Method for preparing tilpotide

Abstract

The invention belongs to the field of medicines, particularly relates to preparation of medical substances, and more particularly relates to a method for preparing tiall. The preparation method comprises the following steps: firstly, carrying out a condensation reaction on a fragment [1-21] and a fragment [22-39] by adopting a solid-phase synthesis method to obtain a fragment [1-39], then cracking the fragment [1-39] by using an acid solution to remove side chain protecting groups OtBu, Trt, Boc and tBu to obtain the tierpotide, and adding a trapping agent in the cracking process. The invention provides a method for preparing tilpotide, which has the advantages of simple post-treatment, low impurity level, no need of special equipment, low production cost and facilitation of industrial production. Compared with the prior art, the method has obvious advantages.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the preparation of a medicine substance, and more specifically relates to a method for preparing telpotide. Background Art

[0002] Tirzepatide is a new type of dual agonist of glucose-dependent insulinotropic polypeptide (GIP) receptor and glucagon-like peptide-1 (GLP-1) receptor developed by Eli Lilly. Both GIP and GLP-1 are hormones secreted by the intestine and can promote insulin secretion. Therefore, tirzepatide can be used clinically to treat type 2 diabetes and obesity. Its peptide sequence is as follows:

[0003]

[0004] There are several patents describing methods for preparing telpotide:

[0005] Patent CN112592387 uses special protected amino acids: Boc-Tyr(tBu)-Aib-OH, Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, Fmoc-Tyr-Ser[Psi(Me,Me)Pro]-OH and Fmoc-(Dmb)Gly-OH, and other amino acids are conventional protected amino acids. The peptide resin is obtained by solid phase synthesis of peptides, and the crude telpotide is obtained by acid cleavage. The finished telpotide is purified by high performance liquid chromatography.

[0006] The method in patent CN110903355 is similar to that in patent CN112592387, except that special protected amino acids are used: Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH and Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, and other amino acids are conventional protected amino acids.

[0007] Patent CN113330024 introduces 7 methods for preparing telpotide:

[0008] Method 1 is to first solid phase synthesize fragments [1-14], [15-29] and [30-39], except for Lys 20 Fmoc-Lys(Alloc)-OH was used, and conventional protected amino acids were used at other sites. The fragments were then condensed in liquid phase, Alloc was removed by tetrakistriphenylphosphine catalysis, and the side chain tetrapeptide Eic(OtBu)-Glu(AEEA-AEEA-OH)-OtBu was coupled. Finally, trifluoroacetic acid cleavage was performed to obtain the crude telpotide.

[0009] Method 2 is to first solid phase synthesize fragments [1-13], [14-21], [22-30] and [31-39], except for Lys 20 The side chain pentapeptide Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, Tyr 1 -Aib 2 -Glu 3 -Gly 4 Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH was used, and conventional protected amino acids were used at other sites; the fragments were then condensed in liquid phase, and finally trifluoroacetic acid cleavage was performed to obtain crude telpotide.

[0010] Methods 3 and 4 both first synthesize fragments [1-14], [15-21], [22-29], and [30-39] on solid phase, except for Lys 20 The side chain pentapeptide Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, Tyr 1 -Aib 2 -Glu 3 -Gly 4 Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH is used, and conventional protected amino acids are used at other sites; then the fragments are condensed in liquid phase, and finally trifluoroacetic acid is cleaved to obtain crude telopoietin. Methods 3 and 4 are similar, except for the treatment method of removing Fmoc after fragment condensation: Method 3 uses conventional liquid phase method (extraction or crystallization); Method 4 uses nanofiltration method to separate and purify the intermediate product.

[0011] Method 5 is to use the O-acyl isomeric peptide method, firstly, the solid phase synthesis method is used to couple the protected amino acids one by one, except for Lys 20 Using Fmo c-Lys(ivDde)-OH, Thr 7 -Ser 8 Boc-Ser[Fmoc-Thr(tBu)]-OH was used, and conventional protected amino acids were used at other sites; ivDde was then removed with hydrazine hydrate, and the side chain tetrapeptide Eic(OtBu)-Glu(AEEA-AEEA-OH)-OtBu was coupled, and finally trifluoroacetic acid cleavage and O-acyl isomeric peptide conversion under alkaline conditions were performed to obtain the crude telpotide.

