Preparation method of Pegcetacoplan

Abstract

The present invention relates to the technical field of drug synthesis, and in particular to a method for preparing Pegcetacoplan. The method for preparing Pegcetacoplan comprises the following steps: a Pegcetacoplan intermediate cyclic peptide and a PEG derivative are subjected to a coupling reaction in a solvent under the action of a base to obtain the Pegcetacoplan. The present invention couples the Pegcetacoplan intermediate cyclic peptide with a specific PEG derivative, and explores the coupling reaction conditions under the system, thereby improving the coupling efficiency, reducing side reactions, and improving the yield, making it more suitable for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of drug synthesis, and in particular to a method for preparing Pegcetacoplan. Background Art

[0002] Pegcetacoplan is a C3 complement inhibitor used to treat paroxysmal nocturnal hemoglobinuria (PNH) and retinal geographic atrophy. In May 2021, Pegcetacoplan was approved by the FDA for the treatment of adult PNH and PNH patients previously treated with C5 inhibitors. In December of the same year, the drug was approved by the European Union for the treatment of adult PNH patients with anemia after receiving C5 inhibitors for at least 3 months, under the trade name "Aspaveli". In December 2023, the drug was approved by the FDA for the treatment of dry age-related macular degeneration (AMD) and macular geographic atrophy (GA).

[0003] Pegcetacoplan is a compound consisting of a cyclic peptide consisting of 15 amino acids and a macromolecular polyethylene glycol compound PEG40K coupled thereto; the cyclic peptide has the following structure:

[0004]

[0005] The existing method for preparing Pegcetacoplan is not only high in cost but also low in yield.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a method for preparing Pegcetacoplan, which improves the yield while reducing the cost and better meets the needs of mass production.

[0008] To achieve the above-mentioned object of the present invention, the present invention provides a method for preparing Pegcetacoplan, comprising the following steps:

[0009] The intermediate cyclic peptide of Pegcetacoplan and the PEG derivative are subjected to a coupling reaction in a solvent under the action of a base to obtain the Pegcetacoplan;

[0010] The structural formulas of the Pegcetacoplan intermediate cyclic peptide and the PEG derivative are shown in Formula I and Formula II, respectively:

[0011] (I);

[0012] (II);

[0013] Here, n is an integer between 800 and 900.

[0014] In a specific embodiment of the present invention, the solvent comprises at least one of acetonitrile, DMF, 1,4-dioxane and THF. Further, the solvent is acetonitrile.

[0015] In a particular embodiment of the invention, the base comprises DIEA.

[0016] In a specific embodiment of the present invention, the molar ratio of the base to the Pegcetacoplan intermediate cyclic peptide is (20-30):1.

[0017] In a specific embodiment of the present invention, the molar ratio of the Pegcetacoplan intermediate cyclic peptide to the PEG derivative is (2-3) : 1, preferably (2.3-2.5) : 1.

[0018] In a specific embodiment of the present invention, the temperature of the coupling reaction is 20-35°C.

[0019] In a specific embodiment of the present invention, the coupling reaction time is 1 to 5 hours, preferably 1 to 3 hours.

[0020] In a specific embodiment of the present invention, the Pegcetacoplan intermediate cyclic peptide is dissolved in a solvent, and the base is added and mixed evenly to obtain a first solution; the PEG derivative is dissolved in a solvent to obtain a second solution; and the second solution is added to the first solution to perform a coupling reaction.

[0021] In a specific embodiment of the present invention, in the first solution, the concentration of the Pegcetacoplan intermediate cyclic peptide is 4.0 to 7.0 mg / mL.

[0022] In a specific embodiment of the present invention, in the second solution, the concentration of the PEG derivative is 30-40 mg / mL.

[0023] In a specific embodiment of the present invention, the second solution is added to the first solution at a rate of 15 to 25 mL / min.

[0024] In a specific embodiment of the present invention, the Pegcetacoplan intermediate cyclic peptide is prepared by a solid phase synthesis method.

