A method for synthesizing antibody-drug conjugate linker SET0568

Abstract

The invention discloses a method for synthesizing an antibody-drug conjugate linker SET0568. The method comprises: preparing MC-VC-PAB from compounds VC-PAB and MC-OSu; preparing MC-VC-PAB-PNP from compounds MC-VC-PAB, DNPC and DIPEA; preparing PNU-159682 from compounds PNU-TBS and TBAF; preparing PNU-DMEA from PNU-159682, DNPC, triethylamine and N,N′-dimethylethylenediamine. Under nitrogen protection, the compound SET0568 is prepared from PNU-DMEA and MC-VC-PAB-PNP. The method is easy to scale up and is convenient for commercial production.

Description

Technical Field

[0001] The present invention relates to a method for synthesizing an antibody-drug conjugate linker, in particular to a method for synthesizing SET0568, and belongs to the technical field of medicinal chemistry. Background Art

[0002] Antibody drug conjugate (ADC) is a new type of anti-tumor drug. Its principle is to connect cytotoxins to antibodies. Through the recognition of specific antigens on the surface of cancer cells by antibodies, the antibodies enter cancer cells through endocytosis, thereby transporting the cytotoxins to the target site, thereby achieving the purpose of targeted treatment of malignant tumors.

[0003] Compared with traditional small molecule anti-tumor drugs, ADC is more specific and effective because it can take advantage of the targeted recognition of antibodies and the high activity of toxins.

[0004] ADC consists of three different components, namely antibodies, linkers and cytotoxins. Antibodies achieve targeting, linkers ensure the stability of ADC during blood transport, and after reaching the target site, the toxins kill cancer cells. According to different mechanisms of action, toxins suitable for ADC are divided into microtubule inhibitors, DNA damaging agents, RNA polymerase inhibitors, etc.

[0005] At present, the toxins used in ADCs sold on the market and in clinical trials are mainly microtubule inhibitors, including compounds designed with DNA topoisomerase I inhibitors, such as Dxd, and compounds designed based on maytansine, such as DM1 and DM4; compounds designed with DNA topoisomerase II inhibitors, such as PNU-159682, a highly efficient anthracycline neomycin metabolite, which has excellent cytotoxicity. Anthracyclines are one of the most widely used chemotherapy drugs, especially in the treatment of aggressive non-Hodgkin's lymphoma (NHL).

[0006] In terms of linkers, the main ones used are non-cleavable types, such as valine-citriline and cyclohexanecarboxylic acid (MCC). After lysosomal hydrolysis, the drug is still active and is combined with a certain amino acid residue through the linker region.

[0007] There are many ways to form antibody-drug conjugates. They can be coupled chemically through the amino or thiol groups on the antibody and the drug linker, or the antibody can be modified to introduce specific functional groups on the antibody and then coupled chemically or by enzyme-catalyzed reaction with the drug linker. Summary of the invention

[0008] In view of this, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for synthesizing a linker SET0568 (MC-VC-PAB-DMEA-PNU159682) for antibody-drug conjugates, which successfully achieves efficient synthesis of SET0568, is easy to scale up, and is convenient for commercial production.

[0009] To achieve the above object, the technical solution adopted by the present invention is: a method for synthesizing an antibody-drug conjugate linker SET0568, the method comprising the following steps:

[0010] The compound VC-PAB (SET0568-1) was dissolved in the first solvent, stirred for 30-60 min, MC-OSu was added and stirred for 1-2 h, and the second solvent was added for post-treatment to obtain (SET0568-2) MC-VC-PAB;

[0011] The compound MC-VC-PAB is dissolved in the first solvent and stirred. When the internal temperature drops to 15°C to 20°C, DNPC is added and stirred for 10min to 20min. DIPEA is added and stirred for 15h to 18h. The second solvent is added for post-treatment. The solid is precipitated, filtered and dried to obtain (SET0568-3) MC-VC-PAB-PNP.

