Method for preparing amylin analog

Abstract

The present invention relates to the technical field of polypeptide drugs, and in particular, to a method for preparing an amylin analog. The method for preparing an amylin analog comprises: (a) sequentially coupling, from the C-terminus to the N-terminus according to a peptide sequence of the amylin analog, various Fmoc-protected amino acids and 20-(tert-butoxy)-20-oxoicosanoic acid to a resin by using a solid-phase synthesis method to obtain a linear peptide resin; and (b) removing side-chain protecting groups of Asp at position 3 and Lys at position 8 of the linear peptide resin, performing a cyclization reaction to form a lactam bridge, and performing a cleavage treatment to obtain the amylin analog. When coupling Gly at position 4 in the peptide sequence, the Fmoc-protected amino acid used is selected from any one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH. In the method for preparing an amylin analog, the probability of side reactions is significantly reduced, the product purity is improved, and the purification difficulty is lowered without increasing the number of reaction steps.

Description

Process for preparing amylin analogs

[0001] Cross-reference to related applications

[0002] This application claims priority to the Chinese patent application No. 202411677614.3, filed on November 22, 2024, and entitled “Process for preparing amylin analogs”, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the technical field of polypeptide drugs, in particular to a process for preparing amylin analogs. BACKGROUND

[0004] Amylin, also known as islet amyloid polypeptide, is a small hormone secreted by islet beta cells. It is another hormone with hypoglycemic effect in addition to insulin in the body. Amylin is composed of 37 amino acid residues, with a relative molecular weight of 3.85 KD, and will form aggregates under metabolic conditions of insulin deficiency, which is one of the pathological characteristics of type 2 diabetes.

[0005] Amylin analogs are drugs that change the amino acid sequence or spatial conformation of amylin to fully retain its physiological effects and improve its physical properties. Developing different types of amylin analogs provides new drug candidates for the treatment of related diseases.

[0006] An existing amylin analog has the following peptide sequence:

[0007] Eicosadioic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24-N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2, forming a disulfide bridge or a lactam bridge between Asp at position 3 and Lys at position 8 (icosadioic acid-γGlu-R-D-G-T-A-T-K-A-T-E-R-L-A-Aad-F-L-Q-R-S-S-F-Sar-A-N-Me-Ile-L-S-S-T-E-V-G-S-N-T-HyP-NH2). Replacing the disulfide bridge with a lactam bridge can significantly improve the stability of the amylin analogue and retain other beneficial properties of the amylin analogue. However, using the existing method for preparing the amylin analogue, the crude product is complex and difficult to purify, and the yield is low.

[0008] Therefore, the present application is provided. SUMMARY

[0009] To solve any one or more of the above deficiencies in the synthesis of amylin analogues, the present application provides a method for preparing an amylin analogue.

[0010] In one aspect, the present application provides a method for preparing an amylin analogue, comprising the following steps:

[0011] (a) using a solid-phase synthesis method, coupling each Fmoc-protected amino acid and icosadioic acid mono-tert-butyl ester to a resin from C-terminal to N-terminal according to the peptide sequence of the amylin analogue to obtain a straight-chain peptide resin;

[0012] (b) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the straight-chain peptide resin, forming a lactam bridge by cyclization reaction, and obtaining the amylin analogue by cleavage treatment;

[0013] The peptide sequence of the amylin analogue is as follows:

[0014] icosadioic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2, forming a lactam bridge between Asp at position 3 and Lys at position 8;

[0015] wherein, when the Gly at position 4 in the coupling peptide sequence, the Fmoc-protected amino acid used is at least one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH.

[0016] In the detailed description of the present application, the cleavage treatment is carried out in a cleavage reagent; the cleavage reagent is mainly prepared from TFA, DODT, TIS and water. Preferably, in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92):(4-6):(2-3):(2-3).

[0017] In the detailed description of the present application, the ratio of the amount of the cleavage reagent to the resin is (75-85)mL:1mmol.

[0018] In the detailed description of the present application, the reaction time of the cleavage treatment is 2.5-3h.

[0019] In the detailed description of the application, when coupling the Asp at the 3rd position in the peptide sequence, any one of Fmoc-Asp(OAll)-OH, Fmoc-Asp(OMpe)-OH and Fmoc-Asp(OPp)-OH is used as the Fmoc-protected amino acid; when coupling the Lys at the 8th position in the peptide sequence, any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH is used as the Fmoc-protected amino acid.

[0020] In the detailed description of the application, the Fmoc-protected amino acids are respectively: Fmoc-Hyp(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Dmb)Gly-OH, Fmoc-Asp(OAll)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Glu-OtBu.

[0021] In the detailed description of the application, the resin comprises at least one of Rink Amide-AM resin (CAS No.: 183599-10-2), Rink Amide MBHA resin (CAS No.: 431041-83-7) and Sieber resin (CAS No.: 915706-90-0). Preferably, the degree of substitution of the resin is 0.3-0.7 mmol / g.

[0022] In the detailed description of the application, in each coupling, a deprotection reaction and a coupling reaction are included; the deprotection reaction is carried out by using a deprotection reagent, the deprotection reagent comprises an aprotic organic solvent containing an organic base, the organic base comprises pip, and the aprotic organic solvent comprises at least one of DCM, DMF and NMP. Preferably, in the deprotection reagent, the volume fraction of the organic base is 15%-25%.

[0023] In the detailed description of the application, the deprotection reaction is carried out for 0.5-1h at 20-30℃.

[0024] In the detailed description of the application, the ratio of the amount of the deprotection reagent to the resin is (38-50)mL:1mmol. Preferably, after the deprotection reaction, a washing step is further included; the ratio of the amount of the solvent for single washing to the resin is (19-27)mL:1mmol; and the number of washing is 5-6 times.

