Method for synthesizing Cagrilintide by large fragment SPPS-LPPS hybrid method

Abstract

The present invention provides a method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method, comprising cutting Cagrilintide into four polypeptide fragments: sequence 1-14 is fragment I, sequence 15-26 is fragment II, sequence 27-35 is fragment III, and sequence 36-39 is fragment IV; fully protected fragment I, fragment II and fragment III are synthesized by SPPS one by one by coupling the protected amino acid monomers at the corresponding sequence positions, and fragment IV is synthesized by solid phase or liquid phase; the fragments are subjected to LPPS method, and condensation / Fmoc removal reaction is repeated to obtain fully protected Cagrilintide. The present invention synthesizes Cagrilintide with a total yield of about 45%, a purity greater than 99.0%, a single impurity less than 0.1%, a simple process, greatly improves production efficiency, is more suitable for industrial production, and has a wide range of application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug synthesis, relates to the synthesis of Cagrilintide, and specifically relates to a method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method. Background Art

[0002] English name: Cagrilintide, peptide sequence: Eicosanedioic acidγ-Glu-Lys-Cys-Asn-Thr-Ala

[0003] -Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Glu-Phe-Leu-Arg-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Pro-NH 2 (Disulfidebridge:Cys4-Cys9).

[0004] Cagrilintide is an amylin analogue being developed by Novo Nordisk. Cagrilintide has previously been shown to be effective in treating obesity in Phase 2 clinical trials. Cagrilintide is a long-acting amylin analogue with a plasma half-life of approximately 7-8 days. Compared with pramlintide (an amylin analogue with a very short plasma half-life and therefore must be injected 2-3 times a day), it has significantly improved plasma stability.

[0005] Chinese patent, publication number: CN114249808A, publication date: March 29, 2022, title: A method for synthesizing Cagrilintide, Hangzhou Nuotai Aosinuo Pharmaceutical Technology Development Co., Ltd. reported a step-wise solid phase coupling method for synthesizing Cagrilintide, in which three pseudo-pro dipeptide fragments were used in the coupling process. They are: Fmoc-Ala-Thr(Pro-me-me)-OH pseudo-pro dipeptide at positions 7-8; Fmoc-Ala-Thr(Pro-me-me)-OH pseudo-pro dipeptide at positions 10-11; Fmoc-Ser-Ser(Pro-me-me)-OH pseudo-pro dipeptide at positions 21-22; other sequences use commonly used amino acid monomers. After coupling-cleavage-cyclization-purification one by one, pure Cagrilintide was obtained.

[0006] Chinese patent, publication number: CN117986347A, publication date: May 7, 2024, named a method for synthesizing Cagrilintide, Hangzhou Sinoda Pharmaceutical Technology Co., Ltd. reported a step-wise solid phase coupling method for synthesizing Cagrilintide, and two pseudo-pro dipeptide fragments were used in the coupling process. They are: Fmoc-Ala-Thr(Pro-me-me)-OH pseudo-pro dipeptide is used at positions 7-8; Fmoc-Ala-Thr(Pro-me-me)-OH pseudo-pro dipeptide is used at positions 10-11; other sequences use commonly used amino acid monomers. After coupling-cleavage-cyclization-purification one by one, pure Cagrilintide is obtained.

[0007] Step-wise SPPS is often used for the synthesis of shorter peptide drugs (usually less than 30 amino acids). As the peptide chain on the solid phase resin grows, the reactivity of the amino group at the nitrogen end of the peptide chain on the resin will decrease due to the steric effect and the folding effect of the long peptide chain. At this time, in order to reduce the missing peptide impurities, it is necessary to increase the feed equivalent of the peptide monomer and extend the coupling reaction time (the difference phase peptide impurities will increase), which will increase the material cost, reduce the purity of the crude peptide, and reduce the yield; if the reaction is still not completed, it needs to be cleaned and re-fed for coupling, resulting in a decrease in production efficiency. Cagrilintide is composed of 38 amino acids and an eicosanedioic acid fatty chain. The applicant found in the Step-wise SPPS synthesis of Cagrilintide that when coupled one by one to Fmoc-Gln(Trt)-OH at the 12th position, the coupling reaction time needs to be extended to more than 3 hours; coupling to Fmoc-Glu-OtBu at the 2nd position and eicosanedioic acid mono-tert-butyl ester at the 1st position requires a second feed to complete the reaction. Summary of the invention

[0008] In order to overcome the many defects of the Step-wise SPPS method for synthesizing Cagrilintide, the present invention provides a method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method. The present invention cuts Cagrilintide into four polypeptide fragments: sequence 1-14 is fragment I (tetradecapeptide), sequence 15-26 is fragment II (twelve peptides), sequence 27-35 is fragment III (nonapeptide), and sequence 36-39 is fragment IV (tetradecapeptide); the fully protected fragment I / fragment II / fragment III can be used as a solid phase carrier with 2-Cl-CTC resin, and the protected amino acid monomers at the corresponding sequence positions are coupled one by one, and finally obtained by cleavage under 1%-5% trifluoroacetic acid dichloromethane solution / hexafluoroisopropanol dichloromethane solution / trifluoroethanol dichloromethane solution or other weakly acidic conditions, and fragment IV can be synthesized by liquid phase synthesis or using Sieber Resin as a solid phase carrier.

[0009] The invention improves the atomic economy of synthesizing Cagrilintide, reduces the impurity content in the synthesis, improves the purity of the synthesized crude peptide, and improves the production efficiency.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] The invention provides a method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method. The method comprises the following steps: cutting Cagrilintide into four polypeptide fragments: sequence 1-14 is fragment I, sequence 15-26 is fragment II, sequence 27-35 is fragment III, and sequence 36-39 is fragment IV; synthesizing fully protected fragment I, fragment II and fragment III by coupling protected amino acid monomers at corresponding sequence positions one by one by a solid phase synthesis method; synthesizing fragment IV with exposed nitrogen end by a solid phase synthesis method or synthesizing fragment IV with exposed nitrogen end by a liquid phase synthesis method; and finally obtaining fully protected Cagrilintide by repeating condensation / Fmoc removal reaction.

