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WO2019047941A1 - 鹅膏毒肽类抗体偶联物 - Google Patents

鹅膏毒肽类抗体偶联物 Download PDF

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WO2019047941A1
WO2019047941A1 PCT/CN2018/104712 CN2018104712W WO2019047941A1 WO 2019047941 A1 WO2019047941 A1 WO 2019047941A1 CN 2018104712 W CN2018104712 W CN 2018104712W WO 2019047941 A1 WO2019047941 A1 WO 2019047941A1
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compound
synthesis
drugs
drug
pharmaceutically acceptable
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PCT/CN2018/104712
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English (en)
French (fr)
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朱义
李�杰
万维李
余永国
卓识
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Sichuan Baili Pharmaceutical Co Ltd
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Sichuan Baili Pharmaceutical Co Ltd
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Priority to EP18852932.5A priority Critical patent/EP3735987A4/en
Priority to NZ766904A priority patent/NZ766904B2/en
Priority to AU2018329066A priority patent/AU2018329066B2/en
Priority to CA3095211A priority patent/CA3095211A1/en
Priority to SG11202008017UA priority patent/SG11202008017UA/en
Priority to JP2020552290A priority patent/JP7296396B2/ja
Priority to US16/970,597 priority patent/US20210113707A1/en
Publication of WO2019047941A1 publication Critical patent/WO2019047941A1/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6831—Fungal toxins, e.g. alpha sarcine, mitogillin, zinniol or restrictocin
    • 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
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/52—Cyclic peptides containing at least one abnormal peptide link with only normal peptide links in the ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention belongs to the field of biopharmaceutical technology, and in particular relates to an amanita peptide antibody conjugate.
  • Amanitin is one of the amanita peptide toxoids isolated from highly toxic mushrooms. It is a bicyclic peptide consisting of 8 amino acids. There are 9 kinds of natural amanita peptides that have been isolated and purified. --Amanita, ⁇ -Amanita, ⁇ -Amanita, ⁇ -Amanita, aminin, amininamide, amanita Amanullin, amanullinic acid and proamanullin, wherein a-amanita and beta-amanita are the major toxins leading to death.
  • Amanita is a class of slow-acting toxins that inhibit the transcription of eukaryotic RNA polymerase II and RNA polymerase III, leading to protein deletion and cell death.
  • the toxin is highly inhibitory to RNA polymerase II, and the KD can reach 3 nM. It is repeatedly absorbed in the body due to intestinal and hepatic circulation in the gastrointestinal tract, and it has persistent and severe damage to the liver, kidney, heart and lungs of the human body.
  • amanita peptide When the amanita peptide is coupled with a large biomolecule carrier (such as an antibody molecule), its toxicity is greatly reduced or even relatively non-toxic. Only after the biomolecule carrier is excised in a specific physiological environment, the amanita peptide will appear. Cytotoxicity.
  • the object of the present invention is to provide a bicyclic octapeptide amanita peptide derivative and a biomacromolecule conjugate which are stable in the blood circulation system and are cleaved by the target cells after being swallowed.
  • An amanita peptide derivative that is an RNA polymerase inhibitor is released and produces strong toxicity to cells by specific inhibition of eukaryotic mRNA synthesis.
  • the technical scheme adopted by the present invention is: a toxin conjugate of the formula (I) or a pharmaceutically acceptable salt thereof:
  • n 0, 1 or 2
  • R 1 is -H or -OH
  • R 2 is -H or -OH
  • R 3 is -H or -OH or C 1-6 alkyl
  • R 4 is -NH 2 or -OH
  • R 5 is -LA
  • A is a part of the biomacromolecules that binds to the target
  • L is any chemical structure that links amanita peptide derivatives and biological macromolecules.
  • the chemical structure of the L comprises a cleavable or non-cleavable structure.
  • the A comprises an antibody or antigen-binding fragment thereof, an antigen-binding polypeptide.
  • the coupling of the L to the target binding biomolecule A comprises the following structure:
  • the L is linked to the toxin via an ester or ether
  • the present invention also encompasses a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described toxin conjugate or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention also encompasses the use of a toxin conjugate as described above or a pharmaceutically acceptable salt thereof for the preparation of an antitumor or anticancer drug.
  • the anti-tumor drug or anti-cancer drug is an anti-lung cancer drug, an anti-kidney cancer drug, an anti-urinary cancer drug, an anti-colorectal cancer drug, an anti-prostate cancer drug, an anti-glioma cell drug, an anti-ovarian cancer drug, Anti-pancreatic cancer drugs, anti-breast cancer drugs, anti-melanoma drugs, anti-hepatocarcinoma drugs, anti-bladder cancer drugs, anti-malignant lymphoma drugs, anti-leukemia drugs, anti-gastric drugs or anti-cancer drugs.
  • the hetero (atom, alkyl, aryl, cyclo) of the present invention refers to a corresponding chemical structure containing atoms other than carbon atoms.
