Preparation method of amylin analogs

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) using a solid phase synthesis method, sequentially coupling each Fmoc-protected amino acid and mono-tert-butyl eicosane diacid to a resin from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog, to obtain a linear peptide resin; (b) removing the side chain protecting groups of the 3-position Asp and the 8-position Lys of the linear peptide resin, cyclizing to form a lactam bridge, and performing cleavage treatment to obtain an amylin analog; wherein, when coupling the 4th position Gly 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 of the present invention, without increasing the number of reaction steps, the probability of side reactions is significantly reduced, the purity of the product is improved, and the difficulty of purification is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a method for preparing an amylin analog. Background Art

[0002] Amylin, also known as islet amyloid polypeptide, is a small hormone secreted by pancreatic β cells. In addition to insulin, it is another hormone with hypoglycemic effect in the body. Amylin is composed of 37 amino acid residues and has a relative molecular weight of 3.85KD. It will form aggregates under the metabolic conditions of insulin deficiency, which is one of the pathological characteristics of type 2 diabetes.

[0003] 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. The development of different types of amylin analogs provides new drug candidates for the treatment of related diseases.

[0004] An existing amylin analog has the following peptide sequence:

[0005] 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 -Thr35 -HyP 36 -NH2, a disulfide bridge or a lactam bridge is formed between Asp at position 3 and Lys at position 8 (eicosanedioic acid-γGlu-RDGTATKATERLA-Aad-FLQRSSF-Sar-AN-Me-Ile-LSSTEVGSNT-HyP-NH2). Replacing the disulfide bridge with a lactam bridge can significantly improve the stability of the amylin analog and retain other beneficial properties of the amylin analog. However, the crude product obtained by the existing method for preparing the amylin analog has complex components, is difficult to purify, and has a low yield.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] In order to solve any one or more of the above defects in the synthesis of amylin analogs, the present invention provides a method for preparing amylin analogs.

[0008] In one aspect, the present invention provides a method for preparing an amylin analog, comprising the following steps:

[0009] (a) using a solid phase synthesis method, coupling each Fmoc-protected amino acid and mono-tert-butyl eicosanedioate to a resin in sequence from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to obtain a linear peptide resin;

[0010] (b) removing the side chain protecting groups of the Asp at position 3 and the 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 an amylin analog;

[0011] The peptide sequence of the amylin analog is as follows:

[0012] 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 -Ser20 -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;

[0013] When coupling the 4th Gly in the peptide sequence, the Fmoc-protected amino acid used is at least one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH.

[0014] In a specific embodiment of the present invention, the cleavage treatment is carried out in a cleavage reagent; the cleavage reagent is mainly made of TFA, DODT, TIS and water. Further, in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92) : (4-6) : (2-3) : (2-3).

[0015] In a specific embodiment of the present invention, the ratio of the amount of the cleavage reagent to the amount of the resin is (75-85) mL: 1 mmol.

[0016] In a specific embodiment of the present invention, the reaction time of the cleavage treatment is 2.5 to 3 hours.

[0017] In a specific embodiment of the present invention, when coupling the third Asp in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Asp(OAll)-OH, Fmoc-Asp(OMpe)-OH and Fmoc-Asp(OPp)-OH; when coupling the eighth Lys in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH.

[0018] In a specific embodiment of the present invention, the Fmoc-protected amino acids are: 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, Fm oc-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.

[0019] In a specific embodiment of the present invention, the resin comprises at least one of Rink Amide-AM resin, Rink Amide MBHA resin and Sieber resin. Further, the substitution degree of the resin is 0.3 to 0.7 mmol / g.

[0020] In a specific embodiment of the present invention, each coupling includes a deprotection reaction and a coupling reaction; the deprotection reaction is carried out using a deprotection reagent, the deprotection reagent includes an aprotic organic solvent containing an organic base, the organic base includes pip, and the aprotic organic solvent includes at least one of DCM, DMF and NMP. Further, in the deprotection reagent, the volume fraction of the organic base is 15% to 25%.

[0021] In a specific embodiment of the present invention, the deprotection reaction time is 0.5 to 1 h, and the deprotection reaction temperature is 20 to 30°C.

[0022] In a specific embodiment of the present invention, the ratio of the amount of the deprotection reagent to the resin is (38-50) mL: 1 mmol. Furthermore, after the deprotection reaction, a washing step is also included; the ratio of the amount of the solvent used in a single wash to the resin is (19-27) mL: 1 mmol; the number of washes is 5-6 times.