[0012] The compound (SET0568-5) PNU-TBS is dissolved in a third solvent, TBAF is added, the temperature is lowered to -5 to 0°C, stirred, reacted for 20 min to 30 min, separated and extracted, dried and concentrated to obtain (SET0568-6) PNU-159682;

[0013] PNU-159682 and DNPC are dissolved in the fourth solvent, stirred with ice water and cooled to 0°C to 5°C, triethylamine is added, and the mixture is kept at 0°C to 5°C and stirred for 30min to 60min to obtain the intermediate (SET0568-7) PNU-PNP, N,N′-dimethylethylenediamine is added, and the mixture is stirred at about 0 to 5°C for 1 to 2h, and the reaction solution is concentrated, purified by a medium-pressure reverse phase column, and freeze-dried to obtain the compound (SET0568-8) PNU-DMEA;

[0014] Under nitrogen protection, the compound PNU-DMEA was dissolved in the first solvent, MC-VC-PAB-PNP was added, the temperature was cooled to 0-10°C in an ice-water bath, stirred for 10-15 min, DIPEA was added, the reaction was continued in an ice-water bath for 2-3 h, post-treated, and lyophilized to obtain the compound SET0568.

[0015] The synthesis method of the antibody-drug conjugate linker SET0568 (MC-VC-PAB-DMEA-PNU159682) provided by the present invention provides SET0568, which is an anthracycline derivative and has the following structure.

[0016]

[0017] The synthesis method of the toxin linker molecule SET0568 (MC-VC-PAB-DMEA-PNU159682) for antibody-drug conjugation has the following preparation steps:

[0018]

[0019] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing MC-VC-PAB.

[0020] Preferably, when preparing MC-VC-PAB, the mixing molar ratio of VC-PAB to MC-OSu is 1.0 eq: 1.0 eq-1.5 eq.

[0021] The synthesis method of the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing MC-VC-PAB-PNP.

[0022] Preferably, when preparing MC-VC-PAB-PNP, the mixing molar ratio of the MC-VC-PAB to the DNPC is 1.0 eq: 1.5 eq-2.5 eq.

[0023] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing PNU-159682.

[0024] Preferably, when preparing PNU-159682, the mixing molar ratio of the PNU-TBS to the TBAF is 1.0 eq: 1.0 eq-1.5 eq.

[0025] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing PNU-DMEA.

[0026] Preferably, when preparing PNU-DMEA, the mixing molar ratio of PNU-159682, DNPC, triethylamine and N,N′-dimethylethylenediamine is 1.0 eq: 1.5 eq-3.0 eq: 2.0 eq-3.5 eq: 1.0 eq-2.5 eq.

[0027] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing SET0568.

[0028] Preferably, when preparing SET0568, the mixing molar ratio of PNU-DMEA, MC-VC-PAB-PNP and DIPEA is 1.0eq: 1.0-1.5eq: 2.0eq-3.0eq, for example, 1.0eq: 1.0-1.4eq: 2.0eq-2.5eq, 1.0eq: 1.0-1.3eq: 2.0eq-2.2eq, and preferably 1.0eq: 1.1eq: 2.0eq.

[0029] Preferably, the first solvent is DMF or DMA; the second solvent is one or a mixed solvent of DCM, EA, PE, MTBE, diethyl ether; the third solvent is DCM or THF; and the fourth solvent is DCM, THF or DMF.

[0030] It should be noted here that the first solvent, the second solvent, the third solvent and the fourth solvent may be the same or different in each step. For example, when preparing MC-VC-PAB, the compound VC-PAB is dissolved in the first solvent, and when preparing MC-VC-PAB-PNP, the compound MC-VC-PAB is dissolved in the first solvent. The first solvents in these two steps may be the same or different. DMF or DMA may be used for both steps, or DMF may be used to dissolve VC-PAB and DMA may be used to dissolve MC-VC-PAB.

[0031] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: the synthesis method of the antibody-drug conjugate linker SET0568 of the present invention is novel and efficient, and is easy to scale up and commercialize.

[0032] Description of the main figures

[0033] Figure 1 This is the HPLC spectrum of compound MC-VC-PAB.

