Preparation method of retaglutide

Abstract

The invention provides a preparation method of retaglutide, which comprises the following steps: by adopting Sieber resin as a solid-phase carrier according to an Fmoc method, sequentially coupling protected amino acids or protected fragments on the solid-phase carrier according to the sequence of the retaglutide to obtain fully-protected peptide resin; the preparation method comprises the following steps: carrying out primary cracking on fully-protected peptide resin, cutting off resin of the fully-protected peptide resin to obtain fully-protected peptide, then carrying out secondary cracking on the fully-protected peptide, and removing a side chain protecting group of the fully-protected peptide to obtain retaglutide; wherein in the process of sequentially coupling the protected amino acids or the protected fragments, the coupling of a Gln-Aib-Ala-Phe-Ile-Glu (Ot Bu)-Tyr-Leu sequence segment is completed by adopting an 8-peptide full protection peptide fragment Fmoc-Gln (Trt)-Aib-Ala-Phe-Ile-Glu (tBu)-Leu-OH; after protected amino acids or protected fragments are sequentially coupled to Fmoc-Ile-alpha MeLeu-OH amino acids at the 13th site of the sequence, side chain protecting groups of amino acids Fmoc-Lys (Alloc)-OH at the 17th site of the sequence are removed, side chains are coupled, then Fmoc protecting groups at the tail ends are removed, and the rest protected amino acids or protected fragments are continuously coupled. By adopting the process, the production efficiency is improved, and the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to a method for preparing Retatrutide. Background Art

[0002] Retatrutide (LY-3437943) is a peptide developed by Eli Lilly and Company for weight loss and control. It is a triple hormone receptor agonist for GLP-1, GIP and GCGR receptors. The Retatrutide peptide is a 39 amino acid long peptide molecule with a 20-carbon fatty acid modification at the 17th position of Lys. The molecular formula is C 221 H 342 N 46 O 68 , with a molecular weight of 4731.4Da. Its structure is as follows Figure 1 shown.

[0003] In a Phase 2 trial, the study showed that in overweight and pre-diabetic adults, the 12 mg dose group achieved a weight loss effect of 17.5% at week 24 and 24.2% at week 48. Currently, Retatrutide is undergoing Phase 3 clinical trials and is the fastest-developed triple hormone receptor agonist.

[0004] Patent CN117024528A uses protected polypeptide fragments (Fmoc-Pro-Pro-Pro-OH, Fmoc-Ser(tBu)-Gly-OH, Fmoc-Gly-Gly-OH, Fmoc-Leu-Leu-OH, Fmoc-Gln(Trt)-Aib-OH, Fmoc-Lys(C20-OtBu-γGlu(Ot Bu)-AEEA)-OH, Fmoc-Ile-αMeLeu-OH, Fmoc-Thr(tBu)-Phe-OH or Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-OH) to replace single amino acids for coupling to obtain Retatrutide.

[0005] Patent CN117903284A uses Dde-Lys(Fmoc)-OH for coupling at the 17th position, and after coupling to the 17th amino acid in sequence from the carbon end to the nitrogen end, the Dde protecting group is selectively removed, and the remaining amino acids are coupled in sequence, wherein fragments such as Boc-Tyr(tBu)-Aib-OH; Fmoc-Ile-{α-Me-Leu}-Leu-OH; and Fmoc-Gly-Gly-OH are introduced to replace the amino acids at the corresponding positions for coupling to obtain Retatrutide.