[0012] Method 6 is to use the natural chemical ligation (NCL) method, firstly, the fragment [1-16] hydrazide is synthesized in solid phase, and the [1-16] thioester is obtained by liquid phase modification, and then the fragment Cys is synthesized in solid phase. 17-[17-39], followed by liquid phase condensation of the fragments and final desulfurization (converting Cys to Ala) to obtain crude telportipeptide.

[0013] Method 7 also uses the natural chemical ligation (NCL) method, which is similar to method 6. First, the fragment [1-20] hydrazide is synthesized on the solid phase, and the [1-20] thioester is obtained by liquid phase modification. At the same time, the fragment Cys is synthesized on the solid phase. 21 -[21-39], followed by liquid phase condensation of the fragments and finally desulfurization (converting Cys to Ala) to obtain crude telportipeptide.

[0014] Among them, the methods for preparing telpotide introduced in patents CN112592387 and CN110903355 are both peptide solid phase synthesis methods. In addition to using conventional protected amino acids, special protected amino acids and fragments are also used in the coupling process. The production scale is limited, and the production capacity is difficult to reach 100 kilograms. At the same time, a large amount of waste liquid is generated, which is not conducive to environmental protection.

[0015] Lys selected in method 1 of patent CN113330024 20 When using Fmoc-Lys(Alloc)-OH, the reaction is incomplete when removing Alloc, resulting in a low crude peptide content and affecting the yield. The C-terminal amino acid of the fragment selected by methods 2, 3 and 4 is easy to racemize, and the coupling temperature needs to be strictly controlled to reduce racemic impurities, which is not conducive to industrial production. At the same time, method 4 uses a nanofiltration method to separate and purify the intermediate product, which requires special nanofiltration equipment, has a high cost and the domestic manufacturing level cannot meet the requirements. Method 5 uses a solid phase synthesis method to couple the protected amino acids one by one, and the production scale is limited; at the same time, there are few commercial manufacturers of Boc-Ser[Fmoc-Thr(tBu)]-OH, and the price is expensive, which is not conducive to industrialization. Methods 6 and 7 both use the natural chemical connection (NCL) method, and multiple intermediates need to be purified by HPLC and freeze-dried before entering the next step. The yield is low and is not conducive to industrialization. Summary of the invention

[0016] The invention provides a method for preparing telpotide, which has simple post-treatment, low impurity level, no need for special equipment, low production cost and is conducive to industrial production.

[0017] The method for preparing telpotide of the present invention comprises the following steps:

[0018] The present invention provides a method for preparing telpotide, which is characterized by comprising the following steps:

[0019] 1) Solid phase synthesis fragment [1-21]: 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[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-Ala-OH;

[0020] 2) Solid phase synthesis fragments [22-39]: NH2-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2;

[0021] 3) Synthesis of fragment [1-39]: Fragment [1-21] and fragment [22-39] were condensed to obtain fragment [1-39]: 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[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-Ala-Phe-Val-Gln(Trt)-Tr p(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2;

[0022] 4) Synthesis of tirpotide: The fragment [1-39] is cleaved with an acidic solution to remove the side chain protecting groups OtBu, Trt, Boc and tBu to obtain tirpotide, and a capture agent is added during the cleavage process.

[0023] In a preferred embodiment, the specific operation in step 1) is to use a solid phase resin as a carrier, sequentially couple the amino acids or amino acid residues from the C-terminus to the N-terminus of the fragment [1-21] with a coupling agent to obtain a peptide resin, remove the Fmoc protecting group, and cleave the peptide from the resin with an acidic solution to obtain a fragment [1-21];

[0024] More preferably, 20% piperidine / DMF solution is used to remove the Fmoc protecting group;

[0025] The amino acid or amino acid residue has N-terminal Fmoc protection and side chain protection;

[0026] The resin is 2-chlorotriphenyl chloride resin. More preferably, the substitution degree of the resin is 0.4 to 1.6 mmol / g; and more preferably, the substitution degree is 1.4 mmol / g.

[0027] In a specific embodiment, the coupling agent is one or more of DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA and PyAop / HOAt / DIPEA, preferably DIPCDI / HOBt.

