WO2023078230A1 - 一种包含sn38的抗体药物偶联物中间体及其制备方法 - Google Patents

一种包含sn38的抗体药物偶联物中间体及其制备方法 Download PDF

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WO2023078230A1
WO2023078230A1 PCT/CN2022/128912 CN2022128912W WO2023078230A1 WO 2023078230 A1 WO2023078230 A1 WO 2023078230A1 CN 2022128912 W CN2022128912 W CN 2022128912W WO 2023078230 A1 WO2023078230 A1 WO 2023078230A1
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reaction
solvent
stir
appropriate time
spin
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French (fr)
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黄长江
熊就凯
闫新新
于红霞
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烟台迈百瑞国际生物医药股份有限公司
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Priority to KR1020247006361A priority Critical patent/KR20240040099A/ko
Priority to AU2022383308A priority patent/AU2022383308A1/en
Priority to EP22836026.9A priority patent/EP4201429A1/en
Priority to CA3228214A priority patent/CA3228214A1/en
Priority to CN202280005164.6A priority patent/CN116018160A/zh
Priority to JP2024516810A priority patent/JP2024533562A/ja
Publication of WO2023078230A1 publication Critical patent/WO2023078230A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to the field of antibody-drug conjugates, in particular to an antibody-drug conjugate intermediate and a preparation method thereof.
  • ADC Antibody-Drug Conjugate
  • ADC Antibody-Drug Conjugate
  • the three components of ADC together form a targeted drug delivery system, in which the antibody achieves targeting, and the linker ensures the stability of the ADC during blood transport, and after reaching the target,
  • the toxin exerts its killing effect on cancer cells.
  • ADC drugs are advancing clinical trials for anti-tumor treatment, most of which are tubulin inhibitors, and a small part are DNA inhibitors.
  • DNA inhibitors Compared with tubulin inhibitors, DNA inhibitors have two advantages: 1) DNA inhibitors (picomolar IC 50 values) have higher activity than tubulin inhibitors (sub-nanomolar IC 50 values), and are less effective in the treatment of antigens. 2) It can kill cancer cells that are not in the division stage, and has advantages in the treatment of solid tumors.
  • Camptothecin compounds are clinically used topoisomerase 1 (TOP1) inhibitors, and have good effects on clinically slow-growing solid tumors.
  • TOP1 topoisomerase 1
  • ADC drugs with camptothecin as warhead provide a new solution to solve the above defects.
  • Enhertu trastuzumab deruxtecan
  • Trodelvy Stacituzumab govitecan
  • Enhertu developed by AstraZeneca/Daiichi Sankyo uses the GGFG tetrapeptide activated by cathepsin B as a linker, and introduces a small self-cleavage structure, which releases the derivative Dxd of Exatecan.
  • the objective response rate of 99 patients treated with Enhertu was 54.5%, and the disease control rate was 93.9%.
  • the linker of sacituzumab govitecan is Mcc-triazole spacer-PEG7-x-lysine-PABC, which decomposes and releases camptothecin (SN38) in the lysosome (pH around 5) in the cell.
  • the clinical phase II results of sacituzumab govitecan for triple-negative breast cancer showed that its effective rate was as high as 30%, and 69.5% of the patients’ tumors shrank. However, there is almost no cure for triple-negative breast cancer clinically.
  • Sacituzumab govitecan can shrink the tumors of 60% of patients; for non-small cell lung cancer that failed chemotherapy, targeted and PD-1 therapy, the control rate of Sacituzumab govitecan was as high as 43%.
  • ADC toxicity such as hematological toxicity, neurotoxicity, pulmonary toxicity, skin toxicity, liver toxicity, eye toxicity, metabolic abnormalities, and cardiotoxicity. Therefore, improving the safety of ADC drugs is a problem that needs to be highly concerned and solved in current drug development.
  • Linker plays a crucial role in the structure of ADC, which will affect the pharmacokinetic parameters, therapeutic index and efficacy of ADC.
  • Linker can maintain the stability of the ADC complex in the blood stream.
  • the design of the linker should first consider the stability, because the half-life of the ADC drug is relatively long, so the linker needs to be stable in order to prevent the linker from being destroyed in the blood. Breaks down to release toxins early. If the linker is not stable enough in the blood, it will cause the ADC to be decomposed before entering the tumor cells, and the drug's effect on the tumor will be reduced, and even other cells will be killed by mistake.
  • the Linker should be able to rapidly release the cytotoxic drug during the process of ADC internalization by tumor cells.
  • the linker plays a significant role in determining the safety and effectiveness of the developed ADC drug. And depending on the structure of the linker, it also greatly affects the difficulty factor, production cost and environmental friendliness of the ADC preparation process, and greatly affects the quality of the ADC drug, such as the stability of the ADC, which in turn will affect the ADC drug. security.
  • the invention provides an antibody-drug conjugate intermediate containing SN38 and a preparation method thereof.
  • the preparation method has simple steps, saves costs, does not introduce heavy metal ions in the reaction, and has higher environmental friendliness.
  • the antibody-drug conjugate intermediate synthesized by the preparation method has good stability, thereby significantly improving the safety of ADC drugs.
  • the present invention relates to an antibody-drug conjugate intermediate as shown in formula (I):
  • X 1 is an alkane chain or a PEG chain
  • X 2 is H or -C(O)NR 1 R 2 , and,
  • R 1 is selected from hydrogen, halogen, hydroxyl, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted
  • R is selected from hydrogen and substituted or unsubstituted C 1-6 alkyl; wherein each substituted C 1-6 alkyl is optionally substituted by 1-5 substituents independently selected from: halogen, hydroxyl, amino , carbonyl, carboxyl, 5-10 membered heterocyclic group and C 1-6 haloalkyl, wherein the 5-10 membered heterocyclic group has 1-3 heteroatoms selected from oxygen, nitrogen and sulfur.
  • said X 1 is selected from: -(CH 2 ) m -or -(CH 2 CH 2 O) p -, wherein said m is selected from 1, 2, 3, 4, 5, 6, preferably Wherein, m is 5; wherein said p is selected from 1, 2, 3, 4, 5, 6, preferably, p is 2.
  • the X is selected from:
  • the R 1 or R 2 is selected from: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, Cl, Br,
  • described X 2 is:
  • the structure of the antibody drug conjugate intermediate is shown in formulas (1)-(16):
  • the invention also provides a preparation method of an antibody drug conjugate intermediate.
  • the R 1 is selected from hydrogen, substituted or unsubstituted C 1-6 alkyl, substituted or unsubstituted C 1-6 hydroxyalkyl, substituted or unsubstituted C 1-6 aminoalkyl, substituted or unsubstituted Substituted C 1-6 alkoxy, substituted or unsubstituted C 1-6 alkanoyl and substituted or unsubstituted C 1-6 alkanal;
  • the R 2 is selected from hydrogen and substituted or unsubstituted C 1-6 alkyl; wherein each substituted C 1-6 alkyl is optionally substituted by 1-5 substituents independently selected from the following: halogen, Hydroxy, amino, carbonyl, carboxyl, 5-10 membered heterocyclic group and C 1-6 haloalkyl, wherein the 5-10 membered heterocyclic group has 1-3 heteroatoms selected from oxygen, nitrogen and sulfur.
  • R 1 or R 2 are selected from: H, methyl, ethyl, propyl, butyl, pentyl, heptyl, methoxy, ethoxy, Cl, Br,
  • Reaction A Dissolve compound a and compound b in a solvent, stir at room temperature for an appropriate time, then add a reducing agent at low temperature, stir for an appropriate time, and then stir at room temperature overnight. After the reaction, spin the solvent and extract , drying and purification;
  • Reaction B Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, spin the solvent to dry after the reaction, and perform beating and filtration;
  • Reaction C Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and spin the solvent to dry after the reaction and perform purification;
  • Reaction D Dissolve the product of Reaction C in the solvent, add acid, and stir for an appropriate time under low temperature conditions. time, add an organic base, and after the reaction is completed, the solvent is spin-dried and purified.
