Synthetic method of antibody coupling drug connexon with aldehyde group
The synthesis of antibody-conjugated drug linkers with aldehyde groups by para-hydroxybenzaldehyde as the starting material has solved the problem of inaccurate drug release and poor stability in the lesion area, and the stability of the linker has been improved and a variety of release methods have been achieved, which is suitable for the development of antibody-conjugated drugs.
Patent Information
- Application Number
- CN202510583967.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-29
AI Technical Summary
The existing antibody-conjugated drug linkers have inaccurate drug release in the lesion area and poor stability of the linker, resulting in rapid drug release and a single connection method, which cannot keep up with the speed of drug development.
Using para-hydroxybenzaldehyde as the starting material, antibody-coupled drug linkers with aldehyde groups are synthesized through substitution, amidation, deprotection and transesterification reactions. The specific steps include the synthesis of compounds 1a, 1b, 1c, 1d and 1e, and the use of FDA-approved non-cleavable linkers to bind the aldehyde group.
The stability of antibody-conjugated drug linker has been improved, the drug connection and release methods have been expanded, and the in vivo circulation stability and multiple release abilities have been improved.
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Figure CN120554271A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of pharmaceutical chemical synthesis, and particularly relates to a method for synthesizing an antibody-drug conjugate linker having an aldehyde group. Background Art
[0002] Antibody-Drug Conjugates (ADC) are innovative targeted biological drugs that connect monoclonal antibodies and biologically active cytotoxic drugs through linkers. Through the targeting effect of monoclonal antibodies, the drugs are directly delivered into tumor cells to kill tumor cells.
[0003] Antibody-drug conjugates generally consist of three parts: a monoclonal antibody used to guide the drug into tumor cells through endocytosis, a linker used to maintain the stability of the entire ADC drug in the blood circulation and to release the drug under appropriate conditions, and a drug used to directly induce apoptosis of cancer cells.
[0004] Existing linkers have numerous issues, such as the inability to precisely release drugs at the lesion site, poor linker stability, rapid drug release in a short time, and relatively limited linker-drug connection methods. As research on ADCs continues to grow, the variety of drugs is also rapidly increasing, but the number of linkers is relatively small, unable to keep up with the pace of drug development. Therefore, the research and development of new antibody-drug conjugate linkers has become an important and urgent research topic. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide a method for synthesizing an antibody-drug conjugate linker having an aldehyde group. The present invention uses p-hydroxybenzaldehyde as a starting material and synthesizes an antibody-drug conjugate linker having an aldehyde group through substitution, amidation, deprotection, ester exchange and other reactions, providing a basis for expanding the types of antibody-drug conjugate linkers.
[0006] The object of the present invention is to achieve the following goals:
[0007] In a first aspect, the present invention provides a method for synthesizing an antibody-drug conjugate linker having an aldehyde group, and the synthetic route is as follows:
[0008]
[0009] Based on the above technical solution, the synthesis method further comprises the following steps:
[0010] (1) Compound 1a was synthesized using p-hydroxybenzaldehyde and ethyl 4-bromobutyrate as starting materials;
[0011] (2) removing the ethyl group from compound 1a obtained in step (1) to synthesize compound 1b;
[0012] (3) Compound 1b obtained in step (2) is reacted with Boc-ethylenediamine to generate compound 1c through amidation reaction;
[0013] (4) subjecting compound 1c obtained in step (3) to an acidic deprotection reaction to remove the Boc group to obtain compound 1d;
[0014] (5) Compound 1d obtained in step (4) is subjected to a condensation reaction with 6-(maleimido)hexanoic acid succinimidyl ester to generate 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(4-(4-formylphenoxy)butyramido)ethyl]hexanamide in one pot, thereby obtaining an antibody-drug conjugate linker having an aldehyde group.
[0015] Based on the above technical solution, further, in step (1), the molar ratio of p-hydroxybenzaldehyde to ethyl 4-bromobutyrate is 1:(1.1-1.5), the solvent is N,N-dimethylformamide, the reaction temperature is 40-60° C., and the reaction time is 4-10 hours.
[0016] Based on the above technical solution, further, the activation reagent for the amidation reaction in step (3) is 1-hydroxybenzotriazole (HOBt), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and N,N-diisopropylethylamine (DIPEA), the reaction solvent is N,N-dimethylformamide, the reaction temperature is 10-40°C, and the reaction time is 10-14 hours.