[0034] Figure 2 This is the HPLC spectrum of compound MC-VC-PAB-PNP.

[0035] Figure 3 This is the LCMS spectrum of compound MC-VC-PAB-PNP.

[0036] Figure 4 This is the HNMR spectrum of the compound MC-VC-PAB-PNP.

[0037] Figure 5 This is the LCMS spectrum of compound SET0568-7.

[0038] Figure 6 This is the LCMS spectrum of compound PNU-DMEA.

[0039] Figure 7 This is the HPLC spectrum of compound MC-VC-PAB-DMEA-PNU159682.

[0040] Figure 8 This is the LCMS spectrum of compound MC-VC-PAB-DMEA-PNU159682.

[0041] Fig. 9 This is the HNMR spectrum of compound MC-VC-PAB-DMEA-PNU159682.

[0042] Fig.10 It is the HPLC spectrum of the compound of Comparative Example 1.

[0043] Fig.11 It is the HPLC spectrum of SET0568-4 of Comparative Example 1. DETAILED DESCRIPTION

[0044] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing SET0568-2 (MC-VC-PAB). Specifically, the method is as follows:

[0045]

[0046] In a specific embodiment of the present invention, when preparing MC-VC-PAB, the mixing molar ratio of VC-PAB to MC-OSu is 1.0 eq: 1.0-1.5 eq, for example, 1.0 eq: 1.2 eq.

[0047] In one embodiment of the present invention, the compound VC-PAB is dissolved in the first solvent and stirred for 30 to 60 minutes. For example, the stirring may be for 33 minutes, 35 minutes, 38 minutes, 40 minutes, 45 minutes, 52 minutes, 55 minutes, and 58 minutes.

[0048] In one embodiment of the present invention, the post-treatment during the preparation of MC-VC-PAB comprises the following steps:

[0049] DCM was added to the filtrate, and the mixture was stirred for 30 to 35 minutes and then directly filtered. The filter cake was slurried with DCM for 30 to 35 minutes, filtered again, and dried to obtain a yellow solid.

[0050] The synthesis method of the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing (SET0568-3) MC-VC-PAB-PNP. Specifically, the method is as follows:

[0051]

[0052] In a specific embodiment of the present invention, when preparing MC-VC-PAB-PNP, the mixing molar ratio of MC-VC-PAB to DNPC is 1.0eq:1.5-2.5eq, such as 1.0eq:1.0eq-1.3eq, 1.0eq:1.1eq-1.3eq, 1.0eq:1.1eq-1.2eq, preferably 1.0eq:1.2eq.

[0053] In one embodiment of the present invention, when preparing MC-VC-PAB-PNP, the post-treatment comprises the following steps:

[0054] The reaction solution was filtered, DCM was added to the filtrate, and after stirring for 30 to 35 minutes, MTBE was added and stirred for 30 to 35 minutes, and then suction filtered. The filter cake was stirred with DCM for 20 to 30 minutes, MTBE was added and stirred for 30 minutes, and then suction filtered. The filter cake was dried to obtain a solid.

[0055] When preparing MC-VC-PAB-PNP, the mixing molar ratio of the MC-VC-PAB to the DNPC is 1.0eq:1.5eq-2.5eq, for example, 1.0eq:1.5eq-2.3eq, 1.0eq:1.8eq-2.3eq, 1.0eq:1.8eq-2.1eq, and preferably 1.0eq:2.0eq.

[0056] The synthesis method of the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing (SET0568-6) PNU-159682. The method is specifically carried out in the following manner:

[0057]

[0058] In a specific embodiment of the present invention, when preparing PNU-159682, the mixing molar ratio of the PNU-TBS to the TBAF is 1.eq: 1.0-1.5eq, such as 1.0eq: 1.0eq-1.4eq, 1.0eq: 1.1eq-1.4eq, 1.0eq: 1.2eq-1.4eq, and preferably 1.0eq: 1.3eq.