[0025] In the detailed description of the application, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid or the eicosadioic acid mono-tert-butyl ester to the resin is (2-4):1.

[0026] In the detailed description of the application, in the coupling reaction, the condensing agent used comprises at least one of Oxyma / DIC, HOBt / DIC and PyBop / DIEA.

[0027] In the detailed description of the application, the coupling reaction is carried out for 1-2h at 25-35℃.

[0028] In the detailed description of the application, after the coupling reaction, a washing step is further included; the ratio of the amount of the solvent for single washing to the resin is (19-27)mL:1mmol; and the number of washing is 3-4 times.

[0029] In the detailed description of the application, in step (b), a DCM solution containing tetrakis(triphenylphosphine)palladium, phenylsilane and NMM is used to remove the side chain protecting groups of Asp at position 3 and Lys at position 8 of the linear peptide resin. Preferably, the molar ratio of the tetrakis(triphenylphosphine)palladium, the phenylsilane and the NMM is 0.1:(5.5-6.5):(6.5-7).

[0030] In the detailed description of the application, in step (b), the cyclization reaction comprises: under the action of PyBop and DIEA, the side chain of the deprotection group of Asp at position 3 reacts with the side chain of the deprotection group of Lys at position 8 to form a lactam bridge.

[0031] In the detailed description of the application, it further comprises collecting the solution after the cleavage treatment, adding a poor solvent to precipitate and collect the solid and wash it; the poor solvent comprises MTBE.

[0032] In the detailed description of the application, it further comprises purifying the solid by reverse phase chromatography. Preferably, in the reverse phase chromatography, mobile phase A is 0.1% TFA in water and mobile phase B is acetonitrile.

[0033] In the detailed description of the application, in the reverse phase chromatography, the gradient elution comprises increasing the volume fraction of mobile phase B from 20% to 50% in 60 min.

[0034] Another aspect of the application provides a preparation method of amylin analogues, comprising the following steps:

[0035] (A) using solid phase synthesis, coupling each Fmoc-protected amino acid to the resin from C-terminal to N-terminal according to the peptide sequence of the amylin analogue to form a polypeptide resin fragment as shown in formula I below:

[0036] (B) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the polypeptide resin fragment, and forming a lactam bridge by cyclization reaction;

[0037] (C) continuing to complete the coupling of the remaining Fmoc-protected amino acids and eicosadioic acid mono-tert-butyl ester according to the peptide sequence of the amylin analogue, and then cleaving to obtain the amylin analogue;

[0038] Fmoc-Asp 3 (OAll)-Gly 4 (Dmb / Hmb)-Thr 5 (tBu)-Ala 6 -Thr 7 (tBu)-Lys 8 (Alloc)-Ala 9 -Thr 10 (tBu)-Glu 11 (OtBu)-Arg 12 (Pbf)-Leu 13 -Ala 14 -Aad 15 (OtBu)-Phe 16 -Leu 17 -Gln 18 (Trt)-Arg 19 (Pbf)-Ser 20 (tBu)-Ser 2 (tBu)1 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 (tBu)-Ser 28 (tBu)-Thr 29 (tBu)-Glu 30 (OtBu)-Val 31 -Gly 32 (Dmb / Hmb)-Ser 33 (tBu)-Asn 34 (Trt)-Thr 35 (tBu)-Hyp 36 (tBu)-resin (I);

[0039] The peptide sequence of the amylin analogue is as follows:

[0040] Eicosadioic acid - γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35-HyP 36 -NH2, forming a lactam bridge between the 3rd Asp and the 8th Lys.

[0041] Compared with the prior art, the application has the following advantages:

[0042] (1) In the preparation method of the amylin analogue of the application, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used for coupling of Gly 4 , and then the side chain protection groups of Asp 3 and Lys 8 are removed and cyclization is carried out, and then the side chain protection group of Gly 4 is removed and the resin is removed in the cleavage process, to obtain the amylin analogue. In this way, the probability of side reactions is significantly reduced, the purity of the product is improved, and the difficulty of purification is reduced without increasing the reaction steps.

[0043] (2) The preparation method of the amylin analogue of the application can be scaled up for production, which can reduce the production cost and has important significance. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions in the specific embodiments or the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings described below are some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0045] Figure 1 is the HPLC spectrum of the crude product obtained by the preparation method of the amylin analogue provided in Example 1 of the application without reverse phase chromatography purification;

[0046] Figure 2 is the HPLC spectrum of the crude product obtained by the preparation method of the amylin analogue provided in Comparative Example 1 without reverse phase chromatography purification. DETAILED DESCRIPTION

[0047] The technical solutions of the application will be described clearly and completely in combination with the drawings and specific embodiments, but those skilled in the art will understand that the following described embodiments are part of the embodiments of the application, not all the embodiments, and should not be regarded as limiting the scope of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application. The specific conditions are not specified in the embodiments, and are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be purchased on the market.

[0048] The meanings of the English abbreviations involved in the present application are shown in Table 1.

[0049] Table 1 meanings of English abbreviations

[0050] In the prior art synthesis method of the amylin analogue of the present application, the whole peptide chain is usually coupled by using a solid-phase synthesis strategy. After complete coupling, the special protecting group is removed, and then cyclization is performed to form an amide bond connection. Then, the protecting group and the resin are cleaved to obtain the peptide chain. However, this synthesis method has many by-products, and the obtained crude product has complex components, is not easy to purify, and has a low yield.