[0017] Based on the above technical solution, further, the molar ratio of compound 1b described in step (3) to 1-hydroxybenzotriazole (HOBt), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropylethylamine (DIPEA), and Boc-ethylenediamine is 1:(1-1.2):(1.2-1.6):(1.2-1.6):(1-1.2).
[0018] Based on the above technical solution, further, the acidic deprotection reaction in step (4) adopts 5-7M hydrochloric acid, the reaction temperature is 10-40°C, and the reaction time is 3-6 hours.
[0019] Based on the above technical solution, further, the condensation agent of the condensation reaction in step (5) is N,N-diisopropylethylamine (DIPEA), the reaction solvent is N,N-dimethylformamide, the reaction temperature is 10-40°C, and the reaction time is 10-14 hours.
[0020] Based on the above technical solution, further, in step (5), the molar ratio of compound 1d, 6-(maleimido)hexanoic acid succinimide ester and N,N-diisopropylethylamine (DIPEA) is 1:(1.0-1.2):(1.1-1.3).
[0021] In a second aspect, the present invention provides an antibody-drug conjugate linker having an aldehyde group obtained by the above-mentioned synthesis method, the structure of which is shown in formula (1):
[0022]
[0023]
[0024] The present invention has the following beneficial effects compared to the prior art:
[0025] The present invention uses an FDA-approved non-cleavable linker to bind an aldehyde group, which gives it better stability in the body's circulation. Its aldehyde group can expand various drug connection and release methods. For example, the aldehyde group can be converted into a thioacetal group to enable it to have the ability to respond to ROS, which has very good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.
[0027] Figure 1 This is the 1H NMR (500 MHz, DMSO-d6) spectrum of compound 1a.
[0028] Figure 2 For compound 1a 13 C NMR (126 MHz, DMSO-d6) spectrum.
[0029] Figure 3 1H NMR (500 MHz, DMSO-d6) spectrum of compound 1b.
[0030] Figure 4 Compound 1b 13 C NMR (126 MHz, DMSO-d6) spectrum.
[0031] Figure 5 This is the 1H NMR (500 MHz, DMSO-d6) spectrum of compound 1c.
[0032] Figure 6 For compound 1c 13 C NMR (126 MHz, DMSO-d6) spectrum.
[0033] Figure 7This is the 1H NMR (500 MHz, DMSO-d6) spectrum of compound 1e.
[0034] Figure 8 For compound 1e 13 C NMR (126 MHz, DMSO-d6) spectrum. DETAILED DESCRIPTION
[0035] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.
[0036] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.
[0037] Example 1
[0038] This embodiment provides a method for synthesizing an antibody-drug conjugate linker having an aldehyde group, comprising the following steps:
[0039] (1) Synthesis of ethyl 4-(4-formylphenoxy)butyrate. The synthetic route is as follows:
[0040]
[0041] 1.4 g (11 mmol, 1 eq) of p-hydroxybenzoic acid was weighed and placed in a 100 ml single-necked flask containing 30 ml of N,N-dimethylformamide. After thorough stirring to dissolve, 1.96 ml (13 mmol, 1.3 eq) of ethyl 4-bromobutyrate was stirred again to disperse. Finally, 3.15 g (22 mmol, 2 eq v / v) of anhydrous potassium carbonate was added. After thorough stirring, the mixture was placed in an oil bath at 50°C for reaction. During this time, the reaction progress was monitored by TLC (EA:PE = 1:10). The results showed that the raw material was completely reacted after 6 hours. The reaction was stopped and cooled to room temperature. Post-treatment: The mixture was poured into 150 ml of distilled water and stirred thoroughly. The aqueous phase was extracted three times with ethyl acetate (50 ml) in batches. The organic phases were combined and dried over anhydrous sodium sulfate. The ethyl acetate in the organic phase was then distilled off under reduced pressure in vacuo and dried to obtain the desired product.
[0042] Ethyl 4-(4-formylphenoxy)butanoate (1a), light yellow oily liquid, 2.47 g, yield 95%.
[0043] 1H NMR (500MHz, DMSO) δ9.86 (d, J = 16.8 Hz, 1H), 7.86 (s, 2H), 7.11 (s, 2H), 4.48 -3.83 (m, 4H), 2.60 - 2.22 (m, 2H), 2.00 (m, 2H), 1.50 -0.76 (m, 3H).