[0028] In some embodiments, the amino acids or amino-terminal residues used in step 1) are:

[0029] Fmoc-Ala-OH, Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc- Ile-Aib-OH, 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-Gly-OH, Fmoc-Glu(Ot Bu)-OH and Boc-Tyr(tBu)-Aib-OH.

[0030] In some other embodiments, the amino acids or amino terminal residues used in step 1) are:

[0031] Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, 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-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH.

[0032] In a specific embodiment, the specific operation in step 2) is: using a solid phase as a carrier, sequentially coupling the amino acids from the C-terminus to the N-terminus of the fragment [22-39] with a coupling agent to obtain a peptide resin, removing the Fmoc protecting group, and cleaving the peptide from the resin with an acidic solution to obtain a fragment [22-39];

[0033] Preferably, the reagent for removing the Fmoc protecting group is a 20% piperidine / DMF solution; the solid phase resin is a Sieber resin, and the substitution degree of the solid phase resin is 0.4-1.0 mmol / g, more preferably, the solid phase resin is a Sieber resin, and the substitution degree is 0.7 mmol / g;

[0034] Preferably, the amino acids used are: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc- Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH and Fmoc-Phe-OH; the coupling agent is one or more of DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA and PyAop / HOAt / DIPEA, preferably DIPCDI / HOB.

[0035] Preferably, the acidic lysis solution is one or more of trifluoroethanol, hexafluoroisopropanol or trifluoroacetic acid solution with dichloromethane as solvent, preferably a trifluoroethanol / dichloromethane solution with a volume ratio of 20%.

[0036] In a preferred embodiment, the acidic lysing agent used in step 4) is TFA / H2O, and the volume ratio of TFA / H2O is 87.5:5:5:2.5

[0037] Specifically, the capture agent in step 4) is one or more of water, triisopropylsilane, ethanedithiol, and dithiothreitol, preferably a mixture of triisopropylsilane and dithiothreitol, and the volume ratio of the two is 5:2.5.

[0038] In a preferred embodiment, the volume ratio of the acidic lysing agent to the capturing agent in step 4) is 92.5:7.5.

[0039] Optionally, the method further comprises a step of purifying tepoxetine, for example, by purifying using high performance liquid chromatography.

[0040] The present invention provides a method for preparing tepote, which does not require nanofiltration to remove impurities, has simple post-treatment, is easy to condense the selected fragments, has low impurity levels, does not require special equipment, has low production costs, and has high total synthesis yields, and is conducive to industrial production. Compared with the prior art, the present invention has obvious advantages. For example, the methods for preparing tepote introduced in CN112592387 and CN110903355 are all polypeptide solid phase synthesis methods, which are limited in production scale and difficult to reach the hundred kilogram level in production capacity. Methods 1, 2 and 6 in CN113330024 have low yields; the impurity Fmoc protecting group in method 3 is difficult to remove, resulting in low purity of the obtained product; method 4 requires special nanofiltration equipment, which is relatively expensive; method 5 requires the use of special materials, which is expensive. The specific total synthesis yields are shown in the following table:

[0041] Purity of crude telpotide Total yield of synthesis Embodiments of the present invention 79％ 72％ CN113330024 Method 1 Undisclosed About 25% CN113330024 Method 2 69.7％ 26.5％ CN113330024 Method 3 About 30% About 60% CN113330024 Method 4 Undisclosed 44.5％ CN113330024 Method 5 64％ 47％ CN113330024 Method 6 Undisclosed 20.8％

[0042] It can be seen from this that the total synthesis yield of the present invention has obvious advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The present invention discloses a route map for preparing telpotide.

[0044] Figure 2 and 3 is the chromatogram of fragment [1-21].

[0045] Figure 4 The fragment is the chromatogram of [22-39].

[0046] Figure 5 and6 is the chromatogram of fragment [1-39].

[0047] Figure 7 This is the chromatogram of crude tepoxetine peptide.

[0048] Figure 8 This is the chromatogram of tirpotide.

