阅读说明：本技术 一种制备利拉鲁肽的方法 (Method for preparing liraglutide ) 是由 王孝花 马健 于 2018-08-21 设计创作，主要内容包括：本发明属于多肽药物制备方法技术领域,特别涉及一种制备利拉鲁肽的方法。包括以下步骤：通过Fmoc固相合成法将Fmoc-Glu-2-R侧链羧酸连接到氨基类型树脂A上得Fmoc-Glu(Resin)-R氨基酸树脂I；再通过多步骤固相偶联反应合成利拉鲁肽肽树酯,最后在酸性条件下裂解、色谱纯化、冷冻干燥得到高纯度利拉鲁肽。本发明解决了利拉鲁肽序列中Gln难耦合的难题,且将Glu侧链连接到氨基树脂上,Glu侧链相对较长,增加了肽链与树脂之间的柔性,使肽链更容易变形而降低反应位阻,该方法不需要使用钯催化体系,避免了钯金属的引入,从而提高合成反应效率和利拉鲁肽产品质量。(The invention belongs to the technical field of preparation methods of polypeptide medicaments, and particularly relates to a method for preparing liraglutide. The method comprises the following steps: connecting Fmoc-Glu-2-R side chain carboxylic acid to amino resin A by Fmoc solid phase synthesis to obtain Fmoc-Glu (resin) -R amino acid resin I; and synthesizing the liraglutide peptide resin through multi-step solid-phase coupling reaction, and finally cracking, chromatographic purification and freeze drying under an acidic condition to obtain the high-purity liraglutide. The invention solves the problem that Gln in a liraglutide sequence is difficult to couple, and the Glu side chain is connected to the amino resin, and is relatively longer, so that the flexibility between the peptide chain and the resin is increased, the peptide chain is easier to deform, and the reaction steric hindrance is reduced.)

1. A method for preparing liraglutide, characterized by comprising the following processes:

connecting Fmoc-Glu-2-R side chain carboxylic acid to amino resin A by Fmoc solid phase synthesis to obtain Fmoc-Glu (resin) -R amino acid resin I; and then synthesizing the liraglutide peptide resin through a solid-phase coupling reaction:

Boc-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (tBu) -Val-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Glu (Resin) -Ala-Ala-Lys (Glu (N α -Palmitoyl) -OtBu) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc-Leu-Val-Arg pbf) -Gly-Arg (pbf) -Gly-R, finally cleaved under acidic conditions, chromatographed, lyophilized to obtain high purity liraglutide.

2. The method for preparing liraglutide according to claim 1, wherein the amino-type Resin A is selected from one of Rinkamide MBHA Resin and Rink Amide AM Resin, and the substitution degree of the amino-type Resin A is preferably 0.3-0.5 mmol/g; Fmoc-Glu-2-R is Fmoc-Glu-2-phenylisoproyl easter; when Fmoc-Glu-2-R side chain carboxylic acid is connected with the amino resin A, the coupling reagent combination is selected from one of PyBOP/DIPEA/HOBt and DEPBT/DIEA.

3. The method for preparing liraglutide according to claim 1, wherein Fmoc-Glu (resin) -OH is prepared by removing a protecting group R with a lysing solution I before coupling Fmoc-Glu (resin) -R amino acid resin I; the lysis solution i is preferably a DCM solution containing 1.5-2.5% of TFA by volume fraction, the acidolysis time is 5min, and the acidolysis times are 2-3.

4. The process for the preparation of liraglutide according to claim 1, wherein the liraglutide resin is prepared by Fmoc-Glu (resin) -R amino acid resin I sequentially with polypeptide fragment NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂VII-R, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (Trt) -OH.

5. The process for the preparation of liraglutide according to claim 4, wherein Fmoc-Glu (resin) -R amino acid resin I is sequentially reacted with polypeptide fragment NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂And when the-VII-R is coupled, the coupling reagent combination is selected from one of PyBOP/DIPEA/HOBt, HATU/DIPEA/HOBt, HBTU/DIPEA/HOBt and DIC/HOBt.

6. The method of claim 4, wherein the polypeptide fragment NH is₂-V-R is NH₂-Ala-Ala-Lys(Glu(N^α-Palmitoyl)-OtBu) -R, polypeptide fragment NH₂VI-R is NH₂-Glu (OtBu) -Phe-Ile-Ala-R, polypeptide fragment NH₂VII to R are NH₂-trp (boc) -Leu-Val-arg (pbf) -Gly-R; preferably, R is phenylisoproyl easter.

