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WO2022078279A1 - Antibody-drug conjugate and use thereof - Google Patents

Antibody-drug conjugate and use thereof Download PDF

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Publication number
WO2022078279A1
WO2022078279A1 PCT/CN2021/123020 CN2021123020W WO2022078279A1 WO 2022078279 A1 WO2022078279 A1 WO 2022078279A1 CN 2021123020 W CN2021123020 W CN 2021123020W WO 2022078279 A1 WO2022078279 A1 WO 2022078279A1
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WIPO (PCT)
Prior art keywords
antibody
pharmaceutically acceptable
acceptable salt
formula
stereoisomer
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PCT/CN2021/123020
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French (fr)
Chinese (zh)
Inventor
丁会
柯天一
于海勇
劳芳
徐云雷
张西东
刘岩
荣鹏飞
王闯
范墨林
李凡
欧阳芳幸
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昆山新蕴达生物科技有限公司
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Priority to CN202180069652.9A priority Critical patent/CN116472064A/en
Publication of WO2022078279A1 publication Critical patent/WO2022078279A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to a new type of linker structure, a drug-linker compound including the linker structure, and an antibody-drug conjugate including the drug-linker compound, the above-mentioned drugs - Preparation methods and applications of linker compounds and antibody-drug conjugates.
  • ADCs Antibody-drug conjugates
  • conjugates have shown unique advantages over pure antibody drugs, by combining them with tumor cell surface antigen binding specificity.
  • the monoclonal antibody is linked to a biologically active cytotoxin, thereby combining the tumor recognition targeting of the antibody with the high-efficiency killing effect of the cytotoxin. big flaw.
  • ADC can accurately target tumor cells while reducing the impact on normal cells, greatly improving the effectiveness and safety of anti-tumor drugs.
  • ADC generally consists of three parts: antibody, linker and toxin.
  • Antibodies are targeted functional macromolecules of ADCs, which play the role of enriching toxins near the tumor tissue site to improve the killing efficiency of toxins.
  • major popular targets such as HER-2, Trop-2, PDL-1, CD30, etc.
  • ADC linkers are divided into two types: cleavable and non-cleavable.
  • the ideal linker should meet the requirements of "good stability and high release efficiency", that is, the ADC remains stable in the blood circulation and can be quickly released after reaching the tumor cells. toxins, killing tumor cells.
  • the linker is crucial for the ADC to function. An unstable linker will lead to off-target ADC and increase the safety risk, while an overly stable linker will affect the release rate of the toxin, thereby affecting the efficacy of the drug.
  • the toxin part of ADC is a small drug molecule that plays a killing role, and generally kills tumor cells by inhibiting DNA or protein synthesis, inhibiting cell mitosis, and the like.
  • the toxins currently used for ADC development mainly include microtubule inhibitors maytansinoids (see EP0425235, US5208020, US5416064, US7276497) and auristatin (MMAE/MMAF, see US2016304621A).
  • the representative drugs currently on the market are T-DM1 developed by Genetech.
  • T-DM1 is a compound formed by a stable thioether linker MCC (4-[N-maleimidomethyl]cyclohexane-1 - carboxylate) conjugated to an ADC consisting of trastuzumab conjugated to the maytansinoid toxoid DM1 (US8337856).
  • MCC stable thioether linker
  • ADC maytansinoid toxoid DM1
  • Other classes of cytotoxins include Calicheamicin (see US5606040), benzodipyrrole derivatives (duocarmycin, see US7129261), pyrrolobenzodiazepines (PBDs, see WO2005/040170) and Derivatives of tree alkaloids.
  • camptothecin derivatives include SN-38, CPT-11, ixatecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like.
  • ADC using camptothecin toxoid is DS-8201 developed by Daiichi Sankyo Co., Ltd. It uses ixatecan, which is highly cytotoxic, as the toxin part.
  • T-DM1 and DS-8201 still have the following shortcomings:
  • T-DM1 As far as T-DM1 is concerned, first of all, the efficacy of T-DM1 is insufficient, one is because its DAR is low, only 3-4, and the other is because it uses the linker of SMCC to connect with DM-1, and SMCC is non-degradable. linker, which reduces the efficacy of T-DM1; secondly, T-DM1 uses DM-1 as a toxin, which is a microtubule inhibitor, and the permeability of cell membranes is weak; thirdly, the presence of T-DM1 reduces white blood cells Serious side effects.
  • ixatecan is 10 times more toxic than SN-38, it cannot be used as a single drug due to its strong cell-killing activity. There is also only one enzymatic cleavage site, which also prolongs the onset time of ADC in cells to some extent. In addition, ixatecan has a short half-life in the blood, which reduces toxic side effects, but faces the risk of a short half-life of the drug.
  • camptothecin-based ADCs there is still a need to develop more effective and safe camptothecin-based ADCs in this field.
  • the preparation has a faster onset time, longer drug half-life, and at the same time, it has the advantages of stability, hydrophilicity and hydrophobicity, and anti-aggregation. Camptothecin ADCs with superior safety indicators are imminent.
  • the inventors designed a linker structure suitable for camptothecin derivatives, and used it as a linking structure between camptothecin derivatives and antibodies, so as to form a linker structure with faster onset time, longer drug half-life, and better drug resistance.
  • this ADC has excellent anti-tumor effect.
  • a first aspect of the present invention provides a compound represented by formula (I),
  • R 11 is a carboxy-substituted C 1 -C 6 alkyl group
  • R 12 is a cyano-substituted C 2 -C 6 alkynyl group, and 1-2 C atoms in X, Y, X' and Y' are replaced by N Atom substitution; preferably, R 11 is a carboxy-substituted C 1 -C 3 alkyl group, and R 12 is a cyano-substituted C 2 -C 3 alkynyl group.
  • X, Y, X' and Y&apos have and only 1 C atom substituted with a N atom.
  • 2 C atoms in X, Y, X' and Y' are substituted with N atoms, and only 1 C atom in X, Y is substituted with N atoms, and X', Y' There is one and only one C atom replaced by N atom.
  • the compound structure is shown below,
  • the compound is linked to the antibody as the linking unit in the antibody-drug conjugate through the formation of a thioether bond between the alkynyl carbon of R12 and the disulfide moiety present in the hinge portion of the antibody.
  • a second aspect of the present invention provides a method for preparing a compound represented by formula (I), comprising the steps of:
  • step (1) The reaction product of step (1) reacts with Pd(PPh 3 ) 2 Cl 2 , triethylamine, and propyn-3-ol in tetrahydrofuran;
  • step (2) reacts with TEMPO, PhI(OAC) 2 , and NH 4 OAC in a solution of CH 3 CN/H 2 O in a ratio of 9:1;
  • step (3) The product of step (3) is produced under the action of TFA/DCM.
  • the third aspect of the present invention provides an antibody-drug conjugate represented by formula (IV), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof
  • the solvate of the isomer or a pharmaceutically acceptable salt thereof characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and an antibody ( AB) connected via a linker represented by the following formula (III):
  • R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkane substituted by -NR 7 R 8 group or C 1 -C 6 alkyl substituted by C 2 -C 6 alkenyl;
  • R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
  • R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
  • R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
  • R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
  • each occurrence of R7 and R8 is independently selected from hydrogen, methyl, isopropyl, Alternatively R7 and R8 may together with the attached N atom form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R9 ;
  • R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
  • each occurrence of R 9 is independently selected from methyl, -NR 7 R 8 , piperidinyl;
  • R 13 represents a carboxy-substituted C 1 -C 6 alkyl group
  • L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
  • n 3 represents an integer from 1 to 4
  • n 4 represents an integer from 1 to 4
  • Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
  • W is a single key or Wherein, position 1 means connecting with -NR 10 -, and position 2 means connecting with (CH 2 CH 2 -O-)n 1 -;
  • Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 to form an amide bond, and the alkynyl carbon of R 12 and the disulfide of the hinge part of the antibody are connected. bond to form a thioether bond,
  • R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 or C 2 -C 4 alkenyl substituted C 1 -C 4 alkyl.
  • R 2 represents hydrogen, nitro, amino
  • R 3 represents hydrogen, halogen, hydroxy, C 1 -C 4 alkyl
  • R 3 represents hydrogen, F, hydroxyl, methyl
  • R 4 represents hydrogen or halogen.
  • R 4 represents hydrogen or F.
  • R 1 and R 2 are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • R 1 and R 2 are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • R and R are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • the compound represented by formula (IV) is gimatecan or gimitecan, more preferably gimatecan:
  • L 2 represents a lysine residue
  • n 4 represents an integer from 1 to 2
  • R 10 represents hydrogen or C 1 -C 4 Alkyl (preferably methyl)
  • Aryl represents a benzene ring group, preferably, the -NR 10 - group and the -(CH 2 )n 4 - group are located in the para position of the benzene ring.
  • La represents
  • the linker represented by formula (III) is a group selected from the group consisting of:
  • the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8, such as 3.3, 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
  • the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 Antibodies, CD30 antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
  • the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably,
  • the amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody
  • the nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2 composed of sequence, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the fourth aspect of the present invention provides a linker-drug intermediate compound represented by formula (VI), characterized in that T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following The joints represented by formula (V) are connected:
  • R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention.
  • L 1 , L 2 and La are as described in the specification of the present invention.
  • Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula L 1 to form an amide bond, and the compound represented by the formula (II) is represented by the 19-position hydroxyl group.
  • the compound of formula (II) is as previously described.
  • the linker-drug intermediate compound is a compound selected from the group consisting of,
  • the fifth aspect of the present invention provides the linker structure shown in general formula (III):
  • the sixth aspect of the present invention provides a method for preparing the antibody-drug conjugate of the third aspect of the present invention, the method comprising:
  • linker-drug intermediate compound represented by the formula (VI) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (VI) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody
  • the compound is linked to the antibody;
  • R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention.
  • T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site.
  • AB-SH represents an antibody carrying a sulfhydryl group
  • AB represents an antibody
  • the seventh aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the fourth aspect of the present invention, the method comprising:
  • N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN- CMTC-1) and PABOH were dissolved in dichloromethane: methanol solution, reacted under the action of EEDQ, recrystallized and purified to obtain the product;
  • step (2) treating the product of step (1) with a solution of piperidine in acetonitrile, and then purifying the product;
  • step (3) reacts with the product of step (2) to generate compound
  • Gematecan-Boc or Gematecan-Boc is treated with triphosgene, DMAP, and dichloromethane to form a formyl chloride compound, and then the reaction compound of step (4) is added, and then deprotected with TFA/DCM. ;
  • step (5) The product of step (5) is subjected to Click reaction with the compound described in the first aspect of the present invention, and the final product is obtained after being treated with TFA/DCM;
  • step (6) can also be replaced by the following steps: the product of step (5) and SM-1 are added to a solution of DMSO/H 2 O, then CuBr is added to catalyze the reaction, the reaction is complete, and after purification, TFA/DCM is added for deprotection , the final product is obtained;
  • the SM-1 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-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-N-(2-aminoethyl)-2-aminoethyl-N
  • the eighth aspect of the present invention provides an antibody-drug conjugate represented by formula (VIII), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof
  • the solvate of the isomer or a pharmaceutically acceptable salt thereof characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and an antibody ( AB) connected via a linker represented by the following formula (VII):
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention.
  • n 5' represents an integer of 1 to 3
  • each n 5 independently represents an integer of 1 to 8
  • W and W' represent an integer Or a single bond
  • the position 1 of W indicates that it is connected to Z
  • the position 2 indicates that it is connected to (CH 2 CH 2 -O-)n 5 -
  • the position 1 of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected
  • position 2 indicates that it is connected to -CH 2- of L 4
  • W and W' are not at the same time
  • Cyclo represents a cyclohexane group
  • L 3 represents or a single bond, each n independently represents an integer from 1 to 6 (eg 2);
  • LP represents a peptide residue consisting of 1 to 7 amino acids
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy (preferably methyl),
  • Q 2 represents -(succinimide-3-yl-N)-, or -Q 1 -NR 10 -, Q 1 is defined as the compound described in the first aspect of the present invention, and Q 1 passes through the carboxyl group of R 11 and - NR 10 - forms an amide bond and connects with L 3 ,
  • -Q 1 -NR 10 - represented by Q 2 is connected by forming a thioether bond between the alkynyl carbon of R 12 and the disulfide bond of the antibody hinge portion,
  • the compound represented by formula (II) is as previously described.
  • the peptide residue of LP is selected from the group consisting of alanine, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues formed from amino acids.
  • the peptide residues of LP are formed from amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues.
  • LP is a peptide residue consisting of 1-5 amino acids.
  • LP is a peptide residue selected from the group consisting of:
  • LP is a peptide residue selected from the group consisting of:
  • L 4 represents
  • L b represents
  • the -NR 10 - group and the -(CH 2 )n 8 - group are located in the para position of the benzene ring.
  • the linker represented by formula (VII) is a group selected from the group consisting of the following, wherein each n independently represents an integer of 1-8:
  • the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8, such as 3.3, 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
  • the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 Antibodies, CD30 antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
  • the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably,
  • the amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody
  • the nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2 composed of sequence, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the ninth aspect of the present invention provides a linker-drug intermediate compound represented by formula (X), characterized in that T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following The joints represented by formula (IX) are connected:
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention.
  • Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -, and Q 1 is defined as the compound described in the first aspect of the present invention,;
  • L 3 , L 4 , L P and L b are as described in the specification of the present invention.
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
  • the nitrogen atom at the 1-position is connected to the methylene group in the linker containing this structure, or Q 1 -NR 10 - represented by Q' 2 , Q 1 forms an amide with -NR 10 - through the carboxyl group of R 11 key and connect with L3 ;
  • the compound of formula (II) is as previously described.
  • the linker-drug intermediate compound is a compound selected from the group consisting of, wherein each n independently represents an integer from 1 to 8:
  • a tenth aspect of the present invention provides a linker structure shown in the general formula (VII):
  • the eleventh aspect of the present invention provides a method for preparing the antibody-drug conjugate of the eighth aspect of the present invention, the method comprising:
  • linker-drug intermediate compound represented by the formula (X) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (X) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody
  • the compound is linked to the antibody;
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention.
  • T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R3 or R4 is a hydroxyl group, the compound represented by the formula (II) uses the oxygen in the hydroxyl group of R3 or R4 as a hydroxyl group .
  • AB-SH represents an antibody carrying a sulfhydryl group
  • AB represents an antibody
  • the twelfth aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the ninth aspect of the present invention.
  • Boc-GGFG and PABOH are generated under the action of EEDQ, using dichloromethane and methanol as solvents, and stirring overnight at room temperature to generate Boc-GGFG-PABOH;
  • N 3 -PEGn-GGFG was condensed with N-Boc-N-methylethylenediamine, and then Boc was removed with TFA/DCM to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH -CH 3 ;
  • the thirteenth aspect of the present invention provides intermediate compounds of formula (XI), (XII):
  • the fourteenth aspect of the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate of the third aspect and the eighth aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the The antibody-drug conjugate, a solvate of a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and an optional pharmaceutically acceptable carrier.
  • the fifteenth aspect of the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the third aspect and the eighth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  • the sixteenth aspect of the present invention provides the antibody-drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates of the third and eighth aspects of the present invention.
  • the antibody-drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates, stereoisomers thereof according to the third aspect and the eighth aspect of the present invention
  • the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
  • the seventeenth aspect of the present invention provides a method for preventing or treating tumor or cancer, which comprises administering an effective amount of the antibody-drug couple of the third aspect and the eighth aspect of the present invention to a subject in need thereof.
  • the conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as described in the fourteenth aspect The pharmaceutical composition or the pharmaceutical preparation of the fifteenth aspect.
  • the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
  • the eighteenth aspect of the present invention provides the antibody-drug conjugate of the third aspect and the eighth aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, Use of a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the pharmaceutical preparation of the fifteenth aspect for the preparation of a reagent for use in Inhibits cancer cell growth, proliferation or migration.
  • the nineteenth aspect of the present invention provides the antibody-drug conjugate of the third aspect and the eighth aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, A solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the pharmaceutical preparation of the fifteenth aspect, which are used for inhibiting the growth, proliferation or migrate.
  • the twentieth aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug conjugates of the third aspect, the eighth aspect of the present invention, Its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the drug described in the fourteenth aspect
  • the composition or the pharmaceutical preparation of the fifteenth aspect comprising administering to the cancer cells an effective amount of the antibody-drug conjugates of the third aspect, the eighth aspect of the present invention, Its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the drug described in the fourteenth aspect.
  • the twenty-first aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, which comprises the antibody-drug conjugates and stereoisomers thereof according to the third and eighth aspects of the present invention or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the tenth
  • a kit for inhibiting the growth, proliferation or migration of cancer cells which comprises the antibody-drug conjugates and stereoisomers thereof according to the third and eighth aspects of the present invention or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the tenth
  • a kit for inhibiting the growth, proliferation or migration of cancer cells which comprises the antibody-drug conjugates and stereoisomers thereof according to the third and eighth aspects of the present invention or a pharmaceutically acceptable salt thereof, or the antibody
  • a twenty-second aspect of the present invention provides an antibody-drug conjugate represented by formula (IV), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof
  • R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 substituted by -NR 7 R 8 alkyl;
  • R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
  • R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
  • R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
  • R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
  • R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
  • R 13 represents a carboxy-substituted C 1 -C 6 alkyl group
  • L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxyl groups, n 3 represents an integer of 1 to 4, and n 4 represents an integer of 1 to 4;
  • Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
  • W is a single key or Wherein, position 1 means connecting with -NR 10 -, and position 2 means connecting with (CH 2 CH 2 -O-)n 1 -;
  • Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 to form an amide bond, and the alkynyl carbon of R 12 and the disulfide of the hinge part of the antibody are connected. bond to form a thioether bond,
  • R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, or C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 .
  • R 3 represents hydrogen, halogen, hydroxyl, or
  • R 4 represents hydrogen or halogen.
  • R 1 and R 2 are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • R and R are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • the compound represented by formula (II) is gimatecan or gimitecan, more preferably gimatecan:
  • L 2 represents a lysine residue
  • n 4 represents an integer from 1 to 2
  • R 10 represents hydrogen or C 1 -C 4
  • Aryl represents a benzene ring group, preferably, the -NR 10 - group and the -(CH 2 )n 4 - group are located in the para position of the benzene ring.
  • the linker represented by formula (III) is a group selected from the group consisting of:
  • the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8.
  • the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of anti-He-r2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody.
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody; preferably, the humanized antibody is a fully human antibody.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
  • the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably,
  • the amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody
  • the nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the twenty-third aspect of the present invention provides a linker-drug intermediate compound represented by formula (VI), wherein T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following formula ( The joints indicated by V) are connected:
  • R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention.
  • L 1 , L 2 and La are as described in the twenty-second aspect of the present invention.
  • Q 1 is the compound described in the first aspect of the present invention, which is connected by forming an amide bond between the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 ,
  • the compound of formula (II) is described in the twenty-second aspect of the present invention.
  • the linker-drug intermediate compound is a compound selected from the group consisting of,
  • a twenty-fourth aspect of the present invention provides a linker, wherein it is represented by the following formula (III),
  • the linker is a linker selected from the twenty-second aspect of the present invention.
  • a twenty-fifth aspect of the present invention provides an antibody-drug conjugate represented by formula (VIII), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof
  • R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention.
  • L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'- or a single bond
  • n 5 represents an integer from 1 to 8
  • W and W' represent Or a single bond
  • the position 1 of W indicates that it is connected to Z
  • the position 2 indicates that it is connected to (CH 2 CH 2 -O-)n 5 -
  • the position 1 of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected
  • position 2 indicates that it is connected to -CH 2- of L 4
  • W and W' are not at the same time
  • Cyclo represents a cyclohexane group
  • LP represents a peptide residue consisting of 2-7 amino acids
  • Aryl represents a C 6 -C 10 aryl group optionally substituted by R 9 , n 7 represents an integer of 1-4, and n 8 represents an integer of 1-4;
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
  • Q 2 represents -(succinimide-3-yl-N)-, or -Q 1 -NR 10 -, Q 1 is the compound described in the first aspect of the present invention, and Q 1 passes through the carboxyl group of R 11 and -NR 10 - form an amide bond to connect with L 3 ;
  • Q 2 is -(succinimide-3-yl-N)-, with the following formula:
  • the compound represented by formula (II) is as described in the twenty-second aspect of the present invention.
  • the peptide residues of LP are formed from amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues.
  • LP is a peptide residue consisting of 2-5 amino acids.
  • LP is a peptide residue selected from the group consisting of:
  • the -NR 10 - group and the -(CH 2 )n 8 - group are located in the para position of the benzene ring.
  • the linker represented by formula (VII) is a group selected from the group consisting of:
  • the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8.
  • the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody , CD19 antibody, CD33 antibody.
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody; preferably, the humanized antibody is a fully human antibody.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
  • the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably,
  • the amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody
  • the nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the twenty-sixth aspect of the present invention provides a linker-drug intermediate compound represented by formula (X), wherein T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following formula ( IX) is formed by connecting the joints indicated:
  • R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention.
  • Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -, and Q 1 is the compound described in the first aspect of the present invention,;
  • L 3 , L 4 , L P and L b are as described in the twenty-fifth aspect of the present invention.
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
  • Q' 2 represents (maleimide-N)-, the following formula structure:
  • the nitrogen atom at the 1-position is connected with the methylene group in the linker containing this structure, or Q' 2 represents Q 1 -NR 10 -, and Q 1 forms an amide bond with -NR 10 - through the carboxyl group of R 11 And connect with L3 ;
  • the compound of formula (II) is described in the twenty-second aspect of the present invention.
  • the linker-drug intermediate compound is a compound selected from the group consisting of:
  • a twenty-seventh aspect of the present invention provides a linker, wherein it is represented by the following formula (VII)
  • the linker is a structure selected from the twenty-fifth aspect of the present invention.
  • a twenty-eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof according to the twenty-second or twenty-fifth aspect of the present invention.
  • a twenty-ninth aspect of the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof.
  • the thirtieth aspect of the present invention provides the antibody-drug conjugate of the twenty-second aspect or the twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody - a drug conjugate, a solvate of a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention for the prevention and/or treatment of tumors or cancers.
  • the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer.
  • the thirty-first aspect of the present invention provides a method of preventing or treating cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the method of the twenty-second or twenty-fifth aspect of the present invention
  • the antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention.
  • the thirty-second aspect of the present invention provides the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the Antibody-drug conjugate, solvate of its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the drug of the twenty-ninth aspect of the present invention Use of a formulation for inhibiting cancer cell growth, proliferation or migration.
  • the thirty-third aspect of the present invention provides the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the Antibody-drug conjugate, solvate of its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the drug of the twenty-ninth aspect of the present invention Agents for inhibiting the growth, proliferation or migration of cancer cells.
  • the thirty-fourth aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug according to the twenty-second or twenty-fifth aspect of the present invention
  • the conjugate, its stereoisomer or its pharmaceutically acceptable salt, or the solvate of said antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, the second of the present invention The pharmaceutical composition of the eighteenth aspect and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention.
  • the thirty-fifth aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, its A stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the drug of the twenty-eighth aspect of the present invention.
  • the thirty-sixth aspect of the present invention provides the antibody-drug conjugate of the twenty-second aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate , the preparation method of the solvate of its stereoisomer or its pharmaceutically acceptable salt, described method comprises:
  • linker-drug intermediate compound represented by the formula (VI) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (VI) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody
  • the compound is linked to the antibody;
  • R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention.
  • T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site.
  • AB-SH represents an antibody carrying a sulfhydryl group
  • AB represents an antibody
  • the thirty-seventh aspect of the present invention provides a method for preparing a linker-drug intermediate compound of the twenty-third aspect of the present invention, the method comprising:
  • N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN- CMTC-1) and PABOH were dissolved in dichloromethane: methanol solution, reacted under the action of EEDQ, recrystallized and purified to obtain the product;
  • step (2) treating the product of step (1) with a solution of piperidine in acetonitrile, and then purifying the product;
  • step (3) reacts with the product of step (2) to generate compound
  • Gematecan-Boc or Gematecan-Boc is treated with triphosgene, DMAP, and dichloromethane to form a formyl chloride compound, and then the reaction compound of step (4) is added, and then deprotected with TFA/DCM. ;
  • step (5) The product of step (5) is subjected to Click reaction with the compound described in the first aspect of the present invention, and the final product is obtained after being treated with TFA/DCM;
  • step (6) can also be replaced by the following steps: the product of step (5) and SM-1 are added to a solution of DMSO/H 2 O, and then CuBr is added to catalyze the reaction to complete the reaction. After purification, TFA/DCM is added for deprotection , the final product is obtained;
  • the SM-1 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-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-N-(2-aminoethyl)-2-aminoethyl-N
  • the thirty-eighth aspect of the present invention provides the antibody-drug conjugate of the twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate , the preparation method of the solvate of its stereoisomer or its pharmaceutically acceptable salt, described method comprises:
  • linker-drug intermediate compound represented by the formula (X) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (X) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody
  • the compound is linked to the antibody;
  • R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention.
  • Q 2 , Q' 2 , L 3 , L 4 , L P and La are as described in the twenty-fifth or twenty-sixth aspect of the present invention.
  • T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site.
  • AB-SH represents an antibody carrying a sulfhydryl group
  • AB represents an antibody
  • the thirty-ninth aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the twenty-sixth aspect of the present invention, the method comprising:
  • Boc-GGFG and PABOH are generated under the action of EEDQ, using dichloromethane and methanol as solvents, and stirring overnight at room temperature to generate Boc-GGFG-PABOH;
  • N 3 -PEGn-GGFG was condensed with N-Boc-N-methylethylenediamine, and then Boc was removed with TFA/DCM to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH -CH 3 ;
  • N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 and gimatecan-PNP (or gimatecan-PNP, SN-38-PNP) in the condition of TEA, DMF The following reaction is carried out to obtain the compound N 3 -PEGn-GGFG-NH-C 2 H 4 -N(CH 3 )-C(O)-gemitecan (or SN-38, or gematecan),
  • FIG. 1 shows the SEC-HPLC results of ADC-1.
  • Figure 2 is the SEC-HPLC profile of ADC-5.
  • Figure 3 is the SEC-HPLC profile of ADC-6.
  • Figure 4 is the SEC-HPLC profile of ADC-8.
  • Figure 5 is the SEC-HPLC profile of ADC-10.
  • Figure 6 is the SEC-HPLC profile of ADC-11.
  • Figure 7 is the SEC-HPLC profile of ADC-12.
  • Figure 8 is the SEC-HPLC profile of ADC-13.
  • Figure 9 is the SEC-HPLC profile of ADC-14.
  • Figure 10 is the SEC-HPLC profile of ADC-15.
  • Figure 11 is the SEC-HPLC profile of ADC-16.
  • Figure 12 is the SEC-HPLC profile of ADC-17.
  • Figure 13 is a release-peak localization map of ADC-5 small molecules.
  • Figure 14 is a release-line graph of ADC-5 small molecule.
  • Figure 15 is the percent change in the area of the main peak in the CL2A-CM sample.
  • Figure 16 is the CM peak area percent change in CL2A-CM samples.
  • Figure 17 is the percent change in the area of the main peak in ADC-5 samples.
  • Figure 18 is the CM peak area percent change in ADC-5 samples.
  • Figure 19 shows the results of inhibition of BXPC-3 cell activity by four ADCs.
  • Figure 20 shows the IC50 of the test drug on BxPC-3.
  • Figure 21 shows the IC50 of the test drug on COLO 205.
  • Figure 22 shows the IC50 of the tested drugs on Calu-3.
  • Figure 23 shows the IC50 of the tested drugs on Calu-6.
  • Figure 24 shows the IC50 of the tested drugs on NCI-N87.
  • Figure 25 is the antitumor activity of ADC-5 in the BxPC-3 tumor model.
  • Figure 26 shows the effect of ADC-5 on the body weight of the BxPC-3 model.
  • Figure 27 shows the anti-tumor activity of ADC-137 and ADC-5 in COLO 205 tumor model.
  • Figure 28 shows the effects of ADC-137 and ADC-5 on the body weight of the COLO 205 model.
  • Figure 29 shows the antitumor activity of ADC-137 and ADC-5 in BxPC-3 tumor model.
  • Figure 30 shows the effects of ADC-137 and ADC-5 on the body weight of the BxPC-3 model.
  • Figure 31 shows the antitumor activity of ADC-137 and ADC-5 in Calu-3 tumor model.
  • Figure 32 shows the effect of ADC-137 and ADC-5 on the body weight of Calu-3 model.
  • Figure 33 shows the antitumor activity of ADC-137 and ADC-5 in Capan-1 tumor model.
  • Figure 34 shows the effects of ADC-137 and ADC-5 on the body weight of the Capan-1 model.
  • Figure 35 shows the effect of ADC-16, ADC-5 and ADC-17 on body weight of the COLO 205 model.
  • Figure 36 is the anti-tumor activity of ADC-16, ADC-5 and ADC-17 in the COLO 205 tumor model.
  • Figure 37 is the antitumor activity of ADC-8, ADC-11, ADC-5 and ADC-12 in the BxPC-3 tumor model.
  • Figure 38 is the effect of ADC-8, ADC-11, ADC-5 and ADC-12 on body weight of the BxPC-3 model.
  • antibody refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3).
  • Antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable region of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 107-108 M -1 ) to an antigen .
  • a "functional fragment” or “analog of an anti-Her-2 antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • a functional fragment generally has the same meaning as an "antibody fragment” and, with respect to an antibody, may refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2 , and so on.
  • the "Fv" fragment consists of a dimer ( VH - VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain.
  • the three CDRs of each variable domain interact to define the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies.
  • the six CDRs collectively confer the target-binding specificity of the intact antibody.
  • a single variable domain or half of an Fv that includes only 3 target-specific CDRs
  • BsAb Bispecific antibody
  • a bispecific antibody and/or an antigen-binding molecule contains Two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.
  • monoclonal antibody or “monoclonal antibody” refers to a population of the same antibody, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by a number of well-known methods. For example, monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • a full-length antibody has two full-length heavy chains (eg VH-CH 1 -CH 2 -CH 3 or VH-CH 1 -CH 2 -CH 3 -CH 4 ) and two full-length light chains ( VL-CL) and hinge region antibodies, such as antibodies naturally produced by antibody-secreting B cells as well as synthetically produced antibodies having the same domains.
  • two full-length heavy chains eg VH-CH 1 -CH 2 -CH 3 or VH-CH 1 -CH 2 -CH 3 -CH 4
  • VL-CL full-length light chains
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1x107 M -1 or 1x108 M -1 or greater (or 1x10-7 M or 1x A dissociation constant (Kd) of 10 ⁇ 8 M or lower binds the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody response, eg, immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for detecting and monitoring binding rates in real time are known and commercially available.
  • nucleic acid molecules refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • expression refers to the process of producing a polypeptide by transcription and translation of a polynucleotide.
  • Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell for receiving, maintaining, replicating and amplifying a vector.
  • Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector” is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • the vector also includes "viral vector” or "viral vector”.
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • drug (drug compound) in the present invention, that is, “toxin” refers to a cytotoxic drug, that is, a compound represented by formula (I) (anti-tumor compound), which can strongly disrupt the normal growth of tumor cells. chemical molecules.
  • cytotoxic drugs can kill tumor cells at a high enough concentration, but due to the lack of specificity, they can also lead to normal cell apoptosis while killing tumor cells.
  • the term includes toxins, such as small molecule toxins or enzymatically active toxins of fungal, bacterial, plant or animal origin, radioisotopes (eg I 131 , Y 90 , Re 186 , I 125 ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic agents
  • the enzyme is preferably a toxic drug, more preferably a camptothecin derivative, more preferably gimitecan and gimatecan.
  • C a -C b (a and b represent an integer of 1 or more, a ⁇ b) includes any specific case of a to b carbons, for example, C 1 -C 6 includes C 1 and C 2 , C 3 , C 4 , C 5 , C 6 , also including any one range of a to b, for example, C 1 -C 6 includes C 1 -C 3 , C 1 -C 4 , C 1 -C 5 , C 2 -C 5 , C 2 -C 4 , C 3 -C 6 , etc.; similarly, "ab-membered ring” (a and b represent an integer of 1 or more, a ⁇ b) represents that the number of ring atoms is a to b
  • the ring structure for example, 3-6 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, and also includes any range from a to b, for
  • halogen refers to fluorine, chlorine, bromine and iodine.
  • C 1 -C 6 hydrocarbon group refers to a straight-chain or branched alkyl group derived from an alkane moiety containing 1-6 carbon atoms by removing one hydrogen atom
  • C 1 -C 6 hydrocarbyl may be saturated, ie "C 1 -C 6 alkyl”
  • C 1-6 alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl , 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl,
  • the "C 1 -C 6 hydrocarbyl” in the present invention may be unsaturated, such as “C 2 -C 6 alkenyl”, “C 3 -C 4 alkenyl”, and “C 3 -C 4 alkenyl” including but not limited to Not limited to propenyl, 1-butenyl, 2-butenyl, and the like.
  • C 1-6 alkoxy refers to a group in which "C 1-6 alkyl” as defined above is connected to the rest of the molecule via an oxygen atom, namely "C 1-6 alkyl-O"-” groups, specifically, include, but are not limited to, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy group, neopentyloxy, n-hexyloxy, etc.; the "C 1-4 alkoxy” refers to a group in which the above-defined "C 1-4 alkyl” is connected to the rest of the molecule through an oxygen atom , namely "C 1-4 alkyl-O-” group, specifically, including but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy group, sec-butoxy, tert-butoxy.
  • 5-6 membered ring refers to a non-aromatic cyclic structure with 5-6 ring atoms, and the ring atoms can be all carbon atoms, thereby forming a carbocyclic ring; it can also contain 1- 3 ring heteroatoms each independently selected from N, O, or S, thereby forming a heterocycle (eg, oxygen-containing heterocycle, nitrogen-containing heterocycle, sulfur-containing heterocycle); the 5-6 membered ring may be saturated
  • the structure can also be an unsaturated structure containing 1 or 2 carbon-carbon double bonds or carbon-carbon triple bonds.
  • 5-6 membered nitrogen-containing heterocycle includes but is not limited to piperidine and piperazine, preferably piperidine.
  • C 6 -C 10 aryl group refers to an aromatic cyclic hydrocarbon group having 6-10 ring-forming carbon atoms, which can be a monovalent group or a divalent or higher group as required, including Monocyclic aryl group and fused-ring aryl group, "fused-ring aryl group” refers to an aryl group containing multiple rings (eg, containing 2) in which each ring in the group shares an adjacent pair of ring carbon atoms with other rings. .
  • the "C 6 -C 10 -membered aryl group” specifically includes a phenyl group and a naphthyl group.
  • linker refers to a chemical structural fragment or bond that is connected to an antibody at one end and a drug (drug compound) at the other end. Other linkers can also be connected. It is then linked to the drug compound.
  • linker structure of the present invention can be synthesized by methods known in the art, or can be synthesized using the methods described in the present invention.
  • the "antibody-drug conjugate" of the present invention refers to a ligand linked to a biologically active drug through a stable linking unit. In the present invention, it refers to linking the monoclonal antibody or fragment with the biologically active toxic drug through the linker structure.
  • salts refer to relatively nontoxic acid addition salts or base addition salts of the conjugates of the present invention.
  • the acid addition salts are salts formed by the conjugates of the present invention with suitable inorganic or organic acids, and these salts can be prepared by subjecting the conjugates of the present invention with suitable organic or inorganic acids in a suitable solvent reaction to prepare.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearic acid Salt, laurosilicate, borate, benzoate, lactate, nitrate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleic acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalene sulfonate, tartrate, benzoate, mesylate, p-toluene Sulfonate, gluconate, lactobionate and lauryl sulfonate, etc.
  • the base addition salts are the salts formed by the conjugates of the present invention and suitable inorganic or organic bases, and these salts can be carried out by making the conjugates of the present invention and suitable inorganic or organic bases in a suitable solvent. reaction to prepare.
  • Representative base addition salts include, for example, salts formed with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium salts, etc.; amine salts, including salts formed with ammonia (NH 3 ), primary, secondary or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, and the like.
  • quaternary ammonium cations such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium,
  • the conjugates of the present invention may exist in specific geometric or stereoisomeric forms.
  • the chiral center may exist in the antitumor compound (the compound represented by formula (I)), or may exist in the
  • the linker structure (the linker represented by formula (II)) may also exist in antibodies and derivatives thereof.
  • Optically active (R)- and (S)-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of the conjugates of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide Pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art
  • the diastereoisomers were resolved and the pure enantiomers recovered.
  • separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
  • solvates eg, hydrates
  • suitable solvates include solvates of the conjugate of the present invention with acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, and the like. Hydrates or ethanolates can also be cited.
  • treating an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any pharmaceutical use of the provided ADCs as well as the pharmaceutical compositions, pharmaceutical formulations provided herein.
  • therapeutic effect means the effect resulting from the treatment of an individual, which alters, generally ameliorates or ameliorates the symptoms of a disease or disease condition, or cures a disease or disease condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have a desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses.
  • a prophylactically effective amount can be administered in one or more administrations.
  • the antitumor compound is not particularly limited as long as it is a compound having an antitumor effect or a compound having a substituent capable of being linked to a linker structure.
  • a part or the whole of the linker is cleaved in tumor cells to free the antitumor compound part, thereby exhibiting an antitumor effect.
  • the linker is cleaved with the linking part of the drug, the antitumor compound is released in its original structure, and its original antitumor effect is exerted.
  • the antitumor compound in the present invention is a compound represented by the following formula (II).
  • R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 substituted by -NR 7 R 8 alkyl;
  • R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
  • R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
  • R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
  • R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
  • R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
  • R 13 represents a carboxy-substituted C 1 -C 6 alkyl group.
  • R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, or C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 .
  • R 3 represents hydrogen, halogen, hydroxyl, or
  • R4 represents hydrogen or halogen.
  • R1 and R2 are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • R3 and R4 are joined together to form the group shown below in A moiety represents a bond to the parent group.
  • the compound represented by formula (I) is a compound selected from the group consisting of:
  • the compound represented by formula (I) is gimatecan or gimatecan:
  • the number of linker-drug linkages (drug loading (DAR, drug load ratio)) connected to one molecule of the antibody affects the effectiveness and safety of the conjugate.
  • the production of antibody-drug conjugates can be carried out by specifying reaction conditions such as the amount of raw materials and reagents used for the reaction in order to make the number of linker-drug linkages constant, but it is different from the chemical reaction of low-molecular-weight compounds. , usually obtained as a mixture of linked different numbers of drugs. Therefore, in the present invention, the number of linker-drug linkages linked to the antibody per molecule is represented by the average value, that is, the average number of drug linkages.
  • the The number of connections refers to the average.
  • the number of antitumor compounds linked to the antibody molecule can be controlled, and as the average number of drug linkages per antibody, about 1 to 10 antitumor compounds can be linked, preferably 2 to 8, more preferably 4 to 8, More preferably, it is 6-8. It should be noted that those skilled in the art can design the reaction of linking the necessary number of drugs on the antibody according to the description of the examples of the present application, and can obtain the antibody with the number of links of the anti-tumor compound controlled.
  • the number of free sulfhydryl groups attached to each antibody molecule is not actually measured.
  • the average number m of sulfhydryl groups attached to each antibody molecule is 6- 8.
  • the present invention provides a compound represented by formula (I),
  • R 11 is a C 1 -C 6 carboxyalkyl group
  • R 12 is a C 2 -C 6 cyanoalkynyl group
  • 1-2 C atoms in X, Y, X' and Y' are substituted by N atoms; preferably Typically, R 11 is C 1 -C 3 carboxyalkyl, and R 12 is C 2 -C 3 cyanoalkynyl.
  • X, Y, X' and Y' have one and only one C atom replaced by a N atom;
  • X, Y, X' and Y' have 2 C atoms substituted with N atoms, and X, Y have only 1 C atom substituted with N atoms, and X' , Y' has and only one C atom is replaced by N atom;
  • the structures of the compounds are shown below, designated CN-A, CN-B, CN-C and CN-D, respectively
  • the compound of formula (I) is used as the linking unit in the antibody-drug conjugate through a thioether formed by the alkynyl carbon of R12 and the disulfide moiety present in the hinge portion of the antibody bond to the antibody, that is, the alkynyl group of R 12 reacts with the disulfide bond of the hinge part of the antibody so that the alkynyl carbon of R 12 is connected to the reduced sulfhydryl group (-SH-) of the hinge part of the antibody, through the carboxyl group of R 11 It forms an amide bond with the amino group present at the end of the linker to connect with other linking units in the linker.
  • a preparation method of the compound of formula (I) (1) Effect of SM-A (5-bromopyridine-2-carboxylic acid) on Boc 2 O, DMAP, t-BuOH (2) A-1 reacted with Pd(PPh 3 ) 2 Cl 2 , triethylamine and propyn-3-ol in tetrahydrofuran at 70 °C for 12 hours to obtain the compound A-2(3) A-2 reacted with TEMPO, PhI(OAC) 2 , NH 4 OAC in a solution of CH 3 CN/H 2 O 9:1 at room temperature for 12 hours to obtain compound A-3; (4) A-3 generates compound CN-A under the action of TFA/DCM.
  • CN-B and CN-C can be prepared by replacing SM-A with SM-B (6-bromonicotinic acid) or SM-C (5-bromopyrimidine-2-carboxylic acid).
  • the linker of the present invention has the structure shown in the following formula (III):
  • L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxyl groups, n 3 represents an integer of 1 to 4, and n 4 represents an integer of 1 to 4;
  • Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
  • W is a single key or Wherein, position 1 means connecting with -NR 10 -, and position 2 means connecting with (CH 2 CH 2 -O-)n 1 -;
  • Q 1 is linked through the formation of an amide bond between the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 .
  • n 1 represents 6, 7, 8, 9, 10, 11 or 12 and n 2 represents 1 or 2.
  • R 10 represents hydrogen or C 1 -C 4 alkyl.
  • L 2 represents a single amino acid residue, such as valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue base, aspartic acid residue.
  • L2 represents a lysine residue
  • the compound represented by formula (II) uses the oxygen in the hydroxyl group at position 19 as the linking site, or when R3 or R4 is a hydroxyl group, the oxygen in the hydroxyl group of R3 or R4 is used as the linking site .
  • R 3 or R 4 when R 3 or R 4 is a hydroxyl group, the compound represented by formula (II) is connected to the above-mentioned La represented by the oxygen in the hydroxyl group of R 3 or R 4 as a linking site.
  • La represents a structure derived from 4-aminobenzyl alcohol.
  • the C-terminus of the amino acid represented by L 2 is attached to the terminal amino group of the group represented by L a .
  • the linker represented by formula (III) is a group selected from the group consisting of:
  • the linker of the present invention has the structure shown in the following formula (VII):
  • L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'- or a single bond
  • n 5 represents an integer of 1 to 8
  • W and W' represent Or a single bond
  • the position 1 of W indicates that it is connected to Z
  • the position 2 indicates that it is connected to (CH 2 CH 2 -O-)n 5 -
  • the position 1 of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected
  • position 2 indicates that it is connected to -CH 2- of L 4
  • W and W' are not at the same time
  • Cyclo represents a cyclohexane group
  • LP represents a peptide residue consisting of 2 to 7 amino acids
  • Aryl represents a C 6 -C 10 aryl group optionally substituted by R 9 ,
  • n 7 represents an integer of 1-4, n 8 represents an integer of 1-4,
  • R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy groups,
  • Q 2 represents -(succinimide-3-yl-N)-, with the following formula:
  • the alkynyl carbon of R 12 and the disulfide bond of the antibody hinge part form a thioether bond to connect, that is, the alkynyl group of R 12 reacts with the disulfide bond of the antibody hinge part to connect the alkynyl carbon of R 12 It is linked to L 3 by forming an amide bond between the carboxyl group of R 11 and -NR 10 - on the reduced sulfhydryl group (-SH-) of the antibody hinge.
  • LP represents a peptide residue consisting of 2 to 7 amino acids. That is, it consists of oligopeptide residues in which 2-7 amino acids are linked by peptide bonds.
  • the amino acid constituting LP is not particularly limited, and is, for example, an L- or D-amino acid, preferably an L -amino acid.
  • amino acids with structures such as ⁇ -alanine, ⁇ -aminocaproic acid, and ⁇ -aminobutyric acid may be used, and, for example, non-natural amino acids such as N-methylated amino acids may be used. type of amino acid.
  • the amino acid sequence of the LP moiety is not particularly limited, and the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L ), and glycine (Gly). ; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit; C), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D) and the like.
  • phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid are preferably used.
  • the pattern of drug release can be controlled according to the type of amino acid.
  • the number of amino acids can be 2-7.
  • the peptide residue represented by LP is linked to the L2 moiety at the N-terminus and to the La moiety at the C-terminus.
  • LP is a peptide residue consisting of 2-5 amino acids.
  • LP is a peptide residue selected from the group consisting of:
  • the compound represented by formula (II) uses the oxygen in the hydroxyl group at position 19 as the linking site, or when R3 or R4 is a hydroxyl group, the oxygen in the hydroxyl group of R3 or R4 is used as the linking site .
  • R 3 or R 4 when R 3 or R 4 is a hydroxyl group, the compound represented by formula (II) is connected to the above-mentioned L represented by the oxygen in the hydroxyl group of R 3 or R 4 as a linking site.
  • L represented by the oxygen in the hydroxyl group of R 3 or R 4 as a linking site.
  • L b represents a structure derived from 4-aminobenzyl alcohol.
  • the disabled C-terminus of the peptide represented by LP is attached to the group represented by Lb , more specifically, the C-terminus is attached to the terminal amino group in the group represented by Lb .
  • the linker represented by formula (VII) is a group selected from the group consisting of:
  • linker intermediate compound of the present invention is shown in the following formulas (XI) and (XII):
  • R 1 , R 2 , R 3 , R 4 , Q 1 , Q′ 2 L 1 , L 2 , L 3 , L 4 , L P , La and L b are as described in the specification of the present invention.
  • the linker-drug intermediate compound of the present invention is a compound represented by the formula (II) represented by T, and the intermediate compound is formed by connecting the compound (T) and the linker represented by the following formula (V):
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention.
  • the linker-drug intermediate compound is a compound selected from the group consisting of,
  • the linker-drug intermediate compound of the present invention is that T represents the compound represented by the formula (II), and the intermediate compound is formed by connecting the compound (T) and the linker represented by the following formula (IX):
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention.
  • Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -,
  • Q 1 -NR 10 - represented by Q 2 , Q 1 is connected to L 3 through the carboxyl group of R 11 and -NR 10 - forming an amide bond;
  • Q 1 , R 10 , L 3 , L 4 , L P and L b are as described in the specification of the present invention.
  • the linker-drug intermediate compound is a compound selected from the group consisting of,
  • gemnotecan when the compound represented by the formula (II) is gemnotecan, when L a and L b represent -NR 10 -(CH 2 )n 3 -, gemnotecan as an antitumor compound and the formula (III) ) and (VII) shown in the linker structure are connected by the structure -N-CH 2 -O-, and gemitecan has two connection sites at the 19th position and the 10th position, which provides more choices for ADC.
  • the antibody-drug conjugate (IV) of the present invention is formed by connecting the compound (T) and the antibody (AB) via a linker represented by the following formula (III):
  • R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention; the definitions of Q 1 , L 1 , L 2 and La are as described in the specification of the present invention; AB represents an antibody.
  • the antibody-drug conjugate (VIII) of the present invention is formed by connecting the compound (T) and the antibody (AB) via a linker represented by the following formula (VII):
  • R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention; the definitions of Q 2 , L 3 , L 4 , LP and L b are as described in the description of the present invention; AB represents an antibody.
  • an antibody-drug conjugate in which an antibody and a linker structure are linked via a thioether can be produced, for example, by the following method.
  • AB-SH represents an antibody carrying a sulfhydryl group
  • AB represents an antibody
  • the compounds represented by formulas (VI) and (X) are the above-mentioned linker-drug intermediate compounds of the present invention.
  • the compound shown in VIII) is the antibody-drug conjugate of the present invention.
  • the compounds represented by formulae (IV) and (VIII) are described in the form of a structure in which one structural moiety from the drug to the end of the linker is linked to one antibody, but in fact, relative to In many cases, a plurality of these structural moieties are linked to one antibody molecule.
  • 2 to 8, preferably 4 to 8, more preferably 6 to 8 linker-drug intermediate compounds are linked to one antibody molecule.
  • the average number of linker-drugs linked to each molecule of antibody is expressed as the average number of drug linkages.
  • the antibody-drug conjugate represented by formula (IV) can be produced by reacting the above-mentioned linker-drug intermediate compound of the present invention with the antibody AB-SH having a thiol group.
  • Antibodies having thiol groups can be obtained by methods known to those skilled in the art (Hermanson, G.T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, the following methods are exemplified: allowing Traut to react with the amino group of the reagent and the antibody; and allowing N-succinimidyl S-acetylthioalkanoate to react with the amino group of the antibody After reacting with hydroxylamine; after reacting N-succinimidyl 3-(pyridyldithio)propionate, reacting reducing agent; reacting dithiothreitol, 2-mercaptoethanol, tri( A reducing agent such as 2-carboxyethyl) phosphine hydrochloride (TCEP) acts on the antibody to reduce the disulfide bond in the hinge portion of the antibody to generate a sulfhydryl group; etc., but not limited to these methods.
  • TCEP partial or A sulfhydryl group-carrying antibody
  • a chelating agent ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), etc. are mentioned, for example. They can be used in concentrations of 1 mM to 20 mM.
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • the buffer solution sodium phosphate, sodium borate, sodium acetate solution or the like can be used.
  • an antibody AB-SH partially or completely reduced to have a thiol group can be obtained.
  • 2-20 molar equivalents of the compounds represented by the formulae (VI) and (X) can be used for each antibody AB-SH having a thiol group to produce an antibody-drug in which 2 to 8 drugs are linked to one antibody.
  • Conjugates (IV), (VIII). Specifically, to the buffer solution containing the antibody AB-SH having a thiol group, a solution in which the compounds represented by the formulae (VI) and (X) are dissolved is added and allowed to react.
  • the buffer solution sodium acetate solution, sodium phosphate, sodium borate, or the like can be used as the buffer solution.
  • the pH during the reaction is 5 to 9, and the reaction is more preferably near pH 7.
  • the compound (2) that is, the compound represented by the formula (II)
  • the organic solvent solution in which the compounds represented by formula (VI) and (X) are dissolved can be added to the buffer solution containing the thiol group-containing antibody AB-SH at 1 to 20% v/v, and the reaction can be carried out.
  • the reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours.
  • the reaction can be terminated by inactivating the reactivity of the unreacted compounds represented by the formulae (VI) and (X) with a thiol-containing reagent.
  • Thiol-containing reagents are, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, 1 to 2 molar equivalents of NAC are added to the compounds represented by the formulae (VI) and (X) to be used, and the reaction is completed by incubating at room temperature for 10 to 30 minutes.
  • the container put the antibody or antibody-drug conjugate solution, use a centrifuge for centrifugation (for example, centrifuge at 2000G-3800G for 5-20 minutes), and concentrate the antibody or antibody-drug conjugate solution.
  • a centrifuge for centrifugation for example, centrifuge at 2000G-3800G for 5-20 minutes
  • Antibody concentration was measured using a UV analyzer according to the manufacturer's instructions.
  • Phosphate buffer eg, 10 mM, pH 6.0
  • sodium chloride eg, 137 mM
  • EDTA ethylenediaminetetraacetic acid
  • PBS6.0/EDTA ethylenediaminetetraacetic acid
  • Phosphate buffer eg, 50 mM, pH 6.5, also referred to herein as PBS6.5/EDTA
  • sodium chloride eg, 50 mM
  • EDTA eg, 2 mM
  • PBS6.5/EDTA Phosphate buffer
  • the NAP-25 column using Sephadex G-25 vector was equilibrated.
  • One of the NAP-25 columns was packed with 2.5 mL of aqueous antibody solution, and then a fraction (3.5 mL) eluted with PBS6.5/EDTA 3.5 mL was obtained by separation. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 5 mg/mL using PBS6.5/EDTA.
  • phosphate buffer eg, PBS7.4
  • sodium phosphate buffer eg, 10 mM, pH 6.0; also referred to in this specification as PBS6.0
  • sodium chloride eg, 137 mM
  • the NAP-25 column is equilibrated with any of the acetic acid buffers (eg, 10 mM, pH 5.5; also referred to herein as ABS) containing sorbitol (eg, 5%).
  • An antibody-drug conjugate reaction aqueous solution for example, about 1.5 mL
  • the antibody fraction is obtained by separation by eluting with an amount of buffer specified by the manufacturer.
  • phosphate buffer eg, PBS7.4
  • sodium phosphate buffer eg, 10 mM, pH 6.0; also referred to in this specification as PBS6.0
  • sodium chloride eg, 137 mM
  • An AKTA column filler: sephadex G 25
  • any of the acetate buffers eg, 10 mM, pH 5.5; also referred to as ABS in this specification
  • sorbitol eg, 5%
  • the injector is loaded with an antibody-drug conjugate reaction aqueous solution (eg, about 2 mL), eluted with an amount of buffer specified by the manufacturer, and the antibody fractions are obtained by separation.
  • the unconnected drug linker low molecular compound (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide) antibody-drug conjugates.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • NAC N-acetyl-L-cysteine
  • dimethyl sulfoxide dimethyl sulfoxide
  • the preparation method of the linker-drug intermediate compound comprises the following steps:
  • CN-CMTC-1 N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine
  • EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
  • the target product with a purity of more than 95% is obtained after preparation and purification.
  • Gemitecan-Boc (or SN-38-Boc, or Gematecan) is reacted with triphosgene, DMAP, and dichloromethane to form the formyl chloride compound of Gemitecan, and then CN-CMTC-5 is added to react After about 5 mins, after quenching with methanol, the column was shorted to obtain the crude product of CN-CMTC-6 compound. After Pre-HPLC purification, pure CN-CMTC-6 was obtained, which was then treated with TFA/DCM for deprotection to obtain CN-CMTC-7, which was then subjected to Click reaction with CN-A, B, C, and D to obtain the compound CN-CMTC-8. , Compound 8 was treated with TFA/DCM to obtain the final product CN-CMTC (see the figure below).
  • CMTC represents gemnotecan.
  • gemnotecan can be replaced by SN-38 Or camptothecin derivatives such as gimatecan (GMTC).
  • the preparation method of the linker-drug intermediate compound comprises the following steps:
  • CN-CMTC-7 and SM-1 were added to DMSO/H2O solution, and then CuBr was added, and then stirred at room temperature for about 30 mins. HPLC detected that the reaction was complete, resulting in SMCC-PEG8-Lys(MMt)-PABC-CMTC, Pre-HPLC After purification, TFA/DCM was added for deprotection to obtain the target product SMCC-PEG8-Lys-PABC-CMTC.
  • the preparation method of the linker-drug intermediate compound comprises the following steps:
  • the preparation method of the linker-drug intermediate compound comprises the following steps:
  • Boc-GGFG and PABOH, dichloromethane and methanol were used as solvents, and stirred overnight at room temperature to generate Boc-GGFG-PABOH;
  • the preparation method of the linker-drug intermediate compound comprises the following steps:
  • DIEA is a base to obtain the compound N 3 -PEGn-GGFG, which is purified by Pre-HPLC and then condensed with N-Boc-N-methylethylenediamine.
  • the condensing agent is HATU, pyridine is used as a base, and DMF is used as a solvent to obtain the target product;
  • CMTC germane-maleimide
  • GMTC gematecan
  • the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof.
  • the antibody is selected from the group consisting of Her-2 antibody, anti-Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER- 4 antibody, CD20, CD30 antibody, CD19 antibody, CD33 antibody.
  • TROP-2 antibody belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), and gastrointestinal tumor-associated antigen. (GA733-1), Surface Marker 1 (M1S1). TROP-2 is overexpressed in various malignant tumors and is an oncogene related to the occurrence, invasion and metastasis of malignant tumors.
  • TACSTD2 tumor-associated calcium signal transducer 2
  • EGP-1 epidermal glycoprotein 1
  • M1S1 Surface Marker 1
  • TROP-2 of the natural sequence in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or a combination thereof.
  • the antibody used in the present invention is preferably an anti-human TROP-2 antibody.
  • the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human TROP-2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.
  • the anti-human TROP-2 antibody may be a humanized antibody or a fully human antibody.
  • the complementarity determining regions (CDRs) of the light chain variable region of the anti-Trop-2 antibody include CDR1 consisting of the amino acid sequence of KASQDVSIAVA, CDR2 consisting of the amino acid sequence of SASYRYT, and consisting of the amino acid sequence of QQHYITPLT CDR3 composed of;
  • the CDRs of the heavy chain variable region include CDR1 composed of the NYGMN amino acid sequence, CDR2 composed of the WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of the GGFGSSYWYFDV amino acid sequence;
  • the light chain of the anti-Trop-2 antibody and the amino acid sequences of the heavy chain are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;
  • the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody are respectively shown in SEQ ID NO: 3 and SEQ ID NO: shown in 4;
  • Her-2 antibodies or antigen-binding fragments thereof are Her-2 antibodies or antigen-binding fragments thereof, including bispecific antibodies and antibody functional derivatives.
  • Her-2 is also known as human epidermal growth factor receptor-2 (human epidermal growth factor receptor 2), or receptor tyrosine protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent) or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans.
  • Her-2 overexpression has been shown to play an important role in the development and progression of certain aggressive types of breast cancer.
  • Her-2 overexpression occurs in approximately 15-30% of breast cancers. In recent years, this protein has become an important biomarker and therapeutic target in about 30% of breast cancer patients.
  • Her-2 overexpression also occurs in ovarian cancer, intestinal gastric cancer, and invasive forms of uterine cancer such as serous endometrial cancer.
  • the natural sequence Her-2 in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or a combination thereof.
  • the antibody used in the present invention is preferably an anti-human Her-2 antibody.
  • the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human Her-2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.
  • the anti-human Her-2 antibody may be a humanized antibody or a fully human antibody.
  • the Her-2 antibody is the trastuzumab antibody described in US5821337, and the complementarity determining region (CDR) of its light chain variable region includes CDR1 consisting of the amino acid sequence of RASQDVNTAVA; consisting of the amino acid sequence of SASFLYS and CDR3 composed of the QQHYTTPPT amino acid sequence, and the CDRs of its heavy chain variable region include CDR1 composed of the DTYIH amino acid sequence; CDR2 composed of the RIYPTNGYTRY amino acid sequence; and CDR3 composed of the WGGDGFYAMDY amino acid sequence.
  • the light chain sequence and heavy chain sequence of the trastuzumab antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Also included are those antibodies that retain Her-2 binding activity after conservative amino acid substitutions to the above-mentioned antibodies.
  • the antibody-drug conjugates of the present invention can be preferably administered to mammals, more preferably humans.
  • the substance to be used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention can be appropriately selected from formulation additives or others commonly used in the art in consideration of the dose and concentration to be administered.
  • the antibody-drug conjugates of the present invention may be administered in the form of a pharmaceutical composition or pharmaceutical formulation containing one or more pharmaceutically suitable ingredients.
  • the above-mentioned pharmaceutical compositions or pharmaceutical preparations may typically contain one or more pharmaceutically acceptable carriers (such as sterile liquids (such as water and oils (including petroleum, animal, vegetable, or synthetic origin) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))).
  • sterile liquids such as water and oils (including petroleum, animal, vegetable, or synthetic origin) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.)
  • Oil such as peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • water is a more representative carrier.
  • saline solution as well as aqueous dextrose and glycerol solutions are also Can be used as liquid carrier, especially can be used for injection solution.Appropriate pharmaceutical excipients are known in this field.As required, the above-mentioned composition can also contain a trace amount of wetting agent or emulsifying agent, or pH buffering agent Examples of suitable pharmaceutical carriers are described in "Examples of W. Martin Carriers Parmaceutical Sciences" by E.W. Martin. The prescription corresponds to the mode of administration.
  • the introduction method includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration, for example, can be by infusion or bolus injection. In certain preferred embodiments, the administration of the antibody-drug conjugates described above is performed by infusion. Parenteral administration is the preferred route of administration.
  • the above-mentioned pharmaceutical composition is formulated into a pharmaceutical composition for intravenous administration to humans, and is formulated according to conventional procedures.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the above-mentioned pharmaceutical composition may further contain a solubilizer and a local anesthetic (eg, lidocaine) for relieving pain at the injection site, as required.
  • a solubilizer eg, lidocaine
  • the above ingredients may be supplied either as a dry freeze-dried powder or anhydrous concentrate in a sealed container (eg, an ampule or sachet, etc., which indicates the amount of active agent), respectively, Or mixed together in unit dosage form.
  • the above-mentioned medicine When the above-mentioned medicine is intended to be administered by infusion, for example, the above-mentioned medicine may be put into an infusion bottle containing sterilized pharmaceutical-grade water or saline.
