Synthetic method of antibody coupling drug connexon

Abstract

The invention provides a synthetic method of an antibody-coupled drug connexon, which comprises the following steps: taking Val-Cit-Pab as an initial raw material, and reacting with 1, 5-glutaric acid mono-tert-butyl ester under the action of a condensing agent to generate OBoc-Glu-Val-Cit-Pab; removing a tert-butyl protecting group from the OBocc-Glu-Val-Cit-Pab under the action of an acid, so as to generate HO-Glu-Val-Cit-Pab; and further reacting the product without the tert-butyl protection with an activating reagent under the action of a condensing agent to obtain the compound OSu-Glu-Val-Cit-Pab. The synthesis process is mild in condition, the product in each reaction step is high in yield and good in purity, the purity requirement of a medicine product is met, and the method is suitable for industrial mass production.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemical industry, and particularly relates to a method for synthesizing an antibody-drug coupling linker OSu-Glu-Val-Cit-Pab. Background Art

[0002] In recent years, scientists have made great efforts to improve the therapeutic activity of mAbs through various modifications. A promising method is to integrate antibodies and small molecule chemical drugs into one molecule, called antibody-drug conjugate (ADC). ADC is structurally composed of three parts: antibody, linker and small molecule cytotoxin. The antibody part can specifically recognize the target expressing tumor antigens, and relies on the linker as a bridge to couple with the small molecule cytotoxin with strong lethality to form a targeted delivery system that only targets tumor cells. This method can achieve a strong combination of the advantages of antibody drugs and small molecule chemical drugs, use antibodies to achieve tumor targeting, and release small molecule cytotoxins with strong lethality in target tissues to effectively kill tumor cells. It has the advantages of high activity, low toxic side effects, and long action time, which greatly improves the therapeutic index of small molecule chemical drugs. At the same time, it also solves the problems of low activity and drug resistance of antibody drugs to a certain extent. It has now become a hot direction for the research and development of anti-tumor drugs, and at the same time, it also has very broad application prospects in other fields such as immunity and anti-infection.

[0003] One of the biggest challenges in the development of ADCs is to provide appropriate linkers for antibody-drug conjugation. Linkers are crucial for the preclinical, clinical efficacy and safety of ADC drugs. Ideal linkers must be sufficiently stable in the circulatory system, because premature release of small molecule cytotoxins in the circulatory system can produce non-targeted and unexpected toxicity. However, these linkers must be able to effectively release cytotoxins in a highly active form in the cytoplasm of target cells after the ADC drug is internalized and transported to specific intracellular bodies. There are many important issues to consider when designing linkers, including the antibody connection site and internalization, the nature of the toxin, the polarity of the linker, etc. ADC linkers that are currently on the market and in clinical trials can be divided into two main categories, namely cleavable linkers and non-cleavable linkers. Cleavable linkers release cytotoxic substances through physiological conditions in cells, and can be further subdivided into acid-sensitive, protease-sensitive or glutathione-sensitive cleavable linkers according to different intracellular environments. Non-cleavable linkers can only be degraded with the help of lysosomes in cells.

[0004] OSu-Glu-Val-Cit-Pab is an enzyme-cleavable peptide linker. Its OSu linker can be quickly connected with different structural fragments to form enzyme-cleavable peptide linkers of different lengths and types. This type of linker has a certain stability in human plasma and can be cleaved by cathepsin B to release toxins after reaching the tumor site. Its molecular formula is: C 27 H 38 N6O9, its structural formula is as follows: .

[0005] The existing OSu-Glu-Val-Cit-Pab has problems such as low yield, high cost, poor purity, and unknown impurity types during the synthesis process. Summary of the invention

[0006] The present invention provides a method for synthesizing an antibody-drug conjugate linker OSu-Glu-Val-Cit-Pab, which is prepared by three-step reaction using Val-Cit-Pab as a starting material. The synthetic route is as follows: .

[0007] The preferred preparation process of an antibody-drug conjugate linker OSu-Glu-Val-Cit-Pab provided by the present invention is: (1) Using Val-Cit-Pab as the starting material, stirring at 0±5°C in a reaction medium containing a condensation agent and a condensation aid. Then adding 1,5-pentanedioic acid mono-tert-butyl ester, raising the temperature to carry out a condensation reaction. After the reaction is completed, DCM is dripped into the reaction solution, and then MTBE and n-heptane are dripped into the reaction solution to precipitate crystals, which are filtered and dried to obtain compound 1, namely OBoc-Glu-Val-Cit-Pab; (2) Compound 1 is subjected to acidic conditions to remove the tert-butyl protecting group, and then directly concentrated under reduced pressure to obtain compound 2, namely HO-Glu-Val-Cit-Pab; (3) Compound 2 is reacted with an activating reagent in a reaction medium containing a condensing agent. After the reaction is completed, a mixed solvent of acetonitrile and MTBE is added dropwise to the reaction solution for crystallization. The mixture is filtered and the filter cake is dried to obtain compound 3, i.e., OSu-Glu-Val-Cit-Pab.