[0059] In one embodiment of the present invention, when preparing PNU-159682, the separation extraction comprises the following steps:

[0060] Water (eg 20 mL) and DCM (eg 50 mL) are added, wherein the volume ratio of water to DCM is 20:50, the aqueous phase is back-extracted with DCM (eg 50 ml), and the organic phases are combined to complete the extraction.

[0061] In a specific embodiment of the present invention, when preparing PNU-159682, anhydrous sodium sulfate can be used for drying and concentration.

[0062] The synthesis method of the antibody-drug conjugate linker SET0568 of the present invention includes the steps of preparing PNU-DMEA (SET0568-8). Specifically, the steps include:

[0063]

[0064] In a specific embodiment of the present invention, when preparing PNU-DMEA, the mixing molar ratio of PNU-159682, DNPC, triethylamine, and N,N′-dimethylethylenediamine is 1.0 eq: 1.5 eq-3.0 eq: 2.0 eq-3.5 eq: 1.0 eq-2.5 eq. For example, it can be 1.0 eq: 1.8 eq-2.5 eq: 2.8 eq-3.2 eq: 1.2 eq-1.8 eq. Preferably, it is 1.0 eq: 2.0 eq: 3.0 eq: 1.5 eq.

[0065] In one embodiment of the present invention, when preparing PNU-DMEA, the reaction solvent is removed by rotary evaporation after treatment. Then the crude product is purified by medium pressure reverse phase preparation of SET0568-8, 120g reverse phase column, wavelength 234nm, water / acetonitrile system purification, 40% to 50% acetonitrile ratio.

[0066] The method for synthesizing the antibody-drug conjugate linker SET0568 of the present invention includes the step of preparing SET0568.

[0067]

[0068] In a specific embodiment of the present invention, when preparing SET0568, the mixing molar ratio of PNU-DMEA, MC-VC-PAB-PNP and DIPEA is 1.0eq: 1.0-1.5eq: 2.0-3.0eq. For example, it can be 1.0eq: 1.0-1.4eq: 2.0eq-2.5eq, 1.0eq: 1.0-1.3eq: 2.0eq-2.2eq; preferably 1.0eq: 1.1eq: 2.0eq.

[0069] In one embodiment of the present invention, when preparing SET0568, the post-treatment is medium pressure reverse phase preparation purification, wavelength 234nm, flow rate 50mL / min, 0.05% NH 4 HCO 3 Water / acetonitrile, 40-50% acetonitrile ratio to produce the product.

[0070] In a specific embodiment of the present invention, the first solvent is DMF or DMA; the second solvent is one or a mixed solvent of DCM, EA, PE, MTBE, ether; the third solvent is DCM or THF; the fourth solvent is DCM, THF or DMF.

[0071] The preferred embodiments of the present invention will be described in detail below.

[0072] Example 1

[0073] Synthesis of Compound (SET0568-2) MC-VC-PAB

[0074] SET0568-1 (VC-PAB) 131g (1.0eq) was added to a 2L three-necked flask, and DMF 1.5L was added. If the solution was not clarified after stirring for 30-60 minutes, the solution was filtered through a Buchner funnel to remove the insoluble matter. The filtrate was transferred to a 2L reaction flask, and Mc-OSu 127.2g (1.2eq) was added. After stirring for one hour, the reaction was monitored by HPLC to be complete. Post-treatment: The filtrate was transferred to a 10L plastic barrel, and DCM (6L) was added. After stirring for 30-35 minutes, the solution was directly filtered by suction. The filter cake was slurried with DCM (2L) for 30-35 minutes, and then filtered again by suction and dried to obtain 135g of a yellow solid. Yield: 68.2%. The HPLC spectrum of compound (SET0568-2) MC-VC-PAB is shown in FIG. Figure 1 shown.