[0012] As a preferred embodiment of the present invention, the steps of the method are:

[0013] Step 1) Synthesis of fully protected fragments Fragment I, Fragment II, Fragment III and Fragment IV:

[0014] 1) Synthesis of fully protected fragment I: Add dichloromethane to a solid phase reactor containing 2-Chlorotrityl Chloride resin to swell it, drain the solvent, add a dry dichloromethane solution of Fmoc-Leu-OH and DIPEA, connect the first amino acid to the resin, add DCM / MeOH / DIPEA to block the unreacted or difficult-to-react chlorinated sites on the resin; wash the resin with solvent, add a deprotection reagent to remove the Fmoc protecting group, add an activated Fmoc-Arg(Pbf)-OH / HOBt / DIC mixture to carry out the coupling reaction of the second amino acid monomer, and after the indene test result is negative, drain and wash to complete the coupling reaction; repeat the above steps, as shown in The following sequence was used for the solid phase synthesis reaction of fragment I: Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu-OtBu, 20-(tBu)-20-oxoicosanoic acid; after the coupling, the resin was washed with dichloromethane and methanol; the resin was dried under vacuum at room temperature; the fully protected fragment I was obtained by cleavage under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution;

[0015] 2) Synthesis of fully protected fragment II: According to the synthesis method of fragment I, the following amino acid monomers Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH are sequentially connected to 2-ChlorotritylChloride resin; after coupling, the resin is washed with dichloromethane and methanol; vacuum dried at room temperature; cleaved under fluorinated alcohol solution or 1%-5% TFA dichloromethane solution to obtain fully protected fragment II;

[0016] 3) Synthesis of fully protected fragment III: According to the synthesis method of fragment I, the following amino acid monomers Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Pro-OH are sequentially connected to 2-ChlorotritylChloride resin; after the coupling, the resin is washed with dichloromethane and methanol; the resin is dried under vacuum at room temperature; and the fully protected fragment III is cleaved under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution;

[0017] 4) Solid phase synthesis of fragment IV with exposed nitrogen end: After Sieber resin is swollen by adding dichloromethane, the solvent is drained, and a deprotection reagent is added to remove the Fmoc protecting group. The resin is drained and washed. The indene test result is positive; the activated Fmoc-Pro-OH / HOBt / DIC mixed solution is added to carry out the coupling reaction of the first amino acid monomer. After the indene test result is negative, the reaction is drained and washed to complete the coupling reaction; the above steps are repeated to carry out the solid phase synthesis reaction of fragment IV according to the following sequence: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH; after the coupling is completed, a deprotection reagent is added to remove Fmoc, and the resin is washed with dichloromethane and methanol; vacuum drying at room temperature; cleavage is carried out under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution to obtain fragment IV with exposed nitrogen end;

[0018] Alternatively, 4-1) Liquid phase synthesis of the nitrogen-terminally exposed fragment IV: Dissolve Fmoc-Thr(tBu)-OH / HOBt in dichloromethane, cool to below 15°C, add DCC for activation, and add H-Pro-NH 2 After the coupling is completed, DCU is removed by filtration, the organic phase is washed with sodium bicarbonate aqueous solution, separated, dried, and concentrated under reduced pressure, and a deprotection reagent is added to remove Fmoc. After the reaction is completed, the reaction is concentrated under reduced pressure and slurried twice with n-heptane; the above coupling and deprotection steps are repeated to couple two monomers, Fmoc-Asn(Trt)-OH and Fmoc-Ser(tBu)-OH; Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 Then, it was purified by column chromatography using a normal phase silica gel column in a dichloromethane-methanol system to obtain Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 Pure product; add deprotection reagent to remove Fmoc, concentrate under reduced pressure, then slurry with n-heptane twice, filter under reduced pressure, collect the filter cake, and dry under reduced pressure to obtain fragment IV with exposed nitrogen end;

[0019] Step 2: Liquid phase coupling of fully protected fragment I, fragment II, fragment III and fragment IV:

[0020] The fully protected fragment III, HOBt or HOAt, and DIPEA obtained in step 1 are dissolved in DMSO, cooled to below 15°C, activated by adding HATU, and the nitrogen-terminally exposed fragment IV obtained in step 1 is added, and a coupling reaction is carried out at 20-35°C. After the coupling is completed, the reaction solution is added dropwise to ice water for crystallization, and the solid is collected by filtration. The filter cake is slurried with a sodium bicarbonate aqueous solution, and the filter cake is collected by filtration. After vacuum drying, a deprotection reagent is added to remove Fmoc. After the reaction is completed, the mixture is concentrated under reduced pressure and slurried twice with isopropyl ether. The coupling and deprotection steps are repeated to sequentially couple the fully protected fragment II obtained in step 1 and the fully protected fragment I obtained in step 1 to obtain fully protected Cagrilintide.

[0021] Step 3: Remove all protecting groups in one step:

[0022] The fully protected Cagrilintide obtained in step 2 is subjected to one-step removal of all protecting groups under the conditions of the lysate, precipitated in an ether solvent, and the solid is collected by centrifugation. The solid is washed twice with an ether solvent, and the solid is collected by centrifugation to obtain the Cagrilintide peptide;

[0023] Step 4: dissolving the Cagrilintide peptide obtained in step 3 in a mixed solvent containing water, adding a cyclization reagent to form a disulfide bond, and after the reaction is completed, quenching the excess cyclization reagent with a vitamin C aqueous solution to obtain a Cagrilintide crude peptide solution;

[0024] Step 5: The crude Cagrilintide peptide solution obtained in step 4 is purified by reverse silica gel chromatography under eluent conditions to prepare Cagrilintide refined peptide.

[0025] As a preferred embodiment of the present invention, the structure of the fully protected fragment I is as follows:

[0026] 20-(tBu)C20-(Glu-OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Cys(Trt)-Ala-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-OH;

[0027] The structure of the fully protected fragment II is as follows:

[0028] Fmoc-Ala-Glu(OtBu)-Phe-Leu-Arg(Pbf)-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-OH;

[0029] The structure of the fully protected fragment III is as follows:

[0030] Fmoc-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-OH;

[0031] The structure of the nitrogen-terminally exposed fragment IV is as follows: H-Ser(tBu)-Asn (Trt)-Thr(tBu)-Pro-NH 2 .

[0032] As a preferred embodiment of the present invention, the deprotection reagent is a mixture of piperidine and an organic solvent or a mixture of pyrrolidine and an organic solvent. When it is a mixture of piperidine and an organic solvent, the mass percentage of piperidine is 10%-50%; when it is a mixture of pyrrolidine and an organic solvent, the mass percentage of pyrrolidine is 20%-50%; the organic solvent is one or more of dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or 2-methyltetrahydrofuran.

[0033] As a preferred embodiment of the present invention, in step 1, the amount of protected amino acid monomer is 2-5 times the total molar number of the starting resin.

[0034] As a preferred embodiment of the present invention, in step 1, the fluorinated alcohol solution is a 15%-50% (volume fraction) hexafluoroisopropanol dichloromethane solution or a 20%-50% (volume fraction) trifluoroethanol dichloromethane solution.

[0035] As a preferred embodiment of the present invention, in step three, the lysis solution is a combination of trifluoroacetic acid and phenol, water, ethanedithiol, triisopropylsilane, dimethyl sulfide, thioanisole, anisole, dithiothreitol or 3,6-dioxo-1,8-octanedithiol, and the volume percentage of trifluoroacetic acid in the lysis solution is not less than 80%.

[0036] As a preferred embodiment of the present invention, in step 3, the mass ratio of the cleavage solution to the fully protected Cagrilintide is 5:1-10:1, the reaction temperature is 10°C-30°C, and the reaction time is 1.5-5h.

[0037] As a preferred embodiment of the present invention, in step three, the ether solvent is a mixture of one or more of isopropyl ether, methyl tert-butyl ether or diethyl ether.

