分离的核酸分子包含：(i)分别编码本公开的第二方面的抗体或其抗原结合片段的重链可变区和轻链可变区的第一核酸和第二核酸，或(ii)分别编码本公开的第二方面 的抗体或其抗原结合片段的重链可变区和重链恒定区的第一核酸，和轻链可变区和轻链恒定区的第二核酸，或(iii)分别编码本公开的第二方面的抗体或其抗原结合片段的重链和轻链的第一核酸和第二核酸。在某些实施方案中，所述第一核酸和第二核酸包含与上述(i)-(iii)中任一第一核酸和第二核酸的简并序列或基本上相同序列的核酸。在某些实施方案中，所述简并序列或基本上相同序列指与(i)-(iii)中所述核酸分子相比具有至少大约85％、90％、95％、99％或更高序列同一性的序列或具有一个或更多个核苷酸取代的序列，或相差不超过3、6、15、30或45个核苷酸的序列。
第四方面，提供了一种载体(例如克隆载体或表达载体)，其包含本公开第三方面的分离的核酸分子。在某些实施方案中，本公开的载体是克隆载体或表达载体。在某些实施方案中，本公开的载体是例如质粒，粘粒，噬菌体，慢病毒等。在某些实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本公开的抗体或其抗原结合片段。
第五方面，提供了一种宿主细胞，其包含本公开第三方面的分离的核酸分子或本公开的第四方面载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、SP2/0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本公开的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、CHO DG44)。
第六方面，提供了制备本公开的第二方面抗体或其抗原结合片段，或本公开的多特异性抗体的方法，其包括，在允许所述抗体或其抗原结合片段、或所述多特异性抗体表达的条件下，培养本公开的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
抗体-药物偶联物
第七方面，提供一种抗Napi2b抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其中抗体为上述结合Napi2b的抗体或其抗原结合片段。
在一些实施方案中，所述抗体药物偶联物选自：


其中，
Tb1为抗Napi2b抗体或其抗原结合片段，例如：lifastuzumab，upifitamab，60A12B3-hz1、22H9C4-hz1、66C12D12-hz1、34F2A10-hz1、C1，16G5B3-hz2和16G5B3-hz1抗体。
q选自0.1-16.0之间的任意数值，优选2-8之间的任意数值。在一些实施方案中，q为1、2、3、4、5、6、7、8、9或10。在一些优选地实施方案中，q为2、4、6或8。
抗体-药物偶联物制备
在本公开的第八方面，本公开提供了制备前述抗体药物偶联物的方法，其包括：
将Tb与式III所示的药物连接体偶联物
在合适的溶剂和条件下进行偶联反应；
其中：
Tb具有上文以及本文具体叙述的任何实施方案中所提供的含义；
R1、R2、R3、X、W、L1、L2、L3、L4具有上文以及本文具体叙述的任何实施方案中所提供的含义。
Lg为离去基团，Lg选自卤素、砜基、三级胺盐基(Me3N+、Et3N+)、重氮盐基、-OMs、MeSO2-、CF3SO3-。
在一些实施方案中，Lg选自F、Cl、MeSO2-。
在一些实施方案中，Lg选自F、MeSO2-。
在一些实施方案中，所述方法包括将Tb与式III所示的药物连接体偶联物在合适的溶剂和条件下进行偶联反应形成C-S键的步骤。
在一些实施方案中，所述Tb与所述药物连接体偶联物的物质的量的比为1:(1-20)，如1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10、1:12、1:14、1:16、1:18、1:(10-20)、1:(12-20)、1:(14-20)、1:(16-20)或1:(18-20)。
在一些实施方案中，所述偶联反应在水和/或有机溶剂中进行。
在一些实施方案中，所述有机溶剂选自N,N-二甲基甲酰胺、二甲基亚砜、N-甲基吡咯烷酮、腈类(例如乙腈)、醇类(例如甲醇、乙醇)或其任意组合。
在一些实施方案中，所述方法进一步包括将偶联产物进行纯化的步骤。
在一些实施方案中，通过层析方法对所述偶联产物进行纯化。
在一些实施方案中，所述层析方法包括离子交换层析、疏水层析、反相层析或亲和层析中的一种或多种。
在本公开的第九方面，本公开提供了一种抗体药物偶联物的群，其包含本公开上前述的抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其中所述的抗体药物偶联物具有一种，两种或多种q值。
在一些实施方案中，当所述群中的的抗体药物偶联物具有一种q值时，q值和DAR值相等。
在一些实施方案中，当所述群中的的抗体药物偶联物具有两种或多种q值时，其中一个特定q值的抗体药物偶联物中占群中所有抗体药物偶联物的比例大于50％，60％,70％,80％,90％,95％，96％，97％，98％，99％。
在一些实施方案中，所述群中抗体药物偶联物中的药物与抗体的比例(DAR)选自1-10中的整数或小数。
在一些实施方案中，所述群的药物与抗体的比例(DAR)选自1.5-2.5、3.5-4.5、5.5-6.5和7.5-8.5；
在一些实施方案中，所述群的药物与抗体的比例(DAR)选自约2.0、4.0、6.0和8.0；
在一些实施方案中，所述群中抗体药物偶联物中的药物与抗体的比例(DAR)选自2、2.5、3、3.5、4、4.5,5、5.5、6、6.5、7、7.2、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.7、8.9和9。
在一些实施方案中，所述的群含有具有1至8的DAR的分布的ADC，例如，1.5、2、4、6、和8(即，1.5、2、4、6、和8的载药种类)。值得注意的是，可以产生降解产物，使得所述的群中也可以包含1、3、5和7的DAR。此外，所述的群也可具有大于8的DAR。所述的抗体药物偶联物由链间二硫化物还原然后偶联产生。在一些实施方案中，所述的抗体药物偶联物包含如下两者：DAR为4或更低(即，载药种类为4或更低)的所述的抗体药物偶联物以及DAR为6或更高(即，载药种类为6或更高)的所述的抗体药物偶联物。
在本公开的第十方面，本公开提供了一种药物组合物，其包含上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第二方面抗Napi2b抗体或其抗原结合片段，第三方面所述核酸，第四方面所述载体，第五方面所述宿主细胞，或者第九方面的抗体药物偶联物群，以及任选的一种或多种药用辅料。
在某些实施方案中，所述药物组合物包含的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第二方面抗Napi2b抗体或其抗原结合片段，第三方面所述核酸，第四方面所述载体，第五方面所述宿主细胞，或者第九方面的抗体药物偶联物群的剂量均为治疗有效量的。
在某些实施方案中，所述药物组合物包含的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本公开的药物组合物包含第九方面的抗体药物偶联物群，及药学上可接受的载体和/或赋形剂。在某些优选地实施方案中，本公开的药物组合物包含上述抗体药物偶联物群，或包含抗体药物偶联物群和缓冲剂。在某些进一步优选地实施方案中，本公开的药物组合物还包括赋形剂和/或表面活性剂。
在某些实施方案中，本公开的药物组合物包含本公开的抗Napi2b抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。在某些优选的实施方案中，本公开的药物组合物包含抗Napi2b抗体或其抗原结合片段，和缓冲剂。在某些进一步优选的实施方案中，本公开的药物组合物还包括赋形剂和/或表面活性剂。
在一些实施方案中，所述缓冲剂为组氨酸缓冲液。在某些优选的实施方案中，所述缓冲剂为pH为6.0的20mM组氨酸缓冲液。
在某些实施方案中，本公开的药物组合物包含本公开的宿主细胞，以及药学上可接受的载体和/或赋形剂，其中所述宿主细胞包含如前所述的分离的核酸分子或载体。
在某些实施方案中，本公开的药物组合物包含本公开的多特异性抗体，以及药学上可接受的载体和/或赋形剂。
在一些实施方案中，所述药物组合物中药物与抗体的比例(DAR)选自1-10中的整数或小数。
在一些实施方案中，所述药物组合物中药物与抗体的比例(DAR)选自1.5-2.5、3.5-4.5、5.5-6.5和7.5-8.5；
在一些实施方案中，所述药物组合物中药物与抗体的比例(DAR)选自约2.0、4.0、6.0和8.0；
在一些实施方案中，所述药物组合物中抗体药物偶联物中的药物与抗体的比例(DAR)选自2、2.5、3、3.5、4、4.5,5、5.5、6、6.5、7、7.2、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.7、8.9和9。
在第十一方面，提供了本公开的抗体或其抗原结合片段在制备试剂盒中的用途，所述试剂盒用于检测Napi2b在样品中的存在或其水平。在另一个方面，本公开提供了诊断性或治疗性试剂盒，其包括一个或多个以下物质：本公开所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、抗体药物偶联物，抗体药物偶联物群或者药物组合物。可选地，所述诊断性或治疗性试剂盒还包括使用说明书。
在本公开的第十二方面，本公开提供了前述的抗体药物偶联物组合物或者前述的药物组合物在制备治疗和/或预防与细胞活动异常相关的疾病(例如癌症疾病)的药物中的用途。所述的抗体药物偶联物组合物或者前述的药物组合物可为治疗有效量的。
在一些实施例中，提供本公开的抗Napi2b抗体或其抗原结合片段、核酸、载体、宿主细胞，或者多特异性抗体在制备药物中的用途，所述药物用于调整(抑制或阻断)Napi2b的活性。
在一些实施例中，提供本公开的抗Napi2b抗体或其抗原结合片段、核酸、载体、宿主细胞，或者多特异性抗体在制备药物中的用途，所述药物用于治疗或预防与Napi2b的活性有关的疾病。
在一些实施例中，提供本公开的抗Napi2b抗体或其抗原结合片段、核酸、载体、宿主细胞、抗体药物偶联物，或者多特异性抗体在制备药物中的用途，所述药物用于治疗或预防与Napi2b的活性有关的肿瘤。
在一些实施例中，提供本公开的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、 其药学上可接受的盐或其药学上可接受的溶剂合物，第二方面抗Napi2b抗体或其抗原结合片段，第三方面所述核酸，第四方面所述载体，第五方面所述宿主细胞，第九方面的抗体药物偶联物群，或第十方面的药物组合物在制备药物中的用途，所述药物用于治疗或预防肿瘤。
在一些实施例中，提供本公开的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第九方面的抗体药物偶联物群，或第十方面的药物组合物在制备药物中的用途，所述药物用于治疗或预防与Tb的靶点相关的肿瘤。
在一些实施例中，提供本公开的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第九方面的抗体药物偶联物群，或第十方面的药物组合物在制备药物中的用途，所述药物用于治疗或预防与NaPi2b相关的肿瘤。
在一些优选地实施例中，提供本公开的上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第二方面抗Napi2b抗体或其抗原结合片段，第三方面所述核酸，第四方面所述载体，第五方面所述宿主细胞，第九方面的抗体药物偶联物群，第十方面的药物组合物在制备药物中的用途，所述药物用于治疗或预防与Napi2b相关的肿瘤。
在本公开的第十三方面，本公开提供了一种将上述第一方面或第七方面抗体药物偶联物，其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，第二方面抗Napi2b抗体或其抗原结合片段，第三方面所述核酸，第四方面所述载体，第五方面所述宿主细胞，第九方面的抗体药物偶联物群，或第十方面的药物组合物，用于治疗和/或预防与细胞活动异常相关的疾病(例如肿瘤)的方法。
在上述第十二和十三方面的中，所述肿瘤选自食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌、非小细胞性肺癌或肺腺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子***、子宫内膜癌、结肠癌(例如人结肠腺癌)、直肠癌、结直肠癌、肝癌、肾癌、尿路上皮癌、表皮癌、非霍奇金淋巴瘤、中枢神经***肿瘤(例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤)、***癌或甲状腺癌。
在一些实施方案中，所述肿瘤与NaPi2b相关的肿瘤。
在一些实施方案中，所述肿瘤为与Napi2b相关的肿瘤。
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文所使用，术语“药学上可接受的盐”的例子是由形成药学上可以接受的阴离子的有机酸形成的有机酸加合盐，包括但不限于甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、α-酮戊二酸盐、α-甘油磷酸盐、烷基磺酸盐或芳基磺酸盐；优选地，所述烷基磺酸盐为甲基磺酸盐或乙基磺酸盐；所述芳基磺酸盐为苯磺酸盐或对甲苯磺酸盐。也可形成合适的无机盐，包括但不限于盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸氢盐和碳酸盐、硫酸盐或磷酸盐等。
如本文所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical  Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。
药学上可接受的盐可使用本领域熟知的标准程序获得，例如，通过将足量的碱性化合物和提供药学上可以接受的阴离子的合适的酸反应。
本公开中，所述药用辅料是指生产药品和调配处方时，使用的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。药用辅料除了赋型、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。根据其来源可分为天然物、半合成物和全合成物。根据其作用与用途可分为：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等；根据其给药途径可分为口服、注射、黏膜、经皮或局部给药、经鼻或口腔吸入给药和眼部给药等。同一药用辅料可用于不同给药途径的药物制剂，且有不同的作用和用途。
所述药物组合物可根据给药途径制成各种适宜的剂型。例如片剂、胶囊剂、颗粒剂、口服溶液剂、口服混悬剂、口服乳剂、散剂、酊剂、糖浆剂、注射剂、栓剂、软膏剂、乳膏剂、糊剂、眼用制剂、丸剂、植入剂、气雾剂、粉雾剂、喷雾剂等。其中，所述的药物组合物或适宜的剂型可以含有0.01mg至1000mg的本公开的化合物或其药学上可接受的盐或偶联物，适宜含有0.1mg至800mg，优选含有0.5-500mg，优选含有0.5至350mg，特别优选1-250mg。
所述药物组合物可以注射剂形式用药，包括注射液、注射用无菌粉末与注射用浓溶液。其中，可使用的载体和溶剂包括水、林格氏溶液和等渗氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
在本公开中，术语“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本公开中术语“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
本公开中，术语“有效剂量”指被给药后会在一定程度上缓解所治疗病症的一种或多种症状的化合物的量。
本公开中，术语“生物活性物”、“生物活性分子”或“药物分子”指抑制或防止细胞的功能和/或 引起细胞死亡或破坏的物质，在本公开的部分实施方案中，偶联物中的生物活性物、生物活性分子或药物分子为具有抗肿瘤生物活性的分子。例如：放射性同位素，例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212和Lu的放射性同位素；金属配合物，例如金属铂配合物、金属金配合物，奥沙利铂等；糖肽类抗生素，例如博来霉素、平阳霉素；DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康，拓扑异构酶II抑制剂，放线菌素D、阿霉素、多柔比星、多卡米星，柔红霉素、米托蒽醌、鬼臼毒素、依托泊苷等；干扰DNA合成药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨、奈拉滨等；作用于结构蛋白的药物，例如微管蛋白抑制剂，长春花生物碱类、长春新碱、长春碱、紫杉醇、多西他赛、卡巴他赛等；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂等；还包括蛋白酶体抑制剂、组蛋白去乙酰化酶抑制剂、肿瘤新生血管生成抑制剂、细胞周期蛋白抑制剂、美登素衍生物、卡里奇霉素衍生物、奥瑞他汀衍生物、Pyrrolobenzodiazepines(PBD)衍生物、美法仑、丝裂霉素C、苯丁酸氮芥、或其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡和坏死的活性物质；酶及其片段，诸如核溶酶；抗生素；毒素，诸如小分子毒素或者细菌、真菌、植物或动物起源的酶活性毒素，包括其片段和/或变体；生长抑制剂；药物模块。术语“毒素”指能够对细胞的生长或增殖产生有害效果的物质。
本公开中，术语“连接体”是指将生物活性分子(药物分子)与靶向部分连接起来的片段。
本公开中，术语“靶向部分”是指偶联物中能够与细胞表面的靶标(或靶标的部分)特异性结合的部分。通过靶向部分与靶标的相互作用，偶联物可以被递送至特定的细胞群。
本公开中，抗体或其抗原结合片段包括衍生化的抗体或其抗原结合片段，例如具有巯基的抗体或其抗原结合片段，其中所述衍生化使得抗体具有与药物连接体偶联物反应的基团或能力。所述巯基-SH可以打开二硫键(例如，通过还原剂TCEP还原)衍生获得。
本文中使用的术语“癌症”和“肿瘤”以相同的含义使用。
本文中使用的术语“基因”不仅包括DNA，而且包括其mRNA、其cDNA和其cRNA。
本文中使用的术语“多核苷酸”以与核酸相同的含义使用，并且还包括DNA、RNA、探针、寡核苷酸和引物。
本文中使用的术语“多肽”和“蛋白”无区别地使用。
本文中使用的术语“细胞”也包括动物个体内的细胞和培养的细胞。
本公开中所述的抗Napi2b抗体中涉及的Napi2b可为本领域常规的Napi2b，当然还表示Napi2b变体。
术语“NaPi2b”，NaPi2b由SLC34A2基因编码，由690个氨基酸组成，结构上8次跨膜，存在4个胞外环。钠离子依赖性磷酸转运蛋白2B(NaPi2b)通过选择性转运二价Pi(HPO4 2-)以维持体内Pi内稳态。人NaPi2b与大鼠同源性为85.3％，与食蟹猴同源性为95.8％。NaPi2b与该家族NaPi2a/2c氨基酸序列相似度高(和NaPi2a同源性为51.29％，和NaPi2c同源性为44.64％)，特别在跨膜区同源性＞80％。
KD指获得自Kd(具体结合分子-靶蛋白相互作用的解离速率)与Ka(具体结合分子-靶蛋白相互作用的结合速率)之比(或Kd/Ka，以摩尔浓度(M)表示)的解离常数。可使用本领域充分建立的方法测定KD值。 测定结合分子的KD的优选方法是通过使用表面等离子共振，例如生物传感器***，如Biacore TM(GE Healthcare Life Sciences)***。
如本文所用，两个氨基酸序列之间的百分比同源性等于两个序列之间的百分比同一性(identity)。两个序列之间的序列百分比同一性是序列共享的相同位置的数目的函数(即，％同源性＝相同位置的数目/位置总数目X100)，其中考虑缺口(gap)的数目和每一缺口的长度，需要将其引入用于两个序列的最优对比。可用本领域通常所知的方法进行序列比较和确定序列间的百分比同一性，可用数学算法实现这种序列比较和百分比同一性的确定。例如，可用Meyers和Miller，1988Comput.Appl.Biosci.4：11-17的算法(已整合入ALIGN程序(版本2.0))来确定氨基酸序列之间和/或核苷酸序列之间的百分比同一性。此外，可用从Accelrys在线获得的GCG软件包中的GAP程序(使用其缺省参数)来确定氨基酸序列之间或核苷酸序列之间的百分比同一性。在一个实施方案中，所述两个序列是等长的。
术语“表位”是指在动物体优选哺乳动物体内具有抗原性或免疫原性活性的抗原多肽或蛋白的一部分。本公开的抗体或其抗原结合片段的表位可以通过现有技术测定，例如合成肽法，免疫信息学预测，多肽活性的测定，表位肽扫描法，噬菌体展示技术，X射线衍射和核磁共振分析，抗体同源建模蛋白对接预测法。本文中使用的短语“结合相同表位的抗体”表示，结合共同表位的不同抗体。如果第二抗体结合第一抗体所结合的部分肽或部分三级结构，那么可以确定，所述第一抗体和所述第二抗体结合相同表位。
本公开中，术语“抗体”取其最广义的解释，包括完整的单克隆抗体、多克隆抗体以及由至少两个完整抗体形成的多特异性抗体(例如双特异性抗体)，只要它们具有所需的生物学活性。在本公开中，“抗体”和“免疫球蛋白”可以互换使用。如本文所述的“抗体分子”或“抗体”指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有免疫特异性结合抗原的抗原结合位点的分子。因此，术语抗体广义上不仅涵盖完整抗体分子，还包括所述抗体的片段以及所述抗体和抗体片段的变体(包括衍生物)。当“抗体分子”或“抗体”与抗原结合片段在同一语境下使用时，“抗体分子”或“抗体”指完整抗体分子或全长抗体。在本说明书中所述术语抗体分子例如包括但非限于单链Fv(scFv)，Fab片段，Fab’片段，F(ab’)2，二硫键连接的Fv(sdFv)，Fv，及完整抗体或全长抗体。术语“单链Fv”或“scFv”是指一种多肽，其包含与抗体VH结构域连接的抗体的VL结构域。例如免疫特异性结合Napi2b的抗体可以与其它抗原发生交叉反应。优选地，免疫特异性结合Napi2b的抗体与其它抗原不发生交叉反应。免疫特异性结合可以例如通过免疫测定或其它本领域技术人员已知的方法鉴别。“完整”抗体或“全长”抗体指包含两条重链(H)和两条轻链(L)的蛋白，所述重链和轻链通过二硫键相互连接，所述蛋白包含：(1)就重链而言，包含可变区(本文缩写为“VH”)和含有三个结构域CH1、CH2、CH3的重链恒定区；和(2)就轻链而言，包含轻链可变区(本文缩写为″VL″)和含有一个结构域CL的轻链恒定区。本公开的抗体包括但非限于单克隆，多特异性，人或嵌合抗体，单链抗体，Fab片段，F(ab′)片段，抗独特型(抗-Id)抗体(包括例如本公开抗体的抗-Id抗体)，和上述任何抗体的表位结合片段。本公开的免疫球蛋白分子可以是免疫球蛋白的任何类型(例如IgG，IgE，IgM，IgD，IgA和IgY)，类别(例如IgG1，IgG2，IgG3，IgG4，IgA1和IgA2)或亚类。优选地，本公开的抗体包含或由具有序列及其具体信息表所述任一氨基酸序列或其片段或变体的VH结构域，VH CDR(本文中多用HCDR表示)，VL结构域，或VL CDR(本文中多用LCDR 表示)组成。
在本公开中，术语“单克隆抗体”指抗体来自一群基本均一的抗体，即构成该集群的各抗体完全相同，除了可能存在的少量天然突变。单克隆抗体具有针对抗原的一个决定簇(表位)的高特异性，而与其相对的多克隆抗体则包含针对不同决定簇(表位)的不同抗体。除了特异性之外，单克隆抗体的优点还在于合成时可以不受其他抗体的污染。此处修饰语“单克隆”表示该抗体的特征在于来自一个基本均一的抗体群，而不应理解成需由特殊方法制得。
在本公开的部分实施方案中，单克隆抗体还特别包括嵌合抗体，即重链和/或轻链的一部分与某种、某类或某亚类抗体相同或同源，其余部分则与另一种、另一类或另一亚类抗体相同或同源，只要它们具有所需的生物学活性(参见例如US 4,816,567；和Morrison等人，1984，PNAS，81:6851-6855)。可用于本公开的嵌合抗体包括灵长类化(primatized)抗体，其包含来自非人灵长类(例如古猴、猩猩等)的可变区抗原结合序列和人恒定区序列。
术语“抗原结合片段”是指抗体的一部分，优选是抗原结合区或可变区。抗体片段的实例包括Fab、Fab′、F(ab′)2、Fd、Fv、dAb和互补决定区片段，二抗体(diabody)，线性抗体和单链抗体分子。本文中使用的术语“抗原结合片段”表示抗体的具有抗原结合活性的部分片段，其中所述片段具有抗体的完全或部分功能，包括非限于单链Fv(scFv)，Fab，Fab’，F(ab’)2，二硫键连接的Fv(sdFv)，Fv，di-scFv等。该术语也包括Fab’，其为在还原条件下处理F(ab’)2得到的抗体的可变区的单价片段。但是，该术语不限于这些分子，只要所述片段具有与抗原的结合亲和力即可。此外，这些功能片段不仅包括用适当的酶处理抗体蛋白的全长分子得到的片段，而且包括使用遗传修饰的抗体基因在适当宿主细胞中生产的蛋白。
本文中使用的术语“Fab’”表示如上所述在还原条件下处理F(ab’)2得到的抗体的可变区的单价片段。但是，本公开的Fab’也包括使用遗传修饰的抗体基因生产的Fab’。
如本文中所使用的，术语“scFv”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)或直接相连(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。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
如本文所用，在所述抗体或其抗原结合片段中的即使含有变体、氨基酸的置换、缺失或添加，也仍然具有结合所述抗原的活性。
术语“双特异性抗体”亦称为“双功能抗体偶联物”，是指由第一抗体(片段)和第二抗体(片段)通 过偶联臂所形成的偶联物，该偶联物保留了各自抗体的活性，故具有双功能和双特异性。
术语“多特异性抗体”包括例如三特异性抗体和四特异性抗体，前者是具有三种不同抗原结合特异性的抗体，而后者是具有四种不同抗原结合特异性的抗体。
术语“完整抗体”或“全长抗体”指包含抗原结合可变区和轻链恒定区(CL)、重链恒定区(CH1、CH2和CH3)的抗体。恒定区可以是天然序列(例如人天然恒定区序列)或其氨基酸序列变体。完整抗体优选是具有一种或多种效应功能的完整抗体。
术语“前抗(Probody)”是一种修饰的抗体，包括一种抗体或一种抗体片段，能专门与其靶点结合，能够与掩蔽基团耦合，其中掩蔽基团指对抗体或抗体片段与其靶点的结合能力的裂解常数比没有耦合掩蔽基团的抗体或抗体片段与其靶点的结合能力的裂解常数至少大100倍或1000倍、或者10000倍。
在本公开中，非人(例如鼠)抗体的“人源化”形式指包含最少量非人免疫球蛋白序列的嵌合抗体。大多数人源化抗体是人接受者免疫球蛋白的超变区残基被置换成具有所需特异性、亲和力和功能的非人(例如小鼠、大鼠、兔或非人灵长类)超变区残基(供者抗体)。在一些实施方案中，人免疫球蛋白的框架区(FR)残基也被置换成非人残基。而且，人源化抗体还可以包含受者抗体或供者抗体中没有的残基。这些修饰是为了进一步优化抗体的性能。人源化抗体一般包含至少一个，通常是两个可变区，其中所有或几乎所有超变环(hypervanable loops)与非人免疫球蛋白的相对应，而FR则完全或几乎完全是人免疫球蛋白的序列。人源化抗体还可以包含免疫球蛋白恒定区(Fc，通常是人免疫球蛋白Fc)的至少一部分。有关细节参见例如Jones等人，1986，Nature，321:522-525；Riechmann等人，1988，Nature，332:323-329；和Presta，1992，Curr Op Struct Bwl 2:593-596。
完整抗体可根据重链恒定区的氨基酸序列分为不同的“类”。主要的五类是IgA、IgD、IgE、IgG和IgM，其中几类还可以分为不同的“亚类”(同种型)，例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2。抗体不同类的重链恒定区分别称为α、β、ε、γ和μ。免疫球蛋白不同类的亚基结构和三维构型是本领域中公知的。
在本文中，本公开的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号***确定。在某些实施方案中，本公开的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或，AbM或IMGT编号***确定。
如本文中所使用的，术语“构架残基区”或“FR残基”是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“胚系抗体基因”是非淋巴细胞表达的免疫球蛋白的编码基因，它没有经历导致表达特异免疫球蛋白的遗传学重排及成熟的成熟过程。本公开的各种实施方案所提供的一个优点来源于一种认识，那就是胚系抗体基因编码的氨基酸序列比成熟抗体基因编码的氨基酸序列更多地保留了动物物种个体的特征性的重要氨基酸序列结构。因此当被治疗性应用于该物种时，更少地被该物种识别为外源物质。
作为氨基酸取代，在本说明书中，保守的氨基酸取代是优选的。保守的氨基酸取代表示，在与氨基酸侧链有关的氨基酸集合内发生的取代。优选的氨基酸集合如下：酸性集合(天冬氨酸和谷氨酸)；碱性集合(赖氨酸、精氨酸和组氨酸)；非极性集合(丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨 酸、蛋氨酸和色氨酸)；和不带电荷的极性家族(甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸和酪氨酸)。更优选的氨基酸集合如下：脂族羟基(丝氨酸和苏氨酸)；含有酰胺的集合(天冬酰胺和谷氨酰胺)；脂族集合(丙氨酸、缬氨酸、亮氨酸和异亮氨酸)；和芳族集合(苯丙氨酸、色氨酸和酪氨酸)。这样的氨基酸取代优选地在不会损害具有原始氨基酸序列的物质的性能的集合内进行。
此外，已知的是，缺失在培养的哺乳动物细胞中生产的抗体的重链的羧基端的赖氨酸残基(Journal of Chromatography A,705:129-134(1995))，还已知的是，缺失在培养的哺乳动物细胞中生产的抗体的重链的羧基端的2个氨基酸残基(甘氨酸和赖氨酸)，并且新位于羧基端处的脯氨酸残基被酰胺化(Analytical Biochemistry,360:75-83(2007))。但是，重链序列的这样的缺失和修饰不会影响抗体的抗原结合亲和力和效应子功能(补体的活化、抗体依赖性的细胞的细胞毒性等)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本文中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本公开中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示；精氨酸可用R或Arg表示；甘氨酸可用G或Gly表示；谷氨酰胺可用Q或Gln表示。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤和传染病)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本公开的目的，有益或所需的临床结果包括但不限于，减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如非人灵长类哺乳动物或人。在某些实施方式中，所述受试者(例如人)肿瘤和传染病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤和传染病)有效量是指，足以预防，阻止，或延迟疾病(例如，肿瘤和传染病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“效应子功能(effector function)”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体同种型而变化。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。
术语“抗体药物偶联物群”是指一组或一群本发明的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐、其互变异构体、或其药学上可接受的溶剂合物组成的混合物，其中抗体药物偶联物 的q可以相同或不同。另外，也可将其称为“抗体药物偶联物混合物”。
使用实验动物可以确定抗体和抗体-药物偶联物对癌症的体内治疗效果，例如，将抗体施用给植入了表达Napi2b的肿瘤细胞系的裸鼠，并测量癌细胞的任何变化。
在本公开中，尽管大多数情况下抗体中的氨基酸取代是被L-氨基酸取代，但也不限于此。在一些实施方案中，抗体肽链中可以包括一个或多个D-氨基酸。包含D-氨基酸的肽在口腔、肠道或血浆中比仅包含L-氨基酸的肽更加稳定而不易降解。
本公开所用的单克隆抗体可以由许多方法生产。例如，用于本公开的单克隆抗体可以通过杂交瘤方法，使用许多物种(包括小鼠、仓鼠、大鼠和人的细胞)获得(参见例如Kohler等人，1975，Nature，256:495)，或者通过重组DNA技术制得(参见例如US 4,816,567)，或者从噬菌体抗体库中分离得到(参见例如Clackson等人，1991，Nature，352:624-628；和Marks等人，1991，Journal of Molecular Biology，222:581-597)。
本文中，除非以其他方式明确指出，在本文中通篇采用的描述方式“各…独立地选自”和“…各自独立地选自”可以互换，均应做广义理解，其既可以是指在不同基团中，相同或不同的符号之间所表达的具体选项之间互相不影响，也可以表示在相同的基团中，相同或不同的符号之间所表达的具体选项之间互相不影响。
本文中，术语“直接键”表示其两侧的基团直接相连，例如，式II所示的化合物中，若X为直接键，则其结构式为其余直接键可参照前述内容进行理解。
本文中，术语“不存在”表示该基团不存在，例如，式II所示的化合物中，若W不存在，则其结构式为
式II所示的化合物中，R1和R2和与其相连的碳原子形成 “虚线”键表示所述杂环与苯环稠合的位置，例如，形成
本文中，式III所示的药物连接体偶联物中，L4为时，其中的标号1、2表示L4与其余基团的连接位置，具体地，1位与L3相连，2位与药物分子即相连时，形成其余标号1、2可参照前述内容进行理解。
本文中，式II所示的化合物中，R3和X和与其相连的碳原子一起形成 虚线表示所述碳环与苯环和吡啶环稠合的位置，形成
本文中，X的定义例如为，“X选自任选取代的所述取代基选自1个或2个C1-4烷基(如甲基)”，则X例如可以为X其余类似的定义可参照前述内容进行理解。
本文中，X的定义例如为，“X选自任选取代的所述取代基选自2个C1-4烷基(如甲基)和与它们同时相连的碳原子一起形成C3-6环烷基(如环丙基)”，则X例如可以为X其余类似的定义可参照前述内容进行理解。
AA1所示氨基酸残基的结构中，若r为0，则本领域技术人员可以理解的是，AA1所示氨基酸残基的结构将变为
AA1所示氨基酸残基的结构中，若Ra与Rb和与它们共同相连的碳原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0所取代，其中，术语“所述4-10元杂环任选地被一个或多个R0所取代”的含义为，所述4-10元杂环可以不被取代，也可以被一个或多个R0所取代，且所述多个R0中，各R0的定义可以相同，也可以不同。其余类似的定义可以参照前述内容进行理解。
本文中，例如，L3选自Lys、Val-Cit、Ala-Ala-Asn、Ala-Ala-Asp、Gly-Gly-Phe-Gly、Val-Lys-Gly、Val-Ala、Lys-Ala-Asn时，“所述赖氨酸(Lys)的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代”的含义为，所述L3各选项中的Lys的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代。其中，“Lys的远 端氨基”指的是赖氨酸残基中裸露的氨基-NH2。“所述赖氨酸(Lys)的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代”表示所述赖氨酸(Lys)的远端氨基可以不被取代，也可以被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代，例如，可以被1个叔丁氧羰基取代，即变为或者，可以被2个甲基取代，即变为或者被2个甲基和1个O同时取代，即变为需要说明的是，“所述赖氨酸(Lys)的远端氨基被O取代”指的是所述赖氨酸(Lys)的远端氨基被氧代，即变为若该远端氨基进一步被两个甲基所取代，则其变为
在本说明书的各部分，本公开化合物的取代基按照基团种类或范围公开。特别指出，本公开包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C1-6烷基”特别指独立公开的甲基、乙基、C3烷基、C4烷基、C5烷基和C6烷基。
在本文中，术语“C1-6烷基”表示直链或支链的含有1-6个碳原子的烷基，包括例如“C1-3烷基”或“C1-4烷基”，甲基，乙基等，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、戊基、己基。
在本文中，术语“C1-4烷基”表示直链或支链的含有1-4个碳原子的烷基，包括例如“C1-3烷基”，甲基，乙基等，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。
在本文中，术语“C2-6烯基”是指含有至少一个双键且碳原子数为2-6的直链、支链或环状的烯基，包括例如“C2-4烯基”等。其实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、 1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基、环戊烯基、1,3-环戊二烯基、环己烯基、1,4-环己二烯基等。
在本文中，术语“C2-6炔基”是指含有至少一个三键且碳原子数为2-6的直链或支链的炔基，包括例如“C2-4炔基”等。其实例包括但不限于：乙炔基、丙炔基、2-丁炔基、2-戊炔基、3-戊炔基、4-甲基-2-戊炔基、2-己炔基、3-己炔基、5-甲基-2-己炔基等。
在本文中，术语“卤素”包括氟、氯、溴、碘。
在本文中，术语“3-6元环烷基”或“C3-6环烷基”是指含有3-6个碳原子的饱和环状烷基，包括环丙烷基(即环丙基)、环丁烷基(即环丁基)、环戊烷基(即环戊基)、环己基。
在本文中，术语“3-7元碳环烷基”或“C3-7环烷基”是指含有3-7个碳原子的饱和环状烷基，包括环丙烷基、环丁烷基、环戊烷基、环己基、环庚基。
在本文中，术语“C1-6烷氧基”是指通过氧原子连接至母体分子部分的如上文所定义的烷基。具体实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基等。
在本文中，术语“C1-4烷氧基”是指通过氧原子连接至母体分子部分的如上文所定义的烷基。具体实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基等。
在本文中，术语“4-10元杂环基”是指含有4-10个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环状基团。术语“4-6元杂环基”是指含有4-6个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环状基团。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。“4-8元杂环基”包括例如“4-8元含氮杂环基”、“4-8元含氧杂环基”、“4-7元杂环基”、“4-7元含氧杂环基”、“4-7元杂环基”、“4-6元杂环基”、“5-7元杂环基”、“5-6元杂环基”、“5-6元含氮杂环基”，包括但不限于氧代环丁烷基、吡咯烷基、四氢呋喃基、哌啶基、哌嗪基、四氢吡喃基、高哌嗪基等。
在本文中，术语“4-10元杂环”是指含有4-10个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环。术语“5-6元杂环”是指含有5-6个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环，包括但不限于吡咯烷、四氢呋喃、哌啶、哌嗪、四氢吡喃等环。
在本文中，术语“芳基”是指具有芳香性的单环或多环烃基，例如6-10元芳基、5-8元芳基等。具体的实例包括但不限于苯基、萘基、蒽基、菲基等。所述“6-10元芳基”是指含有6-10个环原子的芳基。所述“C6-10芳基”是指含有6-10个碳原子的芳基。
在本文中，术语“杂芳基”是指具有芳香性的环状基团，其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。具体实例包括但不限于5-10元杂芳基、5-6元杂芳基、5-10元含氮杂芳基、6-10元含氧杂芳基、6-8元含氮杂芳基、5-8元含氧杂芳基等，例如呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1,2,3-***基、1,2,4-***基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-噁二唑基、吡啶基、2-吡啶酮基、4-吡啶酮基、嘧啶基、1,4-二氧杂环 己二烯基、2H-1,2-噁嗪基、4H-1,2-噁嗪基、6H-1,2-噁嗪基、4H-1,3-噁嗪基、6H-1,3-噁嗪基、4H-1,4-噁嗪基、哒嗪基、吡嗪基、1,2,3-三嗪基、1,3,5-三嗪基、1,2,4,5-四嗪基、氮杂环庚三烯基、1,3-二氮杂环庚三烯基、氮杂环辛四烯基等。
本文用波浪线“～～”表示的结构式中的键意在表示，该结构表示顺式或反式异构体，或任意比例的顺式和反式异构体的混合物。
术语“药物与抗体比”或“DAR”是指在群(或混合物)或组合物或ADC分子中药物和抗体的比例，例如，与ADC的抗体附接的小分子毒素。ADC的DAR可以在1到16的范围内，但是取决于抗体上的连接位点的数量，更高的负载(例如20)也是可能的。提及负载到单个抗体上的药物的数量时，或可替代地，提及一组ADC的平均或均值DAR时，可以使用术语DAR。应当理解，后者通常称为平均DAR。在质谱法测定DAR值的过程中，抗体已经被还原为分离的重轻链，DAR1代表包含轻链或重链偶联1个毒素分子的偶联物；DAR2代表包含轻链或重链偶联2个毒素分子的偶联物；DAR3代表包含轻链或重链偶联3个毒素分子的偶联物。
发明的有益效果
本公开通过大量研究多个靶点抗体偶联药物(ADC)，实现对肿瘤微环境的富集、连接子特有的体内酶切特性以及与靶向部分的偶联方式、结合大量体内体外药效的筛选验证，获得一类新颖的抗体生物活性分子偶联物。利用上述方式获得的偶联物，能够实现多种下述的惊奇的技术效果：
根据上述方式得到的偶联物具有更好的溶解度和优异的化学稳定性，如不发生传统ADC中马来酰亚胺连接方式引起的可逆Michael加成反应，因此可以获得高药物-抗体比，在一些实施方案中，所述偶联物的DAR值能够达到6-8；
具有极高的偶联效率，在一些实施方案中，所述偶联效率能够达到或超过90％；
通过大量研究发现了一类具有高血浆稳定性，但同时又能在肿瘤微环境中(肿瘤细包内和肿瘤细胞外均可)裂解的链接子，因此可以在抗原低表达或者抗原不表达肿瘤中产生好的抗肿瘤效果；
根据上述方式得到的偶联物(ADC)通过对连接子和整体ADC分子的理化性质的调整，提高了整个ADC分子在相对酸性肿瘤环境中的暴露量，因而ADC具有更好的肿瘤组织靶向性，即在肿瘤微环境中的富集能力，增加了生物活性分子在瘤内和血液浓度比，降低了ADC分子的机理相关的毒性(ADC结合非肿瘤组织中细胞表面抗原并内吞后产生的毒性，或者又叫“on-target毒性”)，因此具有更高的治疗指数；
根据上述方式得到的偶联物具有很高的在体内循环中的稳定性，减少了药物分子在非靶组织中的脱落，减少了非靶组织中毒素脱落引起的“off-target”毒性；
所述偶联物的生物活性分子具有更高的抗肿瘤细胞活性，因此具有优异的旁观者效应(by-stander effect)，ADC能够更有效地杀死抗原高表达肿瘤细胞以及肿瘤组织中抗原低表达或抗原不表达的肿瘤细胞；
本公开的毒素-连接子，利用其连接子在肿瘤微环境中的胞外裂解能力，可以和没有细胞内吞能力的抗体组成抗体偶联药物，这类抗体偶联药物依然具有高抗肿瘤活性；
本公开的毒素-连接子，利用其连接子在肿瘤微环境中的胞外裂解能力以及其在肿瘤微环境中的富集能力，可以和没有细胞内吞能力的抗体以及没有肿瘤细胞外抗原结合能力的抗体组成抗体偶联药物，这 类抗体偶联药物依然具有高抗肿瘤活性；
本公开提供的抗Napi2b抗体具有极高的人源化程度或者全人源抗体，从而可安全地施用给人受试者，而不引发免疫原性反应。
综上，本发明ADC具有重大的临床价值。
附图说明
图1.抗Napi2b人源化抗体流式亲和力评价
图2.抗Napi2b人源化抗体内吞活性检测
图3A.66C12D12-hz1抗体在血清中的亲和力检测
图3B.16G5B3-hz2抗体在血清中的亲和力检测
图4.抗人Napi2b ADC在HT29-h.Napi2b CDX模型中体内药效测试
图5.抗人Napi2b ADC在HT29-h.Napi2b CDX模型给药过程中小鼠体重变化
图6.抗人Napi2b ADC在IGR-OV1CDX模型中体内药效测试
图7.抗人Napi2b ADC在IGR-OV1CDX模型给药过程中小鼠体重变化
具体实施方式
以下通过具体实施方式的描述对本公开作进一步说明，但这并非是对本公开的限制。本领域技术人员根据本公开的教导，可以做出各种修改或改进，而不脱离本公开的基本思想和范围。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本公开中的缩写具有以下含义：