[0049] Fig. 9 This is the mass spectrum of tepoxetine. DETAILED DESCRIPTION

[0050] The relevant abbreviations or abbreviations used in the present invention are as follows:

[0051]

[0052]

[0053] The specific route for preparing telpotide of the present invention is as follows Figure 1 The process is to first synthesize fragments [1-21] and fragments [22-39] by a solid phase method, then condense the two fragments by a liquid phase method, and finally remove the amino acid side chain protecting groups in the fragments by a trifluoroacetic acid solution to obtain crude telportin peptide.

[0054] The present invention is further described below with reference to the following specific examples, but they do not constitute limitations of the present invention.

[0055] Example 1: Synthesis of fragment [1-21]

[0056] Weigh 2-chlorotriphenyl chloride resin (8.57 g, Sub = 1.4 mmol / g) and load it into the solid phase reaction column, wash it twice with DMF, swell it with DMF for 30 minutes, and then remove the solution. Weigh 7.47 g (24.0 mmol) of Fmoc-Ala-OH, 6.2 g (48.0 mmol) of DIPEA and 40 ml of DMF, add them into the reaction column, react at room temperature for 4 hours, then add 50 ml of methanol, react at room temperature for 1 hour, remove the solution, and wash it with DMF three times. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir and react at room temperature for 5 minutes, remove the solution, repeat the operation once, and wash it with DMF six times.

[0057] Weigh 29.39 g (24.0 mmol) of Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, 3.89 g (28.8 mmol) of HOBt and 40 ml of DMF, add the solution into the reaction column, slowly add 3.64 g (28.8 mmol) of DIPCDI and react at room temperature for 2 hours. Drain the solution, wash three times with DMF, add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir at room temperature for 5 minutes, drain the solution, repeat the operation once, and wash six times with DMF. Repeat the previous operation, sequentially couple Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, 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-Gly-OH, Fmoc-Glu(OtBu)-OH and Boc-Tyr(tBu)-Aib-OH, and monitor the reaction progress by Kaiser method. Shrink with methanol four times, and dry under reduced pressure to obtain peptide resin.

[0058] Weigh 400 mL of 20% trifluoroethanol / dichloromethane solution and the above peptide resin into a glass bottle, react at room temperature for 2 hours, filter, and wash the resin with 60 mL of dichloromethane. The combined filtrate is concentrated under reduced pressure to an oily substance, dissolved in 40 mL of DMSO, added to 400 mL of water to precipitate a solid, filtered, and dried at 40°C to obtain 39.4 g of fragment [1-21] with a purity of 90.68%. The results are shown in Figure 2 As shown, the yield is 80%.

[0059] Example 2: Synthesis of fragment [1-21]

[0060] Weigh 2-chlorotriphenyl chloride resin (8.57 g, Sub = 1.4 mmol / g) and load it into the solid phase reaction column, wash it twice with DMF, swell it with DMF for 30 minutes, and then remove the solution. Weigh 7.47 g (24.0 mmol) of Fmoc-Ala-OH, 6.2 g (48.0 mmol) of DIPEA and 40 ml of DMF, add them into the reaction column, react at room temperature for 4 hours, then add 50 ml of methanol, react at room temperature for 1 hour, remove the solution, and wash it with DMF three times. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir and react at room temperature for 5 minutes, remove the solution, repeat the operation once, and wash it with DMF six times.

[0061] Weigh 29.39 g (24.0 mmol) of Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, 3.89 g (28.8 mmol) of HOBt and 40 ml of DMF, add the solution into the reaction column, slowly add 3.64 g (28.8 mmol) of DIPCDI and react at room temperature for 2 hours. Drain the solution, wash three times with DMF, add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir at room temperature for 5 minutes, drain the solution, repeat the operation once, and wash six times with DMF. The previous operation was repeated to couple Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, 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-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH in sequence, and the reaction progress was monitored by the Kaiser method. The peptide resin was obtained by methanol shrinkage four times and drying under reduced pressure.

[0062] Weigh 400 mL of 20% trifluoroethanol / dichloromethane solution and the above peptide resin into a glass bottle, react at room temperature for 2 hours, filter, and wash the resin with 60 mL of dichloromethane. The combined filtrate is concentrated under reduced pressure to an oily substance, dissolved in 40 ml of DMSO, added to 400 ml of water to precipitate a solid, filtered, and dried at 40°C to obtain 34.5 g of fragment [1-21] with a purity of 60.29%. The results are shown in Figure 3 As shown, the yield is 70%.