  • Reaction A Dissolve compound a and compound b in a solvent, stir at room temperature for an appropriate time, place at low temperature, add a reducing agent, stir for an appropriate time, then place at room temperature and stir overnight, spin the solvent to dry after the reaction, extract and dry and purification;
  • Reaction B Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, spin the solvent to dry after the reaction, and perform beating and filtration;
  • Reaction C Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and spin the solvent to dry after the reaction and perform purification;
  • Reaction D Dissolve the product of reaction C in the solvent, add acid, and stir for an appropriate time under low temperature conditions. After the reaction is completed, spin the solvent and dissolve it in the solvent with MP2-VC-PAB-PNP, and stir for an appropriate time under low temperature conditions , adding an organic base, and after the reaction, the solvent was spin-dried and purified.
  • Reaction A Dissolve compound a and compound b in a solvent, stir at room temperature for an appropriate time, place at low temperature, add a reducing agent, stir for an appropriate time, and then stir at room temperature overnight. After the reaction, spin the solvent to dry and perform extraction. drying and purification;
  • Reaction B Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, spin the solvent to dry after the reaction, and perform beating and filtration;
  • Reaction C Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and spin the solvent to dry after the reaction and perform purification;
  • Reaction D Dissolving the product of Reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, stirring at constant temperature for an appropriate time, spinning the solvent to dry after the reaction and purifying;
  • Reaction E Dissolve the product of Reaction D and the amine compound in a solvent, add an organic base, and stir for an appropriate time under low temperature conditions. After the reaction, spin the solvent to dry and perform purification;
  • Reaction F Dissolve the product of reaction E in the solvent, add acid, and stir for an appropriate time under low temperature conditions. After the reaction is completed, spin the solvent and dissolve it in the solvent with Mc-VC-PAB-PNP, and stir for an appropriate time under low temperature conditions , adding an organic base, and after the reaction, the solvent was spin-dried and purified.
  • Reaction A Dissolve compound a and compound b in a solvent, stir at room temperature for an appropriate time, place at low temperature, add a reducing agent, stir for an appropriate time, and then stir at room temperature overnight. After the reaction, spin the solvent to dry and perform extraction. drying and purification;
  • Reaction B Dissolve SN38 and DNPC in a solvent, add an organic base, stir at room temperature for an appropriate time, spin the solvent to dry after the reaction, and perform beating and filtration;
  • Reaction C Dissolve the product of reaction B in a solvent, add the product of reaction A and an organic base, stir at room temperature for an appropriate time, and spin the solvent to dry after the reaction and perform purification;
  • Reaction D Dissolving the product of Reaction C in a solvent, adding bis(p-nitrophenyl)carbonate and an organic base, stirring at constant temperature for an appropriate time, spinning the solvent to dry after the reaction and purifying;
  • Reaction E Dissolve the product of Reaction D and the amine compound in a solvent, add an organic base, and stir for an appropriate time under low temperature conditions. After the reaction, spin the solvent to dry and perform purification;
  • Reaction F Dissolve the product of Reaction E in the solvent, add acid, and stir for an appropriate time under low temperature conditions. After the reaction is completed, spin the solvent and dissolve it in the solvent with MP2-VC-PAB-PNP, and stir for an appropriate time under low temperature conditions , adding an organic base, and after the reaction, the solvent was spin-dried and purified.
  • compound (I-1) is selected from:
  • Described compound (I-2) is selected from:
  • the solvents described in any of the above reaction processes can be independently polar solvents and/or non-polar solvents, and the polar solvents are one or more of THF, DMF, DMA, NMP;
  • the non-polar solvent described is one or more of dichloromethane and carbon tetrachloride.
  • organic base described in any of the above reaction processes can be independently one or more of N, N-diisopropylethylamine, triethylamine, pyridine, preferably N, N-diisopropyl One or both of ethylethylamine and pyridine.
  • the acid is one or both of hydrochloric acid, trifluoroacetic acid and citric acid.
  • amine compounds are primary or secondary amines.
  • reaction B one or more combinations of ethyl acetate, n-hexane, and dichloromethane are used for beating.
  • reaction C column chromatography is used for purification, and the eluents are dichloromethane and methanol.
  • reaction D column chromatography is used for purification, and the eluents are dichloromethane and methanol.
  • reaction E column chromatography is used for purification, and the eluents are dichloromethane and methanol.
  • the mobile phase A is MeCN, 0.1% HCOOH, and the mobile phase B is H 2 O, 0.1% HCOOH.
  • the method for preparing an antibody-drug conjugate intermediate (specifically, a linker-SN38 covalent conjugate) provided by the present invention has simple steps in the preparation method, reduces safety problems caused by heavy metal residues, and uses the antibody Antibody-drug conjugates prepared from drug conjugate intermediates will have higher stability in vivo.
  • the inventors surprisingly found that the antibody-drug conjugate prepared by combining the linker of the present invention with SN38 can produce a significant tumor-inhibiting effect.
  • Fig. 1 is the change of body weight of mice
  • Figure 2 shows the change of tumor volume
  • an Antibody Drug Conjugate includes combinations of two or more Antibody Drug Conjugates, and the like.
  • antibody-drug conjugate refers to a compound in which an antibody/antibody functional fragment, a linker, and a drug part are linked together through a chemical reaction, and its structure usually consists of three parts: antibody or antibody-like ligand, The drug part, and the linker (linker) that couples the antibody or antibody-like ligand and the drug.
  • the preparation of antibody-drug conjugates is usually divided into two steps: the first step is to chemically react the linker and the drug part to form a "linker-drug" conjugate; the second step is to combine the "linker-drug"
  • the linker part in the conjugate is covalently coupled with the antibody/antibody functional fragment through sulfhydryl or amino groups.
  • antibody-drug conjugate intermediate used in the present invention refers to the above-mentioned “linker-drug” conjugate, and further, the “antibody-drug conjugate intermediate” referred to in the present invention generally refers to " Linker” and SN38 covalent conjugates.
  • the "antibody-drug conjugate” used in the present invention is prepared by a general preparation method in the field.
  • HIC-HPLC hydrophobic chromatography
  • the present invention aims to provide a new drug conjugate intermediate, and exemplarily prepare multiple ADCs with Heceptin to verify the technical effect.
  • the selection of antibodies does not limit this patent, and exemplarily gives The structure of the specific ADC is shown, wherein the linker and toxin in ADC-1 correspond to the compound of formula (1) used and Heceptin prepared by using the above-mentioned general preparation method of "antibody drug conjugate" in this field (the same below , the linker and toxin in ADC-2 correspond to the compound of formula (2); the linker and toxin in ADC-3 correspond to the compound of formula (3); the linker and toxin in ADC-4 correspond to the formula (4) Compound; Linker and toxin in ADC-5 are corresponding to formula (5) compound; Linker and toxin in ADC-6 are corresponding to formula (6) compound; Linker and toxin in ADC-7 are corresponding to formula (7) ) compound; the linker and toxin in ADC-8 correspond to formula (8); the linker and
  • linking group in the present invention refers to a molecule with bifunctional or multifunctional groups, which can react with protein/antibody molecule and SN38 respectively, thus serving as a "bridge” to connect protein/antibody to SN38.
  • the linking groups involved in the present invention specifically refer to those groups containing acyl structures in their structures.
  • Compound g (10-(2-N-methylethylethylene glycol)-camptothecin trifluoroacetate) was prepared according to the synthetic route of Example 1: Compound d (73.0 mg) was dissolved in 3 mL of dichloromethane and 3 mL of trifluoroacetic acid, placed in an ice-water bath, and stirred at constant temperature for 1 h. Detected by LC-MS method, the reaction ended. After the reaction was completed, the solvent was spin-dried, and then 2 mL of dichloromethane was added, spin-dried, and stripped twice. The collected product was monitored by LC-MS to obtain compound g (70 mg), which was directly put into the next reaction. LC-MS: (M+H) + 580.6.
  • the compound of formula (4) maleimide caproic acid-L-valyl-L-citrulline-p-aminobianyl-(2-N-methylethylethylenedimethoxy) Alcohol)-camptothecin: Mc-VC-PAB-PNP (maleimide caproic acid-L-valyl-L-citrulline-p-aminobianyl-p-nitrophenyl carbonate) (58mg), compound g (42mg) was dissolved in 2.5mL N,N-dimethylformamide, stirred in an ice-water bath for 30mins, DIPEA (42mg) was added dropwise, and stirred in an ice-water bath for 1h.