[0038] As a preferred embodiment of the present invention, in the steps 1) to 4) or in the steps 1) to 4-1), in the mixed solution of resin / protected amino acid monomer / HOBt / DIC, the molar ratio of resin, protected amino acid monomer, HOBt and DIC is 1:2-5:2-5:2-5.

[0039] As a preferred embodiment of the present invention, in step 4, the aqueous mixed solvent is 5%-30% (volume fraction) of acetic acid aqueous solution, 10%-50% (volume fraction) of methanol aqueous solution, 10%-50% (volume fraction) of ethanol aqueous solution or 10%-50% (volume fraction) of acetonitrile aqueous solution.

[0040] As a preferred embodiment of the present invention, in step 4, the cyclization reagent is hydrogen peroxide, DMSO and air or iodine; when hydrogen peroxide is used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the hydrogen peroxide concentration is 1%-10%, and the cyclization temperature is 20-35°C; when DMSO and air are used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the DMSO dosage is 1-10%, and the cyclization temperature is 20-35°C; when iodine is used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the iodine is 0.1-1.0 mol / L methanol, ethanol or acetonitrile solution, and the cyclization temperature is 20-35°C.

[0041] As a preferred embodiment of the present invention, in step 5, the eluent is an aqueous phase with a 0.05-0.2% TFA aqueous solution and an organic phase with acetonitrile; or the aqueous phase is a 10-50 mmol / L potassium dihydrogen phosphate aqueous solution and the organic phase is acetonitrile; or the aqueous phase is a 10-50 mmol / L sodium dihydrogen phosphate aqueous solution and the organic phase is acetonitrile.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] 1) The present invention realizes the synthesis of Cagrilintide by a large fragment solid-liquid hybrid method; firstly, fully protected fragment I (tetradecapeptide) / fragment II (twelve-peptide) / fragment III (nonapeptide) are synthesized by a solid-phase synthesis method; fragment IV (tetradecapeptide) can be synthesized by either a solid-phase method or a full-liquid-phase method. The four oligopeptide fragments can be synthesized by repeating a simple condensation / Fmoc removal reaction to obtain fully protected Cagrilintide.

[0044] 2) The amount of coupling monomer used in each step of the solid phase synthesis of the fragment peptide of the present invention is 2.0 equivalents, which can complete the reaction, has high atom economy and low cost; in addition, the synthesis of each fragment peptide can be carried out in parallel, which greatly shortens the batch synthesis time and has higher production efficiency; in the coupling and docking reaction of the fragment peptide, the equivalent ratio of the fragment peptide is between 1:1-1:1.1, which can not only complete the reaction, but also the coupling and docking reaction of the fragment peptide is carried out in a liquid phase reactor, which is easier to scale up production.

[0045] 3) In addition, the total yield of Cagrilintide synthesized by the present invention is about 45%, which is more than 10% higher than the reported data. The purity of the prepared Cagrilintide is greater than 99.0%, and the single impurity is less than 0.1%. The process is simple, the production efficiency is greatly improved, and it is more suitable for industrial production and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0047] Figure 1 is the fragment cleavage strategy of the present invention.

[0048] Figure 2 It is the synthesis route diagram of the present invention.

[0049] Figure 3 This is the liquid phase synthesis route of fragment IV.

[0050] Figure 4 It is the liquid phase coupling diagram of fragment IV and fragment III.

[0051] Figure 5 It is the liquid phase coupling diagram of fragment II and fragment (III+IV).

[0052] Figure 6 It is the liquid phase coupling diagram of fragment I and fragment (II+III+IV).

[0053] Figure 7 This is the HPLC spectrum of Cagrilintide prepared by purification method one.

[0054] Figure 8 This is the HPLC spectrum of Cagrilintide prepared by purification method 2. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] The present invention provides a method for synthesizing Cagrilintide by a large fragment SPPS-LPPS hybrid method. First, referring to Figure 1 In the present invention, Cagrilintide is cut into four polypeptide fragments, wherein sequences 1-14 are fragment I (tetradecapeptide), sequences 15-26 are fragment II (dodecapeptide), sequences 27-35 are fragment III (nonapeptide), and sequences 36-39 are fragment IV (tetradecapeptide); fully protected fragment I / fragment II / fragment III can be obtained by using 2-Cl-CTC resin as a solid phase carrier, coupling the protected amino acid monomers at the corresponding sequence positions one by one, and finally cleaving them under 1% TFA / hexafluoroisopropanol / pentafluoroethanol or other weakly acidic conditions; fragment IV can be synthesized by liquid phase synthesis or using Sieber Resin as a solid phase carrier.

[0057] See also Figure 2 Fragment I / fragment II / fragment III / fragment IV can be obtained by amidation reaction in liquid phase (THF, DMF or DMSO as solvent), Fmoc protecting group removal, deprotection, disulfide bond synthesis, preparation and purification, etc. to obtain Cagrilintide.

[0058] Example 1

[0059] Solid phase synthesis of fully protected fragment I (tetradecapeptide):

[0060] Synthesis of peptide resin:

[0061] Weigh 20.00 g of 2-Chlorotrityl Chloride resin (the degree of substitution of the resin is 0.76 mmol / g), add it to a solid phase reactor, add 200 mL of dichloromethane to swell the resin for 30 minutes, and drain the solvent; dissolve Fmoc-Leu-OH (10.74 g, 30.40 mmol) and diisopropylethylamine (7.86 g, 60.80 mmol) in 300 mL of dry dichloromethane; add the above solution to the solid phase reactor, control the temperature at 20°C-30°C, react for 3 hours, drain, wash the resin with DMF 5 times, using 200 mL each time; add 300 mL of a mixture of DCM / MeOH / DIPEA (volume ratio 80:15:5), and react at room temperature for 30 minutes (to block the unreacted chloro groups on the resin). Drain and wash the resin with DMF 5 times, 200 mL each time; add 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF=1:4), react at room temperature for 30 minutes, drain and wash the resin with DMF 5 times, 200 mL each time. The indene test result is positive.

[0062] Weigh Fmoc-Arg(Pbf)-OH (19.72 g, 30.40 mmol), HOBt (4.11 g, 30.40 mmol), dissolve in 300 mL DMF, cool to 10-15 ° C, add DIC (3.84 g, 30.40 mmol), continue to activate at 10-15 ° C for 15 minutes, add the resulting activated solution to the solid phase reactor, control the temperature at 20 ° C-30 ° C, take a small amount of resin every 1 hour for indene test, until the indene test is negative, drain, wash the resin with DMF 5 times, each time with 200 mL; add 250 mL 20% piperidine DMF solution (v / v, piperidine:DMF=1:4), temperature controlled at 20°C-30°C for reaction for 30 minutes, drained, and the resin was washed 5 times with DMF, each time with 200 mL, and the indene test result was positive (it should be noted that the numerical value of the above operation is only an embodiment, as long as the molar ratio of 2-ChlorotritylChloride resin / protected amino acid monomer / HOBt / DIC is 1:2-5:2-5:2-5, it is applicable to the present invention; the piperidine in the above operation is an Fmoc protecting group removal agent, and other deprotection agents, such as 20% pyrrolidine DMF solution, 1% DBU DMF solution or 50% diethylamine DMF solution, can also be used to remove Fmoc).