[0025] In a specific embodiment of the present invention, the preparation method further comprises: adding the material after the coupling reaction into a MTBE solution for sedimentation and filtration, then slurrying and washing with MTBE, and then drying.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention couples the intermediate cyclic peptide of Pegcetacoplan with a specific PEG derivative and explores the coupling reaction conditions under the system, which not only improves the coupling efficiency, but also reduces side reactions and improves the yield;

[0028] (2) The material obtained by the coupling reaction of the present invention reduces side reactions, reduces the difficulty of subsequent purification, and is more suitable for large-scale production. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention will be clearly and completely described below in conjunction with specific embodiments, but it will be appreciated by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work, all belong to the scope of protection of the present invention. If specific conditions are not indicated in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not indicated in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] The Chinese meanings of some English abbreviations involved in the present invention are shown in Table 1.

[0031] Table 1 Meaning of English abbreviations

[0032]

[0033] PEG modification of proteins and peptides can improve drug stability, increase drug solubility, reduce drug immunogenicity, enhance resistance to enzymatic hydrolysis, prolong drug half-life, and improve in vivo pharmacokinetic properties. PEG modification of the intermediate cyclic peptide of Pegcetacoplan is also an effective way to solve its application problems.

[0034] In the preparation of existing Pegcetacoplan, the coupling of the intermediate cyclic peptide of Pegcetacoplan with PEG usually uses a solid phase step-by-step coupling method, which is costly; if the intermediate cyclic peptide purified solution is used for direct coupling, the PEG activated substance will be inactivated when it encounters water, which will also lead to a decrease in yield. In order to avoid the introduction of water, the intermediate cyclic peptide can be reacted with PEG in an organic solvent. However, the inventors of the present invention have found that the conventional condensation system may not only cause cyclization between the Asp and Lys side chains in the intermediate cyclic peptide, greatly reducing the utilization rate of the intermediate cyclic peptide, but also increase the by-products, resulting in an increase in the difficulty of separation and purification; in addition, in such a reaction system, the disubstituted product is reduced, and the presence of the monosubstituted substance further increases the difficulty of separation and purification. The present invention is aimed at the structural characteristics of Pegcetacoplan, and adopts a specific reaction system for coupling reaction to reduce side reactions and ensure coupling efficiency and improve yield.

[0035] Based on this, the present invention provides a method for preparing Pegcetacoplan, comprising the following steps:

[0036] The intermediate cyclic peptide of Pegcetacoplan and the PEG derivative are subjected to coupling reaction in a solvent under the action of a base to obtain Pegcetacoplan;

[0037] The structural formulas of the intermediate cyclic peptide and PEG derivative of Pegcetacoplan are shown in Formula I and Formula II, respectively:

[0038] (I);

[0039] (II);

[0040] Here, n is an integer between 800 and 900.

[0041] Through research, it was found that the PEG derivative shown in Formula II was used for liquid phase coupling with the Pegcetacoplan intermediate cyclic peptide. The PEG derivative reacted chemically with the amino group in the Pegcetacoplan intermediate cyclic peptide through its activated group to form an amide bond, which not only reduced side reactions and the difficulty of product purification, but also ensured or even improved the coupling efficiency and increased the yield.

[0042] In a specific embodiment of the present invention, the solvent includes at least one of acetonitrile, DMF, 1,4-dioxane and THF. Further, the solvent is acetonitrile.

[0043] Based on the reaction system of Pegcetacoplan intermediate cyclic peptide and specific PEG derivative, the use of the above-mentioned solvent can take into account both coupling efficiency and product yield and reduce side reactions. In particular, when acetonitrile is used as a solvent, it can not only ensure high coupling efficiency but also significantly improve product yield.

[0044] In a particular embodiment of the invention, the base comprises DIEA.

[0045] The present invention adopts DIEA as a base to catalyze the coupling reaction, which can ensure the coupling reaction and significantly avoid other side reactions, thereby improving the product yield.

[0046] In a specific embodiment of the present invention, the molar ratio of the base to the Pegcetacoplan intermediate cyclic peptide is (20-30):1.

[0047] Regulating the molar ratio of the base to the intermediate cyclic peptide of Pegcetacoplan within the above range is more helpful to ensure the catalytic activity and inhibit the occurrence of side reactions. For example, in different embodiments, the molar ratio of the base to the intermediate cyclic peptide of Pegcetacoplan can be 20:1, 22:1, 25:1, 28:1, 30:1 or any two thereof.

