Synthesis method of linker MC-Gly-Gly-Phe-Gly-OH

Abstract

The invention discloses a synthesis method of a linker MC-Gly-Gly-Phe-Gly-OH, and the synthesis method comprises the following steps: taking Fmoc-Phe-OH and glycine tert-butyl ester hydrochloride as raw materials, and sequentially synthesizing a first intermediate Fmoc-Phe-Gly-OBu and a second intermediate H-Phe-Gly-OBu; the second intermediate and Boc-Gly-Gly-OH are sequentially synthesized into a third intermediate Boc-Gly-Gly-Phe-Gly-OBu and a fourth intermediate H-Gly-Gly-Phe-Gly-OH, and the fourth intermediate and 6-(maleimido) hexanoic acid succinimide ester are coupled to obtain the target product. The conditions of each step reaction are mild, the yield and purity of the intermediate are high, the HPLC purity of the obtained target product reaches 98% or above, the yield reaches 91% or above, the purity requirement of a medicine product is met, the method is suitable for industrial mass production, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody-drug conjugate synthesis, and in particular to a method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH. Background Art

[0002] Antibody-drug conjugates (ADCs) are relatively complex molecules composed of targeted antibodies and small toxin molecules (payloads) with cytotoxic activity connected together through chemical linkers. In recent years, research on payloads and linkers has gradually deepened, and breakthrough progress has been made. ADCs represented by Enhertu (DS-8201) have broken through people's understanding of traditional ADCs and greatly improved clinical treatment effects, becoming blockbuster products. The linker of ADC must not only connect antibodies and toxins, but also be able to cleave and release toxins at the tumor cells. These two functions determine that the linker must have two characteristics: first, it must be stable in the body's circulatory system; second, it must be able to specifically release active ingredients when it reaches the tumor cell site.

[0003] The linkers used in ADCs are divided into two types: cleavable and non-cleavable. For non-cleavable linkers, after entering the lysosome, the monoclonal antibody (mAb) is metabolized by proteolytic mechanism, and the released metabolites include payload, linker and amino acid appendages; if the key pharmacophore of the payload is not affected, a substantial modification of the payload can also produce an effective ADC, as is the case with Kadcyla. However, due to the lack of cell permeability of the charged amino acid appendages, non-cleavable linkers are usually unable to exert a bystander effect, so the application range of ADCs containing non-cleavable linkers is limited, and they are mainly used to treat blood cancers or tumors with high antigen expression. Compared with non-cleavable linkers, cleavable linkers release drugs at target cells under specific conditions.

[0004] Cleavable linkers can be further divided into chemically cleavable or enzymatically cleavable. MC-Gly-Gly-Phe-Gly-OH is an enzymatically cleavable polypeptide linker that has a certain degree of stability in human plasma. Its molecular formula is: 25 H 31 N5O8, the structural formula is as follows:

[0005]

[0006] However, in the process of realizing the technical solution of the invention in the examples of the present application, the inventors of the present application found that the prior art has the following disadvantages in the process of preparing MC-Gly-Gly-Phe-Gly-OH: hydrogenation treatment is required to remove the benzyl group after benzyl protection, which is difficult to operate and not conducive to industrial production; there are many side reactions and impurities, and the purity of intermediates and final products is low, and the yield is low, which affects the product quality and application. Summary of the invention

[0007] The present invention can solve the above problems existing in the prior art by providing a method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH.

[0008] In order to solve the above technical problems, the present invention provides a method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH, comprising the following steps:

[0009] (1) Preparation of the first intermediate: Using Fmoc-Phe-OH and glycine tert-butyl ester hydrochloride as raw materials, under the action of a condensation reagent, the first intermediate Fmoc-Phe-Gly-OBu is synthesized.

