WO2022051963A1 - Method for preparing plecanatide - Google Patents

Abstract

A method for preparing plecanatide. The method comprises the following steps: 1) coupling a Y resin with X, Linker and Fmoc-Leu-OH, respectively, to obtain a Fmoc-Leu-Linker-X-Y resin, wherein X is selected from an amino acid or Pro having an N-terminal H protected by Dmb/Hmb/Tmb; 2) successively linking corresponding protecting amino acids or fragments from a sequence to the Fmoc-Leu-Linker-X-Y resin by means of a solid-phase coupling synthesis method, to prepare a plecanatide linear peptide resin; and 3) cracking the plecanatide linear peptide resin to obtain a linear plecanatide, and subjecting same to cyclization, to obtain plecanatide. By using the Fmoc-Leu-Linker-X-Y resin in the preparation of plecanatide, the phenomenon of polycondensation in a synthesis process is effectively inhibited.

Description

A kind of preparation method of plecanatide technical field

The invention relates to the field of polypeptide drug preparation, in particular to a preparation method of plecanatide.

Background technique

Plecanatide, developed by Synergy Pharmaceuticals in the United States, is an analog of uroguanylin, a cyclic polypeptide containing 16 amino acids, and a guanylate cyclase receptor agonist that promotes natriuresis. It can regulate the acid-base ions in the gastrointestinal tract, induce liquid transport into the gastrointestinal tract, increase the peristalsis of the gastrointestinal tract, and is suitable for the treatment of adult chronic idiopathic constipation. The U.S. Food and Drug Administration (FDA) approved it on January 19, 2017 under the trade name Trulance. Its structural formula is as follows:

At present, the synthesis methods of plecanatide main chain are mainly divided into fragment method and solid-phase synthesis method. Patent (CN104628827A), adopts solid-phase coupling to synthesize peptide resin, then adopts solid-phase oxidation on the resin to obtain plecanatide fully protected peptide resin, and obtains plecanatide crude product after cleavage. This method adopts solid-phase oxidation to cyclize two pairs of disulfide bonds, which has poor oxidation effect, low crude product purity and great difficulty in industrial production. The patent (CN201280021221) adopts the liquid phase fragment method to synthesize plecanatide, which has long synthesis cycle, complicated operation steps and high cost, which is not conducive to commercial production. Patent (CN103694320B), using tBu or Acm as the side chain protecting group of cysteine to obtain plecanatide linear peptide resin, and cleavage to obtain plecanatide linear crude peptide; take plecanatide linear crude peptide, The crude plecanatide is obtained through the first cyclization and the second cyclization. This process cannot avoid the shrinkage of the peptide resin during the coupling process, which makes the subsequent amino acid coupling of the linear peptide difficult, and the crude product after oxidation is low in purity and difficult to purify. In the actual production process, the conventional chemical synthesis method, due to the hydrophobicity of the 6-16 amino acids of the plecanatide sequence, is easy to form a β-sheet conformation, which will cause the amino group to be wrapped in the folded peptide sequence and not easily exposed. The shrinkage phenomenon was most obvious when the 9th amino acid Asn was coupled, which made the coupling of subsequent amino acids more difficult, resulting in incomplete coupling of the peptide resin, more impurities, and difficulty in purification, resulting in low sample purity.

SUMMARY OF THE INVENTION

Aiming at the problem of difficulty in synthesis caused by resin polycondensation in the synthesis process of plecanatide, the present invention aims to provide a preparation method of plecanatide. The invention has stable process, low impurity content and high yield, and is suitable for for mass production.

The preparation method of plecanatide provided by the present invention comprises the following steps:

(1) Y resin is coupled with X, Linker, Fmoc-Leu-OH, respectively, to obtain Fmoc-Leu-Linker-X-Y resin, wherein X is selected from the amino acid or Pro whose N-terminal H is protected by Dmb/Hmb/Hnb;

(2) on the Fmoc-Leu-Linker-X-Y resin, the corresponding protected amino acids or fragments in the sequence are successively accessed by the solid-phase coupling synthesis method to obtain the plecanatide linear peptide resin;

(3) The plecanatide linear peptide resin is cleaved to obtain linear plecanatide, which is cyclized to obtain plecanatide.

