A solid phase synthesis method for antibody-drug conjugate linker

Abstract

The invention discloses a solid phase synthesis method of an antibody-drug conjugate linker, comprising the following steps: (1) coupling Asp, Ala and Ala protected amino acids one by one on an amino resin to obtain a first peptide resin; the N-terminus of the Asp and the C-terminus of the main chain are respectively coupled with a protecting group; (2) removing the protecting group of the Asp carboxyl end on the first peptide resin, coupling Pab, and obtaining a second peptide resin; 3) cracking the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab. The synthesis process of the invention has mild conditions, high yield of the product, good purity, the total yield of the product Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab reaches more than 90%, and the HPLC purity reaches more than 97%, which meets the purity requirements of pharmaceutical products and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a solid phase synthesis method for an antibody-drug conjugate linker. Background Art

[0002] Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab is a protein-degradable (cleavable) peptide prodrug linker and a commonly used linker for antibody-drug conjugates (ADCs). It has a certain stability in human plasma. The structural formulas of Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab are as follows:

[0003]

[0004] Chinese patent CN 106456799 A discloses a liquid phase synthesis method of Fmoc-Ala-Ala-Asn(Trt)-Pab, which first synthesizes dipeptides Fmoc-Ala-Ala-OH and H-Asn(Trt)-Pab separately, and couples the two dipeptides to obtain Fmoc-Ala-Ala-Asn(Trt)-Pab, with a total yield of 67.7%. Chinese patent CN 113274507A discloses a liquid phase synthesis method of H-Ala-Ala-Asn-Pab, which, like patent CN 106456799A, first synthesizes dipeptide H-Asn(Trt)-Pab, couples dipeptide Boc-Ala-Ala-OH to obtain Boc-Ala-Ala-Asn(Trt)-Pab, and after column purification, acid-cuts the protective agent to obtain H-Ala-Ala-Asn-Pab, with a total yield of 11.37%.

[0005] The liquid phase synthesis linker Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab has a long synthesis route and cycle, low overall yield, high cost, and is difficult to achieve industrial production. Summary of the invention

[0006] In view of this, the present invention provides a solid phase synthesis method for antibody-drug conjugate linkers. The present invention adopts a solid phase synthesis method combined with a special synthesis strategy, starting from Asn, first synthesizing an Ala-Ala-Asp fragment, and finally coupling Pab, which effectively improves the total yield and purity of the linker, and the total yield can reach more than 90%, and the HPLC purity can reach more than 97%, solving the above-mentioned shortcomings in the prior art.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A solid phase synthesis method for antibody-drug conjugate linkers comprises the following steps:

[0009] (1) Asp, Ala and protected amino acids of Ala are coupled one by one on an amino resin to obtain a first peptide resin; the N-terminus of Asp and the C-terminus of the main chain are coupled with protecting groups respectively, and the N-termini of the two Alas are coupled with protecting groups.

[0010] (2) removing the protecting group at the carboxyl end of Asp on the first peptide resin and coupling with Pab to obtain a second peptide resin;

[0011] (3) Cleavage of the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab.

[0012] In the process of the second peptide resin cleavage of the present invention, Asp is converted into Asn.

[0013] In the present invention, the main chain carboxyl group of the Asp is coupled with a protecting group, and the protecting group is OAll or OBzl, preferably OAll.

[0014] In some embodiments, the protected amino acid of Asp is Fmoc-Asp-OAll; the protected amino acid of Ala is Fmoc-Ala-OH.

[0015] In some embodiments, the amino resin includes a benzylamine linker type resin; the benzylamine linker type resin is Rink Amide-AM Resin, MBHA Resin or Sieber Resin; the degree of substitution of the Rink Amide-AM Resin is 0.5-1.0 mmol / g, preferably 0.6-0.8 mmol / g, specifically 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g.

[0016] In the present invention, the coupling reaction medium is one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone and dimethyl sulfoxide, preferably N,N-dimethylformamide or dichloromethane.

