阅读说明：本技术 一种替莫瑞林的制备方法 (Preparation method of temeprelin ) 是由 曾德志 董华建 文永均 于 2019-10-31 设计创作，主要内容包括：本发明提供了一种替莫瑞林的制备方法,方法中采用了特殊的保护氨基酸片段：(E)-3-Hexenoicacid-Tyr(tBu)-Ala-Asp(OtBu)-Ala-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu-OH,缩短了制备工艺周期,提高了规模化制备中产品的纯度和收率。(The invention provides a preparation method of tesamorelin, which adopts special protective amino acid fragments: (E) 3-Hexenolic acid-Tyr (tBu) -Ala-Asp (OtBu) -Ala-Ile-Phe-Thr (tBu) -Asn (Trt) -Ser (tBu) -Tyr (tBu) -Arg (Pbf) -Lys (Boc) -Val-Leu-OH, shortens the preparation process period and improves the purity and yield of products in large-scale preparation.)

1. A method for preparing temsirolimus, comprising: amino resin is adopted as initial resin, a solid-phase polypeptide synthesis method is used for preparing temsirolimus peptide resin, the temsirolimus peptide resin is subjected to acidolysis to obtain a crude temsirolimus product, and finally, the crude temsirolimus product is purified and freeze-dried to obtain a pure temsirolimus product:

(E)-3-Hexenoic acid-Tyr-Ala-Asp-Ala⁵-Ile-Phe-Thr-Asn-Ser¹⁰-

Tyr-Arg-Lys-Val-Leu¹⁵-Gly-Gln-Leu-Ser-Ala²⁰-Arg-Lys-Leu-

Leu-Gln²⁵-Asp-Ile-Met-Ser-Arg³⁰-Gln-Gln-Gly-Glu-Ser³⁵-Asn-

Gln-Glu-Arg-Gly⁴⁰-Ala-Arg-Ala-Arg-Leu⁴⁵-NH2。

2. the process for the preparation of tesamorelin according to claim 1, characterized in that: when the 1 st (E) -3-Hexenoic acid is connected to the 15 th Leu, the corresponding protective amino acid fragment is (E) -3-Hexenoic-Tyr (tBu) -Ala-Asp (OtBu) -Ala-Ile-Phe-Thr (tBu) -Asn (Trt) -Ser (tBu) -Tyr (tBu) -Arg (Pbf) -Lys (Boc) -Val-Leu-OH.

3. The method for preparing tesamorelin according to claim 1, wherein the amino resin has an amino substitution value of 0.3 to 1.0mmol/g resin, preferably a substitution value of 0.3 to 0.5mmol/g resin.

4. The preparation method of temsirorelin 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. The process for the preparation of tesamorelin according to any of claims 1 to 4, characterized in that: and (3) carrying out acidolysis on the temorelin peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain a temorelin crude product.

6. The process for the preparation of tesamorelin according to claim 1, characterized in that: and purifying the crude temsirolimus product by high performance liquid chromatography and freeze-drying to obtain a pure temsirolimus product.

Technical Field

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

Background

Temorelin is a new therapeutic drug for abdominal adiposity in Human Immunodeficiency Virus (HIV) infected patients with lipodystrophy, is a GRF synthetic analogue. Similar to endogenous GRF action, it acts in vitro by binding to and activating GRF receptors. GRF is a hypothalamic regulatory polypeptide that stimulates pituitary growth hormone cells to synthesize and release endogenous Growth Hormone (GH). GH exerts pharmacological effects by interacting with specific receptors on target cells, including chondrocytes, osteoblasts, cardiomyocytes, hepatocytes and adipocytes.

The absolute bioavailability of healthy adults after subcutaneous injection of 2mg of tesamorelin is less than 4%. After a single dose of 2mg is injected into healthy people without lipodystrophy and HIV infected patients subcutaneously, the area under the curve (AUC) is respectively (634.6 +/-72.4) and (852.8 +/-91.9) pg.h.mL^-1Blood peak concentration (C)_max) Are respectively as(2874.6. + -. 43.9) and (2822.3. + -. 48.9) pg.mL^-1The peak reaching time is 1.5h, and the apparent distribution volumes are (9.4 +/-3.1) and (10.5 +/-6.1) L.kg^-1Average elimination half-life (t) after continuous application of 14d_1/2) 26 and 38min, respectively.