序列及其具体信息：







现参照下列意在举例说明本公开(而非限定本公开)的实施例来描述本公开。
除非特别指明，本公开中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行。本领域技术人员知晓，实施例以举例方式描述本公开，且不意欲限制本公开所要求保护的范围。
制备方案
以下的实施例中记载的化合物的结构通过核磁共振(1H NMR)或质谱(MS)来确定。
核磁共振(1H NMR)的测定仪器使用Bruker 400MHz核磁共振仪；测定溶剂为氘代甲醇(CD3OD)、氘代氯仿(CDCl3)或六氘代二甲基亚砜(DMSO-d6)；内标物质为四甲基硅烷(TMS)。
实施例中使用的核磁共振(NMR)图谱中的缩写示于以下。
s：单峰(singlet)、d：二重峰(doublet)、t：三重峰(triplet)、q：四重峰(quartet)、dd：双二重峰(double doublet)、qd：四二重峰(quartet doublet)、ddd：双双二重峰(double double doublet)、ddt：双双三重峰(double double triplet)、dddd：双双双二重峰(double double double doublet)、m：多重峰(multiplet)、br：宽峰(broad)、J：偶合常数、Hz：赫兹、DMSO-d6：氘化二甲基亚砜。δ值用ppm值表示。
质谱(MS)的测定仪器使用Agilent(ESI)质谱仪，型号为Agilent 6120B。
一、生物活性分子和合成“药物-连接体化合物”过程使用的中间体的合成
A、生物活性分子的合成
实施例A1.1：(S)-4-乙基-8-氟-4-羟基-9-甲基-11-(1H-吡唑-4-基)-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.1)的合成
步骤一：4-溴-1-(四氢-2H-吡喃-2-基)-1H-吡唑(4.6g)溶于无水四氢呋喃中(50ml)，上述溶液在氮气保护下干冰-丙酮冷却到-78℃，然后滴加正丁基锂(12ml,2M),反应液在-78℃搅拌20分钟，然后加入2-氨基-4-氟-5-甲基苯甲酸甲酯(1.83g),加完后，反应自然升至室温，并连续搅拌反应5小时。反应甲甲醇(3ml)猝灭后加入乙酸乙酯(200ml)，溶液用水洗(100ml x 3)，有机相干燥后除去有机溶剂，用硅胶柱色谱分离得到目标产物(2-氨基-4-氟-5-甲基苯基)(1-(四氢-2H-吡喃-2-基)-1H-吡唑-4-基)甲酮，ESI-MS(m/z):304[M+H]+。
步骤二：(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃[3,4-f]中氮茚并-3,6,10(4H)-三酮(2.63g),(2-氨基-4-氟-5-甲基苯基)(1-(四氢-2H-吡喃-2-基)-1H-吡唑-4-基)甲酮(3.03g),对甲苯磺酸(1.74g)溶于二氯甲烷(50ml)中，然后除去溶剂，混合物在氮气保护下加热至120℃反应4小时。混合物溶于乙酸乙酯(300ml)，有机相水洗(100ml x 2)，干燥后除去有机溶剂，残留物用硅胶柱层析分离得到目标产物(S)-4-乙基-8-氟-4-羟基-9-甲基-11-(1H-吡唑-4-基)-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.1)。ESI-MS(m/z):447[M+H]+。
实施例A1.2：(S)-7-乙基-7-羟基-14-(1H-吡唑-4-基)-10,13-二氢-11H-[1,3]二氧并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.2)的合成
用实施例A1.1相同的方法和反应条件，用6-氨基苯并[d][1,3]二氧环戊烷-5-羧酸甲酯代替2-氨基-4-氟-5-甲基苯甲酸甲酯，得到目标产物(S)-7-乙基-7-羟基-14-(1H-吡唑-4-基)-10,13-二氢-11H-[1,3]二氧并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.2)。ESI-MS(m/z):459[M+H]+。
实施例A1.3：(S)-14-(3-氨基苯基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.3)的合成
化合物(A1.3-A)(1.4g)，化合物(A1.3-B)(2.2g)，Pd2(DBA)3(300mg),三环己基磷(300mg)，醋酸钾(1.1g)依次加入二氧六环(30ml)和水(5ml)的混合溶剂中，混合物在氮气保护下100℃加热搅拌反应12小时。冷却后加入乙酸乙酯(200ml)，水洗(100ml)，干燥后，硅胶柱层析分离得到目标产物(S)-14-(3-氨基苯基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.3)。ESI-MS(m/z):484[M+H]+。
实施例A1.4：(S)-14-(4-氨基苯基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.4)的合成
化合物(A1.4-A)(1.4g)，化合物(A1.4-B)(2.2g)，Pd2(DBA)3(300mg),三环己基磷(300mg)，醋酸钾(1.1g)依次加入二氧六环(30ml)和水(5ml)的混合溶剂中，混合物在氮气保护下100℃加热搅拌反应12小时。冷却后加入乙酸乙酯(200ml)，水洗(100ml)，干燥后，硅胶柱层析分离得到目标产物(S)-14-(4-氨基苯基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并 [3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.4)。
ESI-MS(m/z):484[M+H]+；
1H NMR(400MHz,DMSO)δ7.56(s,1H),7.34(d,J＝8.0Hz,2H),7.27(s,1H),7.14(s,1H),6.89(d,J＝7.8Hz,2H),6.26(s,2H),5.40(s,2H),5.05(s,2H),1.96–1.78(m,2H),0.88(t,J＝7.2Hz,3H)。
实施例A1.5：(S)-14-(3-氨基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.5)的合成
步骤一：(S)-14-(3-氯丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成
冰浴条件下，向化合物(S)7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]茚并[1,2-b]喹啉-8,11(7H)-二酮(A1.5-A，500mg)的75％硫酸溶液(5mL)中加入七水合硫酸亚铁(570mg七水合硫酸亚铁溶于1mL水中)，和4,4-二甲氧基氯丁烷(3.89g)，反应液搅拌三分钟后滴加双氧水(29％，2.5mL)。反应液在0℃下搅拌反应5分钟后升至室温，并搅拌反应3小时。反应液加入水(50mL)稀释，乙酸乙酯(80mL×2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗产品，粗产品用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化得到目标化合物(黄色固体，400mg，收率：67％)。
LCMS(ESI)[M+H]+：468.9；
1H NMR(400MHz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.42(s,2H),5.26(s,2H),3.81(d,J＝5.9Hz,2H),3.22(s,2H),1.98(d,J＝6.7Hz,4H),0.88(t,J＝7.2Hz,3H)。
步骤二：(S)-14-(3-叠氮基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成
向化合物(S)-14-(3-氯丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(200mg)的N,N-二甲基甲酰胺溶液(3mL)中，加入叠氮化钠(284mg)，反应液在100℃下搅拌反应1小时。向反应液中加入水(30mL)，乙酸乙酯(60mL×2)萃取，有机相用经饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到目标化合物(S)-14-(3-叠氮基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(黄色固体，180mg，收率：88％)。
LCMS(ESI)[M+H]+：476；
1H NMR(400MHz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.48(s,1H),6.29(s,2H),5.42(s,2H),5.25(s,2H),3.53–3.49(m,2H),3.16–3.12(m,2H),1.93–1.80(m,4H),0.88(t,J＝7.2Hz,3H)。
步骤三：(S)-14-(3-氨基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成
向化合物(S)-14-(3-叠氮基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成(130mg，0.274mmol)的四氢呋喃(3mL)和水(1mL)混合溶液中加入三苯基膦(108mg，0.411mmol)，在55℃下反应16小时。LCMS显示反应完全。向反应液中加入水(5mL)，用2N盐酸(3mL)调至酸性，用乙酸乙酯(10mL)萃取。水相经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物(S)-14-(3-氨基丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮的合成(A1.5，黄色固体，15mg,收率：12％)。
LCMS(ESI)[M+H]+：449.9；
1H NMR(400MHz,DMSO-d6)δ7.72(s,3H),7.53(s,1H),7.25(s,1H),6.50(s,1H),6.30(s,2H),5.43(s,2H),5.24(s,2H),3.15(d,J＝6.4Hz,2H),3.03(t,J＝6.9Hz,2H),1.96–1.81(m,4H),0.88(t,J＝7.3Hz,3H)。
实施例A1.6：(S)-N-乙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环 戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基乙酰胺(A1.6)的合成
步骤一：(S)-2-(乙基(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧醇[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)氨基)-2-氧乙基乙酸酯(A1.6-B)的制备：
将化合物(S)-7-乙基-14-(2-(乙胺基)乙基)-7-羟基-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-8,11(7H)-二酮(A1.6-A，50mg)，乙酰氧基乙酰氯，(73mg)，三乙胺(50mg)依次加入二氯甲烷(5mL)中，室温搅拌反应1小时。反应液旋蒸除去溶剂，得到粗品油状化合物75mg。
LCMS(ESI)[M+H]+：564.2。
步骤二：(S)-N-乙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基乙酰胺(A1.6)的合成
将粗品化合物(S)-2-(乙基(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧醇[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)氨基)-2-氧乙基乙酸酯(75mg，0.13mmol)溶在浓盐酸和无水乙醇混合溶液(体积比为1/2，3mL)中，然后反应液85℃回流反应1小时。LCMS显示反应完成。反应液浓缩，粗产品经制备色谱(0.01％三氟乙酸的水溶液，乙腈)纯化得到目标化合物(S)-N-乙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基乙酰胺(A1.6)(15mg，收率：26％)为白色固体。
LCMS(ESI)[M+H]+：522.0；
1H NMR(400MHz,DMSO)δ7.92(s,1H),7.53(m,1H),7.25(s,1H),6.50(s,1H),6.31(s,2H),5.43(s,2H),5.34(m,2H),4.65(m,1H),4.07(m,2H),3.52(m,2H),3.41(m,2H), 2.00(m,2H),1.88(m,2H),1.16–1.04(m,3H),0.89–0.83(m,3H)。
实施例A1.7：(S)-N-甲基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基乙酰胺(A1.7)的合成
步骤一：化合物甲基苄基(3-(6-硝基苯并[d][1,3]二氧杂环-5-基)-3-氧丙基)氨基甲酸酯的制备
将化合物1-(6-硝基苯并[d][1,3]二恶英-5-基)乙烷-1-酮(A1.7-A，500mg)，甲胺盐酸盐(1.6g)和多聚甲醛(714mg)溶在乙醇(8mL)中，100℃下闷罐反应16小时。反应液降至室温并减压浓缩，所得残渣用二氯甲烷(80mL)溶解，有机相经水(50mL×3)萃取。所得水相用碳酸氢钠调至PH＝9，然后加入氯甲酸苄酯(513mg,3.0mmol)，在室温下反应16小时。反应液用乙酸乙酯(30mL×3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，浓缩得到粗品，粗产品经C18分离纯化(乙腈/水含0.05％甲酸＝5～95％)得到目标化合物(A1.7-B，180mg，收率23％)。
LCMS(ESI)[M+H]+：387.1.
1H NMR(400MHz,CDCl3)δ7.56(s,1H),7.35(m,5H),7.32(m,1H),6.17(s,2H),5.13(s,2H),3.71(m,2H),3.03(m,3H),2.99–2.86(m,2H)。
步骤二：化合物苄基(3-(6-氨基苯并[d][1,3]二恶英-5-基)-3-氧丙基)(甲基)氨基甲酸酯(A1.7-C)的制备
将化合物A1.7-B甲基苄基(3-(6-硝基苯并[d][1,3]二氧杂环-5-基)-3-氧丙基)氨基甲酸酯(180mg,0.47mmol)溶于饱和氯化铵水溶液(8mL)和乙醇(8mL)混合溶液中，然后向反应液中加入铁粉(130mg)，反应在80℃下搅拌2小时。将反应液过滤，滤液减压浓缩成固体，粗产品经TLC分离纯化(石油醚：乙酸乙酯＝2/1)得到目标化合物A1.7-C(76mg)。
LCMS(ESI)[M+H]+：357.0。
步骤三：苄基(S)-(2-(7-乙基-7-羟基-8,11-二氧基-7,8,11,13-四氢-10H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(甲基)氨基甲酸酯(A1.7-D)的制备
室温下，将化合物A1.7-C苄基(3-(6-氨基苯并[d][1,3]二恶英-5-基)-3-氧丙基)(甲基)氨基甲酸酯(38mg)，化合物(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(29mg)和对甲苯磺酸(23mg)溶于二氯甲烷(5mL)溶液中，溶液澄清混匀后减压浓缩，用油泵抽至真空，反应在120℃真空下反应2小时。将反应液降至室温，加入水(30mL)并用二氯甲烷(30mL×3)萃取。合并有机相依次经无水硫酸钠干燥、过滤，滤液减压浓缩得到目标化合物A1.7-D(50mg)。
LCMS(ESI)[M+H]+＝584.0。
步骤四：(S)7-乙基-7-羟基-14-(2-(甲胺基)乙基)-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-8,11(7H)-二酮(A1.7-E)的制备
室温下，将化合物A1.7-D苄基(S)-(2-(7-乙基-7-羟基-8,11-二氧基-7,8,11,13-四氢-10H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)(甲基)氨基甲酸酯(50mg)溶于二氯甲烷(5mL)溶液中，于0℃下加入三甲基碘硅烷(51mg，0.26mmol)，反应3小时。将反应液浓缩除去溶剂，经高效液相制备纯化(乙腈/水含0.05％甲酸)得到褐色固体化合物A1.7-E(15mg)。
LCMS(ESI)[M+H]+：450.0。
步骤五：化合物(S)-2-((2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧醇[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)(甲基)氨基)-2-氧乙基乙酸酯(A1.7-F)的制备
将化合物A1.7-E(S)7-乙基-7-羟基-14-(2-(甲胺基)乙基)-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(15mg)和三乙胺(15mg)溶于二氯甲烷(5mL)溶液中，于0℃下加入乙酰氧基乙酰氯(20mg)反应1小时。将反应液浓缩除去溶剂得到粗品，所得固体直接用于下一步，未做进一步纯化。
LCMS(ESI)[M5+H]+＝550.1。
步骤六：化合物(S)-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)-2-羟基-N-甲基乙酰胺(A1.7)的制备
将化合物A1.7-F(S)-2-((2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧醇[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)(甲基)氨基)-2-氧乙基乙酸酯(15mg)溶于乙醇(5mL)溶液中，加入浓盐酸(1.5mL)，于80℃下反应2小时。将反应液浓缩除去溶剂，经高效液相制备纯化(乙腈/水含0.05％甲酸)得到白色固体化合物A1.7(1.6mg)。
LCMS(ESI)[M+H]+：508.2。
实施例A1.8：(S)-N-异丙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基乙酰胺(A1.8)的合成
步骤一：化合物异丙基苄基(3-(6-硝基苯并[d][1,3]二恶英-5-基)-3-氧丙基)氨基甲酸酯(A1.8-B)的制备
向化合物(A1.8-A)3-(异丙胺基)-1-(6-硝基苯并[d][1,3]二恶英-5-基)丙-1-酮(900mg)的二氯甲烷溶液(10mL)中，依次加入三乙胺(1.8g)和苄氧基碳酰氯(734mg，4.3mmol)，在室温下反应2小时。反应液加水(50mL)稀释，用二氯甲烷(50mL×3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗品，粗产品经柱层析分离纯化(石油醚：乙酸乙酯＝3/1)得到目标化合物(A1.8-B)(600mg)。
LCMS(ESI)[M+H]+：414.9；
1H NMR(400MHz,DMSO-d6)δ7.72(s,1H),7.34(br s,6H),6.31(s,2H),5.08(s,2H),4.14–4.10(m,1H),3.48(d,J＝7.9Hz,2H),3.03(s,2H),1.13-1.11(m,6H)。
步骤二：化合物苄基(3-(6-氨基苯并[d][1,3]二恶英-5-基)-3-氧丙基)(异丙基)氨基甲酸酯(A1.8-C)的制备
将化合物(A1.8-B)异丙基苄基(3-(6-硝基苯并[d][1,3]二恶英-5-基)-3-氧丙基)氨基甲酸酯(200mg,0.48mmol)溶于饱和氯化铵(3mL)和乙醇(3mL)混合溶液中，然后向反应液中加入铁粉(135mg)，反应在室温下搅拌2小时。将反应液过滤，滤液减压浓缩成固体，粗产品经柱层析分离纯化(石油醚：乙酸乙酯＝3/1)得到目标化合物(A1.8-C)(65mg)。
LCMS(ESI)[M+H]+：395.2。
步骤三：(S)7-乙基-7-羟基-14-(2-(异丙胺基)乙基)-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-8,11(7H)-二酮(A1.8-D)的制备
室温下，将化合物(A1.8-C)苄基(3-(6-氨基苯并[d][1,3]二恶英-5-基)-3-氧丙基)(异丙基)氨基甲酸酯(65mg)，化合物(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(45mg)和对甲苯磺酸(33mg，0.17mmol)溶于二氯甲烷(10mL)溶液中，溶液澄清混匀后减压浓缩，用油泵抽至真空，反应在120℃真空下反应2小时。将反应液降至室温，加入水(50mL)，用二氯甲烷(30mL×3)萃取，合并有机相依次并无水硫酸钠干燥、过滤，滤液减压浓缩，粗产品经柱层 析分离纯化(二氯甲烷/甲醇＝10/1)得到目标化合物(A1.8-D)(40mg)。
LCMS(ESI)[M+H]+：478.0；
1H NMR(400MHz,DMSO-d6)δ8.46(s,1H),7.67(s,1H),7.57(s,1H),7.27(s,1H),6.52(s,1H),6.33(s,2H),5.44(s,2H),5.35(s,2H),3.41(br s,2H),3.23(br s,3H),1.94–1.78(m,2H),1.25(d,J＝6.4Hz,6H),0.88(t,J＝7.3Hz,3H)。
步骤四：化合物((S)-2-((2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)(异丙基)氨基)-2-氧代乙酸乙酯(A1.8-E)的制备
冰浴下，向化合物(S)-7-乙基-7-羟基-14-(2-(异丙胺基)乙基)-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-8,11(7H)-二酮(A1.8-D)(40mg)的二氯甲烷溶液(2mL)中加入三乙胺(35mg)，2-氯-2-氧乙基乙酸酯(57mg)，在零度下反应30分钟。将反应液减压浓缩得到化合物A1.8-E，直接应用于下一步。
LCMS(ESI)[M+H]+：578.0。
步骤五：化合物(S)-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧并[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)-2-羟基-N-异丙基乙酰胺(A1.8)的制备
向化合物(S)-2-((2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)乙基)(异丙基)氨基)-2-氧代乙酸乙酯(A1.8-E，50mg)的乙醇溶液(3mL)中，加入浓盐酸(0.5mL)，在70℃下反应1小时。将反应液浓缩得到粗产品。再经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物(A1.8，3mg)。
LCMS(ESI)[M+H]+：464.0；
1H NMR(400MHz,DMSO-d6)δ7.99(s,1H),7.53(s,1H),7.26(s,1H),6.31(s,2H),5.43(s,2H),5.36(s,2H),4.22(s,2H),3.99–3.94(m,1H),3.44(dd,J＝16.7,7.8Hz,2H),3.33–3.21(m,2H),1.95–1.79(m,2H),1.19(dd,J＝16.4,5.8Hz,6H),0.88(t, J＝7.2Hz,3H)。
实施例A1.9：(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.9)的合成
将化合物(S)-7-乙基-7-羟基-14-(3-氯丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(100mg，0.213mmol)溶于10％硫酸(5mL)溶液中，反应在110℃下反应48小时。向反应液中加入饱和碳酸氢钠(30mL)溶液，用二氯甲烷(10mL X 5)萃取，无水硫酸钠干燥，抽滤，减压浓缩得到粗产品。经高效液相制备纯化(乙腈/水含0.05％甲酸)得到(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.9，1.78mg)。
LCMS(ESI)[M+H]+：451.0；
1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.47–5.37(m,2H),5.32–5.19(m,2H),3.51–3.46(m,2H),3.17–3.13(m,2H),1.92–1.76(m,4H),0.90–0.84(m,3H)。
实施例A1.10：(S)-4-乙基-8-氟-4-羟基-11-(3-羟基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.10)的合成
步骤一：
0℃下，向化合物A1.10-A(10g)的1,2-二氯乙烷(200mL)溶液，滴加1mol/L三氯化硼(96mL)，4-氯丁腈(9.9g)。80℃下搅拌反应2小时。反应液降至室温，加入2mol/L盐酸(90mL)并在80℃下回流搅拌0.5小时。反应液降至室温,加入少量水稀释，用二氯甲烷(200mL X 3)萃取，有机相用无水硫酸钠干燥，抽滤，浓缩得到粗品，粗产品经柱层析分离纯化(石油醚：乙酸乙酯＝10/1)得到目标化合物A1.10-B(4g)。
LCMS(ESI)[M+H]+：230.0。
步骤二：
向化合物A1.10-B(50mg)中加入(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪- 3,6,10(4H)-三酮(35mg)，一水合对甲苯磺酸(41.4mg),溶于二氯甲烷(30mL)溶液中，溶液澄清混匀后减压浓缩，用油泵抽至真空，反应在120℃真空下反应3小时。LCMS显示反应完全。将反应液降至室温，加入水(20mL)，用二氯甲烷(20mL X 3)萃取。合并有机相依次并无水硫酸钠干燥、过滤，滤液减压浓缩得到粗产品。粗产品经柱层析分离纯化(二氯甲烷比甲醇＝20比1)得到目标化合物A1.10-C(80mg)为白色固体。
LCMS(ESI)[M+H]+：457.0。
步骤三：
将化合物A1.10-C(75mg)溶于六甲基磷酰三胺，加入纯水(0.8mL)反应液在100℃下搅拌72小时。LCMS检测反应完成。经制备色谱(0.01％TFA in water，MeCN)纯化得到目标化合物(10mg)。
LCMS(ESI)[M+H]+：439.2；
1H NMR(400MHz,DMSO-d6)δ8.22(d,J＝8.4Hz,1H),7.87(d,J＝10.9Hz,1H),7.31(s,1H),6.50(s,1H),5.43(s,2H),5.30(s,2H),4.67(t,J＝4.9Hz,1H),3.55–3.47(m,2H),3.28–3.20(m,2H),2.51(s,3H),1.93–1.81(m,4H),0.88(t,J＝7.3Hz,3H)。
实施例A1.11：(S)-4-乙基-8-氟-4-羟基-11-(3-氨基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.11)的合成
步骤一：
向A1.10-C(395mg)的N’N-二甲基甲酰胺(10mL)溶液中加入叠氮化钠(432mg)，反应液80℃反应16小时，然后冷却至室温，加入水(50mL)稀释，用乙酸乙酯(80mL*3)萃取，合并有机相，无水硫酸钠干燥，抽滤浓缩得到目标产物A1.11-A(290mg)。
LCMS(ESI)[M+H]+：464.0。
步骤二：
将化合物A1.11-A(93mg)溶于四氢呋喃(5mL)中，加入三苯基膦(78mg)，室温反应4小时。然后向反应液中加入盐酸(4M，1mL)，反应液升至55℃反应16小时，LCMS监测反应完全。反应液直接浓缩，粗品经过反相柱(流动相A为0.05％甲酸水溶液，B为乙腈)提纯得到目标产物A1.11(28mg，收率：36％)为白色固体。
LCMS(ESI)[M+H]+：438.4；
1H NMR(400MHz,CD3OD)δ8.52(s,1H),8.16(d,J＝7.8Hz,1H),7.76(d,J＝10.7Hz,1H),7.63(s,1H),5.49(ABq,J＝78.9,16.3Hz,2H),5.31(br s,2H),3.35–3.32(m,2H),3.22–3.10(m,2H),2.55(s,3H),2.13–2.11(m,2H),1.96–1.94(m,2H),1.01(t,J＝7.4Hz,3H)。
实施例A1.12：(S,E)-14-(3-氨基-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.12)的合成
步骤一：
将化合物A1.12-A(200mg，0.39mmol)，化合物A1.12-B(112mg，0.39mmol)，氟化铯(152mg，0.975mmol)和四三苯基膦钯(45mg，0.039mmol)加入1,4-二氧六环溶液(8mL)。在氮气气氛下120℃微波反应0.5小时。LCMS显示反应完全。向反应液加入二氯甲烷(20mL)和甲醇(10mL)混合溶液稀释，过滤。滤液浓缩，粗品通过制备TLC纯化(二氯甲烷：甲醇＝30:1)得到目标化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-7-基乙酸酯(A1.12-C，60mg，收率：26％)为棕色固体。LCMS(ESI)[M+H]+＝590.3；
1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.49(s,1H),7.35(s,1H),7.09(d,J＝16.7Hz,1H),6.93(s,1H),6.45(d,J＝16.6Hz,1H),6.30(s,2H),5.47(s,2H),5.34–5.24(m,2H),3.94(s,2H),2.21(br s,3H),2.03–1.96(m,2H),1.45(s,9H),0.91(t,J＝6.7Hz,3H).
步骤二：
向化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-7-基乙酸酯(A1.12-C，50mg，0.085mmol)的甲醇溶液(15mL)和加入甲醇钠(9.2mg，0.17mmol)，在50℃下搅拌反应2小时。LCMS显示反应完全。将反应液浓缩得到目标化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.12-D，50mg)粗品为棕色固体。LCMS(ESI)[M+H]+＝548；
步骤三：
向化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.12-D，50mg， 0.091mmol)的二氯甲烷溶液(2mL)中，加入三氟乙酸(1mL)，在室温下搅拌反应30分钟。LCMS显示反应完全。将反应液浓缩，粗品经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物(S,E)-14-(3-氨基-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.12)(8.1mg，收率21％)为棕色固体。
LCMS(ESI)[M+H]+＝448.3；
1H NMR(400MHz,DMSO-d6)δ8.20(s,2H),7.72(s,1H),7.54(s,1H),7.40(d,J＝16.5Hz,1H),7.27(s,1H),6.52–6.46(m,2H),6.31(s,2H),5.42(s,2H),5.27(s,2H),3.86(br s,2H),1.90–1.83(m,2H),0.88(t,J＝7.1Hz,3H).
实施例A1.13：(S,E)-14-(3-羟基-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.13)的合成
步骤一：
将化合物A1.9(200mg,0.444mmol)溶在二甲亚砜(2mL)中，加入IBX(311mg,1.11mmol)，室温下搅拌2小时，然后补加入IBX(186mg,0.666mmol)于反应液中，再向反应液中加入四氢吡咯(6.3mg,0.089mmol)和乙腈(3mL),反应液在室温下搅拌过夜。LCMS检测反应完成,用乙酸乙酯萃取(20mL x 3)，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，旋干，得到粗产品，粗产品通过硅胶柱纯化(DCM:MeOH＝50:1to 10:1)得到目标化合物(S,E)-14-(3-氧代-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.13-A，100mg,纯度50.0％,收率25.0％)为黄色固体。LCMS(ESI)[M+H]+＝447.1。
步骤二：
将化合物A1.13-A(40mg,0.09mmol)溶在四氢呋喃(1mL)中,然后向反应液中加入氰基硼氢化钠(28mg,0.448mmol),在室温下搅拌过夜。LCMS显示反应完成。将反应液浓缩得粗产品，粗产品纯化通过prep-HPLC(HCl in water/MeCN)得目标化合物(3.56mg,纯度93.6％,收率9.0％)为淡黄色固体。
LCMS(ESI)[M+H]+＝449.2；
1H NMR(400MHz,DMSO-d6)δ7.61(s,1H),7.50(s,1H),7.24(s,1H),7.20(d,J＝16.4Hz,1H),6.69–6.59(m,1H),6.49(s,1H),6.30(s,2H),5.42(s,2H),5.26(s,2H),5.16(br s,1H),4.34(br s,2H),1.93–1.81(m,2H),0.88(t,J＝7.3Hz,3H).
实施例A1.14：(S,E)-4-乙基-8-氟-4-羟基-11-(3-羟基-1-丙烯-1-基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉--3,14(4H)-二酮(A1.14)的合成
步骤一：
向25mL的单口瓶中加入化合物(S)-4-乙基-8-氟-4-羟基-11-(3-羟丙基)-9-甲基-1,12-二氢-14H-吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-3,14(4H)-二酮(A1.10，100mg,0.228mmol)，DMSO(0.5mL)和乙腈(0.75mL)，依次加入IBX(160mg,0.571mmol)和(R)-二苯基(吡咯烷-2-基)甲醇(12mg,0.047mmol)，室温下继续搅拌反应16小时，然后向反应液中加入甲醇和二氯甲烷混合溶剂(200mL)(DCM:MeOH＝10:1)，饱和食盐水(300mL)萃取，合并有机相，水洗、干燥、过滤，滤液减压蒸干溶剂得粗品，粗品用快速Flash纯化(DCM:MeOH＝10:1)得到目标化合物(S)-4-乙基-8-氟-4-羟基-11-(3-氧代丙基)-9-甲基-1,12-二氢-14H-吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-3,14(4H)-二酮(A1.14-A,40mg，收率40％)。
LCMS(ESI)[M+H]+＝435.1.
步骤二：
向50mL的三口瓶中加入化合物(S)-4-乙基-8-氟-4-羟基-11-(3-氧代丙基)-9-甲基-1,12-二氢-14H-吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-3,14(4H)-二酮(A1.14-A,40mg,0.228mmol)，无水THF(5mL)，N2保护下降温至-78℃，缓慢滴加三仲丁基硼氢化锂的THF溶液(0.11mL，1N)，保持-78℃继续搅拌1小时。反应完成后加入饱和氯化铵水溶液淬灭反应，升至室温，用饱和食盐水稀释(50mL)，用乙酸乙酯萃取(20mL X 3)，合并有机相水洗、干燥、过滤，减压蒸干溶剂得到(S,E)-4-乙基-8-氟-4-羟基-11-(3-羟基-1-丙烯-1-基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉--3,14(4H)-二酮(A1.14，28mg)为类白色固体。
LCMS(ESI)[M+H]+＝437.1。
实施例A1.15：(S)-7-乙基-7-羟基-14-(2-(正丙基氨基)乙基)-10,13-二氢-11H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1.15a)和(S)-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)-2-羟基-N-正丙基乙酰胺(A1.15b)的合成
步骤一：
将化合物A1.15-A(5.0g，23.9mmol)，丙基胺盐酸盐(22.8g，239mmol)和多聚甲醛(7.18g，239mmol)溶在乙醇(100mL)中，110℃下闷罐反应12小时。LCMS检测反应完全。反应液降至室温并减压浓缩，向反应固体中加入二氯甲烷(100mL)，用水(80mL X 3)洗涤，水相用碳酸氢钠调至pH＝9。向水溶液中加入苄氧基碳酰氯(3.7g，21.8mmol)，在室温下反应2小时。LCMS检测反应完全。用二氯甲烷(100mL X 3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，浓缩得到粗品，粗产品经柱层析分离纯化(石油醚：乙酸乙酯＝10/1)得到目标化合物A1.15-B(1.5g，收率20.1％)为黄色油状液体。
LCMS(ESI)[M+H]+＝415.1；
1H NMR(400MHz,CDCl3)δ7.56(s,1H),7.35(m,6H),6.18(s,2H),5.17–5.12(m,2H),3.69–3.67(m,2H),3.32–3.30(m,2H),3.09–2.90(m,2H),1.59(m,2H),0.90(m,3H)。
步骤二：
将化合物A1.15-B(1.5g,3.63mmol)溶在饱和氯化铵(20mL)和乙醇(20mL)混合溶液中，然后向反应液中加入铁粉(1.02g，18.1mmol)，反应在80℃下搅拌1小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩成固体，粗产品用C18柱反相分离纯化(乙腈/0.05％FA的水溶液:5％到55％)得到目标化合物A1.15-C(600mg,收率43.0％)为黄色固体
LCMS(ESI)[M+H]+＝385.2,tR＝1.329min.
步骤三：
室温下，将化合物A1.15-C(350mg，0.91mmol)，化合物(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(A1.15-D，218mg，0.83mmol)和对甲苯磺酸(170mg，0.87mmol)溶于二氯甲烷(5mL)溶液中，溶液澄清混匀后减压浓缩，用油泵抽至真空，反应在120℃真空下反应2小时。LCMS显示反应完全。将反应液降至室温，加入水(50mL)，用二氯甲烷(30mL X 3)萃取，合并有机相依次并无水硫酸钠干燥、过滤，滤液减压浓缩得到化合物A1.15-E(390mg，收率76％)为褐色固体。
LCMS(ESI)[M+H]+＝612.0。
步骤四：
室温下，将化合物A1.15-E(390mg，0.638mmol)溶于二氯甲烷(5mL)溶液中，0℃氮气保护下滴加三甲基碘硅烷(510mg，2.55mmol)，反应体系室温搅拌2小时。向反应液中加入***(2mL)和浓盐酸(4mL)，搅拌30分钟，用饱和碳酸氢钠将反应体系调至H＝9，用二氯甲烷(50mL X 5)萃取，合并有机相经无水硫酸钠干燥、过滤，滤液减压浓缩得到粗产品。经高效液相制备纯化(乙腈/水含0.05％甲酸)得到化合物A1.15a(200mg，收率66.1％)为米白色固体。
LCMS(ESI)[M+H]+＝478.2；
1H NMR(400MHz,DMSO-d6)δ8.30(s,1H,HCO2H),7.67(s,1H),7.51(s,1H),7.23(s,1H),6.62–6.41(m,1H),6.29(s,2H),5.42(s,2H),5.28(s,2H),3.27–3.25(m,2H),2.91–2.79(m,2H),2.58–2.56(m,2H),1.91–1.79(m,2H),1.44–1.42(m,2H),0.90–0.84(m,6H)。
步骤五：
室温下，将化合物A1.15a(120mg，0.251mmol)溶于二氯甲烷(5mL)中，0℃下滴加化合物乙酰氧基乙酰氯(167mg，1.25mmol)和三乙胺(133mg，1.25mmol)，反应在0℃下搅拌反应30分钟。LCMS检测反应完全。将反应液减压浓缩得到粗品化合物A1.15-F(120mg，收率82.8％)为黄色固体。LCMS(ESI)[M+H]+＝578.3。
步骤六：
室温下，将化合物A1.15-F(120mg，0.207mmol)溶于乙醇(4mL)中，然后向反应液中加入浓盐酸(2mL)，反应在70℃下搅拌反应1小时。LCMS检测反应完全。向反应液加入水(20mL)，用二氯甲烷(30mL X 5)萃取，合并有机相依次并无水硫酸钠干燥、过滤，滤液减压浓缩得到粗产品。经高效液相制备纯化(乙腈/水含0.05％甲酸)得到化合物A1.15b(20mg，收率18.1％)为米白色固体。
LCMS(ESI)[M+H]+＝536.2；
1H NMR(400MHz,DMSO-d6)δ7.93(s,0.7H),7.69(s,0.3H),7.51(s,1H),7.24(s,1H),6.49(s,1H),6.30(s,2H),5.42(s,2H),5.35(s,1.5H),5.29(s,0.5H),4.65–4.62(m,1H),4.13-3.97(m,2H),3.51–3.49(m,2H),3.33–3.30(m,2H),3.24–3.20(m,2H),1.86–1.84(m,2H),1.60–1.52(m,2H),0.89–0.86(m,6H)。
实施例A1.16：(S)-4-乙基-8-氟-4-羟基-11-(3-异丙基氨基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.16)的合成
向A1.10-C(200mg，0.439mmol)和异丙基胺(50mg，0.877mmol)的DMF溶液(10mL)中加入二异丙基乙胺(170mg，1.32mmol)和碘化钠(99mg，0.659mmol)，反应在50℃封管下反应2小时。向反应液中加入乙酸乙酯，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩得到粗品。粗品经pre-TLC分离纯化(二氯甲烷:甲醇＝10:1)后得到黄色固体，化合物继续经高效液相制备纯化(乙腈/水含0.05％甲酸)得到目标化合物(S)-4-乙基-8-氟-4-羟基-11-(3-异丙基氨基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.16，2.03mg)。
LCMS(ESI)[M+H]+＝480.2；
1H NMR(400MHz,DMSO-d6)δ8.28(d,J＝8.0Hz,1H),8.27(s,1H,HCO2H)7.92(d,J＝11.0Hz,1H),7.33(s,1H),6.53(s,1H),5.45(s,2H),5.30(s,2H),33.30–3.22(m,3H),3.18–3.10(m,2H),2.54(s,3H),2.06–1.94(m,2H),1.93–1.80(m,2H),1.26–1.20(m,6H),0.88(t,J＝7.3Hz,3H)。
实施例A1.17：(S)-4-乙基-8-氟-4-羟基-11-(3-环丙基氨基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.17)的合成
向A1.10C(200mg，0.439mmol)和环丙基胺(52mg，0.881mmol)的DMF溶液(10mL)中加入二异丙基乙胺(165mg，1.28mmol)和碘化钠(96mg，0.640mmol)，反应在50℃封管下反应2小时。LCMS监测反应完全。向反应液中加入乙酸乙酯，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩得到粗品。粗品经TLC分离纯化(二氯甲烷:甲醇＝10:1)后继续经高效液相制备纯化(乙腈/水含0.05％甲酸)得到目标化合物(S)-4-乙基-8-氟-4-羟基-11-(3-环丙基氨基丙基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.17，5.12mg)。
LCMS(ESI)[M+H]+＝478.2；
1H NMR(400MHz,DMSO-d6)δ8.22(br s,2H,one is HCO2H),7.86(br s,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.28(s,2H),3.24–3.19(m,2H),2.75–2.68(m,2H),2.49(s,3H),2.16–2.09(m,1H),1.95–1.76(m,4H),0.91–0.83(t,J＝7.3Hz,3H),0.43–0.33(m,2H),0.31–0.21(m,2H)。
实施例A1.18：(S)-4-乙基-8-氟-4-羟基-11-(4-氨基苯基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.18)的合成
步骤一：
氮气保护下，将3-氟-4-甲基苯胺(A1.18A，2.0g，16.3mmol)溶在1,2-二氯乙烷(40mL)中，在0℃下向其中滴加三氯化硼(19.2mL,19.2mmol)后，再缓慢加入对硝基苯腈(2.8g,19.2mmol)，加入完毕将反应液升至80℃搅拌过夜。将反应液冷却至室温向其中加入盐酸(40mL,2M)，后升温至80℃继续搅拌30分钟，经LCMS检测反应完全后，向反应液中加入水(80mL)，用二氯甲烷萃取(100mLⅹ3)，合并有机相经无水硫酸钠干燥，过滤，旋干，粗产品经硅胶柱色谱(PE:EA＝10:1)纯化得目标化合物(A1.18B，2.0g,收率46.5％)。
LCMS(ESI)[M+H]+＝275.1；
1H NMR(400MHz,DMSO)δ8.34(d,J＝8.6Hz,2H),7.77(d,J＝8.6Hz,2H),7.39(s,2H),7.11(d,J＝8.8Hz,1H),6.63(d,J＝12.4Hz,1H),2.00(s,3H)。
步骤二：
将化合物A1.18B(620mg，2.28mmol)，(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]中氮茚-3,6,10(4H)-三酮(600mg,2.28mmol),对甲苯磺酸(560mg,2.96mmol)加入到反应瓶中,用二氯甲烷将其溶解均匀，将二氯甲烷旋干，抽真空，在真空状态下将其加热到120℃，在此温度下保持六个小时，经LCMS检测反应完成后，向体系中加入甲醇(10mL)，再加入水(80mL)有大量沉淀析出，沉淀过滤后干燥得到目标化合物(A1.18C，800mg,收率80.0％)为黄色固体。
LCMS(ESI)[M+H]+＝502.2；
1H NMR(400MHz,DMSO)δ8.52(d,J＝8.2Hz,2H),8.13–7.89(m,3H),7.64(d,J＝8.4Hz,1H),7.37(d,J＝13.4Hz,1H),6.54(s,1H),5.41(s,2H),5.06(m,2H),2.40(s,3H),1.94–1.84(m,2H),0.87(m,3H)。
步骤三：
将雷尼镍(609mg,10.5mmol)加入到化合物A1.18C(1.0g，2.1mmol)的甲醇(25mL)和四氢呋喃(50mL)混合溶液中，反应液在室温氢气氛围下搅拌5小时。LCMS检测反应完全，将反应液过滤，滤液旋干得到目标化合物(S)-4-乙基-8-氟-4-羟基-11-(4-氨基苯基)-9-甲基-1,12-二氢-14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A1.18，650mg，收率67％)为黄色固体。
LCMS(ESI)[M+H]+＝472.3；
1H NMR(400MHz,DMSO)δ7.93–7.81(m,2H),7.40–7.25(m,3H),6.81(d,J＝8.2Hz,2H),6.51(s,1H),5.62(s,2H),5.41(s,2H),5.10(s,2H),2.41(s,3H),1.93–1.80(m,2H),0.88(t,J＝7.2Hz,3H)。
B、含生物活性分子片段中间体的合成
实施例B1.1：(S)-2-氨基-N-((4-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丁氧基)甲基)乙酰胺(B1.1)的合成
化合物(B1.1-A)(368mg)，化合物(B1.1-B)(452mg)和对甲苯磺酸吡啶盐(PPTS)(25mg)在二氯甲烷(20ml)中回流20小时，然后用碳酸氢钠水溶液和盐酸水溶液分别洗涤，减压除去有机溶剂，残留物溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶于减压除去大部分低沸点组分，残留物制备HPLC分离得到目标产物(S)-2-氨基-N-((4-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丁氧基)甲基)乙酰胺(B1.1)。
ESI-MS(m/z):539[M+H]+。
实施例B1.2：(S)-2-((2-氨基乙酰胺基)甲氧基)-N-乙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)乙酰胺(B1.2)的合成
步骤一：
依次将化合物B1.2-A(274mg)，二异丙基乙胺(334mg)和HBTU(369mg)加入N,N-二甲基甲酰胺(10mL)中，随后加入化合物B1.2-B(300mg)。反应液在室温下搅拌2小时。向反应液中加入乙酸乙酯(50mL)，用饱和食盐水(30mL X 3)洗涤，无水硫酸钠干燥，抽滤，滤液减压浓缩成固体，粗产 品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物B1.2-C(300mg)。
LCMS(ESI)[M+H]+：830.2；
1H NMR(400MHz,DMSO-d6)δ8.74(s,1H),7.91–7.84(m,2H),7.80–7.67(m,2H),7.65–7.54(m,2H),7.53–7.46(m,1H),7.45–7.37(m,2H),7.36–7.27(m,2H),7.25–7.23(m,1H),6.57–6.44(m,1H),6.29(s,2H),5.50–5.19(m,4H),4.71–4.57(m,2H),4.32–3.97(m,7H),3.80–3.53(m,4H),3.20–3.14(m,2H),1.92–1.80(m,2H),1.28–1.22(m,3H),0.87–0.82(m,3H).
步骤二：
向化合物B1.2-C(300mg)的N,N-二甲基甲酰胺(4mL)溶液加入哌啶(1mL)，反应液在室温下搅拌20分钟。反应液除去低沸点组分后得到目标产物，直接用于下步合成。
LCMS(ESI)[M+H]+＝608.0。
实施例B1.3：(S)-2-氨基-N-((2-(((4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)氨基)-2-氧代乙氧基)甲基)乙酰胺(B1.3)的合成
用实施例B1.1相同的方法和反应条件，用化合物(B1.3-A)代替(B1.2-B)，得到目标产物(S)-2-氨基-N-((2-(((4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)氨基)-2-氧代乙氧基)甲基)乙酰胺(B1.3)。
ESI-MS(m/z):554[M+H]+。
实施例B1.4：(S)-2-氨基-N-((4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)乙酰胺(B1.4)的合成
向化合物(B1.4-A)(175mg)，化合物(B1.3-A)(409mg)的DMF(5ml)溶液中加入DIPEA(200ul),HBTU(420mg)。化合物室温小搅拌反应20小时，混合物加入乙酸乙酯(100ml),水洗(100ml x 3)，减压除去有机溶剂，然后向残留物中加入1:1DCM/TFA(10ml),室温放置20分钟。减压除去低沸点组分，残留物用制备HPLC分离得到目标产物(S)-2-氨基-N-((4-乙基-8-氟-4-羟基-9-甲 基-3,14-二氧-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)甲基)乙酰胺(B1.4)。
ESI-MS(m/z):467[M+H]+。
实施例B1.5：(S)-2-氨基-N-((7-乙基-7-羟基-8,11-二氧-7,8,11,13-四氢-10H-[1,3]二氧环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)乙酰胺(B1.4)的合成
将B1.5-A(200mg)溶于N,N-二甲基甲酰胺(5mL)中，依次加入N,N-二异丙基乙胺(154.80mg)，2,5-二氧吡咯烷-1-基(叔丁氧羰基)甘氨酸(157mg)于反应液中，反应液在25℃下搅拌60分钟。向反应液中加入水(30mL)，抽滤，干燥滤饼得到白色固体产物(ESI-MS(m/z):579.4[M+H]+)。将上述白色固体产物溶于三氟乙酸和二氯甲烷混合溶液(体积比为1：3；4mL)中,反应液室温下搅拌1小时。然后将反应液浓缩得粗产品，粗产品用C18柱反相分离纯化(乙腈/0.05％甲酸水溶液:5％到50％)得到目标产物(S)-2-氨基-N-((7-乙基-7-羟基-8,11-二氧-7,8,11,13-四氢-10H-[1,3]二氧环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)乙酰胺(B1.5，110mg)。
ESI-MS(m/z):479.3[M+H]+。
实施例B1.6：(S)-2-氨基-N-((4-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-1H-吡唑-1-基)甲基)乙酰胺(B1.6)的合成
用实施例B1.1相同的方法和反应条件，用化合物(B1.6-A)代替化合物(B1.1-B)，得到目标产物(S)-2-氨基-N-((4-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-1H-吡唑-1-基)甲基)乙酰胺(B1.6)。
ESI-MS(m/z):545[M+H]+。
实施例B1.7：(S)-2-氨基-N-((2-(((7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙氧基)甲基)乙酰胺(B1.7)的合成
用实施例B1.2相同的方法和反应条件，用化合物(B1.5-A)代替化合物(B1.2-B)，得到含目标产物(S)-2-氨基-N-((2-(((7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙氧基)甲基)乙酰胺(B1.7)的混合物，上述混合物直接用于下面合成反应。
ESI-MS(m/z):566[M+H]+。
实施例B1.8：(S)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)苯基)乙酰胺(B1.8)的合成
用实施例B1.4相同的方法和反应条件，用化合物(A1.3)代替化合物(B1.3-A)，得到目标产物(S)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]-二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)苯基)乙酰胺(B1.8)。
ESI-MS(m/z):541[M+H]+。
实施例B1.9：(S)-2-氨基-N-(4-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)苯基)乙酰胺(B1.9)的合成
用实施例B1.4相同的方法和反应条件，用化合物(A1.4)代替化合物(B1.3-A)，得到目标产物(S)-2-氨基-N-(4-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]-二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)苯基)乙酰胺(B1.9)。或者采用下列反应条件得到目标产 物。
将化合物A1.4(60mg，0.12mmol)溶在N,N-二甲基甲酰胺(3mL)，依次加入(叔丁氧羰基)甘氨酸(26mg，0.15mmol)、HATU(56mg,0.15mmol)，N,N-二异丙基乙胺(48mg,0.37mmol)，室温下搅拌1小时，TLC检测反应完成。然后直接向上述反应液中加入TFA(1.0mL)。在室温下继续搅拌1小时，LCMS检测反应完成，将反应液浓缩除去三氟乙酸得粗产物，粗产物经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标产物(S)-2-氨基-N-(4-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]-二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)苯基)乙酰胺(B1.9)(26.3mg,收率38％)为黄色固体。
ESI-MS(m/z):541[M+H]+；
1H NMR(400MHz,DMSO)δ10.71(s,1H),8.16(s,2H),7.86(d,J＝8.6Hz,2H),7.67-7.58(m,3H),7.29(s,1H),7.04(s,1H),6.50(s,1H),6.28(s,2H),5.40(s,2H),5.05(s,2H),3.87(s,2H),1.93-1.81(m,2H),0.88(t,J＝7.3Hz,3H)。
实施例B1.10：(S)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]-二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)乙酰胺(B1.10)的合成
步骤一：
室温下，向化合物A1.5(200mg)的N,N-二甲基甲酰胺溶液(5mL)中加入三乙胺(67mg)和2,5-二氧吡咯烷-1-基(叔丁氧羰基)甘氨酸(182mg)，在室温下反应1小时。反应液加入水(30mL)稀释，乙酸乙酯(30mL×2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗产品，粗产品用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化得到目标化合物(B1.10-A，100mg)。
LCMS(ESI)[M+H]+：607。
步骤二：
向化合物B1.10-A(100mg)的二氯甲烷溶液(4mL)中，加入三氟乙酸(2mL)，在室温下搅拌反应1小时。将反应液浓缩，粗品加入N,N-二甲基甲酰胺溶液(3mL)，用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化得到目标化合物(S)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]-二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)乙酰胺(B1.10，80mg)。
LCMS(ESI)[M+H]+：507；
1H NMR(400MHz,DMSO-d6)δ8.26(s,1H),7.64(s,1H),7.51(s,1H),7.24(s,1H),6.51(s,1H),6.29(s,2H),5.42(s,2H),5.23(s,2H),3.31(s,2H),3.30–3.27(m,2H), 3.13–3.07(m,2H),2.04–1.76(m,4H),0.87(t,J＝7.3Hz,3H)。
实施例B1.11：(S)-2-((2-氨基乙酰胺基)甲氧基)-N-异丙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)乙酰胺(B1.11)的合成
步骤一：
将化合物1-(9H-芴-9-基)-3,6-二氧-2,9-二氧-4,7-二氮杂环-11-甲酸(242mg)，二异丙基乙胺(330mg)和N,N,N`,N`-四甲基脲六氟磷酸酯(359mg)溶于N,N-二甲基甲酰胺(10mL)溶液中。随后加入化合物(S)-7-乙基-7-羟基-14-(2-(异丙胺基)乙基)-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-8,11(7H)-二酮(300mg)，反应在室温下反应2小时。向反应液中加入乙酸乙酯(50mL)，用饱和食盐水(30mL X 3)洗涤，无水硫酸钠干燥，抽滤，滤液减压浓缩成固体，粗产品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物化合物B1.11B(220mg)。
LCMS(ESI)[M+H]+：844.0。
步骤二：(S)-2-((2-氨基乙酰胺基)甲氧基)-N-异丙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)乙酰胺的制备
向化合物B1.11B(220mg，0.261mmol)的N,N-二甲基甲酰胺(4mL)溶液中加入哌啶(1mL)，反应在室温下搅拌20分钟。LCMS检测反应反应完全。反应液减压浓缩得到目标化合物(220mg)为褐色固体，上述产物未经纯化直接用于下步合成。
LCMS(ESI)[M+H]+：622.1。
实施例B1.12：(S)-2-氨基-N-((4-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙氧基)甲基)乙酰胺(B1.12)的合成
步骤一：
将化合物A1.10(160mg,0.365mmol)溶在N,N-二甲基甲酰胺(3mL)中，加入(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯((B1.1-A，672mg,1.83mmol)，然后加入盐酸乙酸 乙酯(0.073mL,3M)于反应液中，反应液在室温下搅拌过夜。LCMS检测反应完成。该反应液直接经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物(80mg,收率29.0％)为白色固体。
LCMS(ESI)[M+H]+＝747.4；
1H NMR(400MHz,DMSO-d6)δ8.74(t,J＝6.4Hz,1H),8.28–8.17(m,1H),7.99–7.88(m,3H),7.73(d,J＝7.3Hz,2H),7.63(t,J＝5.7Hz,1H),7.44(t,J＝7.4Hz,2H),7.35(t,J＝7.2Hz,3H),6.58(s,1H),5.48(s,2H),5.29(s,2H),4.66(d,J＝6.3Hz,2H),4.32(d,J＝6.9Hz,2H),4.26(d,J＝6.1Hz,1H),3.71(d,J＝5.8Hz,2H),3.57(t,J＝5.7Hz,2H),3.29–3.20(m,2H),2.55(s,3H),2.00–1.86(m,4H),0.93(t,J＝7.2Hz,3H).
步骤二：B1.12-A(240mg)溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶于减压除去低沸点组分，残留物直接用于下步合成。
ESI-MS(m/z):525.2[M+H]+。