[0023] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid or the mono-tert-butyl eicosanedioate to the resin is (2-4):1.

[0024] In a specific embodiment of the present invention, in the coupling reaction, the condensing agent used includes at least one of Oxyma / DIC, HOBt / DIC and PyBop / DIEA.

[0025] In a specific embodiment of the present invention, the coupling reaction time is 1 to 2 hours; the coupling reaction temperature is 25 to 35°C.

[0026] In a specific embodiment of the present invention, after the coupling reaction, a washing step is further included; the ratio of the amount of solvent used in a single wash to the resin is (19-27) mL: 1 mmol; and the number of washes is 3-4 times.

[0027] In a specific embodiment of the present invention, in step (b), a DCM solution containing tetrakistriphenylphosphine 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. Furthermore, the molar ratio of the tetrakistriphenylphosphine palladium, the phenylsilane and the NMM is 0.1: (5.5-6.5): (6.5-7).

[0028] In a specific embodiment of the present invention, in step (b), the cyclization reaction comprises: under the action of PyBop and DIEA, the side chain of the deprotected Asp at position 3 reacts with the side chain of the deprotected Lys at position 8 to form a lactam bridge.

[0029] In a specific embodiment of the present invention, the process further comprises: collecting the solution after the cracking treatment, adding a poor solvent for precipitation, collecting the solid and washing it; the poor solvent comprises MTBE.

[0030] In a specific embodiment of the present invention, the method further comprises: purifying the solid by reverse phase chromatography. Further, in the reverse phase chromatography, mobile phase A is a 0.1% by volume TFA aqueous solution, and mobile phase B is acetonitrile.

[0031] In a specific embodiment of the present invention, in the reverse phase chromatography, the gradient elution comprises: the volume fraction of mobile phase B increases from 20% to 50% within 60 minutes.

[0032] Another aspect of the present invention provides a method for preparing an amylin analog, comprising the following steps:

[0033] (A) using a solid phase synthesis method, sequentially coupling each Fmoc-protected amino acid to a resin from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to form a polypeptide resin fragment as shown in the following formula I;

[0034] (B) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the polypeptide resin fragment, and performing a cyclization reaction to form a lactam bridge;

[0035] (C) continuing to complete the coupling of the remaining Fmoc-protected amino acids and mono-tert-butyl eicosanedioate according to the peptide sequence of the amylin analog, and then performing cleavage treatment to obtain the amylin analog;

[0036] 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 -Gly32 (Dmb / Hmb)-Ser 33 (tBu)-Asn 34 (Trt)-Thr 35 (tBu)-Hyp 36 (tBu)-resin (Ⅰ);

[0037] The peptide sequence of the amylin analog is as follows:

[0038] 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, forming a lactam bridge between Asp at position 3 and Lys at position 8.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) In the method for preparing amylin analogs of the present invention, Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used to carry out Gly 4 On this basis, Asp 3and Lys 8 The side chain protecting group is then cyclized and then the Gly 4 The side chain protecting groups and resin mounting are used to obtain amylin analogs, which significantly reduces the probability of side reactions without increasing the reaction steps, improves product purity, and reduces the difficulty of purification;

[0041] (2) The preparation method of the amylin analog of the present invention can be scaled up for production and reduce production costs, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 HPLC spectrum of the crude product obtained by the preparation method of the amylin analog provided in Example 1 of the present invention without reverse phase chromatography purification;

[0044] Figure 2 This is the HPLC spectrum of the crude product obtained by the preparation method of the amylin analog provided in Comparative Example 1 without reverse phase chromatography purification. DETAILED DESCRIPTION

[0045] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0046] The Chinese meanings of some English abbreviations involved in the present invention are shown in Table 1.

[0047] Table 1 Meaning of English abbreviations

[0048]

[0049] In the existing synthesis method of the amylin analogue of the present invention, a solid phase synthesis strategy is usually used to couple the entire peptide chain, and after the coupling is complete, the special protecting group is removed and then cyclized to form an amide bond connection, and then the protecting group is cleaved and removed and the peptide chain is mounted on the resin. However, this synthesis method has many by-products, and the crude product obtained has complex components, is difficult to purify, and has a low yield.

[0050] The present invention has found through research that the existing synthetic route is adopted to design Asp 3 and Lys 8 During the cyclization (i.e., DK cyclization), Asp 3 and Gly 4 The peptide chain obtained by DG cyclization has no biological activity. The occurrence of by-products makes the composition of the crude product very complicated, and because the molecular weight obtained by DG cyclization is the same as that of the target product, it is not easy to be detected by analysis, resulting in great difficulty in purification, and further reducing the yield.