[0006] In the methods of patents CN117024528A and CN117903284A, during the solid phase synthesis process, there is an obvious resin shrinkage phenomenon after the coupling of Glu at position 24, which makes it difficult to couple Ile at position 23. It is also very difficult to couple at positions 20 and 19 using Fmoc-Gln(Trt)-Aib-OH dipeptide, which takes several days and has low production efficiency. Moreover, in the presence of a fatty chain on the side chain of the peptide sequence, due to steric hindrance, the subsequent sequence coupling is very difficult, whether it is Fmoc-Ile-αMeLeu-OH or Fmoc-Ile-{α-Me-Leu}-Leu-OH, which takes several days and affects the quality of the crude peptide. In addition, the synthesis methods of Retatrutide provided in the prior art often have the problem of high production costs. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for preparing retaglutide.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] The invention provides a preparation method of retaglutide. The method adopts a Sieber resin as a solid phase carrier according to an Fmoc solid phase synthesis method, sequentially couples protected amino acids or protected fragments on the solid phase carrier according to the sequence order of retaglutide to obtain a fully protected peptide resin; the fully protected peptide resin is cracked once to remove the resin of the fully protected peptide resin to obtain a fully protected peptide; then the fully protected peptide is cracked twice to remove the side chain protecting group of the fully protected peptide to obtain the retaglutide; wherein, in the process of sequentially coupling the protected amino acids or protected fragments, an octapeptide fully protected peptide fragment Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-OH is used to complete the synthesis of Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu u sequence segment; then coupled to the Fmoc-Ile-αMeLeu-OH amino acid at the 13th position of the sequence through the protected amino acid or the protected fragment, the side chain protecting group of the 17th amino acid of the sequence is removed, and the side chain is coupled, and then the terminal Fmoc protecting group is removed, and the remaining protected amino acids or protected fragments are coupled; wherein the 17th amino acid adopts Fmoc-Lys(Alloc)-OH, and the side chain protecting group adopts Alloc protecting group.

[0010] Furthermore, the method specifically comprises: step 1, according to the Fmoc solid phase synthesis method, using Sieber resin as a solid phase carrier, and sequentially coupling protected amino acids or protected fragments on the solid phase carrier according to the sequence order of retaglutide to obtain a fully protected peptide resin; wherein the fully protected peptide resin is Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(C20-OtBu-γGl u(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Se r(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Resin; step 2, using reagent A to cut the peptide chain of the fully protected peptide resin to obtain a solution of the fully protected peptide, filtering to obtain a filtrate, and decompressing and concentrating the filtrate to obtain the fully protected peptide; wherein the fully protected peptide is Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(C20-OtBu-γGlu(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Ser(tBu)-Se r(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-OH; the reagent A is a DCM solution with a volume fraction of 3% to 5% TFA; step 3, adding TFA cleavage reagent to the fully protected peptide to remove the side chain protecting groups, then adding pre-cooled methyl tert-butyl ether for precipitation, filtering and vacuum drying the precipitate to obtain the retaglutide.

[0011] Furthermore, in step 1, the sequence of peptide coupling of the protected amino acids or protected fragments on the solid phase carrier is specifically: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly- OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu -OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmo c-Ile-αMeLeu-OH, C20-OtBu-γGlu(OtBu)-AEEA, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-Gly-OH and Boc-Tyr(tBu)-Aib-OH; wherein the side chain portion is C20-OtBu-γGlu(OtBu)-AEEA.

[0012] Furthermore, the step 1 is specifically as follows: using Siber resin as a solid phase carrier, swelling the resin with a solvent, and sequentially coupling protected amino acids or protected fragments on the solid phase carrier according to the sequence order of retaglutide until the reaction is terminated; after sequentially coupling from Fmoc-Ser(tBu)-OH to Fmoc-Ile-αMeLeu-OH, using tetrakistriphenylphosphine palladium / phenylsilane in the reaction solvent to remove the Alloc protecting group; then completing the coupling of the side chain part C20-OtBu-γGlu(OtBu)-AEEA, and then removing Fmoc, and continuing to sequentially couple from Fmoc-Ser(tBu)-OH to Boc-Tyr(tBu)-Aib-OH to obtain the fully protected peptide resin.

[0013] Furthermore, the Siber resin is used as a solid phase carrier, and the resin substitution degree is 0.3-0.8 mmol / g, preferably 0.5-0.6 mmol / g; optionally, the selected solvent includes one or more of DMF, NMP, and dichloromethane, preferably DMF; optionally, the amount of tetrakistriphenylphosphine palladium / phenylsilane is 0.1-0.3 times the total molar number of the resin, and the amount of phenylsilane is 10-20 eqv of the total molar number of the resin; the reaction solvent can be selected from one or more of DMF, NMP, and dichloromethane, preferably DCM; optionally, the de-Alloc protection time is 0.5-2 hours.