[0048] In a specific embodiment of the present invention, the molar ratio of the Pegcetacoplan intermediate cyclic peptide to the PEG derivative is (2-3) : 1, preferably (2.3-2.5) : 1.

[0049] By regulating the molar ratio of the intermediate cyclic peptide of Pegcetacoplan to the PEG derivative within the above range, in the coupling system of the present invention, the formation of the disubstituted product can be further ensured, while avoiding the waste of raw materials caused by excessive intermediate cyclic peptide of Pegcetacoplan. For example, in different embodiments, the molar ratio of the intermediate cyclic peptide of Pegcetacoplan to the PEG derivative can be 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.8:1, 2.9:1, 3:1 or a range consisting of any two of them, especially when the molar ratio of the intermediate cyclic peptide of Pegcetacoplan to the PEG derivative is 2.4:1, in the reaction system of the present invention, the coupling efficiency is the highest; and when the intermediate cyclic peptide of Pegcetacoplan is further increased, there is no obvious effect on the improvement of the yield of the Pegcetacoplan product.

[0050] In a specific embodiment of the present invention, the temperature of the coupling reaction is 20-35°C.

[0051] The coupling reaction in the system of the present invention has good tolerance to temperature, and can achieve good coupling efficiency in the range of 20 to 35°C, which can be adjusted according to actual needs in actual operation. For example, in different embodiments, the temperature of the coupling reaction can be 20°C, 22°C, 25°C, 28°C, 30°C, 32°C, 35°C, or a range consisting of any two of them.

[0052] In a specific embodiment of the present invention, the coupling reaction time is 1 to 5 hours, preferably 1 to 3 hours.

[0053] For example, in different embodiments, the coupling reaction time can be 1 h, 2 h, 3 h, 4 h, 5 h, or a range consisting of any two of them. The coupling reaction in the system of the present invention is highly efficient and can complete the reaction within 1 h.

[0054] In a specific embodiment of the present invention, the intermediate cyclic peptide of Pegcetacoplan is dissolved in a solvent, and a base is added and mixed uniformly to obtain a first solution; a PEG derivative is dissolved in a solvent to obtain a second solution; and the second solution is added to the first solution to perform a coupling reaction.

[0055] In a specific embodiment of the present invention, in the first solution, the concentration of the intermediate cyclic peptide of Pegcetacoplan is 4.0-7.0 mg / mL.

[0056] For example, in different embodiments, in the first solution, the concentration of the Pegcetacoplan intermediate cyclic peptide can be 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL or a range consisting of any two thereof.

[0057] In a specific embodiment of the present invention, in the second solution, the concentration of the PEG derivative is 30-40 mg / mL.

[0058] For example, in different embodiments, in the second solution, the concentration of the PEG derivative can be 30 mg / mL, 32 mg / mL, 35 mg / mL, 38 mg / mL, 40 mg / mL, or a range consisting of any two thereof.

[0059] In a specific embodiment of the present invention, the second solution is added to the first solution at a rate of 15 to 25 mL / min.

[0060] For example, in different embodiments, the rate of adding the second solution to the first solution can be 15 mL / min, 18 mL / min, 20 mL / min, 22 mL / min, 25 mL / min, or a range consisting of any two thereof.

[0061] Regulating the concentration of the Pegcetacoplan intermediate cyclic peptide and the PEG derivative in the first solution and / or the second solution and the addition rate of the second solution within the above range helps to ensure or even improve the yield of the product while improving the efficiency of the coupling reaction.

[0062] In a specific embodiment of the present invention, the intermediate cyclic peptide of Pegcetacoplan is prepared by a solid phase synthesis method. The intermediate cyclic peptide of Pegcetacoplan can be prepared by a conventional solid phase synthesis method, which will not be described in detail herein. In addition, the present invention does not limit the synthesis method of the intermediate cyclic peptide of Pegcetacoplan, and the intermediate cyclic peptide of Pegcetacoplan prepared by any method is applicable to the coupling method of the present invention.

[0063] In a specific embodiment of the present invention, the preparation method further comprises: adding the material after the coupling reaction to an MTBE solution for sedimentation and filtration, and then using MTBE for pulping and washing, and then performing a drying treatment. The volume of MTBE used for sedimentation and filtration can be 6 to 10 times the total volume of the material after the coupling reaction; the volume of MTBE used for pulping and washing can be 4 to 6 times the total volume of the material after the coupling reaction. After the drying treatment, conventional preparation-grade HPLC purification can be performed.

