Synthesis method of tilpotide

Abstract

The invention discloses a synthesis method of tilpotide, and belongs to the technical field of pharmacy. According to the method, 2-Cl CTC resin is adopted as initial resin, a solid-phase polypeptide synthesis method is adopted to obtain a polypeptide fragment 1 (1-10 amino acids), then a liquid-phase reaction is performed to prepare salicylaldehyde ester of the full-protection polypeptide fragment 1, and the salicylaldehyde ester of the fragment 1 is obtained after acidolysis; rinkAmide AM resin is used as initial resin, a polypeptide solid-phase synthesis method is used for preparing a polypeptide fragment 2 (the 11th amino acid to the 39th amino acid), then salicylaldehyde ester of a polypeptide fragment 1 and the fragment 2 are subjected to STL chemical connection, then acidolysis is performed to obtain a crude product of the tilpotide, a finished product of the tilpotide is obtained after purification, the purity reaches 99.4% or above, and the yield reaches 47%. According to the method, a two-stage method is adopted, and an STL chemical connection method is used for chemical connection, so that the method has the characteristics of low cost, high yield, high crude peptide purity and the like, and is more suitable for industrial production compared with a current main four-stage process.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a method for synthesizing tirzepatide. Background Art

[0002] Tirzepatide is a GLP and GLP-1 dual agonist, and its peptide sequence structure is as follows: H-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Ile-Aib-Leu-Asp-Lys-Ile-Ala-Gln-Lys (eicosanedioic acid-rGlu-AEEA-AEEA)-Ala-Phe-Val-Gln-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-NH2.

[0003] Tirzepatide can improve β-cell function and enhance insulin sensitivity, showing dual improvements in efficacy and tolerance for patients with initially low doses and small subsequent dose increments. Clinical data of tirzepatide indicate that tirzepatide can bring a meaningful reduction in A1C and body weight levels in type II diabetes patients, and at the same time, this drug can also treat other metabolic diseases.

[0004] Since tirzepatide consists of 39 amino acids, has a relatively long sequence and contains many hydrophobic amino acids in the peptide sequence, when synthesized by the method of stepwise condensation of single amino acids, the production cycle is long. As the peptide chain grows continuously during the synthesis process, deletion peptides are likely to be produced, leading to an increase in impurities. Moreover, the properties of these deletion peptide impurities are relatively similar to those of the product, resulting in difficulties in the subsequent purification process of the synthesized crude tirzepatide. This causes a low yield.

[0005] Therefore, it is of great practical significance to develop a method for synthesizing Tzepatide with high yield, short cycle and few impurities. Summary of the Invention

[0006] The object of the present invention is to solve the production problems of many impurities, low purity and yield in the existing synthesis process, which are not conducive to subsequent further industrial scale-up, through a new method for synthesizing tirzepatide.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a method for synthesizing tirzepatide, comprising the following steps:

[0009] Step S1: Synthesize Fragment 2 by solid-phase synthesis: NH2-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys[C 20 -OtBu-Glu(OtBu)-AEEA-AEEA-OtBu]-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;

[0010] Step S2: Synthesize Fragment 1 by solid-phase synthesis: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-OH;

[0011] Step S3: Condense Fragment 1 with salicylaldehyde containing an aldehyde protecting group for 2 - 6 h to obtain Fragment 1 salicylaldehyde ester: NH2-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-OPh(CHO);

[0012] Step S4: Dissolve the Fragment 1 salicylaldehyde ester obtained in Step S3 and Fragment 2 obtained in Step S1 in a mixed solution of organic base / organic acid in a certain ratio for STL chemical ligation and react for 2 - 6 h to obtain crude telotristat peptide;

[0013] Step S5: Purify, concentrate, and lyophilize the crude peptide to obtain pure telotristat peptide.

[0014] Furthermore, in Step S1, an amino resin is used as the resin carrier, and the amino resin is selected from any one of RinkAmide resin, RinkAmide AM resin, Rink MBHA resin, and Sieber resin; the substitution value of the amino resin is 0.2 - 1.2 mmol / g; the Fmoc-protected amino acid corresponding to the amino acid is added to the above amino resin for coupling reaction, and the dosage of the Fmoc-protected amino acid corresponding to the amino acid is 2.0 - 3.0 equivalents of the total molar amount of the charged amino resin.