[0063] Example 3: Synthesis of fragment [22-39]

[0064] Weigh Sieber resin (17.1 g, Sub = 0.7 mmol / g) and load it into the solid phase reaction column, wash it twice with DMF, swell it with DMF for 30 minutes, and then remove the solution. Add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir at room temperature for 5 minutes, remove the solution, repeat the operation once, and wash it with DMF six times.

[0065] Weigh 9.20 g (24.0 mmol) of Fmoc-Ser(tBu)-OH, 3.89 g (28.8 mmol) of HOBt and 40 ml of DMF, add the solution into the reaction column, slowly add 3.64 g (28.8 mmol) of DIPCDI and react at room temperature for 2 hours. Drain the solution, wash three times with DMF, add 20% piperidine / DMF solution to remove the Fmoc protecting group, stir at room temperature for 5 minutes, drain the solution, repeat the operation once, and wash six times with DMF. Repeat the previous operation, sequentially couple Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH and Fmoc-Phe-OH, and monitor the reaction progress by Kaiser method. Shrink with methanol four times, and dry under reduced pressure to obtain peptide resin.

[0066] Weigh 400 mL of 5% trifluoroacetic acid / dichloromethane solution and the above peptide resin into a glass bottle, react at room temperature for 2 hours, filter, and wash the resin with 60 mL of dichloromethane. Combine the filtrates, add 3.1 g of DIPEA to the filtrate, add 50 ml of ethanol to reconstitute, precipitate solids with 200 ml of water, filter, and air dry at 40°C to obtain 26.0 g of fragment [22-39] with a purity of 93.32%. The results are shown in Figure 4 As shown, the yield is 95%.

[0067] Example 4: Synthesis of fragment [1-39]

[0068] Weigh 41.1 g (10 mmol) of fragment [1-21], 27.3 g (12 mmol) of fragment [22-39], 1.6 g (12 mmol) of HOAt, 3.8 g (10 mmol) of HATU and 400 ml of DMF, cool to 0°C, add 2.59 g (20 mmol) of DIPEA, warm to room temperature for 4 hours, add 800 ml of 5% sodium bicarbonate solution to precipitate solid, filter, and dry at 40°C to obtain 60.5 g of fragment [1-39] with a purity of 85.20%. Figure 5 As shown, the yield is 95%.

[0069] Example 5: Synthesis of fragment [1-39]

[0070] Weigh 41.1 g (10 mmol) of fragment [1-21], 27.3 g (12 mmol) of fragment [22-39], 1.6 g (12 mmol) of HOBt, 5.2 g (10 mmol) of PyBop and 400 ml of DMF, cool to 0°C, add 2.59 g (20 mmol) of DIPEA, warm to room temperature and react for 4 hours, add 800 ml of 5% sodium bicarbonate solution to precipitate solid, filter, and dry at 40°C to obtain 57.3 g of fragment [1-39] with a purity of 79.74%. The results are shown in FIG. Figure 6 As shown, the yield is 90%.

[0071] Example 6: Synthesis of Telportide

[0072] Prepare 300 ml lysis buffer (TFA / H 2 O / DTT / TIS=87.5:5:5:2.5) cooled to 0°C, added 60.0 g of fragment [1-39], heated to room temperature for 2 hours, filtered, added the filtrate to 1.2 L cold MTBE, filtered, washed with MTBE, and dried in vacuo at 30°C to obtain 46 g of crude telpotide with a purity of 79.25%. The results are shown in Figure 7 As shown, the yield is 95%.

[0073] The crude product was dissolved in 1% ammonia water and purified by high performance liquid chromatography, with butane bonded silica gel filler as the stationary phase, 0.1% TFA solution as the mobile phase A, acetonitrile as the mobile phase B, the monitoring wavelength was 220nm, and the target peak fraction was collected by gradient elution, concentrated and freeze-dried to obtain 19.2g of telpotide peptide, with a total yield of 40% and a purity of 99.42%. Figure 8 As shown; mass spectrometry characterization is consistent with the theory, such as Fig. 9 shown.

