Preparation method of tilpotide

Abstract

The preparation method comprises the following steps: according to an Fmoc solid-phase synthesis method, adopting Sieber resin as a solid-phase synthesis carrier, and sequentially coupling to obtain full-protection peptide resin; cutting the fully-protected peptide resin by using a DCM solution of TFA with a volume fraction of 3-5%, filtering to remove waste resin, and concentrating the obtained filtrate into a viscous solid; and adding a cracking reagent into the viscous solid, carrying out cracking, adding an ether solvent after the cracking is finished, carrying out sedimentation, and filtering and drying a precipitate to obtain a crude product of the tilpotide. By adopting the method disclosed by the invention, the use amount of TFA can be reduced by 70%, the use amount of ether as a required settling solvent is also reduced by 90%, precipitated particles are easily filtered by using filter cloth or filter paper, the limitation of the volume of a settling kettle on cracking batches is solved, the problem of solid-liquid separation of settling liquid is solved, the production efficiency is greatly improved, and the production cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide synthesis, and specifically relates to a preparation method of tirzepatide. Background Art

[0002] Tirzepatide is an agonist that simultaneously activates two incretin receptors developed by Eli Lilly. The FDA approved it for the treatment of type 2 diabetes on May 13, 2022 (trade name Mounjaro), and approved tirzepatide for the treatment of adult obesity on November 8, 2023 (trade name Zepbound). Tirzepatide is a polypeptide molecule 39 amino acids long, with a 20-carbon fatty acid modification at Lys 20, and its molecular formula is C 225 H 348 N 48 O 68 , with a molecular weight of 4,813.53 Da. Its peptide sequence is shown as follows:

[0003]

[0004] In the head-to-head trial with semaglutide, this drug showed more excellent hypoglycemic and weight loss effects, and the market demand is strong. According to the analysis of SVB Securities, by 2030, the sales of this drug may reach $24.6 billion. Therefore, the demand for its raw material drug is huge.

[0005] The original patent CN113330024A adopted a four-fragment synthesis process, with a crude peptide purity of 70% and a total synthesis yield of 44.4%. After fragment splicing, nanofiltration technology is required to remove by-products. Nanofiltration has very high requirements for the filter membrane (200 Da and 400 Da), and domestic filter membranes cannot reach this fine level. Therefore, this technology is difficult to implement in China. The original patent CN113330024A also adopted a stepwise coupling process, using an amino resin for stepwise coupling to complete the synthesis of the 39-peptide main chain, then removing the side-chain protecting group ivDde of Lys(ivD de), and then coupling the fragment (AEEA)2-γ-Glu-C 20Diacid, with a crude peptide purity of 64% and a total synthesis yield of 47.0%. Patents CN112592387B, CN115651075B, and CN110903355A use the fragment Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-OH as the starting material or intermediate for synthesis, with a crude peptide purity between 70% and 80%. However, the dosage of the TFA cleavage solution in these patents is relatively high (8 - 10 mL / g of resin for the cleavage reagent), and the dosage of the ether solvent is as high as 80 - 100 mL / g 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 relatively high solubility in polypeptides. Using a large amount of TFA for cleavage is not conducive to the precipitation of polypeptides. Otherwise, a large amount of ether solvent is required for precipitation, which will require a large settling tank, otherwise the cleavage batch will be limited. In addition, the concentration of telotristat in the cleavage solution is relatively low. During the sedimentation process using the ether solvent, it is not conducive to the formation of sedimentation particles with a larger particle size. The obtained sedimentation liquid is like milk and cannot be filtered with a filter cloth or filter paper. Only a cup centrifuge (the principle is to separate using the density difference between telotristat and the sedimentation reagent) can be used for separation, and the crude peptide cannot be effectively separated by filtration, which also greatly limits large-scale commercial production. Summary of the Invention