By adding Linker and X between the resin and the target peptide sequence, on the one hand, the spatial distance between the target peptide sequence and the resin is increased, and the spatial structure during the coupling process of the peptide sequence is changed; on the other hand, the N-terminal H is determined by Dmb/Hmb The addition of /Tmb-protected amino acids or Pro inhibits the formation of intermolecular hydrogen bonds, thereby inhibiting the formation of β-sheets, reducing the difficulty of coupling and greatly reducing the generation of impurities.

Preferably, the Y resin is: MBHA resin or AM resin. The above resins are commonly used resins for solid-phase synthesis and can be obtained commercially. Using the above resins, the peptide sequence coupling ends the acidolysis step, and the linker and X can be retained on the resin without affecting the quality of the target compound.

Preferably, the Linker is: HMP, and this structure can ensure that after acid hydrolysis of the peptide resin, the C-terminal of the peptide sequence is a carboxylic acid, and does not affect the carbon-terminal structure of plecanatide.

As a preferred solution, the amino acids described in step (1) are selected from Leu, Val, Ala, and Gly. These amino acids have simple structures and are all monoprotected amino acids, with small steric hindrance and improved coupling efficiency.

In some specific embodiments, the plecanatide linear peptide resin described in step (2) is:

R1-Asn(Trt)-Asp(OtBu)-Glu(OtBu)-Cys(R2)-Glu(OtBu)-Leu-Cys(R3)-Val-Asn(Trt)-Val-Ala-Cys(R2)- Thr(tBu)-Gly-Cys(R3)-Leu-HMP-XY resin, wherein R1 is Fmoc or Boc; R2 is Trt, and R3 is Acm.

Preferably, the peptide resin cracking system in step (3) is a mixed solution of TFA, m-cresol, TIS and Mpr.

Preferably, the cyclization in step (3) is completed by two-step cyclization, the first cyclization adopts air (O ₂ ) oxidation or hydrogen peroxide (H ₂ O ₂ ) oxidation; the second step cyclization adopts I ₂ oxidation. Through the two-step cyclization, without purification in the intermediate process, a product with high purity and yield and accurate positioning of the disulfide bond can be obtained, and the reaction conditions are mild, which is favorable for industrial production.

In the present invention, the Fmoc-Leu-Linker-XY resin is used in the preparation of plecanatide, and the condensation polymerization phenomenon in the synthesis process is effectively suppressed. The yield of the prepared plecanatide is greater than 50%, and the purity is greater than 99.5%. Impurities are less than 0.1%. Compared with the prior art, the process of the invention reduces the synthesis difficulty of plecanatide, improves the product quality, and has great production value.

Description of drawings

FIG. 1 is a chromatogram of the plecanatide peptide prepared in Example 20. FIG.

FIG. 2 is a chromatogram of the plecanatide peptide prepared in Comparative Example 1. FIG.

FIG. 3 is a chromatogram of the plecanatide peptide prepared in Comparative Example 2. FIG.

detailed description

Example 1: Preparation of Fmoc-Leu-HMP-Gly(Dmb)-MBHA resin

20.0 g of MBHA resin (0.50 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Gly(Dmb)-OH: 8.7g, HOBT: 4.5g, DIC: 5.2g were dissolved in 50ml DMF, added to the above solid phase reaction column and reacted for 3.0h, the reaction was over, and washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5min and 15min respectively, and then HMP linker and Fmoc-leu-OH were coupled in turn to obtain Fmoc-Leu-HMP-Gly(Dmb)-MBHA resin.

Example 2; Preparation of Fmoc-Leu-HMP-Gly(Hmb)-AM resin

10.0 g AM resin (1.0 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Gly(Hmb)-OH: 8.7g, HOBT: 4.1g, DIC: 5.1g were dissolved in 50ml DMF, added to the above solid-phase reaction column and reacted for 2.5h, the reaction was completed, and washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5min and 15min respectively, followed by coupling HMP linker and Fmoc-leu-OH to obtain Fmoc-Leu-HMP-Gly(Hmb)-AM resin.

