Compretastatin derivative and antibody drug conjugate thereof

A technology of compretin and antibody drugs, which can be used in drug combinations, anti-tumor drugs, pharmaceutical formulations, etc., and can solve problems such as poor water solubility and difficulty in direct preparation

Active Publication Date: 2021-01-12
SHANGHAI TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CA4 has a strong tumor inhibitory effect, but its water solu

Method used

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  • Compretastatin derivative and antibody drug conjugate thereof
  • Compretastatin derivative and antibody drug conjugate thereof
  • Compretastatin derivative and antibody drug conjugate thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Preparation of antibody-drug conjugate

[0034] (1) Synthesis of Compound 50-5

[0035]

[0036] Synthesis of Compound 50-1

[0037] Comprestatin (316.35 mg, 1 mmol) was dissolved in DCM (10 mL), followed by the addition of p-nitrophenyl chloroformate (403.12 mg, 2 mmol) and pyridine (237.3 mg, 3 mmol). Reaction at room temperature for 4h. The reaction was checked for completion by TLC thin layer chromatography. The crude product was obtained after direct decompression rotary evaporation to remove the solvent, and 5 mL of CH was added 2 Cl 2 and 1g of 60-100 mesh silica gel, mix well and spin dry. The crude product was subjected to silica gel column chromatography (petroleum ether: ethyl acetate = 3:1) to obtain white solid 50-1 (480 mg, 0.997 mmol, 99%). ESI-HRMS calculation result: C 25 h 24 NO 9 [M+H] + :482.1451, actual result: 482.1410.

[0038] Synthesis of Compound 50-2

[0039] Compound 50-1 (0.480 g, 1 mmol), N-Boc-N,N'-dimethylethy...

Embodiment 2

[0110] Example 2: Affinity and Endocytosis Assays of Antibody-Drug Conjugates

[0111] In this example, the affinity and endocytosis of Cetuximab, Cex-50-5, Cex-59, and Cex-70 on tumor cell lines were studied. The cells can be endocytized normally at 37°C, and most of the physiological activities of the cells are inhibited at 4°C, which is regarded as non-endocytosis. The test substance and the cells were incubated at 4°C and 37°C for a period of time, and the internal endocytosis during this time period = (4°C cell surface antibody amount - 37°C cell surface antibody amount) / 4°C cell surface antibody amount × 100% .

[0112] The reagents and consumables used in the following experiments come from: Cetuximab (Shanghai Huanyao Biotechnology Co., Ltd., article number: 205923-56-4), Goat Anti-Human IgG H&L (FITC) was purchased from Abcam Company , DAPI was purchased from Cell Signaling Company, face washing solution (PBS+1%BSA). Instrument: CytoFLEX Flow Cytometer.

[0113] I...

Embodiment 3

[0121] Example 3: In Vitro Cell Proliferation Biological Activity Determination of Antibody-Drug Conjugates

[0122] In this example, the effect of Cetuximab, ADCs Cex-50-5, Cex-59, Cex-70 on the proliferation of tumor cell lines was investigated.

[0123] The reagents and consumables used in the following experiments come from: RPMI1640 medium, trypsin-EDTA, fetal calf serum, recombinant human insulin, 100×penicillin, 1×PBS (pH7.4), CCK8 color reagent purchased from Gibco Company; 10cm petri dish (Corning) and 96-well cell culture plate (Corning); multifunctional microplate reader (SpectraMax i3).

[0124] In this example, the CCK8 colorimetric method was used to evaluate the anti-proliferation effect of the test substance. Cell Counting Kit-8 (referred to as CCK-8) reagent contains WST-8 [chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5 -(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt]. It is reduced to a highly water-soluble yellow formazan produ...

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PUM

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Abstract

The invention discloses a combretastatin derivative and an antibody drug conjugate thereof, and a preparation method and application thereof. The combretastatin derivative and the antibody drug conjugate thereof comprise a compound shown in a formula I or a pharmaceutically acceptable salt or solvate thereof, wherein in the formula I, Ab represents a targeting ligand; L represents a linker for connecting an antibody with CA4 or a derivative thereof; n represents a ratio of a drug to the targeting ligand; and X is NR, wherein R is an alkane chain or a PEG chain. The invention provides preparation of the antibody drug conjugate by taking CA4 as a drug molecule and the derivative thereof, and application of the antibody drug conjugate in treatment of high-proliferative diseases or lesions. The diseases are characterized in that cells generate an antigen or target, and the antigen can be specifically combined with the antibody.

Description

technical field [0001] The invention belongs to the fields of biopharmaceuticals and biotechnology, and in particular relates to a derivative of comprestatin (CA4), which is used for the preparation of antibody-drug conjugates and its application in targeted cancer therapy. Background technique [0002] Antibody-drug conjugates (ADCs) link active drug molecules to antibodies through a chemical link, and antibodies serve as carriers for targeted delivery of drug molecules to target cells. The three parts of ADCs, namely drug molecule, linker and antibody part are very important. The drug molecules used in the early development of ADCs tend to be extremely toxic molecules, but such ADCs will produce large toxic side effects. Recently, Enhertu and Trodelvy will be launched in 2019 and 2020 respectively, both of which use moderately toxic camptothecin derivatives as drug molecules. The research and development of the new generation of ADCs began to pay attention to and pay att...

Claims

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Application Information

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IPC IPC(8): C07K5/062C07K5/103A61K47/68A61K31/09A61P35/00
CPCC07K5/06052C07K5/1008A61K47/6845A61K31/09A61P35/00
Inventor 姜标陈红莉黄容盛耀
Owner SHANGHAI TECH UNIV
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