[0051] The present invention is coupled with Gly 4 When Fmoc-(Dmb)Gly-OH / Fmoc-(Hmb)Gly-OH is used as the reaction material, the DG cyclization side reaction is suppressed; and the present invention does not add additional reaction steps, thereby ensuring production efficiency.

[0052] Based on this, the present invention provides a method for preparing an amylin analog, comprising the following steps:

[0053] (a) using a solid phase synthesis method, coupling each Fmoc-protected amino acid and mono-tert-butyl eicosanedioate to a resin in sequence from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to obtain a linear peptide resin;

[0054] (b) removing the side chain protecting groups of the Asp at position 3 and the Lys at position 8 of the linear peptide resin, cyclizing to form a lactam bridge, and cleaving to obtain an amylin analog;

[0055] The peptide sequence of the amylin analog is as follows:

[0056] 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 -Leu13 -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;

[0057] When coupling the 4th Gly in the peptide sequence, the Fmoc-protected amino acid used is at least one of Fmoc-(Dmb)Gly-OH and Fmoc-(Hmb)Gly-OH.

[0058] The inventors of the present invention have found in their research that the crude product obtained by stepwise coupling and cyclization according to the conventional method has complex components, the main component is not easy to purify, and the yield is low. However, at present, there is no research on the reasons for the complex components of the crude product of amylin analogs. The inventors of the present invention have found through research on the crude product components obtained by cleavage according to the conventional method that the conventional method of coupling Gly 4 When Fmoc-Gly-OH is used for coupling, after removing the special protecting groups of Asp at position 3 and Lys at position 8, the exposed carboxyl group of Asp is more likely to attack Gly 4 The formation of DG cyclization products, on the one hand, leads to a significant reduction in product yield and purity, and on the other hand, the DG cyclization products formed have the same molecular weight and similar structure as the target product after cleavage, which increases the difficulty of separation and purification. Based on the discovery of this problem, the present invention, in the process of preparing amylin analogs, couples Gly 4When Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the reaction material, the Dmb or Hmb protecting group occupies the secondary amine hydrogen on Gly, significantly reducing its cyclization activity. The cleavage reagent used in the cleavage step can effectively remove the protecting group and the mounting, significantly improving the yield and purity.

[0059] The present invention is coupled with Gly 4 When Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH is used as the reaction material, 4 A special protective group is introduced to protect the α-amino group, which inhibits the DG cyclization side reaction, reduces the DG cyclization byproducts, improves the purity and yield of the crude product, and simplifies the subsequent purification operation. Further research found that compared with Fmoc-(Hmb)Gly-OH, the Dmb in Fmoc-(Dmb)Gly-OH has a larger spatial structure than Hmb, which can better inhibit the DG cyclization side reaction, and the crude product obtained by cleavage has a higher purity. In addition, the present invention does not add additional reaction steps, thereby ensuring production efficiency.

[0060] In a specific embodiment of the present invention, the cleavage treatment is carried out in a cleavage reagent; the cleavage reagent is mainly prepared from TFA, DODT, TIS and water. Further, in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92) : (4-6) : (2-3) : (2-3).

[0061] 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 any two thereof; the volume ratio of DODT to water can be 4:2, 5:2, 6:2, 4:3, 5:3 or any two thereof; 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 any two thereof. 4 When using Fmoc-(Dmb)Gly-OH or Fmoc-(Hmb)Gly-OH as the reaction material, combined with the cleavage reagent used in the cleavage step, the protecting group and the mounting can be effectively removed, and the yield and purity can be significantly improved.

[0062] In some embodiments, in the lysis reagent, the volume ratio of TFA, DODT, TIS and water is 90:5:2.5:2.5.

[0063] In a specific embodiment of the present invention, the ratio of the amount of the cleavage reagent to the resin is (75-85) mL: 1 mmol.

[0064] Here, (75-85) mL: 1 mmol means that the amount of the cleavage reagent used can be 75-85 mL compared to 1 mmol of the resin, which is a limitation on the usage ratio of the cleavage reagent and the resin, rather than a limitation on the single usage. In some embodiments, the amount of the cleavage reagent used 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 the resin.

[0065] In a specific embodiment of the present invention, the reaction time of the cleavage treatment is 2.5 to 3 hours.