  • Mc-VC-PAB-PNP maleimide caproic acid-L-valyl-L-citrulline-p-aminobianyl-p-nitrophenyl carbonate
  • Mc-VC-PAB-PNP maleimide caproic acid-L-valyl-L-citrulline-p-aminobianyl-p-nitrophenyl carbonate
  • 35 mg of compound j 35 mg
  • 129 mg of DIPEA dropwise, and stir in an ice-water bath for 0.5 h.
  • the reaction was not complete as detected by LC-MS method.
  • 129 mg of DIPEA stir at room temperature for 0.5 h, the reaction solution turns yellow, and the reaction is completed by LC-MS detection.
  • the compound of formula (9) maleimidediethoxy-L-valyl-L-citrulline-p-aminobianyl-(N-methylethyldiethyl) Oxy-N,N,N-trimethylethylenediamine)-camptothecin: MP2-VC-PAB-PNP (maleimidediethoxy-L-valyl-L-citrul Amino acid-p-aminobianyl-p-nitrophenyl carbonate) (52mg), compound j (34mg) was dissolved in 2.5mL N,N-dimethylformamide, stirred in an ice-water bath for 30mins, and DIPEA ( 36mg), stirred in an ice-water bath for 0.5h.
  • MP2-VC-PAB-PNP maleimidediethoxy-L-valyl-L-citrul Amino acid-p-aminobianyl-p-nitrophenyl carbonate
  • the compound of formula (1) and the CL2A-SN38 intermediate were subjected to thiol coupling with Heceptin by conventional methods in the art (for example, see Example 12 in Chinese Patent Application Publication No. CN102448494A), and an ADC with an average DAR value of 8 was prepared- 1 and HER2-CL2A-SN38 (ADC-17) antibody-drug conjugate.
  • NCI-N87 cells were digested with trypsin and adjusted to a cell density of 50,000 cells/ml, 100 ⁇ L/well was added to a cell culture plate, and incubated in a 5% CO 2 incubator at 37° C. for 14-20 h.
  • the sample group and the control group were serially diluted with the basal medium, and transferred to a cell culture plate with cells at 100 ⁇ L/well; at 37 ° C, 5% CO 2 Incubate in the incubator for 70-74h. Dilute CCK-8 10 times with medium, suck out the old medium in the 96-well plate, add 100 ⁇ L of diluted CCK-8 solution to each well, develop color for 2-4 hours under 5% CO 2 conditions, and centrifuge to remove air bubbles The measurement wavelength 450nm/655nm was selected on the microplate reader for reading, and the results are shown in Table 2.
  • Embodiment 6 Stability experimental research
  • the compound of formula (1) prepared by the present invention has higher stability under weakly acidic or neutral conditions in vivo, which is expected to significantly improve the safety of ADC drugs.
  • the BT474 cell line was resuscitated and propagated for 1-2 passages. After the cells grew in a stable state, they were expanded and cultured to prepare a tumor cell suspension. 0.2* 107 BT474 cells were inoculated into the nude mouse cream pad, and a total of 60 female BALB/c nude mice were inoculated. When the average tumor volume reached about 70 mm 3 , 42 mice were selected and randomly divided into 6 groups according to the tumor volume: the negative control group was control group 1 (normal saline group), control group 2 (ADC-17), experimental group 1 (ADC-4 ), experimental group 2 (ADC-12), experimental group 3 (ADC-1), experimental group 4 (ADC-9), 7 rats in each group. After the model is created successfully.
  • control group 1 was given intravenous injection of normal saline, and the rest of the groups were given intravenous injection of drugs. 3 5mg/kg, experimental group 4 5mg/kg, once a week for 21 weeks. Tumor volume was weighed and measured 3 times a week, and the tumor volume was weighed and measured on the 21st day.
  • each group was compared with the control group 1, and the control group 2
  • the tumor volume of control group 1 reached 107 ⁇ 19.30 mm 3
  • the tumor volumes of control group 2, experimental group 1, experimental group 2, experimental group 3, and experimental group 4 were 49.17 ⁇ 13.29 and 57.12 mm respectively. ⁇ 20.28, 66.26 ⁇ 21.37, 61.32 ⁇ 12.82, 75.76 ⁇ 46.18, 75.19 ⁇ 25.99mm 3 (see Table 6), in which the control group 2, the experimental group 1, the experimental group 2, the experimental group 3 four groups and the control group 1 (physiological Saline group) the statistically significant difference (P ⁇ 0.01), the statistically significant difference between the experimental group 4 and the control group 1 (P ⁇ 0.05).
  • the ADCs constructed using the intermediates of the drug conjugates of the present invention exhibit significant antitumor activity on the breast cancer BT474 cell model, and preferred ADC-1, ADC-4, and ADC-12 have obvious advantages, and are expected to Under the condition that the safety will be significantly improved under in vivo conditions, it can also have a tumor inhibitory effect comparable to that of the ADC-17 group.

