CN117343125B - Synthesis method of antibody-coupled drug linker - Google Patents

Synthesis method of antibody-coupled drug linker Download PDF

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CN117343125B
CN117343125B CN202311661502.4A CN202311661502A CN117343125B CN 117343125 B CN117343125 B CN 117343125B CN 202311661502 A CN202311661502 A CN 202311661502A CN 117343125 B CN117343125 B CN 117343125B
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compound
reaction
action
antibody
condensing agent
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CN117343125A (en
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徐峰
江文
唐智豪
蔡锦
马佳琪
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Zhejiang Jiuzhou Pharmaceutical Co Ltd
Ruibo Suzhou Pharmaceutical Co Ltd
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Zhejiang Jiuzhou Pharmaceutical Co Ltd
Ruibo Suzhou Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

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  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Molecular Biology (AREA)
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Abstract

The invention discloses a method for synthesizing an antibody-coupled drug linker, which comprises the following steps: fmoc-Val-OH is taken as a starting material and reacts with an activating reagent under the action of a condensing agent to generate a compound 1; under the action of alkali and condensing agent, the compound 1 and L-citrulline are subjected to coupling reaction to generate a compound 2; reacting the compound 2 with 4-amino benzyl alcohol under the action of a condensing agent to generate a compound 3; and reacting the compound 3 with a PNP reagent under the action of alkali to generate an antibody coupling drug linker. The synthesis process has mild condition, high product yield and high purity, and the Fmoc-Val-Cit-PAB-PNP product has total yield over 58% and HPLC purity over 98%, and is suitable for industrial production.