Example 3: Preparation of Fmoc-Leu-HMP-Leu(Tmb)-MBHA resin

13.5 g of MBHA resin (0.75 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Leu(Tmb)-OH: 12.6g, HOBT: 4.5g, DIC: 5.2g were dissolved in 50ml DMF, added to the above solid-phase reaction column and reacted for 3.0h, the reaction was over, and washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5min and 15min, respectively, followed by coupling HMP linker and Fmoc-leu-OH to obtain Fmoc-Leu-HMP-Leu(Tmb)-MBHA resin.

Example 4: Preparation of Fmoc-Leu-HMP-Leu(Hmb)-MBHA resin

25.0 g of MBHA resin (0.40 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Leu(Hmb)-OH: 11.5g, HOBT: 4.5g, DIC: 5.2g were dissolved in 50ml DMF, added to the above solid phase reaction column and reacted for 3.0h, the reaction was over, and washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5 min and 15 min respectively, followed by coupling HMP linker and Fmoc-leu-OH to obtain Fmoc-Leu-HMP-Leu(Hmb)-MBHA resin.

Example 5: Preparation of Fmoc-Leu-HMP-Val(Dmb)-AM resin

10.0 g AM resin (1.0 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Val(Dmb)-OH: 11.9g, HOBT: 4.1g, DIC: 5.1g were dissolved in 50ml DMF, added to the above solid phase reaction column for 2.5h reaction, the reaction was over, and washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5 min and 15 min, respectively, and then HMP linker and Fmoc-leu-OH were coupled in turn to obtain Fmoc-Leu-HMP-Val(Dmb)-AM resin.

Example 6: Preparation of Fmoc-Leu-HMP-Ala(Tmb)-AM resin

15.0 g of AM resin (0.65 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Ala(Tmb)-OH: 10.2g, HOBT: 4.1g, DIC: 5.1g were dissolved in 50ml DMF, added to the above solid-phase reaction column and reacted for 2.5h, after the reaction was completed, washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5 min and 15 min, respectively, followed by coupling HMP linker and Fmoc-leu-OH to obtain Fmoc-Leu-HMP-Ala(Tmb)-AM resin.

Example 7: Preparation of Fmoc-Leu-HMP-Pro-MBHA resin

18.0 g of MBHA resin (0.55 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Pro-OH: 7.5g, HOBT: 4.5g, DIC: 5.2g were dissolved in 50ml DMF, added to the above solid phase reaction column and reacted for 3.0h, the reaction was over, washed 4 times with DMF. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5 min and 15 min, respectively, and then HMP linker and Fmoc-leu-OH were coupled in turn to obtain Fmoc-Leu-HMP-Pro-MBHA resin.

Example 8: Preparation of plecanatide resin 1

The Fmoc-Leu-HMP Linker-Gly(Dmb)-MBHA resin in Example 1 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected with ninhydrin. The color of the resin indicated that Fmoc was completely removed, Dissolve Fmoc-Cys(Acm)-OH: 8.3g and HOBt 4.9g in 50ml DMF, add DIC 5.9g to activate for 3-5 minutes, add the activated solution to the above solid-phase reaction column, stir with nitrogen for reaction 2 -4h, the reaction ends when the ninhydrin test is negative.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH were coupled to obtain 42.5 g of plecanatide linear peptide resin.

Example 9: Preparation of plecanatide resin 2

The Fmoc-Leu-HMP-Gly(Hmb)-AM resin in Example 2 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Fmoc-Cys(Acm)-OH: 8.8g and HOBt 4.7g are dissolved in 45ml DMF, add DIC 5.9g to activate for 3-5 minutes, add the activated solution to the above solid phase reaction column, stir with nitrogen to react for 2- 4h, the reaction ended when the ninhydrin test was negative.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH were coupled to obtain 39.5 g of plecanatide linear peptide resin.

Example 10: Preparation of plecanatide resin 3

Preparation of Fmoc-Val-Ala-OH:

50 g of CTC resin (1.0 mmol/g) was loaded into the solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Ala-OH: 30.5g, DIEA: 17.8g were dissolved in 500ml DMF, added to the solid phase reaction column for 4.0h reaction, the reaction was over, washed 4 times with DMF, and added methanol: 50ml, DIEA: 8.9g , end-capping reaction 1.0h. Then, 20% piperidine/DMF was used for deprotection twice, and the deprotection time was 5 min and 15 min respectively, and the coupling of Fmoc-Val-OH was continued to obtain 76.3 g of Fmoc-Val-Ala-CTC resin.

