Synthesis method of tilpotide

Abstract

The synthesis method comprises the following steps: firstly, respectively synthesizing a fragment I (1-13), a fragment II (14-21) and a fragment III (22-39), then synthesizing a fragment (14-39) in a liquid phase by using the fragment II (14-21) and the fragment III (22-39), and finally, condensing, cracking and purifying the fragment I (1-13) and the fragment (14-39) in the liquid phase to prepare the tilpotide. According to the invention, synthesis is carried out through three fragments, so that the synthesis steps are simplified, the racemization problem is avoided, + / -Gly impurities are reduced, and DKP side reaction is avoided and reduced, thereby improving the whole yield.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a method for synthesizing telpotide. Background Art

[0002] Tirzepatide is a long-acting GLP-1 analog developed by Eli Lilly and Company. It was approved by the FDA on December 5, 2017 and is available in the United States. The synthesis processes reported in the existing literature can be roughly divided into two categories:

[0003] The first type is the Fmoc solid phase step-by-step coupling method, which generally follows the following route: a) according to the peptide sequence requirements, each amino acid is coupled in sequence according to the standard Fmoc solid phase synthesis method; an orthogonal protecting group is used to protect the Lys side chain at the Lys20 position, such as Alloc or ivDde; b) after the coupling is completed, the Lys side chain protecting group is removed and the side chain (C 20 Modification coupling of diacid-γ-Glu-(AEEA)2; c) cleavage to obtain crude peptides, and finally purification and freeze-drying. For example, patents CN113330024A and WO2020159949A1, due to the long peptide sequence (39 peptides, excluding side chain synthesis), the production cycle is long, the efficiency of industrial production is low, and the risk is high. Once a reaction site is difficult to couple, the entire synthesis will fail. The various impurities of the crude peptide obtained are difficult to separate (the purity of the crude peptide is 50-60%). At the same time, due to various difficult sites and multiple removal of Fmoc under alkaline conditions, the C-terminal Pro-Pro-Pro sequence is easy to fall off, resulting in a low overall yield and many other problems. For example, patents CN112592387B, CN115651075B, CN110903355A, CN116120403A, CN115991742A, CN116178523A, CN114736271A, CN115181173A, CN115181174A, CN115181174A, etc., based on the above-mentioned related patents, for reaction sites that are difficult to couple, such as Tyr1, At positions Aib2, Iie12, Gly29, etc., strategies such as dipeptide fragments Tyr-Aib dipeptide, pseudopro dipeptide, Gly(Dmb), Iie-Aib dipeptide, and Tyr-Aib-Glu-Gly tetrapeptide were used to solve the coupling difficulties at some sites. Although the synthesis effect of these sites can be improved, the overall cost is increased, and the effect of long peptide coupling is not fundamentally improved. There are still problems such as long production cycle, low efficiency, high risk, and difficulty in separating various impurities.

[0004] The second category is fragment condensation: for example, Eli Lilly patent CN113330024A, Suzhou Tianma CN115991742A and other reports use four fragments (1-14), (15-21), (22-29), and (30-39). First, (22-29) + (30-39) are coupled to obtain (22-39), which is then condensed with (15-21) to obtain (15-39), and finally condensed with (1-14) to obtain a crude peptide, which is then purified to obtain the product. Although this route improves the difficulty of coupling long peptides, it still has the following problems: ① There are many liquid phase condensation fragments, and the fragments will inevitably produce racemic impurities during liquid phase synthesis; the introduction of too many fragments will increase the cost of industrial production, the difficulty of fragment research and quality control, and the production process and post-processing are more complicated than solid phase synthesis; ② Asp15, as the N-terminus of the coupling, has a large steric hindrance and is more difficult to condense, resulting in low yields when (1-14) and (15-21) are condensed; ③ Gly29 has ±Gly impurities; (30-39): Gly30 has ±Gly impurities.

[0005] In addition, Eli Lilly's patent CN113330024A also reported the use of fragments (1-17) and (18-39) and the use of NCL method for condensation, and the reported total yield was low (8%). 13 The position is sterically hindered and coupling is difficult. 21 -Phe 22 -Val 23 The site may cause peptide resin contraction and coupling difficulties, Gly 29 -Gly 30 There is a risk of ±Gly impurities, Pro38 and Pro37 are located in the first three positions of the C-terminus, and during solid-phase coupling, there is a side reaction of DPK impurities after multiple alkaline couplings, which leads to peptide breakage and reduced yield.

