Reduction-oxidation system method for preparing eptifibatide

Abstract

The invention provides a reduction-oxidation system method for preparing eptifibatide, which comprises the following steps: dissolving crude eptifibatide, and adding TCEP for reaction to obtain a crude peptide solution; performing linear peptide purification on the crude peptide solution by using reversed-phase preparative chromatography to obtain a high-purity fine peptide solution; the refined peptide solution is oxidized to the end point through an oxidizing agent, and a cyclopeptide solution is obtained; the cyclic peptide solution is subjected to purification, rotary evaporation and freeze-drying, and the eptifibatide acetate is obtained. According to the method, TCEP is added for a reduction reaction, and a conjugate and a polymer in crude peptide are reduced, so that the content of the eptifibatide is increased. And because of linear peptide purification, TCEP is prevented from hindering oxidation, the oxidation conversion rate is improved, filtration after oxidation is easy, the subsequent purification difficulty is reduced, and the yield is greatly improved. The whole process is simple to operate, short in production period and beneficial to large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a reduction-oxidation system method for preparing eptifibatide. Background Art

[0002] Eptifibatide, also known as Eptifibatide, Eptifibatide, English name: Eptifibatide, molecular formula: C 35 H 49 N ll O9S2, whose structure is Figure 1 shown.

[0003] Eptifibatide is an antiplatelet aggregation agent, developed jointly by COR Therapeutics and Schering-Plough in the United States. It was first launched in the United States by Schering-Plough in July 1998 under the trade name Intrifiban, and in Europe in 1999 under the trade name Intrifiban. Synonyms: Intrifiban TM, Sch-60936, C68-22, SB-1. Eptifibatide is a cyclic peptide, a platelet glycoprotein GPIIb / IIIa receptor antagonist, which can block platelet aggregation reactions caused by various activators.

[0004] Eptifibatide is mainly used to prevent myocardial oxygen supply artery occlusion, heart attack, unstable angina, Q-wave-free myocardial infarction, and sudden death caused by coronary intervention. It can also be used for percutaneous coronary intervention, including angioplasty or atherectomy. It is generally not used for angioplasty of inorganic coronary syndrome. Under the supervision of a dedicated person, it can be used with aspirin or heparin. Advantages of Eptifibatide: ① The blockade of platelet protein IIb / IIIa receptors is reversible. Once adverse reactions occur, the drug can be stopped immediately, and the adverse reactions are mild. ② High selectivity and strong effect. ③ It is almost non-antigenic and will not cause allergic reactions.

[0005] In the prior art, both the original research and references of eptifibatide are synthesized by solid phase synthesis, which will produce a large amount of impurities and solvents, most of which are cleavage residues and multimeric impurities such as dimers, resulting in problems such as difficulty in filtration, high difficulty in purification, and low yield. Summary of the invention

[0006] In view of this, in order to solve the above-mentioned problem, the present invention provides a reduction-oxidation system method for preparing eptifibatide.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The invention provides a reduction-oxidation system method for preparing eptifibatide, which comprises: dissolving crude eptifibatide peptide, adding TCEP [tris(2-carboxyethyl)phosphine] to react, and obtaining a crude peptide solution; performing linear peptide purification on the crude peptide solution by reverse phase preparative chromatography, and obtaining a high-purity refined peptide solution; oxidizing the refined peptide solution to an end point by an oxidant, and obtaining a cyclic peptide solution; purifying the cyclic peptide solution, rotary evaporating, and then freeze-drying, and obtaining eptifibatide acetate.

[0009] Furthermore, the method specifically comprises the following steps: (1) dissolving crude eptifibatide peptide with a dissolving solution, adding TCEP, and filtering after reaching the reaction endpoint time to obtain a crude peptide solution; (2) equilibrating a preparation column with mobile phase A and mobile phase B, and then loading the crude peptide solution onto the preparation column; (3) gradient eluting with mobile phase A and mobile phase B, collecting fractions, and obtaining a refined peptide solution; (4) diluting the refined peptide solution with a dissolving solution to obtain a solution to be oxidized of a certain concentration; (5) adjusting the pH of the solution to be oxidized with a reagent, and reacting after adding hydrogen peroxide in batches or in a single batch, and adding acetic acid to terminate the reaction to obtain an oxidized solution; (6) filtering the oxidized solution to obtain a clear colorless cyclic peptide solution, and continuing to purify, collect, vacuum rotary evaporate, and freeze-dry to obtain the eptifibatide acetate.