[0066] In actual operation, after the cleavage treatment, filtration treatment is performed, a small amount of TFA is used to wash the filter cake, and the filtrate is combined.

[0067] In a specific embodiment of the present invention, when coupling the third Asp 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 the eighth Lys in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH.

[0068] In a specific embodiment of the present invention, 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- moc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Aad(OtBu)-OH, Fm oc-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.

[0069] According to the main chain sequence of the amylin analog, the amino acids protected by Fmoc are coupled in sequence, and then eicosanedioic acid mono-tert-butyl ester is coupled to complete the synthesis of the main chain of the amylin analog. A protecting group different from Fmoc is set on the side chains of the 3rd Asp and the 8th Lys. After the main chain is completed, OAll and Alloc are removed, and then subsequent cyclization is performed.

[0070] In a specific embodiment of the present invention, the resin comprises at least one of Rink Amide-AM resin, Rink Amide MBHA resin and Sieber resin. Further, the substitution degree of the resin is 0.3-0.7 mmol / g.

[0071] In some embodiments, the degree of substitution of the resin may 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 thereof.

[0072] In some embodiments, the resin is pre-swelled when coupling with the first Fmoc-protected amino acid, and specifically, DMF can be used for swelling, but is not limited thereto.

[0073] In a specific embodiment of the present invention, each coupling includes a deprotection reaction and a coupling reaction; a deprotection reagent is used for the deprotection reaction, and the deprotection reagent includes an aprotic organic solvent containing an organic base, the organic base includes pip, and the aprotic organic solvent includes at least one of DCM, DMF and NMP. Further, in the deprotection reagent, the volume fraction of the organic base is 15% to 25%. In some embodiments, the deprotection reagent is a DMF solution containing pip, and the volume fraction of pip is 20%.

[0074] In a specific embodiment of the present invention, the deprotection reaction time is 0.5 to 1 h, and the deprotection reaction temperature is 20 to 30°C.

[0075] In a specific embodiment of the present invention, the ratio of the amount of the deprotection reagent to the resin is (38-50) mL: 1 mmol. Furthermore, after the deprotection reaction, a washing step is also included; the ratio of the amount of the solvent used in a single wash to the resin is (19-27) mL: 1 mmol; the number of washes is 5-6 times.

[0076] In some embodiments, compared to 1 mmol of resin, the amount of the deprotection reagent can be 38 mL, 40 mL, 42 mL, 45 mL, 48 mL, 50 mL or a range consisting of any two thereof; in a single wash after the deprotection reaction, compared to 1 mmol of resin, the amount of the solvent for a single wash can be 19 mL, 20 mL, 22 mL, 24 mL, 25 mL, 27 mL or a range consisting of any two thereof.

[0077] In actual operation, the amount of deprotection reagent and washing solvent is adjusted according to the number of coupled amino acids to ensure both purity and yield. For example, when the number of amino acids in the peptide chain increases by 10, the amount of deprotection reagent increases by 4 mL and the amount of washing solvent increases by 2 mL compared to 1 mmol of resin.

[0078] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid or mono-tert-butyl eicosanedioate to the resin is (2-4):1.

[0079] In some embodiments, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid or mono-tert-butyl eicosanedioate to the resin 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.

[0080] In a specific embodiment of the present invention, in the coupling reaction, the condensing agent used includes at least one of Oxyma / DIC, HOBt / DIC and PyBop / DIEA.

[0081] In a specific embodiment of the present invention, the coupling reaction time is 1 to 2 hours; the coupling reaction temperature is 25 to 35°C.

[0082] In a specific embodiment of the present invention, after the coupling reaction, a washing step is further included; the ratio of the amount of solvent used in a single wash to the resin is (19-27) mL: 1 mmol; and the number of washes is 3-4 times.

[0083] In some embodiments, the amount of solvent used for a single wash can be 19 mL, 20 mL, 22 mL, 24 mL, 25 mL, 27 mL, or a range consisting of any two thereof, compared to 1 mmol of resin. In actual operation, the amount of solvent used for washing is adjusted according to the number of coupled amino acids to ensure both purity and yield. For example, when the number of amino acids in the peptide chain increases by 10, the amount of solvent used for washing increases by 2 mL compared to 1 mmol of resin.