[0014] Further, in step 1, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 2 to 4 times the total molar number of the resin fed, preferably 3 times; optionally, the coupling agent used for the coupling is a combination of DIPCDI and compound A or a combination of DIPEA, compound A and compound B; wherein compound A is HOAt or HOBt or Oxy ma pure, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and compound A; optionally, the Fmoc removal protection agent used for removing Fmoc is a 20% piperidine / DMF solution.

[0015] Furthermore, in step 2, the dosage of the reagent A is 8-10 mL / g of fully protected peptide resin, the reaction temperature is 20-35° C., and the reaction time is 2-4 hours.

[0016] Furthermore, in step 3, the TFA cleavage reagent is a mixed solvent of TFA and one or more of EDT or DTT or DODT, TIS, and H2O, preferably a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O=91:3:3:3.

[0017] Furthermore, in step 3, the amount of the TFA cleavage reagent is 2 to 2.5 mL / g of fully protected peptide resin, the reaction temperature is 20 to 30° C., and the reaction time is 2 to 4 hours.

[0018] Furthermore, in step 3, the amount of methyl tert-butyl ether used for precipitation is 8-10 mL / g of fully protected peptide resin.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0020] The present invention provides a method for preparing retaglutide. In order to avoid the obvious resin shrinkage phenomenon after the coupling of Glu at position 24, which leads to difficulty in amino acid coupling, an 8-peptide fully protected peptide fragment Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-OH is used to complete the coupling of the contraction sequence segment (Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu). In order to avoid the problem of difficulty in coupling Fmoc-Ile-αMeLeu-OH, after coupling Fmoc-Ile-αMeLeu-OH, the Alloc protecting group of Fmoc-Lys(Alloc)-OH at position 17 is selectively removed to complete the coupling of the side chain. Finally, Fmoc is removed to complete the coupling of the remaining amino acids behind Fmoc-Ile-αMeLeu-OH. Through this process, the problem of difficult coupling sequences and sites is solved, defective impurities are reduced, and the purity of the product is improved.

[0021] The present invention adopts Siber resin as the starting solid phase carrier, sequentially completes peptide chain synthesis according to the Fmoc solid phase synthesis method, and then uses a DCM solution with a volume fraction of 3% to 5% TFA to cut the peptide chain, perform a primary cleavage, filter and remove the waste resin, and the obtained filtrate is decompressed and concentrated to a viscous solid to obtain a fully protected peptide. The obtained fully protected peptide is directly added with a TFA cleavage reagent to remove the side chain protecting group and perform a secondary cleavage. Then, methyl tert-butyl ether is added for precipitation, and the precipitate can be filtered to obtain a crude product of Retatrutide. By adopting this method, the amount of TFA can be reduced by 70%, thereby increasing the concentration of Retatrutide in the lysate, and the amount of ether required for precipitation solvent is also reduced by 90%, and the precipitated particles are easy to filter using filter cloth or filter paper. Therefore, not only the limitation of the volume of the sedimentation kettle on the cleavage batch is solved, but also the problem of solid-liquid separation of the sedimentation liquid is solved. The problem of crude peptide sedimentation filtration is solved by adopting a step-by-step cleavage method, which greatly improves production efficiency and reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structural diagram of the Retatrutide peptide;

[0023] Figure 2 It is the synthetic route diagram of Retatrutide provided by the present invention;

[0024] Figure 3 is the HPLC spectrum of Retatrutide prepared in the embodiment of the present invention;

[0025] Figure 4 It is the HPLC spectrum of Retatrutide prepared in the control example. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with specific implementation modes.

[0027] Continue to refer Figure 1 In order to overcome the problems existing in the prior art and avoid the obvious resin shrinkage phenomenon after the coupling of Glu at position 24, the 8-peptide fully protected peptide fragment Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-OH is used to complete the coupling of the contracted sequence segment (Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu, i.e., sequence positions 19 to 26). In order to avoid the problem of difficult coupling of Fmoc-Ile-αMeLeu-OH, Fmoc-Lys(Alloc)-OH is used at position 17. After the coupling of Fmoc-Ile-αMeLeu-OH, the Alloc protecting group at position 17 is selectively removed to complete the coupling of the side chain, and then Fmoc is removed to complete the coupling of the remaining amino acids behind Fmoc-Ile-αMeLeu-OH, so that the Ile defect impurities and Ile-αMeLeu are correspondingly less, and the product purity is improved. Among them, other fragments introduced include Boc-Tyr(tBu)-Aib-OH, Fmoc-Gly-Gly-OH and Fmo c-Pro-Pro-OH. The synthetic route is as follows Figure 2 shown.