[0063] Repeat the steps described in the previous paragraph to carry out the solid phase synthesis reaction of fragment I (tetradecapeptide) according to the following sequence: Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu-OtBu, 20-(tBu)-20-oxoicosanoic acid; after the coupling, wash the resin with dichloromethane three times, 300 mL each time; then wash the resin with methanol three times, 300 mL each time; and dry under vacuum at room temperature.

[0064] Obtain the peptide resin of fragment I (tetradecapeptide):

[0065] 20-(OtBu)C20-(Glu-OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Cys(Trt)-Ala-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-CTC Resin.

[0066] Lysis:

[0067] Prepare 300 mL of lysis solution with hexafluoroisopropanol: dichloromethane = 1:4 (volume ratio), add the peptide resin of fragment I (tetradecapeptide) under ice bath conditions, stir for 30 minutes under ice bath, and then continue to react at room temperature for 2 hours. After the reaction is completed, filter out the resin under reduced pressure, and concentrate the filtrate under reduced pressure at 25-35°C to obtain fully protected fragment I (tetradecapeptide):

[0068] 20-(OtBu)C20-(Glu-OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Cys(Trt)-Ala-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-OH (45.53g, yield 89.0%).

[0069] Example 2

[0070] Solid phase synthesis of fully protected fragment II (dodecapeptide):

[0071] Synthesis of peptide resin:

[0072] Weigh 20.00 g of 2-Chlorotrityl Chloride resin (the degree of substitution of the resin is 0.76 mmol / g), add it to a solid phase reactor, add 200 mL of dichloromethane to swell the resin for 30 minutes, and drain the solvent; dissolve Fmoc-Gly-OH (9.04 g, 30.40 mmol) and diisopropylethylamine (7.86 g, 60.80 mmol) in 300 mL of dry dichloromethane; add the above solution to the solid phase reactor, control the temperature at 20°C-30°C, react for 3 hours, drain, wash the resin with DMF 5 times, using 200 mL each time; add 300 mL of a mixture of DCM / MeOH / DIPEA (volume ratio 80:15:5), and react at room temperature for 30 minutes (to block the unreacted chloro groups on the resin). Drain and wash the resin with DMF 5 times, 200 mL each time; add 250 mL of 20% piperidine DMF solution (v / v, piperidine:DMF=1:4), react at room temperature for 30 minutes, drain and wash the resin with DMF 5 times, 200 mL each time. The indene test result is positive.

[0073] Weigh Fmoc-Phe-OH (11.78 g, 30.40 mmol), HOBt (4.11 g, 30.40 mmol), dissolve in 300 mL DMF, cool to 10-15 ° C, add DIC (3.84 g, 30.40 mmol), continue to activate at 10-15 ° C for 15 minutes, add the resulting activated solution to the solid phase reactor, control the temperature at 20 ° C-30 ° C, take a small amount of resin every 1 hour for indene test, until the indene test is negative, drain, wash the resin with DMF 5 times, each time with 200 mL; add 250 mL 20% piperidine DMF solution (v / v, piperidine:DMF=1:4), temperature controlled at 20°C-30°C for reaction for 30 minutes, drained, and the resin was washed 5 times with DMF, each time with 200 mL, and the indene test result was positive (it should be noted that the numerical value of the above operation is only an embodiment, as long as the molar ratio of 2-ChlorotritylChloride resin / protected amino acid monomer / HOBt / DIC is 1:2-5:2-5:2-5, it is applicable to the present invention; the piperidine in the above operation is an Fmoc protecting group removal agent, and other deprotection agents, such as 20% pyrrolidine DMF solution, 1% DBU DMF solution or 50% diethylamine DMF solution, can also be used to remove Fmoc).

[0074] Repeat the steps described in the previous paragraph to carry out the solid phase synthesis reaction of fragment II (dodecapeptide) according to the following sequence: Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH; after the coupling, wash the resin with dichloromethane three times, 300 mL each time; then wash the resin with methanol three times, 300 mL each time; and dry under vacuum at room temperature.

[0075] Obtain the peptide resin of fragment II (dodecapeptide):

[0076] Fmoc-Ala-Glu(OtBu)-Phe-Leu-Arg(Pbf)-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-CTC Resin.

[0077] Lysis:

[0078] Prepare 300 mL of lysis solution with hexafluoroisopropanol: dichloromethane = 1:19 (volume ratio), add the peptide resin of fragment II (dodecapeptide) under ice bath conditions, stir under ice bath for 30 minutes, and then continue to react at room temperature for 2 hours. After the reaction is completed, filter out the resin under reduced pressure, slowly add the filtrate dropwise to 1.5 liters of pre-cooled isopropyl ether for crystallization, and collect the solid by centrifugation to obtain the fully protected fragment II (dodecapeptide):

[0079] Fmoc-Ala-Glu(OtBu)-Phe-Leu-Arg(Pbf)-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-OH (38.22g, yield 91.5%).

[0080] Example 3

[0081] Solid phase synthesis of fully protected fragment III (nonapeptide):

[0082] Synthesis of peptide resin:

[0083] Weigh 20.00 g of 2-Chlorotrityl Chloride resin (the degree of substitution of the resin is 0.76 mmol / g), add it to a solid phase reactor, add 200 mL of dichloromethane to swell the resin for 30 minutes, and drain the solvent; dissolve Fmoc-Gly-OH (9.04 g, 30.40 mmol) and diisopropylethylamine (7.86 g, 60.80 mmol) in 300 mL of dry dichloromethane; add the above solution to the solid phase reactor, control the temperature at 20°C-30°C, react for 3 hours, drain, wash the resin with DMF 5 times, using 200 mL each time; add 300 mL of a mixture of DCM / MeOH / DIPEA (volume ratio 80:15:5), and react at room temperature for 30 minutes (to block the unreacted chloro groups on the resin). Drain and wash the resin with DMF 5 times, 200 mL each time; add 250 mL of 20% piperidine DMF solution (v / v, piperidine: DMF = 1:4), control the temperature at 20℃-30℃ to react for 30 minutes, drain and wash the resin with DMF 5 times, 200 mL each time. The indene test result is positive.