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Abstract

一种包含SN38的抗体药物偶联物中间体及其制备方法，在其连接子结构上引入具有生物活性的水溶性基团PEG并在PEG上进行结构修饰，该制备方法步骤简单，节约成本，在反应中不会引入重金属离子，具有更高的环境友好性，并且采用该制备方法合成的抗体药物偶联物中间体与现有技术中毒物为SN38的抗体药物偶联物中间体相比具有更高的稳定性和药效。

Description

一种包含SN38的抗体药物偶联物中间体及其制备方法 技术领域
本发明涉及抗体药物偶联物领域，具体涉及一种抗体药物偶联物中间体及其制备方法。
背景技术
抗体偶联药物(Antibody-Drug Conjugate，ADC)作为一种新型的生物靶向药物，实现了单抗靶向作用和小分子药物细胞毒性的强强联合，现已成为肿瘤靶向治疗发展最快的领域之一。ADC的三个组成单元(抗体、细胞毒素和连接子)共同组成靶向药物递送系统，其中抗体实现靶向性，连接子保证ADC在血液转运过程中的稳定性，而到达作用靶点后，毒素发挥对癌细胞的杀伤作用。目前有60多种ADC药物正在推进抗肿瘤治疗临床试验，其中绝大多数毒素为微管蛋白抑制剂，少部分为DNA抑制剂。DNA抑制剂相比较微管蛋白抑制剂有两个优势：1)DNA抑制剂(picomolar IC 50 values)比微管蛋白抑制剂(sub-nanomolar IC 50 values)有更高活性，在治疗较少抗原的肿瘤中起到更好的作用；2)能够杀死不在分裂期的癌症细胞，在进行实体瘤治疗方面具有优势。
喜树碱类化合物是临床中应用的拓扑异构酶1(TOP1)抑制剂，对临床上生长缓慢的实体瘤有良好的效果。但是喜树碱的特殊结构导致其水溶性及脂溶性都很差，必须进行水溶性改造。以喜树碱为弹头的ADC药物为解决上述缺陷提供了新的方案。
目前已有2款以喜树碱衍生物为弹头的ADC药物获批上市，为Enhertu(trastuzumab deruxtecan)和Trodelvy(Sacituzumab govitecan)，它们在治疗肿瘤尤其是恶性肿瘤方面满足了极大的临床需求。阿斯利康/第一三共研发的Enhertu以组织蛋白酶B活化的GGFG四肽作为linker，并引入一小段自裂解结构，释放出来的是Exatecan的衍生物Dxd。在HER2阳性转移性乳腺癌中，99例患者使用Enhertu的客观缓解率为54.5％，疾病控制率为93.9％。Sacituzumab govitecan的连接子为Mcc-triazole spacer-PEG7-x-lysine-PABC，在细胞中溶酶体(pH在5左右)中分解释放喜树碱(SN38)。Sacituzumab govitecan的三阴乳腺癌临床二期结果显示，其有效率高达30％，69.5％的患者肿瘤都出现了缩小，而三阴乳腺癌临床几乎是“无药可治”。此外，针对多线治疗失败的小细胞 肺癌，Sacituzumab govitecan可以使60％的患者肿瘤缩小；针对化疗、靶向和PD-1治疗都失败的非小细胞肺癌，Sacituzumab govitecan的控制率高达43％。但临床试验中，尽管治疗效果显著，但同时也存在较为严重的ADC毒性引发的安全性问题，如血液系统毒性、神经毒性、肺毒性、皮肤毒性、肝毒性、眼毒性、代谢异常、心脏毒性等，因此改善ADC药物的安全性是目前药物开发中需要高度关注和解决的问题。
此外，部分ADC的连接子及载荷的合成中也存在金属离子反应产物不便于去除问题，如Sacituzumab govitecan所用到的CL2A-SN38在合成过程中涉及到点击化学反应(Click反应)(参见中国专利申请公开号CN102448494A，说明书第50页第[0273]段)，因此会导致该反应产物中含有Cu 2+，且不易去除。
连接子(Linker)在ADC结构中起着至关重要的作用，其会影响ADC的药代动力学参数、治疗指数和药效。Linker可维持ADC复合物在血流中的稳定性，连接子的设计首先要考虑到稳定性，因为ADC药物的半衰期比较长，所以需要连接子的性质稳定，这样才能防止连接子在血液中被分解提前释放毒素。如果连接子在血液中不够稳定，就会导致ADC在进入肿瘤细胞之前就被分解，药物对肿瘤的打击作用就会降低，甚至会误杀其他细胞。而且，在肿瘤细胞内化ADC的过程中，Linker应能够快速释放细胞毒性药物。由此，连接子对于开发的ADC药品的安全性、有效性具有显著的决定作用。并且根据连接子结构的不同，也极大的影响ADC的制备过程的难度系数、生产成本以及环境友好性，并且极大的影响ADC药品的质量，如ADC的稳定性等，进而会影响ADC药物的安全性。
发明内容
本发明提供了一种包含SN38的抗体药物偶联物中间体及其制备方法，该制备方法步骤简单，节约成本，在反应中不会引入重金属离子，具有更高的环境友好性，并且采用该制备方法合成的抗体药物偶联物中间体具有良好的稳定性，从而显著提升ADC药物的安全性。
本发明涉及一种如式(Ⅰ)所示的抗体药物偶联物中间体：
Figure PCTCN2022128912-appb-000001
其中：
X 1为烷烃链或PEG链，
X 2为H或-C(O)NR 1R 2，且，
R 1选自氢、卤素、羟基、取代或非取代的C 1-6烷基、取代或非取代的
C 1-6羟烷基、取代或非取代的C 1-6氨基烷基、取代或非取代的C 1-6烷氧基、取代或非取代的C 1-6烷酰基和取代或非取代的C 1-6烷醛基；
R 2选自氢和取代或非取代的C 1-6烷基；其中各取代的C 1-6烷基任选被1-5个独立地选自以下的取代基取代：卤素、羟基、氨基、羰基、羧基、5-10元杂环基和C 1-6卤代烷基，其中所述5-10元杂环基具有1-3个选自氧、氮和硫的杂原子。
优选的，所述的X 1选自：-(CH 2) m-或-(CH 2CH 2O) p-，其中所述的m选自1、2、3、4、5、6，优选的，m为5；其中所述的p选自1、2、3、4、5、6，优选的，p为2。
优选的，所述X 1选自：
Figure PCTCN2022128912-appb-000002
优选的，所述的R 1或R 2选自：H、甲基、乙基、丙基、丁基、戊基、庚基、Cl、Br、
Figure PCTCN2022128912-appb-000003
优选的，所述的X 2为：
Figure PCTCN2022128912-appb-000004
优选的，所述的抗体药物偶联物中间体的结构如式(1)-(16)所示：
Figure PCTCN2022128912-appb-000005
Figure PCTCN2022128912-appb-000006
Figure PCTCN2022128912-appb-000007
Figure PCTCN2022128912-appb-000008
Figure PCTCN2022128912-appb-000009
本发明还提供了一种抗体药物偶联物中间体的制备方法。
进一步的，所述的抗体药物偶联物中间体的结构为：
Figure PCTCN2022128912-appb-000010
Figure PCTCN2022128912-appb-000011
所述的R 1选自氢、取代或非取代的C 1-6烷基、取代或非取代的C 1-6羟烷基、取代或非取代的C 1-6氨基烷基、取代或非取代的C 1-6烷氧基、取代或非取代的C 1-6烷酰基和取代或非取代的C 1-6烷醛基；
所述的R 2选自氢和取代或非取代的C 1-6烷基；其中各取代的C 1-6烷基任选被1-5个独立地选自以下的取代基取代：卤素、羟基、氨基、羰基、羧基、5-10元杂环基和C 1-6卤代烷基，其中所述5-10元杂环基具有1-3个选自氧、氮和硫的杂原子。
优选的，R 1或R 2选自：H、甲基、乙基、丙基、丁基、戊基、庚基、甲氧基、乙氧基、Cl、Br、
Figure PCTCN2022128912-appb-000012
Figure PCTCN2022128912-appb-000013
进一步的，所述的式(4)化合物的制备方法为：
反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低 温条件下加入还原剂，再搅拌适当时间后，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应D：将反应C的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与Mc-VC-PAB-PNP一起溶于溶剂中，低温条件下再搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
反应进程1：
Figure PCTCN2022128912-appb-000014
进一步的，所述的式(12)化合物的制备方法为：
反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应D：将反应C的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与MP2-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行 纯化。
反应进程2：
Figure PCTCN2022128912-appb-000015
进一步的，所述的化合物(I-1)的制备方法为：
反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下中加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应D：将反应C的产物溶于溶剂中，加入二(对硝基苯)碳酸酯和有机碱，恒温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应E：将反应D的产物和胺类化合物溶于溶剂中，加入有机碱，低温条件下搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应F：将反应E的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与Mc-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
