CN117683079B - A solid phase synthesis method for antibody-drug conjugate linker - Google Patents

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

Solid phase synthesis method of antibody coupling drug connector

Technical Field

The invention relates to the technical field of biological medicine, in particular to a solid phase synthesis method of an antibody coupling drug connector.

Background

Fmoc-Ala-Ala-Asn-Pab/H-Ala-Ala-Asn-Pab is a protein-degradable (cleavable) polypeptide prodrug linker, is a common linker for antibody-coupled drugs (ADC), and has a certain stability in human plasma. Fmoc-Ala-Ala-Asn-Pab/H-Ala-Ala-Asn-Pab, structural formulae are shown below, respectively:

Chinese patent CN 106456799A discloses a liquid phase synthesis method of Fmoc-Ala-Asn (Trt) -Pab, which synthesizes first dipeptide Fmoc-Ala-OH and H-Asn (Trt) -Pab, respectively, and couples the two dipeptides to obtain Fmoc-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 is the same as patent CN 106456799A, dipeptide H-Asn (Trt) -Pab is obtained by first synthesizing and coupling dipeptide Boc-Ala-Ala-Ala-OH to obtain Boc-Ala-Ala-Asn (Trt) -Pab, and after column purification, acid-cutting protective agent to obtain H-Ala-Ala-Asn-Pab, the total yield is 11.37%.

The liquid phase synthesis linker Fmoc-Ala-Ala-Asn-Pab/H-Ala-Ala-Asn-Pab has long synthetic route and synthetic period, low comprehensive yield and high cost, and is difficult to realize industrialized production.

Disclosure of Invention

In view of this, the present invention provides a solid phase synthesis method of an antibody-conjugated drug linker. The invention combines a special synthesis strategy by adopting a solid phase synthesis method, firstly synthesizes Ala-Ala-Asp fragments from Asn, and finally couples Pab, thereby effectively improving the total yield and purity of the linker, ensuring that the total yield can reach more than 90 percent and the HPLC purity can reach more than 97 percent, and solving the defects in the prior art.

In order to achieve the above object, the present invention provides the following technical solutions:

a solid phase synthesis method of an antibody-coupled drug linker, comprising the steps of:

(1) The method comprises the steps of coupling Asp, ala and protection amino acids of Ala one by one on amino resin to obtain first peptide resin, wherein the N end of Asp and the C end of a main chain are respectively coupled with a protecting group, and the N ends of the two Ala are coupled with protecting groups.

(2) Removing the protecting group of the Asp carboxyl end on the first peptide resin, and coupling Pab to obtain a second peptide resin;

(3) Cleaving the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab.

In the present invention Asp is converted to Asn during cleavage of the second peptide resin.

In the present invention, the backbone carboxyl group of the Asp is coupled with a protecting group, which is OAll or OBzl, preferably OAll.

In some embodiments, the protected amino acid of Asp is Fmoc-Asp-OAll and the protected amino acid of Ala is Fmoc-Ala-OH.

In some embodiments, the amino Resin comprises a benzylamine Linker type Resin, wherein the benzylamine Linker type Resin is RINK AMIDE-AM Resin, MBHA Resin or Sieber Resin, and the substitution degree of RINK AMIDE-AM Resin is 0.5-1.0mmol/g, preferably 0.6-0.8mmol/g, and particularly can be 0.6mmol/g, 0.7mmol/g and 0.8mmol/g.

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

In the present invention, the coupling and deprotection temperatures are 25-45 ℃, preferably 30-40 ℃, and may be 30 ℃, 35 ℃, 40 ℃ in particular.

In the invention, any one or more of the condensation reagents adopted in the coupling in the step (1) and the step (2) are A, B, C, D, wherein A is any one of DIC, DCC, EDCI, B is any one of HOOBT, HOBT, BOAT, C is any one of DIPEA and NMM, and D is any one of DMAP, HBTU, HATU, TBTU, pyBOP, EEDQ.