[0084] In actual operation, in each coupling of step (a), the step of deprotection reaction may include: placing the resin in a deprotection reagent, mixing at 20-30°C for 0.5-1h, and washing with a solvent; the solvent used for washing may include but is not limited to DMF. In each coupling of step (b), the step of coupling reaction may include: mixing the Fmoc-protected amino acid and Oxyma in a solvent, then adding DIC, activating for 5-10min, and obtaining an activation solution; adding the activation solution to the resin after the deprotection reaction, reacting at 25-35°C for 1-2h, and washing with a solvent; the solvent used for washing may include but is not limited to DMF.

[0085] In a specific embodiment of the present invention, in step (b), a DCM solution containing tetrakistriphenylphosphine 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. Furthermore, the molar ratio of tetrakistriphenylphosphine palladium, phenylsilane and NMM is 0.1: (5.5-6.5): (6.5-7). The amount of DCM used is 25-35 mL for every 1 mmol of resin.

[0086] In a specific embodiment of the present invention, in step (b), the cyclization reaction comprises: under the action of PyBop and DIEA, the side chain of the deprotected Asp at position 3 reacts with the side chain of the deprotected Lys at position 8 to form a lactam bridge.

[0087] In some embodiments, the usage 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.

[0088] In actual operation, the cyclization reaction to form the lactam bridge may include: adding PyBop to a reaction vessel, adding DMF to mix with the resin, adding DIEA, mixing at 20-30° C. for 1.5-2.5 hours, and washing.

[0089] In some embodiments, washing after the cyclization reaction includes: washing with DMF for 3 to 4 times, and then washing with DCM and MTBE alternately for 2 to 3 times. Wherein, compared with 1 mmol of resin, the amount of DMF used in a single wash can be 25 to 35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL or a range consisting of any two thereof; compared with 1 mmol of resin, the amount of DCM used in a single wash can be 25 to 35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL or a range consisting of any two thereof; compared with 1 mmol of resin, the amount of MTBE used in a single wash can be 25 to 35 mL, such as 25 mL, 28 mL, 30 mL, 32 mL, 35 mL or a range consisting of any two thereof.

[0090] In a specific embodiment of the present invention, in step (a) and step (b), the progress of the coupling reaction, cyclization reaction, etc. is monitored by ninhydrin detection. The reaction time is adjusted according to the ninhydrin detection result.

[0091] In a specific embodiment of the present invention, the process further comprises: collecting the solution after the cracking treatment, adding a poor solvent for precipitation, collecting the solid and washing it; the poor solvent comprises MTBE.

[0092] In some embodiments, the volume ratio of the solution after the cleavage treatment to the poor solvent is 1:(7-10), such as 1:7, 1:8, 1:9, 1:10 or a range consisting of any two of them.

[0093] In a specific embodiment of the present invention, the method further comprises: purifying the solid by reverse phase chromatography. Further, in the reverse phase chromatography, mobile phase A is a 0.1% by volume TFA aqueous solution, and mobile phase B is acetonitrile.

[0094] In a specific embodiment of the present invention, in reverse phase chromatography, gradient elution comprises: the volume fraction of mobile phase B increases from 20% to 50% within 60 minutes.

[0095] The preparation method of the amylin analogue of the present invention significantly reduces the probability of side reactions, improves product purity, reduces the difficulty of purification, and can obtain a product with purity that meets the requirements through a simple purification method.

[0096] The present invention provides another method for preparing an amylin analog, comprising the following steps:

[0097] (A) using a solid phase synthesis method, sequentially coupling each Fmoc-protected amino acid to a resin from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to form a polypeptide resin fragment as shown in the following formula I;

[0098] (B) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the peptide resin fragment, and performing a cyclization reaction to form a lactam bridge;

[0099] (C) continuing to complete the coupling of the remaining Fmoc-protected amino acids and mono-tert-butyl eicosanedioate according to the peptide sequence of the amylin analog, and then performing cleavage treatment to obtain the amylin analog;

[0100] 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);

[0101] The peptide sequence of the amylin analog is as follows:

[0102] 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, forming a lactam bridge between Asp at position 3 and Lys at position 8.

[0103] The method for preparing the amylin analogue of the present invention can also be partially coupled, such as coupling at least to the cyclization site according to the main chain, and then firstly performing Asp 3and Lys 8 The side chain protecting groups are removed and cyclized, and then the subsequent coupling is carried out to obtain a complete peptide chain.

[0104] The partial coupling-cyclization-continued coupling method of the present invention is different from the aforementioned preparation method in terms of the timing of cyclization, while the other conditions such as deprotection, coupling, cyclization, and cleavage are consistent with the aforementioned preparation method, and will not be described in detail herein.