  • Boc tert-butoxycarbonyl
  • CO 2 carbon dioxide
  • DIPEA diisopropylethylamine
  • DMSO dimethyl sulfoxide
  • DPBS Dunsen's phosphate buffer
  • DTPA diethyltriamine pentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • FBS fetal bovine serum
  • HATU O-(7-nitrobenzotriazole)-N,N,N,N-tetramethyluronium hexafluorophosphate
  • HOBt 1-hydroxybenzotriazole
  • mAb monoclonal antibody
  • MEM the lowest basal medium
  • MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethyl)-2-(4-sulfophenyl)-2H-tetrazole, internal salt;
  • PAB p-aminobenzyloxy
  • PBS phosphate buffer
  • Tris trishydroxymethylaminomethane
  • Trt-Cl triphenylchloromethane
  • TFA trifluoroacetic acid
  • TBS-Cl tert-butyldimethylchlorosilane
  • EDCI 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.
  • the diseased cells can be sheared to release conjugates of toxic molecules, and it is surprisingly found that such a connection method is simple and efficient, and greatly meets the demand for industrialized large-scale production of such products.
  • the present invention discloses bicyclic octapeptide derivatives which are coupled with a corresponding target binding group by a specific chemical structure, and whose structure is stable in plasma and cleaved into an active ingredient in a specific biological environment, In order to maximize the killing of target cells and minimize the side effects of non-target cells, it can be used for the treatment of various malignant tumors.
  • Figure 1 is a graph showing the results of experiment in Example 13 in cell line BT474.
  • Figure 2 is a graph showing the results of an experiment in Example 13 in the cell line SKBR3.
  • Figure 3 is a graph showing the results of experiment in Example 13 in cell line N87.
  • Post-treatment Add 2 L of isopropyl ether to the reaction mixture, stir the bottle to separate the viscous oil, pour off the supernatant, add 1 L of isopropyl ether, stir vigorously, dip the supernatant again, and add 500 ml of isopropyl The ether was stirred overnight and suction filtered to give 27.2 g, m. MS: [M+H] + 816.73.
  • Synthesis of Compound 25 Synthesis by reference to Liang Zhao, et al. Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation. Chem Bionchem 2015, 16, 1420-1425.
  • Synthesis of Compound 47 The crude product of Compound 46 was dissolved in 1 ml of 10% TFA in dichloromethane, and the mixture was stirred under nitrogen for 1 hour at room temperature, and then the reaction was completely detected, and the solution was spun off, and the reaction was carried out without further purification.
  • Synthesis of Compound 48 The synthesis of Reference Compound 32 was carried out according to the above step 100% yield. After purification, the target peak was lyophilized to give a pale yellow solid of about 20.6 mg, and the total yield in three steps was about 16%. MS: [M+H] + 1466.7436.
  • the sample was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was taken for injection analysis.
  • Mobile phase A: 50 mM PB, 300 mM NaCl, 200 mM Arg, 5% IPA, pH 6.5;
  • the mobile phase A was isocratically eluted for 30 min, the flow rate was 0.714 ml/min, the column temperature was 25 ° C, and the detection wavelength was 280 nm.
  • the sample was centrifuged at 14,000 rpm for 5 minutes, and the supernatant was taken for injection analysis.
  • Mobile phase A: 1.5M ammonium sulfate, 0.025M anhydrous sodium phosphate, pH 7.0
  • A0301 The structure of A0301 is as follows:
  • Tumor cell culture medium Gibco
  • the biosafety cabinet was turned on for 30 minutes in advance, and then ventilated for 3 minutes.
  • the growth medium, test medium, D-PBS and trypsin were preheated in a 37 ° C constant temperature water bath, and then the surface was sterilized with alcohol and placed in a biosafety cabinet.
  • the supernatant was aspirated, mixed with 4 ml of test medium, and counted as 100 ul (50 ul of cell solution was taken, 50 ul of Trypan Blue Stain was added and mixed, and mixed and counted). Plated according to the number of cells set before, 80 ul / well was placed in a 96-well plate, holes E11, F11, G11 were only added 80 ul of detection medium, edge holes were added, 150 ul of DPBS. Dilution of the antibody solution: Prepare the test solution with a starting concentration of 5 uM and 300 ul in the first column of the V-type 96-well plate with the test medium, and add 210 ul of the test medium to the second to the next column, respectively.

