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WO2018033127A1 - 一种低消旋杂质利拉鲁肽的合成方法 - Google Patents

一种低消旋杂质利拉鲁肽的合成方法 Download PDF

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Publication number
WO2018033127A1
WO2018033127A1 PCT/CN2017/097970 CN2017097970W WO2018033127A1 WO 2018033127 A1 WO2018033127 A1 WO 2018033127A1 CN 2017097970 W CN2017097970 W CN 2017097970W WO 2018033127 A1 WO2018033127 A1 WO 2018033127A1
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liraglutide
thr
phe
fmoc
ala
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English (en)
French (fr)
Inventor
付玉清
马洪季
李新宇
张利香
支钦
吴丽芬
刘自成
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Shenzhen JYMed Technology Co Ltd
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Shenzhen JYMed Technology Co Ltd
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Priority to CN201780001586.5A priority Critical patent/CN107960079B/zh
Priority to EP17841099.9A priority patent/EP3505533B1/en
Priority to US16/326,265 priority patent/US11518794B2/en
Publication of WO2018033127A1 publication Critical patent/WO2018033127A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis

Definitions

  • the invention relates to a method for synthesizing a polypeptide, in particular to a method for synthesizing a low-race impurity liraglutide.
  • diabetes With the development of society, the incidence of diabetes in the world has increased significantly. It was estimated to be 2.8% in 2000, 4.3% in 2025, and the number of diabetic patients will increase from 171 million in 2000 to 2025. 380 million. Diabetes is divided into gestational diabetes, specific diabetes, type I diabetes, and type 2 diabetes. Among them, type II diabetes, also known as non-insulin-dependent diabetes, is characterized by the fact that the body itself can produce insulin, but the cells can not respond to it, so that the effect of insulin is greatly reduced. There are many types of hypoglycemic agents for type 2 diabetes, including metformin, sulfonylureas, and receptor kinetin of glucagon-like peptide-1 (GLP-1). GLP-1 receptor kinetin is a recent study. Hot spot.
  • Liraglutide one of the human glucagon-like peptide-1 (GLP-1) analogues, known in English as Liraglutide, is a drug developed by Novo Nordisk of Denmark for the treatment of type 2 diabetes. The injection was approved by the FDA on January 25, 2010 (trade name Victoza), and on March 4, 2011, it was approved by the SFDA. Liraglutide acts as a GLP-1 receptor agonist and can play a good role in lowering blood sugar.
  • GLP-1 human glucagon-like peptide-1
  • the synthesis of liraglutide mainly uses genetic recombination technology and stepwise coupling.
  • genetic synthesis technology is used to synthesize liraglutide, the technical difficulty is relatively high and the cost is relatively high.
  • the intermediate GLP-1(7-37)-OH needs repeated purification by HPLC, and then in the liquid phase with N ⁇ -alkanoyl-Glu. (ONSu)-OtBu reaction, and due to the unprotected N-terminus of GLP-1(7-37)-OH and the complete removal of the side chain protecting group, many impurities are produced, which is difficult to purify.
  • the Fmoc-Gly-resin is obtained by condensation reaction with Fmoc-Gly-OH using a resin as a solid phase carrier;
  • the larupeptide main chain peptide sequence condenses amino acids with N-terminal Fmoc protection and side chain protection, wherein lysine adopts Fmoc-Lys(X)-OH (X is a Lys side chain protecting group) or Fmoc-Lys (N - ⁇ -(N- ⁇ -Palmitoyl-L- ⁇ -glutamyl))-OH, each condensation reaction for 30-35 min; the lysine side chain of liraglutide resin is modified to cleave or directly cleave; purified, lyophilized Liraglutide is obtained.
  • racemic by-products are structurally very close to liraglutide, making the purification and separation of the crude liraglutide peptide very difficult, and no good separation results can be achieved by using various purification separation systems. If multiple separations are carried out, it is foreseeable that separation will cause significant loss of the product; on the other hand, the presence of racemic impurities has an adverse effect on the quality of the drug, affecting not only the stability and efficacy of the drug, but also harmful to human health. Therefore, in the production process of synthesizing liraglutide, the generation of racemic by-products should be minimized.
  • a method for synthesizing a low-rotation impurity liraglutide comprises the following steps:
  • the 2-5 peptide containing Thr-Phe is selected from the group consisting of Thr-Phe, Gly-Thr-Phe, Thr-Phe-Thr, Gly-Thr-Phe-Thr, Glu-Gly-Thr-Phe , Thr-Phe-Thr-Ser, Ala-Glu-Gly-Thr-Phe, Glu-Gly-Thr-Phe-Thr, Gly-Thr-Phe-Thr-Ser, Thr-Phe-Thr-Ser-Asp.
  • the pentapeptide Glu-Phe-Ile-Ala-Trp is used in the process of synthesizing the propeptide.
  • the dipeptide fragment is selected from the group consisting of His-Ala, Ala-Glu, Glu-Gly, Thr-Ser, Ala-Ala;
  • the tripeptide fragment is selected from the group consisting of Glu-Phe-Ile, Ser-Asp-Val, and Thr-Ser-Asp;
  • the tetrapeptide fragment is selected from the group consisting of Lys-Glu-Phe-Ile and Glu-Phe-Ile-Ala;
  • the pentapeptide fragment is selected from the group consisting of Glu-Phe-Ile-Ala-Trp, Ala-Lys-Glu-Phe-Ile, Lys-Glu-Phe-Ile-Ala, Ala-Trp-Leu-Val-Arg;
  • Peptide fragments containing the same amino acid at the beginning and the end are not used in combination.
  • the combination includes Ala-Trp-Leu-Val-Arg, Ala-Lys-Glu-Phe-Ile, Ser-Asp-Val, Glu-Gly.
  • the combination includes Glu-Phe-Ile-Ala-Trp, Ala-Ala, Ser-Asp-Val, Ala-Glu.
  • the combination includes Ala-Trp-Leu-Val-Arg, Glu-Phe-Ile, Thr-Ser-Asp, Ala-Glu.
  • Thr-Phe 2 to 5 peptides containing Thr-Phe are used in the process of synthesizing liraglutide, especially the dipeptides Thr-Phe, the tripeptide Gly-Thr-Phe, and the Thr-Phe- One of Thr can significantly reduce the production of liraglutide racemic impurity D-Thr 5 liraglutide while ensuring that the yield is not lowered.
  • the method for synthesizing liraglutide provided by the invention effectively inhibits or reduces the production of the racemic impurity D-Thr 5 liraglutide which is very similar to the product property, and the racemic impurity D in the prepared crude liraglutide peptide
  • the content of -Thr 5 liraglutide is less than 0.8% (w/w), which is advantageous for the purification of the crude peptide of liraglutide.
  • the higher yield is ensured, and the production cost is greatly reduced; in the synthesis of liraglutide, the synthesis of the dipeptide fragment, the tripeptide fragment, the tetrapeptide fragment, the pentapeptide fragment or a combination thereof and the Gly-resin can be simultaneously It also shortens the certain synthesis time.