[0015] Further, in step S2, a carboxyl resin is used as the resin carrier, and the carboxyl resin is any one of CTC resin and Wang resin; the substitution value of the carboxyl resin is 0.3 - 1.8 mmol / g; the Fmoc-protected amino acid corresponding to the amino acid is added to the above amino resin for coupling reaction, and the dosage of the Fmoc-protected amino acid corresponding to the amino acid is 2.0 - 3.0 equivalents of the total molar amount of the carboxyl resin fed.

[0016] Further, in step S3, the salicylaldehyde containing an aldehyde protecting group is acetone-acetal protected salicylaldehyde.

[0017] Further, in step S4, the base in the organic base / organic acid mixed solution is selected from any one or more of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine; the acid is selected from any one or more of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid.

[0018] Further, the organic base / organic acid mixed solution is selected from a pyridine / acetic acid mixed solution, wherein the volume ratio of pyridine to acetic acid is 6:1 - 1:6.

[0019] Further, after the polypeptide fragment coupling is completed in step S4, acidolysis is carried out, and the acid used in the acidolysis process is selected from organic acids or inorganic acids.

[0020] Further, the condensing agent used in the coupling reaction is one or more of HOBT / DIC, HOBT / EDCI, HBTU / DIEA, and Pybop / DIEA.

[0021] Further, the peptide resin obtained in step S1 is acidolyzed with TFA / water / 1,2-ethanedithiol / TIPS, and the volume ratio of the dosage of TFA / water / 1,2-ethanedithiol / TIPS is 92.5:2.5:2.5:2.5.

[0022] Further, the fully protected peptide resin obtained in step S2 is acidolyzed with a 1% TFA / DCM solution or a 20% trifluoroethanol / DCM solution, or a 20% hexafluoroisopropanol / dichloromethane solution.

[0023] Further, during the connection process of the serine polypeptide, the concentration of fragment 1 during the reaction process is 5 mM - 100 mM.

[0024] The present invention discloses a method for synthesizing tirzepatide. This method uses RinkAmide AM resin as the starting resin and prepares polypeptide fragment 2 by the solid-phase peptide synthesis method. At the same time, starting from 2-Cl trityl resin, the fully protected polypeptide fragment 1 is synthesized by the solid-phase peptide synthesis method, and then through a liquid-phase condensation reaction, acidolysis is carried out to obtain the salicylaldehyde ester of fragment 1. Finally, the salicylaldehyde ester of fragment 1 and fragment 2 are subjected to STL chemical ligation in a liquid-phase buffer salt system to obtain a crude product of tirzepatide with high purity. This crude product is purified by high-pressure preparation once to obtain a qualified finished product of tirzepatide.

[0025] The present invention uses the serine (Ser) site as the chemical ligation site of the polypeptide, not limited to the Ser at position 11, and can include the Ser binding sites at positions 8, 32, and 33.

[0026] At the same time, in the process of synthesizing fragment 2, in addition to the use of conventional protected amino acids, two dipeptide structures are also used to reduce the generation of missing peptides due to difficult coupling during the coupling process, greatly improving the crude peptide purity of fragment 2 and the purification efficiency. The dipeptide structures include: Fmoc-Pro-Pro-OH, Fmoc-Gly-Gly-OH.

[0027] CTC resin is used as the starting resin for synthesizing fragment 1, with a substitution degree between 0.3 - 1.8 mmol / g. The amount of the Fmoc-protected amino acid described in the amino acid is 2.0 - 3.0 equivalents of the molar amount of the fed CTC resin; the condensing agent used in the synthesis of fragment 1 and fragment 2 is one or more of HOBT / DIC, HBTU / DIEA, Pybop / DIEA, and the reaction solvent used is one or more combinations of DCM, DMF, and NMP; the Fmoc deprotection reagent used is 20% piperidine / DMF solution (v / v); when the fully protected peptide resin is acidolyzed, 1% TFA / DCM solution (v / v) or 20% trifluoroethanol / DCM solution is used.