[0051] The present application found through research that, in the cyclization of Asp 3 and Lys 8 (i.e., D-K cyclization) designed by using the existing synthesis route, Asp 3 and Gly 4 cyclization (i.e., D-G cyclization) is more likely to occur. The peptide chain obtained by D-G cyclization does not have biological activity. The occurrence of by-products leads to very complex components of the crude product. Since the molecular weight of the product obtained by D-G cyclization is the same as that of the target product, it is not easy to be found by analysis and detection, resulting in great difficulty in purification, and further reducing the yield.

[0052] In the present application, Fmoc-(Dmb)Gly-OH / Fmoc-(Hmb)Gly-OH is used as the reaction material when coupling Gly 4 , which inhibits the D-G cyclization side reaction. Moreover, the present application does not increase additional reaction steps, thereby ensuring production efficiency.

[0053] Based on this, one aspect of the present application provides a preparation method of an amylin analogue, comprising the following steps:

[0054] (a) using a solid-phase synthesis method, coupling each Fmoc-protected amino acid and eicosadioic acid mono-tert-butyl ester to the resin from the C-terminal to the N-terminal according to the peptide sequence of the amylin analogue, to obtain a straight-chain peptide resin;

[0055] (b) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the straight-chain peptide resin, performing cyclization reaction to form a lactam bridge, and performing cleavage treatment to obtain the amylin analogue;

[0056] The peptide sequence of the amylin analogue is as follows:

[0057] Eicosadioic acid-γGlu 1 -Arg 2 - Asp 3 -Gly 4 -Thr 8 -Ala6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2, forming a lactam bridge between Asp at position 3 and Lys at position 8;

[0058] wherein, when the Gly at position 4 in the coupling peptide sequence, the Fmoc-protected amino acid used is at least one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH.

[0059] The inventors of the present application found in the research that the crude product obtained by stepwise coupling and cyclization according to the conventional method and then cleavage has complex components, and the main component is not easy to purify and has low yield. However, at present, there is no exploration on the reason why the crude product of the amylin analogue has complex components. The present application found through the research on the components of the crude product obtained by cleavage according to the conventional method that when the conventional method is used to couple Gly 4 , Fmoc-Gly-OH is used for coupling, and after removing the special protecting groups of Asp at position 3 and Lys at position 8, the naked carboxyl group of Asp is more likely to attack the secondary amine group of Gly 4 to obtain the D-G cyclization product. The formation of the D-G cyclization product leads to a significant decrease in product yield and purity on the one hand, and on the other hand, the D-G cyclization product formed after cleavage has the same molecular weight as the target product and similar structure, which increases the difficulty of separation and purification. Based on the discovery of this problem, the present application found that in the process of preparing the amylin analogue, when Gly4 When Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the reactant, the Dmb or Hmb protecting group occupies the secondary amine hydrogen on Gly, significantly reducing its cyclization activity, and cooperating with the cleavage reagent used in the cleavage step, the protecting group and the pendant can be effectively removed, significantly improving the yield and purity.

[0060] In the specific embodiments of the present application, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the reactant when coupling Gly 4 When Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the reactant, the Dmb or Hmb protecting group occupies the secondary amine hydrogen on Gly 4 , introduces a special protecting group at Gly , protects the alpha amino group, inhibits the D-G cyclization side reaction, reduces the D-G cyclization byproduct, improves the purity and yield of the crude product, and simplifies the subsequent purification operation. Further research shows that compared with Fmoc-(Hmb)Gly-OH, Dmb in Fmoc-(Dmb)Gly-OH has a larger spatial structure than Hmb, which can better inhibit the D-G cyclization side reaction, and the crude product obtained by cleavage has higher purity. Moreover, the present application does not increase additional reaction steps, ensuring production efficiency.

[0061] In the specific embodiments of the present application, the cleavage treatment is carried out in a cleavage reagent; the cleavage reagent is mainly prepared from TFA, DODT, TIS and water. Preferably, in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92):(4-6):(2-3):(2-3).

[0062] In some embodiments, in the cleavage reagent, the volume ratio of TFA to water can be 88:2, 90:2, 92:2, 88:3, 90:3, 92:3, or a range consisting of any two of them; the volume ratio of DODT to water can be 4:2, 5:2, 6:2, 4:3, 5:3, or a range consisting of any two of them; the volume ratio of TIS to water can be 2:2, 2.5:2, 3:2, 2:2.5, 3:2.5, 2:3, 2.5:3, or a range consisting of any two of them. In the specific embodiments of the present application, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the 4 reactant when coupling Gly , and cooperating with the cleavage reagent used in the cleavage step, the protecting group can be effectively removed, and the yield and purity can be significantly improved.

[0063] In some embodiments, in the cleavage reagent, the volume ratio of TFA, DODT, and water is 90:5:2.5.

[0064] In the specific embodiments of the present application, the ratio of the amount of cleavage reagent to resin is (75-85) mL:1 mmol.

[0065] Herein (75-85) mL: 1 mmol means that the amount of cleavage reagent can be 75-85 mL compared to 1 mmol of resin, which is a limitation on the ratio of the amount of cleavage reagent and resin, rather than a limitation on the amount of single use. In some embodiments, the amount of cleavage reagent can be 75 mL, 78 mL, 80 mL, 82 mL, 85 mL, or a range consisting of any two of them compared to 1 mmol of resin.

[0066] In the specific embodiments of the present application, the reaction time of the cleavage treatment is 2.5-3 h.

[0067] In actual operation, after the cleavage treatment, a filtration treatment is performed, the filter cake is washed with a small amount of TFA, and the filtrates are combined.

[0068] In the specific embodiments of the present application, when coupling Asp at the 3rd position in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Asp(OAll)-OH and Fmoc-Asp(OPp)-OH; when coupling Lys at the 8th position in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH.