  • the above-mentioned drugs When the above-mentioned drugs are administered by injection, ampoules of sterile water for injection or saline may be provided so that, for example, the above-mentioned components are mixed before administration.
  • the pharmaceutical composition or pharmaceutical preparation of the present invention may be a pharmaceutical composition or pharmaceutical preparation containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition or pharmaceutical preparation containing the antibody-drug conjugate and at least one other cancer therapeutic agent pharmaceutical composition.
  • the antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, whereby the anticancer effect can be enhanced.
  • Other anticancer agents used for this purpose may be administered to the individual simultaneously, separately or sequentially with the antibody-drug conjugate, or may be administered at varying intervals.
  • cancer therapeutic agents include albumin-bound paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, or international publications.
  • Such pharmaceutical compositions can be formulated in the form of freeze-dried preparations or liquid preparations as preparations having a selected composition and necessary purity.
  • a preparation When a preparation is formed as a freeze-dried preparation, it may be a preparation containing appropriate preparation additives available in the art.
  • a preparation can be formed as a liquid preparation containing various preparation additives usable in the art.
  • composition and concentration of the pharmaceutical composition vary depending on the administration method, but the affinity of the antibody-drug conjugate contained in the pharmaceutical composition of the present invention for the antigen of the antibody-drug conjugate, that is, the affinity for the antigen, varies. Considering the dissociation constant (Kd value), when the affinity is higher (the Kd value is lower), the drug effect can be exhibited even with a small dose. Therefore, when determining the administration amount of the antibody-drug conjugate, the administration amount can also be set based on the state of the affinity of the antibody-drug conjugate with the antigen.
  • the antibody-drug conjugate of the present invention When the antibody-drug conjugate of the present invention is administered to a human, for example, it may be administered once at about 0.001 to 100 mg/kg, or may be administered multiple times at intervals of 1 to 180 days.
  • the antibody-drug conjugates, pharmaceutical compositions, and pharmaceutical preparations of the present invention can be used to prevent and/or treat tumors or cancers.
  • the tumor or cancer targeted for prevention and/or treatment may be any cancer cell that expresses a protein recognized by the antibody in the antibody-drug conjugate.
  • the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma tumor, liver cancer, bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer.
  • a prophylactically or therapeutically effective amount of an antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the invention is administered to a subject in need thereof for inhibiting the growth, proliferation or migrate.
  • kits for inhibiting the growth, proliferation or migration of cancer cells comprising the antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the present invention.
  • the antibody-drug conjugate of the present invention has fast and efficient tumor cell killing activity, and at the same time has good biocompatibility, low immunogenicity, biological safety and stability.
  • the linker structure of the present invention such as formula (II) has the following advantages: (1) the linker structure of the present invention has suitable molecular weight and hydrophobicity, and has a higher drug loading (DAR, drug to antibody ratio)>7; (2) (3) the present invention The release of the linker structure is especially suitable for the preferred toxins gimatecan, jimitecan, and more preferably jimitecan, which are matched with their cytotoxicity, pharmacokinetics, and tumor inhibitory properties, and the linker is self-cleaving.
  • the speed is faster, which is conducive to the rapid release of toxin molecules, and greatly enhances the drug efficacy; (4)
  • the size, physicochemical properties and coupling sites of the joint designed by the present invention will not affect the physiological activity of the antibody; (5)
  • the present invention The synthesis method of the linker compound is simple and suitable for industrial production.
  • the anti-tumor compound of the present invention selects gimatecan and gimatecan.
  • the toxicity of gimatecan is about 10 times that of SN-38, which is comparable to that of ixatecan, but its safety is much better than that of ixatecan.
  • Satecan which is available alone as an oral preparation.
  • gemnotecan belongs to the camptothecin toxoid, which is substituted at the 10-position hydroxyl group and the 9-position allyl group, and has good antitumor activity.
  • the present invention connects gimatecan and gimatecan with a certain hydrophilic linker by optimizing the design of the linker, so as to increase the targeted drug delivery and solve the problem of water solubility of the toxin molecule. problem.
  • the linker molecule of formula (I) designed by the present invention has a more stable connection with the antibody, reduces the possibility of falling off at the non-target position, improves the safety, and improves the safety. Can achieve high drug loading.
  • the linker gimatecan has a strong ability to penetrate cell membranes, allowing them to kill nearby cancer cells after killing the cancer cells that engulfed the ADC.
  • trasstuzumab antibody (Herceptin antibody) was purchased from Genentech Inc.
  • ADC-137 was only used as a control, and was purchased from Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0.
  • hRS7 antibody was produced in CHO cells.
  • the expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods, and the nucleotide sequences of the light chain and heavy chain of the hRS7 antibody were shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Insert the above two sequences into the same expression vector, extract and prepare transfection plasmids in large quantities, and transfect them into CHO-K1 cells (ATCC CCL-61).
  • the specific transfection and antibody preparation processes are as follows:
  • Cell culture CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium at 37°C, 7% CO 2 , 140 rpm, and 90% relative humidity;
  • the highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity (GE, Mab Select SuRe) and ion exchange (GE, Capto S).
  • the molecular weight and purity of the purified antibodies were analyzed by SDS-PAGE and SEC-HPLC.
  • SDS-PAGE showed that the molecular weight of the prepared hRS7 was in line with expectations, and the purity of the antibody was 99.1% measured by SEC-HPLC.
  • tert-butyl 5-(cyanoethynyl)picolinate A-3 (1.2 g, 5.3 mmol) in DCM (80 mL) was added TFA (80 mL). The mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The residue was triturated with ether (150 ml). The mixture was filtered and the filter cake was washed with ether (50 ml). The filter cake was dried in vacuo to give tert-butyl 5-(cyanoethynyl)picolinate target A (600 mg, 66% yield) as a white solid.
  • tert-Butyl 6-bromonicotinate B-1 (5 g, 19.46 mmol) and 2-yn-1-propanol (3.26 g, 25.3 mmol) were added to the flask followed by Pd( PPh3 ) 2Cl2 ( 0.98 g, 1.4 mmol) and CuI (0.45 g, 2.3 mmol). After purging the flask with nitrogen 3 times to remove oxygen. DMF (64 mL) and TEA (10 mL) were added via syringe. The reaction was stirred at 80°C overnight and monitored by LCMS. After the reaction was consumed, the reaction was quenched with saturated ammonium chloride (300 mL).
  • the hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound CN-C-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 3 hours to obtain the conjugated crude product ADC-1.
  • the coupling reaction crude product is detected by SEC, and the SEC chromatographic conditions are as follows:
  • the purified ADC-1 is shown in Figure 1.
  • the samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
  • the higher peak represents the antibody part
  • the lower peak represents the camptothecin compound part.
  • the retention time positions of the two peaks are the same, and it can be seen that the two have formed ADC conjugates.
  • the absorbance values of conjugate and naked antibody at 280nm and 363nm were measured by UV spectrophotometer.
  • the concentration of gemnotecan in the conjugate was calculated from the absorbance at 363 nm according to the standard curve.
  • the concentration of antibody in the conjugate was calculated by subtracting the absorbance of gemitecan at 280 from the absorbance at 280 nm.
  • the DAR value was calculated from the ratio of these two concentrations to be 6.5. That is, n is 6.5.
  • CN-C in this embodiment can be replaced by CN-A, CN-B, CN-D; gemitecan (CMTC) can be replaced by SN-38, gematecan (GMTC).
  • CMTC gemitecan
  • GMTC gematecan
  • the hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound SMCC-PEG2-GGFG-PABC-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-2.
  • the detection method is as described in step IV-3 of Example 4.
  • the purified ADC-2 was similar to ADC-1 of Figure 1 .
  • the samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
  • DAR was determined as described in Example 4, Step IV-5.
  • the DAR value of ADC-2 is 7.0. That is, n is 7.0.
  • CMTC Gematecan
  • GMTC gematecan
  • the hRS7 antibody of Example 1 was first reduced with 6 times the amount of TCEP in a 5 mg/mL solution of pH 7.2 PBS/EDTA for 2 hours at room temperature. Next, a 16-fold amount of the compound CN-C-PEG 2 -GGFG-PABC-CMTC dissolved in DMSO was added to the antibody solution (final DMSO concentration 10%). The reaction was stirred at room temperature and protected from light for 3 hours to obtain the conjugated crude product ADC-3.
  • the detection method is as described in step IV-3 of Example 4.
  • the purified ADC-3 is similar to ADC-1 of Figure 1 .
  • the samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
  • DAR was determined as described in Example 4, Step IV-5.
  • the DAR value of ADC-3 is 6.8. That is, n is 6.8.
  • CN-C in this embodiment can be replaced by CN-A, CN-B, CN-D; gemitecan (CMTC) can be replaced by SN-38, gematecan (GMTC).
  • CMTC gemitecan
  • GMTC gematecan
  • the hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound MC-GGFG-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-4.
  • the detection method is as described in step IV-3 of Example 4.
  • the purified ADC-4 is similar to ADC-1 of Figure 1 .
  • the samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
  • DAR was determined as described in Example 4, Step IV-5.
  • the DAR value of ADC-4 is 7.4. That is, n is 7.4.
  • the hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound A dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the coupled crude product ADC-X-a.
  • the Herceptin antibody was first reduced in 6 mg/mL pH 7.2 PBS solution with 9.5 times the amount of TCEP for 30 minutes at room temperature. Next, a 12-fold substance amount of Compound A dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-X-b.
  • the coupling reaction crude product is detected by SEC, and the SEC chromatographic conditions are as follows:
  • the absorbance values of conjugate and naked antibody at 280nm and 363nm were measured by UV spectrophotometer.
  • the concentration of Gimatecan in the conjugate was calculated from the absorbance at 363 nm according to the standard curve.
  • the concentration of antibody in the conjugate was calculated by subtracting the absorbance of gimatecan at 280 from the absorbance at 280 nm. The result is as shown.
  • the DAR value of ADC-X-a was calculated from the ratio of these two concentrations to be 2.7, ie, n was 2.7.
  • Herceptin antibody When the antibody is Herceptin antibody:
  • the DAR value of ADC-X-b was calculated from the ratio of these two concentrations to be 7.3, ie, n was 7.3.
  • the hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 7 times the amount of TCEP at 37°C for 2 hours. Next, a 16-fold amount of Compound G dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 20 minutes to obtain the conjugated crude product ADC-5.
  • the detection method is as described in step I-3 of Example 9.
  • DAR was determined as described in Example 9, steps 1-5.
  • the SEC-HPLC profile of ADC-5 is shown in FIG. 2 .
  • the DAR value of ADC-5 is 7.8. That is, n is 7.8.
  • the DAR value of ADC-6 is 5.5. That is, n is 5.5.
  • ADC-8 has a DAR of 3.3. That is, n is 3.3.
  • the DAR value of ADC-10 is 3.5. That is, n is 3.5.
  • the DAR value of ADC-11 is 7.1. That is, n is 7.1.
  • the DAR value of ADC-12 is 6.2. That is, n is 6.2.

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Abstract

An antibody-drug conjugate, a preparation method therefor, and the use thereof. The antibody-drug conjugate is prepared by means of connecting compound (T) with antibody (AB) via a linker represented by the following formula (III) or (VII), wherein T represents a compound represented by formula (II). The antibody-drug conjugate has a faster onset time, a longer drug half-life, a more moderate stability, a good biocompatibility, a low immunogenicity and is safe, and can prevent aggregation and has excellent anti-tumor effects. AB-S-Q1-L1-L2-La-T (IV); AB-S-Q2-L3-L4-LP-Lb-T(VIII); -Q1-L1-L2-La- (III); -Q2-L3-L4-LP-Lb- (VII)

Description

抗体-药物偶联物及其应用Antibody-drug conjugates and their applications 技术领域technical field
本发明涉及生物医药领域,具体地说,本发明涉及一类全新结构的接头结构、包括该接头结构的药物-接头化合物,以及包括所述药物-接头化合物的抗体-药物偶联物、上述药物-接头化合物和抗体-药物偶联物的制备方法及应用。The present invention relates to the field of biomedicine, in particular, the present invention relates to a new type of linker structure, a drug-linker compound including the linker structure, and an antibody-drug conjugate including the drug-linker compound, the above-mentioned drugs - Preparation methods and applications of linker compounds and antibody-drug conjugates.
背景技术Background technique
抗体-药物偶联物(antibody-drug conjugate,下文简称“ADC”或“偶联物”)相对于单纯的抗体药物而言已经显示出独特的优势,其通过将具有肿瘤细胞表面抗原结合特异性的单克隆抗体与具有生物活性的细胞毒素相连,从而将抗体的肿瘤识别靶向性和细胞毒素的高效杀伤作用相结合,巧妙的同时解决了抗体疗效偏低和毒素缺乏靶向性导致毒性过大的缺陷。这使得ADC与传统的抗肿瘤药物相比,在准确的靶向肿瘤细胞的同时,降低对正常细胞的影响,大幅提高了抗肿瘤药物的有效性和安全性。Antibody-drug conjugates (hereinafter referred to as "ADCs" or "conjugates") have shown unique advantages over pure antibody drugs, by combining them with tumor cell surface antigen binding specificity. The monoclonal antibody is linked to a biologically active cytotoxin, thereby combining the tumor recognition targeting of the antibody with the high-efficiency killing effect of the cytotoxin. big flaw. Compared with traditional anti-tumor drugs, ADC can accurately target tumor cells while reducing the impact on normal cells, greatly improving the effectiveness and safety of anti-tumor drugs.
ADC一般包括三个部分:抗体、接头和毒素。抗体为ADC的靶向功能大分子,发挥将毒素富集到肿瘤组织部位附近以提高毒素的杀伤效率的作用。目前针对各大热门靶点如HER-2、Trop-2、PDL-1、CD30等的抗体开发如火如荼,同时也带动了针对这些靶点的ADC开发。ADC generally consists of three parts: antibody, linker and toxin. Antibodies are targeted functional macromolecules of ADCs, which play the role of enriching toxins near the tumor tissue site to improve the killing efficiency of toxins. At present, the development of antibodies against major popular targets such as HER-2, Trop-2, PDL-1, CD30, etc. is in full swing, and it also drives the development of ADCs against these targets.
ADC接头分为可裂解和不可裂解两种类型,理想的接头应符合“稳定性好、释放效率高”的要求,即使得ADC在血液循环中保持稳定,并在到达肿瘤细胞内后能快速释放毒素、杀伤肿瘤细胞。接头对于ADC是否能发挥作用至关重要,不稳定的接头会导致ADC脱靶,增加安全性风险,而过于稳定的接头则影响毒素释放速度,从而影响药效。另外,如何根据特定抗体和特定毒素的理化性质、空间结构、靶细胞生理环境等因素选择适用于具体ADC的接头结构,是目前ADC研发中的热点问题,仍有迫切的研发需求。ADC linkers are divided into two types: cleavable and non-cleavable. The ideal linker should meet the requirements of "good stability and high release efficiency", that is, the ADC remains stable in the blood circulation and can be quickly released after reaching the tumor cells. toxins, killing tumor cells. The linker is crucial for the ADC to function. An unstable linker will lead to off-target ADC and increase the safety risk, while an overly stable linker will affect the release rate of the toxin, thereby affecting the efficacy of the drug. In addition, how to select a linker structure suitable for a specific ADC according to the physicochemical properties, spatial structure, and target cell physiological environment of specific antibodies and specific toxins is a hot issue in the current ADC research and development, and there is still an urgent need for research and development.
ADC的毒素部分为发挥杀伤作用的药物小分子,一般通过抑制DNA或蛋白合成、抑制细胞有丝分裂等方式来杀伤肿瘤细胞。目前用于ADC开发的毒素主要包括微管抑制剂美登素类(参见EP0425235、US5208020、US5416064、US7276497)和奥瑞他汀(MMAE/MMAF,参见US2016304621A)。目前上市的代表性药物有Genetech开发的T-DM1,T-DM1是一种由通过稳定的硫醚连接子MCC(4-[N-顺丁烯二酰亚胺甲基]环己烷-1-羧酸酯)缀合至类美登素类毒素DM1的曲妥株单抗组成的ADC(US8337856)。其他种类的细胞毒素还包括卡奇霉素类(Calicheamicin,参见US5606040),苯并二吡咯类衍生物(duocarmycin,参见US7129261),咯并苯二氮卓类(PBDs,参见WO2005/040170)和喜树碱类衍生物。其中喜树碱类衍生物包括SN-38、CPT-11、依沙替康、9-硝基喜树碱、10-羟基喜树碱等。目前采用喜树碱类毒素的ADC是第一三共株式会社开发的DS-8201,它采用的是细胞毒性较强的依沙替康作为毒素部分。The toxin part of ADC is a small drug molecule that plays a killing role, and generally kills tumor cells by inhibiting DNA or protein synthesis, inhibiting cell mitosis, and the like. The toxins currently used for ADC development mainly include microtubule inhibitors maytansinoids (see EP0425235, US5208020, US5416064, US7276497) and auristatin (MMAE/MMAF, see US2016304621A). The representative drugs currently on the market are T-DM1 developed by Genetech. T-DM1 is a compound formed by a stable thioether linker MCC (4-[N-maleimidomethyl]cyclohexane-1 - carboxylate) conjugated to an ADC consisting of trastuzumab conjugated to the maytansinoid toxoid DM1 (US8337856). Other classes of cytotoxins include Calicheamicin (see US5606040), benzodipyrrole derivatives (duocarmycin, see US7129261), pyrrolobenzodiazepines (PBDs, see WO2005/040170) and Derivatives of tree alkaloids. The camptothecin derivatives include SN-38, CPT-11, ixatecan, 9-nitrocamptothecin, 10-hydroxycamptothecin and the like. Currently, the ADC using camptothecin toxoid is DS-8201 developed by Daiichi Sankyo Co., Ltd. It uses ixatecan, which is highly cytotoxic, as the toxin part.
然而,由无论是T-DM1,还是DS-8201,都仍旧存在以下不足:However, both T-DM1 and DS-8201 still have the following shortcomings:
就T-DM1而言,首先,T-DM1的药效不足,一是因为其DAR低,只有3-4,二是因为其采用了SMCC的接头与DM-1连接,而SMCC为不可降解的接头,这降低了T-DM1的药效;其次,T-DM1使用DM-1作为毒素,该毒素为微管抑制剂,细胞膜的透过性较弱;再次,T-DM1存在降低白细胞等较严重的毒副作用。As far as T-DM1 is concerned, first of all, the efficacy of T-DM1 is insufficient, one is because its DAR is low, only 3-4, and the other is because it uses the linker of SMCC to connect with DM-1, and SMCC is non-degradable. linker, which reduces the efficacy of T-DM1; secondly, T-DM1 uses DM-1 as a toxin, which is a microtubule inhibitor, and the permeability of cell membranes is weak; thirdly, the presence of T-DM1 reduces white blood cells Serious side effects.
就DS-8201而言,依沙替康虽然毒性高于SN-38 10倍,但因依沙替康细胞杀伤活性强,无法单独成药,其对接头的稳定性要求较高,其ADC的接头也仅有一个酶裂解位点,这一定程度上也延长了ADC在胞内的起效时间。此外,依沙替康在血液中半衰期短,这虽然降低了毒副作用,但面临着药物半衰期短的风险。As far as DS-8201 is concerned, although ixatecan is 10 times more toxic than SN-38, it cannot be used as a single drug due to its strong cell-killing activity. There is also only one enzymatic cleavage site, which also prolongs the onset time of ADC in cells to some extent. In addition, ixatecan has a short half-life in the blood, which reduces toxic side effects, but faces the risk of a short half-life of the drug.
由此可见,本领域仍有开发更为有效,兼顾安全的喜树碱类ADC的需求,制备具有更快的起效时间,更长的药物半衰期,同时在稳定性、亲疏水性、防聚集作用等安全性指标方面具有优势的喜树碱类ADC迫在眉睫。It can be seen that there is still a need to develop more effective and safe camptothecin-based ADCs in this field. The preparation has a faster onset time, longer drug half-life, and at the same time, it has the advantages of stability, hydrophilicity and hydrophobicity, and anti-aggregation. Camptothecin ADCs with superior safety indicators are imminent.
发明内容SUMMARY OF THE INVENTION
为解决上述问题,本发明人设计了适用于喜树碱衍生物的接头结构,将其作为喜树碱衍生物与抗体的连接结构,从而形成具有更快起效时间、更长药物半衰期、更适中的稳定性和良好安全性、防聚集的抗体-药物偶联物,该ADC具有优异的抗肿瘤效果。In order to solve the above problems, the inventors designed a linker structure suitable for camptothecin derivatives, and used it as a linking structure between camptothecin derivatives and antibodies, so as to form a linker structure with faster onset time, longer drug half-life, and better drug resistance. Moderate stability and good safety, anti-aggregation antibody-drug conjugate, this ADC has excellent anti-tumor effect.
本发明的第一方面,提供一种式(I)表示的化合物,A first aspect of the present invention provides a compound represented by formula (I),
Figure PCTCN2021123020-appb-000001
Figure PCTCN2021123020-appb-000001
其中,R 11为羧基取代的C 1-C 6烷基,R 12为氰基取代的C 2-C 6炔基,X、Y、X’和Y’中有1-2个C原子被N原子取代;优选地,R 11为羧基取代的C 1-C 3烷基,R 12为氰基取代的C 2-C 3炔基。 Wherein, R 11 is a carboxy-substituted C 1 -C 6 alkyl group, R 12 is a cyano-substituted C 2 -C 6 alkynyl group, and 1-2 C atoms in X, Y, X' and Y' are replaced by N Atom substitution; preferably, R 11 is a carboxy-substituted C 1 -C 3 alkyl group, and R 12 is a cyano-substituted C 2 -C 3 alkynyl group.
在一些实施方案中,X、Y、X’和Y’中有且只有1个C原子被N原子取代。In some embodiments, X, Y, X' and Y' have and only 1 C atom substituted with a N atom.
在一些实施方案中,X、Y、X’和Y’中有2个C原子被N原子取代,且X、Y中有且只有1个C原子被N原子取代,以及X’、Y’中有且只有1个C原子被N原子取代。In some embodiments, 2 C atoms in X, Y, X' and Y' are substituted with N atoms, and only 1 C atom in X, Y is substituted with N atoms, and X', Y' There is one and only one C atom replaced by N atom.
在一些实施方案中,该化合物结构如下所示,In some embodiments, the compound structure is shown below,
Figure PCTCN2021123020-appb-000002
Figure PCTCN2021123020-appb-000002
在一些实施方案中,该化合物作为抗体-药物偶联物中的连接单元,通过R 12的炔基碳与存在于抗体的铰链部的二硫键部分形成硫醚键而与抗体连接。 In some embodiments, the compound is linked to the antibody as the linking unit in the antibody-drug conjugate through the formation of a thioether bond between the alkynyl carbon of R12 and the disulfide moiety present in the hinge portion of the antibody.
本发明的第二方面,提供一种式(I)表示的化合物的制备方法,其包括如下步骤:A second aspect of the present invention provides a method for preparing a compound represented by formula (I), comprising the steps of:
(1)让5-溴吡啶-2-羧酸在Boc 2O,DMAP,t-BuOH存在下反应;所述5-溴吡啶-2-羧酸可替换为6-溴烟酸或5-溴嘧啶-2-羧酸; (1) Let 5-bromopyridine-2-carboxylic acid react in the presence of Boc 2 O, DMAP, t-BuOH; the 5-bromopyridine-2-carboxylic acid can be replaced with 6-bromonicotinic acid or 5-bromonicotinic acid Pyrimidine-2-carboxylic acid;
(2)步骤(1)的反应产物与Pd(PPh 3) 2Cl 2,三乙胺,丙炔-3-醇在四氢呋喃中反应; (2) The reaction product of step (1) reacts with Pd(PPh 3 ) 2 Cl 2 , triethylamine, and propyn-3-ol in tetrahydrofuran;
(3)步骤(2)的反应产物与TEMPO,PhI(OAC) 2,NH 4OAC在CH 3CN/H 2O为9:1的溶液中反应; (3) The reaction product of step (2) reacts with TEMPO, PhI(OAC) 2 , and NH 4 OAC in a solution of CH 3 CN/H 2 O in a ratio of 9:1;
(4)步骤(3)产物在TFA/DCM的作用下生成产物。(4) The product of step (3) is produced under the action of TFA/DCM.
本发明的第三方面,提供一种式(IV)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其特征在于,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下式(III)表示的接头连接而成的:The third aspect of the present invention provides an antibody-drug conjugate represented by formula (IV), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof The solvate of the isomer or a pharmaceutically acceptable salt thereof, characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and an antibody ( AB) connected via a linker represented by the following formula (III):
AB-S-Q 1-L 1-L 2-L a-T     (IV) AB-SQ 1 -L 1 -L 2 -L a -T (IV)
Figure PCTCN2021123020-appb-000003
Figure PCTCN2021123020-appb-000003
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
其中,in,
式(II)中,In formula (II),
R 1选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、被-OC(=O)R 13或-NR 7R 8取代的C 1-C 6烷基、被-SiMe 3取代的C 1-C 6烷基、或-CH=N-O-(C 1-C 6烷基); R 1 is selected from hydrogen, halogen, hydroxy, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C substituted by -OC(=O)R 13 or -NR 7 R 8 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with -SiMe 3 , or -CH=NO-(C 1 -C 6 alkyl);
R 2选自氢、卤素、羟基、硝基、氨基、饱和或不饱和C 1-C 6烷基、C 1-C 6烷氧基、被-NR 7R 8取代的C 1-C 6烷基或被C 2-C 6烯基取代的C 1-C 6烷基; R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkane substituted by -NR 7 R 8 group or C 1 -C 6 alkyl substituted by C 2 -C 6 alkenyl;
R 3选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、-NR 7R 8C(=O)O-基或5-6元含氮杂环基-C(=O)-C 1-C 6烷氧基; R 3 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, -NR 7 R 8 C(=O)O-yl or 5-6 membered nitrogen-containing heterocyclyl-C(=O)-C 1 -C 6 alkoxy;
R 4选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、或C 1-C 6烷氧基; R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
或者R 1和R 2可以连接在一起与母体部分形成任选被R 9取代的5-6元环; Alternatively R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
或者R 3和R 4可以连接在一起与母体部分形成任选被R 9取代的5-6元含氧杂环; Alternatively R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
R 7和R 8每次出现时各自独立地选自氢、C 1-C 6烷基、被羟基或氨基取代的C 1-C 6酰基;或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环; Each occurrence of R 7 and R 8 is independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
优选地,R 7和R 8每次出现时各自独立地选自氢、甲基、异丙基、
Figure PCTCN2021123020-appb-000004
或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环;
Preferably, each occurrence of R7 and R8 is independently selected from hydrogen, methyl, isopropyl,
Figure PCTCN2021123020-appb-000004
Alternatively R7 and R8 may together with the attached N atom form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R9 ;
R 9每次出现时各自独立地选自卤素、羟基、硝基、-NR 7R 8、C 1-C 6烷基、C 1-C 6烷氧基、任选被C 1-C 6烷基取代的哌啶基; Each occurrence of R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
优选地,R 9每次出现时各自独立地选自甲基、-NR 7R 8、哌啶基; Preferably, each occurrence of R 9 is independently selected from methyl, -NR 7 R 8 , piperidinyl;
R 13表示羧基取代的C 1-C 6烷基; R 13 represents a carboxy-substituted C 1 -C 6 alkyl group;
式(III)中,In formula (III),
L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)-CH 2OCH 2-(C=O)-,n 1表示1~24的整数,n 2表示1~4的整数; L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)-CH 2 OCH 2 -(C=O)-,n 1 represents an integer from 1 to 24, and n 2 represents an integer from 1 to 4;
L 2表示缬氨酸残基、胍氨酸残基、苯丙氨酸残基、赖氨酸残基、D-缬氨酸残基、甘氨酸残基、丙氨酸残基、天冬氨酸残基; L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
L a表示-NR 10-(CH 2)n 3-、-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-; L a represents -NR 10 -(CH 2 )n 3 -, -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 4 -O -(C=O)-;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
n 3表示1~4的整数,n 4表示1~4的整数; n 3 represents an integer from 1 to 4, and n 4 represents an integer from 1 to 4;
Aryl表示任选地被R 9取代的C 6-C 10芳基; Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
W为单键或
Figure PCTCN2021123020-appb-000005
其中,位置①表示与-NR 10-相连,位置②表示与(CH 2CH 2-O-)n 1-相连;
W is a single key or
Figure PCTCN2021123020-appb-000005
Wherein, position ① means connecting with -NR 10 -, and position ② means connecting with (CH 2 CH 2 -O-)n 1 -;
式(IV)中,In formula (IV),
Q 1为本发明第一方面所述化合物,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 to form an amide bond, and the alkynyl carbon of R 12 and the disulfide of the hinge part of the antibody are connected. bond to form a thioether bond,
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(III)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C( = O)- or -CH2- moiety of La in the linker represented by III).
在一些实施方案中,R 1表示氢、C 1-C 4烷基、被-NH(C 1-C 4烷基)取代的C 1-C 4烷基、被
Figure PCTCN2021123020-appb-000006
取代的C 1-C 4烷基、被-SiMe 3取代的C 1-C 4烷基、-CH=N-O-(C 3-C 6烷基)或-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH。
In some embodiments, R 1 represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -NH(C 1 -C 4 alkyl), C 1 -C 4 alkyl substituted by
Figure PCTCN2021123020-appb-000006
Substituted C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -SiMe 3 , -CH=NO-(C 3 -C 6 alkyl) or -(CH 2 ) 2 O(C=O )( CH2 ) 2 (C=O)OH.
在一些实施方案中,R 1表示氢、乙基、
Figure PCTCN2021123020-appb-000007
取代的甲基、
Figure PCTCN2021123020-appb-000008
-CH=NO-叔丁基、-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH、。
In some embodiments, R 1 represents hydrogen, ethyl,
Figure PCTCN2021123020-appb-000007
substituted methyl,
Figure PCTCN2021123020-appb-000008
-CH=NO-tert-butyl, -(CH 2 ) 2 O(C=O)(CH 2 ) 2 (C=O)OH,.
在一些实施方案中,R 2表示氢、C 3-C 4烯基、硝基、氨基、被-N(C 1-C 4烷基) 2取代的C 1-C 4烷基或被C 2-C 4烯基取代的C 1-C 4烷基。 In some embodiments, R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 or C 2 -C 4 alkenyl substituted C 1 -C 4 alkyl.
在一些实施方案中,R 2表示氢、硝基、氨基、
Figure PCTCN2021123020-appb-000009
In some embodiments, R 2 represents hydrogen, nitro, amino,
Figure PCTCN2021123020-appb-000009
在一些实施方案中,R 3表示氢、卤素、羟基、C 1-C 4烷基、
Figure PCTCN2021123020-appb-000010
In some embodiments, R 3 represents hydrogen, halogen, hydroxy, C 1 -C 4 alkyl,
Figure PCTCN2021123020-appb-000010
在一些实施方案中,R 3表示氢、F、羟基、甲基、
Figure PCTCN2021123020-appb-000011
In some embodiments, R 3 represents hydrogen, F, hydroxyl, methyl,
Figure PCTCN2021123020-appb-000011
在一些实施方案中,R 4表示氢或卤素。 In some embodiments, R 4 represents hydrogen or halogen.
在一些实施方案中,R 4表示氢或F。 In some embodiments, R 4 represents hydrogen or F.
在一些实施方案中,R 1和R 2连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000012
其中
Figure PCTCN2021123020-appb-000013
部分表示连接于母体基团的键。
In some embodiments, R 1 and R 2 are joined together to form the group shown below
Figure PCTCN2021123020-appb-000012
in
Figure PCTCN2021123020-appb-000013
A moiety represents a bond to the parent group.
在一些实施方案中,R 1和R 2连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000014
其中
Figure PCTCN2021123020-appb-000015
部分表示连接于母体基团的键。
In some embodiments, R 1 and R 2 are joined together to form the group shown below
Figure PCTCN2021123020-appb-000014
in
Figure PCTCN2021123020-appb-000015
A moiety represents a bond to the parent group.
在一些实施方案中,R 3和R 4连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000016
其中
Figure PCTCN2021123020-appb-000017
部分表示连接于母体基团的键。
In some embodiments, R and R are joined together to form the group shown below
Figure PCTCN2021123020-appb-000016
in
Figure PCTCN2021123020-appb-000017
A moiety represents a bond to the parent group.
在一些实施方案中,式(II)表示的化合物如下所示:In some embodiments, the compound represented by formula (II) is shown below:
Figure PCTCN2021123020-appb-000018
Figure PCTCN2021123020-appb-000018
Figure PCTCN2021123020-appb-000019
Figure PCTCN2021123020-appb-000019
Figure PCTCN2021123020-appb-000020
Figure PCTCN2021123020-appb-000020
在一些实施方案中,式(IV)表示的化合物为吉马替康或吉咪替康,更优选为吉咪替康:In some embodiments, the compound represented by formula (IV) is gimatecan or gimitecan, more preferably gimatecan:
Figure PCTCN2021123020-appb-000021
Figure PCTCN2021123020-appb-000021
在一些实施方案中,L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)CH 2-O-CH 2-(C=O)-,n 1表示4~12的整数(优选8),n 2表示1~2的整数(优选2),R 10表示氢或C 1-C 4烷基(优选甲基)。 In some embodiments, L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)CH 2 -O-CH 2 - (C=O)-, n 1 represents an integer of 4-12 (preferably 8), n 2 represents an integer of 1-2 (preferably 2), R 10 represents hydrogen or C 1 -C 4 alkyl (preferably methyl) .
在一些实施方案中,L 2表示赖氨酸残基。 In some embodiments, L 2 represents a lysine residue.
在一些实施方案中,L a表示-NR 10-Aryl-(CH 2)n 4-O-(C=O)-,n 4表示1~2的整数,R 10表示氢或C 1-C 4烷基(优选甲基),Aryl表示苯环基团,优选地,-NR 10-基团和-(CH 2)n 4-基团位于苯环的对位。 In some embodiments, L a represents -NR 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, n 4 represents an integer from 1 to 2, and R 10 represents hydrogen or C 1 -C 4 Alkyl (preferably methyl), Aryl represents a benzene ring group, preferably, the -NR 10 - group and the -(CH 2 )n 4 - group are located in the para position of the benzene ring.
在一些实施方案中,L a表示
Figure PCTCN2021123020-appb-000022
In some embodiments, La represents
Figure PCTCN2021123020-appb-000022
在一些实施方案中,式(III)表示的接头为选自以下所示的基团:In some embodiments, the linker represented by formula (III) is a group selected from the group consisting of:
Figure PCTCN2021123020-appb-000023
Figure PCTCN2021123020-appb-000023
Figure PCTCN2021123020-appb-000024
Figure PCTCN2021123020-appb-000024
在一些实施方案中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个,例如3.3、3.5、5.5、6.2、6.5、6.6、6.8、7.0、7.1、7.2、7.4、7.5或7.8个。In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8, such as 3.3, 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
在一些实施方案中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段。In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
在一些实施方案中,所述抗体选自抗Her-2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER-4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体;优选地,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体。In some embodiments, the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 Antibodies, CD30 antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies.
在一些实施方案中,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv。 In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
在一些实施方案中,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。In some embodiments, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody The nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
在一些实施方案中,所述抗体为抗Her-2抗体,其中,所述抗Her-2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH 氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的CDR3;优选地,所述抗Her-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。In some embodiments, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2 composed of sequence, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
本发明的第四方面,提供一种式(VI)表示的接头-药物中间体化合物,其特征在于,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(V)表示的接头连接而成的:The fourth aspect of the present invention provides a linker-drug intermediate compound represented by formula (VI), characterized in that T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following The joints represented by formula (V) are connected:
Q 1-L 1-L 2-L a-T     (VI) Q 1 -L 1 -L 2 -L a -T (VI)
Figure PCTCN2021123020-appb-000025
Figure PCTCN2021123020-appb-000025
Q 1-L 1-L 2-L a-     (V) Q 1 -L 1 -L 2 -L a - (V)
其中,in,
R 1、R 2、R 3、R 4的定义如的定义如本发明说明书所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention;
L 1、L 2、L a的定义如本发明说明书所述; The definitions of L 1 , L 2 and La are as described in the specification of the present invention;
Q 1为本发明第一方面所述化合物,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula L 1 to form an amide bond, and the compound represented by the formula (II) is represented by the 19-position hydroxyl group. Oxygen is used as a linking site, or when R3 or R4 is a hydroxyl group, the oxygen in the hydroxyl group of R3 or R4 is used as a linking site, and is connected to the right end - C( = O)- or -CH2- moiety.
在一些实施方案中,所述式(II)所示的化合物如前所述。In some embodiments, the compound of formula (II) is as previously described.
在一些实施方案中,所述接头-药物中间体化合物是选自以下的化合物,In some embodiments, the linker-drug intermediate compound is a compound selected from the group consisting of,
Figure PCTCN2021123020-appb-000026
Figure PCTCN2021123020-appb-000026
Figure PCTCN2021123020-appb-000027
Figure PCTCN2021123020-appb-000027
Figure PCTCN2021123020-appb-000028
Figure PCTCN2021123020-appb-000028
Figure PCTCN2021123020-appb-000029
Figure PCTCN2021123020-appb-000029
Figure PCTCN2021123020-appb-000030
Figure PCTCN2021123020-appb-000030
本发明的第五方面,提供通式(III)所示的接头结构:The fifth aspect of the present invention provides the linker structure shown in general formula (III):
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
其中,Q 1、L 1、L 2、L a的定义的定义如本发明说明书所述。 The definitions of Q 1 , L 1 , L 2 , and La are as described in the specification of the present invention.
本发明的第六方面,提供本发明第三方面的抗体-药物偶联物的制备方法,所述方法包括:The sixth aspect of the present invention provides a method for preparing the antibody-drug conjugate of the third aspect of the present invention, the method comprising:
Figure PCTCN2021123020-appb-000031
Figure PCTCN2021123020-appb-000031
使式(VI)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(VI)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (VI) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (VI) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
其中,R 1、R 2、R 3、R 4的定义如的定义如本发明说明书所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention;
Q 1、L 1、L 2、L a的定义如的定义如本发明说明书所述; The definitions of Q 1 , L 1 , L 2 , and La are as described in the specification of the present invention;
T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site. The oxygen of the above-mentioned formula (V) is connected to the right end -C( = O)- or -CH 2 - part of La in the linker represented by the above formula (V);
AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
本发明的第七方面,提供本发明第四方面的接头-药物中间体化合物的制备方法,所述方法包括:The seventh aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the fourth aspect of the present invention, the method comprising:
(1)将N-[(9H-芴-9-基甲氧基)羰基]-N'-[(4-甲氧基苯基)二苯基甲基]-L-赖氨酸(CN-CMTC-1)和PABOH溶解在二氯甲烷:甲醇的溶液中,在EEDQ的作用下反应,重结晶纯化得到产物;(1) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN- CMTC-1) and PABOH were dissolved in dichloromethane: methanol solution, reacted under the action of EEDQ, recrystallized and purified to obtain the product;
(2)用哌啶乙腈溶液处理步骤(1)产物,而后纯化产物;(2) treating the product of step (1) with a solution of piperidine in acetonitrile, and then purifying the product;
(3)用DCC,NHS与O-(2-叠氮乙基)-O-[2-(二羟乙酰基-氨基)乙基]七聚乙二醇(CN-CMTC-4)在DMF溶液中反应生成CN-CMTC-4活性酯;(3) Using DCC, NHS and O-(2-azidoethyl)-O-[2-(diglycolyl-amino)ethyl]heptaethylene glycol (CN-CMTC-4) in DMF solution The middle reaction generates CN-CMTC-4 active ester;
(4)步骤(3)的活性酯与步骤(2)的产物反应生成化合物;(4) the active ester of step (3) reacts with the product of step (2) to generate compound;
(5)吉咪替康-Boc或吉马替康-Boc用三光气,DMAP,与二氯甲烷作用生成甲酰氯化合物,再加入步骤(4)的反应化合物,后用TFA/DCM脱保护处理;(5) Gematecan-Boc or Gematecan-Boc is treated with triphosgene, DMAP, and dichloromethane to form a formyl chloride compound, and then the reaction compound of step (4) is added, and then deprotected with TFA/DCM. ;
(6)步骤(5)的产物与本发明第一方面所述的化合物进行Click反应,用TFA/DCM处理后得最终产物;(6) The product of step (5) is subjected to Click reaction with the compound described in the first aspect of the present invention, and the final product is obtained after being treated with TFA/DCM;
任选地,步骤(6)还可用以下步骤替代:步骤(5)的产物与SM-1加入DMSO/H 2O的溶液中,再加入CuBr催化,反应完全,纯化后加入TFA/DCM脱保护,得最终产物; Optionally, step (6) can also be replaced by the following steps: the product of step (5) and SM-1 are added to a solution of DMSO/H 2 O, then CuBr is added to catalyze the reaction, the reaction is complete, and after purification, TFA/DCM is added for deprotection , the final product is obtained;
所述SM-1为The SM-1 is
Figure PCTCN2021123020-appb-000032
Figure PCTCN2021123020-appb-000032
本发明的第八方面,提供一种式(VIII)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其特征在于,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下式(VII)表示的接头连接而成的:The eighth aspect of the present invention provides an antibody-drug conjugate represented by formula (VIII), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof The solvate of the isomer or a pharmaceutically acceptable salt thereof, characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and an antibody ( AB) connected via a linker represented by the following formula (VII):
AB-S-Q 2-L 3-L 4-L P-L b-T     (VIII) AB-SQ 2 -L 3 -L 4 -L P -L b -T (VIII)
Figure PCTCN2021123020-appb-000033
Figure PCTCN2021123020-appb-000033
-Q 2-L 3-L 4-L P-L b-     (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
其中,in,
式(II)中,In formula (II),
R 1、R 2、R 3、R 4的定义如本发明说明书所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention;
式(VII)中,In formula (VII),
L 3表示-Z-W-(CH 2CH 2-O)n 5-W’-、-(CH 2)n 5’-C(=O)-NR 10-(CH 2CH 2-O)n 5-或单键,n 5’表示1~3的整数,各n 5独立地表示1~8的整数,W、W’表示
Figure PCTCN2021123020-appb-000034
或单键,其中,W的位置①表示与Z相连,位置②表示与(CH 2CH 2-O-)n 5-相连,W’的位置①表示与(CH 2CH 2-O-)n 5-相连,位置②表示与L 4的-CH 2-相连,且W、W’不同时为
Figure PCTCN2021123020-appb-000035
Z表示-CH 2-Cyclo-C(=O)-NR 10-或单键,Cyclo表示环己烷基团,
L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'-, -(CH 2 )n 5' -C(=O)-NR 10 -(CH 2 CH 2 -O)n 5 - or a single bond, n 5' represents an integer of 1 to 3, each n 5 independently represents an integer of 1 to 8, and W and W' represent an integer
Figure PCTCN2021123020-appb-000034
Or a single bond, wherein, the position ① of W indicates that it is connected to Z, the position ② indicates that it is connected to (CH 2 CH 2 -O-)n 5 -, and the position ① of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected, position ② indicates that it is connected to -CH 2- of L 4 , and W and W' are not at the same time
Figure PCTCN2021123020-appb-000035
Z represents -CH 2 -Cyclo-C(=O)-NR 10 - or a single bond, Cyclo represents a cyclohexane group,
优选地,L 3表示
Figure PCTCN2021123020-appb-000036
Figure PCTCN2021123020-appb-000037
或单键,各n独立地表示1-6的整数(例如2);
Preferably, L 3 represents
Figure PCTCN2021123020-appb-000036
Figure PCTCN2021123020-appb-000037
or a single bond, each n independently represents an integer from 1 to 6 (eg 2);
L 4表示-(CH 2)n 6-C(=O)-或-(CH 2)n 6a-NR 10-C(=O)-(CH 2)n 6b-O-(CH 2)n 6b-C(=O)-,n 6表示1~6的整数,n 6a表示1~4的整数,n 6b表示1~3的整数, L 4 represents -(CH 2 )n 6 -C(=O)- or -(CH 2 )n 6a -NR 10 -C(=O)-(CH 2 )n 6b -O-(CH 2 )n 6b -C(=O)-, n 6 represents an integer from 1 to 6, n 6a represents an integer from 1 to 4, and n 6b represents an integer from 1 to 3,
L P表示由1~7个氨基酸构成的肽残基, LP represents a peptide residue consisting of 1 to 7 amino acids,
L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 7-O-、-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-(CH 2)n 8-O-(C=O)-、-NR 10-(CH 2)n 8-O-(CH 2)n 8-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-Aryl-(CH 2)n 8-O-(C=O)-、-NR 10-Aryl-(CH 2)n 8-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-,各Aryl独立地表示任选地被R 9取代的C 6-C 10芳基,各n 7独立地表示1~4的整数,各n 8独立地表示1~4的整数, L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 7 -O-, -NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -(CH 2 )n 8 -O-(C=O)-, -NR 10 -(CH 2 )n 8 -O-(CH 2 )n 8 -(C=O)-NR 10 -( CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 - or -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, each Aryl independently represents optional C 6 -C 10 aryl group substituted by R 9 , each n 7 independently represents an integer of 1-4, and each n 8 independently represents an integer of 1-4,
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基(优选甲基), Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy (preferably methyl),
Q 2表示-(琥珀酰亚胺-3-基-N)-、或-Q 1-NR 10-,Q 1的定义如本发明第一方面所述化合物,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接, Q 2 represents -(succinimide-3-yl-N)-, or -Q 1 -NR 10 -, Q 1 is defined as the compound described in the first aspect of the present invention, and Q 1 passes through the carboxyl group of R 11 and - NR 10 - forms an amide bond and connects with L 3 ,
式(VIII)中,In formula (VIII),
Q 2表示的-(琥珀酰亚胺-3-基-N)-,如下式结构: -(Succinimide-3-yl-N)- represented by Q 2 has the following structure:
Figure PCTCN2021123020-appb-000038
Figure PCTCN2021123020-appb-000038
以该结构的3位与抗体连接,在1位的氮原子上与包含该结构的接头内的亚甲基连接,It is linked to the antibody at the 3-position of the structure, and is linked to the methylene group in the linker containing the structure at the nitrogen atom of the 1-position,
或者Q 2表示的-Q 1-NR 10-,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, Alternatively, -Q 1 -NR 10 - represented by Q 2 is connected by forming a thioether bond between the alkynyl carbon of R 12 and the disulfide bond of the antibody hinge portion,
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(VII)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)-, -O- or -CH2- moiety of L b in the linker represented by VII).
在一些实施方案中,式(II)表示的化合物如前所述。In some embodiments, the compound represented by formula (II) is as previously described.
在一些实施方案中,L P的肽残基为由选自丙氨酸、苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的肽残基。 In some embodiments, the peptide residue of LP is selected from the group consisting of alanine, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues formed from amino acids.
在一些实施方案中,L P的肽残基为由选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的肽残基。 In some embodiments, the peptide residues of LP are formed from amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues.
在一些实施方案中,L P为由1-5个氨基酸构成的肽残基。 In some embodiments, LP is a peptide residue consisting of 1-5 amino acids.
在一些实施方案中,L P为选自以下的肽残基: In some embodiments, LP is a peptide residue selected from the group consisting of:
-VA-;-va-;
-K-;-K-;
-GGFG-;-ggfg-;
-VC-;-vc-;
-EVC-;-evc-;
-DVC;-DVC;
-EGGFG-;-EGGFG-;
-DGGFG-。-DGGFG-.
在一些实施方案中,L P为选自以下的肽残基: In some embodiments, LP is a peptide residue selected from the group consisting of:
-K-;-K-;
-GGFG-;-ggfg-;
-VC-;-vc-;
-EVC-;-evc-;
-DVC;-DVC;
-EGGFG-;-EGGFG-;
-DGGFG-。-DGGFG-.
在一些实施方案中,L 4表示-(CH 2)n 6-C(=O)-或-(CH 2)n 6a-NR 10-C(=O)-(CH 2)n 6b-O-(CH 2)n 6b-C(=O)-,n 6表示2~5的整数,n 6a表示1~3的整数,n 6b表示1~2的整数,R 10表示氢或C 1-C 4烷基(优选甲基)。 In some embodiments, L 4 represents -(CH 2 )n 6 -C(=O)- or -(CH 2 )n 6a -NR 10 -C(=O)-(CH 2 )n 6b -O- (CH 2 )n 6b -C(=O)-, n 6 represents an integer of 2-5, n 6a represents an integer of 1-3, n 6b represents an integer of 1-2, R 10 represents hydrogen or C 1 -C 4 alkyl (preferably methyl).
在一些实施方案中,L 4表示
Figure PCTCN2021123020-appb-000039
In some embodiments, L 4 represents
Figure PCTCN2021123020-appb-000039
在一些实施方案中,L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 7-O-、-NR 10-(CH 2)n 8-NR 10-C(=O)-、、-NR 10-(CH 2)n 8-O-(C=O)-、-NR 10-(CH 2)n 8-O-(CH 2)n 8-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-Aryl-(CH 2)n 8-O-C(=O)-、-NR 10-Aryl-(CH 2)n 8-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-NR 10-(CH 2)n 8-NR 10- (C=O)-,其中各R 10独立地表示氢或C 1-C 4烷基(优选甲基),各n 7独立地表示1~2的整数,各n 8独立地表示1~2的整数,Aryl表示苯环基团。 In some embodiments, L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 7 -O-, -NR 10 -(CH 2 )n 8 -NR 10 -C (=O)-, -NR 10 -(CH 2 )n 8 -O-(C=O)-, -NR 10 -(CH 2 )n 8 -O-(CH 2 )n 8 -(C= O)-NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 -OC(=O)-, -NR 10 -Aryl-( CH 2 )n 8 -or -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, where each R 10 independently represents hydrogen or C 1 -C 4 alkyl (preferably methyl), each n 7 independently represents an integer of 1-2, each n 8 independently represents an integer of 1-2, and Aryl represents a benzene ring group .
在一些实施方案中,L b表示
Figure PCTCN2021123020-appb-000040
Figure PCTCN2021123020-appb-000041
In some embodiments, L b represents
Figure PCTCN2021123020-appb-000040
Figure PCTCN2021123020-appb-000041
在一些实施方案中,-NR 10-基团和-(CH 2)n 8-基团位于苯环的对位。 In some embodiments, the -NR 10 - group and the -(CH 2 )n 8 - group are located in the para position of the benzene ring.
在一些实施方案中,式(VII)表示的接头为选自以下所示的基团,其中,各n独立地表示1-8的整数:In some embodiments, the linker represented by formula (VII) is a group selected from the group consisting of the following, wherein each n independently represents an integer of 1-8:
Figure PCTCN2021123020-appb-000042
Figure PCTCN2021123020-appb-000042
Figure PCTCN2021123020-appb-000043
Figure PCTCN2021123020-appb-000043
Figure PCTCN2021123020-appb-000044
Figure PCTCN2021123020-appb-000044
Figure PCTCN2021123020-appb-000045
Figure PCTCN2021123020-appb-000045
在一些实施方案中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个,例如3.3、3.5、5.5、6.2、6.5、6.6、6.8、7.0、7.1、7.2、7.4、7.5或7.8个。In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8, such as 3.3, 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
在一些实施方案中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段。In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
在一些实施方案中,所述抗体选自抗Her-2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER-4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体;优选地,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体。In some embodiments, the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 Antibodies, CD30 antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies.
在一些实施方案中,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv。 In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
在一些实施方案中,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。In some embodiments, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody The nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
在一些实施方案中,所述抗体为抗Her-2抗体,其中,所述抗Her-2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的CDR3;优选地,所述抗Her-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。In some embodiments, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2 composed of sequence, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
本发明的第九方面,提供一种式(X)表示的接头-药物中间体化合物,其特征在于,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(IX)表示的接头连接而成的:The ninth aspect of the present invention provides a linker-drug intermediate compound represented by formula (X), characterized in that T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following The joints represented by formula (IX) are connected:
Q’ 2-L 3-L 4-L P-L b-T     (X) Q' 2 -L 3 -L 4 -L P -L b -T (X)
Figure PCTCN2021123020-appb-000046
Figure PCTCN2021123020-appb-000046
Q’ 2-L 3-L 4-L P-L b-     (IX) Q' 2 -L 3 -L 4 -L P -L b - (IX)
其中,in,
R 1、R 2、R 3、R 4的定义如本发明说明书所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention;
Q’ 2表示(马来酰亚胺-N)-或Q 1-NR 10-,Q 1的定义如本发明第一方面所述化合物,; Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -, and Q 1 is defined as the compound described in the first aspect of the present invention,;
L 3、L 4、L P、L b的定义如本发明说明书所述; The definitions of L 3 , L 4 , L P and L b are as described in the specification of the present invention;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
式(IX)中,In formula (IX),
Q’ 2表示的(马来酰亚胺-N)-,如下式结构: (maleimide-N)- represented by Q' 2 has the following formula structure:
Figure PCTCN2021123020-appb-000047
Figure PCTCN2021123020-appb-000047
以该结构在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q’ 2表示的Q 1-NR 10-,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; With this structure, the nitrogen atom at the 1-position is connected to the methylene group in the linker containing this structure, or Q 1 -NR 10 - represented by Q' 2 , Q 1 forms an amide with -NR 10 - through the carboxyl group of R 11 key and connect with L3 ;
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(IX)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)-, -O- or -CH2- moiety of L b in the linker represented by IX).
在一些实施方案中,所述式(II)所示的化合物如前所述。In some embodiments, the compound of formula (II) is as previously described.
在一些实施方案中,所述接头-药物中间体化合物是选自以下的化合物,其中,各n独立地表示1-8的整数:In some embodiments, the linker-drug intermediate compound is a compound selected from the group consisting of, wherein each n independently represents an integer from 1 to 8:
Figure PCTCN2021123020-appb-000048
Figure PCTCN2021123020-appb-000048
Figure PCTCN2021123020-appb-000049
Figure PCTCN2021123020-appb-000049
Figure PCTCN2021123020-appb-000050
Figure PCTCN2021123020-appb-000050
Figure PCTCN2021123020-appb-000051
Figure PCTCN2021123020-appb-000051
Figure PCTCN2021123020-appb-000052
Figure PCTCN2021123020-appb-000052
Figure PCTCN2021123020-appb-000053
Figure PCTCN2021123020-appb-000053
Figure PCTCN2021123020-appb-000054
Figure PCTCN2021123020-appb-000054
本发明的第十方面,提供一种提供通式(VII)所示的接头结构:A tenth aspect of the present invention provides a linker structure shown in the general formula (VII):
-Q 2-L 3-L 4-L P-L b-     (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
其中,Q 2、L 3、L 4、L P、L b的定义如本发明说明书所述。 The definitions of Q 2 , L 3 , L 4 , LP and L b are as described in the specification of the present invention.
本发明的第十一方面,提供本发明第八方面的抗体-药物偶联物的制备方法,所述方法包括:The eleventh aspect of the present invention provides a method for preparing the antibody-drug conjugate of the eighth aspect of the present invention, the method comprising:
Figure PCTCN2021123020-appb-000055
Figure PCTCN2021123020-appb-000055
使式(X)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(X)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (X) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (X) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
其中,R 1、R 2、R 3、R 4的定义如本发明说明书所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention;
Q 2、Q’ 2、L 3、L 4、L P、L b的定义如本发明说明书所述; The definitions of Q 2 , Q' 2 , L 3 , L 4 , L P and L b are as described in the description of the present invention;
T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(IX)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R3 or R4 is a hydroxyl group, the compound represented by the formula (II) uses the oxygen in the hydroxyl group of R3 or R4 as a hydroxyl group . The oxygen of the above-mentioned formula (IX) is connected to the right end -C(=O)-, -O- or -CH 2 - part of L b in the linker represented by the above formula (IX);
AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
本发明的第十二方面,提供本发明第九方面的接头-药物中间体化合物的制备方法。The twelfth aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the ninth aspect of the present invention.
在某些实施方式中,包括如下步骤:In certain embodiments, the following steps are included:
(1)Boc-GGFG与PABOH在EEDQ的作用下,二氯甲烷与甲醇作溶剂,室温搅拌过夜,生成Boc-GGFG-PABOH;(1) Boc-GGFG and PABOH are generated under the action of EEDQ, using dichloromethane and methanol as solvents, and stirring overnight at room temperature to generate Boc-GGFG-PABOH;
(2)Boc-GGFG-PABOH在TFA/DCM的作用下脱掉Boc生成GGFG-PABOH;(2) Boc-GGFG-PABOH strips off Boc under the action of TFA/DCM to generate GGFG-PABOH;
(3)GGFG-PABOH与N 3-PEGn-NHS活性酯反应生成N 3-PEGn-GGFG-PABOH,n=0,2,4,6或8; (3) GGFG-PABOH reacts with N 3 -PEGn-NHS active ester to generate N 3 -PEGn-GGFG-PABOH, n=0, 2, 4, 6 or 8;
(4)吉咪替康-Boc、SN-38-Boc或吉马替康与DMAP,三光气在二氯甲烷的溶剂中反应5mins,加入步骤(3)的N 3-PEGn-GGFG-PABOH,生成N 3-PEGn-GGFG-PABC-吉咪替康-Boc,或N 3-PEGn-GGFG-PABC-SN-38-Boc,或N 3-PEGn-GGFG-PABC-吉马替康, (4) Gematecan-Boc, SN-38-Boc or Gematecan and DMAP, triphosgene are reacted in the solvent of dichloromethane for 5mins, the N 3 -PEGn-GGFG-PABOH of step (3) is added, Generate N 3 -PEGn-GGFG-PABC-Gimantecan-Boc, or N 3 -PEGn-GGFG-PABC-SN-38-Boc, or N 3 -PEGn-GGFG-PABC-Gimantecan,
(5)步骤(4)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click的条件得最终化合物,m=2,4,6,8。(5) The product of step (4) and alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) were obtained under the condition of Click Final compound, m=2,4,6,8.
在另一些实施方式中,包括如下步骤:In other embodiments, the following steps are included:
(1)Boc-GGFG在TFA/DCM的作用下脱掉Boc,除掉TFA与二氯甲烷后,与N 3-PEGn-NHS在二氯甲烷中反应,用DIEA做碱,得到化合物N 3-PEGn-GGFG,n=0,2,4,6或8; (1) Boc-GGFG removes Boc under the action of TFA/DCM, removes TFA and dichloromethane, reacts with N 3 -PEGn-NHS in dichloromethane, and uses DIEA as a base to obtain compound N 3 - PEGn-GGFG, n=0, 2, 4, 6 or 8;
(2)N 3-PEGn-GGFG与N-Boc-N-甲基乙二胺缩合,再用TFA/DCM脱掉Boc后,得化合物N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3(2) N 3 -PEGn-GGFG was condensed with N-Boc-N-methylethylenediamine, and then Boc was removed with TFA/DCM to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH -CH 3 ;
(3)N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3与吉咪替康-PNP(或吉马替康-PNP,SN-38-PNP)在TEA,DMF的条件下反应得化合物N 3-PEGn-GGFG-NH-C 2H 4-N(CH 3)-C(O)-吉咪替康(或SN-38,或吉马替康-PNP), (3) N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 and gimatecan-PNP (or gimatecan-PNP, SN-38-PNP) under the conditions of TEA, DMF Reaction to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -N(CH 3 )-C(O)-gemitecan (or SN-38, or gematecan-PNP),
(4)步骤(3)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click的条件得最终化合物,m=2,4,6,8。(4) The product of step (3) and alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) were obtained under the condition of Click Final compound, m=2,4,6,8.
本发明的第十三方面,提供式(XI)、(XII)的中间体化合物:The thirteenth aspect of the present invention provides intermediate compounds of formula (XI), (XII):
Q 1-L 1-L 2-L a-H     (XI) Q 1 -L 1 -L 2 -L a -H (XI)
Q 2-L 3-L 4-L P-L b-H     (XII) Q 2 -L 3 -L 4 -L P -L b -H (XII)
本发明的第十四方面,提供一种药物组合物,其包含本发明第三方面、第八方面的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,以及任选的药学上可接受的载体。The fourteenth aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate of the third aspect and the eighth aspect of the present invention, its stereoisomer or a pharmaceutically acceptable salt thereof, or the The antibody-drug conjugate, a solvate of a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and an optional pharmaceutically acceptable carrier.
本发明的第十五方面,提供一种药物制剂,其包含本发明第三方面、第八方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物。The fifteenth aspect of the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the third aspect and the eighth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, Or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
本发明的第十六方面,提供本发明第三方面、第八方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂,其用于预防和/或治疗肿瘤或癌症;The sixteenth aspect of the present invention provides the antibody-drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates of the third and eighth aspects of the present invention. The combination, a solvate of a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition according to the fourteenth aspect or the pharmaceutical preparation according to the fifteenth aspect, for prevention and/or treatment tumor or cancer;
或者,提供本发明第三方面、第八方面的所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂在制备预防和/或治疗肿瘤或癌症的药物中的用途。Alternatively, provide the antibody-drug conjugates, stereoisomers or pharmaceutically acceptable salts thereof, or the antibody-drug conjugates, stereoisomers thereof according to the third aspect and the eighth aspect of the present invention Use of a solvate of a isomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the pharmaceutical preparation of the fifteenth aspect in the preparation of a medicament for preventing and/or treating tumors or cancer .