[0010] Furthermore, in step (1) and step (4), the dissolving solution is one or a mixture of two of dimethylsulfoxide (DMSO), acetonitrile, methanol or water.

[0011] Furthermore, in step (1), the amount of TCEP added is 0.1 to 2 equivalents of the crude eptifibatide peptide; preferably, the amount of TCEP added is 0.2 to 0.8 equivalents of the crude eptifibatide peptide.

[0012] Furthermore, in step (1), the reaction endpoint time is 0.25h to 6h; preferably, the reaction endpoint time is 1h to 3h.

[0013] Furthermore, in step (1), the filtering method is any one of diatomaceous earth, rapid qualitative filter paper, 0.22 μm or 0.45 μm filter membrane, or a combination thereof.

[0014] Furthermore, the mobile phase A is an acetate buffer; wherein the acetate in the acetate buffer is selected from any one of ammonium acetate, sodium acetate or potassium acetate, or a mixture of the above buffer salts in any proportion, or a mixture of acetic acid and ammonia water, acetic acid and sodium hydroxide aqueous solution, acetic acid and potassium hydroxide aqueous solution; the concentration of the acetate is 5mmol / L to 500mmol / L; preferably, the concentration of the acetate is 20mmol / L to 200mmol / L; the mobile phase B is acetonitrile.

[0015] Furthermore, in step (4), the concentration of the solution to be oxidized is 0.2 mg / mL to 4.0 mg / mL; preferably, the concentration of the solution to be oxidized is 0.5 mg / mL to 2.0 mg / mL.

[0016] Furthermore, in step (5), the reagent is acetic acid, ammonia water, sodium hydroxide or potassium hydroxide.

[0017] Furthermore, in step (5), the amount of hydrogen peroxide added in batches is 1 / 8 to 2 equivalents of the crude peptide amount of eptifibatide, and stirring is continued for 0.25h to 2h, and added 1 to 5 times in total; preferably, the amount of hydrogen peroxide added in batches is 1 / 8 to 1 / 2 equivalents of the crude peptide amount of eptifibatide, and stirring is continued for 0.5h to 1h, and added 2 to 4 times in total.

[0018] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0019] For crude eptifibatide line peptide solution, adding TCEP can increase the content but cannot be used directly for oxidation. The combination of adding TCEP and line peptide purification process to the line peptide solution can not only increase the line peptide content, but also solve the problems of content loss and process flow difficulties caused by filtration difficulties. The technical solution provided by the present invention is to add TCEP to the linear peptide and column purification, which can increase the total content of eptifibatide by 10% to 15%, reduce the content of by-products produced during synthesis and cleavage, and reduce the difficulty of purification. At the same time, the method has a high oxidation conversion rate and reduces the loss consequences caused by the use of strong adsorbents due to filtration difficulties, making it easy to filter before and after oxidation. The final oxidation conversion rate is above 98%, the total yield is above 50%, and the process flow is simplified. It is used to solve the filtration difficulties and excessive losses that occur after amplification during the preparation process of cyclic peptides; a certain amount of EDT-bound line peptides and polypeptide polymers will be produced during the cleavage treatment, which reduces the content of line peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] One or more embodiments are exemplarily described by pictures in the corresponding drawings, and these exemplifications do not constitute limitations on the embodiments. Unless otherwise stated, the pictures in the drawings do not constitute proportional limitations.

[0021] Figure 1 It is a structural diagram of eptifibatide;

[0022] Figure 2 It is the superimposed chromatogram before and after adding TCEP in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The inventors found that the preparation of eptifibatide can be prepared by conventional solid phase synthesis or by fragment condensation in liquid phase. Chinese patent CN 105037496A discloses a method for preparing eptifibatide, including using Fmoc-Cys (Trt) resin as a coupling resin carrier, sequentially coupling and accessing Fmoc-protected amino acids and protected amino acid tripeptide fragments; cracking the eptifibatide resin using a cleavage agent; oxidizing the reduced eptifibatide in an acetonitrile aqueous solution using an oxidant, and concentrating after oxidation; purifying the eptifibatide crude concentrate, converting salts, and freeze-drying to obtain eptifibatide fine products. Although this method is simple, the process operation after the scale-up operation is difficult and the yield is low.

