Preparation method of Tirzeptide

Abstract

The invention discloses a preparation method of Tirzepine, which specifically comprises the following steps: preparing a cracking reagent and pre-cooling, adding Tirzepine peptide resin for reaction, filtering and collecting a cracking solution and diluting with water, then adding an organic solvent for extraction and liquid separation, then collecting a water phase, adjusting the pH value, standing and collecting a precipitate, dissolving the precipitate and then filtering, purifying the filtrate, and freeze-drying fractions. According to the method, the use of ether is avoided, a water dilution method is used for replacing an ether sedimentation method, a protective group falling off during cracking is removed through an extraction step, subsequent purification is easier, the use of ether is not involved, the method is short in production period, small in waste liquid amount, low in cost, environment-friendly, few in impurity and high in yield, and continuous industrial large-scale production is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide drug synthesis, and particularly relates to a preparation method of Tirzepatide. Background Art

[0002] Patients with type 2 diabetes mellitus (T2DM) frequently suffer from a variety of comorbidities, including cognitive problems such as cognitive decline, cognitive impairment, or dementia. Patients with diabetes are 1.5 to 2 times more likely to develop cognitive decline, mild cognitive impairment, or dementia than those without the disease. This relationship is independent of other risk factors for cognitive dysfunction and accounts for a prevalence of 13% in patients with diabetes aged 65 to 74 years and 24% in patients aged 75 years or older. No single cause for the increased risk of cognitive dysfunction in patients with diabetes has been identified. It has been proposed and is being studied that certain diabetes therapies, including glucagon-like peptide-1 (GLP-1) receptor agonists, may have a beneficial effect on cognitive function.

[0003] Patients with type 2 diabetes (T2DM) are at risk for developing heart failure with preserved ejection fraction (HFpEF) as well as heart failure with reduced ejection fraction (HFrEF). See, e.g., J. Ho et al., Predictors of New-Onset Heart Failure; Circulation: Heart Failure 6:279-286 (2013). Current treatments for HFpEF may include lifestyle changes to induce weight loss and administration of symptom-relieving agents for comorbid conditions. Bariatric surgery has shown some benefits for patients with HFpEF. See, e.g., Mikhalkova et al., Obesity (2018). Despite the increasing incidence of HFpEF, there are currently few successful and effective treatments.

[0004] Tirzepatide is a dual agonist of GIP and GLP-1. Tirzepatide has demonstrated efficacy by improving beta cell function and insulin sensitivity. Tirzepatide has shown dual improvements in efficacy and tolerability in patients who initially use a lower dose and have smaller subsequent dose increments. After 8 weeks of treatment with Tirzepatide, Japanese patients with type 2 diabetes showed a significant reduction in AIC and body weight. Tirzepatide can also improve markers of non-alcoholic steatohepatitis (NASH, liver inflammation and cell damage caused by liver fat) in patients with type 2 diabetes. These new Tirzepatide data build on the positive results of studies conducted to date in patients with type 2 diabetes, providing additional evidence that Tirzepatide can bring meaningful reductions in AIC and weight levels in patients with type 2 diabetes, and that the drug can also treat other metabolic diseases.

[0005] However, in the existing Tirzepatide synthesis process (such as Chinese invention patents CN 112592387A, CN110903355 A, CN 115651075 A, CN 113330024 A, etc.), a large amount of ether is needed to precipitate the peptide in the lysate during the cleavage stage, which has the following disadvantages: (1) ether is flammable and explosive, and the operation process is dangerous; (2) the amount of ether used is more than 10 times the volume of the lysate, which is costly and seriously pollutes the environment; (3) the ether precipitation process is a heterogeneous system, and the protective groups cleaved and detached are partially wrapped in solid particles, making the subsequent purification difficult; (4) the obtained Tirzepatide has more impurities and a lower yield. The crude peptide precipitated from the ether needs to be dried, dissolved and filtered again for purification, which is not only time-consuming but also not easy. Summary of the invention