The above-mentioned Fmoc-Val-Ala-CTC resin was cleaved with 20% TFE/DCM for 2.0 h, the cracking was completed, the resin was filtered, and the filtrate was concentrated to obtain 24.1 g of Fmoc-Val-Ala-OH weight.

Preparation of Fmoc-Cys(Trt)-Glu(OtBu)-OH:

70 g of CTC resin (0.7 mmol/g) was loaded into the solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Glu(OtBu)-OH: 46.5g, DIEA: 18.8g were dissolved in 500ml DMF, added to the solid phase reaction column for 4.0h reaction, the reaction was over, washed 4 times with DMF, added methanol: 50ml, DIEA : 9.1g, end-capping reaction for 1.0h. After deprotection with 20% piperidine/DMF twice, the deprotection time was 5min and 15min respectively, and the coupling of Fmoc-Cys(Trt)-OH was continued to obtain Fmoc-Cys(Trt)-Glu(OtBu)-CTC resin 95.1 g.

The above Fmoc-Cys(Trt)-Glu(OtBu)-CTC resin was cracked with 20% TFE/DCM for 2.0h, the cracking was completed, the resin was filtered, and the filtrate was concentrated to obtain the weight of Fmoc-Cys(Trt)-Glu(OtBu)-OH 41.6g.

The Fmoc-Leu-HMP-Leu(Tmb)-MBHA resin in Example 3 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Fmoc-Cys(Acm)-OH: 7.3g and HOBt 6.7g were dissolved in 45ml DMF, and DIC 8.1g was added to activate for 5 minutes. The reaction ended when the ninhydrin test was negative.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Val-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-Glu(OtBu)-OH, Fmoc-Glu(OtBu) -OH, Fmoc-Asp(OtBu)-OH, Boc-Asn(Trt)-OH were coupled to obtain 39.5 g of plecanatide linear peptide resin.

Example 11: Preparation of plecanatide resin 4

Preparation of Fmoc-Leu-Cys(Acm)-Val-OH:

50 g of CTC resin (1.0 mmol/g) was loaded into the solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Val-OH: 33.9g, DIEA: 19.5g were dissolved in 500ml DMF, added to the above solid-phase reaction column to react for 4.0h, the reaction was over, washed 4 times with DMF, and added methanol: 50ml, DIEA: 8.1g , end-capping reaction 1.0h. After deprotection with 20% piperidine/DMF twice, the deprotection time was 5min and 15min respectively, and the coupling of Fmoc-Cys(Acm)-OH and Fmoc-Leu-OH was continued to obtain Fmoc-Leu-Cys(Acm)- Val-CTC resin 91.1 g.

The above Fmoc-Leu-Cys(Acm)-Val-CTC resin was cracked with 20% TFE/DCM for 2.0h, the cracking was completed, the resin was filtered, and the filtrate was concentrated to obtain Fmoc-Leu-Cys(Acm)-Val-OH weight 38.3g .

Preparation of Fmoc-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-OH:

80g of CTC resin (0.6mmol/g) was loaded into the solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Fmoc-Glu(OtBu)-OH: 33.5g, DIEA: 16.3g were dissolved in 500ml DMF, added to the solid phase reaction column for 4.0h reaction, the reaction was over, washed 4 times with DMF, added methanol: 50ml, DIEA : 10.3g, end-capping reaction for 1.0h. After deprotection with 20% piperidine/DMF twice, the deprotection time was 5min and 15min, respectively, and the coupling of Fmoc-Cys(Trt)-OH and Fmoc-Glu(OtBu)-OH was continued to obtain Fmoc-Glu(OtBu) -Cys(Trt)-Glu(OtBu)-CTC resin 110.5 g.

