Preparation method of tilpotide

Abstract

The invention relates to the technical field of medicine preparation, and discloses a preparation method of tirpotide, which comprises the following steps: S1, taking 10mmol of RinkAmide-AmResin, adding into a reaction column, and swelling for 30 minutes by using DMF (Dimethyl Formamide); s2, a deprotection mode is as follows: DBLK deprotection is performed twice for 5 min and 15 min respectively, and the resin is washed once with DMF in the middle; and S3, after deprotection, washing the resin with DMF for 5 times, taking a small amount of resin, and determining the completion of Fmoc deprotection through a Kaiser color test. The method effectively solves the problems that the preparation process is more, the process is tedious and the cost is high when a fragment method is adopted for synthesis, and the purification effect is influenced by most impurities difficult to remove; the purification process is simple, and a sample with the purity larger than 99.0% and the single impurity smaller than 0.2% can be obtained through two-step HPLC purification; a novel salt exchange method is provided, after a purified qualified sample solution is subjected to sedimentation centrifugation and washing centrifugation desalination, various salts can be added into the product, and then the product in other salt forms of the tilpotide is obtained through freeze-drying.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and in particular relates to a method for preparing telpotide. Background Art

[0002] (Telpotide) is a dual agonist of the gastric inhibitory polypeptide glucagon-like peptide-1 receptor. It is the world's first and currently the only dual agonist of the GIP (glucose-dependent insulinotropic polypeptide) / GLP-1 (glucagon-like peptide-1) receptor. The drug integrates the effects of two incretin hormones, GIP and GLP-1, and is injected once a week to improve blood sugar control and assist in weight management. Telpotide has demonstrated excellent blood sugar-lowering and weight-reducing effects in multiple clinical trials, especially in head-to-head comparisons with similar drugs.

[0003] Patent CN201911063432.6 introduces that sites 1 to 4 use the fully protected peptide fragment Boc-Tyr(tBu)-Aib-Glu(OtBu)-Gly-OH, and site K20 uses the fully protected peptide fragment Fmoc-Lys((AEEA-AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu))-OH; the crude telotriol peptide is cleaved and purified by high performance liquid chromatography. The chromatographic filler used for purification is 10 μm reverse phase C18, and two flow The mobile phase system was alternately purified, the first mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution, the second mobile phase system was 50mmol ammonium acetate / water solution-acetonitrile, gradient system elution was adopted, cyclic injection purification was adopted, high performance liquid chromatography was used for salt exchange, the mobile phase system was 1% acetic acid / water solution-acetonitrile, the chromatographic filler for purification was 10μm reverse phase C18, gradient elution was adopted, the main peak of salt exchange was collected, and it was concentrated under reduced pressure to obtain Tirzepatide acetic acid aqueous solution, which was freeze-dried to obtain pure Tirzepatide.

[0004] Patent CN202110359421.3, Patent CN202110359495.7 and Patent CN202110359422.8 respectively introduce the use of a 2-6 peptide fragment containing Pro for coupling, a 2-6 peptide fragment containing Aib for coupling, and a 5-7 peptide fragment containing Phe-Val-Gln-Trp-Leu for coupling; cleavage to obtain crude telpotide peptide, which is then dissolved in acetonitrile aqueous solution, and HPLC gradient elution is performed on the crude telpotide peptide solution using octadecyl bonded silica gel as the stationary phase and TFA aqueous solution and acetonitrile as the mobile phase, the telpotide fractions are collected, and part of the acetonitrile is removed by rotary evaporation to obtain a primary purified solution of telpotide. The primary purified solution of telpotide is subjected to HPLC linear elution using octadecyl bonded silica gel as the stationary phase and NaClO4 salt solution and acetonitrile as the mobile phase, and the telpotide fractions are collected, and the acetonitrile and most of the water are removed by rotary evaporation, and telpotide is obtained by freeze-drying.