[0069] ​In the detailed description of the application, each Fmoc-protected amino acid is Fmoc-Hyp(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Hmb)Gly-OH or Fmoc-(Dmb)Gly-OH, Fmoc-Asp(OAll)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Glu-OtBu, respectively.

[0070] In the detailed description of the application, each Fmoc-protected amino acid is Fmoc-Hyp(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Hmb)Gly-OH or Fmoc-(Dmb)Gly-OH, Fmoc-Asp(OAll)-OH, Fmoc-Arg-Pbf)-OH and Fmoc-Glu-OtBu, respectively.

[0071] In the detailed description of the application, the resin comprises at least one of Rink Amide-AM resin, Rink Amide MBHA resin and Sieber resin. Preferably, the substitution degree of the resin is 0.3-0.7 mmol / g.

[0072] In some embodiments, the substitution degree of the resin can be 0.3 mmol / g, 0.4 mmol / g, 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g or a range consisting of any two of them.

[0073] In some embodiments, the resin is swollen before coupling with the first Fmoc-protected amino acid. DMF can be used for swelling, but is not limited thereto.

[0074] In the specific embodiments of the present application, each coupling includes a deprotection reaction and a coupling reaction; the deprotection reaction is performed using a deprotection reagent, which includes an aprotic organic solvent containing an organic base, and the organic base includes pip, and the aprotic organic solvent includes at least one of DCM, DMF and NMP. Preferably, the volume fraction of the organic base in the deprotection reagent is 15%-25%. In some embodiments, the deprotection reagent is a DMF solution containing pip, and the volume fraction of pip is 20%.

[0075] In the specific embodiments of the present application, the deprotection reaction is performed for 0.5-1 h at a temperature of 20-30°C.

[0076] In the specific embodiments of the present application, the ratio of the amount of the deprotection reagent to the resin is (38-50) mL: 1 mmol. Preferably, after the deprotection reaction, a washing step is further included; the ratio of the amount of the solvent used for single washing to the resin is (19-27) mL: 1 mmol; and the number of washing times is 5-6.

[0077] In some embodiments, the amount of the deprotection reagent can be 38 mL, 40 mL, 42 mL, 45 mL, 48 mL, 50 mL or a range formed by any two of them, compared to 1 mmol of the resin; and the amount of the solvent used for single washing after the deprotection reaction can be 19 mL, 20 mL, 22 mL, 24 mL, 25 mL, 27 mL or a range formed by any two of them, compared to 1 mmol of the resin.

[0078] In actual operation, the amount of the deprotection reagent and the amount of the solvent used for washing are adjusted according to the number of the coupled amino acids, so as to ensure the purity and yield. For example, when the number of the amino acids in the peptide chain increases by 10, the amount of the deprotection reagent is increased by 4 mL and the amount of the solvent used for washing is increased by 2 mL, compared to 1 mmol of the resin.

[0079] In the specific embodiments of the present application, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid or dodecanedioic acid mono-tert-butyl ester to the resin is (2-4): 1.

[0080] In some embodiments, the molar ratio of Fmoc-protected amino acid or eicosanedioic mono-tert-butyl ester to resin in the coupling reaction can be 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1, 3.2:1, 3.5:1, 3.8:1, 4:1, or a range consisting of any two of them.

[0081] In the specific embodiments of the present application, the condensing agent used in the coupling reaction includes at least one of Oxyma / DIC, HOBt / DIC, and PyBop / DIEA.

[0082] In the specific embodiments of the present application, the time of the coupling reaction is 1-2 h; and the temperature of the coupling reaction is 25-35°C.

[0083] In the specific embodiments of the present application, after the coupling reaction, a washing step is further included; the ratio of the amount of solvent used in single washing to the resin is (19-27) mL:1 mmol; and the number of washing is 3-4 times.

[0084] In some embodiments, the amount of solvent used in single washing can be 19 mL, 20 mL, 22 mL, 24 mL, 25 mL, 27 mL, or a range consisting of any two of them, compared to 1 mmol of resin. In actual operation, the amount of solvent used in washing is adjusted according to the number of coupled amino acids, so as to ensure the purity and yield. For example, when the number of amino acids in the peptide chain increases by 10, the amount of solvent used in washing increases by 2 mL compared to 1 mmol of resin.

[0085] In actual operation, in each coupling of step (a), the step of deprotection reaction can include: placing the resin in a deprotection reagent, mixing at 20-30°C for 0.5-1 h, and then washing with a solvent; and the washing solvent can include but is not limited to DMF. In each coupling of step (b), the step of coupling reaction can include: mixing Fmoc-protected amino acid and Oxyma in a solvent, then adding DIC, and activating for 5-10 min to obtain an activation solution; adding the activation solution to the resin after the deprotection reaction, and reacting at 25-35°C for 1-2 h, and then washing with a solvent; and the washing solvent can include but is not limited

[0086] In the specific embodiments of the present application, in step (b), a DCM solution containing tetrakis(triphenylphosphine)palladium, phenylsilane, and NMM is used to remove the side chain protection groups of Asp at position 3 and Lys at position 8 of the linear peptide resin. Preferably, the molar ratio of tetrakis(triphenylphosphine)palladium, phenylsilane, to NMM is 0.1:(5.5-6.5):(6.5-7). Wherein, the amount of DCM used is 25-35 mL per 1 mmol of resin.

[0087] In a specific embodiment of this application, step (b) includes the cyclization reaction in which, under the action of PyBop and DIEA, the 3-position Asp side chain with the protection group removed reacts with the 8-position Lys side chain with the protection group removed to form a lactam bridge.