[0075] Synthesis of compound (SET0568-3) MC-VC-PAB-PNP

[0076] Take SET0568-2 (MC-VC-PAB, 135g, 1.0eq) and dissolve it in DMF. When the internal temperature drops to 15-20°C, add DNPC (143.4g, 2.0eq), stir for 15min, then add DIPEA (3.1g, 0.1eq), and stir for 15-18h. Take samples for HPLC monitoring. If there is no raw material, the reaction is complete. Post-treatment: Filter the reaction liquid, transfer the filtrate to a 20L plastic bucket, add DCM (5.8L), stir for 30-35min, add MTBE (3.9L), stir for 30-35min, and then filter. Stir the filter cake with 1.5L DCM for 20-30min, then add 3L MTBE, stir for 30min, and then filter. Dry the filter cake to obtain 147g of solid, yield: 84.5%, LCMS: 737.77, test result: 738.31 [M+H] +.

[0077] HPLC spectrum of compound (SET0568-3) MC-VC-PAB-PNP ( Figure 2 )、LCMS spectrum( Figure 3 ) and HNMR spectra ( Figure 4 ) as shown.

[0078] Synthesis of compound (SET0568-6) PNU-159682

[0079] Compound SET0568-5500 mg (PNU-TBS, 1.0 eq) was placed in a 50 mL single-necked bottle, 5 mL of THF was added, the temperature was lowered to -5 to 0 °C with stirring, 0.86 mL of TBAF (1 M in THF, 1.3 eq) was added dropwise to the reaction bottle, and the reaction was carried out for about 20 min. LCMS detected that the reaction of the raw material was complete, 20 mL of water and 50 mL of DCM were added, and the aqueous phase was back-extracted with 50 ml of DCM. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain (SET0568-6) PNU-159682, which was directly used in the next step.

[0080] Synthesis of compound SET0568-8PNU-DMAE

[0081] SET0568-6 (PNU-159682 crude product, 1.0 eq) obtained in the previous step and DNPC 400 mg (2.0 eq) were added with DCM 10 mL, and the mixture was stirred with ice water and cooled to about 0-5°C. Triethylamine / TEA 200 mg (3.0 eq) was added to the reaction system, and the mixture was stirred at 0-5°C for 30-60 min. LCMS detection showed that the raw material was completely converted to obtain the intermediate SET0568-7, LCMS: 806.73, and the detection result was 807.52 [M+H]+, and the reaction was directly carried out to the next step.

[0082] Take 86.5 mg of N, N'-dimethylethylenediamine (DMEA, 1.5 eq) and add it to the reaction system, stir at about 0-5°C for 1-2 hours. HPLC / LC-MS detection shows that the raw material conversion is complete. Post-treatment: remove the reaction solvent by rotary evaporation. The crude product SET0568-8 is purified by medium-pressure reverse phase preparation, 120 g reverse phase column, wavelength 234 nm, water / acetonitrile system purification, and the product is obtained at a ratio of 40-50% acetonitrile. The product is collected and freeze-dried to obtain (SET0568-8) PNU-DMEA, 231 mg, HPLC: 94%, LCMS: 755.77, detection result: 756.81 [M+H] +.

[0083] The LCMS spectrum of SET0568-7 is as follows Figure 5 The LCMS spectrum of SET0568-8 is shown in Figure 6 shown.

[0084] Synthesis of compound (SET0568) MC-VC-PAB-DMEA-PNU159682

[0085] At room temperature and under nitrogen protection, 205 mg SET0568-8 (PNU-DMAE, 1.0 eq) was dissolved in 2 ml DMF, 220 mg SET0568-3 (MC-VC-PAB-PNP, 1.1 eq) was added, and then cooled to an internal temperature of 0-10°C in an ice-water bath, stirred for 10-15 min, and DIPEA (2.0 eq, 70 mg) was added, and the reaction was continued in an ice-water bath for 2-3 h, and the reaction was complete. Post-treatment: medium pressure reverse phase preparation and purification, wavelength 234 nm, flow rate 50 mL / min, 0.05% NH4HCO3 water / acetonitrile, 40-50% acetonitrile ratio to produce the product, collect the product, and freeze-dry to obtain SET0568 (MC-VC-PAB-DMEA-PNU159682) 54 mg, HPLC: 92.2%, LCMS: 1354.43, detection result 1355.31 [M+H] +.