在一些实施方案中,所述的肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌、肺癌、胰腺癌、结直肠癌。In some embodiments, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
本发明的第十七方面,提供一种预防或治疗肿瘤或癌症的方法,其包括向有此需要的受试者施用有效量的本发明第三方面、第八方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂。The seventeenth aspect of the present invention provides a method for preventing or treating tumor or cancer, which comprises administering an effective amount of the antibody-drug couple of the third aspect and the eighth aspect of the present invention to a subject in need thereof. The conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, as described in the fourteenth aspect The pharmaceutical composition or the pharmaceutical preparation of the fifteenth aspect.
在一些实施方案中,所述的肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌、肺癌、胰腺癌、结直肠癌。In some embodiments, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
本发明的第十八方面,提供第三方面、第八方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂用于制备试剂的用途,所述试剂用于抑制癌细胞生长、增殖或迁移。The eighteenth aspect of the present invention provides the antibody-drug conjugate of the third aspect and the eighth aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, Use of a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the pharmaceutical preparation of the fifteenth aspect for the preparation of a reagent for use in Inhibits cancer cell growth, proliferation or migration.
本发明的第十九方面,提供第三方面、第八方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂,其用于抑制癌细胞的生长、增殖或迁移。The nineteenth aspect of the present invention provides the antibody-drug conjugate of the third aspect and the eighth aspect, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, A solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the pharmaceutical preparation of the fifteenth aspect, which are used for inhibiting the growth, proliferation or migrate.
本发明的第二十方面,提供一种抑制癌细胞生长、增殖或迁移的方法,其包括给癌细胞施用有效量的本发明第三方面、第八方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂。The twentieth aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug conjugates of the third aspect, the eighth aspect of the present invention, Its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the drug described in the fourteenth aspect The composition or the pharmaceutical preparation of the fifteenth aspect.
本发明的第二十一方面,提供一种抑制癌细胞生长、增殖或迁移的试剂盒,其包括本发明第三方面、第八方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、第十四方面所述的药物组合物或第十五方面所述的药物制剂。The twenty-first aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, which comprises the antibody-drug conjugates and stereoisomers thereof according to the third and eighth aspects of the present invention or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the fourteenth aspect or the tenth The pharmaceutical preparation described in the fifth aspect.
本发明的第二十二方面,提供式(IV)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下 式(III)表示的接头连接而成的:A twenty-second aspect of the present invention provides an antibody-drug conjugate represented by formula (IV), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof The solvate of the isomer or a pharmaceutically acceptable salt thereof, wherein AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a combination of compound (T) and antibody (AB) Connected via a linker represented by the following formula (III):
AB-S-Q 1-L 1-L 2-L a-T     (IV) AB-SQ 1 -L 1 -L 2 -L a -T (IV)
Figure PCTCN2021123020-appb-000056
Figure PCTCN2021123020-appb-000056
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
其中,in,
式(II)中,In formula (II),
R 1选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、被-OC(=O)R 13或-NR 7R 8取代的C 1-C 6烷基、被-SiMe 3取代的C 1-C 6烷基、或-CH=N-O-(C 1-C 6烷基); R 1 is selected from hydrogen, halogen, hydroxy, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C substituted by -OC(=O)R 13 or -NR 7 R 8 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with -SiMe 3 , or -CH=NO-(C 1 -C 6 alkyl);
R 2选自氢、卤素、羟基、硝基、氨基、饱和或不饱和C 1-C 6烷基、C 1-C 6烷氧基、或被-NR 7R 8取代的C 1-C 6烷基; R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 substituted by -NR 7 R 8 alkyl;
R 3选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、或-NR 7R 8C(=O)O-基; R 3 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or -NR 7 R 8 C(=O)O-yl;
R 4选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、或C 1-C 6烷氧基; R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
或者R 1和R 2可以连接在一起与母体部分形成任选被R 9取代的5-6元环; Alternatively R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
或者R 3和R 4可以连接在一起与母体部分形成任选被R 9取代的5-6元含氧杂环; Alternatively R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
R 7和R 8每次出现时各自独立地选自氢、C 1-C 6烷基、被羟基或氨基取代的C 1-C 6酰基;或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环; Each occurrence of R 7 and R 8 is independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
R 9每次出现时各自独立地选自卤素、羟基、硝基、-NR 7R 8、C 1-C 6烷基、C 1-C 6烷氧基、任选被C 1-C 6烷基取代的哌啶基; Each occurrence of R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
R 13表示羧基取代的C 1-C 6烷基; R 13 represents a carboxy-substituted C 1 -C 6 alkyl group;
式(III)中,In formula (III),
L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)-CH 2OCH 2-(C=O)-,n 1表示1~24的整数,n 2表示1~4的整数; L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)-CH 2 OCH 2 -(C=O)-,n 1 represents an integer from 1 to 24, and n 2 represents an integer from 1 to 4;
L 2表示缬氨酸残基、胍氨酸残基、苯丙氨酸残基、赖氨酸残基、D-缬氨酸残基、甘氨酸残基、丙氨酸残基、天冬氨酸残基; L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
L a表示-NR 10-(CH 2)n 3-、-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-; L a represents -NR 10 -(CH 2 )n 3 -, -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 4 -O -(C=O)-;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基,n 3表示1~4的整数,n 4表示1~4的整数; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxyl groups, n 3 represents an integer of 1 to 4, and n 4 represents an integer of 1 to 4;
Aryl表示任选地被R 9取代的C 6-C 10芳基; Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
W为单键或
Figure PCTCN2021123020-appb-000057
其中,位置①表示与-NR 10-相连,位置②表示与(CH 2CH 2-O-)n 1-相连;
W is a single key or
Figure PCTCN2021123020-appb-000057
Wherein, position ① means connecting with -NR 10 -, and position ② means connecting with (CH 2 CH 2 -O-)n 1 -;
式(IV)中,In formula (IV),
Q 1为本发明第一方面所述化合物,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, Q 1 is the compound described in the first aspect of the present invention, which is connected by the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 to form an amide bond, and the alkynyl carbon of R 12 and the disulfide of the hinge part of the antibody are connected. bond to form a thioether bond,
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3 或R 4的羟基中的氧作为连接部位,连接于上述式(III)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C( = O)- or -CH2- moiety of La in the linker represented by III).
在一些实施方案中,R 1表示氢、C 1-C 4烷基、被-NH(C 1-C 4烷基)取代的C 1-C 4烷基、被
Figure PCTCN2021123020-appb-000058
取代的C 1-C 4烷基、被-SiMe 3取代的C 1-C 4烷基、-CH=N-O-(C 3-C 6烷基)或-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH。
In some embodiments, R 1 represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -NH(C 1 -C 4 alkyl), C 1 -C 4 alkyl substituted by
Figure PCTCN2021123020-appb-000058
Substituted C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -SiMe 3 , -CH=NO-(C 3 -C 6 alkyl) or -(CH 2 ) 2 O(C=O )( CH2 ) 2 (C=O)OH.
在一些实施方案中,R 2表示氢、C 3-C 4烯基、硝基、氨基、或被-N(C 1-C 4烷基) 2取代的C 1-C 4烷基。 In some embodiments, R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, or C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 .
在一些实施方案中,R 3表示氢、卤素、羟基、或
Figure PCTCN2021123020-appb-000059
In some embodiments, R 3 represents hydrogen, halogen, hydroxyl, or
Figure PCTCN2021123020-appb-000059
在一些实施方案中,R 4表示氢或卤素。 In some embodiments, R 4 represents hydrogen or halogen.
在一些实施方案中,R 1和R 2连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000060
其中
Figure PCTCN2021123020-appb-000061
部分表示连接于母体基团的键。
In some embodiments, R 1 and R 2 are joined together to form the group shown below
Figure PCTCN2021123020-appb-000060
in
Figure PCTCN2021123020-appb-000061
A moiety represents a bond to the parent group.
在一些实施方案中,R 3和R 4连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000062
其中
Figure PCTCN2021123020-appb-000063
部分表示连接于母体基团的键。
In some embodiments, R and R are joined together to form the group shown below
Figure PCTCN2021123020-appb-000062
in
Figure PCTCN2021123020-appb-000063
A moiety represents a bond to the parent group.
在一些实施方案中,式(II)表示的化合物如下所示:In some embodiments, the compound represented by formula (II) is shown below:
Figure PCTCN2021123020-appb-000064
Figure PCTCN2021123020-appb-000064
Figure PCTCN2021123020-appb-000065
Figure PCTCN2021123020-appb-000065
优选地,式(II)表示的化合物为吉马替康或吉咪替康,更优选为吉咪替康:Preferably, the compound represented by formula (II) is gimatecan or gimitecan, more preferably gimatecan:
Figure PCTCN2021123020-appb-000066
Figure PCTCN2021123020-appb-000066
在一些实施方案中,L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)CH 2-O-CH 2-(C=O)-,n 1表示4~12的整数,n 2表示1~2的整数,R 10表示氢或C 1-C 4烷基。 In some embodiments, L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)CH 2 -O-CH 2 - (C=O)-, n 1 represents an integer of 4-12, n 2 represents an integer of 1-2, and R 10 represents hydrogen or C 1 -C 4 alkyl.
在一些实施方案中,L 2表示赖氨酸残基。 In some embodiments, L 2 represents a lysine residue.
在一些实施方案中,L a表示-NR 10-Aryl-(CH 2)n 4-O-(C=O)-,n 4表示1~2的整数,R 10表示氢或C 1-C 4烷基,Aryl表示苯环基团,优选地,-NR 10-基团和-(CH 2)n 4-基团位于苯环的对位。 In some embodiments, L a represents -NR 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, n 4 represents an integer from 1 to 2, and R 10 represents hydrogen or C 1 -C 4 The alkyl group, Aryl represents a benzene ring group, preferably, the -NR 10 - group and the -(CH 2 )n 4 - group are located in the para position of the benzene ring.
在一些实施方案中,式(III)表示的接头为选自以下所示的基团:In some embodiments, the linker represented by formula (III) is a group selected from the group consisting of:
Figure PCTCN2021123020-appb-000067
Figure PCTCN2021123020-appb-000067
Figure PCTCN2021123020-appb-000068
Figure PCTCN2021123020-appb-000068
在一些实施方案中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个。In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8.
在一些实施方案中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段。In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
在一些实施方案中,所述抗体选自抗He-r2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体。In some embodiments, the antibody is selected from the group consisting of anti-He-r2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER4 antibody, CD20 antibody, CD30 antibody, CD19 antibody, CD33 antibody.
在一些实施方案中,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体。In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody; preferably, the humanized antibody is a fully human antibody.
在一些实施方案中,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv。 In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
在一些实施方案中,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。In some embodiments, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody The nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
在一些实施方案中,所述抗体为抗Her-2抗体,其中,所述抗Her2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的CDR3;优选地,所述抗Her2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。In some embodiments, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
本发明的第二十三方面,提供式(VI)表示的接头-药物中间体化合物,其中,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(V)表示的接头连接而成的:The twenty-third aspect of the present invention provides a linker-drug intermediate compound represented by formula (VI), wherein T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following formula ( The joints indicated by V) are connected:
Q 1-L 1-L 2-L a-T     (VI) Q 1 -L 1 -L 2 -L a -T (VI)
Figure PCTCN2021123020-appb-000069
Figure PCTCN2021123020-appb-000069
Q 1-L 1-L 2-L a-     (V) Q 1 -L 1 -L 2 -L a - (V)
其中,in,
R 1、R 2、R 3、R 4的定义如本发明第二十二方面所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention;
L 1、L 2、L a的定义如本发明第二十二方面所述; The definitions of L 1 , L 2 and La are as described in the twenty-second aspect of the present invention;
Q 1为本发明第一方面所述化合物,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接, Q 1 is the compound described in the first aspect of the present invention, which is connected by forming an amide bond between the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 ,
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C( = O)- or -CH2- moiety of La in the linker represented by V).
在一些实施方案中,所述式(II)所示的化合物如本发明第二十二方面所述。In some embodiments, the compound of formula (II) is described in the twenty-second aspect of the present invention.
在一些实施方案中,所述接头-药物中间体化合物是选自以下的化合物,In some embodiments, the linker-drug intermediate compound is a compound selected from the group consisting of,
Figure PCTCN2021123020-appb-000070
Figure PCTCN2021123020-appb-000070
Figure PCTCN2021123020-appb-000071
Figure PCTCN2021123020-appb-000071
Figure PCTCN2021123020-appb-000072
Figure PCTCN2021123020-appb-000072
Figure PCTCN2021123020-appb-000073
Figure PCTCN2021123020-appb-000073
本发明的第二十四方面,提供接头,其中,其为下式(III)表示,A twenty-fourth aspect of the present invention provides a linker, wherein it is represented by the following formula (III),
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
其中,Q 1、L 1、L 2、L a的定义如本发明第二十二方面所述。 Wherein, the definitions of Q 1 , L 1 , L 2 , and La are as described in the twenty-second aspect of the present invention.
在一些实施方案中,所述接头为选自本发明第二十二方面中的接头。In some embodiments, the linker is a linker selected from the twenty-second aspect of the present invention.
本发明的第二十五方面,提供式(VIII)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下式(VII)表示的接头连接而成的:A twenty-fifth aspect of the present invention provides an antibody-drug conjugate represented by formula (VIII), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof The solvate of the isomer or a pharmaceutically acceptable salt thereof, wherein AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a combination of compound (T) and antibody (AB) Connected via a linker represented by the following formula (VII):
AB-S-Q 2-L 3-L 4-L P-L b-T     (VIII) AB-SQ 2 -L 3 -L 4 -L P -L b -T (VIII)
Figure PCTCN2021123020-appb-000074
Figure PCTCN2021123020-appb-000074
-Q 2-L 3-L 4-L P-L b-     (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
其中,in,
式(II)中,In formula (II),
R 1、R 2、R 3、R 4的定义如本发明第二十二方面所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention;
式(VII)中,In formula (VII),
L 3表示-Z-W-(CH 2CH 2-O)n 5-W’-或单键,n 5表示1~8的整数,W、W’表示
Figure PCTCN2021123020-appb-000075
或单键,其中,W的位置①表示与Z相连,位置②表示与(CH 2CH 2-O-)n 5-相连,W’的位置①表示与(CH 2CH 2-O-)n 5-相连,位置②表示与L 4的-CH 2-相连相连,且W、W’不同时为
Figure PCTCN2021123020-appb-000076
Z表示-CH 2-Cyclo-C(=O)-NR 10-或单键,Cyclo表示环己烷基团;
L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'- or a single bond, n 5 represents an integer from 1 to 8, and W and W' represent
Figure PCTCN2021123020-appb-000075
Or a single bond, wherein, the position ① of W indicates that it is connected to Z, the position ② indicates that it is connected to (CH 2 CH 2 -O-)n 5 -, and the position ① of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected, position ② indicates that it is connected to -CH 2- of L 4 , and W and W' are not at the same time
Figure PCTCN2021123020-appb-000076
Z represents -CH 2 -Cyclo-C(=O)-NR 10 - or a single bond, and Cyclo represents a cyclohexane group;
L 4表示-(CH 2)n 6-C(=O)-,n 6表示1~6的整数; L 4 represents -(CH 2 )n 6 -C(=O)-, and n 6 represents an integer from 1 to 6;
L P表示由2~7个氨基酸构成的肽残基; LP represents a peptide residue consisting of 2-7 amino acids;
L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 8-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-,Aryl表示任选地被R 9取代的C 6-C 10芳基,n 7表示1~4的整数,n 8表示1~4的整数; L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 8 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 8 -O -(C=O)-, Aryl represents a C 6 -C 10 aryl group optionally substituted by R 9 , n 7 represents an integer of 1-4, and n 8 represents an integer of 1-4;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
Q 2表示-(琥珀酰亚胺-3-基-N)-、或-Q 1-NR 10-,Q 1为本发明第一方面所述化合物,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; Q 2 represents -(succinimide-3-yl-N)-, or -Q 1 -NR 10 -, Q 1 is the compound described in the first aspect of the present invention, and Q 1 passes through the carboxyl group of R 11 and -NR 10 - form an amide bond to connect with L 3 ;
式(VIII)中,In formula (VIII),
Q 2为-(琥珀酰亚胺-3-基-N)-,如下式结构: Q 2 is -(succinimide-3-yl-N)-, with the following formula:
Figure PCTCN2021123020-appb-000077
Figure PCTCN2021123020-appb-000077
以该结构的3位与抗体连接,在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q 2为-Q 1-NR 10-,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, It is linked to the antibody at the 3-position of the structure, and the nitrogen atom at the 1-position is linked to the methylene group in the linker containing the structure, or Q 2 is -Q 1 -NR 10 -, and the alkynyl carbon of R 12 is linked to The disulfide bond in the hinge of the antibody forms a thioether bond to connect,
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(VII)表示的接头中L b的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)- or -CH2- moiety of L b in the linker represented by VII).
在一些实施方案中,式(II)表示的化合物如本发明第二十二方面中所述。In some embodiments, the compound represented by formula (II) is as described in the twenty-second aspect of the present invention.
在一些实施方案中,L P的肽残基为由选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的肽残基。 In some embodiments, the peptide residues of LP are formed from amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid peptide residues.
在一些实施方案中,L P为由2-5个氨基酸构成的肽残基。 In some embodiments, LP is a peptide residue consisting of 2-5 amino acids.
在一些实施方案中,L P为选自以下的肽残基: In some embodiments, LP is a peptide residue selected from the group consisting of:
-GGFG-;-ggfg-;
-VC-;-vc-;
-EVC-;-evc-;
-DVC;-DVC;
-EGGFG-;-EGGFG-;
-DGGFG-。-DGGFG-.
在一些实施方案中,L 4表示-(CH 2)n 6-C(=O)-,n 6表示2~5的整数。 In some embodiments, L 4 represents -(CH 2 )n 6 -C(=O)-, and n 6 represents an integer of 2-5.
在一些实施方案中,L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 8-NR 10-C(=O)-、-NR 10-Aryl-(CH 2)n 8-O-C(=O)-,其中R 10表示氢或C 1-C 4烷基,n 7表示1~2的整数,n 8表示1~2的整数,Aryl表示苯环基团。 In some embodiments, L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 8 -NR 10 -C(=O)-, -NR 10 -Aryl-(CH 2 ) n 8 -OC(=O)-, wherein R 10 represents hydrogen or C 1 -C 4 alkyl group, n 7 represents an integer of 1-2, n 8 represents an integer of 1-2, and Aryl represents a benzene ring group .
在一些实施方案中,-NR 10-基团和-(CH 2)n 8-基团位于苯环的对位。 In some embodiments, the -NR 10 - group and the -(CH 2 )n 8 - group are located in the para position of the benzene ring.
在一些实施方案中,式(VII)表示的接头为选自以下所示的基团:In some embodiments, the linker represented by formula (VII) is a group selected from the group consisting of:
Figure PCTCN2021123020-appb-000078
Figure PCTCN2021123020-appb-000078
Figure PCTCN2021123020-appb-000079
Figure PCTCN2021123020-appb-000079
在一些实施方案中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个。In some embodiments, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8.
在一些实施方案中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段。In some embodiments, the antibody (AB) is a full-length antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof.
在一些实施方案中,所述抗体选自抗Her-2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER3、HER4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体。In some embodiments, the antibody is selected from the group consisting of anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER3, HER4 antibody, CD20 antibody, CD30 antibody , CD19 antibody, CD33 antibody.
在一些实施方案中,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体。In some embodiments, the antibody is a murine antibody, a chimeric antibody, a humanized antibody; preferably, the humanized antibody is a fully human antibody.
在一些实施方案中,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv。 In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv.
在一些实施方案中,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。In some embodiments, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises CDR1 consisting of the amino acid sequence of KASQDVSIVA, consisting of the amino acid sequence of SASYRYT CDR2 composed of sequence, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; preferably, The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding of the light chain and the heavy chain of the anti-Trop-2 antibody The nucleotide sequences are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively.
在一些实施方案中,所述抗体为抗Her-2抗体,其中,所述抗Her2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的CDR3;优选地,所述抗Her2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。In some embodiments, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her2 antibody comprises CDR1 consisting of the amino acid sequence of RASQDVNTAVA, consisting of the amino acid sequence of SASFLYS CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
本发明的第二十六方面,提供式(X)表示的接头-药物中间体化合物,其中,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(IX)表示的接头连接而成的:The twenty-sixth aspect of the present invention provides a linker-drug intermediate compound represented by formula (X), wherein T represents a compound represented by formula (II), and the intermediate compound is compound (T) with the following formula ( IX) is formed by connecting the joints indicated:
Q’ 2-L 3-L 4-L P-L b-T     (X) Q' 2 -L 3 -L 4 -L P -L b -T (X)
Figure PCTCN2021123020-appb-000080
Figure PCTCN2021123020-appb-000080
Q’ 2-L 3-L 4-L P-L b-     (IX) Q' 2 -L 3 -L 4 -L P -L b - (IX)
其中,in,
R 1、R 2、R 3、R 4的定义如本发明第二十二方面所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention;
Q’ 2表示(马来酰亚胺-N)-或Q 1-NR 10-,Q 1为本发明第一方面所述化合物,; Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -, and Q 1 is the compound described in the first aspect of the present invention,;
L 3、L 4、L P、L b的定义如本发明第二十五方面所述; The definitions of L 3 , L 4 , L P and L b are as described in the twenty-fifth aspect of the present invention;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
式(IX)中,In formula (IX),
Q’ 2表示(马来酰亚胺-N)-,如下式结构: Q' 2 represents (maleimide-N)-, the following formula structure:
Figure PCTCN2021123020-appb-000081
Figure PCTCN2021123020-appb-000081
以该结构在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q’ 2表示Q 1-NR 10-,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; In this structure, the nitrogen atom at the 1-position is connected with the methylene group in the linker containing this structure, or Q' 2 represents Q 1 -NR 10 -, and Q 1 forms an amide bond with -NR 10 - through the carboxyl group of R 11 And connect with L3 ;
式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(IX)表示的接头中L b的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)- or -CH2- moiety of L b in the linker represented by IX).
在一些实施方案中,所述式(II)所示的化合物如本发明第二十二方面所述。In some embodiments, the compound of formula (II) is described in the twenty-second aspect of the present invention.
在一些实施方案中,所述接头-药物中间体化合物是选自以下的化合物:In some embodiments, the linker-drug intermediate compound is a compound selected from the group consisting of:
Figure PCTCN2021123020-appb-000082
Figure PCTCN2021123020-appb-000082
Figure PCTCN2021123020-appb-000083
Figure PCTCN2021123020-appb-000083
Figure PCTCN2021123020-appb-000084
Figure PCTCN2021123020-appb-000084
Figure PCTCN2021123020-appb-000085
Figure PCTCN2021123020-appb-000085
本发明的第二十七方面,提供接头,其中,其为下式(VII)表示A twenty-seventh aspect of the present invention provides a linker, wherein it is represented by the following formula (VII)
-Q’ 2-L 3-L 4-L P-L b-     (VII) -Q' 2 -L 3 -L 4 -L P -L b - (VII)
其中,Q’ 2、L 3、L 4、L P、L b的定义如本发明第二十五方面或第二十六方面所述。 Wherein, the definitions of Q' 2 , L 3 , L 4 , LP , and L b are as described in the twenty-fifth or twenty-sixth aspect of the present invention.
在一些实施方案中,所述接头为选自本发明第二十五方面的结构。In some embodiments, the linker is a structure selected from the twenty-fifth aspect of the present invention.
本发明的第二十八方面,提供一种药物组合物,其包含本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,以及任选的药学上可接受的载体。A twenty-eighth aspect of the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof according to the twenty-second or twenty-fifth aspect of the present invention. An acceptable salt, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.
本发明的第二十九方面,提供一种药物制剂,其包含本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物。A twenty-ninth aspect of the present invention provides a pharmaceutical preparation comprising the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof, or a pharmaceutically acceptable compound thereof. An accepted salt, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
本发明的第三十方面,提供本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂的用途,其用于预防和/或治疗肿瘤或癌症。The thirtieth aspect of the present invention provides the antibody-drug conjugate of the twenty-second aspect or the twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody - a drug conjugate, a solvate of a stereoisomer thereof or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention for the prevention and/or treatment of tumors or cancers.
在一些实施方案中,所述的肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌。In some embodiments, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer , bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer.
本发明的第三十一方面,提供一种预防或治疗癌症的方法,其包括向有此需要的受试者施用预防或治疗有效量的本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂。The thirty-first aspect of the present invention provides a method of preventing or treating cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the method of the twenty-second or twenty-fifth aspect of the present invention The antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof, or a solvate of the antibody-drug conjugate, its stereoisomer or a pharmaceutically acceptable salt thereof , the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention.
本发明的第三十二方面,提供本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂的用途,所述试剂用于抑制癌细胞生长、增殖或迁移。The thirty-second aspect of the present invention provides the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the Antibody-drug conjugate, solvate of its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the drug of the twenty-ninth aspect of the present invention Use of a formulation for inhibiting cancer cell growth, proliferation or migration.
本发明的第三十三方面,提供本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂,其用于抑制癌细胞的生长、增殖或迁移。The thirty-third aspect of the present invention provides the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the Antibody-drug conjugate, solvate of its stereoisomer or pharmaceutically acceptable salt thereof, the pharmaceutical composition of the twenty-eighth aspect of the present invention and/or the drug of the twenty-ninth aspect of the present invention Agents for inhibiting the growth, proliferation or migration of cancer cells.
本发明的第三十四方面,提供一种抑制癌细胞生长、增殖或迁移的方法,其包括给癌细胞施用有效量本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂。The thirty-fourth aspect of the present invention provides a method for inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug according to the twenty-second or twenty-fifth aspect of the present invention The conjugate, its stereoisomer or its pharmaceutically acceptable salt, or the solvate of said antibody-drug conjugate, its stereoisomer or its pharmaceutically acceptable salt, the second of the present invention The pharmaceutical composition of the eighteenth aspect and/or the pharmaceutical preparation of the twenty-ninth aspect of the present invention.
本发明的第三十五方面,提供一种抑制癌细胞生长、增殖或迁移的试剂盒,其包括本发明第二十二方面或第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、本发明第二十八方面的药物组合物和/或本发明第二十九方面所述的药物制剂。The thirty-fifth aspect of the present invention provides a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate of the twenty-second or twenty-fifth aspect of the present invention, its A stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of a stereoisomer or a pharmaceutically acceptable salt thereof, the drug of the twenty-eighth aspect of the present invention The composition and/or the pharmaceutical preparation according to the twenty-ninth aspect of the present invention.
本发明的第三十六方面,提供本发明第二十二方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物的制备方法,所述方法包括:The thirty-sixth aspect of the present invention provides the antibody-drug conjugate of the twenty-second aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate , the preparation method of the solvate of its stereoisomer or its pharmaceutically acceptable salt, described method comprises:
Figure PCTCN2021123020-appb-000086
Figure PCTCN2021123020-appb-000086
使式(VI)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(VI)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (VI) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (VI) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
其中,R 1、R 2、R 3、R 4的定义如本发明第二十二方面所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention;
Q 1、L 1、L 2、L a的定义如本发明第二十二方面所述; The definitions of Q 1 , L 1 , L 2 , and La are as described in the twenty-second aspect of the present invention;
T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site. The oxygen of the above-mentioned formula (V) is connected to the right end -C( = O)- or -CH 2 - part of La in the linker represented by the above formula (V);
AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
本发明的第三十七方面,提供本发明第二十三方面接头-药物中间体化合物的制备方法,所述方法包括:The thirty-seventh aspect of the present invention provides a method for preparing a linker-drug intermediate compound of the twenty-third aspect of the present invention, the method comprising:
(1)将N-[(9H-芴-9-基甲氧基)羰基]-N'-[(4-甲氧基苯基)二苯基甲基]-L-赖氨酸(CN-CMTC-1)和PABOH溶解在二氯甲烷:甲醇的溶液中,在EEDQ的作用下反应,重结晶纯化得到产物;(1) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN- CMTC-1) and PABOH were dissolved in dichloromethane: methanol solution, reacted under the action of EEDQ, recrystallized and purified to obtain the product;
(2)用哌啶乙腈溶液处理步骤(1)产物,而后纯化产物;(2) treating the product of step (1) with a solution of piperidine in acetonitrile, and then purifying the product;
(3)用DCC,NHS与O-(2-叠氮乙基)-O-[2-(二羟乙酰基-氨基)乙基]七聚乙二醇(CN-CMTC-4)在DMF溶液中反应生成CN-CMTC-4活性酯;(3) Using DCC, NHS and O-(2-azidoethyl)-O-[2-(diglycolyl-amino)ethyl]heptaethylene glycol (CN-CMTC-4) in DMF solution The middle reaction generates CN-CMTC-4 active ester;
(4)步骤(3)的活性酯与步骤(2)的产物反应生成化合物;(4) the active ester of step (3) reacts with the product of step (2) to generate compound;
(5)吉咪替康-Boc或吉马替康-Boc用三光气,DMAP,与二氯甲烷作用生成甲酰氯化合物,再加入步骤(4)的反应化合物,后用TFA/DCM脱保护处理;(5) Gematecan-Boc or Gematecan-Boc is treated with triphosgene, DMAP, and dichloromethane to form a formyl chloride compound, and then the reaction compound of step (4) is added, and then deprotected with TFA/DCM. ;
(6)步骤(5)的产物与本发明第一方面所述的化合物进行Click反应,用TFA/DCM处理后得最终产物;(6) The product of step (5) is subjected to Click reaction with the compound described in the first aspect of the present invention, and the final product is obtained after being treated with TFA/DCM;
任选地,步骤(6)还可用以下步骤替代:步骤(5)的产物与SM-1加入DMSO/H 2O的溶液中,再加入CuBr催化,反应完全,纯化后加入TFA/DCM脱保护,得最终产物; Optionally, step (6) can also be replaced by the following steps: the product of step (5) and SM-1 are added to a solution of DMSO/H 2 O, and then CuBr is added to catalyze the reaction to complete the reaction. After purification, TFA/DCM is added for deprotection , the final product is obtained;
所述SM-1为The SM-1 is
Figure PCTCN2021123020-appb-000087
Figure PCTCN2021123020-appb-000087
本发明的第三十八方面,提供本发明第二十五方面所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂 合物的制备方法,所述方法包括:The thirty-eighth aspect of the present invention provides the antibody-drug conjugate of the twenty-fifth aspect of the present invention, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate , the preparation method of the solvate of its stereoisomer or its pharmaceutically acceptable salt, described method comprises:
Figure PCTCN2021123020-appb-000088
Figure PCTCN2021123020-appb-000088
使式(X)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(X)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (X) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (X) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
其中,R 1、R 2、R 3、R 4的定义如本发明第二十二方面所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the twenty-second aspect of the present invention;
Q 2、Q’ 2、L 3、L 4、L P、L a的定义如本发明第二十五或第二十六方面所述; The definitions of Q 2 , Q' 2 , L 3 , L 4 , L P and La are as described in the twenty-fifth or twenty-sixth aspect of the present invention;
T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site. The oxygen of the above-mentioned formula (V) is connected to the right end -C( = O)- or -CH 2 - part of La in the linker represented by the above formula (V);
AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
本发明的第三十九方面,提供本发明第二十六方面的接头-药物中间体化合物的制备方法,所述方法包括:The thirty-ninth aspect of the present invention provides a method for preparing the linker-drug intermediate compound of the twenty-sixth aspect of the present invention, the method comprising:
方案A:Option A:
(1)Boc-GGFG与PABOH在EEDQ的作用下,二氯甲烷与甲醇作溶剂,室温搅拌过夜,生成Boc-GGFG-PABOH;(1) Boc-GGFG and PABOH are generated under the action of EEDQ, using dichloromethane and methanol as solvents, and stirring overnight at room temperature to generate Boc-GGFG-PABOH;
(2)Boc-GGFG-PABOH在TFA/DCM的作用下脱掉Boc生成GGFG-PABOH;(2) Boc-GGFG-PABOH strips off Boc under the action of TFA/DCM to generate GGFG-PABOH;
(3)GGFG-PABOH与N 3-PEGn-NHS活性酯反应生成N 3-PEGn-GGFG-PABOH,n=0,2,4,6或8; (3) GGFG-PABOH reacts with N 3 -PEGn-NHS active ester to generate N 3 -PEGn-GGFG-PABOH, n=0, 2, 4, 6 or 8;
(4)吉咪替康-Boc、SN-38-Boc或吉马替康与DMAP,三光气在二氯甲烷的溶剂中反应,加入步骤(3)的N 3-PEGn-GGFG-PABOH,生成N 3-PEGn-GGFG-PABC-吉咪替康-Boc,或N 3-PEGn-GGFG-PABC-SN-38-Boc,或N 3-PEGn-GGFG-PABC-吉马替康, (4) Gematecan-Boc, SN-38-Boc or Gematecan is reacted with DMAP, triphosgene in a solvent of dichloromethane, and N 3 -PEGn-GGFG-PABOH of step (3) is added to generate N 3 -PEGn-GGFG-PABC-Gimantecan-Boc, or N 3 -PEGn-GGFG-PABC-SN-38-Boc, or N 3 -PEGn-GGFG-PABC-Gimantecan,
(5)步骤(4)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click反应得最终化合物,m=2,4,6,8;或者,(5) The product of step (4) reacts with alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) to obtain the final Compound, m=2, 4, 6, 8; or,
方案B:Option B:
(1)Boc-GGFG在TFA/DCM的作用下脱掉Boc,除掉TFA与二氯甲烷后,与N 3-PEGn-NHS在二氯甲烷中反应,用DIEA做碱,得到化合物N 3-PEGn-GGFG,n=0,2,4,6或8; (1) Boc-GGFG removes Boc under the action of TFA/DCM, removes TFA and dichloromethane, reacts with N 3 -PEGn-NHS in dichloromethane, and uses DIEA as a base to obtain compound N 3 - PEGn-GGFG, n=0, 2, 4, 6 or 8;
(2)N 3-PEGn-GGFG与N-Boc-N-甲基乙二胺缩合,再用TFA/DCM脱掉Boc后,得化合物N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3(2) N 3 -PEGn-GGFG was condensed with N-Boc-N-methylethylenediamine, and then Boc was removed with TFA/DCM to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH -CH 3 ;
(3)N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3与吉咪替康-PNP(或吉马替康-PNP,SN-38-PNP)在有TEA,DMF的条件下反应得化合物N 3-PEGn-GGFG-NH-C 2H 4-N(CH 3)-C(O)-吉咪替康(或SN-38,或吉马替康), (3) N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 and gimatecan-PNP (or gimatecan-PNP, SN-38-PNP) in the condition of TEA, DMF The following reaction is carried out to obtain the compound N 3 -PEGn-GGFG-NH-C 2 H 4 -N(CH 3 )-C(O)-gemitecan (or SN-38, or gematecan),
(4)步骤(3)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click反应得最终化合物,m=2,4,6,8。(4) The product of step (3) reacts with alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) to obtain the final Compound, m=2,4,6,8.
附图说明Description of drawings
图1为ADC-1的SEC-HPLC结果。Figure 1 shows the SEC-HPLC results of ADC-1.
图2为ADC-5的SEC-HPLC图谱。Figure 2 is the SEC-HPLC profile of ADC-5.
图3为ADC-6的SEC-HPLC图谱。Figure 3 is the SEC-HPLC profile of ADC-6.
图4为ADC-8的SEC-HPLC图谱。Figure 4 is the SEC-HPLC profile of ADC-8.
图5为ADC-10的SEC-HPLC图谱。Figure 5 is the SEC-HPLC profile of ADC-10.
图6为ADC-11的SEC-HPLC图谱。Figure 6 is the SEC-HPLC profile of ADC-11.
图7为ADC-12的SEC-HPLC图谱。Figure 7 is the SEC-HPLC profile of ADC-12.
图8为ADC-13的SEC-HPLC图谱。Figure 8 is the SEC-HPLC profile of ADC-13.
图9为ADC-14的SEC-HPLC图谱。Figure 9 is the SEC-HPLC profile of ADC-14.
图10为ADC-15的SEC-HPLC图谱。Figure 10 is the SEC-HPLC profile of ADC-15.
图11为ADC-16的SEC-HPLC图谱。Figure 11 is the SEC-HPLC profile of ADC-16.
图12为ADC-17的SEC-HPLC图谱。Figure 12 is the SEC-HPLC profile of ADC-17.
图13为ADC-5小分子的释放-出峰定位图。Figure 13 is a release-peak localization map of ADC-5 small molecules.
图14为ADC-5小分子的释放-折线图。Figure 14 is a release-line graph of ADC-5 small molecule.
图15为CL2A-CM样品中主峰面积百分比变化。Figure 15 is the percent change in the area of the main peak in the CL2A-CM sample.
图16为CL2A-CM样品中CM峰面积百分比变化。Figure 16 is the CM peak area percent change in CL2A-CM samples.
图17为ADC-5样品中主峰面积百分比变化。Figure 17 is the percent change in the area of the main peak in ADC-5 samples.
图18为ADC-5样品中CM峰面积百分比变化。Figure 18 is the CM peak area percent change in ADC-5 samples.
图19为4种ADC对BXPC-3细胞活性的抑制结果。Figure 19 shows the results of inhibition of BXPC-3 cell activity by four ADCs.
图20为待测药在BxPC-3上的IC50。Figure 20 shows the IC50 of the test drug on BxPC-3.
图21为待测药在COLO 205上的IC50。Figure 21 shows the IC50 of the test drug on COLO 205.
图22为待测药在Calu-3上的IC50。Figure 22 shows the IC50 of the tested drugs on Calu-3.
图23为待测药在Calu-6上的IC50。Figure 23 shows the IC50 of the tested drugs on Calu-6.
图24为待测药在NCI-N87上的IC50。Figure 24 shows the IC50 of the tested drugs on NCI-N87.
图25为ADC-5在BxPC-3肿瘤模型中的抗肿瘤活性。Figure 25 is the antitumor activity of ADC-5 in the BxPC-3 tumor model.
图26为ADC-5对BxPC-3模型体重的影响。Figure 26 shows the effect of ADC-5 on the body weight of the BxPC-3 model.
图27为ADC-137、ADC-5在COLO 205肿瘤模型中的抗肿瘤活性。Figure 27 shows the anti-tumor activity of ADC-137 and ADC-5 in COLO 205 tumor model.
图28为ADC-137、ADC-5对COLO 205模型体重的影响。Figure 28 shows the effects of ADC-137 and ADC-5 on the body weight of the COLO 205 model.
图29为ADC-137、ADC-5在BxPC-3肿瘤模型中的抗肿瘤活性。Figure 29 shows the antitumor activity of ADC-137 and ADC-5 in BxPC-3 tumor model.
图30为ADC-137、ADC-5对BxPC-3模型体重的影响。Figure 30 shows the effects of ADC-137 and ADC-5 on the body weight of the BxPC-3 model.
图31为ADC-137、ADC-5在Calu-3肿瘤模型中的抗肿瘤活性。Figure 31 shows the antitumor activity of ADC-137 and ADC-5 in Calu-3 tumor model.
图32为ADC-137、ADC-5对Calu-3模型体重的影响。Figure 32 shows the effect of ADC-137 and ADC-5 on the body weight of Calu-3 model.
图33为ADC-137、ADC-5在Capan-1肿瘤模型中的抗肿瘤活性。Figure 33 shows the antitumor activity of ADC-137 and ADC-5 in Capan-1 tumor model.
图34为ADC-137、ADC-5对Capan-1模型体重的影响。Figure 34 shows the effects of ADC-137 and ADC-5 on the body weight of the Capan-1 model.
图35为ADC-16、ADC-5和ADC-17对COLO 205模型体重的影响。Figure 35 shows the effect of ADC-16, ADC-5 and ADC-17 on body weight of the COLO 205 model.
图36为ADC-16、ADC-5和ADC-17在COLO 205肿瘤模型中的抗肿瘤活性。Figure 36 is the anti-tumor activity of ADC-16, ADC-5 and ADC-17 in the COLO 205 tumor model.
图37为ADC-8、ADC-11、ADC-5和ADC-12在BxPC-3肿瘤模型中的抗肿瘤活性。Figure 37 is the antitumor activity of ADC-8, ADC-11, ADC-5 and ADC-12 in the BxPC-3 tumor model.
图38为ADC-8、ADC-11、ADC-5和ADC-12对BxPC-3模型体重的影响。Figure 38 is the effect of ADC-8, ADC-11, ADC-5 and ADC-12 on body weight of the BxPC-3 model.
具体实施方式Detailed ways
定义definition
为了更容易理解本发明,以下具体定义了某些技术和科学术语。除非在本文中另有明确定义,本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
本发明中,“抗体”指免疫球蛋白,是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同,故其抗原性也不同。据此,可将免疫球蛋白分为五类,或称为免疫球蛋白的同种型,即IgM、IgD、IgG、IgA和IgE,其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别,又可分为不同的亚类,如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。In the present invention, "antibody" refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are μ, δ, and γ chains, respectively. , alpha chains, and epsilon chains. The same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified into kappa chains or lambda chains by the difference in the constant region. Each of the five classes of Ig can have a kappa chain or a lambda chain.
本发明所述的抗体轻链可进一步包含轻链恒定区,所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。The antibody light chain of the present invention may further comprise a light chain constant region comprising human or murine κ, λ chains or variants thereof.
本发明所述的抗体重链可进一步包含重链恒定区,所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。The antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大,为可变区(Fv区);靠近C端的其余氨基酸序列相对稳定,为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性,又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区和4个FR区组成,从氨基端到羧基端依次排列的顺序为:FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3;重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3,HCDR1-3)。The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, and is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3).
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体,优选人源化抗体。Antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).
本发明中,抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分,其少于全长,但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点),并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此,抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物,以及合成产生的衍生物,例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段,包括修饰的片段(参见,例如,Methods in Molecular Biology,Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003);Chapter 1;p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链,例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸,并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段,其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M -1的Ka)抗原的抗体。“功能片段”或“抗Her-2抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用,功能片段一般与“抗体片段”含义相同,且就抗体而论,可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段,例如Fv、Fab、F(ab') 2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域以非共价结合方式而形成的二聚体(V H-V L二聚体)组成。在该构型中,每个可变结构域的三个CDRs相互作用,以确定V H-V L二聚体表面上的靶结合位点,与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是,即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半),仍可具有识别和结合靶的能力。 In the present invention, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable region of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers. Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 107-108 M -1 ) to an antigen . A "functional fragment" or "analog of an anti-Her-2 antibody" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment" and, with respect to an antibody, may refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2 , and so on. The "Fv" fragment consists of a dimer ( VH - VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.
本发明中,术语“双特异性”(Bispecific antibody,BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇,通常,双特异性抗体和/或抗原结合分子包含两种抗原结合位点,其中每种特异于不同的抗原性决定簇。在某些实施方案中,所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇,特别是在两种不同的细胞上表达的两种抗原性决定簇。In the present invention, the term "Bispecific antibody" (BsAb) refers to an antibody and/or an antigen-binding molecule that can specifically bind to two different antigenic determinants. Generally, a bispecific antibody and/or an antigen-binding molecule contains Two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.
本发明中,“单克隆抗体”或“单抗”指相同抗体的群体,表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反,所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备。例如,单克隆抗体可以通过永生化B细胞来制备,例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。In the present invention, "monoclonal antibody" or "monoclonal antibody" refers to a population of the same antibody, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods. For example, monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
本发明中,全长抗体是具有两条全长重链(例如VH-CH 1-CH 2-CH 3或VH-CH 1-CH 2-CH 3-CH 4)和两条全长轻链(VL-CL)和铰链区的抗体,例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。 In the present invention, a full-length antibody has two full-length heavy chains (eg VH-CH 1 -CH 2 -CH 3 or VH-CH 1 -CH 2 -CH 3 -CH 4 ) and two full-length light chains ( VL-CL) and hinge region antibodies, such as antibodies naturally produced by antibody-secreting B cells as well as synthetically produced antibodies having the same domains.
术语“嵌合抗体”是指这样的抗体,其中可变区序列源自一个物种,恒定区序列源自另一物种,如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
“人源化”抗体是指非人(例如小鼠)抗体形式,其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab') 2或者抗体的其它抗原结合亚序列),含有源自非人免疫球蛋白 的最小序列。优选地,人源化抗体是人免疫球蛋白(接受者抗体),其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。 "Humanized" antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
此外,在人源化中,还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变,由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变,其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常,引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外,CDR内的突变通常不超过一个或两个。因此,本发明所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。In addition, in humanization, it is also possible to mutate amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody . For example, PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions. In addition, there are usually no more than one or two mutations within a CDR. Accordingly, the humanized antibodies of the present invention also encompass antibodies comprising 1 or 2 two amino acid mutations within the CDRs.
本发明中,关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用,并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常,免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1x10 8M -1或更大的亲和常数Ka(或者1x10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定,例如,免疫测定、表面等离子共振(SPR)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定。用于实时检测和监测结合速率的仪器和方法是已知的,并且可商购。 In the present invention, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof is used interchangeably herein and refers to the passage of an antibody or antigen-binding fragment between the antibody and antigen's antibody binding sites The ability of non-covalent interactions to form one or more non-covalent bonds with alloantigens. The antigen may be an isolated antigen or present in tumor cells. Typically, an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1x107 M -1 or 1x108 M -1 or greater (or 1x10-7 M or 1x A dissociation constant (Kd) of 10 −8 M or lower binds the antigen. Affinity constants can be determined by standard kinetic methods of antibody response, eg, immunoassays, surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art. Instruments and methods for detecting and monitoring binding rates in real time are known and commercially available.
本发明中,术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物,包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用,术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链,且可为cDNA。In the present invention, the terms "polynucleotide" and "nucleic acid molecule" refer to oligomers or polymers comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
本发明中,分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基,或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。In the present invention, an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. An "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
本发明中,关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如,启动子可以可操作地连接至编码多肽的核酸,从而所述启动子调控或介导所述核酸的转录。In the present invention, "operably linked" with respect to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
本发明中,“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价,包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽,凝胶电泳之后考马斯蓝染色,Lowry蛋白测定以及Bradford蛋白测定。In the present invention, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
本发明中,“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时,载体中所含的核酸复制,从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。In the present invention, a "host cell" is a cell for receiving, maintaining, replicating and amplifying a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
本发明中,“载体”是可复制的核酸,当载体转化入适当的宿主细胞时,可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞,用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离,但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体,例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。In the present invention, a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell. References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. References to vectors also include those that contain nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
本发明中,载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒,其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。In the present invention, the vector also includes "viral vector" or "viral vector". A viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
本发明中,“表达载体”包括能够表达DNA的载体,所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列,并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。 表达载体一般来源于质粒或病毒DNA,或者可以包含这两者的元件。因此,表达载体指重组DNA或RNA构建体,例如质粒、噬菌体、重组病毒或其他载体,当引入适当的宿主细胞时,导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的,并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。In the present invention, an "expression vector" includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
本发明所述的“药物(药物化合物)”,即“毒素”,指细胞毒性药物,即式(I)所示的化合物(抗肿瘤化合物),能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞,但是由于缺乏特异性,在杀伤肿瘤细胞的同时,也会导致正常细胞凋亡。该术语包括毒素,如真菌、细菌、植物或动物来源的小分子毒素或酶活性毒素,放射性同位素(例如I 131、Y 90、Re 186、I 125),毒性药物,化疗药物,抗生素和核溶酶,优选为毒性药物,更优选喜树碱类衍生物,更优选吉咪替康、吉马替康。 The "drug (drug compound)" in the present invention, that is, "toxin", refers to a cytotoxic drug, that is, a compound represented by formula (I) (anti-tumor compound), which can strongly disrupt the normal growth of tumor cells. chemical molecules. In principle, cytotoxic drugs can kill tumor cells at a high enough concentration, but due to the lack of specificity, they can also lead to normal cell apoptosis while killing tumor cells. The term includes toxins, such as small molecule toxins or enzymatically active toxins of fungal, bacterial, plant or animal origin, radioisotopes (eg I 131 , Y 90 , Re 186 , I 125 ), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic agents The enzyme is preferably a toxic drug, more preferably a camptothecin derivative, more preferably gimitecan and gimatecan.
本发明中,“C a-C b”(a和b表示1以上的整数,a<b)包括a至b个碳的任何一种具体情况,例如C 1-C 6包括C 1、C 2、C 3、C 4、C 5、C 6,也包括a至b中的任何一个范围,例如C 1-C 6包括C 1-C 3、C 1-C 4、C 1-C 5、C 2-C 5、C 2-C 4、C 3-C 6等;同理,“a-b元环”(a和b表示1以上的整数,a<b)表示成环原子数为a至b个的环结构,例如3-6元环包括3元环、4元环、5元环、6元环,也包括a至b中的任何一个范围,例如3-6元环包括3-4元环、3-5元环、4-6元环、4-5元环等。 In the present invention, "C a -C b " (a and b represent an integer of 1 or more, a<b) includes any specific case of a to b carbons, for example, C 1 -C 6 includes C 1 and C 2 , C 3 , C 4 , C 5 , C 6 , also including any one range of a to b, for example, C 1 -C 6 includes C 1 -C 3 , C 1 -C 4 , C 1 -C 5 , C 2 -C 5 , C 2 -C 4 , C 3 -C 6 , etc.; similarly, "ab-membered ring" (a and b represent an integer of 1 or more, a<b) represents that the number of ring atoms is a to b The ring structure, for example, 3-6 membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, and also includes any range from a to b, for example, 3-6 membered ring includes 3-4 membered ring , 3-5-membered ring, 4-6-membered ring, 4-5-membered ring, etc.
本发明中,“卤素”是指氟、氯、溴、碘。In the present invention, "halogen" refers to fluorine, chlorine, bromine and iodine.
本发明中,“C 1-C 6烃基”是指从含有1-6个碳原子的烷烃部分去除一个氢原子而衍生得到的直链或支链的烷基,具体地,本发明中的“C 1-C 6烃基”可以是饱和的,即“C 1-C 6烷基”,C 1-6烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、异戊基、2-甲基丁基、新戊基、1-乙基丙基、正己基、异己基、4-甲基戊基、3-甲基戊基、2-甲基戊基、1-甲基戊基、3,3-二甲基丁基、2,2-二甲基丁基、1,1-二甲基丁基、1,2-二甲基丁基、1,3-二甲基丁基、2,3-二甲基丁基、2-乙基丁基、1-甲基-2-甲基丙基等;所述“C 1-4烷基”是指从含有1-4个碳原子的烷烃部分去除一个氢原子而衍生得到的直链或支链的烷基,具体地,C 1-4烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基。本发明中的“C 1-C 6烃基”可以是不饱和的,例如“C 2-C 6烯基”,“C 3-C 4烯基”,“C 3-C 4烯基”包括但不限于丙烯基、1-丁烯基、2-丁烯基等。 In the present invention, "C 1 -C 6 hydrocarbon group" refers to a straight-chain or branched alkyl group derived from an alkane moiety containing 1-6 carbon atoms by removing one hydrogen atom, specifically, in the present invention " C 1 -C 6 hydrocarbyl" may be saturated, ie "C 1 -C 6 alkyl", C 1-6 alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl , isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 4-methylpentyl , 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethyl Butyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1-methyl-2-methylpropane base, etc.; the "C 1-4 alkyl" refers to a straight-chain or branched alkyl group derived by removing one hydrogen atom from an alkane moiety containing 1-4 carbon atoms, specifically, C 1-4 Alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl. The "C 1 -C 6 hydrocarbyl" in the present invention may be unsaturated, such as "C 2 -C 6 alkenyl", "C 3 -C 4 alkenyl", and "C 3 -C 4 alkenyl" including but not limited to Not limited to propenyl, 1-butenyl, 2-butenyl, and the like.
本发明中,“C 1-6烷氧基”是指上文所定义的“C 1-6烷基”经由氧原子与分子其余部分连接的基团,即“C 1-6烷基-O-”基团,具体的,包括但不限于,例如甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、仲丁氧基、叔丁氧基、新戊氧基、正己氧基等;所述的“C 1-4烷氧基”是指上文所定义的“C 1-4烷基”通过氧原子与分子其余部分连接的基团,即“C 1-4烷基-O-”基团,具体地,包括但不限于,甲氧基、乙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、仲丁氧基、叔丁氧基。 In the present invention, "C 1-6 alkoxy" refers to a group in which "C 1-6 alkyl" as defined above is connected to the rest of the molecule via an oxygen atom, namely "C 1-6 alkyl-O"-" groups, specifically, include, but are not limited to, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy group, neopentyloxy, n-hexyloxy, etc.; the "C 1-4 alkoxy" refers to a group in which the above-defined "C 1-4 alkyl" is connected to the rest of the molecule through an oxygen atom , namely "C 1-4 alkyl-O-" group, specifically, including but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy group, sec-butoxy, tert-butoxy.
本发明中,“5-6元环”是指具有5-6个成环原子的非芳香性环状结构,所述成环原子可以均为碳原子,从而形成碳环;还可以含有1-3个各自独立地选自N、O或S的环杂原子,从而形成杂环(例如,含氧杂环、含氮杂环、含硫杂环);所述5-6元环可以为饱和结构,还可以为含有1或2个碳碳双键或碳碳三键的不饱和结构。In the present invention, "5-6 membered ring" refers to a non-aromatic cyclic structure with 5-6 ring atoms, and the ring atoms can be all carbon atoms, thereby forming a carbocyclic ring; it can also contain 1- 3 ring heteroatoms each independently selected from N, O, or S, thereby forming a heterocycle (eg, oxygen-containing heterocycle, nitrogen-containing heterocycle, sulfur-containing heterocycle); the 5-6 membered ring may be saturated The structure can also be an unsaturated structure containing 1 or 2 carbon-carbon double bonds or carbon-carbon triple bonds.
本发明中,“5-6元含氮杂环”包括但不限于哌啶、哌嗪,优选哌啶。In the present invention, "5-6 membered nitrogen-containing heterocycle" includes but is not limited to piperidine and piperazine, preferably piperidine.
本发明中,“C 6-C 10芳基”是指具有6-10个成环碳原子的芳香性环状烃基,其可以是一价基团或根据需要的二价以上的基团,包括单环芳基和稠环芳基,“稠环芳基”是指基团中的每个环与其他环共用相邻一对环碳原子的含有多个环(例如含有2个)的芳基。本发明中,“C 6-C 10元芳基”可以具体列举出苯基、萘基。 In the present invention, "C 6 -C 10 aryl group" refers to an aromatic cyclic hydrocarbon group having 6-10 ring-forming carbon atoms, which can be a monovalent group or a divalent or higher group as required, including Monocyclic aryl group and fused-ring aryl group, "fused-ring aryl group" refers to an aryl group containing multiple rings (eg, containing 2) in which each ring in the group shares an adjacent pair of ring carbon atoms with other rings. . In the present invention, the "C 6 -C 10 -membered aryl group" specifically includes a phenyl group and a naphthyl group.
本发明中,结构式中的
Figure PCTCN2021123020-appb-000089
表示作为该部分或取代基与核心或骨架结构的连接点的键。
In the present invention, in the structural formula
Figure PCTCN2021123020-appb-000089
Represents the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.
本发明所述“接头”、“接头结构”或“连接子”或“连接单元”是指一端与抗体连接而另一端与药物(药物化合物)相连的化学结构片段或键,也可以连接其它接头后再与药物化合物相连。本发 明的接头结构可以通过本领域已知方法合成,也可使用本发明所述的方法进行合成。The "linker", "linker structure" or "linker" or "linking unit" in the present invention refers to a chemical structural fragment or bond that is connected to an antibody at one end and a drug (drug compound) at the other end. Other linkers can also be connected. It is then linked to the drug compound. The linker structure of the present invention can be synthesized by methods known in the art, or can be synthesized using the methods described in the present invention.
本发明所述“抗体-药物偶联物”,即ADC,指配体通过稳定的连接单元与具有生物活性的药物相连。在本发明中是指将单克隆抗体或者片段通过接头结构与具有生物活性的毒性药物相连。The "antibody-drug conjugate" of the present invention, namely ADC, refers to a ligand linked to a biologically active drug through a stable linking unit. In the present invention, it refers to linking the monoclonal antibody or fragment with the biologically active toxic drug through the linker structure.
本发明中,“药学上可接受的盐”是指相对无毒的本发明的偶联物的酸加成盐或碱加成盐。所述酸加成盐为本发明的偶联物与合适的无机酸或者有机酸形成的盐,这些盐可通过使本发明的偶联物与适宜的有机酸或无机酸在适当的溶剂中进行反应来制备。代表性酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、亚硫酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月硅酸盐、硼酸盐、苯甲酸盐、乳酸盐、硝酸盐、磷酸盐、磷酸氢盐、碳酸盐、碳酸氢盐、甲苯甲酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、苹果酸盐、抗坏血酸盐、鞣酸盐、扑酸盐、藻酸盐、萘磺酸盐、酒石酸盐、苯甲酸盐、甲磺酸盐、对甲苯磺酸盐、葡萄糖酸盐、乳糖酸盐和月桂基磺酸盐等。所述碱加成盐为本发明的偶联物与合适的无机碱或者有机碱形成的盐,这些盐可通过使本发明的偶联物与适宜的无机碱或者有机碱在适当的溶剂中进行反应来制备。代表性碱加成盐包括例如与碱金属、碱土金属、季铵阳离子形成的盐,例如钠盐、锂盐、钾盐、钙盐、镁盐、四甲基季铵盐、四乙基季铵盐等;胺盐,包括与氨(NH 3)、伯胺、仲胺或叔胺形成的盐,如甲胺盐、二甲胺盐、三甲胺盐、三乙胺盐、乙胺盐等。 In the present invention, "pharmaceutically acceptable salts" refer to relatively nontoxic acid addition salts or base addition salts of the conjugates of the present invention. The acid addition salts are salts formed by the conjugates of the present invention with suitable inorganic or organic acids, and these salts can be prepared by subjecting the conjugates of the present invention with suitable organic or inorganic acids in a suitable solvent reaction to prepare. Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, sulfite, acetate, oxalate, valerate, oleate, palmitate, stearic acid Salt, laurosilicate, borate, benzoate, lactate, nitrate, phosphate, hydrogen phosphate, carbonate, bicarbonate, toluate, citrate, maleic acid Salt, fumarate, succinate, malate, ascorbate, tannate, pamoate, alginate, naphthalene sulfonate, tartrate, benzoate, mesylate, p-toluene Sulfonate, gluconate, lactobionate and lauryl sulfonate, etc. The base addition salts are the salts formed by the conjugates of the present invention and suitable inorganic or organic bases, and these salts can be carried out by making the conjugates of the present invention and suitable inorganic or organic bases in a suitable solvent. reaction to prepare. Representative base addition salts include, for example, salts formed with alkali metal, alkaline earth metal, quaternary ammonium cations, such as sodium, lithium, potassium, calcium, magnesium, tetramethylquaternary ammonium, tetraethylquaternary ammonium salts, etc.; amine salts, including salts formed with ammonia (NH 3 ), primary, secondary or tertiary amines, such as methylamine salts, dimethylamine salts, trimethylamine salts, triethylamine salts, ethylamine salts, and the like.
本发明的偶联物可以存在特定的几何或立体异构体形式,本发明的偶联物中,手性中心可以存在于抗肿瘤化合物(式(I)所示的化合物)中,可以存在于接头结构(式(II)所示的接头)中,还可以存在于抗体及其衍生物中。本发明中,所有的这类化合物,包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体,及其外消旋混合物和其他混合物,例如对映异构体或非对映体富集的混合物,均包括在本发明的范围之内。The conjugates of the present invention may exist in specific geometric or stereoisomeric forms. In the conjugates of the present invention, the chiral center may exist in the antitumor compound (the compound represented by formula (I)), or may exist in the The linker structure (the linker represented by formula (II)) may also exist in antibodies and derivatives thereof. In the present invention, all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and racemic mixtures thereof and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, are included in the present invention within the range.
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明的偶联物的一种对映体,可以通过不对称合成或者具有手性助剂的衍生作用来制备,其中将所得非对映体混合物分离,并且辅助基团裂开以提供纯的所需对映异构体。或者,当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时,与适当的光学活性的酸或碱形成非对映异构体的盐,然后通过本领域所公知的常规方法进行非对映异构体拆分,然后回收得到纯的对映体。此外,对映异构体和非对映异构体的分离通常是通过使用色谱法完成的,所述色谱法采用手性固定相,并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of the conjugates of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to provide Pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, followed by conventional methods known in the art The diastereoisomers were resolved and the pure enantiomers recovered. In addition, separation of enantiomers and diastereomers is usually accomplished by the use of chromatography employing a chiral stationary phase, optionally in combination with chemical derivatization (eg, from amines to amino groups) formate).