  • Example 1 is an HPLC chart of the crude liraglutide peptide prepared in Example 1;
  • Example 2 is an HPLC chart of liraglutide spermatopeptide prepared in Example 9;
  • the protecting group is a protecting group which is commonly used in the field of amino acid synthesis to protect the amino acid main chain and the amino group, carboxyl group and the like which interfere with the synthesis of the side chain, and prevents the amino group and the carboxyl group from reacting during the preparation of the target product to form impurities.
  • the amino acid of the side chain is protected, and the side chain structure is known and the common protecting group is used to protect the amino group, carboxyl group and the like on the side chain of the amino acid.
  • the present invention is protected by the Trt protecting group.
  • a side chain of histidine or glutamine protecting the side chain of glutamic acid and aspartic acid by OtBu protecting group; protecting the side chain of tryptophan by Boc; protecting threonine, serine, tyrosine by tBu protecting group
  • the side chain of the acid; the side chain of lysine is protected by Alloc; the side chain of arginine is protected by the Pbf protecting group.
  • the N-terminus of the amino acid is preferably protected by an Fmoc protecting group, and histidine is also protected by a Boc protecting group.
  • amino acids or peptides used in the present invention may be protected with a protecting group depending on the needs of the synthesis.
  • the propeptide refers to the synthesis of liraglutide, and the polypeptide fragment of the 2-5 peptide of Thr-Phe is not included in the synthesis sequence of the C-terminus-N-terminus of the liraglutide peptide sequence, and the side chain of the propeptide is A protecting group can be coupled.
  • the propeptide can be obtained by enzymatic synthesis (purchase) or synthesized by a known method. In particular, propeptides are obtained using polypeptide solid phase synthesis.
  • the structure of the racemic impurity in the crude peptide of liraglutide in the present specification is NH 2 -His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu -Gly-Gln-Ala-Ala-Lys(N- ⁇ -(N- ⁇ -Palmitoyl-L- ⁇ -glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly-COOH, expressed as D-Thr 5 liraglutide.
  • Boc-His(Trt)-Ala-OH (2.89 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF, and DIC (0.49 mL, 3.15 mmol) was added for 5 minutes under ice bath.
  • the activated solution was added to the above solid phase reaction column, stirred under nitrogen for 2 h, the ninhydrin test was negative, the DMF was washed 4 times, and the DCM was washed twice;
  • H-Arg(pbf)-Gly-Wang resin was synthesized by the method of Example 1, and Fmoc-Gly-OH and Fmoc-Arg (pbf) were sequentially coupled to H-Arg(pbf)-Gly-Wang resin.
  • Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF and activated by adding DIC (0.49 mL, 3.15 mmol) for 5 minutes under ice bath. A good solution was added to the above solid phase reaction column, and the reaction was stirred for 2 hours under nitrogen, and the ninhydrin test was negative to obtain Fmoc-Arg(pbf)-Gly-2-CTC resin.
  • reaction solution was drained, DMF was washed 3 times, DBLK was deprotected (5+7 min), DMF was washed 6 times, and ninhydrin was negative, and H-Arg(pbf)-Gly-2-CTC resin was obtained.
  • H-Arg(pbf)-Gly-2-CTC resin was synthesized by the method of Example 3, and Fmoc-Gly-OH and Fmoc-Ala- were sequentially coupled to H-Arg(pbf)-Gly-2-CTC resin.
  • H-Arg(pbf)-Gly-2-CTC resin was synthesized by the method of Example 3, and Fmoc-Gly-OH and Fmoc-Arg were sequentially coupled to H-Arg(pbf)-Gly-2-CTC resin.
  • H-Arg(pbf)-Gly-2-CTC resin was synthesized by the method of Example 3, and Fmoc-Gly-OH and Fmoc-Ala- were sequentially coupled to H-Arg(pbf)-Gly-2-CTC resin.
  • H-Arg(pbf)-Gly-Wang resin was synthesized by the method of Example 1, and Fmoc-Gly-OH and Fmoc-Ala-Trp (Boc) were sequentially coupled to H-Arg(pbf)-Gly-Wang resin.
  • H-Arg(pbf)-Gly-2-CTC resin was synthesized by the method of Example 3, and Fmoc-Gly-OH and Fmoc-Ala- were sequentially coupled to H-Arg(pbf)-Gly-2-CTC resin.
  • Example 1 1) The crude liraglutide obtained in Example 1 was stirred, stirred with water, adjusted to pH 8.5 with ammonia water to completely dissolve, and the solution was filtered through a 0.45 ⁇ m microporous membrane filter, and purified;
  • High-performance liquid chromatography was used for salt exchange.
  • the mobile phase system was 1% acetic acid/water solution-acetonitrile.
  • the chromatographic packing with purification was 10 ⁇ m reverse phase C18, and the flow rate of 77mm*250mm column was 90mL/min. Take off, cycle the sample method, load on the column, start the mobile phase elution, collect the spectrum, observe the change of the absorbance, collect the main peak of the salt change and check the purity with the analytical liquid phase, combine the salt peak solution, concentrate under reduced pressure, An aqueous solution of liraglutide acetate was obtained and used.
  • the column is a column with octasilane-bonded silica as the stationary phase.
  • the diameter and length of the column are: 150mm ⁇ 250mm, the aqueous solution of 0.06% ammonia is phase A, and the chromatographic pure acetonitrile is B.
  • the pure product of Liraglutide is 1.17g, the purity of pure liraglutide is 99.27%, the total yield is 25.12%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, Immediately adjacent to the main peak, the relative retention time is about 1.0 and the content is 0.27%.
  • the HPLC chromatogram is shown in Figure 2. The peak retention time and peak area results are shown in Table 3.
  • Example 2 The crude liraglutide peptide prepared in Example 2 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.15g, the purity is 99.31%, the total yield is 23.14%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.25%.
  • the HPLC profile is similar to Figure 2.
  • Example 3 The crude liraglutide peptide obtained in Example 3 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.16g, the purity is 99.23%, the total yield is 24.14%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.26%.
  • the HPLC profile is similar to Figure 2.
  • Example 4 The crude liraglutide peptide prepared in Example 4 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.19g, the purity is 99.36%, the total yield is 24.36%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.27%.
  • the HPLC profile is similar to Figure 2.
  • Example 5 The crude liraglutide peptide prepared in Example 5 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.23g, the purity is 99.48%, the total yield is 25.14%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.26%.
  • the HPLC profile is similar to Figure 2.
  • Example 6 The crude liraglutide peptide prepared in Example 6 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.21g, the purity is 99.50%, the total yield is 24.65%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.27%.
  • the HPLC profile is similar to Figure 2.
  • Example 7 The crude liraglutide peptide prepared in Example 7 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.14g, the purity is 99.51%, the total yield is 23.46%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.26%.
  • the HPLC profile is similar to Figure 2.