[0010]

[0011] (2) Preparation of the second intermediate: removing the Fmoc protecting group from the first intermediate under the action of a base to obtain the second intermediate H-Phe-Gly-OBu,

[0012]

[0013] (3) Preparation of the third intermediate: The second intermediate and Boc-Gly-Gly-OH are reacted with a condensing agent to synthesize the third intermediate Boc-Gly-Gly-Phe-Gly-OBu.

[0014]

[0015] (4) Preparation of the fourth intermediate: removing the Boc protecting group from the third intermediate Boc-Gly-Gly-Phe-Gly-OBu under the action of acid to obtain the fourth intermediate H-Gly-Gly-Phe-Gly-OH.

[0016]

[0017] (5) Preparing the target linker: coupling the fourth intermediate with 6-(maleimido)hexanoic acid succinimidyl ester under the action of a base to obtain the MC-Gly-Gly-Phe-Gly-OH,

[0018]

[0019] In a preferred embodiment of the present invention, in steps (1) and (3), the synthesis temperature is -5 to 5°C.

[0020] In a preferred embodiment of the present invention, in steps (1) and (3), the condensing agent includes at least one of DCC, DIC or EDCI.

[0021] In a preferred embodiment of the present invention, in steps (2) and (5), the base comprises at least one of piperidine, sodium carbonate, sodium bicarbonate, DIEA, DIPEA, triethylamine or diethylamine.

[0022] In a preferred embodiment of the present invention, in step (4), the acid comprises TFA, hydrochloric acid or acetic acid.

[0023] In a preferred embodiment of the present invention, in steps (2) and (4), the temperature for removing the protecting group is 40-50°C.

[0024] In a preferred embodiment of the present invention, in step (5), the coupling temperature is 20-30°C.

[0025] The invention has the beneficial effects that: the invention provides a method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH, using Fmoc-Phe-OH, glycine tert-butyl ester hydrochloride, Boc-Gly-Gly-OH and 6-(maleimido)hexanoic acid succinimide ester as raw materials, preparing a target product through step-by-step reactions, the synthesis conditions of each step-by-step reaction are mild, and the amino group and carboxyl group protection are removed simultaneously under acidic conditions, the operation is simple, the yield of the intermediate is improved, and the method is suitable for industrialized scale-up production, the yield and purity of each intermediate are high, the HPLC purity of the obtained target product reaches more than 98%, and the yield reaches more than 91%, thereby meeting the purity requirements of pharmaceutical products and being suitable for industrialized large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the HPLC spectrum of the pure MC-Gly-Gly-Phe-Gly-OH synthesized by the present invention;

[0027] Figure 2 It is the LC-MS spectrum of the pure MC-Gly-Gly-Phe-Gly-OH synthesized by the present invention. DETAILED DESCRIPTION

[0028] 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.

[0029] Table 1

[0030]

[0031]

[0032] The present invention provides a method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH, using Fmoc-Phe-OH and glycine tert-butyl ester hydrochloride as starting materials. The starting materials are simple, cheap, easy to purchase, and of high quality, which is conducive to reducing costs and improving product yield and purity. The specific synthesis route is as follows:

[0033]

[0034] The above synthesis process is carried out in an organic solvent, and the organic solvent includes at least one of DCM, DMF, EA, TCM, THF, toluene, DMAC or THFDCM.

[0035] Specifically, in the above synthesis route, step (1) is carried out under the action of a condensation reagent at a temperature of -5 to 5° C. The condensation reagent includes at least one of DCC, DIC or EDCI.

[0036] Step (2) is carried out under the action of a base at a temperature of 40-50° C. The base comprises at least one of piperidine, sodium carbonate, sodium bicarbonate, DIEA, DIPEA, triethylamine or diethylamine.

[0037] Step (3) is carried out under the action of a condensation reagent at a temperature of -5 to 5° C. The condensation reagent includes at least one of DCC, DIC or EDCI.