[0005] In summary, the problems existing in the prior art are: 1. The liquid-to-liquid connection in the existing solid-liquid mixing method is time-consuming and laborious, and impurities between the fragments are difficult to remove and separate, which affects the next step of connection. Thus, the purity and yield of the entire product are affected.

[0006] 2. The existing process has a relatively simple control on impurities and fails to effectively control most of the difficult-to-remove impurities that appear during the synthesis process.

[0007] 3. The existing purification method has simple steps and significantly improves product yield and purity. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a method for preparing telpotide which can overcome the above problems or at least partially solve the above problems.

[0009] The present invention is achieved by a method for preparing telpotide, comprising: S1, add 10mmol RinkAmide-AmResin into the reaction column and swell in DMF for 30 minutes; S2, deprotection method: DBLK deprotection twice, the time is 5min and 15min respectively, and the resin is washed once with DMF in between; S3. After deprotection, the resin was washed 5 times with DMF, and a small amount of resin was taken to confirm the completion of Fmoc deprotection by Kaiser color test; S4, coupling method: take 30mmol protected amino acid and 36mmol HOBt, dissolve them in appropriate amount of DMF, add 36mmol DIC to stir and activate, add them to the reaction column and react at 25-35℃ for 2-5 hours, monitor the completion of the reaction by Kaiser color test, after the reaction is completed, drain the solvent and wash the resin 3 times with DMF; S5, using the same method as above, sequentially coupling the protected amino acids at positions 1 to 39 or the raw materials to obtain the main chain peptide resin of telpotide; S6. Take telpotide peptide resin, add TFA mixed solution, the mixed solution ratio is TFA:EDT / Tis / phenol / H2O=90:2.5:2.5:2.5:2.5, 10 ml / gram of resin of cleavage reagent; stir and react at 20-30°C for 2-4 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, add MTBE to precipitate, wash the precipitate with MTBE 3 times, and dry to obtain an off-white powder, which is 46.5 g of crude telpotide, with a crude purity of 81.06%.

[0010] Preferably in the present invention, the degree of substitution of 10 mmol RinkAmide-AmResin in S1 is 0.40 mmol / g.

[0011] Preferably, in the present invention, DBLK in S2 is 20% piperidine / DMF.

[0012] Preferably, in S5, positions 36 to 38 are made of Fmoc-Pro-Pro-Pro-OH, positions 29 to 30 are made of Fmoc-Gly-Gly-OH, positions 20 are made of Fmoc-Lys((AEEA-AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu))-OH, positions 15 to 16 are made of Fmoc-Asp(OtBu)-Lys(Boc)-OH, positions 12 to 13 are made of Fmoc-Ile-Aib-OH, positions 3 to 4 are made of Fmoc-Glu(OtBu)-Gly-OH, and positions 1 to 2 are made of Boc-Tyr(tBu)-Aib-OH.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention effectively solves the problem of adopting fragment method for synthesis, which has many preparation processes, complicated process, high cost, and each fragment needs to be purified, which makes it difficult to control impurities and has a low degree of industrialization. It also effectively solves the problem that most of the difficult-to-remove impurities affect the purification effect.

[0014] 2. The purification process of the present invention is simple, and a sample with a purity of >99.0% and a single impurity of <0.2% can be obtained by two-step HPLC purification.

[0015] 3. The present invention provides a new salt exchange method. After the purified qualified sample solution is desalted by sedimentation centrifugation and washing centrifugation, various salts can be added to the product, and then freeze-dried to obtain other salt forms of telpotide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a flowchart of a method provided by an embodiment of the present invention; Figure 2 It is a schematic diagram of crude product data of the method provided by an embodiment of the present invention; Figure 3 It is a schematic diagram of finished product data of the method provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0017] In order to further understand the content, features and effects of the present invention, the following embodiments are given as examples and described in detail with reference to the accompanying drawings.

[0018] The structure of the present invention is described in detail below in conjunction with the accompanying drawings.

[0019] like Figure 1~Figure 3 As shown, a method for preparing telpotide provided in an embodiment of the present invention.