  • Example 8 The crude liraglutide peptide prepared in Example 8 was purified by the same purification method as in Example 9.
  • Liraglutide Derivative of Liraglutide is 1.21g, the purity is 99.48%, the total yield is 24.12%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, relative retention time It is 1.0 and the content is 0.25%.
  • the HPLC profile is similar to Figure 2.
  • Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF and activated by adding DIC (0.49 mL, 3.15 mmol) for 5 minutes under ice bath. A good solution was added to the above solid phase reaction column, and the reaction was stirred for 2 hours under nitrogen, and the ninhydrin test was negative to obtain Fmoc-Arg(pbf)-Gly-Wang resin.
  • reaction solution was drained, DMF was washed 3 times, DBLK was deprotected (5+7 min), DMF was washed 6 times, and ninhydrin was negative, and H-Arg(pbf)-Gly-Wang resin was obtained.
  • Fmoc-Arg(pbf)-OH coupling method Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc- are sequentially coupled on H-Arg(pbf)-Gly-Wang resin.
  • the crude peptide of liraglutide yield is 80.5%, the purity is 68.63%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, the relative retention time is about 1.0, the content is 2.39%.
  • the HPLC spectrum is shown in Figure 3.
  • the peak retention time and peak area results are shown in Table 4.
  • Fmoc-Arg(pbf)-OH (3.245 g, 5 mmol) and HOBt (0.426 g, 3.15 mmol) were dissolved in 15 mL of DMF and activated by adding DIC (0.49 mL, 3.15 mmol) for 5 minutes under ice bath. A good solution was added to the above solid phase reaction column, and the reaction was stirred for 2 hours under nitrogen, and the ninhydrin test was negative to obtain Fmoc-Arg(pbf)-Gly-2-CTC resin.
  • reaction solution was drained, DMF was washed 3 times, DBLK was deprotected (5+7 min), DMF was washed 6 times, and ninhydrin was negative, and H-Arg(pbf)-Gly-2-CTC resin was obtained.
  • the crude peptide yield of liraglutide is 79.8%, the purity is 68.61%, the racemic impurity similar to the structure of liraglutide, D-Thr 5 liraglutide, close to the main peak, the relative retention time is about 1.0, the content is 2.40%.
  • the HPLC profile is similar to Figure 3.
  • Example 1 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.74%, respectively, which was relatively reduced by 69.04% and 69.17%, respectively.
  • Example 2 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.71%, respectively, which was relatively reduced by 70.29% and 70.42%, respectively.
  • Example 3 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.70%, respectively, which was relatively reduced by 70.71% and 70.83%, respectively.
  • Example 4 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.69%, respectively, and decreased by 71.13% and 71.25%, respectively.
  • Example 5 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.70%, respectively, which was relatively reduced by 70.71% and 70.93%, respectively.
  • Example 6 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.71%, respectively, which was relatively reduced by 70.29% and 70.42%, respectively.
  • Example 7 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.70%, respectively, which was relatively reduced by 70.71% and 70.93%, respectively.
  • Example 8 reduced the D-Thr 5 liraglutide content from 2.39%, 2.40% to 0.71%, respectively, which was relatively reduced by 70.29% and 70.42%, respectively.
  • the method for synthesizing liraglutide provided by the present invention can greatly reduce the amount of the racemic impurity D-Thr 5 liraglutide produced in the crude peptide synthesis of liraglutide, D-Thr 5 liraglutide The content is less than 0.8%, which is very advantageous for purification.
  • the yield of liraglutide in Example 1 was 91.2%, and the purity was 70.61%, which was 10.7% and 1.98% higher than that of Comparative Example 1, respectively, and was increased by 11.4% and 2.0, respectively, compared with Comparative Example 2. %.
  • the yield of liraglutide in Example 2 was 90.2%, and the purity was 71.68%, which was increased by 9.7% and 3.05%, respectively, compared with Comparative Example 1, and was increased by 10.4% and 3.07%, respectively, compared with Comparative Example 2.
  • the yield of liraglutide in Example 3 was 90.5%, and the purity was 72.02%, which was increased by 10.0% and 3.39%, respectively, compared with Comparative Example 1, and was increased by 10.7% and 3.41%, respectively, compared with Comparative Example 2.
  • the yield of liraglutide in Example 4 was 91.5%, and the purity was 72.72%, which was increased by 11.0% and 4.09%, respectively, compared with Comparative Example 1, and was increased by 11.7% and 4.11%, respectively, compared with Comparative Example 2.
  • the yield of liraglutide in Example 5 was 91.8%, and the purity was 70.72%, which was 11.3% and 2.09% higher than that of Comparative Example 1, respectively, and was increased by 12.0% and 2.11%, respectively, compared with Comparative Example 2.
  • the yield of liraglutide in Example 6 was 91.5%, and the purity was 71.53%, which was 11.0% and 2.9% higher than that of Comparative Example 1, respectively, and was 11.7% and 2.92% higher than Comparative Example 2, respectively.
  • the yield of liraglutide in Example 7 was 90.6%, and the purity was 72.03%, which was 10.1% and 3.40% higher than that of Comparative Example 1, respectively, and was increased by 10.8% and 3.42%, respectively, compared with Comparative Example 2.
  • the yield of liraglutide in Example 8 was 90.8%, and the purity was 70.36%, which was 10.3% and 1.73% higher than that of Comparative Example 1, respectively, and was increased by 11.0% and 1.75%, respectively, compared with Comparative Example 2. It can be seen that the method for synthesizing liraglutide provided by the present invention can improve the yield and purity of the crude peptide of liraglutide and is advantageous for purification.
  • the method of the invention can greatly reduce the production of the racemic impurity D-Thr 5 liraglutide in the synthesis of liraglutide, while ensuring that the yield is not lowered, and is advantageous for purifying the crude peptide of liraglutide to obtain a pedipeptide.
  • the crude liraglutide peptide prepared in Comparative Example 1 was purified according to the purification method of the above Example 9, and the pure liraglutide product was 0.47 g, the purity was 98.36%, and the total yield was 11.34%, with Lilaru.
  • the racemic impurity with similar peptide structure, D-Thr 5 liraglutide is adjacent to the main peak with a relative retention time of about 1.0 and a content of 1.23%.
  • the HPLC chromatogram is shown in Figure 4.
  • the peak retention time and peak area results are shown in Table 5.
  • the crude peptide of liraglutide of Comparative Example 2 was purified by the purification method of the above Example 9, and the pure product of liraglutide was 0.48 g, the purity was 97.53%, and the total yield was 12.58%, which was similar to the structure of liraglutide. Racemic impurities, D-Thr 5 liraglutide, immediately adjacent to the main peak, with a relative retention time of about 1.0 and a content of 1.23%.
  • the HPLC profile is similar to Figure 4.
  • the method of the present invention substantially reduces the production of the racemic impurity D-Thr 5 liraglutide in the synthesis of liraglutide, and at the same time ensures the yield of liraglutide, which is favorable for purification to obtain low D- liraglutide fine peptide thr 5 liraglutide content.