[0006] In view of the shortcomings of the above patents, it is urgent to design a process route that can reduce side reactions such as racemization, simplify the synthesis reaction steps, avoid peptide resin shrinkage, and ultimately significantly improve product yield. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for synthesizing telpotide, and the specific route is as follows:

[0008] The synthesis steps were simplified by synthesizing according to three fragments, namely (1-13), (14-21), and (22-39). The C-terminus of fragment (1-13) is Aib (chiral, no racemization problem), and the C-terminus of fragment (14-21) is Ala 21 Low racemization, and Leu at the N-terminus 14The position is relatively low, which is convenient for liquid phase condensation with (1-13); at the same time, when synthesizing fragment (22-39), a dipeptide (Gly 29 -Gly 30 ), reducing ±Gly impurities; using tripeptide (Pro 36 -Pro 37 -Pro 38 ) Avoid and reduce the DKP side reaction, thereby increasing the yield of the synthesis. The specific technical solution is as follows:

[0009] A method for synthesizing telpotide comprises the following steps:

[0010] First, fragment I (1-13), fragment II (14-21), and fragment III (22-39) are synthesized separately, and then fragment II (14-21) and fragment III (22-39) are used to synthesize fragment (14-39) in liquid phase. Finally, fragment I (1-13) and fragment (14-39) are condensed, cracked, and purified in liquid phase to prepare telportide.

[0011] Preferably, the method specifically comprises the following steps:

[0012] (1) Synthetic fragment I (1-13):

[0013] Fmoc-Iie-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH were sequentially coupled to 2-CTC Resin and cleaved to prepare fragment I (1-13);

[0014] (2) Synthetic fragment II (14-21):

[0015] Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Leu-OH were sequentially coupled to 2-CTC Resin and cleaved to prepare fragment II (14-21);

[0016] Alternatively, Alloc-Lys(Fmoc)-OH is coupled to 2-CTC Resin, and then Fmoc is removed. After washing, 2-3 times of Eic(OtBu)-γGlu(AEEA-AEEA)-OtBu, 2.4-3.6 times of PyBOP / HOAt, and 4-6 times of DIPEA system are added. After coupling, Alloc is removed, and after washing, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, tBu-C 20 –diacid-Glu(AEEA-AEEA-OH)-OtBu, remove Alloc, wash and then sequentially couple with Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, and cleave to produce fragment II (14-21);

[0017] (3) Synthetic fragment III (22-39):

[0018] Fmoc-Ser(tBu)-OH, Fmoc-Pro-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-Ala-OH, Fmoc-Iie-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, and Fmoc-Phe-OH were sequentially coupled to Sieber Resin and cleaved to prepare fragment III (22-39);

[0019] (4) Synthetic fragments (14-39):

[0020] Dissolve fragment II (14-21) with a solvent, add a coupling reagent for pre-reaction, then add fragment III (22-39) and an alkali reagent, and stir to react; after the reaction, add diethylamine, stir, extract, and concentrate to obtain fragment (14-39);

[0021] (5) Synthesis of tepote

[0022] Dissolve fragment I (1-13) in a solvent, add a coupling reagent and stir to react, wash, collect the organic layer and dry it after the reaction is completed; then add a DMF solution of fragment (14-39), stir to clarify, add DIPEA, stir to react, extract, concentrate, cleave, precipitate, purify and freeze-dry to obtain tepoxetine.

[0023] Preferably, in step (1), compared with 2-CTC Resin, 5 times the feed is used for coupling Fmoc-Iie-OH and Fmoc-Aib-OH, 6 times PyBOP / HOAt and 6.5 times DIPEA, and the coupling is carried out for 4 to 6 hours, preferably 5 hours; when coupling the remaining amino acids, 3 times the amino acids and 3.6 times the HOBt / DIC system are used, and the coupling is carried out for 1 to 3 hours, preferably 2 hours.

[0024] Preferably, in step (2), compared with 2-CTC Resin, 2 times the feed is used for coupling Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH, 2.4 times PyBOP / HOAt and 4 times DIPEA, and the coupling is carried out for 4 to 6 hours, preferably 5 hours; when coupling the remaining amino acids, 3 times the amino acids and 3.6 times the HOBt / DIC system are used, and the coupling is carried out for 1 to 3 hours, preferably 2 hours.

[0025] Preferably, in step (2), the synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH is specifically as follows:

[0026] Dissolve Eic(OtBu)-COOH in a solvent and add an activating agent and a condensing agent to react at room temperature for 2 hours to prepare an Eic(OtBu)-OSu solution, and then condense with H-Glu-OtBu and DIPEA for 4 hours to obtain Eic(OtBu)-Glu-OBtu; wash, dry, filter, and obtain a filtrate, add an activating agent and a condensing agent, stir and react for 3 hours, and then add NH2-AEEA-AEEA-COOH to condense for 8 hours; wash, dry, filter, and concentrate to obtain an oily substance, dissolve it in a solvent, add a condensing agent to react, and then dropwise add it into a sodium carbonate aqueous solution of Fmoc-Lys-OH to react overnight, and purify to obtain Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH;

[0027] The activating agent is selected from HONb or HOSu, preferably HOSu; the condensing agent is selected from DCC or DIC, preferably DIC; the solvent is selected from THF, DCM or DMF, preferably DCM;

[0028] During the condensation, the base is selected from pyridine, triethylamine or DIPEA, preferably DIPEA; the condensation is carried out for 2 to 4 hours.