The above Fmoc-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-CTC resin was cracked with 20% TFE/DCM for 2.0h, the cracking was completed, the resin was filtered, and the filtrate was concentrated to obtain Fmoc-Glu(OtBu)-Cys( Trt)-Glu(OtBu)-OH weight 53.8 g.

The Fmoc-Leu-Linker-Val(Dmb)-AM resin in Example 5 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Fmoc-Cys(Acm)-OH: 8.4g and HOBt: 5.2g were dissolved in 45ml DMF, DIC: 5.7g was added to activate for 5 minutes, the activated solution was added to the above solid phase reaction column, and nitrogen was stirred to react for 2- 4h, the reaction ended when the ninhydrin test was negative.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-Cys(Acm)-Val-OH, Fmoc-Glu(OtBu)-Cys(Trt)-Glu(OtBu)-OH, Fmoc-Asp(OtBu )-OH and Fmoc-Asn(Trt)-OH were coupled to obtain 45.3 g of plecanatide peptide resin.

Example 12: Preparation of Plecanatide Resin 5

The Fmoc-Leu-HMP-Ala(Tmb)-AM resin in Example 6 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Dissolve Fmoc-Cys(Acm)-OH: 9.3g and HOBt: 5.7g in 45ml DMF, add DIC: 6.3g to activate for 5 minutes, add the activated solution to the above solid phase reaction column, stir with nitrogen to react 2- 4h, the reaction ended when the ninhydrin test was negative.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH were coupled to obtain 43.1 g of plecanatide peptide resin.

Example 13: Preparation of Plecanatide Resin 6

The Fmoc-Leu-HMP-Pro-MBHA resin in Example 7 was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. (Acm)-OH: 7.5g and HOBt: 4.3g were dissolved in 48ml DMF, DIC: 5.4g was added to activate for 5 minutes, the activated solution was added to the above solid-phase reaction column, and the reaction was stirred for 2-4h with nitrogen. The ninhydrin test was negative, indicating the end of the reaction.

Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH were coupled to obtain 42.8 g of plecanatide peptide resin.

Example 14: Preparation of crude plecanatide peptide 1

Using 42 g of plecanatide resin obtained in Example 8, add a lysate (amount of 10 ml/gram of plecanatide resin) with a volume ratio of TFA/TIS/MPR/m-Cresol=92.5/2.5/2.5/2.5, and stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was drained to obtain an off-white powder, which was dried under vacuum to constant weight to obtain 16.9 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 86.4%.

Example 15: Preparation of crude plecanatide peptide 2

Take 39 g of plecanatide resin obtained in Example 9, add a lysate (amount of 10 ml/gram of plecanatide resin) with a volume ratio of TFA/TIS/MPR/m-Cresol=91/3/3/3, and stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was pumped to dryness to obtain an off-white powder, which was dried under reduced pressure to constant weight to obtain 17.1 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 81.8%.

Example 16: Preparation of crude plecanatide peptide 3

Take 39 g of plecanatide resin obtained in Example 10, add a lysate (amount of 10 ml/gram of plecanatide resin) with a volume ratio of TFA/TIS/MPR/m-Cresol=88/4/5/3, and stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was drained to obtain an off-white powder, which was dried under vacuum to constant weight to obtain 14.9 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 87.8%.

Example 17: Preparation of crude plecanatide peptide 4

Take 45 g of plecanatide resin obtained in Example 11, add a lysis solution with a volume ratio of TFA/TIS/MPR/m-Cresol=90/4/2/4 (amount of 10 ml/gram of plecanatide resin), stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was pumped to dryness to obtain an off-white powder, which was dried under reduced pressure to constant weight to obtain 17.8 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 85.6%.

Example 18: Preparation of crude plecanatide peptide 5

Take 43 g of plecanatide resin obtained in Example 12, add a lysis solution with a volume ratio of TFA/TIS/MPR/m-Cresol=90/2/2/6 (amount of 10 ml/gram of plecanatide resin), stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was pumped to dryness to obtain an off-white powder, which was dried under vacuum to constant weight to obtain 15.8 g of linear crude peptide.

Take the plecanatide linear crude peptide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 86.6%.

