Intermediates of antibody-drug conjugates and preparation methods thereof

Abstract

The present disclosure provides intermediates of antibody-drug conjugates and preparation methods therefor. Specifically, the present disclosure provides a method for preparing a compound of formula (I) or a salt thereof, comprising a step of subjecting compound 1-4 or a salt thereof and compound 2-4 or a salt thereof to an amide condensation reaction in the presence of a condensing agent. By optimizing the synthesis process and switching to column chromatography purification with higher throughput, the present disclosure provides a linker preparation method having mild reaction conditions and a simple workup procedure, which can meet the requirements of large-scale industrial production.

Description

Intermediates for antibody-drug conjugates and their preparation methods

[0001] This disclosure is based on and claims priority to Chinese Patent Application No. 202411857793.9, filed on December 17, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to the field of drug synthesis technology, specifically to intermediates for antibody-drug conjugates and their preparation methods. Background Technology

[0003] With the continuous breakthroughs in biotechnology, antibody-drug conjugates (ADCs) have become one of the representatives of targeted therapies. Due to their advantages of high targeting, high specificity, and high activity, they are known in the industry as "biological missiles." ADCs consist of three parts: a monoclonal antibody, an effector molecule, and a linker connecting the two. They utilize the targeting and high specificity of antibodies to achieve precise delivery of effector molecules to tumor sites, making it possible to drug highly active effector molecules that were traditionally undruggable.

[0004] The preparation process of small molecules such as linkers and intermediates used in antibody-drug conjugates (ADCs) has a significant impact on the product, such as the stability of the target product and subsequent process scale-up. Process scale-up differs from laboratory operations and is quite challenging. WO2019114666A1 discloses a method for preparing the compound shown in TL033, using high-performance liquid chromatography (HPLC) for purification. This method is only suitable for gram-level laboratory production and not for industrial-scale production. Developing a simple process with mild reaction conditions suitable for large-scale industrial production of linkers and intermediates is of great significance for the development and application of ADC drugs. Summary of the Invention

[0005] This disclosure provides a compound of formula (I) or a salt thereof, and a method for its preparation. By optimizing the synthesis process and using column chromatography with higher throughput, this disclosure provides a preparation method with mild reaction conditions, simple post-processing, and suitability for large-scale industrial production. The compound of formula (I) can be used to prepare linkers for ADCs, adapting to the requirements of large-scale ADC production processes, providing an efficient and feasible process route for the batch preparation of ADCs, and contributing to the industrial production of ADCs.

[0006] On the one hand, this disclosure provides a method for preparing a compound of formula (I) or a salt thereof suitable for industrial production.

[0007] It includes the following steps:

[0008] Compounds 1-4 or their salts undergo an amide condensation reaction with compounds 2-4 or their salts in the presence of a condensing agent to give compounds of formula (I) or their salts.

[0009] PG1 is selected from amino protecting groups.

[0010] In some embodiments, PG1 is selected from 4-methoxytriphenylmethyl (MMT), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl, or ethoxycarbonyl. In some specific embodiments, PG1 is 4-methoxytriphenylmethyl (MMT).

[0011] In some implementations, the compound of formula (I) is

[0012] In some implementations, compounds 2-4 are

[0013] In some embodiments, the condensing agent is selected from one or more of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and N,N'-carbonyldiimidazole (CDI). In some specific embodiments, the condensing agent is 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ).

[0014] In some embodiments, the solvent for the amide condensation reaction is selected from one or more of dichloromethane, N,N-dimethylformamide, tetrahydrofuran, dimethyltetrahydrofuran, and 1,4-dioxane. In some specific embodiments, the solvent is dichloromethane.

[0015] In some embodiments, the molar ratio of compounds 1-4 to compounds 2-4a is 1:(0.5-1.5). In some specific embodiments, the molar ratio of compounds 1-4 to compounds 2-4a is 1:(0.6-1.4), 1:(0.8-1.2), 1:(0.9-1.1), or 1:1.

[0016] In some embodiments, the molar ratio of compound 2-4a to the condensing agent is 1:(1-2). In some specific embodiments, the molar ratio of compound 2-4a to the condensing agent is 1:(1.1-1.9), 1:(1.2-1.8), 1:(1.3-1.7), 1:(1.4-1.6), or 1:1.57.