[0020] Embodiment 1 S1, add 10mmol RinkAmide-AmResin into the reaction column and swell in DMF for 30 minutes; S2, deprotection method: DBLK deprotection twice, the time is 5min and 15min respectively, and the resin is washed once with DMF in between; S3. After deprotection, the resin was washed 5 times with DMF, and a small amount of resin was taken to confirm the completion of Fmoc deprotection by Kaiser color test; S4, coupling method: take 30mmol protected amino acid and 36mmol HOBt, dissolve them in appropriate amount of DMF, add 36mmol DIC to stir and activate, add them to the reaction column and react at 25-35℃ for 2-5 hours, monitor the completion of the reaction by Kaiser color test, after the reaction is completed, drain the solvent and wash the resin 3 times with DMF; S5, using the same method as above, sequentially coupling the protected amino acids at positions 1 to 39 or the raw materials to obtain the main chain peptide resin of telpotide; S6. Take tulpoic acid peptide resin, add TFA mixed solution, the mixed solution ratio is TFA:EDT / Tis / phenol / H2O=90:2.5:2.5:2.5:2.5, 10 ml / g resin of cleavage reagent; stir and react at 20-30°C for 2-4 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, add MTBE to precipitate, wash the precipitate with MTBE 3 times, and dry to obtain an off-white powder, which is 46.5 g of crude tulpoic acid, with a crude purity of 81.06%. Figure 2 shown.

[0021] The degree of substitution of 10mmol RinkAmide-AmResin in S1 is 0.40mmol / g.

[0022] DBLK in S2 is 20% piperidine / DMF.

[0023] In S5, positions 36 to 38 use the raw material Fmoc-Pro-Pro-Pro-OH, positions 29 to 30 use the raw material Fmoc-Gly-Gly-OH, positions 20 use Fmoc-Lys((AEEA-AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu))-OH, positions 15 to 16 use the raw material Fmoc-Asp(OtBu)-Lys(Boc)-OH, positions 12 to 13 use the raw material Fmoc-Ile-Aib-OH, positions 3 to 4 use the raw material Fmoc-Glu(OtBu)-Gly-OH, and positions 1 to 2 use the raw material Boc-Tyr(tBu)-Aib-OH.

[0024] Embodiment 2 Take 46.5 g of crude telpotide, add 10 L of 10% acetonitrile / pure water solution and stir evenly, adjust the pH of the crude solution to 7.0 with ammonia water to completely dissolve the sample; heat the sample solution in a 45°C water bath for 2 h, and then filter with a 0.45 um filter membrane to obtain a crude telpotide solution; take the crude telpotide solution and purify it using a preparative high performance liquid chromatograph, and the purification conditions are as follows: One Pure: Instrument: Preparative HPLC Chromatographic column: 150mm dynamic axial compression column Packing: Kromasil C8 10μm 100Å Mobile phase: Phase A: 1.1% phosphoric acid, 1.4% triethylamine aqueous solution (1L water plus 11ml phosphoric acid, 14ml triethylamine) Phase B: Acetonitrile Detection wavelength: λ=230nm Flow rate: 400ml / min Gradient elution conditions: Time (min) Phase A (%, V / V) Phase B (%, V / V) 0 90 10 7 90 10 8 68 42 48 63 47 Collect samples from different time periods of the main peak, use HPLC analysis to detect, and combine the collected solutions with purity ≥95% and single impurity <1%; Second pure: Instrument: Preparative HPLC Chromatographic column: 150mm dynamic axial compression column Packing: Kromasil C8 10μm 100Å Mobile phase: Phase A: 50mmol / L ammonium formate aqueous solution (adjust pH to 7.8 with ammonia water) Phase B: Acetonitrile Detection wavelength: λ=230nm Flow rate: 400ml / min Gradient elution conditions: Time (min) Phase A (%, V / V) Phase B (%, V / V) 0 90 10 7 90 10 8 70 30 48 60 40 Samples from different time periods of the main peak were collected and tested using HPLC. The collected solutions with a combined purity of ≥99% and a single impurity of <0.2% were considered qualified solutions.