少量粗产物经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物。
ESI-MS(m/z):525.1[M+H]+；
1H NMR(400MHz,DMSO)δ9.13(t,J＝6.6Hz,1H),8.21(d,J＝8.1Hz,1H),8.02(brs,2H),7.89(d,J＝10.8Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H),5.28(s,2H),4.66(d,J＝6.5Hz,2H),3.64(s,2H),3.53(t,J＝6.1Hz,2H),3.25-3.18(m,2H),2.52(s,3H),1.98-1.84(m,4H),0.88(t,J＝7.3Hz,3H)。
实施例B1.13：(S)-2-氨基-N-(3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)乙酰胺(B1.13)的合成
步骤一：
室温下，向化合物A1.11(200mg)的N,N-二甲基甲酰胺溶液(5mL)中加入三乙胺(67mg)和2,5-二氧吡咯烷-1-基(叔丁氧羰基)甘氨酸(182mg)，在室温下反应1小时。反应液加入水(30mL)稀释，乙酸乙酯(30mL×2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗产品，粗产品用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化得到目标化合物(B1.13-A，104mg)。
LCMS(ESI)[M+H]+：595。
步骤二：
向化合物B1.13-A(100mg)的二氯甲烷溶液(4mL)中，加入三氟乙酸(2mL)，在室温下搅拌反应1小时。将反应液浓缩，粗品加入N,N-二甲基甲酰胺溶液(3mL)，用C18柱(乙腈/0.05％甲酸的水溶 液：5％-60％)进一步纯化得到目标化合物(S)-2-氨基-N-(3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)乙酰胺(B1.13，88mg)。
LCMS(ESI)[M+H]+：495。
实施例B1.14：(S)-2-氨基-N-((3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙氧基)甲基)乙酰胺(B1.14)的合成
步骤一：化合物(B1.1-A)(368mg)，化合物(A1.9)(440mg)和对甲苯磺酸吡啶盐(PPTS)(25mg)在二氯甲烷(20ml)中回流20小时，然后用碳酸氢钠水溶液和盐酸水溶液分别洗涤，减压除去有机溶剂，得到粗产物，粗产品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物化合物B1.14A(240mg)。
LCMS(ESI)[M+H]+：759.5。
步骤二：B1.14-A(240mg)溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶于减压除去低沸点组分，残留物直接用于下步合成。少量粗产物经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物。
ESI-MS(m/z):537.4[M+H]+；
1H NMR(400MHz,DMSO-d6)δ9.13(t,1H)，8.04(br,2H),7.58(s，1H)，7.51(s，1H)，7.25(s，1H)，6.29(s，2H)，5.43(S，2H)，5.21(s,2H)，4.65(d，2H)，3.63(m，2H)，3.53(m，2H)，3.11(m，2H)，1.87(m，4H)，0.88(t，3H)。
实施例B1.15：(S,E)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)乙酰胺
步骤一：
室温下，向化合物A1.12(200mg，0.447mmol)的N,N-二甲基甲酰胺溶液(5mL)中加入三乙胺(135mg，1.341mmol)和N-羟基-2,5-二氧吡咯烷叔丁氧羰基甘氨酸酯(183mg，0.671mmol)，在室温下反应1小时。LCMS显示反应完全。向反应液中加入水(20mL)，用乙酸乙酯(20mL×3)萃取，有 机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到目标化合物B1.15A(130mg，收率：48％)为棕色固体。
LCMS(ESI)[M+H]+＝605.6。
步骤二：
室温下，将化合物B1.15-A(130mg，0.215mmol)用盐酸1，4-二氧六环溶液(5mL)溶解，在室温下反应30分钟。LCMS显示反应完全。反应液通过反向纯化得到目标化合物(60mg，收率：52％)为棕色固体。
LCMS(ESI)[M+H]+＝505.2；
1H NMR(400MHz,DMSO-d6)δ8.85(s,1H),8.15(s,2H),7.70(s,1H),7.54(s,1H),7.26(s,1H),7.21(d,J＝16.2Hz,1H),6.51(d,J＝16.2Hz,1H),6.31(s,2H),5.42(s,2H),5.28(s,2H),4.19(s,2H),1.86–1.82(m,4H),0.87(t,J＝7.3Hz,3H).
实施例B1.16：(S,E)-2-氨基-N-(((3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)乙酰胺
步骤一：
在氮气保护下，将原料A1.13(150mg,0.335mmol)溶在甲苯(3mL)中，加入(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯(B1.1-A，308mg,0.84mmol)，然后将醋酸锌(123mg,0.67mmol)加入到反应液中，反应液在100℃下搅拌过夜。LCMS检测反应完成。该反应液直接经反相色谱(乙腈/0.05％甲酸的水溶液:5％到50％)纯化后得目标化合物(9H-芴-9-基)甲基(S，E)-(2-(((3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环[4,5-g]吡喃[3'，4'：6,7]吲哚嗪[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯(B1.16-A，80mg,收率31％)为白色固体。
LCMS(ESI)[M+H]+＝757.4。
步骤二：
将化合物B1.16-A(80mg,0.11mmol)溶在N,N-二甲基甲酰胺(2mL)中,然后向反应液中加入哌啶(0.05mL)，在室温下搅拌1小时。LCMS显示反应完成。将反应液浓缩得粗产品，粗产品用C18柱反相分离纯化(乙腈/0.05％氨水的水溶液:5％到50％)得到目标化合物(35mg,收率62％)为棕色固体。
LCMS(ESI)[M+H]+＝535.2,tR＝1.070min.
实施例B1.17：(S,E)-2-氨基-N-(((3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-氧代-3,4,12,14-四氢- 1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)乙酰胺
步骤一：
将化合物A1.14(80mg,0.183mmol)和化合物(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯(B1.1-A，135mg,0.367mmol)溶于甲苯(4mL),加入醋酸锌(190mg,1.03mmol)，反应液在100摄氏度下搅拌48小时。冷却至室温，向其中加入水(10mL)，用乙酸乙酯(10mL×3)萃取。合并有机相经无水硫酸钠干燥，过滤，浓缩得到粗产物，粗产物经反相色谱(0.05％FA的水溶液/乙腈:5％到100％)纯化后得到目标化合物(S,E)-2-(Fmoc氨基)-N-(((3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)乙酰胺(B1.17-A，30mg)为白色固体。
LCMS(ESI)[M+H]+＝745.2；
1H NMR(400MHz,DMSO)δ8.82(m 1H),8.21(m,1H),7.89(m,2H),7.84(d,J＝7.8Hz,2H),7.68(m,1H),7.61(m,1H),7.42(m,1H),7.37(m,1H),7.33–7.28(m,4H),6.72–6.55(m,1H),6.53(s,1H),5.42(s,2H),5.28(s,2H),4.75(m,2H),4.34(m,2H),4.28–4.24(m,2H),4.20(m,1H),3.70(d,J＝6.1Hz,2H),2.47(s,3H),1.90–1.82(m,2H),0.88(t,J＝7.3Hz,3H)。
步骤二：
将化合物4(25mg,0.034mmol)，溶于N,N-二甲基甲酰胺(2mL)中,然后加入二乙胺(0.2mL)，加毕，反应液室温搅拌1小时，LCMS检测反应完成后，将反应液直接减压蒸干溶剂得到粗产品目标化合物(S,E)-2-氨基-N-(((3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)乙酰胺为黄色粘稠状产品。
LCMS(ESI)[M+H]+＝523.2；
LCMS(ESI)[M+H]+＝523.1,tR＝0.446min,1.311min.
1H NMR(400MHz,DMSO)δ9.26(t,J＝6.5Hz,1H),8.23(d,J＝8.2Hz,1H),8.06(s,2H),7.91(m,1H),7.40(d,J＝16.3Hz,1H),7.34(s,1H),6.70–6.60(m,1H),6.54(s,1H),5.44(s,2H),5.35(s,2H),4.80(d,J＝6.6Hz,2H),4.38(d,J＝3.9Hz,2H),3.68(d,2H),2.52(s,3H),1.90–1.84(m,2H),0.88(m,3H)。
C、含偶联连接段分子片段中间体的合成
实施例C1.1：N6-(叔丁氧基羰基)-N2-((1-(2-(甲硫基)嘧啶-5-基)-1-氧代- 5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.1)
步骤一：29-叠氮基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯
向26-叠氮基-3,6,9,12,15,18,21,24-八氧杂-26-烷-1-醇(4.39g)的DMF(40ml)溶液中加入NaH(0.6g，60％)。上述混合物搅拌30分钟后，加入溴乙酸叔丁酯(2.4g)。混合物在干燥条件下搅拌20小时。然后向混合物中加入乙酸乙酯(200ml)和水(200ml，开始时慢慢加入)，有机相水洗(100ml x 3)，无水硫酸钠干燥，减压除去溶剂，残留物经过硅胶柱色谱分离得到目标产物，29-叠氮基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯。
ESI-MS(m/z):554[M+H]+。
步骤二：29-氨基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯
向29-叠氮基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯(1.16g)的乙酸乙酯(20ml)溶液中加入钯碳催化剂(Pd/C，10％，100mg)。上述溶液在氢气气氛下搅拌5小时，然后滤去钯碳后，减压除去溶剂得到目标产物。
ESI-MS(m/z):528[M+H]+。
步骤三：1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酸
29-氨基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯(527mg)和2-甲硫基-嘧啶-5-羧酸(170mg)加入干燥DMF(10ml),然后在冰浴冷却下向上述溶液中依次加入DIPEA(0.2ml)，HBTU(420mg)。上述混合物室温小搅拌20小时。然后用乙酸乙酯(100ml)稀释，水洗(100ml x 4),有机相无水硫酸钠干燥后，减压除去有机溶剂，残留物溶于DCM(10ml)，然后加入TFA(10ml),混合物室温下搅拌1小时，然后减压除去低沸点组分，残留物经过制备HPLC分离得到1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酸。
ESI-MS(m/z):624[M+H]+。
步骤四：N6-(叔丁氧基羰基)-N2-((1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酰基)-L-缬氨酸)-L-赖氨酸
1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酸(623mg)溶于DMF(10ml)，0℃下加入DIPEA(300ul)，HBTU(420mg)。混合物搅拌30分钟，然后加入缬氨酸-(Boc)赖氨酸二肽化合物(345mg)。反应搅拌20小时，然后加入乙酸乙酯(100ml)，稀盐酸(20ml x3)洗，有机相干燥后减压除去有机溶剂，粗产物通过制备HPLC分离得到目标化合物N6-(叔丁氧基羰 基)-N2-((1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.1)。
ESI-MS(m/z):951[M+H]+。
实施例C1.2：N6-(叔丁氧基羰基)-N2-((29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.2)
步骤一：29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯
29-叠氮基-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯(1.7g)，2-甲硫基-5-乙炔基嘧啶(450mg)溶于DMSO-水(20ml，4:1)中，向混合物中加入溴化亚铜(50mg)。反应液室温下搅拌2小时，然后加入乙酸乙酯(100ml)，水洗(100ml x 3)，干燥后减压除去有机溶剂，粗产物用硅胶柱层析分离得到目标产物29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯。
MS(m/z):704[M+H]+。
步骤二：29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸
29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸叔丁酯(1g)溶于二氯甲烷(10ml)，然后加入TFA(5ml)。混合物室温静置1小时，然后减压除去低沸点组分得到目标产物29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸。
MS(m/z):648[M+H]+。
步骤三：N6-(叔丁氧基羰基)-N2-((29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酰基)-L-缬氨酸)-L-赖氨酸
29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酸(647mg)溶于DMF(10ml)，0℃下加入DIPEA(300ul)，HBTU(420mg)。混合物搅拌30分钟，然后加入缬氨酸-赖氨酸二肽化合物(345mg)。反应搅拌20小时，然后加入乙酸乙酯(100ml)，稀盐酸(20ml x 3)洗，有机相干燥后减压除去有机溶剂，粗产物通过制备HPLC分离得到目标化合物N6-(叔丁氧基羰基)-N2-((29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂-29-烷酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.2)。
ESI-MS(m/z):975[M+H]+。
实施例C1.3：(1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酰基)-甘胺酸-甘氨酸-L-苯丙氨酸(化合物C1.3)
用实施例C1.1步骤四相同的方法和反应条件，用相应的原料，得到目标产物(1-(2-(甲硫基)嘧啶-5-基)-1-氧代-5,8,11,14,17,20,23,26,29-九氧杂-2-氮杂-31-烷酰基)-甘胺酸-甘氨酸-L-苯丙氨酸(化合物C1.3)。
ESI-MS(m/z):885[M+H]+。
实施例C1.4：(36-(2-(甲硫基)嘧啶-5-基)-31-氧代-3,6,9,12,15,18,21,24,27-九氧杂-30-氮杂-三十六-(35-炔)-酰基)甘氨酸甘氨酸苯丙氨酸(化合物C1.4)
用实施例C1.1步骤四相同的方法和反应条件，用反应式所示的原料，得到目标产物(36-(2-(甲硫基)嘧啶-5-基)-31-氧代-3,6,9,12,15,18,21,24,27-九氧杂-30-氮杂-三十六-(35-炔)-酰基)甘氨酸甘氨酸苯丙氨酸(化合物C1.4)。
ESI-MS(m/z):951[M+H]+。
实施例C1.5：N6-(叔丁氧基羰基)-N2-((36-(2-(甲硫基)嘧啶-5-基)-31-氧代-3,6,9,12,15,18,21,24,27-九氧杂-30-氮杂-三十六-(35-炔)-酰基)-L-缬氨酸)-L-赖氨酸(C1.5)
用实施例C1.1步骤四相同的方法和反应条件，用反应式所示的化合物作为原料，得到目标产物N6-(叔丁氧基羰基)-N2-((36-(2-(甲硫基)嘧啶-5-基)-31-氧代-3,6,9,12,15,18,21,24,27-九氧杂-30-氮杂-三十六-(35-炔)-酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.5)。
ESI-MS(m/z):1017[M+H]+。
实施例C1.6：N6-(叔丁氧基羰基)-N2-((6-(2-(甲硫基)嘧啶-5-基)-己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.6)
将6-(2-(甲硫基)嘧啶-5-基)己-5-炔酸(920mg)和N-羟基丁二酰亚胺(537mg)溶于二氯甲烷(50mL)中，然后加入二环己基碳二亚胺(963mg)。反应液室温搅拌1小时，然后减压除去二氯甲烷，残留物溶于N,N-二甲基甲酰胺(50mL)，然后加入N2-L-缬氨酸-N6-(Boc)-L-赖氨酸(1.5g)，反应液室温下搅拌反应16小时，将反应液倒入200mL水中，用饱和碳酸氢钠溶液调节pH至11，上述水溶液用乙酸乙酯萃取两次，弃掉有机相，将水相用柠檬酸调节pH到4-5，用乙酸乙酯(100mL x 3)萃取三次，有机相用饱和食盐水(100mL)洗涤，无水硫酸钠干燥，抽滤，旋蒸，得到粗产品2.8g，粗产品用快速色谱法分离纯化(DCM:MeOH＝30:1)得到目标化合物N6-(叔丁氧基羰基)-N2-((6-(2-(甲硫基)嘧啶-5-基)-己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(1.0g)(化合物C1.6)。
ESI-MS(m/z):564.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ12.45(s,1H),8.68(s,2H),8.12(d,J＝7.3Hz,1H),7.88(d,J＝8.9Hz,1H),6.76(s,1H),4.27–4.19(m,1H),4.11(d,J＝4.9Hz,1H),2.88(d,J＝7.9Hz,3H),2.73(s,1H),2.59(s,1H),2.52(s,4H),2.40–2.24(m,3H),2.03–1.88(m,2H),1.77(d,J＝3.2Hz,2H),1.68(d,J＝7.0Hz,1H),1.60–1.49(m,1H),1.36(s,15H),0.85(dd,J＝14.8,6.7Hz,7H)。
实施例C1.7：N6-(叔丁氧基羰基)-N2-((6-(2-(甲磺酰基)嘧啶-5-基)-己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.7)
向化合物C1.6(260mg)的四氢呋喃(3mL)和水(3mL)混合溶液中加入过氧单磺酸钾(1.414g)，在室温下搅拌反应1小时。将反应液过滤，滤液用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)纯化得到目标化合物N6-(叔丁氧基羰基)-N2-((6-(2-(甲磺酰基)嘧啶-5-基)-己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.7)(120mg)。
LCMS(ESI)[M+H]+:596；
1H NMR(400MHz,DMSO-d6)δ12.48(s,1H),9.13(s,2H),8.14(d,J＝7.0Hz,1H),7.90(d,J＝9.1Hz,1H),6.77(s,1H),4.24(t,J＝7.7Hz,1H),4.12(br s,1H),3.41(s,3H),2.89(d,J＝6.0Hz,2H),2.43–2.29(m,2H),2.03–1.93(m,2H),1.82(br s,2H),1.63–1.60(m,4H),1.37(s,12H),0.88–0.83(m,6H)。
实施例C1.8：N6-(叔丁氧基羰基)-N2-((6-(2-(甲硫基)嘧啶-5-甲酰胺基)己酰基)-L-缬氨酸)-L-赖 氨酸(化合物C1.8)
用实施例C1.6相同的方法和反应条件，用反应式所示的已知化合物作为原料，得到目标产物N6-(叔丁氧基羰基)-N2-((6-(2-(甲硫基)嘧啶-5-甲酰胺基)己酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.8)。
ESI-MS(m/z):611[M+H]+。
实施例C1.9：N6-(叔丁氧基羰基)-N2-((6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.9)
步骤一：6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸
室温下，将2-甲硫基-5-乙炔基嘧啶(1.5g)、6-叠氮基己酸甲酯(1.71g)溶于叔丁醇与水的混合溶剂(20mL/25mL)中，加入维生素C钠(5.7g),溴化亚铜(2.9g)搅拌反应10h。反应加入乙酸乙酯(200ml)，水洗(100ml x 5)，干燥后除去有机溶剂，硅胶柱层析得到6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸甲酯(2.8g)，ESI-MS(m/z):321.9[M+H]+。上述产物溶于四氢呋喃(100ml),加入氢氧化锂(1M，20ml)，混合物搅拌3小时，然后加入盐酸至反应液pH 2，然后加入乙酸乙酯(200ml)，水洗(100ml x 3)，干燥，除去有机溶剂得到目标产物6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸。ESI-MS(m/z):308.4[M+H]+。
步骤二：N6-(叔丁氧基羰基)-N2-((6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰)-L-缬氨酸)-L-赖氨酸
将化合物6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(370mg,)和N-羟基丁二酰亚胺(152mg)溶在二氯甲烷(5mL)中搅拌，再加入二环乙基碳二亚胺(273mg)，然后反应液在室温下搅拌1小时。向反应液中加入10mL水，用乙酸乙酯萃取(20mL x 2)，有机相用饱和食盐水洗，用无水硫酸钠干燥，过滤，滤液减压浓缩得到粗品。将此粗品溶解在N,N-二甲基甲酰胺(10mL)搅拌，加入化合物N2-L-缬氨酸-N6-(Boc)-L-赖氨酸，反应液在室温下反应16小时，向反应液中缓慢加入柠檬酸将PH调至5左右，加入水，再用乙酸乙酯萃取(3x10mL)，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩除去乙酸乙酯得到目标产物N6-(叔丁氧基羰基)-N2-((6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基) 己酰基)-L-缬氨酸)-L-赖氨酸(化合物C1.9，400mg)。
ESI-MS(m/z):635.3[M+H]+；
1H NMR(400MHz,DMSO-d6)δ12.46(s,1H),9.06(s,2H),9.06(s,1H),8.70(s,1H),8.07(d,J＝7.4Hz,1H),7.77(d,J＝9.0Hz,1H),6.76(t,J＝5.6Hz,1H),4.41(t,J＝7.0Hz,2H),4.19(dd,J＝8.9,7.0Hz,1H),4.14–4.05(m,1H),2.92–2.85(m,2H),2.56(s,3H),2.24–2.10(m,2H),1.97–1.81(m,3H),1.71–1.64(m,1H),1.61–1.50(m,3H),1.36(s,9H),1.28–1.22(m,6H),0.88–0.76(m,6H)。
实施例C1.10：(6-(2-(甲硫基)嘧啶-5-基)己-(5-炔)-酰基)甘氨酸甘氨酸-L-苯丙氨酸(化合物C1.10)
用实施例C1.6相同的方法和反应条件，上述反应式所示的已知原料，得到目标产物(6-(2-(甲硫基)嘧啶-5-基)己-5-炔酰基)甘氨酸甘氨酸-L-苯丙氨酸(化合物C1.10)。ESI-MS(m/z):498[M+H]+。
用实施例C1.6相同的方法和反应条件，利用不同的反应原料，得到下表中目标产物。
实施例C1.13：(29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂29烷酰基)甘氨酸甘氨酸-L-苯丙氨酸(化合物C1.13)
用实施例C1.1步骤四相同的方法和反应条件，利用已知原料，得到目标产物(29-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-3,6,9,12,15,18,21,24,27-九氧杂29烷酰基)甘氨酸甘氨酸-L-苯丙氨酸(化合物C1.13)。
ESI-MS(m/z):909[M+H]+。
实施例C1.14：N6-(叔丁氧基羰基)-N2-((39-(2-(甲硫基)嘧啶-5-基)-5,34-二氧代-3,9,12,15,18,21,24,27,30-九氧杂-6,33-二氮杂三十九烷-38-炔酰基)-L-缬氨酸)-L-赖氨酸(C1.14)
步骤一：
将化合物C1.14-A(336mg)溶于二氯甲烷(5mL)中，加入N-羟基丁二酰亚胺(98mg，0.85mmol)，二环己基碳二亚胺(175mg，0.85mmol)室温搅拌反应1小时，然后将化合物N2-L-缬氨酸-N6-(Boc)-L-赖氨酸(230mg)加入反应液，室温搅拌反应过夜。浓缩除去溶剂得到粗产品，粗产品用C18柱反相分离纯化(乙腈/0.01％FA的水溶液：5％-50％)得到无色油状目标化合物(C1.14B，290mg)。
LCMS(ESI)[M+H]+＝882.3。
步骤二：
将化合物C1.14-B(300mg)溶于无水甲醇(5mL)中，加入Pd/C(10％,60mg)，氢气置换三次，室温搅拌反应过夜，抽滤，浓缩得到目标化合物C1.14-C(293mg)为无色油状物。
LCMS(ESI)[M+H]+：856.1。
步骤三：
将6-(2-(甲硫基)嘧啶-5-基)己-5-炔酸(84mg)溶于N,N-二甲基甲酰胺(3mL)中，然后依次加入N,N-二异丙基乙胺(61mg)，HATU(108mg)。反应液室温搅拌20分钟，然后加入化合物C1.14-C(196mg)，40℃搅拌反应3小时，粗产品用C18柱反相分离纯化(乙腈/0.01％FA的水溶液：5％-65％)得到目标化合物N6-(叔丁氧基羰基)-N2-((39-(2-(甲硫基)嘧啶-5-基)-5,34-二氧代-3,9,12,15,18,21,24,27,30-九氧杂-6,33-二氮杂三十九烷-38-炔酰基)-L-缬氨酸)-L-赖氨酸(C1.14)(170mg)。
LCMS(ESI)[M+H]+：1074.0；
1H NMR(400MHz,DMSO-d6)δ8.68(s,2H),8.23(t,J＝5.7Hz,1H),8.13(d,J＝8.9Hz,1H),7.99(t,J＝5.6Hz,1H),7.43(d,J＝6.5Hz,1H),6.69(s,1H),4.09(dd,J＝8.8,6.5Hz,1H),4.02(s,2H),3.98(d,J＝2.9Hz,2H),3.74–3.70(m,1H),3.50(s,30H),3.45(d,J＝6.2Hz,2H),3.39(d,J＝5.8Hz,2H),3.26(d,J＝6.1Hz,2H),3.20(q,J＝5.9Hz,2H),2.81(t,J＝6.6Hz,2H),2.52(s,3H),2.24(t,J＝7.4Hz,2H),2.12–2.06 (m,1H),1.79–1.74(m,2H),1.67–1.59(m,1H),1.50(d,J＝5.3Hz,1H),1.36(s,9H),1.24(s,2H),1.20–1.09(m,2H),0.85(t,J＝6.3Hz,6H)。
实施例C1.15：N6-(叔丁氧基羰基)-N2-((32-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-5-氧代-3,9,12,15,18,21,24,27,30-九氧杂-6-氮杂三十二烷酰基)-L-缬氨酸)-L-赖氨酸(C1.15)
将化合物C1.14-B(130mg)，5-乙炔基-2-甲硫基嘧啶(44mg)，维生素C钠(3mg)和硫酸铜(5mg)加入到叔丁醇(2mL)和水(2mL)的混合溶液中，反应在室温氮气下反应3小时。向反应液中加入水(10mL)，用二氯甲烷(30mL×3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，浓缩得到粗品，粗产品TLC分离纯化(二氯甲烷：甲醇＝10比1)得到目标化合物N6-(叔丁氧基羰基)-N2-((32-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)-5-氧代-3,9,12,15,18,21,24,27,30-九氧杂-6-氮杂三十二烷酰基)-L-缬氨酸)-L-赖氨酸(C1.15)(90mg)。
LCMS(ESI)[M+H]+＝1032.3。
实施例C1.16：N6,N6-二甲基-N2-((6-(2-(甲硫基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
向化合物C1.16-A(10.0g)加入氯化氢-二氧六环(100mL)溶液，在室温下反应2小时。反应液减压浓缩得到白色固体目标化合物C1.16-B(8.0g)。
LCMS(ESI)[M+H]+：380.1。
1H NMR(400MHz,DMSO)δ8.18(d,J＝7.4Hz,1H),7.40–7.30(m,5H),7.26(d,J＝8.8Hz,1H),5.08–4.99(m,2H),4.23–4.11(m,1H),3.94–3.88(m,1H),2.78–2.73(m,2H),2.03–1.94(m,1H),1.77–1.51(m,4H),1.44–1.31(m,2H),0.87(dd,J＝17.3,6.6Hz,6H)。
步骤二：
将化合物C1.16-B(3.0g)和醋酸钠(1.90g)溶在甲醇(100mL)溶液中，室温下反应10分钟， 向反应液中加入多聚甲醛(2.8g)，室温下搅拌反应30分钟，再向反应液中加入氰基硼氢化钠(1.0g)，反应在室温下搅拌反应16小时。反应液过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.16-C(1.70g)。
LCMS(ESI)[M+H]+：408.1；
1H NMR(400MHz,DMSO)δ7.86(d,J＝7.3Hz,1H),7.40–7.26(m,6H),5.03(s,2H),4.08–4.06(m,1H),3.90–3.85(m,1H),2.50–2.45(m,2H),2.35(s,6H),2.05–1.93(m,1H),1.73–1.55(m,2H),1.51–1.40(m,2H),1.33–1.22(m,2H),0.85(dd,J＝16.5,6.8Hz,6H)。
步骤三：
室温下，将化合物C1.16-C(1.6g)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(10％，0.16g)，氢气下室温下搅拌反应12小时。将反应液过滤，滤液减压浓缩得到目标化合物C1.16-D(680mg)。
LCMS(ESI)[M+H]+：274.2。
步骤四：
将6-(2-(甲硫基)嘧啶-5-基)己-5-炔酸(944mg)溶于N,N-二甲基甲酰胺(30mL)中，然后依次加入N,N-二异丙基乙胺(1.3g)，2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU，1.8g)。反应液室温搅拌20分钟，然后加入化合物C1.16-D(1.1g)，40℃搅拌反应3小时，粗产品用C18柱反相分离纯化(乙腈/0.01％甲酸的水溶液：5％-65％)得到白色固体目标化合物N6,N6-二甲基-N2-((6-(2-(甲硫基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸C1.16(1.2g)。
LCMS(ESI)[M+H]+：492.1；
1H NMR(400MHz,DMSO)δ8.68(s,2H),8.00(d,J＝7.6Hz,1H),7.91(d,J＝9.0Hz,1H),4.20(t,1H),4.08(dd,J＝12.7,7.7Hz,1H),2.47–2.40(m,4H),2.37–2.31(m,2H),2.30(s,6H),2.01–1.92(m,1H),1.82–1.73(m,2H),1.71–1.56(m,2H),1.49–1.37(m,2H),1.33–1.23(m,2H),0.88–0.82(m,6H).
实施例C1.17：N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸C1.17
将6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg),化合物C1.16-D(328mg)，三乙胺(322mg)溶于N,N-二甲基甲酰胺(5mL)。然后再加入1-羟基苯并***(HOBT，162mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，229mg),
反应液室温搅拌16小时，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到 目标化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1.17，白色固体，327mg)。
LCMS(ESI)[M+H]+：524.4。
1H NMR(400MHz,)δ9.13(s,2H),7.95(t,J＝8.8Hz,2H),4.21(dd,J＝8.8,6.9Hz,1H),4.08–4.03(m,1H),3.41(s,3H),2.55(t,J＝7.0Hz,2H),2.42–2.32(m,4H),2.27(s,6H),1.98(dd,J＝13.6,6.8Hz,1H),1.86–1.77(m,2H),1.74–1.55(m,2H),1.47–1.37(m,2H),1.31–1.23(m,2H),0.85(dd,J＝12.8,6.8Hz,6H)。
实施例C1.18：N2-(叔丁氧基羰基)-N6-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
化合物C1.18-B(3g)溶于二氯甲烷(30ml)，加入DIPEA(4ml)，然后加入化合物C1.18-A(3.48g)，混合物室温下搅拌反应20小时。向反应液中加入乙酸乙酯(200ml)，混合物依次用盐酸(0.1M,30ml x 3)、水(30ml x 3)洗涤，无水硫酸钠干燥，减压浓缩后得到目标混合物C1.18-C(4.7g)。
步骤二：
室温下，将化合物C1.18-C(4.7g)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(10％，0.6g)，在氢气下室温下搅拌反应12小时。将反应液过滤，滤液减压浓缩得到目标化合物C1.18-D(3.4g)。LCMS(ESI)[M+H]+：346.2。
步骤三：
将6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(2.68g),溶于N,N-二甲基甲酰胺(50mL),然后再加入三乙胺(3ml),HBTU(3.8g),混合物室温下搅拌10分钟，然后加入乙酸乙酯(200ml)，混合物用饱和碳酸氢钠(20ml x 2)，盐酸(0.1M，50ml x 2)，水(50ml x 2)洗，无水硫酸钠干燥，减压浓缩得到粗产物中间体，上述粗产物溶于DMF(30ml)，加入DIPEA(1.6ml)，然后加入C1.18-D(3.4g)，反应液室温搅拌3小时，然后加入乙酸乙酯(200ml)，用盐酸(0.1M，50ml x 2)，水(50ml x 2)洗，无水硫酸钠干燥，减压浓缩得到粗产物，经硅胶柱色谱分离提纯得到目标化合物N2-(叔丁氧基羰基)-N6-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1.18，白色固体，3.8g)。
LCMS(ESI)[M+H]+：596.4。
实施例C1.19：N6,N6-二甲基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
将化合物6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(1.0g，3.3mmol)溶在四氢呋喃和水(40mL，3:1)混合溶液中，加入过氧单磺酸钾(10.0g，16.3mmol)。室温搅拌反应3小时，LCMS检测反应完成。反应液过滤，滤饼用DMSO洗涤，合并滤液。经反相纯化(C18，乙腈：0.1％甲酸＝5％-55％)纯化，得到6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(750mg，收率67.9％)白色固体。
LCMS(ESI)[M+H]+＝340.1；
1H NMR(400MHz,DMSO-d6)δ9.48(s,2H),8.95(s,1H),4.49(t,J＝7.0Hz,2H),3.45(s,3H),2.22(t,J＝7.3Hz,2H),1.95–1.84(m,2H),1.61–1.50(m,2H),1.36–1.26(m,2H)。
步骤二：
将化合物6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(300mg，0.88mmol)溶于N,N-二甲基甲酰胺(6mL)，加入HATU(337mg，0.88mmol)，DIPEA(286mg，2.21mmol)，室温搅拌反应30分钟，然后加入二肽C1.16-D(243mg，0.88mmol)，室温搅拌反应2小时。LCMS检测反应完成，反应液通过C18柱分离纯化(乙腈/0.01％FA的水溶液：5％-50％)得到目标化合物N6,N6-二甲基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(300mg，收率57％)白色固体。
LCMS(ESI)[M+H]+＝595.5,tR＝2.024min。
1H NMR(400MHz,DMSO-d6)δ9.49(s,2H),9.01(s,1H),7.85(br s,1H),7.84(d,J＝8.9Hz,1H),4.48(t,J＝6.8Hz,2H),4.17–4.15(m,1H),4.02(br s,1H),3.44(s,3H),2.42(br s,2H),2.30(s,6H),2.18–2.14(m,2H),1.99–1.96(m,1H),1.88(dd,J＝14.6,7.1Hz,2H),1.67(br s,1H),1.59–1.53(m,2H),1.43(br s,2H),1.28–1.26(m,5H),0.83–0.89(m,6H).
实施例C1.20：N6,N6-二乙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
将化合物C1.16-B(5.0g,12.05mmol)溶在二氯甲烷(100mL)中，向反应液中加入乙醛(3.2g,72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g,60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.20-A(4.57g,收率82.0％)为白色固体。
LCMS(ESI)[M+H]+＝436.4；
1H NMR(400MHz,DMSO-d6)δ7.70(d,J＝7.0Hz,1H),7.41(d,J＝9.0Hz,1H),7.38–7.26(m,5H),5.08–4.99(m,2H),4.00(dd,J＝12.6,6.5Hz,1H),3.86(dd,J＝8.6,6.8Hz,1H),2.74(dd,J＝14.0,6.9Hz,4H),2.64–2.54(m,2H),2.05–1.94(m,1H),1.72–1.52(m,2H),1.52–1.38(m,2H),1.38–1.18(m,2H),1.04(t,J＝7.1Hz,6H),0.87–0.81(m,6H)。
步骤二：
室温下，将化合物C1.20-A(1.6g，3.68mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C1.20-B(900mg,收率82％)为米白色固体。
1H NMR(400MHz,DMSO-d6)δ8.04(br s,1H),4.02–3.99(m,1H),3.10(d,J＝4.5Hz,1H),2.65(q,J＝7.1Hz,4H),2.55–2.51(m,2H),2.06–1.93(m,1H),1.73–1.54(m,2H),1.47–1.38(m,2H),1.30–1.21(m,2H),1.01(t,J＝7.1Hz,6H),0.89(d,J＝6.9Hz,3H),0.79(d,J＝6.8Hz,3H)。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg,1mmol)溶于DMF(8mL)中，依次加入HATU(380mg,1mmol)和三乙胺(322mg,2.5mmol)，室温搅拌20分钟后加入化合物C1.20-B(301mg,1mmol)，室温继续搅拌30分钟。LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物(280mg，收率51％)为白色固体。
LCMS(ESI)[M+H]+＝552.3；
1H NMR(400MHz,DMSO-d6)δ9.13(s,2H),7.95(d,J＝8.9Hz,1H),7.86(d,J＝7.2Hz,1H),4.18(dd,J＝8.8,6.8Hz,1H),4.02(dd,J＝12.8,7.2Hz,1H),3.41(s,3H),2.74–2.69(m,4H),2.62–2.52(m,4H),2.44–2.29(m,2H),2.04–1.94(m,1H),1.86–1.77(m,2H),1.72–1.54(m,2H),1.51–1.39(m,2H),1.33–1.23(m,2H),1.02(t,J＝7.2Hz,6H),0.87–0.82(m,6H)。
实施例C1.21：N6,N6-二乙基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸
将化合物6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(500mg,1.475mmol)，溶于N,N-二甲基甲酰胺(10mL),然后加入HATU(560mg,1.475mmol)，N,N-二异丙基乙胺(476mg,3.688mmol)，搅拌30分钟，然后加入化合物C1.20-B(444mg，1.475mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二乙基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(330mg，收率34％)为白色固体。
LCMS(ESI)[M+H]+＝623.4；
1H NMR(400MHz,DMSO)δ9.49(s,2H),9.04(s,1H),7.84(d,J＝8.9Hz,1H),7.80(d,J＝7.1Hz,1H),4.47(t,J＝7.0Hz,2H),4.14(dd,J＝8.8,6.6Hz,1H),4.06–3.95(m,1H),3.44(s,3H),2.67–2.64(m,4H),2.55(t,J＝7.4Hz,2H),2.21–2.10(m,2H),2.02–1.94(m,1H),1.92–1.84(m,2H),1.72–1.63(m,1H),1.72–1.63(m,3H),1.59–1.54(m,2H),1.32–1.21(m,4H),1.01(t,J＝7.1Hz,6H),0.81(dd,J＝9.6,6.8Hz,6H)。
实施例C1.22：N6,N6-二丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
将化合物C1.16-B(5.0g，12mmol)溶在二氯甲烷(100mL)中，向反应液中加入正丙醛(4.2g，72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g，60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.22-A(4.57g,收率82.0％)为白色固体。
LCMS(ESI)[M+H]+＝464.0；
1H NMR(400MHz,DMSO-d6)δ7.81(d,J＝7.3Hz,1H),7.38–7.28(m,5H),5.04(d,J＝1.7Hz,2H),4.12–4.02(m,1H),3.94–3.82(m,1H),2.65–2.52(m,6H),2.06–1.94(m,1H),1.76–1.64(m,1H),1.64–1.53(m,1H),1.52–1.40(m,6H),1.34–1.18(m,2H),0.92–0.80(m,12H)。
步骤二：
室温下，将化合物C1.22-A(2.0g，4.32mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C1.22-B(1.2g,收率85.5％)为白色固体。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(100mg,0.373mmol)，溶于N,N-二甲基甲酰胺(1mL),然后加入HATU(142mg,0.373mmol)，N,N-二异丙基乙胺(120mg,0.93mmol)，该体系搅拌30分钟，然后加入化合物C1.22-B(122mg，0.371mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(50mg，收率28％)为淡黄色固体。
LCMS(ESI)[M+H]+＝580.0；
1H NMR(400MHz,DMSO-d6)δ8.24(s,2H),7.98–7.93(m,2H),4.24–4.16(m,1H),4.10(d,J＝5.2Hz,1H),3.41(s,3H),2.79–2.64(m,6H),2.55(t,J＝7.1Hz,2H),2.45–2.26(m,2H),2.06–1.91(m,1H),1.89–1.78(m,2H),1.76–1.66(m,1H), 1.64–1.57(m,1H),1.57–1.42(m,6H),1.37–1.24(m,2H),0.93–0.78(m,12H)。
实施例C1.23：N6,N6-二丙基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸
将化合物6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(500mg,1.475mmol)，溶于N,N-二甲基甲酰胺(10mL),然后加入HATU(560mg,1.475mmol)，N,N-二异丙基乙胺(476mg,3.688mmol)，该体系搅拌30分钟，然后加入化合物C1.22-B(485mg，1.475mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二丙基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(320mg，收率33％)为白色固体。
LCMS(ESI)[M+H]+＝651.5；
1H NMR(400MHz,DMSO)δ9.49(s,2H),9.00(s,1H),7.95(d,J＝7.2Hz,1H),7.79(d,J＝8.9Hz,1H),4.47(t,J＝6.9Hz,2H),4.21–4.11(m,1H),4.09–4.01(m,1H),3.44(s,3H),2.45–2.35(m,6H),2.23–2.09(m,2H),1.97–1.86(m,3H),1.72–1.64(m,1H),1.61–1.50(m,3H),1.43–1.34(m,6H),1.32–1.22(m,4H),0.86–0.77(m,12H)。
实施例C1.24：(S)-(6-((4-(羟甲基)苯基)氨基)-5-(6-(2-(甲硫基)嘧啶-5-基)己-5-炔酰胺基)-6-氧代己基)氨基甲酸叔丁酯
步骤一：
将化合物C1.24-A(3.70g，15.7mmol)，二异丙基乙胺(11.0mL,62.8mmol)和HBTU(8.90g,23.6mmol)溶于N,N-二甲基甲酰胺(30mL)中，反应在室温下反应30分钟。随后加入化合物Boc保护的赖氨酸(3.86g，15.7mmol)，反应在室温下反应2小时。LCMS检测反应完全。向反应液中加入柠檬酸水溶液(30mL)调至PH＝5，用二氯甲烷(50mL X 3)萃取水溶液，合并有机相并用无水硫酸钠干燥，抽滤，减压浓缩得到目标化合物C1.24-B(7.0g,粗品)为黄色油状物。LCMS(ESI)[M+H]+＝465.1,tR＝1.751min.
步骤二：
将化合物C1.24-B(7.00g，5.60mmol)，二异丙基乙胺(10.7mL,60.4mmol)和HBTU(8.60g, 22.7mmol)溶于N,N-二甲基甲酰胺(100mL)中，随后加入对氨基苄醇(3.71g，30.2mmol)，反应在室温下反应2小时。LCMS检测反应完全。向反应液中加入乙酸乙酯(300mL)，用饱和食盐水(100mL X3)洗涤，无水硫酸钠干燥，抽滤，滤液减压浓缩成固体，粗产品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物(S)-(6-((4-(羟甲基)苯基)氨基)-5-(6-(2-(甲硫基)嘧啶-5-基)己-5-炔酰胺基)-6-氧代己基)氨基甲酸叔丁酯(6.00g,收率：69.9％)为黄色油状物。
LCMS(ESI)[M+H]+＝570.1；
1H NMR(400MHz,CDCl3)δ9.24(s,1H),8.46(s,2H),7.51–7.47(m,2H),7.31–7.23(m,3H),7.02–6.95(m,1H),4.83(br s,1H),4.63–4.60(m,2H),3.19–2.96(m,4H),2.57(s,3H),2.52–2.48(m,2H),2.47–2.41(m,2H),1.99–1.92(m,2H),1.87–1.77(m,2H),1.76–1.63(m,2H),1.43(s,9H)。
实施例C1.25：(S)-(6-((4-(羟甲基)苯基)氨基)-5-(6-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)-6-氧代己基)氨基甲酸叔丁酯
步骤一：
将化合物C1.25-A(1.60g，5.21mmol)，二异丙基乙胺(2.68mL,20.8mmol)和HBTU(2.97g,7.82mmol)溶于N,N-二甲基甲酰胺(30mL)，反应在室温下反应30分钟。随后加入Boc保护的赖氨酸(1.28g，5.21mmol)，反应在室温下反应2小时。LCMS检测反应完全。向反应液中加入柠檬酸水溶液(30mL)调至PH＝5，用二氯甲烷(50mL X 3)萃取水溶液，合并有机相并用无水硫酸钠干燥，抽滤，减压浓缩得到目标化合物C1.25-B(3.0g,粗品)为黄色油状物。
LCMS(ESI)[M+H]+＝536.0；
步骤二：
将化合物C1.25-B(3.00g，5.60mmol)，二异丙基乙胺(2.89g,22.4mmol)和HBTU(3.19g,8.40mmol)溶于N,N-二甲基甲酰胺(30mL)。随后加入对氨基苄醇(1.38g，11.2mmol)，反应在室温下反应2小时。LCMS检测反应完全。向反应液中加入乙酸乙酯(200mL)，用饱和食盐水(80mL X 3)洗涤，无水硫酸钠干燥，抽滤，滤液减压浓缩成固体，粗产品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物(S)-(6-((4-(羟甲基)苯基)氨基)-5-(6-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)-6-氧代己基)氨基甲酸叔丁酯(2.90g)为黄色油状物。
LCMS(ESI)[M+H]+＝641.1；
1H NMR(400MHz,CDCl3)δ9.13(s,1H),8.95(s,2H),7.94(s,1H),7.47(d,J＝8.4Hz,2H),7.24(d,J＝8.4Hz,2H),6.83–6.77(m,1H),4.93–4.80(m,1H),4.64–4.58(m,2H),4.40–4.29(m,2H),3.12–2.97(m,4H),2.60(s,3H),2.31–2.23(m,2H),1.96–1.83(m,4H),1.76–1.61(m,4H),1.41(s,9H),1.37–1.29(m,4H)。
实施例C1.26：N6,N6-二丁基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸
步骤一：
将化合物C1.16-B(10.0g)溶在二氯甲烷(200mL)溶液中，向反应液中加入正丁醛(10.4g)，室温下搅拌反应10分钟，再向反应液中分批次加入三乙酰氧基硼氢化钠(25.6g)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.26-A(4.9g)为白色固体。
LCMS(ESI)[M+H]+＝492.7；
1H NMR(400MHz,CDCl3)δ7.35–7.30(m,5H),5.14–5.08(m,2H),4.33–4.30(m,1H),4.14–4.12(m,1H),2.94–2.80(m,6H),2.13–2.11(m,1H),1.85–1.82(m,2H),1.58–1.55(m,4H),1.40–1.22(m,8H),0.98–0.87(m,12H)。
步骤二：
室温下，将化合物C1.26-A(5.0g)溶于甲醇(100mL)溶液中，然后向反应液中加入Pd/C(10％，1g)，氢气下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C1.26-B(3.68g)为白色固体。
LCMS(ESI)[M+H]+＝358.3。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(1.0g)溶于N,N-二甲基甲酰胺(15mL),然后依次加入N,N-二异丙基乙胺(1.2g)，HATU(1.4g)，反应搅拌30分钟，然后加入化合物C1.26-B(1.3g)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物N6,N6-二丁基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1.26，500mg)为黄色固体。
LCMS(ESI)[M+H]+＝608.7；
1H NMR(400MHz,DMSO)δ9.37(s,1H),9.13(s,2H),8.19(d,J＝7.3Hz,1H),7.92(d,J＝8.8Hz,1H),4.25–4.20(m,1H),4.20–4.13(m,1H),3.42(s,3H),3.06–2.99(m, 6H),2.57–2.53(m,2H),2.41–2.30(m,2H),1.99–1.95(m,2H),1.87–1.79(m,2H),1.78–1.71(m,1H),1.62–1.56(m,4H),1.34–1.32(m,8H),0.94–0.85(m,12H)。
实施例C1.27：(S)-1-乙基-4-(3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)哌啶-4-羧酸
步骤一：
将化合物C1.27-A(5.0g，20.49mmol)溶在DMF(100mL)中，向反应液中加入N-((苄氧基)羰基)-L-缬氨酸2,5-二氧吡咯烷-N-羟基酯(7.13g，20.49mmol)，100℃下搅拌反应48小时。LCMS显示反应完全。反应液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.27-B(1.3g,收率13.3％)为黄色固体。
LCMS(ESI)[M-boc]+＝378.2；
1H NMR(400MHz,DMSO)δ7.38–7.33(m,5H),7.32–7.27(m,1H),7.20(d,J＝9.0Hz,1H),5.14–5.02(m,2H),3.97–3.87(m,1H),3.70–3.59(m,2H),3.08–2.95(m,2H),2.04–1.97(m,1H),1.95–1.87(m,2H),1.76–1.69(m,2H),1.40(s,9H),0.91–0.84(m,6H)。
步骤二：
室温下，将化合物C1.27-B(1.3g，2.71mmol)溶于乙酸乙酯中(10mL)溶液中，然后向反应液中加入二氧六环-HCl(3N，10mL)，室温下搅拌反应1小时。LCMS显示反应完成。将反应液减压浓缩得到目标化合物C1.27-C(1.06g,收率94.4％)为黄色固体。
LCMS(ESI)[M+H]+＝378.4。
步骤三：
将化合物C1.27-C(1.06g，2.81mmol)溶在二氯甲烷(10mL)中，向反应液中加入乙醛(0.75g，16.87mmol)，室温下搅拌反应10分钟，再向反应液中发批次加入三乙酰氧基硼氢化钠(3g，14.05mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，减压蒸馏除去二氯甲烷，剩余水相经反相分离(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C1.27-D(1.13g,收率99.23％)为白色固体。
LCMS(ESI)[M+H]+＝406.1；
1H NMR(400MHz,MeOD)δ7.42–7.25(m,5H),5.22–5.06(m,2H),3.91(d,J＝7.0 Hz,1H),3.52–3.36(m,2H),3.25–2.93(m,4H),2.66–2.54(m,1H),2.47–2.36(m,1H),2.27–2.14(m,2H),2.13–2.06(m,1H),1.35–1.29(m,4H),0.98–0.90(m,6H)。
步骤四：
室温下，将化合物C1.27-D(1.13g，2.78mmol)溶于甲醇(20mL)溶液中，然后向反应液中加入Pd/C(0.5g)，氢气下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C1.27-E(0.58g,收率76.18％)为白色固体。LCMS(ESI)[M+H]+＝272.4。
步骤五：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(1g,3.73mmol)，溶于N,N-二甲基甲酰胺(5mL),然后依次加入N,N-二异丙基乙胺(1.2g,9.32mmol)，HATU(1.42g,3.73mmol)，反应液搅拌30分钟，然后加入化合物C1.27-E(1.01g，3.73mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物(S)-1-乙基-4-(3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)哌啶-4-羧酸(C1.27，500mg,收率28.7％)为黄色固体。
LCMS(ESI)[M+H]+＝522.5；
1H NMR(400MHz,DMSO)δ9.12(s,2H),8.08(s,1H),7.88(d,J＝9.0Hz,1H),4.28–4.25(m,1H),3.41(s,3H),2.89–2.78(m,2H),2.57–2.52(m,4H),2.45–2.30(m,4H),2.07–2.01(m,2H),1.99–1.96(m,1H),1.95–1.88(m,2H),1.85–1.79(m,2H),1.04(t,J＝7.1Hz,3H),0.88–0.83(m,6H)。
二、药物-连接体化合物合成
实施例2.1：N-((S)-1-(((S)-6-氨基-1-((2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-001)
步骤一：
将化合物C1.6(50mg,0.10mol),化合物B1.5(71mg，0.12mol)，N,N-二异丙基乙胺(39 mg,0.30mol)溶于DMF(5mL)。然后依次加入1-羟基苯并***(16mg,0.12mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(23mg,0.12mmol),上述反应液室温搅拌16小时，加入50mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到粗品，粗产品用快硅胶柱层析分离纯化(DCM:MeOH＝100:0到90:10)得到目标化合物2.1A，(62mg，黄色固体，收率58％)。
LCMS(ESI)[M+H]+：1024.8。
步骤二：
将化合物2.1A(40mg,0.04mmol)溶于四氢呋喃和水的混合溶液(体积比为1/1；4mL)中，加入过氧单磺酸钾(246mg,0.4mmol)，反应液在室温下搅拌5小时，加入30mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到目标化合物2.1B(30mg，黄色固体,收率74％)。
LCMS(ESI)[M+H]+＝1055.9。
步骤三：
将化合物2.1B(30mg,0.03mmol)溶于三氟乙酸和二氯甲烷混合溶液(体积比为1：3；4mL)中,反应液在室温下搅拌1小时。然后减压浓缩得粗产品，粗产品经制备色谱(0.01％TFAin water,MeCN)纯化得到目标化合物N-((S)-1-(((S)-6-氨基-1-((2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(9mg,白色固体，收率33％)。
LCMS(ESI)[M+H]+：955.8；
1H NMR(400MHz,DMSO-d6)δ9.09(s,2H),8.54(s,1H),8.15(m,2H),7.84(m,1H),7.80(s,1H),7.64(br s,3H),7.53(s,1H),7.26(s,1H),6.51(s,1H),6.30(d,J＝2.9Hz,2H),5.59–5.46(m,2H),5.43(s,2H),4.87–4.78(m,1H),4.70–4.60(m,1H),4.17–4.03(m,2H),3.82–3.58(m,2H),3.41(s,3H),2.80–2.70(m,2H),2.40–2.22(m,2H),1.93–1.68(m,6H),1.65–1.57(m,1H),1.56–1.45(m,3H),1.36–1.22(m,2H),0.87(t,J＝7.3Hz,3H),0.80(d,J＝5.2Hz,6H)。
用实施例2.1相同的方法和反应条件，用所示的不同起始原料，得到下表中实施例2.2到实施例2.8所示的目标产物。


