Preparation method of ADC (Analog to Digital Converter) linker
By simplifying the preparation method of ADC linkers, the problems of long steps and high-temperature reactions in the prior art are solved, and high-yield and low-cost industrial production is achieved.
Patent Information
- Application Number
- CN202510716131.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-29
- Publication Date
- 2025-09-05
AI Technical Summary
Existing methods for preparing ADC linkers have long steps, which affects the overall yield. They also involve high-temperature reflux reactions, resulting in high costs and a high number of impurities, making them unsuitable for industrial production.
The compound represented by Formula II is reacted with the compound represented by Formula III to obtain the compound represented by Formula IV, which is then reacted with the compound represented by Formula V without separation or purification to prepare the ADC linker represented by Formula I, thereby simplifying the steps and controlling the conditions.
The simplified preparation process of the ADC linker is achieved, which is convenient for operation and control, improves the yield, is conducive to industrial production, and reduces costs.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing an ADC linker. Background Art
[0002] Antibody-Drug Conjugates (ADCs) are a class of targeted biopharmaceuticals consisting of an antibody, a linker, and a cytotoxic drug. They are designed to couple a target-specific monoclonal antibody with a highly potent cytotoxic drug via a specific linker. ADCs combine highly specific targeting with a highly potent killing effect, enabling accurate and efficient elimination of cancer cells, and have become a hot topic in anticancer drug research and development.
[0003] The design of ADC is relatively complex, and it is necessary to consider the three components of antibodies, linkers, and small molecule drugs, as well as the reasonable combination between them. It can be seen that the selection and preparation of linkers play an important role in the design and production process of ADC drugs. Derutecan is a conjugate of linkers and payloads used in the marketed ADC drug Trastuzumab Deruxtecan. In addition to its use in deruxtecan, deruxtecan can also be combined with other antibodies to explore different combinations of antibody-drug conjugates for use in different therapeutic areas. Datopotamab deruxtecan, an ADC drug first approved in 2024, is an application of deruxtecan combined with different antibodies. Antibody-drug conjugates such as Patritumab Deruxtecan, which have entered the Phase III clinical trial stage, also use deruxtecan as a linker and toxin drug part.
[0004] The linker portion of drutecan is MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH (CASE No.: 1599440-25-1, structure shown in Formula I). Currently available literature provides limited information on the preparation of this linker. The existing preparation route uses a Fomc-protected dipeptide as the starting material and proceeds through approximately six steps to obtain the compound shown in Formula I. This lengthy process affects overall yield, and the preparation process involves high-temperature reactions such as reflux. Therefore, further research into the preparation of this ADC linker is crucial to improve yield, reduce impurity generation, and lower costs, thereby facilitating expanded production and increasing product revenue.
[0005] Summary of the Invention
[0006] In view of this, the present invention provides a method for preparing an ADC linker. The present invention provides a method for preparing the ADC linker MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH, comprising reacting a compound represented by Formula II (H-Gly-Gly-Phe-OH) with a compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) to obtain a compound represented by Formula IV; and then reacting with a compound represented by Formula V without separation and purification to obtain a linker represented by Formula I (MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH). This method has a short preparation process, is easy to operate, has controllable conditions, and is conducive to industrial production.
[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0008] The present invention provides a method for preparing an ADC linker, comprising the following steps:
[0009] Step 1: Using the compound H-Gly-Gly-Phe-OH of Formula II as a starting material, reacting it with the compound of Formula III (6-(maleimido)hexanoic acid succinimidyl ester) in the presence of an acid-binding agent to obtain a compound of Formula IV;
[0010] Step 2: activating the compound of Formula IV obtained in Step 1 in a condensing agent, and reacting the activated compound with the compound of Formula V in the presence of an acid-binding agent to obtain the compound of Formula I, which is the ADC linker;
[0011]
[0012]
[0013] In some specific embodiments of the present invention, the condensing agent includes any one or more of HATU, TBTU, HBT U or HOBT.
[0014] In some specific embodiments of the present invention, the acid-binding agent comprises an organic weak base, preferably any one or more of triethylamine, pyridine or N-methylmorpholine.
[0015] In some specific embodiments of the present invention, the solvent for the compound represented by Formula II, the compound represented by Formula III, and the compound represented by Formula V comprises an organic solvent or a mixture of an organic solvent and water; the organic solvent comprises one or more of acetonitrile, dichloromethane, DMF, or DMAc.