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Abstract

二环八肽类衍生物以特殊的化学结构与相应的靶结合基团偶联，其结构在血浆中稳定而在特定的生物环境中裂解成为活性成份的药物，以达到对靶细胞杀伤性的最大化和对非靶细胞毒害副作用的最小化，可用于多种恶性肿瘤的治疗。

Description

鹅膏毒肽类抗体偶联物 技术领域
本发明属于生物制药技术领域，具体涉及一种鹅膏毒肽类抗体偶联物。
背景技术
鹅膏毒肽(amanitin)是由剧毒蘑菇中分离出来的鹅膏肽类毒素中的一种，为8个氨基酸组成的双环肽，已分离纯化的天然鹅膏毒肽有9种，分别是α-鹅膏毒肽、β-鹅膏毒肽、γ-鹅膏毒肽、ε-鹅膏毒肽、鹅膏素(amanin)、三羟毒伞肽酰胺(amaninamide)、鹅膏无毒环肽(amanullin)、一羟鹅膏毒肽羧酸(amanullinic acid)和前鹅膏无毒环肽(proamanullin)，其中α-鹅膏毒肽和β-鹅膏毒肽是导致死亡的主要毒素。鹅膏毒肽是一类慢作用毒素，可抑制真核RNA聚合酶II和RNA聚合酶III的转录，导致蛋白质缺失和细胞死亡。该类毒素对RNA聚合酶II的抑制性极高，KD可达3nM，在肠胃道中因肠肝循环而反复在体内被吸收，对人体的肝肾心肺等器官能产生持续性的严重损害。
将鹅膏毒肽与大的生物分子载体(如抗体分子)偶联后，其毒性大大降低甚至相对无毒，仅在特定生理环境中生物分子载体被切除之后，鹅膏毒肽才会显现其细胞毒性。
近年来，世界上一些研究机构和公司对鹅膏毒肽分子进行了结构改造，以获得具有药用价值的抗体-毒素偶联物，例如：德国海德堡医药有限责任公司将天然鹅膏毒肽分子的1-位、3-δ-位或6’-酚羟基以连接子与单抗偶联；美国Agensys公司对α-鹅膏毒肽的吲哚环上苯基进行结构修饰，从6’-酚羟基或7’-位处以连接子与抗体相连；杭州多禧生物科技有限公司将鹅膏毒肽衍生物的5’-、7’-位硝化后引出带酰胺结构的连接子与抗体偶联；韩国Leg℃hem Biosciences公司也研究了从6’-酚羟基和3-δ-位处以连接子与抗体相连的方法。在以上的合成方法中，从6’-酚羟基处接出连接子的方法需要对鹅膏毒肽分子其它位点的羟基进行多步保护和脱保护，合成路线繁杂，苯环上5’-、7’-位的取代反应定位不确定易导致产品分离有一定难度，收率较低。
发明内容
基于以上现有技术，本发明的目的在于提供一种双环八肽类鹅膏毒肽衍生物和生物大分子的偶合物，它们在血液循环系统内稳定，被目标细胞内吞后被剪切，释放出作为RNA聚合酶抑制剂的鹅膏毒肽衍生物，并通过对真核生物mRNA合成的专一抑制产生对细胞的强烈毒性。
为了实现以上目的，本发明采用的技术方案为：结构式(I)的毒素偶联物或其药学上可接受的盐：
Figure PCTCN2018104712-appb-000001
其中：
n＝0，1或2，
R 1为-H或-OH，
R 2为-H或-OH，
R 3为-H或-OH或C 1-6烷基，
R 4为-NH 2或-OH，
R 5为-L-A，
A为与靶点结合的生物大分子部分，
L为连接鹅膏毒肽衍生物和生物大分子的任意化学结构。
作为优选，所述L的化学结构中，包含可裂解型或不可裂解型的结构。
作为优选，所述A包括抗体或其抗原结合片段、抗原结合多肽。
作为优选，所述L与靶结合生物分子A偶联处包含以下结构：
Figure PCTCN2018104712-appb-000002
作为优选，所述L与与毒素通过酯或者醚连接
本发明还包括一种含有包括上述毒素偶联物或其药学上可接受的盐作为活性成分的药物组合物。
本发明还包括一种上述毒素偶联物或其药学上可接受的盐在制备抗肿瘤药物或抗癌药物中的用途。
进一步的，所述抗肿瘤药物或抗癌药物为抗肺癌药物、抗肾癌药物、抗尿道癌药物、抗结直肠癌药物、抗前列腺癌药物、抗胶质细胞瘤药物、抗卵巢癌药物、抗胰腺癌药物、抗乳腺癌药物、抗黑色素瘤药物、抗肝癌药物、抗膀胱癌药物、抗恶性淋巴瘤药物、抗白血病药物、抗胃癌药物或抗食道癌药物。
定义：按照本领域的标准惯例，本发明用于式和表格中的符号
Figure PCTCN2018104712-appb-000003
表示作为部分或取代基与化合物结构的核心或核的连接点的键。
按照本领域的标准惯例，本发明杂(原子、烷基、芳基、环基)，是指含有除碳原子以外的其它原子的相应化学结构。
缩写和符号：
Boc：叔丁氧羰基；
PyBOP：1H-苯并三唑-1-基氧三吡咯烷基鏻六氟磷酸盐；
Cit：瓜氨酸；
CO 2：二氧化碳；
DCM：二氯甲烷；
DIPEA：二异丙基乙胺；
DMF：二甲基甲酰胺；
DMSO：二甲基亚砜；
DPBS：杜氏磷酸盐缓冲液；
DTPA：二乙基三胺五乙酸；
DTT：二硫苏糖醇；
EA：乙酸乙酯；
EDTA：乙二胺四乙酸；
FBS：胎牛血清；
HATU：O-(7-氮苯并三氮唑)-N,N,N,N-四甲基脲六氟磷酸酯；
H 2O：水；
HOBt：1-羟基苯并三唑；
mAb：单克隆抗体；