[0017] In some embodiments, the reaction temperature is 15-40°C. In some specific embodiments, the reaction temperature is 15-35°C, 15-30°C, 20-35°C, or 20-30°C.

[0018] In some implementations, the reaction time is 1-6 hours. In some specific implementations, the reaction temperature is 1-5 hours, 1-4 hours, 1-3 hours, 1-2 hours, 2-6 hours, 2-5 hours, 2-4 hours, 2-3 hours, 3-6 hours, 3-5 hours, 3-4 hours, 4-5 hours, or 4-6 hours.

[0019] In some embodiments, the reaction product is purified by column chromatography to obtain a compound of formula (I) or a salt thereof.

[0020] In some embodiments, the mobile phase used for column chromatography purification is a mixture of ethyl acetate and methanol. In some specific embodiments, the mobile phase used for column chromatography purification is a mixture of ethyl acetate and methanol at a volume ratio of 20:1.

[0021] In some embodiments, the method for preparing the compound of formula (I) or a salt thereof further includes the following one or more steps:

[0022] (a) Compound 1-1 or its salt reacts in the presence of an azide reagent and a base to give compound 1-2 or its salt.

[0023] (b) Compounds 1-2 or their salts react in the presence of a reducing agent and an acid to give compounds 1-3 or their salts.

[0024] (c) Compounds 1-3 or their salts react with diethylene glycol anhydride via an amide condensation reaction to yield compounds 1-4 or their salts.

[0025] In some embodiments, the azide reagent in step (a) is selected from one or more of sodium azide and diphenyl azidophosphate (DPPA). In some specific embodiments, the azide reagent in step (a) is diphenyl azidophosphate (DPPA).

[0026] In some embodiments, the base in step (a) is selected from one or more of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylamine, and N,N-diisopropylethylamine (DIPEA). In some specific embodiments, the base in step (a) is 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0027] In some embodiments, the reaction solvent in step (a) is selected from one or more of tetrahydrofuran, dichloromethane, and dimethyl sulfoxide. In some specific embodiments, the reaction solvent in step (a) is tetrahydrofuran.

[0028] In some embodiments, the molar ratio of compound 1-1 or its salt to the azide reagent in step (a) is 1:(1-5). In some specific embodiments, the molar ratio of compound 1-1 or its salt to the azide reagent in step (a) is 1:(1-4), 1:(2-4), 1:(2-3), or 1:2.5.

[0029] In some embodiments, the molar ratio of compound 1-1 or its salt to the base in step (a) is 1:(1-10). In some specific embodiments, the molar ratio of compound 1-1 or its salt to the base in step (a) is 1:(2-8), 1:(3-7), 1:(4-6), or 1:5.

[0030] In some implementations, the temperature is controlled to not exceed 40°C during the feeding process in step (a). In some specific implementations, the temperature is controlled to not exceed 35°C, 30°C, 25°C, or 20°C during the feeding process in step (a).

[0031] In some embodiments, in step (a), compound 1-1 or its salt and an azide reagent are first added to the reaction solvent, the temperature is lowered to 0-30°C, and then a base is added. In some specific embodiments, the temperature is first lowered to 0-25°C, then to 0-20°C, then to 0-15°C, or then to 0-10°C, and then a base is added.

[0032] In some implementations, the reflux reaction time of step (a) is 8-24 hours. In some specific implementations, the reflux reaction time of step (a) is 12-24 hours, 16-24 hours, 20-24 hours, 12-20 hours, 16-20 hours, or 12-16 hours.

[0033] In some embodiments, the reflux reaction temperature of step (a) is 50-70°C. In some specific embodiments, the reflux reaction temperature of step (a) is 55-70°C, 60-70°C, 55-65°C, or 60-65°C. In some specific embodiments, the reflux reaction temperature of step (a) is 60°C.

[0034] In some implementations, in step (a), after the reaction is complete, the reaction solution is extracted with an organic solvent, washed with an acidic solution, and dried to obtain the crude product.

[0035] In some embodiments, the organic phase used in step (a) is ethyl acetate.

[0036] In some embodiments, the acidic solution used for washing in step (a) is dilute sulfuric acid. In some specific embodiments, the dilute sulfuric acid is 0.5N dilute sulfuric acid.