[0028] In addition, the methods of patents CN117024528A and CN117903284A use solid phase carrier resins selected from Rink Amide Resin, Rink Amide AM Resin or Rink Amide MBHA. These resins require high concentrations of TFA lysate, resulting in a high dosage (8-10 mL / gram of resin for lysate), and the dosage of ether solvents is as high as 80-100 mL / gram of resin, which will bring a large amount of ether waste liquid to production. These ether waste liquids cannot be recycled by distillation because they contain a high concentration of trifluoroacetic acid. TFA has a high solubility for polypeptides. Using a large amount of TFA for lysing is not conducive to the precipitation of polypeptides. Otherwise, a large amount of ether solvents are required for precipitation, which will require a large sedimentation kettle, otherwise the lysing batch will be limited. In addition, the concentration of Retatrutide in the lysate is low, which is not conducive to the formation of larger sedimentation particles during the sedimentation process with ether solvents. The resulting sedimentation liquid is like milk and cannot be filtered with filter cloth or filter paper. It can only be separated by a cup centrifuge (the principle is to use the difference in density of Retatrutide and the density of the sedimentation reagent to separate). It is impossible to use filtration to effectively separate the crude peptide, which also greatly limits large-scale commercial production.

[0029] Based on this, the present invention adopts the Fmoc solid phase synthesis method, uses Sieber resin as a solid phase carrier, sequentially couples to obtain a fully protected peptide resin, then uses a DCM solution with a volume fraction of 3% to 5% TFA to cut the fully protected peptide, and then uses a TFA cleavage reagent to treat the fully protected peptide. After ether solvent precipitation, it is filtered and dried to obtain the Retatrutide crude peptide.

[0030] The specific steps are as follows:

[0031] Step 1: According to the Fmoc solid phase synthesis method, Sieber resin was used as a solid phase carrier to sequentially couple to obtain the fully protected peptide resin Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(C20-OtBu-γGlu(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Resin. The peptide sequence is as follows:

[0032] Table 1 Amino acid coupling process

[0033]

[0034]

[0035] Step 2: Use a DCM solution with a volume fraction of 3% to 5% TFA to remove the peptide chain to obtain the entire protective peptide Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(C20-OtBu-γGlu(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu) u)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-OH, filtered, and the filtrate was concentrated under reduced pressure to a viscous state to obtain the fully protected peptide.

[0036] Step 3: The fully protected peptide obtained in step 2 is directly added with a cleavage reagent having a mass fraction of TFA to remove the side chain protecting groups. Then, 8 to 10 mL / g of fully protected peptide resin is added to precooled methyl tert-butyl ether for precipitation, the precipitate is filtered, and the filter cake is vacuum dried to obtain the crude product of Retatrutide.

[0037] The Fmoc solid phase synthesis method described in step 1 is: using Siber resin as a solid phase carrier, the resin substitution degree is 0.3-0.8 mmol / g, preferably 0.5-0.6 mmol / g; the resin is swollen with a solvent, and the selected solvent includes DMF, NMP, dichloromethane, etc., preferably DMF; various amino acids and fragments are coupled in sequence until the reaction is terminated by the ninhydrin detection method. After the protected amino acids or fragments are coupled in the order of 1-18 in Table 1, the Alloc protecting group is removed by using tetrakistriphenylphosphine palladium / phenylsilane. The amount of tetrakistriphenylphosphine palladium / phenylsilane is 0.1-0.3 times the total molar number of the resin, the amount of phenylsilane is 10-20eqv of the total molar number of the resin, and the reaction solvent can be selected from DMF, NMP, dichloromethane, etc., preferably DCM, and the Alloc protection time is removed. Then complete the coupling of the side chain part C20-OtBu-γGlu(OtBu)-AEEA, then remove Fmoc, and continue to complete the coupling of the protected amino acids or fragments in the order of 22-30 in Table 1.