[0025] Embodiment 3 Concentration: Take the qualified solution of telpotide and use a rotary evaporator to concentrate it under reduced pressure, with a water bath temperature of 30°C-35°C, and vortex until the volume is 1 / 3 of the qualified solution to obtain a qualified sample concentrate.

[0026] Sedimentation centrifugation: Take the qualified sample concentrate and adjust the pH to 4.6±0.1 with phosphoric acid, stir evenly, transfer to a centrifuge cup, centrifuge at 3600r / min for 3min, and discard the upper liquid.

[0027] Washing and centrifugation: Transfer the centrifuged solid to the sedimentation tank again, add purified water to stir, adjust the pH to 4.6±0.1 with dilute phosphoric acid, stir and centrifuge at 3600r / min for 3min, discard the upper liquid. Repeat the above washing and centrifugation steps 3-4 times to obtain the desalted sample of tepoxetine.

[0028] Dissolution: After the last centrifugation, the solid was washed with purified water and the pH of the sample was adjusted to 7.2±0.1 with 20 g / L sodium hydroxide aqueous solution to completely dissolve the sample to obtain a sodium salt solution of tepol.

[0029] Lyophilization: The tepote sodium salt solution was lyophilized to remove water to obtain 20.1 g of tepote sodium salt sample with a purity of 99.85% and a single impurity of 0.07%. Figure 3 shown.

[0030] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0031] The above description is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this patent will not depart from the scope of the technical solution of the present invention.

Claims

1. A method for preparing telpotide, Features: include: S1, add 10mmol RinkAmide-AmResin into the reaction column and swell in DMF for 30 minutes; S2, deprotection method: DBLK deprotection twice, the time is 5min and 15min respectively, and the resin is washed once with DMF in between; S3. After deprotection, the resin was washed 5 times with DMF, and a small amount of resin was taken to confirm the completion of Fmoc deprotection by Kaiser color test; S4, coupling method: take 30mmol protected amino acid and 36mmol HOBt, dissolve them in appropriate amount of DMF, add 36mmol DIC to stir and activate, add them to the reaction column and react at 25-35℃ for 2-5 hours, monitor the completion of the reaction by Kaiser color test, after the reaction is completed, drain the solvent and wash the resin 3 times with DMF; S5, using the same method as above, sequentially coupling the protected amino acids at positions 1 to 39 or the raw materials to obtain the main chain peptide resin of telpotide; S6. Take telpotide peptide resin, add TFA mixed solution, the mixed solution ratio is TFA:EDT / Tis / phenol / H2O=90:2.5:2.5:2.5:2.5, 10 ml / gram of resin of cleavage reagent; stir and react at 20-30°C for 2-4 hours, filter the reaction mixture with a sand core funnel, collect the filtrate, add MTBE to precipitate, wash the precipitate with MTBE 3 times, and dry to obtain an off-white powder, which is 46.5 g of crude telpotide, with a crude purity of 81.06%.

2. A method for preparing telpotide according to claim 1, Features: The degree of substitution of 10mmol RinkAmide-AmResin in S1 is 0.40mmol / g.

3. A method for preparing telpotide according to claim 1, Features: DBLK in S2 is 20% piperidine / DMF.

4. The method for preparing telpotide according to claim 1, Features: In S5, positions 36 to 38 use the raw material Fmoc-Pro-Pro-Pro-OH, positions 29 to 30 use the raw material Fmoc-Gly-Gly-OH, positions 20 use Fmoc-Lys((AEEA-AEEA-γ-Glu-(OtBu)-Eicosanedioic acid-OtBu))-OH, positions 15 to 16 use the raw material Fmoc-Asp(OtBu)-Lys(Boc)-OH, positions 12 to 13 use the raw material Fmoc-Ile-Aib-OH, positions 3 to 4 use the raw material Fmoc-Glu(OtBu)-Gly-OH, and positions 1 to 2 use the raw material Boc-Tyr(tBu)-Aib-OH.

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