Example 19: Preparation of crude plecanatide peptide 6

Take 42 g of plecanatide resin obtained in Example 13, add a lysing solution with a volume ratio of TFA/TIS/MPR/m-cresol=93/2/4/1 (amount of 10 ml/gram of plecanatide resin), stir evenly , the reaction mixture was stirred at room temperature for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, the resin was washed three times with a small amount of TFA, the filtrate was combined and concentrated under reduced pressure, and anhydrous ether was added to precipitate, and then the precipitate was washed with anhydrous ether for 3 Next, the product was drained to obtain an off-white powder, which was dried under vacuum to constant weight to obtain 16.9 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 84.6%.

Example 20: Purification of crude plecanatide peptide 1

Using the crude peptide of plecanatide in Example 14, using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification to obtain 10.3 g of plecanatide refined peptide , the yield is 61.0%, the purity is 99.7%, and the maximum mono-impurity is 0.04%. The HPLC chromatogram is shown in Figure 1, and the characteristic peak retention time and peak area results are shown in Table 1.

Table 1 The retention time and peak area results of the characteristic peaks of plecanatide spermatozoa in Example 20

<Peak Table>

Detector A210nm

Peak#	Ret.Time	Area	Height	Area%	Tailing Factor	Resolution(USP)	Number of Theoretical Plate(USP)
1	10.653	3405	167	0.03	--	--	5133
2	10.992	882	80	0.01	--	0.537	4333
3	11.193	847	78	0.01	--	0.324	6103
4	11.663	1104	53	0.01	--	0.821	6670
5	12.779	6753	219	0.07	0.681	1.827	6165
6	14.064	9853914	910744	99.71	0.928	2.755	39667
7	17.267	8358	251	0.08	--	5.617	6484
8	18.027	1410	98	0.01	--	1.123	21067
9	18.231	1017	82	0.01	--	0.325	9317
10	19.425	1575	130	0.02	1.005	2.177	51756
11	20.381	3679	106	0.04	0.929	1.384	6137
Total		9882945	912008	100.00

Example 21: Purification of crude plecanatide peptide 2

Using the crude peptide of plecanatide in Example 15, using high-efficiency preparative liquid phase, Daiso C18 filler, and sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification to obtain 9.9 g of plecanatide refined peptide , the yield is 59.3%, the purity is 99.5%, and the maximum mono-impurity is 0.1%. Its HPLC chromatogram is similar to that in Figure 1.

Example 22: Purification of crude plecanatide peptide 3

Using the crude peptide of plecanatide in Example 16, using high-efficiency preparative liquid phase, Daiso C18 filler, and sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification to obtain 9.8 g of plecanatide refined peptide , the yield is 58.3%, the purity is 99.4%, and the maximum single heterogeneity is 0.07%. Its HPLC chromatogram is similar to that in Figure 1.

Example 23: Purification of crude plecanatide peptide 4

Using the crude peptide of plecanatide in Example 17, using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification to obtain 10.1 g of plecanatide refined peptide , the yield was 60.1%, the purity was 99.6%, and the maximum single heterogeneity was 0.06%, and its HPLC chromatogram was similar to that in Figure 1.

Example 24: Purification of crude plecanatide peptide 5

Using the crude peptide of plecanatide in Example 18, using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification to obtain 9.5 g of plecanatide refined peptide , the yield was 56.5%, the purity was 99.7%, and the maximum single heterogeneity was 0.04%. Its HPLC chromatogram was similar to that in Figure 1.

Example 25: Purification of crude plecanatide peptide 6

Using the crude peptide of plecanatide in Example 19, using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, the crude peptide was purified, and lyophilized after purification, 10.8 g of plecanatide refined peptide, The yield is 64.2%, the purity is 99.5%, the maximum mono-heteropoly is 0.05%, and its HPLC chromatogram is similar to that in Figure 1.

Comparative Example 1:

20.0 g of wang resin (1.0 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Dissolve Fmoc-Leu-OH: 14.12g, HOBT: 5.5g, DIC: 5.2g, DMAP: 0.48g in 50ml DMF, add it to the above solid-phase reaction column and react for 4.0-6.0h, the reaction ends, wash with DMF 4 times. Take an appropriate amount of resin for substitution value determination to obtain Fmoc-Leu-wang resin.