[0006] Based on the above problems, the present invention provides a new preparation method of Tirzepatide, which solves many problems in the cracking process, is environmentally friendly, has few impurities, high yield, and is conducive to continuous industrial scale-up production. The specific technical scheme is as follows:

[0007] A method for preparing Tirzepatide comprises the following steps:

[0008] (1) Prepare the cleavage reagent and pre-cool it, then add the Tirzepatide peptide resin and stir to react;

[0009] (2) collecting the lysate in (1) by filtration and diluting it with water to form an aqueous solution, then adding an organic solvent to perform extraction and separation, and collecting the aqueous phase;

[0010] (3) adjusting the pH of the aqueous phase, allowing the aqueous phase to stand and collecting the precipitated precipitate, re-dissolving and adjusting the pH of the precipitate, filtering, reversing the phase to purify the filtrate, and freeze-drying the qualified fraction to obtain Tirzepatide.

[0011] Preferably, in step (1), the cleavage reagent consists of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of (70-95)%: (0.5-10)%: (0.5-10)%: (0.5-10)%.

[0012] Preferably, in step (1), the precooling temperature is -25 to 5°C, preferably -20 to -10°C.

[0013] Preferably, in step (1), the reaction time is 1 to 5 hours, preferably 2 to 3 hours; the reaction temperature is 10 to 50°C, preferably 25 to 35°C.

[0014] Preferably, in step (1), the usage ratio of Tirzepatide peptide resin to cleavage reagent is 1 g: (8-20) mL, preferably 1 g: (8-12) mL.

[0015] Preferably, in step (2), the ratio of lysate to water is 1 L: (10-1000) L, preferably 1 L: (50-100) L.

[0016] Preferably, in step (2), the organic solvent is selected from any one of dichloromethane and ethyl acetate or a mixture of the two in any proportion.

[0017] Preferably, in step (2), the ratio of the aqueous solution to the organic solvent is 1L:(0.1-100)L, preferably 1L:(0.5-10)L.

[0018] Preferably, in step (3), the alkali solution for adjusting the pH of the aqueous phase is selected from at least one of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, and aqueous ammonia;

[0019] Preferably, the pH of the aqueous phase is adjusted to 3.0 to 6.0, preferably 4.0 to 5.0.

[0020] Preferably, in step (3), the alkaline solution for adjusting the pH of the precipitate is selected from at least one of an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution, and aqueous ammonia;

[0021] Preferably, the pH of the precipitate is adjusted to 6.5 to 9.5, preferably 7.5 to 8.5.

[0022] Compared with the prior art, the advantages of the present invention are:

[0023] The present invention provides a novel preparation method of Tirzepatide, which can avoid the use of ether, replace the ether precipitation method with a water dilution method, and remove the cleaved and detached protecting group through an extraction step. Subsequent purification is easier and the total yield is higher. The production cycle is short, the amount of waste liquid is small, the cost is reduced, the environment is friendly, and the impurities are small, the yield is high, and continuous industrialized amplification production is conducive. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention, in which:

[0025] Figure 1 The refined peptide chromatogram of Tirzepatide prepared in Example 1.

[0026] FIG. 2 is a refined peptide chromatogram of Tirzepatide obtained in Example 2.

[0027] Figure 3 The refined peptide chromatogram of Tirzepatide prepared in Example 3.

[0028] Figure 4 This is the refined peptide chromatogram of Tirzepatide prepared in Comparative Example 1.

[0029] Figure 5 This is the refined peptide chromatogram of Tirzepatide prepared in Comparative Example 2.

[0030] Figure 6 This is the refined peptide chromatogram of Tirzepatide prepared in Comparative Example 3. DETAILED DESCRIPTION

[0031] The disclosure below provides many different embodiments or examples to implement different structures of the present invention. In order to simplify the disclosure of the present invention, the parts and settings of specific examples are described below. Of course, they are only examples, and the purpose is not to limit the present invention. In addition, the present invention can repeat reference numbers and / or reference letters in different examples, and this repetition is for the purpose of simplification and clarity, which itself does not indicate the relationship between the various embodiments and / or settings discussed.