[0086] HPLC spectrum of compound MC-VC-PAB-DMEA-PNU159682 ( Figure 7 )、LCMS spectrum( Figure 8 ) and HNMR spectra ( Fig. 9 ).

[0087] 1H NMR (400MHz, DMSO) δ13.99 (s, 1H), 13.23 (s, 1H), 9.96 (s, 1H), 8.05 (d, J=7.3Hz, 1H), 7.92- 7.86 (m, 2H), 7.79 (d, J=8.6Hz, 1H), 7.66-7.54 (m, 4H), 7.27 (s, 2H), 6.99 (s, 2H), 5.97 (t, J =5.7Hz, 1H), 5.47 (s, 1H), 5.39 (s, 2H), 5.20 (d, J = 20.3Hz, 2H), 5.06 (d, J = 17.7Hz, 1H), 4.9 5 (d, J=15.8Hz, 3H), 4.58 (d, J=1.7Hz, 1H), 4.38 (dd, J=13.3, 7.9Hz, 1H), 4.23 (d, J=1.8Hz, 1 H), 4.21-4.14 (m, 2H), 3.98 (s, 4H), 3.92 (s, 1H), 3.66 (t, J=9.0Hz, 1H), 3.50 (d, J=11.7Hz, 1H), 3.43-3.37(m, 4H), 3.31(s, 3H), 3.06-2.81(m, 12H), 2.69(dd, J=32.4, 20.5Hz, 4H), 2. 35(d, J=14.3Hz, 1H), 2.13 (ddd, J=35.0, 17.5, 10.3Hz, 4H), 1.96 (dd, J=13.4, 6.6Hz, 1H), 1 .68 (s, 3H), 1.57-1.36 (m, 8H), 1.19 (dd, J=18.5, 10.7Hz, 8H), 0.83 (dd, J=12.2, 6.7Hz, 8H).

[0088] Comparative Example 1

[0089] This comparative example provides a method for synthesizing an antibody-drug conjugate linker SET0568, which specifically comprises the following steps:

[0090]

[0091] Synthesis of compound (SET0568-4) MC-VC-PAB-DMEA

[0092] Take SET0568-3 (MC-VC-PAB-PNP, 1.5g, 1.0eq) and dissolve it in 20ml DMF. When the internal temperature is 20-25℃, add 168mg N,N′-dimethylethylenediamine (DMEA, 1.01eq), stir for 15min, then add 381mg 1-hydroxybenzotriazole (HOBt, 1.5eq), stir for 5-10min. Add DIPEA (2.0eq, 480mg) to the system, stir at room temperature, and the reaction is complete when there is no raw material detected by HPLC, as shown in the HPLC spectrum ( Fig.10 ), the system has dimer and SET0568-3 byproducts. Post-treatment: Add 100ml of MTBE to the reaction solution, stir for 30-60min, filter with a Buchner funnel, SET0568-4 see HPLC spectrum ( Fig.11 ), the HPLC purity was only 54.2%. It can be seen that a large amount of by-products continued to be generated in the reaction system, the product was not stable, and the yield was obviously low.

[0093] Comparative Example 2

[0094] This comparative example provides a method for synthesizing an antibody-drug conjugate linker SET0568, which specifically comprises the following steps:

[0095]

[0096] The synthesis yield of compound SET0568-4 is very low, and there are many side reactions in the reaction, such as hydrolysis ring opening, dimerization, etc. Therefore, we changed the synthesis strategy and used comparative example 2, DMEA (N, N'-dimethylethylenediamine) SET0568-4A protected by a protecting group, and then obtained SET0568-4 with a high yield by deprotection.