[0064] The molecular weight of the PEG derivative (SC-PEG40K-SC) shown in Formula II used in the following specific examples is 40 KDa; the intermediate cyclic peptide of Pegcetacoplan is prepared by conventional solid phase synthesis method with a purity of ≥99%.

[0065] Example 1

[0066] This embodiment provides a method for preparing Pegcetacoplan, comprising the following steps:

[0067] (1) Weigh 11.41 g of the intermediate cyclic peptide of Pegcetacoplan into 2000 mL of acetonitrile, sonicate to fully dissolve it, then add 13.1 mL of DIEA and mix well to obtain a first solution;

[0068] (2) Weigh 100 g of the PEG derivative shown in formula II, add it into 3000 mL of acetonitrile, and sonicate to fully dissolve it to obtain a second solution;

[0069] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 20 °C for 1 h.

[0070] (4) The coupled material is added to MTBE for sedimentation and filtration, and then washed twice with MTBE by slurrying, and then dried in a vacuum drying oven, and purified by conventional preparative HPLC. The volume of MTBE used for sedimentation is 8 times the total volume of the coupled material, and the volume of MTBE used for slurrying and washing is 5 times the total volume of the coupled material.

[0071] Example 2

[0072] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0073] In step (1) and step (2) of this example, equal volumes of DMF are used to replace acetonitrile in step (1) and step (2) of example 1, respectively.

[0074] Example 3

[0075] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0076] In step (1) and step (2) of this example, acetonitrile in step (1) and step (2) of example 1 is replaced by equal volumes of 1,4-dioxane.

[0077] Example 4

[0078] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0079] In step (1) and step (2) of this example, equal volumes of THF are used to replace acetonitrile in step (1) and step (2) of example 1, respectively.

[0080] Example 5

[0081] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0082] In step (1) and step (2) of this embodiment, acetonitrile in step (1) and step (2) of embodiment 1 is replaced by equal volumes of PBS buffer with a pH of 7.0.

[0083] Example 6

[0084] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0085] In step (1) and step (2) of this embodiment, the acetonitrile in step (1) and step (2) of embodiment 1 is replaced by equal volumes of purified water, respectively.

[0086] Example 7

[0087] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that different solvents are used in step (1) and step (2).

[0088] In step (1) and step (2) of this embodiment, equal volumes of methanol are used to replace acetonitrile in step (1) and step (2) of embodiment 1, respectively.

[0089] The materials after the reaction in step (3) in the preparation methods of Examples 1 to 7 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 2.

[0090] Table 2 HPLC test results of the materials after the reaction in different embodiments

[0091]

[0092] From the above test results, it can be seen that when acetonitrile is used as a solvent, the effect is the best, followed by DMF, 1,4-dioxane and THF. When purified water or PBS buffer is used as a solvent, the PEG derivative shown in Formula II will be hydrolyzed and lose its activity, resulting in a decrease in product yield. When MeOH is used as a solvent, the PEG derivative shown in Formula II will undergo an ester exchange reaction, causing the terminal group to become a methyl group and lose its activity in reacting with the Pegcetacoplan intermediate cyclic peptide, resulting in a decrease in product yield.

[0093] Example 8

[0094] This embodiment provides a method for preparing Pegcetacoplan, comprising the following steps:

[0095] (1) Weigh 9.50 g of the intermediate cyclic peptide of Pegcetacoplan into 1665 mL of acetonitrile, sonicate to fully dissolve it, then add 13.1 mL of DIEA and mix well to obtain a first solution;

[0096] (2) Weigh 100 g of the PEG derivative shown in formula II, add it into 3000 mL of acetonitrile, and sonicate to fully dissolve it to obtain a second solution;

[0097] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 25°C for 1 h.

[0098] (4) The coupled material is added to MTBE for sedimentation and filtration, and then washed twice with MTBE by beating and slurrying, and then transferred to a vacuum drying oven for drying and purification. The volume of MTBE used for sedimentation is 8 times the total volume of the coupled material, and the volume of MTBE used for beating and washing is 5 times the total volume of the coupled material.