反应进程3：
Figure PCTCN2022128912-appb-000016
进一步的，所述的化合物(I-2)的制备方法为：
反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下中加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应D：将反应C的产物溶于溶剂中，加入二(对硝基苯)碳酸酯和有机碱，恒温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应E：将反应D的产物和胺类化合物溶于溶剂中，加入有机碱，低温条件下搅拌适当时间，反应结束后旋干溶剂并进行纯化；
反应F：将反应E的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与MP2-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
反应进程4：
Figure PCTCN2022128912-appb-000017
进一步的，所述的化合物(I-1)选自：
Figure PCTCN2022128912-appb-000018
Figure PCTCN2022128912-appb-000019
Figure PCTCN2022128912-appb-000020
所述的化合物(I-2)选自：
Figure PCTCN2022128912-appb-000021
Figure PCTCN2022128912-appb-000022
进一步的，上述任一反应进程中所述的“低温条件下”为冰水浴下。
进一步的，上述任一反应进程中所述的溶剂可独立地为极性溶剂和/或非极性溶剂，所述的极性溶剂为THF、DMF、DMA、NMP的一种或几种；所述的非极性溶剂为二氯甲烷、四氯化碳的一种或几种。
进一步的，上述任一反应进程中所述的有机碱可独立地为N，N-二异丙基乙胺、三乙胺、吡啶的一种或几种，优选为N，N-二异丙基乙胺以及吡啶中的一种或两种。
进一步的，所述的酸为盐酸、三氟乙酸、柠檬酸中的一种或两种。
进一步的，所述的胺类化合物为伯胺或仲胺。
进一步的，所述的反应A中使用乙酸乙酯进行萃取，所述的纯化采用柱层析法，洗脱剂为二氯甲烷和甲醇。
进一步的，所述的反应B中使用乙酸乙酯、正己烷、二氯甲烷中的一种或多种组合进行打浆。
进一步的，所述的反应C中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
进一步的，所述的反应D中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
进一步的，所述的反应E中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
进一步的，所述的反应F中采用制备液相法进行纯化，流动相A为MeCN，0.1％HCOOH，流动相B为H 2O，0.1％HCOOH。
进一步的，所述反应均在氮气保护下进行。
本发明提供的制备抗体药物偶联物中间体(具体为连接基团-SN38共价偶联物)的方法，除制备方法步骤简单，降低了由于重金属残留导致的安全性问题，且使用该抗体药物偶联物中间体制备的抗体药物偶联物在体内将具有更高的稳定性。
同时，发明人惊奇的发现，本发明中的连接子与SN38组合，所制备的抗体药物偶联物能够产生显著的肿瘤抑制效果。
附图说明
图1为小鼠体重变化情况；
图2为肿瘤体积变化情况；
图3相对肿瘤体积情况。
具体实施方式
[缩写]
除非另有说明，本发明使用的所有缩写具有本领域普通技术人员所理解的相同含义。如本发明所用，常用的缩写及其定义如下所示：
缩写 定义
DCC N,N'-二环己基碳二亚胺
EDCI 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
DIC N,N'-二异丙基碳二亚胺
HATU 2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
HBTU 苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
HBPIPU (苯并三氮唑-1-基氧基)二哌啶碳六氟磷酸盐
HBPyU O-(苯并三唑-1-基)-N,N,N',N'-二吡咯基脲六氟磷酸盐
HCTU 6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯
HDMA 1-[(二甲基氨基)(吗啡啉)甲基]-3-氧代-1H-[1,2,3]三氮唑[4,5-b]并吡啶3-六氟磷酸盐
TATU 2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲四氟硼酸盐
TBTU O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸盐
TCTU O-(6-氯-1H-苯并三唑-1-基)-N,N,N',N'-四甲基脲四氟硼酸盐
TCFH N,N,N',N'-四甲基氯甲脒六氟磷酸盐
TDBTU N,N,N',N'-四甲基-O-(4-羰基-3,4-二氢-1,2,3-苯并三嗪-3-基)脲四氟硼酸盐
TFFH 氟代-N,N,N',N'-四甲基脲六氟磷酸盐
BTFFH N,N,N',N'-双(四亚甲基)氟代甲脒六氟磷酸盐
TSTU 2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯
PyBOP 1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐
PyCIOP 氯代三吡咯烷基六氟磷酸盐
PyAOP (3H-1,2,3-三唑并[4,5-b]吡啶-3-氧基)三-1-吡咯烷基六氟磷酸盐
PyCIU 1-(氯-1-吡咯烷基亚甲基)吡咯烷六氟磷酸盐
DEPBT 3-(二乙氧基邻酰氧基)-1,2,3-苯并三嗪-4-酮
EEDQ 2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉
Mc 马来酰亚胺基己酰基
SN38 Camptothecin
DMF N,N-二甲基甲酰胺
DMA 二甲基乙酰胺
DNPC 二(对硝基苯)碳酸酯
DMSO 二甲基亚砜
THF 四氢呋喃
DCM 二氯甲烷
DIPEA N,N-二异丙基乙胺
TFA 三氟乙酸
[定义]
与说明书的各方面相关的各种术语在说明书和权利要求书中通篇使用。除非另外指明，否则此类术语被赋予本领域的普通含义。其它具体定义的术语应按照与本文所提供的定义相符的方式理解。
如本文所用，术语“一个”和“一种”和“所述”是按照标准惯例使用的并且意指一个或多个，除非上下文另有指示。因此例如，对“一种抗体药物偶联物”的提及包括两个或更多个抗体药物偶联物的组合等等。
应当理解，无论何处在本文中用语言“包含”描述方面，除此之外还提供了以“由......组成”和/或“基本上由......组成”描述的类似方面。
本发明所使用的术语“抗体药物偶联物”表示抗体/抗体功能性片段、连接子、药物部分经过化学反应连接在一起的化合物，其结构通常由三部分组成：抗体或抗体类配体、药物部分、以及将抗体或抗体类配体及药物偶联起来的连接子(linker)。目前抗体药物偶联物的制备通常分为两步：第一步是将连接子与药物部分通过化学反应形成“连接子-药物”偶联物，第二步是将“连接子-药物”偶联物中的连接子部分与抗体/抗体功能性片 段通过巯基或者氨基共价偶合在一起。本发明所使用的术语“抗体药物偶联物中间体”是指上述所述的“连接子-药物”偶联物，进一步的，本发明涉及的“抗体药物偶联物中间体”泛指“连接基团”和SN38共价偶联物。
本发明所用的“抗体药物偶联物”采用本领域通用制备方法制备，示例性的，本发明所用的抗体药物偶联物的制备方法为:用pH＝7.4的PBS缓冲液，把抗体配制为10mg/mL的溶液，加入适量当量的TCEP，振荡混匀1小时，再加入5.0摩尔当量的连接子-毒素(即式1-式16所示的化合物)，振荡混匀，反应1h，反应结束后，超滤除去残留的小分子，加载至疏水色谱(HIC-HPLC)进行DAR、药物分布、裸抗比例检测。
可以理解的是，本发明旨在提供一种新的药物偶联物中间体，并示例性的以Heceptin制备多例ADC对技术效果进行验证，抗体的选择不对本专利造成限制，示例性地给出具体ADC的结构，其中，ADC-1中的连接子和毒素对应于所用的式(1)化合物与Heceptin利用上述本领域制备“抗体药物偶联物”的通用制备方法制备而成(下同，ADC-2中的连接子和毒素对应于式(2)化合物；ADC-3中的连接子和毒素对应于式(3)化合物；ADC-4中的连接子和毒素对应于式(4)化合物；ADC-5中的连接子和毒素对应于式(5)化合物；ADC-6中的连接子和毒素对应于式(6)化合物；ADC-7中的连接子和毒素对应于式(7)化合物；ADC-8中的连接子和毒素对应于式(8)；化合物ADC-9中的连接子和毒素对应于式(9)；化合物ADC-10中的连接子和毒素对应于式(10)化合物；ADC-11中的连接子和毒素对应于式(11)化合物；ADC-12中的连接子和毒素对应于式(12)化合物；ADC-13中的连接子和毒素对应于式(13)化合物；ADC-14中的连接子和毒素对应于式(14)化合物；ADC-15中的连接子和毒素对应于式(15)化合物；ADC-16中的连接子和毒素对应于式(16)化合物；ADC-17中的连接子和毒素对应于CL2A-SN38)与：其中，q可选自1、2、3、4、5、6、7、8、9、10。
Figure PCTCN2022128912-appb-000023
Figure PCTCN2022128912-appb-000024
Figure PCTCN2022128912-appb-000025
Figure PCTCN2022128912-appb-000026
Figure PCTCN2022128912-appb-000027
