Preparation Method Of Cagrilintide

Preparation Method Of Cagrilintide
Background technology
Cagrilintide is a novel long-acting acylated amylin analogue that acts as a non-selective amylin receptor (AMYR) and calcitonin G protein-coupled receptor (CTR) agonist, which is a cyclic polypeptide composed of 38 amino acids with 38 amino acids in a sequence and a pair of disulfide bonds. Cagliptide can significantly reduce body weight and reduce food intake, which has potential in obesity research. Amylin is another hormone associated with hunger and satiety in addition to the GLP-1 signaling pathway.
Cagliptide can reduce energy intake, regulate food choices and preferences, and co-secrete with insulin to regulate glucose, inhibit postprandial glucagon release, and delay gastric emptying. It has obvious advantages in the treatment of diabetes, obesity, metabolic syndrome and cardiovascular disease, and has a wide range of application prospects.

Basic information
Chinese name: 卡格列肽
English name: Cagrilintide
CAS Number: 1415456-99-3
Sequence: γ-glu-lys-cys-asn-thr-ala-thr-cys-ala-thr-gln-arg-leu-ala-glu-phe-leu-arg-his-ser-ser-asn-asn-phe-gly-ile-leu-pro-thr-asn-val-gly-ser-asn-thr-pro-NH2 ( Disulfidebridge:cys4-cys9)
Formula: C194H312N54O59S2
Molecular Weight: 4409.01

Raw materials for Cagrilintide

According to the relevant patents of cagrilintide, a synthesis method of cagrilintide can be obtained. The 7th-8th amino acids of the cagrilintide sequence were selected as the pseudoprodipeptide Fmoc-Ala-Thr(pro-me-me)-OH, the 10th-11th amino acids of the sequence were used as the pseudopredipeptide Fmoc-Ala-Thr(pro-me-me)-OH, and the 21st-22nd amino acids of the sequence were used as the pseudoprodipeptide Fmoc-Ser-Ser(pro-me-me)-OH. The remaining sites were coupling according to the amino acid sequence. The peptide resin was lysed to obtain canagliptide linear peptide. Then the crude product of canagliptide was obtained by liquid phase cyclization, and the canagliptide refined peptide was obtained by purification, salting, concentration and lyophilization. This method uses three pseudo-codipeptides for coupling, which solves the problem of increasing the difficulty of coupling due to resin condensation, but increases the production cost, and the purity of the refined peptides prepared by this process is low, which does not meet the quality requirements of the API.

The flowchart is as below.

(1) Preparation of canaglitide peptide resin
Take 1.5mmol of the first protective amino acid and 1.5mmol HOBT, dissolve it with an appropriate amount of DMF and cool to 0~15°C; Another 1.5mmol DIC is taken, slowly added to the DMF solution of the protective amino acid after stirring, and stirred for 10 minutes in an environment of 0~15°C to obtain the activated protective amino acid solution, set aside.
Take 0.5 mmol of amino resin (substitution degree 0.42 mmol/g), swell with DMF solution for 60 minutes, use 20% Pip/DMF solution to protect for 30 minutes, wash and filter to obtain the resin to be reacted.
The first protective amino acid solution after activation is added to the resin to be reacted, and the coupling reaction is carried out at 25~35°C for 120~300 minutes, filtered and washed, and a resin containing 1 protective amino acid is obtained: Fmoc-Pro-Resin.
The same method was used to access the 2~38th protective amino acids or fragments corresponding to the above: Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH· H2O，Fmoc-Pro-OH· H2O，Fmoc-Leu-OH，Fmoc-Ile-OH，Fmoc-Pro-OH· H2O，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-Pro-OH· H2O，Fmoc-Glu(OtBu)-OH，Fmoc-Ala-OH· H2O，Fmoc-Leu-OH，Fmoc-Arg(Pbf)-OH，Fmoc-Gln(Trt)-OH，Fmoc-Ala-Thr(psi(Me,Me)pro)-OH，Fmoc-Cys(Trt)-OH，Fmoc-Ala-Thr(psi(Me,Me)pro)-OH，Fmoc-Thr(tBu)-OH，Fmoc-Asn(Trt)-OH， Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu-OtBu, mono-tert-butyl eicosanedioate, and decanaglitide peptide peptide resin 4.98g.

(2) Preparation of Cagrilintide linear peptide crude peptide
Take 4.49g of canagliptide peptide resin and add TFA mixed solution, the ratio of the mixed solution is TFA:EDT/Tis/phenol/H2O=87.5:2.5:5:5:2.5:2.5, and the dosage is 8ml/gram of resin. Stirring reaction at 20~30°C for 3 hours, the reaction mixture is filtered with a sand core funnel, the filtrate is collected, the resin is washed 3 times with a small amount of TFA, the filtrate is combined and then added to the MTBE precipitate, and then washed and precipitated with MTBE 3 times, and the white powder is 2.08g of canaglitide linear peptide crude peptide.

(3) Preparation of cagrilintide cyclic peptide crude peptide solution
0.50g of canagliptide linear peptide crude peptide was configured into linear peptide crude peptide solution at a concentration of 5.0mg/ml, and 5ml of DMSO solution was added drop under the stirred state, and the reaction was 20~30°C for 20h to obtain canaglitide cyclic peptide crude peptide solution.

(4) Preparation of cagrilintide spermatide
The crude peptide solution of cagrilintide cyclic peptide was taken and filtered with a 0.45μm mixed microporous filter membrane.
High performance liquid chromatography was used for purification and preparation, and canagliptide fractions were collected.
High performance liquid chromatography was used to change the salt, the main peak of the salt exchange was collected and the purity was detected by analyzing the liquid phase, and the solution of the main peak of the salt exchange was combined with the solution of the main peak of salt exchange, and the aqueous solution of canagliptide acetate was obtained by freeze-drying, and the purity was 99.58%, the maximum single impurity was 0.09%, the total yield was 31.87%, and the molecular weight was 4409.0.


The above embodiment shows that compared with the step-by-step coupled synthesis process, the purity of the product obtained by the method provided by the present invention is greater than 99.0%, and the single impurity is less than 0.1%, which improves the product quality and total yield, the process is simple and easy to control, the degree of industrialization is high, and it has a wide range of practical value and application prospects.