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Abstract

一种低消旋杂质利拉鲁肽的合成方法，包括如下步骤：合成得到前肽，使用固相合成法在前肽上偶联含有Thr-Phe的2～5肽；进一步固相合成得到利拉鲁肽树脂，经侧链修饰后裂解或直接裂解、纯化、冻干，得到利拉鲁肽。提供的利拉鲁肽的合成方法有效抑制或减少了与产品性质极为相似的消旋杂质D-Thr5的产生，有利于利拉鲁肽粗肽的纯化，同时保证其较高的收率，大大降低了生产成本；在利拉鲁肽合成过程中，二肽片段、三肽片段、四肽片段、五肽片段或其组合与Gly-树脂的合成可同时进行，也缩短了一定的合成时间。

Description

一种低消旋杂质利拉鲁肽的合成方法 技术领域
本发明涉及一种多肽的合成方法，特别涉及一种低消旋杂质利拉鲁肽的合成方法。
背景技术
随着社会的发展，世界范围内糖尿病的发病率呈明显增加的趋势，2000年曾估计为2.8％，到2025年预计为4.3％，糖尿病患者人数也将从2000年的1.71亿增加到2025年的3.8亿。糖尿病分为妊娠期糖尿病、特异性糖尿病、I型糖尿病和II型糖尿病。其中，II型糖尿病又名非胰岛素依赖型糖尿病，特点是人体自身能够产生胰岛素，但细胞无法对其作出反应，使胰岛素的效果大打折扣。针对II型糖尿病的降糖药种类很多，包括二甲双胍、磺脲类药物和胰高血糖样肽-1(GLP-1)的受体激动素等，GLP-1的受体激动素是近年来研究的热点。
利拉鲁肽，人胰高糖素样肽-1(GLP-1)类似物中的一种，英文名为Liraglutide，是丹麦诺和诺德公司研制出的一种治疗II型糖尿病的药物，其注射剂分别于2010年1月25日获得FDA批准上市(商品名为Victoza)、2011年3月4日获得SFDA批准上市。利拉鲁肽作为GLP-1受体激动剂能起到良好的降低血糖作用。
目前，利拉鲁肽的合成，主要采用基因重组技术和逐步偶联的方法。采用基因重组技术合成利拉鲁肽时，技术难度较大，成本相对较高，中间体GLP-1(7-37)-OH需要HPLC反复纯化，再在液相条件下与Nα-alkanoyl-Glu(ONSu)-OtBu反应，并且由于GLP-1(7-37)-OH N端未保护以及侧链保护基全部脱除，会导致产生许多杂质，难以纯化。采用逐步偶联的方法进行利拉鲁肽合成时，本领域技术人员周知，以树脂为固相载体，与Fmoc-Gly-OH缩合反应获得Fmoc-Gly-树脂；通过固相合成法，按照利拉鲁肽主链肽序依次缩合具有N端Fmoc保护且侧链保护的氨基酸，其中赖氨酸采用Fmoc-Lys(X)-OH(X为Lys侧链保护基团)或Fmoc-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-OH，每次缩合反应30-35min；利拉鲁肽树脂赖氨酸侧链经修饰后裂解或直接裂解；纯化，冻干得到利拉鲁肽。由于利拉鲁肽的序列较长且有较多的疏水氨基酸，逐步偶联时易形成β折叠，导致树脂收缩严重，延长反应时间，进而粗肽中产生较多与产品性质极为接近的消旋杂质NH2-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH(D-Thr5利拉鲁肽)，纯化比较困难；同时树脂收缩严重，反应不完全，导致收率偏低。
一方面，消旋体副产物在结构上与利拉鲁肽非常接近，使得利拉鲁肽粗肽的纯化分离十分困难，使用多种纯化分离体系尝试皆不能达到良好的分离效果。如果进行多次分离，则可以预见分离会导致产品的显著损失；另一方面，消旋杂质的存在，对药品质量有着不利影响，不仅影响药物的稳定性和疗效，甚至对人体健康有害。因此，在合成利拉鲁肽的制造工艺中，应当尽可能地减少消旋体副产物的产生。
利拉鲁肽主链氨基酸有31个，采用片段方法进行合成存在很多种形式，但只有合适的片段方法才能保证产生较少的消旋体副产物，又能降低合成工艺的复杂程度，同时保证利拉鲁肽的收率、纯度。发明人通过长期的实验，意外发现使用本发明的方法合成利拉鲁肽，可以大幅降低利拉鲁肽消旋杂质NH2-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH(D-Thr5利拉鲁肽)的产生，方法简单同时保证收率不会降低。
发明内容
本发明的目的在于提供一种低消旋杂质利拉鲁肽的合成方法。
本发明所采取的技术方案是：
一种低消旋杂质利拉鲁肽的合成方法，包括如下步骤：
1)合成得到前肽，之后使用固相合成法在前肽上偶联含有Thr-Phe的2～5肽；
2)进一步固相合成得到利拉鲁肽树脂，经侧链修饰后裂解或直接裂解、纯化、冻干，得到利拉鲁肽。
作为上述合成方法的进一步改进，含有Thr-Phe的2～5肽选自Thr-Phe、Gly-Thr-Phe、Thr-Phe-Thr、Gly-Thr-Phe-Thr、Glu-Gly-Thr-Phe、Thr-Phe-Thr-Ser、Ala-Glu-Gly-Thr-Phe、Glu-Gly-Thr-Phe-Thr、Gly-Thr-Phe-Thr-Ser、Thr-Phe-Thr-Ser-Asp。
作为上述合成方法的进一步改进，在合成前肽的过程中使用五肽Glu-Phe-Ile-Ala-Trp。
作为上述合成方法的进一步改进，在合成利拉鲁肽的过程中，采用二肽片段、三肽片段、四肽片段、五肽片段或其组合与氨基酸、Fmoc-Gly-树脂偶联，得到利拉鲁肽树脂；其中：
作为上述合成方法的进一步改进，二肽片段选自His-Ala、Ala-Glu、Glu-Gly、Thr-Ser、Ala-Ala；
三肽片段选自Glu-Phe-Ile、Ser-Asp-Val、Thr-Ser-Asp；
四肽片段选自Lys-Glu-Phe-Ile、Glu-Phe-Ile-Ala；
五肽片段选自Glu-Phe-Ile-Ala-Trp、Ala-Lys-Glu-Phe-Ile、Lys-Glu-Phe-Ile-Ala、 Ala-Trp-Leu-Val-Arg；
首尾含相同氨基酸的肽片段不组合使用。
作为上述合成方法的进一步改进，组合包括Ala-Trp-Leu-Val-Arg、Ala-Lys-Glu-Phe-Ile、Ser-Asp-Val、Glu-Gly。
作为上述合成方法的进一步改进，组合包括Glu-Phe-Ile-Ala-Trp、Ala-Ala、Ser-Asp-Val、Ala-Glu。
作为上述合成方法的进一步改进，组合包括Ala-Trp-Leu-Val-Arg、Glu-Phe-Ile、Thr-Ser-Asp、Ala-Glu。
本发明的有益效果是：
发明人通过长期的实验，意外发现在合成利拉鲁肽的过程中使用含有Thr-Phe的2～5肽，特别是使用二肽Thr-Phe、三肽Gly-Thr-Phe、Thr-Phe-Thr中的一个，可以大幅降低利拉鲁肽消旋杂质D-Thr5利拉鲁肽的产生，同时保证收率不会降低。
本发明提供的利拉鲁肽的合成方法有效抑制或减少了与产品性质极为相似的消旋杂质D-Thr5利拉鲁肽产生，制备得到的利拉鲁肽粗肽中，消旋杂质D-Thr5利拉鲁肽的含量低于0.8％(w/w)，有利于利拉鲁肽粗肽的纯化。同时保证其较高的收率，大大降低了生产成本；在利拉鲁肽合成过程中，二肽片段、三肽片段、四肽片段、五肽片段或其组合与Gly-树脂的合成可同时进行，也缩短了一定的合成时间。
附图说明
图1是实施例1制备得到的利拉鲁肽粗肽的HPLC图；
图2是实施例9制备得到的利拉鲁肽精肽的HPLC图；
图3是对比例1制备得到的利拉鲁肽粗肽的HPLC图；
图4是对比例3制备得到的利拉鲁肽精肽的HPLC图。
具体实施方式
下面结合具体实施例对本发明的利拉鲁肽的合成方法作进一步详细的说明以便于本领域的技术人员进一步地理解本发明。实施例不应限定为对保护范围的限制。
使用的英文缩写具体含义如表1所示。
表1说明书和权利要求书中所使用的英文缩写具体含义
Figure PCTCN2017097970-appb-000001
Figure PCTCN2017097970-appb-000002
