阅读说明：本技术 一种Setmelanotide的制备方法 (Preparation method of Setmelanotide ) 是由 董华建 郭德文 文永均 于 2020-07-06 设计创作，主要内容包括：本发明提供了一种制备Setmelanotide的方法,方法中采用氨基树脂为起始树脂,按Setmelanotide的氨基序列反相依次接入相应的保护氨基酸得到Setmelanotide肽树脂,Setmelanotide保护肽树脂经酸解后和再经氧化环化后获得Setmelanotide粗品,Setmelanotide粗品再经过纯化和冻干得到Setmelanotide纯品。本发明工艺操作简单,产品总收率高,适合规模化生产。(The invention provides a method for preparing Setmelonide, which comprises the steps of taking amino resin as starting resin, sequentially adding corresponding protective amino acids according to the reversed phase of an amino sequence of Setmelonide to obtain Setmelonide peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelonide protective peptide resin to obtain a Setmelonide crude product, and purifying and freeze-drying the Setmelonide crude product to obtain a pure Setmelonide product. The method has the advantages of simple process operation and high total product yield, and is suitable for large-scale production.)

1. A method for preparing Setmelanotide, comprising: taking amino resin as starting resin, sequentially inoculating corresponding protective amino acids according to the reverse phase of the amino sequence of Setmelactone to obtain Setmelactone peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelactone protective peptide resin to obtain a Setmelactone crude product, and purifying and freeze-drying the Setmelactone crude product to obtain a Setmelactone pure product:

Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2。

2. the method for producing Setmelantotide according to claim 1, characterized in that: the substitution value of the amino resin is 0.3-1.5 mmol/g resin, and the preferable substitution value is 0.6-1.0 mmol/g resin.

3. The amino resin according to claim 2 is selected from Rink Amide-MBHA resin, Rink Amide-BHA resin and Rink Amide resin, preferably Rink Amide-MBHA resin.

4. The method for producing Setmelantotide according to claim 1, characterized in that: the Setmelanotide peptide resin comprises the following components:

Ac-Arg (Pbf) -Cys (R1) -D-Ala-His (Trt) -D-Phe-Arg (Pbf) -Trp (R2) -Cys (R1) -amino resin

Wherein: r1 is Trt or Acm

R2 is Boc or H.

5. The method for producing Setmelantotide according to claim 1, characterized in that: the dosage of the Fmoc-protected amino acid is 1.2-6 times of the total mole number of the amino groups of the initial resin; preferably 2.5 to 4.0 times.

6. The method for producing Setmelanotide according to any one of claims 1 to 5, wherein:

the Setmelactone peptide resin is subjected to acidolysis, resin and side chain protecting groups are removed at the same time, and then a Setmelactone crude product is obtained after oxidation.

7. The method for producing Setmelantotide according to claim 1, characterized in that: and purifying the crude product of Setmelanotide by high performance liquid chromatography and freeze-drying to obtain a pure product of Setmelanotide.

Technical Field

The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a preparation method of Setmelanotide.

Background

Setmelactone is an oligopeptide melanocortin-4 receptor (MC4R) agonist, and research results show that subcutaneous daily injection of setelactone can reduce the excessive diet and body weight of three LEPR-deficient obesity patients. Setmelanotide was well tolerated and no serious adverse events were reported.

Setmelanotide has the following structure:

Ac-Arg-c(Cys-D-Ala-His-D-Phe-Arg-Trp-Cys)-NH2

the preparation method of Setmelanotide is rarely reported, Chinese patent CN201080051643 reports a liquid phase fragment condensation method, the method is complex and cumbersome, and large-scale production is not convenient, and the invention provides an efficient preparation method of Setmelanotide to meet the medical application.

Disclosure of Invention

The invention provides an efficient preparation method, which adopts amino resin as initial resin, and has the advantages of simple preparation process operation, high product yield and high purity.

The invention provides a preparation method of Setmelanotide, which comprises the following steps: the method comprises the following steps of taking amino resin as starting resin, sequentially inoculating corresponding protective amino acids according to the reverse phase of an amino sequence of Setmelactone to obtain Setmelactone peptide resin, carrying out acidolysis and oxidative cyclization on the Setmelactone protective peptide resin to obtain a Setmelactone crude product, and purifying and freeze-drying the Setmelactone crude product to obtain a pure Setmelactone product.