7. The method of claim 6, wherein the polypeptide fragment is NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂The preparation method of the-VII-R comprises the following steps:

1) Fmoc-Lys (Glu (N) was prepared by Fmoc solid phase synthesis method^αCoupling of carboxyl groups of- -Palmitoyl) -OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH to CTC resin B preparation of Fmoc- -Lys (Glu (N)^α-Palmitoyl) -OtBu) -ctcreesin amino acid Resin II, Fmoc-Ala-CTC Resin amino acid Resin III, Fmoc-Gly-CTC Resin amino acid Resin IV;

2) respectively preparing full-protection polypeptide fragment V peptide resin, full-protection polypeptide fragment VI peptide resin and full-protection polypeptide fragment VII peptide resin on amino acid resin II, amino acid resin III and amino acid resin IV by Fmoc solid-phase synthesis;

3) respectively acidolyzing the fully-protected polypeptide fragment V peptide resin, the fully-protected polypeptide fragment VI peptide resin and the fully-protected polypeptide fragment VII peptide resin by using a lysis solution i to obtain a fully-protected polypeptide fragment Fmoc-V-OH, a fully-protected polypeptide fragment Fmoc-VI-OH and a fully-protected polypeptide fragment Fmoc-VII-OH;

4) respectively protecting carboxyl groups of a full-protection polypeptide fragment Fmoc-V-OH, a full-protection polypeptide fragment Fmoc-VI-OH and a full-protection polypeptide fragment Fmoc-VII-OH by using a carboxyl protection reagent to prepare a full-protection polypeptide fragment Fmoc-V-phenylisopyrophosphate, a full-protection polypeptide fragment Fmoc-VI-phenylisopyrophosphate and a full-protection polypeptide fragment Fmoc-VII-phenylisopyrophosphate easter;

5) removing Fmoc from the fully protected polypeptide fragment Fmoc-V-phenylisoproyl easter, the fully protected polypeptide fragment Fmoc-VI-phenylisoproyl easter and the fully protected polypeptide fragment Fmoc-VII-phenylisoproyl easter to obtain the polypeptide fragment NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂-VII-R。

8. The method for preparing liraglutide according to claim 7, wherein the CTC resin B in the step 1) is selected from one of 2-Chlorotrityl chloride resin, 4-Methyl-Trityl-C1 resin and 4-Methoxy-Trityl-Cl resin, and the substitution degree of the CTC resin B is preferably 0.5-0.9 mmol/g.

9. The process for preparing liraglutide according to claim 7, wherein the carboxyl protecting agent in step 4) is 2-phenylprop-2-yl 2, 2, 2-trichloroacetimidate.

10. The method for preparing liraglutide according to claim 7, wherein the molar ratio of the carboxylic acid to the carboxyl protecting agent in the step 4) is 1: 1.5-2.5.

Technical Field

The invention belongs to the field of medicine synthesis, and particularly relates to a method for preparing liraglutide.

Background

Liraglutide, the name liraglutide in England, is a glucagon-like peptide-1 (GLP-1) analogue, and has a sequence of H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys (N-His-Glu-Gly-Asp-Val-Ser-Ser-Tyr-Leu-Ala-Ala-Lys)^α-PA L-gamma-Glu) -Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, formula: c₁₇₂H₂₆₅N₄₃O₅₁As a subcutaneous injection preparation, it has a good hypoglycemic action, and can improve glycemic control by lowering fasting and postprandial blood glucose in type 2 diabetic patients, and can also lower the body weight of the patients.

The prior synthesis methods of liraglutide include patent CN103087181, patent CN 102286092, patent CN103145828, document J.Med.chem.2000, 43, 1664-1669, document Chin.J.org.chem.2016, 36, 218-221 and document Chinese Journal of Pharmaceuticals 2013, 44(2), 121-124, which are sequentially connected and synthesized by Fmoc strategy solid phase method, then Lys side chain protecting groups are removed, and Lys side chain amino groups are modified to obtain the liraglutide resin. When the method is used for sequentially synthesizing the liraglutide main chain, incomplete peptide with similar polarity to the liraglutide is easily generated, so that the purification is difficult. In addition, the method modifies Lys side chain amino after the synthesis of the liraglutide main chain is finished, incomplete reaction or over reaction is easily caused due to high steric hindrance of the Lys side chain, defective peptide is generated, the total yield is low, meanwhile, more impurities exist, and the purification is difficult.