Temorelin has the following structure:

(E)-3-Hexenoic acid-Tyr-Ala-Asp-Ala⁵-Ile-Phe-Thr-Asn-Ser¹⁰-Tyr-Arg-Lys-Val-Leu¹⁵-Gly-Gln-Leu-Ser-Ala²⁰-Arg-Lys-Leu-Leu-Gln²⁵-Asp-Ile-Met-Ser-Arg³⁰-Gln-Gln-Gly-Glu-Ser³⁵-Asn-Gln-Glu-Arg-Gly⁴⁰-Ala-Arg-Ala-Arg-Leu⁴⁵-NH2

the preparation method of the temorelin is reported, and the invention provides an efficient temorelin preparation method, which improves the purity and yield of high products so as to meet medical application.

Disclosure of Invention

The invention provides a novel high-efficiency preparation method, which adopts special protected amino acid, shortens the preparation process period and improves the product purity and yield in a large-scale preparation method.

The invention provides a preparation method of tesamorelin, which comprises the following steps: amino resin is adopted as initial resin, the starting resin is prepared by a solid-phase polypeptide synthesis method, the temmorelin resin is obtained by the polypeptide solid-phase synthesis method, the temmorelin resin is acidolyzed to obtain a crude temmorelin product, and finally the crude temmorelin product is purified to obtain a pure temmorelin product.

In addition to other conventional protected amino acids, the following special protected amino acid fragments are used in the synthesis process of the temorelin resinoid:

(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu-OH。

temsirolin peptide resin:

(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala⁵-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)¹⁰-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu¹⁵-Gly-Gln(Trt)-Leu-Ser(tBu)-Ala²⁰-Arg(Pbf)-Lys(Boc)-Leu-Leu-Gln(Trt)²⁵-Asp(OtBu)-Ile-Met-Ser(tBu)-Arg(Pbf)³⁰-Gln(Trt)-Gln(Trt)-Gly-Glu(OtBu)-Ser(tBu)³⁵-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Arg(Pbf)-Gly⁴⁰-Ala-Arg(Pbf)-Ala-Arg(Pbf)-Leu⁴⁵-amino resins

In the preparation method of the temsirolimus, the amino resin has an amino substitution value of 0.3 to 1.0mmol/g resin, and preferably a substitution value of 0.3 to 0.5mmol/g resin.

In the preparation method of the temsirorelin, 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 the temorelin, 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.

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

Further, an acidolysis agent adopted in acidolysis of the temorelin 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 each gram of the tirmorelin resin, and preferably, 9-11 ml of acidolysis agent required by each gram of the tirmorelin resin. The time for cracking by using the acidolysis agent is 1-5 hours, preferably 2 hours at room temperature.

Further, the crude temsirolimus product is purified by high performance liquid chromatography and freeze-dried to obtain a pure temsirolimus product, and the specific method comprises the following steps:

adding water into the crude temeprelin product, stirring, adjusting the pH value to 8.5 by using ammonia water until the solution is completely dissolved, filtering the solution by using a 0.45-micrometer mixed microporous filter membrane, and purifying for later use;

purifying by adopting a high performance liquid chromatography, wherein a chromatographic filler for purification 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 in 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 purified intermediate concentrated solution of the tesamorelin;

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 a gradient elution and circulation loading method, loading a 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 temorelin acetic acid aqueous solution, and freeze-drying to obtain a pure temorelin product.

The method of the invention directly uses the following special protected amino acids:

(E)-3-Hexenoic acid-Tyr(tBu)-Ala-Asp(OtBu)-Ala-Ile-Phe-Thr(tBu)-Asn(Trt)-Ser(tBu)-Tyr(tBu)-Arg(Pbf)-Lys(Boc)-Val-Leu-OH。

the production period is shortened, the purity of the crude product is greatly improved, the product yield is improved, the purity of the obtained product is more than 99.0 percent, and compared with the prior art, the process has wide practical value and application prospect.

Detailed Description

The invention discloses a method for synthesizing tesamorelin, which can be realized by appropriately improving process parameters by a person skilled in the art with reference 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

The invention is further illustrated by the following examples.