Claims

1. A method for preparing tepote, characterized in that The following steps are involved: 1) Solid phase synthesis fragments [1-21]: 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[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-Ala-OH; 2) Fragments synthesized using the solid phase method [22-39]: NH2-Phe-Val-Gln(Trt)-Trp(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2; 3) Synthesis of fragment [1-39]: Fragment [1-21] and fragment [22-39] were condensed to obtain fragment [1-39]: 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[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-Ala-Phe-Val-Gln(Trt)-Tr p(Boc)-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-NH2; 4) Synthesis of tirpotide: The fragment [1-39] was cleaved with an acidic solution to remove the side chain protecting groups OtBu, Trt, Boc and tBu to obtain tirpotide, and a capture agent was added during the cleavage process.

2. The method according to claim 1, characterized in that The specific operation in step 1) is to use a solid phase resin as a carrier, use a coupling agent to sequentially couple the amino acids or amino acid residues from the C-terminus to the N-terminus of the fragment [1-21] to obtain a peptide resin, remove the Fmoc protecting group, and cleave the peptide from the resin with an acidic solution to obtain a fragment [1-21]; Preferably, 20% piperidine / DMF solution is used to remove the Fmoc protecting group; the amino acid or amino acid residue has N-terminal Fmoc protection and side chain protection; the resin is 2-chlorotriphenyl chloride resin, more preferably, the substitution degree of the resin is 0.4~1.6mmol / g; further preferably, the substitution degree is 1.4mmol / g.

3. The method according to claim 2, characterized in that The coupling agent is one or more of DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA and PyAop / HOAt / DIPEA, preferably DIPCDI / HOBt.

4. The method according to claim 2, characterized in that The amino acids or amino terminal residues used in step 1) are: Fmoc-Ala-OH, Fmoc-Lys[Eic(OtBu)-Glu(AEEA-AEEA)-OtBu]-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala- OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Ile-Aib-OH, Fmo c-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-O H, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH and Boc-Tyr(tBu)-Aib-OH.

5. The method according to claim 2, characterized in that The amino acids or amino terminal residues used in step 1) are: Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Aib-OH, Fmoc-Ile-OH, 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-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH.

6. The method according to claim 1, characterized in that The specific operation in step 2) is as follows: using the solid phase as a carrier, sequentially coupling the amino acids from the C-terminus to the N-terminus of the fragment [22-39] with a coupling agent to obtain a peptide resin, removing the Fmoc protecting group, and cleaving the peptide from the resin with an acidic solution to obtain the fragment [22-39]; Preferably, the reagent for removing the Fmoc protecting group is a 20% piperidine / DMF solution; the solid phase resin is a Sieber resin, and the substitution degree of the solid phase resin is 0.4-1.0 mmol / g, more preferably, the solid phase resin is a Sieber resin, and the substitution degree is 0.7 mmol / g; The amino acids used are: Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmo c-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Gly-Gly-OH, Fmoc-Ala-OH, Fmoc-IIe-OH, Fmoc-Le u-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH and Fmoc-Phe-OH; the coupling agent is one or more of DIPCDI / HOBt, PyBop / HOBt / DIPEA, HBTU / HOBt / DIPEA, DIPCDI / HOAt, HATU / HOAt / DIPEA and PyAop / HOAt / DIPEA, preferably DIPCDI / HOB.

7. The method according to claim 2 and claim 6, characterized in that The acidic lysis solution is one or more of trifluoroethanol, hexafluoroisopropanol or trifluoroacetic acid solutions with dichloromethane as solvent, preferably a trifluoroethanol / dichloromethane solution with a volume ratio of 20%.

8. The method according to claim 1, characterized in that The acidic lysing agent used in step 4) is TFA / H2O, and the volume ratio of TFA / H2O is 87.5:5:5:2.

5.

9. The method according to claim 1, characterized in that The capture agent in step 4) is one or more of water, triisopropylsilane, ethanedithiol, and dithiothreitol, preferably a mixture of triisopropylsilane and dithiothreitol, and the volume ratio of the two is 5:2.

5.

10. The method according to claim 1, characterized in that The volume ratio of the acidic cleavage agent to the capture agent in step 4) is 92.5:7.5; optionally, the method further comprises a step of purifying tepoxetine.

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