MEM：最低基础培养基；
MTS：3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲酯基)-2-(4-磺苯基)-2H-四唑，内盐；
MTT：3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐；
PAB：对氨基苯甲氧基；
PBS：磷酸盐缓冲液；
Sodium Pyruvate：丙酮酸钠；
THF：四氢呋喃；
TLC：薄层色谱法；
Tris：三羟甲基氨基甲烷；
Val：缬氨酸；
Trt-Cl：三苯基氯甲烷；
TBTU：O-(1H-苯并三唑-1-基)-N,N,N’,N’-四甲基异脲四氟化硼；
HOBT：1-羟基苯并三唑；
TFA：三氟醋酸；
TBS-Cl：叔丁基二甲基氯硅烷；
HOSu：N-羟基丁二酰亚胺；
Na 2CO 3：碳酸钠；
EDCI：1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。
本发明的有益效果如下：
在本发明中，我们从鹅膏毒肽或其衍生物分子的2-位脯氨酸羟基处通过生物药学上可接受的连接结构与抗体分子偶联，能够获得在血浆中稳定无毒而在病变细胞内可被剪切释放出毒性分子的偶联物，并且惊喜地发现，此类连接方法简单高效，极大地满足了对此类产品进行工业化大生产的需求。
本发明公开了二环八肽类衍生物，该类化合物以特殊的化学结构与相应的靶结合基团偶联，其结构在血浆中稳定而在特定的生物环境中裂解成为活性成份的药物，以达到对靶细胞杀伤性的最大化和对非靶细胞毒害副作用的最小化，可用于多种恶性肿瘤的治疗。
附图说明
图1是实施例13在细胞系BT474中实验结果图。
图2是实施例13在细胞系SKBR3中实验结果图。
图3是实施例13在细胞系N87中实验结果图。
具体实施方式
下面结合对本发明提供作进一步详细的说明。
实施例1：小分子payload ama-1的合成
Figure PCTCN2018104712-appb-000004
Figure PCTCN2018104712-appb-000005
固相合成中间体08：
以N-芴甲氧羰基-O-叔丁基-L-羟脯氨酸预装树脂为起始原料，经20％哌啶(1g树脂加入20ml 20％哌啶)脱去保护基Fmoc以后，DMF为溶液(20ml/g)，依次加入Fmoc-N-三苯甲基-L-天冬酰胺(Fmoc-Asn(Trt)-OH)(3eq)，TBTU(2.5eq)，HOBT(1.8eq)，DIPEA(6eq)，于室温(28℃)下反应2h后，用DMF(每次1g树脂20mlDMF)洗涤3次，再按前面的操作连接后面的氨基酸，最后连接完全以后，用1％TFA(每次1g树脂20ml，1％TFA 5min，重复三次)从树脂上切割下来，旋去溶液，用甲基叔丁基醚搅拌析晶，就得到化合物08，收率约为54％，高效纯度为76％。MS：[M+H] +1417.6123。
化合物09合成：
取10g化合物08粗品，用TFA(10ml/g)溶解后,于室温下搅拌反应5h，50℃减压除去TFA,制备液相色谱纯化，得纯品化合物09约4.3g，收率为43％，纯度为95.6％。MS：[M+H] +760.2144。
化合物13的合成：
于250ml单口瓶中加入(4S)-羟基异亮氨酸2.94g，1,4-二氧六环40ml，饱和碳酸钠溶液ml，搅拌均匀，并分批加入Fmoc-OSu，10min后，继续于室温下搅拌反应12h，原料反应完全，向反应液中加入50ml水，并用5％的柠檬酸溶液调PH至4左右，乙酸乙酯萃取3次(每次50ml)，收集有机层，饱和食盐水50ml洗涤一次，无水硫酸钠干燥，浓缩得淡黄色油状物。无需纯化直接投下一步。收率＞100％。
化合物14的合成：
将上述化合物13的粗品用40mlDMF溶解后，加入咪唑2.68g(2eq)，分批加入TBS-Cl，加毕，于室温下搅拌12h，原料反应完全，加入水50ml，乙酸乙酯50ml，搅拌，分出有机层，水层再用乙酸乙酯萃取2次(每次50ml)，收集有机层，无水硫酸钠干燥，过滤浓缩得淡黄 色油状物，硅胶柱层析(洗脱液：PE:EA＝5:1)，得油状物4.5g。两步收率约为46.6％。
化合物15的合成：
于250ml单口瓶中依次加入化合物14，HOSu(1.23g，1.15eq)，DCC(2.23g，1.15eq)，THF50ml，氮气保护下，室温搅拌6h，反应完全后，加水50ml，乙酸乙酯50ml，搅拌10min后，分出有机层，水层再用乙酸乙酯萃取2次，每次50ml，合并有机层，无水硫酸钠干燥，过滤，浓缩得淡黄色油状物，制备液相色谱纯化得白色泡沫状固体约3.24g，收率60％。1H-NMR(400MHz，DMSO-d6)：0.08(s,6H),0.86(s,9H),0.98(d,3H,J＝8.0Hz)，1.06(d,3H,J＝5.6),1.95(t,J＝10.8),2.83(s,4H),4.21(dd,1H,J＝16.8Hz,8.0Hz),4.34(dd,1H,J＝12Hz,4Hz),4.67～4.73(m,1H),7.31(d,2H,J＝8.0Hz),7.34～7.46(m,2H),7.70～7.76(m,2H),7.89(t,2H,J＝12.0),8.24(d,1H,J＝8.8Hz)；MS:[M+H] +581.34。