[0037] In some embodiments, the crude product in step (a) is purified by column chromatography to obtain compounds 1-2 or their salts.

[0038] In some implementations, the elution solvent used in step (a) for column chromatography purification is ethyl acetate.

[0039] In some embodiments, the reducing agent in step (b) is selected from one or more of triphenylphosphine, trimethylphosphine (Me3P), tri-tert-butylphosphine (Bu3P), and 1,2-bis(diphenylphosphine)ethane (DPPE). In some specific embodiments, the reducing agent in step (b) is triphenylphosphine.

[0040] In some embodiments, the acid in step (b) is selected from one or more of hydrochloric acid, sulfuric acid, acetic acid, and trifluoroacetic acid. In some specific embodiments, the acid in step (b) is sulfuric acid. In some specific embodiments, the acid in step (b) is a 5% sulfuric acid solution.

[0041] In some embodiments, the reaction solvent in step (b) is selected from toluene, tetrahydrofuran, dichloromethane, dimethyltetrahydrofuran, 1,4-dioxane, or methyl tert-butyl ether. In some specific embodiments, the reaction solvent in step (b) is toluene.

[0042] In some embodiments, the molar ratio of compound 1-2 or its salt to the reducing agent in step (b) is 1:(0.5-2). In some specific embodiments, the molar ratio of compound 1-2 or its salt to the reducing agent in step (b) is 1:(0.6-1.5), 1:(0.8-1.4), 1:(0.9-1.3), 1:(1-1.2), or 1:1.1.

[0043] In some embodiments, the reaction time of compounds 1-2 or their salts with the reducing agent in step (b) is 1-8 hours. In some specific embodiments, the reaction time of compounds 1-2 or their salts with the reducing agent in step (b) is 2-6 hours, 2-5 hours, 2-4 hours, 3-6 hours, 3-5 hours, or 3-4 hours.

[0044] In some embodiments, the reaction temperature of compounds 1-2 or their salts with the reducing agent in step (b) is controlled at 15-40°C. In some specific embodiments, the reaction temperature of compounds 1-2 or their salts with the reducing agent in step (b) is 20-40°C, 25-40°C, or 30-40°C.

[0045] In some embodiments, in step (b), the reaction solution of compound 1-2 or its salt with the reducing agent is allowed to stand and separated. The lower aqueous phase is washed with an organic solvent, the aqueous phase is adjusted to alkali, and extracted with an organic solvent. The resulting organic phase is dried to obtain compound 1-3 or its salt.

[0046] In some implementations, the lower aqueous phase in step (b) is washed with ethyl acetate.

[0047] In some implementations, the aqueous phase in step (b) is alkali-adjusted with ammonia.

[0048] In some implementations, step (b) uses dichloromethane to extract the aqueous phase after alkali adjustment.

[0049] In some embodiments, the molar ratio of compounds 1-3 or their salts to diethylene glycol anhydride in step (c) is 1:(0.5-2). In some specific embodiments, the molar ratio of compounds 1-3 or their salts to diethylene glycol anhydride in step (c) is 1:(0.6-1.5), 1:(0.8-1.4), 1:(0.9-1.3), 1:(1-1.2), or 1:1.1.

[0050] In some embodiments, the reaction solvent in step (c) is selected from dichloromethane, tetrahydrofuran, dimethyltetrahydrofuran, or 1,4-dioxane. In some specific embodiments, the reaction solvent in step (c) is dichloromethane.

[0051] In some implementations, the reaction time for step (c) is 1-12 hours. In some specific implementations, the reaction time for step (c) is 2-10 hours, 3-8 hours, or 4-6 hours.

[0052] In some embodiments, the reaction temperature of step (c) is controlled at 15-40°C. In some specific embodiments, the reaction temperature of step (c) is controlled at 20-40°C, 25-40°C, 30-40°C, 35-40°C, 15-35°C, 20-35°C, 25-35°C, 30-35°C, 15-30°C, 20-30°C, or 25-30°C.

[0053] In some embodiments, the reaction solution in step (c) is added to purified water for liquefaction, the organic phase is washed with purified water, and the organic phase is dried to obtain compounds 1-4 or their salts.