[0029] Preferably, in step (1) and step (2), 2-CTC Resin is Fmoc-Aib-2CTC or Fmoc-Aib-2CTC, and the degree of substitution is 0.2 to 0.6 mmol / g, preferably 0.3 to 0.5 mmol / g;

[0030] In step (3), the degree of substitution of Sieber Resin is 0.3 to 0.7 mmol / g, preferably 0.4 to 0.6 mmol / g.

[0031] Preferably, in step (3), compared with Sieber Resin, 3 times the feed is used to couple Fmoc-Pro-Pro-Pro-OH and Fmoc-Gly-Gly-OH, 3.6 times the HOBt / DIC, and the coupling is carried out for 2 to 4 hours, preferably 3 hours; when coupling the remaining amino acids, 3 times the amino acid and 3.6 times the HOBt / DIC system are used, and the coupling is carried out for 1 to 3 hours, preferably 2 hours.

[0032] Preferably, in step (4), the solvent is selected from any one of DMF, DCM, THF, and DMSO, preferably DMF;

[0033] The coupling reagent is selected from any one of HOAt / DIC, PyBOP / HOBt / DIPEA, HATU / HOBt / DIPEA, HATU / HOAt / DIPEA, HATU / HOBt / pyridine, preferably HATU / HOBt / DIPEA;

[0034] The base reagent is selected from any one of pyridine, triethylamine and DIPEA, preferably DIPEA;

[0035] The solvent is selected from any one of DMF, THF, DMSO, and DCM, preferably DMF;

[0036] The feeding multiple of the solvent is 1.05 to 1.2 equivalents, preferably 1.05 equivalents.

[0037] Preferably, in step (5), the coupling reagent is HOSu / DIC or HONb / DIC, preferably HONb / DIC;

[0038] The solvent is selected from any one of DMF, THF, DMSO, and DCM, preferably DMF;

[0039] The feeding multiple of the solvent is 1.05 to 1.2 equivalents, preferably 1.05 equivalents.

[0040] Preferably, in step (1) and step (2), the lysis is specifically as follows: the volume ratio of the lysis solution is TFA:DCM=1:99-5:95 or TFE:DCM=1:3-1:4, preferably TFE:DCM=1:3; lysis is carried out at room temperature for 3h;

[0041] In step (3), the lysis is specifically as follows: the volume ratio of the lysis solution is TFA:DCM=5:95-15:85 or TFE:DCM=1:3-1:4, preferably TFA:DCM=10:95; lysis is carried out at room temperature for 3 hours;

[0042] In step (5), the cleavage is specifically as follows: the cleavage solution is a composition of TFA, TIS, H2O, and PhOMe, preferably with a volume ratio of TFA:TIS:H2O:PhOMe=85:5:5:5; the cleavage solution is 4-10 mL / g, preferably 5 ml / g; the cleavage is performed for 2-3 h, preferably 2 h.

[0043] Compared with the prior art, the advantages of the present invention are:

[0044] (1) The step-by-step coupling process has many problems, such as long cycle, high synthesis risk, low efficiency and difficulty in separating various impurities (the purity of crude peptide is between 50 and 60%). The present invention cleverly uses three fragments, which on the one hand reduces the process production cycle, is conducive to large-scale amplification, and reduces the risk of synthesis; on the other hand, fragmentation is used at difficult sites to reduce the difficulty of coupling and obtain a crude peptide with higher purity (65-70%); at the same time, the impurity spectrum of the obtained crude peptide is better separated from the main peak, which is convenient for purification and preparation, thereby improving the overall yield of the entire process.

[0045] (2) Original process design (15-21): Asp 15 As the N-terminal of the coupling, the steric hindrance is large and the condensation is difficult, resulting in low yields when (1-14) and (15-21) are condensed. 29 ±Gly impurities are present; (30-39): After multiple removal of Fmoc under alkaline conditions, 38 、Pro 37 The side reaction of DPK impurity at the position leads to peptide fragmentation and reduces the yield. 13 The position is sterically hindered and coupling is difficult. 21 -Phe 22 -Val 23 The site will have peptide resin contraction and coupling difficulties. At the same time, the peptide sequence of this fragment is long, and Lys 20 Side chain coupling (AEEA-AEEA-γGlu-C 20 The present invention avoids the above problems by using (1-13) and (14-21) fragments, and cleverly avoids the side reactions of ±Gly impurities and DPK impurities in the synthesis of (22-39) fragments, which lead to peptide breakage and reduce the yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention, in which:

[0047] Figure 1 is the synthetic route of fragment I (1-13) of Example 1;

[0048] Figure 2 is the HPLC chromatogram of fragment I (1-13) in Example 1;