保护基是在氨基酸合成领域常用的保护氨基酸主链以及侧链上氨基、羧基等干扰合成的基团的保护基团，防止氨基、羧基等在制备目标产物过程中发生反应，生成杂质，对于本发明中需要保护侧链的氨基酸来说，本领域技术人员公知其侧链结构以及知晓采用常用保护基来保护氨基酸侧链上的氨基、羧基等基团，作为优选，本发明通过Trt保护基保护组氨酸、谷氨酰胺的侧链；通过OtBu保护基保护谷氨酸、天冬氨酸的侧链；通过Boc保护色氨酸的侧链；通过tBu保护基保护苏氨酸、丝氨酸、酪氨酸的侧链；通过Alloc保护赖氨酸的侧链；通过Pbf保护基保护精氨酸的侧链。此外，在本发明所述方法涉及的氨基酸中，氨基酸N端均优选通过Fmoc保护基进行保护，而组氨酸也可通过Boc保护基进行保护。
根据合成需要，本发明所使用的氨基酸或肽，特别是二肽、三肽、四肽、五肽等可以使用保护基保护。
本说明书中，前肽指利拉鲁肽合成中，按照利拉鲁肽肽序列C端-N端的合成顺序，不包含有Thr-Phe的2～5肽的多肽片段，前肽的侧链上可以偶联保护基。前肽可以委托合成(购买)获得，或按公知的方法合成得到。特别的，前肽使用多肽固相合成法得到。
本说明书中利拉鲁肽粗肽中的消旋杂质结构为NH2-His-Ala-Glu-Gly-D-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-COOH，用D-Thr5利拉鲁肽表示。
实施例1
1)取4.25g Fmoc-Gly-Wang树脂(0.279mmol/g)装入固相反应柱中，DMF洗两次，DMF溶胀30分钟，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阳性；
2)将Fmoc-Arg(pbf)-OH(3.245g，5mmol)和HOBt(0.426g，3.15mmol)溶解在15mLDMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到上述固相反应柱中，氮气搅拌反应2h，茚三酮检测呈阴性，得到Fmoc-Arg(pbf)-Gly-Wang树脂。抽干反应液，DMF洗涤3次，DBLK脱保护(5+7min)， DMF洗涤6次，茚三酮检测呈阴性，得到H-Arg(pbf)-Gly-Wang树脂；
3)在H-Arg(pbf)-Gly-Wang树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Ala-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-Asp(OtBu)-Val-OH、Fmoc-Thr(tBu)-OH、Fmoc-Thr(tBu)-Phe-OH、Fmoc-Glu(OtBu)-Gly-OH；
4)将Boc-His(Trt)-Ala-OH(2.89g，5mmol)和HOBt(0.426g，3.15mmol)溶解于15mLDMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到上述固相反应柱中，氮气搅拌2h，茚三酮检测呈阴性，DMF洗涤4次，DCM洗涤2次；
5)向上述固相反应柱中加入15mL DCM和1.08g苯硅烷，氮气搅拌1分钟后加入0.289g四(三苯基膦)钯，反应0.5h，抽干，DCM洗涤6次，茚三酮检测呈阳性；
6)将Fmoc-Glu-OtBu(2.128g，5mmol)、HOBt(0.709g，5.25mmol)和PyBOP(2.602g，5mmol)溶解在25mL DMF中，冰浴条件下加入DIPEA(1.75mL，10mmol)活化3分钟，将活化好的溶液加入上述固相反应柱，氮气搅拌反应2h，茚三酮检测呈阴性，抽干，DMF洗涤4次，DCM洗涤2次；
7)向上述固相反应柱中加入20mL DCM和DIPEA(1.75mL，10mmol)，氮气搅拌均匀后缓慢滴加棕榈酰氯(1.374g，5mmol)，滴加完毕后继续反应2h，茚三酮检测呈阴性，抽干，DCM洗涤6次，MeOH收缩，真空干燥后得到肽树脂9.43g；
8)取所得利拉鲁肽树脂9.43g，加入体积比为TFA：水：EDT＝90：5：5的混合酸解液(用量10mL/克利拉鲁肽树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末，真空减压干燥至恒重。
得到4.25g利拉鲁肽粗肽，收率为91.2％，纯度为70.61％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.74％。HPLC图谱如图1所示，特征峰保留时间与峰面积结果如表2所示。
表2、实施例1的利拉鲁肽粗肽的特征峰保留时间与峰面积结果
Figure PCTCN2017097970-appb-000003
Figure PCTCN2017097970-appb-000004
实施例2
1)参照实施例1的方法合成得到H-Arg(pbf)-Gly-Wang树脂，在H-Arg(pbf)-Gly-Wang树脂上依次偶联Fmoc-Gly-OH、Fmoc-Arg(pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、、Fmoc-Glu(OtBu)-Phe-Ile-Ala-Trp(Boc)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-Ala-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-Asp(OtBu)-Val-OH、Fmoc-Thr(tBu)-OH、Fmoc-Thr(tBu)-Phe-OH、Fmoc-Gly-OH、Fmoc-Ala-Glu(OtBu)-OH；
2)将Boc-His(Trt)-OH(2.49g，5mmol)和HOBt(0.426g，3.15mmol)溶解于15mL DMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到固相反应柱中，氮气搅拌2h，茚三酮检测呈阴性，DMF洗涤4次，DCM洗涤2次；余下方法步骤参照实施例1中步骤5-8，真空减压干燥至恒重。
得到4.34g利拉鲁肽粗肽，利拉鲁肽收率为90.2％，纯度为71.68％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.71％。HPLC图谱与实施例图1相似。
实施例3
1)取3.58g Fmoc-Gly-2-CTC树脂(0.279mmol/g)装入固相反应柱中，DMF洗两次，DMF溶胀30分钟，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阳性；
2)将Fmoc-Arg(pbf)-OH(3.245g，5mmol)和HOBt(0.426g，3.15mmol)溶解在15mLDMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到上述固相反应柱中，氮气搅拌反应2h，茚三酮检测呈阴性，得到Fmoc-Arg(pbf)-Gly-2-CTC树脂。抽干反应液，DMF洗涤3次，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阴性，得到H-Arg(pbf)-Gly-2-CTC树脂；
3)在H-Arg(pbf)-Gly-2-CTC树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-Phe-Ile-OH、Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu))、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Thr(tBu)-Ser(tBu)-Asp(OtBu)-OH、Fmoc-Gly-Thr(tBu)-Phe-OH、Fmoc-Ala-Glu(OtBu)-OH、Boc-His(Trt)-OH；
4)每克树脂10mL 50％TFA/DCM溶液脱Boc保护基20min，用DCM洗涤6次，真空减压干燥得到肽树脂9.21g；