[0006] To solve the above problems, the present invention aims to provide a preparation method for telotristat. The present invention uses Siber resin as the starting solid-phase carrier, sequentially completes the peptide chain synthesis according to the standard Fmoc solid-phase synthesis operation, and then uses a DCM solution with a volume fraction of 3% - 5% TFA for peptide chain cleavage (the dosage is 8 - 10 mL / g of fully protected peptide resin), filters to remove the waste resin, and concentrates the obtained filtrate under reduced pressure to a viscous solid. The obtained viscous solid is directly added to the cleavage reagent (the dosage is 2 - 2.5 mL / g of fully protected peptide resin) to remove the side-chain protecting groups. Then, it is added to the ether reagent (the dosage is 8 - 10 mL / g of fully protected peptide resin) for precipitation, and the precipitate is filtered to obtain the crude telotristat. By adopting this process, the dosage of TFA can be reduced by 70%, thereby increasing the concentration of telotristat in the cleavage solution. Further, the dosage of the ether used as the sedimentation solvent is also reduced by 90%, and the precipitated particles are easily filtered using a filter cloth or filter paper. Thus, not only the limitation of the cleavage batch by the volume of the settling tank is solved, but also the problem of solid-liquid separation of the sedimentation liquid is solved, greatly improving the production efficiency and reducing the production cost.

[0007] The specific technical solutions are as follows:

[0008] The present invention provides a preparation method for telotristat, including the following steps:

[0009] Step 1: According to the conventional Fmoc solid-phase synthesis method, using Sieber resin as the solid-phase synthesis carrier, sequentially couple to obtain the fully protected peptide resin Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Resin;

[0010] Step 2: Cut the fully protected peptide resin described in Step 1 with a DCM solution of TFA with a volume fraction of 3% - 5% to obtain a solution of the fully protected peptide Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2, filter to remove the waste resin, and concentrate the obtained filtrate to a viscous solid;

[0011] Step 3: Add a cleavage reagent to the viscous solid obtained in Step 2 for cleavage to remove the side-chain protecting groups. After the cleavage is completed, add an ether solvent for sedimentation. The precipitate is filtered and dried to obtain the crude telotristat.

[0012] Further, in the synthesis process of the peptide resin in Step 1, in addition to using conventional protected amino acids, the following fragments were also used: Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, Boc-Tyr(tBu)-Aib-OH, Fmoc-Ile-Aib-OH, and Fmoc-Gly-Gly-OH, and they were coupled in sequence according to the peptide sequence shown in the following table:

[0013]

[0014]

[0015] Further, the Fmoc conventional solid-phase synthesis method in Step 1 is as follows: Using Siber resin as the solid-phase carrier, the resin substitution degree is 0.3 - 0.8 mmol / g, preferably 0.5 - 0.6 mmol / g; swelling the resin with a solvent, and the solvent is selected from DMF, NMP, dichloromethane, etc., preferably DMF; sequentially coupling various amino acids and fragments until the reaction is detected to terminate by the ninhydrin detection method. The dosage of the Fmoc-protected amino acid or protected amino acid fragment is 2 - 4 times the total molar amount of the charged resin, preferably 3 times; the coupling agent is a composition of DIPCDI and Compound A or a composition of DIPEA, Compound A, and Compound B, where Compound A is HOAt, HOBt, or Oxymapure, and Compound B is PyAOP, PyBOP, HATU, HBTU, or TBTU, preferably a composition of DIPCDI and Compound A. The Fmoc deprotection reagent is a 20% piperidine / DMF solution.

[0016] Further, the dosage of the DCM solution of TFA with a volume fraction of 3% - 5% in Step 2 is 8 - 10 mL / g of the fully protected peptide resin;

[0017] Further, the reaction conditions for the cleavage in Step 2 are: the reaction temperature is 20 - 35 °C, and the reaction time is 2 - 4 hours.

[0018] Further, the filtrate obtained in Step 2 is concentrated to a viscous solid under vacuum.

[0019] Further, the dosage of the cleavage reagent in Step 3 is 2 - 2.5 mL / g of the fully protected peptide resin.

[0020] Further, the cleavage reagent in step 3 is a mixed solvent of one or more of EDT, DTT, DODT and TFA, TIS, H2O; preferably, the cleavage reagent is a mixed solvent of TFA, DTT, TIS, H2O; preferably, the mass ratio of TFA:DTT:TIS:H2O is 91:3:3:3.