[0044] (2) Synthesis of 4-(4-formylphenoxy)butyric acid. The synthetic route is as follows:
[0045]
[0046] The specific steps of the experiment are as follows:
[0047] Weigh 2.47g of 1a into a 100ml single-necked flask, add 20ml of methanol, stir thoroughly to dissolve, and slowly add 20ml of 15% potassium hydroxide solution dropwise. After the addition is complete, heat to 80°C and incubate for reaction. Monitor the reaction by TLC (EA:PE = 1:5 v / v). After 3 hours, the starting material disappears from the reaction solution, stop the reaction, and cool the reaction solution to room temperature. Prepare 20ml of 3M HCl solution and slowly add it dropwise to the cooled reaction solution. Stir while checking the pH of the reaction solution with pH paper. Stop adding when the solution reaches pH = 1. Stir rapidly to precipitate a large amount of solid. Isolate the solid by filtration and wash it with deionized water several times to remove residual acid and solvent. Vacuum dry to obtain the desired product.
[0048] 4-(4-Formylphenoxy)butanoic acid (1b), pale yellow solid, 2.01 g, yield 92%.
[0049] 1 H NMR (500MHz, DMSO) δ12.04(s,1H),9.86(d,J=2.0Hz,1H),7.93-7.70(m,2H),7.12(m,2H),4.17-3.98(m,2H),2.39(m,2H),1.97(m,2H).
[0050] (3) Synthesis of 4-(4-formylphenoxy)-N-(2-pivalamidoethyl)butyramide (Compound 1c). The synthetic route is as follows:
[0051]
[0052] The specific steps of the experiment are as follows:
[0053] Weigh 1.84 g (8.8 mmol, 1 eq) of 1b into a single-necked flask, add 30 ml of N,N-dimethylformamide and stir to fully dissolve it, then add 1.3 g (9.68 mmol, 1.1 eq) of 1-hydroxybenzotriazole and 2.53 g (13.2 mmol, 1.5 eq) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.3 ml (13.2 mmol, 1.5 eq) of N,N-diisopropylethylamine, adding each compound one by one. After thorough stirring to dissolve, 1.84 ml (9.68 mmol, 1.1 eq) of Boc-ethylenediamine was added and allowed to react at 25°C. The reaction was monitored by TLC (MeOH:DCM = 1:30 v / v). After 12 hours, the reaction completely ceased. Post-treatment: Most of the DMF was removed by vacuum distillation. 60 ml of deionized water was then added to the mixture. The aqueous phase was extracted with ethyl acetate (40 ml x 3). The organic phase was collected, silica gel was added, and the solvent was removed by vacuum distillation. Purification was performed by column chromatography (MeOH:DCM = 1:30) and vacuum drying to obtain the desired product.
[0054] 4-(4-Formylphenoxy)-N-(2-pivalamidoethyl)butanamide (1c), white solid, 1.9 g, yield 65%.
[0055] 1 H NMR(500MHz, CDCl3)δ9.88(s,1H),7.86-7.80(m,2H),7.02-6.96(m,2H),6.35(s,1 H),4.91(s,1H),4.10(m,3H),3.37(m,2H),2.40(m,2H),2.16(m,2H),1.43(s,9H).
[0056] HRMS:[M+Na] + :357.1790,found:357.1792.
[0057] (4) Synthesis of 4-(4-formylphenoxy)-N-(2-aminoethyl)butanamide (Compound 1d). The synthetic route is as follows:
[0058]
[0059] The specific steps of the experiment are as follows:
[0060] 1 g (3 mmol) of 1c was placed in a single-necked flask containing 30 ml of methanol. 20 ml of 6 M HCl was slowly added dropwise through a constant pressure dropping funnel. The reaction was allowed to react at room temperature. The reaction progress was monitored by TLC. After 5 h, the starting material spot disappeared. The reaction solution was removed by vacuum distillation and dried in vacuo to obtain a dark red oily liquid, which was used directly in the next reaction without further treatment.