[0028] In the method for synthesizing tirzepatide of the present invention, during the synthesis of fragment 2, the amino resin is one of Rink AM Resin or Rink MBHA Resin; the substitution degree of the selected resin is 0.2 - 1.2 mmol / g; the dosage of the Fmoc-protected amino acid or protected amino acid fragment is 2.0 - 3.0 times the total molar amount of the charged resin; the condensing agent used for synthesizing each amino acid is one or more of DIC / HOBT, HBTU / DIEA, Pybop / DIEA; the selected solvent is one or a combination of DCM, NMP, and DMF; the Fmoc deprotecting reagent is a piperidine / DMF (v / v) solution, and the preferred ratio is a 20% piperidine / DMF solution; the obtained Trizepatide peptide resin is acidolyzed with TFA / water / 1,2-ethanedithiol / TIPS, and the preferred ratio is TFA / water / 1,2-ethanedithiol / TIPS = 92.5% / 2.5% / 2.5% / 2.5% to obtain polypeptide fragment 2.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The method of the present invention uses 2-Cl CTC resin as the starting resin, and uses solid-phase peptide synthesis to obtain polypeptide fragment 1 (the 1st to 10th amino acids), and then prepares the salicylaldehyde ester of the fully protected polypeptide fragment 1 by liquid-phase reaction, and obtains fragment 1 salicylaldehyde ester after acidolysis. At the same time, using RinkAmide AM resin as the starting resin, the polypeptide solid-phase synthesis method is used to prepare polypeptide fragment 2 (the 11th to 39th amino acids), and then the salicylaldehyde ester of polypeptide fragment 1 and fragment 2 are subjected to STL chemical ligation (serine-threonine chemical ligation method), and then acidolysis to obtain the crude product of tirzepatide, and the purity of the crude product can reach 88% or more. Subsequently, the crude product of tirzepatide is purified by one-time high-pressure preparation to obtain the final qualified tirzepatide product, and the purity of one-time purification can be as high as more than 99.4%, and the overall yield reaches 47%. This method adopts a two-stage method and uses the STL chemical ligation method for chemical ligation, which has the characteristics of low cost, scalable production, high yield, and high purity of the crude peptide, and is more suitable for industrial production than the current main four-stage process. Description of the Drawings

[0031] Figure 1 It is the flow chart for preparing tirzepatide of the present invention;

[0032] Figure 2 It is the NMR spectrum of acetonide-protected salicylaldehyde 2 prepared in Example 1;

[0033] Figure 3 It is the HPLC spectrum of fragment 1 in Example 2;

[0034] Figure 4Mass spectrum of Fragment 1 in Example 2;

[0035] Figure 5 HPLC spectrum of the salicylaldehyde ester of Fragment 1 in Example 3;

[0036] Figure 6 Mass spectrum of the salicylaldehyde ester of Fragment 1 in Example 3;

[0037] Figure 7 HPLC spectrum of the crude Fragment 2 in Example 4;

[0038] Figure 8 HPLC spectrum of the purified Fragment 2 in Example 4;

[0039] Figure 9 Mass spectrum of the purified Fragment 2 in Example 4;

[0040] Figure 10 HPLC spectrum of the coupled intermediate peptide in Example 5;

[0041] Figure 11 Mass spectrum of the coupled intermediate peptide in Example 5;

[0042] Figure 12 HPLC spectrum of the coupled intermediate peptide in Example 6;

[0043] Figure 13 HPLC spectrum of the coupled intermediate peptide in Example 7;

[0044] Figure 14 HPLC spectrum of the crude tirzepatide in Example 8;

[0045] Figure 15 Mass spectrum of the crude tirzepatide in Example 8;

[0046] Figure 16 HPLC spectrum of the pure tirzepatide in Example 9; Detailed implementation manners

[0047] For better illustration of the present invention, the following examples are specifically listed. Obviously, the described examples are only a part of the present invention, rather than all the examples. Based on the examples in the present invention, other examples obtained by those skilled in the art without creative work all fall within the scope of protection of the present invention.