实施例2.9：(S)-6-氨基-N-(2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-009)
步骤一：
将化合物B1.5(30mg),化合物C1.9(48mg)，N,N-二异丙基乙胺(23mg)溶于DMF(5mL)。向上述溶液中加入1-羟基苯并***(9mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(13mg),反应室温搅拌16小时，加入50mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到粗品，粗产品用快速色谱法分离纯化(DCM:MeOH＝100:0to 90:10)得到目标化合物2.9A(35mg，淡黄色固体，收率43％)。
LCMS(ESI)[M+H]+：1095.8
步骤二：
将化合物2.9A(35mg)溶于四氢呋喃和水的混合溶液(体积比为1/1；4mL)中，加入过氧单磺酸钾(184mg)，反应液室温下搅拌5小时，加入30mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到目标化合物2.9B(25mg，淡黄色固体，收率71％)。
LCMS(ESI)[M+H]+：1126.9。
步骤三：
将化合物2.9B(25mg)溶于三氟乙酸和二氯甲烷混合溶液(体积比为1：3；4mL)中,反应液在室温下搅拌1小时。液浓缩得粗产品，经prep-HPLC(0.01％TFA in water,MeCN)纯化得到目标化合物(S)-6-氨基-N-(2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺三氟乙酸盐(5.1mg)。
LCMS(ESI)[M+H]+：1026.9；
1H NMR(400MHz,DMSO)δ9.46(s,2H),8.91(s,1H),8.53(s,1H),8.18(s,1H),8.11–8.10(m,1H),7.79(s,1H),7.73–7.72(m,1H),7.63(s,3H),7.53(s,1H),7.26(s,1H),6.52(s,1H),6.29(m,2H),5.61–5.46(m,2H),5.43(s,2H),4.85–4.83(m,1H),4.63–4.60(m 1H),4.44(s,2H),4.06–4.03(m,3H),2.75(s,2H),2.12–2.10(m,2H),1.89–1.85(m,6H),1.56–1.53(m,8H),1.24(br s,4H),0.87–0.84(m,3H),0.77(d,J＝6.1Hz,6H).
实施例2.10：N-((7S,10S)-7-(4-氨基丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-10-异丙基-3,6,9,12,16-五氧代-14,20,23,26,29,32,35,38,41-九氧杂-2,5,8,11,17-五氮杂四十三烷-43-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-010)
步骤一：
将化合物C1.14(112mg)溶在N,N-二甲基甲酰胺(2mL)中，依次加入三乙胺(32mg，0.31mmol)，1-羟基苯并***(17mg，0.13mmol)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(24mg，0.13mmol)，搅拌20分钟，然后将化合物B1.5(50mg)加入于反应液中，反应液在40℃下搅拌过夜。向反应液中加入10mL水，二氯甲烷萃取，无水硫酸钠干燥，抽滤，旋干得到粗品。粗产品用C18柱反相分离纯化(乙腈/0.01％FA的水溶液：0％-55％)得到白色固体目标化合物2.10A(75mg，收率46％)。
LCMS(ESI)[M+H]+：1534.0。
步骤二：
将化合物2.10A(75mg)溶在四氢呋喃和水(8mL，1:1)溶液中，加入过氧单磺酸钾(211mg)室温搅拌反应10小时。向反应液中加入10mL二氯甲烷，萃取，分液，无水硫酸钠干燥，抽滤旋干得到目标产物2.10B(50mg，收率65％)。
LCMS(ESI)[M+H]+：1566.2。
步骤三：
将化合物2.10B(50mg)溶在三氟乙酸和二氯甲烷混合溶液(体积比为1：2；4mL)中，然后反应液在室温下搅拌1小时。将反应液浓缩得粗产品，粗产品经制备色谱(0.01％TFAin water，MeCN)得到目标化合物N-((7S,10S)-7-(4-氨基丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-10-异丙基-3,6,9,12,16-五氧代-14,20,23,26,29,32,35,38,41-九氧杂-2,5,8,11,17-五氮杂四十三烷-43-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(10.5mg，收率21％)。
LCMS(ESI)[M+H]+：1466.9；
1H NMR(400MHz,DMSO-d6)δ9.12(s,2H),8.62(t,J＝5.7Hz,1H),8.26–8.11(m,2H),8.03(t,J＝5.8Hz,1H),7.93(t,J＝5.3Hz,1H),7.85(d,J＝8.7Hz,1H),7.79(d, J＝5.6Hz,1H),7.61(s,3H),7.54(s,1H),7.26(s,1H),6.50(s,1H),6.31(d,J＝1.7Hz,2H),5.46(d,J＝17.2Hz,4H),4.76(dt,J＝14.8,8.7Hz,2H),4.20–4.15(m,4H),4.00(s,3H),3.95(d,J＝5.5Hz,3H),3.72–3.67(m,2H),3.50(d,J＝3.9Hz,30H),3.41(s,3H),3.25–3.19(m,4H),2.76(d,J＝5.6Hz,2H),2.56(t,J＝4.9Hz,2H),2.27(t,J＝7.3Hz,2H),1.90(dd,J＝16.1,7.4Hz,2H),1.84–1.78(m,2H),1.63(s,1H),1.53–1.48(m,2H),1.30(d,J＝7.7Hz,2H),0.87–0.84(m,4H),0.78–0.75(m,6H)。
实施例2.11：N-((S)-1-(((S)-6-氨基-1-((2-((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-011)
步骤一：
向化合物B1.10(75mg)的N,N-二甲基甲酰胺溶液(3mL)中依次加入化合物C1.7(107mg)，三乙胺(45mg)，HOBT(25mg)和EDCI(35mg)，在35℃下反应4小时。向反应液中加入水(30mL)，乙酸乙酯(30mL×2)萃取，有机相用无水硫酸钠干燥，浓缩得到目标化合物2.11A(60mg，收率：37％)。
LCMS(ESI)[M+H]+：1084；
步骤二：
向化合物2.11A(60mg)的二氯甲烷溶液(4mL)中加入三氟乙酸和二氯甲烷混合溶液(体积比为1：2；4mL)，在室温下搅拌反应1小时。将反应液浓缩，粗品经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物N-((S)-1-(((S)-6-氨基-1-((2-((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(9mg，收率18％)。
LCMS(ESI)[M+H]+：983.9；
1H NMR(400MHz,DMSO-d6)δ9.08(s,2H),8.34(s,1H),8.24(s,1H),8.19(d,J＝7.1Hz,1H),8.00(d,J＝8.3Hz,1H),7.95(s,1H),7.63(s,1H),7.49(d,J＝2.8Hz,1H),7.24(s,1H),6.50(s,1H),6.28(d,J＝1.9Hz,2H),5.42(s,2H),5.24(s,2H),4.25–4.07(m,2H),3.74–3.70(m,3H),3.25(d,J＝12.4Hz,3H),3.08(d,J＝7.6Hz,2H),2.73(s,2H),2.40–2.19(m,3H),2.01–1.64(m,10H),1.52(br s,4H),1.34(br s,2H),0.87(t,J＝7.3Hz,3H),0.83–0.75(m,6H)。
实施例2.12：(S)-6-氨基-N-(2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)-2-((S)-2-异丙基-35-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五酰胺)己酰胺(DL-012)
步骤一：
将化合物C1.15(80mg),化合物B1.5(37mg)，三乙胺(23mg，0.231mo,)溶于DMF(5mL)。然后再加入1-羟基苯并***(12mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(17mg)，反应液室温搅拌16小时，加入50mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到粗品，粗产品用快速色谱法分离纯化(DCM:MeOH＝100:0到90:10)得到目标化合物2.12A(75mg，收率65％)。
LCMS(ESI)[M+H]+：1492.0。
步骤二：
将化合物2.12A(50mg)溶于四氢呋喃和水的混合溶液(体积比为1/1；4mL)中，加入过氧单磺酸钾(202mg)，反应液在室温下搅拌5小时，加入30mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到目标化合物2.12B(40mg，收率80％)。
LCMS(ESI)[M+H]+：1524.1。
步骤三：
将化合物2.12B(30mg)溶在三氟乙酸和二氯甲烷混合溶液(体积比为1比3；4mL)中,然后反应液在室温下搅拌1小时。将反应液浓缩得粗产品，粗产品经prep-HPLC(水含0.01％三氟乙酸,乙腈)纯化得到目标化合物(S)-6-氨基-N-(2-((((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)甲基)氨基)-2-氧代乙基)-2-((S)-2-异丙基-35-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五酰胺)己酰胺(9mg,收率30％)。
LCMS(ESI)[M+H]+：1423.9。
实施例2.13：N-((13S,16S)-13-(4-(二甲基氨基)丁基)-3-乙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-17-甲基-4,9,12,15-四氧代-6-氧杂-3,8,11,14-四氮杂十八烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-013)
将化合物C1.17(50mg),化合物B1.2(58mg)，三乙胺(24mg)溶于N,N-二甲基甲酰胺(2mL)。然后依次加入1-羟基苯并***(16mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(22mg)，反应液室温搅拌16小时。反应液直接经制备色谱(0.01％甲酸水溶液,乙腈)纯化得到目标化合物(21mg,收率10％)。
LCMS(ESI)[M+H]+：1113.0；
1H NMR(400MHz,MeOD)δ8.93(s,2H),8.40–8.33(m,1H),8.30–8.25(m,1H),8.25–8.21(m,1H),7.65(s,1H),7.48(s,1H),7.34(s,1H),6.22(s,2H),5.47(dd,J＝78.6,16.4Hz,2H),5.28–5.13(m,2H),4.73–4.59(m,2H),4.30–4.05(m,4H),3.94–3.82(m,2H),3.70–3.57(m,2H),3.54–3.44(m,1H),3.34(s,6H),3.16–3.08(m,2H),2.90(s,6H),2.58–2.40(m,4H),2.04–1.83(m,6H),1.80–1.68(m,3H),1.54–1.36(m,2H),1.24–1.15(m,3H),1.03–0.86(m,9H)。
实施例2.14：N-((13S,16S)-13-(4-(N-氧代-N,N-二甲基氨基)丁基)-3-乙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b] 喹啉-14-基)-17-甲基-4,9,12,15-四氧代-6-氧杂-3,8,11,14-四氮杂十八烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-014)
步骤一：
将化合物C1.16(49mg),化合物B1.2(71mg)，N,N-二异丙基乙胺(39mg)溶于DMF(5mL)。然后依次加入1-羟基苯并***(16mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(23mg),加完后反应液室温搅拌16小时，加入50mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到粗品，粗产品用快硅胶柱层析分离纯化(DCM:MeOH＝100:0到90:10)得到目标化合物2.14A(61mg，收率60％)。
LCMS(ESI)[M+H]+：1081.6；
步骤二：
将化合物2.14A(40mg)溶于四氢呋喃和水的混合溶液(体积比为1/1；4mL)中，加入过氧单磺酸钾(227mg,0.37mmol)，反应液在室温下搅拌5小时，LCMS显示反应完毕,加入30mL乙酸乙酯稀释，有机相用水洗涤三次(25mL x 3)，无水硫酸钠干燥，浓缩除去乙酸乙酯得到粗品化合物，粗产品经制备色谱(0.01％甲酸水溶液,乙腈)纯化得到目标化合物N-((13S,16S)-13-(4-(N-氧代-N,N-二甲基氨基)丁基)-3-乙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-17-甲基-4,9,12,15-四氧代-6-氧杂-3,8,11,14-四氮杂十八烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(9mg,收率33％)。
LCMS(ESI)[M+H]+＝1129.0。
实施例2.15：N-((13S,16S)-13-(4-(N,N-二甲基氨基)丁基)-3-异丙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-17-甲基-4,9,12,15-四氧代-6-氧杂-3,8,11,14-四氮杂十八烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-015)
向化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(42mg，C1.17)和化合物(S)-2-((2-氨基乙酰胺基)甲氧基)-N-异丙基-N-(2-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)乙酰胺(50mg，B1.11)，的N,N-二甲基甲酰胺(10mL)溶液中，依次加入HBTU(31mg)和二异丙基乙胺(21mg)。反应在室温下搅拌2小时。反应液过滤后经高效液相制备纯化(乙腈/水含0.05％三氟乙酸)得到目标化合物N-((13S,16S)-13-(4-(N,N-二甲基氨基)丁基)-3-异丙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-17-甲基-4,9,12,15-四氧代-6-氧杂-3,8,11,14-四氮杂十八烷-16-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(15.2mg)。
LCMS(ESI)[M+H]+＝1127.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.73(t,J＝8.7Hz,1H),8.23–8.17(m,1H),8.10(d,J＝7.5Hz,1H),7.97–7.93(m,2H),7.52(s,1H),7.25(s,1H),6.50(s,1H),6.30(s,2H),5.42(s,2H),5.36(s,2H),4.66(d,J＝6.6Hz,2H),4.33–4.21(m,4H),4.20–4.13(m,2H),3.76–3.74(m,2H),3.40(s,3H),3.40–3.38(m,1H),3.30–3.21(m,2H),3.04–2.95(m,2H),2.76(s,3H),2.74(s,3H),2.39–2.31(m,2H),2.00–1.74(m,6H),1.68–1.49(m,4H),1.37–1.27(m,2H),1.23–1.16(m,6H),0.89–0.82(m,9H)。
实施例2.16：N-((10S,13S)-10-(4-(N,N-二甲基氨基)丁基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-14-甲基-1,6,9,12-四氧代-3-氧杂-5,8,11-三氮杂十五烷-13-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-016)
将化合物N6，N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)十六烷基-5-炔酰基)-L-丙酰基)-L-赖氨酸(C1.17，52.3mg)溶于N,N-二甲基甲酰胺(2mL)，然后再依次加入HBTU(38mg,0.10mmol)和N,N-二异丙基乙胺(26mg,0.20mmol)，加完后反应液室温搅拌30分钟，然后加入化合物2-氨基-N-((2-(((1S，9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H，12H-苯并[de]吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)乙酰胺 (2.16A，根据CN104755494A中第147-148页报道的相同方法制备的，63mg),LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物N-((10S,13S)-10-(4-(N,N-二甲基氨基)丁基)-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-14-甲基-1,6,9,12-四氧代-3-氧杂-5,8,11-三氮杂十五烷-13-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(8.1mg,收率7％)。
LCMS(ESI)[M+H]+＝1085.5；
1H NMR(400MHz,DMSO-d6)δ9.31(s,1H),9.11(s,2H),8.72(t,J＝6.8Hz,1H),8.51(d,J＝8.8Hz,1H),8.19(t,J＝6.8Hz,1H),8.10(d,J＝7.1Hz,1H),7.91(d,J＝8.6Hz,1H),7.79(d,J＝10.9Hz,1H),7.32(s,1H),6.53(s,1H),5.63–5.54(m,1H),5.42(s,2H),5.20(s,2H),4.68–4.58(m,2H),4.26–4.12(m,2H),4.01(s,2H),3.73(d,J＝5.4Hz,2H),3.20–3.10(m,2H),3.05–2.95(m,2H),2.76(d,J＝4.7Hz,6H),2.55–2.53(m,2H),2.42–2.28(m,6H),2.21–2.13(m,2H),2.02–1.77(m,6H),1.76–1.62(m,2H),1.62–1.51(m,3H),1.37–1.21(m,2H),0.90–0.80(m,9H)。
实施例2.17：叔丁基((S)-3-乙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-13-((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)-4,9,12-三氧代-6-氧杂-3,8,11-三氮杂十七烷-17-基)氨基甲酸酯(DL-017)
将化合物C1.7(90.0mg)，二异丙基乙胺(68.0mg)和HATU(100mg)溶于N,N-二甲基甲酰胺(5mL)中。随后加入化合物B1.2(80mg)，室温下反应2小时。LCMS检测反应完全。向反应液中加入乙酸乙酯(50mL)，用饱和食盐水(30mL X 3)洗涤，无水硫酸钠干燥，抽滤，滤液减压浓缩成固体，粗产品经高效液相制备纯化(乙腈/水含0.05％甲酸)得到黄色固体化合物叔丁基((S)-3-乙基-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-13-((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)-4,9,12-三氧代-6-氧杂-3,8,11-三氮杂十七烷-17-基)氨基甲酸酯(2.06mg，收率10.6％)。
LCMS(ESI)[M+H]+：1184.9。
实施例2.18：N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-018)
将化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(126mg)(C1.17)，B1.12(150mg)，1-羟基苯并***(49mg)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(69mg)和二异丙基乙胺(156mg,1.21mmol)溶在N,N-二甲基甲酰胺(10mL)溶液中，反应在40℃下搅拌12小时。LCMS显示反应完成。反应液过滤经高效液相制备纯化(乙腈/水含0.05％甲酸)得到黄色固体化合物N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-018)(12mg，收率6.0％)。
LCMS(ESI)[M+H]+＝1030.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.62(t,J＝6.4Hz,1H),8.23–8.13(m,2H),8.07(d,J＝7.2Hz,1H),7.95–7.83(m,2H),7.31(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.64–4.53(m,2H),4.28–4.20(m,1H),4.19–4.11(m,1H),3.76–3.69(m,2H),3.50(t,J＝5.7Hz,2H),3.41(s,3H),3.23–3.19(m,2H),2.67–2.62(m,2H),2.57–2.54(m,2H),2.54(s,3H),2.42–2.26(m,3H),2.02–1.77(m,6H),1.68–1.66(m,1H),1.58–1.45(m,3H),1.36–1.25(m,2H),0.88(t,J＝7.4Hz,3H),0.82(t,J＝6.3Hz,6H)。
实施例2.19：N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-019)
将化合物B1.14(100mg,0.186mmol),化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(100mg，0.191mmol)溶于DMF(2mL)。然后再加入1-羟基苯并***(38mg,0.280mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(53mg,0.280mmol),然后再加入三乙胺(56mg,0.559mmol)加完后反应液室温搅拌16小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并 [3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(20mg,收率10％)为黄色固体。
LCMS(ESI)[M+H]+＝1042.5；
1H NMR(400MHz,DMSO-d6)δ9.28(s,1H,TFA),9.10(s,2H),8.64(br s,1H),8.19(br s,1H),8.09(d,J＝6.4Hz,1H),7.92(d,J＝7.8Hz,1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.29(s,2H),5.43(s,2H),5.24(s,2H),4.67–4.52(m,2H),4.30–4.10(m,2H),3.74(br s,2H),3.50(br s,2H),3.41(s,3H),3.17–3.07(m,2H),3.04–2.91(m,2H),2.75(s,6H),2.46–2.24(m,3H),2.04–1.66(m,9H),1.63–1.46(m,3H),1.32–1.29(m,2H),0.87–0.82(m,9H)。
实施例2.20：N-((S)-1-(((S)-6-氨基-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-020)
步骤一：
向化合物B1.13(75mg)的N,N-二甲基甲酰胺溶液(3mL)中依次加入化合物C1.7(107mg)，三乙胺(45mg)，HOBT(25mg)和EDCI(35mg)，在35℃下反应4小时。向反应液中加入水(30mL)，乙酸乙酯(30mL×2)萃取，有机相用无水硫酸钠干燥，浓缩得到目标化合物2.20A(76mg)。
LCMS(ESI)[M+H]+：1072.5；
步骤二：
向化合物2.20A(60mg，0.06mmol)的二氯甲烷溶液(2mL)中，加入三氟乙酸(1mL)，在室温下搅拌反应1小时。LCMS显示反应完全。将反应液浓缩，经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((S)-1-(((S)-6-氨基-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(43mg，收率80％)为黄色固体。
LCMS(ESI)[M+H]+：972.9；
1H NMR(400MHz,DMSO-d6)δ9.09(s,2H),8.24–8.17(m,2H),8.12(d,J＝7.0Hz,1H),7.99–7.93(m,1H),7.88–7.84(m,2H),7.64(s,3H,NH2-TFA),7.31(s,1H),6.52(s,1H),5.44(s,2H),5.30(s,2H),4.25–4.18(m,1H),4.15–4.11(m,1H),3.76–3.71(m,2H),3.41(s,3H),3.32–3.26(m,2H),3.21–3.17(m,1H),2.79–2.70(m,2H),2.52(s,3H),2.40–2.24(m,3H),1.94–1.82(m,7H),1.80–1.73(m,2H),1.58–1.49(m,3H),1.36–1.31(m,1H),1.18–1.14(m,1H),0.87(t,J＝7.4Hz,3H),0.80–0.76(m,6H)。
实施例2.21：N-((S)-1-(((S)-5-氨基-6-((2-((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)氨基)-2-氧代乙基)氨基)-6-氧代己基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺
步骤一：
将化合物C1.18(430mg)溶于N,N-二甲基甲酰胺(5mL)中，然后加入EDCI(160mg)，HOBT(115mg)和三乙胺(214mg)。加完毕后反应液室温搅拌20分钟，然后加入化合物B1.10(314mg)，室温搅拌反应3小时，LCMS检测反应完全，加水稀释，乙酸乙酯萃取，有机相浓缩的粗品2.21A(350mg)。
LCMS(ESI)[M+H]+：1084.3；
步骤二：
向化合物2.21A(350mg)的二氯甲烷溶液(4mL)中加入三氟乙酸和二氯甲烷混合溶液(体积比为1：2；4mL)，在室温下搅拌反应1小时。将反应液浓缩，粗品经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物N-((S)-1-(((S)-5-氨基-6-((2-((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基)氨基)-2-氧代乙基)氨基)-6-氧代己基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(100mg)。
LCMS(ESI)[M+H]+：984.4；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.69(t,J＝6.1Hz,1H),8.14(t,J＝5.4Hz,1H),8.08(s,3H,NH2-TFA),7.95(t,J＝4.9Hz,1H),7.89(d,J＝8.9Hz,1H),7.66(s, 1H),7.51(s,1H),7.25(s,1H),6.48(s,1H),6.29(s,2H),5.42(s,2H),5.25(s,2H),4.09(t,J＝8.1Hz,1H),3.83(d,J＝5.7Hz,2H),3.40(s,3H),3.33–3.27(m,2H),3.15–3.06(m,3H),2.97–2.94(m,1H),2.57–2.53(m,2H),2.43–2.28(m,3H),1.95–1.68(m,9H),1.46–1.28(m,4H),0.88(t,J＝7.3Hz,3H),0.82(d,J＝6.7Hz,6H)。
实施例2.22：N-((S)-1-(((S)-5-氨基-6-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-6-氧代己基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺DL-022)
步骤一：
将化合物C1.18(136mg，0.23mmol)溶于N,N-二甲基甲酰胺(5mL)中，然后加入EDCI(48mg，0.25mmol)，HOBT(34mg,0.25mmol)和三乙胺(64mg,0.63mmol)。加完毕后反应液室温搅拌20分钟，然后加入化合物B1.13(3105mg，0.19mmol)，室温搅拌反应3小时，LCMS检测反应完全，加水稀释，乙酸乙酯萃取，有机相浓缩的粗品。所得化合物溶于二氯甲烷(4mL)中，然后加入三氟乙酸(2mL)，反应1小时后，浓缩，粗品经制备提纯得目标化合物N-((S)-1-(((S)-5-氨基-6-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-6-氧代己基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-022)(88mg)。
LCMS(ESI)[M+H]+＝972.9；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.72(t,J＝5.5Hz,1H),8.23(d,J＝8.4Hz,1H),8.17(t,J＝5.4Hz,1H),8.11(s,3H,NH2-TFA),7.97(t,J＝5.6Hz,1H),7.93–7.86(m,2H),7.32(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.14–4.02(m,2H),3.85–3.83(m,2H),3.41(s,3H),3.32(d,J＝6.2Hz,2H),3.24–3.16(m,2H),3.11–2.94(m,2H),2.55(s,3H),2.35–2.30(m,3H),1.92–1.69(m,10H),1.43–1.29(m,4H),0.88(t,J＝7.3Hz,3H),0.82(d,J＝6.7Hz,6H)。
实施例2.23：(S)-6-氨基-N-(2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)-2-((S)-2-异丙基- 35-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)己酰胺(DL-023)
步骤一：
向化合物B1.13(46mg)的N,N-二甲基甲酰胺溶液(3mL)中依次加入化合物2.23A(100mg)，三乙胺(50ul)，HOBT(16mg)和EDCI(23mg)，在35℃下反应4小时。向反应液中加入水(30mL)，乙酸乙酯(30mL×2)萃取，有机相用无水硫酸钠干燥，浓缩得到目标化合物2.23B(105mg)。
LCMS(ESI)[M+H]+：1358.4；
步骤二：
化合物2.23B(105mg)，2-甲磺酰基-5-乙炔基嘧啶(45mg)溶于DMSO-水(2ml，4:1)中，向混合物中加入溴化亚铜(50mg)。反应液室温下搅拌2小时，然后加入乙酸乙酯(100ml)，水洗(100ml x 3)，干燥后减压除去有机溶剂，粗产物用硅胶柱层析分离得到目标产物2.23C(84m mg)，MS(m/z):1540.8[M+H]+；
步骤三：
将化合物2.23C溶在三氟乙酸和二氯甲烷混合溶液(体积比为1：2；4mL)中，然后反应液在室温下搅拌30分钟。LCMS显示反应完成。将反应液浓缩得粗产品，粗产品经制备色谱(0.01％TFA in water,MeCN)得到目标化合物(S)-6-氨基-N-(2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)-2-((S)-2-异丙基-35-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)-4,8-二氧代-6,12,15,18,21,24,27,30,33-九氧杂-3,9-二氮杂三十五烷酰胺基)己酰胺(DL-023,8mg，三氟乙酸盐，收率5％)。
LCMS(ESI)[M+H]+＝1440.8；
1H NMR(400MHz,DMSO-d6)δ9.49(s,2H),8.91(s,1H),8.24–8.18(m,3H),8.01(t,J＝5.6Hz,1H),7.96(t,J＝5.5Hz,1H),7.90–7.84(m,2H),7.63(s,3H,TFA salt),7.32(s,1H),5.44(s,2H),5.30(s,2H),4.66(t,J＝5.1Hz,2H),4.21(dd,J＝15.2,7.9Hz,2H),3.97(s,2H),3.93(s,2H),3.89(t,J＝5.1Hz,2H),3.75–3.72(m,2H),3.56–3.54(m,2H),3.54–3.38(m,30H),3.33–3.13(m,5H),2.75(d,J＝7.1Hz,2H),2.52(s,3H),2.02–1.94(m,1H),1.91–1.81(m,3H),1.71–1.65(m,1H),1.58–1.46(m,3H),1.38–1.29(m,2H),0.88(t,J＝7.3Hz,3H),0.81–0.75(m,6H)。
实施例2.24：N-((S)-1-(((S)-6-(二甲基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-024)
向化合物B1.13(40mg，0.081mmol)和化合物C1.17(42mg，0.081mmol)的N,N-二甲基甲酰胺溶液(2mL)溶解中依次加入PyBOP(41mg，0.081)，DIPEA(20mg，0.16mmol)，反应液在室温下搅拌2小时。LCMS显示反应完全。反应液经高效液相制备纯化(乙腈/0.05％三氟乙酸的水溶液)得到化合物N-((S)-1-(((S)-6-(二甲基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(11.54mg，收率14％)为黄色固体。
LCMS(ESI)[M+H]+＝1000.6；
1H NMR(400MHz,DMSO-d6)δ9.42(s,1H),9.08(s,2H),8.27–8.18(m,2H),8.14(d,J＝6.9Hz,1H),7.96(t,J＝6.9Hz,1H),7.91–7.77(m,2H),7.31(s,1H),5.44(s,2H),5.29(s,2H),4.15–4.12(m,1H),3.81–3.65(m,3H),3.41(s,3H),3.33–3.25(m,2H),3.22–3.15(m,2H),3.03–2.95(m,2H),2.77–2.71(m,6H),2.38–2.23(m,3H),1.92–1.81(m,7H),1.78–1.73(m,2H),1.62–1.56(m,3H),1.38–1.29(m,2H),0.92–0.84(m,3H),0.84–0.72(m,6H)。
实施例2.25：N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代 乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-025)
将化合物C1.20(30mg,0.054mmol),化合物B1.13(27mg，0.054mmol)溶于N,N-二甲基甲酰胺(2mL)。然后再依次加入HBTU(22mg,0.054mmol)以及N,N-二异丙基乙胺(17mg,0.135mmol，加完毕后反应液室温搅拌30分钟。LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(23mg,收率42％)为黄色固体。
LCMS(ESI)[M+H]+＝1028.5；
1H NMR(400MHz,DMSO-d6)δ9.08(s,2H),9.02(s,1H),8.21(d,J＝7.4Hz,2H),8.13(d,J＝7.1Hz,1H),7.97(t,J＝5.4Hz,1H),7.87(d,J＝10.6Hz,2H),7.31(s,1H),6.52(s,1H),5.44(s,2H),5.30(s,2H),4.28–4.18(m,1H),4.14(t,J＝7.7Hz,1H),3.80–3.65(m,2H),3.41(s,3H),3.31–3.24(m,2H),3.22–3.15(m,2H),3.12–3.06(m,4H),3.02–2.91(m,2H),2.36–2.23(m,2H),1.95–1.68(m,9H),1.68–1.49(m,4H),1.38–1.28(m,2H),1.13(t,J＝7.2Hz,6H),0.86(t,J＝7.2Hz,3H),0.79–0.76(m,6H)。
实施例2.26：N-((S)-1-(((S)-6-(二丙基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-026)
将化合物C1.22(31mg,0.054mmol,化合物B1.13(27mg，0.054mmol)溶于N,N-二甲基甲酰胺(2mL)。然后再依次加入HBTU(22mg,0.054mmol)以及N,N-二异丙基乙胺(17mg,0.135mmol，反应液室温搅拌30分钟。LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈) 纯化得到目标化合物N-((S)-1-(((S)-6-(二丙基氨基)-1-((2-((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(25mg,收率45％)为黄色固体。
LCMS(ESI)[M+H]+＝1056.6；
H NMR(400MHz,DMSO-d6)δ9.09(s,2H),8.27–8.19(m,2H),8.13(d,J＝7.3Hz,1H),7.97(t,J＝5.3Hz,1H),7.91–7.84(m,2H),7.31(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.24(d,J＝6.7Hz,1H),4.13(t,J＝7.8Hz,1H),3.73(dd,J＝14.7,5.9Hz,2H),3.41(s,3H),3.28(d,J＝6.4Hz,2H),3.19(t,J＝7.6Hz,2H),2.99(br,6H),2.33–2.27(m,2H),1.95–1.70(m,9H),1.62–1.57(m,8H),1.33–1.30(m,2H),0.96–0.82(m,9H),0.82–0.73(m,6H)。
实施例2.27：N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-(((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺
向化合物B1.15(40mg，0.074mmol)和化合物C1.20(40mg，0.072mmol)的N,N-二甲基甲酰胺溶液(4mL)溶解中依次加入HBTU(30mg，0.079mmol)、DIPEA(26mg，0.2mmol)。反应液室温下反应1小时。LCMS显示反应完全。反应液经高效液相制备纯化(乙腈/0.05％三氟乙酸水溶液)得到化合物N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-(((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(11.1mg，收率14％)。
LCMS(ESI)[M+H]+＝1038.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),9.02(s,1H,TFA),8.33–8.25(m,2H),8.12(d,J＝7.3Hz,1H),7.92(d,J＝8.4Hz,1H),7.68(d,J＝4.1Hz,1H),7.52(d,J＝2.8Hz,1H),7.26(s,1H),7.15(d,J＝16.2Hz,1H),6.52–6.50(m,2H),6.30(s,2H),5.43(s,2H),5.28–5.24(m,2H),4.27(d,J＝6.2Hz,2H),4.12(br s,2H),3.81(br s,2H),3.41(s,3H),3.11–3.07(m,6H),2.97(br s,2H),2.53–2.50(m,1H),2.39–2.24(m,4H),1.85–1.79(m,8H),1.16(t,J＝6.4Hz,6H),0.88(t,J＝6.4Hz,3H),0.78(d,J＝6.4Hz,6H)。
实施例2.28：(S)-6-(二甲基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.19(40mg，0.06mmol)溶于N,N-二甲基甲酰胺(1.5mL)，依次加入HBTU(26mg，0.06mmol)、B1.14(36mg，0.06mmol)和DIPEA(66mg，0.17mmol，室温搅拌反应1小时。LCMS检测反应完成，反应液通过制备(乙腈/0.05％三氟乙酸水溶液)分离纯化得到目标化合物(S)-6-(二甲基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(13.88mg，TFA salt收率16％)为米黄色固体。
LCMS(ESI)[M+H]+＝1113.7；
1H NMR(400MHz,DMSO-d6)δ9.46(s,2H),9.29(s,1H,TFA salt),8.92(s,1H),8.63(t,J＝6.6Hz,1H),8.18(br s,1H),8.03(d,J＝7.6Hz,1H),7.81(d,J＝8.3Hz,1H),7.59(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.42(s,2H),5.24(s,2H),4.64–4.55(m,2H),4.46(t,J＝7.0Hz,2H),4.28–4.20(m,1H),4.15–4.09(m,1H),3.76–3.71(m,4H),3.53–3.48(m,4H),3.44(s,3H),3.10–3.08(m,2H),2.97–2.95(m,2H),2.75(d,J＝4.9Hz,6H),2.20–2.09(m,2H),1.92–1.83(m,5H),1.68–1.65(m,1H),1.63–1.50(m,5H),0.90–0.74(m,9H)。
实施例2.29：(S)-6-(二乙基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.21(58mg,0.09mmol),化合物B1.14(50mg,0.09mmol)溶于DMF(1mL),然后再加入HBTU(35mg,0.09mmol)和N,N-二异丙基乙胺(30mg,0.23mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFAin water,MeCN)纯化得到目标化合物(S)-6-(二乙基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(14mg,收率13％)为黄色固体。
LCMS(ESI)[M+H]+＝1141.7；
1H NMR(400MHz,DMSO)δ9.46(s,2H),8.98(s,1H,TFA),8.93(s,1H),8.64(t,J＝6.5Hz,1H),8.18(d,J＝5.7Hz,1H),8.03(d,J＝7.4Hz,1H),7.81(d,J＝8.5Hz,1H),7.58(s,1H),7.50(d,J＝3.1Hz,1H),7.24(s,1H),6.52(s,1H),6.28(s,2H),5.42(s,2H),5.23(s,2H),4.62–4.55(m,2H),4.46(t,J＝6.9Hz,2H),4.27–4.23(m,1H),4.11–4.09(m,1H),3.74(d,J＝5.8Hz,2H),3.49(t,J＝5.8Hz,2H),3.44(s,3H),3.13–3.06(m,6H),3.01–2.93(m,2H),2.22–2.07(m,2H),1.91–1.83(m,6H),1.70–1.51(m,5H),1.33–1.21(m,6H),1.16(t,J＝7.2Hz,6H),0.87(t,J＝7.4Hz,3H),0.82–0.76(m,6H)。
实施例2.30：(S)-6-(二丙基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.23(61mg,0.09mmol),化合物B1.14(50mg,0.09mmol)溶于DMF(1mL),然后再加入HBTU(35mg,0.09mmol)和N,N-二异丙基乙胺(30mg,0.23mmol),加完后反应液室温搅拌1小 时，LCMS检测反应完成后，将反应液浓缩得粗产品，粗产品经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物(S)-6-(二丙基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(21mg,收率19％)为黄色固体。
LCMS(ESI)[M+H]+＝1169.8；
1H NMR(400MHz,DMSO)δ9.46(s,2H),9.01(s,1H,TFA),8.93(s,1H),8.64(t,J＝6.4Hz,1H),8.19(t,J＝5.4Hz,1H),8.02(d,J＝7.3Hz,1H),7.81(d,J＝8.3Hz,1H),7.58(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.28(s,2H),5.42(s,2H),5.23(s,2H),4.60–4.58(m,2H),4.46(t,J＝6.9Hz,2H),4.24(d,J＝6.0Hz,1H),4.13–4.09(m,1H),3.74(d,J＝5.7Hz,2H),3.49(t,J＝5.8Hz,2H),3.44(s,3H),3.10(br s,2H),3.02–2.95(m,6H),2.19–2.15(m,2H),1.88–1.86(m,6H),1.71–1.69(m,1H),1.61–1.56(m,8H),1.28–1.26(m,6H),0.88–0.83(m,9H),0.81–0.78(m,6H)。
实施例2.31：(S)-6-(二甲基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.19(113mg,0.19mmol),化合物B1.12(100mg,0.19mmol)溶于DMF(1mL),然后加入HBTU(72mg,0.19mmol)和N,N-二异丙基乙胺(61mg,0.48mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA的水溶液，乙腈)纯化得到目标化合物(S)-6-(二甲基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(16mg,收率8％)为白色固体。
LCMS(ESI)[M+H]+＝1101.6；
1H NMR(400MHz,DMSO)δ9.46(s,2H),8.93(s,1H),8.63(t,J＝6.3Hz,1H),8.20–8.28(m,2H),8.04(d,J＝7.2Hz,1H),7.88(d,J＝10.8Hz,1H),7.81(d,J＝8.3Hz,1H),7.31(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.63–4.54(m,2H),4.46(t,J＝ 7.0Hz,2H),4.29–4.19(m,1H),4.15–4.08(m,1H),3.73(d,J＝6.9Hz,2H),3.50(t,J＝5.8Hz,2H),3.44(s,3H),3.24–3.14(m,2H),3.01–2.91(m,2H),2.72(s,6H),2.53(s,3H),2.23–2.07(m,2H),1.93–1.84(m,6H),1.72–1.64(m,1H),1.61–1.45(m,6H),1.34–1.22(m,4H),0.87(t,J＝7.4Hz,3H),0.82–0.76(m,6H)。
实施例2.32：(S)-6-(二乙基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.21(59mg,0.10mmol),化合物B1.12(50mg,0.10mmol)，溶于DMF(1mL),然后再加入HBTU(36mg,0.10mmol)和N,N-二异丙基乙胺(31mg,0.24mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％甲酸溶液,乙腈)纯化得到目标化合物(S)-6-(二乙基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(33.8mg,收率32％)为白色固体。
LCMS(ESI)[M+H]+＝1129.7；
1H NMR(400MHz,DMSO)δ9.46(s,2H),8.93(s,1H),8.61(t,J＝6.4Hz,1H),8.29(s,1H),8.24–8.15(m,2H),8.00(d,J＝7.2Hz,1H),7.85(d,J＝10.5Hz,2H),7.31(s,1H),5.44(s,2H),5.26(s,2H),4.63–4.55(m,2H),4.46(t,J＝6.8Hz,2H),4.23–4.18(m,1H),4.11(d,J＝7.9Hz,1H),3.77–3.69(m,2H),3.50(t,J＝5.9Hz,2H),3.44(s,3H),3.17–3.15(m,3H),2.61(q,J＝6.7Hz,4H),2.22–2.10(m,2H),1.92–1.84(m,6H),1.68(d,J＝6.7Hz,1H),1.57–1.48(m,3H),1.39(d,J＝5.9Hz,2H),1.30–1.22(m,4H),0.97(t,J＝6.9Hz,6H),0.88(t,J＝7.3Hz,3H),0.82–0.77(m,6H)。
实施例2.33：(S)-6-(二丙基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物C1.23(62mg,0.10mmol),化合物B1.12(50mg,0.10mmol)溶于DMF(1mL),然后加入HBTU(36mg,0.10mmol)和N,N-二异丙基乙胺(31mg,0.24mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFAin water,MeCN)纯化得到目标化合物(S)-6-(二丙基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(35.2mg,收率32％)为黄色固体。
LCMS(ESI)[M+H]+＝1157.8；
1H NMR(400MHz,DMSO)δ9.46(s,2H),9.05(s,1H,TFA),8.93(s,1H),8.64(t,J＝6.7Hz,1H),8.20(d,J＝7.4Hz,2H),8.03(d,J＝7.2Hz,1H),7.88(d,J＝10.8Hz,1H),7.81(d,J＝8.4Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H),5.28(s,2H),4.62–4.55(m,2H),4.46(t,J＝7.0Hz,2H),4.24(m,1H),4.13–4.07(m,1H),3.74(m,2H),3.50(m,2H),3.44(s,3H),3.23–3.16(m,2H),3.01–2.95(m,6H),2.50(s,3H),2.22–2.09(m,2H),1.95–1.84(m,8H),1.67–1.56(m,9H),1.33–1.23(m,6H),0.91–0.86(m,9H),0.82–0.77(m,6H)。
实施例2.34：N-((11S,14S)-11-(4-(二乙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-034)
将化合物(S)-2-氨基-N-((3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧基-3,4,12,14-四氢-1H-吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-11-基)丙氧基)甲基)乙酰胺(B1.12，30mg,0.057mmol),N6，N6-二乙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)十六烷基-5-炔酰基)-L-丙酰基)-L-赖氨酸(C1.20，31mg，0.057mmol)溶于DMF(1mL)中。然后依次加入HBTU(22mg,0.057mmol)以及N,N-二异丙基乙胺(18mg,0.143mmol)，加完毕后反应液室温搅拌一小时。LCMS检测反应完成后，将 反应液经制备色谱(0.01％的三氟乙酸水溶液,乙腈)纯化得到目标化合物三氟乙酸盐N-((11S,14S)-11-(4-(二乙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(3.18mg,收率5％)为黄色固体。
LCMS(ESI)[M+H]+＝1058.7；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.65(t,J＝6.5Hz,1H),8.21-8.19(m,2H),8.09(d,J＝7.3Hz,1H),7.92(d,J＝8.6Hz,1H),7.88(d,J＝10.8Hz,1H),7.32(s,1H),6.53(s,1H),5.44(s,2H),5.28(s,2H),4.64–4.56(m,2H),4.29–4.22(m,1H),4.15(t,J＝7.4Hz,1H),3.74(d,J＝4.6Hz,2H),3.51–3.49(m,2H),3.24–3.18(m,2H),3.13–3.07(m,4H),3.01–2.95(m,2H),2.55(s,3H),2.40–2.31(m,3H),1.99–1.74(m,8H),1.65–1.49(m,4H),1.38–1.25(m,2H),1.16(t,J＝7.2Hz,6H),0.89-0.79(m,9H)。
实施例2.35：N-((11S,14S)-11-(4-(二丙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-035)
将化合物(S)-2-氨基-N-((3-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧基-3,4,12,14-四氢-1H-吡喃并[3'，4'：6,7]吲哚并[1,2-b]喹啉-11-基)丙氧基)甲基)乙酰胺(B1.12，30mg,0.057mmol),N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-丙酰基)-N6，N6-二丙基-L-赖氨酸(C1.22，33mg，0.057mmol)溶于DMF(1mL)中。然后再依次加入HBTU(22mg,0.057mmol)以及N,N-二异丙基乙胺(18mg,0.143mmol)，加完毕后反应液室温搅拌一小时。LCMS检测反应完成后，将反应液经制备色谱(0.01％的三氟乙酸水溶液,乙腈)纯化得到目标化合物N-((11S,14S)-11-(4-(二丙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-035)(10.65mg,收率16％)为黄色固体。
LCMS(ESI)[M+H]+＝1086.7；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.65(t,J＝6.2Hz,1H),8.28–8.16(m,2H),8.08(d,J＝6.8Hz,1H),7.92(d,J＝8.1Hz,1H),7.88(d,J＝10.9Hz,1H),7.32(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.63–4.52(m,2H),4.31–4.19(m,1H),4.19–4.08(m,1H),3.74(d,J＝4.9Hz,2H),3.50(t,J＝5.2Hz,2H),3.41(s,3H),3.24–3.18(m,2H),3.03–2.94(m,6H),2.55(s,3H),2.46–2.24(m,3H),2.05– 1.67(m,8H),1.66–1.51(m,8H),1.36–1.22(m,4H),0.91–0.80(m,15H)。
实施例2.36：N-((11S,14S)-11-(4-(二乙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-036)
将化合物C1.20(40mg,0.075mmol),化合物B1.14(41mg,0.075mmol)溶于DMF(1mL)，然后再加入HOBt(15.2mg,0.113mmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(21.5mg,0.113mmol)，然后再加入三乙胺(23mg,0.225mmol)加完后反应液室温搅拌16小时，LCMS检测反应完成后，将反应液浓缩得粗产品，粗产品经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((11S,14S)-11-(4-(二乙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(16mg,收率20％)为黄色固体。
LCMS(ESI)[M+H]+＝1070.6；
1H NMR(400MHz,DMSO-d6)δ9.13–9.08(m,2H),9.01(s,1H),8.64(t,J＝6.5Hz,1H),8.20(t,J＝5.7Hz,1H),8.09(d,J＝7.4Hz,1H),7.92(d,J＝8.4Hz,1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.29(s,2H),5.43(s,2H),5.24(s,2H),4.65–4.53(m,2H),4.26(d,J＝6.5Hz,1H),4.20–4.10(m,1H),3.74(d,J＝5.5Hz,2H),3.50(t,J＝5.8Hz,2H),3.41(s,3H),3.14–3.07(m,6H),2.98(s,2H),2.55(d,J＝7.3Hz,2H),2.41–2.29(m,2H),2.03–1.89(m,2H),1.89–1.77(m,7H),1.61–1.56(m,2H),1.32(s,2H),1.16(t,J＝7.2Hz,6H),0.91–0.78(m,9H)。
实施例2.37：N-((11S,14S)-11-(4-(二正丙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-037)
将化合物C1.22(43mg,0.074mmol),化合物B1.14(40mg，0.075mmol)，溶于N,N-二甲基甲 酰胺(1mL)，然后依次加入HBTU(28mg,0.075mmol)，N,N-二异丙基乙胺(24mg,0.187mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物的三氟乙酸盐(8.5mg,收率10％)为黄色固体。
LCMS(ESI)[M+H]+＝1098.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),9.03(s,1H),8.64(t,J＝6.4Hz,1H),8.19(t,J＝5.9Hz,1H),8.08(d,J＝7.4Hz,1H),7.92(d,J＝8.5Hz,1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),6.29(s,2H),5.42(s,2H),5.24(s,2H),4.66–4.52(m,2H),4.31–4.21(m,1H),4.19–4.10(m,1H),3.74(d,J＝5.5Hz,2H),3.49–3.48(m,2H),3.40(s,3H),3.15–3.06(m,2H),3.02–2.95(m,6H),2.59–2.52(m,3H),2.41–2.29(m,2H),2.05–1.90(m,2H),1.91–1.77(m,6H),1.63–1.57(m,6H),1.31–1.29(m,2H),0.92–0.80(m,15H).
采用本实施例中同样的操作，反应液直接经制备色谱(0.1％甲酸水溶液，乙腈)纯化，冻干，得到目标化合物的甲酸盐(32.6mg,收率39％)，为白色固体。
LCMS(ESI)[M+H]+＝1098.6；
1H NMR(400MHz,DMSO-d6)δ9.09(s,2H),8.56(t,J＝6.5Hz,1H),8.17(s,1H),8.14(t,J＝5.7Hz,1H),7.97(d,J＝7.3Hz,1H),7.89(d,J＝8.6Hz,1H),7.58(s,1H),7.50(s,1H),7.24(s,1H),6.45(s,1H),6.28(s,2H),5.48–5.35(m,2H),5.24(s,2H),4.64–4.54(m,2H),4.23–4.07(m,2H),3.79–3.66(m,2H),3.50(t,J＝5.9Hz,2H),3.40(s,3H),3.14–3.08(t,J＝8.2Hz,2H),2.59–2.52(m,2H),2.36–2.27(m,8H),2.00–1.76(m,7H),1.72–1.62(m,1H),1.58–1.48(m,1H),1.40–1.20(m,8H),0.88–0.77(m,15H).
取DL-037甲酸盐(16mg)，通过制备HPLC纯化(0.1％碳酸氢铵水，乙腈)，冻干，得游离态的目标化合物(DL-037，10mg，收率65％)，为白色固体。
LCMS(ESI)[M+H]+＝1098.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.61(t,J＝6.4Hz,1H),8.21(t,J＝5.6Hz,1H),8.04(d,J＝7.2Hz,1H),7.95(d,J＝8.5Hz,1H),7.53(s,1H),7.48(s,1H),7.22(s,1H),6.50(s,1H),6.26(s,2H),5.48–5.35(m,2H),5.16(s,2H),4.63–4.51(m,2H),4.25–4.10(m,2H),3.82–3.63(m,2H),3.49(t,J＝5.8Hz,2H),3.40(s,3H),3.06(t,J＝8.0Hz,2H),2.56–2.51(m,2H),2.41–2.23(m,8H),1.96–1.76(m,7H),1.71–1.57(m,1H),1.57–1.48(m,1H),1.37–1.21(m,8H),0.88–0.78(m,15H).
实施例2.38：(S)-6-(二丙基氨基)-N-(2-(((((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺
将化合物B1.16(30mg,0.06mmol),化合物C1.23(37mg，0.06mmol)溶于DMF(1mL)。然后再加入HBTU(21mg,0.06mmol)和N,N-二异丙基乙胺(16mg,0.12mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物(S)-6-(二丙基氨基)-N-(2-(((((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(4mg,收率6％)为黄色固体。
LCMS(ESI)[M+H]+＝1167.6；
1H NMR(400MHz,DMSO)δ9.45(s,2H),8.98(s,1H,TFA),8.92(s,1H),8.74(t,J＝6.8Hz,1H),8.25(t,J＝5.8Hz,1H),8.05(d,J＝7.3Hz,1H),7.82(d,J＝8.4Hz,1H),7.63(s,1H),7.53(s,1H),7.26(d,J＝12.0Hz,1H),7.26(s,1H),6.58–6.52(m,1H),6.50(s,1H),6.30(s,2H),5.42(s,2H),5.31(s,2H),4.74–4.67(m,2H),4.46(m,2H),4.30(m,1H),4.10(m,1H),3.77(m,2H),3.44(s,3H),3.01–2.95(m,8H),2.23–2.18(m,1H),2.13(m,1H),2.00(m,2H),1.90–1.84(m,6H),1.63–1.57(m,9H),0.88(m,9H),0.81–0.78(m,6H)。
实施例2.39：4-((S)-6-氨基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺)己酰胺基)苄基N-乙基-N-(2-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)胺基甲酸酯
步骤一：
在0℃冰浴条件下，向C1.24(150mg，0.264mmol)和三乙胺(89mg，0.88mmol)的二氯甲烷(10mL)溶液中加入N，N-琥珀酰亚胺基碳酸酯(DSC，90mg，0.352mmol)，反应在0℃氮气下反应30分钟，随后滴加化合物A1.6-A(82mg，0.176mmol)，反应在室温下反应12小时。LCMS监测反应完全。反应液减压浓缩成粗产品，经高效液相制备纯化(乙腈/水含0.05％甲酸)得到黄色油状化合物DL-039-A(27mg，收率：14.4％)为黄色油状物。
LCMS(ESI)[M+H]+＝1059.2；
步骤二：
向化合物DL-039-A(60mg，0.057mmol)的四氢呋喃(5mL)和水(1mL)的混合溶液中加入过氧单磺酸钾(Oxone，349mg，0.567mmol)，反应在室温下搅拌12小时。TLC检测反应完全。向反应液中加入水(10mL)，用二氯甲烷(10mL X 3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩得到目标化合物DL-039-B(60mg，粗品)为黄色固体。
LCMS(ESI)[M+H]+＝1091.0；
步骤三：
将化合物DL-039-B(60mg,0.055mmol)溶在二氯甲烷(1mL)溶液中，0℃下向反应液中加入二氯乙酸(1mL)，反应在室温下搅拌3小时。LCMS显示反应完成。滤液减压浓缩成粗产品，经高效液相制备纯化(乙腈/水含0.05％三氟乙酸)得到黄色固体化合物4-((S)-6-氨基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺)己酰胺基)苄基N-乙基-N-(2-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)胺基甲酸酯(2.44mg，收率3.69％)。
LCMS(ESI)[1/2M+H]+＝496.3,tR＝2.252min。
实施例2.40：4-((S)-6-氨基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)己酰胺基)苄基N-ethyl-N-(2-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)胺基甲酸酯(DL-040)
步骤一：
在0℃冰浴条件下，向C1.25(300mg，0.469mmol)和三乙胺(126mg，1.25mmol)的二氯甲烷(10mL)中加入N，N-琥珀酰亚胺基碳酸酯(DSC，160mg，0.626mmol)，反应在0℃反应30分钟，随后滴加化合物A1.6-A(145mg，0.313mmol)，然后反应在室温下反应12小时。LCMS监测反应完全。反应液减压浓缩成粗产品，经高效液相制备纯化(乙腈/水含0.05％甲酸)得到黄色油状化合物DL-040-A(70mg，收率：19.8％)为黄色油状物。LCMS(ESI)[M+H]+＝1130.1。
步骤二：
向化合物DL-040-A(70mg，0.062mmol)的四氢呋喃(5mL)和水(1mL)的混合溶液中加入过氧单磺酸钾(Oxone，381mg，0.619mmol)，反应在室温下搅拌12小时。TLC检测反应反应完全。向反应液中加入水(10mL)，用二氯甲烷(10mL x 3)萃取，有机相经饱和食盐水洗涤，无水硫酸钠干燥，抽滤，减压浓缩得到目标化合物DL-040-B(70mg，粗品)为黄色固体。LCMS(ESI)[M+H]+＝1162.6,tR＝1.710min。
步骤三：
将化合物DL-040-B(70mg,0.060mmol)溶在二氯甲烷(1mL)溶液中，0℃下向反应液中加入二氯乙酸(1mL)，反应在室温下搅拌3小时。LCMS显示反应完成。滤液减压浓缩成粗产品，经高效液相制备纯化(乙腈/水含0.05％三氟乙酸)得到黄色固体化合物4-((S)-6-氨基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)己酰胺基)苄基N-ethyl-N-(2-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)乙基)胺基甲酸酯(5.54mg，收率8.66％)。
LCMS(ESI)[1/2M+H]+＝1062.5,tR＝4.052min；
1H NMR(400MHz,DMSO-d6)δ10.22(br s,1H),9.46(s,2H),8.93(s,1H),8.22–8.03(m,1H),7.85(br s,3H,NH2-TFA),7.61–7.45(m,3H),7.32–7.26(m,1H),7.26–7.17(m,2H),6.54(s,1H),6.35(s,2H),5.91(s,2H),5.47(s,2H),5.37–5.30(m,1H),5.27–5.20(m,1H),5.10–5.00(m,1H),4.97–4.78(m,1H),4.52–4.26(m,3H),3.56–3.46(m,2H),3.44(s,3H),2.83–2.71(m,2H),2.21–2.11(m,2H),1.98–1.78(m,4H),1.67–1.48(m,6H),1.37–1.22(m,4H),1.09–0.96(m,3H),0.93–0.78(m,3H)。
实施例2.41：(S)-6-(二甲基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-041)
将化合物B1.17(30mg),化合物C1.19(37mg)溶于DMF(1mL)。然后再加入HBTU(21mg)和N,N-二异丙基乙胺(16mg),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物(S)-6-(二甲基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(11mg)。
LCMS(ESI)[M+H]+＝1099.5。
实施例2.42：(S)-6-(二乙基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-042)
将化合物B1.17(30mg),化合物C1.21(37mg)溶于DMF(1mL)。然后再加入HBTU(21mg)和N,N-二异丙基乙胺(16mg),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物(S)-6-(二乙基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(7mg)。
LCMS(ESI)[M+H]+＝1127.5。
实施例2.43：(S)-6-(二丙基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-043)
将化合物B1.17(17.5mg,0.033mmol)和化合物C1.23(22mg,0.034mmol)溶于N,N-二甲基甲 酰胺(2mL)中，然后依次加入HOBT(7mg,0.051mmol)，EDCI(10mg,0.051mmol)，DIPEA(9mg,0.068mmol)，加毕，反应液室温搅拌1小时，反应液直接经制备高效液相色谱(0.01％三氟乙酸水溶液,乙腈)纯化，制备液冷冻干燥得到目标化合物(S)-6-(二乙基氨基)-N-(2-(((((E)-3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(7mg,收率18％)为淡黄色固体产品。
LCMS(ESI)[M+H]+＝1155.5.
实施例2.44：N-((11S,14S,E)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-044)
将化合物B1.17(60mg),化合物C1.17(80mg)溶于DMF(2mL)。然后再加入HBTU(45mg)和N,N-二异丙基乙胺(35mg),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((11S,14S,E)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(21mg)。
LCMS(ESI)[M+H]+＝1028.5。
实施例2.45：N-((11S,14S,E)-11-(4-(二乙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-045)
将化合物B1.17(60mg),化合物C1.20(80mg)溶于DMF(2mL)。然后再加入HBTU(45mg)和N,N-二异丙基乙胺(35mg),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((11S,14S,E)-11-(4-(二乙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(29mg)。
LCMS(ESI)[M+H]+＝1056.5。
实施例2.46：N-((11S,14S,E)-11-(4-(二丙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-046)
将化合物B1.17(60mg),化合物C1.22(80mg)溶于DMF(2mL)。然后再加入HBTU(45mg)和N,N-二异丙基乙胺(35mg),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA in water,MeCN)纯化得到目标化合物N-((11S,14S,E)-11-(4-(二乙基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(30mg)。
LCMS(ESI)[M+H]+＝1084.2；
1H NMR(400MHz,DMSO)δ9.09(s,2H),8.73(d,J＝6.3Hz,1H),8.30–8.24(m,2H),8.05(m,1H),7.94–7.88(m,2H),7.38-7.33(m,2H),6.67–6.60(m,1H),6.53(s,1H),5.44(s,2H),5.37(s,2H),4.73(t,J＝6.2Hz,2H),4.34(d,J＝4.2Hz,2H),4.26–4.20(m,1H),4.17–4.13(m,1H),3.86–3.69(m,4H),3.40(s,3H),2.41–2.30(m,8H),2.02–1.94(m,2H),1.89–1.85(m,2H),1.82–1.77(m,2H),1.74–1.63(m,2H),1.60–1.50(m,2H),1.47–1.39(m,8H),0.90–0.86(m,6H),0.85–0.80(m,9H)。
实施例2.47：N-((11S,14S,E)-11-(4-(二丙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-047)
将化合物B1.16(45mg,0.08mmol),化合物C1.22(49mg，0.08mmol),溶于DMF(1mL)。然后再加入HBTU(32mg,0.08mmol)和N,N-二异丙基乙胺(27mg,0.2mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％三氟乙酸的水溶液,乙腈)纯化得到目标化合物N-((11S,14S,E)-11-(4-(二丙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13- 氧代-4-氧杂-6,9,12-三氮杂十六烷-1-烯-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-047，10mg)为黄色固体。
LCMS(ESI)[M+H]+＝1096.6；
1H NMR(400MHz,DMSO)δ9.10(s,2H),9.05(s,1H,TFA),8.75(t,J＝5.3Hz,1H),8.26(t,J＝6.4Hz,1H),8.11(d,J＝7.6Hz,1H),7.93(d,J＝8.4Hz,1H),7.63(s,1H),7.53(s,1H),7.26–2.24(m,2H),6.65–6.43(m,2H),6.30(s,2H),5.42(s,2H),5.30(s,2H),4.74–4.69(m,1H),4.31–4.29(m,2H),4.26-424(m,1H),4.19–4.12(m,1H),3.79–2.76(m,1H),3.40(s,3H),3.02–2.95(m,8H),2.55(m,2H),2.39–2.32(m,2H),2.01–1.93(m,2H),1.92–1.77(m,5H),1.62–1.58(m,6H),1.36–1.30(m,2H),0.91–0.83(m,15H)。
实施例2.48：N-((11S,14S)-11-(4-(二丁基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-048)
将化合物C1.26(50mg，0.08mmol)，化合物B1.12(43mg，0.08mmol)溶于N,N-二甲基甲酰胺(1.5mL)，然后依次加入DIPEA(26mg，0.21mmol)，HBTU(31mg，0.08mmol)，室温搅拌反应1小时。LCMS检测反应完成，反应液直接通过高效制备(0.01％三氟乙酸的水溶液,乙腈)分离纯化得到目标化合物(13.57mg)为黄色固体。
LCMS(ESI)[M+H]+＝1114.6；
1H NMR(400MHz,DMSO)δ9.09(s,2H),9.05(s,1H,TFA),8.64(t,J＝7.2Hz,1H),8.23–8.16(m,2H),8.07(d,J＝7.3Hz,1H),7.93–7.85(m,2H),7.32(s,1H),6.52(s,1H),5.44(s,2H),5.29(s,2H),4.61-4.59(m,2H),4.24–4.22(m,1H),4.19–4.10(m,1H),3.73–3.70(m,2H),3.50(s,3H),3.21–3.18(m,6H),3.00–2.98(m,8H),2.54(s,3H),1.97–1.78(m,7H),1.56–1.52(m,8H),1.36–1.25(m,6H),0.93–0.78(m,15H)。
实施例2.49：(S)-6-(二甲基氨基)-N-(2-(((((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-049)
将化合物B1.16(45mg,0.08mmol),化合物C1.19(50mg，0.08mmol),溶于DMF(1mL)。然后依次加入N,N-二异丙基乙胺(27mg,0.2mmol)，HBTU(32mg,0.08mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液直接经制备色谱(0.01％的甲酸水溶液，乙腈)纯化得到目标化合物(S)-6-(二甲基氨基)-N-(2-(((((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烷并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-049，3mg)为白色固体。
LCMS(ESI)[M+H]+＝1111.1；
1H NMR(400MHz,DMSO)δ9.45(s,2H),8.92(s,1H),8.69(t,J＝6.6Hz,1H),8.25(t,J＝5.9Hz,1H),8.19(s,1H,FA),7.99(d,J＝7.0Hz,1H),7.83(d,J＝8.5Hz,1H),7.64(s,1H),7.52(s,1H),7.27(m,2H),6.59–6.48(m,2H),6.29(s,2H),5.42(s,2H),5.31(s,2H),4.74–4.67(m,2H),4.44(m,2H),4.30(d,J＝4.8Hz,2H),4.21(m,1H),4.12(m,1H),3.83–3.70(m,4H),3.43(s,3H),2.25(m,2H),2.15(s,6H),2.04–1.97(m,2H),1.90–1.85(m,4H),1.55–1.51(m,3H),1.39–1.34(m,6H),0.90–0.83(m,3H),0.80(t,J＝7.2Hz,6H)。
实施例2.50：1-乙基-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙氧基)甲基)氨基)-2-氧代乙基)-4-((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)哌啶-4-甲酰胺(DL-050)
将化合物B1.12(50mg,0.10mmol),化合物C1.27(50mg，0.10mmol),溶于DMF(1mL)。然后依次加入N,N-二异丙基乙胺(30mg,0.24mmol)和HBTU(36mg,0.10mmol)，加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％FA in water,MeCN)纯化得到目标化合物1-乙基-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙氧基)甲基)氨基)-2-氧代乙基)-4-((S)-3-甲基-2-(6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺基)丁酰胺基)哌啶-4-甲酰胺(DL-050，10mg)为白色固体。
LCMS(ESI)[M+H]+＝1028.6；
1H NMR(400MHz,DMSO)δ9.08(s,2H),8.42(s,1H,FA),8.18–8.15(m,4H),7.98(t, J＝5.6Hz,1H),7.86(d,J＝10.9Hz,1H),7.30(s,1H),6.51(s,1H),5.43(s,2H),5.28(s,2H),4.72–4.63(m,1H),4.53–4.45(m,1H),4.07–4.06(m,1H),3.79–3.69(m,1H),3.58–3.55(m,2H),3.55–3.48(m,5H),3.21–3.16(m,3H),2.55(s,3H),2.66–2.64(m,2H),2.40–2.26(m,5H),1.99–1.71(m,12H),0.96(t,J＝7.1Hz,3H),0.93–0.85(m,9H)。
实施例2.51：(R)-6-(二甲基氨基)-N-(4-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)苯基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-051)和(S)-6-(二甲基氨基)-N-(4-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)苯基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-052)
将化合物A1.18(40mg，0.08mmol)和C1.19(71mg，0.12mmol)溶于N,N-二甲基甲酰胺(3mL)。再加入T3P(254mg,0.40mmol,50％w/w乙酸乙酯的混合物)，将反应液于室温下搅拌1小时。LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物(R)-6-(二甲基氨基)-N-(4-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)苯基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-051，8.1mg)和(S)-6-(二甲基氨基)-N-(4-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)苯基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-052，8.8mg)均为白色固体。
DL-051：
LCMS(ESI)[M+H]+＝1048.2,tR＝1.258min；
1H NMR(400MHz,DMSO)δ10.12(s,1H),9.40(s,2H),8.84(s,1H),8.55(d,J＝7.4Hz,1H),8.26(s,1H),8.05(d,J＝7.8Hz,1H),7.99–7.89(m,3H),7.73(d,J＝8.4Hz,1H),7.57(d,J＝8.7Hz,2H),7.36(s,1H),5.41(s,2H),5.10(s,2H),4.40(m,3H),4.22–4.10(m,1H),3.44(s,3H),2.40(s,3H),2.30–2.25(m,2H),2.24–2.20(m,2H),2.19–2.15(m,6H),2.03–1.93(m,2H),1.92–1.78(m,6H),1.72–1.66(m,1H),1.61– 1.54(m,2H),1.48–1.42(m,2H),1.30–1.26(m,2H),0.91–0.86(m,9H)。
DL-052：
LCMS(ESI)[M+H]+＝1048.2,tR＝1.283min；
1H NMR(400MHz,DMSO)δ10.34(s,1H),9.45(s,2H),8.94(s,1H),8.20(s,1H),7.99–7.85(m,4H),7.75(d,J＝7.4Hz,1H),7.59(m,2H),7.36(s,1H),6.55(s,1H),5.42(s,2H),5.10(s,2H),4.52–4.42(m,3H),4.25–4.17(m,1H),3.44(s,3H),2.41(s,3H),2.30–2.13(m,8H),2.05–1.97(m,2H),1.95–1.81(m,7H),1.60–1.55(m,2H),1.50–1.44(m,2H),1.33–1.28(m,4H),0.95–0.83(m,9H)。
三、抗体制备与鉴定
实施例3、抗Napi2b抗体制备
3.1抗原的制备
1)抗原制备：人Napi2b(h.Napi2b)的氨基酸序列参考UniProt蛋白数据库中的O95436，其第二个胞外环为234aa-362aa；人Napi2a的氨基酸序列参考UniProt蛋白数据库中的Q06495；人Napi2c的氨基酸序列参考UniProt蛋白数据库中的Q8N130。食蟹猴Napi2b(c.Napi2b)的氨基酸序列参考UniProt蛋白数据库中的A0A2K5UHY1，其第二个胞外环为234aa-362aa。大鼠Napi2b(r.Napi2b)的氨基酸序列参考UniProt蛋白数据库中的Q9JJ09，其第二个胞外环为235aa-363aa。以上氨基酸序列经由密码子优化，全长序列合成于慢病毒载体pLVX上或者pCDNA3.4载体上，第二个胞外环序列合成于pTT5-hFc(hFc:SEQ ID No：85)及pTT5-mFc(mFc:SEQ ID No：86)上。
将包含第二个胞外环的pTT5-hFc或pTT5-mFc质粒通过PEImax(Polysciences，24765-1)瞬时转染HEK293-EBNA细胞，表达7天后通过ProA填料(GE，Mabselect XL)纯化获得人(h.)Napi2b-mFc/Napi2b-hFc、食蟹猴(c.)Napi2b-mFc/Napi2b-hFc及大鼠(r.)Napi2b-mFc/Napi2b-hFc抗原。
2)细胞系构建：通过慢病毒包装***制备病毒，获得病毒后感染CHO细胞、HT29细胞或HEK293T细胞，通过嘌呤霉素筛选，获得分别过表达人Napi2a/b/c的稳定细胞系，分别为CHO-h.Napi2b细胞、HT29-h.Napi2b细胞、HEK293T-h.Napi2a细胞、HEK293T-h.Napi2c细胞。
3.2小鼠免疫及免疫效价检测
选用CD1(维通利华)、KM(维通利华)、Balb/c(集萃药康)品系各4只小鼠，均为雌性，6周龄左右入组。采用CHO-h.Napi2b细胞及pCDNA3.4-h.Napi2b质粒进行一次初始免疫和两次加强免疫，结束后对小鼠眼眶静脉采血。
2次加强免疫过后，通过ELISA检测血清效价。检测方法如下：实验前一晚用CBS包被液包被人Napi2b-hFc抗原，包被浓度1ug/mL，体积100uL，4℃包被过夜；第二天去除包被液，每孔用300uL PBS洗一次，每孔加入100uL 2％BSA(Biotopped，A6020)(in PBS)，37℃封闭2小时；用2％BSA(in PBS)稀释血清，500倍起始，3倍稀释，11个浓度点，稀释后取100uL加入板中，37℃孵育2小时；去除上清，每孔用300uL PBST洗三次，每孔加入100uL抗小鼠HRP二抗(Jackson，115-035-003)(in 2％BSA)，37℃孵育1小时；去除二抗，每孔用300uL PBST洗五次，随后每孔加入100uL TMB底 物(湖州英创，TMB-S-004)，显色5-20分钟；每孔加入50uL 2N HCl终止反应，酶标仪(厂家：MD，型号：SpectraMax)读取OD450nM读数，导入到GraphPad软件进行拟合，根据检测结果对CD1、KM、Balb/c各选取一只小鼠进行杂交瘤融合。
3.3杂交瘤融合筛选
杂交瘤融合：取小鼠脾脏、***制备细胞悬液，与SP2/0小鼠骨髓瘤细胞1：1比例进行混合，采用细胞电融合缓冲液进行重悬，使用BTX-ECM2001进行电融合反应。完全融合培养基(RPMI160+15％FBS+1×HAT)重悬，2万-2.5万每孔分至96孔细胞培养板中，37℃、5％CO2培养7天后进行筛选。共进行2轮融合，第一轮融合85板，第二轮融合40板。
母克隆筛选：通过人Napi2b-hFc蛋白进行ELISA初筛，实验方法如上所述，初筛后挑选出阳性杂交瘤克隆。通过包被人、食蟹猴、大鼠Napi2b-hFc进行ELISA复筛，并采用Napi2b阳性肿瘤细胞OVCAR3进行细胞ELISA复筛。最后选出阳性克隆用于亚克隆。通过上述方法，第一轮融合选出22个母克隆进行亚克隆，第二轮融合选出16个母克隆进行亚克隆。
亚克隆筛选：先后通过包被人、食蟹猴、大鼠Napi2b-hFc进行ELISA筛选及通过进行细胞ELISA筛选，实验方法如上文所述。每板选取1-3个共43个阳性克隆用无血清培养基放大培养。
3.4鼠源抗体评价和序列钓取
采用无血清培养基培养杂交瘤细胞，收集的单克隆上清，通过ProA填料进行纯化，获得鼠源抗体。先后通过包被人Napi2b-hFc、食蟹猴Napi2b-hFc、大鼠Napi2b-hFc及OVCAR3进行ELISA和细胞ELISA评价，实验方法如上文所述，鼠源抗体按照10ug/mL起始，3倍稀释，11个浓度点，共评价39个鼠源抗体。部分高亲和力的单克隆鼠源抗体进行Elisa和细胞亲和力测定结果如表1所示。
表1.部分鼠源抗体的亲和力测定
对选取亲和力高的单克隆钓取序列，方法为：候选杂交瘤细胞收集约1×105个，通过Trizol提取RNA，然后使用PrimeScript RT reagent试剂盒通过PolyA进行反转录获得cDNA；分别于重链、轻链上游设计上游引物，重链CH1区域、轻链CL区域设计下游引物。通过PCR扩增产物，通过琼脂糖胶回收试剂盒进行片段回收；送样北京擎科生物技术有限公司进行测序。其中5克隆号为16G5B3、60A12B3、22H9C4、66C12D12和34F2A10，对它们的鼠源抗体16G5B3、60A12B3、22H9C4、66C12D12和34F2A10分别测序，鼠源抗体可变区及CDR具体序列见序列信息表。
3.5抗体人源化
采用CDR grafting的方法，首先将通过常规BLAST方法，找到同原始鼠源序列同源性最高的人germline序列，作为模板；将鼠源抗体的CDR移植到人源模板上，构建成嵌合体；根据结构分析鼠源抗体中能保留其原始构象的FR氨基酸，将嵌合体中相应的氨基酸回复突变为鼠源氨基酸以保持原有亲和力；将构建的人源化抗体进行计算及免疫原性分析，找到高免疫原性片段，进行低免疫原性片段的替换。对鼠源抗体16G5B3进行人源化获得人源化可变区16G5B3-hz1和16G5B3-hz2。对鼠源抗体60A12B3、22H9C4、66C12D12和34F2A10分别进行人源化获得人源化可变区60A12B3-hz1、22H9C4-hz1、66C12D12-hz1和34F2A10-hz1。各人源化抗体可变区及CDR具体序列见序列信息表，其中CDR按IMGT和Kabat定义方式提供。
3.6抗Napi2b人源化抗体表达
将各人源化抗体的重链可变区分别与重链IgG1恒定区(SEQ NO:70)连接，各人源化抗体的轻链可变区分别与Kappa恒定区(SEQ NO:80)连接，抗体C1(简称C1，重、轻可变区序列来自于IMGT，ID：478)的重轻链可变区分别与IgG1恒定区(SEQ NO:70)、Kappa恒定区(SEQ NO:80)连接)，连接获得的氨基酸序列提交给通用生物进行基因合成，进行密码子优化后，构建到PTT5载体上，质粒合成好后，通过PEImax转染HEK293E细胞，表达7天左右，离心收集上清。上清采用ProA填料进行纯化。纯化好的抗体全部超滤到PBS缓冲液，测定浓度，-20度保存。
人源化可变区16G5B3-hz1、16G5B3-hz2，60A12B3-hz1、22H9C4-hz1、66C12D12-hz1和34F2A10-hz1连接后表达获得的抗体分别命名为16G5B3-hz1抗体、16G5B3-hz2抗体，60A12B3-hz1抗体、22H9C4-hz1抗体、66C12D12-hz1抗体和34F2A10-hz1抗体。各人源化抗体恒定区及重轻链序列见序列信息表。
实施例4、抗Napi2b抗体的鉴定
4.1人源化抗体ELISA亲和力评价
1)ELISA法：按1ug/ml用CBS包被抗原蛋白h.Napi2b-mFc、r.Napi2b-mFc及c.Napi2b-mFc，2％BSA(in PBS)37℃封闭2小时，加入梯度稀释的抗体(10ug/mL起始，3倍稀释，11个浓度点)，37℃孵育2小时，加入HRP标记的抗人二抗(Jackson，109-035-088)，37℃孵育1小时，加入TMB底物显色，用2M HCl终止后上机读取450nM吸光值。
2)细胞ELISA法：胰酶消化离心收集OVCAR3细胞，培养基重悬细胞，每孔5^104个细胞铺至96孔板中过夜，4％多聚甲醛固定细胞，2％BSA(in PBS)37℃封闭2小时，加入梯度稀释的抗体(10ug/mL起始，3倍稀释，11个浓度点)，37℃孵育2小时，加入HRP标记的抗人二抗(Jackson，货号109-035-088)，37℃孵育1小时，加入TMB底物显色，用2M HCl终止后上机读取450nM吸光值。
实验结果如表2所示，16G5B3-hz2抗体、22H9C4-hz1抗体、66C12D12-hz1抗体与人、大鼠、食蟹猴Napi2b蛋白及内源性表达Napi2b的OVCAR3细胞均具有较高亲和力。
表2.人源化抗体ELISA亲和力评价