本发明中,本发明的偶联物的溶剂合物(例如水合物)也在本发明的范围内。作为适当的溶剂合物,具体而言,可以列举本发明的偶联物与丙酮、2-丁醇、2-丙醇、乙醇、乙酸乙酯、四氢呋喃、二乙醚等形成的溶剂合物。还可以列举出水合物或乙醇化物。In the present invention, solvates (eg, hydrates) of the conjugates of the present invention are also within the scope of the present invention. Specific examples of suitable solvates include solvates of the conjugate of the present invention with acetone, 2-butanol, 2-propanol, ethanol, ethyl acetate, tetrahydrofuran, diethyl ether, and the like. Hydrates or ethanolates can also be cited.
本发明中,“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解,或者在治疗后保持不变。因此,治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的ADC以及本文所提供的药物组合物、药物制剂的任何药学用途。In the present invention, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment and/or cure. Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease. Treatment also includes any pharmaceutical use of the provided ADCs as well as the pharmaceutical compositions, pharmaceutical formulations provided herein.
本发明中,“疗效”表示由个体的治疗所导致的效果,其改变、通常改良或改善疾病或疾病状况的症状,或者治愈疾病或疾病状况。In the present invention, "therapeutic effect" means the effect resulting from the treatment of an individual, which alters, generally ameliorates or ameliorates the symptoms of a disease or disease condition, or cures a disease or disease condition.
本发明中,“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此,其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
本发明中,“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量,例如,防止或延迟疾病或症状的发生或复发,减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生,并且可以仅在施用一系列剂量之后发生。因此,预防有效量可以在一次或多次施用中施用。In the present invention, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material or composition comprising a compound that, when administered to a subject, will have a desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
本文中提及的文献均以其整体援引加入本文中。Documents mentioned herein are incorporated by reference in their entirety.
[抗肿瘤化合物][Anti-tumor compound]
以下,对于连接于本发明的抗体-药物偶联物的抗肿瘤化合物进行说明。作为抗肿瘤化合物,只要是具有抗肿瘤效果的化合物、具有能连接于接头结构的取代基的化合物即可,没有特别限制。对于抗肿瘤化合物而言,接头的一部分或全部在肿瘤细胞内被切断而游离出抗肿瘤化合物部分从而显示抗肿瘤效果。在与药物的连接部分切断接头时,以抗肿瘤化合物的本来的结构游离出抗肿瘤化合物,发挥其本来的抗肿瘤效果。Hereinafter, the antitumor compounds linked to the antibody-drug conjugates of the present invention will be described. The antitumor compound is not particularly limited as long as it is a compound having an antitumor effect or a compound having a substituent capable of being linked to a linker structure. For antitumor compounds, a part or the whole of the linker is cleaved in tumor cells to free the antitumor compound part, thereby exhibiting an antitumor effect. When the linker is cleaved with the linking part of the drug, the antitumor compound is released in its original structure, and its original antitumor effect is exerted.
本发明中的抗肿瘤化合物为下式(II)表示的化合物。The antitumor compound in the present invention is a compound represented by the following formula (II).
Figure PCTCN2021123020-appb-000090
Figure PCTCN2021123020-appb-000090
式(II)中,In formula (II),
R 1选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、被-OC(=O)R 13或-NR 7R 8取代的C 1-C 6烷基、被-SiMe 3取代的C 1-C 6烷基、或-CH=N-O-(C 1-C 6烷基); R 1 is selected from hydrogen, halogen, hydroxy, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C substituted by -OC(=O)R 13 or -NR 7 R 8 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with -SiMe 3 , or -CH=NO-(C 1 -C 6 alkyl);
R 2选自氢、卤素、羟基、硝基、氨基、饱和或不饱和C 1-C 6烷基、C 1-C 6烷氧基、或被-NR 7R 8取代的C 1-C 6烷基; R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or C 1 -C 6 substituted by -NR 7 R 8 alkyl;
R 3选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、或-NR 7R 8C(=O)O-基; R 3 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, or -NR 7 R 8 C(=O)O-yl;
R 4选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、或C 1-C 6烷氧基; R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
或者R 1和R 2可以连接在一起与母体部分形成任选被R 9取代的5-6元环; Alternatively R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
或者R 3和R 4可以连接在一起与母体部分形成任选被R 9取代的5-6元含氧杂环; Alternatively R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
R 7和R 8每次出现时各自独立地选自氢、C 1-C 6烷基、被羟基或氨基取代的C 1-C 6酰基;或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环; Each occurrence of R 7 and R 8 is independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
R 9每次出现时各自独立地选自卤素、羟基、硝基、-NR 7R 8、C 1-C 6烷基、C 1-C 6烷氧基、任选被C 1-C 6烷基取代的哌啶基; Each occurrence of R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
R 13表示羧基取代的C 1-C 6烷基。 R 13 represents a carboxy-substituted C 1 -C 6 alkyl group.
在某些优选实施方案中,R 1表示氢、C 1-C 4烷基、被-NH(C 1-C 4烷基)取代的C 1-C 4烷基、被
Figure PCTCN2021123020-appb-000091
取代的C 1-C 4烷基、被-SiMe 3取代的C 1-C 4烷基、-CH=N-O-(C 3-C 6烷基)或-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH。
In certain preferred embodiments, R 1 represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -NH(C 1 -C 4 alkyl), C 1 -C 4 alkyl substituted by
Figure PCTCN2021123020-appb-000091
Substituted C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -SiMe 3 , -CH=NO-(C 3 -C 6 alkyl) or -(CH 2 ) 2 O(C=O )( CH2 ) 2 (C=O)OH.
在某些优选实施方案中,R 2表示氢、C 3-C 4烯基、硝基、氨基、或被-N(C 1-C 4烷基) 2取代的C 1-C 4烷基。 In certain preferred embodiments, R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, or C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 .
在某些优选实施方案中,R 3表示氢、卤素、羟基、或
Figure PCTCN2021123020-appb-000092
In certain preferred embodiments, R 3 represents hydrogen, halogen, hydroxyl, or
Figure PCTCN2021123020-appb-000092
在某些优选实施方案中,R 4表示氢或卤素。 In certain preferred embodiments, R4 represents hydrogen or halogen.
在某些优选实施方案中,R 1和R 2连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000093
其中
Figure PCTCN2021123020-appb-000094
部分表示连接于母体基团的键。
In certain preferred embodiments, R1 and R2 are joined together to form the group shown below
Figure PCTCN2021123020-appb-000093
in
Figure PCTCN2021123020-appb-000094
A moiety represents a bond to the parent group.
在某些优选实施方案中,R 3和R 4连接在一起形成以下所示的基团
Figure PCTCN2021123020-appb-000095
其中
Figure PCTCN2021123020-appb-000096
部分表示连接于母体基团的键。
In certain preferred embodiments, R3 and R4 are joined together to form the group shown below
Figure PCTCN2021123020-appb-000095
in
Figure PCTCN2021123020-appb-000096
A moiety represents a bond to the parent group.
在某些优选实施方案中,式(I)表示的化合物为选自以下的化合物:In certain preferred embodiments, the compound represented by formula (I) is a compound selected from the group consisting of:
Figure PCTCN2021123020-appb-000097
Figure PCTCN2021123020-appb-000097
Figure PCTCN2021123020-appb-000098
Figure PCTCN2021123020-appb-000098
在某些优选实施方案中,式(I)表示的化合物为吉马替康或吉咪替康:In certain preferred embodiments, the compound represented by formula (I) is gimatecan or gimatecan:
Figure PCTCN2021123020-appb-000099
Figure PCTCN2021123020-appb-000099
本发明中,在抗体-药物偶联物中,连接于1分子的抗体的接头-药物的连接数(载药量(DAR,durg to antibody ratio))影响偶联物的有效性、安全性。对于抗体-药物偶联物的制造而言,为了使接头-药物的连接数为定数,可规定反应的原料·试剂的使用量等的反应条件而进行实施,但与低分子化合物的化学反应不同,通常以连接不同数目的药物的混合物的形式获得。因此,本发明中,用平均值即平均药物连接数表示连接于每一个分子抗体的接头-药物的连接数。本发明中,原则上只要没有特别说明,除了表示具有不同的药物连接数的抗体-药物偶联物混合物中包含的具有特定的药物连接数的抗体-药物偶联物的情况之外,药物的连接数是指平均值。可控制连接于抗体分子的抗肿瘤化合物的连接数,作为每抗体的药物平均连接数,可连接1~10个左右的抗肿瘤化合物,优选为2~8个,更优选为4~8个,更优选为6~8。需要说明的是,本领域技术人员可根据本申请的实施例的记载,来设计在抗体上连接必要数目的药物的反应,可得到控制了抗肿瘤化合物的连接数的抗体。在本申请实施例中,未实际测定每一个抗体分子上连接的游离巯基的数目,通过控制反应物摩尔比、反应条件,可以预期每一个抗体分子上所连接的巯基的平均数目m为6-8。In the present invention, in the antibody-drug conjugate, the number of linker-drug linkages (drug loading (DAR, drug load ratio)) connected to one molecule of the antibody affects the effectiveness and safety of the conjugate. The production of antibody-drug conjugates can be carried out by specifying reaction conditions such as the amount of raw materials and reagents used for the reaction in order to make the number of linker-drug linkages constant, but it is different from the chemical reaction of low-molecular-weight compounds. , usually obtained as a mixture of linked different numbers of drugs. Therefore, in the present invention, the number of linker-drug linkages linked to the antibody per molecule is represented by the average value, that is, the average number of drug linkages. In the present invention, unless otherwise specified, in principle, except for the case where antibody-drug conjugates with a specific number of drug linkages contained in antibody-drug conjugate mixtures with different numbers of drug linkages, the The number of connections refers to the average. The number of antitumor compounds linked to the antibody molecule can be controlled, and as the average number of drug linkages per antibody, about 1 to 10 antitumor compounds can be linked, preferably 2 to 8, more preferably 4 to 8, More preferably, it is 6-8. It should be noted that those skilled in the art can design the reaction of linking the necessary number of drugs on the antibody according to the description of the examples of the present application, and can obtain the antibody with the number of links of the anti-tumor compound controlled. In the examples of this application, the number of free sulfhydryl groups attached to each antibody molecule is not actually measured. By controlling the molar ratio of reactants and reaction conditions, it can be expected that the average number m of sulfhydryl groups attached to each antibody molecule is 6- 8.
[接头结构][Joint structure]
对于本发明的抗体-药物偶联物中将抗肿瘤化合物与抗体连接的接头结构进行说明。In the antibody-drug conjugate of the present invention, the linker structure for connecting the antitumor compound and the antibody will be described.
本发明提供一种式(I)表示的化合物,The present invention provides a compound represented by formula (I),
Figure PCTCN2021123020-appb-000100
Figure PCTCN2021123020-appb-000100
其中,R 11为C 1-C 6羧基烷基,R 12为C 2-C 6氰基炔基,X、Y、X’和Y’中有1-2个C原子被N原子取代;优选地,R 11为C 1-C 3羧基烷基,R 12为C 2-C 3氰基炔基。 Wherein, R 11 is a C 1 -C 6 carboxyalkyl group, R 12 is a C 2 -C 6 cyanoalkynyl group, and 1-2 C atoms in X, Y, X' and Y' are substituted by N atoms; preferably Typically, R 11 is C 1 -C 3 carboxyalkyl, and R 12 is C 2 -C 3 cyanoalkynyl.
在本发明的某些实施方案中,X、Y、X’和Y’中有且只有1个C原子被N原子取代;In certain embodiments of the present invention, X, Y, X' and Y' have one and only one C atom replaced by a N atom;
在本发明的某些实施方案中,X、Y、X’和Y’中有2个C原子被N原子取代,且X、Y中有且只有1个C原子被N原子取代,以及X’、Y’有且只有1个C原子被N原子取代;In certain embodiments of the invention, X, Y, X' and Y' have 2 C atoms substituted with N atoms, and X, Y have only 1 C atom substituted with N atoms, and X' , Y' has and only one C atom is replaced by N atom;
在本发明的某些实施方案中,该化合物结构如下所示,分别命名为CN-A,CN-B,CN-C和CN-DIn certain embodiments of the present invention, the structures of the compounds are shown below, designated CN-A, CN-B, CN-C and CN-D, respectively
Figure PCTCN2021123020-appb-000101
Figure PCTCN2021123020-appb-000101
在本发明的某些实施方案中,式(I)化合物作为抗体-药物偶联物中的连接单元,通过R 12的炔基碳与存在于抗体的铰链部的二硫键部分形成的硫醚键而与抗体相连,也即R 12的炔基与抗体铰链部的二硫键反应使得R 12的炔基碳连接到抗体铰链部的还原型巯基(-SH-)上,通过R 11的羧基与存在于接头末端的氨基形成酰胺键而与接头内的其他连接单元相连。 In certain embodiments of the invention, the compound of formula (I) is used as the linking unit in the antibody-drug conjugate through a thioether formed by the alkynyl carbon of R12 and the disulfide moiety present in the hinge portion of the antibody bond to the antibody, that is, the alkynyl group of R 12 reacts with the disulfide bond of the hinge part of the antibody so that the alkynyl carbon of R 12 is connected to the reduced sulfhydryl group (-SH-) of the hinge part of the antibody, through the carboxyl group of R 11 It forms an amide bond with the amino group present at the end of the linker to connect with other linking units in the linker.
在本发明的某些实施方式中,提供了式(I)化合物的制备方法:(1)SM-A(5-溴吡啶-2-羧酸)在Boc 2O,DMAP,t-BuOH的作用下,50℃反应12小时,生成A-1;(2)A-1与Pd(PPh 3) 2Cl 2,三乙胺,丙炔-3-醇在四氢呋喃中70℃反应12小时,得化合物A-2(3)A-2与TEMPO,PhI(OAC) 2,NH 4OAC在CH 3CN/H 2O为9:1的溶液中室温反应12小时,得化合物A-3;(4)A-3在TFA/DCM的作用下生成化合物CN-A。步骤(1)中,将SM-A替换为SM-B(6-溴烟酸)或SM-C(5-溴嘧啶-2-羧酸)可制备获得CN-B和CN-C。 In certain embodiments of the present invention, there is provided a preparation method of the compound of formula (I): (1) Effect of SM-A (5-bromopyridine-2-carboxylic acid) on Boc 2 O, DMAP, t-BuOH (2) A-1 reacted with Pd(PPh 3 ) 2 Cl 2 , triethylamine and propyn-3-ol in tetrahydrofuran at 70 ℃ for 12 hours to obtain the compound A-2(3) A-2 reacted with TEMPO, PhI(OAC) 2 , NH 4 OAC in a solution of CH 3 CN/H 2 O 9:1 at room temperature for 12 hours to obtain compound A-3; (4) A-3 generates compound CN-A under the action of TFA/DCM. In step (1), CN-B and CN-C can be prepared by replacing SM-A with SM-B (6-bromonicotinic acid) or SM-C (5-bromopyrimidine-2-carboxylic acid).
本发明的接头具有下式(III)所示的结构:The linker of the present invention has the structure shown in the following formula (III):
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
式(III)中,L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)-CH 2OCH 2-(C=O)-,n 1表示1~24的整数,n 2表示1~4的整数; In formula (III), L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)-CH 2 OCH 2 -(C =O)-, n 1 represents an integer from 1 to 24, and n 2 represents an integer from 1 to 4;
L 2表示缬氨酸残基、胍氨酸残基、苯丙氨酸残基、赖氨酸残基、D-缬氨酸残基、甘氨酸残基、丙氨酸残基、天冬氨酸残基; L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
L a表示-NR 10-(CH 2)n 3-、-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-; L a represents -NR 10 -(CH 2 )n 3 -, -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 4 -O -(C=O)-;
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基,n 3表示1~4的整数,n 4表示1~4的整数; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxyl groups, n 3 represents an integer of 1 to 4, and n 4 represents an integer of 1 to 4;
Aryl表示任选地被R 9取代的C 6-C 10芳基; Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
W为单键或
Figure PCTCN2021123020-appb-000102
其中,位置①表示与-NR 10-相连,位置②表示与(CH 2CH 2-O-)n 1-相连;
W is a single key or
Figure PCTCN2021123020-appb-000102
Wherein, position ① means connecting with -NR 10 -, and position ② means connecting with (CH 2 CH 2 -O-)n 1 -;
[L 1部分] [L 1 part]
L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)-CH 2OCH 2-(C=O)-,n 1表示1~24的整数, n 2表示1~4的整数,其中,-(CH 2CH 2-O-)n 1部分表示聚乙二醇部分,通过聚乙二醇部分,使得药物的溶解度得到增强。 L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)-CH 2 OCH 2 -(C=O)-,n 1 represents an integer from 1 to 24, and n 2 represents an integer from 1 to 4, wherein -(CH 2 CH 2 -O-)n 1 part represents a polyethylene glycol part, and the solubility of the drug is improved by the polyethylene glycol part be enhanced.
在本发明的某些实施方案中,Q 1通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接。 In certain embodiments of the present invention, Q 1 is linked through the formation of an amide bond between the carboxyl group of R 11 and the left-terminal amino group -NR 10 - in the formula of L 1 .
在本发明的某些实施方案中,n 1表示6、7、8、9、10、11或12,n 2表示1或2。 In certain embodiments of the invention, n 1 represents 6, 7, 8, 9, 10, 11 or 12 and n 2 represents 1 or 2.
在本发明的某些实施方案中,R 10表示氢或C 1-C 4烷基。 In certain embodiments of the present invention, R 10 represents hydrogen or C 1 -C 4 alkyl.
[L 2部分] [Part L 2 ]
L 2表示单个氨基酸残基,例如缬氨酸残基、胍氨酸残基、苯丙氨酸残基、赖氨酸残基、D-缬氨酸残基、甘氨酸残基、丙氨酸残基、天冬氨酸残基。 L 2 represents a single amino acid residue, such as valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue base, aspartic acid residue.
在本发明的某些实施方案中,L 2表示赖氨酸残基。 In certain embodiments of the invention, L2 represents a lysine residue.
[L a部分] [L a part]
L a表示-NR 10-(CH 2)n 3-、-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-;R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基,n 3表示1~4的整数,n 4表示1~4的整数。 L a represents -NR 10 -(CH 2 )n 3 -, -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 4 -O -(C=O)-; each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy groups, n 3 represents an integer from 1 to 4, n 4 Indicates an integer of 1 to 4.
在本发明的某些实施方案中,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(III)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 In certain embodiments of the present invention, the compound represented by formula (II) uses the oxygen in the hydroxyl group at position 19 as the linking site, or when R3 or R4 is a hydroxyl group, the oxygen in the hydroxyl group of R3 or R4 is used as the linking site . Oxygen is used as a linking site and is linked to the right-end -C( = O)- or -CH 2 - moiety of La in the linker represented by the above formula (III).
在本发明的某些实施方案中,式(II)表示的化合物以19位的羟基中的氧作为连接部位,连接于上述L a表示的-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-右端-C(=O)-部分。 In certain embodiments of the present invention, the compound represented by the formula (II) is linked to -NR 10 -(CH 2 ) n 4 -NR 10 - represented by La above using the oxygen in the hydroxyl group at position 19 as a linking site (C=O)- or -NR10 -Aryl-( CH2 )n4 - O-(C=O)-right-C(=O)-moiety.
在本发明的某些实施方案中,式(II)表示的化合物当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述L a表示的-NR 10-(CH 2)n 3-右端-CH 2-部分。 In certain embodiments of the present invention, when R 3 or R 4 is a hydroxyl group, the compound represented by formula (II) is connected to the above-mentioned La represented by the oxygen in the hydroxyl group of R 3 or R 4 as a linking site. NR 10 -(CH 2 )n 3 -right-CH 2 -moiety.
在本发明的某些实施方案中,L a表示-NR 10-Aryl-(CH 2)n 4-O-(C=O)-,其中n 4表示1或2;R 10每次出现时各自独立地表示氢、任选被1个羟基取代的甲基、乙基、丙基、异丙基。 In certain embodiments of the invention, L a represents -NR 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, wherein n 4 represents 1 or 2; each occurrence of R 10 is individually independently represents hydrogen, methyl, ethyl, propyl, or isopropyl optionally substituted with one hydroxyl group.
在本发明的某些实施方案中,L a表示由4-氨基苄醇衍生得到的结构。 In certain embodiments of the present invention, La represents a structure derived from 4-aminobenzyl alcohol.
在本发明的某些实施方案中,L 2所示的氨基酸C端连接于L a所示的基团的末端氨基。 In certain embodiments of the present invention, the C-terminus of the amino acid represented by L 2 is attached to the terminal amino group of the group represented by L a .
在本发明的某些实施方案中,式(III)表示的接头为选自以下所示的基团:In certain embodiments of the present invention, the linker represented by formula (III) is a group selected from the group consisting of:
Figure PCTCN2021123020-appb-000103
Figure PCTCN2021123020-appb-000103
Figure PCTCN2021123020-appb-000104
Figure PCTCN2021123020-appb-000104
本发明的接头具有下式(VII)所示的结构:The linker of the present invention has the structure shown in the following formula (VII):
-Q 2-L 3-L 4-L P-L b-     (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
式(VII)中,L 3表示-Z-W-(CH 2CH 2-O)n 5-W’-或单键,n 5表示1~8的整数,W、W’表示
Figure PCTCN2021123020-appb-000105
或单键,其中,W的位置①表示与Z相连,位置②表示与(CH 2CH 2-O-)n 5-相连,W’的位置①表示与(CH 2CH 2-O-)n 5-相连,位置②表示与L 4的-CH 2-相连,且W、W’不同时为
Figure PCTCN2021123020-appb-000106
Z表示-CH 2-Cyclo-C(=O)-NR 10-或单键,Cyclo表示环己烷基团,
In formula (VII), L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'- or a single bond, n 5 represents an integer of 1 to 8, and W and W' represent
Figure PCTCN2021123020-appb-000105
Or a single bond, wherein, the position ① of W indicates that it is connected to Z, the position ② indicates that it is connected to (CH 2 CH 2 -O-)n 5 -, and the position ① of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected, position ② indicates that it is connected to -CH 2- of L 4 , and W and W' are not at the same time
Figure PCTCN2021123020-appb-000106
Z represents -CH 2 -Cyclo-C(=O)-NR 10 - or a single bond, Cyclo represents a cyclohexane group,
L 4表示-(CH 2)n 6-C(=O)-,n 6表示1~6的整数, L 4 represents -(CH 2 )n 6 -C(=O)-, n 6 represents an integer of 1 to 6,
L P表示由2~7个氨基酸构成的肽残基, LP represents a peptide residue consisting of 2 to 7 amino acids,
L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 8-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-,Aryl表示任选地被R 9取代的C 6-C 10芳基,n 7表示1~4的整数,n 8表示1~4的整数, L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 8 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 8 -O -(C=O)-, Aryl represents a C 6 -C 10 aryl group optionally substituted by R 9 , n 7 represents an integer of 1-4, n 8 represents an integer of 1-4,
R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基, Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy groups,
Q 2表示-(琥珀酰亚胺-3-基-N)-,如下式结构: Q 2 represents -(succinimide-3-yl-N)-, with the following formula:
Figure PCTCN2021123020-appb-000107
Figure PCTCN2021123020-appb-000107
以该结构的3位与抗体连接,在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q 2为-Q 1-NR 10-,Q 1的定义如本发明的定义所述,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接,也即R 12的炔基与抗体铰链部的二硫键反应使得R 12的炔基碳连接到抗体铰链部的还原型巯基(-SH-)上,通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接。 It is connected to the antibody at the 3-position of the structure, and is connected to the methylene group in the linker comprising the structure at the nitrogen atom of the 1-position, or Q 2 is -Q 1 -NR 10 -, and Q 1 is as defined in the present invention. According to the definition, the alkynyl carbon of R 12 and the disulfide bond of the antibody hinge part form a thioether bond to connect, that is, the alkynyl group of R 12 reacts with the disulfide bond of the antibody hinge part to connect the alkynyl carbon of R 12 It is linked to L 3 by forming an amide bond between the carboxyl group of R 11 and -NR 10 - on the reduced sulfhydryl group (-SH-) of the antibody hinge.
[L 4部分] [Part L 4 ]
在本发明的某些实施方案中,L 4表示-(CH 2)n 6-C(=O)-,n 6表示2、3、4或5。 In certain embodiments of the invention, L 4 represents -(CH 2 )n 6 -C(=O)-, and n 6 represents 2, 3, 4 or 5.
[L P部分] [ LP part]
L P表示由2~7个氨基酸构成的肽残基。即,通过2-7个氨基酸以肽键连接而成的寡肽的残基而构成。对于构成L P的氨基酸没有特别限制,例如为L-或D-氨基酸,优选为L-氨基酸。另外,除了α-氨基酸之外,也可以是β-丙氨酸、ε-氨基己酸、γ-氨基丁酸等结构的氨基酸,此外,可以是例如经N-甲基化的氨基酸等非天然型的氨基酸。 LP represents a peptide residue consisting of 2 to 7 amino acids. That is, it consists of oligopeptide residues in which 2-7 amino acids are linked by peptide bonds. The amino acid constituting LP is not particularly limited, and is, for example, an L- or D-amino acid, preferably an L -amino acid. In addition to α-amino acids, amino acids with structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid may be used, and, for example, non-natural amino acids such as N-methylated amino acids may be used. type of amino acid.
对于L P部分的氨基酸序列没有特别限制,作为构成的氨基酸,可举出苯丙氨酸(Phe;F)、酪氨酸(Tyr;Y)、亮氨酸(Leu;L),甘氨酸(Gly;G)、丙氨酸(Ala;A)、缬氨酸(Val;V)、赖氨酸(Lys;K)、瓜氨酸(Cit;C)、丝氨酸(Ser;S)、谷氨酸(Glu;E)、天冬氨酸(Asp;D)等。这些中可优选举出苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸。可根据氨基酸的种类,来控制药物游离的模式。氨基酸的数目可以是2-7个。 The amino acid sequence of the LP moiety is not particularly limited, and the constituent amino acids include phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L ), and glycine (Gly). ; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit; C), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D) and the like. Among these, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid, and aspartic acid are preferably used. The pattern of drug release can be controlled according to the type of amino acid. The number of amino acids can be 2-7.
在本发明的某些实施方案中,L P所示的肽残基在N末端与L 2部分连接,在C末端与L a部分连接。 In certain embodiments of the invention, the peptide residue represented by LP is linked to the L2 moiety at the N-terminus and to the La moiety at the C-terminus.
在本发明的某些实施方案中,L P为由2-5个氨基酸构成的肽残基。 In certain embodiments of the invention, LP is a peptide residue consisting of 2-5 amino acids.
在本发明的某些实施方案中,L P为选自以下的肽残基: In certain embodiments of the invention, LP is a peptide residue selected from the group consisting of:
-GGFG-;-ggfg-;
-VC-;-vc-;
-EVC-;-evc-;
-DVC-;-dvc-;
-EGGFG-;-EGGFG-;
-DGGFG-。-DGGFG-.
[L b部分] [Part L b ]
在本发明的某些实施方案中,L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 8-NR 10-C(=O)-、-NR 10-Aryl-(CH 2)n 8-O-C(=O)-,其中R 10表示氢或C 1-C 4烷基,n 7表示1~2的整数,n 8表示1~2的整数,Aryl表示苯环基团。 In certain embodiments of the invention, L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 8 -NR 10 -C(=O)-, -NR 10 - Aryl-(CH 2 )n 8 -OC(=O)-, wherein R 10 represents hydrogen or C 1 -C 4 alkyl, n 7 represents an integer of 1-2, n 8 represents an integer of 1-2, and Aryl represents phenyl ring group.
在本发明的某些实施方案中,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(VII)表示的接头中L b的右端-C(=O)-或-CH 2-部分。 In certain embodiments of the present invention, the compound represented by formula (II) uses the oxygen in the hydroxyl group at position 19 as the linking site, or when R3 or R4 is a hydroxyl group, the oxygen in the hydroxyl group of R3 or R4 is used as the linking site . Oxygen is used as a linking site, and is linked to the right -C(=O)- or -CH 2 - moiety of L b in the linker represented by the above formula (VII).
在本发明的某些实施方案中,式(II)表示的化合物以19位的羟基中的氧作为连接部位,连接于上述L a表示的-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-右端-C(=O)-部分。 In certain embodiments of the present invention, the compound represented by the formula (II) is linked to -NR 10 -(CH 2 ) n 4 -NR 10 - represented by La above using the oxygen in the hydroxyl group at position 19 as a linking site (C=O)- or -NR10 -Aryl-( CH2 )n4 - O-(C=O)-right-C(=O)-moiety.
在本发明的某些实施方案中,式(II)表示的化合物当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述L b表示的-NR 10-(CH 2)n 3-右端-CH 2-部分。 In certain embodiments of the present invention, when R 3 or R 4 is a hydroxyl group, the compound represented by formula (II) is connected to the above-mentioned L represented by the oxygen in the hydroxyl group of R 3 or R 4 as a linking site. NR 10 -(CH 2 )n 3 -right-CH 2 -moiety.
在本发明的某些实施方案中,L b表示-NR 10-(CH 2)n 4-NR 10-(C=O)-,n 4表示1或2;R 10每次出现时各自独立地表示氢、任选被1个羟基取代的甲基、乙基、丙基、异丙基。 In certain embodiments of the invention, L b represents -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)-, n 4 represents 1 or 2; each occurrence of R 10 is independently Represents hydrogen, methyl, ethyl, propyl, or isopropyl optionally substituted with one hydroxyl group.
在本发明的某些实施方案中,L b表示-NR 10-Aryl-(CH 2)n 4-O-(C=O)-,其中n 4表示1或2;R 10每次出现时各自独立地表示氢、任选被1个羟基取代的甲基、乙基、丙基、异丙基。 In certain embodiments of the invention, L b represents -NR 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, wherein n 4 represents 1 or 2; each occurrence of R 10 is individually independently represents hydrogen, methyl, ethyl, propyl, or isopropyl optionally substituted with one hydroxyl group.
在本发明的某些实施方案中,L b表示由4-氨基苄醇衍生得到的结构。 In certain embodiments of the present invention, L b represents a structure derived from 4-aminobenzyl alcohol.
在本发明的某些实施方案中,L P所示的肽残疾的C末端连接于L b所示的基团,更具体而言,C末端连接于L b所示的基团中的末端氨基。 In certain embodiments of the invention, the disabled C-terminus of the peptide represented by LP is attached to the group represented by Lb , more specifically, the C-terminus is attached to the terminal amino group in the group represented by Lb .
在本发明的某些实施方案中,式(VII)表示的接头为选自以下所示的基团:In certain embodiments of the present invention, the linker represented by formula (VII) is a group selected from the group consisting of:
Figure PCTCN2021123020-appb-000108
Figure PCTCN2021123020-appb-000108
Figure PCTCN2021123020-appb-000109
Figure PCTCN2021123020-appb-000109
[接头中间体化合物][Linker Intermediate Compound]
本发明的接头中间体化合物,如下式(XI)、(XII)所示:The linker intermediate compound of the present invention is shown in the following formulas (XI) and (XII):
Q 1-L 1-L 2-L a-H     (XI) Q 1 -L 1 -L 2 -L a -H (XI)
Q’ 2-L 3-L 4-L P-L b-H     (XII) Q' 2 -L 3 -L 4 -L P -L b -H (XII)
R 1、R 2、R 3、R 4、Q 1、Q’ 2L 1、L 2、L 3、L 4、L P、L a、L b的定义如本发明说明书所述。 The definitions of R 1 , R 2 , R 3 , R 4 , Q 1 , Q′ 2 L 1 , L 2 , L 3 , L 4 , L P , La and L b are as described in the specification of the present invention.
[接头-药物中间体化合物][Linker-drug intermediate compound]
本发明的接头-药物中间体化合物是将T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(V)表示的接头连接而成的:The linker-drug intermediate compound of the present invention is a compound represented by the formula (II) represented by T, and the intermediate compound is formed by connecting the compound (T) and the linker represented by the following formula (V):
Q 1-L 1-L 2-L a-T     (VI) Q 1 -L 1 -L 2 -L a -T (VI)
Figure PCTCN2021123020-appb-000110
Figure PCTCN2021123020-appb-000110
Q 1-L 1-L 2-L a-     (V) Q 1 -L 1 -L 2 -L a - (V)
其中,R 1、R 2、R 3、R 4的定义如本发明说明书所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention;
Q 1、L 1、L 2、L a的定义如本发明说明书所述。 The definitions of Q 1 , L 1 , L 2 and La are as described in the specification of the present invention.
在本发明的某些实施方案中,接头-药物中间体化合物是选自以下的化合物,In certain embodiments of the invention, the linker-drug intermediate compound is a compound selected from the group consisting of,
Figure PCTCN2021123020-appb-000111
Figure PCTCN2021123020-appb-000111
Figure PCTCN2021123020-appb-000112
Figure PCTCN2021123020-appb-000112
Figure PCTCN2021123020-appb-000113
Figure PCTCN2021123020-appb-000113
Figure PCTCN2021123020-appb-000114
Figure PCTCN2021123020-appb-000114
Figure PCTCN2021123020-appb-000115
Figure PCTCN2021123020-appb-000115
本发明的接头-药物中间体化合物是将T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(IX)表示的接头连接而成的:The linker-drug intermediate compound of the present invention is that T represents the compound represented by the formula (II), and the intermediate compound is formed by connecting the compound (T) and the linker represented by the following formula (IX):
Q’ 2-L 3-L 4-L P-L b-T     (X) Q' 2 -L 3 -L 4 -L P -L b -T (X)
Figure PCTCN2021123020-appb-000116
Figure PCTCN2021123020-appb-000116
Q’ 2-L 3-L 4-L P-L b-     (IX) Q' 2 -L 3 -L 4 -L P -L b - (IX)
其中,R 1、R 2、R 3、R 4的定义如本发明说明书所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention;
Q’ 2表示(马来酰亚胺-N)-或Q 1-NR 10-, Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -,
Q’ 2表示的(马来酰亚胺-N)-,如下式结构: (maleimide-N)- represented by Q' 2 has the following formula structure:
Figure PCTCN2021123020-appb-000117
Figure PCTCN2021123020-appb-000117
以该结构在1位的氮原子上与包含该结构的接头内的亚甲基连接,is attached to the methylene group in the linker comprising the structure at the nitrogen atom at position 1 with this structure,
或者Q 2表示的Q 1-NR 10-,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; Or Q 1 -NR 10 - represented by Q 2 , Q 1 is connected to L 3 through the carboxyl group of R 11 and -NR 10 - forming an amide bond;
Q 1、R 10、L 3、L 4、L P、L b的定义如本发明说明书所述。 The definitions of Q 1 , R 10 , L 3 , L 4 , L P and L b are as described in the specification of the present invention.
在本发明的某些实施方案中,接头-药物中间体化合物是选自以下的化合物,In certain embodiments of the invention, the linker-drug intermediate compound is a compound selected from the group consisting of,
Figure PCTCN2021123020-appb-000118
Figure PCTCN2021123020-appb-000118
Figure PCTCN2021123020-appb-000119
Figure PCTCN2021123020-appb-000119
Figure PCTCN2021123020-appb-000120
Figure PCTCN2021123020-appb-000120
在式(II)表示的化合物为吉马替康或吉咪替康的情况下,在L a表示-NR 10-(CH 2)n 4-NR 10--(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-时,L b表示-NR 10-(CH 2)n 8-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-时,作为抗肿瘤化合物的吉马替康或吉咪替康与式(III)、(VII)所示的接头结构以carbamate的结构(-NC(=O)O-)进行连接,在血液中更稳定,毒副作用更小。 When the compound represented by the formula (II) is gimatecan or gimitecan , La represents -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR When 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, L b represents -NR 10 -(CH 2 )n 8 -NR 10 -(C=O)- or -NR 10 -Aryl When -(CH 2 )n 8 -O-(C=O)-, the linker structures of gimatecan or gimitecan as anti-tumor compounds and the linkers represented by formulae (III) and (VII) have the structure of carbamate (-NC(=O)O-) is connected, which is more stable in blood and has less toxic and side effects.
在式(II)表示的化合物为吉咪替康的情况下,在L a、L b表示-NR 10-(CH 2)n 3-时,作为抗肿瘤化合物的吉咪替康与式(III)、(VII)所示的接头结构以结构-N-CH 2-O-进行连接,吉咪替康有19位和10位两个连接位点,为ADC提供了更多样的选择。 When the compound represented by the formula (II) is gemnotecan, when L a and L b represent -NR 10 -(CH 2 )n 3 -, gemnotecan as an antitumor compound and the formula (III) ) and (VII) shown in the linker structure are connected by the structure -N-CH 2 -O-, and gemitecan has two connection sites at the 19th position and the 10th position, which provides more choices for ADC.
[抗体-药物偶联物][Antibody-Drug Conjugates]
本发明的抗体-药物偶联物(IV)是将化合物(T)与抗体(AB)经由下式(III)表示的接头连接而成的:The antibody-drug conjugate (IV) of the present invention is formed by connecting the compound (T) and the antibody (AB) via a linker represented by the following formula (III):
AB-S-Q 1-L 1-L 2-L a-T     (IV) AB-SQ 1 -L 1 -L 2 -L a -T (IV)
Figure PCTCN2021123020-appb-000121
Figure PCTCN2021123020-appb-000121
-Q 1-L 1-L 2-L a-     (III) -Q 1 -L 1 -L 2 -L a - (III)
R 1、R 2、R 3、R 4的定义如本发明说明书所述;Q 1、L 1、L 2、L a的定义如本发明说明书所述;AB表示抗体。 The definitions of R 1 , R 2 , R 3 and R 4 are as described in the specification of the present invention; the definitions of Q 1 , L 1 , L 2 and La are as described in the specification of the present invention; AB represents an antibody.
本发明的抗体-药物偶联物(VIII)是将化合物(T)与抗体(AB)经由下式(VII)表示的接头连接而成的:The antibody-drug conjugate (VIII) of the present invention is formed by connecting the compound (T) and the antibody (AB) via a linker represented by the following formula (VII):
AB-S-Q 2-L 3-L 4-L P-L b-T     (VIII) AB-SQ 2 -L 3 -L 4 -L P -L b -T (VIII)
Figure PCTCN2021123020-appb-000122
Figure PCTCN2021123020-appb-000122
-Q 2-L 3-L 4-L P-L b-     (VII)。 -Q 2 -L 3 -L 4 -L P -L b - (VII).
R 1、R 2、R 3、R 4的定义如本发明说明书所述;Q 2、L 3、L 4、L P、L b的定义如本发明说明书所述;AB表示抗体。 The definitions of R 1 , R 2 , R 3 and R 4 are as described in the description of the present invention; the definitions of Q 2 , L 3 , L 4 , LP and L b are as described in the description of the present invention; AB represents an antibody.
[抗体-药物偶联物的制造方法][Manufacturing method of antibody-drug conjugate]
下面,对于本发明的抗体-药物偶联物或其制造中间体的代表性的制造方法进行说明。Next, a representative production method of the antibody-drug conjugate of the present invention or its production intermediate will be described.
具体而言,本发明中,经由硫醚而将抗体和接头结构连接的抗体-药物偶联物例如可通过下述的方法来制造。Specifically, in the present invention, an antibody-drug conjugate in which an antibody and a linker structure are linked via a thioether can be produced, for example, by the following method.
Figure PCTCN2021123020-appb-000123
Figure PCTCN2021123020-appb-000123
或者,or,
Figure PCTCN2021123020-appb-000124
Figure PCTCN2021123020-appb-000124
即,使式(VI)、(X)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(VI)、(X)所示的接头-药物中间体化合物与抗体连接;制备得到式(IV)、(VIII)表示的抗体-药物偶联物。其中,R 1、R 2、R 3、R 4、Q 1、Q 2、Q’ 2、L 1、L 2、L 3、L 4、L P、L a、L b的定义如本发明说明书所述。 That is, by reacting the linker-drug intermediate compounds represented by the formulae (VI) and (X) with AB-SH, the compounds of the formulae (VI) and ( The linker-drug intermediate compound shown in X) is connected with the antibody; the antibody-drug conjugate represented by formula (IV) and (VIII) is prepared. Wherein, R 1 , R 2 , R 3 , R 4 , Q 1 , Q 2 , Q' 2 , L 1 , L 2 , L 3 , L 4 , L P , La and L b are as defined in the description of the present invention said.
式中,AB-SH表示携带巯基的抗体,AB表示抗体,式(VI)、(X)所示的化合物即为本发明上述的接头-药物中间体化合物,作为产物的式(IV)、(VIII)所示的化合物即为本发明的抗体-药物偶联物。In the formula, AB-SH represents an antibody carrying a sulfhydryl group, AB represents an antibody, and the compounds represented by formulas (VI) and (X) are the above-mentioned linker-drug intermediate compounds of the present invention. The compound shown in VIII) is the antibody-drug conjugate of the present invention.
为了便于说明,式(IV)、(VIII)所示的化合物中,以1个从药物至接头末端的结构部分与1个抗体连接而成的结构的形式进行了记载,但实际上,相对于1个抗体分子而言连接有多个该结构部分的情况较多。例如,如上所述,在一个抗体分子上连接2~8个、优选为4~8个、更优选为6~8个接头-药物中间体化合物。该情况在以下的制造方法的说明中也同样。实际上,如上所述,本发明中,以平均药物连接数表示连接于每一个分子抗体的接头-药物的平均数目。For convenience of explanation, the compounds represented by formulae (IV) and (VIII) are described in the form of a structure in which one structural moiety from the drug to the end of the linker is linked to one antibody, but in fact, relative to In many cases, a plurality of these structural moieties are linked to one antibody molecule. For example, as described above, 2 to 8, preferably 4 to 8, more preferably 6 to 8 linker-drug intermediate compounds are linked to one antibody molecule. This is also the same in the following description of the manufacturing method. In fact, as described above, in the present invention, the average number of linker-drugs linked to each molecule of antibody is expressed as the average number of drug linkages.
即,如上所示,通过使本发明上述的接头-药物中间体化合物与具有巯基的抗体AB-SH反应,可制造式(IV)所示的抗体-药物偶联物。That is, as described above, the antibody-drug conjugate represented by formula (IV) can be produced by reacting the above-mentioned linker-drug intermediate compound of the present invention with the antibody AB-SH having a thiol group.
具有巯基的抗体可利用本领域技术人员公知的方法获得(Hermanson,G.T、Bioconjugate  Techniques、pp.56-136、pp.456-493、Academic Press(1996))。例如,可举出以下方法:使Traut举出试剂与抗体的氨基作用;使N-琥珀酰亚胺基S-乙酰硫代链烷酸酯(N-succinimidyl S-acetylthioalkanoate)类与抗体的氨基作用后,与羟基胺作用;在使N-琥珀酰亚胺基3-(吡啶基二硫代)丙酸酯作用后,使还原剂作用;使二硫苏糖醇、2-巯基乙醇、三(2-羧基乙基)膦盐酸盐(TCEP)等还原剂与抗体作用而将抗体内铰链部的二硫键还原从而产生巯基;等等,但不限于这些方法。Antibodies having thiol groups can be obtained by methods known to those skilled in the art (Hermanson, G.T, Bioconjugate Techniques, pp.56-136, pp.456-493, Academic Press (1996)). For example, the following methods are exemplified: allowing Traut to react with the amino group of the reagent and the antibody; and allowing N-succinimidyl S-acetylthioalkanoate to react with the amino group of the antibody After reacting with hydroxylamine; after reacting N-succinimidyl 3-(pyridyldithio)propionate, reacting reducing agent; reacting dithiothreitol, 2-mercaptoethanol, tri( A reducing agent such as 2-carboxyethyl) phosphine hydrochloride (TCEP) acts on the antibody to reduce the disulfide bond in the hinge portion of the antibody to generate a sulfhydryl group; etc., but not limited to these methods.
具体而言,作为还原剂,相对于每一个抗体内铰链部二硫键,使用0.3~3摩尔当量TCEP,在含有螯合剂的缓冲液中,使其与抗体反应,由此,可得到部分或完全地将抗体内铰链部二硫醚还原而得到的携带巯基的抗体(AB-SH)。作为螯合剂,可举出例如乙二胺四乙酸(EDTA)、二亚乙基三胺五乙酸(DTPA)等。可以以1mM~20mM的浓度使用它们。作为缓冲液,可使用磷酸钠、硼酸钠、乙酸钠溶液等。在具体的例子中,于4℃~37℃使抗体与TCEP反应1~4小时,由此,可得到部分或完全还原而具有巯基的抗体AB-SH。Specifically, using 0.3 to 3 molar equivalents of TCEP per disulfide bond in the hinge portion of the antibody as a reducing agent, and reacting it with the antibody in a buffer containing a chelating agent, partial or A sulfhydryl group-carrying antibody (AB-SH) obtained by completely reducing the disulfide in the hinge part of the antibody. As a chelating agent, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), etc. are mentioned, for example. They can be used in concentrations of 1 mM to 20 mM. As the buffer solution, sodium phosphate, sodium borate, sodium acetate solution or the like can be used. In a specific example, by reacting the antibody with TCEP at 4°C to 37°C for 1 to 4 hours, an antibody AB-SH partially or completely reduced to have a thiol group can be obtained.
相对于每一个具有巯基的抗体AB-SH,可使用2~20摩尔当量的式(VI)、(X)所示的化合物,制造1个抗体连接2个-8个药物而成的抗体-药物偶联物(IV)、(VIII)。具体而言,在含有具有巯基的抗体AB-SH的缓冲液中,添加溶解有式(VI)、(X)所示的化合物的溶液并使其进行反应。此处,作为缓冲液,可使用乙酸钠溶液、磷酸钠、硼酸钠等。反应时的pH为5~9,更优选在pH7附近反应。作为溶解化合物(2)(即式(II)所示化合物)的溶剂,可使用二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)、二甲基乙酰胺(DMA)、N-甲基-2-吡啶酮(NMP)等有机溶剂。可以以1~20%v/v将溶解有式(VI)、(X)所示的化合物的有机溶剂溶液添加到含有具有巯基的抗体AB-SH的缓冲液中并进行反应。反应温度为0~37℃、更优选为10~25℃,反应时间为0.5~2小时。可通过利用含硫醇试剂使未反应的式(VI)、(X)所示的化合物的反应性失活而结束反应。含硫醇试剂例如为半胱氨酸或N-乙酰-L-半胱氨酸(NAC)。更具体而言,添加相对于使用的式(VI)、(X)所示的化合物而言1~2摩尔当量的NAC,于室温孵育10~30分钟,由此使反应结束。2-20 molar equivalents of the compounds represented by the formulae (VI) and (X) can be used for each antibody AB-SH having a thiol group to produce an antibody-drug in which 2 to 8 drugs are linked to one antibody. Conjugates (IV), (VIII). Specifically, to the buffer solution containing the antibody AB-SH having a thiol group, a solution in which the compounds represented by the formulae (VI) and (X) are dissolved is added and allowed to react. Here, as the buffer solution, sodium acetate solution, sodium phosphate, sodium borate, or the like can be used. The pH during the reaction is 5 to 9, and the reaction is more preferably near pH 7. As a solvent for dissolving the compound (2) (that is, the compound represented by the formula (II)), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N- Methyl-2-pyridone (NMP) and other organic solvents. The organic solvent solution in which the compounds represented by formula (VI) and (X) are dissolved can be added to the buffer solution containing the thiol group-containing antibody AB-SH at 1 to 20% v/v, and the reaction can be carried out. The reaction temperature is 0 to 37°C, more preferably 10 to 25°C, and the reaction time is 0.5 to 2 hours. The reaction can be terminated by inactivating the reactivity of the unreacted compounds represented by the formulae (VI) and (X) with a thiol-containing reagent. Thiol-containing reagents are, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, 1 to 2 molar equivalents of NAC are added to the compounds represented by the formulae (VI) and (X) to be used, and the reaction is completed by incubating at room temperature for 10 to 30 minutes.
对于制造的抗体-药物偶联物(IV)、(VIII),可利用以下的共通操作,进行浓缩、缓冲液交换、纯化等的操作。For the produced antibody-drug conjugates (IV) and (VIII), operations such as concentration, buffer exchange, and purification can be performed by the following common operations.
共通操作A:抗体或抗体-药物偶联物水溶液的浓缩Common Procedure A: Concentration of Aqueous Antibody or Antibody-Drug Conjugates
在容器内,放入抗体或抗体-药物偶联物溶液,使用离心机进行离心操作(例如,以2000G~3800G离心5~20分钟),将抗体或抗体-药物偶联物溶液浓缩。In the container, put the antibody or antibody-drug conjugate solution, use a centrifuge for centrifugation (for example, centrifuge at 2000G-3800G for 5-20 minutes), and concentrate the antibody or antibody-drug conjugate solution.
共通操作B:抗体的浓度测定Common operation B: Determination of antibody concentration
使用UV测定器,按照制造商规定的方法,进行抗体浓度的测定。Antibody concentration was measured using a UV analyzer according to the manufacturer's instructions.
此时,使用随着抗体不同而显示不同的280nm吸光系数(1.3mLmg -1cm -1~1.8mLmg -1cm -1)。 At this time, the absorbance at 280 nm (1.3 mLmg -1 cm -1 to 1.8 mL mg -1 cm -1 ) which differs depending on the antibody was used.
共通操作C-1:抗体的缓冲液交换Common operation C-1: Buffer exchange of antibodies
按照制造商说明书的方法,用含有氯化钠(例如,137mM)及乙二胺四乙酸(EDTA,例如,5mM)的磷酸缓冲液(例如,10mM,pH6.0)(本说明书中也称为PBS6.0/EDTA)。将使用了Sephadex G-25载体的NAP-25柱平衡化。针对一根该NAP-25柱,装填2.5mL抗体水溶液,然后分离用PBS6.0/EDTA3.5mL洗脱的级分(3.5mL)。利用共通操作A将该级分浓缩,使用共通操作B,进行抗体浓度的测定,然后使用PBS6.0/EDTA,将抗体浓度调整为10mg/mL。Phosphate buffer (eg, 10 mM, pH 6.0) containing sodium chloride (eg, 137 mM) and ethylenediaminetetraacetic acid (EDTA, eg, 5 mM) (also referred to in this specification as PBS6.0/EDTA). The NAP-25 column using Sephadex G-25 vector was equilibrated. One of the NAP-25 columns was packed with 2.5 mL of aqueous antibody solution, and then the fraction (3.5 mL) eluted with PBS6.0/EDTA 3.5 mL was separated. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 10 mg/mL using PBS6.0/EDTA.
共通操作C-2:抗体的缓冲液交换Common operation C-2: Buffer exchange of antibodies
按照制造商规定的方法,用含有氯化钠(例如,50mM)及EDTA(例如,2mM)的磷酸缓冲液(例如,50mM,pH6.5,本说明书中也称为PBS6.5/EDTA)。将使用了Sephadex G-25载体的NAP-25柱平衡化。针对一根该NAP-25柱,装填2.5mL抗体水溶液,然后分离获取用PBS6.5/EDTA 3.5mL洗脱的级分(3.5mL)。利用共通操作A将该级分浓缩,使用共通操作B进行抗体浓度的测定,然后使用PBS6.5/EDTA,将抗体浓度调整为5mg/mL。Phosphate buffer (eg, 50 mM, pH 6.5, also referred to herein as PBS6.5/EDTA) containing sodium chloride (eg, 50 mM) and EDTA (eg, 2 mM) was used according to the manufacturer's protocol. The NAP-25 column using Sephadex G-25 vector was equilibrated. One of the NAP-25 columns was packed with 2.5 mL of aqueous antibody solution, and then a fraction (3.5 mL) eluted with PBS6.5/EDTA 3.5 mL was obtained by separation. This fraction was concentrated by common procedure A, and the antibody concentration was measured by common procedure B, and then the antibody concentration was adjusted to 5 mg/mL using PBS6.5/EDTA.
共通操作D-1:抗体-药物偶联物的纯化Common Operation D-1: Purification of Antibody-Drug Conjugates
使用市售的磷酸缓冲液(例如,PBS7.4)、含有氯化钠(例如,137mM)的磷酸钠缓冲液(例如,10mM,pH6.0;本说明书中也称为PBS6.0),或含有山梨糖醇(例如,5%)的乙酸缓冲液(例如,10mM,pH5.5;本说明书中也称为ABS)中的任一种缓冲液将NAP-25柱平衡化。 在该NAP-25柱中装填抗体-药物偶联物反应水溶液(例如,约1.5mL),用制造商规定的量的缓冲液洗脱,由此分离获取抗体级分。将该分离获取的级分再次装填至NAP-25柱,用缓冲液洗脱,进行凝胶过滤纯化操作,反复操作计2~3次,由此,得到了除去了未连接的药物接头、低分子化合物(三(2-羧基乙基)膦盐酸盐(TCEP),N-乙酰-L-半胱氨酸(NAC),二甲基亚砜)的抗体-药物偶联物。Use commercially available phosphate buffer (eg, PBS7.4), sodium phosphate buffer (eg, 10 mM, pH 6.0; also referred to in this specification as PBS6.0) containing sodium chloride (eg, 137 mM), or The NAP-25 column is equilibrated with any of the acetic acid buffers (eg, 10 mM, pH 5.5; also referred to herein as ABS) containing sorbitol (eg, 5%). An antibody-drug conjugate reaction aqueous solution (for example, about 1.5 mL) is packed in the NAP-25 column, and the antibody fraction is obtained by separation by eluting with an amount of buffer specified by the manufacturer. The fractions obtained by this separation were re-packed on a NAP-25 column, eluted with a buffer, and subjected to a gel filtration purification operation. The operation was repeated 2 to 3 times, whereby unconnected drug linkers were removed. Antibody-drug conjugates of molecular compounds (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide).
共通操作D-2:抗体-药物偶联物的纯化Common Procedure D-2: Purification of Antibody-Drug Conjugates
使用市售的磷酸缓冲液(例如,PBS7.4)、含有氯化钠(例如,137mM)的磷酸钠缓冲液(例如,10mM,pH6.0;本说明书中也称为PBS6.0),或含有山梨糖醇(例如,5%)的乙酸缓冲液(例如,10mM,pH5.5;本说明书中也称为ABS)中的任一种缓冲液将AKTA柱(填料:sephadex G 25)平衡化。进样器装载抗体-药物偶联物反应水溶液(例如,约2mL),用制造商规定的量的缓冲液洗脱,由此分离获取抗体级分。由此,得到了除去了未连接的药物接头、低分子化合物(三(2-羧基乙基)膦盐酸盐(TCEP),N-乙酰-L-半胱氨酸(NAC),二甲基亚砜)的抗体-药物偶联物。Use commercially available phosphate buffer (eg, PBS7.4), sodium phosphate buffer (eg, 10 mM, pH 6.0; also referred to in this specification as PBS6.0) containing sodium chloride (eg, 137 mM), or An AKTA column (filler: sephadex G 25) was equilibrated with any of the acetate buffers (eg, 10 mM, pH 5.5; also referred to as ABS in this specification) containing sorbitol (eg, 5%) . The injector is loaded with an antibody-drug conjugate reaction aqueous solution (eg, about 2 mL), eluted with an amount of buffer specified by the manufacturer, and the antibody fractions are obtained by separation. Thus, the unconnected drug linker, low molecular compound (tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide) antibody-drug conjugates.
[接头-药物中间体化合物的制造方法][Production method of linker-drug intermediate compound]
在某些具体实施方式中,接头-药物中间体化合物的制备方法包括如下步骤:In certain specific embodiments, the preparation method of the linker-drug intermediate compound comprises the following steps:
将N-[(9H-芴-9-基甲氧基)羰基]-N'-[(4-甲氧基苯基)二苯基甲基]-L-赖氨酸(CN-CMTC-1)和PABOH溶解在1:1的二氯甲烷:甲醇的溶液中,加入2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ),避光室温搅拌过夜,生成CN-CMTC-2。过柱纯化后,用5%的哌啶乙腈溶液处理,生成CN-CMTC-3。过柱纯化后,用DCC,NHS与CN-CMTC-4在DMF溶液中反应生成CN-CMTC-4的活性酯,CN-CMTC-4的活性酯与CN-CMTC-3反应生成化合物CN-CMTC-5,制备纯化后得纯度95%以上的目标产物。N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN-CMTC-1 ) and PABOH were dissolved in a 1:1 solution of dichloromethane:methanol, added 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), and stirred overnight at room temperature in the dark. Generate CN-CMTC-2. After column purification, it was treated with 5% piperidine in acetonitrile to generate CN-CMTC-3. After column purification, react with DCC, NHS and CN-CMTC-4 in DMF solution to generate the active ester of CN-CMTC-4, and the active ester of CN-CMTC-4 reacts with CN-CMTC-3 to generate the compound CN-CMTC -5, the target product with a purity of more than 95% is obtained after preparation and purification.
吉咪替康-Boc(或SN-38-Boc,或吉马替康)用三光气,DMAP,与二氯甲烷作用生成吉咪替康的甲酰氯化合物,再加入CN-CMTC-5,反应约5mins后,用甲醇猝灭后,过短柱,得CN-CMTC-6化合物的粗品。Pre-HPLC纯化后得纯品CN-CMTC-6,再用TFA/DCM处理脱保护得CN-CMTC-7,再与CN-A,B,C,D进行Click反应得化合物CN-CMTC-8,化合物8用TFA/DCM处理后得最终产物CN-CMTC(见下图)。Gemitecan-Boc (or SN-38-Boc, or Gematecan) is reacted with triphosgene, DMAP, and dichloromethane to form the formyl chloride compound of Gemitecan, and then CN-CMTC-5 is added to react After about 5 mins, after quenching with methanol, the column was shorted to obtain the crude product of CN-CMTC-6 compound. After Pre-HPLC purification, pure CN-CMTC-6 was obtained, which was then treated with TFA/DCM for deprotection to obtain CN-CMTC-7, which was then subjected to Click reaction with CN-A, B, C, and D to obtain the compound CN-CMTC-8. , Compound 8 was treated with TFA/DCM to obtain the final product CN-CMTC (see the figure below).
其中,CN表示本申请的连接子接头CN-A,CN-B,CN-C或CN-D,CMTC表示吉咪替康,上述反应中,吉咪替康(CMTC)可以替换为SN-38或吉马替康(GMTC)等喜树碱衍生物。Wherein, CN represents the connector connector CN-A, CN-B, CN-C or CN-D of the present application, and CMTC represents gemnotecan. In the above reaction, gemnotecan (CMTC) can be replaced by SN-38 Or camptothecin derivatives such as gimatecan (GMTC).
Figure PCTCN2021123020-appb-000125
Figure PCTCN2021123020-appb-000125
在某些具体实施方式中,接头-药物中间体化合物的制备方法包括如下步骤:In certain specific embodiments, the preparation method of the linker-drug intermediate compound comprises the following steps:
将CN-CMTC-7与SM-1加入DMSO/H2O的溶液中,再加入CuBr后,常温搅拌约30mins,HPLC检测反应完全,生成SMCC-PEG8-Lys(MMt)-PABC-CMTC,Pre-HPLC纯化后,再加入TFA/DCM脱保护,得目标产物SMCC-PEG8-Lys-PABC-CMTC。CN-CMTC-7 and SM-1 were added to DMSO/H2O solution, and then CuBr was added, and then stirred at room temperature for about 30 mins. HPLC detected that the reaction was complete, resulting in SMCC-PEG8-Lys(MMt)-PABC-CMTC, Pre-HPLC After purification, TFA/DCM was added for deprotection to obtain the target product SMCC-PEG8-Lys-PABC-CMTC.
Figure PCTCN2021123020-appb-000126
Figure PCTCN2021123020-appb-000126
在另一些具体实施方式中,接头-药物中间体化合物的制备方法包括如下步骤:In other specific embodiments, the preparation method of the linker-drug intermediate compound comprises the following steps:
将CN-CMTC-7-NH 2加入CN-C(或CN-A,CN-B,CN-D)的活性酯的DMF溶液中,反应约2小时后,HPLC显示原料反应完全,有目标产物CN-C(CN-A,CN-B,CN-D)-PEG 8-Lys(MMt)-PABC-CMTC的生成,纯化后,用TFA/DCM处理后,得目标产物。 Add CN-CMTC-7-NH 2 to the DMF solution of the active ester of CN-C (or CN-A, CN-B, CN-D), after about 2 hours of reaction, HPLC showed that the reaction of the raw materials was complete, and there was the target product The formation of CN-C(CN-A, CN-B, CN-D)-PEG 8 -Lys(MMt)-PABC-CMTC, after purification, after treatment with TFA/DCM, the target product was obtained.