[0032] The Tirzepatide peptide resin in the present invention is prepared by using one of Rink MBHA resin, Rink Amide resin, RinkAmide AM resin or Sieber amide resin as a starting material, and coupling Fmoc-protected amino acids, or Fmoc-protected diamino acids, or Fmoc-protected peptide fragments one by one in the order from N-terminus to C-terminus of the Tirzepatide peptide sequence through a standard SPPS (solid phase peptide synthesis) method to obtain the Tirzepatide peptide resin.

[0033] Embodiment 1:

[0034] Prepare 100 mL of cleavage reagent, mix according to the volume ratio of TFA (trifluoroacetic acid): EDT (1,2-ethanedithiol): Tis (triisopropylsilane): water = 88:5:2:5, and precool to -20°C. Weigh 10.0 g of Tirzepatide peptide resin and add it to the cleavage reagent, stir at 25°C for 2.5 hours. Filter and collect the cleavage solution, wash the Tirzepatide peptide resin after the reaction with 10 mL TFA, and combine the washing solution with the filtrate. Add the collected filtrate to 5000 mL of water for dilution, add 2500 mL of dichloromethane, stir for 2 hours, and collect the aqueous phase. Adjust the pH of the aqueous phase to 4.5 with 1M sodium hydroxide solution, let it stand and collect the lower precipitation layer, and discard the upper aqueous solution. Adjust the pH of the precipitation layer to 8.0 with 1M sodium hydroxide solution to make it a clear solution, filter with a 0.45μm filter membrane, and collect the filtrate. The combined filtrate was purified by reverse phase chromatography and converted to ammonium salt, and lyophilized to obtain 3.1 g of Tirzepatide, with a total yield of 65%, a purity of 99.46%, and a maximum single impurity of 0.13%. The test results of the Tirzepatide sample prepared in Example 1 are as follows: Figure 1 As shown, the purity of the finished product is ≥99%, the maximum single impurity is ≤0.15%, the total yield is equal to 65%, no ether is used in the whole process, the operation is safer, and the pollution to the environment is less.

[0035] Embodiment 2:

[0036] Prepare 1000mL of cleavage reagent, mix according to the volume ratio of TFA: EDT: Tis: water = 88: 1: 1: 10, and pre-cool to -20℃. Weigh 100.0g of Tirzepatide peptide resin, add it to the cleavage reagent, and stir at 25℃ for 3h. Filter and collect the lysate, wash the Tirzepatide peptide resin after the reaction with 100mL TFA, and combine the washing liquid with the filtrate. Add the collected filtrate to 50L water for dilution, add 30L dichloromethane + 3L ethyl acetate, stir for 3h, and collect the aqueous phase. Adjust the pH of the aqueous phase to 4.6 with 1M potassium hydroxide solution, let it stand and collect the lower precipitation layer, and discard the upper aqueous solution. Adjust the pH of the precipitation layer to 7.8 with 1M potassium hydroxide solution to become a clear solution, filter with 0.45μm filter membrane, and collect the filtrate. The combined filtrate was purified by reverse phase chromatography and converted into ammonium salt, and lyophilized to obtain 34 g of Tirzepatide, with a total yield of 70.7%, a purity of 99.78%, and a maximum single impurity of 0.15%. The test results of the Tirzepatide sample prepared in Example 2 are shown in Figure 2. The purity of the finished product is ≥99%, the maximum single impurity is ≤0.15%, and the total yield is greater than 65%. No ether is used in the whole process, the operation is safer, and the pollution to the environment is less.