[0048] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0049] Among them, the 1st to 10th amino acids, and the 11th to 39th amino acids refer to each amino acid of tirzepatide from the N-terminus to the CONH2 terminus at the C-terminus in sequence.

[0050] Synthesis of acetonide-protected salicylaldehyde 2 in Example 1

[0051]

[0052] At room temperature, salicylaldehyde (50 g, 409.4 mmol) and lithium tetrafluoroborate (1.1 g, 12.3 mmol) were dissolved in anhydrous methanol (500 ml). Subsequently, under nitrogen protection, trimethyl orthoformate (43.4 g, 409.4 mmol) was added. Then the reaction solution was stirred and refluxed at 80 °C overnight for 8 - 10 h, and the reaction was monitored to be complete by TLC plate. After the reaction was complete, saturated aqueous NaHCO3 solution (200 ml) was added to quench the reaction, methanol was removed by concentration, and then it was diluted with ethyl acetate (1 L). The organic layer was separated and collected, and this organic layer was washed twice with saturated sodium bicarbonate (200 ml) and saturated brine (200 ml) respectively. After washing, it was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain a pale yellow liquid (67.2 g, yield 97.6%). This pale yellow liquid can be directly used in the next step without further purification. The NMR is as follows Figure 2 as shown.

[0053] Synthesis of fully protected fragment 1 in Example 2

[0054] Fully protected fragment 1: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-OH (SEQ ID NO.1);

[0055]

[0056] Weigh 60.6 g of CTC resin (substitution degree: 1.65 mmol / g) and add it to a solid-phase synthesis reactor. Add 200 ml of DMF to swell the resin for 10 min. Additionally, weigh 68.9 g (150 mmol) of Fmoc-Tyr(tBu)-OH and dissolve it in 300 ml of DMF. After adding DIEA (38.7 g, 300 mmol), mix well and add it to the above solid-phase reactor. React at room temperature for 3 h, drain, and wash with DMF 4 times. Add 300 ml of DMF, then continue to add methanol (16 g, 500 mmol) and DIEA (38.7 g, 300 mmol), and continue to react at room temperature for 1.0 h. Drain and wash with DMF 5 times. Deprotect Fmoc from the peptide resin in the solid-phase reactor once with 20% piperidine / DMF (V / V) solution (25 min). After completion, wash with DMF 5 times, and the ninhydrin test result is positive.

[0057] Weigh 82.29 g (200 mmol) of Fmoc-Asp(OtBu)-OH and 27.0 g (200 mmol) of HOBT, dissolve them in 300 ml of DMF at 0 °C, add DIC (25.2 g, 200 mmol), activate for 5 min, then add the solution to the solid-phase reactor, react for 2.0 h, the ninhydrin test result is negative, drain, and wash with DMF 4 times.

[0058] Repeat the above steps, couple the remaining amino acids according to the peptide sequence to obtain Fragment 1: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-OH. The weight of the peptide resin is: 212.6 g. React the peptide resin with 300 ml of TFA / DCM (V / V, the volume ratio of TFA:DCM = 1:99) solution for 2.0 h, filter, collect the filtrate, extract twice with an appropriate amount of purified water to obtain the organic layer filtrate, add an appropriate amount of anhydrous sodium sulfate for drying, filter the filtrate and concentrate it to dryness to obtain Fragment 1: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-OH (SEQ ID NO.1), and the fully protected peptide segment is 152.4 g. The HPLC chromatogram is as follows Figure 3 shown, and the Mass chromatogram is as follows Figure 4 shown.