In the above method for producing Setmelanotide, the substitution value of the amino resin is 0.3 to 1.5mmol/g resin, and preferably 0.6 to 1.0mmol/g resin.

Further, the amino resin is selected from Rink Amide-MBHA resin, Rink Amide-BHA resin and Rink Amide resin, preferably Rink Amide-MBHA resin.

In the preparation method of Setmelanotide, the dosage of Fmoc-protected amino acid is 1.2-6 times of the total mole number of the amino groups of the initial resin; preferably 2.5 to 4.0 times.

In a preferred embodiment of the present invention, the solid-phase coupling synthesis method comprises: and (3) after the Fmoc protecting group of the protected amino acid-resin obtained in the previous step is removed, carrying out coupling reaction with the next protected amino acid. The coupling reaction time is 60-300 minutes, and preferably 100-140 minutes.

The Setmelanotide peptide resin is:

ac-arg (pbf) -Cys (R1) -D-Ala-his (trt) -D-Phe-arg (pbf) -Trp (R2) -Cys (R1) -amino resin wherein: r1 is Trt or Acm

R2 is Boc or H

Preferably, the Setmelactone peptide resin is subjected to acidolysis, resin and side chain protecting groups are removed at the same time, and then a crude Setmelactone product is obtained after oxidative cyclization.

More preferably, the acidolysis agent used in acidolysis of the Setmelanotide peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water; wherein the volume ratio of the mixed solvent is as follows: 80-95% of TFA, 1-10% of EDT and the balance of water.

More preferably, the volume ratio of the mixed solvent is: 89-91% of TFA, 4-6% of EDT and the balance of water. Optimally, the volume ratio of the mixed solvent is as follows: TFA 90%, EDT 5%, balance water.

The dosage of the acidolysis agent is 4-15 mL per gram of Setmelanotide peptide resin; preferably, 9-11 mL of acidolysis agent is needed per gram of the Setmelanotide peptide resin. The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.

Further, oxidizing agent used in the oxidative cyclization is iodine and H₂O₂Or DMSO, preferably iodine. The oxidant is added in a titration mode, and the oxidant is stopped adding when the oxidation end point is reached.

Further, the method for purifying the crude Setmelactone by high performance liquid chromatography and freeze-drying to obtain the pure Setmelactone comprises the following steps:

purifying by adopting a high performance liquid chromatography, wherein a chromatographic filler is 10 mu m reverse phase C18, alternately purifying by adopting two mobile phase systems, the first mobile phase system is 0.1% TFA/aqueous solution-0.1% TFA/acetonitrile solution, the second mobile phase system is 50mmol ammonium acetate/aqueous solution-acetonitrile, the flow rate of a 77mm 250mm chromatographic column is 90mL/min, eluting by adopting a gradient system, circularly injecting and purifying, sampling a crude product solution into the chromatographic column, starting mobile phase elution, collecting a main peak to evaporate acetonitrile, and filtering by using a 0.45 mu m filter membrane to obtain a Setmelantotide purified intermediate concentrated solution;

performing salt exchange by high performance liquid chromatography, wherein the mobile phase system is 1% acetic acid/water solution-acetonitrile, the chromatographic packing for purification is reversed phase C18 with 10 μm, and the flow rate of 77mm × 250mm chromatographic column is 90 mL/min; adopting a gradient elution and circulation loading method, loading the sample into a chromatographic column, starting mobile phase elution, collecting a map, observing the change of the absorbance, collecting a main salt exchange peak, detecting the purity by using an analysis liquid phase, combining main salt exchange peak solutions, concentrating under reduced pressure to obtain a Setmelanotide acetic acid aqueous solution, and freeze-drying to obtain a pure Setmelanotide product.

The method has the advantages of simple process operation and high total product yield, and is suitable for large-scale production.

Detailed Description

The invention discloses a method for synthesizing Setmelanotide, which can be realized by appropriately improving process parameters by referring to the content in the text. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as appropriate variations and combinations of the methods described herein, may be made and the techniques of the present invention employed without departing from the spirit and scope of the invention.

In the specific embodiment of the present invention, the English abbreviations used in the application documents have the corresponding Chinese meanings as shown in the following table.

The invention is further illustrated by the following examples.