[0049] Figure 3 is the synthesis route diagram of side chain A in Example 2;

[0050] Figure 4 is the HPLC chromatogram of side chain A in Example 2;

[0051] Figure 5 is the synthetic route diagram of fragment II (14-21) of Example 3;

[0052] Figure 6 is the HPLC chromatogram of Fragment II (14-21) of Example 3;

[0053] Figure 7 is the synthetic route diagram of fragment II (14-21) of Example 4;

[0054] Figure 8 is the HPLC chromatogram of Fragment II (14-21) of Example 4;

[0055] Fig. 9 is the synthetic route diagram of fragment III (22-39) of Example 5;

[0056] Fig.10 is the HPLC chromatogram of fragment III (22-39) of Example 5;

[0057] Fig.11 is the synthetic route of crude telportin peptide in Example 7;

[0058] Fig.12 is the HPLC chromatogram of the crude telpotide peptide in Example 7;

[0059] Fig.13 is the HPLC chromatogram of the refined telpotide peptide in Example 7;

[0060] Fig.14 is the mass spectrum of the refined telpotide peptide in Example 7;

[0061] Fig.15 is the HPLC chromatogram of the crude telpotide peptide in Example 8;

[0062] Fig.16 It is the HPLC chromatogram of the refined telpotide peptide in Example 8. DETAILED DESCRIPTION

[0063] The disclosure below provides many different embodiments or examples to implement different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0064] Example 1

[0065] Synthesis of fragment I (1-13):

[0066] Boc-Tyr(tBu)-Aib-Glu(tBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Iie-Aib-COOH

[0067] Take 100g 2-CTC Resin (Sub = 1.2mmol / g), add DMF to swell for 30 minutes, drain, add Fmoc-Aib-OH (32.5g, 100mmol), DIPEA52ml, react for 4 hours, add 300ml methanol to seal, shrink to obtain Fmoc-Aib-2CTC, substitution degree 0.43mmol / g; select 23.25g Fmoc-Aib-2CTC Resin (10mmol), using the classic Fmoc synthesis method, the following amino acid raw materials were coupled in sequence: Fmoc-Iie-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH, Boc-Tyr(tBu)-OH, among which when coupling the first amino acid Fmoc-Iie-OH and the tenth amino acid Fmoc-Aib-OH, 50mmol of feed, 60mmol of PyBOP, 60mmol of HOAt and 65mmol of DIPEA were used, and the reaction was carried out for 5h. The coupling of other amino acids was carried out with 30 mmol amino acid, 36 mmol HOBt and 36 mmol DIC, and the reaction was carried out for 2 h. After the coupling was completed, the mixture was washed with DMF 5 times, DCM 3 times, and then 1 L of methanol was used for 2 times. After drying, 42.7 g of peptide resin was obtained.

[0068] Take 42g of peptide resin, add 500ml of cleavage reagent (TFE: DCM = 1:3 (volume ratio); room temperature, stir and react for 3h, then concentrate to 1 / 4 of the original volume, about 140ml, add 1200 times water, stir overnight, and precipitate solid to obtain fragment I (1-13) 19.2g, purity 92.32% (see Figure 2 ).

[0069] Example 2

[0070] Synthesis of side chain A:

[0071] (Fmoc-Lys[AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu)]-OH)

[0072] Weigh 10 mmol Eic(OtBu)-COOH, add 10.5 mmol HOSu into 200 ml DCM, stir to dissolve, add 10.5 mmol DIC, react at room temperature for 2 h, and obtain Eic(OtBu)-Osu solution for use.

[0073] Add 10.05 mmol H-Glu-OtBu, stir to dissolve, then add 20 mmol DIPEA, react at room temperature for 4 hours to obtain Eic(OtBu)-Glu-OBtu solution, wash with 5% citric acid aqueous solution and saturated sodium chloride aqueous solution three times each, dry with anhydrous sodium sulfate, filter, and the filtrate is set aside.

[0074] Add 10.5mmol HOSu to the above filtrate, add 10.5mmol DIC and stir to react for 3h. Then add 10.5mmol NH2-AEEA-AEEA-COOH and 20mmol DIPEA to react for 8 hours. After the reaction, wash with 5% citric acid aqueous solution and saturated sodium chloride aqueous solution 3 times each, dry with anhydrous sodium sulfate, filter, and concentrate to dryness to obtain an oily substance: Eic(OtBu)-γGlu(AEEA-AEEA)-OtBu.

[0075] 21 g of the above oil was added to 100 ml of DMF to dissolve, and then CDI was added in 5 times, 4 mmol CDI was added each time, and the addition was repeated 5 minutes apart. After the addition was completed, the reaction was stirred for 20 minutes. The pre-activated reaction solution was then added dropwise to a 10.5 mmol sodium carbonate aqueous solution of Fmoc-Lys-OH, and the reaction was carried out at room temperature overnight to obtain a crude product of side chain A: Fmoc-Lys[AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu)]-OH. The crude product of side chain A was purified to obtain a purity of 99.58%. Its HPLC chromatogram is shown in Figure 4 .