The method is characterized in that the method is synthesized by adopting a solid phase fragment condensation method in the patents CN102875665, CN104045705 and CN103864918, each fragment input by the solid phase fragment condensation is 1.5-3.5 times excessive, peptide fragments are seriously wasted, and the synthesis cost is very high; meanwhile, the resin substitution value of solid phase segment condensation is limited, so that the material flux is reduced, the solvent is wasted, and a large amount of waste liquid is generated.

Patent CN103275208 and patent CN103304659 firstly synthesize a polypeptide fragment, and then gradually couple the rest amino acids according to the amino acid sequence of liraglutide to prepare liraglutide peptide resin, and modification of Lys side chain amino is carried out on the liraglutide peptide resin, which easily causes incomplete reaction or over reaction, generates defective peptide, and has low total yield, more impurities and difficult purification, and the problem of difficult dissolution and difficult coupling exists because the sequence of the polypeptide fragment synthesized by patent CN103304659 is too long.

Patent CN104650219 adopts the method of liquid phase segment condensation to synthesize full protection liraglutide main chain polypeptide, then goes through four-step reaction in the liquid phase reaction system and obtains full protection liraglutide crude, and the aftertreatment is loaded down with trivial details, is unfavorable for the industrial production to the modification of Lys side chain amino is gone on full protection liraglutide main chain polypeptide, and the steric hindrance is big, the solubility is low, the reaction is difficult, produces defective peptide easily, causes the material extravagant.

Patent CN103980358 first of all synthesized the fragment Fmoc-Lys (Glu (N)^α-Palmitoyl) -OtBu) -OH, and then coupling the amino acids one by adopting a solid-phase synthesis method to prepare the liraglutide without modifying Lys side chain amino on the backbone of the liraglutide. However, this method couples the Fmoc-Lys (Glu (N)^αThe amount of-Palmitoyl) -OtBu) -OH is large, the cost is high, and a peptide chain is long, the steric hindrance is large and the coupling is difficult during the coupling reaction; the 17 th Gln is difficult to couple, so that-Gln polypeptide impurities are easily generated, and the content of single impurities is high.

CN 107880111 discloses that firstly, Glu side chain carboxyl is supported on hydroxyl type resin by Fmoc solid phase method to obtain Fmoc-Glu (resin) -OAll amino acid resin, then coupled with Fmoc-Lys (Pg1) -OH, and then coupled with 10 peptide fragments at the C end of liraglutide to obtain the liraglutide peptide resin through multi-step coupling reaction to His at the N end of liraglutide, after removing protecting group All in palladium catalytic system, coupled with 10 peptide fragments at the C end of liraglutide in the presence of activating agent system. The method is easy to introduce palladium metal into the liraglutide, and has the problems of overlong fragment, difficult dissolution and low coupling efficiency.

Other patents disclose the recombinant production of liraglutide by gene, however, the gene expression has the disadvantages of large workload, serious three wastes, large technical difficulty, high production cost and the like.

Disclosure of Invention

Aiming at the problems, the invention provides a method for preparing liraglutide, which comprises the steps of firstly connecting amino resin to the 17 th Gln position of liraglutide through Fmoc-Glu-2-phenylisopyroester ester side chain carboxylic acid, and then sequentially coupling NH₂-Ala-Ala-Lys(Glu(N^α-palmityl) -OtBu) -phenylisoproyl ester fragment, NH₂-Glu (OtBu) -Phe-Ile-Ala-phenylisopropylester fragment, NH₂Preparing liraglutide (17-31) -phenylisopropylcopy ester peptide resin from Trp (Boc) -Leu-Val-Arg (pbf) -Gly-phenylcopy ester fragment, and preparing Boc-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (tBu) -Val-Ser (tBu) -Ser (Ser) (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Glu (Resin) -Ala-Lys (Glu (N-Glu)^α-Palmitoyl) -OtBu) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-phenylisoproyl easter (liraglutide peptide resin).

The method solves the problem that Gln is difficult to couple in the conventional method, the Glu side chain is connected to the solid-phase amino resin, the Glu side chain is relatively long, the flexibility between the peptide chain and the resin is increased, the peptide chain is easier to deform, the reaction steric hindrance is reduced, a palladium catalytic system is not needed in the method, the pollution of palladium metal to a product is avoided, and the synthesis reaction efficiency and the quality of the liraglutide product are improved.