[0099] Example 9

[0100] This embodiment provides a method for preparing Pegcetacoplan, comprising the following steps:

[0101] (1) Weigh 10.45 g of the intermediate cyclic peptide of Pegcetacoplan into 1832 mL of acetonitrile, sonicate to fully dissolve it, then add 13.1 mL of DIEA and mix well to obtain a first solution;

[0102] (2) Weigh 100 g of the PEG derivative shown in formula II, add it into 3000 mL of acetonitrile, and sonicate to fully dissolve it to obtain a second solution;

[0103] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 25°C for 1 h.

[0104] (4) The coupled material is added to MTBE for sedimentation and filtration, and then washed twice with MTBE by beating and slurrying, and then transferred to a vacuum drying oven for drying and purification. The volume of MTBE used for sedimentation is 8 times the total volume of the coupled material, and the volume of MTBE used for beating and washing is 5 times the total volume of the coupled material.

[0105] Example 10

[0106] This embodiment provides a method for preparing Pegcetacoplan, comprising the following steps:

[0107] (1) Weigh 12.34 g of the intermediate cyclic peptide of Pegcetacoplan into 2163 mL of acetonitrile, sonicate to fully dissolve it, then add 13.1 mL of DIEA and mix well to obtain a first solution;

[0108] (2) Weigh 100 g of the PEG derivative shown in formula II, add it into 3000 mL of acetonitrile, and sonicate to fully dissolve it to obtain a second solution;

[0109] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 25°C for 1 h.

[0110] (4) The coupled material is added to MTBE for sedimentation and filtration, and then washed twice with MTBE by beating and slurrying, and then transferred to a vacuum drying oven for drying and purification. The volume of MTBE used for sedimentation is 8 times the total volume of the coupled material, and the volume of MTBE used for beating and washing is 5 times the total volume of the coupled material.

[0111] Embodiment 11

[0112] This embodiment provides a method for preparing Pegcetacoplan, comprising the following steps:

[0113] (1) Weigh 14.25 g of the intermediate cyclic peptide of Pegcetacoplan into 2498 mL of acetonitrile, sonicate to fully dissolve it, then add 13.1 mL of DIEA and mix well to obtain a first solution;

[0114] (2) Weigh 100 g of the PEG derivative shown in formula II, add it into 3000 mL of acetonitrile, and sonicate to fully dissolve it to obtain a second solution;

[0115] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 25°C for 1 h.

[0116] (4) The coupled material is added to MTBE for sedimentation and filtration, and then washed twice with MTBE by beating and slurrying, and then transferred to a vacuum drying oven for drying and purification. The volume of MTBE used for sedimentation is 8 times the total volume of the coupled material, and the volume of MTBE used for beating and washing is 5 times the total volume of the coupled material.

[0117] The materials after the reaction in step (3) in the preparation methods of Examples 1 and 8 to 11 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 3.

[0118] Table 3 HPLC test results of the materials after the reaction in different embodiments

[0119]

[0120] From the above test results, it can be seen that when the reaction equivalent of the intermediate cyclic peptide of Pegcetacoplan is 2.4 equivalents, the reaction coupling efficiency is optimal, and further increasing the equivalent of the intermediate cyclic peptide of Pegcetacoplan has no obvious effect on the formation of Pegcetacoplan. Therefore, controlling the reaction equivalent of the intermediate cyclic peptide of Pegcetacoplan to 2.4 equivalents can not only ensure the reaction efficiency and product yield, but also reduce the material cost.

[0121] Example 12

[0122] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that the reaction temperature in step (3) is different.

[0123] In step (3) of this embodiment, the reaction temperature is 25°C.

[0124] Example 13

[0125] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that the reaction temperature in step (3) is different.

[0126] In step (3) of this embodiment, the reaction temperature is 30°C.

[0127] Embodiment 14

[0128] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that the reaction temperature in step (3) is different.

[0129] In step (3) of this embodiment, the reaction temperature is 35°C.

[0130] The materials after the reaction in step (3) in the preparation methods of Example 1 and Examples 12 to 14 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 4.

[0131] Table 4 HPLC test results of the materials after the reaction in different embodiments

[0132]

[0133] It can be seen from the above test results that the reaction of the present invention has good tolerance to the reaction temperature.