[0038] Step (4) is carried out under the action of an acid at a temperature of 40 to 50° C. The acid comprises at least one of TFA, hydrochloric acid or acetic acid. In this step, acid removal of Boc protection is adopted, which is easy to operate and suitable for industrial scale-up production; and this reaction step can remove amino and carboxyl protection at the same time, simplifying the reaction steps, reducing the process flow, saving costs, and improving the yield of the intermediate, and the purity of the obtained product is higher.

[0039] In addition, deprotection before the last step of the reaction avoids side reactions and reduces impurities.

[0040] Step (5) is carried out under the action of a base at a temperature of 20 to 30° C. The base comprises at least one of piperidine, sodium carbonate, sodium bicarbonate, DIEA, DIPEA, triethylamine or diethylamine.

[0041] The synthesis process of the linker MC-Gly-Gly-Phe-Gly-OH of the present invention is described in detail below through specific examples.

[0042] Example 1

[0043] Synthesis of the first intermediate Fmoc-Phe-Gly-OBu

[0044]

[0045] Weigh 15.0g of Fmoc-Phe-OH and 200mL of THF in a 500mL jacketed bottle, start stirring, and control the temperature at 20±5°C under nitrogen protection. After the reaction liquid is dissolved, add 7.14g of glycine tert-butyl ester hydrochloride to the above jacketed bottle; after the addition is completed, cool the reaction to 0±5°C. After the cooling is completed, weigh 20.2g of HATU and add it to the above jacketed bottle; after the addition is completed, weigh 13.8g of DIEA and add it dropwise to the above jacketed bottle. After the addition is completed, keep stirring at 0±5°C for 17-20h. If the raw material is <0.5% through HPLC control, the reaction is over.

[0046] After the reaction is completed, the reaction solution is evaporated to remove the solvent at 32±5°C, and the crude product obtained by rotary evaporation is transferred to a 500mL single-mouth bottle, 150mL DCM is added, and the solution is stirred at 20±5°C. 100mL 1N dilute hydrochloric acid is added to the above-mentioned single-mouth bottle, stirred for 3-5min, and the organic phase obtained by separation is added with 1N dilute hydrochloric acid again, stirred for 3-5min. Separation obtains an organic phase, adds 100mL of saturated sodium bicarbonate aqueous solution, stirs for 3-5min, separates the organic phase, adds 100mL of 10% sodium chloride solution, stirs for 3-5min, separates the organic phase obtained by separation, adds 100mL of drinking water, stirs for 2-3min, separates the organic phase. The organic phase obtained by separation is dried and filtered by anhydrous sodium sulfate. The filtered organic phase is transferred to a 1000mL single-mouth bottle, stirring is turned on, the reaction liquid temperature is maintained at 25±5°C, 450mL MTBE is added dropwise, and after the addition is completed, 25±5°C is maintained and stirred for 1-2h. After stirring, the reaction solution was filtered, and the filter cake was rinsed with 40 mL of MTBE. The obtained solid was placed in a vacuum oven and dried at 35±2° C. for 3-5 h to finally obtain 17.6 g of Fmoc-Phe-Gly-OBu with a purity of 98% and a yield of 91%.

[0047] Example 2

[0048] The second intermediate H-Phe-Gly-OBu

[0049]

[0050] Weigh 8.7g of Fmoc-Phe-Gly-OBu into a 500mL single-mouth bottle, stir and dissolve with 90mL of THF, and protect with nitrogen; weigh 26.8g of TEA and add it into the above single-mouth bottle. After the addition is complete, the reaction solution is heated to 45±5℃ and stirred for 2-3h. The reaction is completed if the raw material is <0.5% by HPLC.

