CN111217901A

CN111217901A - Preparation method of Somalutide

Info

Publication number: CN111217901A
Application number: CN201911063508.5A
Authority: CN
Inventors: 郭德文; 曾德志; 文永均
Original assignee: Chengdu Shengnuo Biopharm Co ltd
Current assignee: Chengdu Shengnuo Biopharm Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-06-02

Abstract

the invention provides a preparation method of somaglutide, which adopts special protective amino acid fragments including Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH and Fmoc-Lys (AEEA-AEEA-gamma Glu (α -OtBu) -octaneedioic acid (mon-tBu)) -OH, and solves the problem of low product purity in a large-scale preparation method.

Description

Preparation method of Somalutide

Technical Field

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

Background

unlike GLP-1, the pharmacokinetic and pharmacodynamic characteristics of the Somalutide in human are suitable for once-a-week dosing regimens, after subcutaneous injection, the action time of the Somalutide is prolonged by a mechanism comprising a self-association action which slows absorption, binding with albumin, and higher enzyme stability to dipeptidyl peptidase IV (DPP-IV) and Neutral Endopeptidase (NEP), thereby having a longer plasma half-life.

The somaglutide has the following structure:

His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γGlu-Octadecanedioic acid)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

the preparation method of the Somaloutide has been reported, and the invention provides a high-efficiency preparation method of the Somaloutide to produce a high-purity product so as to meet the medical application.

Disclosure of Invention

The invention provides a novel high-efficiency preparation method, which adopts special protected amino acid and solves the problem of low product purity in a large-scale preparation method.

The invention provides a preparation method of Somalutide, which comprises the following steps: the method is characterized in that Fmoc-Gly resin is used as starting resin, the starting resin is prepared by a solid-phase polypeptide synthesis method, the SomaluU peptide resin is obtained by the polypeptide solid-phase synthesis method, the SomaluU peptide resin is acidolyzed to obtain a crude product of SomaluU peptide, and finally the crude product of SomaluU peptide is purified to obtain a pure product of Somalu peptide.

In addition to other conventional protected amino acids, the following special protected amino acids and fragments are used in the synthesis process of the somaglutide multi-resin:

(1)Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH

(2)Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioic acid(mon-tBu))-OH

somalutide peptide resin:

Boc-His (Boc) -Aib-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (AEEA-AEEA-Gamma Glu (α -OtBu) -Octadecaneedioic acid (mon-tBu)) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (Pbf) -Gly-Arg (Pbf) -Gly-resin

In the preparation method of the Somaloutide, 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 the preparation method of the Somaloutide, the Fmoc-Gly resin is obtained by coupling carrier resin and Fmoc-Gly-OH; wherein the substitution value of the Fmoc-Gly-resin is 0.2-1.0 mmol/g resin, and the preferable substitution value is 0.3-0.5 mmol/g resin.

Further preferably, the carrier resin is a Trityl-Cl type resin or a hydroxyl type resin, wherein the Trityl-Cl type resin is preferably a Trityl-Cl resin, a 4-methylitrityl-Cl resin, a 4-Methoxytrityl-Cl (4-Methoxytrityl chloride) resin or a 2-Cl Trity-Cl (2-chlorotrityl chloride) resin; the hydroxyl resin is preferably Wang resin or p-hydroxymethylphenoxymethyl polystyrene (HMP) resin.

As a preferable embodiment of the present invention, when the support resin is trityl chloride resin, the coupling method of Fmoc-Gly-OH and the support resin is as follows: the carboxyl of Fmoc-Gly-OH and Cl-substituted alkyl in the resin are subjected to esterification reaction under the action of alkali to access protected amino acid.

In a preferred embodiment of the present invention, when the support resin is a hydroxyl-type resin, the coupling method of Fmoc-Gly-OH to the support resin is as follows: the carboxyl of Fmoc-Gly-OH and the hydroxyl in the resin are subjected to esterification reaction under the action of a coupling agent, an activating agent and an alkali catalyst to access and protect amino acid.

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.