Figure PCTCN2021123020-appb-000127
Figure PCTCN2021123020-appb-000127
在某些具体实施方式中,接头-药物中间体化合物的制备方法包括如下步骤:In certain specific embodiments, the preparation method of the linker-drug intermediate compound comprises the following steps:
Boc-GGFG与PABOH在EEDQ的作用下,二氯甲烷与甲醇作溶剂,室温搅拌过夜,生成Boc-GGFG-PABOH;Under the action of EEDQ, Boc-GGFG and PABOH, dichloromethane and methanol were used as solvents, and stirred overnight at room temperature to generate Boc-GGFG-PABOH;
Boc-GGFG-PABOH在TFA/DCM的作用下脱掉Boc生成GGFG-PABOH;再与N 3-PEGn-NHS活性酯反应生成N 3-PEGn-GGFG-PABOH,n=0,2,4,6,8; Boc-GGFG-PABOH was removed under the action of TFA/DCM to generate GGFG-PABOH; then reacted with N 3 -PEGn-NHS active ester to generate N 3 -PEGn-GGFG-PABOH, n=0, 2, 4, 6 ,8;
吉咪替康(SN-38,吉马替康)与DMAP,三光气在二氯甲烷的溶剂中反应5mins后,再加入N 3-PEGn-GGFG-PABOH继续反应5mins后生成N 3-PEGn-GGFG-PABC-CMTC(SN-38,GMTC),Pre-HPLC纯化后再与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0时)用Click反应条件得最终目标产物,m=2,4,6,8。该步骤中的吉咪替康(CMTC)可替换为SN-38、吉马替康(GMTC)。 Gematecan (SN-38, Gematecan) reacted with DMAP and triphosgene in dichloromethane solvent for 5mins, then added N 3 -PEGn-GGFG-PABOH and continued to react for 5mins to generate N 3 -PEGn- GGFG-PABC-CMTC (SN-38, GMTC), purified by Pre-HPLC and then combined with alkyne-maleimide (n=2, 4, 6, 8) or alkyne-PEGm-maleimide The amine (when n=0) was used to obtain the final target product using Click reaction conditions, m=2, 4, 6, 8. Gematecan (CMTC) in this step can be replaced by SN-38, gematecan (GMTC).
在某些具体实施方式中,接头-药物中间体化合物的制备方法包括如下步骤:In certain specific embodiments, the preparation method of the linker-drug intermediate compound comprises the following steps:
Boc-GGFG在TFA/DCM的作用下脱掉Boc,除掉TFA与二氯甲烷后,与N3-PEGn-NHS在二氯甲烷中反应,n=0,2,4,6,8;Boc-GGFG removes Boc under the action of TFA/DCM, removes TFA and dichloromethane, and reacts with N3-PEGn-NHS in dichloromethane, n=0, 2, 4, 6, 8;
用DIEA做碱,得到化合物N 3-PEGn-GGFG,Pre-HPLC纯化后再与N-Boc-N-甲基乙二胺缩合,缩合剂使用HATU,吡啶做碱,DMF作溶剂得到目标产物; Use DIEA as a base to obtain the compound N 3 -PEGn-GGFG, which is purified by Pre-HPLC and then condensed with N-Boc-N-methylethylenediamine. The condensing agent is HATU, pyridine is used as a base, and DMF is used as a solvent to obtain the target product;
用TFA/DCM脱掉Boc后,Pre-HPLC纯化得化合物N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3After removing Boc with TFA/DCM, the compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 was purified by Pre-HPLC.
N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3和CMTC-PNP在TEA做碱,DMF做溶剂的条件下反应30mins后,得化合物N 3-PEGn-GGFG-NH-C 2H 4-N(CH 3)-C(O)-CMTC,再与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0时)用Click反应条件得最终目标产物,m=2,4,6,8。该步骤中的吉咪替康(CMTC)可替换为SN-38、吉马替康(GMTC)。 N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 and CMTC-PNP were reacted with TEA as base and DMF as solvent for 30 mins to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -N(CH 3 )-C(O)-CMTC, and then with alkyne-maleimide (n=2, 4, 6, 8) or alkyne-PEGm-maleimide ( When n=0), the final target product was obtained with Click reaction conditions, m=2, 4, 6, 8. Gematecan (CMTC) in this step can be replaced by SN-38, gematecan (GMTC).
本发明的抗体-药物偶联物中,所述的抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段。在本发明的某些实施方案中,所述抗体选自Her-2抗体、抗Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER-4抗体、CD20、CD30抗体、CD19抗体、CD33抗体。In the antibody-drug conjugate of the present invention, the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof. In certain embodiments of the invention, the antibody is selected from the group consisting of Her-2 antibody, anti-Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER- 4 antibody, CD20, CD30 antibody, CD19 antibody, CD33 antibody.
本发明优选的抗体为抗TROP-2抗体,或其抗原结合片段,包括双特异性抗体和抗体功能性衍生物。TROP-2抗体属于TACSTD家族,是由TACSTD2基因编码表达的细胞表面糖蛋白,又名肿瘤相关钙离子信号转导子2(TACSTD2)、表皮糖蛋白1(EGP-1)、胃肠肿瘤相关抗原(GA733-1)、表面标志物1(M1S1)。TROP-2在多种恶性肿瘤中过表达,是一种与恶性肿瘤发生、侵袭和转移有关的癌基因。Preferred antibodies of the present invention are anti-TROP-2 antibodies, or antigen-binding fragments thereof, including bispecific antibodies and antibody functional derivatives. TROP-2 antibody belongs to the TACSTD family and is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1), and gastrointestinal tumor-associated antigen. (GA733-1), Surface Marker 1 (M1S1). TROP-2 is overexpressed in various malignant tumors and is an oncogene related to the occurrence, invasion and metastasis of malignant tumors.
本发明中的天然序列的TROP-2可以从自然界分离得到,也可以通过重组DNA技术、化学合成法或它们的组合制备得到。The TROP-2 of the natural sequence in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or a combination thereof.
本发明中所用的抗体优选为抗人TROP-2抗体。The antibody used in the present invention is preferably an anti-human TROP-2 antibody.
在某些优选实施方案中,所述抗人TROP-2抗体中的重链和轻链的CDR1、CDR2和/或CDR3分别为RS7单抗重链和轻链的CDR1、CDR2和/或CDR3。In certain preferred embodiments, the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human TROP-2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.
在某些优选实施方案中,所述抗人TROP-2抗体可以为人源化抗体或全人源抗体。In certain preferred embodiments, the anti-human TROP-2 antibody may be a humanized antibody or a fully human antibody.
在某些优选实施方案中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示;In certain preferred embodiments, the complementarity determining regions (CDRs) of the light chain variable region of the anti-Trop-2 antibody include CDR1 consisting of the amino acid sequence of KASQDVSIAVA, CDR2 consisting of the amino acid sequence of SASYRYT, and consisting of the amino acid sequence of QQHYITPLT CDR3 composed of; the CDRs of the heavy chain variable region include CDR1 composed of the NYGMN amino acid sequence, CDR2 composed of the WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of the GGFGSSYWYFDV amino acid sequence; Preferably, the light chain of the anti-Trop-2 antibody and the amino acid sequences of the heavy chain are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding nucleotide sequences of the light chain and heavy chain of the anti-Trop-2 antibody are respectively shown in SEQ ID NO: 3 and SEQ ID NO: shown in 4;
本发明优选的抗体为Her-2抗体或其抗原结合片段,包括双特异性抗体和抗体功能性衍生物。Her-2也称为人表皮生长因子受体-2(human epidermal growth factor receptor 2),或受体酪氨酸蛋白激酶erbB-2,也称为CD340(分化簇340)、原癌基因Neu、Erbb2(啮齿动物)或ERBB2(人类),是一种人类中由ERBB2基因编码的蛋白质。Preferred antibodies of the present invention are Her-2 antibodies or antigen-binding fragments thereof, including bispecific antibodies and antibody functional derivatives. Her-2 is also known as human epidermal growth factor receptor-2 (human epidermal growth factor receptor 2), or receptor tyrosine protein kinase erbB-2, also known as CD340 (cluster of differentiation 340), proto-oncogene Neu, Erbb2 (rodent) or ERBB2 (human), is a protein encoded by the ERBB2 gene in humans.
已显示Her-2过表达在乳腺癌的某些侵袭性类型的发展和进展中起重要作用。大约15-30%的乳腺癌中发生Her-2过表达。近年来,该蛋白质已成为大约30%乳腺癌患者的重要生物标志物和治疗靶标。Her-2过表达还发生在卵巢癌、肠胃癌和侵袭形式的子宫癌例如浆液性子宫内膜癌中。Her-2 overexpression has been shown to play an important role in the development and progression of certain aggressive types of breast cancer. Her-2 overexpression occurs in approximately 15-30% of breast cancers. In recent years, this protein has become an important biomarker and therapeutic target in about 30% of breast cancer patients. Her-2 overexpression also occurs in ovarian cancer, intestinal gastric cancer, and invasive forms of uterine cancer such as serous endometrial cancer.
可用于本发明的Her-2抗体的实例包括但不限于:记载于US5821337的曲妥珠单抗(trastuzumab),记载于CN101981056B的帕妥珠单抗(pertuzumab),可用于本发明的抗体还可以通过CN103476941A中公开的载体设计、构建和构建展示抗体的抗体库的方法筛选获得,也可以索伦托医疗公司(Sorrento Therapeutics,Inc.)的文库进行筛选获得。Examples of Her-2 antibodies that can be used in the present invention include, but are not limited to: trastuzumab described in US5821337, pertuzumab described in CN101981056B, and the antibodies that can be used in the present invention can also It can be obtained by screening the method of designing, constructing and constructing an antibody library for displaying antibodies by the vector disclosed in CN103476941A, or it can be obtained by screening the library of Sorrento Therapeutics, Inc.
本发明中的天然序列的Her-2可以从自然界分离得到,也可以通过重组DNA技术、化学合成法或它们的组合制备得到。The natural sequence Her-2 in the present invention can be isolated from nature, and can also be prepared by recombinant DNA technology, chemical synthesis method or a combination thereof.
本发明中所用的抗体优选为抗人Her-2抗体。The antibody used in the present invention is preferably an anti-human Her-2 antibody.
在某些优选实施方案中,所述抗人Her-2抗体中的重链和轻链的CDR1、CDR2和/或CDR3分别为RS7单抗重链和轻链的CDR1、CDR2和/或CDR3。In certain preferred embodiments, the CDR1, CDR2 and/or CDR3 of the heavy and light chains in the anti-human Her-2 antibody are CDR1, CDR2 and/or CDR3 of the heavy and light chains of the RS7 mAb, respectively.
在某些优选实施方案中,所述抗人Her-2抗体可以为人源化抗体或全人源抗体。In certain preferred embodiments, the anti-human Her-2 antibody may be a humanized antibody or a fully human antibody.
在某些优选实施方案中,所述Her-2抗体为US5821337所述曲妥珠抗体,其轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1;由SASFLYS氨基酸序列组成的CDR2;和由QQHYTTPPT氨基酸序列组成的CDR3,并且其重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1;由RIYPTNGYTRY氨基酸序列组成的CDR2;和由WGGDGFYAMDY氨基酸序列组成的CDR3。曲妥珠抗体的轻链序列及重链序列分别如SEQ ID NO:5和SEQ ID NO:6所示。还可包括那些对上述抗体进行保守氨基酸取代后,保留了Her-2结合活性的抗体。In certain preferred embodiments, the Her-2 antibody is the trastuzumab antibody described in US5821337, and the complementarity determining region (CDR) of its light chain variable region includes CDR1 consisting of the amino acid sequence of RASQDVNTAVA; consisting of the amino acid sequence of SASFLYS and CDR3 composed of the QQHYTTPPT amino acid sequence, and the CDRs of its heavy chain variable region include CDR1 composed of the DTYIH amino acid sequence; CDR2 composed of the RIYPTNGYTRY amino acid sequence; and CDR3 composed of the WGGDGFYAMDY amino acid sequence. The light chain sequence and heavy chain sequence of the trastuzumab antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. Also included are those antibodies that retain Her-2 binding activity after conservative amino acid substitutions to the above-mentioned antibodies.
本发明的抗体-药物偶联物可优选向哺乳动物给予,更优选人。The antibody-drug conjugates of the present invention can be preferably administered to mammals, more preferably humans.
作为含有本发明的抗体-药物偶联物的药物组合物中使用的物质,可考虑给予量、给予浓度,从本领域中通常使用的制剂添加物或其他物中适当选择使用。The substance to be used in the pharmaceutical composition containing the antibody-drug conjugate of the present invention can be appropriately selected from formulation additives or others commonly used in the art in consideration of the dose and concentration to be administered.
在本发明的某些实施方案中,本发明的抗体-药物偶联物可以以含有1种以上的药学上的适应性成分的药物组合物或药物制剂的形式给予。例如,上述药物组合物或药物制剂中,代表性地,可以含有1种以上的药学上可接受的载体(例如灭菌的液体(例如水及油(包含石油、动物、植物、或合成来源的油(例如花生油、大豆油、矿物油、芝麻油等)))。在静脉内给予上述药物组合物的情况下,水是更具有代表性的载体。另外,食盐水溶液、以及葡萄糖水溶液及甘油水溶液也可作为液体载体,尤其是可用于注射用溶液。适当药学赋形剂在该领域中是已知的。根据需要,上述组合物中还可以含有微量的润湿剂或乳化剂、或pH缓冲化剂。适当的药学载体的例子记载于E.W.Martin的“W.Martin载体的例子Parmaceutical Sciences”中。其处方与给予的方式相对应。In certain embodiments of the present invention, the antibody-drug conjugates of the present invention may be administered in the form of a pharmaceutical composition or pharmaceutical formulation containing one or more pharmaceutically suitable ingredients. For example, the above-mentioned pharmaceutical compositions or pharmaceutical preparations may typically contain one or more pharmaceutically acceptable carriers (such as sterile liquids (such as water and oils (including petroleum, animal, vegetable, or synthetic origin) Oil (such as peanut oil, soybean oil, mineral oil, sesame oil, etc.))). In the case of intravenous administration of the above-mentioned pharmaceutical composition, water is a more representative carrier. In addition, saline solution, as well as aqueous dextrose and glycerol solutions are also Can be used as liquid carrier, especially can be used for injection solution.Appropriate pharmaceutical excipients are known in this field.As required, the above-mentioned composition can also contain a trace amount of wetting agent or emulsifying agent, or pH buffering agent Examples of suitable pharmaceutical carriers are described in "Examples of W. Martin Carriers Parmaceutical Sciences" by E.W. Martin. The prescription corresponds to the mode of administration.
各种输送系统是已知的,可以为了给予本发明的抗体-药物偶联物而使用。作为导入方法,可举出皮内、肌肉内、腹腔内、静脉内、及皮下路径,但不限于这些。例如可以利用输液或快速浓注进行给予。在特定的优选实施方式中,上述抗体-药物偶联物的给予利用输液进行。肠胃外的给予是优选的给予路径。Various delivery systems are known and can be used for administration of the antibody-drug conjugates of the invention. The introduction method includes, but is not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration, for example, can be by infusion or bolus injection. In certain preferred embodiments, the administration of the antibody-drug conjugates described above is performed by infusion. Parenteral administration is the preferred route of administration.
代表性的实施方式中,将上述药物组合物形成向人静脉内给予的药物组合物,按照常规步骤形成处方。代表性地,用于静脉内给予的组合物是灭菌的等张性的水性缓冲液中的溶液。根据需要,上述药物组合物还可以含有增溶剂及用于缓解注射部位的疼痛的局麻剂(例如利多卡因)。通常,上述成分可以通过以下任一方式供给:以经密封的容器(例如将显示活性剂的量的安瓿或药囊等密封)中的干燥冷冻干燥粉末或无水的浓缩物的形式,分别地或在单位剂型中一起混合地供给。预定通过输液来给予上述药物时,例如可将上述药物放入到含有灭菌的制药级的水或食盐水的输液瓶中。当通过注射来给予上述药物时,可提供注射用灭菌水或食盐水的安瓿,以使得例如在给予前混合上述成分。In a representative embodiment, the above-mentioned pharmaceutical composition is formulated into a pharmaceutical composition for intravenous administration to humans, and is formulated according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The above-mentioned pharmaceutical composition may further contain a solubilizer and a local anesthetic (eg, lidocaine) for relieving pain at the injection site, as required. Generally, the above ingredients may be supplied either as a dry freeze-dried powder or anhydrous concentrate in a sealed container (eg, an ampule or sachet, etc., which indicates the amount of active agent), respectively, Or mixed together in unit dosage form. When the above-mentioned medicine is intended to be administered by infusion, for example, the above-mentioned medicine may be put into an infusion bottle containing sterilized pharmaceutical-grade water or saline. When the above-mentioned drugs are administered by injection, ampoules of sterile water for injection or saline may be provided so that, for example, the above-mentioned components are mixed before administration.
本发明的药物组合物或药物制剂,可以是仅含有本发明的抗体-药物偶联物的药物组合物或药物制剂,也可以是含有抗体-药物偶联物及至少一种其他癌治疗剂的药物组合物。也可将本发明的抗体-药物偶联物与其他癌治疗剂一同给予,由此,可增强抗癌效果。出于这种目的使用的其他抗癌剂,可与抗体-药物偶联物同时地、分别地或连续地向个体给予,也可改变给予间隔而进行给予。作为这样的癌治疗剂,可举出白蛋白结合型紫杉醇、卡铂、顺铂、吉西他滨、伊立替康(CPT-11)、紫杉醇、培美曲塞、索拉非尼、长春碱或国际公开第WO2003/038043号小册子中记载的药剂、以及LH-RH类似物(亮丙瑞林、戈舍瑞林等)、磷酸雌二醇氮芥、雌激素拮抗剂(他莫昔芬、雷洛昔芬等)、芳香酶抑制剂(阿那曲唑、来曲唑、依西美坦)等,但只要是具有抗肿瘤活性的药剂,就不受限制。The pharmaceutical composition or pharmaceutical preparation of the present invention may be a pharmaceutical composition or pharmaceutical preparation containing only the antibody-drug conjugate of the present invention, or may be a pharmaceutical composition or pharmaceutical preparation containing the antibody-drug conjugate and at least one other cancer therapeutic agent pharmaceutical composition. The antibody-drug conjugate of the present invention can also be administered together with other cancer therapeutic agents, whereby the anticancer effect can be enhanced. Other anticancer agents used for this purpose may be administered to the individual simultaneously, separately or sequentially with the antibody-drug conjugate, or may be administered at varying intervals. Examples of such cancer therapeutic agents include albumin-bound paclitaxel, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine, or international publications. The agents described in the pamphlet No. WO2003/038043, as well as LH-RH analogs (leuprolide, goserelin, etc.), estradiol phosphate mustard, estrogen antagonists (tamoxifen, ranloxacin, etc.) Xifen, etc.), aromatase inhibitors (anastrozole, letrozole, exemestane), etc., but they are not limited as long as they have antitumor activity.
这样的药物组合物可以作为具有选定的组成和必要的纯度的制剂,以冷冻干燥制剂或液状制剂的形式形成制剂。作为冷冻干燥制剂而形成制剂时,可以是含有本领域中可使用的适当的制剂添加物的制剂。另外,在液体制剂的情况也同样,可以作为含有本领域中可使用的各种的制剂添加物的液状制剂而形成制剂。Such pharmaceutical compositions can be formulated in the form of freeze-dried preparations or liquid preparations as preparations having a selected composition and necessary purity. When a preparation is formed as a freeze-dried preparation, it may be a preparation containing appropriate preparation additives available in the art. In addition, also in the case of a liquid preparation, a preparation can be formed as a liquid preparation containing various preparation additives usable in the art.
药物组合物的组成及浓度根据给予方法的不同而变化,但从本发明的药物组合物中包含的抗体-药物偶联物针对抗体-药物偶联物的抗原的亲和性、即针对抗原的解离常数(Kd值)方面考虑,亲和性越高(Kd值越低)时,即使为少量的给予量,也能发挥药效。因此,当确定抗体-药物偶联物的给予量时,也可基于抗体-药物偶联物与抗原的亲和性的状况来设定给予量。当将本发明的抗体-药物偶联物向人给予时,例如,可以以约0.001~100mg/kg给予1次,或以1~180天1次的间隔多次给予。The composition and concentration of the pharmaceutical composition vary depending on the administration method, but the affinity of the antibody-drug conjugate contained in the pharmaceutical composition of the present invention for the antigen of the antibody-drug conjugate, that is, the affinity for the antigen, varies. Considering the dissociation constant (Kd value), when the affinity is higher (the Kd value is lower), the drug effect can be exhibited even with a small dose. Therefore, when determining the administration amount of the antibody-drug conjugate, the administration amount can also be set based on the state of the affinity of the antibody-drug conjugate with the antigen. When the antibody-drug conjugate of the present invention is administered to a human, for example, it may be administered once at about 0.001 to 100 mg/kg, or may be administered multiple times at intervals of 1 to 180 days.
本发明的抗体-药物偶联物、药物组合物、药物制剂可以用于预防和/或治疗肿瘤或癌症。关于作为预防和/或治疗对象的肿瘤或癌症,只要是表达抗体-药物偶联物中的抗体可识别的蛋白的癌细胞即可。在本发明的某些实施方案中,所述肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌。The antibody-drug conjugates, pharmaceutical compositions, and pharmaceutical preparations of the present invention can be used to prevent and/or treat tumors or cancers. The tumor or cancer targeted for prevention and/or treatment may be any cancer cell that expresses a protein recognized by the antibody in the antibody-drug conjugate. In certain embodiments of the invention, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma tumor, liver cancer, bladder cancer, gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer.
在本发明的某些实施方案中,向需要的受试者施用预防或治疗有效量的本发明的抗体-药物偶联物、药物组合物或药物制剂,用于抑制癌细胞的生长、增殖或迁移。In certain embodiments of the invention, a prophylactically or therapeutically effective amount of an antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the invention is administered to a subject in need thereof for inhibiting the growth, proliferation or migrate.
在本发明的某些实施方案中,提供一种抑制癌细胞生长、增殖或迁移的试剂盒,其包括本发明的抗体-药物偶联物、药物组合物或药物制剂。In certain embodiments of the present invention, there is provided a kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate, pharmaceutical composition or pharmaceutical formulation of the present invention.
本发明的技术效果:Technical effect of the present invention:
本发明的抗体-药物偶联物具有快速高效的肿瘤细胞杀伤活性,同时具有良好的生物相容性、低免疫原性、生物安全性和稳定性。The antibody-drug conjugate of the present invention has fast and efficient tumor cell killing activity, and at the same time has good biocompatibility, low immunogenicity, biological safety and stability.
本发明的如式(II)的接头结构具有如下的优势:(1)本发明的接头结构分子量和亲疏水性合适,具有较高的载药量(DAR,durg to antibody ratio)>7;(2)本发明的接头结构能够提高接头-药物化合物的抗聚集能力;有助于提高抗肿瘤化合物中吉马替康、吉咪替康的亲水性,增加ADC的生物安全性;(3)本发明的接头结构释放的情况尤其适用于优选毒素吉马替康、吉咪替康,更优选吉咪替康,与其细胞毒性、药代动力学,以及肿瘤抑制性等特性匹配,该接头自裂解的速度更快,有助于毒素分子快速释放,大大增强了药效;(4)本发明设计的接头的大小、理化性质,以及偶联位点不会影响抗体的生理活性;(5)本发明的接头合物合成方法简易, 适合工业化生产。The linker structure of the present invention such as formula (II) has the following advantages: (1) the linker structure of the present invention has suitable molecular weight and hydrophobicity, and has a higher drug loading (DAR, drug to antibody ratio)>7; (2) (3) the present invention The release of the linker structure is especially suitable for the preferred toxins gimatecan, jimitecan, and more preferably jimitecan, which are matched with their cytotoxicity, pharmacokinetics, and tumor inhibitory properties, and the linker is self-cleaving. The speed is faster, which is conducive to the rapid release of toxin molecules, and greatly enhances the drug efficacy; (4) The size, physicochemical properties and coupling sites of the joint designed by the present invention will not affect the physiological activity of the antibody; (5) The present invention The synthesis method of the linker compound is simple and suitable for industrial production.
优选的是,本发明的抗肿瘤化合物选择吉马替康、吉咪替康,吉马替康毒性是SN-38的10倍左右,与依沙替康相当,但其安全性大大优于依沙替康,其可单独作为口服制剂成药。吉咪替康与SN-38,伊立替康等一样,都属于喜树碱类毒素,其是在10位羟基取代和9位烯丙基取代,有很好的抗肿瘤活性,吉咪替康(10-羟基-9-烯丙基喜树碱)抗各种肿瘤细胞的体外活性实验结果表明,它对各种肿瘤细胞的IC50大部分都在几个到十几个nM水平,总体来说活性要好于拓扑替康、9-硝基喜树碱和9-羟基喜树碱,且对大部分细胞株的活性也好于SN-38(CN1903201A)。SN-38目前已被临床试验证明其是优良的ADC毒素分子,吉咪替康在结构上虽与SN-38非常接近,但目前仍未见有成功将其作为ADC毒素分子的临床报道,可见其用于ADC药物具有技术难度。且吉咪替康水溶性仍旧不好,因此无法设计为直接口服或采用通常的注射剂。Preferably, the anti-tumor compound of the present invention selects gimatecan and gimatecan. The toxicity of gimatecan is about 10 times that of SN-38, which is comparable to that of ixatecan, but its safety is much better than that of ixatecan. Satecan, which is available alone as an oral preparation. Like SN-38, irinotecan, etc., gemnotecan belongs to the camptothecin toxoid, which is substituted at the 10-position hydroxyl group and the 9-position allyl group, and has good antitumor activity. (10-Hydroxy-9-allylcamptothecin) in vitro activity experiments against various tumor cells showed that its IC50 for various tumor cells was mostly at the level of several to a dozen nM. The activity is better than topotecan, 9-nitrocamptothecin and 9-hydroxycamptothecin, and the activity on most cell lines is also better than SN-38 (CN1903201A). SN-38 has been proven by clinical trials to be an excellent ADC toxin molecule. Although gemitecan is very close to SN-38 in structure, there is still no clinical report of successfully using it as an ADC toxin molecule. It can be seen that Its use in ADC drugs is technically difficult. In addition, gemitecan is still poorly water-soluble, so it cannot be designed for direct oral administration or conventional injections.
基于此,本发明通过对连接子的优化设计,将吉咪替康和吉马替康与具有一定亲水性的连接子连接,以在增加靶向给药的同时,解决毒素分子水溶性的难题。同时,本发明自行设计的式(I)接头分子与目前的马来酰亚胺相比,其与抗体的连接更稳定,减少了在非靶点位置脱落的可能性,提高了安全性,并能达到高载药量。此外,连接子的吉马替康具有很强渗透细胞膜的能力,让它们在杀伤吞入ADC的癌细胞之后,能够杀死附近的癌细胞。Based on this, the present invention connects gimatecan and gimatecan with a certain hydrophilic linker by optimizing the design of the linker, so as to increase the targeted drug delivery and solve the problem of water solubility of the toxin molecule. problem. At the same time, compared with the current maleimide, the linker molecule of formula (I) designed by the present invention has a more stable connection with the antibody, reduces the possibility of falling off at the non-target position, improves the safety, and improves the safety. Can achieve high drug loading. In addition, the linker gimatecan has a strong ability to penetrate cell membranes, allowing them to kill nearby cancer cells after killing the cancer cells that engulfed the ADC.
实施例Example
下面结合具体实施例,对本发明的方案进行解释。应理解,这些实施例仅用于举例说明本发明而不用于限制本发明的范围。下列实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。The solution of the present invention will be explained below with reference to specific embodiments. It should be understood that these examples are only intended to illustrate the present invention and not to limit the scope of the present invention. If no specific technique or condition is indicated in the following examples, the technique or condition described in the literature in this field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.
曲妥珠抗体(Herceptin抗体)购自Genentech Inc.。Trastuzumab antibody (Herceptin antibody) was purchased from Genentech Inc.
以下实施例中,ADC-137仅作为对照,购自常州辰鸿生物科技有限公司,CAS#:1279680-68-0。In the following examples, ADC-137 was only used as a control, and was purchased from Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0.
实施例1:hRS7抗体的制备和检测Example 1: Preparation and detection of hRS7 antibody
1.基因合成、转染和抗体制备1. Gene synthesis, transfection and antibody preparation
hRS7抗体在CHO细胞产生。含hRS7抗体基因的表达载体分别用常规的分子生物学方法构建,hRS7抗体轻链和重链核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示。将上述两序列插入到同一表达载体中,大量提取制备转染质粒,并转染到CHO-K1细胞(ATCC CCL-61)中,具体转染和抗体制备的过程如下:hRS7 antibody was produced in CHO cells. The expression vectors containing the hRS7 antibody gene were constructed by conventional molecular biology methods, and the nucleotide sequences of the light chain and heavy chain of the hRS7 antibody were shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Insert the above two sequences into the same expression vector, extract and prepare transfection plasmids in large quantities, and transfect them into CHO-K1 cells (ATCC CCL-61). The specific transfection and antibody preparation processes are as follows:
(1)细胞培养:CHO-K1细胞悬浮生长于ActiPro(GE HyClone)培养基,于37℃,7%CO 2,140rpm,90%相对湿度进行培养; (1) Cell culture: CHO-K1 cells were grown in suspension in ActiPro (GE HyClone) medium at 37°C, 7% CO 2 , 140 rpm, and 90% relative humidity;
(2)转染:在进入对数生长期后,取细胞离心,重悬于新鲜的ActiPro培养基,计数并调节细胞密度到1.35×10 7个/毫升,转500μl细胞悬液到电击杯中,然后加入40μg构建好的质粒,将细胞与质粒混匀,然后用电转方式导入质粒(Bio-rad电转仪); (2) Transfection: After entering the logarithmic growth phase, centrifuge the cells, resuspend them in fresh ActiPro medium, count and adjust the cell density to 1.35×10 7 cells/ml, and transfer 500 μl of the cell suspension into the electroporation cup , then add 40 μg of the constructed plasmid, mix the cells with the plasmid, and then introduce the plasmid by electroporation (Bio-rad electroporator);
(3)亚克隆:电转后的细胞用37℃的ActiPro培养基重悬,每孔100μl分装于96孔板。测定细胞上清以测定抗体的表达水平。将表达水平较高的克隆从96孔板转移到24孔板培养,而后再转入6孔板培养,测定细胞的抗体产量和产率,选择表达量最高的3个克隆进行亚克隆,而后转入摇瓶,放在培养箱中继续培养。(3) Subcloning: The electroporated cells were resuspended in ActiPro medium at 37°C, and 100 μl per well was dispensed into a 96-well plate. Cell supernatants were assayed to determine antibody expression levels. The clones with higher expression levels were transferred from the 96-well plate to the 24-well plate for culture, and then transferred to the 6-well plate for culture. The antibody production and yield of the cells were determined, and the 3 clones with the highest expression levels were selected for subcloning, and then transferred to into a shaker flask and placed in an incubator to continue culturing.
2抗体的纯化2 Purification of antibodies
收集摇瓶培养的高表达的细胞液,用蛋白A亲和纯化(GE,Mab Select SuRe)和离子交换纯化(GE,Capto S)。采用SDS-PAGE和SEC-HPLC对纯化后的抗体进行分子量和纯度分析。SDS-PAGE测定结果表明制备的hRS7分子量符合预期,用SEC-HPLC法测得抗体纯度为99.1%。The highly expressed cell fluid cultured in shake flasks was collected and purified by protein A affinity (GE, Mab Select SuRe) and ion exchange (GE, Capto S). The molecular weight and purity of the purified antibodies were analyzed by SDS-PAGE and SEC-HPLC. The results of SDS-PAGE showed that the molecular weight of the prepared hRS7 was in line with expectations, and the purity of the antibody was 99.1% measured by SEC-HPLC.
实施例2:接头化合物A的合成Example 2: Synthesis of Linker Compound A
Figure PCTCN2021123020-appb-000128
Figure PCTCN2021123020-appb-000128
I-1化合物5-溴吡啶甲酸叔丁酯A-1的制备Preparation of compound I-1 tert-butyl 5-bromopicolinate A-1
Figure PCTCN2021123020-appb-000129
Figure PCTCN2021123020-appb-000129
向5-溴吡啶甲酸SM-A(8.0g,39.60mmol)在t-BuOH(50mL)中的溶液中加入(Boc) 2O(12.96g,59.40mmol)和DMAP(0.484g,3.96mmol)。将混合物在50℃下搅拌12小时。LCMS监测反应。将该溶液减压浓缩。残余物通过硅胶色谱法纯化(石油醚/乙酸乙酯=12∶1),得到5-溴吡啶甲酸叔丁酯A-1(7.96g,78.04%收率),为白色固体。LCMS:M-56+H +=202,纯度:99.18%. 1H NMR(400MHz,DMSO)δ8.83(d,J=2.0Hz,1H),8.24(dd,J=8.4,2.3Hz,1H),7.93(d,J=8.2Hz,1H),1.56(s,9H). To a solution of 5 -bromopicolinic acid SM-A (8.0 g, 39.60 mmol) in t-BuOH (50 mL) was added (Boc)2O (12.96 g, 59.40 mmol) and DMAP (0.484 g, 3.96 mmol). The mixture was stirred at 50°C for 12 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 12:1) to give tert-butyl 5-bromopicolinate A-1 (7.96 g, 78.04% yield) as a white solid. LCMS: M-56+H + =202, Purity: 99.18%. 1 H NMR (400 MHz, DMSO) δ 8.83 (d, J=2.0 Hz, 1H), 8.24 (dd, J=8.4, 2.3 Hz, 1H ),7.93(d,J=8.2Hz,1H),1.56(s,9H).
I-2化合物5-(3-羟基丙-1-炔-1-基)吡啶甲酸叔丁酯A-2的制备Preparation of I-2 Compound 5-(3-hydroxyprop-1-yn-1-yl)picolinate tert-butyl ester A-2
Figure PCTCN2021123020-appb-000130
Figure PCTCN2021123020-appb-000130
在N 2下,向5-溴吡啶甲酸叔丁酯A-1(7.46g,28.9mmol)在THF(80mL)中的溶液中添加TEA(8.77g,86.7mmol),prop-2-yn-1-ol SM2(2.3ml,37.6mmol)和Pd(PPh 3) 2Cl 2(1.0g,1.45mmol)。将混合物在70℃下搅拌12小时。LCMS监测反应。将该溶液减压浓缩。残余物通过硅胶色谱纯化(石油醚/乙酸乙酯=1:1),得到5-(3-羟基丙-1-炔-1-基)吡啶甲酸叔丁酯A-2(5.8g,86.7%产率)。黄色固体。LCMS:M-56+H +=178,纯度:93.52%. 1H NMR(400MHz,CDCl 3)δ8.80(d,J=1.4Hz,1H),8.00(dd,J=8.1,0.7Hz,1H),7.82(dd,J=8.1,2.1Hz,1H),4.54(d,J=6.3Hz,2H),2.39(t,J=6.3Hz,1H),1.64(s,9H). To a solution of tert-butyl 5-bromopicolinate A-1 (7.46 g, 28.9 mmol) in THF (80 mL) under N2 was added TEA (8.77 g, 86.7 mmol), prop-2-yn-1 -ol SM2 (2.3 ml, 37.6 mmol) and Pd( PPh3 ) 2Cl2 (1.0 g , 1.45 mmol). The mixture was stirred at 70°C for 12 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1:1) to give tert-butyl 5-(3-hydroxyprop-1-yn-1-yl)picolinate A-2 (5.8 g, 86.7%) Yield). Yellow solid. LCMS: M-56+H + =178, purity: 93.52%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.80 (d, J=1.4 Hz, 1H), 8.00 (dd, J=8.1, 0.7 Hz, 1H), 7.82(dd, J=8.1, 2.1Hz, 1H), 4.54(d, J=6.3Hz, 2H), 2.39(t, J=6.3Hz, 1H), 1.64(s, 9H).
I-3 5-(氰基乙炔基)吡啶甲酸叔丁酯A-3的制备Preparation of I-3 tert-butyl 5-(cyanoethynyl)picolinate A-3
Figure PCTCN2021123020-appb-000131
Figure PCTCN2021123020-appb-000131
向5-(3-羟基丙-1-炔-1-基)吡啶甲酸叔丁酯A-2(3.2g,13.72mmol)的CH 3CN(45mL)和H 2O(5ml)溶液中加入TEMPO(107.2mg,0686mmol),PhI(OAC) 2(9.7g,30.2mmol)和NH 4OAc(4.2g,54.87mmol)。将混合物在室温搅拌12小时。LCMS监测反应。将该溶液减压浓缩。加入H 2O(100ml),并将混合物用EtOAc(50ml)萃取。有机层用盐水(50ml)洗涤,经Na 2SO 4干燥,并将滤液浓缩至干。残余物通过硅胶色谱法纯化(石油醚/乙酸乙酯=10∶1),得到5-(氰基乙炔基)吡啶甲酸叔丁酯A-3(1.2g,收率38.7%),为白色固体。 To a solution of tert-butyl 5-(3-hydroxyprop-1-yn-1-yl)picolinate A-2 (3.2 g, 13.72 mmol) in CH3CN (45 mL) and H2O (5 mL) was added TEMPO (107.2 mg, 0686 mmol), PhI(OAC) 2 (9.7 g, 30.2 mmol) and NH4OAc (4.2 g, 54.87 mmol). The mixture was stirred at room temperature for 12 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. H2O (100ml) was added and the mixture was extracted with EtOAc (50ml). The organic layer was washed with brine (50 ml), dried over Na2SO4 , and the filtrate was concentrated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10:1) to give tert-butyl 5-(cyanoethynyl)picolinate A-3 (1.2 g, yield 38.7%) as a white solid .
LCMS:M-56+H +=173,纯度:99.67%. 1H NMR(400MHz,CDCl 3)δ8.94(dd,J=2.0,0.7Hz,1H),8.10(dd,J=8.1,0.8Hz,1H),8.03(dd,J=8.1,2.1Hz,1H),1.65(s,9H). LCMS: M-56+H + =173, Purity: 99.67%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.94 (dd, J=2.0, 0.7 Hz, 1H), 8.10 (dd, J=8.1, 0.8 Hz,1H),8.03(dd,J=8.1,2.1Hz,1H),1.65(s,9H).
I-5 5-(氰基乙炔基)吡啶甲酸叔丁酯目标A的制备Preparation of I-5 tert-butyl 5-(cyanoethynyl)picolinate target A
Figure PCTCN2021123020-appb-000132
Figure PCTCN2021123020-appb-000132
向5-(氰基乙炔基)吡啶甲酸叔丁酯A-3(1.2g,5.3mmol)在DCM(80mL)中的溶液中添加TFA(80ml)。将混合物在室温搅拌3小时。LCMS监测反应。将该溶液减压浓缩。将残余物用乙醚(150ml)研磨。过滤混合物,滤饼用乙醚(50ml)洗涤。将滤饼真空干燥,得到5-(氰基乙炔基)吡啶甲酸叔丁酯目标A(600mg,66%产率),为白色固体。To a solution of tert-butyl 5-(cyanoethynyl)picolinate A-3 (1.2 g, 5.3 mmol) in DCM (80 mL) was added TFA (80 mL). The mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The residue was triturated with ether (150 ml). The mixture was filtered and the filter cake was washed with ether (50 ml). The filter cake was dried in vacuo to give tert-butyl 5-(cyanoethynyl)picolinate target A (600 mg, 66% yield) as a white solid.
LCMS:MH +=173,纯度:99.03%. 1H NMR(400MHz,DMSO)δ13.71(s,1H),9.09(dd,J=2.0,0.7Hz,1H),8.42(dd,J=8.1,2.1Hz,1H),8.21-8.05(m,1H). LCMS: MH + =173, purity: 99.03%. 1 H NMR (400 MHz, DMSO) δ 13.71 (s, 1H), 9.09 (dd, J=2.0, 0.7 Hz, 1H), 8.42 (dd, J=8.1 ,2.1Hz,1H),8.21-8.05(m,1H).
实施例3:接头化合物B的合成Example 3: Synthesis of Linker Compound B
Figure PCTCN2021123020-appb-000133
Figure PCTCN2021123020-appb-000133
II-1 6-溴烟酸叔丁酯B-1的制备Preparation of II-1 tert-butyl 6-bromonicotinic acid B-1
Figure PCTCN2021123020-appb-000134
Figure PCTCN2021123020-appb-000134
在室温和N 2条件下,向6-溴烟酸SM-B(5g,25mmol)在t-BuOH(50mL)的溶液中搅拌加入Boc 2O(11g)和DMAP(300mg)。将反应在60℃下搅拌过夜。TLC监测反应。将反应混合物用氯化铵水溶液(50mL)稀释,并用EtOAc(100mL)萃取。合并的有机层经无水 硫酸钠干燥,并真空蒸发至干。残余物通过柱纯化(己烷/乙酸乙酯=10∶1),得到6-溴烟酸叔丁酯B-1(5g,产率78%),为白色固体。 To a solution of 6-bromonicotinic acid SM-B (5 g, 25 mmol) in t-BuOH (50 mL) was added Boc 2 O (11 g) and DMAP (300 mg) with stirring at room temperature under N2 . The reaction was stirred at 60°C overnight. The reaction was monitored by TLC. The reaction mixture was diluted with aqueous ammonium chloride (50 mL) and extracted with EtOAc (100 mL). The combined organic layers were dried over anhydrous sodium sulfate and evaporated to dryness in vacuo. The residue was purified by column (hexane/ethyl acetate=10:1) to give tert-butyl 6-bromonicotinate B-1 (5 g, yield 78%) as a white solid.
LCMS:[M+H] +=259.9,纯度:99%. LCMS: [M+H] + =259.9, purity: 99%.
II-2 6-(3-羟基丙-1-炔-1-基)烟酸酯叔丁酯B-2的制备Preparation of II-2 6-(3-hydroxyprop-1-yn-1-yl)nicotinate tert-butyl ester B-2
Figure PCTCN2021123020-appb-000135
Figure PCTCN2021123020-appb-000135
将6-溴烟酸叔丁酯B-1(5g,19.46mmol)和2-炔-1-丙醇(3.26g,25.3mmol)加入烧瓶中,然后添加Pd(PPh 3) 2Cl 2(0.98g,1.4mmol)和CuI(0.45g,2.3mmol)。用氮气吹扫烧瓶3次以除去氧气后。通过注射器加入DMF(64mL)和TEA(10mL)。将反应在80℃下搅拌过夜,并通过LCMS监测。反应消耗后,将反应用饱和氯化铵(300mL)淬灭。然后将反应物用水稀释并用乙酸乙酯(50mL)洗涤3次。然后将有机相用无水硫酸钠干燥。浓缩并通过柱子(石油醚/乙酸乙酯=2:1)纯化,得到所需的6-(3-羟基丙-1-炔-1-基)烟酸酯叔丁酯B-2(3.1g,收率68%)为棕色油状。LCMS:[M+H] +=234,纯度:93%. 1H NMR(400MHz,CDC1 3)δ9.08(d,J=1.3Hz,1H),8.21(dd,J=8.1,2.1Hz,1H),7.48(d,J=8.4Hz,1H),4.55(s,2H),1.61(d,J=7.3Hz,9H). tert-Butyl 6-bromonicotinate B-1 (5 g, 19.46 mmol) and 2-yn-1-propanol (3.26 g, 25.3 mmol) were added to the flask followed by Pd( PPh3 ) 2Cl2 ( 0.98 g, 1.4 mmol) and CuI (0.45 g, 2.3 mmol). After purging the flask with nitrogen 3 times to remove oxygen. DMF (64 mL) and TEA (10 mL) were added via syringe. The reaction was stirred at 80°C overnight and monitored by LCMS. After the reaction was consumed, the reaction was quenched with saturated ammonium chloride (300 mL). The reaction was then diluted with water and washed three times with ethyl acetate (50 mL). The organic phase was then dried over anhydrous sodium sulfate. Concentration and purification by column (petroleum ether/ethyl acetate=2:1) gave the desired tert-butyl 6-(3-hydroxyprop-1-yn-1-yl)nicotinate B-2 (3.1 g , yield 68%) as brown oil. LCMS: [M+H] + = 234, purity: 93%. 1 H NMR (400MHz, CDC1 3 ) δ 9.08 (d, J=1.3 Hz, 1H), 8.21 (dd, J=8.1, 2.1 Hz, 1H), 7.48(d, J=8.4Hz, 1H), 4.55(s, 2H), 1.61(d, J=7.3Hz, 9H).
II-3 6-(3-羟基丙-1-炔-1-基)烟酸叔丁酯B-3的制备Preparation of II-3 tert-butyl 6-(3-hydroxyprop-1-yn-1-yl)nicotinate B-3
Figure PCTCN2021123020-appb-000136
Figure PCTCN2021123020-appb-000136
向6-(3-羟基丙-1-炔-1-基)烟酸酯叔丁酯B-2(3.1g,13.3mmol),TEMPO(105mg,0.67mmol),PhI(OAc) 2(9.42g,29.26mmol)和NH 4OAc(4.096g,53.2mmol)加入CH 3CN/H 2O(45mL/5mL)。将反应混合物在室温搅拌4小时。LCMS监测反应。将反应真空浓缩。然后将反应物用水稀释并用乙酸乙酯洗涤。有机层用无水硫酸钠干燥。并通过快速柱浓缩纯化(石油醚/乙酸乙酯=10/1),得到6-(丙-1-炔-1-基)烟酸叔丁酯B-3(900mg,收率30%),为绿色固体。LCMS:[M+H] +=229,纯度:97%; 1H NMR(400MHz,CDCl 3)δ9.51-9.02(m,1H),8.31(dd,J=8.1,2.1Hz,1H),7.70(dd,J=8.1,0.8Hz,1H),1.62(s,10H). To 6-(3-hydroxyprop-1-yn-1-yl)nicotinate tert-butyl ester B-2 (3.1 g, 13.3 mmol), TEMPO (105 mg, 0.67 mmol), PhI(OAc) 2 (9.42 g , 29.26 mmol) and NH4OAc (4.096 g, 53.2 mmol) were added CH3CN / H2O (45 mL/5 mL). The reaction mixture was stirred at room temperature for 4 hours. The reaction was monitored by LCMS. The reaction was concentrated in vacuo. The reaction was then diluted with water and washed with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate. And purified by flash column concentration (petroleum ether/ethyl acetate=10/1) to obtain tert-butyl 6-(prop-1-yn-1-yl)nicotinate B-3 (900 mg, yield 30%), as a green solid. LCMS: [M+H] + =229, purity: 97%; 1 H NMR (400 MHz, CDCl 3 ) δ 9.51-9.02 (m, 1H), 8.31 (dd, J=8.1, 2.1 Hz, 1H), 7.70(dd,J=8.1,0.8Hz,1H),1.62(s,10H).
II-4 6-(氰基乙炔基)烟酸靶标B的制备Preparation of II-4 6-(cyanoethynyl)nicotinic acid target B
Figure PCTCN2021123020-appb-000137
Figure PCTCN2021123020-appb-000137
在室温下,向6-(丙-1-炔-1-基)烟酸酯叔丁基B-3(900mg,3.95mmol)在CH 2Cl 2(50mL)的溶液中缓慢加入TFA(50mL)。然后将其在室温搅拌3小时。LCMS监测反应。然后将溶液真空浓缩。向混合物中加入水(100mL)和乙酸乙酯(100mL)。然后将反应物用水稀释并用乙酸乙酯洗涤。有机相用无水硫酸钠干燥。通过快速柱浓缩(石油醚/乙酸乙酯=2/1)纯化,得到6-(氰基乙炔基)烟酸目标B(463mg,收率68%),其为棕色固体。LCMS:[M+H] +=172.9,纯度:99%; 1H NMR(400MHz,DMSO)δ13.86(s,1H),9.14(d,J=2.0Hz,1H),8.41(dd,J=8.1,2.0Hz,1H),8.11(d,J=8.0Hz,1H). To a solution of 6-(prop-1-yn-1-yl)nicotinate tert-butyl B-3 (900 mg, 3.95 mmol) in CH 2 Cl 2 (50 mL) was slowly added TFA (50 mL) at room temperature . It was then stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The solution was then concentrated in vacuo. To the mixture were added water (100 mL) and ethyl acetate (100 mL). The reaction was then diluted with water and washed with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate. Purification by flash column concentration (petroleum ether/ethyl acetate=2/1) afforded 6-(cyanoethynyl)nicotinic acid target B (463 mg, 68% yield) as a brown solid. LCMS: [M+H] + =172.9, purity: 99%; 1 H NMR (400 MHz, DMSO) δ 13.86 (s, 1H), 9.14 (d, J=2.0 Hz, 1H), 8.41 (dd, J =8.1,2.0Hz,1H),8.11(d,J=8.0Hz,1H).
实施例4:接头化合物C的合成Example 4: Synthesis of Linker Compound C
Figure PCTCN2021123020-appb-000138
Figure PCTCN2021123020-appb-000138
III-1 5-溴嘧啶-2-羧酸叔丁酯C-1的制备Preparation of III-1 tert-butyl 5-bromopyrimidine-2-carboxylate C-1
Figure PCTCN2021123020-appb-000139
Figure PCTCN2021123020-appb-000139
向5-溴嘧啶-2-羧酸SM-C(4.5g,22.2mmol)的t-BuOH(45mL)的溶液中,加入(Boc) 2O(9.67g,44.40mmol)和DMAP(0.270g,2.22mmol)。将混合物在50℃下搅拌12小时。TLC监测反应。将该溶液减压浓缩。残余物通过硅胶色谱法纯化(石油醚/乙酸乙酯=12∶1),得到5-溴嘧啶-2-羧酸叔丁酯C-1(4.8g,84.20%收率),为白色固体。LCMS:M-56+H +=203,纯度:99.41%. 1H NMR(400MHz,CDCl 3)δ8.95(s,2H),1.66(s,9H). To a solution of 5-bromopyrimidine-2-carboxylic acid SM-C (4.5 g, 22.2 mmol) in t-BuOH (45 mL) was added (Boc)2O (9.67 g , 44.40 mmol) and DMAP (0.270 g, 2.22 mmol). The mixture was stirred at 50°C for 12 hours. The reaction was monitored by TLC. The solution was concentrated under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=12:1) to give tert-butyl 5-bromopyrimidine-2-carboxylate C-1 (4.8 g, 84.20% yield) as a white solid. LCMS: M-56+H + =203, purity: 99.41%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.95 (s, 2H), 1.66 (s, 9H).
III-2 5-(3-羟基丙-1-炔-1-基)嘧啶-2-羧酸叔丁酯C-2的制备Preparation of III-2 5-(3-hydroxyprop-1-yn-1-yl)pyrimidine-2-carboxylate tert-butyl ester C-2
Figure PCTCN2021123020-appb-000140
Figure PCTCN2021123020-appb-000140
向5-溴嘧啶-2-羧酸叔丁酯C-1(4.8g,18.6mmol)在THF(60mL)的溶液中加入TEA(9.6ml),2-炔-1-丙醇SM2 3.3ml,55.8mmol),CuI(0.425g,2.23mmol)和Pd(PPh 3) 2Cl 2(0.94g,1.34mmol)。将混合物在70℃下搅拌12h。LCMS监测反应。用饱和氯化铵溶液(300ml)淬灭该溶液。用水(300ml)稀释混合物,并用EtOAc(500ml)洗涤3次。然后将有机相用无水硫酸钠干燥,将滤液减压浓缩。残余物通过硅胶色谱法纯化(石油醚/乙酸乙酯=1:1),得到5-(3-羟基丙-1-炔-1-基)嘧啶-2-羧酸叔丁酯C-2(3.12g,收率为72.6%),为白色固体。LCMS:M-56+H +=179,纯度:98.03%. 1H NMR(400MHz,CDCl 3)δ8.92(s,2H),4.57(d,J=6.4Hz,2H),2.33(t,J=6.4Hz,1H),1.67(s,9H). To a solution of tert-butyl 5-bromopyrimidine-2-carboxylate C-1 (4.8 g, 18.6 mmol) in THF (60 mL) was added TEA (9.6 ml), 2-yn-1-propanol SM2 3.3 ml, 55.8 mmol), CuI (0.425 g, 2.23 mmol) and Pd( PPh3 )2Cl2 ( 0.94 g , 1.34 mmol). The mixture was stirred at 70 °C for 12 h. The reaction was monitored by LCMS. The solution was quenched with saturated ammonium chloride solution (300 ml). The mixture was diluted with water (300ml) and washed 3 times with EtOAc (500ml). The organic phase was then dried over anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1:1) to give tert-butyl 5-(3-hydroxyprop-1-yn-1-yl)pyrimidine-2-carboxylate C-2( 3.12 g, 72.6% yield) as a white solid. LCMS: M-56+H + =179, purity: 98.03%. 1 H NMR (400 MHz, CDCl 3 ) δ 8.92 (s, 2H), 4.57 (d, J=6.4 Hz, 2H), 2.33 (t, J=6.4Hz, 1H), 1.67(s, 9H).
III-3 5-(氰基乙炔基)嘧啶-2-羧酸叔丁酯C-3的制备Preparation of III-3 5-(cyanoethynyl)pyrimidine-2-carboxylate tert-butyl ester C-3
Figure PCTCN2021123020-appb-000141
Figure PCTCN2021123020-appb-000141
向5-(3-羟基丙-1-炔-1-基)嘧啶-2-羧酸叔丁酯C-2(2.13g,9mmol)的CH 3CN(36mL)和H 2O(4ml)溶液中,加入Tempo(141mg,0.9mmol),PhI(OAC) 2(6.42g,19.9mmol)和NH 4OAc(2.79g,36.2mmol)。将混合物在室温搅拌12h。LCMS监测反应。将该溶液减压浓缩。加入H 2O(100毫升)。用EA(50ml)萃取混合物3次。有机层用盐水(50mL)洗涤,经无水硫酸钠干燥,并将滤液浓缩至干。残余物通过硅胶色谱纯化(石油醚/乙酸乙酯=10∶1),得到5-(氰基乙炔基)嘧啶-2-羧酸叔丁酯C-3(1.5g,75%产率),为黄色固体。LCMS:M-56+H +=174,纯度:97.73%. 1H NMR(400MHz,CDCl 3)δ9.10(s,2H),1.67(s,9H). To a solution of 5-(3-hydroxyprop-1-yn-1-yl)pyrimidine-2-carboxylate tert-butyl ester C-2 (2.13 g, 9 mmol) in CH3CN (36 mL) and H2O (4 mL) To this, Tempo (141 mg, 0.9 mmol), PhI(OAC) 2 (6.42 g, 19.9 mmol) and NH4OAc (2.79 g, 36.2 mmol) were added. The mixture was stirred at room temperature for 12 h. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. H2O (100 mL) was added. The mixture was extracted 3 times with EA (50 ml). The organic layer was washed with brine (50 mL), dried over anhydrous sodium sulfate, and the filtrate was concentrated to dryness. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 10:1) to give tert-butyl 5-(cyanoethynyl)pyrimidine-2-carboxylate C-3 (1.5 g, 75% yield), For the yellow solid. LCMS: M-56+H + =174, purity: 97.73%. 1 H NMR (400 MHz, CDCl 3 ) δ 9.10 (s, 2H), 1.67 (s, 9H).
III-4 5-(氰乙基)嘧啶-2-羧酸靶C的制备Preparation of III-4 5-(cyanoethyl)pyrimidine-2-carboxylic acid target C
Figure PCTCN2021123020-appb-000142
Figure PCTCN2021123020-appb-000142
向5-(氰基乙炔基)嘧啶-2-羧酸叔丁酯C-3(1.5g,6.5mmol)在DCM(80mL)中的溶液中添加TFA(80ml)。将混合物在室温搅拌3小时。LCMS监测反应。将该溶液减压浓缩。将残余物在碱性条件下通过制备型HPLC纯化,得到5-(氰基乙炔基)嘧啶-2-羧酸目标C(500mg,44.2%产率),为黄色固体。LCMS:M+H +=174,纯度:96.53%. 1H NMR(400MHz,DMSO)δ14.02(s,1H),9.41(d,J=27.6Hz,2H). To a solution of tert-butyl 5-(cyanoethynyl)pyrimidine-2-carboxylate C-3 (1.5 g, 6.5 mmol) in DCM (80 mL) was added TFA (80 mL). The mixture was stirred at room temperature for 3 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The residue was purified by preparative HPLC under basic conditions to give 5-(cyanoethynyl)pyrimidine-2-carboxylic acid target C (500 mg, 44.2% yield) as a yellow solid. LCMS: M+H + =174, purity: 96.53%. 1 H NMR (400 MHz, DMSO) δ 14.02 (s, 1H), 9.41 (d, J=27.6 Hz, 2H).
实施例5:偶联物ADC-1的制备Example 5: Preparation of conjugate ADC-1
Figure PCTCN2021123020-appb-000143
Figure PCTCN2021123020-appb-000143
IV-1中间体CN-C-CMTC的制备Preparation of IV-1 Intermediate CN-C-CMTC
Figure PCTCN2021123020-appb-000144
Figure PCTCN2021123020-appb-000144
IV-1.1将CN-CMTC-1(640mg,1.0mmol),PABOH(123mg,1.0mmol)与EEDQ(370mg,1.5mmol)溶解在DCM/MeOH(10mL/10mL)中,在室温条件下反应过夜,HPLC检测有新峰生成,原料CN-CMTC-1完全消失,向反应液中加入适量的乙醚沉降后,有固体析出,离心得固体,固体重复用乙醚洗3次,离心,干燥后,得白色固体粗品CN-CMTC-2 650mg左右,产率:87.2%。LCMS:[M+1] +=746.5。 IV-1.1 CN-CMTC-1 (640 mg, 1.0 mmol), PABOH (123 mg, 1.0 mmol) and EEDQ (370 mg, 1.5 mmol) were dissolved in DCM/MeOH (10 mL/10 mL) and reacted at room temperature overnight, HPLC detected a new peak, and the raw material CN-CMTC-1 completely disappeared. After adding an appropriate amount of diethyl ether to the reaction solution, a solid was precipitated. Centrifuge to obtain a solid. The solid crude CN-CMTC-2 is about 650 mg, the yield: 87.2%. LCMS: [M+1] + =746.5.
Figure PCTCN2021123020-appb-000145
Figure PCTCN2021123020-appb-000145
IV-1.2将CN-CMTC-2(640mg,0.86mmol)用二乙胺(10mL)溶解后反应3小时,TLC检测,反应完全,溶液旋干后,用50mL的乙酸乙酯溶解,再用饱和氯化钠溶液洗三次后,有机层用无水硫酸钠干燥,过滤,旋干后得油状物,用MeOH:DCM=50:1的洗脱液过柱后得白色固体约240mg,产率:53.4%;LCMS:524.3.IV-1.2 CN-CMTC-2 (640 mg, 0.86 mmol) was dissolved in diethylamine (10 mL) and reacted for 3 hours. TLC detected that the reaction was complete. After the solution was spin-dried, it was dissolved in 50 mL of ethyl acetate, and then saturated with After washing with sodium chloride solution three times, the organic layer was dried with anhydrous sodium sulfate, filtered, and spin-dried to obtain an oily substance, which was passed through the column with an eluent of MeOH:DCM=50:1 to obtain a white solid of about 240 mg. Yield: 53.4%; LCMS: 524.3.
Figure PCTCN2021123020-appb-000146
Figure PCTCN2021123020-appb-000146
IV-1.3将CN-CMTC-4(230mg,0.42mmol),DCC(104mg,0.50mmol)与NHS(53mg,0.46mmol)溶解在DMF(5mL)中,在室温条件下2小时后,TLC检测CN-CMTC-4完全消失,反 应液加入CN-CMTC-3(219mg,0.42mmol)的DMF(2mL)溶液,室温条件下反应过夜,HPLC检测有新峰生成,Pre-HPLC纯化后得目标产物CN-CMTC-5约210mg.产率:47.8%。LCMS:[M+1] +=1060.7. IV-1.3 CN-CMTC-4 (230 mg, 0.42 mmol), DCC (104 mg, 0.50 mmol) and NHS (53 mg, 0.46 mmol) were dissolved in DMF (5 mL), after 2 hours at room temperature, CN was detected by TLC -CMTC-4 disappeared completely, the reaction solution was added with CN-CMTC-3 (219mg, 0.42mmol) in DMF (2mL) solution, reacted overnight at room temperature, a new peak was detected by HPLC, and the target product CN was obtained after Pre-HPLC purification - CMTC-5 about 210 mg. Yield: 47.8%. LCMS: [M+1] + = 1060.7.