The specific scheme of the invention is as follows:

a method of preparing liraglutide comprising the steps of:

connecting Fmoc-Glu-2-R side chain carboxylic acid to amino resin A by Fmoc solid phase synthesis to obtain Fmoc-Glu (resin) -R amino acid resin I; and then synthesizing the liraglutide peptide resin through a solid-phase coupling reaction: Boc-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (tBu) -Phe-Thr (tBu) -Ser (tBu) -Asp (tBu) -Val-Ser (tBu) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Glu (resin) -Ala-Ala-Lys (Glu (N)^α-Palmitoyl) -OtBu) -Glu (OtBu) -Phe-Ile-Ala-Trp (Boc) -Leu-Val-Arg (pbf) -Gly-R, finally cracking under acidic condition, chromatographic purification, freeze-drying to obtain high-purity liraglutide.

Preferably, the amino Resin A is selected from one of Rink Amide MBHA Resin and Rink Amide AM Resin; more preferably, the substitution degree of the amino resin A is 0.3 to 0.5 mmol/g.

Preferably, Fmoc-Glu-2-R is Fmoc-Glu-2-phenylisoproyl easter.

Preferably, when Fmoc-Glu-2-R side chain carboxylic acid is attached to the amino type resin A, the coupling reagent combination is one selected from PyBOP/DIPEA/HOBt and DEPBT/DIEA.

Preferably, before coupling reaction of Fmoc-Glu (resin) -R amino acid resin I, a protecting group R is removed by using a lysate I to prepare Fmoc-Glu (resin) -OH; more preferably, the lysis solution i is a DCM solution containing 1.5-2.5% by volume of TFA, the acidolysis time is 5min, and the acidolysis times is 2-3.

Preferably, the liraglutide peptide resin is prepared by Fmoc-Glu (resin) -R amino acid resin I and polypeptide fragment NH in sequence₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂-VII-R、Fmoc-Gly-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Leu-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Val-OH, Fmoc-Asp (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Phe-OH, Fmoc-Thr (tBu) -OH, Fmoc-Gl-OH, Fmoc-Glu (OtBu) -OH, Fmoc-Ala-OH, Boc-His (trt) -OH.

Preferably, Fmoc-Glu (resin) -R amino acid resin I is sequentially reacted with polypeptide fragment NH₂-V-R, polypeptide fragment

NH₂-VI-R, polypeptide fragment NH₂And when the-VII-R is coupled, the coupling reagent combination is selected from one of PyBOP/DIPEA/HOBt, HATU/DIPEA/HOBt, HBTU/DIPEA/HOBt and DIC/HOBt.

Preferably, the polypeptide fragment NH₂-V-R is NH₂-Ala-Ala-Lys(Glu(N^α-palmitoyl) -OtBu) -R, polypeptide fragment NH₂VI-R is NH₂-Glu (OtBu) -Phe-Ile-Ala-R, polypeptide fragment NH₂VII to R are NH₂-trp (boc) -Leu-Val-arg (pbf) -Gly-R; more preferably, R is phenylisoproyl easter.

Preferably, the polypeptide fragment NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂The preparation method of the-VII-R comprises the following steps:

step 1, Fmoc-Lys (Glu (N) was prepared by Fmoc solid phase synthesis^αCoupling of carboxyl groups of- -Palmitoyl) -OtBu) -OH, Fmoc-Ala-OH, Fmoc-Gly-OH to CTC resin B preparation of Fmoc- -Lys (Glu (N)^α-Palmitoyl) -OtBu) -CTC Resin amino acid Resin II, Fmoc-Ala-CTC Resin amino acid Resin III, Fmoc-Gly-ctcresesin amino acid Resin IV;

step 2, preparing full-protection polypeptide fragment V peptide resin, full-protection polypeptide fragment VI peptide resin and full-protection polypeptide fragment VII peptide resin on amino acid resin II, amino acid resin III and amino acid resin IV respectively through Fmoc solid phase synthesis;

step 3, respectively acidolyzing the fully-protected polypeptide fragment V peptide resin, the fully-protected polypeptide fragment VI peptide resin and the fully-protected polypeptide fragment VII peptide resin by using a lysis solution i to obtain a fully-protected polypeptide fragment Fmoc-V-OH, a fully-protected polypeptide fragment Fmoc-VI-OH and a fully-protected polypeptide fragment Fmoc-VII-OH;