[0084] Weigh Fmoc-Val-OH (10.32 g, 30.40 mmol), HOBt (4.11 g, 30.40 mmol), dissolve in 300 mL DMF, cool to 10-15 ° C, add DIC (3.84 g, 30.40 mmol), continue to activate at 10-15 ° C for 15 minutes, add the resulting activated solution to the solid phase reactor, control the temperature at 20 ° C-30 ° C, take a small amount of resin every hour for indene test until the indene test is negative, drain, wash the resin with DMF 5 times, each time with 200 mL; add 250 mL 20% piperidine DMF solution (v / v, piperidine: DMF = 1: 4), react at 20 ° C-30 ° C for 30 minutes, drain, wash the resin with DMF 5 times, each time with 200 mL, and the indene test result is positive (it should be noted that the numerical value of the above operation is only an example, as long as the 2-Chlorotrityl The molar ratio of Chloride resin / protected amino acid monomer / HOBt / DIC in the range of 1:2-5:2-5:2-5 is suitable for the present invention; the piperidine in the above operation is a Fmoc protecting group removing agent, and other deprotecting agents, such as 20% pyrrolidine DMF solution, 1% DBU DMF solution or 50% diethylamine DMF solution, can also be used to remove Fmoc).

[0085] Repeat the steps described in the previous paragraph to carry out the solid phase synthesis reaction of fragment III (nonapeptide) according to the following sequence: Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Leu-OH (tetrachlorobenzoquinone is used as a detector in this coupling step), Fmoc-Ile-OH, Fmoc-Pro-OH; after the coupling, wash the resin with dichloromethane three times, 300 mL each time; then wash the resin with methanol three times, 300 mL each time; and dry under vacuum at room temperature.

[0086] Obtain the peptide resin of fragment III (nonapeptide):

[0087] Fmoc-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-CTC Resin.

[0088] Lysis:

[0089] 300 mL of lysis solution was prepared with hexafluoroisopropanol: dichloromethane = 1:4 (volume ratio), and the peptide resin of fragment III (nonapeptide) was added under ice bath conditions. The mixture was stirred for 30 min under ice bath, and then the reaction was continued at room temperature for 2 hours. After the reaction was completed, the resin was filtered off under reduced pressure, and the filtrate was concentrated under reduced pressure at 25-35 ° C to obtain fully protected fragment III (nonapeptide): Fmoc-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-OH (19.05 g, yield 87.8%).

[0090] Example 4

[0091] Solid phase synthesis of the N-terminally exposed fragment IV (tetrapeptide):

[0092] Synthesis of peptide resin:

[0093] Weigh 20.00 g of Sieber resin (the degree of substitution of the resin is 0.60 mmol / g), add it to a solid phase reactor, add 200 mL of dichloromethane to swell the resin for 30 minutes, and drain the solvent; add 250 mL of 20% piperidine DMF solution (v / v, piperidine: DMF = 1:4), control the temperature at 20℃-30℃ to react for 30 minutes, drain, wash the resin with DMF 5 times, using 200 mL each time, and the indene test result is positive.

[0094] Weigh Fmoc-Pro-OH (8.10 g, 24.00 mmol), HOBt (3.24 g, 24.00 mmol), dissolve in 300 mL DMF, cool to 10-15 ° C, add DIC (3.03 g, 24 mmol), continue to activate at 10-15 ° C for 15 minutes, add the resulting activated solution to the solid phase reactor, control the temperature at 20 ° C-30 ° C, take a small amount of resin every 1 hour for indene test, until the indene test is negative, drain, wash the resin with DMF 5 times, each time with 200 mL; add 250 mL 20% piperidine DMF solution (v / v, piperidine:DMF=1:4), temperature controlled at 20°C-30°C for 30 minutes, drained, and the resin was washed 5 times with DMF, each time with 200 mL, and the indene test result was positive (it should be noted that the numerical value of the above operation is only an embodiment, as long as the molar ratio of Sieber resin / protected amino acid monomer / HOBt / DIC is 1:2-5:2-5:2-5, it is applicable to the present invention; the piperidine in the above operation is an Fmoc protecting group removal agent, and other deprotection agents, such as 20% pyrrolidine DMF solution, 1% DBU DMF solution or 50% diethylamine DMF solution, can also be used to remove Fmoc.).

[0095] Repeat the steps described in the previous paragraph to carry out the solid phase synthesis reaction of fragment IV (tetrapeptide) according to the following sequence: Fmoc-Thr(tBu)-OH (tetrachlorobenzoquinone is used as a detector for coupling in this step), Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH; after the coupling, wash the resin with dichloromethane three times, 300 mL each time; then wash the resin with methanol three times, 300 mL each time; dry under vacuum at room temperature; obtain the peptide resin of fragment IV (tetrapeptide): H-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-Sieber Resin.

[0096] Lysis:

[0097] Prepare a 2.5% (volume fraction) trifluoroacetic acid solution in dichloromethane, add the peptide resin of fragment IV (tetrapeptide) under ice bath conditions, stir for 30 minutes under ice bath conditions, and then continue to react at room temperature for 2 hours. After the reaction is completed, adjust the pH to neutral with diisopropylethylamine, filter out the resin under reduced pressure, and concentrate the filtrate under reduced pressure at 25-35°C to obtain fragment IV (tetrapeptide) with exposed nitrogen end: H-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 (8.63g, yield 93.3%).

[0098] Example 5

[0099] The nitrogen-terminally exposed fragment IV (tetrapeptide) can also be obtained by liquid phase synthesis. The specific synthesis route is as follows: Figure 3 As shown:

[0100] Weigh Fmoc-Thr(tBu)-OH (12.08 g, 30.40 mmol) and HOBt (4.52 g, 33.44 mmol) in a 500 mL round-bottom flask, add 200 mL of DCM, cool in an ice bath for 10 minutes, add DCC (6.90 g, 33.44 mmol), continue to activate in an ice bath for 15 minutes, and add H-Pro-NH 2 (5.21 g, 45.60 mmol), remove the ice bath, react at room temperature until the reaction of Fmoc-Thr(tBu)-OH is complete as monitored by HPLC, filter out the solid (DCU) in the system, wash the filtrate with sodium bicarbonate aqueous solution three times, 30 mL each time; dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the product Fmoc-Thr(tBu)-Pro-NH 2 Crude; Fmoc-Thr(tBu)-Pro-NH 2 The crude product was added to a 500 mL round-bottom flask, and 150 mL of 20% pyrrolidine dichloromethane solution (volume ratio 1:4) was added under ice bath. After 10 min, the ice bath was removed, and the mixture was reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and then slurried with n-heptane twice, 100 mL each time, filtered, and the filter cake was washed with a small amount of n-heptane. The filter cake was collected and dried under reduced pressure to obtain H-Thr(tBu)-Pro-NH 2 The crude product was directly used in the next reaction.

[0101] Weigh Fmoc-Asn(Trt)-OH (18.14 g, 30.40 mmol) and HOBt (4.52 g, 33.44 mmol) in a 500 mL round-bottom flask, add 200 mL of DCM, cool in an ice bath for 10 minutes, add DCC (6.90 g, 33.44 mmol), continue to activate in an ice bath for 15 minutes, and add the H-Thr(tBu)-Pro-NH obtained in the previous step. 2 Remove the ice bath and react at room temperature until the reaction of Fmoc-Asn(Trt)-OH is complete as monitored by HPLC. Filter to remove the solid (DCU) in the system, wash the filtrate with sodium bicarbonate aqueous solution three times, 30 mL each time; dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the product Fmoc-Asn(Trt)-Thr(tBu)-Pro-NH 2 Crude; Fmoc-Asn(Trt)-Thr(tBu)-Pro-NH 2The crude product was added to a 500 mL round-bottom flask, and 150 mL of 20% pyrrolidine dichloromethane solution (volume ratio 1:4) was added under ice bath. After 10 min, the ice bath was removed, and the mixture was reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and then slurried with n-heptane twice, 100 mL each time, filtered, and the filter cake was washed with a small amount of n-heptane. The filter cake was collected and dried under reduced pressure to obtain H-Asn(Trt)-Thr(tBu)-Pro-NH 2 The crude product was directly used in the next reaction.