5)取肽树脂加入体积比为TFA：Anisole：TIS：H2O：EDT＝92：2：2：2：2的混合酸解液(用量10mL/克利拉鲁肽树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末，真空减压干燥至恒重。
得到4.31g利拉鲁肽粗肽，利拉鲁肽粗肽收率为90.5％，纯度为72.02％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.70％。HPLC 图谱与图1相似。
实施例4
参照实施例3的方法合成得到H-Arg(pbf)-Gly-2-CTC树脂，在H-Arg(pbf)-Gly-2-CTC树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-Phe-Ile-OH、Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu))、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-Asp(OtBu)-Val-OH、Fmoc-Thr(tBu)-Phe-Thr(tBu)-OH、Fmoc-Glu(OtBu)-Gly-OH、Boc-His(Trt)-Ala-OH；余下方法步骤参照实施例3中步骤4-5，真空减压干燥至恒重。
得到4.41g利拉鲁肽粗肽，利拉鲁肽粗肽收率为91.5％，纯度为72.72％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.69％。HPLC图谱与图1相似。
实施例5
参照实施例3的方法合成得到H-Arg(pbf)-Gly-2-CTC树脂，在H-Arg(pbf)-Gly-2-CTC树脂上依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Lys(Alloc)-Glu(OtBu)-Phe-Ile-Ala-OH、Fmoc-Ala-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、moc-Asp(OtBu)-OH、Fmoc-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-OH、Fmoc-Glu(OtBu)-Gly-OH、Boc-His(Trt)-Ala-OH；余下方法步骤参照实施例3中步骤4-5，真空减压干燥至恒重。
得到4.50g利拉鲁肽粗肽，利拉鲁肽粗肽收率为91.8％，纯度为70.72％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.70％。HPLC图谱与图1相似。
实施例6
参照实施例3的方法合成得到H-Arg(pbf)-Gly-2-CTC树脂，在H-Arg(pbf)-Gly-2-CTC树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Lys(Alloc)-Glu(OtBu)-Phe-Ile-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、 Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-Thr(tBu)-Phe-Thr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Boc-His(Trt)-Ala-OH；余下方法步骤参照实施例1中步骤5-8，真空减压干燥至恒重。
得到4.50g利拉鲁肽粗肽，利拉鲁肽粗肽收率为91.5％，纯度为71.53％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.71％。HPLC图谱与图1相似。
实施例7
参照实施例1的方法合成得到H-Arg(pbf)-Gly-Wang树脂，在H-Arg(pbf)-Gly-Wang树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-Phe-Ile-OH、Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu))、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Glu(OtBu)Gly-Thr(tBu)-Phe-Thr(tBu)-OH、Boc-His(Trt)-Ala-OH；余下方法步骤参照实施例3中步骤4-5，真空减压干燥至恒重。
得到4.40g利拉鲁肽粗肽，利拉鲁肽粗肽收率为90.6％，纯度为72.03％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.70％。HPLC图谱与图1相似。
实施例8
参照实施例3的方法合成得到H-Arg(pbf)-Gly-2-CTC树脂，在H-Arg(pbf)-Gly-2-CTC树脂上依次偶联Fmoc-Gly-OH、Fmoc-Ala-Trp(Boc)-Leu-Val-Arg(Pbf)-OH、Fmoc-Glu(OtBu)-Phe-Ile-OH、Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu))、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-OH、Fmoc-Glu(OtBu)-OH、Boc-His(Trt)-Ala-OH；余下方法步骤参照实施例3中步骤4-5，真空减压干燥至恒重。
得到4.52g利拉鲁肽粗肽，利拉鲁肽粗肽收率为90.8％，纯度为70.36％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.71％。HPLC图谱与图1相似。
实施例9、利拉鲁肽精肽的制备
1)取实施例1所得利拉鲁肽粗品，加水搅拌，用氨水调pH8.5至完全溶解，溶液用0.45μm微孔滤膜过滤，纯化备用；
2)采用高效液相色谱法进行纯化，纯化用色谱填料为10μm的反相C18，流动相系统为0.1％TFA/水溶液-0.1％TFA/乙腈溶液，50mm*250mm的色谱柱流速为90mL/min，采用梯度系统洗脱，循环进样纯化，取粗品溶液上样于色谱柱中，启动流动相洗脱，收集主峰蒸去乙腈后，得利拉鲁肽纯化中间体浓缩液。取利拉鲁肽纯化中间体浓缩液，用0.45μm滤膜滤过备用。
3)采用高效液相色谱法进行换盐，流动相系统为1％醋酸/水溶液-乙腈，纯化用色谱填料为10μm的反相C18，77mm*250mm的色谱柱流速为90mL/min，采用梯度洗脱，循环上样方法，上样于色谱柱中，启动流动相洗脱，采集图谱，观测吸收度的变化，收集换盐主峰并用分析液相检测纯度，合并换盐主峰溶液，减压浓缩，得到利拉鲁肽醋酸水溶液，备用。
4)采用HPLC进行脱盐纯化，色谱柱为以八烷基硅烷键合硅胶为固定相的色谱柱，柱子直径和长度为：150mm×250mm，0.06％氨水的水溶液为A相，色谱纯乙腈为B相，流速：480ml/min，梯度：32％B-65％B，检测波长：275nm，旋转蒸发浓缩，冷冻干燥。
得利拉鲁肽纯品1.17g，利拉鲁肽纯品的纯度为99.27％，总收率为25.12％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.27％。HPLC图谱如图2所示，特征峰保留时间与峰面积结果如表3所示。
表3实施例9的利拉鲁肽特征峰保留时间与峰面积结果
Figure PCTCN2017097970-appb-000005
实施例10、利拉鲁肽精肽的制备
实施例2制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.15g，纯度为99.31％，总收率为23.14％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.25％。HPLC图谱与图 2相似。
实施例11、利拉鲁肽精肽的制备