[0021] Further, the reaction conditions for the cleavage in step 3 are: the reaction temperature is 20-30 °C, and the reaction time is 2-4 hours.

[0022] Further, the dosage of the ether reagent in step 3 is 8-10 mL / g of the fully protected peptide resin;

[0023] Preferably, the ether reagent in step 3 is methyl tert-butyl ether;

[0024] Preferably, the ether reagent in step 3 is precooled to -8 to -5 °C.

[0025] Further, the drying in step 3 is vacuum drying.

[0026] The beneficial effects of the present invention are:

[0027] The present invention uses Siber resin as the starting solid-phase carrier, and according to the conventional Fmoc solid-phase synthesis method, the fully protected peptide resin is sequentially coupled. Then, the peptide chain is cleaved with a DCM solution containing 3%-5% by volume of TFA, the waste resin is filtered off, the obtained filtrate is concentrated under reduced pressure to a viscous solid, the obtained viscous solid is directly added with a cleavage reagent to remove the side-chain protecting groups, then an ether reagent is added for precipitation, and the precipitate is filtered and dried to obtain the crude telotristat. By using the telotristat preparation process of the present invention, the dosage of TFA can be reduced by 70%, so that the concentration of telotristat in the cleavage solution is increased. Further, the dosage of the ether used as the sedimentation solvent is also reduced by 90%, and the precipitated particles are easy to filter with a filter cloth or filter paper. Finally, 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, greatly improving the production efficiency and reducing the production cost. Description of the Drawings

[0028] Figure 1 It is the HPLC chromatogram of Example 2;

[0029] Figure 2 It is the HPLC chromatogram of Example 3;

[0030] Figure 3 It is the HPLC chromatogram of Example 4;

[0031] Figure 4 It is the HPLC chromatogram of the comparative example. Detailed Embodiments

[0032] To better understand the present invention, the present invention will be further described below with reference to the following embodiments and accompanying drawings. The embodiments are only for explanation and do not limit the present invention in any way. In the embodiments, all the original reagent materials are commercially available, and the experimental methods without specific conditions are the conventional methods and conventional conditions well-known in the art, or the conditions recommended by the instrument manufacturer.

[0033]

[0034]

[0035] Example 1: Peptide resin synthesis

[0036] According to the conventional Fmoc solid-phase synthesis method, Sieber resin was used as the solid-phase synthesis carrier, and the fully protected peptide resin Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Resin was obtained by sequential coupling. In the synthesis process of the peptide resin, in addition to using conventional protected amino acids, the following fragments Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioic acid(mon-tBu))-OH, Boc-Tyr(tBu)-Aib-OH, Fmoc-Ile-Aib-OH and Fmoc-Gly-Gly-OH were also used, and the coupling was carried out in sequence according to the peptide sequence shown in Table 1 below:

[0037] Table 1

[0038]

[0039]

[0040] Specifically, the synthesis of the peptide resin includes: adding the starting amino Sieber resin (192.3 g, Sub0 = 0.52 mmol / g) into a solid-phase reactor with a synthesis scale of 100 mmol, swelling with an appropriate amount of DMF for 30 minutes, pumping out the solvent, and washing twice with an appropriate amount of DMF. Add an appropriate amount of 20% Pip / DMF solution to the reactor for deprotection, with the deprotection temperature at 25 ± 5 °C and the deprotection time of 5 + 7 min. Wash with DMF 6 times, and the Kaiser Test shows a positive result. The feeding ratio of Fmoc-AA-OH (or fragment): DIC: HOBt = 1:1.2:1.2. Dissolve with an appropriate amount of DMF, pre-activate with DIC at room temperature for 5 ± 0.5 min, then pour it into the reactor. The coupling temperature is 30 ± 5 °C, and the Kaiser Test is started after 60 min of reaction time, with a negative result as the criterion. After the reaction is completed, wash with DMF 3 times. Coupling cycle: Couple from the C-terminus to the N-terminus in sequence, and repeat the above steps until the coupling is completed. Finally, wash the resin 3 times with DCM and 3 times with methanol, and vacuum dry to obtain 788.0 g of peptide resin, with a resin weight gain rate of 96.7%.