[0102] Weigh Fmoc-Ser(tBu)-OH (11.66 g, 30.40 mmol) and HOBt (4.52 g, 33.44 mmol) in a 500 mL round-bottom flask, add 200 mL of DCM, cool in an ice bath for 10 minutes, add DCC (6.90 g, 33.44 mmol), continue to activate in an ice bath for 15 minutes, and add the H-Asn(Trt)-Thr(tBu)-Pro-NH 2 , remove the ice bath, react at room temperature until the reaction of Fmoc-Ser(tBu)-OH is complete as monitored by HPLC, filter out the solid (DCU) in the system, wash the filtrate with sodium bicarbonate aqueous solution three times, 30 mL each time; dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure to obtain the product Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 Crude product.

[0103] Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 Purify by normal phase silica gel column chromatography with an elution gradient of DCM / MeOH 10:1 (V / V) to 5:1 (V / V) to obtain Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 The pure product was added to a 500 mL round-bottom flask, and 150 mL of 20% pyrrolidine dichloromethane solution (volume ratio 1:4) was added under ice bath. After 10 minutes, the ice bath was removed, and the mixture was reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and then slurried twice with n-heptane, 100 mL each time, filtered, and the filter cake was washed with a small amount of n-heptane. The filter cake was collected and dried under reduced pressure to obtain the fragment IV (tetrapeptide) with exposed nitrogen end: H-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH 2 (20.44 g, yield 87.2%) The crude product was directly used in the next reaction.

[0104] Example 6

[0105] Solution phase coupling of the nitrogen-terminally exposed fragment IV (tetrapeptide) and the fully protected fragment III (nonapeptide) was performed as follows Figure 4 As shown:

[0106] Weigh the fully protected fragment III (nonapeptide) (14.27 g, 10.00 mmol) and HOBt (1.49 g, 11.00 mmol) in a 500 mL round-bottom flask, add 150 mL of DMSO to dissolve, then cool in an ice bath for 10 minutes, add DIPEA (1.55 g, 12.00 mmol), HATU (3.99 g, 10.50 mmol) and continue to activate in an ice bath for 10 minutes, and add the nitrogen-terminally exposed fragment IV (tetrapeptide) (8.09 g, 10.50 mmol), remove the ice bath, react at room temperature until the reaction of fragment III (nonapeptide) monitored by HPLC is complete, pour the reaction solution into ice water for crystallization, filter and collect the solid, slurry the solid with 100 ml of 5% sodium bicarbonate aqueous solution, and collect the solid by filtration under reduced pressure; add the above crude product to a 500 mL round-bottom flask, add 150 ml of 20% pyrrolidine dichloromethane solution (volume ratio 1:4) under ice bath, remove the ice bath after 10 minutes, react at room temperature for 1 hour, concentrate under reduced pressure, then slurry twice with isopropyl ether, 100 ml each time, filter, wash the filter cake with a small amount of n-heptane, collect the filter cake, and dry under reduced pressure to obtain the crude fragment III+IV (thirteen peptide) with exposed nitrogen end, which is directly used for the next reaction.

[0107] Example 7

[0108] Liquid phase coupling of fully protected fragment II (dodecapeptide) and fragment (III+IV) (tridecapeptide) was carried out as follows Figure 5 As shown:

[0109] The fully protected fragment II (12-peptide) (27.48 g, 10.00 mmol) and HOBt (1.49 g, 11.00 mmol) were weighed into a 500 mL round-bottom flask, 150 mL of DMSO was added to dissolve, and then cooled in an ice bath for 10 minutes, DIPEA (1.55 g, 12.00 mmol) and HATU (3.99 g, 10.50 mmol) were added, and continued to activate in an ice bath for 15 min, and fragment (III+IV) (13-peptide) (20.55 g, 10.50 mmol) was added, the ice bath was removed, and the reaction was carried out at room temperature until the reaction of fragment II (12-peptide) was complete as monitored by HPLC, and the reaction solution was added dropwise to 750 mL of ice water for crystallization, and the solid was collected by filtration, and the solid was precipitated with 5% carbon The mixture was slurried with 100 mL of sodium bicarbonate aqueous solution and the solid was collected by filtration under reduced pressure. The crude product was added to a 500 mL round-bottom flask, and 150 mL of 20% pyrrolidine dichloromethane solution (volume ratio 1:4) was added under ice bath. The ice bath was removed after 10 minutes, and the mixture was reacted at room temperature for 1 hour. The mixture was concentrated under reduced pressure, and then slurried with isopropyl ether twice, 100 mL each time, filtered, and the filter cake was washed with a small amount of n-heptane. The filter cake was collected and dried under reduced pressure to obtain the crude fragment (II+III+IV) (twenty-pentapeptide) with exposed nitrogen end, which was directly used for the next step reaction.

[0110] Example 8

[0111] Fully protected Cagrilintide can be obtained by liquid phase coupling of fragment I (tetradecapeptide) and fragment (II+III+IV) (twenty-pentapeptide), as follows: Figure 6 As shown:

[0112] Weigh the fully protected fragment I (tetradecapeptide) (26.92 g, 8.00 mmol) and HOBt (1.19 g, 8.80 mmol) in a 500 mL round-bottom flask, add DMSO 150 mL to dissolve, then cool in an ice bath for 10 minutes, add DIPEA (1.55 g, 12.00 mmol) and HATU (3.19 g, 8.40 mmol) and continue to activate in an ice bath for 15 minutes, add fragment (II+III+IV) (twenty-pentapeptide) (37.52 g, 8.40 mmol), remove the ice bath, react at room temperature until the reaction of fragment I (tetradecapeptide) is complete as monitored by HPLC, add the reaction solution dropwise into 750 mL ice water for crystallization, filter and collect the solid, slurry the solid with 100 mL of 5% sodium bicarbonate aqueous solution, and filter and collect the solid under reduced pressure to obtain fully protected Cagrilintide.

[0113] One-step deprotection of fully protected Cagrilintide: TFA / EDT / TIS / H 2O=87.5:5:2.5:5 (V / V) lysate 500mL, ice bath cooling for 30min, add the cooled lysate to the reaction bottle containing fully protected Cagrilintide, naturally heat and react for 2-3 hours, concentrate the reaction solution under reduced pressure at 20-30°C to 200 ml, slowly add the obtained concentrate dropwise to 1.8 liters of pre-cooled isopropyl ether for crystallization, collect the solid by centrifugation, wash the solid twice with new pre-cooled isopropyl ether, 500mL each time, collect the solid by centrifugation, dry under reduced pressure at room temperature for 1 hour to obtain the crude Cagrilintide peptide.