[0024] First, a linear peptide resin of eptifibatide is synthesized in a solid phase, and then the linear peptide is cleaved and directly oxidized in a liquid phase to form a disulfide bond, as disclosed in patents CN 1858060 and CN101838308A. However, a large amount of dimer impurities are present in the crude peptide obtained by such methods. These dimer impurities not only cause a large reduction in the content, but also make filtration difficult and result in large losses, thus affecting subsequent purification and product yield.

[0025] Patent CN 105001304A provides a novel reduction-oxidation system for preparing eptifibatide by adding a reducing agent to remove dimer impurities during the synthesis process. Although the total yield of eptifibatide is improved, there are still cleavage residues and eptifibatide-bound byproducts after cleavage, and it is difficult to filter during subsequent purification pretreatment, resulting in large losses.

[0026] The effect of the above-mentioned patented technology is not ideal, which is manifested in that the crude product has a low content and is difficult to dissolve due to the formation of by-products by eptifibatide dimers or combination with cleavage residues. At the same time, the participation of impurities after oxidation leads to a low oxidation conversion rate and difficulty in purification.

[0027] Based on this, the present invention provides a technical solution for preparing eptifibatide using a novel reduction-oxidation system to address the problems that various impurities are easily generated during the synthesis of eptifibatide, especially the generation of dimer impurities (two linear peptides connected by a disulfide bond), which leads to high difficulty in purifying the crude peptide and low yield.

[0028] The oxidation of eptifibatide linear peptide by this system can not only greatly reduce or even completely remove the dimer impurities in the crude product, but also reduce the difficulty of pre-purification treatment, thereby reducing the difficulty of purification. As a result, the total yield of the product is improved, the process flow is simple to operate, and it is easy to scale up production.

[0029] The present invention provides a reduction-oxidation system method for preparing eptifibatide, which comprises: after dissolving crude eptifibatide peptide, adding TCEP (tris(2-chloroethyl)phosphate) to react to obtain a crude peptide solution; performing linear peptide purification on the crude peptide solution by reverse phase preparative chromatography to obtain a high-purity refined peptide solution; oxidizing the refined peptide solution to an end point by an oxidant to obtain a cyclic peptide solution; purifying the cyclic peptide solution, rotary evaporating and then freeze-drying to obtain eptifibatide acetate. The present invention reduces the conjugates and polymers in the crude peptide by adding TCEP for reduction reaction, thereby increasing the content of eptifibatide. In addition, because the linear peptide purification is performed, TCEP is avoided from hindering the oxidation, and the oxidation conversion rate is also improved, the filtration after oxidation is easy, the difficulty of subsequent purification is reduced, and the yield is greatly improved. The whole process is simple to operate, the production cycle is short, and it is conducive to large-scale industrial production.

[0030] The method specifically comprises the following steps:

[0031] (1) dissolving crude eptifibatide peptide in a dissolving solution, adding TCEP, and filtering after reaching the reaction endpoint time to obtain a crude peptide solution;

[0032] (2) After equilibrating the preparative column with mobile phase A and mobile phase B, the crude peptide solution is loaded onto the preparative column;

[0033] (3) performing gradient elution with mobile phase A and mobile phase B, collecting fractions, and obtaining a refined peptide solution;

[0034] (4) diluting the refined peptide solution with a dissolving solution to obtain a solution to be oxidized at a certain concentration;

[0035] (5) In the solution to be oxidized, the pH is adjusted with a reagent, and hydrogen peroxide is added in batches or in a single batch to react, and acetic acid is added to terminate the reaction to obtain an oxidized solution;

[0036] (6) Filtering the oxidation solution to obtain a clear colorless cyclopeptide solution, further purifying, collecting, vacuum evaporating, and freeze-drying to obtain eptifibatide acetate.

[0037] The present invention is described in detail below in conjunction with specific implementation modes.

[0038] Embodiment 1:

[0039] Take 20g of crude linear peptide of eptifibatide, of which the content of eptifibatide is 9.7g. Dissolve in 5L of 20% volume ratio acetonitrile / water, the oxidation concentration of linear peptide is 1.94g / L, add 2.0g TCEP and stir for 1h to obtain a linear peptide solution with a content of 10.8g; filter the linear peptide solution through a 0.25μm nylon filter membrane with a pump pressure of ≤0.1MPa to obtain a sample solution to be purified; phase A is 0.5% acetic acid adjusted to pH 5.5 with ammonia water, phase B is acetonitrile, and eluted with a gradient of 10% to 30% B / 40min. Wait for the sample solution to be loaded and eluted to obtain a 10.7g content linear peptide refined peptide solution. Add a certain amount of methanol and water to the above linear peptide refined peptide solution to make it 5000mL of 5% volume ratio methanol / water, and the linear peptide concentration is 2.0g / L. Add 1 / 2 equivalent of hydrogen peroxide to each barrel of peptide solution and stir continuously for 1.0h, add twice in total, and finally add acetic acid to adjust the pH to 5.0 to terminate the reaction. Finally, 10.8g of eptifibatide cyclopeptide solution was obtained, and the oxidation conversion rate (eptifibatide content in cyclopeptide solution / eptifibatide content in peptide essence) was 100.9%. The cyclopeptide solution was clear and colorless, and the sample solution to be purified was obtained after filtering through a 0.22μm filter membrane. After cyclopeptide purification, 10.5g of lyophilized product with a purity of 99.80% was finally obtained.