[0041] Example 2: Preparation of crude tezepelumab

[0042] Take 100 g of the peptide resin prepared in Example 1, add 900 mL of a 5% TFA / DCM solvent by volume ratio, and cleave at 25 °C for 2 hours. Filter, and concentrate the filtrate under vacuum at 30 °C to a viscous state. Add 200 mL of a cleavage reagent with a mass ratio of TFA: DTT: TIS: H2O = 91:3:3:3 (the cleavage reagent is pre-cooled to -3 °C), cleave at 25 °C for 2 hours, then add it to 800 mL of methyl tert-butyl ether (pre-cooled to -5 °C), and filter with a Buchner funnel. The filter paper is fast qualitative filter paper (standard number GB / T1914-2017, pore size 10 - 12 μm). Wash the filter cake three times with methyl tert-butyl ether, with each amount being 100 mL. Vacuum pump the filter cake under nitrogen protection until it is cracked, crush it, and vacuum dry for 5 hours to obtain 68.0 g of crude tezepelumab, with a purity of 90.48%, a content of 70.93%, and a content yield of 79.0%. (The HPLC chromatogram is shown in the appendix Figure 1 )

[0043] Example 3: Preparation of crude tezepelumab

[0044] Take 50 g of the peptide resin prepared in Example 1, add 400 mL of a 3% (v / v) TFA / DCM solvent, and cleave at 25 °C for 3 hours. Filter, and concentrate the filtrate under vacuum at 30 °C to a viscous state. Add 125 mL of a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O = 91:3:3:3 (the cleavage reagent is pre-cooled to -5 °C), and cleave at 25 °C for 3 hours. Then add it to 500 mL of methyl tert-butyl ether (pre-cooled to -8 °C), filter using a Buchner funnel with a fast qualitative filter paper (standard number GB / T 1914-2017, pore size 10 - 12 μm). Wash the filter cake three times with methyl tert-butyl ether, with each usage of 50 mL. Vacuum pump the filter cake under nitrogen protection until it is cracked and dry, crush it, and then dry it under vacuum for 5 hours to obtain 33.6 g of crude telotristat, with a purity of 88.98%, a content of 67.11%, and a content yield of 73.9%. (The HPLC chromatogram is shown in the appendix Figure 2 )

[0045] Example 4: Preparation of crude telotristat

[0046] Take 50 g of the peptide resin prepared in Example 1, add 500 mL of a 4% (v / v) TFA / DCM solvent, and cleave at 25 °C for 3 hours. Filter, and concentrate the filtrate under vacuum at 30 °C to a viscous state. Add 125 mL of a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O = 91:3:3:3 (the cleavage reagent is pre-cooled to -5 °C), and cleave at 25 °C for 4 hours. Then add it to 600 mL of methyl tert-butyl ether (pre-cooled to -7 °C), filter using a Buchner funnel with a fast qualitative filter paper (standard number GB / T 1914-2017, pore size 10 - 12 μm). Wash the filter cake three times with methyl tert-butyl ether, with each usage of 50 mL. Vacuum pump the filter cake under nitrogen protection until it is cracked and dry, crush it, and then dry it under vacuum for 5 hours to obtain 33.8 g of crude telotristat, with a purity of 85.61%, a content of 65.71%, and a content yield of 73.5%. (The HPLC chromatogram is shown in the appendix Figure 3 )

[0047] Comparative Example: Preparation of crude telotristat

[0048] Take 100 g of the peptide resin prepared in Example 1, add 800 mL of a cleavage reagent with a mass ratio of TFA:DTT:TIS:H2O = 91:3:3:3 (the cleavage reagent is pre-cooled to -5 °C), and cleave at 25 °C for 2 hours. Then add it to 8000 mL of methyl tert-butyl ether (pre-cooled to -8 °C), centrifuge using a 2 L cup centrifuge, wash three times with methyl tert-butyl ether, with each time of 150 mL, blow it to a powder state with nitrogen, and then dry it under vacuum for 5 hours to obtain 70.2 g of crude telotristat, with a purity of 89.54%, a content of 67.34%, and a content yield of 78.2% (The HPLC chromatogram is shown in the appendix Figure 4 )