[0054] In some embodiments, the method for preparing the compound of formula (I) or a salt thereof further includes the following one or more steps:

[0055] (d) Compound 2-1 or its salt reacts with compound 2-2 or its salt in the presence of a condensing agent to undergo an amide condensation reaction, yielding compound 2-3 or its salt.

[0056] (e) Reaction of compound 2-3 or its salt with a deprotecting agent yields compound 2-4 or its salt.

[0057] In some embodiments, PG2 is selected from 4-methoxytriphenylmethyl (MMT), benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), allyloxycarbonyl (Alloc), trimethylsilylethoxycarbonyl (Teoc), methoxycarbonyl, or ethoxycarbonyl. In some specific embodiments, PG2 is 9-fluorenylmethoxycarbonyl (Fmoc).

[0058] In some embodiments, compound 2-1 is

[0059] In some implementations, compounds 2-3 are

[0060] In some implementations, compounds 2-4 are

[0061] In some embodiments, the condensing agent in step (d) is selected from one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and N,N'-carbonyldiimidazole (CDI). In some specific embodiments, the condensing agent in step (d) is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU).

[0062] In some embodiments, the solvent for the amide condensation reaction in step (d) is selected from N,N-dimethylformamide, dichloromethane, tetrahydrofuran, dimethyltetrahydrofuran, and 1,4-dioxane. In some specific embodiments, the solvent for the amide condensation reaction in step (d) is N,N-dimethylformamide.

[0063] In some embodiments, the molar ratio of compound 2-1 or its salt to compound 2-2 or its salt in step (d) is 1:(0.5-1.5). In some specific embodiments, the molar ratio of compound 2-1 or its salt to compound 2-2 or its salt in step (d) is 1:(0.6-1.4), 1:(0.7-1.3), 1:(0.8-1.2), 1:(0.9-1.1), or 1:1.

[0064] In some embodiments, the reaction temperature of compound 2-1 or its salt with compound 2-2 or its salt in step (d) is 15-40°C. In some specific embodiments, the reaction temperature of compound 2-1 or its salt with compound 2-2 or its salt in step (d) is 20-40°C, 25-40°C, 30-40°C, 15-35°C, 20-35°C, 25-35°C, 30-35°C, 15-30°C, 20-30°C, or 25-30°C.

[0065] In some embodiments, the reaction time of compound 2-1 or its salt with compound 2-2 or its salt in step (d) is 0.5-8 hours. In some specific embodiments, the reaction time of compound 2-1 or its salt with compound 2-2 or its salt in step (d) is 1-6 hours, 2-5 hours, 2-4 hours, or 2-3 hours.

[0066] In some embodiments, the reaction solution of compound 2-1 or its salt with compound 2-2 or its salt in step (d) is further extracted with an extraction solvent, and the resulting organic phase is washed with a solution.

[0067] In some embodiments, the extraction solvent used in step (d) comprises one or more organic solvents selected from isopropyl acetate, ethyl acetate, tetrahydrofuran, and 2-methyltetrahydrofuran. In some specific embodiments, the extraction solvent used in step (d) is isopropyl acetate.

[0068] In some embodiments, the extraction solvent used in step (d) further comprises an alkaline solution. In some specific embodiments, the alkaline solution is a sodium bicarbonate solution, a 1-10% sodium bicarbonate solution, or a 5% sodium bicarbonate solution.

[0069] In some implementations, the solution used to wash the organic phase in step (d) is a sodium chloride solution.

[0070] In some embodiments, the deprotecting agent in step (e) is selected from one or more of ethylenediamine, triethylamine, and N,N-diisopropylethylamine (DIPEA). In some specific embodiments, the deprotecting agent in step (e) is ethylenediamine.

[0071] In some embodiments, the molar ratio of compound 2-3 or its salt to the deprotecting agent in step (e) is 1:(1-5). In some specific embodiments, the molar ratio of compound 2-3 or its salt to the deprotecting agent in step (e) is 1:(1-4), 1:(2-4), 1:(2-3), 1:(2.4-2.8), 1:(2.5-2.7), or 1:2.6.