[0037] Embodiment 3:

[0038] Prepare 2000mL of cleavage reagent, mix according to the volume ratio of TFA: EDT: Tis: water = 85: 2.5: 2.5: 10, and pre-cool to -20℃. Weigh 240.0g of Tirzepatide peptide resin, add it to the cleavage reagent, and stir for 3h at 25℃. Filter and collect the cleavage solution, wash the Tirzepatide peptide resin after the reaction with 200mL TFA, and combine the washing solution with the filtrate. Add the collected filtrate to 100L water for dilution, add 50L dichloromethane + 10L ethyl acetate, stir for 3h, and collect the aqueous phase. Adjust the pH of the aqueous phase to 4.4 with a 10% dilute ammonia solution, let it stand and collect the lower precipitation layer, and discard the upper aqueous solution. Adjust the pH of the precipitation layer to 8.2 with a 1M sodium hydroxide solution to become a clear solution, filter with a 0.45μm filter membrane, and collect the filtrate. The filtrate was purified by reverse phase chromatography and converted to ammonium salt, and lyophilized to obtain 83 g of Tirzepatide, with a total yield of 72.2%, a purity of 99.83%, and a maximum single impurity of 0.09%. The test results of the Tirzepatide sample prepared in Example 3 are as follows: Figure 3 As shown, the purity of the finished product is ≥99%, the maximum single impurity is ≤0.15%, the total yield is greater than 65%, no ether is used in the whole process, the operation is safer, and the pollution to the environment is less.

[0039] Comparative Example 1:

[0040] Prepare 100 mL of cleavage reagent, mix according to the volume ratio of TFA: EDT: Tis: water = 88: 5: 2: 5, and pre-cool to -20°C. Weigh 10.0 g of Tirzepatide peptide resin, add it to the cleavage reagent, and stir at 25°C for 2.5 hours. Filter and collect the lysate, wash the reacted Tirzepatide peptide resin with 10 mL of TFA, and combine the washing liquid with the filtrate. Add the filtrate to 1 L of anhydrous ether for precipitation, and then wash the precipitate with anhydrous ether 3 times. Centrifuge to obtain the precipitate, and drain to obtain an off-white powder, which is the crude Tirzepatide. The crude product is dissolved in dilute ammonia water to form a clear solution, filtered through a 0.45 μm filter membrane, and then purified by reverse phase chromatography and converted to ammonium salt. After freeze-drying, 2.1 g of Tirzepatide is obtained, with a total yield of 43.7%, a purity of 99.57%, and a maximum single impurity of 0.13%. The test results of the Tirzepatide sample prepared in Comparative Example 1 are as follows. Figure 4 As shown, although the purity of the finished product is ≥99% and the maximum single impurity is ≤0.15%, the total yield is less than 50%, and a large amount of ether is used in the process, the operation risk factor is high, and the environmental pollution is serious.

[0041] Comparative Example 2:

[0042] Prepare 1000mL of cleavage reagent, mix according to the volume ratio of TFA: EDT: Tis: water = 88: 1: 1: 10, and pre-cool to -20℃. Weigh 100.0g of Tirzepatide peptide resin, add it to the cleavage reagent, and stir at 25℃ for 3h. Filter and collect the lysate, wash the Tirzepatide peptide resin after the reaction with 100mL TFA, add the filtrate to 10L of methyl tert-butyl ether for precipitation, and then wash the precipitate with methyl tert-butyl ether 3 times, centrifuge to obtain the precipitate, and dry the off-white powder, which is the crude Tirzepatide. The crude product is dissolved in dilute ammonia water to obtain a clear solution, filtered with a 0.45μm filter membrane, and the filtrate is collected. The combined filtrate was purified by reverse phase chromatography and converted to ammonium salt, and then freeze-dried to obtain 22 g of Tirzepatide, with a total yield of 45.7%, a purity of 99.82%, and a maximum single impurity of 0.12%. The test results of the Tirzepatide sample prepared in Comparative Example 2 are as follows: Figure 5 As shown, although the purity of the finished product is ≥99% and the maximum single impurity is ≤0.15%, the total yield is less than 50%, and a large amount of methyl tert-butyl ether is used in the process, the operation risk factor is high, and the environmental pollution is serious.