[0040] Embodiment 2:

[0041] Take 235g of crude linear peptide of eptifibatide, of which the content of eptifibatide is 118.4g. Dissolve it in 59L of 15% volume ratio acetonitrile / water, the oxidation concentration of linear peptide is 2.0g / L, add 16.1g of TCEP and stir to react for 2h to obtain a linear peptide solution with a content of 130.2g; filter the linear peptide solution through a 0.45μm nylon filter membrane with a pump pressure of ≤0.1MPa to obtain a sample solution to be purified; phase A is 0.1% acetic acid adjusted to pH 5.0 with ammonia water, and phase B is acetonitrile, eluted with a gradient of 10% to 50% B / 40min. The above sample solution is divided into two batches for sample loading, purification and elution to obtain a 125.6g linear peptide solution. The above peptide solution was divided into two barrels, and a certain amount of acetonitrile was added so that each barrel was 62.5L of 5% acetonitrile / water by volume, and the peptide concentration was 1.0g / L. 1 / 4 equivalent of hydrogen peroxide of the crude peptide in each barrel of peptide solution was added in batches and stirred continuously for 0.5h, added 4 times in total, and finally acetic acid was added to adjust the pH to 4.7 when the reaction was terminated. Finally, 122.93g of eptifibatide cyclic peptide solution was obtained, and the oxidation conversion rate (the content of eptifibatide in the cyclic peptide solution / the content of eptifibatide in the peptide solution) was 100.9%. The cyclic peptide solution was clear and colorless, and the sample solution to be purified was obtained after filtering with a filter through a 0.45μm filter membrane. After cyclic peptide purification, 95.2g of freeze-dried product with a purity of 99.75% was finally obtained.

[0042] Embodiment 3:

[0043] The difference from Implementation 1 is that the parameters of the purification process are shown in Table 1.

[0044] Comparative Example 1

[0045] Take 160g of crude linear peptide of eptifibatide, of which the content of eptifibatide is 79.8g. Dissolve it in 160L of 15% volume ratio acetonitrile / water to make the concentration of linear peptide 0.5g / L. Add 1 / 4 equivalent hydrogen peroxide of the barrel of crude peptide and stir continuously for 0.5h, add 4 times in total, and finally add acetic acid to adjust the pH to 4.9 to terminate the reaction. The oxidized cyclic peptide is turbid, and the oxidized cyclic peptide solution is filtered through 30 0.45μm filter membranes to obtain the sample solution to be purified; after the cyclic peptide is purified, the sample is freeze-dried, and finally 53.8g of freeze-dried product with a purity of 99.67% is obtained.

[0046] Comparative Example 2: The difference from Comparative Example 1 is that the various parameters of the purification process are shown in Table 1.

[0047] Comparative Example 3: The difference from Comparative Example 1 is that the parameters of the purification process are as shown in Table 1, and iodine is used instead of hydrogen peroxide when oxidizing the crude peptide.

[0048] Table 1 Information table of various conditions of embodiments and comparative examples

[0049]

[0050] It can be seen from Table 2 that the addition of TCEP can effectively increase the content of refined peptides, and the final yield of refined peptides obtained in Examples 1-3 is greater than that in Comparative Examples 1-3; and the amount and type of filtration membranes used in Examples 1-3 are less than those in Comparative Examples 1-3, and the pump pressure used is less than that in Comparative Examples 1-3, indicating that the filtration difficulty of Implementation 1-3 is greatly reduced relative to Comparative Examples 1-3; By comparing the oxidation conversion rates of the Examples and Comparative Examples, it can be seen that the addition of TCEP is beneficial to the subsequent oxidation process.