化合物10的合成
将180mg化合物09用干燥的DMF1.5ml溶解后，加入化合物15(825mg，6eq)，DIPEA(0.25ml，6eq)氮气保护下，于室温反应5h，HPLC监控反应，原料09基本反应完全。无需后处理直接投下一步。
化合物11的合成
向上述反应液中加入哌啶0.3ml，继续于室温下搅拌反应2h，HPLC检测原料反应完全，停止反应，用制备液相色谱纯化(中性，乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干得到白色粉末状固体136mg，两步收率约为52％，MS:[M+H] +1003.5654。
化合物12的合成
将136mg化合物11用干燥的DMF溶解后，加入EDCI(130mg,5eq)，HOBT(367mg,20eq)，DIPEA(0.12ml，5eq)，于室温下搅拌4h后，HPLC检测反应完全后，制备液相色谱纯化(中性，乙腈/纯水体系)，收集目标峰，减压除去乙腈以后，冻干得到白色粉末状固体60mg，收率约45％，MS：[M+H] +985.5421。
Figure PCTCN2018104712-appb-000006
化合物17的合成
在500ml的圆底烧瓶中，加入30g的化合物16(49.92mmol，1.0eq)，再加入240ml的DMF搅拌溶解，之后加入22.76g二(对硝基苯)碳酸酯(74.87mmol，1.5eq)，并滴入9.68g的DIPEA(74.87mmol，1.5eq)，室温搅拌反应1.5h，TLC检测原料反应完全，停止反应。后处理：剧烈搅拌下向反应瓶中加入异丙醚1.5L，搅拌2h倾去上层液体，残余物中加入800ml异丙醚继续剧烈搅拌1h，抽滤，滤饼再置于600ml的异丙醚中搅拌过夜，抽滤，得土黄色粉末状固体31.2g，收率为81.6％。MS:[M+H] +767.62。
化合物18的合成
在500ml的单口瓶中，加入31.2g的化合物17(40.73mmol，1.0eq)，再加入150ml的DMF搅拌将其溶解，加入用50ml的DMF溶解的8.1g甲基(2-(甲基氨基)乙基)氨基甲酸叔丁酯(36.66mmol，1.1eq)，室温搅拌反应3.5h，TLC检测化合物17反应完全，停止反应。后处理：反应液中加入2L异丙醚搅拌，瓶璧析出粘稠油状物，倾去上清液，再加入1L的异丙醚，剧烈搅拌，再次倾去上清液，再加入500ml异丙醚搅拌过夜，抽滤，得27.2g土黄色粉 末状固体，收率81.9％。MS：[M+H] +816.73。
化合物19的合成
在500ml的圆底烧瓶中，称入20g化合物18(24.53mmol)，再加入100ml的DMF将其溶解，于冰浴下10分钟内滴入20ml二乙胺，之后室温搅拌反应2h，TLC检测原料化合物18反应完全，停止反应。后处理：减压下浓缩除去DMF和二乙胺备用，产品未经纯化直接投下一步。
化合物20的合成
在500ml的圆底烧瓶中，加入150ml的DMF将前一步的化合物19的粗品溶解，再加入11.7g 6-(马来酰亚胺基)己酸琥珀酰亚胺酯(37.98mmol，1.55eq)，随后滴加8.4ml的DIEA(50.63mmol，2.06eq)，滴加完毕后，室温搅拌反应2h，TLC检测，原料化合物19反应完全，停止反应。后处理：反应液中加入1.5L异丙醚搅拌析晶，析出固体和粘于瓶璧的类油状粘稠物，倾去上清液，再加入500ml甲叔醚和500mlml异丙醚各搅洗一次，抽滤，滤饼于45℃的真空干燥箱中减压干燥，得20g土黄色粉末状固体粗品。MS：[M+H] +787.63。
化合物21的合成
取0.4g化合物20，用2ml 50％TFA二氯甲烷溶液溶解后，于室温下搅拌2h，TLC检测，反应完全，旋去溶液备用，无需纯化，直接投下一步。
Figure PCTCN2018104712-appb-000007
化合物22的合成：
20mL棕色瓶中加入100mg(0.10mmol,1.0eq)化合物12，二(对硝基苯)碳酸酯304mg(1.0mmol,10eq)，DMF 3mL，溶清后加入DIPEA(260mg，20eq)，N 2保护，升至28℃反应24h，HPLC监控反应，原料012，NPC反应完全，粗品不用处理，直接投下一步反应。
化合物23的合成
向化合物22的反应液中加入化合物21的粗品，加入适量DIPEA，保持反应液PH在8～9，氮气保护下，于室温下搅拌3h，HPLC监测反应完全后，制备液相色谱纯化目标产品，得浅黄色固体30mg。MS：[M+H] +1697.9150。
化合物ama-1的合成