[0076] Example 3

[0077] Synthetic fragment II (14-21):

[0078] Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Iie-Ala-Gln(Trt)-Lys(AEEA-AEE A-Glu-C 20 )-Ala-OH

[0079] Weigh Fmoc-Ala-2CTC Resin (sub = 0.45mmol / g, 22.2g, 10mmol); sequentially couple the following amino acid raw materials: Alloc-Lys (Fmoc) -OH as the raw material, 3 times (30mmol) of the raw material, 3.6 times (36mmol) of HOBt / DIC system for coupling, after 2h of coupling reaction, use 20% DBLK solution to remove Fmoc, each time for 7 minutes, remove twice, wash and sequentially couple the following amino acid raw materials: 30mmol Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, tBu-C20-diacid-Glu (AEEA-AEEA-OH) -OtBu, 36mmol HOBt, 36mmol DIC, 4 times (40 mmol) DIPEA system, coupling reaction for 4 hours, after coupling, DMF washing, using 0.2 times (2 mmol) tetrakis triphenylphosphine palladium to remove Alloc, DCM, DMF washing 5 times, sequentially coupled with the following amino acid raw materials: Fmoc-Gln (Trt) -OH, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys (Boc) -OH, Fmoc-Asp (OtBu) -OllH, Fmoc-Leu-OH coupling all use 3 times (30 mmol) amino acid, using 3.6 times (36 mmol) HOBt / DIC system, reaction for 2 hours, and finally 38.5 grams of peptide resin were obtained.

[0080] 38 g of peptide resin was added with 400 ml of lysis solution (TFE:DCM=1:3 V / V), and the mixture was lysed for 3 h at room temperature (20-25° C.), then concentrated to 1 / 4 of the original volume (i.e., about 100 ml), and 800 ml of n-heptane was added. The mixture was stirred overnight, and a solid was precipitated to obtain 18.5 g of fragment II (14-21) with a purity of 89.58%. The HPLC chromatogram is shown in FIG. Figure 6 .

[0081] Example 4

[0082] Synthetic fragment II (14-21):

[0083] Fmoc-Leu-Asp(OtBu)-Lys(Boc)-Iie-Ala-Gln(Trt)-Lys(AEEA-AEE A-Glu-C 20 )-Ala-OH

[0084] Weigh Fmoc-Ala-2CTC Resin (sub = 0.45mmol / g, 22.2g, 10mmol); sequentially couple the following amino acid raw materials: side chain A obtained in Example 2, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, wherein at the second amino acid site of the coupling fragment: Fmoc-Lys(AEEA-AEEA-Glu-C 20 When OtBu)-OH, 2 times the feed (20 mmol), 2.4 times PyBOP / HOAt (24 mmol) and 4 times DIPEA (40 mmol) were used, and the coupling reaction was carried out for 5 hours. The coupling of other amino acids was carried out with 3 times (30 mmol) of amino acid, 3.6 times HOBt / DIC (36 mmol) system, and the reaction was carried out for 2 hours. Finally, 39.2 grams of peptide resin were obtained.

[0085] 39 g of peptide resin was added with 400 ml of lysis solution (TFE:DCM=1:3 V / V), and the mixture was lysed for 3 h at room temperature (20-25° C.), then concentrated to 1 / 4 of the original volume (i.e., about 100 ml), and 800 ml of n-heptane was added. The mixture was stirred overnight, and a solid was precipitated to obtain 17.6 g of fragment II (14-21) with a purity of 89.99%. The HPLC chromatogram is shown in FIG. Figure 8 .

[0086] Example 5

[0087] Synthetic fragment III (22-39):

[0088] NH2-Phe-Val-Trp(Boc)-Leu-Iie-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-CONH2

[0089] Weigh Sieber Resin resin (substitution degree 0.5 mmol / g, 20 g, 10 mmol), remove Fmoc and couple the following amino acid raw materials in sequence according to the classical solid phase synthesis strategy: Fmoc-Ser(tBu)-OH, Fmoc-Pro-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-Ala-OH, Fmoc-Iie-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, and Fmoc-Phe-OH.

[0090] When coupling the tripeptide Fmoc-Pro-Pro-Pro-OH of the 2nd to 4th amino acids of the fragment and the dipeptide Fmoc-Gly-Gly-OH of the 10th to 11th positions of the fragment, 3 times (30 mmol) of feed and 3.6 times (36 mmol) of HOBt / DIC were used, and the coupling reaction was carried out for 3 hours. The coupling of other amino acids was carried out with 3 times (30 mmol) of amino acids and 3.6 times (36 mmol) of HOBt / DIC system, and the reaction was carried out for 2 hours. 44.1 g of peptide resin was obtained.