实施例3制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.16g，纯度为99.23％，总收率为24.14％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.26％。HPLC图谱与图2相似。
实施例12、利拉鲁肽精肽的制备
实施例4制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.19g，纯度为99.36％，总收率为24.36％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.27％。HPLC图谱与图2相似。
实施例13、利拉鲁肽精肽的制备
实施例5制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.23g，纯度为99.48％，总收率为25.14％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.26％。HPLC图谱与图2相似。
实施例14、利拉鲁肽精肽的制备
实施例6制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.21g，纯度为99.50％，总收率为24.65％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.27％。HPLC图谱与图2相似。
实施例15、利拉鲁肽精肽的制备
实施例7制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.14g，纯度为99.51％，总收率为23.46％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.26％。HPLC图谱与图2相似。
实施例16、利拉鲁肽精肽的制备
实施例8制得的利拉鲁肽粗肽使用与实施例9同样的纯化方法进行纯化。
得利拉鲁肽纯品1.21g，纯度为99.48％，总收率为24.12％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为0.25％。HPLC图谱与图2相似。
对比例1、利拉鲁肽粗肽的制备
1)取4.25g Fmoc-Gly-Wang树脂(0.279mmol/g)装入固相反应柱中，DMF洗两次，DMF溶胀30分钟，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阳性；
2)将Fmoc-Arg(pbf)-OH(3.245g，5mmol)和HOBt(0.426g，3.15mmol)溶解在15mLDMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到上述固相反应柱中，氮气搅拌反应2h，茚三酮检测呈阴性，得到Fmoc-Arg(pbf)-Gly-Wang树脂。抽干反应液，DMF洗涤3次，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阴性，得到H-Arg(pbf)-Gly-Wang树脂；
3)按照上述Fmoc-Arg(pbf)-OH偶联的方法，在H-Arg(pbf)-Gly-Wang树脂上依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys(Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-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
4)向上述固相反应柱中加入15mL DCM和1.08g苯硅烷，氮气搅拌1分钟后加入0.289g四(三苯基膦)钯，反应0.5h，抽干，DCM洗涤6次，茚三酮检测呈阳性；
5)将Fmoc-Glu-OtBu(2.128g，5mmol)、HOBt(0.709g，5.25mmol)和PyBOP(2.602g，5mmol)溶解在25mL DMF中，冰浴条件下加入DIPEA(1.75mL，10mmol)活化3分钟，将活化好的溶液加入上述固相反应柱，氮气搅拌反应2h，茚三酮检测呈阴性，抽干，DMF洗涤4次，DCM洗涤2次；
6)向上述固相反应柱中加入20mL DCM和DIPEA(1.75mL，10mmol)，氮气搅拌均匀后缓慢滴加棕榈酰氯(1.374g，5mmol)，滴加完毕后继续反应2h，茚三酮检测呈阴 性，抽干，DCM洗涤6次，MeOH收缩，真空干燥后得到肽树脂7.46g；
7)向装有利拉鲁肽肽树脂7.46g的圆底烧瓶中加入体积比为TFA：水：EDT＝90：5：5的混合酸解液(用量10mL/克利拉鲁肽树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末，真空减压干燥至恒重后得到3.68g利拉鲁肽粗肽。
利拉鲁肽粗肽收率为80.5％，纯度为68.63％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为2.39％。HPLC图谱如图3所示，特征峰保留时间与峰面积结果如表4所示。
表4对比例1的利拉鲁肽粗肽的特征峰保留时间与峰面积结果
Figure PCTCN2017097970-appb-000006
Figure PCTCN2017097970-appb-000007
对比例2、利拉鲁肽粗肽的制备
1)取3.58g Fmoc-Gly-2-CTC树脂(0.279mmol/g)装入固相反应柱中，DMF洗两次，DMF溶胀30分钟，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阳性；
2)将Fmoc-Arg(pbf)-OH(3.245g，5mmol)和HOBt(0.426g，3.15mmol)溶解在15mLDMF中，冰浴条件下加入DIC(0.49mL，3.15mmol)活化5分钟，将活化好的溶液加入到上述固相反应柱中，氮气搅拌反应2h，茚三酮检测呈阴性，得到Fmoc-Arg(pbf)-Gly-2-CTC树脂。抽干反应液，DMF洗涤3次，DBLK脱保护(5+7min)，DMF洗涤6次，茚三酮检测呈阴性，得到H-Arg(pbf)-Gly-2-CTC树脂；
3)在H-Arg(pbf)-Gly-Wang树脂上依次偶联Fmoc-Gly-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Val-OH、Fmoc-Leu-OH、Fmoc-Trp(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys(N-ε-(Nα-Palmitoyl-L-γ-glutamyl-OtBu))、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Val-OH、Fmoc-Asp(OtBu)-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；
4)每克树脂10mL 50％TFA/DCM溶液脱Boc保护基20min，用DCM洗涤6次，真空减压干燥得到肽树脂8.52g；
5)向装有利拉鲁肽肽树脂8.52g的圆底烧瓶中加入体积比为TFA：Anisole：TIS：H2O：EDT＝92：2：2：2：2的混合酸解液(用量10mL/克利拉鲁肽树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末，真空减压干燥至恒重后得到3.68g利拉鲁肽粗肽。
利拉鲁肽粗肽收率为79.8％，纯度为68.61％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为2.40％。HPLC图谱与图3相似。