[0097] In actual synthetic practice, the reaction conversion rate and yield of Boc-protected SET0568-4 (SET0568-5) were very high, but in the deprotection stage, different deprotection reagents such as HCl / dioxane, HCl / EtOAc, and HCl / Et 2 O, TFA, TsOH / THF-CH2C12, Me3SiI / CHCl3 or CH3CN, the yields are low and many side reactions occur;

[0098] After the protecting group was replaced with Fmoc, the reaction conversion rate and yield of Fmoc-protected SET0568-4 (SET0568-5) were also very high. However, in the deprotection stage, when different deprotection reagents such as diethylamine, diisopropylamine, 1,8-diazobispiro[5.4.0]undec-7-ene (DBU), piperidine, ethanolamine, cyclohexylamine, morpholine, pyrrolidone 2, etc. were tried, many side reactions occurred, such as hydrolysis and chain scission, and the yield was low.

[0099] Therefore, the final total yield of Comparative Example 2 is still very low, making it difficult to scale up production and commercialize.

[0100] The above examples and comparative examples illustrate that (SET0568) MC-VC-PAB-DMEA-PNU159682 and the synthesis method of the present invention can be efficiently synthesized, easily scaled up, and convenient for commercial production.

[0101] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for synthesizing an antibody-drug conjugate linker SET0568, wherein the chemical structure of SET0568 is: ； The synthesis method comprises the following steps: Dissolve the compound VC-PAB in the first solvent, stir for 30 min to 60 min, add MC-OSu and stir for 1 h to 2 h, add the second solvent for post-treatment to obtain MC-VC-PAB; The compound MC-VC-PAB is dissolved in a first solvent and stirred, the internal temperature is lowered to 15°C to 20°C, DNPC is added, stirred for 10min to 20min, DIPEA is added, stirred for 15h to 18h, and a second solvent is added for post-treatment to obtain MC-VC-PAB-PNP; The compound PNU-TBS is dissolved in a third solvent, TBAF is added, the temperature is lowered to -5-0°C with stirring, the reaction is carried out for 20-30 minutes, the liquids are separated and extracted, and the mixture is dried and concentrated to obtain PNU-159682; The PNU-159682 and DNPC are dissolved in a fourth solvent, cooled to 0°C to 5°C by stirring with ice water, triethylamine is added, and the mixture is kept at 0°C to 5°C and stirred for 30min to 60min to obtain an intermediate PNU-PNP, N,N'-dimethylethylenediamine is added, and the mixture is stirred at about 0 to 5°C for 1 to 2h, and post-processed. The reaction solution is concentrated, purified by a medium-pressure reverse phase column, and freeze-dried to obtain a compound PNU-DMEA; Under nitrogen protection, the compound PNU-DMEA was dissolved in the first solvent, MC-VC-PAB-PNP was added, the temperature was cooled to 0-10°C in an ice-water bath, stirred for 10-15 minutes, DIPEA was added, the reaction was continued in an ice-water bath for 2-3 hours, post-processed, and freeze-dried to obtain the SET0568; The first solvent is DMF or DMA, the second solvent is one or a mixed solvent of DCM, EA, PE, MTBE, and diethyl ether, the third solvent is DCM or THF, and the fourth solvent is DCM, THF, or DMF.

2. The synthesis method according to claim 1, wherein When preparing MC-VC-PAB, the mixing molar ratio of the VC-PAB to the MC-OSu is 1.0eq:1.0eq-1.5eq.

3. The synthesis method according to claim 1, wherein When preparing MC-VC-PAB-PNP, the mixing molar ratio of the MC-VC-PAB to the DNPC is 1.0eq:1.5eq-2.5eq.

4. The synthesis method according to claim 1, wherein When preparing PNU-159682, the mixing molar ratio of the PNU-TBS to the TBAF is 1.0eq:1.0eq-1.5eq.

5. The synthesis method according to claim 1, wherein When preparing PNU-DMEA, the mixing molar ratio of PNU-159682, DNPC, triethylamine and N,N'-dimethylethylenediamine is 1.0eq: 1.5eq-3.0eq: 2.0eq-3.5eq: 1.0eq-2.5eq.

6. The synthesis method according to claim 1, wherein When preparing SET0568, the mixing molar ratio of PNU-DMEA, MC-VC-PAB-PNP and DIPEA is 1.0eq: 1.0-1.5eq: 2.0eq-3.0eq.

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