In the present invention, the protecting group deprotection agent is a piperidine N, N-dimethylformamide solution, preferably, the concentration of the piperidine N, N-dimethylformamide solution is 20% -50%, and specifically, may be 20%, 30%, 40%, 50%, and preferably 20% thereof. The deprotecting agent OAll in the step (2) is a dichloromethane solution containing Pd (PPh ₃)₄ and Ph-SiH ₃), preferably, the molar ratio of the peptide resin to Pd (PPh ₃)₄ to Ph-SiH ₃) is 1 (0.1-1): (1-10), specifically, may be 1:0.5:1:0.5:10, 1:0.5:5, 1:0.1:5, 1:1:5, more preferably 1:0.5:5.

In the present invention, the cracking solution used in the step (3) is a dichloromethane solution of trifluoroethanol, and the concentration of the dichloromethane solution of trifluoroethanol is preferably 30% -80%, specifically may be 30%, 40%, 50%, 60%, 70% or 80%, and more preferably is 50%.

In some embodiments, the method comprises the following steps:

a) Using benzylamine Linker type Resin as solid phase synthesis carrier, coupling protective amino acid Fmoc-Asp-PG to obtain initial peptide Resin Fmoc-Asp (-RINK AMIDE-AM Resin) -OAll;

b) Fmoc-solid phase synthesis method is adopted to sequentially couple Fmoc-Ala-OH and Fmoc-Ala-OH to obtain a first peptide Resin, namely Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -OAll;

c) Removing the protecting group PG at the carboxyl end of the Asp on the Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -PG, adding H-Pab-OH for coupling to obtain a second peptide Resin, namely 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 invention has the beneficial effects that the invention discloses a solid phase synthesis method of an antibody coupling drug linker (Fmoc-Ala-Ala-Asn-Pab/H-Ala-Ala-Asn-Pab), the synthesis process condition is mild, the product yield is high, 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%, the purity requirement of medical products is met, and the invention is suitable for industrial mass production.

Drawings

FIG. 1 shows the synthetic route of Fmoc-Ala-Ala-Asn-Pab/H-Ala-Ala-Asn-Pab synthesized according to the present invention;

FIG. 2 shows an HPLC chromatogram of the synthesized H-Ala-Ala-Asn-Pab of the present invention;

FIG. 3 shows the LC-MS spectrum of the H-Ala-Ala-Asn-Pab synthesized according to the invention;

FIG. 4 shows an HPLC chromatogram of Fmoc-Ala-Ala-Asn-Pab synthesized according to the present invention;

FIG. 5 shows the LC-MS spectrum of Fmoc-Ala-Ala-Asn-Pab synthesized according to the present invention.

Detailed Description

The invention provides a solid phase synthesis method of an antibody coupled drug linker. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

Unless otherwise specified, the test materials adopted in the invention are all common commercial products and can be purchased in the market.

In the present invention, the names and english abbreviations of the reagents and instruments used are shown in table 1.

Table 1 reagents and apparatus

The invention is further illustrated by the following examples:

EXAMPLE 1 Synthesis of starting peptide Resin Fmoc-Asp (-RINK AMIDE-AM Resin) -OAll

RINK AMIDE-AM Resin swelling to a solid phase reactor, 16.1g Rink Amide-AM Resin (degree of substitution: 0.62mmol/g;10 mmol) and 150mL DMF were added. The jacket cold/hot medium switch of the solid phase reactor is opened, the temperature is adjusted to be within T=30+/-3 ℃, and swelling is carried out for 1-2h. After swelling, the resin was suction filtered and washed 2 times with 150ml of LDMF, each time with suction.

Deprotection 150mL of deprotected solution (20% pip/80% dmf) was added to the solid phase reactor, the nitrogen valve and solid phase reactor jacket cold/hot medium switch were opened, the temperature was adjusted to T-in = 30 ± 3 ℃ and the reaction was carried out for 0.5h. After the reaction was completed, the mixture was suction-filtered and washed with 150mL of DMF 6 times, each time with suction. Sampling for NT detection, and ending the deprotection reaction after the NT detection is positive.