[0072] In some embodiments, the solvent for the deprotection reaction in step (e) is selected from N,N-dimethylformamide, dichloromethane, tetrahydrofuran, dimethyltetrahydrofuran, and 1,4-dioxane. In some specific embodiments, the solvent for the deprotection reaction in step (e) is N,N-dimethylformamide.

[0073] In some implementations, the reaction time for step (e) is 4-24 hours. In some specific implementations, the reaction time for step (e) is 6-20 hours, 8-16 hours, 10-16 hours, or 12-16 hours.

[0074] In some embodiments, the reaction product of step (e) is concentrated under reduced pressure, and the crude product is purified by column chromatography to obtain compounds 2-4 or their salts.

[0075] In some embodiments, the vacuum concentration temperature in step (e) is 30-60°C. In some specific embodiments, the vacuum concentration temperature in step (e) is 35-55°C, 40-55°C, 45-50°C, or 40-45°C.

[0076] In some embodiments, the mobile phase used for column chromatography purification in step (e) is a mixture of ethyl acetate and methanol. In some specific embodiments, the mobile phase used for column chromatography purification in step (e) is a mixture of ethyl acetate and methanol at a volume ratio of 20:1.

[0077] On the other hand, this disclosure provides a method for preparing compounds 1-4 or their salts, the method comprising the following steps:

[0078] (a) Compound 1-1 or its salt reacts in the presence of an azide reagent and a base to give compound 1-2 or its salt.

[0079] (b) Compounds 1-2 or their salts react in the presence of a reducing agent and an acid to give compounds 1-3 or their salts.

[0080] (c) Compounds 1-3 or their salts react with diethylene glycol anhydride via an amide condensation reaction to yield compounds 1-4 or their salts.

[0081] In some implementations, step (a) is as described above.

[0082] In some implementations, step (b) is as described above.

[0083] In some implementations, step (c) is as described above.

[0084] On the other hand, this disclosure provides a method for preparing compounds 2-4 or their salts, the preparation comprising the following steps:

[0085] (d) Compound 2-1 or its salt reacts with compound 2-2 or its salt in the presence of a condensing agent to undergo an amide condensation reaction, yielding compound 2-3 or its salt.

[0086] (e) Reaction of compound 2-3 or its salt with a deprotecting agent yields compound 2-4 or its salt.

[0087] In some implementations, step (d) is as described above.

[0088] In some implementations, step (e) is as described above.

[0089] On the other hand, this disclosure provides a method for improving the conversion of compound 2-1 to compound 2-3, the method comprising extracting an organic phase by using an extraction solvent to extract a reaction solution of compound 2-1 or its salt with compound 2-2 or its salt; the extraction solvent comprising one or more organic solvents selected from isopropyl acetate, ethyl acetate, tetrahydrofuran, and 2-methyltetrahydrofuran. In some specific embodiments, the extraction solvent is isopropyl acetate.

[0090] In some embodiments, the extraction solvent further comprises an alkaline solution. In some specific embodiments, the alkaline solution is a sodium bicarbonate solution, a 1-10% sodium bicarbonate solution, or a 5% sodium bicarbonate solution.

[0091] In some embodiments, the method includes washing the organic phase with a solution. In some embodiments, the solution used to wash the organic phase is a sodium chloride solution.

[0092] On the other hand, this disclosure provides a method for improving the conversion of compound 2-3 to compound 2-4, the method comprising deprotecting compound 2-3 or its salt in an organic solvent selected from one or more of isopropyl acetate, ethyl acetate, tetrahydrofuran, and 2-methyltetrahydrofuran. In some specific embodiments, the organic solvent is isopropyl acetate.

[0093] On the other hand, this disclosure provides a method for improving the chemical stability of compounds 2-4, the method comprising dissolving compounds 2-4 in an organic solvent in a chemical reaction, said organic solvent being selected from one or more of isopropyl acetate, ethyl acetate, tetrahydrofuran, and 2-methyltetrahydrofuran. In some specific embodiments, the organic solvent is isopropyl acetate. On the other hand, this disclosure provides compounds for preparing antibody-drug conjugates or their drug-linker payloads, selected from:

[0094] On the other hand, this disclosure provides the use of compounds in the preparation of antibody-drug conjugates or their drug-linker payloads, said compounds being selected from:

[0095] On the other hand, this disclosure provides intermediates for preparing compounds of formula (Ia), which are selected from:

[0096] The compound of formula (Ia) is shown below:

[0097] On the other hand, this disclosure provides the use of intermediates in the preparation of compounds of formula (Ia), said intermediates being selected from:

[0098] The compound of formula (Ia) is shown below:

[0099] Beneficial effects of the invention

[0100] 1. The synthetic route disclosed herein can meet the needs of large-scale industrial production. The raw materials are readily available in the market, the reaction conditions are mild, the post-processing is simple, and the purification can be carried out by a higher throughput forward column chromatography process. The production capacity of the compound using the method of formula (I) disclosed herein can reach more than 10 kg / batch.

[0101] 2. The synthetic route disclosed herein has a high overall yield. In some embodiments, the overall yield of the reaction producing compound (Ia) from compound 1-1 as a starting material is as high as 40% or more.

[0102] 3. In the synthetic route disclosed herein, the introduction of the azide group uses non-explosive diphenyl azidophosphate (DPPA) instead of the traditional inorganic azide reagent sodium azide, which makes the conditions milder and safer. At the same time, the conventional two-step reaction of hydroxy azidation (activation followed by azidation) is reduced to direct azidation using DPPA, which reduces the number of synthetic steps and improves the yield. The yield of this step is >95%. Detailed Implementation

[0103] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0104] The abbreviations used in this disclosure have the following meanings:

[0105] Example 1: Preparation of compounds 1-4

[0106] Step 1: Synthesis of Compounds 1-2

[0107] 14 kg of compound 1-1 and 23.38 kg of DPPA were added to 63 kg of THF solvent, and the temperature was lowered to 0-10 °C. 25.76 kg of DBU was added in batches, and the temperature was controlled not to exceed 20 °C during the addition process. After the addition was completed, the mixture was refluxed at 60 °C for 16 h, and the reaction was confirmed to be complete by LC-MS.

[0108] The reaction solution was cooled to below 10°C, and the organic phase was extracted using ethyl acetate. The extracted organic phase was washed with 0.5N dilute sulfuric acid solution, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent, yielding 15.4 kg of product (yield: 98.7%). ESI-MS (m / z): 482.3 [M+H2O] +

[0109] Step 2: Synthesis of compounds 1-3

[0110] 15.4 kg of compounds 1-2 and 9.70 kg of PPh3 obtained in the previous step were added to 80.08 kg of toluene, and 92.80 kg of 5% sulfuric acid solution was added dropwise. The reaction was carried out at 30-40℃ for 3 h, and the reaction was confirmed to be complete by LC-MS. The reaction solution was allowed to stand and separated. The lower aqueous phase was washed with ethyl acetate, and the aqueous phase was adjusted with ammonia water to obtain an alkali. It was then extracted with dichloromethane, and the resulting organic phase was dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure, yielding 13.86 kg of product, 95.6% of which was directly added to the next reaction. ESI-MS (m / z): 439.3 [M+H] +

[0111] Step 3: Synthesis of compounds 1-4

[0112] The 13.86 kg of compounds 1-3 and 4.06 kg of diethylene glycol anhydride obtained in the previous step were added to 69.30 kg of DCM and reacted at 20–30 °C for 6 h. The reaction was confirmed to be complete by LC-MS. Purified water was added to the mixture for liquefaction, and the organic phase was washed with 98.41 kg of purified water. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by distillation under reduced pressure to obtain 14 kg of product, with a yield of 80.0%.

[0113] 1H NMR(400MHz,Chloroform-d)δ7.86-7.76(m,1H),4.18(s,2H),4.16(s,2H),3.74-3.61(m,31H),3. 58(dd,J=5.3,4.0Hz,2H),3.53(t,J=5.5Hz,2H),3.39(t,J=5.1Hz,2H).ESI-MS(m / z):555.3[M+H] +

[0114] Example 2, Preparation of compound 2-4a

[0115] 32.0 kg of compound 2-1a, 6.10 kg of compound 2-2, and 22.70 kg of HATU were dissolved in 160 kg of DMF. The reaction solution was heated to 20-30 °C and stirred for 2 hours. The reaction was confirmed to be complete by TLC. The reaction solution was extracted with isopropyl acetate and 5% sodium bicarbonate solution. The organic phase was washed with sodium chloride solution and stirred for 30 minutes. After standing for 30 minutes, the mixture was separated, dried over anhydrous sodium sulfate, and filtered. 10.60 kg of diethylamine was added to the filtrate, and the mixture was stirred at room temperature for 12 hours. The reaction was confirmed to be complete by TLC. The mixture was concentrated under reduced pressure at 40-45 °C. The obtained solid was purified by column chromatography (EA / MeOH = 20 / 1) to give compound 2-4a as 15.4 kg of off-white solid, yield: 59.23%.