[0134] Embodiment 15

[0135] This example refers to the preparation method of Pegcetacoplan in Example 1, with the only difference being that the reaction time in step (3) is different.

[0136] In step (3) of this embodiment, the reaction time is 2 h.

[0137] Example 16

[0138] This example refers to the preparation method of Pegcetacoplan in Example 1, with the only difference being that the reaction time in step (3) is different.

[0139] In step (3) of this embodiment, the reaction time is 3 hours.

[0140] Embodiment 17

[0141] This example refers to the preparation method of Pegcetacoplan in Example 1, with the only difference being that the reaction time in step (3) is different.

[0142] In step (3) of this embodiment, the reaction time is 5 hours.

[0143] The materials after the reaction in step (3) in the preparation methods of Examples 1 and 15 to 17 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 5.

[0144] Table 5 HPLC test results of the materials after the reaction in different embodiments

[0145]

[0146] It can be seen from the above test results that the coupling reaction system of the present invention has a high coupling reaction efficiency and can complete the reaction within 1 hour.

[0147] Embodiment 18

[0148] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the amount of DIEA used in step (1) is different.

[0149] In step (1) of this example, the amount of DIEA used is 17.4 mL.

[0150] Embodiment 19

[0151] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the amount of DIEA used in step (1) is different.

[0152] In step (1) of this example, the amount of DIEA used is 8.6 mL.

[0153] Embodiment 20

[0154] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the amount of DIEA used in step (1) is different.

[0155] In step (1) of this example, the amount of DIEA used is 4.3 mL.

[0156] The materials after the reaction in step (3) in the preparation methods of Examples 1 and 18 to 20 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 6.

[0157] Table 6 HPLC test results of the materials after the reaction in different embodiments

[0158]

[0159] From the above test results, it can be seen that in the coupling reaction system of the present invention, the base has a certain influence on the reaction. If the equivalent is insufficient, a suitable alkaline environment cannot be provided, resulting in the inability to completely carry out the reaction; if the equivalent is too much, the degradation or racemization of the cyclic peptide of Pegcetacoplan will be caused, resulting in a decrease in the utilization rate of raw materials. Therefore, the present invention further regulates the molar ratio of the base to the intermediate cyclic peptide of Pegcetacoplan to (20-30): 1, which is more conducive to taking into account the degree of reaction, avoiding the degradation or racemization of the raw materials, improving the conversion rate and ensuring the utilization rate of the raw materials.

[0160] Embodiment 21

[0161] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the type of base in step (1) is different.

[0162] In step (1) of this example, an equal molar amount of NMM is used to replace DIEA in step (1) of Example 1.

[0163] Embodiment 22

[0164] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the type of base in step (1) is different.

[0165] In step (1) of this example, an equal molar amount of TEA is used to replace DIEA in step (1) of Example 1.

[0166] Embodiment 23

[0167] This example is based on the preparation method of Pegcetacoplan of Example 1, except that the type of base in step (1) is different.

[0168] In step (1) of this example, an equal molar amount of NaHCO3 (added in solid form) is used to replace DIEA in step (1) of Example 1.

[0169] The materials after the reaction in step (3) in the preparation methods of Examples 1 and Examples 21 to 23 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 7.

[0170] Table 7 HPLC test results of the materials after the reaction in different embodiments

[0171]

[0172] From the above test results, it can be seen that in the coupling reaction system of the present invention, when DIEA, NMM, and TEA organic bases are used, a more suitable alkaline environment can be provided to ensure the coupling reaction. In particular, when DIEA is used, the raw material conversion rate and product yield in the coupling reaction are better.

[0173] Embodiment 24

[0174] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (1), the amount of acetonitrile used is different.

[0175] In step (1) of this embodiment, the amount of acetonitrile used is 3800 mL.

[0176] Embodiment 25

[0177] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (1), the amount of acetonitrile used is different.

[0178] In step (1) of this embodiment, the amount of acetonitrile used is 2850 mL.

[0179] Embodiment 26

[0180] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (1), the amount of acetonitrile used is different.

[0181] In step (1) of this embodiment, the amount of acetonitrile used is 1630 mL.

[0182] Embodiment 27

[0183] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (1), the amount of acetonitrile used is different.

[0184] In step (1) of this embodiment, the amount of acetonitrile used is 1270 mL.