[0008] Wherein, the reaction medium in step (1) is at least one of DMF, DMAc, THF, EA, DCM, toluene, N-methylpyrrolidone or dimethyl sulfoxide.

[0009] Wherein, the acid in step (2) is at least one of TFA, hydrochloric acid and acetic acid, and preferably, the acid is TFA.

[0010] Wherein, the molar ratio of compound 1 to acid in step (2) is in the range of 1:3-5.

[0011] Wherein, the reaction medium in step (2) is one of DCM, DMF, EA and TCM.

[0012] Wherein, the reaction temperature of step (2) is 10-30°C.

[0013] Wherein, the activation reagent in step (3) is N-hydroxysuccinimide.

[0014] Wherein, the reaction temperature of step (3) is -5~5°C.

[0015] Wherein, the condensing agent in steps (1) to (3) includes at least one of DIC, DCC, EDCI, HOBT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP. Preferably, the condensing agent in step (1) is N,N'-dicyclohexylcarbocyanine (DCC), and the condensing agent in step (3) is EDCI.

[0016] Beneficial effects of the present invention: The present invention provides a method for synthesizing an antibody-drug conjugate linker. The entire synthesis process has mild conditions, and the post-treatment method of each reaction step is simple and has high yield and good purity. The total yield of the reaction product OSu-Glu-Val-Cit-Pab reaches more than 81%, and the HPLC purity reaches more than 98%, which meets the purity requirements of pharmaceutical products and is suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the HPLC spectrum of the pure product OSu-Glu-Val-Cit-Pab of Example 1 of the present invention.

[0018] Figure 2 This is the LC-MS spectrum of the pure product OSu-Glu-Val-Cit-Pab of Example 1 of the present invention. DETAILED DESCRIPTION

[0019] In order to further understand the present invention, the following is a detailed description of a method for synthesizing an antibody-drug conjugate linker provided by the present invention in conjunction with the examples. It should be understood that these examples are only for further describing the features of the present invention in detail, and are not intended to limit the scope of the present invention or the scope of the claims of the present invention.

[0020] The technical scheme of the present invention is further described below in conjunction with the accompanying drawings and examples. The names and English abbreviations of the reagents and instruments used in this application are shown in Table 1.

[0021] Table 1 Reagents and instruments used Abbreviations Chinese meaning DIC Diisopropylcarbodiimide DCC N,N'-Dicyclohexylcarbodiimide EDCI 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride HOBT 1-Hydroxy-benzotriazole DIPEA Triisopropylethylamine NMM N-Methylmorpholine DMAP 4-Dimethylaminopyridine HBTU O-Benzotriazole-tetramethyluronium hexafluorophosphate HATU 2-(7-Oxybenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate TBTU 2-(1H-Benzotriazol L-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate PyBOP 1H-Benzotriazol-1-yl-oxytripyrrolidino hexafluorophosphate HOPO 2-Hydroxypyridine-N-oxide Val Valine Cit Citrulline Glu Glutamine Pab p-Aminobenzyl alcohol Boc tert-Butyloxycarbonyl EA Ethyl acetate DMF N,N'-Dimethylformamide DMAc N,N'-Dimethylacetamide DCM Dichloromethane TCM Chloroform THF Tetrahydrofuran MTBE Methyl tert-butyl ether TFA Trifluoroacetic acid HOSu N-Hydroxysuccinamide HPLC High performance liquid chromatography

[0022] Example 1 Step (1): Synthesis of Compound 1: OBoc-Glu-Val-Cit-Pab Weigh 5.95 g of 1,5-pentanedioic acid mono-tert-butyl ester, add it to a 1000 mL jacketed bottle, and dissolve it with 100 mL DMF. Add 6.30 g EDCI and 3.90 g HOPO to the jacketed bottle, maintain the temperature at 0±5°C, and stir the reaction for 30-45 minutes. Weigh 10.0 g Val-Cit-Pab, dissolve it with 50 mL DMF, add it to the reaction solution, then raise the temperature to 20±5°C, and the reaction time is greater than 12 hours. The reaction is completed by HPLC control, 300 mL DCM is added to the reaction solution, and then 150 mL MTBE and 150 mL n-heptane are added dropwise, the solid is filtered out, and 13.10 g of white solid powder is obtained, with a yield of 90.44% and a purity of 97.0%.