[0091] Take 44g of peptide resin, add 450g of cleavage solution (TFA:DCM=10:90, V / V); 0-5℃, gradually warm to room temperature, cleave for 3h, then slowly add the reaction solution to 500g of 5% sodium carbonate aqueous solution, separate; wash 3 times with 250 times DCM, combine the DCM layers, concentrate to 20% of the original volume, i.e. 1 / 5 volume (i.e. about 100ml), add to 1000ml of n-heptane, stir, cool to 0℃, precipitate a large amount of solid, filter, wash the filter cake 3 times, 30ml of n-heptane each time. Drain to obtain fragment III (22-39) solid: 21.7g, the purity of the purified fragment III (22-39) is 94.28%, and its HPLC chromatogram is shown in Fig.10 .

[0092] Example 6

[0093] Synthetic fragments (14-39):

[0094] NH2-Leu-Asp(OtBu)-Lys(Boc)-Iie-Ala-Gln(Trt)-Lys(AEEA-AEE A-Glu-C 20)-Ala-Phe-Val-Trp(Boc)-Leu-Iie-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-CONH2

[0095] Take the fragment II (14-21) obtained in Example 3 or Example 4, dissolve 5 mmol in 100 mL of DMF solvent, stir to dissolve, add coupling reagent 6 mmol HATU and 6 mmol HOBt, pre-react for 30 min, then add 50 mL of the fragment III (22-39) (5.05 mmol) obtained in Example 6 DMF solution, add alkaline reagent DIPEA (8mmol), stir and react for 2h, perform HPLC to monitor the reaction, when the fragment (14-21) peak is less than 1%, judge that the reaction is basically completed, add 15mL of 20% piperidine-DMF solution, stir for 20min, obtain fragment (14-39) solution, freeze the reaction solution, then add it into 200ml citric acid aqueous solution, extract with ethyl acetate several times, wash the obtained organic phase with citric acid aqueous solution, sodium carbonate aqueous solution, saturated sodium chloride aqueous solution, dry the organic phase with anhydrous sodium sulfate, concentrate to about 50ml, add 500ml of n-heptane dropwise, stir to precipitate solid, filter, wash the filter cake with n-heptane 3 times (20ml / time), and dry under reduced pressure at 40℃ to obtain about 17.2g of solid.

[0096] Example 7: Synthesis of Telportide

[0097] The fragment I (1-13) (5.5 mmol) obtained in Example 1 was dissolved in 100 ml of DCM solution, and HONb (6 mmol) and DIC (6 mmol) were added. After stirring for 2 hours, HPLC monitoring was performed. The chromatographic peak of (1-13) was less than 1%. A 5% citric acid aqueous solution and a saturated sodium chloride aqueous solution were added to wash twice. The organic layer was collected and dried over anhydrous sodium sulfide. The DCM layer was taken and the DCM solution of the fragment (14-39) (5 mmol) obtained in Example 6 was added. The mixture was stirred to clarify. DIPEA was added and the mixture was stirred for 2 hours. HPLC monitoring was performed. The chromatographic peak of (14-39) was less than 1%. After the reaction was completed, a 5% citric acid aqueous solution and a saturated sodium chloride aqueous solution were added to wash twice. The organic layer was collected and dried over anhydrous sodium sulfide. The DCM layer was concentrated. After concentration, 5 mL / g of cleavage solution was added for cleavage (the cleavage solution was TFA::TIS:H2O:PhOMe=85:5:5:5 (V:V:V:V).

[0098] After 2 hours of cleavage reaction, the mixture was added to 10 times the volume of tertiary methyl ether to precipitate a solid. The solid was dried under reduced pressure at 30°C to obtain about 18.7 g of crude peptide, with a weight yield of about 77.7%, a content of about 80%, a content yield of 62.16%, and a crude peptide purity of 72.65%. The HPLC chromatogram is shown in Fig.12 .

[0099] The crude peptide was purified by reverse phase high performance liquid chromatography and freeze dried to obtain refined telpotide peptide, the purity of which was 99.68%. Fig.13 , and its mass spectrum is shown in Fig.14 .

[0100] The crude peptide synthesis diagram of telportopeptide is shown in Figure 2. Fig.11 As shown in the figure, fragments I, II and III are all replaced by simplified formulas. Among them, the simplified formula of fragment I (1-13) is: Boc-Y-Aib-EGTFTSDYSI-Aib-COOH; the simplified formula of fragment II (14-21) is: Fomc-LDKIANK(X)-A-COOH; the simplified formula of fragment III (22-39) is: NH2-FVWLIAGGPSSGAPPPS-CONH2.