[0059] Synthesis of Fragment 1 salicylaldehyde ester in Example 3

[0060] Fragment 1 salicylaldehyde ester: NH2-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-OPh(CHO)

[0061]

[0062] The polypeptide fragment 1 (152.4 g, 92.7 mmol) and the acetonide-protected salicylaldehyde compound 2 (23.36 g, 139.1 mmol) were dissolved in DCM (800 ml), and then HOBt (18.7 g, 139.1 mmol) and EDCI (26.6 g, 139.1 mmol) were added. The reaction solution was stirred at room temperature for 4 h. After the reaction was monitored by LCMS to be complete, the reaction solution was washed 3 times with 1 mol / L hydrochloric acid aqueous solution to obtain an organic layer filtrate. An appropriate amount of anhydrous sodium sulfate was added for drying. After the filtrate was filtered, it was concentrated to dryness to obtain the crude product of the fully protected fragment 1 salicylaldehyde ester. This crude product was subjected to a cleavage reaction with 600 ml of TFA / H2O / phenol (v / v / v, where the volume ratio of TFA:H2O:phenol is 95:2.5:2.5) at room temperature for 2 h. After the reaction was detected by LCMS to be complete, 4 L of ice-cold methyl tert-butyl ether was added to precipitate a white solid, which was centrifuged and dried 3 times to obtain the crude product of white fragment 1 salicylaldehyde ester. The purity of this crude product was determined by high performance liquid chromatography to be 83%. After purification by high performance liquid chromatography once and freeze-drying, a white pure product powder of fragment 1 salicylaldehyde ester (63.5 g, yield 53.9%) was obtained. The HPLC chromatogram is as follows Figure 5 shown, and the Mass chromatogram is as follows Figure 6 shown.

[0063] Synthesis of Fragment 2 in Example 4

[0064]

[0065] Take 166.7 g of Fmoc-RinkAmide AM resin (substitution degree 0.6 mmol / g). First, it was swollen with 1 L of DMF for 5 min. After filtration, the resin was deprotected with 1 L of 20% piperidine / DMF for 25 min. The result detected by ninhydrin was positive. After filtration, it was washed 5 times with DMF.

[0066] Take 200 mmol of Fmoc-Ser(tBu)-OH and 200 mmol of HOBt, dissolve them in 600 ml of DMF; separately take 200 mmol of DIC, and slowly add it to the above DMF solution under stirring. After stirring and reacting at room temperature for 5 min, slowly add the above RinkAmide AM resin after removing Fmoc, and carry out the coupling reaction for 2 h. After the ninhydrin test result shows negative, the coupling is complete. After filtration, the resin is washed 5 times with 500 ml of DMF to obtain Fmoc-Ser(tBu)-amino resin. Then, it is deprotected with 600 ml of 20% piperidine / DMF for 20 min. After the ninhydrin test result shows positive, it is filtered and washed 5 times with DMF to obtain NH2-Ser(tBu)-amino resin.

[0067] Prepare the Fmoc-protected amino acids corresponding to the 2nd - 27th amino acids for use first, and the structures are shown in Table 1.

[0068] The activation method of the Fmoc-protected amino acid corresponding to the 2nd amino acid is as follows:

[0069] Take 300 mmol of Fmoc amino acid and 300 mmol of HOBt, dissolve them in 800 ml of DMF; separately take 300 mmol of DIC, and slowly add it to the above DMF solution under stirring. After stirring and reacting at room temperature for 5 min, slowly add the above RinkAmide AM resin after removing Fmoc, and carry out the coupling reaction for 2 h. After the ninhydrin test result shows negative, the coupling is complete. After filtration, the resin is washed 5 times with 500 ml of DMF to obtain Fmoc-Pro-Ser(tBu)-amino resin. Then, it is deprotected with 600 ml of 20% piperidine / DMF for 20 min. After the ninhydrin test result shows positive, it is filtered and washed 5 times with DMF to obtain NH2-Pro-Ser(tBu)-amino resin.

[0070] Adopt the same method as above, and sequentially introduce the Fmoc-protected amino acids corresponding to the 3rd - 27th amino acids, that is, after deprotecting the Fmoc of the Fmoc-[1~(n - 1)] amino acid-amino resin obtained in the previous step, couple it with the activated Fmoc-protected amino acid (the nth) for 60 - 300 min, n = 1~2. After all the protected amino acids are introduced, wash it 3 times with DMF, 3 times with methanol, drain, and vacuum dry to obtain 491 g of polypeptide resin.