Figure PCTCN2021123020-appb-000147
Figure PCTCN2021123020-appb-000147
IV-1.4将CMTC-Boc(50mg,0.1mmol),DMAP(61mg,0.5mmol)与三光气(24mg,0.08mmol)称量好后加入圆底烧瓶。加入DCM(1mL)后,搅拌大约5分钟,TLC显示,CMTC-Boc已经反应完全,再加入CN-CMTC-5(105mg,0.1mmol),继续搅拌10分钟左右,LCMS显示大部分为目标产物,部分掉Boc的产物,反应液过一个短的硅胶柱,用DCM:MeOH=10:1的洗脱剂洗脱后,浓缩,Pre-HPLC纯化后得产物CN-CMTC-6大约45mg,产率:28.3%。LCMS:[1/2M+1]+=794.9.IV-1.4 CMTC-Boc (50 mg, 0.1 mmol), DMAP (61 mg, 0.5 mmol) and triphosgene (24 mg, 0.08 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that CMTC-Boc had reacted completely, then CN-CMTC-5 (105 mg, 0.1 mmol) was added, and the stirring was continued for about 10 minutes. LCMS showed that most of the products were the target product, The product of Boc was partially removed, the reaction solution was passed through a short silica gel column, eluted with DCM:MeOH=10:1 eluent, concentrated, and purified by Pre-HPLC to obtain the product CN-CMTC-6, about 45 mg, with a yield of about 45 mg. : 28.3%. LCMS: [1/2M+1]+=794.9.
Figure PCTCN2021123020-appb-000148
Figure PCTCN2021123020-appb-000148
IV-1.5将化合物CN-CMTC-6(40mg,0.025mmol)溶解在TFA/DCM(1mL/1mL)的溶液中,室温条件20mins小时后,HPLC检测,CN-CMTC-6已经完全消失,减压蒸馏除掉TFA/DCM,加入适量的NaHCO 3溶液,调节pH为中性,Pre-HPLC纯化后得目标产物约25mg.产率:66.8%;LCMS:[1/2M+1]+=745.7. IV-1.5 Compound CN-CMTC-6 (40 mg, 0.025 mmol) was dissolved in a solution of TFA/DCM (1 mL/1 mL). After 20 mins at room temperature, HPLC detected that CN-CMTC-6 had completely disappeared. TFA/DCM was distilled off, an appropriate amount of NaHCO 3 solution was added, and the pH was adjusted to neutral. After Pre-HPLC purification, about 25 mg of the target product was obtained. Yield: 66.8%; LCMS: [1/2M+1]+=745.7.
Figure PCTCN2021123020-appb-000149
Figure PCTCN2021123020-appb-000149
IV-1.6将CN-CMTC-7(20mg,0.013)与CN-C(3mg,0.014)溶解在DMSO/H2O(0.4/0.4mL)中,加入CuBr(2mg),反应液在室温条件下搅拌1小时,HPLC显示反应完全,并有新峰生成,Pre-HPLC纯化后得产物CN-CMTC-8大约(15mg).产率:72.1%;LCMS:[1/2M+1]+=850.9。IV-1.6 CN-CMTC-7 (20 mg, 0.013) and CN-C (3 mg, 0.014) were dissolved in DMSO/H2O (0.4/0.4 mL), CuBr (2 mg) was added, and the reaction solution was stirred at room temperature for 1 After hours, HPLC showed that the reaction was complete and a new peak was formed. After Pre-HPLC purification, the product CN-CMTC-8 was obtained (15 mg). Yield: 72.1%; LCMS: [1/2M+1]+=850.9.
Figure PCTCN2021123020-appb-000150
Figure PCTCN2021123020-appb-000150
IV-1.7将CN-CMTC-8(10mg)溶解在DCM(1mL)中,反应液中加入TFA(0.3mL),反应约30mins,HPLC显示反应完全,Pre-HPLC纯化,得CN-C-CMTC的三氟乙酸盐约5mg,产率:59.5%;LCMS:[1/2M+1]+=714.9.IV-1.7 CN-CMTC-8 (10 mg) was dissolved in DCM (1 mL), TFA (0.3 mL) was added to the reaction solution, and the reaction was carried out for about 30 mins. HPLC showed that the reaction was complete. Purified by Pre-HPLC, CN-C-CMTC was obtained. about 5 mg of trifluoroacetate, yield: 59.5%; LCMS: [1/2M+1]+=714.9.
IV-2偶联粗产物ADC-1的合成Synthesis of IV-2 Coupling Crude Product ADC-1
Figure PCTCN2021123020-appb-000151
Figure PCTCN2021123020-appb-000151
将实施例1的hRS7抗体在5mg/mL的pH 7.2 PBS/EDTA溶液中首先用TCEP 6倍的物质的量在室温进行还原2小时。接着向抗体溶液中加入16倍的物质的量的溶解在DMSO中的化合物CN-C-CMTC(DMSO终浓度10%)。在室温条件下搅拌避光反应3小时,得到偶联粗产物ADC-1。The hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound CN-C-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 3 hours to obtain the conjugated crude product ADC-1.
IV-3偶联粗产物ADC-1的SEC检测SEC detection of IV-3 conjugated crude product ADC-1
偶联反应粗产品用SEC检测,SEC色谱条件如下:The coupling reaction crude product is detected by SEC, and the SEC chromatographic conditions are as follows:
色谱柱型号:TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
流动相条件:0.1MPB+0.2MNaCL+5%IPAMobile phase conditions: 0.1MPB+0.2MNaCl+5%IPA
检测器波长:280nm/363nmDetector wavelength: 280nm/363nm
柱温:30℃Column temperature: 30℃
流速:0.4mL/minFlow rate: 0.4mL/min
洗脱方式:等度洗脱Elution method: isocratic elution
进样体积:10μLInjection volume: 10 μL
运行时间:40minRunning time: 40min
通过SEC的检测,进行280nm与363nm对比,蛋白出峰位置的363nm吸收明显增强,表明小分子已经偶联到蛋白上。Through the detection of SEC, 280nm and 363nm were compared, and the absorption at 363nm at the peak position of the protein was significantly enhanced, indicating that the small molecule has been coupled to the protein.
IV-4偶联反应产品纯化:Purification of IV-4 coupling reaction products:
通过AKTA(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/海藻糖5%的溶液中,SEC检测,小分子已经完全除掉。纯化后的ADC-1见图1。样品超滤浓缩到5mg/mL,冻干保存。图1中,较高的峰表示抗体部分,较低的峰表示喜树碱化合物部分,二者峰保留时间位置相同,可见二者已形成了ADC偶联物。After desalting and purification by AKTA (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into a 5% solution of PBS/trehalose, and the small molecules were completely removed by SEC detection. The purified ADC-1 is shown in Figure 1. The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage. In Figure 1, the higher peak represents the antibody part, and the lower peak represents the camptothecin compound part. The retention time positions of the two peaks are the same, and it can be seen that the two have formed ADC conjugates.
IV-5 DAR的测定Determination of IV-5 DAR
用紫外分光光度计测定偶联物与裸抗在280nm与363nm吸光度值。偶联物中吉咪替康的浓度由363nm的吸光值按照标准曲线计算而得。偶联物中抗体的浓度由280nm的吸光值减去吉咪替康在280的吸光值计算而得。DAR值由这两个浓度的比值计算而得为6.5。即,n为6.5。The absorbance values of conjugate and naked antibody at 280nm and 363nm were measured by UV spectrophotometer. The concentration of gemnotecan in the conjugate was calculated from the absorbance at 363 nm according to the standard curve. The concentration of antibody in the conjugate was calculated by subtracting the absorbance of gemitecan at 280 from the absorbance at 280 nm. The DAR value was calculated from the ratio of these two concentrations to be 6.5. That is, n is 6.5.
本实施例中的CN-C可以替换为CN-A,CN-B,CN-D;吉咪替康(CMTC)可以替换为SN-38,吉马替康(GMTC)。CN-C in this embodiment can be replaced by CN-A, CN-B, CN-D; gemitecan (CMTC) can be replaced by SN-38, gematecan (GMTC).
实施例6:偶联物ADC-2的制备Example 6: Preparation of conjugate ADC-2
Figure PCTCN2021123020-appb-000152
Figure PCTCN2021123020-appb-000152
V-1中间体SMCC-PEG 2-GGFG-PABC-CMTC的制备 Preparation of V-1 Intermediate SMCC-PEG 2 -GGFG-PABC-CMTC
Figure PCTCN2021123020-appb-000153
Figure PCTCN2021123020-appb-000153
V-1.1将N 3-PEG-NHS(300mg,1.0mmol)与GGFG-PABOH(441mg,1.0mmol)溶解在NMP(5mL)中,在室温条件下反应过夜,HPLC显示有新峰生成,Pre-HPLC纯化后得N 3-PEG 2-GGFG-PABOH大约410mg,产率大约65.5%.LCMS:[M+1] +=627.5; V-1.1 Dissolve N 3 -PEG-NHS (300 mg, 1.0 mmol) and GGFG-PABOH (441 mg, 1.0 mmol) in NMP (5 mL) and react at room temperature overnight, HPLC showed that a new peak was generated, Pre- After HPLC purification, about 410 mg of N 3 -PEG 2 -GGFG-PABOH was obtained, and the yield was about 65.5%. LCMS: [M+1] + =627.5;
Figure PCTCN2021123020-appb-000154
Figure PCTCN2021123020-appb-000154
V-1.2将CMTC-Boc(50mg,0.1mmol),DMAP(61mg,0.5mmol)与三光气(24mg,0.08mmol)称量好后加入圆底烧瓶。加入DCM(1mL)后,搅拌大约5分钟,TLC显示,CMTC-Boc已经反应完全,再加入N 3-PEG 2-GGFG-PABOH(63mg,0.1mmol),继续搅拌10分钟左右,LCMS显示大部分为目标产物,部分掉Boc的产物,反应液过硅胶柱,用DCM:MeOH=10:1的洗脱剂洗脱后,浓缩,Pre-HPLC纯化后得产物N 3-PEG 2-GGFG-PABC-CMTC-Boc大约48mg,产率:41.9%。LCMS:[M+1] +=1157.8. V-1.2 CMTC-Boc (50 mg, 0.1 mmol), DMAP (61 mg, 0.5 mmol) and triphosgene (24 mg, 0.08 mmol) were weighed and added to a round bottom flask. After adding DCM (1 mL), stirring for about 5 minutes, TLC showed that CMTC-Boc had reacted completely, then N 3 -PEG 2 -GGFG-PABOH (63 mg, 0.1 mmol) was added, and stirring was continued for about 10 minutes, LCMS showed that most of the As the target product, part of the Boc product was removed, the reaction solution was passed through a silica gel column, eluted with DCM:MeOH=10:1 eluent, concentrated, and purified by Pre-HPLC to obtain the product N 3 -PEG 2 -GGFG-PABC -CMTC-Boc about 48 mg, yield: 41.9%. LCMS: [M+1] + = 1157.8.
Figure PCTCN2021123020-appb-000155
Figure PCTCN2021123020-appb-000155
V-1.3将SMCC-YNE(8mg,0.03)与N 3-PEG 2-GGFG-PABC-CMTC-Boc(30mg,0.03)溶解在DMSO/H 2O(0.4/0.4mL)中,加入CuBr(2mg)反应液在室温条件下搅拌1小时,HPLC显示反应完全,并有新峰生成,Pre-HPLC纯化后得产物大约(20mg),产率:53.9%;LCMS:[1/2M+1]+=716.5。 V-1.3 SMCC-YNE (8 mg, 0.03) and N 3 -PEG 2 -GGFG-PABC-CMTC-Boc (30 mg, 0.03) were dissolved in DMSO/H 2 O (0.4/0.4 mL), and CuBr (2 mg) was added. ) The reaction solution was stirred at room temperature for 1 hour. HPLC showed that the reaction was complete and a new peak was formed. After Pre-HPLC purification, the product was about (20 mg), yield: 53.9%; LCMS: [1/2M+1]+ = 716.5.
Figure PCTCN2021123020-appb-000156
Figure PCTCN2021123020-appb-000156
V-1.4将SMCC-PEG 2-GGFG-PABC-CMTC-Boc(15mg)溶解在DCM(1mL)中,反应液中加入TFA(0.3mL),反应约30mins,HPLC显示反应完全,Pre-HPLC纯化,得SMCC-PEG 2-GGFG-PABC-CMTC约8mg,产率:57.5%;LCMS:[M+1] +=1331.7。 V-1.4 Dissolve SMCC-PEG 2 -GGFG-PABC-CMTC-Boc (15 mg) in DCM (1 mL), add TFA (0.3 mL) to the reaction solution, and react for about 30 mins, HPLC showed that the reaction was complete, and purified by Pre-HPLC , about 8 mg of SMCC-PEG 2 -GGFG-PABC-CMTC was obtained, yield: 57.5%; LCMS: [M+1] + =1331.7.
V-2偶联粗产物ADC-2的合成Synthesis of V-2 Coupling Crude Product ADC-2
Figure PCTCN2021123020-appb-000157
Figure PCTCN2021123020-appb-000157
将实施例1的hRS7抗体在5mg/mL的pH 7.2 PBS/EDTA溶液中首先用TCEP 6倍的物质的量在室温进行还原2小时。接着向抗体溶液中加入16倍的物质的量的溶解在DMSO中的化合物SMCC-PEG2-GGFG-PABC-CMTC(DMSO终浓度10%)。在室温条件下搅拌避光反应1小时,得到偶联粗产物ADC-2。The hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound SMCC-PEG2-GGFG-PABC-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-2.
V-3偶联粗产物ADC-2的SEC检测SEC detection of V-3 conjugated crude product ADC-2
检测方法如实施例4步骤IV-3所述。The detection method is as described in step IV-3 of Example 4.
V-4偶联反应产品纯化:Product purification of V-4 coupling reaction:
通过AKTA(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/海藻糖5%的溶液中,SEC检测,小分子已经完全除掉。纯化后的ADC-2类似图1的ADC-1。样品超滤浓缩到5mg/mL,冻干保存。After desalting and purification by AKTA (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into a 5% solution of PBS/trehalose, and the small molecules were completely removed by SEC detection. The purified ADC-2 was similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
V-5 DAR的测定Determination of V-5 DAR
DAR的测定如实施例4步骤IV-5所述。ADC-2的DAR值为7.0。即,n为7.0。DAR was determined as described in Example 4, Step IV-5. The DAR value of ADC-2 is 7.0. That is, n is 7.0.
本实施例中的吉咪替康(CMTC)可以替换为SN-38,吉马替康(GMTC)。Gematecan (CMTC) in this example can be replaced by SN-38, gematecan (GMTC).
实施例7:偶联物ADC-3的制备Example 7: Preparation of conjugate ADC-3
Figure PCTCN2021123020-appb-000158
Figure PCTCN2021123020-appb-000158
VI-1中间体CN-C-PEG 2-GGFG-PABC-CMTC的制备 Preparation of VI-1 Intermediate CN-C-PEG 2 -GGFG-PABC-CMTC
Figure PCTCN2021123020-appb-000159
Figure PCTCN2021123020-appb-000159
VI-1.1将CN-C(6mg,0.03mmol)与N 3-PEG 2-GGFG-PABC-CMTC-Boc(30mg,0.03mmol)溶解在DMSO/H 2O(0.4/0.4mL)中,加入CuBr(2mg)反应液在室温条件下搅拌1小时,HPLC显示反应完全,并有新峰生成,Pre-HPLC纯化后得产物大约(18mg).产率:50.8%;LCMS:[M+1] +=1367.9。 VI-1.1 CN-C (6 mg, 0.03 mmol) and N 3 -PEG 2 -GGFG-PABC-CMTC-Boc (30 mg, 0.03 mmol) were dissolved in DMSO/H 2 O (0.4/0.4 mL), CuBr was added (2mg) The reaction solution was stirred at room temperature for 1 hour. HPLC showed that the reaction was complete and a new peak was formed. After Pre-HPLC purification, the product was about (18mg). Yield: 50.8%; LCMS: [M+1] + = 1367.9.
Figure PCTCN2021123020-appb-000160
Figure PCTCN2021123020-appb-000160
VI-1.2将CN-C-PEG 2-GGFG-PABC-CMTC-Boc(15mg)溶解在DCM(1mL)中,反应液中加入TFA(0.3mL),反应约30mins,HPLC显示反应完全,Pre-HPLC纯化,得CN-C-PEG 2-GGFG-PABC-CMTC约6mg,产率:43.1%;LCMS:[M+1] +=1267.6. VI-1.2 Dissolve CN-C-PEG 2- GGFG-PABC-CMTC-Boc (15mg) in DCM (1mL), add TFA (0.3mL) to the reaction solution, react for about 30mins, HPLC shows that the reaction is complete, Pre- Purified by HPLC, about 6 mg of CN-C-PEG 2 -GGFG-PABC-CMTC was obtained, yield: 43.1%; LCMS: [M+1] + =1267.6.
VI-2偶联粗产物ADC-3的合成Synthesis of VI-2 Coupling Crude Product ADC-3
Figure PCTCN2021123020-appb-000161
Figure PCTCN2021123020-appb-000161
将实施例1的hRS7抗体在5mg/mL的pH 7.2 PBS/EDTA溶液中首先用TCEP 6倍的物质的量在室温进行还原2小时。接着向抗体溶液中加入16倍的物质的量的溶解在DMSO中的化合物CN-C-PEG 2-GGFG-PABC-CMTC(DMSO终浓度10%)。在室温条件下搅拌避光反应3小时,得到偶联粗产物ADC-3。 The hRS7 antibody of Example 1 was first reduced with 6 times the amount of TCEP in a 5 mg/mL solution of pH 7.2 PBS/EDTA for 2 hours at room temperature. Next, a 16-fold amount of the compound CN-C-PEG 2 -GGFG-PABC-CMTC dissolved in DMSO was added to the antibody solution (final DMSO concentration 10%). The reaction was stirred at room temperature and protected from light for 3 hours to obtain the conjugated crude product ADC-3.
VI-3偶联粗产物ADC-3的SEC检测SEC detection of VI-3 conjugated crude product ADC-3
检测方法如实施例4步骤IV-3所述。The detection method is as described in step IV-3 of Example 4.
VI-4偶联反应产品纯化:Purification of VI-4 coupling reaction product:
通过AKTA(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/海藻糖5%的溶液中,SEC检测,小分子已经完全除掉。纯化后的ADC-3类似图1的ADC-1。样品超滤浓缩到5mg/mL,冻干保存。After desalting and purification by AKTA (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into a 5% solution of PBS/trehalose, and the small molecules were completely removed by SEC detection. The purified ADC-3 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
VI-5 DAR的测定Determination of VI-5 DAR
DAR的测定如实施例4步骤IV-5所述。ADC-3的DAR值为6.8。即,n为6.8。DAR was determined as described in Example 4, Step IV-5. The DAR value of ADC-3 is 6.8. That is, n is 6.8.
本实施例中的CN-C可以替换为CN-A,CN-B,CN-D;吉咪替康(CMTC)可以替换为SN-38,吉马替康(GMTC)。CN-C in this embodiment can be replaced by CN-A, CN-B, CN-D; gemitecan (CMTC) can be replaced by SN-38, gematecan (GMTC).
实施例8:偶联物ADC-4的制备Example 8: Preparation of conjugate ADC-4
Figure PCTCN2021123020-appb-000162
Figure PCTCN2021123020-appb-000162
VII-1中间体MC-GGFG-CMTC的制备Preparation of VII-1 Intermediate MC-GGFG-CMTC
Figure PCTCN2021123020-appb-000163
Figure PCTCN2021123020-appb-000163
VII-1.1将Boc-GGFG-OH(436mg,1.0mmol),DCC(247mg,1.2mmol)与NHS(148mg,1.1mmol)溶解在DCM(15mL)后,搅拌大约1小时,HPLC显示,Boc-GGFG-OH已经反应完全,并有Boc-GGFG-NHS的峰生成,再加入甘氨酸(75mg,10mmol),继续搅拌5小时后,HPLC显示,Boc-GGFG-NHS的峰完全消失,并有新峰生成,LCMS确认为目标产物,浓缩,Pre-HPLC纯化后得产物Boc-GGFGG大约380mg,产率:77.1%。LCMS:[M+1] +=494.2. VII-1.1 After dissolving Boc-GGFG-OH (436 mg, 1.0 mmol), DCC (247 mg, 1.2 mmol) and NHS (148 mg, 1.1 mmol) in DCM (15 mL), and stirring for about 1 hour, HPLC showed that Boc-GGFG -OH has been reacted completely, and the peak of Boc-GGFG-NHS has been generated, then glycine (75mg, 10mmol) was added, and after stirring for 5 hours, HPLC showed that the peak of Boc-GGFG-NHS completely disappeared, and a new peak was generated , LCMS confirmed as the target product, concentrated and purified by Pre-HPLC to obtain about 380 mg of product Boc-GGGFG, yield: 77.1%. LCMS: [M+1] + = 494.2.
Figure PCTCN2021123020-appb-000164
Figure PCTCN2021123020-appb-000164
VII-1.2向Boc-GGFGG-OH(200mg,0.4mmol),四氢呋喃(6mL),甲苯(2mL)的溶液中滴加吡啶(4.7mL,0.48mmol)及四乙酸铅(212mg,0.48mmol),加热回流5小时后,反应液冷却至室温,用硅藻土过滤后,有机相减压蒸馏除去,再用乙酸乙酯溶解,用饱和食盐水洗两次,有机层用无水硫酸钠干燥后,有机相减压蒸馏除去,用石油醚:乙酸乙酯=10:1过柱,得目标产物约150mg,产率:73.9%,LCMS:[M+1] +=508.3。 VII-1.2 Pyridine (4.7 mL, 0.48 mmol) and lead tetraacetate (212 mg, 0.48 mmol) were added dropwise to a solution of Boc-GGGFG-OH (200 mg, 0.4 mmol), tetrahydrofuran (6 mL), and toluene (2 mL), and heated After refluxing for 5 hours, the reaction solution was cooled to room temperature, filtered through celite, the organic phase was distilled off under reduced pressure, dissolved in ethyl acetate and washed twice with saturated brine. The phase was distilled off under reduced pressure and passed through the column with petroleum ether:ethyl acetate=10:1 to obtain about 150 mg of the target product, yield: 73.9%, LCMS: [M+1] + =508.3.
Figure PCTCN2021123020-appb-000165
Figure PCTCN2021123020-appb-000165
VII-1.3在0℃条件下向Boc-GGFG-AC(140mg,0.27mmol)和吉咪替康(218mg,0.54mmol)的四氢呋喃(15mL)溶液中加入叔丁醇钾(60.48mg,0.54mmol),反应液在室温条件下搅拌15mins后,HPLC检测有新峰生成,在0℃的条件下,加入乙酸乙酯和水,用乙酸乙酯与氯仿进行萃取,有机层用无水硫酸钠干燥后,有机层减压蒸馏除去,得粗品约120mg,可直接用于下一步反应。VII-1.3 To a solution of Boc-GGFG-AC (140 mg, 0.27 mmol) and gemitecan (218 mg, 0.54 mmol) in tetrahydrofuran (15 mL) at 0°C was added potassium tert-butoxide (60.48 mg, 0.54 mmol) , the reaction solution was stirred at room temperature for 15 mins, and a new peak was detected by HPLC. At 0 °C, ethyl acetate and water were added, and extracted with ethyl acetate and chloroform. The organic layer was dried with anhydrous sodium sulfate. , the organic layer was distilled off under reduced pressure to obtain about 120 mg of crude product, which could be directly used in the next reaction.
Figure PCTCN2021123020-appb-000166
Figure PCTCN2021123020-appb-000166
VII-1.4将Boc-GGFG-CMTC(80mg)溶解在DCM(2mL)中,反应液中加入TFA(0.6mL),反应约30mins,HPLC显示反应完全,Pre-HPLC纯化,得GGFG-CMTC约50mg,产率:70.6%;LCMS:[M+1] +=752.5. VII-1.4 Boc-GGFG-CMTC (80 mg) was dissolved in DCM (2 mL), TFA (0.6 mL) was added to the reaction solution, and the reaction was carried out for about 30 mins. HPLC showed that the reaction was complete. Purified by Pre-HPLC, about 50 mg of GGFG-CMTC was obtained. , Yield: 70.6%; LCMS: [M+1] + =752.5.
Figure PCTCN2021123020-appb-000167
Figure PCTCN2021123020-appb-000167
VII-1.5将GGFG-CMTC(30mg,0.04mmol)与MC-NHS(13mg,0.04mmol)溶解在NMP(3 mL)中,在室温条件下反应过夜,HPLC显示有新峰生成,Pre-HPLC纯化后得MC-GGFG-CMTC大约18mg,产率大约47.7%.LCMS:[M+1] +=945.5; VII-1.5 GGFG-CMTC (30 mg, 0.04 mmol) and MC-NHS (13 mg, 0.04 mmol) were dissolved in NMP (3 mL) and reacted overnight at room temperature. HPLC showed that a new peak was generated, and purified by Pre-HPLC The obtained MC-GGFG-CMTC was about 18 mg, and the yield was about 47.7%. LCMS: [M+1] + =945.5;
VII-2偶联粗产物ADC-4的合成Synthesis of VII-2 Coupling Crude Product ADC-4
Figure PCTCN2021123020-appb-000168
Figure PCTCN2021123020-appb-000168
将实施例1的hRS7抗体在5mg/mL的pH 7.2 PBS/EDTA溶液中首先用TCEP 6倍的物质的量在室温进行还原2小时。接着向抗体溶液中加入16倍的物质的量的溶解在DMSO中的化合物MC-GGFG-CMTC(DMSO终浓度10%)。在室温条件下搅拌避光反应1小时,得到偶联粗产物ADC-4。The hRS7 antibody of Example 1 was first reduced in 5 mg/mL pH 7.2 PBS/EDTA solution with 6 times the amount of TCEP at room temperature for 2 hours. Next, a 16-fold amount of the compound MC-GGFG-CMTC dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the conjugated crude product ADC-4.
VII-3偶联粗产物ADC-4的SEC检测SEC detection of VII-3 conjugated crude product ADC-4
检测方法如实施例4步骤IV-3所述。The detection method is as described in step IV-3 of Example 4.
VII-4偶联反应产品纯化:Purification of VII-4 coupling reaction product:
通过AKTA(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/海藻糖5%的溶液中,SEC检测,小分子已经完全除掉。纯化后的ADC-4类似图1的ADC-1。样品超滤浓缩到5mg/mL,冻干保存。After desalting and purification by AKTA (filler: sephadex G 25), the conjugate was obtained, and the conjugate was changed into a 5% solution of PBS/trehalose, and the small molecules were completely removed by SEC detection. The purified ADC-4 is similar to ADC-1 of Figure 1 . The samples were concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
VII-5 DAR的测定Determination of VII-5 DAR
DAR的测定如实施例4步骤IV-5所述。ADC-4的DAR值为7.4。即,n为7.4。DAR was determined as described in Example 4, Step IV-5. The DAR value of ADC-4 is 7.4. That is, n is 7.4.
实施例9:偶联物ADC-X的制备Example 9: Preparation of conjugate ADC-X
Figure PCTCN2021123020-appb-000169
Figure PCTCN2021123020-appb-000169
I-1中间体A的制备Preparation of I-1 Intermediate A
Figure PCTCN2021123020-appb-000170
Figure PCTCN2021123020-appb-000170
I-1.1将A1(170mg,0.4mmol)与VC-PABOH(150mg,0.4mmol)溶解在DMF(5mL)中,在室温条件下反应过夜,HPLC显示有新峰生成,Pre-HPLC纯化后得A2纯品大约120mg,产率大约43.5%.LCMS:[M+1] +=690.6。 I-1.1 Dissolve A1 (170 mg, 0.4 mmol) and VC-PABOH (150 mg, 0.4 mmol) in DMF (5 mL), react at room temperature overnight, HPLC shows that a new peak is generated, and A2 is obtained after Pre-HPLC purification Pure about 120 mg, about 43.5% yield. LCMS: [M+1] + = 690.6.
Figure PCTCN2021123020-appb-000171
Figure PCTCN2021123020-appb-000171
I-1.2.将吉马替康(Gimatecan)(44.7mg,0.1mmol)、DMAP(36.6mg,0.3mmol)与三光气(14.8mg,0.05mmol)称量好后加入圆底烧瓶。加入DCM(2mL)后,搅拌大约5分钟,TLC显示,Gimatecan已经反应完全,再加入A2(68.9mg,0.1mmol),继续搅拌4分钟左右,反应液过一个短的硅胶柱,用DCM:MeOH=10:1的洗脱剂洗脱后,浓缩,Pre-HPLC纯化后得产物A 10mg左右,产率:8.6%。LCMS:[M+1] +=1164.3。 I-1.2. Gimatecan (44.7 mg, 0.1 mmol), DMAP (36.6 mg, 0.3 mmol) and triphosgene (14.8 mg, 0.05 mmol) were weighed and added to a round bottom flask. After adding DCM (2 mL), stirring for about 5 minutes, TLC showed that Gimatecan had reacted completely, then A2 (68.9 mg, 0.1 mmol) was added, and stirring was continued for about 4 minutes, the reaction solution was passed through a short silica gel column, and DCM:MeOH was used. After eluting with the eluent of =10:1, concentrating, and purifying by Pre-HPLC to obtain about 10 mg of product A, the yield: 8.6%. LCMS: [M+1] + =1164.3.
I-2偶联粗产物ADC-X的合成Synthesis of I-2 Coupling Crude Product ADC-X
Figure PCTCN2021123020-appb-000172
Figure PCTCN2021123020-appb-000172
将实施例1制备的hRS7抗体在5mg/mL的pH 6.5 10mM磷酸盐溶液中首先用TCEP 5倍的物质的量在室温进行还原2小时。接着向抗体溶液中加入8倍的物质的量的溶解在DMF中的化合物A(DMF终浓度15%)。在室温条件下搅拌避光反应1小时,得到偶联粗产物ADC-X-a。The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 5 times the amount of TCEP at room temperature for 2 hours. Next, an 8-fold amount of Compound A dissolved in DMF (final DMF concentration 15%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 1 hour to obtain the coupled crude product ADC-X-a.
或者:or:
将Herceptin抗体在6mg/mL的pH 7.2 PBS溶液中首先用TCEP 9.5倍的物质的量在室温进行还原30分钟。接着向抗体溶液中加入12倍的物质的量的溶解在DMF中的化合物A(DMF 终浓度10%)。在4℃条件下搅拌避光反应3小时,得到偶联粗产物ADC-X-b。The Herceptin antibody was first reduced in 6 mg/mL pH 7.2 PBS solution with 9.5 times the amount of TCEP for 30 minutes at room temperature. Next, a 12-fold substance amount of Compound A dissolved in DMF (final DMF concentration 10%) was added to the antibody solution. The reaction was stirred and protected from light at 4°C for 3 hours to obtain the coupled crude product ADC-X-b.
I-3偶联粗产物ADC-X的检测Detection of I-3 Coupling Crude Product ADC-X
偶联反应粗产品用SEC检测,SEC色谱条件如下:The coupling reaction crude product is detected by SEC, and the SEC chromatographic conditions are as follows:
色谱柱型号:TSKgel G4000SWxl 7.8mmI.D.*30cm,8μmColumn model: TSKgel G4000SWxl 7.8mmI.D.*30cm, 8μm
检测器波长:280nm/363nmDetector wavelength: 280nm/363nm
柱温:30℃Column temperature: 30℃
流速:1mL/minFlow rate: 1mL/min
洗脱方式:等度洗脱Elution method: isocratic elution
进样体积:10μLInjection volume: 10 μL
运行时间:55minRunning time: 55min
通过SEC的检测,进行280nm与363nm对比,蛋白出峰位置的363nm吸收明显增强,表明小分子已经偶联到蛋白上。Through the detection of SEC, 280nm and 363nm were compared, and the absorption at 363nm at the peak position of the protein was significantly enhanced, indicating that the small molecule has been coupled to the protein.
I-4偶联反应产品纯化:I-4 coupling reaction product purification:
通过脱盐柱PD-10(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/蔗糖5%的溶液中,SEC检测,小分子已经完全除掉。纯化后的ADC-X样品超滤浓缩到5mg/mL,冻干保存。After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained. The conjugate was then changed into a PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed. The purified ADC-X sample was concentrated to 5 mg/mL by ultrafiltration and lyophilized for storage.
I-5 DAR的测定Determination of I-5 DAR
用紫外分光光度计测定偶联物与裸抗在280nm与363nm吸光度值。偶联物中Gimatecan的浓度由363nm的吸光值按照标准曲线计算而得。偶联物中抗体的浓度由280nm的吸光值减去gimatecan在280的吸光值计算而得。结果如所示。The absorbance values of conjugate and naked antibody at 280nm and 363nm were measured by UV spectrophotometer. The concentration of Gimatecan in the conjugate was calculated from the absorbance at 363 nm according to the standard curve. The concentration of antibody in the conjugate was calculated by subtracting the absorbance of gimatecan at 280 from the absorbance at 280 nm. The result is as shown.
抗体为实施例1制备的hRS7抗体时:When the antibody is the hRS7 antibody prepared in Example 1:
ADC-X-a的DAR值由这两个浓度的比值计算而得为2.7,即,n为2.7。The DAR value of ADC-X-a was calculated from the ratio of these two concentrations to be 2.7, ie, n was 2.7.
抗体为Herceptin抗体时:When the antibody is Herceptin antibody:
ADC-X-b的DAR值由这两个浓度的比值计算而得为7.3,即,n为7.3。The DAR value of ADC-X-b was calculated from the ratio of these two concentrations to be 7.3, ie, n was 7.3.
实施例10偶联物ADC-5的制备Example 10 Preparation of conjugate ADC-5
Figure PCTCN2021123020-appb-000173
Figure PCTCN2021123020-appb-000173
偶联物ADC-5Conjugate ADC-5
VII-1中间体A的制备Preparation of VII-1 Intermediate A
Figure PCTCN2021123020-appb-000174
Figure PCTCN2021123020-appb-000174
VII-1.1将G1(475mg,1.18mmol)溶解在DCM(3.0mL)的反应液中,加入TBSCl(401mg,2.95mmol),再加入DIPEA(481mg,3.7mmol)后反应1小时,HPLC检测已经反应完全,加入甲醇猝灭后,过硅胶柱后得目标产物G2 510mg,产率:86.1%.LCMS:[M+1] +=505.6. VII-1.1 G1 (475 mg, 1.18 mmol) was dissolved in the reaction solution of DCM (3.0 mL), TBSCl (401 mg, 2.95 mmol) was added, DIPEA (481 mg, 3.7 mmol) was added, and the reaction was performed for 1 hour, and the reaction was detected by HPLC. Complete, after adding methanol to quench, and passing through a silica gel column to obtain the target product G2 510mg, yield: 86.1%. LCMS: [M+1] + =505.6.
Figure PCTCN2021123020-appb-000175
Figure PCTCN2021123020-appb-000175
VII-1.2将G2(40mg,0.08mmol),DMAP(44mg,0.36mmol)与三光气(14mg,0.05mmol)称量好后加入圆底试管。加入DCM(0.5mL)后,搅拌大约7分钟,TLC显示,G2已经反应完全,再加入CL2-L(60mg,0.06mmol),继续搅拌10分钟左右,加入1d甲醇猝灭,旋干后,Pre-HPLC纯化后得目标产物G3 39mg左右,产率31.2%.LCMS:[M+1] +=1575.6. VII-1.2 G2 (40 mg, 0.08 mmol), DMAP (44 mg, 0.36 mmol) and triphosgene (14 mg, 0.05 mmol) were weighed and added to a round-bottomed test tube. After adding DCM (0.5 mL), stirring for about 7 minutes, TLC showed that G2 had reacted completely, then CL2-L (60 mg, 0.06 mmol) was added, stirring was continued for about 10 minutes, 1d methanol was added to quench, and after spin-drying, Pre -After HPLC purification, the target product G3 is about 39mg, the yield is 31.2%. LCMS: [M+1] + =1575.6.
Figure PCTCN2021123020-appb-000176
Figure PCTCN2021123020-appb-000176
VII-1.3将G3(39mg,0.025mmol)溶解在TBAF/DMF(1mL)的反应液中,30mins后,Pre-HPLC纯化后得到目标产物,冻干后得G4 25mg左右,产率:68.5%.LCMS:[M+1]+=1475.6.VII-1.3 G3 (39mg, 0.025mmol) was dissolved in the reaction solution of TBAF/DMF (1mL), after 30mins, the target product was obtained after Pre-HPLC purification, and after lyophilization, G4 was obtained about 25mg, yield: 68.5%. LCMS: [M+1]+=1475.6.
Figure PCTCN2021123020-appb-000177
Figure PCTCN2021123020-appb-000177
VII-1.4将G4(40mg,0.026mmol)溶解在DMF/H2O(0.8mL/1.2mL)中,室温条件下加入G5(13mg,0.052mmol),反应液在室温条件下搅拌5mins,HPLC显示有新峰生成,LCMS显示有目标产物生成,Pre-HPLC纯化后得产物G6大约25mg,产率:54.7%.LCMS:[1/2M+1] +=1749.9. VII-1.4 G4 (40mg, 0.026mmol) was dissolved in DMF/H2O (0.8mL/1.2mL), G5 (13mg, 0.052mmol) was added at room temperature, the reaction solution was stirred at room temperature for 5mins, HPLC showed that there was a new The peak was generated, and LCMS showed that the target product was generated. After Pre-HPLC purification, about 25 mg of product G6 was obtained, yield: 54.7%. LCMS: [1/2M+1] + =1749.9.
Figure PCTCN2021123020-appb-000178
Figure PCTCN2021123020-appb-000178
VII-1.5将G6(20mg,0.011mmol)溶解在DCM/DCA/苯甲醚(1mL/0.1mL/0.1mL)的溶液中, 在室温条件下反应0.5h,HPLC显示有新峰生成,原料已经全部消失,加入适量乙醚沉降后,Pre-HPLC纯化,冻干得目标产物G约12mg,产率:70.6%.LCMS:[M-1] -=1492.8. VII-1.5 G6 (20 mg, 0.011 mmol) was dissolved in a solution of DCM/DCA/anisole (1 mL/0.1 mL/0.1 mL), reacted at room temperature for 0.5 h, HPLC showed that a new peak was generated, and the raw material had been All disappeared, after adding appropriate amount of ether for sedimentation, Pre-HPLC purification, lyophilization to obtain the target product G about 12 mg, yield: 70.6%. LCMS: [M-1] - = 1492.8.
VI-2偶联粗产物ADC-5的合成Synthesis of VI-2 Coupling Crude Product ADC-5
Figure PCTCN2021123020-appb-000179
Figure PCTCN2021123020-appb-000179
将实施例1制备的hRS7抗体在5mg/mL的pH 6.5 10mM磷酸盐溶液中首先用TCEP 7倍的物质的量在37℃进行还原2小时。接着向抗体溶液中加入16倍的物质的量的溶解在DMSO中的化合物G(DMSO终浓度10%)。在室温条件下搅拌避光反应20分钟,得到偶联粗产物ADC-5。The hRS7 antibody prepared in Example 1 was first reduced in 5 mg/mL pH 6.5 10 mM phosphate solution with 7 times the amount of TCEP at 37°C for 2 hours. Next, a 16-fold amount of Compound G dissolved in DMSO (final DMSO concentration 10%) was added to the antibody solution. The reaction was stirred at room temperature and protected from light for 20 minutes to obtain the conjugated crude product ADC-5.
VI-3偶联粗产物ADC-5的检测Detection of VI-3 conjugated crude product ADC-5
检测方法如实施例9步骤I-3所述。The detection method is as described in step I-3 of Example 9.
VI-4偶联反应产品纯化:Purification of VI-4 coupling reaction product:
通过脱盐柱PD-10(填料:sephadex G 25)脱盐纯化后,得偶联物,偶联物再换液到PBS/蔗糖5%的溶液中,SEC检测,小分子已经完全除掉,样品超滤浓缩到5mg/mL,冻干保存。After desalting and purification by desalting column PD-10 (filler: sephadex G 25), the conjugate was obtained, and the conjugate was then replaced with PBS/sucrose 5% solution. SEC detected that the small molecules had been completely removed, and the sample was over It was concentrated to 5 mg/mL by filtration and lyophilized for storage.
VI-5 DAR的测定Determination of VI-5 DAR
DAR的测定如实施例9步骤1-5所述。ADC-5的SEC-HPLC图谱如图2所示。DAR was determined as described in Example 9, steps 1-5. The SEC-HPLC profile of ADC-5 is shown in FIG. 2 .
ADC-5的DAR值为7.8。即,n为7.8。The DAR value of ADC-5 is 7.8. That is, n is 7.8.
实施例11:ADC-6的合成Example 11: Synthesis of ADC-6
Figure PCTCN2021123020-appb-000180
Figure PCTCN2021123020-appb-000180
合成方法:resolve resolution:
1.1将S1(16mg,0.032mmol),DMAP(17mg,0.144mmol)与三光气(6mg,0.02mmol)称量好后加入圆底试管中。加入DCM(0.5mL)后,搅拌大约3分钟,TLC显示,S1大部分消失,再加入A1(20mg,0.03mmol),继续搅拌4分钟左右,将反应液加入乙醚中沉降两次后,Pre- HPLC纯化后得产物大约4.5mg,产率:15.5%.LCMS:[M+1] +=1165.6. 1.1 S1 (16 mg, 0.032 mmol), DMAP (17 mg, 0.144 mmol) and triphosgene (6 mg, 0.02 mmol) were weighed and added to a round-bottomed test tube. After adding DCM (0.5 mL), stirring for about 3 minutes, TLC showed that most of S1 disappeared, then A1 (20 mg, 0.03 mmol) was added, and stirring was continued for about 4 minutes. After the reaction solution was added to ether and settled twice, Pre- After HPLC purification, the product was about 4.5 mg, yield: 15.5%. LCMS: [M+1] + =1165.6.
Figure PCTCN2021123020-appb-000181
Figure PCTCN2021123020-appb-000181
1.2将A2(4.5mg)溶解在TFA/DCM(0.5mL/0.5mL)的反应液中,3mins后,加入乙醚稀释,猝灭,旋干后,Pre-HPLC纯化后得到目标产物,冻干后得A 2.5mg左右,产率:60.9%.LCMS:[M+1] +=1065.6. 1.2 A2 (4.5 mg) was dissolved in the reaction solution of TFA/DCM (0.5 mL/0.5 mL), after 3 mins, diethyl ether was added to dilute, quenched, spin-dried, and purified by Pre-HPLC to obtain the target product. After lyophilization Obtained A about 2.5mg, yield: 60.9%. LCMS: [M+1] + =1065.6.
1.3 A与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,获得的ADC-6的SEC-HPLC结果如图3所示。1.3 The coupling, purification, and detection steps of A and the hRS7 antibody prepared in Example 1 are the same as those of Steps VI-2-VI-5 in Example 10, and the SEC-HPLC results of the obtained ADC-6 are shown in Figure 3.
ADC-6的DAR值为5.5。即,n为5.5。The DAR value of ADC-6 is 5.5. That is, n is 5.5.
实施例12:ADC-7的合成Example 12: Synthesis of ADC-7
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000182
Figure PCTCN2021123020-appb-000182
1.1将B1(475mg,1.18mmol)溶解在DCM(3.0mL)的反应液中,加入TBSCl(401mg,2.95mmol),再加入DIPEA(481mg,3.7mmol)后反应1小时,HPLC检测已经反应完全,加入甲醇猝灭后,过硅胶柱后得目标产物510mg,产率:86.1%.LCMS:[M+1] +=505.6. 1.1 Dissolve B1 (475 mg, 1.18 mmol) in the reaction solution of DCM (3.0 mL), add TBSCl (401 mg, 2.95 mmol), add DIPEA (481 mg, 3.7 mmol) and react for 1 hour, HPLC detects that the reaction has been completed, After adding methanol for quenching, the target product 510mg was obtained after passing through silica gel column, yield: 86.1%. LCMS: [M+1] + =505.6.
Figure PCTCN2021123020-appb-000183
Figure PCTCN2021123020-appb-000183
1.2将B2(40mg,0.08mmol),DMAP(44mg,0.36mmol)与三光气(14mg,0.05mmol)称量好后加入圆底试管。加入DCM(0.5mL)后,搅拌大约7分钟,TLC显示,B2已经反应完全,再加入N3-PEG8-Lys(MMT)-PABOH(60mg,0.06mmol),继续搅拌10分钟左右,加入1d甲醇猝灭,旋干后,Pre-HPLC纯化后得目标产物39mg左右,产率31.2%.LCMS:[M+1] +=1575.6. 1.2 B2 (40 mg, 0.08 mmol), DMAP (44 mg, 0.36 mmol) and triphosgene (14 mg, 0.05 mmol) were weighed and added to a round-bottomed test tube. After adding DCM (0.5 mL), stirring for about 7 minutes, TLC showed that B2 had reacted completely, then N3-PEG8-Lys(MMT)-PABOH (60 mg, 0.06 mmol) was added, stirring was continued for about 10 minutes, and 1d methanol was added 39mg of the target product was obtained after being purified by Pre-HPLC, and the yield was 31.2%. LCMS: [M+1] + =1575.6.
Figure PCTCN2021123020-appb-000184
Figure PCTCN2021123020-appb-000184
1.3将B3(39mg,0.025mmol)溶解在TBAF/DMF(1mL)的反应液中,30mins后,Pre-HPLC纯化后得到目标产物,冻干后得25mg左右,产率:68.5%.LCMS:[M+1] +=1475.6. 1.3 B3 (39mg, 0.025mmol) was dissolved in the reaction solution of TBAF/DMF (1mL), after 30mins, the target product was obtained after Pre-HPLC purification, and about 25mg was obtained after lyophilization, yield: 68.5%. LCMS: [ M+1] + = 1475.6.
Figure PCTCN2021123020-appb-000185
Figure PCTCN2021123020-appb-000185
1.4向B4(19mg,0.013mmol)与炔乙胺(5mg,0.1mmol)溶解在DMSO/H2O(1mL/1mL)的溶液中,加入CuBr(0.2mg,0.001mmol)反应1.5小时后,HPLC显示目标产物生成,Pre-HPLC纯化得目标产物约15mg产率:76.1%.LCMS:[M+1] +=1529.7. 1.4 Dissolve B4 (19mg, 0.013mmol) and propargylamine (5mg, 0.1mmol) in a solution of DMSO/H2O (1mL/1mL), add CuBr (0.2mg, 0.001mmol) and react for 1.5 hours, HPLC shows the target The product was generated and purified by Pre-HPLC to obtain about 15 mg of the target product. Yield: 76.1%. LCMS: [M+1] + =1529.7.
Figure PCTCN2021123020-appb-000186
Figure PCTCN2021123020-appb-000186
1.5将B5(7mg,0.0046)溶解在DMF/H2O(0.8mL/1.2mL)中,室温条件下加入CN-B-4F(3mg,0.0071mmol),反应液在室温条件下搅拌5mins,HPLC显示有新峰生成,LCMS显示有目标产物生成,Pre-HPLC纯化后得产物B6大约4mg,产率:54.05%.LCMS:[1/2M+1] +=843.8. 1.5 B5 (7mg, 0.0046) was dissolved in DMF/H2O (0.8mL/1.2mL), CN-B-4F (3mg, 0.0071mmol) was added at room temperature, the reaction solution was stirred at room temperature for 5mins, HPLC showed that there were A new peak was formed, and LCMS showed that the target product was formed. After Pre-HPLC purification, about 4 mg of product B6 was obtained, yield: 54.05%. LCMS: [1/2M+1] + =843.8.
Figure PCTCN2021123020-appb-000187
Figure PCTCN2021123020-appb-000187
将B6(4mg,0.0024mmol)溶解在DCM/DCA/苯甲醚(1mL/0.1mL/0.1mL)的溶液中,在室温条件下反应0.5h,HPLC显示有新峰生成,原料已经全部消失,加入适量乙醚沉降后,Pre-HPLC纯化,冻干得目标产物约2mg,产率:59.0%.LCMS:[M-1] -=1425.8. B6 (4mg, 0.0024mmol) was dissolved in a solution of DCM/DCA/anisole (1mL/0.1mL/0.1mL), and the reaction was carried out at room temperature for 0.5h. HPLC showed that a new peak was generated, and the raw materials had all disappeared. After adding an appropriate amount of ether for sedimentation, Pre-HPLC purification, lyophilization to obtain about 2 mg of the target product, yield: 59.0%. LCMS: [M-1] - = 1425.8.
1.6 B与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-7。1.6 The coupling, purification and detection steps of B and the hRS7 antibody prepared in Example 1 are the same as steps VI-2-VI-5 in Example 10, and finally ADC-7 is obtained.
实施例13:ADC-8的合成Example 13: Synthesis of ADC-8
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000188
Figure PCTCN2021123020-appb-000188
1.1将C1(80mg,0.1mmol),C2(52mg,0.1mmol)与DIEA(25mg.0.2mmol)溶解在DMF(2mL)的反应液中,在室温条件下搅拌2h后,HPLC检测C2完全消失,Pre-HPLC纯化后得目标产物约30mg,产率:50.9%.LCMS:[M+1] +=1179.2. 1.1 Dissolve C1 (80mg, 0.1mmol), C2 (52mg, 0.1mmol) and DIEA (25mg.0.2mmol) in the reaction solution of DMF (2mL), after stirring at room temperature for 2h, HPLC detects that C2 disappears completely, After Pre-HPLC purification, about 30 mg of the target product was obtained, the yield: 50.9%. LCMS: [M+1] + =1179.2.
1.2 C与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-8,其SEC-HPLC结果如图4所示。The coupling, purification and detection steps of 1.2 C and the hRS7 antibody prepared in Example 1 are the same as steps VI-2-VI-5 in Example 10, and finally ADC-8 is obtained, and its SEC-HPLC results are shown in Figure 4.
ADC-8的DAR值为3.3。即,n为3.3。ADC-8 has a DAR of 3.3. That is, n is 3.3.
实施例14:ADC-9的合成Example 14: Synthesis of ADC-9
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000189
Figure PCTCN2021123020-appb-000189
1.1将D2(51.8mg,0.1mmol),DMAP(36.6mg,0.3mmol)与三光气(14.8mg,0.05mmol)称量好后加入圆底烧瓶。加入DCM(2mL)后,搅拌大约5分钟,TLC显示,D2已经反应完全,再加入D1(87mg,0.1mmol),继续搅拌4分钟左右,反应液过一个短得硅胶柱,用DCM:MeOH=10:1的洗脱剂洗脱后,浓缩,Pre-HPLC纯化后得产物D3 40mg左右,产率:28.3%。LCMS:[M+1] +=1119.6. 1.1 D2 (51.8 mg, 0.1 mmol), DMAP (36.6 mg, 0.3 mmol) and triphosgene (14.8 mg, 0.05 mmol) were weighed and added to a round bottom flask. After adding DCM (2 mL), stirring for about 5 minutes, TLC showed that D2 had reacted completely, then D1 (87 mg, 0.1 mmol) was added, and stirring was continued for about 4 minutes, the reaction solution was passed through a short silica gel column, and DCM:MeOH= After eluting with 10:1 eluent, concentration, and Pre-HPLC purification to obtain about 40 mg of product D3, yield: 28.3%. LCMS: [M+1] + = 1119.6.
Figure PCTCN2021123020-appb-000190
Figure PCTCN2021123020-appb-000190
1.2将D3(19mg,0.015mmol),溶解在TFA/DCM(0.2mL/0.8mL)的反应液中,反应2h后,HPLC显示原料已基本消失,Pre-HPLC纯化新峰得目标化合物约(10mg),产率为:56.8%。LCMS:[M+1] +=1249.6. 1.2 Dissolve D3 (19 mg, 0.015 mmol) in the reaction solution of TFA/DCM (0.2 mL/0.8 mL), after 2 h of reaction, HPLC showed that the raw material had basically disappeared, and the new peak was purified by Pre-HPLC to obtain the target compound (about (10 mg). ), yield: 56.8%. LCMS: [M+1] + = 1249.6.
1.3 D与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,获得ADC-9。1.3 The coupling, purification and detection steps of D and the hRS7 antibody prepared in Example 1 are the same as steps VI-2-VI-5 in Example 10 to obtain ADC-9.
实施例15:ADC-10的合成Example 15: Synthesis of ADC-10
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000191
Figure PCTCN2021123020-appb-000191
1.1将E1(404mg,1.0mmol),E2(285mg,1.0mmol)与K 2CO3(207mg,1.5mmol)溶解在DMF(15mL)的反应液中,室温条件下反应5h后,HPLC显示有部分原料剩余,新峰生成后,加入适量的NH4Cl调节pH为中性后,柱层析纯化得目标产物约140mg,产率:22.9%. 1.1 Dissolve E1 (404 mg, 1.0 mmol), E2 (285 mg, 1.0 mmol) and K 2 CO3 (207 mg, 1.5 mmol) in the reaction solution of DMF (15 mL), after 5 h of reaction at room temperature, HPLC showed that there were some raw materials The remainder, after the new peak was generated, after adding an appropriate amount of NH4Cl to adjust the pH to neutral, the target product was purified by column chromatography to obtain about 140 mg of the target product, the yield: 22.9%.
LCMS:[M+1] +=610.6. LCMS: [M+1] + = 610.6.
Figure PCTCN2021123020-appb-000192
Figure PCTCN2021123020-appb-000192
1.2将E3(61mg,0.1mmol)溶解在TFA/DCM(0.5mL/0.5mL)的反应液中反应30mins后,HPLC显示有新峰生成,粗品得目标化合物约40mg,产率:78.4%。LCMS:[M+1] +=510.6. 1.2 After dissolving E3 (61 mg, 0.1 mmol) in the reaction solution of TFA/DCM (0.5 mL/0.5 mL) and reacting for 30 mins, HPLC showed that a new peak was formed, and the crude product obtained about 40 mg of the target compound, yield: 78.4%. LCMS: [M+1] + = 510.6.
Figure PCTCN2021123020-appb-000193
Figure PCTCN2021123020-appb-000193
1.3将E5(26mg,0.05mmol)与EDCl(19mg,1.0mmol),HOBt(13mg,1.0mmol)在DMF(3mL)的反应液中搅拌3h后,加入E4(25mg,0.05mmol)反应1h后,E4完全消失后,有新峰生成,纯化后得目标产物约4mg,产率7.8%。LCMS:[M+1] +=1021.3。 1.3 After stirring E5 (26 mg, 0.05 mmol) with EDCl (19 mg, 1.0 mmol), HOBt (13 mg, 1.0 mmol) in the reaction solution of DMF (3 mL) for 3 h, adding E4 (25 mg, 0.05 mmol) and reacting for 1 h, After E4 disappeared completely, a new peak was formed. After purification, about 4 mg of the target product was obtained, and the yield was 7.8%. LCMS: [M+1] + =1021.3.
1.4E与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,获得的ADC-10的SEC-HPLC结果如图5所示。The coupling, purification and detection steps of 1.4E and the hRS7 antibody prepared in Example 1 are the same as steps VI-2-VI-5 in Example 10, and the obtained SEC-HPLC results of ADC-10 are shown in FIG. 5 .
ADC-10的DAR值为3.5。即,n为3.5。The DAR value of ADC-10 is 3.5. That is, n is 3.5.
实施例16:ADC-11的合成Example 16: Synthesis of ADC-11
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000194
Figure PCTCN2021123020-appb-000194
1.1将F1(26mg,0.5mmol)与EDCl(19mg,1.0mmol),HOBt(13mg,1.0mmol)在DMF(3mL)的反应液中搅拌3h后,加入F2(25mg,0.05mmol)反应1h后,F2完全消失后,有新峰生成,纯化后得目标产物约30mg,产率58.8%。LCMS:[M+1] +=1016.3。 1.1 After stirring F1 (26 mg, 0.5 mmol) with EDCl (19 mg, 1.0 mmol), HOBt (13 mg, 1.0 mmol) in the reaction solution of DMF (3 mL) for 3 h, adding F2 (25 mg, 0.05 mmol) and reacting for 1 h, After F2 disappeared completely, a new peak was formed. After purification, about 30 mg of the target product was obtained, and the yield was 58.8%. LCMS: [M+1] + =1016.3.
1.2 F与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-11,其SEC-HPLC结果如图6所示。1.2 The coupling, purification, and detection steps of F and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-11 was obtained, and its SEC-HPLC results were shown in Figure 6.
ADC-11的DAR值为7.1。即,n为7.1。The DAR value of ADC-11 is 7.1. That is, n is 7.1.
实施例17:ADC-12的合成Example 17: Synthesis of ADC-12
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000195
Figure PCTCN2021123020-appb-000195
1.1将G1(30mg,0.041mmol),G2(21mg,0.041mmol)与DIEA(10mg,0.08mmol)溶解在DMF(2mL)的反应液中,在室温条件下搅拌2h后,HPLC检测G2完全消失,Pre-HPLC纯化后得目标产物G约30mg,产率:53.7%.LCMS:[M+1] +=1179.2. 1.1 Dissolve G1 (30mg, 0.041mmol), G2 (21mg, 0.041mmol) and DIEA (10mg, 0.08mmol) in the reaction solution of DMF (2mL), stir at room temperature for 2h, HPLC detects that G2 disappears completely, After Pre-HPLC purification, about 30 mg of the target product G was obtained, the yield: 53.7%. LCMS: [M+1] + =1179.2.
1.2 G与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-12,其SEC-HPLC结果如图7所示。The coupling, purification, and detection steps of 1.2 G and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-12 was obtained, and its SEC-HPLC results were shown in Figure 7.
ADC-12的DAR值为6.2。即,n为6.2。The DAR value of ADC-12 is 6.2. That is, n is 6.2.
实施例18:ADC-13的合成Example 18: Synthesis of ADC-13
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000196
Figure PCTCN2021123020-appb-000196
1.1将H1(404mg,1.0mmol),H2(285mg,1.0mmol)与K 2CO3(207mg,1.5mmol)溶解在DMF(15mL)的反应液中,室温条件下反应5h后,HPLC显示有部分原料剩余,新峰生成后,加入适量的NH4Cl调节pH为中性后,柱层析纯化得目标产物H3约210mg,产率:39.7%. 1.1 Dissolve H1 (404 mg, 1.0 mmol), H2 (285 mg, 1.0 mmol) and K 2 CO3 (207 mg, 1.5 mmol) in the reaction solution of DMF (15 mL), after 5 h of reaction at room temperature, HPLC showed that there were some raw materials The rest, after the new peak was generated, after adding an appropriate amount of NH4Cl to adjust the pH to neutral, the target product H3 was purified by column chromatography to obtain about 210 mg of the target product, the yield: 39.7%.
LCMS:[M+1] +=530.6. LCMS: [M+1] + = 530.6.
Figure PCTCN2021123020-appb-000197
Figure PCTCN2021123020-appb-000197
1.2将H3(53mg,0.1mmol),DMAP(61mg,0.5mmol)与三光气(14.8mg,0.05mmol)称量好后加入圆底烧瓶。加入DCM(2mL)后,搅拌大约15分钟,TLC显示,H3已经反应完全,再加入H4(104mg,0.1mmol),继续搅拌10分钟左右,Pre-HPLC纯化后得产物H5约80mg左右,产率:50.0%。LCMS:[M+1] +=1599.6. 1.2 H3 (53 mg, 0.1 mmol), DMAP (61 mg, 0.5 mmol) and triphosgene (14.8 mg, 0.05 mmol) were weighed and added to a round bottom flask. After adding DCM (2mL), stir for about 15 minutes, TLC shows that H3 has reacted completely, then add H4 (104mg, 0.1mmol), continue stirring for about 10 minutes, after Pre-HPLC purification, product H5 is about 80mg, and the yield is about 80mg. : 50.0%. LCMS: [M+1] + = 1599.6.
Figure PCTCN2021123020-appb-000198
Figure PCTCN2021123020-appb-000198
1.3将H5(48mg,0.03mmol)溶解在DMSO/H 2O(0.8mL/1.2mL)中,室温条件下加入H6(15mg,0.05),反应液在室温条件下搅拌1h后,HPLC显示有新峰生成,LCMS显示有目标产物生成,Pre-HPLC纯化后得产物大约40mg,产率:71.4%.LCMS:[1/2M+1] +=937.7. 1.3 Dissolve H5 (48 mg, 0.03 mmol) in DMSO/H 2 O (0.8 mL/1.2 mL), add H6 (15 mg, 0.05) at room temperature, and stir the reaction solution for 1 h at room temperature. The peak was generated, and LCMS showed that the target product was generated. After Pre-HPLC purification, the product was about 40 mg, yield: 71.4%. LCMS: [1/2M+1] + =937.7.
Figure PCTCN2021123020-appb-000199
Figure PCTCN2021123020-appb-000199
1.4将H7(30mg,0.016mmol)溶解在DCM/DCA/苯甲醚(1mL/0.1mL/0.1mL)的溶液中,在试问条件下反应0.5h,HPLC显示有新峰生成,原料已经全部消失,加入适量乙醚沉降后,Pre-HPLC纯化,冻干得目标产物约18mg,产率:72.1%.LCMS:[M+1] +=1617. 1.4 Dissolve H7 (30mg, 0.016mmol) in a solution of DCM/DCA/anisole (1mL/0.1mL/0.1mL), react under the conditions for 0.5h, HPLC shows that a new peak is generated, and the raw materials have all disappeared , after adding an appropriate amount of ether for sedimentation, Pre-HPLC purification, lyophilization to obtain about 18 mg of the target product, yield: 72.1%. LCMS: [M+1] + =1617.