Pre-activation to the activation flask was added 100mL DMF,11.9g Fmoc-Asp-OAll (30 mmol) and 4.2g HOBt (30 mmol) and the solution was stirred. The temperature was controlled in a cold water bath to T-internal=5±5 ℃, 4.7ml DIC (30 mmol) was added and activated with slow stirring for 10±5min.

And (3) coupling, namely adding the activation solution into a solid phase reactor, washing an activation bottle by using 50mL of DMF, adding the activation bottle into the solid phase reactor, opening a nitrogen valve and a jacket cold/heat medium switch of the solid phase reactor, adjusting the temperature to be within T=30+/-3 ℃, and reacting for 2 hours. After the reaction, the mixture was filtered off with suction and washed 4 times with 150mL of DMF, each time with suction. Sampling for NT detection, wherein the NT detection is negative, and the coupling reaction is finished to obtain Fmoc-Asp (-RINK AMIDE-AM Resin) -OAll.

Example 2 Fmoc-Ala-OH coupling

Deprotection 150mL of deprotected solution (20% PIP/80% DMF) was added to the solid phase reactor, the nitrogen valve and solid phase reactor jacket cold/hot medium switch were opened, the temperature was adjusted to T-in = 30 ± 3 ℃ and the reaction was carried out for 0.5h. After the reaction was completed, the mixture was suction-filtered and washed with 150mL of DMF 6 times, each time with suction. Sampling for NT detection, and ending the deprotection reaction after the NT detection is positive.

Pre-activation, adding 100mL DMF,9.4g Fmoc-Ala-OH (30 mmol) and 4.2g HOBt (30 mmol) to the activation flask, stirring to dissolve. The temperature was controlled in a cold water bath to T-internal=5±5 ℃, 4.7ml DIC (30 mmol) was added and activated with slow stirring for 10±5min.

And (3) coupling, namely adding the activation solution into a solid phase reactor, taking a 50mLDMF washing activation bottle, adding the activation bottle into the solid phase reactor, opening a nitrogen valve and a jacket cold/heat medium switch of the solid phase reactor, regulating the temperature to be within T=30+/-3 ℃, and reacting for 2 hours. After the reaction, the mixture was filtered off with suction and washed 4 times with 150mL of DMF, each time with suction. Sampling for NT detection, and after the coupling reaction, fmoc-Ala-Asp (-RINK AMIDE-AM Resin) -OAll is obtained

Example 3 Fmoc-Ala-OH coupling

Deprotection 150mL of deprotected solution (20% PIP/80% DMF) was added to the solid phase reactor, the nitrogen valve and solid phase reactor jacket cold/hot medium switch were opened, the temperature was adjusted to T-in = 30 ± 3 ℃ and the reaction was carried out for 0.5h. After the reaction was completed, the mixture was suction-filtered and washed with 150mL of DMF 6 times, each time with suction. Sampling for NT detection, and ending the deprotection reaction after the NT detection is positive.

Pre-activation, adding 100mL DMF,9.4g Fmoc-Ala-OH (30 mmol) and 4.2g HOBt (30 mmol) to the activation flask, stirring to dissolve. The temperature was controlled in a cold water bath to T-internal=5±5 ℃, 4.7ml DIC (30 mmol) was added and activated with slow stirring for 10±5min.

And (3) coupling, namely adding the activation solution into a solid phase reactor, taking a 50mLDMF washing activation bottle, adding the activation bottle into the solid phase reactor, opening a nitrogen valve and a jacket cold/heat medium switch of the solid phase reactor, regulating the temperature to be within T=30+/-3 ℃, and reacting for 2 hours. After the reaction, the mixture was filtered off with suction and washed with 150mL of DCM 4 times each time with suction and washed with 150mL of DCM 2 times each time with suction. Sampling for NT detection, wherein the NT detection is negative, and the coupling reaction is finished to obtain Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -OAll.