[0114] Cyclization of Cagrilintide peptide: Dissolve the crude Cagrilintide peptide in 20% acetonitrile aqueous solution, dilute with purified water to a concentration of 1-10g / L; dissolve iodine in acetonitrile (concentration 10-50g / L); slowly add the iodine acetonitrile solution to the diluted Cagrilintide peptide solution under stirring at room temperature, until the solution turns yellow, and does not fade after stirring at room temperature for 5 minutes, indicating that the reaction has reached the end point, and then add 10% vitamin C aqueous solution to the system until the system is colorless and transparent. Obtain a solution of crude Cagrilintide peptide.

[0115] Preparation and purification of crude Cagrilintide cyclic peptide:

[0116] The crude Cagrilintide cyclic peptide solution obtained in the previous step was filtered using a mixed microporous membrane with a pore size of 0.45 μm.

[0117] The purification and preparation were carried out by high performance liquid chromatography, and the Cagrilintide fractions were collected according to the position of the reference substance.

[0118] See Table 1 for preparation and purification method.

[0119] Table 1. Purification method 1

[0120]

[0121] The main parameters of the preparation and purification method 1 are shown in Table 1: 0.1% trifluoroacetic acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B; UniSil 10-120 C18 produced by Changshu Nanomicrobiology Technology Co., Ltd. was used as filler; 15.73 g of lyophilized powder was obtained after purification, with an estimated yield of 44.6% (calculated based on 8 mmol of the crude product) and a purity of 99.79% (see Figure 7 , Figure 7 See Table 2 for data).

[0122] Table 2. Test results

[0123]

[0124] See Table 3 for preparation and purification method 2.

[0125] Table 3. Purification method 2

[0126]

[0127] The main parameters of the preparation and purification method 2 are shown in Table 3: 20 mmol / L potassium dihydrogen phosphate aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B; UniSil 10-120 C18 produced by Changshu Nanomicrobiology Technology Co., Ltd. was used as filler; the purified Cagrilintide fraction was desalted and transsalted and then freeze-dried to obtain 15.34 g of freeze-dried powder, with an estimated yield of 43.5% (calculated based on 8 mmol of the crude product) and a purity of 99.81% (see Figure 8 , Figure 8 See Table 4 for the data).

[0128] Table 4. Test results

[0129]

[0130] The above embodiments show that compared with the one-by-one coupling solid phase synthesis method (CN202111615700.8), the purity of Cagrilintide prepared by the method provided by the present invention is greater than 99.0%, and the single impurity is less than 0.1%. The total yield of Cagrilintide synthesized by the present invention is about 45%, and the product yield is increased by more than 10%.

[0131] The solid phase synthesis of the fragment peptides of the present invention can complete the reaction with an amount of 2.0 equivalents of coupled amino acid monomers in each step, with high atom economy and low cost; in addition, the synthesis of each fragment peptide can be carried out in parallel, which greatly shortens the batch synthesis time and has higher production efficiency; in the coupling and docking reaction of the fragment peptides, the equivalent ratio of the fragment peptides is between 1:1 and 1:1.1, which can complete the reaction, and the coupling and docking reaction of the fragment peptides is carried out in a liquid phase reactor, with a simple process, greatly improved production efficiency, more suitable for industrial production, and has broad application prospects.