1.5 H与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-13,其SEC-HPLC结果如图8所示。The coupling, purification, and detection steps of 1.5 H and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-13 was obtained, and its SEC-HPLC results were shown in Figure 8.
ADC-13的DAR值为7.5。即,n为7.5。The DAR value of ADC-13 is 7.5. That is, n is 7.5.
实施例19:ADC-14的合成Example 19: Synthesis of ADC-14
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000200
Figure PCTCN2021123020-appb-000200
1.1将K2(26mg,0.05mmol)与EDCl(19mg,1.0mmol),HOBt(13mg,1.0mmol)在DMF(3mL)的反应液中搅拌3h后,加入K1(25mg,0.05mmol)反应1h后,K1完全消失后,有新峰生成,纯化后得目标产物约5mg,产率10.0%。LCMS:[M+1] +=959.3. 1.1 After stirring K2 (26 mg, 0.05 mmol) with EDCl (19 mg, 1.0 mmol), HOBt (13 mg, 1.0 mmol) in the reaction solution of DMF (3 mL) for 3 h, adding K1 (25 mg, 0.05 mmol) and reacting for 1 h, After K1 disappeared completely, a new peak was formed. After purification, about 5 mg of the target product was obtained, and the yield was 10.0%. LCMS: [M+1] + = 959.3.
1.2 K与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-14,其SEC-HPLC图谱如图9所示。1.2 The coupling, purification, and detection steps of K and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-14 was obtained, and its SEC-HPLC chromatogram was shown in Figure 9.
ADC-14的DAR值为7.8。即,n为7.8。The DAR value of ADC-14 is 7.8. That is, n is 7.8.
实施例20:ADC-15的合成Example 20: Synthesis of ADC-15
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000201
Figure PCTCN2021123020-appb-000201
1.1在L1(1g,2.82mmol)的THF(30mL)/甲苯(10mL)溶液中加入四乙酸铅(3.75g,8.47mmol)和吡啶(0.56g,7.05mmol),将反应液升温至80℃反应3小时。将反应液用水稀释(100mL),用乙酸乙酯(50mL x 2)萃取,合并有机相后用饱和食盐水洗,无水硫酸钠干燥后过滤,滤液旋干后过硅胶柱得目标产物L2大约811mg白色固体,产率:78.01%。LCMS:[M+1] +=369.3. 1.1 Lead tetraacetate (3.75g, 8.47mmol) and pyridine (0.56g, 7.05mmol) were added to the THF (30mL)/toluene (10mL) solution of L1 (1g, 2.82mmol), and the reaction solution was heated to 80°C for reaction 3 hours. The reaction solution was diluted with water (100 mL), extracted with ethyl acetate (50 mL x 2), the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate and filtered, the filtrate was spin-dried and passed through a silica gel column to obtain about 811 mg of the target product L2 White solid, yield: 78.01%. LCMS: [M+1] + = 369.3.
Figure PCTCN2021123020-appb-000202
Figure PCTCN2021123020-appb-000202
1.2.在L2(200mg,0.543mmol)的THF(8mL)溶液中于冰水浴条件下加入CMTC(219.56mg,0.543mmol)和氢氧化锂(26mg,1.09mmol),将反应液升至室温条件下反应30分钟,HPLC显示有新峰生成。将反应液于冰水浴条件下用柠檬酸淬灭后直接送Pre-HPLC纯化后得目标产物L3大约124mg左右,产率:32.04%。LCMS:[M+1] +=713.7. 1.2. Add CMTC (219.56mg, 0.543mmol) and lithium hydroxide (26mg, 1.09mmol) to the THF (8mL) solution of L2 (200mg, 0.543mmol) under ice-water bath conditions, and the reaction solution was raised to room temperature After 30 minutes of reaction, HPLC showed the formation of a new peak. The reaction solution was quenched with citric acid under ice-water bath conditions, and then directly sent to Pre-HPLC for purification to obtain about 124 mg of the target product L3, yield: 32.04%. LCMS: [M+1] + = 713.7.
Figure PCTCN2021123020-appb-000203
Figure PCTCN2021123020-appb-000203
1.3.在L3(70mg,0.098mmol)的DMF(2mL)溶液中加入哌啶(41.7mg,0.49mmol)。将反应液在室温条件下反应1小时,HPLC显示有新峰生成。将反应液于冰水浴条件下用柠檬酸淬灭后直接送Pre-HPLC纯化后得目标产物L4大约27mg左右,产率:56.05%。LCMS:[M+1] +=491.4. 1.3. To a solution of L3 (70 mg, 0.098 mmol) in DMF (2 mL) was added piperidine (41.7 mg, 0.49 mmol). The reaction solution was reacted at room temperature for 1 hour, and HPLC showed that a new peak was formed. The reaction solution was quenched with citric acid under ice-water bath conditions, and then directly sent to Pre-HPLC for purification to obtain about 27 mg of the target product L4, yield: 56.05%. LCMS: [M+1] + = 491.4.
Figure PCTCN2021123020-appb-000204
Figure PCTCN2021123020-appb-000204
1.4在MC-GGFG-OH(68.9mg,0.0146mm ol)的DMF(4mL)溶液中加入吡啶(21mg,0.26mmol)和HATU(75.58mg,0.2mmol)。将在室温条件下反应半小时后加入化合物L4(65mg,0.132mmol)并于室温条件下搅拌反应5小时。HPLC显示有新峰生成,将反应液直接送Pre-HPLC纯化后得目标产物L大约31mg左右,产率:24.76%。LCMS:[M+1] +=945.8. 1.4 To a solution of MC-GGFG-OH (68.9 mg, 0.0146 mmol) in DMF (4 mL) was added pyridine (21 mg, 0.26 mmol) and HATU (75.58 mg, 0.2 mmol). Compound L4 (65 mg, 0.132 mmol) was added after the reaction at room temperature for half an hour and the reaction was stirred at room temperature for 5 hours. HPLC showed that a new peak was generated, and the reaction solution was directly sent to Pre-HPLC for purification to obtain about 31 mg of the target product L, yield: 24.76%. LCMS: [M+1] + = 945.8.
1.5 L与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-15,其SEC-HPLC结果如图10所示。The coupling, purification, and detection steps of 1.5 L and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-15 was obtained, and its SEC-HPLC results were shown in Figure 10.
ADC-15的DAR值为7.2。即,n为7.2。The DAR value of ADC-15 is 7.2. That is, n is 7.2.
实施例21:ADC-16的合成Example 21: Synthesis of ADC-16
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000205
Figure PCTCN2021123020-appb-000205
1.1在N-(叔丁氧羰基)乙醇胺M1(50mg,0.31mmol)的DCM(3mL)溶液中加入三乙胺(62.77mg,0.62mmol)和二(对硝基苯)碳酸酯(94.36mg,0.31mmol),在室温条件下反应16小时,HPLC显示有新峰生成,将反应液直接旋干后得粗品大约101mg,产率大约99.8%。将粗品M2直接用于下一步反应,不进行进一步纯化。1.1 To a solution of N-(tert-butoxycarbonyl)ethanolamine M1 (50 mg, 0.31 mmol) in DCM (3 mL) was added triethylamine (62.77 mg, 0.62 mmol) and bis(p-nitrobenzene) carbonate (94.36 mg, 0.31 mmol), reacted at room temperature for 16 hours, HPLC showed that a new peak was formed, and the reaction solution was directly spin-dried to obtain about 101 mg of crude product, and the yield was about 99.8%. Crude M2 was used directly in the next reaction without further purification.
Figure PCTCN2021123020-appb-000206
Figure PCTCN2021123020-appb-000206
1.2.在M2(12.1mg,0.037mmol)的DMF(1mL)溶液中加入三乙胺(7.5mg,0.074mmol)和CMTC(15mg,0.037mmol),在室温条件下反应3小时,HPLC显示有新峰生成。将反应液直接送Pre-HPLC纯化后得目标产物M3大约21mg左右,产率:95.7%。LCMS:[M+1] +=592.4. 1.2. Triethylamine (7.5 mg, 0.074 mmol) and CMTC (15 mg, 0.037 mmol) were added to a solution of M2 (12.1 mg, 0.037 mmol) in DMF (1 mL), and the reaction was carried out at room temperature for 3 hours. Peak generation. The reaction solution was directly sent to Pre-HPLC for purification to obtain about 21 mg of the target product M3, yield: 95.7%. LCMS: [M+1] + = 592.4.
Figure PCTCN2021123020-appb-000207
Figure PCTCN2021123020-appb-000207
1.3.在M3(18mg,0.03mmol)的DCM(2mL)溶液中加入三氟乙酸(0.5mL)。将反应液在室温条件下反应半小时,HPLC显示有新峰生成。向反应液中加入乙醚(1mL)后有固体析出,震 荡后离心沉降。出去溶剂后将固体干燥得目标产物M4大约14.9mg左右,产率:99.64%。1.3. To a solution of M3 (18 mg, 0.03 mmol) in DCM (2 mL) was added trifluoroacetic acid (0.5 mL). The reaction solution was reacted at room temperature for half an hour, and HPLC showed that a new peak was formed. Diethyl ether (1 mL) was added to the reaction solution and a solid was precipitated. After shaking, the solution was centrifuged for sedimentation. After removing the solvent, the solid was dried to obtain about 14.9 mg of the target product M4, yield: 99.64%.
Figure PCTCN2021123020-appb-000208
Figure PCTCN2021123020-appb-000208
1.4在MC-GGFG-OH(24.16mg,0.046mmol)的DMF(2mL)溶液中加入吡啶(4.18mg,0.061mmol)和HATU(17.49mg,0.046mmol)。将在室温条件下反应半小时后加入化合物M4(14.95mg,0.030mmol)并于室温条件下搅拌反应3小试。HPLC显示有新峰生成,将反应液直接送Pre-HPLC纯化后得目标产物M大约21mg左右,产率:68.83%。LCMS:[M+1] +=1003.3. 1.4 To a solution of MC-GGFG-OH (24.16 mg, 0.046 mmol) in DMF (2 mL) was added pyridine (4.18 mg, 0.061 mmol) and HATU (17.49 mg, 0.046 mmol). After half an hour of reaction at room temperature, compound M4 (14.95 mg, 0.030 mmol) was added and the reaction was stirred at room temperature for 3 hours. HPLC showed that a new peak was generated, and the reaction solution was directly sent to Pre-HPLC for purification to obtain about 21 mg of the target product M, yield: 68.83%. LCMS: [M+1] + = 1003.3.
1.5 M与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2-VI-5,最后获得ADC-16,其SEC-HPLC结果如图11所示。The coupling, purification, and detection steps of 1.5 M and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-16 was obtained, and its SEC-HPLC results were shown in Figure 11.
ADC-16的DAR值为7.2。即,n为7.2。The DAR value of ADC-16 is 7.2. That is, n is 7.2.
实施例22:ADC-17的合成Example 22: Synthesis of ADC-17
合成方法:resolve resolution:
Figure PCTCN2021123020-appb-000209
Figure PCTCN2021123020-appb-000209
1.1将N1(20mg)溶解在DCM(2mL)中,加入DIEA(38m g)再加入三光气(21mg),反应液搅拌1h后,加入CMTC(40mg),再加入DMAP(36mg)反应5h后,HPLC检测又新峰生成,pre-HPLC纯化后,冻干,得目标产物N2 26mg.LCMS:[M+1] +=619.3. 1.1 Dissolve N1 (20mg) in DCM (2mL), add DIEA (38mg) and then add triphosgene (21mg), after stirring the reaction solution for 1h, add CMTC (40mg), then add DMAP (36mg) and react for 5h, HPLC detected a new peak, and after pre-HPLC purification, lyophilized to obtain the target product N2 26mg. LCMS: [M+1] + =619.3.
Figure PCTCN2021123020-appb-000210
Figure PCTCN2021123020-appb-000210
1.2将N2(26mg)加入T FA/DCM(0.5mL/0.5mL)的反应液中,反应半小时后,旋干,得目标产物N3 16mg,可直接用于下一步反应。LCMS:[M+1] +=519.3 1.2 N2 (26mg) was added to the reaction solution of TFA/DCM (0.5mL/0.5mL), and after half an hour of reaction, spin-dried to obtain 16mg of the target product N3, which can be directly used in the next reaction. LCMS: [M+1] + = 519.3
Figure PCTCN2021123020-appb-000211
Figure PCTCN2021123020-appb-000211
1.3将N3(10mg),N4(8mg)与HATU(9mg)溶解在DMF(1mL)的反应液中,加入DIEA(0.008mL),反应液室温条件下反应2h后,HPLC显示有新峰生成,LCMS显示为目标产物,Pre-HPLC纯化后得目标产物N 8mg.LCMS:[M+1] +=1117.2 1.3 N3 (10mg), N4 (8mg) and HATU (9mg) were dissolved in the reaction solution of DMF (1mL), DIEA (0.008mL) was added, and the reaction solution was reacted at room temperature for 2h, and HPLC showed that a new peak was generated, LCMS showed that it was the target product, and the target product N was obtained after Pre-HPLC purification. 8mg. LCMS: [M+1] + =1117.2
1.4 N与实施例1制备的hRS7抗体的偶联、纯化、检测步骤同实施例10中的步骤VI-2- VI-5,最后获得ADC-17,其SEC-HPLC结果如图12所示。1.4 The coupling, purification, and detection steps of N and the hRS7 antibody prepared in Example 1 were the same as steps VI-2-VI-5 in Example 10, and finally ADC-17 was obtained, and its SEC-HPLC results were shown in Figure 12.
ADC-17的DAR值为6.6。即,n为6.6。The DAR value of ADC-17 is 6.6. That is, n is 6.6.
实施例23 ADC-5的稳定性Example 23 Stability of ADC-5
1.实验目的1. Experimental purpose
考察ADC-5在冻存冻融过程中的稳定性。The stability of ADC-5 during freezing and thawing was investigated.
2.实验设计2. Experimental Design
ADC-5样品在特定的缓冲液中保存,其经过-20℃的冻存与冻融后考察其载药变化和抗体的聚集变化。ADC-5 samples were stored in a specific buffer, and their drug loading changes and antibody aggregation changes were investigated after freezing and thawing at -20°C.
3.实验方法3. Experimental method
3.1 ADC-5样品-20℃冻存稳定性3.1 Storage stability of ADC-5 samples at -20℃
ADC-5(-20℃冻存)湿冰上融解,分装,并直接进样检测RPC4和SEC;分装好的样品-20℃冻存,每间隔至少1天取出一份,湿冰上融解,直接进样分析,RPC4和SEC。同时用ADC-137(常州辰鸿生物科技有限公司,CAS#:1279680-68-0)作为对照。ADC-5 (frozen at -20°C) thawed on wet ice, aliquoted, and directly injected to detect RPC4 and SEC; the aliquoted samples were frozen at -20°C, taken out at least 1 day apart, and placed on wet ice Thaw, Direct Injection Analysis, RPC4 and SEC. At the same time, ADC-137 (Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0) was used as a control.
4.结果处理与分析4. Result processing and analysis
4.1液相方法4.1 Liquid phase method
4.1.1 RP-C4(UPLC)4.1.1 RP-C4 (UPLC)
色谱柱:YMC Triart Bio C4 3μm 3.0*150mm(编号QCC-RP-025)Chromatographic column: YMC Triart Bio C4 3μm 3.0*150mm (No. QCC-RP-025)
流速:0.6mL/min  柱温:70℃  样品盘温度:4℃  进样量:5μLFlow rate: 0.6mL/min Column temperature: 70℃ Sample tray temperature: 4℃ Injection volume: 5μL
紫外检测器波长:280nm、363nm、383nmUV detector wavelength: 280nm, 363nm, 383nm
流动相A:0.1%TFA in water流动相B:0.1%TFA in CANMobile phase A: 0.1% TFA in water Mobile phase B: 0.1% TFA in CAN
表1 RP-C4梯度Table 1 RP-C4 gradient
Time/minTime/min %A%A %B%B
22 8080 2020
44 4040 6060
99 4040 6060
1010 55 9595
1212 55 9595
12.512.5 8080 2020
1515 8080 2020
4.1.2 SEC(HPLC)4.1.2 SEC (HPLC)
色谱柱:TSKgel G3000SWxl 5μm 7.8*300mmChromatographic column: TSKgel G3000SWxl 5μm 7.8*300mm
流速:0.4mL/min  柱温:30℃Flow rate: 0.4mL/min Column temperature: 30℃
样品盘温度:4℃  进样量:30μLSample tray temperature: 4℃ Injection volume: 30μL
紫外检测器波长:280nm、363nm、383nmUV detector wavelength: 280nm, 363nm, 383nm
流动相:0.1M PB&0.2M NaCl pH7.0&5%IPA,等度洗脱40minMobile phase: 0.1M PB&0.2M NaCl pH7.0&5%IPA, isocratic elution 40min
4.2对照ADC-137/ADC-5样品-20℃冻存稳定性4.2 The stability of the control ADC-137/ADC-5 samples at -20 ℃ cryopreservation
4.2.1样品RP分析结果4.2.1 Sample RP analysis results
表2样品RP分析Table 2 Sample RP Analysis
Figure PCTCN2021123020-appb-000212
Figure PCTCN2021123020-appb-000212
Figure PCTCN2021123020-appb-000213
Figure PCTCN2021123020-appb-000213
上表中,Area%/363nm表示指363nm检测条件下,各时间出峰所占总面积百分比。RT3.2指出峰时间为3.2分钟,这个位置是ADC-137中小分子SN-38的出峰时间。后四列的出峰位置均为ADC-137的出峰位置,主峰位置是RT4.67。In the above table, Area%/363nm refers to the percentage of the total area occupied by peaks at each time under the detection condition of 363nm. RT3.2 indicated that the peak time was 3.2 minutes, which was the peak time of small molecule SN-38 in ADC-137. The peak positions of the last four columns are the peak positions of ADC-137, and the main peak position is RT4.67.
Figure PCTCN2021123020-appb-000214
Figure PCTCN2021123020-appb-000214
上表中,RT4.12表示ADC-5偶联的小分子CM(即吉咪替康)的出峰时间,后六列的出峰时间均为ADC-5的出峰时间。RT4.69是ADC-5的主峰位置。In the above table, RT4.12 represents the peak time of ADC-5-conjugated small molecule CM (ie, gemitecan), and the peak time of the last six columns is the peak time of ADC-5. RT4.69 is the main peak position of ADC-5.
在-20℃冻存21天后,ADC-137和ADC-5主峰面积百分比下降不明显,从游离的SN38和CM的变化来看,ADC-137和ADC-5均相对较为稳定。After being frozen at -20℃ for 21 days, the percentage of the main peak area of ADC-137 and ADC-5 did not decrease significantly. From the changes of free SN38 and CM, ADC-137 and ADC-5 were relatively stable.
实施例24 ADC-5在血清和缓冲溶液中小分子的释放Example 24 Small molecule release of ADC-5 in serum and buffer solutions
1.1实验设计:1.1 Experimental Design:
ADC-5在猴血清(pH7.4)和PBS缓冲溶液(pH7.4)经过37℃孵不同的时间后,检测溶液中游离的小分子的浓度。样品经过有机溶剂(甲醇)沉淀蛋白萃取后,含有游离小分子的上清液直接进样分析,并以ADC-5总小分子含量为基础得到释放百分比。After ADC-5 was incubated in monkey serum (pH7.4) and PBS buffer solution (pH7.4) for different times at 37°C, the concentration of free small molecules in the solution was detected. After the sample was extracted with organic solvent (methanol) precipitating protein, the supernatant containing free small molecules was directly injected for analysis, and the release percentage was obtained based on the total small molecule content of ADC-5.
1.2实验方法:1.2 Experimental method:
样品放置:50μL ADC(等体积样品缓冲液做空白)分别加入450μL缓冲溶液或血清(猴混合血清),混匀后37℃放置孵育,间隔取样(0/1/2/4/8/24/48h-即后续每隔24小时取样,视情况调整时间间隔);Sample placement: 50 μL of ADC (with equal volume of sample buffer as blank) were added to 450 μL of buffer solution or serum (monkey mixed serum), mixed and incubated at 37°C, sampling at intervals (0/1/2/4/8/24/ 48h - that is, follow-up sampling every 24 hours, adjust the time interval according to the situation);
样品处理:取30μL样品/空白,加入150μL沉淀剂(含1.2μg/mL SN38内标),涡旋充分混匀,14800rpm 5min 4℃,取上清液进样分析。Sample processing: Take 30 μL of sample/blank, add 150 μL of precipitant (containing 1.2 μg/mL SN38 internal standard), vortex to mix well, 14800 rpm for 5 min at 4 °C, take the supernatant for injection analysis.
1.3样品信息1.3 Sample Information
样品名称sample name 蛋白浓度protein concentration DARDAR
ADC-5ADC-5 2.1622.162 7.87.8
1.4液相方法1.4 Liquid phase method
Figure PCTCN2021123020-appb-000215
Figure PCTCN2021123020-appb-000215
1.5样品进样设计1.5 Sample injection design
样品进样设计Sample injection design
Blank-MeOHBlank-MeOH
IS-SN38IS-SN38
CM-0.2μg/mLCM-0.2μg/mL
CM-2μg/mLCM-2μg/mL
CM-20μg/mLCM-20μg/mL
CM-100μg/mLCM-100μg/mL
ADC-5-缓冲液-0~n hADC-5-buffer-0~n h
ADC-5-Serum-0~n hADC-5-Serum-0~nh
(0/1/2/4/8/24/48h~,即后续每隔24小时取样,视情况调整时间间隔)(0/1/2/4/8/24/48h~, that is, subsequent sampling every 24 hours, adjust the time interval according to the situation)
1.6检测结果1.6 Test results
16.1实验结果(连续监测349h结果)16.1 Experimental results (349h continuous monitoring results)
实验结果如图13、图14和下表3所示。The experimental results are shown in Figure 13, Figure 14 and Table 3 below.
表3:ADC-5小分子的释放(连续监测349h数据统计结果)Table 3: Release of ADC-5 small molecules (statistical results of continuous monitoring 349h data)
Figure PCTCN2021123020-appb-000216
Figure PCTCN2021123020-appb-000216
ADC-5样品,其小分子CM在PBS和猴血清中的半衰期分别为约55小时和75小时。经过349h后,ADC-5在PBS中解离最高达到94.22%,而在血清中,其最高解离比例为79.23%。For ADC-5 samples, the half-lives of small molecule CM in PBS and monkey serum were approximately 55 hours and 75 hours, respectively. After 349h, ADC-5 dissociated up to 94.22% in PBS, while in serum, its highest dissociation ratio was 79.23%.
实施例25:ADC-5在不同pH条件下的稳定性Example 25: Stability of ADC-5 under different pH conditions
1.实验目的1. Experimental purpose
考察ADC-5在不同pH条件中的稳定性,与CM、CL2A-CM对比。The stability of ADC-5 in different pH conditions was investigated and compared with CM and CL2A-CM.
2.实验设计2. Experimental Design
在不同的pH条件中,ADC-5的降解杂质通过RP-C4分析,可以统计ADC-5主峰、CL2A-CM、CM等峰面积变化,同时可以研究其他降解有关物质。In different pH conditions, the degradation impurities of ADC-5 can be analyzed by RP-C4, and the peak area changes of ADC-5 main peak, CL2A-CM, CM can be counted, and other degradation related substances can be studied at the same time.
其中,CM指的是吉咪替康,CL2A-CM的结构式为Among them, CM refers to gemitecan, and the structural formula of CL2A-CM is
Figure PCTCN2021123020-appb-000217
Figure PCTCN2021123020-appb-000217
3.实验方法3. Experimental method
3.1溶液配制3.1 Solution preparation
先配制50mM PB溶液,分别调节pH至6.0/6.5/7.0/7.5/8.0,然后再配制含10%DMSO的PB溶液(50mM PB pH6.0/6.5/7.0/7.5/8.0分别加入10%DMSO即可)。First prepare 50mM PB solution, adjust the pH to 6.0/6.5/7.0/7.5/8.0 respectively, then prepare PB solution containing 10% DMSO (50mM PB pH6.0/6.5/7.0/7.5/8.0 respectively add 10% DMSO to Can).
3.2样品放置条件3.2 Sample placement conditions
先使用DMSO分别配制1mg/mL的CM溶液和CL2A-CM溶液,然后分别取20μL 1mg/mL的CM溶液和CL2A-CM溶液至50mM PB pH6.0/6.5/7.0/7.5/8.0溶液中,DMSO终浓度为10%;接着放置于25℃进样盘,于0h/3h/6h/9h/12h/15h C18-RP进样2μL分析(0h样品混匀后马上直接进样分析)。First use DMSO to prepare 1mg/mL CM solution and CL2A-CM solution respectively, then take 20μL of 1mg/mL CM solution and CL2A-CM solution to 50mM PB pH 6.0/6.5/7.0/7.5/8.0 solution, DMSO The final concentration was 10%; then it was placed in a 25°C injection tray, and 2 μL of C18-RP was injected at 0h/3h/6h/9h/12h/15h for analysis (the sample was directly injected for analysis after mixing at 0h).
取20μL ADC-5溶液至180μL 50mM PB pH6.0/6.5/7.0/7.5/8.0&10%DMSO溶液中,DMSO终浓度为10%;接着放置于25℃进样盘,于0h/3h/6h/9h/12h/15h C4-RP进样25μL分析。(0h样品混匀后马上直接进样分析)。Take 20μL of ADC-5 solution to 180μL of 50mM PB pH6.0/6.5/7.0/7.5/8.0&10% DMSO solution, the final concentration of DMSO is 10%; 9h/12h/15h C4-RP was injected with 25μL for analysis. (The sample was directly injected and analyzed immediately after the 0h sample was mixed).
3.3液相方法3.3 Liquid phase method
C18-RP:C18-RP:
色谱柱:ACQUITY UPLC BEH C18,1.7μm,2.1*100mmChromatographic column: ACQUITY UPLC BEH C18, 1.7μm, 2.1*100mm
流动相:A:0.1%TFA in water;B:0.1%TFA in ACNMobile phase: A: 0.1% TFA in water; B: 0.1% TFA in ACN
DAD:245nm,363nm,383nmDAD: 245nm, 363nm, 383nm
流速:0.4mL/minFlow rate: 0.4mL/min
运行时间:14minRunning time: 14min
进样量:2μL       柱温35℃Injection volume: 2μL Column temperature 35℃
Time/minTime/min A%A% B%B%
00 9595 55
8.48.4 55 9595
1010 00 100100
10.110.1 9595 55
11.511.5 9595 55
C4-RP:C4-RP:
色谱柱:YMC Trait Bio C4,3μm,4.6*150mmChromatographic column: YMC Trait Bio C4, 3μm, 4.6*150mm
流动相:A:0.1%TFA in water;B:0.1%TFA in ACNMobile phase: A: 0.1% TFA in water; B: 0.1% TFA in ACN
DAD:245nm,363nm,383nmDAD: 245nm, 363nm, 383nm
流速:0.6mL/minFlow rate: 0.6mL/min
运行时间:15minRunning time: 15min
进样量:25μL       柱温70℃Injection volume: 25μL Column temperature 70℃
Time/minTime/min A%A% B%B%
00 8080 2020
22 8080 2020
44 4040 6060
99 4040 6060
1010 55 9595
1212 55 9595
12.512.5 8080 2020
1515 8080 22
4.结果处理与分析4. Result processing and analysis
4.1 CL2A-CM的稳定性分析4.1 Stability analysis of CL2A-CM
混合溶液的pH值pH of the mixed solution
表4空白溶液(50mM PB)与10%DMSO混合后pHTable 4 pH of blank solution (50mM PB) mixed with 10% DMSO
PB缓冲液pH值:PB buffer pH: 6.06.0 6.56.5 7.07.0 7.57.5 8.08.0
混合后的pH值:pH after mixing: 6.386.38 6.896.89 7.347.34 7.847.84 8.328.32
上表4以及图15、图16的结果表明:在不同的pH中,CL2A-CM随时间不断降解,且在pH6-8之间pH越高降解越快,相应的CM也随pH增高而加快。The results in Table 4 and Figure 15 and Figure 16 show that: at different pH, CL2A-CM degrades continuously with time, and between pH 6-8, the higher the pH, the faster the degradation, and the corresponding CM also accelerates with the increase of pH. .
4.2 ADC-5样品的稳定性统计4.2 Stability statistics of ADC-5 samples
表5 ADC-5样品统计结果Table 5 Statistical results of ADC-5 samples
Figure PCTCN2021123020-appb-000218
Figure PCTCN2021123020-appb-000218
Figure PCTCN2021123020-appb-000219
Figure PCTCN2021123020-appb-000219
由上表5和图17、图18可知,在pH6.0 PB&10%DMSO中,ADC-5存在溶解度明显降低现象,其面积百分比降低明显。随着时间和pH的增加,CM的绝对量(峰面积)依次增加,其中pH8.0降解最快。It can be seen from the above Table 5 and Figure 17 and Figure 18 that in pH 6.0 PB&10% DMSO, the solubility of ADC-5 is significantly reduced, and its area percentage is significantly reduced. With the increase of time and pH, the absolute amount (peak area) of CM increased sequentially, and pH8.0 degraded the fastest.
5.结果与结论5. Results and Conclusions
在25℃不同pH条件中,CM相对较为稳定,没有产生明显的杂质;CL2A-CM随着时间的增加逐渐释放CM,且pH越高CM释放越快;ADC-5中没有发现较多的CL2A-CM的存在,而是以CM的形式释放,且pH越低释放越快。Under different pH conditions at 25 °C, CM was relatively stable and did not produce obvious impurities; CL2A-CM gradually released CM with time, and the higher the pH, the faster the CM was released; ADC-5 did not find more CL2A -In the presence of CM, it is released in the form of CM, and the lower the pH, the faster the release.
实施例26:ADC细胞杀伤试验Example 26: ADC cell killing assay
将RPMI-1640 Gibco,22400089)培养基与FBS(四季青,13011-8611)按9:1比例混合,配置成完全培养基。培养人原位胰腺腺癌BXPC-3(
Figure PCTCN2021123020-appb-000220
CRL-168 TM)细胞,当细胞覆盖整个培养皿底面积的80%以上时,对细胞进行传代并计数,将细胞浓度调整到0.8×10 5个/mL,取100μL加入到96孔板中继续培养。在37℃,CO 2细胞培养箱中继续培养24h后取出。弃去原培养液后,改用含有1%FBS的培养液继续培养30min。
RPMI-1640 Gibco, 22400089) medium and FBS (Sijiqing, 13011-8611) were mixed in a ratio of 9:1 to prepare a complete medium. Cultured human orthotopic pancreatic adenocarcinoma BXPC-3 (
Figure PCTCN2021123020-appb-000220
CRL- 168TM ) cells, when the cells covered more than 80% of the bottom area of the entire culture dish, the cells were passaged and counted, the cell concentration was adjusted to 0.8×10 5 cells/mL, and 100 μL was added to the 96-well plate to continue nourish. Incubate for 24 h at 37 °C in a CO 2 cell incubator. After discarding the original culture medium, the culture medium containing 1% FBS was used to continue culturing for 30 min.
配置不同浓度的受试品:将实施例5-8制备的ADC1-4,以及实施例1制备的hSR7单抗作为阴性对照,用1%FBS的培养液按10 000nmol/L的起始梯度进行10倍稀释,得到1 000nmol/L、100nmol/L、10nmol/L、1nmol/L、0.1nmol/L、0.01nmol/L、0.001nmol/L和0.0001nmol/L共9个浓度点,每孔三个复孔,弃去96孔板中的原培养液,将上述配制好的不同浓度的受试品加入96孔板中,每孔100μl,放入到CO 2培养箱中继续培养72h。向每孔中加入10μL CCK8试剂(碧云天生物技术Beyotime Biotechnology,C0040),放入到CO 2细胞培养箱中进行孵育2h。然后经SpectraMax M5多功能酶标仪,在450nm波长进行读数,通过检测线粒体内的脱氢酶活性而指试对细胞增殖的抑制作用。 Prepare test products with different concentrations: ADC1-4 prepared in Example 5-8 and hSR7 mAb prepared in Example 1 were used as negative controls, and the culture medium of 1% FBS was used at an initial gradient of 10 000 nmol/L. 10-fold dilution to obtain a total of 9 concentration points of 1 000nmol/L, 100nmol/L, 10nmol/L, 1nmol/L, 0.1nmol/L, 0.01nmol/L, 0.001nmol/L and 0.0001nmol/L, three in each well. Repeat the wells, discard the original culture solution in the 96-well plate, add the above-prepared test substances of different concentrations into the 96-well plate, 100 μl per well, and put them into a CO 2 incubator for 72 hours. Add 10 μL of CCK8 reagent (Beyotime Biotechnology, C0040) to each well, and put it into a CO 2 cell incubator for incubation for 2 h. Then, the SpectraMax M5 multi-function microplate reader was used for reading at a wavelength of 450 nm, and the inhibitory effect on cell proliferation was measured by detecting the dehydrogenase activity in the mitochondria.
本发明的ADC体外抑制实验结果如图19所示,其中,纵坐标具体而言,其为细胞给药后,细胞量减少后,吸光值也会降低,然后用检测的值和空白对照的比值作图。The results of the ADC in vitro inhibition experiment of the present invention are shown in Figure 19, wherein the ordinate is specifically, after the administration of cells, after the cell volume is reduced, the absorbance value will also decrease, and then the ratio of the detected value to the blank control is used. drawing.
相较于空白对照组,4种ADC受试品均显示对BXPC-3活性的抑制作用,并随着受试品的浓度升高,细胞活性明显降低,即呈剂量依赖性。另外,还分别测试4种ADC对HCT-116、Capan-1、NCI-N87、HCC1806、H1975和Calu-3细胞的抑制活性,发现本发明制备的ADC对这几种细胞活性也具有抑制作用(表6)。Compared with the blank control group, the four ADC test products all showed inhibitory effects on the activity of BXPC-3, and with the increase of the concentration of the test products, the cell activity decreased significantly, that is, in a dose-dependent manner. In addition, the inhibitory activities of 4 ADCs on HCT-116, Capan-1, NCI-N87, HCC1806, H1975 and Calu-3 cells were also tested, and it was found that the ADC prepared by the present invention also had inhibitory effects on these cell activities ( Table 6).
表6Table 6
受试品test article IC 50值(nmol/L) IC 50 value (nmol/L) R 2 R 2
ADC-1ADC-1 1.0991.099 0.9720.972
ADC-2ADC-2 0.7480.748 0.9570.957
ADC-3ADC-3 13.1613.16 0.9580.958
ADC-4ADC-4 4.8114.811 0.9800.980
实施例27 ADC-137和ADC-5样品的细胞杀伤试验Example 27 Cell killing assay of ADC-137 and ADC-5 samples
本实验的研究目的是评估4种药物对5个细胞系的细胞增殖影响。通过检测在不同药物浓度处理后的细胞活力,计算50%抑制浓度。具体的是:在5株细胞中测定两个ADC样品ADC-137(作为对照,常州辰鸿生物科技有限公司,CAS#:1279680-68-0)、ADC-5,以及两个其对应的小分子样品SN-38和吉咪替康的细胞杀伤活性,并每株细胞设定一个质控参照对照、一个空白对照和一个溶媒对照。每个化合物9个浓度,3个复孔,72小时后用检测细胞活力,并计算IC50。The purpose of this experiment was to evaluate the effects of 4 drugs on cell proliferation in 5 cell lines. The 50% inhibitory concentration was calculated by detecting cell viability after treatment with different drug concentrations. Specifically: two ADC samples ADC-137 (as a control, Changzhou Chenhong Biotechnology Co., Ltd., CAS#: 1279680-68-0), ADC-5, and two corresponding small The cell killing activity of molecular samples SN-38 and gemnotecan, and each cell line was set with a quality control reference control, a blank control and a vehicle control. Each compound has 9 concentrations and 3 replicate wells. After 72 hours, cell viability was detected and IC50 was calculated.
(1)实验材料(1) Experimental materials
细胞系cell line
序号serial number 细胞系cell line 组织来源tissue source 质控参照对照quality control reference 处理时间processing time
11 BxPC-3BxPC-3 Pancreas Pancreas 顺铂Cisplatin 72h72h
22 Calu-3Calu-3 LungLung 顺铂Cisplatin 72h72h
33 COLO 205 COLO 205 Large intestine/ColorectumLarge testine/Colorectum 顺铂Cisplatin 72h72h
44 NCI-N87NCI-N87 Stomach Stomach 顺铂Cisplatin 72h72h
55 Calu-6Calu-6 LungLung 顺铂Cisplatin 72h72h
所有细胞将置于37℃、5%CO 2和95%湿度条件下培养。用于细胞培养的培养基品牌是Hyclone/Gibco,并加入10-15%的胎牛血清。 All cells will be cultured at 37°C, 5% CO2 and 95% humidity. The brand of media used for cell culture is Hyclone/Gibco with 10-15% fetal bovine serum.
试剂和耗材Reagents and Consumables
1.细胞培养常规培养液和耗材1. Cell culture medium and consumables
2.胎牛血清FBS(ExCell Bio.,Cat#FND500)2. Fetal bovine serum FBS (ExCell Bio., Cat#FND500)
3.CellTiter-Glo Luminescent Cell Viability Assay(Promega,Cat#G7573)3. CellTiter-Glo Luminescent Cell Viability Assay (Promega, Cat#G7573)
4. 96孔黑壁透明平底细胞培养板(Corning,Cat#3340)4. 96-well black-walled clear flat-bottom cell culture plate (Corning, Cat#3340)
待测药Drug to be tested
编号Numbering 名称name 溶剂solvent 体积(μl)Volume (μl) 浓度 concentration
11 ADC-137ADC-137 PBSPBS 0.9mL*20.9mL*2 50μg/mL50μg/mL
22 ADC-5ADC-5 PBSPBS 0.9mL*20.9mL*2 45μg/mL45μg/mL
33 SN-38SN-38 DMSODMSO 5.2mg5.2mg   
44 吉咪替康gemitecan DMSODMSO 6.5mg6.5mg   
质控参照对照药Quality control reference drug
名称name 分子量molecular weight 包装Package 供货商supplier 浓度concentration 保存save
顺铂Cisplatin 300.05300.05 20mg20mg 齐鲁制药Qilu Pharmaceutical 3.33mM3.33mM RTRT
仪器instrument
EnVision多标记微孔板检测仪,PerkinElmer,2104-0010A;EnVision Multi-label Microplate Reader, PerkinElmer, 2104-0010A;
细胞计数仪,Inno-Alliance Biotech,Countstar;Cell counter, Inno-Alliance Biotech, Countstar;
CO2培养箱,Thermo Scientific,Model 3100 Series;CO2 incubator, Thermo Scientific, Model 3100 Series;
生物安全柜,Thermo Scientific,Model 1300 Series A2;Biological Safety Cabinet, Thermo Scientific, Model 1300 Series A2;
倒置显微镜,Olympus,CKX41SF;Inverted microscope, Olympus, CKX41SF;
冰箱,SIEMENS,KK25E76TI。Refrigerator, SIEMENS, KK25E76TI.
(2)实验方法(2) Experimental method
1.收获处于对数生长期的细胞并用培养液稀释成细胞悬液,转移至96孔细胞板中,置于37℃、5%CO 2和95%湿度条件下培养过夜,第二天至细胞密度至80%时开始进行药物处理。 1. Harvest cells in logarithmic growth phase and dilute them with culture medium to form a cell suspension, transfer to a 96-well cell plate, and culture overnight at 37°C, 5% CO 2 and 95% humidity. Drug treatment begins when the density reaches 80%.
2.用相应溶剂溶解被测化合物形成储存液并进行梯度稀释,得到10倍工作浓度溶液;同样制备顺铂阳性药的10倍溶液。2. Dissolve the tested compound with the corresponding solvent to form a stock solution and perform gradient dilution to obtain a 10-fold working concentration solution; also prepare a 10-fold solution of the cisplatin positive drug.
3.在已接种细胞的96孔板中每孔加入10μL药物溶液,每个细胞浓度设置三个复孔(参考附录2)。被测化合物最高浓度为10μM,9个浓度,3.16倍稀释。3. Add 10 μL of drug solution to each well of the 96-well plate that has been seeded with cells, and set up three replicate wells for each cell concentration (refer to Appendix 2). The highest concentration of the tested compound was 10 μM, 9 concentrations, 3.16 times dilution.
4.将已加药的96孔板中的细胞置于37℃、5%CO2、95%湿度条件下继续培养72小时。4. The cells in the medicated 96-well plate were placed under the conditions of 37° C., 5% CO 2 , and 95% humidity for further culturing for 72 hours.
5.72小时后,将CellTiter-Glo试剂和药物处理细胞培养板放置于室温平衡30分钟。5. After 72 hours, equilibrate the CellTiter-Glo reagent and drug-treated cell culture plates for 30 minutes at room temperature.
6.每孔加入50μL的CellTiter-Glo试剂。6. Add 50 μL of CellTiter-Glo reagent to each well.
7.在定轨摇床上振动2分钟使细胞充分裂解。7. Shake for 2 minutes on an orbital shaker to fully lyse the cells.
8.将细胞培养放置于室温平衡10分钟。8. Allow the cell culture to equilibrate at room temperature for 10 minutes.
9.用EnVision读取化学发光值。9. Read the chemiluminescence value with EnVision.
(3)数据处理(3) Data processing
使用GraphPad Prism 5.0软件分析数据,利用非线性S曲线回归来拟合数据得出剂量-效应曲线,并由此计算IC 50值。 Data were analyzed using GraphPad Prism 5.0 software, and a dose-response curve was fitted to the data using nonlinear S-curve regression, from which IC50 values were calculated.
细胞存活率(%)=(Lum 待测药-Lum 培养液对照)/(Lum 细胞对照-Lum 培养液对照)×100%. Cell viability (%)=(Lum test drug- Lum culture solution control )/(Lum cell control -Lum culture solution control )×100%.
Lum 细胞对照-Lum 培养液对照设为100%,Lum Medium control值设为0%. Lum cell control - Lum culture medium control is set to 100%, and Lum Medium control value is set to 0%.
扩增倍数=(第五天Lum None treated-Lum Medium control)/(第二天Lum None treated-Lum Medium control) Amplification fold = (the fifth day Lum None treated -Lum Medium control )/(the second day Lum None treated -Lum Medium control )
实验结果Experimental results
表7-1.化合物在5株细胞系的IC50值Table 7-1. IC50 values of compounds in 5 cell lines
Figure PCTCN2021123020-appb-000221
Figure PCTCN2021123020-appb-000221
表7-2.化合物在5株细胞系的最大浓度抑制率)Table 7-2. The maximum concentration inhibition rate of compounds in 5 cell lines)
Figure PCTCN2021123020-appb-000222
Figure PCTCN2021123020-appb-000222
实验结果如上表7-1、7-2和图20-24所示。The experimental results are shown in Tables 7-1, 7-2 and Figures 20-24 above.
实施例28 ADC-137和ADC-5样品的动物学抑瘤实验结果Example 28 Results of the animal tumor inhibition experiment of ADC-137 and ADC-5 samples
(1)实验方法(1) Experimental method
细胞培养cell culture
将人胰腺腺癌细胞BxPC-3(ATCC CRL-1687)、人结肠癌细胞Colo205(ATCC CCL-222)、人肺腺癌细胞Calu-3(ATCC HTB-55)和人胰腺癌细胞Capan-1(ATCC HTB-79)体外单层培养,培养条件为1640培养基中加10%热灭活胎牛血清并加琼脂,于37℃、含5%CO 2空气的培养箱中培养。一周两次用0.25%胰酶进行消化处理传代。当细胞呈指数生长期时,收取细胞,计数,接种。 Human pancreatic adenocarcinoma cells BxPC-3 (ATCC CRL-1687), human colon cancer cells Colo205 (ATCC CCL-222), human lung adenocarcinoma cells Calu-3 (ATCC HTB-55) and human pancreatic adenocarcinoma cells Capan-1 (ATCC HTB-79) in vitro monolayer culture, the culture conditions are 1640 medium with 10% heat-inactivated fetal bovine serum and agar, and cultured at 37°C in an incubator containing 5% CO 2 air. Digestion and passage were performed twice a week with 0.25% trypsin. When cells are in exponential growth phase, cells are harvested, counted, and seeded.
肿瘤细胞接种及瘤块传代Tumor cell inoculation and tumor mass passage
将5.0×10 6肿瘤细胞悬浮于0.1ml PBS与Matrigel混合物(1:1),接种于5只裸鼠右侧肩胛处(P1代)。待肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到新的一批裸鼠(P2代)。 5.0×10 6 tumor cells were suspended in 0.1 ml of a mixture of PBS and Matrigel (1:1), and inoculated into the right scapula of 5 nude mice (passage P1). When the tumor grows to 500-800mm 3 , the tumor-bearing mice are sacrificed with CO 2 anesthesia, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into 20-30 mm 3 small tumor pieces, Inoculated into a new batch of nude mice (P2 generation).
瘤块接种种及分组给药Tumor mass inoculation and group administration
本试验使用P6代肿瘤组织进行受试品的抗肿瘤活性评价。待P5代肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到正式实验用鼠的右侧肩胛处,一共接种70只鼠。瘤块接种13天后肿瘤平均体积达到约135mm 3时,剔除瘤体积过小或过大的小鼠,将剩余的50只小鼠根据瘤体积随机分组并开始给药。 In this experiment, the P6 generation tumor tissue was used to evaluate the antitumor activity of the test article. When the P5 generation tumor grew to 500-800 mm, the tumor - bearing mice were sacrificed with CO anesthesia, the tumor mass was removed, the surrounding necrotic tissue was removed, and the tumor mass in good condition was cut into 20-30 mm small tumors The block was inoculated to the right scapula of the formal experimental mice, and a total of 70 mice were inoculated. When the average tumor volume reached about 135 mm 3 13 days after tumor mass inoculation, mice with too small or too large tumor volume were eliminated, and the remaining 50 mice were randomly grouped according to tumor volume and started to be administered.
Figure PCTCN2021123020-appb-000223
Figure PCTCN2021123020-appb-000223
实验观察和数据收集Experimental observation and data collection
肿瘤细胞接种后,除了观察肿瘤生长情况,还对药物治疗对动物行为的影响进行监测:实验动物的活动性,摄食和饮水,体重变化(体重每周测量2次),眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算方法为:肿瘤体积(mm 3)=1/2×(a×b 2)(其中a表示长径,b表示短径)。 After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were also monitored: the activity of experimental animals, food and water intake, body weight changes (body weight was measured twice a week), eyes, coat and other abnormal situation. The clinical symptoms observed during the experiment were all recorded in the raw data. The tumor volume was calculated as follows: tumor volume (mm 3 )=1/2×(a×b 2 ) (where a represents the long diameter, and b represents the short diameter).
当单只动物的体重下降超过15%时(BWL>15%),给予相应单只动物停药处理,体重下降恢复到10%以内,恢复给药。当单只小鼠体重下降>20%,按照动物福利对其实施安乐死。When the body weight of a single animal decreased by more than 15% (BWL>15%), the corresponding single animal was given drug withdrawal treatment, and the body weight decreased to less than 10%, and the drug was resumed. When individual mice lost >20% body weight, they were euthanized in accordance with animal welfare.
疗效评价标准Efficacy Evaluation Criteria
相对肿瘤增殖率,T/C%,即在某一时间点,治疗组和对照组的相对肿瘤体积或瘤重的百分比值。计算公式如下:T/C%=T RTV/C RTV×100%(T RTV:治疗组平均RTV;C RTV:溶媒对照组平均RTV;RTV=V t/V 0,V 0为分组时该动物的瘤体积,V t为治疗后该动物的瘤体积);或T/C%=T TW/C TW×100%(T TW:治疗组实验终结时平均瘤重;C TW:溶媒对照组实验终结时平均瘤重)。 The relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C%=T RTV /C RTV × 100% (T RTV : the average RTV of the treatment group; C RTV : the average RTV of the vehicle control group; RTV=V t /V 0 , V 0 is the animal at the time of grouping or T/C%=T TW /C TW × 100% ( T TW : the average tumor weight at the end of the experiment in the treatment group; C TW : the vehicle control group experiment mean tumor weight at termination).
统计分析Statistical Analysis
本实验用one-way ANOVA进行各组间肿瘤均值的比较。方差齐性分析得出F值有显著性差异,在ANOVA分析之后用Dunnet’s T3(方差不齐)法再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。In this experiment, one-way ANOVA was used to compare the mean tumor values among the groups. Homogeneity of variance analysis showed that the F value was significantly different, and Dunnet's T3 (unequal variance) method was used for multiple comparisons after ANOVA analysis. All data analyses were performed with SPSS 17.0. p<0.05 was considered a significant difference.
(2)实验结果(2) Experimental results
实验结果如下表和图25-34所示。The experimental results are shown in the following table and Figures 25-34.
Figure PCTCN2021123020-appb-000224
Figure PCTCN2021123020-appb-000224
Figure PCTCN2021123020-appb-000225
Figure PCTCN2021123020-appb-000225
实施例29 ADC-16、ADC-5和ADC-17的动物学抑瘤实验Example 29 Animal tumor inhibition experiments of ADC-16, ADC-5 and ADC-17
1.实验方法1. Experimental method
1.1细胞培养1.1 Cell Culture
Colo205细胞体外单层培养,培养条件为1640培养基中加10%热灭活胎牛血清并加琼脂,于37℃、含5%CO 2空气的培养箱中培养。一周两次用0.25%胰酶进行消化处理传代。当细胞呈指数生长期时,收取细胞,计数,接种。 Colo205 cells were cultured in monolayer in vitro, cultured in 1640 medium with 10% heat-inactivated fetal bovine serum and agar, and cultured at 37°C in an incubator containing 5% CO 2 air. Digestion and passage were performed twice a week with 0.25% trypsin. When cells are in exponential growth phase, cells are harvested, counted, and seeded.
1.2肿瘤细胞接种及瘤块传代1.2 Inoculation of tumor cells and passage of tumor mass
将5.0×10 6Colo205肿瘤细胞悬浮于0.1ml PBS与Matrigel混合物(1:1),接种于5只裸鼠右侧肩胛处(P1代)。待肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到新的一批裸鼠(P2代)。 5.0×10 6 Colo205 tumor cells were suspended in 0.1 ml of a mixture of PBS and Matrigel (1:1), and inoculated on the right scapula of 5 nude mice (passage P1). When the tumor grows to 500-800mm 3 , the tumor-bearing mice are sacrificed with CO 2 anesthesia, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into 20-30 mm 3 small tumor pieces, Inoculated into a new batch of nude mice (P2 generation).
1.3瘤块接种种及分组给药1.3 Tumor mass inoculation and group administration
本试验使用P6代肿瘤组织进行受试品的抗肿瘤活性评价。待P5代肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到正式实验用鼠的右侧肩胛处,一共接种70只鼠。瘤块接种13天后肿瘤平均体积达到约135mm 3时,剔除瘤体积过小或过大的小鼠,将剩余的50只小鼠根据瘤体积随机分组并开始给药。给药方案见下表。 In this experiment, the P6 generation tumor tissue was used to evaluate the antitumor activity of the test article. When the P5 generation tumor grew to 500-800 mm, the tumor - bearing mice were sacrificed with CO anesthesia, the tumor mass was removed, the surrounding necrotic tissue was removed, and the tumor mass in good condition was cut into 20-30 mm small tumors The block was inoculated to the right scapula of the formal experimental mice, and a total of 70 mice were inoculated. When the average tumor volume reached about 135 mm 3 13 days after tumor mass inoculation, mice with too small or too large tumor volume were eliminated, and the remaining 50 mice were randomly grouped according to tumor volume and started to be administered. The dosing schedule is shown in the table below.
Figure PCTCN2021123020-appb-000226
Figure PCTCN2021123020-appb-000226
备注:IV表示静脉注射,QW*4表示每周给药一次,共4次Remarks: IV means intravenous injection, QW*4 means once a week, a total of 4 times
1.5实验观察和数据收集1.5 Experimental observation and data collection
肿瘤细胞接种后,除了观察肿瘤生长情况,还对药物治疗对动物行为的影响进行监测:实验动物的活动性,摄食和饮水,体重变化(体重每周测量2次),眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算方法为:肿瘤体积(mm 3)=1/2×(a×b 2)(其中a表示长径,b表示短径)。 After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were also monitored: the activity of experimental animals, food and water intake, body weight changes (body weight was measured twice a week), eyes, coat and other abnormal situation. The clinical symptoms observed during the experiment were all recorded in the raw data. The tumor volume was calculated as follows: tumor volume (mm 3 )=1/2×(a×b 2 ) (where a represents the long diameter, and b represents the short diameter).
当单只动物的体重下降超过15%时(BWL>15%),给予相应单只动物停药处理,体重下降恢复到10%以内,恢复给药。当单只小鼠体重下降>20%,按照动物福利对其实施安乐死。When the body weight of a single animal decreased by more than 15% (BWL>15%), the corresponding single animal was given drug withdrawal treatment, and the body weight decreased to less than 10%, and the drug was resumed. When individual mice lost >20% body weight, they were euthanized in accordance with animal welfare.
1.6疗效评价标准1.6 Efficacy evaluation criteria
相对肿瘤增殖率,T/C%,即在某一时间点,治疗组和对照组的相对肿瘤体积或瘤重的百分比值。计算公式如下:T/C%=T RTV/C RTV×100%(T RTV:治疗组平均RTV;C RTV:溶媒对照 组平均RTV;RTV=V t/V 0,V 0为分组时该动物的瘤体积,V t为治疗后该动物的瘤体积);或T/C%=T TW/C TW×100%(T TW:治疗组实验终结时平均瘤重;C TW:溶媒对照组实验终结时平均瘤重)。 The relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C%=T RTV /C RTV × 100% (T RTV : the average RTV of the treatment group; C RTV : the average RTV of the vehicle control group; RTV=V t /V 0 , V 0 is the animal at the time of grouping or T/C%=T TW /C TW × 100% ( T TW : the average tumor weight at the end of the experiment in the treatment group; C TW : the vehicle control group experiment mean tumor weight at termination).
1.7实验终点1.7 Experimental end point
最后一次给药1周后,所有小鼠取肿瘤,并称重、拍照。One week after the last administration, tumors were taken from all mice, weighed, and photographed.
1.8统计分析1.8 Statistical analysis
本实验用one-way ANOVA进行各组间肿瘤均值的比较。方差齐性分析得出F值有显著性差异,在ANOVA分析之后用Dunnet’s T3(方差不齐)法再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。In this experiment, one-way ANOVA was used to compare the mean tumor values among the groups. Homogeneity of variance analysis showed that the F value was significantly different, and Dunnet's T3 (unequal variance) method was used for multiple comparisons after ANOVA analysis. All data analyses were performed with SPSS 17.0. p<0.05 was considered a significant difference.
2.实验结果2. Experimental results
实验结果如下表和图35、图36所示。The experimental results are shown in the following table and Fig. 35 and Fig. 36 .
Figure PCTCN2021123020-appb-000227
Figure PCTCN2021123020-appb-000227
实施例30 ADC-8、ADC-11、ADC-5和ADC-12样品的动物学抑瘤实验结果Example 30 The results of the animal tumor inhibition experiment of ADC-8, ADC-11, ADC-5 and ADC-12 samples
实验方法experimental method
1.1细胞培养1.1 Cell Culture
BxPC-3细胞体外单层培养,培养条件为1640培养基中加10%热灭活胎牛血清并加琼脂,于37℃、含5%CO 2空气的培养箱中培养。一周两次用0.25%胰酶进行消化处理传代。当细胞呈指数生长期时,收取细胞,计数,接种。 BxPC-3 cells were cultured in monolayer in vitro, cultured in 1640 medium with 10% heat-inactivated fetal bovine serum and agar, and cultured at 37°C in an incubator containing 5% CO 2 air. Digestion and passage were performed twice a week with 0.25% trypsin. When cells are in exponential growth phase, cells are harvested, counted, and seeded.
1.2肿瘤细胞接种及瘤块传代1.2 Inoculation of tumor cells and passage of tumor mass
将5.0×10 6BxPC-3肿瘤细胞悬浮于0.1ml PBS与Matrigel混合物(1:1),接种于5只裸鼠右侧肩胛处(P1代)。待肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到新的一批裸鼠(P2代)。 5.0×10 6 BxPC-3 tumor cells were suspended in 0.1 ml of a mixture of PBS and Matrigel (1:1), and inoculated on the right scapula of 5 nude mice (passage P1). When the tumor grows to 500-800mm 3 , the tumor-bearing mice are sacrificed with CO 2 anesthesia, the tumor mass is removed, the surrounding necrotic tissue is removed, and the tumor mass in good condition is cut into 20-30 mm 3 small tumor pieces, Inoculated into a new batch of nude mice (P2 generation).
1.3瘤块接种种及分组给药1.3 Tumor mass inoculation and group administration
本试验使用P6代肿瘤组织进行受试品的抗肿瘤活性评价。待P5代肿瘤长至500-800mm 3时,将荷瘤小鼠用CO 2麻醉处死,取出瘤块,去除周围坏死的组织,将状态较好的将瘤块切成20-30mm 3的小瘤块,接种到正式实验用鼠的右侧肩胛处,一共接种70只鼠。瘤块接种13天后肿瘤平均体积达到约135mm 3时,剔除瘤体积过小或过大的小鼠,将剩余的50只小鼠根据瘤体积随机分组并开始给药。给药方案见下表。 In this experiment, the P6 generation tumor tissue was used to evaluate the antitumor activity of the test article. When the P5 generation tumor grew to 500-800 mm, the tumor - bearing mice were sacrificed with CO anesthesia, the tumor mass was removed, the surrounding necrotic tissue was removed, and the tumor mass in good condition was cut into 20-30 mm small tumors The block was inoculated to the right scapula of the formal experimental mice, and a total of 70 mice were inoculated. When the average tumor volume reached about 135 mm 3 13 days after tumor mass inoculation, mice with too small or too large tumor volume were eliminated, and the remaining 50 mice were randomly grouped according to tumor volume and started to be administered. The dosing schedule is shown in the table below.
Figure PCTCN2021123020-appb-000228
Figure PCTCN2021123020-appb-000228
Figure PCTCN2021123020-appb-000229
Figure PCTCN2021123020-appb-000229
1.5实验观察和数据收集1.5 Experimental observation and data collection
肿瘤细胞接种后,除了观察肿瘤生长情况,还对药物治疗对动物行为的影响进行监测:实验动物的活动性,摄食和饮水,体重变化(体重每周测量2次),眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算方法为:肿瘤体积(mm 3)=1/2×(a×b 2)(其中a表示长径,b表示短径)。 After tumor cell inoculation, in addition to observing tumor growth, the effects of drug treatment on animal behavior were also monitored: the activity of experimental animals, food and water intake, body weight changes (body weight was measured twice a week), eyes, coat and other abnormal situation. The clinical symptoms observed during the experiment were all recorded in the raw data. The tumor volume was calculated as follows: tumor volume (mm 3 )=1/2×(a×b 2 ) (where a represents the long diameter, and b represents the short diameter).
当单只动物的体重下降超过15%时(BWL>15%),给予相应单只动物停药处理,体重下降恢复到10%以内,恢复给药。当单只小鼠体重下降>20%,按照动物福利对其实施安乐死。When the body weight of a single animal decreased by more than 15% (BWL>15%), the corresponding single animal was given drug withdrawal treatment, and the body weight decreased to less than 10%, and the drug was resumed. When individual mice lost >20% body weight, they were euthanized in accordance with animal welfare.
1.6疗效评价标准1.6 Efficacy evaluation criteria
相对肿瘤增殖率,T/C%,即在某一时间点,治疗组和对照组的相对肿瘤体积或瘤重的百分比值。计算公式如下:T/C%=T RTV/C RTV×100%(T RTV:治疗组平均RTV;C RTV:溶媒对照组平均RTV;RTV=V t/V 0,V 0为分组时该动物的瘤体积,V t为治疗后该动物的瘤体积);或T/C%=T TW/C TW×100%(T TW:治疗组实验终结时平均瘤重;C TW:溶媒对照组实验终结时平均瘤重)。 The relative tumor proliferation rate, T/C%, is the percentage value of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C%=T RTV /C RTV × 100% (T RTV : the average RTV of the treatment group; C RTV : the average RTV of the vehicle control group; RTV=V t /V 0 , V 0 is the animal at the time of grouping or T/C%=T TW /C TW × 100% ( T TW : the average tumor weight at the end of the experiment in the treatment group; C TW : the vehicle control group experiment mean tumor weight at termination).
1.7实验终点1.7 Experimental end point
最后一次给药1周后,所有小鼠取肿瘤,并称重、拍照。One week after the last administration, tumors were taken from all mice, weighed, and photographed.
1.8统计分析1.8 Statistical analysis
本实验用one-way ANOVA进行各组间肿瘤均值的比较。方差齐性分析得出F值有显著性差异,在ANOVA分析之后用Dunnet’s T3(方差不齐)法再进行多重比较。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。In this experiment, one-way ANOVA was used to compare the mean tumor values among the groups. Homogeneity of variance analysis showed that the F value was significantly different, and Dunnet's T3 (unequal variance) method was used for multiple comparisons after ANOVA analysis. All data analyses were performed with SPSS 17.0. p<0.05 was considered a significant difference.