[0088] In some embodiments, the ratio of PyBop to DIEA is 14.82-16.38 g : 9.03-9.98 mL. The solvent for the cyclization reaction may include, but is not limited to, DMF.

[0089] In practice, the cyclization reaction to form a lactam bridge may include: adding PyBop to the reaction vessel, adding DMF and mixing with the resin, adding DIEA, mixing at 20-30℃ for 1.5-2.5h, and washing.

[0090] In some embodiments, the washing after the cyclization reaction includes: washing 3-4 times with DMF, followed by washing 2-3 times alternately with DCM and MTBE. The amount of DMF used in a single wash, relative to 1 mmol of resin, can be 25-35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL, or any combination thereof; the amount of DCM used in a single wash, relative to 1 mmol of resin, can be 25-35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL, or any combination thereof; the amount of MTBE used in a single wash, relative to 1 mmol of resin, can be 25-35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL, or any combination thereof.

[0091] In a specific embodiment of this application, in steps (a) and (b), the progress of coupling reactions, cyclization reactions, etc., is monitored by ninhydrin detection. The reaction time is adjusted based on the ninhydrin detection results.

[0092] In a specific embodiment of this application, the method further includes: collecting the solution after pyrolysis, adding a poor solvent to precipitate the precipitate, collecting the solid and washing it; the poor solvent includes MTBE.

[0093] In some embodiments, the volume ratio of the solution after pyrolysis to the undesirable solvent is 1:(7-10), such as 1:7, 1:8, 1:9, 1:10, or any combination thereof.

[0094] In a specific embodiment of this application, the method further includes purifying the solid using reversed-phase chromatography. Preferably, in the reversed-phase chromatography, mobile phase A is an aqueous solution of TFA with a volume fraction of 0.1%, and mobile phase B is acetonitrile.

[0095] In a specific embodiment of this application, in reversed-phase chromatography, gradient elution includes increasing the volume fraction of mobile phase B from 20% to 50% within 60 minutes.

[0096] The method for preparing amylin analogues disclosed in this application significantly reduces the probability of side reactions, improves product purity, and reduces purification difficulty. Products with the required purity can be obtained through a simple purification method.

[0097] This application provides another method for preparing amylin analogues, comprising the following steps:

[0098] (A) Using a solid-phase synthesis method, each Fmoc-protected amino acid is coupled to the resin sequentially from the C-terminus to the N-terminus according to the peptide sequence of the amylin analogue to form a polypeptide resin fragment as shown in Formula I below.

[0099] (B) Remove the side chain protecting groups at the 3-position Asp and the 8-position Lys of the polypeptide resin fragment, and cyclize to form a lactam bridge.

[0100] (C) Continue to complete the coupling of the remaining Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester according to the peptide sequence of the amylin analog, and then cleave to obtain the amylin analog.

[0101] Fmoc-Asp 3 (OAll)-Gly 4 (Hmb / Hmb)-Thr 5 (tBu)-Ala 6 -Thr 7 (tBu)-Lys 8 (Alloc)-Ala 9 -Thr 10 (tBu)-Glu 11 (OtBu)-Arg 12 (Pbf)-Leu 13 -Ala 14 -Aad 15 (OtBu)-Phe 16 -Leu 17 -Gln 18 (Trt)-Arg 19 (Pbf)-Ser 20 (tBu)-Ser 2 (tBu) 1 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 (tBu)-Ser28 (tBu)-Thr 29 (tBu)-Glu 30 (OtBu)-Val 31 -Gly 32 (Dmb / Hmb)-Ser 33 (tBu)-Asn 34 (Trt)-Thr 35 (tBu)-Hyp 36 (tBu)-resin(Ⅰ);

[0102] The peptide sequence of amylin analogues is as follows:

[0103] Eicosanedioic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2 forms a lactam bridge between the 3rd position Asp and the 8th position Lys.

[0104] The method for preparing amylin analogues in this application can also involve partial coupling, such as coupling the main chain to at least the cyclization site, and then performing Asp first. 3and Lys 8 The side chain protecting groups are removed and cyclized, and then subsequent coupling is carried out to obtain the complete peptide chain.

[0105] The partial coupling-cyclization-continuous coupling method of this application differs from the aforementioned preparation method only in the timing of cyclization; the conditions for deprotection, coupling, cyclization, and cleavage are the same as those in the aforementioned preparation method, and will not be elaborated here.

[0106] Example 1

[0107] This embodiment provides a method for preparing amylin analogues, wherein the peptide sequence of the amylin analogues is as follows: eicosanoic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2 forms a lactam bridge between the 3rd Asp and the 8th Lys position;

[0108] The specific preparation method includes the following steps:

[0109] (1) Accurately weigh 18.87g (10mmol) of Rink Amide-AM Resin (0.53mmol / g), add 190mL of DMF to swell for 2h, dry under vacuum, wash the resin with 190mL of DMF twice, and dry under vacuum.

[0110] (2) Add 380 mL of 20% Pip DMF solution to the reactor, react for 30 min, dry the mixture, wash the resin with 190 mL of DMF 6 times, take a sample for ninhydrin detection, and proceed to the next step after ninhydrin shows a positive result.

[0111] (3) Weigh 12.28g Fmoc-Hyp(tBu)-OH and 12.44g Oxyma and add them to a container. Add 50mL DMF and stir to dissolve. Control the temperature of the mixed solution at 5-15℃ and add 4.60mL DIC. React for 10-15min. Add it to the reactor and mix with the resin. Control the temperature at 30℃ and react for 1h. Take a sample for ninhydrin detection. If it is negative, continue coupling for 30min. After coupling is completed, wash with 190mL DMF 4 times.