[0071] Take 1 g of the above resin, add 5 ml of cleavage reagent [TFA / water / 1,2-ethanedithiol / TIPS = 92.5% /

[0072] 2.5% / 2.5% / 2.5% (V / V / V / V), stir evenly, stir and react at room temperature for 3 h. Filter the reaction mixture through a fritted funnel, collect the filtrate, wash the resin with a small amount of TFA three times. After combining the filtrates, add at least five volumes of ice-cold methyl tert-butyl ether to precipitate. Centrifuge to obtain a white solid, and then wash it with methyl tert-butyl ether twice. Drain to obtain a white powder, which is the crude product of polypeptide fragment 2. The purity measured by high-performance liquid chromatography is 80.4%. After purification by high-performance liquid chromatography once and freeze-drying, 209.5 g of white pure product of fragment 2 is obtained, with a purity of 94.6% and a yield of 57.2%. The HPLC crude product spectrum is as follows Figure 7 as shown, the spectrum of the purified pure product is as follows Figure 8 as shown, the Mass spectrum is as follows Figure 9 as shown.

[0073] Table 1 Fmoc amino acids corresponding to amino acids and corresponding resin equivalent numbers

[0074]

[0075] Example 5 Synthesis of Tirzepatide

[0076]

[0077] Dissolve fragment 1 salicylaldehyde ester (18.4 g, 14.2 mmol) and fragment 2 (40 g, 10.9 mmol) in 200 mL of pyridine / acetic acid = 1:1 (V / V) solution, react for 3 h. After HPLC detection shows that fragment 2 is consumed, add ice-cold methyl tert-butyl ether to precipitate a white solid to obtain 56.8 g of the coupled intermediate peptide. The purity detected by HPLC is 92.23%. See the HPLC spectrum as follows Figure 10 as shown, the mass of the coupled intermediate state is as follows Figure 11 as shown.

[0078] Example 6

[0079] At room temperature, dissolve fragment 1 salicylaldehyde ester (18.4 g, 14.2 mmol) and fragment 2 (40 g, 10.9 mmol) in 200 mL of pyridine / acetic acid = 1:3 (V / V) solution, react for 3 h. HPLC detection shows that fragment 2 is not completely reacted. React for 6 h. After HPLC detection shows that fragment 2 is consumed, add ice-cold methyl tert-butyl ether to precipitate a white solid to obtain 54.2 g of the coupled intermediate peptide. The purity detected by HPLC is 90.32%, as follows Figure 12 as shown.

[0080] Example 7

[0081] At room temperature, fragment 1 salicylaldehyde ester (18.4 g, 14.2 mmol) and fragment 2 (40 g, 10.9 mmol) were dissolved in 200 mL of a pyridine / acetic acid / DMF = 1:1:2 (V / V) solution, and the reaction was carried out for 6 h. HPLC detection showed that fragment 2 was not completely reacted. After the reaction was carried out overnight and HPLC detection showed that fragment 2 was consumed completely, frozen methyl tert-butyl ether was added to precipitate a white solid to obtain the coupled intermediate peptide. The purity detected by HPLC was 73.62%, as follows Figure 13 as shown

[0082] Example 8

[0083] 56.8 g of the crude coupled intermediate peptide obtained in Example 5 was dissolved in 350 ml of a cleavage solution of TFA / H2O (95% / 5%). After reacting at room temperature for 20 min, HPLC detection showed that the reaction was complete. Frozen methyl tert-butyl ether was added to precipitate a white solid to obtain the crude telotristat peptide. The purity detected by HPLC was 88.78%. The HPLC detection spectrum is as follows Figure 14 as shown, and the Mass spectrum is as follows Figure 15 as shown

[0084] 56.8 g of the crude telotristat peptide obtained in Example 8 was dissolved and clarified in 280 ml of water with a pH of 8.5, and then filtered. Using octadecyl silica gel as the stationary phase, HPLC gradient elution was carried out with an aqueous sodium acetate solution and acetonitrile as the mobile phase to obtain a qualified fraction of telotristat peptide. The acetonitrile was removed, and freeze-drying was carried out to obtain 43.2 g of the purified telotristat peptide. The HPLC purity was 99.4%. The yield of this step was 82.3%, and the overall yield was 47.0%. The spectrum of the separated qualified pure product is as follows Figure 16 as shown