*：/代表未检测
4.2人源化抗体流式亲和力评价
胰酶消化离心收集OVCAR3细胞，预冷PBS洗三次，1％BSA(in PBS)重悬细胞，每孔1^105个细胞铺至96孔尖底板中，加入梯度稀释抗体(20ug/mL起始，4倍稀释，8个浓度点)，4℃孵育1小时，预冷PBS洗三次后每孔加入1uL抗人APC荧光二抗(BioLegend，货号410712)，4℃孵育0.5小时，预冷PBS洗三次后重悬细胞流式细胞仪(Sony，LE-SA3800GA)检测。
实验结果如图1所示，16G5B3-hz2抗体、66C12D12-hz1抗体与OVCAR3细胞均具有较高亲和力，EC50分别为560.9ng/ml和957.9ng/ml，且均优于C1抗体。
4.3人源化抗体动态亲和力评价
通过ForteBio测定人源化抗体的动态亲和力。实验步骤如下：1.传感器准备：取出ProA传感器，用PBST稀释液(PH7.4)将传感器预湿10min；2.样品稀释：将需要固化的抗体用生物素标记后稀释至5ug/ml，抗原h.Napi2b-mFc和c.Napi2b-mFc从500nM起始，2倍稀释，5个浓度点，设置0浓度点；3.设置程序，放入传感器板和样品板，开始程序，传感器用20mM甘氨酸溶液(pH 1.7)再生；4.使用Octet分析软件分析数据，实验结果如表3所示，16G5B3-hz2抗体、66C12D12-hz1抗体与人、猴Napi2b均有较高亲和力。
表3.人源化抗体动态亲和力检测
4.4人源化抗体非特异性评价
胰酶消化离心收集HEK293T-h.Napi2a、HEK293T-h.Napi2c细胞，预冷PBS洗三次，1％BSA(in PBS)重悬细胞，每孔3^105个细胞铺至96孔尖底板中，加入50ug/mL待检测抗体4℃孵育1小时，预冷PBS洗三次后每孔加入1uL抗人APC荧光二抗，4℃孵育0.5小时，预冷PBS洗三次后重悬细胞，流式细胞 仪上机检测。
实验结果如表4所示，16G5B3-hz2抗体、22H9C4-hz1抗体及66C12D12-hz1抗体均不会非特异识别人Napi2a、Napi2c。
表4.人源化抗体非特异性结合检测
4.5人源化抗体内吞活性评价
胰酶消化离心收集HCC4006细胞(南京科佰)，用浓度为10ug/mL的待检测抗体4℃孵育1小时，PBS洗3次，用RPMI 1640+10％FBS重悬细胞，分成3份分别在37℃孵育0、2、4小时，PBS洗三次后加入1uL抗人APC荧光二抗，4℃孵育0.5小时，PBS洗三次后重悬细胞上机。按照以下公式计算内吞率：内吞率(％)＝【1-(该时间点检测样品平均荧光值-该时间点阴性对照样品平均荧光值)/(0小时检测样品平均荧光值-0小时阴性对照样品平均荧光值)】*100。
实验结果如表5和图2所示，16G5B3-hz2抗体、66C12D12-hz1抗体有很强的内吞活性，并且随着时间延长，内吞率也增加。
表5.人源化抗体内吞率检测
4.6人源化抗体血清稳定性评价
1)样品处理：用人血清将人源化候选抗体16G5B3-hz2、66C12D12-hz1稀释至20ug/mL，0.22um滤器过滤除菌，每个候选设置3个时间点分别为第1天、8天、15天，每管500uL，封口膜封口后置于37℃培养箱，分别于第1天、8天、15天收样至-80℃冰箱保存。
2)ELISA法：按1ug/ml用CBS包被抗原蛋白h.Napi2b-mFc，2％BSA(in PBS)37℃封闭2小时，加入梯度稀释的抗体，37℃孵育2小时，加入HRP标记的抗人二抗，37℃孵育1小时，加入TMB底物显色，用2M HCl终止后上机读取450nM吸光值。
实验结果如图3A和3B所示，16G5B3-hz2抗体与66C12D12-hz1在血浆中第1天、8天、15天亲和力无明显变化，具有良好的的血清稳定性。
四、包含细胞生物活性分子和连接体的化合物与抗体的偶联
实施例5：Napi2b-ADC的制备
实施例5.1 Napi2b-ADC-07的制备
其中Tb为Napi2b抗体(包括下述的66C12D2-hz1，16G5B3-hz2，C1等)，q为1、2、3、4、5、6、7、8、9或10.
5.1.1.Napi2b-ADC-07(DAR8)样品制备
将抗Napi2b抗体66C12D2-hz1 30mg，用稀释液(20mM PB，pH 7.5)稀释，加入终浓度为1mM的依地酸钠溶液，混匀；加入抗体5.75倍当量的TCEP溶液，混匀，室温放置90分钟；向上述溶液体系加入抗体12倍当量的溶解于二甲基亚砜中的DL-037，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为6.0的20mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有抗Napi2b抗体66C12D2-hz1ADC组合物的溶液66C12D2-hz1-ADC07(DAR8)。利用质谱法测定DAR值，
色谱条件：色谱柱：PLRP-S，2.1*50mm,5μm；流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN；柱温：30℃样品室温度：8℃流速：0.6ml/min进样量：2μl
表6.流动相梯度
样品处理：分别取样品50μg，加入1M DTT 2μl，加超纯水至50μl稀释至约1.0mg/ml浓度，混匀，室温还原30min。
LC/MS型号：Agilent 1290-6545XT Q-TOF
质谱条件：Gas temp：320℃，Drying Gas：Nebulizer：35psi；Sheath Gas Temp：350℃；sheath Gas Flow：11l/min；m/z 500～3000。
结果如下所示：
表7 66C12D2-hz1-ADC07(DAR8)理论分子量及实测分子量