[0112] (4) Repeat steps (2) and (3) to couple the corresponding Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester according to the peptide chain sequence of the amylin analogue until the main chain coupling is completed; wherein, for every 10 additional amino acids in the main chain, the amount of 20% Pip DMF solution is increased by 40 mL, and the amount of DMF used for washing is increased by 20 mL; each Fmoc-protected amino acid and eicosanoic acid monotert-butyl ester, Oxyma, and DIC is 3 equivalents of the resin.

[0113] Among them, the Fmoc-protected amino acids in order are: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-S er(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fm oc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Dmb)Gly-OH, Fmoc-Asp(OAll)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Glu-OtBu.

[0114] (5) Weigh 1.25g of tetratriphenylphosphine palladium and dissolve it in 30mL of DCM. Measure 7.5mL of benzylsilane and 7.7mL of NMM and add them to the reaction vessel in turn. Mix with the resin obtained in step (4) and react for 1h. Repeat the above steps twice, dry the mixture, take a sample for ninhydrin detection, and proceed to the next step if positive.

[0115] Wash with 300mL DCM 4 times, then wash with 300mL DMF 4 times, and then dry.

[0116] Weigh 15.6g of PyBop and add it to the reaction vessel. Add 40mL of DMF and mix it evenly with the resin. While stirring, slowly add 9.5mL of DIEA and react for 2 hours. Take a sample for ninhydrin detection. After a negative test, wash with 300mL of DMF 4 times. Then wash twice with 300mL of DCM and 300mL of MTBE alternately. Dry under vacuum and place in an oven until the weight loss is less than 1%.

[0117] (6) Prepare 800 mL of lysis buffer at a volume ratio of TFA:DODT:TIS:H2O of 90:5:2.5:2.5. Mix the lysis buffer with the resin obtained in step (5) and react for 2.5 h. Filter the solution, wash the filter cake with a small amount of TFA, and combine the filtrates to obtain 900 mL of filtrate. Add the filtrate to 7500 mL of MTBE, centrifuge, remove the supernatant, add 3500 mL of MTBE to the solid and slurry twice to obtain the crude product. Dry the crude product in an oven until the weight loss is less than 1%, obtaining 50.27 g of crude amylin analogue. The HPLC chromatogram of the crude product is shown in Figure 1. The yield of the crude amylin analogue is 115.5%, and the HPLC purity is 72.45%.

[0118] (7) The crude product obtained in step (6) was purified by reverse chromatography. In reverse chromatography: the column packing material was C18, the mobile phase A was an aqueous solution of TFA with a volume fraction of 0.1%, the mobile phase B was acetonitrile, and the gradient elution was as follows: the volume fraction of mobile phase B increased from 20% to 50% within 60 min. 15.07 g of lyophilized amylin analogue was obtained, with a yield of 31.4%.

[0119] Example 2

[0120] This embodiment refers to the preparation method of Example 1, the only difference being that the composition of the lysis solution is different in step (6).

[0121] In this embodiment, 800 mL of lysis buffer was prepared according to a volume ratio of TFA:DODT:TIS:H2O of 90:2.5:5:2.5.

[0122] The yield of the crude product obtained in step (6) was 120.5%, and the HPLC purity was 69.2%; the yield of the pure product obtained after purification in step (7) was 30.8%.

[0123] Example 3

[0124] This embodiment refers to the preparation method of Example 1, the only difference being that the composition of the lysis solution is different in step (6).

[0125] In this embodiment, 800 mL of lysis buffer was prepared with a volume ratio of TFA:DODT:H2O of 90:5:5.

[0126] The yield of the crude product obtained in step (6) was 117.3%, and the HPLC purity was 64.3%; the yield of the pure product obtained after purification in step (7) was 29.3%.

[0127] Example 4

[0128] This embodiment refers to the preparation method of Example 1, the only difference being that in steps (3) and (4), the amounts of each Fmoc-protected amino acid, eicosanoic acid monotert-butyl ester, and condensing agent are different.

[0129] In this embodiment, the amount of Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester is 2 equivalents of the resin, and the amount of Oxyma and DIC is 2 equivalents of the resin.

[0130] The yield of the crude product obtained in step (6) was 119.5%, and the HPLC purity was 67.3%; the yield of the pure product obtained after purification in step (7) was 27.3%.

[0131] Example 5

[0132] This embodiment refers to the preparation method of Example 1, the only difference being that in steps (3) and (4), the amounts of each Fmoc-protected amino acid, eicosanoic acid monotert-butyl ester, and condensing agent are different.

[0133] In this embodiment, the amount of Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester is 4 equivalents of the resin, and the amount of Oxyma and DIC is 4 equivalents of the resin.

[0134] The yield of the crude product obtained in step (6) was 122.1%, and the HPLC purity was 68.6%; the yield of the pure product obtained after purification in step (7) was 29.1%.

[0135] Example 6

[0136] This embodiment refers to the preparation method of Example 1, the only difference being that the amount of PyBop and DIEA used in step (5) is different.

[0137] In this embodiment, the amount of PyBop used is 15.60g, and the amount of DIEA used is 4.75mL.

[0138] The yield of the crude product obtained in step (6) was 110.3%, and the HPLC purity was 66.8%; the yield of the pure product obtained after purification in step (7) was 26.4%.

[0139] Example 7

[0140] This embodiment refers to the preparation method of Example 1. Steps (1)-(3) and (6)-(7) are the same as in Example 1, while steps (4)-(5) are different.