[0038] The amount of Fmoc-protected amino acid or protected amino acid fragment is 2 to 4 times, preferably 3 times, the total molar number of the resin fed; the coupling agent is a combination of DIPCDI and compound A or a combination of DIPEA, compound A and compound B, wherein compound A is HOAt or HOBt or Oxyma pure, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and compound A. The Fmoc deprotection agent is a 20% piperidine / DMF solution.

[0039] The amount of the 3% to 5% TFA DCM solution solvent in step 2 is 8 to 10 mL / g of fully protected peptide resin, the reaction temperature is 20 to 35° C., and the reaction time is 2 to 4 hours.

[0040] The cleavage reagent in step 3 is a mixed solvent of TFA and one or more of EDT or DTT or DODT, TIS, and H2O, preferably a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O=91:3:3:3. The amount of the cleavage reagent is 2-2.5 mL / g of fully protected peptide resin, the reaction temperature is 20-30°C, and the reaction time is 2-4 hours. The amount of methyl tert-butyl ether used for precipitation is 8-10 mL / g of fully protected peptide resin.

[0041] Example 1: Peptide resin synthesis

[0042] The starting amino Sieber resin (19.3g, Sub0 = 0.52mmol / g) was added to the solid phase reactor, the synthesis scale was 10mmol, an appropriate amount of DMF was added to swell for 30 minutes, the solvent was removed, and an appropriate amount of DMF was washed twice. An appropriate amount of 20% Pip / DMF solution was added to the reactor for deprotection, the deprotection temperature was 25±5°C, the deprotection time was 5+7min, DMF was washed 6 times, and Kaiser Test was positive. The feed ratio Fmoc-AA-OH (or fragment): DIC: HOBt = 1: 1.2: 1.2, an appropriate amount of DMF was added to dissolve, DIC was added to pre-activate at room temperature for 5±0.5min, and then poured into the reactor, the coupling temperature was 30±5°C, and the reaction time was 120min before starting Kaiser Test detection, which was negative. After the reaction was completed, it was washed 3 times with DMF. Coupling cycle: coupling from the carbon end to the nitrogen end in sequence, repeating the above steps, and completing the coupling of protected amino acids or fragments in the order of 1-18 in Table 1.

[0043] Add 100mL of dichloromethane and phenylsilane (10.8g, 10eqv) to the reactor, then add tetratriphenylphosphine palladium (3.5g, 0.3eqv), and remove Alloc protection for 2 hours. Then complete the coupling of the side chain part C20-OtBu-γGlu(OtBu)-AEEA (11.0g, 1.5eqv), then remove Fmoc, and continue to complete the coupling of the protected amino acids or fragments in the order of 20-28 in Table 1. The feed ratio Fmoc-AA-OH (or fragment): DIC: HOBt = 1: 1.2: 1.2, add an appropriate amount of DMF to dissolve, add DIC to pre-activate at room temperature for 5±0.5min, and then pour it into the reactor. The coupling temperature is 30±5℃, and the reaction time is 120min. Start Kaiser Test detection, negative. After the reaction is completed, wash with DMF 3 times. Coupling cycle: couple from the carbon end to the nitrogen end in sequence.

[0044] Finally, the resin was washed with DCM for 3 times, methanol for 3 times, and vacuum dried to obtain a peptide resin. The peptide resin weighed 84.7 g, and the resin weight gain rate was 105.1%.

[0045] Example 2: Preparation of crude Retatrutide

[0046] Take the peptide resin, add 850mL of 5% TFA / DCM solvent by volume, crack at 25°C for 2 hours, filter, and concentrate the filtrate under vacuum at 30°C until it becomes viscous. Add 200mL of a cracking reagent with a mass ratio of TFA:DTT:TIS:H2O=91:3:3:3 (the cracking reagent is pre-cooled to -3°C), crack at 25°C for 2 hours, then add to 800mL of methyl tert-butyl ether (pre-cooled to -5°C), filter with a Buchner funnel, and use a fast qualitative filter paper (standard number GB / T1914-2017, pore size 10-12μm). The filter cake is washed three times with methyl tert-butyl ether, each time using 100mL. The filter cake is vacuum pumped to dryness under nitrogen protection, crushed and vacuum dried for 5 hours to obtain 49.9g of crude peptide, the weight yield of crude peptide is 105.4%, the HPLC purity is 75.71%, and the HPLC spectrum is shown in Figure 3 .

