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Disubstituted maleamide linker for antibody-drug conjugation and its preparation method and use

A maleamide and linker technology, applied in the field of new disulfide chain bridging and cross-linking reagents, can solve the problems of high R&D and final industrialization costs, time-consuming large-scale preparation and production, and low antibody/protein expression

Active Publication Date: 2022-02-22
MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gene recombination technology requires a lot of work and delicate design to find a suitable site for drug conjugation or polyethylene glycol modification, and the expression level of site-directed modified antibodies / proteins obtained by existing gene recombination technology is low, Therefore, it is very time-consuming in large-scale preparation and production, and the cost of research and development and final industrialization is very high

Method used

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  • Disubstituted maleamide linker for antibody-drug conjugation and its preparation method and use
  • Disubstituted maleamide linker for antibody-drug conjugation and its preparation method and use
  • Disubstituted maleamide linker for antibody-drug conjugation and its preparation method and use

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Experimental program
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preparation example Construction

[0140] Preparation method of antibody-drug conjugate

[0141] The antibody-drug conjugate preparation route is shown below. The disulfide bonds between the antibody chains are reduced to generate a total of 8 sulfhydryl groups, and the maleamide linker drug conjugate is cross-linked with the reduced antibody sulfhydryl group to generate the corresponding antibody drug conjugate, wherein the antibody drug The conjugate exists in one or two forms as in the following reactions.

[0142]

[0143] Dilute the antibody stock solution to 2-10mg / mL with reaction buffer, add 140-200 times excess molar ratio of dithiothreitol (DTT), or add 6.0-20 times excess molar ratio of tris(2-carboxyethyl ) phosphine hydrochloride (TCEP), the reaction solution was stirred at 10-35°C for 2-48 hours; the reaction buffer described here can be a buffer prepared in the following ratio: 50mM potassium dihydrogen phosphate-sodium hydroxide (KH 2 PO 4 -NaOH) / 150mM sodium chloride (NaCl) / 1mM diethyltri...

Embodiment 1

[0168] Embodiment 1 (synthesis and preparation of formula 1-12 compound)

[0169] 1.1 Synthesis of Compound F-1 (Formula 1)

[0170] 1.1.1 Synthesis of intermediate B-1 (step a)

[0171]

[0172] Weigh 4-fluoronitrobenzene (10.0g, 0.071mol), diethylene glycol (75.2g, 0.71mol) and potassium carbonate (14.7g, 0.11mol) into a 250mL round bottom bottle, stir at 80°C under nitrogen protection 22 hours. Slowly cool down to room temperature, extract with dichloromethane, wash with 1mol / L dilute hydrochloric acid, water and saturated brine in sequence, dry over anhydrous sodium sulfate, and spin to dry the solvent. Column chromatography (silica gel, 200-300 mesh, PE / EtOAC 10:1) gave the product as a light yellow transparent liquid (15.1 g, yield 94%). LC-MS (M + ) Theoretical value: 227.08, LC-MS (ESI, M+H + ) measured value: 228.12.

[0173] 1.1.2 Synthesis of intermediate C-1 (step b)

[0174]

[0175] Intermediate compound B-1 (3.0g, 9.52mmol) was dissolved in acetone ...

Embodiment 2

[0241] Embodiment 2 (synthesis and preparation of formula 19-25 compound)

[0242] 2.1 Synthesis of Compound G-1 (Formula 19)

[0243]

[0244] Compound F-3 (200mg, 0.34mmol) and compound D1 (220mg, 0.34mmol) were dissolved in N,N-dimethylformamide (10 mL), and 1-ethyl-(3-dimethyl Aminopropyl) carbodiimide hydrochloride (EDCI) (77 mg, 0.40 mmol) and 1-hydroxybenzotriazole (HOBT) (54 mg, 0.40 mmol), and the reaction mixture was stirred at room temperature overnight. The reaction solution was diluted with ethyl acetate, extracted with water, washed with water and saturated brine successively, dried over anhydrous sodium sulfate, and spun to dry the solvent. The crude product was separated and purified by silica gel chromatography (dichloromethane / methanol), and dried by suction to obtain yellow amorphous solid G-1 (3.5 g, yield 98%). LC-MS (M + ) Theoretical value: 1226.40, LC-MS (ESI, M+H + ) measured value: 1227.42.

[0245] 2.2 Synthesis of Compound G-2 (Formula 20) ...

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Abstract

The invention provides a double-substituted maleamide linker coupled with an antibody and its preparation method and application. Specifically, the invention couples strong cytotoxic active substances and biological macromolecules through a new type of linker couplet. This type of linker can selectively act on the disulfide chain at the same time, thereby greatly improving the material uniformity of the conjugate. The conjugate prepared by the linker of the present invention has high inhibitory activity on cell lines expressing corresponding antigens. The present invention also provides the preparation method and application of the above-mentioned conjugate.

Description

technical field [0001] The invention relates to a novel disulfide chain bridging and crosslinking reagent, a macromolecule, a therapeutic conjugate and a synthesis method thereof. More specifically, the present invention relates to a conjugate obtained by cross-linking cytotoxic drugs and macromolecules with a maleamide-based disulfide chain bridging cross-linking reagent, and a preparation method and use thereof. Background technique [0002] Over the years, a great deal of research has focused on improving the delivery efficiency of drugs or other agents to target cells, tissues or tumors to achieve maximum efficacy and minimum toxicity. Although many efforts have been made to develop efficient methods of delivering biologically active molecules into cells in vitro and in vivo, none have proven to be entirely satisfactory. Optimizing the binding of a drug to its intracellular target molecule while minimizing intracellular redistribution of the drug (eg, into neighboring c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D207/456C07D403/14C07D401/14C07D491/18C07D519/00C07D413/14C07D471/04C07K5/027C07K7/02C07K5/037C07K5/062C07K16/28C07K16/32A61K47/68A61K31/5365A61K38/06A61K38/05A61K31/395A61K31/551A61K31/454A61K31/496A61K31/437A61K31/4745A61P35/00
CPCA61K38/05A61K38/06A61K31/395A61K31/437A61K31/454A61K31/4745A61K31/496A61K31/5365A61K31/551C07D207/456C07D401/14C07D403/14C07D413/14C07D471/04C07D491/18C07D519/00C07K5/02C07K5/0205C07K5/06052C07K7/02C07K16/2863C07K16/32C07K19/00
Inventor 沈竞康孟韬马兰萍王昕彭红丽张永良于霆陈驎
Owner MABWELL (SHANGHAI) BIOSCIENCE CO LTD
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