[0141] Steps (4) and (5) of this embodiment are as follows:

[0142] (4) Following the peptide chain sequence of the amylin analogue, repeat steps (2) and (3) to couple the corresponding Fmoc-protected amino acids until they are coupled to the cyclization site. The materials and operations are the same as in Example 1. The Fmoc-protected amino acids are, in order: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH, Fmoc-Ala-OH, Fmoc-Sar-OH, Fm oc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, F moc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Dmb)Gly-OH;

[0143] (5) Weigh 1.25g of tetratriphenylphosphine palladium and dissolve it in 35mL of DCM. Measure 7.5mL of benzylsilane and 7.7mL of NMM and add them to the reaction vessel in succession. Mix with the resin obtained in step (4) and react for 1h. Repeat the above steps twice, dry the mixture, take a sample for ninhydrin detection, and proceed to the next step if positive.

[0144] Wash with 300mL DCM 4 times, then wash with 300mL DMF 4 times, and then dry.

[0145] Weigh 15.6g of PyBop and add it to the reaction vessel. Add 40mL of DMF and mix it evenly with the resin. Slowly add 9.5mL of DIEA and react for 2 hours. Take a sample for ninhydrin detection. Wash with DMF 4 times, using 300mL of DMF each time.

[0146] Then repeat steps (2) and (3) to couple the remaining Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester until the main chain coupling is complete; wash twice with 300 mL DCM and 300 mL MTBE alternately and then dry.

[0147] The yield of the crude product obtained in step (6) was 122.4%, and the HPLC purity was 60.4%; the yield of the pure product obtained after purification in step (7) was 22.3%.

[0148] Example 8

[0149] This embodiment refers to the preparation method of Example 1, the only difference being that Fmoc-(Dmb)Gly-OH in step (4) is replaced with an equal amount of Fmoc-(Hmb)Gly-OH.

[0150] The yield of the crude product obtained in step (6) was 117.4%, and the HPLC purity was 45.7%; the yield of the pure product obtained after purification in step (7) was 16.3%.

[0151] Comparative Example 1

[0152] Comparative Example 1 refers to the preparation method of the amylin analogue in Example 1, the difference being that the types of amino acids protected by some Fmoc in step (4) are different.

[0153] In Comparative Example 1, the Fmoc-protected amino acid Fmoc-(Dmb)Gly-OH of the fourth Gly in the coupled peptide sequence was replaced with Fmoc-Gly-OH.

[0154] The crude product obtained in step (6) had a yield of 115.4% and an HPLC purity of 12.1%. The HPLC chromatogram of the crude product is shown in Figure 2. The pure product obtained after purification in step (7) had a yield of 4.1%.

[0155] The method for preparing amylin analogues in this application significantly reduces the probability of side reactions, improves product purity, and reduces purification difficulty without increasing the number of reaction steps.

[0156] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application. Industrial applicability

[0157] In the preparation method of the amylin analogue of the present invention, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used to carry out Gly 4 The coupling, and on this basis, the Asp removal 3 and Lys 8The side-chain protecting groups are then cyclized, and then Gly is removed during the cleavage process. 4 By attaching side-chain protecting groups and resin, amylin analogues are obtained. This significantly reduces the probability of side reactions, improves product purity, and simplifies purification without adding reaction steps. The method for preparing amylin analogues in this invention can be scaled up for production, reducing production costs and is of significant importance.

Claims

1. A method for preparing amylin analogues, characterized in that, Includes the following steps: (a) Using a solid-phase synthesis method, each Fmoc-protected amino acid and eicosanoic acid monotert-butyl ester were sequentially coupled to the resin from the C-terminus to the N-terminus according to the peptide sequence of the amylin analogue to obtain a linear peptide resin. (b) Remove the side chain protecting groups at the 3-position Asp and the 8-position Lys of the linear peptide resin, cyclize to form a lactam bridge, and cleave to obtain an amylin analog. The peptide sequence of the amyloid analogue is as follows: Eicosanedioic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2 forms a lactam bridge between the 3rd Asp and the 8th Lys position; When Gly is coupled at position 4 in the peptide sequence, the amino acid protected by Fmoc is at least one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH.

2. The method for preparing the amylin analogue according to claim 1, characterized in that, The pyrolysis process is carried out in a pyrolysis reagent, which is mainly prepared from TFA, DODT, TIS and water.

3. The method for preparing the amylin analogue according to claim 2, characterized in that, The volume ratio of TFA, DODT, TIS and water in the lysis reagent is (88-92):(4-6):(2-3):(2-3).

4. The method for preparing the amylin analogue according to claim 2, characterized in that, The ratio of the amount of the lysis reagent to the amount of the resin is (75-85) mL: 1 mmol.

5. The method for preparing the amylin analogue according to claim 2, characterized in that, The reaction time for the pyrolysis treatment is 2.5-3 hours.

6. The method for preparing the amylin analogue according to claim 1, characterized in that, When Asp is coupled at the 3rd position of the peptide sequence, the amino acid protected by Fmoc is any one of Fmoc-Asp(OAll)-OH, Fmoc-Asp(OMpe)-OH, and Fmoc-Asp(OPp)-OH; when Lys is coupled at the 8th position of the peptide sequence, the amino acid protected by Fmoc is any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH.

7. The method for preparing the amylin analogue according to claim 1, characterized in that, The amino acids protected by each Fmoc are respectively: Fmoc-Hyp(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmo c-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-N-Me-Ile-OH , Fmoc-Ala-OH, Fmoc-Sar-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-A rg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fmoc-A la-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Al a-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH, Fmoc-Asp(OAll)-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Glu-OtBu.

8. The method for preparing the amylin analogue according to claim 1, characterized in that, The resin includes at least one of Rink Amide-AM resin, Rink Amide-MBHA resin, and Sieber resin; Preferably, the resin substitution degree is 0.3-0.7 mmol / g.