[0047] Comparative Example: According to patent CN117903284A

[0048] The starting amino Sieber resin (19.3 g, Sub0 = 0.52 mmol / g) was added to a solid phase reaction kettle, the synthesis scale was 10 mmol, and an appropriate amount of DMF was added to swell for 30 minutes, the solvent was removed, and an appropriate amount of DMF was washed twice. The peptide resin was synthesized according to the method of patent CN117903284A to obtain 77.3 g of peptide resin, and the resin weight gain rate was 95.5%.

[0049] Take the peptide resin, add 800mL of 5% TFA / DCM solvent by volume, crack at 25°C for 2 hours, filter, and concentrate the filtrate under vacuum at 30°C until it becomes viscous. Add 200mL of a cracking reagent with a mass ratio of TFA:DTT:TIS:H2O=91:3:3:3 (the cracking reagent is pre-cooled to -3°C), crack at 25°C for 2 hours, then add to 800mL of methyl tert-butyl ether (pre-cooled to -5°C), filter with a Buchner funnel, and the filter paper is a rapid qualitative filter paper (standard number GB / T1914-2017, pore size 10-12μm). The filter cake is washed three times with methyl tert-butyl ether, each time with an amount of 100mL. The filter cake is vacuum pumped to dryness under nitrogen protection, crushed and vacuum dried for 5 hours to obtain 40.3g of crude peptide, the weight yield of crude peptide is 85.2%, the HPLC purity is 65.47%, and the HPLC spectrum is shown in Figure 4 .

[0050] It can be seen that the crude peptide yield and purity of the present invention are significantly improved by comparing the control example with Example 2. Moreover, compared with the cleavage and sedimentation method of patent CN117903284A, the amount of TFA and methyl tert-butyl ether used in the present invention is significantly reduced, and the precipitated particles are easy to filter, which is convenient for industrial production.

[0051] Appendix: Abbreviations and English meanings in this article

[0052]

[0053]

[0054] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A method for preparing retaglutide, characterized in that: The method uses the Fmoc solid phase synthesis method, uses Sieber resin as a solid phase carrier, and sequentially couples protected amino acids or protected fragments on the solid phase carrier according to the sequence order of retaglutide to obtain a fully protected peptide resin; The fully protected peptide resin is subjected to a primary cleavage to remove the resin of the fully protected peptide resin to obtain the fully protected peptide, and then the fully protected peptide is subjected to a secondary cleavage to remove the side chain protecting groups of the fully protected peptide to obtain the retaglutide; In the process of sequentially coupling the protected amino acids or protected fragments, the 8-peptide fully protected peptide fragment Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-OH is used to complete the coupling of the Gln-Aib-Ala-Phe-Ile-Glu-Tyr-Leu sequence segment; then, the protected amino acids or protected fragments are sequentially coupled to the Fmoc-Ile-αMeLeu-OH amino acid at the 13th position of the sequence, the side chain protecting group of the 17th amino acid of the sequence is removed, and the side chain is coupled, and then the terminal Fmoc protecting group is removed, and the remaining protected amino acids or protected fragments are coupled; wherein, the 17th amino acid adopts Fmoc-Lys(Alloc)-OH, and the side chain protecting group adopts the Alloc protecting group.