表中，mAb表示未偶联的单克隆抗体；LC代表抗体轻链；HC代表抗体重链；DAR1代表包含轻链或重链偶联1个毒素分子的偶联物；DAR2代表包含轻链或重链偶联2个毒素分子的偶联物；DAR3代表包含轻链或重链偶联3个毒素分子的偶联物；其中，单抗理论分子量以G0F糖型计算。下文中mAb、LC、HC、DAR1、DAR2、DAR3如上说明。
66C12D2-hz1-ADC07(DAR8)样品轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，1％，1％，98％)，由此计算66C12D2-hz1-ADC07(DAR8)样品偶联比(DAR值)为8.0。根据重轻链偶联不同个数毒素分子的情况可推知，q可为4,5,6,7,8。
利用上述相同的方法，将66C12D2-hz1替换为16G5B3-hz2，获得含有抗Napi2b抗体16G5B3-hz2ADC组合物的溶液16G5B3-hz2-ADC07(DAR8)。
5.1.2 Napi2b-ADC-07(DAR6)样品制备
将抗Napi2b抗体16G5B3-hz2 30mg，用稀释液(20mM PB，pH 7.5)稀释，加入终浓度为1mM的依地酸钠溶液，混匀；加入抗体5.75倍当量的TCEP溶液，混匀，室温放置90分钟；向上述溶液体系加入抗体7.0倍当量的溶解于二甲基亚砜中的DL-037，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为6.0的20mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有抗Napi2b抗体16G5B3-hz2ADC组合物的溶液16G5B3-hz2-ADC07(DAR6)。采用7.1.1中相同质谱法测定结果如下
表8. 16G5B3-hz2-ADC07(DAR6)理论分子量及实测分子量
16G5B3-hz2-ADC07(DAR6)样品轻链偶联0～1个毒素分子(LC，DAR1比例分别为18％，82％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，11％，28％，60％)，由此计算16G5B3-hz2-ADC07(DAR6)样品DAR值为6.6。根据重轻链偶联不同个数毒素分子的情况可推知，q可为2,3,4,5，6,7,8。
利用上述相同的方法，将16G5B3-hz2替换为66C12D2-hz1，获得含有抗Napi2b抗体66C12D2-hz1ADC组合物的溶液66C12D2-hz1-ADC07(DAR6)。
5.1.3 Napi2b-ADC-07(DAR1)样品制备
将抗Napi2b抗体16G5B3-hz2 30mg，用稀释液(20mM PB，pH 7.5)稀释，加入终浓度为1mM的依地酸钠溶液，混匀；加入抗体5.75倍当量的TCEP溶液，混匀，室温放置90分钟；向上述溶液体系加入 抗体1.5倍当量的溶解于二甲基亚砜中的DL-037，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为6.0的20mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有抗Napi2b抗体16G5B3-hz2ADC组合物的溶液16G5B3-hz2-ADC07(DAR1)。采用7.1.1中相同质谱法测定结果如下：
表9 16G5B3-hz2-ADC07(DAR1)理论分子量及实测分子量
16G5B3-hz2-ADC07(DAR1)样品轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为56％，32％，12％，0％)，由此计算16G5B3-hz2-ADC07(DAR1)样品偶联比(DAR值)为1.1。根据重轻链偶联不同个数毒素分子的情况可推知，q可为2,3，4,5,6,7,8。
利用上述相同的方法，将16G5B3-hz2替换为66C12D2-hz1，获得含有抗Napi2b抗体66C12D2-hz1ADC组合物的溶液66C12D2-hz1-ADC07(DAR1)。
实施例5.2：Napi2b-ADC-04的制备
其中Tb为Napi2b抗体，q为1、2、3、4、5、6、7、8、9或10.
利用5.1.1相同的方法，将DL-037替换为DL-019，制备有抗Napi2b抗体-ADC组合物的溶液。
实施例5.3：Napi2b-ADC-06的制备
其中Tb为Napi2b抗体，q为1、2、3、4、5、6、7、8、9或10.
利用5.1.1相同的方法，将DL-037替换为DL-036，制备有抗Napi2b抗体-ADC组合物的溶液。
五、生物活性分子体外生物活性测定
实施例6.1：生物活性分子对体外细胞活性的抑制活性检测一
首先培养肿瘤细胞；将本公开的生物活性分子与肿瘤细胞进行共培养，然后添加CCK8试剂(东仁化学科技有限公司，Cat:CK04,Lot:JJ744)，用酶标仪(厂家：Molecular Devices,型号：SpectraMax M2)读数(检测波长为450nm)，检测线粒体内的脱氢酶的活性，以评价生物活性分子对细胞增殖的抑制作用。肿瘤细胞来源见表10。
表10.
体外细胞活性检测方法：用对应检测培养基(含2％FBS)稀释生物活性分子(12个浓度梯度)。使用胰酶通过常规方法对肿瘤细胞进行消化，收集管细胞数，用对应的检测培养基(含2％FBS)重悬。将稀释的生物活性分子加入96孔板中，再加入细胞。然后每孔加入CCK8试剂20μL，反应至4小时，酶标仪读数(检测波长为450nm)。实验结果如表11.1,11.2,11.3,11.4,11.5和11.6所示。
参照CN104755494B制备获得DXD，具体结构如下，
表11.1生物活性分子对NCl-H358细胞杀伤结果