Preferably, the somalufide peptide resin is subjected to acidolysis, and simultaneously resin and side chain protecting groups are removed to obtain a crude somalufide product:

more preferably, the acidolysis agent used in the acidolysis of the somalipeptide 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 of acidolysis agent per gram of the somalutide peptide resin; preferably, 9-11 mL of acidolysis agent is required per gram of the somalutide peptide resin. The time for cracking by using the acidolysis agent is 1-6 hours, preferably 3-4 hours at room temperature.

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

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

purifying by high performance liquid chromatography, wherein a chromatographic filler for purification is 10 mu m reverse phase C18, alternately purifying by two mobile phase systems, the first mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, the second mobile phase system is 50mmol ammonium acetate/water solution-acetonitrile, the flow rate of a 77mm 250mm chromatographic column is 90mL/min, eluting by a gradient system, circularly injecting and purifying, sampling a crude product solution into the chromatographic column, starting mobile phase elution, collecting a main peak, evaporating acetonitrile, and filtering by a 0.45 mu m filter membrane to obtain a Somalu peptide 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); the method comprises the steps of 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, carrying out reduced pressure concentration to obtain a Somali peptide acetic acid aqueous solution, and carrying out freeze drying to obtain a Somali peptide pure product.

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

(1)Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH

(2)Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioic acid(mon-tBu))-OH

directly avoids the risk of introducing heavy gold palladium salt during Lys (Alloc) deprotection or the genotoxic substance hydrazine during Lys (IVDde) deprotection, and simultaneously avoids [ D-His ]¹]-Somatoglutide, [ Des-Gly [ ]⁴]-somaglutide and [ Lys (AEEA-AEEA-D-γGlu(α-tBu)-Octadecanedioic acid)²⁶]The production of impurities such as the Somalutide improves the purity of a crude product, reduces the purification difficulty, improves the product yield, and ensures that the purity of the obtained product is more than 99.0 percent.

Detailed Description

The invention discloses a method for synthesizing Somalutide, which can be realized by appropriately improving process parameters by taking the contents of the Somalutide as reference by a person skilled in the art. 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.

Example 1 Synthesis of Somali peptide resin

Somalutide peptide resin:

The starting resin is Fmoc-Gly-resin, and the starting resin is subjected to Fmoc protection removal and coupling reaction, and is sequentially coupled with the protected amino acids shown in the table 2 to prepare the Somali peptide resin. The protected amino acids or fragments corresponding to the protected amino acids used in this example are shown below:

TABLE 2

The peptide sequence n ═	Protected amino acids
		2	Fmoc-Arg(Pbf)
3	Fmoc-Gly-OH
		4	Fmoc-Arg(Pbf)
5	Fmoc-Val
		6	Fmoc-Leu
7	Fmoc-Trp(Boc)
		8	Fmoc-Ala
9	Fmoc-Ile
		10	Fmoc-Phe
11	Fmoc-Glu(OtBu)
		12	Fmoc-Lys(AEEA-AEEA-γGlu(α-OtBu)-Octadecanedioic acid(mon-tBu))
13	Fmoc-Ala
		14	Fmoc-Ala
15	Fmoc-Gln(Trt)
		16	Fmoc-Gly
17	Fmoc-Glu(OtBu)
		18	Fmoc-Leu
19	Fmoc-Tyr(tBu)
		20	Fmoc-Ser(tBu)
21	Fmoc-Ser(tBu)
		22	Fmoc-Val
23	Fmoc-Asp(OtBu)
		24	Fmoc-Ser(tBu)
25	Fmoc-Thr(tBu)
		26	Fmoc-Phe
27	Fmoc-Thr(tBu)
		28	Boc-His(Boc)-Aib-Glu(OtBu)-Gly-OH

1. Introduction of the 2 nd protected amino acid

Dissolving 0.09mol of the 2 nd protected amino acid and 0.09mol of HOBt in a proper amount of DMF; and adding 0.09mol DIC slowly into the protected amino acid DMF solution under stirring, and reacting for 30 minutes under stirring at room temperature to obtain an activated protected amino acid solution for later use.

0.03mol of Fmoc-Gly-resin (substitution value about 0.5mmol/g) was taken, deprotected with 20% PIP/DMF solution for 25 min, washed and filtered to give Fmoc-removed resin.

And adding the activated 2 nd protected amino acid solution into the Fmoc-removed resin, performing coupling reaction for 120-300 minutes, filtering and washing to obtain the resin containing 2 protected amino acids.