[0017] In the present invention, the coupling and deprotection temperature is 25-45°C, preferably 30-40°C, specifically 30°C, 35°C, 40°C.

[0018] In the present invention, the condensation reagent used in the coupling in step (1) and step (2) is any one or more of A, B, C, and D, wherein A is selected from any one of DIC, DCC, and EDCI, B is selected from any one of HOOBT, HOBT, and BOAT, C is selected from any one of DIPEA and NMM, and D is selected from any one of DMAP, HBTU, HATU, TBTU, PyBOP, and EEDQ.

[0019] In the present invention, the deprotecting agent of the protecting group is a N,N-dimethylformamide solution of piperidine. Preferably, the concentration of the N,N-dimethylformamide solution of piperidine is 20% to 50%, specifically 20%, 30%, 40%, 50%, preferably 20%. The deprotecting agent of OAll in step (2) is a dichloromethane solution containing Pd(PPh3)4 and Ph-SiH3; preferably, the molar ratio of the peptide resin, Pd(PPh3)4 and Ph-SiH3 is 1:(0.1-1):(1-10), specifically 1:0.5:1:1:0.5:10, 1:0.5:5, 1:0.1:5, 1:1:5, more preferably 1:0.5:5.

[0020] In the present invention, the lysis solution used for lysis in step (3) is a dichloromethane solution of trifluoroethanol, and the concentration of the dichloromethane solution of trifluoroethanol is preferably 30% to 80%, specifically 30%, 40%, 50%, 60%, 70% or 80%, more preferably 50%.

[0021] In some specific embodiments, the present invention comprises the following steps:

[0022] a) Using benzylamine linker type resin as solid phase synthesis carrier, coupling protected amino acid Fmoc-Asp-PG to obtain the starting peptide resin: Fmoc-Asp(-Rink Amide-AM Resin)-OAll;

[0023] b) using Fmoc-solid phase synthesis method, sequentially coupling Fmoc-Ala-OH and Fmoc-Ala-OH to obtain the first peptide resin: Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-OAll;

[0024] C) removing the protecting group PG at the Asp carboxyl end of the Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-PG, adding H-Pab-OH for coupling, and obtaining the second peptide resin: Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-Pab;

[0025] d) cleaving the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab.

[0026] The invention has the beneficial effects as follows: the invention discloses a solid phase synthesis method for an antibody-drug conjugate linker (Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab); the synthesis process conditions of the invention are mild; the yield of the product is high and the purity is good; the total yield of the product Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab can reach more than 90%; the HPLC purity reaches more than 97%, which meets the purity requirements of pharmaceutical products and is suitable for large-scale industrial production; in the cracking process of the invention, one technical means realizes two technical effects, Asp is also converted into Asn, and an unexpected technical effect is achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The synthetic route of Fmoc-Ala-Ala-Asn-Pab / H-Ala-Ala-Asn-Pab synthesized by the present invention is shown;

[0028] Figure 2 4 shows the HPLC spectrum of H-Ala-Ala-Asn-Pab synthesized by the present invention;

[0029] Figure 3 4 shows the LC-MS spectrum of H-Ala-Ala-Asn-Pab synthesized by the present invention;

[0030] Figure 4 4 shows the HPLC spectrum of Fmoc-Ala-Ala-Asn-Pab synthesized by the present invention;

[0031] Figure 5 4 shows the LC-MS spectrum of Fmoc-Ala-Ala-Asn-Pab synthesized in the present invention. DETAILED DESCRIPTION

[0032] The present invention provides a solid phase synthesis method for antibody-drug conjugate linkers. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0033] Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.

[0034] In the present invention, the names and English abbreviations of the reagents and instruments used are shown in Table 1.

[0035] Table 1 Reagents and instruments used

[0036]

[0037]

[0038] The present invention will be further described below in conjunction with embodiments:

[0039] Example 1 Synthesis of the starting peptide resin Fmoc-Asp (-Rink Amide-AM Resin)-OAll

[0040] Swelling of Rink Amide-AM Resin: Add 16.1 g of Rink Amide-AM Resin (degree of substitution: 0.62 mmol / g; 10 mmol) and 150 mL of DMF to the solid phase reactor. Turn on the cold / hot medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 °C, and swell for 1-2 hours. After the swelling is completed, filter and wash the resin twice with 150 mL of DMF, draining it each time.