表11.2生物活性分子对HT29细胞杀伤结果


表11.3生物活性分子对LS174T细胞杀伤结果
表11.4生物活性分子对NCl-H322M细胞杀伤结果
表11.5生物活性分子对PANC-1细胞杀伤结果
表11.6生物活性分子对HCC1806细胞杀伤结果
测试结果表明，本公开的生物活性分子具有肿瘤细胞杀伤作用。
实施例6.2：生物活性分子对体外细胞活性的抑制活性检测二
首先培养肿瘤细胞NUCG-4，PC-9，HT29，NCI-H358，KYSE520，A431和A549；将本公开的生物活性分子或片段与肿瘤细胞进行共培养，然后添加CCK8试剂(东仁化学科技有限公司，Cat:CK04,Lot:JJ744)，用酶标仪(厂家：Molecular Devices,型号：SpectraMax M2)读数(检测波长为450nm)，检测线粒体内的脱氢酶的活性，以评价生物活性分子对细胞增殖的抑制作用。肿瘤细胞来源见表12。
表12.肿瘤细胞来源
体外细胞活性检测方法：使用胰酶通过常规方法对肿瘤细胞进行消化，收集管细胞数，用对应的检测培养基(含2％FBS)重悬，将5000-10000细胞/孔加至96孔板。用DMSO稀释的生物活性分子，加入100uL至96孔板，浓度从10μM开始，3倍稀释(12个浓度梯度)，37℃5％CO2培养3-4天，然后每孔加 入CCK8试剂20μL，反应至2-6小时，酶标仪读数(检测波长为450nm)。生物活性分子及其检测结果如表13所示。
表13 A1.9和B1.14的活性测定
测试结果表明，生物活性分子A1.9和B1.14具有肿瘤细胞杀伤作用，其中A1.9在多个肿瘤细胞中均具有强于Dxd的肿瘤杀伤作用。A1.9在KYSE520的体外细胞活性的抑制活性检测中，EC50为49.62nM，具有杀伤KYSE520的作用。
实施例7：抗Napi2b抗体药物偶联物体内活性测试
7.1 Napi2b抗体药物偶联物对HT29人结肠癌移植瘤药效测试
为验证抗人Napi2b ADC药物的体内药效，评价受试药物在皮下异种移植雌性Balb/c Nude小鼠模型中的抗肿瘤作用，购买5-6周龄的雌性Balb/c Nude小鼠(维通利华)，将生长至对数生长期的HT29-h.Napi2b细胞采用EDTA消化及PBS重悬，每只小鼠皮下接种5*106细胞，待肿瘤生长至100-200m3大小，采用静脉给药，每周一次，每次1mg/kg或3mg/kg，具体方案如下表。本实验的主要观察指标为：1)TGI(％)，计算公式为：TGI(％)＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积)2)试验终点肿瘤体积大小照片、肿瘤重量。
实验结果如表14、图4所示，16G5B3-hz2-ADC07(DAR8)和66C12-hz1-ADC07(DAR8)，在小鼠体内均有良好的抑瘤效果，3mg/kg剂量下TGI与C1-ADC07(DAR8)相当，1mg/kg剂量下两者均优于C1-ADC07(DAR8)，其中66C12-hz1--ADC07(DAR8)在1mg/kg下TGI达到了97.83％，明显优于另外两者。小鼠体重变化情况如图5所示，表明整个给药过程中2个ADC对小鼠体重无影响。
表14.抗人Napi2b ADC体内药效-HT29-h.Napi2b CDX模型