EXAMPLE 4 coupling H-Pab-OH

Deprotection 150mL of deprotected solution (5.8 g Pd (PPh 3) 4 (5 mmol) +5.4g Ph-SiH3 (50 mmol)/150 mL DCM) was added to the solid phase reactor, the nitrogen valve and solid phase reactor jacket cold/hot media switch were opened, the temperature was adjusted to Tinternal=30.+ -. 3 ℃ and the reaction was carried out for 1.0h. After the reaction was completed, the mixture was filtered off with suction, and washed 6 times with 150mL of DCM, each time with suction.

Coupling 100mL of DMF,3.7g of H-Pab-OH (30 mmol) and 7.4gEEDQ (30 mmol) were added to the activated flask and the solution was stirred. The activation solution is added into a solid phase reactor, another 50mL DMF is taken for washing the activation bottle, the activation bottle is added into the solid phase reactor, a nitrogen valve and a jacket cold/heat medium switch of the solid phase reactor are opened, the temperature is regulated to be within T=30+/-3 ℃, and the reaction is carried out for 5 hours. After the reaction, the mixture was filtered off with suction and washed 4 times with 150mL of DMF, each time with suction. After the coupling reaction, fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -Pab was obtained.

Example 5 peptide Resin Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -Pab sample collection

Washing the peptide resin in the solid phase reactor, washing the peptide resin with 150ml MTBE 2 times and draining each time, washing the peptide resin with 150ml DCM 2 times and draining each time, and washing the peptide resin with 150ml MTBE 2 times and draining each time.

And (3) drying, namely, blowing nitrogen, primarily drying the peptide resin, transferring the peptide resin out of the solid phase reactor, and starting vacuum drying in a vacuum oven with the temperature of 30+/-3 ℃ and the pressure of less than or equal to-0.09 Mpa. After drying, fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -Pab 22.1g was obtained in 100.9% yield.

EXAMPLE 6 preparation of H-Ala-Ala-Asn-Pab

Swelling of peptide Resin to a solid phase reactor, 11.0gFmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -Pab peptide Resin obtained in example 5 and 100mL DMF were added. The jacket cold/hot medium switch of the solid phase reactor is opened, the temperature is adjusted to be within T=30+/-3 ℃, and swelling is carried out for 1-2h. After swelling, the resin was suction filtered and washed 2 times with 100mL DMF, each time with suction.

Deprotection 100mL of deprotection solution (20% PIP/80% DMF) was added to the solid phase reactor, the nitrogen valve and solid phase reactor jacket cold/hot medium switch were opened, the temperature was adjusted to T-in = 30 ± 3 ℃ and the reaction was performed for 0.5h. After the reaction was completed, the mixture was suction-filtered and washed with 100mL of DMF 6 times, each time with suction.

Washing the peptide resin in the solid phase reactor, washing the peptide resin with 100mL MTBE 2 times and draining each time, washing the peptide resin with 100mL DCM 2 times and draining each time, and washing the peptide resin with 100mLMTBE times and draining each time.

And (3) drying, namely, blowing nitrogen, primarily drying the peptide resin, transferring the peptide resin out of the solid phase reactor, and starting vacuum drying in a vacuum oven with the temperature of 30+/-3 ℃ and the pressure of less than or equal to-0.09 Mpa. After drying, 10.1g of H-Ala-Ala-Asp (-RinkAmide-AM Resin) -Pab was obtained.

Cleavage, taking 100mL of a cleavage solution (50% TFAin DCM), adding into a cleavage reactor, and precooling to about T-in=2-8 ℃. 10.0g of peptide resin was slowly added to the lysate, the temperature was controlled to T-in = 30 ± 3 ℃, and the lysate was allowed to lyse for 2-3h. After completion of the cleavage reaction, the mixture was filtered and the filter cake was washed 2 times with 50mL DCM and dried. Combining the filtrates, concentrating under reduced pressure, pulping the concentrated residue with 100mLMTBE, filtering to obtain H-Ala-Ala-Asn-Pab as white powder, weighing 1.75g, total yield 92.5%, HPLC purity 97.93%

The HPLC spectrum of H-Ala-Ala-Asn-Pab is shown in FIG. 2, and the LC-MS spectrum is shown in FIG. 3.