[0041] Deprotection: Add 150 mL of deprotection solution (20% PIP / 80% DMF) to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 0.5 h. After the reaction is completed, filter and wash with 150 mL DMF 6 times, each time draining. Take a sample for NT detection. If the NT test is positive, the deprotection reaction is completed.

[0042] Pre-activation: Add 100 mL DMF, 11.9 g Fmoc-Asp-OAll (30 mmol) and 4.2 g HOBt (30 mmol) to the activation bottle, stir to dissolve. Control the temperature to T = 5 ± 5 ° C in a cold water bath, add 4.7 ml DIC (30 mmol), and slowly stir to activate for 10 ± 5 min.

[0043] Coupling: Add the above activation solution to the solid phase reactor, take another 50mL DMF to wash the activation bottle, and add it to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 2h. After the reaction is completed, filter and wash with 150mL DMF 4 times, each time draining. Take a sample for NT detection, NT test negative, the coupling reaction is completed, and Fmoc-Asp (-Rink Amide-AM Resin) -OAll is obtained.

[0044] Example 2: Coupling of Fmoc-Ala-OH

[0045] Deprotection: Add 150 mL of deprotection solution (20% PIP / 80% DMF) to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 0.5 h. After the reaction is completed, filter and wash with 150 mL DMF 6 times, each time draining. Take a sample for NT detection. If the NT test is positive, the deprotection reaction is completed.

[0046] Pre-activation: Add 100 mL DMF, 9.4 g Fmoc-Ala-OH (30 mmol) and 4.2 g HOBt (30 mmol) to the activation bottle, stir to dissolve. Control the temperature to T = 5 ± 5 ° C in a cold water bath, add 4.7 ml DIC (30 mmol), and slowly stir to activate for 10 ± 5 min.

[0047] Coupling: Add the above activation solution to the solid phase reactor, take another 50mL DMF to wash the activation bottle, and add it to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 2 hours. After the reaction is completed, filter and wash with 150mL DMF 4 times, each time draining. Take a sample for NT detection, NT test is negative, the coupling reaction is completed, and Fmoc-Ala-Asp (-Rink Amide-AM Resin)-OAll

[0048] Example 3: Coupling of Fmoc-Ala-OH

[0049] Deprotection: Add 150 mL of deprotection solution (20% PIP / 80% DMF) to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 0.5 h. After the reaction is completed, filter and wash with 150 mL DMF 6 times, each time draining. Take a sample for NT detection. If the NT test is positive, the deprotection reaction is completed.

[0050] Pre-activation: Add 100 mL DMF, 9.4 g Fmoc-Ala-OH (30 mmol) and 4.2 g HOBt (30 mmol) to the activation bottle, stir to dissolve. Control the temperature to T = 5 ± 5 ° C in a cold water bath, add 4.7 ml DIC (30 mmol), and slowly stir to activate for 10 ± 5 min.

[0051] Coupling: Add the above activation solution to the solid phase reactor, take another 50mL DMF to wash the activation bottle, and add it to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 2 hours. After the reaction is completed, filter and wash with 150mL DMF 4 times, each time draining; wash with 150mL DCM 2 times, each time draining. Take a sample for NT detection, NT test negative, the coupling reaction is completed, and Fmoc-Ala-Ala-Asp (-Rink Amide-AMResin)-OAll is obtained.

[0052] Example 4: Coupling of H-Pab-OH

[0053] Deprotection: Add 150 mL of deprotection solution (5.8 g Pd(PPh3)4 (5 mmol) + 5.4 g Ph-SiH3 (50 mmol) / 150 mL DCM) to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 1.0 h. After the reaction is completed, filter and wash 6 times with 150 mL DCM, drain each time.