[0185] The materials after the reaction in step (3) in the preparation methods of Examples 1 and Examples 24 to 27 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 8.

[0186] Table 8 HPLC test results of the materials after the reaction in different embodiments

[0187]

[0188] From the above test results, it can be seen that regulating the concentration of the Pegcetacoplan intermediate cyclic peptide in the first solution within the range of 4 to 7 mg / mL can ensure the smooth progress of the coupling reaction; when the concentration is too low, the reaction time needs to be extended to ensure the completion of the reaction, and when the concentration is too high, the cyclic peptide is difficult to dissolve and the reaction efficiency cannot be further improved.

[0189] Embodiment 28

[0190] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (2), the amount of acetonitrile used is different.

[0191] In step (2) of this embodiment, the amount of acetonitrile used is 5000 mL.

[0192] Implementation 29

[0193] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (2), the amount of acetonitrile used is different.

[0194] In step (2) of this embodiment, the amount of acetonitrile used is 3333 mL.

[0195] Embodiment 30

[0196] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (2), the amount of acetonitrile used is different.

[0197] In step (2) of this embodiment, the amount of acetonitrile used is 2500 mL.

[0198] Embodiment 31

[0199] This example is based on the preparation method of Pegcetacoplan of Example 1, with the only difference being that in step (2), the amount of acetonitrile used is different.

[0200] In step (2) of this embodiment, the amount of acetonitrile used is 2000 mL.

[0201] The materials after the reaction in step (3) in the preparation methods of Examples 1 and Examples 28 to 31 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 9.

[0202] Table 9 HPLC test results of the materials after the reaction in different embodiments

[0203]

[0204] From the above test results, it can be seen that by regulating the concentration of PEG in the second solution within the range of 30 to 40 mg / mL, coupling can be performed at a better coupling rate.

[0205] Embodiment 32

[0206] This example refers to the method for preparing Pegcetacoplan in Example 1, with the only difference being that in step (3), the speed of adding the second solution is different.

[0207] In step (3) of this embodiment, the second solution is added at a rate of 5 mL / min.

[0208] Implementation 33

[0209] This example refers to the method for preparing Pegcetacoplan in Example 1, with the only difference being that in step (3), the speed of adding the second solution is different.

[0210] In step (3) of this embodiment, the second solution is added at a rate of 15 mL / min.

[0211] Embodiment 34

[0212] This example refers to the method for preparing Pegcetacoplan in Example 1, with the only difference being that in step (3), the speed of adding the second solution is different.

[0213] In step (3) of this embodiment, the addition rate of the second solution is 25 mL / min.

[0214] Embodiment 35

[0215] This example refers to the method for preparing Pegcetacoplan in Example 1, with the only difference being that in step (3), the speed of adding the second solution is different.

[0216] In step (3) of this embodiment, the addition rate of the second solution is 35 mL / min.

[0217] The materials after the reaction in step (3) in the preparation methods of Example 1 and Examples 32 to 35 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 10.

[0218] Table 10 HPLC test results of the materials after the reaction in different embodiments

[0219]

[0220] Comparative Example 1

[0221] Comparative Example 1 provides a method for preparing Pegcetacoplan, comprising the following steps:

[0222] (1) Weigh 11.41 g of the intermediate cyclic peptide of Pegcetacoplan into 2000 mL of acetonitrile, sonicate to fully dissolve it, then add 3 mL of DIEA and mix well to obtain a first solution;

[0223] (2) Weigh 100 g of COOH-PEG40K-COOH and add it to 3000 mL of acetonitrile. Sonicate to fully dissolve it. Then add 2.60 g of PyBop and sonicate to dissolve it to obtain a second solution.

[0224] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 20 °C for 1 h.

[0225] Comparative Example 2

[0226] Comparative Example 2 provides a method for preparing Pegcetacoplan, comprising the following steps:

[0227] (1) Weigh 11.41 g of the intermediate cyclic peptide of Pegcetacoplan into 2000 mL of acetonitrile and dissolve it completely by ultrasonication to obtain a first solution;

[0228] (2) Weigh 100 g of COOH-PEG40K-COOH and add it to 3000 mL of acetonitrile, add 0.68 g of HOBt, cool on ice for 5 min, and then add 0.77 mL of DIC to activate for 15 min to obtain the second solution;

[0229] (3) Add the second solution into the first solution at a rate of 20 mL / min. After the addition is complete, transfer to a shaker and react at 20 °C for 1 h.