本发明中的术语“连接基团”是指一种具有双官能团或多官能团的分子，可分别与蛋白/抗体分子和SN38反应，因此作为一种“桥梁”将蛋白/抗体与SN38连接起来。本发明涉及的连接基团特指那些结构中含有酰基结构的基团。
[具体实施例]
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于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条件的实验方法，通常按照常规条件或按照制造厂商建议的条件进行，未注明具体来源的试剂，为市场购买的常规试剂。除非另外说明，否则所有的百分数、比率、比例、或份数按重量计。
除非另行定义，文中所使用的所有专业与科学用于与本领域熟悉人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明办法中。文中所述的较佳实施方式与材料仅作示范之用。
实施例1 式(12)化合物的制备方法
Figure PCTCN2022128912-appb-000028
1)化合物c(2-Boc-N-甲基乙基乙二醇)的合成方法
将化合物a(氨基乙二醇)0.6g和化合物b(N-Boc-乙醛)1.0g溶于20mL甲醇中，室温搅拌6h。将反应置于冰水浴，加入三乙酰氧基硼氢化钠，冰水浴搅拌1h，恢复至室温搅拌过夜。通过LC-MS法检测，产物没有紫外吸收。将溶剂旋干，用乙酸乙酯萃取2次，无水Na 2SO 4，干燥旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝20：1，冲洗，得产物1.0g，产率66％。LC-MS:(M+H) +262.6。
2)SN38-PNP(10-对硝基苯基碳酸酯-喜树碱)的合成方法
将SN38(喜树碱)1.0g和DNPC(二(对硝基苯)碳酸酯)1.6g溶于100mL四氢呋喃中，加三乙胺2mL，室温搅拌1.5h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，用乙酸乙酯：正己烷＝20mL：100mL打浆，过滤得固体，用二氯甲烷20mL打浆，过滤，LC-MS监测收集产品得SN38-PNP固体700mg，产率49.3％。LC-MS:(M+H) +557.4。
3)化合物d(Boc-N-甲基乙基二醇-喜树碱)的合成方法
将SN38-PNP 250mg溶于30mL N,N-二甲基甲酰胺中，加入235mg Boc-DMEDA-PEG和233uL N,N-二异丙基乙胺，室温搅拌16h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝20：1，冲洗。LC-MS监测收集产品得Boc-DMEDA-PEG-SN38固体213mg。收率70％。LC-MS:(M+H) +680.5。
4)化合物g(10-(2-N-甲基乙基乙二醇)-喜树碱三氟乙酸盐)的合成方法
将化合物d 73.0mg溶于3mL二氯甲烷和3mL三氟乙酸中，置于冰水浴中，恒温搅拌1h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，再加入甲苯2mL和二氯甲烷2mL，旋干，带两次。LC-MS监测收集产品得SN38-DMEDA·TFA，直接投入下一步反应。LC-MS:(M+H) +580.6。
5)式(12)化合物(马来酰亚胺二乙氧基-L-缬氨酰基-L-瓜氨酸-对氨基汴基-(2-N-甲基乙基乙二醇)-喜树碱)的合成方法
将MP2-VC-PAB-PNP 50mg溶于4mL N,N-二甲基甲酰胺中，放入冰水浴恒温，加氮气保护。加入化合物g 38mg，再加入三乙胺42u L，恒温搅拌0.5h后，恢复至室温反应0.5h。LC-MS法检测，反应完成。反应结束后，将溶剂低温旋出，制备液相法纯化，制备柱：Sun
Figure PCTCN2022128912-appb-000029
Prep C18 OBD TM 5um，19*250mm Column；流动相A：MeCN，流动相B：H 2O。LC-MS监测收集纯品得式(12)化合物固体18mg。收率22％。LC-MS:(M+H) +1197.2。
实施例2 式(4)化合物的制备方法
Figure PCTCN2022128912-appb-000030
按照实施例1的合成路线制备化合物g(10-(2-N-甲基乙基乙二醇)-喜树碱三氟乙酸盐)：将化合物d(73.0mg)溶于3mL二氯甲烷和3mL三氟乙酸中，置于冰水浴中，恒温搅拌1h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，再加入二氯甲烷2mL，旋干，带两次。LC-MS监测收集产品得化合物g(70mg)，直接投入下一步反应。LC-MS:(M+H) +580.6。
按照实施例1的合成路线制备式(4)化合物(马来酰亚胺己酸-L-缬氨酰基-L-瓜氨酸-对氨基汴基-(2-N-甲基乙基乙二醇)-喜树碱)：将Mc-VC-PAB-PNP(马来酰亚胺己酸-L-缬氨酰基-L-瓜氨酸-对氨基汴基-对硝基苯基碳酸酯)(58mg),化合物g(42mg)溶于2.5mL N,N-二甲基甲酰胺中后，冰水浴搅拌30mins,滴加DIPEA(42mg)，冰水浴搅拌1h。通过LC-MS法检测，反应未完成。继续加入加三乙胺(10mg)，室温搅拌1h，反应溶液变黄，通过LC-MS法检测，反应完成。反应完成后，将溶剂低温旋干，制备液相法纯化，制备柱：Sun
Figure PCTCN2022128912-appb-000031
Prep C18 OBD TM 5um，19*250mm Column；流动相A：MeCN，0.1％HCOOH，流动相B：H 2O，0.1％HCOOH。LC-MS监测收集纯品得式(4)化合物固体34mg，收率为40.5％。LC-MS:(M+H) +1179.2。
实施例3 式(1)化合物的制备方法
Figure PCTCN2022128912-appb-000032
1)化合物c(2-Boc-N-甲基乙基乙二醇)的合成方法
将化合物a(氨基乙二醇)0.6g和化合物b(N-Boc-(甲胺基)乙醛)1.0g溶于20mL甲醇中，室温搅拌6h。将反应体系置于冰水浴，加入三乙酰氧基硼氢化钠，冰水浴搅拌1h，恢复至室温搅拌过夜。通过LC-MS法检测，产物没有紫外吸收。将溶剂旋干，用乙酸乙酯萃取2次，加入无水Na 2SO 4沉淀，倒出有机相，干燥旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝20：1，冲洗，得产物1.0g，产率66％。LC-MS:(M+H) +262.6。
2)SN38-PNP(10-对硝基苯基碳酸酯-喜树碱)的合成方法
将SN38 1.0g和DNPC(二(对硝基苯)碳酸酯)1.6g溶于100mL四氢呋喃中，加三乙胺2mL，室温搅拌1.5h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，用乙酸乙酯：正己烷＝20mL：100mL打浆，过滤得固体，用二氯甲烷20mL打浆，过滤，LC-MS监测收集产品得SN38-PNP固体700mg，产率49.3％。LC-MS:(M+H) +557.4。
3)化合物d(Boc-N-甲基乙基二醇-喜树碱)的合成方法
将500mg SN38-PNP溶于30mL N,N-二甲基甲酰胺中，加入370mg化合物c和233uL N,N-二异丙基乙胺，室温搅拌3h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝20：1，冲洗。LC-MS监测收集产品得化合物d固体250mg。收率45％。LC-MS:(M+H) +680.5。
4)化合物h(Boc-N-甲基乙基二乙氧基对硝基活性酯-喜树碱)的合成方法
将化合物d 220.0mg溶于5mL二氯甲烷中，加入二(对硝基苯)碳酸酯150mg、DIPEA 86mg，恒温搅拌16h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝50：1。收集产品得化合物h固体220mg，收率68.5％直接投入下一步反应。LC-MS:(M+H) +845.6。
5)化合物i(Boc-N-甲基乙基二乙氧基-N,N,N-三甲基乙二胺-喜树碱)的合成方法
将化合物h(Boc-N-甲基乙基二乙氧基对硝基活性酯-喜树碱)220mg，N,N,N-三甲基乙二胺50mg溶于5mL N,N-二甲基甲酰胺中后，滴加DIPEA(65mg)，冰水浴搅拌1h。通过LC-MS法检测，反应完成后，将溶剂旋干，柱层析法纯化，洗脱剂为二氯甲烷：甲醇＝20：1。收集产品得化合物i固体157mg，收率75％直接投入下一步反应。LC-MS:(M+H) + 808.6。
6)化合物j(N-甲基乙基二乙氧基-N,N,N-三甲基乙二胺-喜树碱-三氟乙酸盐)的合成方法
将化合物i(Boc-N-甲基乙基二乙氧基-N,N,N-三甲基乙二胺-喜树碱)157.0mg溶于3mL二氯甲烷和1.3mL三氟乙酸中，置于冰水浴中，恒温搅拌1h。通过LC-MS法检测，反应结束。反应完成后，将溶剂旋干，再加入二氯甲烷2mL，旋干，带两次。得化合物j 120mg，直接投入下一步反应。LC-MS:(M+H) +708.3。
7)式(1)化合物(马来酰亚胺己酸-L-缬氨酰基-L-瓜氨酸-对氨基汴基-(N-甲基乙基二乙氧基-N,N,N-三甲基乙二胺)-喜树碱)的合成方法
将Mc-VC-PAB-PNP(马来酰亚胺己酸-L-缬氨酰基-L-瓜氨酸-对氨基汴基-对硝基苯基碳酸酯)52mg，化合物j 35mg溶于2.5mL N,N-二甲基甲酰胺中后，冰水浴搅拌30mins，滴加DIPEA 129mg，冰水浴搅拌0.5h。通过LC-MS法检测，反应未完成。继续加入加DIPEA 129mg，室温搅拌0.5h，反应溶液变黄，通过LC-MS法检测，反应完成。反应完成后，将溶剂低温旋干，制备液相法纯化，制备柱：Sun
Figure PCTCN2022128912-appb-000033
Prep C18 OBD TM 5um，19*250mm Column；流动相A：MeCN，0.1％HCOOH，流动相B：H 2O，0.1％HCOOH。LC-MS监测收集纯品得式(1)化合物固体20mg，收率为20.5％。LC-MS:(M+H) +1307.6。
实施例4 式(9)化合物的制备方法
Figure PCTCN2022128912-appb-000034