[0051] After the reaction is completed, the reaction solution is evaporated to remove the solvent at 32±5°C, and the crude product obtained by rotary evaporation is transferred to a 250mL single-mouth bottle, and 40mL DCM and 30mL drinking water are added, and the solution is stirred at 20±5°C to dissolve. 1N dilute hydrochloric acid is added to the above single-mouth bottle to adjust the pH to 2-3. After the adjustment is completed, stir for 3-5min to separate the liquids. The aqueous phase and the organic phase are separated. 25mL of drinking water is added to the organic phase and stirred for 3-5min to separate the liquids. The aqueous phase obtained by separation is combined and 25mL of DCM is added to stir for 3-5min to separate the liquids; the aqueous phase obtained by separation is added with 25mL of DCM again, stirred for 3-5min to separate the liquids; the aqueous phase obtained by separation is added with 25mL of DCM again, stirred for 3-5min to separate the liquids. 80mL of DCM is added to the aqueous phase obtained by separation, stirring is maintained at 20±5°C, and a saturated sodium bicarbonate aqueous solution is added to adjust the pH to 8-9. After adjustment, the organic phase was separated and added with 30 mL of 10% sodium chloride aqueous solution, stirred for 3-5 min to separate; the organic phase was separated and added with 30 mL of drinking water, stirred for 3-5 min to separate. The organic phase was separated and added with anhydrous sodium sulfate to dry, filtered, and the organic phase was evaporated at 32±2°C for 2-5 h to remove the solvent, and finally 4.6 g of H-Phe-Gly-OBu was obtained, with a purity of 95% and a yield of 95%.

[0052] Example 3

[0053] Synthesis of the third intermediate Boc-Gly-Gly-Phe-Gly-OBu

[0054]

[0055] Weigh 4.5g of H-Phe-Gly-OBu into a 100mL jacketed bottle, stir and dissolve with 45mL of DMF, and protect with nitrogen; weigh 3.7g of Boc-Gly-Gly-OH and add it to the above jacketed bottle; after adding, cool the reaction to 0±5℃. After cooling, weigh 1.80g of HOPO and add it to the above jacketed bottle; after adding, weigh 5.2g of EDCI and add it to the jacketed bottle; after adding, keep stirring at 0±5℃ for 17-20h. Through HPLC control, if the raw material is <0.5%, the reaction is over.

[0056] After the reaction is completed, the reaction solution is transferred to a 250 mL single-mouth bottle and stirred at 25±5°C. 132 mL of DCM is added dropwise to the single-mouth bottle, and stirred at 25±5°C for 1-2 hours. After the stirring is completed, the reaction solution is filtered, the filter cake is rinsed with 20 mL of DCM, and then dried by rotary evaporation at 32±5°C for 3-6 hours to remove the solvent, and 7.3 g of Boc-Gly-Gly-Phe-Gly-OBu is obtained, with a purity of 96% and a yield of 92%.

[0057] Example 4

[0058] Synthesis of the fourth intermediate H-Gly-Gly-Phe-Gly-OH

[0059]

[0060] Weigh 5.0g of Boc-Gly-Gly-Phe-Gly-OBu into a 250mL single-mouth bottle, stir and dissolve with 50mL of DCM, and protect with nitrogen. Add 80g of TFA to the above single-mouth bottle, and after the addition is complete, heat to 38±5℃ and stir for 20-22h. If the raw material is <0.5% by HPLC, the reaction is over.

[0061] After the reaction is completed, the reaction solution is cooled to 20±5°C, and 150 mL of MTBE is added dropwise to the reaction solution. After the addition is complete, the solution is stirred for 1-2 hours. After the stirring is completed, the solution is filtered, and the filter cake is rinsed with 20 mL of MTBE. The wet product is dried by rotary evaporation at 32±5°C for 3-6 hours to finally obtain 3.1 g of H-Gly-Gly-Phe-Gly-OH with a purity of 95% and a yield of 90%.

