阅读说明：本技术 一种阿托西班的制备方法 (Preparation method of atosiban ) 是由 董华建 郭德文 文永均 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种阿托西班的制备方法,方法中采用了特殊的保护氨基酸片段：Mpr(R)-D-Tyr(Et),提高了规模化制备中产品的纯度和收率。(The invention provides a preparation method of atosiban, which adopts special protective amino acid segments: mpr (R) -D-Tyr (Et) improves the purity and yield of products in large-scale preparation.)

1. A method for preparing atosiban is characterized by comprising the following steps: adopting amino resin as initial resin, synthesizing atosiban linear peptide resin in a solid phase, obtaining atosiban linear peptide after acidolysis of the atosiban linear peptide resin, dissolving the atosiban linear peptide by adopting an acetic acid solution, then dropwise adding iodine while stirring until complete cyclization to obtain a crude atosiban product, and obtaining a pure product after purifying the crude atosiban product:

2. a process for the preparation of atosiban according to claim 1, wherein: when Mpr at position 1 and D-Tyr at position 2 are grafted together, the corresponding protected amino acid is Mpr (R) -D-Tyr (Et).

3. A process for preparing atosiban according to claim 1, wherein the amino resin has an amino substitution value of 0.3 to 1.5mmol/g resin, preferably a substitution value of 0.5 to 1.0mmol/g resin.

4. A process for preparing atosiban according to claim 1, wherein the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, preferably Rink Amide MBHA resin.

5. A process for the preparation of atosiban according to claim 1, wherein: the dosage of the Fmoc-protected amino acid or the protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

6. A process for the preparation of atosiban according to any one of claims 1 to 5, wherein: and (3) carrying out acidolysis on the atosiban peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain a crude product of the atosiban linear peptide.

7. A process for the preparation of atosiban according to claim 1, wherein: dissolving the atosiban linear peptide crude product by using acetic acid, filtering, and oxidizing and cyclizing by using an oxidizing agent to obtain an atosiban crude product solution. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or H₂O₂Or DMSO, preferably iodine.

8. A process for the preparation of atosiban according to claim 1, wherein: and purifying the atosiban crude product by high performance liquid chromatography and freeze-drying to obtain a pure atosiban product.

Technical Field

The invention relates to the field of medicine synthesis, and particularly relates to a preparation method of atosiban.

Background

Atosiban (Atosiban) is a synthetic peptide substance that produces competitive inhibition of human oxytocin at the receptor level. The results of animal experiments of rats and guinea pigs show that the product can reduce the contraction frequency and tension of uterus and inhibit the contraction of uterus after being combined with oxytocin receptor. The product also binds to vasopressin receptor and inhibits the action of vasopressin.

Atosiban has the following structure:

regarding the preparation method of atosiban, the common method in the prior art at home at present is Fmoc solid phase synthesis, which adopts amino resin as the starting carrier resin, sequentially accesses protective amino acid, and the obtained atosiban iodine is oxidized and then cracked to obtain the atosiban. However, the above-mentioned conventional processes are insufficient in terms of purity of crude peptide and total yield.

In addition, the product has the structure of D-Tyr (Et), and Fmoc-D-Tyr (Et) is easy to generate racemization reaction in the peptide splicing process to generate Tyr (Et)²]-atosiban impurity, the impurity being in combination with atosibanTosiban is similar in polarity and is difficult to completely remove through purification, thereby affecting the quality of the product.

Disclosure of Invention

The invention provides a preparation method of atosiban, aiming at preparing high-purity atosiban, and in order to realize the aim, the invention provides the following technical scheme:

the invention provides a preparation method of atosiban, which comprises the following steps: the method comprises the following steps of adopting amino resin as starting resin, synthesizing atosiban linear peptide resin in a solid phase manner, carrying out acidolysis on the atosiban linear peptide resin to obtain the atosiban linear peptide, dissolving the atosiban linear peptide by adopting an acetic acid solution, then dropwise adding iodine while stirring until complete cyclization is carried out to obtain a crude atosiban product, and purifying the crude atosiban product to obtain a pure product.