按照实施例3的合成路线制备式(9)化合物(马来酰亚胺二乙氧基-L-缬氨酰基-L-瓜氨酸-对氨基汴基-(N-甲基乙基二乙氧基-N,N,N-三甲基乙二胺)-喜树碱)：将MP2-VC-PAB-PNP(马来酰亚胺二乙氧基-L-缬氨酰基-L-瓜氨酸-对氨基汴基-对硝基苯基碳酸酯)(52mg),化合物j(34mg)溶于2.5mL N,N-二甲基甲酰胺中后，冰水浴搅拌30mins,滴加DIPEA(36mg)，冰水浴搅拌0.5h。通过LC-MS法检测，反应未完成。继续加入加DIPEA(8mg)，室温搅拌1.5h，反应溶液变黄，通过LC-MS法检测，反应完成。反应完成后，将溶剂低温旋干，制备液相法纯化，制备柱：S un
Figure PCTCN2022128912-appb-000035
Prep C18 OBD TM 5um，19*250mm Column；流动相A：MeCN，0.1％HCOOH，流动相B：H 2O，0.1％HCOOH。LC-MS监测收集纯品得式(9)化合物固体23mg，收率为35.2％。LC-MS:(M+H) +1325.4。
实施例5 ADC-1对NCI-N87细胞的抑制作用
将式(1)化合物和CL2A-SN38中间体分别通过本领域常规方法与Heceptin进行巯基偶联(例如参见中国专利申请公开号CN102448494A中的实施例12)，制备得到平均DAR值为8的ADC-1和HER2-CL2A-SN38(ADC-17)抗体药物偶联物。将NCI-N87细胞胰酶消化后调整细胞密度为50000个/ml，100μL/孔加入细胞培养板中，37℃，5％CO 2培养箱中孵育14-20h。用基础培养基对分别对样品组和对照组(供试品成分见表1)进行梯度稀释，以100μL/孔转入铺有细胞的细胞培养板中；在37℃条件下，5％CO 2培养箱中孵育70-74h。用培养基将CCK-8稀释10倍，将96孔板中的旧培养基吸出，每孔加入100μL稀释的CCK-8溶液，5％CO 2条件下显色2-4h，离心去气泡后在酶标仪上选择测定波长450nm/655nm进行读数，其结果见表2。
表1 供试品成分
序号 供试品 平均DAR
样品1 ADC-1 8
对照组1 ADC-17 8
对照组2 HER2裸抗体 /
表2 对NCI-N87细胞的抑制作用
序号 供试品 IC50(ng/ml)
样品1 ADC-1 251
对照组1 ADC-17 210
对照组2 HER2裸抗体 NA
实验结果表明，ADC-1组与ADC-17组对NCI-N87细胞的抑制效果相当。
实施例6 稳定性实验研究
1)精密称取化合物式(1)化合物(2.1mg)、CL2A-SN38(2.1mg) 分别溶于DMSO 700μL；
2)分别配制pH为6、7、8的PB(0.2M)缓冲液；
3)取50μL式(1)化合物的DMSO溶液于离心管，加入pH＝6的PB(0.2M)缓冲液950μL，化合物浓度为60μg/mL。放置于37℃恒温体系中；取50μL CL2A-SN38的DMSO溶液于离心管，加入pH＝6的PB(0.2M)缓冲液950μL，化合物浓度为60μg/mL。放置于37℃恒温体系中；
4)分别在时间为0h、2h、4h、6h、24h、48h取样40μL，加入乙腈120μL，离心，取上清液送LC-MS定量检测。
5)结果处理：
以T＝0h的LC-MS浓度为基准，数据＝T/T 0*100％，结果分别如表3和表4所示。
表3 式(1)化合物
时间 pH＝6 pH＝7 pH＝8
0 1 1 1
2 0.988606 0.878866 0.56405
4 0.97612 0.801849 0.284131
6 0.92084 0.821934 0.123407
24 0.86821 0.256709 0.009607
48 0.78862 0.122173 0.012562
在pH为6时，式(1)化合物48小时后分解了21.2％；
在pH为7时，式(1)化合物48小时后分解了87.8％；
在pH为8时，式(1)化合物48小时后分解了98.7％。
表4 CL2A-SN38
时间 pH＝6 pH＝7 pH＝8
0 1 1 1
2 0.747874 0.703812 0.628692
4 0.647065 0.585106 0.434695
6 0.412955 0.571543 0.38435
24 0.035931 0.022606 0.025316
48 0.020243 0.01906 0.019275
在pH为6时，CL2A-SN38 48小时后分解了98.0％；
在pH为7时，CL2A-SN38 48小时后分解了98.1％；
在pH为8时，CL2A-SN38 48小时后分解了98.1％。
由表3-表4中的结果，可以看出：
1)式(1)化合物在弱酸性条件下比CL2A-SN38更稳定。
2)式(1)化合物在中性条件下，6h分解了18％，24h分解了75％，CL2A-SN38在中性条件下，6h分解了43％，24h分解了98％。
3)式(1)化合物在碱性条件下，24h分解了97％，CL2A-SN3824h分解了98％。
因此，采用本发明制备的式(1)化合物与CL2A-SN38相比，在体内弱酸性或中性条件下具有更高的稳定性，预计将显著提高ADC药物安全性。
实施例7评估抗Her2-药物偶联物对原位乳腺癌发展的抑制作用
1.实验方法
BT474细胞株复苏后传1-2代，待细胞生长状态稳定后扩大培养，制备肿瘤细胞悬液，裸鼠乳脂垫接种0.2*10 7个BT474细胞，共接种60只雌性BALB/c裸鼠。平均肿瘤体积达到约70mm 3时，选取42只，按照肿瘤体积随机分成6组：阴性对照组为对照组1(生理盐水组)、对照组2(ADC-17)、实验组1(ADC-4)、实验组2(ADC-12)、实验组3(ADC-1)、实验组4组(ADC-9)，每组7只。模型建立成功后。按上述分组，对照组1给予生理盐水静脉注射给药，其余组别给予静脉注射药物，给药剂量为对照组2 5mg/kg、实验组1 5mg/kg、实验组2 5mg/kg、实验组3 5mg/kg、实验组4 5mg/kg，每周1次，持续21周。每周3次称重并测量肿瘤体积，于第21天称量和测量肿瘤体积。
2.试验结果及分析
1)小鼠体重变化情况：
表5 小鼠的体重情况
Figure PCTCN2022128912-appb-000036
实验过程无小鼠死亡，实验结束时，各组与对照组1相比，对照组2
(ADC-17组)、实验组1(ADC-4)、实验组2(ADC-12)、实验组3
(ADC-1)、实验组4(ADC-9)体重未出现明显差异，具体见表5和图1。
2)小鼠肿瘤体积情况：
表6 小鼠肿瘤体积情况
Figure PCTCN2022128912-appb-000037
注：各组与对照组1生理盐水组相比，“ *”表示P＜0.05；“ **”表示P＜0.01；
各组与对照组2相比，“ #”表示P＜0.05，“ ##”表示P＜0.01
在给药后21天对照组1的肿瘤体积达到107±19.30mm 3，对照组2、实验组1、实验组2、实验组3、实验组4五组的肿瘤体积分别为49.17±13.29、57.12±20.28、66.26±21.37、61.32±12.82、75.76±46.18、75.19±25.99mm 3(见表6)，其中对照组2、实验组1、实验组2、实验组3 四组与对照组1(生理盐水组)在统计学上的差异非常显著(P＜0.01)，实验组4与对照组1在统计学上的差异显著(P＜0.05)。实验组1、实验组2、实验组3四组与对照组2的肿瘤体积在统计学上无显著性差异(P＞0.05)，且其相对肿瘤体积也均无显著性差异(P＞0.05)，即效果相当。其中对照组2、实验组1、实验组2、实验组3、实验组4五组的相对肿瘤增殖率(T/C)分别为49.30％、55.00％、62.58％、62.48％、67.19％和63.20％。由以上数据可知本实验测试的对照组2、实验组1、实验组2、实验组3组对应的四个抗体药物偶联物均能极为显著地抑制BT474肿瘤生长且效果相当，而实验组4也可起到抑制BT474肿瘤生长地作用。在试验过程中各组肿瘤体积变化情况与相对肿瘤体积变化情况分别如图2和图3所示。
综上所述，利用本发明中药物偶联物中间体构建的ADC对乳腺癌BT474细胞模型显示出显著的抗肿瘤活性，优选的ADC-1、ADC-4、ADC-12具有明显优势，预计在体内条件下会显著提高安全性的情况下，还能起到与ADC-17组效果相当的肿瘤抑制效果。
本发明已通过各个具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各个具体实施方式，普通技术人员在本发明的范围内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (23)

  1. 一种如式(Ⅰ)所示的抗体药物偶联物中间体：
    Figure PCTCN2022128912-appb-100001
    其中：
    X 1为烷烃链或PEG链，
    X 2为H或-C(O)NR 1R 2，且，
    R 1选自氢、卤素、羟基、取代或非取代的C 1-6烷基、取代或非取代的C 1-6羟烷基、取代或非取代的C 1-6氨基烷基、取代或非取代的C 1-6烷氧基、取代或非取代的C 1-6烷酰基和取代或非取代的C 1-6烷醛基；
    R 2选自氢和取代或非取代的C 1-6烷基；其中各取代的C 1-6烷基任选被1-5个独立地选自以下的取代基取代：卤素、羟基、氨基、羰基、羧基、5-10元杂环基和C 1-6卤代烷基，其中所述5-10元杂环基具有1-3个选自氧、氮和硫的杂原子。
  2. 根据权利要求1所述的抗体药物偶联物中间体，其特征在于，所述的X 1选自：
    -(CH 2) m-，或
    -(CH 2CH 2O) p-，
    其中：
    所述的m为1、2、3、4、5、6，优选的，m为5；
    所述的p为1、2、3、4、5、6，优选的，p为2；
    优选的，X 1选自：
    Figure PCTCN2022128912-appb-100002
  3. 根据权利要求1所述的抗体药物偶联物中间体，其特征在于，所述的R 1或R 2独立地选自：H、甲基、乙基、丙基、丁基、戊基、庚基、甲氧基、乙氧基、Cl、Br、
    Figure PCTCN2022128912-appb-100003
  4. 根据权利要求1-3任一所述的抗体药物偶联物中间体，其特征在于，所述的X 2选自：