[0105] Example 1

[0106] This example provides a method for preparing an amylin analog, wherein the peptide sequence of the amylin analog 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, forming a lactam bridge between Asp at position 3 and Lys at position 8;

[0107] The specific preparation method comprises the following steps:

[0108] (1) Accurately weigh 18.87 g (10 mmol) of Rink Amide-AM Resin (0.53 mmol / g), add 190 mL of DMF to swell for 2 h, drain, wash the resin with 190 mL of DMF, wash twice, and drain.

[0109] (2) Add 380 mL of 20% Pip in DMF solution to the reactor, react for 30 min, drain, and wash the resin with 190 mL of DMF for 6 times. Take a sample for ninhydrin detection. If ninhydrin is positive, proceed to the next step.

[0110] (3) Weigh 12.28g Fmoc-Hyp(tBu)-OH and 12.44g Oxyma into a container, add 50mL DMF and stir to dissolve, control the temperature of the mixed solution at 5-15℃, add 4.60mL DIC to react for 10-15min, add to the reactor and mix with the resin, control the temperature at 30℃ to react for 1h, take a sample for ninhydrin test, and continue coupling for 30min if it is negative; after coupling, use 190mL DMF to wash 4 times.

[0111] (4) Repeat steps (2) and (3) to couple the corresponding Fmoc-protected amino acids and mono-tert-butyl eicosanedioate according to the peptide chain sequence of the amylin analog, until the main chain coupling is completed; wherein, for every 10 amino acids added to 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, mono-tert-butyl eicosanedioate, Oxyma, and DIC are 3 equivalents of the resin;

[0112] 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.

[0113] (5) Weigh 1.25 g of tetrakistriphenylphosphine palladium and dissolve it in 30 mL of DCM. Measure 7.5 mL of phenylsilane and 7.7 mL of NMM and add them to the reaction container and mix with the resin obtained in step (4) for 1 h. Repeat the above steps twice, drain, take a sample for ninhydrin test, and proceed to the next step if positive.

[0114] Wash with 300 mL DCM, wash 4 times, wash with 300 mL DMF 4 times, and drain;

[0115] Weigh 15.6 g of PyBop and add it to the reaction container. Add 40 mL of DMF and mix it evenly with the resin. Slowly add 9.5 mL of DIEA while stirring and react for 2 hours. Take a sample for ninhydrin detection. After a negative test result, wash it with 300 mL of DMF for 4 times, wash it with 300 mL of DCM and 300 mL of MTBE alternately for 2 times, drain it, and place it in an oven to dry until the weight loss is less than 1%.

[0116] (6) Prepare 800 mL of cleavage solution according to the volume ratio of TFA﹕DODT﹕TIS﹕H2O of 90﹕5﹕2.5﹕2.5, mix the cleavage solution with the resin obtained in step (5) and react for 2.5 hours, filter, wash the filter cake with a small amount of TFA, combine the filtrate 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 beat it twice to obtain a crude product, place it in an oven to dry until the weight loss is less than 1%, and obtain 50.27 g of crude amylin analog. The HPLC spectrum of the crude product is as follows: Figure 1 As shown, the yield of the crude amylin analog was 115.5% and the HPLC purity was 72.45%.

[0117] (7) The crude product obtained in step (6) was purified by reverse chromatography; in the reverse chromatography, the column packing 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: the volume fraction of the mobile phase B increased from 20% to 50% within 60 min; 15.07 g of the pure lyophilized amylin analog was obtained, with a yield of 31.4%.

[0118] Example 2

[0119] This example refers to the preparation method of Example 1, with the only difference being that in step (6), the composition of the lysate is different.

[0120] In this embodiment, 800 mL of lysis solution was prepared according to the volume ratio of TFA﹕DODT﹕TIS﹕H2O of 90﹕2.5﹕5﹕2.5.

[0121] 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%.

[0122] Example 3

[0123] This example refers to the preparation method of Example 1, with the only difference being that in step (6), the composition of the lysate is different.

[0124] In this embodiment, 800 mL of lysis solution was prepared according to the volume ratio of TFA:DODT:H2O of 90:5:5.

[0125] 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%.

[0126] Example 4

[0127] This example is based on the preparation method of Example 1, with the only difference being that in step (3) and step (4), different amounts of the Fmoc-protected amino acids, mono-tert-butyl eicosanedioate, and condensing agent are used.

[0128] In this embodiment, the amount of Fmoc-protected amino acid and mono-tert-butyl eicosanedioate is 2 equivalents of the resin, and the amount of Oxyma and DIC is 2 equivalents of the resin.