[0054] Coupling: Add 100mL DMF, 3.7g H-Pab-OH (30mmol) and 7.4g EEDQ (30mmol) to the activation bottle, stir to dissolve. Add the above activation solution to the solid phase reactor, take another 50mL DMF to wash the activation bottle, and add it to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30±3℃, and react for 5h. After the reaction is completed, filter and wash 4 times with 150mL DMF, drain each time. The coupling reaction is completed to obtain Fmoc-Ala-Ala-Asp (-Rink Amide-AM Resin)-Pab.

[0055] Example 5: Peptide resin Fmoc-Ala-Ala-Asp (-Rink Amide-AM Resin)-Pab collection

[0056] Washing: The peptide resin in the solid phase reactor was washed with 150 ml MTBE twice, each time the solution was pumped dry; washed with 150 ml DCM twice, each time the solution was pumped dry; and then washed with 150 ml MTBE twice, each time the solution was pumped dry.

[0057] Drying: nitrogen was blown on the resin to preliminarily dry it, and it was transferred out of the solid phase reactor and placed in a vacuum oven at a temperature of 30±3°C and a pressure of ≤-0.09 MPa to start vacuum drying. After drying, 22.1 g of Fmoc-Ala-Ala-Asp(-Rink Amide-AMResin)-Pab was obtained with a yield of 100.9%.

[0058] Example 6: Preparation of H-Ala-Ala-Asn-Pab

[0059] Swelling of peptide resin: 11.0 g of Fmoc-Ala-Ala-Asp (-Rink Amide-AM Resin)-Pab peptide resin obtained in Example 5 and 100 mL of DMF were added to the solid phase reactor. The cold / heating medium switch of the solid phase reactor jacket was turned on, and the temperature was adjusted to T = 30 ± 3 ° C. and the resin was swelled for 1-2 h. After the swelling was completed, the resin was filtered and washed twice with 100 mL of DMF, and each time it was drained.

[0060] Deprotection: Add 100 mL of deprotection solution (20% PIP / 80% DMF) to the solid phase reactor, open the nitrogen valve and the cold / heat medium switch of the solid phase reactor jacket, adjust the temperature to T = 30 ± 3 ° C, and react for 0.5 h. After the reaction is completed, filter and wash with 100 mL DMF 6 times, each time draining.

[0061] Washing: The peptide resin in the solid phase reactor was washed with 100 mL MTBE twice, and the solution was dried each time; washed with 100 mL DCM twice, and the solution was dried each time; and washed with 100 mL MTBE twice again, and the solution was dried each time.

[0062] Drying: nitrogen was blown on the resin to preliminarily dry it, and it was transferred out of the solid phase reactor and placed in a vacuum oven at a temperature of 30±3°C and a pressure of ≤-0.09 MPa to start vacuum drying. After drying, 10.1 g of H-Ala-Ala-Asp(-RinkAmide-AMResin)-Pab was obtained.

[0063] Cleavage: Measure 100 mL of cleavage solution (50% TFA in DCM), add it to the cleavage reactor, and precool it to about T = 2-8°C. Slowly add 10.0 g of peptide resin to the cleavage solution, control the temperature to T = 30 ± 3°C, and cleave for 2-3 hours. After the cleavage reaction is completed, filter, wash the filter cake twice with 50 mL of DCM, and drain each time. The filtrate is combined and concentrated under reduced pressure. The concentrated residue is slurried with 100 mL of MTBE and filtered to obtain H-Ala-Ala-Asn-Pab, which is a white powder weighing 1.75 g, with a total yield of 92.5% and a HPLC purity of 97.93%

[0064] The HPLC spectrum of H-Ala-Ala-Asn-Pab is shown in Figure 2 , LC-MS spectrum see Figure 3 .