[0016] In some specific embodiments of the present invention, the molar ratio of the compound represented by formula II to the acid binding agent includes 1: (1.5-5.5); and / or
[0017] The molar ratio of the compound represented by formula II to the condensing agent includes 1: (1.05-3); and / or
[0018] The molar ratio of the compound represented by formula II to the compound represented by formula V is 1:(1-1.1).
[0019] In some embodiments of the present invention, the reaction temperature comprises 20°C ± 5°C; and / or
[0020] The reaction time in step 1 is 30 to 35 minutes; and / or
[0021] The activation time in step 2 is 20 to 60 minutes; and / or
[0022] The reaction time in step 2 is 2 to 4 hours.
[0023] In some specific embodiments of the present invention, the reaction in step 2 further includes the steps of extraction and / or crystallization to obtain the compound represented by formula I.
[0024] In some specific embodiments of the present invention, the extraction step comprises: extracting the solution after the reaction in step 2 with ethyl acetate, washing with water, washing with saturated brine, drying, and concentrating.
[0025] In some specific embodiments of the present invention, the solvent used for the crystallization includes ethylene glycol dimethyl ether; preferably, the crystallization step includes: dispersing the reaction solution in step 2 with ethylene glycol dimethyl ether at 45-55°C, crystallizing at 5-25°C, filtering, and washing.
[0026] The present invention also provides the use of the ADC linker prepared by the preparation method in the preparation of ADC.
[0027] The present invention also provides a method for preparing ADC, comprising connecting an antibody and a drug using the ADC linker prepared by the preparation method.
[0028] The present invention also provides the use of the ADC prepared by the method in preparing anticancer drug preparations.
[0029] The present invention includes but is not limited to providing the following beneficial effects:
[0030] The present invention provides a method for preparing an ADC linker MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH, comprising reacting a compound represented by Formula II (H-Gly-Gly-Phe-OH) with a compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) to obtain a compound represented by Formula IV; and then reacting the compound with a compound represented by Formula V without separation or purification to obtain a linker represented by Formula I (MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH). This method has a short preparation process, is easy to operate, and has controllable conditions, making it suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.
[0032] Figure 1 1 shows the HPLC spectrum of the compound of formula I prepared in Example 1;
[0033] Figure 2 1 shows the mass spectrum of the compound of formula I prepared in Example 1;
[0034] Figure 3 1 is an HPLC spectrum of the compound of formula I prepared in Example 2;
[0035] Figure 4 1 and 2 show the HPLC pattern of the compound of formula I prepared in Example 3. DETAILED DESCRIPTION
[0036] The present invention discloses methods for preparing ADC linkers. Those skilled in the art can draw upon the present disclosure and appropriately modify process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications readily apparent to those skilled in the art are considered encompassed by the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is apparent that those skilled in the art will be able to modify, adapt, and combine the methods and applications described herein to implement and apply the present invention without departing from the content, spirit, and scope of the present invention.
[0037] The present invention provides a method for preparing an ADC linker. The compound represented by Formula II (H-Gly-Gly-Phe-OH) is reacted with the compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) to obtain the compound represented by Formula IV. A condensing agent and the compound represented by Formula V are added to the reaction solution, and the reaction is continued to obtain the linker represented by Formula I (MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH).
[0038]
[0039] The preparation process of the present invention, the complete route is as follows:
[0040]
[0041] The preparation method of compound I includes: adding the compound represented by formula II (H-Gly-Gly-Phe-OH), a solvent, and an acid binding agent to a reaction flask and stirring; dissolving the compound represented by formula III (6-(maleimido)hexanoic acid succinimide ester) in a solvent and adding it dropwise to the reaction flask; stirring the reaction at 20°C ± 5°C; sampling and monitoring until the reaction is complete; and obtaining the compound represented by formula IV.
[0042] A condensing agent is added to the reaction solution and stirred for activation; the compound represented by Formula V is dispersed with a solvent and an acid binding agent is added thereto; the suspension of the compound represented by Formula V is then added to the reaction system, stirred for reaction, and the compound represented by Formula I is obtained after post-treatment / purification.