[0116] Example 3: Preparation of (S)-2-(32-azido-5-oxo-3,9,12,15,18,21,24,27,30-nonazo-6-azatridodecanoamide)-N-(4-(hydroxymethyl)phenyl)-6-(((4-methoxyphenyl)diphenylmethyl)amino)hexanoamide (compound of formula (Ia)).

[0117] 14.0 kg of compounds 1-4 and 14.0 kg of compounds 2-4a were dissolved in 86 kg of DCM, and 10.4 kg of EEDQ was added. The reaction mixture was stirred at 20-30 °C for 2-4 hours. The reaction was confirmed to be complete by LC-MS, and purified by column chromatography (EA / MeOH = 20 / 1) to obtain compound (Ia) as a colorless oil, 15.8 kg, yield: 59.0%.

[0118] Example 4: Solvent stability test of compound 2-4a

[0119] Take a small amount of compound 2-4a and dissolve it in 10 times its volume of various organic solvents. Shake overnight at room temperature. Take samples the next day for TLC detection (developing solvent: DCM / MeOH = 10:1) and observe the stability of the samples. If spots of compounds other than compound 2-4a appear in the thin layer, it indicates that the sample has deteriorated, indicating that compound 2-4a is unstable in the organic solvent. The specific experimental results are shown in Table 1 below.

[0120] Table 1 Solvent stability results of compounds 2-4a

[0121] The above experimental results indicate that compound 2-4a exhibits chemical stability issues when dissolved for extended periods or in acetonitrile or dichloromethane at temperatures above room temperature. Selecting isopropyl acetate or other reaction solvents can significantly improve the stability of compound 2-4a.

[0122] Although specific embodiments of this disclosure have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are all within the scope of this disclosure. The full scope of this disclosure is given by the appended claims and any equivalents thereof.

Claims

1. A method for preparing a compound of formula (I) or a salt thereof suitable for industrial production, wherein, The preparation method comprises that compound 1-4 or a salt thereof and compound 2-4 or a salt thereof are subjected to an amide condensation reaction in the presence of a condensing agent to obtain the compound of formula (I) or a salt thereof, PG1 is selected from amino protecting groups.

2. The production method according to claim 1, wherein The condensing agent is selected from one or more of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and N,N'-carbonyldiimidazole (CDI).

3. The production method according to claim 1 or 2, wherein The method for preparing the compound of formula (I) or its salt further includes the following steps: (a) reacting compound 1-1 or a salt thereof in the presence of an azidation reagent and a base to give compound 1-2 or a salt thereof, (b) reacting compound 1-2 or a salt thereof in the presence of a reducing agent and an acid to give compound 1-3 or a salt thereof, (c) compound 1-3 or a salt thereof is subjected to an amide condensation reaction with diglycolic anhydride to obtain compound 1-4 or a salt thereof, 4. The production method according to claim 3, wherein The azide reagent in step (a) is selected from one or more of sodium azide and diphenyl azidophosphate (DPPA).

5. The production method according to claim 3 or 4, wherein The base in step (a) is selected from one or more of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), triethylamine, and N,N-diisopropylethylamine (DIPEA).

6. The method of making according to any one of claims 3-5, wherein, The reflux reaction temperature in step (a) is 50-70°C.

7. The method of making according to any one of claims 3-6, wherein, The reducing agent in step (b) is selected from one or more of triphenylphosphine, trimethylphosphine (Me3P), tri-tert-butylphosphine (Bu3P), and 1,2-bis(diphenylphosphine)ethane (DPPE).