[0043] Comparative Example 3:

[0044] Prepare 2000mL of cleavage reagent, mix according to the volume ratio of TFA: EDT: Tis: water = 85: 2.5: 2.5: 10, and pre-cool to -20℃. Weigh 240.0g of Tirzepatide peptide resin, add it to the cleavage reagent, and stir at 25℃ for 3h. Filter and collect the lysate, wash the Tirzepatide peptide resin after the reaction with 200mL TFA, add the filtrate to 16L of methyl tert-butyl ether for precipitation, and then wash the precipitate with methyl tert-butyl ether 3 times, centrifuge to obtain the precipitate, and dry the off-white powder, which is the crude Tirzepatide. The crude product is dissolved in dilute ammonia water to obtain a clear solution, filtered with a 0.45μm filter membrane, and the filtrate is collected. The filtrate was purified by reverse phase chromatography and converted to ammonium salt, and lyophilized to obtain 52 g of Tirzepatide, with a total yield of 45.0%, a purity of 99.19%, and a maximum single impurity of 0.12%. The test results of the Tirzepatide sample prepared in Comparative Example 3 are as follows: Figure 6 As shown, although the purity of the finished product is ≥99% and the maximum single impurity is ≤0.15%, the total yield is less than 50%, and a large amount of methyl tert-butyl ether is used in the process, the operation risk factor is high, and the environmental pollution is serious.

[0045] Those skilled in the art will readily appreciate other embodiments of the invention after considering the specification and practicing the invention herein. The invention is intended to cover any variations, uses or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art of the invention. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the claims.

[0046] It should be understood that the present invention is not limited to the exact construction that has been described above and shown in the drawings and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.

Claims

1. A method for preparing Tirzepatide, characterized in that: The following steps are involved: (1) Prepare the cleavage reagent and pre-cool it, then add the Tirzepatide peptide resin and stir to react; (2) collecting the lysate in (1) by filtration and diluting it with water to form an aqueous solution, then adding an organic solvent to perform extraction and separation, and collecting the aqueous phase; (3) adjusting the pH of the aqueous phase, allowing the aqueous phase to stand and collecting the precipitated precipitate, re-dissolving and adjusting the pH of the precipitate, filtering, reverse-phase purifying the filtrate, and freeze-drying the qualified fraction to obtain the Tirzepatide.

2. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (1), the cleavage reagent is composed of trifluoroacetic acid, 1,2-ethanedithiol, triisopropylsilane and water in a volume ratio of (70-95)%: (0.5-10)%: (0.5-10)%: (0.5-10)%.

3. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (1), the precooling temperature is -25 to 5°C, preferably -20 to -10°C.

4. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (1), the reaction time is 1 to 5 hours, preferably 2 to 3 hours; the reaction temperature is 10 to 50°C, preferably 25 to 35°C.

5. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (1), the usage ratio of the Tirzepatide peptide resin to the cleavage reagent is 1 g: (8-20) mL, preferably 1 g: (8-12) mL.

6. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (2), the ratio of the lysate to water is 1 L: (10-1000) L, preferably 1 L: (50-100) L.

7. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (2), the organic solvent is selected from any one of dichloromethane and ethyl acetate or a mixture of the two in any proportion.

8. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (2), the ratio of the aqueous solution to the organic solvent is 1L:(0.1-100)L, preferably 1L:(0.5-10)L.

9. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (3), the alkali solution for adjusting the pH of the aqueous phase is selected from at least one of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, and ammonia water; Preferably, the pH of the aqueous phase is adjusted to 3.0 to 6.0, preferably 4.0 to 5.

0.

10. The method for preparing Tirzepatide according to claim 1, characterized in that: In step (3), the alkaline solution for adjusting the pH of the precipitate is selected from at least one of a sodium hydroxide aqueous solution, a potassium hydroxide aqueous solution, and ammonia water; Preferably, the pH of the precipitate is adjusted to 6.5 to 9.5, preferably 7.5 to 8.5.

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