[0230] Comparative Example 3

[0231] Comparative Example 3 refers to the preparation method of Pegcetacoplan in Example 1, except that the type of PEG in step (2) is different.

[0232] The PEG derivative used in Comparative Example 3 is: CDI-PEG-CDI, the PEG molecular weight is 40K, and the structural formula is as follows:

[0233] .

[0234] Among them, CDI-PEG-CDI can be purchased or prepared by oneself. If prepared by oneself, it can be prepared by reacting N, N-carbonyldiimidazole with COOH-PEG40K-COOH.

[0235] The materials after the reaction in step (3) in the preparation methods of Example 1 and Comparative Examples 1 to 3 were respectively subjected to HPLC detection to record the peak areas of the Pegcetacoplan intermediate cyclic peptide and Pegcetacoplan. The results are shown in Table 11.

[0236] Table 11 HPLC test results of the materials after the reaction of different embodiments and comparative examples

[0237]

[0238] From the above test results, it can be seen that when the PyBop system is used, the activity of PyBop is too high, and it forms a competitive relationship with the COOH of the Asp amino acid side chain of the intermediate cyclic peptide, thereby reducing the utilization rate of the intermediate cyclic peptide. At the same time, it is easy to cause single substitution, and the expected double substitution product cannot be obtained, resulting in greater difficulty in subsequent separation and purification. When the DIC and HOBT systems are used, the method of pre-activation followed by coupling is adopted. Although it can reduce the occurrence of self-cyclization, it is easy to produce single substitution, and the subsequent separation and purification is difficult. When the PEG structure used is CDI-PEG-CDI, since the CDI ester is very active and easily reacts with water to lose its activity, the reaction system needs to strictly ensure an anhydrous environment, which greatly increases the production cost; the reaction system without dehydration has low raw material utilization.

[0239] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing Pegcetacoplan, characterized in that: The method comprises the following steps: dissolving the intermediate cyclic peptide of Pegcetacoplan in a solvent, adding a base and mixing evenly to obtain a first solution; dissolving a PEG derivative in a solvent to obtain a second solution; adding the second solution to the first solution to perform a coupling reaction to obtain the Pegcetacoplan; The structural formulas of the Pegcetacoplan intermediate cyclic peptide and the PEG derivative are shown in Formula I and Formula II, respectively: （Ⅰ）； （Ⅱ）； Wherein, n is an integer between 800 and 900; The solvent is acetonitrile; The base is selected from one of DIEA, NMM and TEA; the molar ratio of the base to the Pegcetacoplan intermediate cyclic peptide is (20-30):1; The molar ratio of the Pegcetacoplan intermediate cyclic peptide to the PEG derivative is (2-3):1; The temperature of the coupling reaction is 20-35°C; The coupling reaction time is 1 to 5 hours; The second solution is added to the first solution at a speed of 15 to 25 mL / min.

2. The method for preparing Pegcetacoplan according to claim 1, characterized in that: The base is DIEA.

3. The method for preparing Pegcetacoplan according to claim 1, characterized in that: The molar ratio of the Pegcetacoplan intermediate cyclic peptide to the PEG derivative is (2.3-2.5):

1.

4. The method for preparing Pegcetacoplan according to claim 1, characterized in that: The coupling reaction time is 1 to 3 hours.

5. The method for preparing Pegcetacoplan according to claim 1, characterized in that: In the first solution, the concentration of the Pegcetacoplan intermediate cyclic peptide is 4.0 to 7.0 mg / mL.

6. The method for preparing Pegcetacoplan according to claim 1, characterized in that: In the second solution, the concentration of the PEG derivative is 30-40 mg / mL.

7. The method for preparing Pegcetacoplan according to claim 1, characterized in that: The Pegcetacoplan intermediate cyclic peptide is prepared by a solid phase synthesis method.

8. The method for preparing Pegcetacoplan according to claim 1, characterized in that: Also includes: The material after the coupling reaction is added into MTBE solution for sedimentation and filtration, and then washed with MTBE pulping and then dried.

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