2. The method for preparing retaglutide according to claim 1, characterized in that: The method specifically includes: Step 1: According to the Fmoc solid phase synthesis method, Sieber resin is used as a solid phase carrier, and protected amino acids or protected fragments are sequentially coupled on the solid phase carrier according to the sequence order of retaglutide to obtain a fully protected peptide resin; wherein the fully protected peptide resin is Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(t Bu)-Ile-αMeLeu-Leu-Asp(OtBu)-Lys(Boc)-Lys(C20-OtBu-γGlu(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-A la-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pr o-Pro-Ser(tBu)-Sieber Resin; Step 2: Use reagent A to cut the peptide chain of the fully protected peptide resin to obtain a solution of the fully protected peptide, filter to obtain a filtrate, and reduce the pressure and concentrate the filtrate to obtain the fully protected peptide; wherein the fully protected peptide is Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-αMeLeu-Leu-Asp(OtBu)-Ly s(Boc)-Lys(C20-OtBu-γGlu(OtBu)-AEEA)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-OH; the reagent A is a DCM solution with a volume fraction of 3% to 5% TFA; Step 3: adding TFA cleavage reagent to the fully protected peptide to remove the side chain protecting groups, then adding it to pre-cooled methyl tert-butyl ether for precipitation, filtering the precipitate and vacuum drying to obtain the retaglutide.

3. The method for preparing retaglutide according to claim 2, characterized in that: In step 1, the sequence of sequentially coupling protected amino acids or protected fragments on the solid phase carrier is specifically: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly- Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-αMeLeu-OH, C20-OtBu-γGlu(OtBu)-AEEA, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH , Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-Gly-OH and Boc-Tyr(tBu)-Aib-OH; Wherein, the C20-OtBu-γGlu(OtBu)-AEEA is a side chain portion.

4. The method for preparing retaglutide according to claim 3, characterized in that: The step 1 specifically comprises: using Siber resin as a solid phase carrier, swelling the resin with a solvent, and sequentially coupling protected amino acids or protected fragments on the solid phase carrier according to the sequence order of retaglutide until the reaction is terminated; after sequentially coupling from Fmoc-Ser(tBu)-OH to Fmo c-Ile-αMeLeu-OH, using tetrakistriphenylphosphine palladium / phenylsilane in a reaction solvent to remove the Alloc protecting group; then completing the coupling of the side chain part C20-OtBu-γGlu(OtBu)-AEEA, and then removing Fmoc, and continuing to sequentially couple from Fmoc-Ser(tBu)-OH to Boc-Tyr(tBu)-Aib-OH to obtain the fully protected peptide resin.

5. The method for preparing retaglutide according to claim 3, characterized in that: The Siber resin is used as the solid phase carrier, and the resin substitution degree is 0.3-0.8 mmol / g, preferably 0.5-0.6 mmol / g; Optionally, the selected solvent includes one or more of DMF, NMP, and dichloromethane, preferably DMF; Optionally, the amount of tetrakistriphenylphosphine palladium / phenylsilane is 0.1 to 0.3 times the total mole of the resin, and the amount of phenylsilane is 10 to 20 eqv of the total mole of the resin; the reaction solvent can be selected from one or more of DMF, NMP, and dichloromethane, preferably DCM; Optionally, the Alloc protection removal time is 0.5 to 2 hours.

6. The method for preparing retaglutide according to claim 3, characterized in that: In step 1, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 2 to 4 times the total molar number of the resin fed, preferably 3 times; Optionally, the coupling agent used for the coupling is a combination of DIPCDI and compound A or a combination of DIPEA, compound A and compound B; wherein compound A is HOAt or HOBt or Oxyma pure, and compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and compound A; Optionally, the Fmoc removal protecting agent used for removing Fmoc is a 20% piperidine / DMF solution.

7. The method for preparing retaglutide according to claim 2, characterized in that: In step 2, the dosage of the reagent A is 8-10 mL / g of fully protected peptide resin, the reaction temperature is 20-35° C., and the reaction time is 2-4 hours.

8. The method for preparing retaglutide according to claim 2, characterized in that: In step 3, the TFA cleavage reagent is a mixed solvent of TFA and one or more of EDT or DTT or DODT, TIS, and H2O, preferably a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O=91:3:3:

3.

9. The method for preparing retaglutide according to claim 2, characterized in that: In step 3, the amount of the TFA cleavage reagent is 2-2.5 mL / g of fully protected peptide resin, the reaction temperature is 20-30° C., and the reaction time is 2-4 hours.

10. The method for preparing retaglutide according to claim 2, characterized in that: In step 3, the amount of methyl tert-butyl ether used for precipitation is 8-10 mL / g of fully protected peptide resin.

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