[0101] Example 8: Synthesis of Telportide

[0102] The fragment I (1-13) (5.5 mmol) obtained in Example 1 was dissolved in 100 ml of DMF solution, and HOSu (6 mmol) and DIC (6 mmol) were added. After stirring for 2 hours, the chromatographic peak of (1-13) was monitored by HPLC. The chromatographic peak of (1-13) was less than 1%. The DMF solution of the fragment (14-39) (5 mmol) obtained in Example 6 was added, stirred to clarify, and DIPEA was added. After stirring for 2 hours, the chromatographic peak of (14-39) was monitored by HPLC. After the reaction was completed, the solid was added to a frozen 5% citric acid aqueous solution, and the solid was precipitated. It was filtered, washed with purified water 3 times, and dried at 40° C. to obtain 42 g.

[0103] Add 350 ml of lysis buffer (lysis buffer is TFA::TIS:H2O:PhOMe=90:4:4:2 (V:V:V:V).

[0104] After 2 hours of cleavage reaction, the mixture was added to 8 times the volume of methyl tert-ether to precipitate a solid. The solid was dried under reduced pressure at 30°C to obtain about 19.1 g of crude peptide, with a weight yield of about 79.4%, a content of about 79%, a content yield of 62.72%, and a crude peptide purity of 76.26%. The HPLC chromatogram is shown in Fig.15 . .

[0105] The crude peptide was purified by reverse phase high performance liquid chromatography and freeze dried to obtain refined telportin peptide, the purity of which was 99.38%. Fig.16 .

[0106] The abbreviations or English meanings of the reagents or raw materials used in the present invention are shown in Table 1.

[0107] Table 1 Abbreviations and English meanings

[0108]

[0109]

[0110]

[0111] Those skilled in the art will readily appreciate other embodiments of the invention after considering the specification and practicing the invention herein. The invention is intended to cover any variations, uses or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art of the invention. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0112] It should be understood that the present invention is not limited to the exact construction that has been described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for synthesizing tepote, characterized in that: The following steps are involved: First, fragment I (1-13), fragment II (14-21), and fragment III (22-39) are synthesized separately, and then fragment II (14-21) and fragment III (22-39) are used to synthesize fragment (14-39) in liquid phase. Finally, fragment I (1-13) and fragment (14-39) are condensed, cracked, and purified in liquid phase to prepare telportipeptide.

2. The method for synthesizing telpotide according to claim 1, characterized in that: The specific steps include: (1) Synthetic fragment I (1-13): Fmoc-Iie-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Aib-OH and Boc-Tyr(tBu)-OH were sequentially coupled to 2-CTC Resin and cleaved to prepare the fragment I (1-13); (2) Synthetic fragment II (14-21): Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Leu-OH were sequentially coupled to 2-CTC Resin, and cleaved to prepare the fragment II (14-21); Alternatively, Alloc-Lys(Fmoc)-OH is coupled to 2-CTC Resin, and then Fmoc is removed. After washing, 2-3 times of Eic(OtBu)-γGlu(AEEA-AEEA)-OtBu, 2.4-3.6 times of PyBOP / HOAt, and 4-6 times of DIPEA system are added. After coupling, Alloc is removed, and after washing, Fmoc-AEEA-OH, Fmoc-AEEA-OH, Fmoc-Glu-OtBu, tBu-C 20 –diacid-Glu(AEEA-AEEA-OH)-OtBu, remove Alloc, wash and then sequentially couple with Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Iie-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Leu-OH, and cleave to produce the fragment II (14-21); (3) Synthetic fragment III (22-39): Fmoc-Ser(tBu)-OH, Fmoc-Pro-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-Ala-OH, Fmoc-Iie-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH and Fmoc-Phe-OH were sequentially coupled to Sieber Resin and cleaved to prepare the fragment III (22-39); (4) Synthetic fragments (14-39): Dissolve the fragment II (14-21) in a solvent, add a coupling reagent for pre-reaction, then add the fragment III (22-39) and an alkali reagent, and stir to react; after the reaction, add diethylamine, stir, extract, and concentrate to obtain the fragment (14-39); (5) Synthesis of tepote The fragment I (1-13) is dissolved in a solvent, a coupling reagent is added, and the reaction is stirred. After the reaction is completed, the organic layer is washed, collected, and dried. Then, a DMF solution of the fragment (14-39) is added, stirred to clarify, and DIPEA is added, and the reaction is stirred. After the reaction is completed, extraction, concentration, cleavage, precipitation, purification, and freeze-drying are performed to prepare the tepoxetine.

3. The method for synthesizing telpotide according to claim 2, characterized in that: In step (1), compared with the 2-CTC Resin, 5 times the feed is used to couple the Fmoc-Iie-OH and the Fmoc-Aib-OH, 6 times the PyBOP / HOAt and 6.5 times the DIPEA, and the coupling is carried out for 4 to 6 hours, preferably 5 hours; when coupling the remaining amino acids, 3 times the amino acids and 3.6 times the HOBt / DIC system are used, and the coupling is carried out for 1 to 3 hours, preferably 2 hours.