[0023] Step (2): Synthesis of Compound 2: HO-Glu-Val-Cit-Pab Weigh 13.00 g of compound 1 into a 500 mL reaction bottle and dissolve it in 50 mL DCM. Add 11.59 g of TFA to the reaction bottle and stir the reaction at 20±5°C for more than 12 hours. After the reaction is completed by HPLC control, the reaction solution is concentrated under reduced pressure at 30±5°C, redissolved in 20 mL DCM, concentrated under reduced pressure again at 30±5°C, and the reaction solution is spin-dried. Repeat three times to obtain 11.3 g of yellow oily liquid with a yield of 96.80% and a purity of 96.3%.

[0024] Step (3): Synthesis of compound 3: OSu-Glu-VC-Pab Weigh 11.0 g of compound 2 into a 500 mL reaction bottle and dissolve it in 100 mL DMF. Add 2.82 g HOSu and 9.20 g DCC, and stir the reaction at 0±5°C for more than 12 hours. After the reaction is completed by HPLC control, a mixed solution of 100 mL DCM and 300 mL MTBE is added dropwise to the reaction solution to precipitate a white solid, which is filtered to obtain 12.30 g of solid compound with a yield of 93.44% and a purity of 98.7%.

Claims

1. A method for synthesizing an antibody-drug conjugate linker OSu-Glu-Val-Cit-Pab, which is prepared by three steps of reaction using Val-Cit-Pab as a starting material. Specifically, Val-Cit-Pab is used as the starting material to prepare OBoc-Glu-Val-Cit-Pab through condensation reaction, and the tert-butyl protecting group is removed under acidic conditions to prepare HO-Glu-Val-Cit-Pab, which is reacted with an activation reagent to prepare OSu-Glu-Val-Cit-Pab. The synthetic route is as follows: 。 2. The synthesis method according to claim 1, wherein the reaction medium in the condensation reaction in step (1) is at least one of DMF, DMAc, THF, EA, DCM, toluene, N-methylpyrrolidone or dimethyl sulfoxide.

3. The synthesis method according to claim 1, wherein step (2) is carried out under acidic conditions, and the acid is at least one of TFA, hydrochloric acid, and acetic acid.

4. The synthesis method according to claim 1 or 3, wherein the molar ratio of compound 1 to acid in step (2) is in the range of 1:3-5.

5. The synthesis method according to claim 1, wherein the reaction medium in step (2) is one of DCM, DMF, EA and TCM.

6. The synthesis method according to claim 1, wherein the reaction temperature in step (2) is 10-30°C.

7. The synthesis method according to claim 1, wherein step (3) is performed in the presence of an activating reagent, and the activating reagent is N-hydroxysuccinimide.

8. The synthesis method according to claim 1, wherein the reaction temperature in step (3) is -5 to 5°C.

9. The synthesis method according to claim 1, wherein the condensing agent in steps (1) to (3) is at least one of DIC, DCC, EDCI, HOBT, DIPEA, NMM, DMAP, HBTU, HATU, TBTU or PyBOP.

10. The synthesis method according to claim 1, characterized in that The specific preparation process is as follows: (1): Using Val-Cit-Pab as the starting material, adding 1,5-pentanedioic acid mono-tert-butyl ester after stirring in a reaction medium containing a condensation agent and a condensation auxiliary agent, raising the temperature to carry out a condensation reaction, and after the reaction is completed, adding DCM dropwise to the reaction solution, and then adding MTBE and n-heptane dropwise to precipitate crystals, filtering and drying to obtain compound 1, namely OBoc-Glu-Val-Cit-Pab; (2): Compound 1 is subjected to acidic conditions to remove the tert-butyl protecting group, and then directly concentrated under reduced pressure to obtain compound 2, namely HO-Glu-Val-Cit-Pab; (3): Compound 2 is reacted with an activating reagent in a reaction medium containing a condensing agent. After the reaction is completed, a mixed solvent of acetonitrile and MTBE is added dropwise to the reaction solution for crystallization. The mixture is filtered and the filter cake is dried to obtain compound 3, i.e., OSu-Glu-Val-Cit-Pab.

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