[0062] Example 5

[0063] Synthesis of MC-Gly-Gly-Phe-Gly-OH

[0064]

[0065] Weigh 0.5g of H-Gly-Gly-Phe-Gly-OH into a 50mL jacketed bottle, stir with 5mL of DMAC to dissolve, and protect with nitrogen. Weigh 0.4g of 6-(maleimido)hexanoic acid succinimidyl ester and add it to the above jacketed bottle. After the addition is complete, the reaction temperature drops to 0±5°C. After the cooling is completed, weigh 0.5g of DIEA and add it to the above jacketed bottle. After the addition is complete, keep stirring at 0±5°C for 18-20h. Through HPLC control, if the raw material is <0.5%, the reaction is over.

[0066] After the reaction is completed, the reaction solution is heated to 25±5℃. After the heating is completed, 35mL MTBE is added to the reaction solution, and the addition is completed and stirred for 1-2h. After the stirring is completed, the reaction solution is filtered, and the filter cake is rinsed with 10mL MTBE. The solid is added to a 100mL single-mouth bottle and 25mL DCM and 25mL MTBE are added to the 100mL single-mouth bottle and stirred for 1-2h. The stirring process is maintained at 25±5℃. After the stirring is completed, the reaction solution is filtered, and the filter cake is rinsed with 10mL DCM to obtain a solid, which is dried by rotary evaporation at 32±5℃ to remove the solvent. Finally, 0.72g of MC-Gly-Gly-Phe-Gly-OH is obtained, with a purity of 98% and a yield of 91%.

[0067] The synthesized MC-Gly-Gly-Phe-Gly-OH was tested, and the spectrum is shown in the attached figure. Figure 1 and 2 shown.

[0068] The synthesis method of the present invention has mild conditions for each step reaction, high yield and high purity of the intermediates obtained by each step reaction, high yield and high purity of the final product, meets the requirements of the pharmaceutical industry, and is suitable for large-scale production; the synthesis method of the present invention has mild and controllable conditions, is conducive to reducing production costs and reducing environmental pollution.

[0069] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH, characterized in that: The steps include: (1) Preparation of the first intermediate: Using Fmoc-Phe-OH and glycine tert-butyl ester hydrochloride as raw materials, under the action of a condensation reagent, the first intermediate Fmoc-Phe-Gly-OBu is synthesized. (2) Preparation of the second intermediate: removing the Fmoc protecting group from the first intermediate under the action of a base to obtain the second intermediate H-Phe-Gly-OBu, (3) Preparation of the third intermediate: The second intermediate and Boc-Gly-Gly-OH are reacted with a condensing agent to synthesize the third intermediate Boc-Gly-Gly-Phe-Gly-OBu. (4) Preparation of the fourth intermediate: removing the Boc protecting group from the third intermediate Boc-Gly-Gly-Phe-Gly-OBu under the action of acid to obtain the fourth intermediate H-Gly-Gly-Phe-Gly-OH. (5) Preparing the target linker: coupling the fourth intermediate with 6-(maleimido)hexanoic acid succinimidyl ester under the action of a base to obtain the MC-Gly-Gly-Phe-Gly-OH, 2. The method for synthesizing the linker MC-Gly-Gly-Phe-Gly-OH according to claim 1, characterized in that: In steps (1) and (3), the synthesis temperature is -5 to 5°C.

3. The method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH according to claim 2, characterized in that: In steps (1) and (3), the condensation agent includes at least one of DCC, DIC or EDCI.

4. The method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH according to claim 1, characterized in that: In steps (2) and (5), the base comprises at least one of piperidine, sodium carbonate, sodium bicarbonate, DIEA, DIPEA, triethylamine or diethylamine.

5. The method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH according to claim 1, characterized in that: In step (4), the acid includes TFA, hydrochloric acid or acetic acid.

6. The method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH according to claim 1, characterized in that: In steps (2) and (4), the temperature for removing the protecting group is 40 to 50°C.

7. The method for synthesizing a linker MC-Gly-Gly-Phe-Gly-OH according to claim 1, characterized in that: In step (5), the coupling temperature is 20-30°C.

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