4. The method for synthesizing telpotide according to claim 2, characterized in that: In step (2), compared with the 2-CTC Resin, the Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH, 2.4 times PyBOP / HOAt and 4 times DIPEA are used for coupling for 4 to 6 hours, preferably 5 hours; when coupling the remaining amino acids, 3 times the amino acids and 3.6 times the HOBt / DIC system are used for coupling for 1 to 3 hours, preferably 2 hours.

5. The method for synthesizing telpotide according to claim 2, characterized in that: In step (2), the synthesis of Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH is specifically as follows: Dissolve Eic(OtBu)-COOH in a solvent and add an activating agent and a condensing agent to react at room temperature for 2 hours to prepare an Eic(OtBu)-OSu solution, and then condense with H-Glu-OtBu and DIPEA for 4 hours to obtain Eic(OtBu)-Glu-OBtu; wash, dry, filter, and obtain a filtrate, add the activating agent and the condensing agent to stir and react for 3 hours, and then add NH2-AEEA-AEEA-COOH to condense for 8 hours; wash, dry, filter, and concentrate to obtain an oily substance, dissolve it in a solvent, add a condensing agent to react, and then dropwise add it into a sodium carbonate aqueous solution of Fmoc-Lys-OH to react overnight, and purify to obtain the Fmoc-Lys(AEEA-AEEA-γ-Glu(OtBu)-Eic(OtBu))-OH; The activation reagent is selected from HONb or HOSu, preferably HOSu; the condensing agent is selected from DCC or DIC, preferably DIC; the solvent is selected from THF, DCM or DMF, preferably DCM; During the condensation, the base is selected from pyridine, triethylamine or DIPEA, preferably DIPEA; the condensation is carried out for 2 to 4 hours.

6. The method for synthesizing telpotide according to claim 2, characterized in that: In step (1) and step (2), the 2-CTC Resin is Fmoc-Aib-2CTC or Fmoc-Aib-2CTC, and the degree of substitution is 0.2 to 0.6 mmol / g, preferably 0.3 to 0.5 mmol / g; In step (3), the substitution degree of Sieber Resin is 0.3 to 0.7 mmol / g, preferably 0.4 to 0.6 mmol / g.

7. The method for synthesizing telpotide according to claim 2, characterized in that: In step (3), compared with the Sieber Resin, the Fmoc-Pro-Pro-Pro-OH and the Fmoc-Gly-Gly-OH are coupled using 3 times the feed, 3.6 times the HOBt / DIC, and the coupling is carried out for 2 to 4 hours, preferably 3 hours; when coupling the remaining amino acids, 3 times the amino acids and 3.6 times the HOBt / DIC system are used, and the coupling is carried out for 1 to 3 hours, preferably 2 hours.

8. The method for synthesizing telpotide according to claim 2, characterized in that: In step (4), the solvent is selected from any one of DMF, DCM, THF, and DMSO, preferably DMF; The coupling reagent is selected from any one of HOAt / DIC, PyBOP / HOBt / DIPEA, HATU / HOBt / DIPEA, HATU / HOAt / DIPEA, HATU / HOBt / pyridine, preferably HATU / HOBt / DIPEA; The alkaline reagent is selected from any one of pyridine, triethylamine and DIPEA, preferably DIPEA; The solvent is selected from any one of DMF, THF, DMSO, and DCM, preferably DMF; The feeding multiple of the solvent is 1.05 to 1.2 equivalents, preferably 1.05 equivalents.

9. The method for synthesizing telpotide according to claim 2, characterized in that: In step (5), the coupling reagent is HOSu / DIC or HONb / DIC, preferably HONb / DIC; The solvent is selected from any one of DMF, THF, DMSO, and DCM, preferably DMF; The feeding multiple of the solvent is 1.05 to 1.2 equivalents, preferably 1.05 equivalents.

10. The method for synthesizing telpotide according to claim 1, characterized in that: In step (1) and step (2), the cleavage is specifically as follows: the volume ratio of the cleavage solution is TFA:DCM=1:99-5:95 or TFE:DCM=1:3-1:4, preferably TFE:DCM=1:3; cleavage is carried out at room temperature for 3h; In step (3), the cleavage is specifically as follows: the volume ratio of the cleavage solution is TFA:DCM=5:95-15:85 or TFE:DCM=1:3-1:4, preferably TFA:DCM=10:95; cleavage at room temperature for 3h; In step (5), the cleavage is specifically as follows: the cleavage solution is a composition of TFA, TIS, H2O, and PhOMe, preferably with a volume ratio of TFA:TIS:H2O:PhOMe=85:5:5:5; the cleavage solution is 4-10 mL / g, preferably 5 ml / g; the cleavage is performed for 2-3 h, preferably 2 h.

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