[0132] The above is only a preferred embodiment of the present invention, and is not any formal or substantial limitation of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention. Any technician familiar with this profession, without departing from the spirit and scope of the present invention, can make some changes, modifications and evolutions of the technical content disclosed above, which are equivalent embodiments of the present invention; at the same time, any changes, modifications and evolutions of any equivalent changes made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for synthesizing Cagrilintide by a large fragment SPPS-LPPS hybrid method, characterized in that: The method comprises cutting Cagrilintide into four polypeptide fragments: sequence 1-14 is fragment I, sequence 15-26 is fragment II, sequence 27-35 is fragment III, and sequence 36-39 is fragment IV; synthesizing fully protected fragment I, fragment II and fragment III by coupling protected amino acid monomers at corresponding sequence positions one by one through a solid phase synthesis method, synthesizing fragment IV with exposed nitrogen end through a solid phase synthesis method or a liquid phase synthesis method; and finally obtaining fully protected Cagrilintide by repeated condensation / Fmoc removal reaction between the polypeptide fragments in a liquid phase; The steps of the method are: Step 1) Synthesis of fully protected fragments I, II, III and IV: 1) Synthesis of fully protected fragment I: Add dichloromethane to a solid phase reactor containing 2-Chlorotrityl Chloride resin to swell it, drain the solvent, add a dry dichloromethane solution of Fmoc-Leu-OH and DIPEA, connect the first amino acid to the resin, add DCM / MeOH / DIPEA to block the unreacted or difficult-to-react chlorinated sites on the resin; wash the resin with solvent, add a deprotection reagent to remove the Fmoc protecting group, add an activated Fmoc-Arg(Pbf)-OH / HOBt / DIC mixture to carry out the coupling reaction of the second amino acid monomer, and after the indene test result is negative, drain and wash to complete the coupling reaction; repeat the above steps, as shown in The following sequence was used for the solid phase synthesis reaction of fragment I: Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu-OtBu, 20-(tBu)-20-oxoicosanoic acid; after the coupling, the resin was washed with dichloromethane and methanol; the resin was dried under vacuum at room temperature; the fully protected fragment I was obtained by cleavage under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution; 2) Synthesis of fully protected fragment II: According to the synthesis method of fragment I, the following amino acid monomers Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH were sequentially connected to 2-Chlorotrityl Chloride resin; after coupling, the resin was washed with dichloromethane and methanol; the resin was dried under vacuum at room temperature; and the fully protected fragment II was obtained by cleavage under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution; 3) Synthesis of fully protected fragment III: According to the synthesis method of fragment I, the following amino acid monomers Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, and Fmoc-Pro-OH are sequentially connected to 2-ChlorotritylChloride resin; after the coupling, the resin is washed with dichloromethane and methanol; the resin is dried under vacuum at room temperature; and the fully protected fragment III is cleaved under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution; 4) Solid phase synthesis of fragment IV with exposed nitrogen end: After Sieber resin is swollen by adding dichloromethane, the solvent is drained, and a deprotection reagent is added to remove the Fmoc protecting group. The resin is drained and washed. The indene test result is positive; the activated Fmoc-Pro-OH / HOBt / DIC mixed solution is added to carry out the coupling reaction of the first amino acid monomer. After the indene test result is negative, the reaction is drained and washed to complete the coupling reaction; the above steps are repeated to carry out the solid phase synthesis reaction of fragment IV according to the following sequence: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH; after the coupling is completed, a deprotection reagent is added to remove Fmoc, and the resin is washed with dichloromethane and methanol; vacuum drying at room temperature; cleavage is carried out under the conditions of fluorinated alcohol solution or 1%-5% TFA dichloromethane solution to obtain fragment IV with exposed nitrogen end; Alternatively, 4-1) Liquid phase synthesis of IV with exposed nitrogen end: Fmoc-Thr(tBu)-OH / HOBt was dissolved in dichloromethane, cooled to below 15°C, activated by adding DCC, and H-Pro-NH2 was added. After coupling, DCU was removed by filtration, and the organic phase was washed with sodium bicarbonate aqueous solution, separated, dried, and concentrated under reduced pressure. A deprotection reagent was added to remove Fmoc. After the reaction was completed, the reaction was concentrated under reduced pressure and slurried twice in n-heptane. The above coupling and deprotection steps were repeated to couple Fmoc-Asn(Trt)-OH, Fm oc-Ser(tBu)-OH two monomers; after obtaining Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH2, use normal phase silica gel column, column chromatography purification in dichloromethane methanol system to obtain pure Fmoc-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH2; add deprotection reagent to remove Fmoc, concentrate under reduced pressure, then beat with n-heptane twice, filter under reduced pressure, collect filter cake, and dry under reduced pressure to obtain fragment IV with exposed nitrogen end; Step 2: Liquid phase coupling of fully protected fragment I, fragment II, fragment III and fragment IV: The fully protected fragment III, HOBt or HOAt, and DIPEA obtained in step 1 are dissolved in DMSO, cooled to below 15°C, activated by adding HATU, and the nitrogen-terminally exposed fragment IV obtained in step 1 is added, and a coupling reaction is carried out at 20-35°C. After the coupling is completed, the reaction solution is added dropwise to ice water for crystallization, and the solid is collected by filtration. The filter cake is slurried with a sodium bicarbonate aqueous solution, and the filter cake is collected by filtration. After vacuum drying, a deprotection reagent is added to remove Fmoc. After the reaction is completed, the mixture is concentrated under reduced pressure and slurried twice with isopropyl ether. The coupling and deprotection steps are repeated to sequentially couple the fully protected fragment II obtained in step 1 and the fully protected fragment I obtained in step 1 to obtain fully protected Cagrilintide. Step 3: Remove all protecting groups in one step: The fully protected Cagrilintide obtained in step 2 is subjected to one-step removal of all protecting groups under the conditions of the lysate, precipitated in an ether solvent, and the solid is collected by centrifugation. The solid is washed twice with an ether solvent, and the solid is collected by centrifugation to obtain the Cagrilintide peptide; Step 4: dissolving the Cagrilintide peptide obtained in step 3 in a mixed solvent containing water, adding a cyclization reagent to form a disulfide bond, and after the reaction is completed, quenching the excess cyclization reagent with a vitamin C aqueous solution to obtain a Cagrilintide crude peptide solution; Step 5: The crude Cagrilintide peptide solution obtained in step 4 is purified by reverse silica gel chromatography under eluent conditions to obtain refined Cagrilintide peptide; The structure of the fully protected fragment I is as follows: 20-(tBu)C20-(Glu-OtBu)-Lys(Boc)-Cys(Trt)-Asn(Trt)-Thr(tBu)-Ala-Thr(tBu)-Cys(Trt)-Ala-Thr(tBu)-Gln(Trt)-Arg(Pbf)-Leu-OH; The structure of the fully protected fragment II is as follows: Fmoc-Ala-Glu(OtBu)-Phe-Leu-Arg(Pbf)-His(Trt)-Ser(tBu)-Ser(tBu)-Asn(Trt)-Asn(Trt)-Phe-Gly-OH; The structure of the fully protected fragment III is as follows: Fmoc-Pro-Ile-Leu-Pro-Pro-Thr(tBu)-Asn(Trt)-Val-Gly-OH; The structure of fragment IV with exposed nitrogen terminus is as follows: H-Ser(tBu)-Asn(Trt)-Thr(tBu)-Pro-NH2.

2. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: The deprotection reagent is a mixture of piperidine and an organic solvent or a mixture of pyrrolidine and an organic solvent. When the mixture is a mixture of piperidine and an organic solvent, the mass percentage of piperidine is 10%-50%; when the mixture is a mixture of pyrrolidine and an organic solvent, the mass percentage of pyrrolidine is 20%-50%; the organic solvent is one or more of dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone or 2-methyltetrahydrofuran.

3. The method for synthesizing Cagrilintide by a large fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 1, the amount of the protected amino acid monomer is 2-5 times the total molar amount of the starting resin.

4. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 1, the fluorinated alcohol solution is a 15%-50% by volume hexafluoroisopropanol dichloromethane solution or a 20%-50% by volume trifluoroethanol dichloromethane solution.

5. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 3, the lysis solution is a combination of trifluoroacetic acid and phenol, water, ethanedithiol, triisopropylsilane, dimethyl sulfide, thioanisole, anisole, dithiothreitol or 3,6-dioxo-1,8-octanedithiol, and the volume percentage of trifluoroacetic acid in the lysis solution is not less than 80%.

6. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 3, the mass ratio of the cleavage solution to the fully protected Cagrilintide is 5:1-10:1, the reaction temperature is 10°C-30°C, and the reaction time is 1.5-5h.

7. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 3, the ether solvent is a mixture of one or more of isopropyl ether, methyl tert-butyl ether or diethyl ether.

8. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 1) to step 4) or in step 1) to step 4-1), in the mixed solution of resin / protected amino acid monomer / HOBt / DIC, the molar ratio of resin, protected amino acid monomer, HOBt and DIC is 1:2-5:2-5:2-5.

9. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 4, the aqueous mixed solvent is 5%-30% acetic acid aqueous solution, 10%-50% methanol aqueous solution, 10%-50% ethanol aqueous solution or 10%-50% acetonitrile aqueous solution.

10. The method for synthesizing Cagrilintide by a large-fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 4, the cyclization reagent is hydrogen peroxide, DMSO and air or iodine; when hydrogen peroxide is used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the hydrogen peroxide concentration is 1%-10%, and the cyclization temperature is 20-35°C; when DMSO and air are used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the DMSO dosage is 1-10%, and the cyclization temperature is 20-35°C; when iodine is used for cyclization, the crude peptide concentration of Cagrilintide linear peptide is 1-10.0 mg / mL, the iodine is 0.1-1.0 mol / L methanol, ethanol or acetonitrile solution, and the cyclization temperature is 20-35°C.

11. The method for synthesizing Cagrilintide by a large fragment SPPS-LPPS hybrid method according to claim 1, characterized in that: In step 5, the eluent is a 0.05-0.2% TFA aqueous solution in the aqueous phase and acetonitrile in the organic phase; or a 10-50 mmol / L potassium dihydrogen phosphate aqueous solution in the aqueous phase and acetonitrile in the organic phase; or a 10-50 mmol / L sodium dihydrogen phosphate aqueous solution in the aqueous phase and acetonitrile in the organic phase.

Images