Description

Synthesis method of antibody-coupled drug linker
Technical Field
The invention relates to the technical field of antibody coupling drug synthesis, in particular to a synthesis method of an antibody coupling drug linker Fmoc-Val-Cit-PAB-PNP.
Background
Antibodies have long been studied and clinically used as therapeutic agents. In 1975, kohler and milstein invented hybridoma technology in mice (Hybridoma technique), and monoclonal antibody therapy began in the corner of the head. In 1986 GREG WINTER opened a monoclonal antibody humanization technique. After this, several therapeutic monoclonal antibody drugs have been developed for the treatment of various cancers. With the gradual rise of antibody technology over the past several decades, antibody conjugated drugs have also entered a rapid development phase. Antibody-conjugated drugs (ADC-drug conjugates) are an emerging class of drugs that combine both Antibody and small molecule cytotoxins. The antibody provides cell targeting, can accurately deliver the drug to target cells, and furthest reduces the toxicity to normal tissue cells, so that the antibody has very broad application prospects in tumor treatment and other fields, such as immunity, anti-infection and the like. The ADC drug consists of three parts, namely a humanized/humanized monoclonal antibody (mAb), a cytotoxicity load (Cytotoxic payload or warhead) and a linker (linker), wherein the antibody is coupled with the cytotoxicity drug through the linker, so that the ADC cell targeting and the effect of killing cells are endowed together.
The Linker (Linker) is a chemical structure that links the cytotoxic load to the monoclonal antibody. The linker is not only easy to attach to the antibody and the load, but the covalent attachment established needs to be stable in the circulatory system, but also to be easily hydrolyzed after endocytosis of the target cell to release the drug active. After the antibody-mediated ADC reaches the cell, the ADC enters the cell by endocytosis, and the junction of the linker and the cytotoxic load is cleaved by hydrolysis or the like, thereby releasing the cytotoxic substance for its function. The linkers can be divided into two types, cleavable and non-cleavable, according to their way of hydrolysis within the cell. The cleavable linker is one which releases cytotoxic substances by physiological conditions in the cell and can be further subdivided into acid-sensitive, protease-sensitive or glutathione-sensitive cleavable linkers depending on the intracellular environment. While the non-cleavable linker can only be degraded by means of the lysosomes of the cells, the non-cleavable linker ADC drug has a longer half-life and lower off-target toxicity in the blood compared to the cleavable linker.
Fmoc-Val-Cit-PAB-PNP is a degradable polypeptide prodrug linker, is a common connecting bridge of antibody coupled drugs (ADC), and has certain stability in human blood plasma. Fmoc-Val-Cit-PAB-PNP has the molecular formula: c 40H42N6O10, the structural formula is as follows:
。
In the process of realizing the technical scheme in the embodiment of the application, the inventor discovers that the existing Fmoc-Val-Cit-PAB-PNP has the problems of low yield, high cost, unknown impurity types and the like in the synthesis process.
Disclosure of Invention
The invention can solve the defects in the prior art by providing a method for synthesizing an antibody-coupled drug linker.
In order to solve the technical problems, the invention provides a synthesis method of an antibody-coupled drug linker, which comprises the following steps:
(1) Fmoc-Val-OH is taken as an initial raw material, reacts with an activating reagent under the action of a condensing agent, is crystallized and purified to obtain a compound 1,
Wherein AE is N-succinimidyl or pentafluorophenol;
(2) Under the action of alkali and condensing agent, the compound 1 is subjected to coupling reaction with L-citrulline at 0-30 ℃, and is extracted and purified to obtain a compound 2;
(3) Reacting the compound 2 with 4-amino benzyl alcohol under the action of a condensing agent, crystallizing and purifying to obtain a compound 3;
(4) Reacting the compound 3 with a PNP reagent under the action of alkali, crystallizing and purifying to obtain a compound 4, namely the antibody coupling drug linker;
In a preferred embodiment of the present invention, in the step (1), the temperature of the reaction is 20 to 40 ℃; the activating reagent is N-hydroxysuccinimide or pentafluorophenol.
In a preferred embodiment of the present invention, in the step (2), the temperature of the reaction is 0 to 10 ℃; the reaction medium of the coupling reaction is at least one of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide, N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide.
In a preferred embodiment of the present invention, in the steps (1) to (3), the condensing agent includes any one of DIC, DCC, EDCI, HOOBT, HOBT, BOAT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP; and (3) adding a condensation auxiliary HOPO.
In a preferred embodiment of the present invention, in the steps (1), (3) and (4), the crystallization method is as follows: methyl tert-butyl ether or a mixture of methyl tert-butyl ether and methylene chloride was added to the reaction mixture, and the mixture was stirred until a solid was precipitated. In the step (2), the extraction method comprises the following steps: and regulating the pH value of the reaction liquid to 3-4, then adding tetrahydrofuran for extraction, and taking an organic phase.
In a preferred embodiment of the present invention, in the step (4), the PNP reagent includes at least one of bis (4-nitrophenyl) carbonate or 4-nitrobenzyl chloroformate.
The beneficial effects of the invention are as follows: the synthesis method of the antibody-coupled drug linker has mild conditions in the whole synthesis process, the post-treatment method of each reaction step is simple, the yield is high, the purity is good, the total yield of the Fmoc-Val-Cit-PAB-PNP product reaches more than 58%, the HPLC purity reaches more than 98%, the purity requirement of the medical product is met, and the method is suitable for industrial mass production.
Drawings
FIG. 1 is an HPLC chart of the Fmoc-Val-Cit-PAB-PNP pure synthesized according to the present invention;
FIG. 2 is a LC-MS spectrum of the Fmoc-Val-Cit-PAB-PNP pure synthesized according to the present invention.
Detailed Description
The technical scheme of the application is further described below with reference to the accompanying drawings and examples. The names and English abbreviations of the reagents and instruments used in the present application are shown in Table 1.
Table 1 reagents and apparatus
The invention provides a method for synthesizing an antibody-coupled drug linker Fmoc-Val-Cit-PAB-PNP, which specifically comprises the following steps:
(1) Fmoc-Val-OH is taken as a starting material, and reacts with N-hydroxysuccinimide or pentafluorophenol serving as an activating reagent in a reaction medium containing a condensing agent at 20-40 ℃, after the reaction is finished, the reaction liquid is dripped into MTBE to crystallize and separate out the compound 1, and the compound 1, namely Fmoc-Val-OAE, is obtained after filtering and drying a filter cake, wherein the structural formula is as follows:
Wherein AE is N-succinimidyl or pentafluorophenol;