    Figure PCTCN2022128912-appb-100004
  5. 权利要求1所述的抗体药物偶联物中间体，其特征在于：所述的抗体药物偶联物中间体的结构如式(1)-(16)所示：
    Figure PCTCN2022128912-appb-100005
    Figure PCTCN2022128912-appb-100006
    Figure PCTCN2022128912-appb-100007
    Figure PCTCN2022128912-appb-100008
    Figure PCTCN2022128912-appb-100009
  6. 一种抗体药物偶联物中间体的制备方法，所述的抗体药物偶联物中间体为：
    Figure PCTCN2022128912-appb-100010
    Figure PCTCN2022128912-appb-100011
    所述的R 1选自氢、取代或非取代的C 1-6烷基、取代或非取代的C 1-6羟烷基、取代或非取代的C 1-6氨基烷基、取代或非取代的C 1-6烷氧基、取代或非取代的C 1-6烷酰基和取代或非取代的C 1-6烷醛基；
    所述的R 2选自氢和取代或非取代的C 1-6烷基；其中各取代的C 1-6烷基任选被1-5个独立地选自以下的取代基取代：卤素、羟基、氨基、羰基、羧基、5-10元杂环基和C 1-6卤代烷基，其中所述5-10元杂环基具有1-3个选自氧、氮和硫的杂原子；
    优选的，R 1或R 2选自：H、甲基、乙基、丙基、丁基、戊基、庚基、甲氧基、乙氧基、Cl、Br、
    Figure PCTCN2022128912-appb-100012
    Figure PCTCN2022128912-appb-100013
    所述制备方法选自以下反应过程：
    反应过程1：
    Figure PCTCN2022128912-appb-100014
    反应过程2：
    Figure PCTCN2022128912-appb-100015
    反应过程3：
    Figure PCTCN2022128912-appb-100016
    反应过程4：
    Figure PCTCN2022128912-appb-100017
  7. 根据权利要求6所述的制备方法，其特征在于，所述的化合物(I-1)选自：
    Figure PCTCN2022128912-appb-100018
    Figure PCTCN2022128912-appb-100019
    或，所述的化合物(I-2)选自：
    Figure PCTCN2022128912-appb-100020
    Figure PCTCN2022128912-appb-100021
  8. 根据权利要求6所述的制备方法，其特征在于，所述反应过程1包括以下条件：
    反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下加入还原剂，再搅拌适当时间后，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
    反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
    反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应D：将反应C的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与Mc-VC-PAB-PNP一起溶于溶剂中，低温条件下再搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
  9. 根据权利要求6所述的制备方法，其特征在于，所述反应过程2包括以下条件：
    反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
    反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
    反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应D：将反应C的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与MP2-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
  10. 根据权利要求6所述的制备方法，其特征在于，所述反应过程3包括以下操作条件：
    反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下中加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
    反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
    反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应D：将反应C的产物溶于溶剂中，加入二(对硝基苯)碳酸酯和有机碱，恒温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应E：将反应D的产物和胺类化合物溶于溶剂中，加入有机碱，低温条件下搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应F：将反应E的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与Mc-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
  11. 根据权利要求6所述的制备方法，其特征在于，所述反应过程4包括以下操作条件：
    反应A：将化合物a和化合物b溶于溶剂中，室温搅拌适当时间后置于低温条件下中加入还原剂后搅拌适当时间，再置于室温搅拌过夜，反应结束后旋干溶剂并进行萃取、干燥和纯化；
    反应B：将SN38和DNPC溶于溶剂中，加入有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行打浆和过滤；
    反应C：将反应B的产物溶于溶剂中，加入反应A的产物及有机碱，室温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应D：将反应C的产物溶于溶剂中，加入二(对硝基苯)碳酸酯和有机碱，恒温搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应E：将反应D的产物和胺类化合物溶于溶剂中，加入有机碱，低温条件下搅拌适当时间，反应结束后旋干溶剂并进行纯化；
    反应F：将反应E的产物溶于溶剂中，加入酸，低温条件下搅拌适当时间，反应结束后旋干溶剂后与MP2-VC-PAB-PNP一起溶于溶剂中，低温条件下搅拌适当时间，加入有机碱，反应结束后旋干溶剂并进行纯化。
  12. 根据权利要求6－11中任一项所述的方法，其中所述的“低温条件下”为冰水浴下。
  13. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的溶剂为极性溶剂和/或非极性溶剂，所述的极性溶剂为THF、DMF、DMA、NMP的一种或几种；所述的非极性溶剂为二氯甲烷、四氯化碳的一种或几种。
  14. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的有机碱为N，N-二异丙基乙胺、三乙胺、吡啶的一种或几种，优选为N，N-二异丙基乙胺以及吡啶中的一种或两种。
  15. 根据权利要6－11中任一项所述的制备方法，其特征在于所述的酸为盐酸、三氟乙酸、柠檬酸中的一种或两种。
  16. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的胺类化合物为伯胺或仲胺。
  17. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应A中使用乙酸乙酯进行萃取，所述的纯化采用柱层析法，洗脱剂为二氯甲烷和甲醇。
  18. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应B中使用乙酸乙酯、正己烷、二氯甲烷中的一种或多种组合进行打浆。
  19. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应C中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
  20. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应D中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
  21. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应E中采用柱层析法进行纯化，洗脱剂为二氯甲烷和甲醇。
  22. 根据权利要求6－11中任一项所述的制备方法，其特征在于所述的反应F中采用制备液相法进行纯化，流动相A为MeCN，0.1％HCOOH，流动相B为H 2O，0.1％HCOOH。
  23. 根据权利要求6－11中任一项所述的制备方法，其特征在于，所述反应均在氮气保护下进行。
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CN110945009A (zh) * 2019-10-23 2020-03-31 烟台迈百瑞国际生物医药有限公司 一种寡肽连接子中间体及其制备方法
CN113710277A (zh) * 2021-07-19 2021-11-26 烟台迈百瑞国际生物医药股份有限公司 一种负载双毒素的抗体药物偶联物及其应用

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