[0129] 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%.

[0130] Example 5

[0131] This example is based on the preparation method of Example 1, with the only difference being that in step (3) and step (4), different amounts of the Fmoc-protected amino acids, mono-tert-butyl eicosanedioate, and condensing agent are used.

[0132] In this embodiment, the amount of Fmoc-protected amino acid and mono-tert-butyl eicosanedioate used is 4 equivalents of the resin, and the amount of Oxyma and DIC used is 4 equivalents of the resin.

[0133] 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%.

[0134] Example 6

[0135] This example is based on the preparation method of Example 1, with the only difference being that in step (5), the amounts of PyBop and DIEA used are different.

[0136] In this embodiment, the amount of PyBop used is 15.60 g, and the amount of DIEA used is 4.75 mL.

[0137] 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%.

[0138] Example 7

[0139] This example refers to the preparation method of Example 1, steps (1) to (3) and (6) to (7) are the same as Example 1, and steps (4) to (5) are different.

[0140] Steps (4) and (5) of this embodiment are as follows:

[0141] (4) According to the peptide chain sequence of the amylin analog, repeat steps (2) and (3) to couple the corresponding Fmoc-protected amino acids until the cyclization site is coupled. The materials and operations are the same as those in Example 1; wherein 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, Fmoc- 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;

[0142] (5) Weigh 1.25 g of tetrakistriphenylphosphine palladium and dissolve it in 35 mL of DCM. Measure 7.5 mL of phenylsilane and 7.7 mL of NMM and add them to the reaction container and mix with the resin obtained in step (4) for 1 hour. Repeat the above steps twice, drain, take a sample for ninhydrin test, and proceed to the next step if positive.

[0143] Wash with 300 mL DCM, wash 4 times, wash with 300 mL DMF 4 times, and drain;

[0144] Weigh 15.6 g of PyBop into a reaction vessel, add 40 mL of DMF and mix well with the resin, slowly add 9.5 mL of DIEA and react for 2 h, take a sample for ninhydrin detection; wash with DMF 4 times, using 300 mL of DMF each time;

[0145] Then repeat steps (2) and (3) to couple the remaining Fmoc-protected amino acids and mono-tert-butyl eicosanedioate until the main chain coupling is completed; wash twice with 300 mL DCM and 300 mL MTBE alternately and drain.

[0146] 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%.

[0147] Example 8

[0148] This example is prepared by referring to the preparation method of Example 1, except that the Fmoc-(Dmb)Gly-OH in step (4) is replaced with an equal amount of Fmoc-(Hmb)Gly-OH.

[0149] 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%.

[0150] Comparative Example 1

[0151] Comparative Example 1 The preparation method of the amylin analog is similar to that of Example 1, except that the types of partially Fmoc-protected amino acids in step (4) are different.

[0152] In Comparative Example 1, the Fmoc-protected amino acid Fmoc-(Dmb)Gly-OH of the 4th Gly in the coupling peptide sequence was replaced with Fmoc-Gly-OH.

[0153] The yield of the crude product obtained in step (6) was 115.4%, and the HPLC purity was 12.1%. The HPLC spectrum of the crude product was as follows: Figure 2 As shown; the yield of the pure product obtained after purification according to step (7) is 4.1%.

[0154] In the preparation method of the amylin analogue of the present invention, the probability of side reactions is significantly reduced without increasing the reaction steps, the purity of the product is improved, and the difficulty of purification is reduced.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an amylin analog, characterized in that: The steps include: (a) using a solid phase synthesis method, coupling each Fmoc-protected amino acid and mono-tert-butyl eicosanedioate to a resin in sequence from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to obtain a linear peptide resin; (b) removing the side chain protecting groups of the Asp at position 3 and the 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 an amylin analog; The peptide sequence of the amylin analog 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, forming a lactam bridge between Asp at position 3 and Lys at position 8; Among them, when coupling the 4th Gly in the peptide sequence, the Fmoc-protected amino acid used is Fmoc-(Dmb)Gly-OH; The cleavage treatment is carried out in a cleavage reagent; in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92): (4-6): (2-3): (2-3); In step (b), the cyclization reaction includes: under the action of PyBop and DIEA, the side chain of the 3-position Asp that has been deprotected reacts with the side chain of the 8-position Lys that has been deprotected to form a lactam bridge; the usage ratio of the PyBop and the DIEA is 14.82-16.38 g: 9.03-9.98 mL.