The reaction medium includes at least one of DCM, TCM, THF, EA or toluene.
(2) Coupling reaction is carried out on the compound 1 with L-citrulline in a reaction medium containing alkali and condensing agent at 0-30 ℃, preferably 0-10 ℃, after the reaction is finished, the pH value of reaction liquid is regulated to 3-4, tetrahydrofuran is used for extraction, an organic phase is collected, and washing, concentrating and drying are carried out on the organic phase to obtain a compound 2, namely Fmoc-Val-Cit-OH, wherein the structural formula is as follows:
wherein the alkali comprises at least one of sodium carbonate, potassium carbonate or triisopropylethylamine, and the dosage of the alkali is 1-3 equivalents of the dosage of the compound 1. Preferably, the base is an inorganic base sodium carbonate or potassium carbonate. The use of inorganic base is beneficial to improving the reaction efficiency and saving the reaction time.
The reaction medium comprises at least one of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide, N-diethylformamide, N-methylpyrrolidone or dimethyl sulfoxide.
In this reaction step, the hydrolysis of the active ester, namely, the compound 1 can be effectively reduced by controlling the reaction temperature to be low in the range of 0 to 30 ℃, preferably 0 to 10 ℃, which contributes to the improvement of the purity and yield of the compound 2.
In addition, the crystallization and purification method can obviously simplify the post-treatment process and improve the yield.
(3) The compound 2 reacts with 4-amino benzyl alcohol in a reaction medium containing condensing agent at 0-30 ℃, after the reaction is finished, DCM and MTBE are added dropwise into the reaction liquid, the compound 3 is crystallized and separated out, and the compound 3 is obtained after filtration, washing and drying, namely the linker precursor Fmoc-Val-Cit-PAB, and the structural formula is as follows:
The reaction medium includes at least one of DCM, TCM, THF, EA or toluene.
(4) The compound 3 reacts with PNP reagent in a reaction medium containing alkali at 0-30 ℃, after the reaction is finished, DCM and MTBE are added dropwise into the reaction liquid to separate out the compound 4, and the compound 4 is obtained after filtration, washing and drying, namely the target product antibody coupling drug linker Fmoc-Val-Cit-PAB-PNP of the invention, and the structural formula is as follows:
Wherein the base comprises at least one of sodium carbonate, potassium carbonate or triisopropylethylamine, and the dosage of the base is 1-3 equivalents of the dosage of the compound 3.
The PNP reagent comprises at least one of 4-nitrophenylcarbonate or 4-nitrobenzyl chloroformate.
In the above steps (1) to (3), the condensing agent includes at least one of DIC, DCC, EDCI, HOOBT, HOBT, BOAT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP. Wherein, in the step (1), the condensing agent is preferably N, N' -Dicyclohexylcarbodiimide (DCC). DCC is used as a condensing agent, the solubility of a byproduct DCU is poor, the DCU is easy to separate out and remove at low temperature, the post-treatment process can be simplified, the method is suitable for industrial large-scale production, and the cost is saved. In the step (3), the condensation reagent is preferably EDCI, and EDCI can be used in combination with a condensation auxiliary agent HOPO to accelerate the reaction speed and reduce racemization, and meanwhile, the method has higher safety, effectively reduces the generation of impurities such as urea and the like, and can obtain a high-purity product after direct crystallization.
Example 1
Synthesis of Compound 1: fmoc-Val-OSu
To a 500mL three-necked flask, 10.0g of Fmoc-Val-OH (29 mmol), 4.1g HOSU (35 mmol) and 100mL of THF were added, and the solution was stirred and dissolved, and after the dissolution, the reaction solution was transferred to an ice-water bath and warmed to Tinterior=0 to 5 ℃. 7.3g DCC (35 mmol) was weighed, the solution was diluted with 10ml THF and added dropwise to the reaction flask via a constant pressure funnel, the process temperature T being =0-5 ℃. After the completion of the dropwise addition, the temperature was maintained, and the reaction was continued for 0.5h. The ice water bath was removed, the hot water bath was warmed to T-in = 30 ± 3 ℃, the reaction was continued and HPLC monitored for progress of the reaction.
After the reaction was completed, the reaction solution was added dropwise to 200mL of MTBE, a white solid was precipitated in the solution, and the filter cake was dried. Fmoc-Val-OSu was obtained as a white powder weighing 12.5g in 97% yield.
Example 2
Synthesis of Compound 2 Fmoc-Val-Cit-OH
To a 500ml three-necked flask, 10.0g of Fmoc-Val-OSu (23 mmol), 4.8g H-Cit-OH (28 mmol) and 100ml of THF were charged, and the mixture was stirred at a temperature of T=0 to 5℃and 5.5g of sodium carbonate (52 mmol) and 60ml of water were added dropwise. After the completion of the dropwise addition, the temperature was maintained, the reaction was continued, and the progress of the reaction was monitored by HPLC.
After the reaction, the reaction mixture was successively adjusted to pH 3-4 with HCl at a concentration of 1 mol/L, extracted with THF, and the organic phase was collected. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, slurried with n-hexane, filtered, and the cake dried. Fmoc-Val-Cit-OH was obtained as a white powder weighing 11.0g in 90% yield (81% overall).
Example 3
Synthesis of Compound 3 Fmoc-Val-Cit-PAB
To a 500mL three-necked flask, 10.0g of Fmoc-Val-Cit-OH (24 mmol), 3.0g of 4-aminobenzyl alcohol (25 mmol) and 100mL of DMF were added, the solution was stirred at a temperature of T=0 to 5℃and after the solution was removed, 5.8g of EDCI (36 mmol) and 3.5g of HOPO (36 mmol) were added, the reaction was continued, and the progress of the reaction was monitored by HPLC.
After the reaction was completed, DCM and MTBE were added dropwise, a white solid precipitated from the solution, filtered, and the filter cake was dried. Fmoc-Val-Cit-PAB was obtained as an off-white powder weighing 10.5g in 90% yield (total yield 72%).
Example 4
Synthesis of Compound 4 Fmoc-Val-Cit-PAB-PNP
To a 500mL three-necked flask, 10.0g of Fmoc-Val-Cit-PAB (16 mmol), 10.2g of bis (4-nitrophenyl) carbonate (32 mmol) and 100mL of DMF were added, and after the dissolution was performed by stirring at a temperature of T=0 to 5℃, 4.2g of DIPEA (32 mmol) was added to continue the reaction, and the progress of the reaction was monitored by HPLC.
After the reaction was completed, DCM and MTBE were added dropwise, a white solid precipitated from the solution, filtered, and the filter cake was dried. Fmoc-Val-Cit-PAB-PNP was obtained as an off-white powder weighing 10.0g in 80.3% yield (total yield 58%) with test patterns shown in FIGS. 1 and 2.
Aiming at the obvious disadvantages of low reaction purity, complicated post-treatment, incapability of large-scale production and the like in the traditional process, the invention improves the process, effectively improves the reaction center control purity by adjusting the reaction reagent and controlling the conditions, simplifies the post-treatment process, and is more suitable for industrialized mass production, thereby being beneficial to saving the cost and improving the reaction efficiency, the product yield and the purity.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims (2)