4mL棕色瓶中加入15mg化合物23，加入1ml 5％TFA/MeOH溶清，氮气保护，升至28℃反应1h，HPLC(纯水)监控反应，原料23反应完，氮气吹干溶剂后HPLC半制备，乙腈浓 度约35％出产品，冻干后得白色固体11mg，收率Y＝42％。MS：[M+H] +1583.7532。
实施例2：小分子payload ama-2的合成
Figure PCTCN2018104712-appb-000008
取ama-1约6mg，用二氯甲烷溶解后，加入1.0eq的mCPBA的二氯甲烷溶液(0.05g/ml)13微升，于室温下搅拌2h，HPLC检测原料反应完全，制备液相制备纯化，冻干，得到白色粉末状固体约3mg。收率：49.5％。MS:[M+H] +1599.8450。
实施例3：小分子payload ama-3的合成
Figure PCTCN2018104712-appb-000009
化合物24的合成：参考化合物09的合成，得到白色固体约1.2g。收率25.7％。MS：[M+H] +790.4254。
化合物25的合成：参考文献Liang Zhao,et al.Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation.ChemBionchem 2015,16,1420-1425进行合成。
化合物26的合成：参考化合物10的合成方法，化合物24投料300mg，无需纯化直接投下一步。
化合物27的合成：参考化合物11的合成方法，制备液相纯化，冻干得到白色固体195mg，两步总收率44.1％。MS：[M+H] +1163.6341。
化合物28的合成：参考化合物12的合成方法，化合物27投料150mg，得到目标化合物白色固体约96mg，收率为65％。MS：[M+H] +1145.6124。
化合物29的合成：参考化合物22的合成，化合物28投料80mg，主点明显，无需纯化，直接投下一步。
化合物30的合成：参考化合物23的合成，制备纯化后得到淡黄色固体约59mg，收率：45.4％。MS：[M+H] +1857.9813。
化合物ama-3的合成：参考化合物ama-1的合成，化合物30投料40mg，制备纯化后得到淡黄色固体约8.4mg。收率：24％。MS：[M+H] +1629.8021。
实施例4：小分子payload ama-4的合成
Figure PCTCN2018104712-appb-000010
化合物30的合成：向化合物29(其中，化合物28重新累料至500mg，本次投料130mg， 未经纯化，按收率100％计算)的反应液中，加入49mg N-叔丁氧羰基-1,6-二氨基己烷(2eq)，滴加DIPEA适量，保持溶液pH在8～9，氮气保护下，室温搅拌4h，高效检测化合物29反应完全后，制备液相纯化，收集目标峰，旋去有机溶剂以后，冻干得到白色固体约83.4mg，收率约53％。[M+H] +1387.7541。
化合物31的合成：取80mg化合物30，用1ml20％TFA的二氯甲烷溶液溶解后，氮气保护下，室温搅拌2h，高效监测化合物30反应完全，减压旋去有机溶剂，待用。
化合物32的合成：将化合物31的粗品用DMF 2mL溶解以后，用DIPEA调pH至8～9，加入6-(马来酰亚胺基)己酸琥珀酰亚胺酯38mg(2eq)，氮气保护下，室温搅拌6h，高效监测化合物31反应完全，制备液相纯化，收集目标峰，旋去有机溶剂以后，冻干得到白色固体约46mg，收率54％，[M+H] +1480.7841。
化合物ama-4的合成：参照化合物ama-3的合成，化合物32投料44mg，冻干得到目标化合物21mg，收率56.4％，[M+H] +1152.5431。
实施例5：小分子payload ama-5的合成
Figure PCTCN2018104712-appb-000011
取12mg ama-4，用二氯甲烷溶解后，加入5.0eq的mCPBA的二氯甲烷溶液(0.05g/ml)165微升，于室温下搅拌2h，HPLC检测原料反应完全，制备液相制备纯化，冻干，得到黄色粉末状固体约4mg。收率：32.9％。MS:[M+H] +1268.5821。
实施例6小分子payload ama-6的合成
Figure PCTCN2018104712-appb-000012
化合物33的合成：参考化合物10的合成，化合物09投料300mg，无需纯化，直接投下一步。
化合物34的合成：参考化合物11的合成，制备液相纯化后，冻干得白色固体约248.9mg，收率为55.6％，MS:[M+H] +1133.6348。
化合物35的合成：参考化合物12的合成，原料34投料220mg，制备液相纯化后，冻干得白色固体约137.3mg，收率为63.4％，MS:[M+H] +1115.6147.
化合物36的合成：参考化合物22的合成，原料35投料120mg，无需纯化直接投下一步。
化合物37的合成：参考化合物23的合成，制备液相纯化后，旋去有机相，冻干得白色固体约112.4mg，收率为：57.1％，MS:[M+H] +1827.9857.