The above Fmoc-Leu-wang resin was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Fmoc-Cys(Acm)-OH: 7.9g and HOBt 4.1g dissolved in 50ml DMF, add DIC 5.3g to activate for 3-5 minutes, add the activated solution to the above solid phase reaction column, stir the reaction with nitrogen for 2-4h, and the ninhydrin test is negative, which is The reaction ends. Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asn(Trt)-OH were coupled to obtain 39.5 g of plecanatide linear peptide resin.

Take 30 g of the plecanatide resin obtained above, add a lysis solution with a volume ratio of TFA/TIS/MPR/m-cresol=93/2/4/1 (amount of 10ml/gram of plecanatide resin), stir evenly, room temperature The reaction was stirred for 2-4 hours, the reaction mixture was filtered with a sand core funnel, the filtrate was collected, and the resin was washed three times with a small amount of TFA. It was drained to obtain an off-white powder, which was dried in vacuo to constant weight to obtain 10.9 g of a linear crude peptide.

Take the linear crude peptide of plecanatide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 68.6%.

The above-mentioned plecanatide crude peptide was purified by using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, purified and freeze-dried to obtain plecanatide refined peptide 5.18g, with a yield of 5.18 g. 38.3%, the purity is 97.1%, and the maximum single impurity is 2.08%. Its HPLC chromatogram is shown in Figure 2, and the characteristic peak retention time and peak area results are shown in Table 2.

Table 2 The retention time and peak area results of the characteristic peaks of plecanatide spermatozoa in Comparative Example 1

<Peak Table>

Detector A210nm

Peak#	Ret.Time	Area	Height	Area%	Tailing Factor	Resolution(USP)	Number of Theoretical Plate(USP)
1	12.258	1455	155	0.03	1.092	--	35753
2	12.659	4023	350	0.07	--	1.361	23614
3	12.877	1176	130	0.02	--	0.317	2428
4	13.639	5278669	510771	97.12	0.941	1.160	41134
5	14.011	112903	10597	2.08	--	0.617	3571
6	14.468	29474	2214	0.54	--	0.706	26594
7	14.874	7450	530	0.14	--	1.067	21477
Total		5435149	524746	100.00

Comparative Example 2:

15.0 g of wang resin (0.65 mmol/g) was loaded into a solid-phase reaction column, washed twice with DCM, and swollen with DCM for 30 minutes. Dissolve Fmoc-Leu-OH: 14.12g, HOBT: 5.5g, DIC: 5.2g, DMAP: 0.48g in 50ml DMF, add it to the above solid-phase reaction column and react for 4.0-6.0h, the reaction ends, wash with DMF 4 times. Take an appropriate amount of resin for substitution value determination to obtain Fmoc-Leu-wang resin.

The above Fmoc-Leu-wang resin was used, deprotected twice with 20% piperidine/DMF, and the color of the resin was detected by ninhydrin. The color of the resin indicated that Fmoc was completely removed. Fmoc-Cys(Acm)-OH: 8.9g and HOBt 4.5g dissolved in 50ml DMF, add DIC 5.3g to activate for 3-5 minutes, add the activated solution to the above solid phase reaction column, stir the reaction with nitrogen for 2-4h, and the ninhydrin test is negative, which is The reaction ends. Repeat the above deprotection and amino acid coupling steps to complete Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ala-OH, Fmoc- Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Leu-Cys(Acm)-Val-OH prepared in Example 11, Fmoc-Glu(OtBu)-Cys(Trt)- prepared in Example 11 Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH were coupled to obtain 41.7 g of plecanatide linear peptide resin.

Take 40 g of plecanatide resin obtained above, add a lysis solution with a volume ratio of TFA/TIS/MPR/m-cresol=93/2/4/1 (amount of 10 ml/gram of plecanatide resin), stir evenly, room temperature The reaction was stirred for 2-4 hours, the reaction mixture was filtered using a sand core funnel, the filtrate was collected, and the resin was washed three times with a small amount of TFA. It was drained to obtain an off-white powder, which was dried under vacuum to constant weight to obtain 14.3 g of a linear crude peptide.