2. Inoculating 3-30 protected amino acids or fragments

And sequentially inoculating the corresponding 3 rd to 30 th protected amino acids or fragments by the same method to obtain the somaglutide peptide resin.

Example 2 preparation of crude Somalutide

The preparation method comprises the following steps of taking the somaltulin peptide resin prepared in example 1, adding a cracking reagent (10 mL/g resin of the cracking reagent) with the volume ratio of TFA, water and EDT (95: 5), uniformly stirring, stirring at room temperature for reaction for 3 hours, filtering a reaction mixture by using a sand core funnel, collecting filtrate, washing the resin with a small amount of TFA for 3 times, combining the filtrates, concentrating under reduced pressure, adding anhydrous ether for precipitation, washing the precipitate with the anhydrous ether for 3 times, and drying to obtain white-like powder, namely a crude somaltulin peptide product, wherein the purity of the crude product is 66.8%.

Example 3 purification of crude Somalutide

Taking the crude product of the Somalutide prepared in the embodiment 2, adding water, stirring, adjusting the pH to 8.5 by using ammonia water until the crude product is completely dissolved, filtering the solution by using a 0.45-micron mixed microporous filter membrane, and purifying for later use;

purification was performed by high performance liquid chromatography using reverse phase C18 with 10 μm chromatography packing and alternating purification with two mobile phase systems, the first being 0.1% TFA/water-0.1% TFA/acetonitrile and the second being 50mmol ammonium acetate/water-acetonitrile. The flow rate of a chromatographic column of 77mm x 250mm is 90mL/min, a gradient system is adopted for elution, the sample is circularly injected and purified, a crude product solution is taken to be loaded in 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-micrometer filter membrane to obtain a Somaloutide 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); the method comprises the steps of 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 Somalou peptide acetic acid aqueous solution, and freeze-drying to obtain a Somalou peptide pure product 31.3g, wherein the purity is 99.6%, the maximum single impurity content is 0.10%, the total yield is 25.4%, and the molecular weight is 4113.6 (100% M + H).

The embodiment shows that the purity of the product obtained by the method provided by the invention is more than 99.0%, and the single impurity is less than 0.15%, so that the product quality is improved, and the method has wide practical value and application prospect.

Claims

1. A method of preparing a somaglutide, comprising: the method comprises the following steps of (1) preparing a SomaluU peptide resin by adopting Fmoc-Gly resin as an initial resin and using a solid-phase polypeptide synthesis method, carrying out acidolysis on the SomaluU peptide resin to obtain a crude product of SomaluU, and finally purifying and freeze-drying to obtain a pure product of SomaluU:

His¹-Aib-Glu-Gly-Thr⁵-Phe-Thr-Ser-Asp-Val¹⁰-Ser-Ser-Tyr-Leu-Glu¹⁵-

Gly-Gln-Ala-Ala-Lys²⁰(AEEA-AEEA-γGlu-Octadecanedioicacid)-Glu-

Phe-Ile-Ala-Trp²⁵-Leu-Val-Arg-Gly-Arg³⁰-Gly-OH。

2. the method for preparing somaglutide according to claim 1, characterized in that: when the 1 st His to the 4 th Gly are accessed together, the corresponding protected amino acid is Boc-His (Boc) -Aib-Glu (OtBu) -Gly-OH.

3. the method for preparing the somaglutide according to claim 1, wherein the protected amino acid is Fmoc-Lys (AEEA-AEEA-gamma Glu (α -OtBu) -octaneedioic acid (mon-tBu)) -OH when the 20 th Lys is inserted.

4. The method for preparing somaglutide according to claim 1, characterized in that: the Fmoc-Gly resin is obtained by coupling carrier resin and Fmoc-Gly-OH; wherein the substitution value of the Fmoc-Gly-resin is 0.2-1.0 mmol/g resin; the preferred substitution value is 0.3 to 0.5mmol/g resin.

5. The method for preparing somaglutide according to claim 1, characterized in that: 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. The method for producing somaglutide according to any one of claims 1 to 5, wherein: and carrying out acidolysis on the Somalutide peptide resin, and simultaneously removing the resin and a side chain protecting group to obtain a crude product of the Somalutide.

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