[0065] Example 7: Preparation of Fmoc-Ala-Ala-Asn-Pab

[0066] Lysis: Take 100 mL of lysate (50% TFA in DCM) and add it to the lysate reactor. Precool it to about T 内 =2-8°C. Slowly add 10.0 g of Fmoc-Ala-Ala-Asp (-Rink Amide-AM Resin)-Pab peptide resin obtained in Example 5 to the cleavage solution, control the temperature to T = 30 ± 3°C, and cleave for 2-3 hours. After the cleavage reaction is completed, filter, wash the filter cake twice with 50 mL of DCM, and drain each time. Combine the filtrates, concentrate under reduced pressure, and slurry the concentrated residue with 100 mL of MTBE, filter, and obtain Fmoc-Ala-Ala-Asn-Pab, which is a white powder weighing 2.82 g, with a total yield of 93.7% and a HPLC purity of 98.10%

[0067] The HPLC spectrum of Fmoc-Ala-Ala-Asn-Pab is shown in Figure 4 , LC-MS spectrum see Figure 5 .

[0068] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A solid phase synthesis method for antibody-drug conjugate linkers, characterized in that: include: (1) Asp, Ala and protected amino acids of Ala are coupled one by one on an amino resin to obtain a first peptide resin; the N-terminus of Asp and the C-terminus of the main chain are coupled with protecting groups respectively; (2) removing the protecting group at the carboxyl end of Asp on the first peptide resin and coupling with Pab to obtain the second peptide resin; (3) cleaving the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab; The cleavage solution used in step (3) is a dichloromethane solution of trifluoroethanol.

2. The solid phase synthesis method according to claim 1, characterized in that The main chain carboxyl group of the Asp is coupled with a protecting group, and the protecting group is OAll or OBzl.

3. The solid phase synthesis method according to claim 1 or 2, characterized in that: The protected amino acid of Asp is Fmoc-Asp-OAll; The protected amino acid of Ala is Fmoc-Ala-OH, and Asp is converted into Asn during the cleavage process.

4. The solid phase synthesis method according to claim 1, characterized in that: The amino resin includes benzylamine linker type resin; the benzylamine linker type resin is Rink Amide-AM Resin, MBHA Resin or Sieber Resin; the substitution degree of the Rink Amide-AM Resin is 0.5-1.0 mmol / g.

5. The solid phase synthesis method according to claim 1, characterized in that The coupling reaction medium is one or more of dichloromethane, chloroform, tetrahydrofuran, N,N-dimethylformamide, N,N-diethylformamide, N-methylpyrrolidone and dimethyl sulfoxide.

6. The solid phase synthesis method according to claim 1, characterized in that The coupling and deprotection temperature is 25-45°C.

7. The solid phase synthesis method according to claim 1, characterized in that The deprotecting agent of the protecting group is an N,N-dimethylformamide solution of piperidine or a dichloromethane solution containing Pd(PPh3)4 and Ph-SiH3.

8. The solid phase synthesis method according to claim 1, characterized in that The condensation reagent used in the coupling in step (1) and step (2) is any one or more of A, B, C, and D, wherein A is selected from any one of DIC, DCC, and EDCI, B is selected from any one of HOOBT, HOBT, and BOAT, C is selected from any one of DIPEA and NMM, and D is selected from any one of DMAP, HBTU, HATU, TBTU, PyBOP, and EEDQ.

9. The solid phase synthesis method according to claim 1, characterized in that: The concentration of the dichloromethane solution of trifluoroethanol in step (3) is 30% to 80%.

10. A solid phase synthesis method of an antibody-drug conjugate linker, characterized in that: include: a) Using benzylamine linker resin as a solid phase synthesis carrier, coupling the protected amino acid Fmoc-Asp-PG to obtain the starting peptide resin: Fmoc-Asp(-Rink Amide-AM Resin)-OAll; b) Using the Fmoc-solid phase synthesis method, Fmoc-Ala-OH and Fmoc-Ala-OH were coupled in sequence to obtain the first peptide resin: Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-OAll; c) removing the protecting group PG at the Asp carboxyl end of the Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-PG, adding H-Pab-OH for coupling, and obtaining the second peptide resin: Fmoc-Ala-Ala-Asp(-Rink Amide-AM Resin)-Pab; d) cleaving the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab; The cleavage solution used for cleavage is a dichloromethane solution of trifluoroethanol.

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