[0051] Application Examples

[0052] The sample HY018-20220816-LP and the sample HY018-20220816-LP+TCEP were tested by the operation of Example 1, wherein HY018-20220816-LP indicated that TCEP was not added to the batch, and the sample HY018-20220816-LP+TCEP indicated that TCEP was added to HY018-20220816-LP; the obtained data are shown in Table 2:

[0053] Table 2 Data analysis before and after adding TCEP

[0054]

[0055] Mass spectrometry analysis showed that the conjugate was a disulfide polymer formed by EDT and the thiol group of peptide and a conjugate between peptide molecules, that is, a conjugated byproduct of the cleavage residue after cleavage and eptifibatide.

[0056] Depend on Figure 2 As can be seen from Table 2, the purity and content of the peptide were significantly improved and the polymer was significantly reduced after adding TCEP.

[0057] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A reduction-oxidation system method for preparing eptifibatide, characterized in that: The method includes: After dissolving the crude eptifibatide peptide, TCEP was added to react to obtain a crude peptide solution; Purifying the crude peptide solution by reverse phase preparative chromatography to obtain a high-purity refined peptide solution; The refined peptide solution is oxidized to an end point by an oxidant to obtain a cyclic peptide solution; The cyclic peptide solution is purified, rotary evaporated and then freeze-dried to obtain eptifibatide acetate.

2. The reduction-oxidation system method for preparing eptifibatide according to claim 1, characterized in that: The method specifically comprises the following steps: (1) dissolving crude eptifibatide peptide in a dissolving solution, adding TCEP, and filtering after reaching the reaction endpoint time to obtain a crude peptide solution; (2) After equilibrating the preparative column with mobile phase A and mobile phase B, the crude peptide solution is loaded onto the preparative column; (3) performing gradient elution with mobile phase A and mobile phase B, collecting fractions, and obtaining a refined peptide solution; (4) diluting the refined peptide solution with a dissolving solution to obtain a solution to be oxidized at a certain concentration; (5) In the solution to be oxidized, the pH is adjusted with a reagent, and hydrogen peroxide is added in batches or in a single batch to react, and acetic acid is added to terminate the reaction to obtain an oxidized solution; (6) Filter the oxidation solution to obtain a clear colorless cyclic peptide solution, and continue to purify, collect, vacuum evaporate, and freeze-dry to obtain the eptifibatide acetate.

3. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (1) and step (4), the dissolving liquid is one or a mixture of two of dimethyl sulfoxide, acetonitrile, methanol or water; Preferably, the dissolving liquid is an acetonitrile aqueous solution or a methanol solution.

4. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (1), the amount of TCEP added is 0.1 to 2 equivalents of the crude eptifibatide peptide; Preferably, the amount of TCEP added is 0.2 to 0.8 equivalents of the crude eptifibatide peptide.

5. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (1), the reaction endpoint time is 0.25h to 6h; Preferably, the reaction endpoint time is 1 h to 3 h.

6. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (1), the filtering method is any one of diatomaceous earth, rapid qualitative filter paper, 0.22 μm or 0.45 μm filter membrane, or a combination thereof.

7. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: The mobile phase A is acetate buffer; Wherein, the acetate in the acetate buffer is selected from any one of ammonium acetate, sodium acetate or potassium acetate, or a mixture of the above buffer salts in any proportion, or a mixture of acetic acid and ammonia water, acetic acid and sodium hydroxide aqueous solution, acetic acid and potassium hydroxide aqueous solution; The concentration of the acetate is 5 mmol / L to 500 mmol / L; preferably, the concentration of the acetate is 20 mmol / L to 200 mmol / L; The mobile phase B is acetonitrile.

8. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (4), the concentration of the solution to be oxidized is 0.2 mg / mL to 4.0 mg / mL; Preferably, the concentration of the solution to be oxidized is 0.5 mg / mL to 2.0 mg / mL.

9. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (5), the reagent is acetic acid, ammonia water, sodium hydroxide or potassium hydroxide.

10. The reduction-oxidation system method for preparing eptifibatide according to claim 2, characterized in that: In step (5), the amount of hydrogen peroxide added in batches is 1 / 8 to 2 equivalents of the crude eptifibatide peptide, and stirring is continued for 0.25h to 2h, and added 1 to 5 times in total; Preferably, the amount of hydrogen peroxide added in batches is 1 / 8 to 1 / 2 equivalent of the crude eptifibatide peptide, and stirring is continued for 0.5h to 1h, and added 2 to 4 times in total.

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