Wherein the synthetic process of the atosiban resin uses the following special protected amino acid fragment Mpr (R) -D-Tyr (Et) besides other conventional protected amino acids.

Atosiban resin was:

mpr (R) -D-Tyr (Et) -Ile-Thr (tBu) -Asn (Trt) -Cys (Trt) -Pro-Orn (Boc) -Gly-amino resin

In the preparation method of atosiban, the amino resin has an amino substitution value of 0.3 to 1.5mmol/g resin, and the preferable substitution value is 0.5 to 1.0mmol/g resin.

In the preparation method of atosiban, the amino resin is one of Rink MBHA resin, Rink Amide resin or Rink Amide AM resin, and Rink Amide MBHA resin is preferred.

In the preparation method of atosiban, the dosage of Fmoc-protected amino acid or protected amino acid fragment is 1.2-6 times of the total mole number of the charged resin; preferably 2.5 to 3.5 times.

In a preferred embodiment of the present invention, the atosiban resin is subjected to acidolysis while removing the resin and the side chain protecting groups to obtain a crude atosiban linear peptide.

Further, an acidolysis agent adopted in the acidolysis of the atosiban resin is a mixed solvent of trifluoroacetic acid (TFA), 1, 2-Ethanedithiol (EDT) and water, and the mixture ratio of the mixed solvent is as follows: the TFA ratio is 80-95% (V/V), the EDT ratio is 1-10% (V/V), and the balance is water. The preferred formulation is 89-91% TFA, 4-6% EDT, and the balance water. Preferably, the mixture ratio is 90%, EDT 5% and the balance of water.

The dosage of the acidolysis agent is 4-15 ml of acidolysis agent required by one gram of atosiban resin, and preferably 9-11 ml of acidolysis agent required by one gram of atosiban resin. The time for cracking by using the acidolysis agent is 1-5 hours, preferably 2 hours at room temperature.

Further, the crude product of the atosiban linear peptide is dissolved by acetic acid, and oxidized and cyclized by an oxidizing agent after being filtered to obtain a solution of the crude atosiban. The volume percentage concentration of the acetic acid is 20-40%, and the preferred concentration is 30%. The oxidant is iodine or 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 atosiban crude product is purified by high performance liquid chromatography and freeze-dried to obtain a pure atosiban product, and the specific method comprises the following steps:

purifying by high performance liquid chromatography, wherein the chromatographic filler for purification is 10 μm reversed phase C18, a mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, a chromatographic column with flow rate of 77mm x 250mm is 90mL/min, a gradient system is adopted for elution, purification is performed by circulating sample injection, a crude product solution is sampled on the chromatographic column, the mobile phase elution is started, a main peak is collected, acetonitrile is evaporated, and then the crude product solution is filtered by a 0.45 μm filter membrane to obtain an atosiban 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 90mL/min (corresponding flow rate can be adjusted according to chromatographic columns with different specifications); adopting gradient elution and circulation sample loading method, loading sample in chromatographic column, starting mobile phase elution, collecting atlas, observing change of absorbance, collecting main peak of salt exchange and analyzing liquid phase to detect purity, combining main peak solution of salt exchange, concentrating under reduced pressure to obtain atosiban acetic acid aqueous solution, and freeze-drying to obtain pure atosiban.

Detailed Description

The invention discloses a method for synthesizing atosiban, which can be realized by appropriately improving process parameters by a person skilled in the art according 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 Chinese meanings corresponding to the English abbreviations used in the application documents are shown in Table 1.

TABLE 1 English abbreviation definitions

English abbreviation	Name of Chinese	English abbreviation	Name of Chinese
				Fmoc	9-fluorenylmethoxycarbonyl group	Acm	Acetamidomethyl
tBu	Tert-butyl radical	Trt	Trityl radical
				Boc	Tert-butyloxycarbonyl radical	Asn	Asparagine and its use
Gly	Glycine	Thr	Threonine
				Orn	Ornithine	Ile	Isoleucine
Pro	Proline	D-Tyr(Et)	O-ethyl-D-tyrosine
				Cys	Cysteine	Mpr	Mercaptopropionic acid

The invention is further illustrated by the following examples.