[0043] In the preparation method, the compounds represented by Formula II and Formula III are reacted, and the solvent used is an organic solvent such as acetonitrile, dichloromethane, DMF, DMAc, or a mixture of an organic solvent and water; preferably acetonitrile, DMF, or a mixed solvent of acetonitrile / water;
[0044] In the preparation method, the reaction of the compounds represented by Formula II and Formula III is carried out at 20°C ± 5°C;
[0045] In the preparation method, the condensing agent used is HATU, TBTU, HBTU, HOBT, etc., and the molar ratio of Formula II to the condensing agent is 1:(1.05-3). In the preparation method shown, the acid binding agent is an organic weak base, preferably triethylamine, pyridine, or N-methylmorpholine; the molar ratio of Formula II to the acid binding agent is 1:(1.5-5.5).
[0046] Unless otherwise specified, the raw materials and reagents used in the preparation method of the ADC linker provided by the present invention can be purchased from the market.
[0047] The present invention will be further described below in conjunction with the embodiments:
[0048] Example 1
[0049] To a reaction flask, add 0.5 g of the compound represented by Formula II (H-Gly-Gly-Phe-OH), 6 mL of DMF, and 0.28 g (1.5 eq) of triethylamine (TEA) and stir. Dissolve 0.6 g (1.1 eq) of the compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) (EMCS) in 1.8 mL of DMF and add dropwise to the reaction flask. Stir and react at approximately 20°C ± 5°C for 35 minutes. Sampling is performed to confirm the reaction is complete.
[0050] To the above reaction solution, 0.67 g (1.05 eq) of HATU (a condensing agent) was added, and the mixture was stirred and activated for 20 min. The compound represented by Formula V was dispersed in 5 mL of DMF, and 0.18 g (1 eq) of triethylamine was added to form a suspension. The suspension containing 0.19 g (1.1 eq) of the compound represented by Formula V was added to the reaction system, and the mixture was stirred at 20°C ± 5°C for 2 h. The reaction was completed after sampling.
[0051] The reaction solution was added dropwise to 38.4 mL of ethylene glycol dimethyl ether precooled to 5°C, stirred and crystallized for 2 hours, and filtered to obtain a solid. The filter cake was washed with ethylene glycol dimethyl ether (12.8 mL). The filter cake was transferred to a reaction flask, dispersed with 15 mL of ethylene glycol dimethyl ether, heated to 45-55°C and stirred for 1-2 hours, cooled to 15-25°C and crystallized for 1-2 hours, filtered to obtain a solid, and washed with ethylene glycol dimethyl ether (5 mL) to obtain 0.72 g of a white powder of the compound represented by Formula I with a yield of 65%. The purity was greater than 98% ( ) by high performance liquid chromatography (detection method: chromatographic column: Welch Xtimate C18 4.6 mm x 150 mm, 5 um; mobile phase A: 0.05% phosphoric acid aqueous solution (V / V); mobile phase B: acetonitrile). Figure 1 ). MS (ESI, Neg.): m / z 615.3(MH), 1231.6(2M-H)( Figure 2 ).
[0052] Example 2
[0053] To a reaction flask, add 5.5 g of the compound represented by Formula II (H-Gly-Gly-Phe-OH), 55 mL of a 50% aqueous acetonitrile solution, and 3.9 g (2.5 eq) of pyridine and stir. Dissolve 9.0 g (1.5 eq) of the compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) (EMCS) in 26.4 mL of acetonitrile and add dropwise to the reaction flask. Stir and react at approximately 20°C ± 5°C for 30 min. Sampling is performed to confirm the reaction is complete.
[0054] To the above reaction solution, 11.3 g (1.8 eq) of TBTU, a condensing agent, was added and activated by stirring for 30 min. 2.1 g (1.05 eq) of the compound represented by formula V was dispersed in 27.5 mL of 50% acetonitrile aqueous solution, and 1.87 g (1.2 eq) of pyridine was added to form a suspension. The suspension containing the compound represented by formula V was added to the reaction system, stirred at 20°C ± 5°C for 1.5 h, and the reaction was complete after sampling.
[0055] The mixture was extracted with ethyl acetate (110 mL * 2), washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated; the crude product was dispersed with 165 mL of ethylene glycol dimethyl ether, heated to 45-55 ° C and stirred for 1-2 h, cooled to 15-25 ° C and crystallized for 1-2 h, and the solid was filtered. The filter cake was washed with ethylene glycol dimethyl ether (55 mL) to obtain 8.25 g of the compound represented by formula I as a white powder, with a yield of 68% and a purity greater than 97.5% ( Figure 3 ).