[0085] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should all fall within the protection scope determined by the claims of the present invention

Claims

1. A method for synthesizing tirzepatide, characterized in that, It includes the following steps: Step S1: Synthesize Fragment 2 by solid-phase synthesis method: NH2-Ser(tBu)-Ile-Aib-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys[C 20 -OtBu-Glu(OtBu)-AEEA-AEEA-OtBu]-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; Step S2: Synthesize fragment 1 by solid-phase synthesis method: Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(tBu)-Tyr(tBu)-OH; Step S3: Condense fragment 1 with salicylaldehyde containing aldehyde protecting group for 2 - 6 h to obtain fragment 1 salicylaldehyde ester: NH2-Tyr-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-OPh(CHO); Step S4: Dissolve the fragment 1 salicylaldehyde ester obtained in step S3 and the fragment 2 obtained in step S1 in a mixed solution of organic base / organic acid in a certain ratio for STL chemical ligation, react for 2 - 6 h, and obtain crude telotristat peptide after acidolysis; Step S5: Purify, concentrate, and lyophilize the crude peptide to obtain pure telotristat peptide.

2. The method according to claim 1, characterized in that In step S1, an amino resin is used as the resin carrier, and the amino resin is selected from any one of RinkAmide resin, RinkAmide AM resin, Rink MBHA resin, and Sieber resin; the substitution value of the amino resin is 0.2 - 1.2 mmol / g; the Fmoc-protected amino acid corresponding to the amino acid is added to the above amino resin for coupling reaction, and the dosage of the Fmoc-protected amino acid corresponding to the amino acid is 2.0 - 3.0 equivalents of the total molar amount of the charged amino resin.

3. The method according to claim 1, characterized in that, In step S2, a carboxyl resin is used as the resin carrier, and the carboxyl resin is any one of CTC resin and Wang resin; the substitution value of the carboxyl resin is 0.3 - 1.8 mmol / g; the Fmoc-protected amino acid corresponding to the amino acid is added to the above amino resin for coupling reaction, and the dosage of the Fmoc-protected amino acid corresponding to the amino acid is 2.0 - 3.0 equivalents of the total molar amount of the charged carboxyl resin.

4. The method according to claim 1, characterized in that, In step S3, the salicylaldehyde containing aldehyde protecting group is acetonide-protected salicylaldehyde.

5. The method according to claim 1, wherein In step S4, the base in the organic base / organic acid mixed solution is selected from any one or more of pyridine, 2-methylpyridine, 2,6-dimethylpyridine, 2,4,6-trimethylpyridine, triethylamine, and N,N-diisopropylethylamine; the acid is selected from any one or more of acetic acid, citric acid, oxalic acid, formic acid, propionic acid, and butyric acid.

6. The method according to claim 4, wherein The organic base / organic acid mixed solution is selected from pyridine / acetic acid mixed solution, wherein the volume ratio of pyridine to acetic acid in the mixed solution is 6:1 - 1:

6.

7. The method according to claim 1, wherein In step S4, acidolysis is carried out after the coupling of polypeptide fragments is completed, and the acid used in the acidolysis process is selected from organic acids or inorganic acids.

8. The method according to claim 2, wherein The condensing agent used in the coupling reaction is one or more of HOBT / DIC, HOBT / EDCI, HBTU / DIEA, and Pybop / DIEA.

9. The method according to claim 1, characterized in that, The peptide resin obtained in the step S1 is acidolyzed with TFA / water / 1,2-ethanedithiol / TIPS, and the volume ratio of the dosage of TFA / water / 1,2-ethanedithiol / TIPS is 92.5:2.5:2.5:2.

5.

10. The method according to claim 1, wherein The fully protected peptide resin obtained in the step S2 is acidolyzed with 1% TFA / DCM solution or 20% trifluoroethanol / DCM solution, 20% hexafluoroisopropanol / dichloromethane solution.

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