7.2 Napi2b抗体药物偶联物对IGR-OV1人源卵巢癌移植瘤药效测试
用相似的方法评价测试药物66C12D2-hz1-ADC07(DAR8)在人源卵巢癌IGR-OV1细胞株皮下异种移植雌性BALB/c裸小鼠动物模型中的抗肿瘤作用。实验结果如表15、图6所示，66C12D2-hz1-ADC07(DAR8)在小鼠体内有良好的抑瘤效果，3mg/kg剂量下TGI与C1-ADC07(DAR8)相当，1mg/kg剂量下均优于C1-ADC07(DAR8)。小鼠体重变化情况如图7所示，表明整个给药过程中66C12D2-hz1-ADC07(DAR8)对小鼠体重无影响。
表15.抗人Napi2b ADC体内药效-IGR-OV1CDX模型
从而本公开ADC有更宽的治疗指数和治疗效果。尽管本公开的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本公开的保护范围之内。本公开的保护范围由所附权利要求及其任何等同物给出。

Claims (42)

  1. 式XV所示的抗体药物偶联物，
    或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，
    其中：
    Tb为抗NaPi2b的抗体或其抗原结合片段；
    q为药物抗体偶联比；
    D为生物活性分子片段；
    L1为延伸单元；
    L2不存在或为连接单元；
    L3选自Val-AA1-Gly、Val-AA1、Ala-AA1、Gly-AA1、AA1-Gly、Ala-AA1-Gly、Gly-AA1-Gly、AA1-Ala-Asn、AA1；所述AA1所示氨基酸残基的结构如下所示，
    其中：Ra、Rb各自独立地选自H、且Ra、Rb不同时为H；
    或者，Ra与Rb和与它们共同相连的碳原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0所取代；
    r、r1各自独立地选自0到20的任意整数；
    Rm1、Rn1各自独立地选自H、C1-6烷基、C3-6环烷基和-COORx1；
    Rx1选自C1-6烷基；
    或者，Rm1与Rn1和与它们共同相连的氮原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0’所取代；
    Rz选自C1-6烷基；
    R0、R0’各自独立地选自C1-6烷基、C3-6环烷基、-NRm2Rn2和任选被C1-6烷基取代的4-10元杂环基；
    Rm2、Rn2各自独立地选自H和C1-6烷基；
    L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连。
  2. 如权利要求1所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)Tb所述抗体或其抗原结合片段包括Fab、Fab'、F(ab')2、Fd、Fv、dAb、互补决定区片段、非人抗体、人源化抗体、嵌合抗体、全人抗体、前抗、单克隆抗体、双特异性抗体或多特异性抗体；
    (2)q选自0.1-16.0之间的任意数值；
    (3)D为具有抗肿瘤生物活性分子片段。
  3. 如权利要求2所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)q选自0.1、1、2、3、4、5、6、7、8、9、10、11、12和之间的任意数值；
    (2)所述的生物活性分子片段中，所述的生物活性分子为DNA拓扑异构酶抑制剂或微管蛋白抑制剂；
    (3)L1选自 各Z独立地选自直接键、碳碳三键、碳碳双键、C6-10芳基、5-10元杂芳基和酰胺基；Rx、Ry各自独立地选自H和C1-4烷基；各m独立地选自0、1、2、3、4、5和6；y1选自1-6之间任意整数；各y2独立地选自0-15之间任意整数；各y3独立地选自1、2和3；各y4独立地选自0和1；1位通过S原子与Tb相连，2位与L2或L3相连；
    (4)L2不存在或存在，L2存在时，L2选自 y1选自1-6之间任意整数，各y2独立地选自0-10之间任意整数，各y3独立地选自1和2，各y4独立地选自0和1，1位与L1相连，2位与L3相连；
    (5)AA1的氨基酸残基中，Ra、Rb中，任一个为H，另一个选自
    或者，AA1的氨基酸残基中，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的5-6元杂环；
    (6)AA1的氨基酸残基中，r、r1各自独立地选自0、1、2、3、4和5；
    (7)AA1的氨基酸残基中，Rm1、Rn1各自独立地选自H、甲基、乙基、正丙基、正丁基、-COOCH3、-COOCH2CH3、-COOCH2CH2CH3、-COOCH(CH3)2、-COOC(CH3)3和-COOCH2CH2CH2CH3；或者，Rm1与Rn1和与它们共同相连的氮原子一起，形成被R0’取代的5-6元杂环；
    (8)AA1的氨基酸残基中，Rz为甲基；
    (9)AA1的氨基酸残基中，R0、R0’各自独立地选自C1-6烷基、-NRm2Rn2和任选被C1-6烷基取代的5-6元杂环基。
  4. 如权利要求3所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)q选自0.1、1、2、3、4、5、6、7、8和之间的任意数值；
    (2)当所述的生物活性分子片段中的生物活性分子为DNA拓扑异构酶抑制剂时，所述的DNA拓扑异构酶抑制剂为喜树碱类生物活性分子；
    (3)当所述的生物活性分子片段中的生物活性分子为微管蛋白抑制剂时，所述的微管蛋白抑制剂为MMAF类微管蛋白抑制剂，MMAE类微管蛋白抑制剂；
    (4)L1中，各Z独立地选自直接键、碳碳三键和碳碳双键；
    (5)L1中，y1为4、5或6；
    (6)L1中，各y2独立地选自6-10之间任意整数；
    (7)L2不存在或存在，L2存在时，L2选自 1位与L1相连，2位与L3相连；
    (8)AA1所示氨基酸残基中，Ra、Rb中，任一个为H，另一个选自或，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的5-6元杂环为被R0取代的哌啶环或哌嗪环；
    (9)AA1的氨基酸残基中，r、r1各自独立地选自0和4；
    (10)AA1的氨基酸残基中，Rm1、Rn1各自独立地选自H、C1-6烷基、C3-6环烷基和叔丁氧羰基；或者，Rm1与Rn1和与它们共同相连的氮原子一起，形成被R0’取代的哌啶环或哌嗪环；
    (11)AA1的氨基酸残基中，R0选自C1-6烷基和被C1-6烷基取代的5-6元杂环基，所述5-6元杂环基选自哌啶基和哌嗪基；
    (12)AA1的氨基酸残基中，R0’选自C1-6烷基和-NRm2Rn2；
    (13)AA1的氨基酸残基中，Rm2、Rn2为甲基；
    (14)L4不存在或存在，L4存在时，L4为1位与L3相连，2位与D相连。
  5. 如权利要求1-4中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)q选自2、3、4、5、6、7、8和之间的任意数值；
    (2)L1选自 m选自2、3和4，y1选自1-6之间任意整数，各y2独立地选自0-10之间任意整数，各y3独立地选自1或2，1位通过S原子与Tb相连，2位与L2或L3相连；
    (3)L3选自AA1、AA1-Gly、Val-AA1-Gly、AA1-Ala-Asn；
    (4)AA1所示氨基酸残基中，Ra与Rb和与它们共同相连的碳原子一起，形成被R0取代的5-6元杂环为被R0取代的哌啶环；
    (5)AA1的氨基酸残基中，r、r1中，任一个为0，另一个为4；
    (6)AA1的氨基酸残基中，Rm1、Rn1各自独立地选自H和C1-6烷基；或，Rm1与Rn1和与它们共同相连的氮原子一起，形成被R0’取代的哌啶环；
    (7)AA1的氨基酸残基中，R0选自甲基、乙基和被甲基取代的5-6元杂环基，所述5-6元杂环基为哌 啶基；优选地，R0选自甲基、乙基和
    (8)AA1的氨基酸残基中，R0’选自甲基和-NRm2Rn2；
    (9)当所述的生物活性分子片段中的生物活性分子为DNA拓扑异构酶抑制剂、所述的DNA拓扑异构酶抑制剂为喜树碱类生物活性分子时，所述的喜树碱类生物活性分子为喜树碱、DXD、取代基被修饰的喜树碱或取代基被修饰的DXD。
  6. 如权利要求1-5中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)q选自3、4、5、6、7、8和之间的任意数值；优选地，q选自4、5、6、7、8和之间的任意数值；优选地，q选自6、7、8和之间的任意数值；
    (2)L1选自1位通过S原子与Tb相连，2位与L2或L3相连；
    (3)L2不存在或
    (4)L3选自AA1、Val-AA1-Gly；优选地，L3选自Val-AA1-Gly；
    (5)AA1所示氨基酸残基中，Ra与Rb和与它们共同相连的碳原子一起，形成1号碳原子为与Ra和Rb共同相连的碳原子；
    (6)r、r1中，r为4，r1为0时，Rm1、Rn1各自独立地选自H和C1-6烷基；r为0，r1为4时，Rm1、Rn1各自独立地选自C1-6烷基，或，Rm1与Rn1和与它们共同相连的氮原子一起，形成1号碳原子为与Ra和Rb共同相连的碳原子。
  7. 如权利要求1-6中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    AA1所示氨基酸残基选自
    最优选地，AA1所示氨基酸残基选自
  8. 如权利要求1-7中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    L3选自 X-选自卤素离子、羧酸根离子、硫酸根离子、硫酸氢根离子和OH-，1位与L1或L2相连，2位与L4或D相连；
    最优选地，L3选自 1位与L1或L2相连，2位与L4或D相连。
  9. 如权利要求1-8中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    的结构选自以下结构片段：
    优选地，的结构选自以下：


    其中，1位与Tb相连，2位与D相连。
  10. 如权利要求1-9中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    其中，所述抗体药物偶联物具有式I所式结构：
    其中：Tb、L1、L2、L3、L4和q的定义如权利要求1-9中任一项所述；
    R1、R2各自独立地选自H、卤素、-OH、任选取代的C1-6烷基和任选取代的C1-6烷氧基，或者，
    R1和R2和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N，羰基，亚砜基或砜基或其任意组合；
    R3选自H、卤素、-OH、-NH2、任选取代的C1-6烷基和任选取代的C1-6烷氧基，或者，
    R3和X和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N，羰基，亚砜基或砜基或其任意组合，或者，
    R3和R2和与其相连的碳原子一起形成5-7元碳环或5-7元杂环，所述杂环含有1个或多个O，S，N，羰基，亚砜基或砜基或其任意组合；
    W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
    X选自直接键，任选取代的-O-(CH2)n3-、-NR4-(CH2)n3-、-S-(CH2)n3-、羰基-(CH2)n3-、-SO2-(CH2)n3-、-(CH2)n1-、C3-6环烷基、C6-10芳基、5-10元杂芳基和4-10元杂环基，1位和母环相连，2位和W或L4相连；所述取代基选自一个或多个C1-4烷基、C3-6环烷基，或者多个C1-4烷基和与它们同时相连的碳原子一起形成C3-6环烷基；
    各M独立地选自直接键和-CR5aR5b-；
    R4、R5、R5a、R5b、R6、R7各自独立地选自H、任选取代的C1-4烷基、任选取代的C1-4烷氧基和任选取代的C3-6环烷基；
    n、n’、n1、n2、n3各自独立地选自0到6之间的任意整数；
    1位与喜树碱母核相连，2位与W或L4相连。
  11. 如权利要求10所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)R1、R2各自独立地选自H、卤素、C1-4烷基；或者，R1和R2和与其相连的碳原子一起形成5-6元杂环，所述杂环含有1个、2个或3个O，S或N或其任意组合；
    (2)R3选自H、C1-4烷基；或，R3和X和与其相连的碳原子一起形成5-6元碳环；
    (3)W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
    (4)X选自任选取代的-(CH2)n1-、C6-10芳基、5-10元杂芳基和4-10元杂环基，1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基，或者2个C1-4烷基和与它们同时相连的碳原子一起形成C3-6环烷基；
    (5)R4、R5各自独立地选自H、C1-4烷基和C3-6环烷基；
    (6)R5a、R5b各自独立地选自H和C1-4烷基；
    (7)各R7独立地选自H和C1-4烷基；
    (8)n选自1、2和3；
    (9)n1选自1、2、3和4；
    (10)n2为1；
    (10)n3为0。
  12. 如权利要求10或11所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)R1选自H和卤素，R2选自H和C1-4烷基；或，R1和R2和与其相连的碳原子形成 虚线表示所述杂环与苯环稠合的位置；
    (2)R3为H；或，R3和X和与其相连的碳原子一起形成虚线表示所述碳环与苯环和吡啶环稠合的位置；
    (3)W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
    (4)各R4独立地选自H、C1-4烷基和C3-6环烷基，R5为H；
    (5)R5a、R5b各自独立地选自H和甲基；
    (6)R7为H；
    (7)n为1。
  13. 如权利要求10-12中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一个或多个：
    (1)R1为H或F，R2为H或甲基；或，R1和R2和与其相连的碳原子一起形成虚线表示所述杂环与苯环稠合的位置；
    (2)W选自-O-、-NR4-和1位X相连，2位与L4或L3相连；
    (3)各R4独立地选自H、甲基、乙基、正丙基、异丙基、叔丁基和环丙基，R5为H；
    (4)X选自任选取代的 1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基，或者2个C1-4烷基和与它们同时相连的碳原子一起形成C3-6环烷基；所述的C1-4烷基如甲基；所述的C3-6环烷基如环丙基。
  14. 如权利要求10-13中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    (1)W选自-O-和-NR4-；
    (2)X选自 1位和母环相连，2位和W或L4相连。
  15. 如权利要求10-14中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    W不存在时，X选自1位和母环相连，2位和L4相连；W存在时，X选自 1位和母环相连，2位和W相连。
  16. 如权利要求10-15中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    的结构选自以下：


    其中，1位与L4相连；当L4不存在时，1位与L3相连。
  17. 如权利要求10-16中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，所述的抗体药物偶联物具有式I-1、式I-2或式I-3所式结构：
    其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4和q如权利要求1-16中任一项所定义；
    其中，Tb、L1、L2、L3、L4、X、R1、R2、R3和q如权利要求1-16中任一项所定义；
    其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4、R5、n和q如权利要求1-16中任一项所定义。
  18. 如权利要求10-17中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，所述抗体药物偶联物具有式I-1A、式I-1B、式I-2A、式I-2B、式I-3A或式I-3B所式结构：
    其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q如权利要求1-16中任一项所定义；
    其中，Tb、L2、L3、L4、X、R1、R2、R3和q如权利要求1-16中任一项所定义；
    其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q如权利要求1-16中任一项所定义。
  19. 权利要求10-18中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其中，所述的抗体药物偶联物具有式I-A或式I-B所式结构：
    其中，Tb、X、R1、R2、R3、Ra、Rb和q如权利要求1-16中任一项所定义；
    其中，Tb、X、R1、R2、R3、Ra、Rb和q如权利要求1-16中任一项所定义。
  20. 权利要求1-19中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其中，所述的抗体药物偶联物选自以下：


















  21. 如权利要求1-20任一所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    Tb为抗Napi2b抗体或其抗原结合片段；优选地，所述的抗Napi2b的抗体为lifastuzumab，upifitamab，60A12B3-hz1、22H9C4-hz1、66C12D12-hz1、34F2A10-hz1、C1，16G5B3-hz2和16G5B3-hz1抗体或其抗原结合片段。
  22. 一种抗Napi2b的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含如下的互补决定区CDR：
    (a)SEQ ID NO:1、17、19、34、36、52、54、71、73、74或33所示的重链可变区VH中含有的HCDR1、HCDR2、以及HCDR3；和/或
    (b)SEQ ID NO:2、18、20、35、37、53、55、72、75或51所示的轻链可变区VL中含有的LCDR1、LCDR2、以及LCDR3。
  23. 权利要求22所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (1)VH和/或VL，其中按IMGT编号***定义：
    (a)所述VH包含：序列为SEQ ID NO:11的HCDR1，序列为SEQ ID NO:12的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:15的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:10的LCDR3；
    (b)所述VH包含：序列为SEQ ID NO:11的HCDR1，序列为SEQ ID NO:13的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:15的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:10的LCDR3；
    (c)所述VH包含：序列为SEQ ID NO:28的HCDR1，序列为SEQ ID NO:29的HCDR2，序列为SEQ ID NO:31的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:32的LCDR1，序列为YTS的LCDR2，序列为SEQ ID NO:27的LCDR3；
    (d)所述VH包含：序列为SEQ ID NO:28的HCDR1，序列为SEQ ID NO:30的HCDR2，序列为SEQ ID NO:31的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:32的LCDR1，序列为YTS的LCDR2，序列为SEQ ID NO:27的LCDR3；
    (e)所述VH包含：序列为SEQ ID NO:46的HCDR1，序列为SEQ ID NO:47的HCDR2，序列为SEQ ID NO:49的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:50的LCDR1，序列为SAS的LCDR2，序列为SEQ ID NO:45的LCDR3；
    (f)所述VH包含：序列为SEQ ID NO:46的HCDR1，序列为SEQ ID NO:48的HCDR2，序列为SEQ ID NO:49的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:50的LCDR1，序列为SAS的LCDR2，序列为SEQ ID NO:45的LCDR3；
    (g)所述VH包含：序列为SEQ ID NO:65的HCDR1，序列为SEQ ID NO:66的HCDR2，序列为SEQ ID NO:68的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:69的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:64的LCDR3；
    (h)所述VH包含：序列为SEQ ID NO:65的HCDR1，序列为SEQ ID NO:67的HCDR2，序列为SEQ ID NO:68的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:69的LCDR1，序列为WAS的LCDR2，序列为SEQ ID NO:64的LCDR3；
    或
    (i)所述VH包含：序列为SEQ ID NO:76的HCDR1，序列为SEQ ID NO:77的HCDR2，序列为SEQ ID NO:78的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:79的LCDR1，序列为HTS的LCDR2，序列为SEQ ID NO:16的LCDR3；
    (2)VH和/或VL，其中按kabat编号***定义：
    (a)所述VH包含：序列为SEQ ID NO:3的HCDR1，序列为SEQ ID NO:4的HCDR2，序列为SEQ ID NO:6的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:7的LCDR1，序列为SEQ ID NO:9的LCDR2，序列为SEQ ID NO:10的LCDR3；
    (b)所述VH包含：序列为SEQ ID NO:3的HCDR1，序列为SEQ ID NO:5的HCDR2，序列为SEQ ID  NO:6的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:8的LCDR1，序列为SEQ ID NO:9的LCDR2，序列为SEQ ID NO:10的LCDR3；
    (c)所述VH包含：序列为SEQ ID NO:21的HCDR1，序列为SEQ ID NO:22的HCDR2，序列为SEQ ID NO:24的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:25的LCDR1，序列为SEQ ID NO:26的LCDR2，序列为SEQ ID NO:27的LCDR3；
    (d)所述VH包含：序列为SEQ ID NO:21的HCDR1，序列为SEQ ID NO:23的HCDR2，序列为SEQ ID NO:24的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:25的LCDR1，序列为SEQ ID NO:26的LCDR2，序列为SEQ ID NO:27的LCDR3；
    (e)所述VH包含：序列为SEQ ID NO:38的HCDR1，序列为SEQ ID NO:39的HCDR2，序列为SEQ ID NO:41的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:42的LCDR1，序列为SEQ ID NO:44的LCDR2，序列为SEQ ID NO:45的LCDR3；
    (f)所述VH包含：序列为SEQ ID NO:38的HCDR1，序列为SEQ ID NO:40的HCDR2，序列为SEQ ID NO:41的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:43的LCDR1，序列为SEQ ID NO:44的LCDR2，序列为SEQ ID NO:45的LCDR3；
    (g)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:57的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:61的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3；
    (h)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:58的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:62的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3；或
    (i)所述VH包含：序列为SEQ ID NO:56的HCDR1，序列为SEQ ID NO:59的HCDR2，序列为SEQ ID NO:60的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:62的LCDR1，序列为SEQ ID NO:63的LCDR2，序列为SEQ ID NO:64的LCDR3；
    优选地，所述抗Napi2b抗体或其抗原结合片段结合人Napi2b、猴Napi2b和/或大鼠Napi2b。
  24. 权利要求22或23所述的抗Napi2b抗体或其抗原结合片段，其中，所述抗Napi2b抗体或其抗原结合片段包含：
    (a)SEQ ID NO:1或17所示的VH，和/或，SEQ ID NO:2或18所示的VL；
    (b)SEQ ID NO:19或34所示的VH，和/或，SEQ ID NO:20或35所示的VL；
    (c)SEQ ID NO:36或52所示的VH，和/或，SEQ ID NO:37或53所示的VL；
    (d)SEQ ID NO:54、71或73所示的VH，和/或，SEQ ID NO:55或72所示的VL；
    (e)SEQ ID NO:74或33所示的VH，和/或，SEQ ID NO:75或51所示的VL；
    (f)与(a)-(e)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(a)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；或者
    (g)与(a)-(e)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(a)中的任一VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合的VL；优选地，所述的置换是保守置换。
  25. 权利要求22-24任一项抗Napi2b所述的抗体或其抗原结合片段，其中，所述抗Napi2b抗体或其抗原结合片段包含：
    (a)SEQ ID NO:1所示序列的VH和SEQ ID NO:2所示序列的VL；
    (b)SEQ ID NO:17所示序列的VH和SEQ ID NO:18所示序列的VL；
    (c)SEQ ID NO:19所示序列的VH和SEQ ID NO:20所示序列的VL；
    (d)SEQ ID NO:34所示序列的VH和SEQ ID NO:35所示序列的VL；
    (e)SEQ ID NO:36所示序列的VH和SEQ ID NO:37所示序列的VL；
    (f)SEQ ID NO:52所示序列的VH和SEQ ID NO:53所示序列的VL；
    (g)SEQ ID NO:54所示序列的VH和SEQ ID NO:55所示序列的VL；
    (h)SEQ ID NO:71所示序列的VH和SEQ ID NO:72所示序列的VL；
    (i)SEQ ID NO:73所示序列的VH和SEQ ID NO:72所示序列的VL；
    (j)SEQ ID NO:74所示序列的VH和SEQ ID NO:75所示序列的VL；
    (k)SEQ ID NO:33所示序列的VH和SEQ ID NO:51所示序列的VL；
    (l)VH和VL，与(a)至(k)任一组中的VH和VL相比，其VH具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，其VL具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者
    (m)VH和VL，与(a)至(k)任一组中的VH和VL相比，其VH具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；和/或，其VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；优选地，所述的置换是保守置换。
  26. 权利要求22-25任一项所述抗Napi2b抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是嵌合抗体、人源化抗体或全人源抗体；
    可选地，所述抗体或其抗原结合片段选自scFv、Fab、Fab’、(Fab’)2、Fv片段、二硫键连接的Fv(dsFv)、双抗体(diabody)和多特异性抗体；
    例如，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
  27. 权利要求22-26任一项所述的抗Napi2b抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含：
    (a)人免疫球蛋白的重链恒定区CH或其变体；和/或
    (b)人免疫球蛋白的轻链恒定区CL或其变体，
    其中，所述变体与其所源自的野生型序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；优选地，所述的置换是保守置换；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；和/或，所述轻链恒定区是κ或λ轻链恒定区；
    更优选地，所述抗体或其抗原结合片段包含人IgG1重链恒定区；和/或所述抗体或其抗原结合片段包含人κ轻链恒定区。
  28. 权利要求27所述的抗Napi2b抗体或其抗原结合片段，其中，
    所述重链恒定区包括SEQ ID NO:70所示的CH或其变体，所述变体与SEQ ID NO:70相比具有至多20个氨基酸的保守置换例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换，例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的保守置换；或者与SEQ ID NO:70相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或
    所述轻链恒定区包括SEQ ID NO:80所示的CL或其变体，所述变体与SEQ ID NO:80相比具有至多20个氨基酸的保守置换例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换，例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的保守置换，或者与SEQ ID NO:80相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:70所示的重链恒定区CH和SEQ ID NO:80所示的轻链恒定区CL。
  29. 权利要求22-28任一项所述的抗Napi2b抗体或其抗原结合片段，其中，所述抗体包括：
    (a)包括SEQ ID NO:71所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:72所示的VL和SEQ ID NO:80所示的CL的轻链；
    (b)包括SEQ ID NO:73所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:72所示的VL和SEQ ID NO:80所示的CL的轻链；优选包括SEQ ID NO:81所示的重链和SEQ ID NO:82所示的轻链；
    (c)包括SEQ ID NO:17所示的VH和SEQ ID NO:70所示的CH的重链，和，包括SEQ ID NO:18所示的VL和SEQ ID NO:80所示的CL的轻链；优选包括SEQ ID NO:83所示的重链和SEQ ID NO:84所示的轻链。
  30. 如权利要求1-21任一所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，Tb选自权利要求22-29任一所述的抗Napi2b抗体或其抗原结合片段，
  31. 权利要求1-21,30中任一项所述的抗体药物偶联物或所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其中，所述抗体药物偶联物选自：


    其中，Tb1为lifastuzumab，upifitamab，60A12B3-hz1、22H9C4-hz1、66C12D12-hz1、34F2A10-hz1、C1，16G5B3-hz2和16G5B3-hz1抗体；q选自0.1-16.0之间的任意数值，优选2-8之间的任意数值，更优选地，q为2、4、6或8。
  32. 制备权利要求1-21,30,31中任一项所述的抗体药物偶联物的方法，其包括：
    将Tb与式III所示的药物连接体偶联物在合适的溶剂和条件下进行偶联反应；
    其中：
    L1、L2、L3、L4、Tb如权利要求1-21中任一项所定义；
    R1、R2、R3、X、W如权利要求1-21所定义；
    Lg为离去基团，Lg选自卤素、砜基、三级胺盐基(Me3N+、Et3N+)、重氮盐基、-OMs、MeSO2-、CF3SO3-。
  33. 权利要求32的方法，其中，所述方法包括将Tb与式III所示的药物连接体偶联物 在合适的溶剂和条件下进行偶联反应形成C-S键的步骤；
    优选地，所述Tb与所述药物连接体偶联物的物质的量的比为1:(1-20)；
    优选地，所述偶联反应在水和/或有机溶剂中进行；优选地，所述有机溶剂选自N,N-二甲基甲酰胺、二甲基亚砜、N-甲基吡咯烷酮、腈类(例如乙腈)、醇类(例如甲醇、乙醇)或其任意组合；所述的腈类可为乙腈，所述的醇类可为甲醇、乙醇；
    优选地，所述方法进一步包括将偶联产物进行纯化的步骤；优选地，通过层析方法对所述偶联产物进行纯化；优选地，所述层析方法包括离子交换层析、疏水层析、反相层析或亲和层析中的一种或多种。
  34. 分离的核酸分子，其编码权利要求22-29任一项所述的抗Napi2b抗体或其抗原结合片段。
  35. 载体，其包含权利要求34所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  36. 宿主细胞，其包含权利要求34所述的分离的核酸分子或权利要求35所述的载体。
  37. 制备权利要求22-29任一项所述的抗Napi2b抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求36所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  38. 一种抗体-药物偶联物，其中抗体为权利要求22-29任一项所述的抗Napi2b抗体或其抗原结合片段，其通过接头与偶联部分连接，所述偶联部分选自：可检测的标记、放射性同位素、荧光物质、发光物质、有色物质、酶、聚乙二醇、核素、核酸、小分子毒素、具有结合活性多肽、蛋白、受体、配体，以及其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡或坏死的活性物质。
  39. 一种抗体药物偶联物的群，其包含权利要求1-21，30，31，38中任一项所述的抗体药物偶联物，所述的抗体药物偶联物具有一种、两种或多种q值；
  40. 一种药物组合物，其包含权利要求1-21，30，31，38中任一项所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、或权利要求22-29任一项所述的抗Napi2b抗体或其抗原结合片，或如权利要求34所述的核酸分子、或如权利要求35所述的载体、或如权利要求36所述的宿主细胞、或如权利要求39所述的抗体药物偶联物的群；优选还包括药学上可接受的载体和/或赋形剂。
  41. 权利要求39所述的群或权利要求40所述的药物组合物，其特征在于，药物与抗体的比例(DAR)选自1-10中的整数或小数；
    优选地，所述群的药物与抗体的比例(DAR)选自1.5-2.5、3.5-4.5、5.5-6.5和7.5-8.5；
    优选地，所述群的药物与抗体的比例(DAR)选自约2.0、4.0、6.0和8.0；
    优选地，所述群的药物与抗体的比例(DAR)选自2、2.5、3、3.5、4、4.5,5、5.5、6、6.5、7、7.2、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.7、8.9和9。
  42. 一种权利要求1-21，30，31，38中任一项所述抗体药物偶联物的立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、或权利要求22-2,9任一项所述的抗Napi2b抗体或其抗原结合片段、或如权利要求34所述的核酸分子、或如权利要求35所述的载体、或如权利要求36所述的宿主细胞、或如权利要求39所述的抗体药物偶联物的群，或者权利要求40所述的药物组合物在制备治疗和/或预防与细胞活动异常相关的疾病的药物中的用途；
    可选地；所述的与细胞活动异常相关的疾病可为癌症疾病；
    优选地，所述癌症疾病选自食管癌、脑瘤、肺癌、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子***、子宫内膜癌、结肠癌、直肠癌、结直肠癌、肝癌、肾癌、尿路上皮癌、表皮癌、非霍奇金淋巴瘤、中枢神经***肿瘤、***癌或甲状腺癌；所述的食管癌例如食管腺癌或食管鳞状细胞癌；所述的肺癌例如小细胞性肺癌、非小细胞性肺癌或肺腺癌；所述的中枢神经***肿瘤例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤；所述结肠癌例如人结肠腺癌；
    优选地，所述癌症疾病选自结肠癌，结直肠癌，结肠腺癌，小细胞肺癌，卵巢癌；
    更优选地，所述癌症疾病为与Napi2b相关的癌症疾病。
PCT/CN2023/107220 2022-07-14 2023-07-13 抗体药物偶联物及其制备方法和用途 WO2024012524A1 (zh)

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