step 4, respectively protecting carboxyl groups of the fully protected polypeptide fragment Fmoc-V-OH, the fully protected polypeptide fragment Fmoc-VI-OH and the fully protected polypeptide fragment Fmoc-VII-OH by using a carboxyl protection reagent to prepare a fully protected polypeptide fragment Fmoc-V-phenylisoproyl easter, a fully protected polypeptide fragment Fmoc-VI-phenylisoproyl easter and a fully protected polypeptide fragment Fmoc-VII-phenylisoproyl easter;

step 5, removing Fmoc on the fully protected polypeptide fragment Fmoc-V-phenylisoproyl easter, the fully protected polypeptide fragment Fmoc-VI-phenylisoproyl easter and the fully protected polypeptide fragment Fmoc-VII-phenylisoproyl easter to obtain a polypeptide fragment NH₂-V-R, polypeptide fragment NH₂-VI-R, polypeptide fragment NH₂-VII-R。

Preferably, the CTC resin B in the step 1 is selected from one of 2-Chlorotrityl chloride resin, 4-Methyl-Trityl-Cl resin and 4-Methoxy-Trityl-Cl resin, and more preferably, the substitution degree of the CTC resin B is 0.5-0.9 mmol/g.

Preferably, the carboxy protecting reagent in step 4 is 2-phenylprop-2-yl 2, 2, 2-trichloroacetimidate.

Preferably, the molar ratio of the carboxylic acid to the carboxyl protecting agent is 1: 1.5-2.5.

Compared with the prior art, the invention has the advantages that:

(1) Fmoc-Glu-2-phenylisopyrol ester side chain carboxylic acid and amino resin are connected to the 17 th Gln position of the liraglutide (the middle position of the liraglutide peptide chain is shown in the specification), so that the flexibility between the peptide chain and the resin is increased, the peptide chain is easier to deform, and the reaction steric hindrance is reduced.

(2) Glu side chain carboxylic acid is connected with amino resin and finally becomes Gln through acidolysis, thereby solving the problem of difficult coupling of Gln in conventional polypeptide synthesis.

(3) By reacting NH with₂-Ala-Ala-Lys(Glu(N^αthe-Palmitoyl) -OtBu) -phenylisopropylpyrrolidone is coupled to Fmoc-Glu (resin) -OH, so that the problems of heavy metal introduction, low synthesis efficiency and easy generation of diacylation impurities caused by modification of Lys side chains on the fully-protected liraglutide main chain peptide resin are avoided.

(4) The synthesis method has simple process, difficult amino acid synthesis and contribution to industrial production, and the yield of the refined liraglutide product is 40.7 percent and the HPLC purity is 99.85 percent.

Detailed Description

The invention discloses a method for preparing liraglutide, which can be realized by a person skilled in the art by appropriately improving process parameters by referring to the content. 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 and applications of this 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 in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Some common abbreviations in the present invention have the following meanings:

the invention is further illustrated by the following examples:

raw materials and reagents used in the method for preparing liraglutide provided by the invention can be obtained from the market, and the preparation method of 2-phenylpropan-2-yl 2, 2, 2-trichloroacetimidate refers to Wessel, H.P; iversen.t.; bundle, D.R.J.chem.Sot.Perkin Trans I, 1985, 2247-. Fmoc-Lys (Glu (N)^α-Palmitoyl) -OtBu) -OH is commercially available or can be prepared on its own, prepared according to the method of preparation I Guryanov; a Bondesan, j.pept.sci.2016, 22 (7): 471-.

Reference example 2 preparation of phenylpropan-2-yl 2, 2, 2-trichloroacetimidate

Adding NaH (0.5g, 21mmol) into a 20ml ether solution, stirring under the protection of nitrogen, dropwise adding a 2-phenyl-2-propanol (210mmol) ether solution (30ml), cooling the solution to 0 ℃ by using a cold salt bath after the solid is dissolved, then dropwise adding trichloroacetonitrile (20ml, 20mmol) within 15min, heating the reaction mixture to 20 ℃ within 60min after the dropwise adding is finished, concentrating to obtain a syrup-like liquid, then adding 20ml of pentane (containing 0.8ml of anhydrous methanol), violently shaking, filtering, washing with pentane (2 x 20ml), concentrating the filtrate to obtain an imido ester transparent liquid, and directly using the imido ester transparent liquid in the next step. The hexane solution of the imidate can be stored at 5 ℃ for up to 2 months.