Take the plecanatide linear crude peptide obtained above, add the linear peptide to 10% acetonitrile/water solution, the linear peptide concentration is 1mg/ml, then add 2.0eq hydrogen peroxide to the solution, react for 2.0 hours, and then add to the reaction system 10% volume of acetic acid was added, iodine was added, and the reaction was continued for 20 hours. HPLC detected the end of the reaction, and the crude peptide purity of plecanatide was 73.1%.

The above-mentioned plecanatide crude peptide was purified by using high-efficiency preparative liquid phase, Daiso C18 filler, sodium dihydrogen phosphate acetonitrile purification system, purified and lyophilized to obtain plecanatide refined peptide 6.8g with a yield of 6.8 g. 40.2%, the purity is 97.69%, the maximum single impurity is 0.96%, its HPLC chromatogram is shown in Figure 3, and the characteristic peak retention time and peak area results are shown in Table 3.

Table 3 The retention time and peak area results of the characteristic peaks of plecanatide spermatozoa in Comparative Example 2

<Peak Table>

Detector A210nm

Peak#	Ret.Time	Area	Height	Area%	Tailing Factor	Resolution(USP)	Number of Theoretical Plate(USP)
1	13.644	10156945	980018	97.69	1.013	--	39655
2	13.996	46456	6332	0.45	--	--	--
3	14.537	99317	9351	0.96	--	--	43897
4	15.145	93971	4125	0.90	1.343	1.393	10361
Total		10396689	999826	100.00

The data show that the purity of the plecanatide crude peptide obtained in Examples 14-19 is higher than that of the comparative examples 1 and 2. It can be seen that the synthetic method of plecanatide provided by the present invention can improve the purity of the plecanatide crude peptide. . The HPLC chromatograms and the corresponding data in Examples 20-25 and Comparative Examples 1 and 2 show that, using the present invention to synthesize plecanatide, after simple purification, the purity and yield of the obtained plecanatide refined peptide are higher than Comparative examples 1 and 2 are high, and the maximum single impurity is also controlled. It can be seen that the synthesis method of plecanatide provided by the present invention can reduce the generation of impurities, reduce the difficulty of purification, and improve the product yield.

Claims (8)

1. A preparation method of plecanatide, characterized in that, comprising the following steps:

   (1) Y resin is coupled with X, Linker, Fmoc-Leu-OH respectively to obtain Fmoc-Leu-Linker-X-Y resin, wherein X is selected from the amino acid or Pro whose N-terminal H is protected by Dmb/Hmb/Tmb;

   (2) sequentially inserting the corresponding protected amino acids or fragments in the sequence on the Fmoc-Leu-Linker-X-Y resin by a solid-phase coupling synthesis method to obtain a plecanatide linear peptide resin;

   (3) The plecanatide linear peptide resin is cleaved to obtain linear plecanatide, which is cyclized to obtain plecanatide.
2. The preparation method of plecanatide according to claim 1, wherein the Y resin is: MBHA resin or AM resin.
3. The preparation method of plecanatide according to claim 1, wherein the Linker is: HMP.
4. The preparation method of plecanatide according to claim 1, wherein the amino acid in step (1) is selected from Leu, Val, Ala, Gly.
5. The preparation method of plecanatide according to claim 1, wherein the linear peptide resin of plecanatide in step (2) is: R1-Asn(Trt)-Asp(OtBu)-Glu(OtBu) -Cys(R2)-Glu(OtBu)-Leu-Cys(R3)-Val-Asn(Trt)-Val-Ala-Cys(R2)-Thr(tBu)-Gly-Cys(R3)-Leu-HMP- XY resin, wherein R1 is Fmoc or Boc; R2 is Trt and R3 is Acm.
6. The preparation method of plecanatide according to claim 1, wherein the peptide resin cracking system in step (3) is a mixed solution of TFA, m-cresol, TIS and Mpr.
7. The preparation method of plecanatide according to claim 1, wherein the cyclization in step (3) is completed by the first step of cyclization and the second step of cyclization.
8. The preparation method of plecanatide according to claim 6, characterized in that, in step (3), the first cyclization adopts air oxidation or hydrogen peroxide oxidation; and the second step cyclization adopts iodine oxidation.

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