9. The method for preparing the amylin analogue according to claim 1, characterized in that, Each coupling reaction includes a deprotection reaction and a coupling reaction; the deprotection reaction is carried out using a deprotection reagent, which includes an aprotic organic solvent containing an organic base, wherein the organic base includes pip, and the aprotic organic solvent includes at least one of DCM, DMF, and NMP.

10. The method for preparing the amylin analogue according to claim 9, characterized in that, The volume fraction of the organic base is 15%-25%.

11. The method for preparing the amylin analogue according to claim 9, characterized in that, The deprotection reaction takes 0.5-1 hour and is carried out at a temperature of 20-30°C.

12. The method for preparing the amylin analogue according to claim 9, characterized in that, The ratio of the amount of the deprotecting agent to the amount of resin is (38-50) mL: 1 mmol.

13. The method for preparing the amylin analogue according to claim 9, characterized in that, After the deprotection reaction, a washing step is also included; the ratio of solvent used in a single wash to resin is (19-27) mL: 1 mmol; the number of washes is 5-6.

14. The method for preparing the amylin analogue according to claim 9, characterized in that, In the coupling reaction, the molar ratio of the Fmoc-protected amino acid or the eicosanoic acid monotert-butyl ester to the resin is (2-4):

1.

15. The method for preparing the amylin analogue according to claim 9, characterized in that, The coupling reaction uses at least one of Oxyma / DIC, HOBt / DIC, and PyBop / DIEA as the condensing agent.

16. The method for preparing the amylin analogue according to claim 9, characterized in that, The coupling reaction takes 1-2 hours and is carried out at a temperature of 25-35°C.

17. The method for preparing the amylin analogue according to claim 9, characterized in that, The coupling reaction is followed by a washing step; the ratio of solvent used in a single wash to resin is (19-27) mL: 1 mmol; the number of washes is 3-4.

18. The method for preparing the amylin analogue according to claim 1, characterized in that, In step (b), a DCM solution containing tetraphenylphosphine palladium, benzyl silane and NMM is used to remove the Asp and Lys side chain protecting groups at the 3-position and 8-position of the linear peptide resin.

19. The method for preparing the amylin analogue according to claim 18, characterized in that, The molar ratio of the tetraphenylphosphine palladium, the benzyl silane, and the NMM is 0.1:(5.5-6.5):(6.5-7).

20. The method for preparing the amylin analogue according to claim 1, characterized in that, In step (b), the cyclization reaction includes: under the action of PyBop and DIEA, the 3-position Asp side chain with the deprotected group reacts with the 8-position Lys side chain with the deprotected group to form a lactam bridge.

21. The method for preparing the amylin analogue according to claim 1, characterized in that, Also includes: Collect the solution after the pyrolysis treatment, add a poor solvent to precipitate it out, collect the solid and wash it; The undesirable solvents include MTBE.

22. The method for preparing the amylin analogue according to claim 21, characterized in that, Also includes: The solid was purified using reversed-phase chromatography.

23. The method for preparing the amylin analogue according to claim 22, characterized in that, In the reversed-phase chromatography, mobile phase A is an aqueous solution of TFA with a volume fraction of 0.1%, and mobile phase B is acetonitrile; In the reversed-phase chromatography, gradient elution includes increasing the volume fraction of mobile phase B from 20% to 50% over 60 minutes.

24. A method for preparing amylin analogues, characterized in that, Includes the following steps: (A) Using a solid-phase synthesis method, each Fmoc-protected amino acid is coupled to the resin sequentially from the C-terminus to the N-terminus according to the peptide sequence of the amylin analogue to form a polypeptide resin fragment as shown in Formula I below. (B) Remove the side chain protecting groups at the 3-position Asp and the 8-position Lys of the polypeptide resin fragment, and form a lactam bridge through cyclization reaction; (C) Continue to complete the coupling of the remaining Fmoc-protected amino acids and eicosanoic acid monotert-butyl ester according to the peptide sequence of the amylin analog, and then cleave to obtain the amylin analog. Fmoc-Asp 3 (OAll)-Gly 4 (Dmb / Hmb)-Thr 5 (tBu)-Ala 6 -Thr 7 (tBu)-Lys 8 (Alloc)-Ala 9 -Thr 10 (tBu)-Glu 11 (OtBu)-Arg 12 (Pbf)-Leu 13 -Ala 14 -Aad 15 (OtBu)-Phe 16 -Leu 17 -Gln 18 (Trt)-Arg 19 (Pbf)-Ser 20 (tBu)-Ser 2 (tBu) 1 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 (tBu)-Ser 28 (tBu)-Thr 29 (tBu)-Glu 30 (OtBu)-Val 31 -Gly 32 (Dmb / Hmb)-Ser 33 (tBu)-Asn 34 (Trt)-Thr 35 (tBu)-HyP 36 (tBu)-resin (Ⅰ); The peptide sequence of the amyloid analogue is as follows: Eicosanedioic acid-γGlu 1 -Arg 2 -Asp 3 -Gly 4 -Thr 5 -Ala 6 -Thr 7 -Lys 8 -Ala 9 -Thr 10 -Glu 11 -Arg 12 -Leu 13 -Ala 14 -Aad 15 -Phe 16 -Leu 17 -Gln 18 -Arg 19 -Ser 20 -Ser 21 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Leu 26 -Ser 27 -Ser 28 -Thr 29 -Glu 30 -Val 31 -Gly 32 -Ser 33 -Asn 34 -Thr 35 -HyP 36 -NH2 forms a lactam bridge between the 3rd position Asp and the 8th position Lys.

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