EXAMPLE 7 preparation of Fmoc-Ala-Ala-Asn-Pab

Cleavage 100mL of the cleavage solution (50% TFAin DCM) was measured and added to the cleavage reactor, and precooled to about T _{Inner part} =2-8 ℃. 10.0g Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -Pab peptide Resin obtained in example 5 was slowly added to the lysate and the temperature was controlled to within T = 30.+ -. 3 ℃ and the lysate was cleaved for 2-3h. After completion of the cleavage reaction, the mixture was filtered and the filter cake was washed 2 times with 50mL DCM and dried. The filtrates were combined, concentrated under reduced pressure, and the concentrated residue was slurried with 100mL MTBE and filtered to give Fmoc-Ala-Ala-Asn-Pab as a white powder weighing 2.82g with a total yield of 93.7% and HPLC purity of 98.10%

The HPLC spectrum of Fmoc-Ala-Ala-Asn-Pab is shown in FIG. 4 and the LC-MS spectrum is shown in FIG. 5.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. A method of solid phase synthesis of an antibody-conjugated drug linker, comprising:

(1) Coupling Asp, ala and protection amino acid of Ala one by one on amino resin to obtain a first peptide resin, wherein the N end of Asp and the C end of a main chain are respectively coupled with a protection group;

(2) Removing the protecting group of the Asp carboxyl end on the first peptide resin, and coupling Pab to obtain a second peptide resin;

(3) Cleaving the second peptide resin to obtain Fmoc-Ala-Ala-Asn-Pab or H-Ala-Ala-Asn-Pab;

the cracking liquid used in the step (3) is methylene chloride solution of trifluoroethanol.

2. The solid phase synthesis method according to claim 1, wherein the backbone carboxyl group of Asp is coupled with a protecting group, which is OAll or OBzl.

3. A solid phase synthesis method according to claim 1 or 2, wherein,

The protecting amino acid of the Asp is Fmoc-Asp-OAll;

The protected amino acid of Ala is Fmoc-Ala-OH, and Asp is converted to Asn during the cleavage.

4. The method according to claim 1, wherein the amino Resin comprises a benzylamine Linker type Resin, wherein the benzylamine Linker type Resin is RINK AMIDE-AM Resin, MBHA Resin or Sieber Resin, and the substitution degree of RINK AMIDE-AM Resin is 0.5-1.0mmol/g.

5. The solid phase synthesis method according to claim 1, wherein the coupled reaction medium is one or more of dichloromethane, chloroform, tetrahydrofuran, N-dimethylformamide, N-diethylformamide, N-methylpyrrolidone and dimethylsulfoxide.

6. The solid phase synthesis method according to claim 1, wherein the coupling and deprotection temperatures are 25-45 ℃.

7. The solid phase synthesis method according to claim 1, wherein the protecting group deprotection agent is piperidine in N, N-dimethylformamide or in dichloromethane containing Pd (PPh ₃)₄ and Ph-SiH ₃).

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

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

10. A method of solid phase synthesis of an antibody-conjugated drug linker, comprising:

a) Using benzylamine Linker type Resin as solid phase synthesis carrier, coupling protective amino acid Fmoc-Asp-PG to obtain initial peptide Resin Fmoc-Asp (-RINK AMIDE-AM Resin) -OAll;

b) Fmoc-solid phase synthesis method is adopted to sequentially couple Fmoc-Ala-OH and Fmoc-Ala-OH to obtain a first peptide Resin, namely Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -OAll;

c) Removing the protecting group PG at the carboxyl end of the Asp on the Fmoc-Ala-Ala-Asp (-RINK AMIDE-AM Resin) -PG, adding H-Pab-OH for coupling to obtain a second peptide Resin, namely 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 cracking liquid for cracking is methylene dichloride solution of trifluoroethanol.