8. The preparation method according to any one of claims 3-7, wherein, The acid in step (b) is selected from one or more of hydrochloric acid, sulfuric acid, acetic acid and trifluoroacetic acid.

9. The method of making according to any one of claims 1-8, wherein, The method for preparing the compound of formula (I) or its salt further includes the following steps: (d) amide condensation reaction of compound 2-1 or a salt thereof with compound 2-2 or a salt thereof in the presence of a condensing agent to obtain compound 2-3 or a salt thereof, (e) reacting compound 2-3 or a salt thereof with a deprotecting group reagent to obtain compound 2-4 or a salt thereof, 10. The production method according to claim 9, wherein The condensing agent in step (d) is selected from one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and N,N'-carbonyldiimidazole (CDI). The solvent for the amide condensation reaction in step (d) is selected from N,N-dimethylformamide, dichloromethane, tetrahydrofuran, dimethyltetrahydrofuran, and 1,4-dioxane; In step (d), the molar ratio of compound 2-1 or its salt to compound 2-2 or its salt is 1:(0.5-1.5); In step (d), the reaction temperature of compound 2-1 or its salt with compound 2-2 or its salt is 15-40℃; or In step (d), the reaction time of compound 2-1 or its salt with compound 2-2 or its salt is 0.5-8 hours.

11. The production method according to claim 9 or 10, wherein In step (d), the reaction solution of compound 2-1 or its salt with compound 2-2 or its salt is further extracted with an extraction solvent, and the organic phase is washed with a solution.

12. The method of making according to claim 11, wherein, The extraction solvent used in step (d) comprises one or more organic solvents selected from isopropyl acetate, ethyl acetate, tetrahydrofuran and 2-methyltetrahydrofuran; Preferably, the extraction solvent used in step (d) further comprises an alkaline solution; Preferably, the solution used to wash the organic phase in step (d) is a sodium chloride solution.

13. The method of making according to any one of claims 9-12, wherein, The deprotecting agent in step (e) is selected from one or more of ethylenediamine, triethylamine, and N,N-diisopropylethylamine (DIPEA); In step (e), the molar ratio of compound 2-3 or its salt to the deprotecting agent is 1:(1-5); The solvent for the deprotection reaction in step (e) is selected from N,N-dimethylformamide, dichloromethane, tetrahydrofuran, dimethyltetrahydrofuran, and 1,4-dioxane; The reaction time for step (e) is 4-24 hours; The reaction product of step (e) was concentrated under reduced pressure, and the crude product was purified by column chromatography to obtain compound 2-4 or its salt. The vacuum concentration temperature in step (e) is 30-60°C; or, In step (e), the mobile phase used for column chromatography purification is a mixture of ethyl acetate and methanol.

14. A process for the preparation of compound 1-4 or a salt thereof, wherein, The preparation method includes the following steps: (a) reacting compound 1-1 or a salt thereof in the presence of an azidation reagent and a base to give compound 1-2 or a salt thereof, (b) reacting compound 1-2 or a salt thereof in the presence of a reducing agent and an acid to give compound 1-3 or a salt thereof, (c) compound 1-3 or a salt thereof is subjected to an amide condensation reaction with diglycolic anhydride to obtain compound 1-4 or a salt thereof, Steps (a)-(c) are as described in any one of claims 3-8.

15. A process for the preparation of compound 2-4 or a salt thereof, wherein, The preparation includes the following steps: (d) amide condensation reaction of compound 2-1 or a salt thereof with compound 2-2 or a salt thereof in the presence of a condensing agent to obtain compound 2-3 or a salt thereof, (e) reacting compound 2-3 or a salt thereof with a deprotecting group reagent to obtain compound 2-4 or a salt thereof, Steps (d)-(e) are as described in any one of claims 9-13.

16. A compound for use in the preparation of an antibody drug conjugate or drug-linker payload thereof, wherein the compound is selected from:

17. Use of a compound in the manufacture of an antibody drug conjugate or a drug- linker-payload thereof, wherein the compound is selected from:

18. An intermediate for the preparation of a compound of formula (la) ###00014### (la) wherein, The intermediate is selected from: The connector of formula (Ia) is shown below:

19. Use of an intermediate in the preparation of a compound of formula (la), wherein the intermediate is selected from: The compounds of formula (Ia) are as follows:

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