化合物ama-6的合成
参考化合物ama-1的合成，化合物37投料20mg，制备纯化后，冻干得到淡黄色固体约7mg，收率为40％。MS:[M+H] +1599.8324。
实施例7：小分子payload ama-7的合成
Figure PCTCN2018104712-appb-000013
化合物38的合成：参考化合物07的合成，计算其loading大约为0.32mmol/g。
化合物39的合成：参考化合物08的合成，旋干得棕色油状物，未经纯化直接投下一步。
化合物40的合成：参考化合物09的合成，制备纯化，冻干得淡黄色固体约526.4mg，收率从化合物38计算的量开始约为34％。MS:[M+H] +866.3512。
化合物41的合成：参考化合物10的合成，化合物40投料500mg，目标峰冻干得类白色固体约531.6mg，收率约为63％。MS:[M+H]+1461.6624。
化合物42的合成：参考化合物11的合成，化合物41投料525mg，制备纯化，收集目标峰，冻干得到淡黄色固体约368.5mg，收率约82.8％。MS:[M+H] +1239.5764。
化合物43的合成：参考化合物12的合成，化合物42投料350mg，冻干目标峰得淡黄色固体约223.4mg，收率约为64.7％。MS:[M+H] +1221.5748。
化合物44的合成：参考化合物22的合成，化合物43投料220mg，未经纯化直接投下一步。
Figure PCTCN2018104712-appb-000014
化合物45的合成：参考化合物30的合成，制备纯化，冻干得类白色固体约130.2mg，收率约49.4％。MS:[M+H] +1463.7524。
化合物46的合成：取化合物45约125mg，用1ml甲醇溶解后，加入12.5mg 10％的Pd/C，于外温40℃，0.5Mpa下，氢化还原反应12h，高效检测反应完全后，过滤除去Pd/C,收集滤液，浓缩得棕色油状物，无需纯化直接投下一步。
化合物47的合成：将化合物46的粗品，用1ml 10％TFA的二氯甲烷溶液溶解后，氮气保护下，室温搅拌反应1h后，高效检测反应完全，旋去溶液，无需纯化直接投下一步。
化合物48的合成：参考化合物32的合成，按上步100％收率计算，制备纯化后，目标峰冻干得到淡黄色固体约20.6mg，三步总收率约为16％。MS:[M+H] +1466.7436。
化合物ama-7的合成
参考化合物ama-1的合成，原料48投料10mg，制备纯化后，收集目标峰，冻干得类白色固体约3.2mg，收率为37.9％。MS:[M+H] +1238.6512。
实施例8：小分子payload ama-8的合成
Figure PCTCN2018104712-appb-000015
化合物49的合成：取α-Amanitin 30mg，用1mlDMF溶解后，加入9mg碳酸钾(3eq)，室温搅拌1h后，加入碘甲烷13ul(10eq)，室温搅拌12h后，制备液相分离纯化，冻干以后，得到目标化合物约9.1mg，收率30％。MS:[M+H] +933.4031。
化合物50的合成：取上述化合物49用1mlDMF溶解后，加入N,N’-二琥珀酰亚胺基碳酸酯25mg(10eq)，加入三乙胺0.14ml(20eq)，室温搅拌12h后，原料反应完全，制备液相纯化，收集目标峰，冻干得白色固体约5.6mg，收率59.9％，MS:[M+H] +959.4501。
化合物51的合成：参照化合物22的合成，将上述5.6mg化合物50全部参与反应，未经纯化直接投下一步。
化合物ama-8的合成：
参照化合物23的合成，制备纯化后，冻干得白色固体约3.2mg，收率32.8％，MS:[M+H] +1671.7211。
实施例9：小分子payload ama-9的合成
Figure PCTCN2018104712-appb-000016
取化合物ama-8约2mg，用0.5ml二氯甲烷溶解后，加入0.6mg(3eq)间氯过氧苯甲酸(m-CPBA)，室温下搅拌反应3h后，制备液相纯化，收集目标峰，冻干得白色固体约0.8mg，收率39.6％，MS:[M+H] +1687.7142。
实施例10：小分子payload ama-10的合成
Figure PCTCN2018104712-appb-000017
取化合物ama-6约4mg，用0.5ml二氯甲烷溶解后，加入0.5mg(1.2eq)间氯过氧苯甲酸(m-CPBA)，室温下搅拌反应2h后，制备液相纯化，收集目标峰，冻干得白色固体约1.2mg，收率29.7％，MS:[M+H] +1615.7320。
实施例11：
本实施例制备了以下化合物：
Figure PCTCN2018104712-appb-000018
Figure PCTCN2018104712-appb-000019
Figure PCTCN2018104712-appb-000020
Figure PCTCN2018104712-appb-000021
实施例12：抗体-药物偶联物制备
1)通用偶联方法：将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/ml。加入10倍于抗体摩尔分子数的TCEP，室温下反应2h。使用超滤离心管换液至pH6.5的磷酸缓冲液中，再加入10倍于抗体摩尔分子数的DHAA，室温下反应2h。然后加入3倍于抗体摩尔分子数的payload，室温下反应4h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload。
2)抗体-药物偶联DAR的检测
单体率检测条件：
样品14000rpm离心5分钟，取上清液进样分析。
仪器：Waters e2695(2489UV/Vis)；
色谱柱：TSKgel G3000SWXL(7.8×300mm，5μm)；
流动相：A：50mM PB，300mM NaCl，200mM Arg，5％IPA，pH6.5；
流动相A等度洗脱30min，流速：0.714ml/min，柱温25℃，检测波长：280nm。
DAR检测条件：
样品14000rpm离心5分钟，取上清液进样分析。