[0049] As can be seen from Example 2 and the comparative example, when the cleavage reagents TFA:DTT:TIS:H2O = 91:3:3:3 and the cleavage time is the same, the crude peptide purity and content yield are close, but the amounts of the cleavage reagent and methyl tert-butyl ether used in Example 2 are much smaller than those in the comparative example.

Claims

1. A method for preparing tirzepatide, characterized in that, It includes the following steps: Step 1: According to the Fmoc solid-phase synthesis method, using Sieber resin as the solid-phase synthesis carrier, sequentially couple to obtain the fully protected peptide resin Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(AEEA-AEEA-γGlu(α-OtBu)-Eicosanedioicacid(mon-tBu))-Ala-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Sieber Resin; Step 2: Use a DCM solution of TFA with a volume fraction of 3% - 5% to cleave the fully protected peptide resin described in Step 1, filter to remove the waste resin, and concentrate the obtained filtrate to a viscous solid; Step 3: Add a cleavage reagent to the viscous solid described in Step 2 for cleavage. After the cleavage is completed, add an ether solvent for precipitation. The precipitate is filtered and dried to obtain the crude telotristat.

2. The preparation method according to claim 1, wherein The Fmoc solid-phase synthesis method described in Step 1 is: Using Sieber resin with a substitution degree of 0.3 - 0.8 mmol / g as the solid-phase synthesis carrier, swell the resin with a solvent, and sequentially couple Fmoc-protected amino acids or protected amino acid fragments until the reaction is detected to terminate by the ninhydrin detection method; Preferably, the solvent is selected from one of DMF, NMP, and dichloromethane; Preferably, the dosage of the Fmoc-protected amino acid or protected amino acid fragment is 2 - 4 times the total molar amount of the charged resin; Preferably, the coupling agent used for the coupling is a composition of DIPCDI and Compound A or a composition of DIPEA, Compound A, and Compound B, where Compound A is HOAt, HOBt, or Oxyma pure, and Compound B is PyAOP, PyBOP, HATU, HBTU, or TBTU.

3. The preparation method according to claim 1, characterized in that, The dosage of the DCM solution of TFA with a volume fraction of 3% - 5% described in Step 2 is 8 - 10 mL / g of the fully protected peptide resin.

4. The preparation method according to claim 1, characterized in that, The reaction conditions for the cleavage described in Step 2 are: the reaction temperature is 20 - 35 °C, and the reaction time is 2 - 4 hours.

5. The preparation method according to claim 1, characterized in that, The filtrate obtained in Step 2 is concentrated to a viscous solid under vacuum.

6. The preparation method according to claim 1, characterized in that, The dosage of the cleavage reagent described in Step 3 is 2 - 2.5 mL / g of the fully protected peptide resin.

7. The preparation method according to claim 1, wherein, The cleavage reagent described in Step 3 is a mixed solvent of one or several of EDT, DTT, DODT and TFA, TIS, H2O; Preferably, the cleavage reagent is a mixed solvent of TFA, DTT, TIS, and H2O; Preferably, the mass ratio of TFA:DTT:TIS:H2O is 91:3:3:

3.

8. The preparation method according to claim 1, characterized in that, The reaction conditions for the cleavage in Step 3 are: the reaction temperature is 20 - 30 °C, and the reaction time is 2 - 4 hours.

9. The preparation method according to claim 1, wherein The dosage of the ether reagent in Step 3 is 8 - 10 mL / g of the fully protected peptide resin; Preferably, the ether reagent in Step 3 is methyl tert-butyl ether; Preferably, the ether reagent in Step 3 is precooled to -8 - -5 °C.

10. The preparation method according to claim 1, characterized in that, The drying in Step 3 is vacuum drying.

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