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

3. The method for preparing an amylin analog according to claim 1, characterized in that: The reaction time of the cracking treatment is 2.5 to 3 hours.

4. The method for preparing an amylin analog according to claim 1, characterized in that: When coupling the third Asp in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Asp(OAll)-OH, Fmoc-Asp(OMpe)-OH and Fmoc-Asp(OPp)-OH; when coupling the eighth Lys in the peptide sequence, the Fmoc-protected amino acid used is any one of Fmoc-Lys(Alloc)-OH and Fmoc-Lys(Dde)-OH.

5. The method for preparing an amylin analog 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, 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-A rg(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)-O H, Fmoc-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.

6. The method for preparing an amylin analog according to claim 1, characterized in that: The resin includes at least one of RinkAmide-AM resin, RinkAmide-MBHA resin and Sieber resin; The substitution degree of the resin is 0.3-0.7 mmol / g.

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

8. The method for preparing an amylin analog according to claim 7, characterized in that: The deprotection reaction time is 0.5 to 1 h, and the deprotection reaction temperature is 20 to 30°C.

9. The method for preparing an amylin analog according to claim 7, characterized in that: The ratio of the amount of the deprotection reagent to the amount of the resin is (38-50) mL: 1 mmol.

10. The method for preparing an amylin analog according to claim 7, characterized in that: In the coupling reaction, the molar ratio of the Fmoc-protected amino acid or the mono-tert-butyl eicosanedioate to the resin is (2-4):

1.

11. The method for preparing an amylin analog according to claim 7, characterized in that: In the coupling reaction, the condensing agent used includes at least one of Oxyma / DIC, HOBt / DIC and PyBop / DIEA.

12. The method for preparing an amylin analog according to claim 7, characterized in that: The coupling reaction time is 1 to 2 hours; the coupling reaction temperature is 25 to 35°C.

13. The method for preparing an amylin analog according to claim 1, characterized in that: In step (b), a DCM solution containing tetrakistriphenylphosphine 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.

14. The method for preparing an amylin analog according to claim 1, characterized in that: Also includes: collecting the solution after the cracking treatment, adding a poor solvent for precipitation, collecting the solid and washing it; The poor solvent includes MTBE.

15. The method for preparing an amylin analog according to claim 14, characterized in that: Also includes: The solid was purified by reverse phase chromatography.

16. The method for preparing an amylin analog according to claim 15, characterized in that: In the reverse 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 reverse phase chromatography, the gradient elution includes: the volume fraction of mobile phase B increases from 20% to 50% within 60 minutes.

17. A method for preparing an amylin analog, characterized in that: The steps include: (A) using a solid phase synthesis method, sequentially coupling each Fmoc-protected amino acid to a resin from the C-terminus to the N-terminus according to the peptide sequence of the amylin analog to form a polypeptide resin fragment as shown in the following formula I; (B) removing the side chain protecting groups of Asp at position 3 and Lys at position 8 of the polypeptide resin fragment, and performing a cyclization reaction to form a lactam bridge; (C) continuing to complete the coupling of the remaining Fmoc-protected amino acids and mono-tert-butyl eicosanedioate according to the peptide sequence of the amylin analog, and then performing cleavage treatment to obtain the amylin analog; Fmoc-Asp 3 (OAll)-Gly 4 (Dmb)-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 -Read 17 -Gln 18 (Trt)-Arg 19 (Pbf)-Ser 20 (tBu)-Ser 2 (tBu) 1 -Phe 22 -Sar 23 -Ala 24 -N-Me-Ile 25 -Read 26 -Ser 27 (tBu)-Ser 28 (tBu)-Thr 29 (tBu)-Glu 30 (OtBu)-Val 31 -Gly 32 (Dmb)-Ser 33 (tBu)-Asn 34 (Trt)-Thr 35 (tBu)-HyP 36 (tBu)-フィック(Ⅰ); The peptide sequence of the amylin analog 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, forming a lactam bridge between Asp at position 3 and Lys at position 8; The cleavage treatment is carried out in a cleavage reagent; in the cleavage reagent, the volume ratio of TFA, DODT, TIS and water is (88-92): (4-6): (2-3): (2-3); In step (B), the cyclization reaction includes: under the action of PyBop and DIEA, the side chain of the 3-position Asp that has been deprotected reacts with the side chain of the 8-position Lys that has been deprotected to form a lactam bridge; the usage ratio of the PyBop and the DIEA is 14.82-16.38 g: 9.03-9.98 mL.

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