[0056] Example 3
[0057] To a reaction flask, add 12g of the compound represented by Formula II (H-Gly-Gly-Phe-OH), 144mL of DMF, and 10.9g (2.5eq) of N-methylmorpholine and stir. Dissolve 19.9g (1.5eq) of the compound represented by Formula III (6-(maleimido)hexanoic acid succinimidyl ester) (EMCS) in 43mL of DMF and add dropwise to the reaction flask. Stir and react at approximately 20°C ± 5°C for 35 minutes. Sampling is performed to confirm the reaction is complete.
[0058] To the above reaction solution, 11.6 g (2 eq) of HOBT and 16.3 g (1 eq) of HBTU were added as condensing agents, and the mixture was stirred and activated for 1 h. The compound represented by Formula V was dispersed in 120 mL of DMF, and 8.7 g (2 eq) of N-methylmorpholine was added to form a suspension. The suspension containing 4.1 g (1 eq) of the compound represented by Formula V was added to the reaction system, and the mixture was stirred at 20°C ± 5°C for 4 h. Sampling was performed to control the reaction, and the reaction was complete.
[0059] The reaction solution was added dropwise to 921.6 mL of ethylene glycol dimethyl ether precooled to 5°C, stirred and crystallized for 2 hours, and filtered to obtain a solid. The filter cake was washed with ethylene glycol dimethyl ether (307.2 mL). The filter cake was transferred to a reaction flask, dispersed with 360 mL of ethylene glycol dimethyl ether, heated to 45-55°C and stirred for 1-2 hours, cooled to 15-25°C and crystallized for 1-2 hours, filtered to obtain a solid, and washed with ethylene glycol dimethyl ether (120 mL) to obtain 17.7 g of a white powder of the compound represented by Formula I, with a yield of 67% and a purity greater than 98% ( Figure 4 ).
[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method for preparing an ADC linker, characterized in that: The steps include: Step 1: Using the compound H-Gly-Gly-Phe-OH of Formula II as a starting material, reacting it with the compound of Formula III (6-(maleimido)hexanoic acid succinimidyl ester) in the presence of an acid-binding agent to obtain a compound of Formula IV; Step 2: activating the compound of Formula IV obtained in Step 1 in a condensing agent, and reacting the activated compound with the compound of Formula V in the presence of an acid-binding agent to obtain the compound of Formula I, which is the ADC linker; 2. The preparation method according to claim 1, wherein The condensing agent includes any one or more of HATU, TBTU, HBTU or HOBT; and / or The acid binding agent includes an organic weak base; preferably any one or more of triethylamine, pyridine or N-methylmorpholine.
3. The preparation method according to claim 1 or 2, wherein The solvent for the compound represented by Formula II, the compound represented by Formula III and the compound represented by Formula V includes an organic solvent or a mixture of an organic solvent and water; the organic solvent includes one or more of acetonitrile, dichloromethane, DMF or DMAc.
4. The preparation method according to any one of claims 1 to 3, characterized in that The molar ratio of the compound represented by formula II to the acid binding agent is 1:(1.5-5.5); and / or The molar ratio of the compound represented by formula II to the condensing agent includes 1: (1.05-3); and / or The molar ratio of the compound represented by formula II to the compound represented by formula V is 1:(1-1.1).
5. The preparation method according to any one of claims 1 to 4, characterized in that The reaction temperature includes 20°C ± 5°C; and / or The reaction time in step 1 is 30 to 35 minutes; and / or The activation time in step 2 is 20 to 60 minutes; and / or The reaction time in step 2 is 2 to 4 hours.
6. The preparation method according to any one of claims 1 to 5, characterized in that After the reaction in step 2, the step of extracting and / or crystallizing to obtain the compound represented by formula I is also included.
7. The preparation method according to any one of claims 1 to 6, characterized in that The extraction step comprises: extracting the solution after the reaction in step 2 with ethyl acetate, washing with water, washing with saturated brine, drying, and concentrating; The solvent used for the crystallization includes ethylene glycol dimethyl ether; preferably, the crystallization step includes: dispersing the solution after the reaction in step 2 with ethylene glycol dimethyl ether at 45-55°C, crystallizing at 5-25°C, filtering, and washing.
8. Use of the ADC linker prepared by the preparation method according to claims 1 to 7 in the preparation of ADC.
9. A method for preparing ADC, characterized in that: The invention comprises connecting an antibody and a drug using an ADC linker prepared by the preparation method according to claims 1 to 7.
10. Use of the ADC prepared by the method according to claim 9 in the preparation of anticancer drug preparations.