仪器：Waters H-class(TUV)；
色谱柱：Proteomix HIC Butyl-NP5(4.6×35mm，5μm)；
流动相：A：1.5M硫酸铵，0.025M无水磷酸钠，pH7.0
B：0.025M无水磷酸钠，25％IPA，pH7.0
流动相A平衡色谱柱，流动相A和B梯度洗脱，流速0.8ml/min；柱温25℃，检测波长：214nm。
A0301结构式如下：
Figure PCTCN2018104712-appb-000022
3)结果
从表1可以得出，从羟脯氨酸位点连出的抗体偶联物有较好的单体率以及较高的DAR。由于结构相似，化合物1-化合物14也有较好的单体率以及较高的DAR。
编号 单体率 DAR
Herceptin-007 93.86％ --
Herceptin-007-A0301 93.47％ 1.81
Herceptin-007-ama-3 95.08％ 1.87
Herceptin-007-ama-4 96.34％ 1.86
Herceptin-007-ama-6 95.44％ 1.7
Herceptin-007-ama-8 95.27％ 1.86
Herceptin-007-ama-9 95.20％ 1.83
Herceptin-007-ama-10 95.61％ 1.86
表1
实施例13：体外活性测试
1)实验材料
细胞：来源于中国科学院细胞库
肿瘤细胞培养基：Gibco
FBS：BIOWEST
2)培养基的配制
生长培养基(with 10％FBS,Penicillin/streptomycin(100U/ml)
检测培养基(with 1％FBS,Penicillin/streptomycin(100U/ml)
3)操作
提前30min开启生物安全柜紫外灯照射，后开通风3min。将生长培养基、检测培养基、D-PBS和胰酶放入37℃恒温水浴锅里预热，之后用酒精对表面进行消毒，放入生物安全柜中。选择汇合率在80％左右的细胞，放于生物安全柜中，吸掉旧培养基，用D-PBS润洗，吸弃，用胰酶消化2～3min，后加入生长培养基中和，离心1200rpm，3min。吸去离心上清，用4ml检测培养基混匀，取100ul计数(其中取出50ul细胞液，加入50ul Trypan Blue Stain并混匀，混匀后计数)。按照之前定好的细胞数铺板，80ul/孔铺于96孔板中，孔E11、F11、G11只加80ul检测培养基，边缘孔加入，150ul的DPBS。抗体溶液的稀释：用检测培养基在V型96孔板第一列中配制起始浓度为5uM，300ul的供试品品溶液，后面第2到10列分别加入210ul的检测培养基，从混匀的第一列中取30ul加入到第二列，用排枪上下混匀10次，弃枪头，后面7个浓度依次操作。铺板24h后加入稀释抗体，每孔20ul和设置对照，第11列只加入20ul的检测培养基，每个浓度设置2个复孔，加入后于细胞涡旋震荡器上混匀，550rpm，3min。
4)检测
4天后取出MTS试剂，常温避光解冻后，充分涡旋混匀后，在生物安全柜中，沿孔侧壁按每100μL细胞培养体积加入20μL CellTiter
Figure PCTCN2018104712-appb-000023
One Solution Reagen MTS试剂，轻轻拍动板面，使MTS溶液混合均匀后，放于细胞培养箱中避光静置孵育2h。反应结束后，取出96孔板，于酶标仪中检测OD490nm吸光值，并进行数据记录、整理、存储。
5)结果
如表2、附图1-3所示，在细胞系BT474/SKBR3/N87中，表现出较好的抗肿瘤活性。由于结构相似，化合物1-化合物14也有较好的抗肿瘤活性。
Figure PCTCN2018104712-appb-000024
表2
以上通过实施例来详细说明本发明的实施方式，借此对本发明如何应用技术手段来解决技术问题，并达成技术效果的实现过程能充分理解并据以实施。需要明确的是，只要不构成冲突，本发明中的各个实施例以及各实施例中的各个特征可以相互结合，所形成的技术方案均在本发明的保护范围之内。

Claims (8)

  1. 结构式(I)的毒素偶联物或其药学上可接受的盐：
    Figure PCTCN2018104712-appb-100001
    其中：
    n＝0，1或2，
    R 1为-H或-OH，
    R 2为-H或-OH，
    R 3为-H或-OH或C 1-6烷基，
    R 4为-NH 2或-OH，
    R 5为-L-A，
    A为与靶点结合的生物大分子部分，
    L为连接鹅膏毒肽衍生物和生物大分子的任意化学结构。
  2. 根据权利要求1所述的毒素偶联物或其药学上可接受的盐，其特征在于，所述L的化学结构中，包含可裂解型或不可裂解型的结构。
  3. 根据权利要求1所述的毒素偶联物或其药学上可接受的盐，其特征在于，所述A包括抗体或其抗原结合片段、抗原结合多肽。
  4. 根据权利要求1所述的毒素偶联物或其药学上可接受的盐，其特征在于，所述L与靶结合生物分子A偶联处包含以下结构：
    Figure PCTCN2018104712-appb-100002
  5. 根据权利要求1所述的毒素偶联物或其药学上可接受的盐，其特征在于，所述L与毒素通过酯或者醚连接。
  6. 一种包括权利要求1-5任一所述的毒素偶联物或其药学上可接受的盐的药物组合物。
  7. 一种权利要求1-5任一所述的毒素偶联物或其药学上可接受的盐在制备抗肿瘤药物或抗癌药物中的用途。
  8. 如权利要求7所述的用途，其特征在于，所述抗肿瘤药物或抗癌药物为抗肺癌药物、 抗肾癌药物、抗尿道癌药物、抗结直肠癌药物、抗前列腺癌药物、抗胶质细胞瘤药物、抗卵巢癌药物、抗胰腺癌药物、抗乳腺癌药物、抗黑色素瘤药物、抗肝癌药物、抗膀胱癌药物、抗恶性淋巴瘤药物、抗白血病药物、抗胃癌药物或抗食道癌药物。
PCT/CN2018/104712 2017-09-08 2018-09-08 鹅膏毒肽类抗体偶联物 Ceased WO2019047941A1 (zh)

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