从实施例1～8与对比例1、2中的HPLC图谱及相应的数据可知，消旋体杂质峰，D-Thr5利拉鲁肽峰与利拉鲁肽主峰紧密相邻，实施例1中保留时间分别为49.288min、49.875min，对比例1中保留时间分别为48.253min、48.835min；D-Thr5利拉鲁肽的相对保留时间约为1.0，远远达不到分离的要求，如果D-Thr5利拉鲁肽含量较大，粗肽的纯化分离将十分困难。然而与对比例1、2相比，实施例1将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.74％，分别相对减少了69.04％和69.17％。与对比例1、2相比，实施例2将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.71％，分别相对减少了70.29％和70.42％。与对比例1、2相比，实施例3将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.70％，分别相对减少了70.71％和70.83％。与对比例1、2相比，实施例4将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.69％，分别相对减少了71.13％和71.25％。与对比例1、2相比，实施例5将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.70％，分别相对减少了70.71％和70.93％。与对比例1、2相比，实施例6将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.71％，分别相对减少了70.29％和70.42％。与对比例1、2相比，实施例7将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.70％，分别相对减少了70.71％和70.93％。与对比例1、2相比，实施例8将D-Thr5利拉鲁肽含量分别从2.39％、2.40％降低到了0.71％，分别相对减少了70.29％和70.42％。由此可知，本发明提供的利拉鲁肽的合成方法可大大减少利拉鲁肽粗肽合成中的消旋杂质D-Thr5利拉鲁肽的生成量，D-Thr5利拉鲁肽的含量少于0.8％，十分有利于纯化。
此外，实施例1中利拉鲁肽的收率达91.2％，纯度为70.61％，与对比例1相比分别提高了10.7％、1.98％，与对比例2相比分别提高了11.4％、2.0％。实施例2中利拉鲁肽的收率达90.2％，纯度为71.68％，与对比例1相比分别提高了9.7％、3.05％，与对比例2相比分别提高了10.4％、3.07％。实施例3中利拉鲁肽的收率达90.5％，纯度为72.02％，与对比例1相比分别提高了10.0％、3.39％，与对比例2相比分别提高了10.7％、3.41％。实施例4中利拉鲁肽的收率达91.5％，纯度为72.72％，与对比例1相比分别提高了11.0％、4.09％，与对比例2相比分别提高了11.7％、4.11％。实施例5中利拉鲁肽的收率达91.8％，纯度为70.72％，与对比例1相比分别提高了11.3％、2.09％，与对比例2相比分别提高了12.0％、2.11％。实施例6中利拉鲁肽的收率达91.5％，纯度为71.53％，与对比例1相比分别提高了11.0％、2.9％，与对比例2相比分别提高了11.7％、2.92％。实施例7中利拉鲁肽的收率达90.6％，纯度为72.03％，与对比例1相比分别提高了10.1％、3.40％，与对比例2相比分别提高了10.8％、3.42％。实施例8中利拉鲁肽的收率达90.8％，纯度为70.36％，与对比例1相比分别提高了10.3％、1.73％，与对比例2相比分别提高了11.0％、1.75％。由此可知，本发明提供的利拉鲁肽的合成方法可提高利拉鲁肽粗肽的收率和纯度，有利于纯化。
本发明的方法可以大幅降低利拉鲁肽合成中消旋杂质D-Thr5利拉鲁肽的产生，同时保证收率不会降低，有利于利拉鲁肽粗肽纯化得到精肽。
对比例3、利拉鲁肽精肽的制备
取对比例1制得的利拉鲁肽粗肽，按上述实施例9的纯化方法纯化，得利拉鲁肽纯品0.47g，纯度为98.36％，总收率为11.34％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为1.23％。HPLC图谱如图4所示，特征峰保留时间与峰面积结果如表5所示。
表5对比例3的利拉鲁肽特征峰保留时间与峰面积结果
Figure PCTCN2017097970-appb-000008
对比例2的利拉鲁肽粗肽按上述实施例9的纯化方法纯化，得利拉鲁肽纯品0.48g，纯度为97.53％，总收率为12.58％，与利拉鲁肽结构相近的消旋杂质，D-Thr5利拉鲁肽，紧邻主峰，相对保留时间约为1.0，含量为1.23％。HPLC图谱与图4相似。
从实施例9～15与对比例3中的HPLC图谱可知，经过简单的纯化步骤，利拉鲁肽中的D-Thr5利拉鲁肽已基本除去，精肽中最大含量仅为0.27％，而对比例中的D-Thr5利拉鲁肽含量则较难除去，精肽中含量高达1.23％，比本发明方法中粗肽中的含量还大，可以预见如果为了减少消旋杂质D-Thr5利拉鲁肽的含量而进一步纯化必然会使原本较低的收率更加降低。
由此可知，本发明的方法本大幅降低了利拉鲁肽合成中消旋杂质D-Thr5利拉鲁肽的产生，同时保证了利拉鲁肽的收率，有利于纯化得到低D-Thr5利拉鲁肽含量的利拉鲁肽精肽。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (9)

  1. 一种低消旋杂质利拉鲁肽的合成方法，包括如下步骤：
    合成得到前肽，之后使用固相合成法在前肽上偶联含有Thr-Phe的2～5肽，
    进一步固相合成得到利拉鲁肽树脂，经侧链修饰后裂解或直接裂解、纯化、冻干，得到利拉鲁肽。
  2. 根据权利要求1所述的合成方法，其特征在于：含有Thr-Phe的2～5肽选自Thr-Phe、Gly-Thr-Phe、Thr-Phe-Thr、Gly-Thr-Phe-Thr、Glu-Gly-Thr-Phe、Thr-Phe-Thr-Ser、Ala-Glu-Gly-Thr-Phe、Glu-Gly-Thr-Phe-Thr、Gly-Thr-Phe-Thr-Ser、Thr-Phe-Thr-Ser-Asp。
  3. 根据权利要求1所述的合成方法，其特征在于：在合成前肽的过程中使用五肽Glu-Phe-Ile-Ala-Trp。
  4. 根据权利要求1或2所述的合成方法，其特征在于：在合成利拉鲁肽的过程中，采用二肽片段、三肽片段、四肽片段、五肽片段或其组合与氨基酸、Fmoc-Gly-树脂偶联，得到利拉鲁肽树脂；其中：
    二肽片段选自His-Ala、Ala-Glu、Glu-Gly、Thr-Ser、Ala-Ala；
    三肽片段选自Glu-Phe-Ile、Ser-Asp-Val、Thr-Ser-Asp；
    四肽片段选自Lys-Glu-Phe-Ile、Glu-Phe-Ile-Ala；
    五肽片段选自Glu-Phe-Ile-Ala-Trp、Ala-Lys-Glu-Phe-Ile、Lys-Glu-Phe-Ile-Ala、Ala-Trp-Leu-Val-Arg；
    首尾含相同氨基酸的肽片段不组合使用。
  5. 根据权利要求4所述的合成方法，其特征在于：组合包括Glu-Phe-Ile、Lys-Glu-Phe-Ile或Ala-Lys-Glu-Phe-Ile中的一个与Ala-Trp-Leu-Val-Arg的组合。
  6. 根据权利要求1所述的合成方法，其特征在于：含有Thr-Phe的2～5肽选自Thr-Phe、三肽Gly-Thr-Phe、Thr-Phe-Thr中的一个。
  7. 根据权利要求4所述的合成方法，其特征在于：组合包括Ala-Trp-Leu-Val-Arg、Ala-Lys-Glu-Phe-Ile、Ser-Asp-Val、Glu-Gly。
  8. 根据权利要求4所述的合成方法，其特征在于：组合包括Glu-Phe-Ile-Ala-Trp、Ala-Ala、Ser-Asp-Val、Ala-Glu。
  9. 根据权利要求4所述的合成方法，其特征在于：组合包括Ala-Trp-Leu-Val-Arg、Glu-Phe-Ile、Thr-Ser-Asp、Ala-Glu。
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