3.实验结果3. Experimental results
实验结果如图37、图38所示。The experimental results are shown in FIGS. 37 and 38 .
产业实用性Industrial Applicability
本发明中,通过包含新型的接头结构的抗体-药物偶联物,实现了高载药量,同时得到起效时间更快、药物半衰期更长、稳定性优异、生物相容性良好、低免疫原性和安全性良好的抗体-药物偶联物。本发明的的抗体-药物偶联物表现出优异的抗肿瘤效果。In the present invention, through the antibody-drug conjugate comprising a novel linker structure, high drug loading is achieved, and at the same time, faster onset time, longer drug half-life, excellent stability, good biocompatibility, and low immunity are obtained. Antibody-drug conjugates with good originality and safety. The antibody-drug conjugate of the present invention exhibits excellent antitumor effect.

Claims (43)

  1. 式(VIII)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其特征在于,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下式(VII)表示的接头连接而成的:Antibody-drug conjugate represented by formula (VIII), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof The solvate is characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and antibody (AB) represented by the following formula (VII) The connector is connected by:
    AB-S-Q 2-L 3-L 4-L P-L b-T    (VIII) AB-SQ 2 -L 3 -L 4 -L P -L b -T (VIII)
    Figure PCTCN2021123020-appb-100001
    Figure PCTCN2021123020-appb-100001
    -Q 2-L 3-L 4-L P-L b-    (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
    其中,in,
    式(II)中,In formula (II),
    R 1选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、被-OC(=O)R 13或-NR 7R 8取代的C 1-C 6烷基、被-SiMe 3取代的C 1-C 6烷基、或-CH=N-O-(C 1-C 6烷基); R 1 is selected from hydrogen, halogen, hydroxy, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C substituted by -OC(=O)R 13 or -NR 7 R 8 1 -C 6 alkyl, C 1 -C 6 alkyl substituted with -SiMe 3 , or -CH=NO-(C 1 -C 6 alkyl);
    R 2选自氢、卤素、羟基、硝基、氨基、饱和或不饱和C 1-C 6烷基、C 1-C 6烷氧基、被-NR 7R 8取代的C 1-C 6烷基或被C 2-C 6烯基取代的C 1-C 6烷基; R 2 is selected from hydrogen, halogen, hydroxy, nitro, amino, saturated or unsaturated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkane substituted by -NR 7 R 8 group or C 1 -C 6 alkyl substituted by C 2 -C 6 alkenyl;
    R 3选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、C 1-C 6烷氧基、NR 7R 8C(=O)O-基或5-6元含氮杂环基-C(=O)-C 1-C 6烷氧基; R 3 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NR 7 R 8 C(=O)O-yl or 5-6 membered containing Azacyclyl-C(=O) -C1 - C6alkoxy;
    R 4选自氢、卤素、羟基、硝基、氨基、C 1-C 6烷基、或C 1-C 6烷氧基; R 4 is selected from hydrogen, halogen, hydroxyl, nitro, amino, C 1 -C 6 alkyl, or C 1 -C 6 alkoxy;
    或者R 1和R 2可以连接在一起与母体部分形成任选被R 9取代的5-6元环; Alternatively R1 and R2 can be linked together with the parent moiety to form a 5-6 membered ring optionally substituted with R9 ;
    或者R 3和R 4可以连接在一起与母体部分形成任选被R 9取代的5-6元含氧杂环; Alternatively R and R can be linked together with the parent moiety to form a 5-6 membered oxygen-containing heterocycle optionally substituted with R ;
    R 7和R 8每次出现时各自独立地选自氢、C 1-C 6烷基、被羟基或氨基取代的C 1-C 6酰基;或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环; Each occurrence of R 7 and R 8 is independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 acyl substituted with hydroxy or amino; or R 7 and R 8 may be combined with the N atom to which they are attached taken together to form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R;
    优选地,R 7和R 8每次出现时各自独立地选自氢、甲基、异丙基、
    Figure PCTCN2021123020-appb-100002
    或者R 7与R 8可以与所连接的N原子一起形成任选被R 9取代的5-6元含氮杂环;
    Preferably, each occurrence of R7 and R8 is independently selected from hydrogen, methyl, isopropyl,
    Figure PCTCN2021123020-appb-100002
    Alternatively R7 and R8 may together with the attached N atom form a 5-6 membered nitrogen-containing heterocycle optionally substituted by R9 ;
    R 9每次出现时各自独立地选自卤素、羟基、硝基、-NR 7R 8、C 1-C 6烷基、C 1-C 6烷氧基、任选被C 1-C 6烷基取代的哌啶基; Each occurrence of R 9 is independently selected from halogen, hydroxy, nitro, -NR 7 R 8 , C 1 -C 6 alkyl, C 1 -C 6 alkoxy, optionally C 1 -C 6 alkane substituted piperidinyl;
    优选地,R 9每次出现时各自独立地选自甲基、-NR 7R 8、哌啶基; Preferably, each occurrence of R 9 is independently selected from methyl, -NR 7 R 8 , piperidinyl;
    R 13表示羧基取代的C 1-C 6烷基; R 13 represents a carboxy-substituted C 1 -C 6 alkyl group;
    式(VII)中,In formula (VII),
    L 3表示-Z-W-(CH 2CH 2-O)n 5-W’-、-(CH 2)n 5’-C(=O)-NR 10-(CH 2CH 2-O)n 5-或单键,n 5’表示1~3的整数,各n 5独立地表示1~8的整数,W、W’表示
    Figure PCTCN2021123020-appb-100003
    或单键,其中,W的位置①表示与Z相连,位置②表示与(CH 2CH 2-O-)n 5-相连,W’的位置①表示与(CH 2CH 2-O-)n 5-相连,位置②表示与L 4的-CH 2-相连,且W、W’不同时为
    Figure PCTCN2021123020-appb-100004
    Z表示-CH 2-Cyclo-C(=O)-NR 10-或单键,Cyclo表示环己烷基团;
    L 3 represents -ZW-(CH 2 CH 2 -O)n 5 -W'-, -(CH 2 )n 5' -C(=O)-NR 10 -(CH 2 CH 2 -O)n 5 - or a single bond, n 5' represents an integer of 1 to 3, each n 5 independently represents an integer of 1 to 8, and W and W' represent an integer
    Figure PCTCN2021123020-appb-100003
    Or a single bond, wherein, the position ① of W indicates that it is connected to Z, the position ② indicates that it is connected to (CH 2 CH 2 -O-)n 5 -, and the position ① of W' indicates that it is connected to (CH 2 CH 2 -O-)n 5 - is connected, position ② indicates that it is connected to -CH 2- of L 4 , and W and W' are not at the same time
    Figure PCTCN2021123020-appb-100004
    Z represents -CH 2 -Cyclo-C(=O)-NR 10 - or a single bond, and Cyclo represents a cyclohexane group;
    优选地,L 3表示
    Figure PCTCN2021123020-appb-100005
    Figure PCTCN2021123020-appb-100006
    或单键,各n独立地表示1-6的整数(例如2);
    Preferably, L 3 represents
    Figure PCTCN2021123020-appb-100005
    Figure PCTCN2021123020-appb-100006
    or a single bond, each n independently represents an integer from 1 to 6 (eg 2);
    L 4表示-(CH 2)n 6-C(=O)-或-(CH 2)n 6a-NR 10-C(=O)-(CH 2)n 6b-O-(CH 2)n 6b-C(=O)-,n 6表示1~6的整数,n 6a表示1~4的整数,n 6b表示1~3的整数; L 4 represents -(CH 2 )n 6 -C(=O)- or -(CH 2 )n 6a -NR 10 -C(=O)-(CH 2 )n 6b -O-(CH 2 )n 6b -C(=O)-, n 6 represents an integer from 1 to 6, n 6a represents an integer from 1 to 4, and n 6b represents an integer from 1 to 3;
    L P表示由1~7个氨基酸构成的肽残基; LP represents a peptide residue consisting of 1-7 amino acids;
    L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 7-O-、-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-(CH 2)n 8-O-(C=O)-、-NR 10-(CH 2)n 8-O-(CH 2)n 8-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-Aryl-(CH 2)n 8-O-(C=O)-、-NR 10-Aryl-(CH 2)n 8-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-,各Aryl独立地表示任选地被R 9取代的C 6-C 10芳基,各n 7独立地表示1~4的整数,各n 8独立地表示1~4的整数; L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 7 -O-, -NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -(CH 2 )n 8 -O-(C=O)-, -NR 10 -(CH 2 )n 8 -O-(CH 2 )n 8 -(C=O)-NR 10 -( CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 - or -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, each Aryl independently represents optional C 6 -C 10 aryl substituted by R 9 , each n 7 independently represents an integer of 1-4, and each n 8 independently represents an integer of 1-4;
    R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基(优选甲基); Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy (preferably methyl);
    Q 2表示-(琥珀酰亚胺-3-基-N)-、或-Q 1-NR 10-,Q 1为以下式(I)所述化合物,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; Q 2 represents -(succinimide-3-yl-N)-, or -Q 1 -NR 10 -, Q 1 is a compound described in the following formula (I), Q 1 passes through the carboxyl group of R 11 and -NR 10 - form an amide bond to connect with L 3 ;
    其中,式(I)表示的化合物如下所示:Wherein, the compound represented by formula (I) is as follows:
    Figure PCTCN2021123020-appb-100007
    Figure PCTCN2021123020-appb-100007
    其中,R 11为羧基取代的C 1-C 6烷基,R 12为氰基取代的C 2-C 6炔基,X、Y、X’和Y’中有1-2个C原子被N原子取代;优选地,R 11为羧基取代的C 1-C 3烷基,R 12为氰基取代的C 2-C 3炔基; Wherein, R 11 is a carboxy-substituted C 1 -C 6 alkyl group, R 12 is a cyano-substituted C 2 -C 6 alkynyl group, and 1-2 C atoms in X, Y, X' and Y' are replaced by N Atom substitution; preferably, R 11 is a carboxy-substituted C 1 -C 3 alkyl group, and R 12 is a cyano-substituted C 2 -C 3 alkynyl group;
    优选地,X、Y、X’和Y’中有且只有1个C原子被N原子取代;Preferably, X, Y, X' and Y' have only 1 C atom substituted by N atom;
    优选地,X、Y、X’和Y’中有2个C原子被N原子取代,且X、Y中有且只有1个C原子被N原子取代,以及X’、Y’中有且只有1个C原子被N原子取代;Preferably, 2 C atoms in X, Y, X' and Y' are substituted by N atoms, and in X, Y, and only 1 C atom is substituted by N atoms, and in X', Y', and only 1 1 C atom is replaced by N atom;
    式(VIII)中,In formula (VIII),
    Q 2为-(琥珀酰亚胺-3-基-N)-,如下式结构: Q 2 is -(succinimide-3-yl-N)-, with the following formula:
    Figure PCTCN2021123020-appb-100008
    Figure PCTCN2021123020-appb-100008
    以该结构的3位与抗体连接,在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q 2为-Q 1-NR 10-,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, It is linked to the antibody at the 3-position of the structure, and the nitrogen atom at the 1-position is linked to the methylene group in the linker containing the structure, or Q 2 is -Q 1 -NR 10 -, and the alkynyl carbon of R 12 is linked to The disulfide bond in the hinge of the antibody forms a thioether bond to connect,
    式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(VII)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)-, -O- or -CH2- moiety of L b in the linker represented by VII).
  2. 根据权利要求1所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 1表示氢、C 1-C 4烷 基、被-NH(C 1-C 4烷基)取代的C 1-C 4烷基、被
    Figure PCTCN2021123020-appb-100009
    取代的C 1-C 4烷基、被-SiMe 3取代的C 1-C 4烷基、-CH=N-O-(C 3-C 6烷基)或-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH;
    The antibody-drug conjugate according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of salts, wherein R 1 represents hydrogen, C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted by -NH(C 1 -C 4 alkyl),
    Figure PCTCN2021123020-appb-100009
    Substituted C 1 -C 4 alkyl, C 1 -C 4 alkyl substituted with -SiMe 3 , -CH=NO-(C 3 -C 6 alkyl) or -(CH 2 ) 2 O(C=O )(CH 2 ) 2 (C=O)OH;
    优选地,R 1表示氢、乙基、
    Figure PCTCN2021123020-appb-100010
    Figure PCTCN2021123020-appb-100011
    取代的甲基、
    Figure PCTCN2021123020-appb-100012
    -CH=NO-叔丁基、-(CH 2) 2O(C=O)(CH 2) 2(C=O)OH、。
    Preferably, R 1 represents hydrogen, ethyl,
    Figure PCTCN2021123020-appb-100010
    quilt
    Figure PCTCN2021123020-appb-100011
    substituted methyl,
    Figure PCTCN2021123020-appb-100012
    -CH=NO-tert-butyl, -(CH 2 ) 2 O(C=O)(CH 2 ) 2 (C=O)OH,.
  3. 根据权利要求1或2所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 2表示氢、C 3-C 4烯基、硝基、氨基、被-N(C 1-C 4烷基) 2取代的C 1-C 4烷基或被C 2-C 4烯基取代的C 1-C 4烷基; The antibody-drug conjugate according to claim 1 or 2, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of the accepted salts wherein R 2 represents hydrogen, C 3 -C 4 alkenyl, nitro, amino, C 1 -C 4 alkyl substituted with -N(C 1 -C 4 alkyl) 2 or C 1 -C 4 alkyl substituted by C 2 -C 4 alkenyl;
    优选地,R 2表示氢、硝基、氨基、
    Figure PCTCN2021123020-appb-100013
    Preferably, R 2 represents hydrogen, nitro, amino,
    Figure PCTCN2021123020-appb-100013
  4. 根据权利要求1-3任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 3表示氢、卤素、羟基、C 1-C 4烷基、
    Figure PCTCN2021123020-appb-100014
    The antibody-drug conjugate according to any one of claims 1-3, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein R 3 represents hydrogen, halogen, hydroxy, C 1 -C 4 alkyl,
    Figure PCTCN2021123020-appb-100014
    优选地,R 3表示氢、F、羟基、甲基、
    Figure PCTCN2021123020-appb-100015
    Preferably, R 3 represents hydrogen, F, hydroxyl, methyl,
    Figure PCTCN2021123020-appb-100015
  5. 根据权利要求1-4任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 4表示氢或卤素; The antibody-drug conjugate according to any one of claims 1-4, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein R 4 represents hydrogen or halogen;
    优选地,R 4表示氢或F。 Preferably, R 4 represents hydrogen or F.
  6. 根据权利要求1-5任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 1和R 2连接在一起形成以下所示的基团
    Figure PCTCN2021123020-appb-100016
    其中
    Figure PCTCN2021123020-appb-100017
    部分表示连接于母体基团的键;
    The antibody-drug conjugate according to any one of claims 1-5, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of pharmaceutically acceptable salts wherein R and R are linked together to form the group shown below
    Figure PCTCN2021123020-appb-100016
    in
    Figure PCTCN2021123020-appb-100017
    moiety represents a bond to the parent group;
    优选地,R 1和R 2连接在一起形成以下所示的基团
    Figure PCTCN2021123020-appb-100018
    其中
    Figure PCTCN2021123020-appb-100019
    部分表示连接于母体基团的键。
    Preferably, R1 and R2 are joined together to form the group shown below
    Figure PCTCN2021123020-appb-100018
    in
    Figure PCTCN2021123020-appb-100019
    A moiety represents a bond to the parent group.
  7. 根据权利要求1-6任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,R 3和R 4连接在一起形成以下所示的基团
    Figure PCTCN2021123020-appb-100020
    其中
    Figure PCTCN2021123020-appb-100021
    部分表示连接于母体基团的键。
    The antibody-drug conjugate according to any one of claims 1-6, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof Solvates of pharmaceutically acceptable salts wherein R and R are linked together to form the group shown below
    Figure PCTCN2021123020-appb-100020
    in
    Figure PCTCN2021123020-appb-100021
    A moiety represents a bond to the parent group.
  8. 根据权利要求1-7任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,式(II)表示的化合物如下所示:The antibody-drug conjugate according to any one of claims 1 to 7, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein the compound represented by the formula (II) is shown below:
    Figure PCTCN2021123020-appb-100022
    Figure PCTCN2021123020-appb-100022
    Figure PCTCN2021123020-appb-100023
    Figure PCTCN2021123020-appb-100023
    优选地,式(II)表示的化合物为吉马替康或吉咪替康,更优选为吉咪替康:Preferably, the compound represented by formula (II) is gimatecan or gimitecan, more preferably gimatecan:
    Figure PCTCN2021123020-appb-100024
    Figure PCTCN2021123020-appb-100024
  9. 根据权利要求1-8任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,式(I)表示的化合物的结构如下所示,The antibody-drug conjugate according to any one of claims 1 to 8, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein the structure of the compound represented by the formula (I) is shown below,
    Figure PCTCN2021123020-appb-100025
    Figure PCTCN2021123020-appb-100025
  10. 根据权利要求1-9任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,式(I)表示的化合物作为抗体-药物偶联物中的连接单元,通过R 12的炔基碳与存在于抗体的铰链部的二硫键部分形成硫醚键而与抗体连接。 The antibody-drug conjugate according to any one of claims 1-9, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein the compound represented by the formula (I) acts as a linking unit in an antibody-drug conjugate through a disulfide bond between the alkynyl carbon of R 12 and the hinge portion of the antibody Some form thioether bonds to link with the antibody.
  11. 根据权利要求1-10任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L P的肽残基为由选自丙氨酸、苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的肽残基; The antibody-drug conjugate according to any one of claims 1-10, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its The solvate of a pharmaceutically acceptable salt, wherein the peptide residue of LP is selected from the group consisting of alanine, phenylalanine, glycine, valine, lysine, citrulline, serine, glutamine Peptide residues formed from amino acids in acid and aspartic acid;
    或者,L P为由1-5个氨基酸构成的肽残基; Alternatively, LP is a peptide residue consisting of 1-5 amino acids;
    或者,L P为选自以下的肽残基: Alternatively, LP is a peptide residue selected from the group consisting of:
    -VA-;-va-;
    -K-;-K-;
    -GGFG-;-ggfg-;
    -VC-;-vc-;
    -EVC-;-evc-;
    -DVC;-DVC;
    -EGGFG-;-EGGFG-;
    -DGGFG-。-DGGFG-.
  12. 根据权利要求1-11任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L 4表示-(CH 2)n 6-C(=O)-或-(CH 2)n 6a-NR 10-C(=O)-(CH 2)n 6b-O-(CH 2)n 6b-C(=O)-,n 6表示2~5的整数,n 6a表示1~3的整数,n 6b表示1~2的整数,R 10表示氢或C 1-C 4烷基(优选甲基); The antibody-drug conjugate according to any one of claims 1-11, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein L 4 represents -(CH 2 )n 6 -C(=O)- or -(CH 2 )n 6a -NR 10 -C(=O)-(CH 2 ) n 6b -O-(CH 2 )n 6b -C(=O)-, n 6 represents an integer of 2 to 5, n 6a represents an integer of 1 to 3, n 6b represents an integer of 1 to 2, R 10 represents hydrogen or C 1 -C 4 alkyl (preferably methyl);
    优选地,L 4表示
    Figure PCTCN2021123020-appb-100026
    Preferably, L 4 represents
    Figure PCTCN2021123020-appb-100026
  13. 根据权利要求1-12任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L b表示-NR 10-(CH 2)n 7-、-NR 10-(CH 2)n 7-O-、-NR 10-(CH 2)n 8-NR 10-C(=O)-、、-NR 10-(CH 2)n 8-O-(C=O)-、-NR 10-(CH 2)n 8-O-(CH 2)n 8-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-、-NR 10-Aryl-(CH 2)n 8-O-C(=O)-、-NR 10-Aryl-(CH 2)n 8-或-NR 10-Aryl-(CH 2)n 8-O-(C=O)-NR 10-(CH 2)n 8-NR 10-(C=O)-,其中各R 10独立地表示氢或C 1-C 4烷基(优选甲基),各n 7独立地表示1~2的整数,各n 8独立地表示1~2的整数,Aryl表示苯环基团; The antibody-drug conjugate according to any one of claims 1-12, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein L b represents -NR 10 -(CH 2 )n 7 -, -NR 10 -(CH 2 )n 7 -O-, -NR 10 -(CH 2 ) n 8 -NR 10 -C(=O)-, -NR 10 -(CH 2 )n 8 -O-(C=O)-, -NR 10 -(CH 2 )n 8 -O-(CH 2 )n 8 -(C=O)-NR 10 -(CH 2 )n 8 -NR 10 -(C=O)-, -NR 10 -Aryl-(CH 2 )n 8 -OC(=O)-, -NR 10 -Aryl-(CH 2 )n 8 -or -NR 10 -Aryl-(CH 2 )n 8 -O-(C=O)-NR 10 -(CH 2 )n 8 -NR 10 -(C =O)-, wherein each R 10 independently represents hydrogen or C 1 -C 4 alkyl (preferably methyl), each n 7 independently represents an integer of 1 to 2, and each n 8 independently represents an integer of 1 to 2 , Aryl represents a benzene ring group;
    优选地,-NR 10-基团和-(CH 2)n 8-基团位于苯环的对位; Preferably, the -NR 10 - group and the -(CH 2 )n 8 - group are located in the para position of the benzene ring;
    优选地,L b表示
    Figure PCTCN2021123020-appb-100027
    Figure PCTCN2021123020-appb-100028
    Preferably, L b represents
    Figure PCTCN2021123020-appb-100027
    Figure PCTCN2021123020-appb-100028
  14. 根据权利要求1所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,式(VII)表示的接头为选自以下所示的基团,其中,各n独立地表示1-8的整数:The antibody-drug conjugate according to claim 1, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a salt, wherein the linker represented by the formula (VII) is a group selected from the group consisting of the following, wherein each n independently represents an integer of 1-8:
    Figure PCTCN2021123020-appb-100029
    Figure PCTCN2021123020-appb-100029
    Figure PCTCN2021123020-appb-100030
    Figure PCTCN2021123020-appb-100030
    Figure PCTCN2021123020-appb-100031
    Figure PCTCN2021123020-appb-100031
    Figure PCTCN2021123020-appb-100032
    Figure PCTCN2021123020-appb-100032
  15. 根据权利要求1-14任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个例如3.3、3.5、5.5、6.2、6.5、6.6、6.8、7.0、7.1、7.2、7.4、7.5或7.8个。The antibody-drug conjugate according to any one of claims 1 to 14, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein, for one antibody molecule, the average number of linker-drug linkages is 2 to 8, preferably 4 to 8, more preferably 6 to 8, such as 3.3, 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
  16. 根据权利要求1-15任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段;The antibody-drug conjugate according to any one of claims 1-15, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer or its A solvate of a pharmaceutically acceptable salt, wherein the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof;
    优选地,所述抗体选自抗Her-2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER-4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体;优选地,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体;Preferably, the antibody is selected from anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 antibody, CD30 antibody Antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies;
    优选地,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv; Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv;
    更优选地,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示;More preferably, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises the CDR1 consisting of the KASQDVSIVA amino acid sequence and the SASYRYT amino acid sequence. CDR2, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; Preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding nucleosides of the light chain and heavy chain of the anti-Trop-2 antibody The acid sequences are shown in SEQ ID NO:3 and SEQ ID NO:4 respectively;
    更优选地,所述抗体为抗Her-2抗体,其中,所述抗Her-2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的 CDR3;优选地,所述抗Her-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。More preferably, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises the CDR1 consisting of the RASQDVNTAVA amino acid sequence and the SASFLYS amino acid sequence. CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  17. 式(X)表示的接头-药物中间体化合物,其特征在于,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(IX)表示的接头连接而成的:A linker-drug intermediate compound represented by formula (X), characterized in that T represents a compound represented by formula (II), and the intermediate compound is formed by connecting compound (T) and a linker represented by formula (IX) below of:
    Q’ 2-L 3-L 4-L P-L b-T    (X) Q' 2 -L 3 -L 4 -L P -L b -T (X)
    Figure PCTCN2021123020-appb-100033
    Figure PCTCN2021123020-appb-100033
    Q’ 2-L 3-L 4-L P-L b-    (IX) Q' 2 -L 3 -L 4 -L P -L b - (IX)
    其中,in,
    R 1、R 2、R 3、R 4的定义如权利要求1-7中任一项所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in any one of claims 1-7;
    Q’ 2表示(马来酰亚胺-N)-或Q 1-NR 10-,Q 1为式(I)所述化合物,式(I)所述化合物如权利要求1、9-10任一项所限定; Q' 2 represents (maleimide-N)- or Q 1 -NR 10 -, Q 1 is the compound of formula (I), and the compound of formula (I) is any of claims 1, 9-10 term limited;
    L 3、L 4、L P、L b的定义如权利要求1、11-13中任一项所述; The definitions of L 3 , L 4 , L P and L b are as described in any one of claims 1, 11-13;
    R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
    式(IX)中,In formula (IX),
    Q’ 2表示(马来酰亚胺-N)-,如下式结构: Q' 2 represents (maleimide-N)-, the following formula structure:
    Figure PCTCN2021123020-appb-100034
    Figure PCTCN2021123020-appb-100034
    以该结构在1位的氮原子上与包含该结构的接头内的亚甲基连接,或者Q’ 2表示Q 1-NR 10-,Q 1通过R 11的羧基与-NR 10-形成酰胺键而与L 3连接; In this structure, the nitrogen atom at the 1-position is connected with the methylene group in the linker containing this structure, or Q' 2 represents Q 1 -NR 10 -, and Q 1 forms an amide bond with -NR 10 - through the carboxyl group of R 11 And connect with L3 ;
    式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(IX)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C(=O)-, -O- or -CH2- moiety of L b in the linker represented by IX).
  18. 权利要求17所述的接头-药物中间体化合物,其中,所述式(II)所示的化合物为权利要求8所述的化合物;优选地,所述接头-药物中间体化合物是选自以下的化合物,其中,各n独立地表示1-8的整数:The linker-drug intermediate compound of claim 17, wherein the compound represented by the formula (II) is the compound of claim 8; preferably, the linker-drug intermediate compound is selected from the following Compounds where each n independently represents an integer from 1 to 8:
    Figure PCTCN2021123020-appb-100035
    Figure PCTCN2021123020-appb-100035
    Figure PCTCN2021123020-appb-100036
    Figure PCTCN2021123020-appb-100036
    Figure PCTCN2021123020-appb-100037
    Figure PCTCN2021123020-appb-100037
    Figure PCTCN2021123020-appb-100038
    Figure PCTCN2021123020-appb-100038
    Figure PCTCN2021123020-appb-100039
    Figure PCTCN2021123020-appb-100039
    Figure PCTCN2021123020-appb-100040
    Figure PCTCN2021123020-appb-100040
    Figure PCTCN2021123020-appb-100041
    Figure PCTCN2021123020-appb-100041
  19. 接头,其特征在于,其为下式(VII)表示A linker, characterized in that it is represented by the following formula (VII)
    -Q 2-L 3-L 4-L P-L b-    (VII) -Q 2 -L 3 -L 4 -L P -L b - (VII)
    其中,Q 2、L 3、L 4、L P、L b的定义如权利要求1、9-13中任一项所述;优选的是,所述接头为选自权利要求14所示的基团。 Wherein, the definitions of Q 2 , L 3 , L 4 , LP and L b are as described in any one of claims 1, 9-13; preferably, the linker is a group selected from the group shown in claim 14 group.
  20. 式(IV)表示的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其特征在于,AB表示抗体,T表示式(II)所示化合物,所述抗体-药物偶联物是将化合物(T)与抗体(AB)经由下式(III)表示的接头连接而成的:Antibody-drug conjugate represented by formula (IV), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof The solvate is characterized in that AB represents an antibody, T represents a compound represented by formula (II), and the antibody-drug conjugate is a compound (T) and antibody (AB) represented by the following formula (III) The connector is connected by:
    AB-S-Q 1-L 1-L 2-L a-T    (IV) AB-SQ 1 -L 1 -L 2 -L a -T (IV)
    Figure PCTCN2021123020-appb-100042
    Figure PCTCN2021123020-appb-100042
    -Q 1-L 1-L 2-L a-    (III) -Q 1 -L 1 -L 2 -L a - (III)
    其中,in,
    式(II)中,In formula (II),
    R 1、R 2、R 3、R 4的定义如权利要求1-7中任一项所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in any one of claims 1-7;
    式(III)中,In formula (III),
    L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)-CH 2OCH 2-(C=O)-,n 1表示1~24的整数,n 2表示1~4的整数; L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)-CH 2 OCH 2 -(C=O)-,n 1 represents an integer from 1 to 24, and n 2 represents an integer from 1 to 4;
    L 2表示缬氨酸残基、胍氨酸残基、苯丙氨酸残基、赖氨酸残基、D-缬氨酸残基、甘氨酸残基、丙氨酸残基、天冬氨酸残基; L 2 represents valine residue, guanidine residue, phenylalanine residue, lysine residue, D-valine residue, glycine residue, alanine residue, aspartic acid Residues;
    L a表示-NR 10-(CH 2)n 3-、-NR 10-(CH 2)n 4-NR 10-(C=O)-或-NR 10-Aryl-(CH 2)n 4-O-(C=O)-; L a represents -NR 10 -(CH 2 )n 3 -, -NR 10 -(CH 2 )n 4 -NR 10 -(C=O)- or -NR 10 -Aryl-(CH 2 )n 4 -O -(C=O)-;
    R 10每次出现时各自独立地选自氢、任选被1或2个羟基取代的C 1-C 6烷基; Each occurrence of R 10 is independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted with 1 or 2 hydroxy;
    n 3表示1~4的整数,n 4表示1~4的整数; n 3 represents an integer from 1 to 4, and n 4 represents an integer from 1 to 4;
    Aryl表示任选地被R 9取代的C 6-C 10芳基; Aryl represents a C 6 -C 10 aryl optionally substituted by R 9 ;
    W为单键或
    Figure PCTCN2021123020-appb-100043
    其中,位置①表示与-NR 10-相连,位置②表示与(CH 2CH 2-O-)n 1-相连;
    W is a single key or
    Figure PCTCN2021123020-appb-100043
    Wherein, position ① means connecting with -NR 10 -, and position ② means connecting with (CH 2 CH 2 -O-)n 1 -;
    式(IV)中,In formula (IV),
    Q 1为式(I)所述化合物,式(I)所述化合物如权利要求1、9-10任一项所限定,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接,通过R 12的炔基碳与抗体铰链部的二硫键形成硫醚键而连接, Q 1 is the compound of formula (I), and the compound of formula (I) is as defined in any one of claims 1, 9-10, which is through the carboxyl group of R 11 and the left-terminal amino group in the formula of L 1 -NR 10 - It is connected by forming an amide bond, and the alkynyl carbon of R 12 and the disulfide bond of the antibody hinge are connected by forming a thioether bond,
    式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(III)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C( = O)- or -CH2- moiety of La in the linker represented by III).
  21. 根据权利要求20的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L 1表示-NR 10-W-(CH 2CH 2-O-)n 1-(CH 2)n 2-NR 10-(C=O)CH 2-O-CH 2-(C=O)-,n 1表示4~12的整数(优选8),n 2表示1~2的整数(优选2),R 10表示氢或C 1-C 4烷基(优选甲基)。 The antibody-drug conjugate according to claim 20, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof Solvate, wherein L 1 represents -NR 10 -W-(CH 2 CH 2 -O-)n 1 -(CH 2 )n 2 -NR 10 -(C=O)CH 2 -O-CH 2 - (C=O)-, n 1 represents an integer of 4-12 (preferably 8), n 2 represents an integer of 1-2 (preferably 2), R 10 represents hydrogen or C 1 -C 4 alkyl (preferably methyl) .
  22. 根据权利要求20-21任一项的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L 2表示赖氨酸残基。 The antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 20 to 21, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof Solvates of acceptable salts wherein L2 represents a lysine residue.
  23. 根据权利要求20-22任一项的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,L a表示-NR 10-Aryl-(CH 2)n 4-O-(C=O)-,n 4表示1~2的整数,R 10表示氢或C 1-C 4烷基(优选甲基),Aryl表示苯环基团,优选地,-NR 10-基团和-(CH 2)n 4-基团位于苯环的对位; The antibody-drug conjugate according to any one of claims 20-22, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof Solvates of acceptable salts, wherein L a represents -NR 10 -Aryl-(CH 2 )n 4 -O-(C=O)-, n 4 represents an integer of 1 to 2, R 10 represents hydrogen or C 1 -C 4 alkyl (preferably methyl), Aryl represents a benzene ring group, preferably, -NR 10 -group and -(CH 2 )n 4 -group are located in the para position of the benzene ring;
    优选地,L a表示
    Figure PCTCN2021123020-appb-100044
    Preferably, La represents
    Figure PCTCN2021123020-appb-100044
  24. 根据权利要求20所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,式(III)表示的接头为选自以下所示的基团:The antibody-drug conjugate according to claim 20, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a salt, wherein the linker represented by the formula (III) is a group selected from the group consisting of:
    Figure PCTCN2021123020-appb-100045
    Figure PCTCN2021123020-appb-100045
    Figure PCTCN2021123020-appb-100046
    Figure PCTCN2021123020-appb-100046
  25. 根据权利要求20-24任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,对于一个抗体分子,所述接头-药物的平均连接数目为2~8个,优选为4~8个、更优选为6~8个,例如3.3、3.5、5.5、6.2、6.5、6.6、6.8、7.0、7.1、7.2、7.4、7.5或7.8个。The antibody-drug conjugate according to any one of claims 20-24, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof Solvates of pharmaceutically acceptable salts, wherein, for one antibody molecule, the average number of linker-drug linkages is 2-8, preferably 4-8, more preferably 6-8, for example 3.3 , 3.5, 5.5, 6.2, 6.5, 6.6, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5 or 7.8.
  26. 根据权利要求20-25任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,其中,所述抗体(AB)为全长抗体或其抗原结合片段,或双特异性抗体或其抗原结合片段;The antibody-drug conjugate according to any one of claims 20 to 25, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof or a pharmaceutically acceptable salt thereof A solvate of a pharmaceutically acceptable salt, wherein the antibody (AB) is a full-length antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof;
    优选地,所述抗体选自抗Her-2抗体、Trop-2抗体、EGFR抗体、B7-H3抗体、PD-1抗体、PD-L1抗体、HER-3、HER-4抗体、CD20抗体、CD30抗体、CD19抗体、CD33抗体;优选地,所述抗体为鼠源抗体、嵌合抗体、人源化抗体;优选地,所述人源化抗体是全人源抗体;Preferably, the antibody is selected from anti-Her-2 antibody, Trop-2 antibody, EGFR antibody, B7-H3 antibody, PD-1 antibody, PD-L1 antibody, HER-3, HER-4 antibody, CD20 antibody, CD30 antibody Antibodies, CD19 antibodies, CD33 antibodies; preferably, the antibodies are murine antibodies, chimeric antibodies, and humanized antibodies; preferably, the humanized antibodies are fully human antibodies;
    优选地,所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv和dsFv; Preferably, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2 , single chain Fv (scFv), Fv and dsFv;
    更优选地,所述抗体为抗TROP-2抗体,其中,所述抗Trop-2抗体的轻链可变区的互补决定区(CDR)包括由KASQDVSIAVA氨基酸序列组成的CDR1,由SASYRYT氨基酸序列组成的CDR2,和由QQHYITPLT氨基酸序列组成的CDR3;重链可变区的CDR包括由NYGMN氨基酸序列组成的CDR1,由WINTYTGEPTYTDDFKG氨基酸序列组成的CDR2,和由GGFGSSYWYFDV氨基酸序列组成的CDR3;优选地,所述抗Trop-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:1和SEQ ID NO:2所示;优选地,所述抗Trop-2抗体的轻链和重链的编码核苷酸序列分别如SEQ ID NO:3和SEQ ID NO:4所示;More preferably, the antibody is an anti-TROP-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Trop-2 antibody comprises the CDR1 consisting of the KASQDVSIVA amino acid sequence and the SASYRYT amino acid sequence. CDR2, and CDR3 composed of QQHYITPLT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of NYGMN amino acid sequence, CDR2 composed of WINTYTGEPTYTDDFKG amino acid sequence, and CDR3 composed of GGFGSSYWYFDV amino acid sequence; Preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Trop-2 antibody are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; preferably, the coding nucleosides of the light chain and heavy chain of the anti-Trop-2 antibody The acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;
    更优选地,所述抗体为抗Her-2抗体,其中,所述抗Her-2抗体的轻链可变区的互补决定区(CDR)包括由RASQDVNTAVA氨基酸序列组成的CDR1,由SASFLYS氨基酸序列组成的CDR2,和由QQHYTTPPT氨基酸序列组成的CDR3;重链可变区的CDR包括由DTYIH氨基酸序列组成的CDR1,由RIYPTNGYTRY氨基酸序列组成的CDR2,和由WGGDGFYAMDY氨基酸序列组成的CDR3;优选地,所述抗Her-2抗体的轻链及重链的氨基酸序列分别如SEQ ID NO:5和SEQ ID NO:6所示。More preferably, the antibody is an anti-Her-2 antibody, wherein the complementarity determining region (CDR) of the light chain variable region of the anti-Her-2 antibody comprises the CDR1 consisting of the RASQDVNTAVA amino acid sequence and the SASFLYS amino acid sequence. CDR2, and CDR3 composed of QQHYTTPPT amino acid sequence; CDRs of heavy chain variable region include CDR1 composed of DTYIH amino acid sequence, CDR2 composed of RIYPTNGYTRY amino acid sequence, and CDR3 composed of WGGDGFYAMDY amino acid sequence; preferably, the The amino acid sequences of the light chain and heavy chain of the anti-Her-2 antibody are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  27. 式(VI)表示的接头-药物中间体化合物,其特征在于,T表示式(II)所示化合物,所述中间体化合物是将化合物(T)与下式(V)表示的接头连接而成的:A linker-drug intermediate compound represented by formula (VI), wherein T represents a compound represented by formula (II), and the intermediate compound is formed by connecting compound (T) and a linker represented by formula (V) below of:
    Q 1-L 1-L 2-L a-T    (VI) Q 1 -L 1 -L 2 -L a -T (VI)
    Figure PCTCN2021123020-appb-100047
    Figure PCTCN2021123020-appb-100047
    Q 1-L 1-L 2-L a-    (V) Q 1 -L 1 -L 2 -L a - (V)
    其中,in,
    R 1、R 2、R 3、R 4的定义如权利要求1-7中任一项所述; The definitions of R 1 , R 2 , R 3 and R 4 are as described in any one of claims 1-7;
    L 1、L 2、L a的定义如权利要求20-23中任一项所述; The definitions of L 1 , L 2 and La are as described in any one of claims 20-23;
    Q 1为式(I)所述化合物,式(I)所述化合物如权利要求1、9-10任一项所限定,其通过R 11的羧基与L 1式中的左端氨基-NR 10-形成酰胺键而连接, Q 1 is the compound of formula (I), and the compound of formula (I) is as defined in any one of claims 1, 9-10, which is through the carboxyl group of R 11 and the left-terminal amino group in the formula of L 1 -NR 10 - connected by forming an amide bond,
    式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分。 The compound represented by the formula (II ) is connected to the above formula ( The right -C( = O)- or -CH2- moiety of La in the linker represented by V).
  28. 权利要求27所述的接头-药物中间体化合物,其中,所述式(II)所示的化合物为权利要求8所述的化合物;优选地,所述接头-药物中间体化合物是选自以下的化合物,The linker-drug intermediate compound of claim 27, wherein the compound represented by the formula (II) is the compound of claim 8; preferably, the linker-drug intermediate compound is selected from the following compound,
    Figure PCTCN2021123020-appb-100048
    Figure PCTCN2021123020-appb-100048
    Figure PCTCN2021123020-appb-100049
    Figure PCTCN2021123020-appb-100049
    Figure PCTCN2021123020-appb-100050
    Figure PCTCN2021123020-appb-100050
    Figure PCTCN2021123020-appb-100051
    Figure PCTCN2021123020-appb-100051
  29. 接头,其特征在于,其为下式(III)表示,A linker is characterized in that it is represented by the following formula (III),
    -Q 1-L 1-L 2-L a-    (III) -Q 1 -L 1 -L 2 -L a - (III)
    其中,Q 1、L 1、L 2、L a的定义如权利要求20-23中任一项所述;优选的是,所述接头为选自权利要求24所示的基团。 Wherein, the definitions of Q 1 , L 1 , L 2 , and La are as described in any one of claims 20 to 23 ; preferably, the linker is selected from the groups shown in claim 24 .
  30. 一种药物组合物,其包含权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物,以及任选的药学上可接受的载体。A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1-16 or 20-26, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug A solvate of a conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable carrier.
  31. 一种药物制剂,其包含权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物。A pharmaceutical preparation comprising the antibody-drug conjugate of any one of claims 1-16 or 20-26, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate A solvate of a conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.
  32. 权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂的用途,其用于预防和/或治疗肿瘤或癌症;The antibody-drug conjugate of any one of claims 1-16 or 20-26, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer Use of a solvate of a body or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or the pharmaceutical preparation of claim 31, for the prevention and/or treatment of tumors or cancer;
    或者,权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要 求30所述的药物组合物和/或权利要求31所述的药物制剂在制备预防和/或治疗肿瘤或癌症的药物中的用途;Or, the antibody-drug conjugate of any one of claims 1-16 or 20-26, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer The solvate of the isomer or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or the pharmaceutical preparation of claim 31 in the preparation of a medicament for preventing and/or treating tumor or cancer use;
    优选地,所述的肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌、肺癌、胰腺癌、结直肠癌。Preferably, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer, bladder cancer , gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
  33. 一种预防或治疗肿瘤或癌症的方法,其包括向有此需要的受试者施用预防或治疗有效量的权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂;A method of preventing or treating tumor or cancer, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-16 or 20-26, Its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a solvate of its stereoisomer or a pharmaceutically acceptable salt thereof, the pharmaceutical combination of claim 30 substance and/or the pharmaceutical formulation of claim 31;
    优选地,所述的肿瘤或癌症选自乳腺癌、结直肠癌、肺癌、胰腺癌、卵巢癌、前列腺癌、宫颈癌、肾癌、尿道癌、胶质细胞瘤、黑色素瘤、肝癌、膀胱癌、胃癌、食道癌;优选地,所述癌症是原位癌或转移癌;优选地,所述乳腺癌为三阴交乳腺癌、肺癌、胰腺癌、结直肠癌。Preferably, the tumor or cancer is selected from breast cancer, colorectal cancer, lung cancer, pancreatic cancer, ovarian cancer, prostate cancer, cervical cancer, kidney cancer, urethral cancer, glioblastoma, melanoma, liver cancer, bladder cancer , gastric cancer, esophageal cancer; preferably, the cancer is carcinoma in situ or metastatic cancer; preferably, the breast cancer is Sanyinjiao breast cancer, lung cancer, pancreatic cancer, and colorectal cancer.
  34. 权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂用于制备试剂的用途,所述试剂用于抑制癌细胞生长、增殖或迁移。The antibody-drug conjugate of any one of claims 1-16 or 20-26, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer Use of a solvate of a body or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or the pharmaceutical formulation of claim 31 for the preparation of an agent for inhibiting the growth of cancer cells , proliferation or migration.
  35. 权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂,其用于抑制癌细胞的生长、增殖或迁移。The antibody-drug conjugate of any one of claims 1-16 or 20-26, its stereoisomer or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, its stereoisomer A solvate of a body or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or the pharmaceutical preparation of claim 31, for use in inhibiting the growth, proliferation or migration of cancer cells.
  36. 一种抑制癌细胞生长、增殖或迁移的方法,其包括给癌细胞施用有效量权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂。A method of inhibiting the growth, proliferation or migration of cancer cells, comprising administering to the cancer cells an effective amount of the antibody-drug conjugate of any one of claims 1-16 or 20-26, a stereoisomer thereof, or a A pharmaceutically acceptable salt, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or claim 31 the pharmaceutical preparation.
  37. 一种抑制癌细胞生长、增殖或迁移的试剂盒,其包括权利要求1-16或20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物、权利要求30所述的药物组合物和/或权利要求31所述的药物制剂。A kit for inhibiting the growth, proliferation or migration of cancer cells, comprising the antibody-drug conjugate of any one of claims 1-16 or 20-26, a stereoisomer thereof or a pharmaceutically acceptable A salt, or a solvate of the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition of claim 30 and/or the pharmaceutical formulation of claim 31 .
  38. 权利要求1-16任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物的制备方法,所述方法包括:The antibody-drug conjugate of any one of claims 1-16, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof A process for the preparation of a solvate of an acceptable salt of the above, comprising:
    Figure PCTCN2021123020-appb-100052
    Figure PCTCN2021123020-appb-100052
    使式(X)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(X)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (X) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (X) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
    其中,R 1、R 2、R 3、R 4的定义如权利要求1-7中任一项所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in any one of claims 1-7;
    Q 2、Q’ 2、L 3、L 4、L P、L b的定义如权利要求1、11-13或17中任一项所述; Q 2 , Q' 2 , L 3 , L 4 , L P , L b are as defined in any one of claims 1 , 11-13 or 17;
    T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(IX)表示的接头中L b的右端-C(=O)-、-O-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site. The oxygen of the above-mentioned formula (IX) is connected to the right end -C(=O)-, -O- or -CH 2 - part of L b in the linker represented by the above formula (IX);
    AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
  39. 权利要求17-18所述的接头-药物中间体化合物的制备方法,所述方法包括:The preparation method of the described linker-drug intermediate compound of claim 17-18, described method comprises:
    方案A:Option A:
    (1)Boc-GGFG与PABOH在EEDQ的作用下,二氯甲烷与甲醇作溶剂,室温搅拌过夜,生成Boc-GGFG-PABOH;(1) Boc-GGFG and PABOH are generated under the action of EEDQ, using dichloromethane and methanol as solvents, and stirring overnight at room temperature to generate Boc-GGFG-PABOH;
    (2)Boc-GGFG-PABOH在TFA/DCM的作用下脱掉Boc生成GGFG-PABOH;(2) Boc-GGFG-PABOH strips off Boc under the action of TFA/DCM to generate GGFG-PABOH;
    (3)GGFG-PABOH与N 3-PEGn-NHS活性酯反应生成N 3-PEGn-GGFG-PABOH,n=0,2,4,6或8; (3) GGFG-PABOH reacts with N 3 -PEGn-NHS active ester to generate N 3 -PEGn-GGFG-PABOH, n=0, 2, 4, 6 or 8;
    (4)吉咪替康-Boc、SN-38-Boc或吉马替康与DMAP,三光气在二氯甲烷的溶剂中反应,加入步骤(3)的N 3-PEGn-GGFG-PABOH,生成N 3-PEGn-GGFG-PABC-吉咪替康-Boc,或N 3-PEGn-GGFG-PABC-SN-38-Boc,或N 3-PEGn-GGFG-PABC-吉马替康, (4) Gematecan-Boc, SN-38-Boc or Gematecan is reacted with DMAP, triphosgene in a solvent of dichloromethane, and N 3 -PEGn-GGFG-PABOH of step (3) is added to generate N 3 -PEGn-GGFG-PABC-Gimantecan-Boc, or N 3 -PEGn-GGFG-PABC-SN-38-Boc, or N 3 -PEGn-GGFG-PABC-Gimantecan,
    (5)步骤(4)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click反应得最终化合物,m=2,4,6,8;或者,(5) The product of step (4) reacts with alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) to obtain the final Compound, m=2, 4, 6, 8; or,
    方案B:Option B:
    (1)Boc-GGFG在TFA/DCM的作用下脱掉Boc,除掉TFA与二氯甲烷后,与N 3-PEGn-NHS在二氯甲烷中反应,用DIEA做碱,得到化合物N 3-PEGn-GGFG,n=0,2,4,6或8; (1) Boc-GGFG removes Boc under the action of TFA/DCM, removes TFA and dichloromethane, reacts with N 3 -PEGn-NHS in dichloromethane, and uses DIEA as a base to obtain compound N 3 - PEGn-GGFG, n=0, 2, 4, 6 or 8;
    (2)N 3-PEGn-GGFG与N-Boc-N-甲基乙二胺缩合,再用TFA/DCM脱掉Boc后,得化合物N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3(2) N 3 -PEGn-GGFG was condensed with N-Boc-N-methylethylenediamine, and then Boc was removed with TFA/DCM to obtain compound N 3 -PEGn-GGFG-NH-C 2 H 4 -NH -CH 3 ;
    (3)N 3-PEGn-GGFG-NH-C 2H 4-NH-CH 3与吉咪替康-PNP(或吉马替康-PNP,SN-38-PNP)在有TEA,DMF的条件下反应得化合物N 3-PEGn-GGFG-NH-C 2H 4-N(CH 3)-C(O)-吉咪替康(或SN-38,或吉马替康), (3) N 3 -PEGn-GGFG-NH-C 2 H 4 -NH-CH 3 and gimatecan-PNP (or gimatecan-PNP, SN-38-PNP) in the condition of TEA, DMF The following reaction is carried out to obtain the compound N 3 -PEGn-GGFG-NH-C 2 H 4 -N(CH 3 )-C(O)-gemitecan (or SN-38, or gematecan),
    (4)步骤(3)产物与炔烃-马来酰亚胺(n=2,4,6,8时)或炔烃-PEGm-马来酰亚胺(n=0)用Click反应得最终化合物,m=2,4,6,8。(4) The product of step (3) reacts with alkyne-maleimide (when n=2, 4, 6, 8) or alkyne-PEGm-maleimide (n=0) to obtain the final Compound, m=2,4,6,8.
  40. 权利要求20-26任一项所述的抗体-药物偶联物、其立体异构体或其药学上可接受的盐,或所述抗体-药物偶联物、其立体异构体或其药学上可接受的盐的溶剂合物的制备方法,所述方法包括:The antibody-drug conjugate of any one of claims 20-26, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or the antibody-drug conjugate, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof A process for the preparation of a solvate of an acceptable salt of the above, comprising:
    Figure PCTCN2021123020-appb-100053
    Figure PCTCN2021123020-appb-100053
    使式(VI)所示的接头-药物中间体化合物与AB-SH反应,以通过由抗体的铰链部的二硫键部分形成的硫醚键将式(VI)所示的接头-药物中间体化合物与抗体连接;The linker-drug intermediate compound represented by the formula (VI) is reacted with AB-SH to connect the linker-drug intermediate represented by the formula (VI) through a thioether bond formed by the disulfide bond moiety of the hinge portion of the antibody The compound is linked to the antibody;
    其中,R 1、R 2、R 3、R 4的定义如权利要求1-7中任一项所述; Wherein, the definitions of R 1 , R 2 , R 3 and R 4 are as described in any one of claims 1-7;
    Q 1、L 1、L 2、L a的定义如权利要求20-23中任一项所述; The definitions of Q 1 , L 1 , L 2 , and La are as described in any one of claims 20-23;
    T表示式(II)所示的化合物,式(II)表示的化合物以19位的羟基中的氧作为连接部位,或者当R 3或R 4为羟基时,以R 3或R 4的羟基中的氧作为连接部位,连接于上述式(V)表示的接头中L a的右端-C(=O)-或-CH 2-部分; T represents the compound represented by the formula (II), and the compound represented by the formula (II) uses the oxygen in the hydroxyl group at the 19th position as the linking site, or when R 3 or R 4 is a hydroxyl group, the oxygen in the hydroxyl group of R 3 or R 4 is used as a linking site. The oxygen of the above-mentioned formula (V) is connected to the right end -C( = O)- or -CH 2 - part of La in the linker represented by the above formula (V);
    AB-SH表示携带巯基的抗体,AB表示抗体。AB-SH represents an antibody carrying a sulfhydryl group, and AB represents an antibody.
  41. 权利要求27-28所述的接头-药物中间体化合物的制备方法,所述方法包括:The preparation method of the described linker-drug intermediate compound of claim 27-28, described method comprises:
    (1)将N-[(9H-芴-9-基甲氧基)羰基]-N'-[(4-甲氧基苯基)二苯基甲基]-L-赖氨酸(CN-CMTC-1)和PABOH溶解在二氯甲烷:甲醇的溶液中,在EEDQ的作用下反应,重结晶纯化得到产物;(1) N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N'-[(4-methoxyphenyl)diphenylmethyl]-L-lysine (CN- CMTC-1) and PABOH were dissolved in dichloromethane: methanol solution, reacted under the action of EEDQ, recrystallized and purified to obtain the product;
    (2)用哌啶乙腈溶液处理步骤(1)产物,而后纯化产物;(2) treating the product of step (1) with a solution of piperidine in acetonitrile, and then purifying the product;
    (3)用DCC,NHS与O-(2-叠氮乙基)-O-[2-(二羟乙酰基-氨基)乙基]七聚乙二醇(CN-CMTC-4)在DMF溶液中反应生成CN-CMTC-4活性酯;(3) Using DCC, NHS and O-(2-azidoethyl)-O-[2-(diglycolyl-amino)ethyl]heptaethylene glycol (CN-CMTC-4) in DMF solution The middle reaction generates CN-CMTC-4 active ester;
    (4)步骤(3)的活性酯与步骤(2)的产物反应生成化合物;(4) the active ester of step (3) reacts with the product of step (2) to generate compound;
    (5)吉咪替康-Boc或吉马替康-Boc用三光气,DMAP,与二氯甲烷作用生成甲酰氯化合物,再加入步骤(4)的反应化合物,后用TFA/DCM脱保护处理;(5) Gematecan-Boc or Gematecan-Boc is treated with triphosgene, DMAP, and dichloromethane to form a formyl chloride compound, and then the reaction compound of step (4) is added, and then deprotected with TFA/DCM. ;
    (6)步骤(5)的产物与式(I)所述的化合物进行Click反应,用TFA/DCM处理后得最终产物,式(I)所述化合物如权利要求1、9-10任一项所限定;(6) The product of step (5) is subjected to Click reaction with the compound of formula (I), and the final product is obtained after treatment with TFA/DCM. The compound of formula (I) is as claimed in any one of claims 1, 9-10 limited;
    任选地,步骤(6)还可用以下步骤替代:步骤(5)的产物与SM-1加入DMSO/H 2O的溶液中,再加入CuBr催化,反应完全,纯化后加入TFA/DCM脱保护,得最终产物; Optionally, step (6) can also be replaced by the following steps: the product of step (5) and SM-1 are added to a solution of DMSO/H 2 O, and then CuBr is added to catalyze the reaction to complete the reaction. After purification, TFA/DCM is added for deprotection , the final product is obtained;
    所述SM-1为The SM-1 is
    Figure PCTCN2021123020-appb-100054
    Figure PCTCN2021123020-appb-100054
  42. 式(I)表示的化合物,A compound represented by formula (I),
    Figure PCTCN2021123020-appb-100055
    Figure PCTCN2021123020-appb-100055
    其中,R 11为羧基取代的C 1-C 6烷基,R 12为氰基取代的C 2-C 6炔基,X、Y、X’和Y’中有1-2个C原子被N原子取代;优选地,R 11为羧基取代的C 1-C 3烷基,R 12为氰基取代的C 2-C 3炔基; Wherein, R 11 is a carboxy-substituted C 1 -C 6 alkyl group, R 12 is a cyano-substituted C 2 -C 6 alkynyl group, and 1-2 C atoms in X, Y, X' and Y' are replaced by N Atom substitution; preferably, R 11 is a carboxy-substituted C 1 -C 3 alkyl group, and R 12 is a cyano-substituted C 2 -C 3 alkynyl group;
    优选地,X、Y、X’和Y’中有且只有1个C原子被N原子取代;Preferably, X, Y, X' and Y' have only 1 C atom substituted by N atom;
    优选地,X、Y、X’和Y’中有2个C原子被N原子取代,且X、Y中有且只有1个C原子被N原子取代,以及X’、Y’中有且只有1个C原子被N原子取代;Preferably, 2 C atoms in X, Y, X' and Y' are substituted by N atoms, and in X, Y, and only 1 C atom is substituted by N atoms, and in X', Y', and only 1 1 C atom is replaced by N atom;
    优选地,式(I)表示的化合物结构如下所示,Preferably, the structure of the compound represented by formula (I) is shown below,
    Figure PCTCN2021123020-appb-100056
    Figure PCTCN2021123020-appb-100056
    优选地,式(I)表示的化合物作为抗体-药物偶联物中的连接单元,通过R 12的炔基碳与存在于抗体的铰链部的二硫键部分形成硫醚键而与抗体连接。 Preferably, the compound represented by formula (I) is linked to the antibody by forming a thioether bond between the alkynyl carbon of R 12 and the disulfide bond moiety present in the hinge portion of the antibody as a linking unit in the antibody-drug conjugate.
  43. 权利要求42所述化合物的制备方法,所述方法包括:The preparation method of the described compound of claim 42, described method comprises:
    (1)让5-溴吡啶-2-羧酸在Boc 2O,DMAP,t-BuOH的作用下反应;所述5-溴吡啶-2-羧酸可替换为6-溴烟酸或5-溴嘧啶-2-羧酸; (1) Let 5-bromopyridine-2-carboxylic acid react under the action of Boc 2 O, DMAP, t-BuOH; the 5-bromopyridine-2-carboxylic acid can be replaced with 6-bromonicotinic acid or 5-bromonicotinic acid bromopyrimidine-2-carboxylic acid;
    (2)步骤(1)的反应产物与Pd(PPh 3) 2Cl 2,三乙胺,丙炔-3-醇在四氢呋喃中反应; (2) The reaction product of step (1) reacts with Pd(PPh 3 ) 2 Cl 2 , triethylamine, and propyn-3-ol in tetrahydrofuran;
    (3)步骤(2)的反应产物与TEMPO,PhI(OAC) 2,NH 4OAC在CH 3CN/H 2O为9:1的溶液中反应; (3) The reaction product of step (2) reacts with TEMPO, PhI(OAC) 2 , and NH 4 OAC in a solution of CH 3 CN/H 2 O in a ratio of 9:1;
    (4)步骤(3)产物在TFA/DCM的作用下生成产物。(4) The product of step (3) is produced under the action of TFA/DCM.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11607459B1 (en) 2020-09-30 2023-03-21 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
US11814394B2 (en) 2021-11-16 2023-11-14 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2024109840A1 (en) * 2022-11-22 2024-05-30 康诺亚生物医药科技(成都)有限公司 Fused ring compound, conjugate thereof and use thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016030791A1 (en) * 2014-08-28 2016-03-03 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
CN106188293A (en) * 2015-04-17 2016-12-07 江苏恒瑞医药股份有限公司 Anti-C-met antibodies and anti-C-met antibodies-cytotoxic drug conjugate and medical usage thereof
CN109562189A (en) * 2016-05-17 2019-04-02 艾伯维生物制药股份有限公司 Anti- cMet antibody drug conjugates and its application method
US20190241649A1 (en) * 2013-10-11 2019-08-08 Asana Biosciences, Llc Protein-polymer-drug conjugates
CN110234357A (en) * 2016-11-23 2019-09-13 梅尔莎纳医疗公司 Contain peptide linker for antibody-drug conjugates
WO2020092385A1 (en) * 2018-10-29 2020-05-07 Mersana Therapeutics, Inc. Cysteine engineered antibody-drug conjugates with peptide-containing linkers

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190241649A1 (en) * 2013-10-11 2019-08-08 Asana Biosciences, Llc Protein-polymer-drug conjugates
WO2016030791A1 (en) * 2014-08-28 2016-03-03 Pfizer Inc. Stability-modulating linkers for use with antibody drug conjugates
CN106188293A (en) * 2015-04-17 2016-12-07 江苏恒瑞医药股份有限公司 Anti-C-met antibodies and anti-C-met antibodies-cytotoxic drug conjugate and medical usage thereof
CN109562189A (en) * 2016-05-17 2019-04-02 艾伯维生物制药股份有限公司 Anti- cMet antibody drug conjugates and its application method
CN110234357A (en) * 2016-11-23 2019-09-13 梅尔莎纳医疗公司 Contain peptide linker for antibody-drug conjugates
WO2020092385A1 (en) * 2018-10-29 2020-05-07 Mersana Therapeutics, Inc. Cysteine engineered antibody-drug conjugates with peptide-containing linkers

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11607459B1 (en) 2020-09-30 2023-03-21 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
US11685742B2 (en) 2020-09-30 2023-06-27 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
US11952384B2 (en) 2020-09-30 2024-04-09 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
US12091418B2 (en) 2020-09-30 2024-09-17 Duality Biologics (Suzhou) Co., Ltd. Anti-tumor compound and preparation method and use thereof
US11814394B2 (en) 2021-11-16 2023-11-14 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
US11999748B2 (en) 2021-11-16 2024-06-04 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2024109840A1 (en) * 2022-11-22 2024-05-30 康诺亚生物医药科技(成都)有限公司 Fused ring compound, conjugate thereof and use thereof

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