[0061] (5) Synthesis of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(4-(4-formylphenoxy)butyramido)ethyl]hexanamide (Compound 1e). The synthetic route is as follows:
[0062]
[0063] The specific steps of the experiment are as follows:
[0064] Compound 1d obtained in the previous step was dissolved in 20 ml of N,N-dimethylformamide. 948 mg (3.3 mmol, 1.1 eq) of succinimidyl 6-(maleimido)hexanoate and 0.58 ml (3.6 mmol, 1.2 eq) of N,N-diisopropylethylamine were added to the solution. The reaction was stirred at room temperature and monitored by TLC (MeOH:DCM = 1:15 v / v). After 12 hours, the reaction was complete. The solvent was removed by vacuum distillation and the product was purified by column chromatography (MeOH:DCM = 1:30 v / v). The solvent was removed by vacuum distillation and the product was washed with methyl tert-butyl ether for 12 hours. The methyl tert-butyl ether was removed by filtration to obtain the desired product.
[0065] 6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(4-(4-formylphenoxy)butyramido)ethyl]hexanamide (1e), white solid, 0.6 g, yield 50%.
[0066] 1 H NMR (500MHz, DMSO) δ9.86 (s, 1H), 7.97-7.69 (m, 4H), 7.11 (d, J = 8.2Hz, 2H), 7.00 (d, J = 1.8Hz, 2H), 4.08 (t, J = 6.3 Hz,2H),3.36(t,J=7.1Hz,2H),3.13-2.92(m,4H),2.23(t,J=7.3Hz,2H),1.97(m,4H),1.45(m,4H),1.15(m,2H).
[0067] HRMS:[M+Na] + :466.1954,found:466.1950.
[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A method for synthesizing an antibody-drug conjugate linker having an aldehyde group, characterized in that: The synthetic route is as follows:
2. The synthesis method according to claim 1, wherein The synthetic method comprises the following steps: (1) Compound 1a was synthesized using p-hydroxybenzaldehyde and ethyl 4-bromobutyrate as starting materials; (2) removing the ethyl group from compound 1a obtained in step (1) to synthesize compound 1b; (3) Compound 1b obtained in step (2) is reacted with Boc-ethylenediamine to generate compound 1c through amidation reaction; (4) subjecting compound 1c obtained in step (3) to an acidic deprotection reaction to remove the Boc group to obtain compound 1d; (5) Compound 1d obtained in step (4) is subjected to a condensation reaction with 6-(maleimido)hexanoic acid succinimidyl ester to generate 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-[2-(4-(4-formylphenoxy)butyramido)ethyl]hexanamide in one pot, thereby obtaining an antibody-drug conjugate linker having an aldehyde group.
3. The synthesis method according to claim 2, characterized in that In step (1), the molar ratio of p-hydroxybenzaldehyde to ethyl 4-bromobutyrate is 1:(1.1-1.5), the solvent is N,N-dimethylformamide, the reaction temperature is 40-60° C., and the reaction time is 4-10 hours.
4. The synthesis method according to claim 2, characterized in that The activating reagents for the amidation reaction in step (3) are 1-hydroxybenzotriazole (HOBt), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and N,N-diisopropylethylamine (DIPEA), the reaction solvent is N,N-dimethylformamide, the reaction temperature is 10-40°C, and the reaction time is 10-14 hours.
5. The synthesis method according to claim 4, characterized in that The molar ratio of compound 1b described in step (3) to 1-hydroxybenzotriazole (HOBt), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), N,N-diisopropylethylamine (DIPEA), and Boc-ethylenediamine is 1:(1-1.2):(1.2-1.6):(1.2-1.6):(1-1.2).
6. The synthesis method according to claim 2, characterized in that The acidic deprotection reaction in step (4) adopts 5-7M hydrochloric acid, the reaction temperature is 10-40°C, and the reaction time is 3-6 hours.
7. The synthesis method according to claim 2, characterized in that The condensation agent of the condensation reaction in step (5) is N,N-diisopropylethylamine (DIPEA), the reaction solvent is N,N-dimethylformamide, the reaction temperature is 10-40° C., and the reaction time is 10-14 hours.
8. The synthesis method according to claim 7, characterized in that The molar ratio of compound 1d, 6-(maleimido)hexanoic acid succinimidyl ester and N,N-diisopropylethylamine (DIPEA) in step (5) is 1:(1.0-1.2):(1.1-1.3).
9. The antibody-drug conjugate linker having an aldehyde group obtained by the synthesis method according to any one of claims 1 to 8, characterized in that: The structure is shown in formula (1):