1. A method for synthesizing an antibody-conjugated drug linker, comprising the steps of:
(1) Fmoc-Val-OH is taken as a starting material, reacts with an activating reagent under the action of a condensing agent DCC, is crystallized and purified to obtain a compound 1,
(2) Under the action of alkali and condensing agent, the compound 1 is subjected to coupling reaction with L-citrulline at 0-10 ℃, and is extracted and purified to obtain a compound 2; the extraction method comprises the following steps: adjusting the pH value of the reaction liquid to 3-4, then adding tetrahydrofuran for extraction, taking an organic phase, washing, concentrating and drying; the reaction medium of the coupling reaction is tetrahydrofuran;
(3) Under the action of a condensing agent EDCI and a condensing aid HOPO, reacting the compound 2 with 4-amino benzyl alcohol in a DMF solvent, crystallizing and purifying to obtain a compound 3;
(4) Reacting the compound 3 with a PNP reagent under the action of alkali, adding DIPEA to continue the reaction after the reaction is completed, crystallizing and purifying to obtain a compound 4, namely the antibody coupling drug linker; the PNP reagent is bis (4-nitrophenyl) carbonate;
In the step (2), the condensing agent comprises any one of DIC, DCC, EDCI, HOOBT, HOBT, BOAT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP;
In the steps (1), (3) and (4), the crystallization method is as follows: and adding methyl tertiary butyl ether or a mixture of methyl tertiary butyl ether and methylene dichloride into the reaction liquid, stirring until solid is separated out, filtering, washing and drying.
2. The method of claim 1, wherein in step (1), the reaction temperature is 20 to 40 ℃.
CN202311661502.4A 2023-12-06 2023-12-06 Synthesis method of antibody-coupled drug linker Active CN117343125B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111560078A (en) * 2020-06-19 2020-08-21 联宁（苏州）生物制药有限公司 Double-arm intermediate with maleimide joint and synthetic method thereof
CN114502200A (en) * 2020-04-15 2022-05-13 深圳康源久远生物技术有限公司 Antibody drug conjugates

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114502200A (en) * 2020-04-15 2022-05-13 深圳康源久远生物技术有限公司 Antibody drug conjugates
CN111560078A (en) * 2020-06-19 2020-08-21 联宁（苏州）生物制药有限公司 Double-arm intermediate with maleimide joint and synthetic method thereof

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