Preparation method of Zilucoplan

Abstract

The present invention relates to the technical field of polypeptide drugs, and in particular to a method for preparing Zilucoplan. The method for preparing Zilucoplan comprises the following steps: coupling Fmoc-protected amino acid compounds to a resin from the C-terminus to the N-terminus in sequence according to the peptide sequence of Zilucoplan; in the coupling, the first Fmoc-protected amino acid compound used is Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH. The present invention provides a novel method for preparing Zilucoplan, which significantly improves the yield and purity of Zilucoplan, shortens the time of each coupling reaction, and significantly shortens the coupling time of the coupling PEG24 site, thereby greatly improving production efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and in particular to a method for preparing Zilucoplan. Background Art

[0002] Zilucoplan (ZILBRYSQ) is a small molecule complement C5 inhibitor developed by UCB Pharmaceuticals. It has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of adult patients with generalized myasthenia gravis (gMG) who are positive for anti-acetylcholine receptor (AChR) antibodies.

[0003] Zilucoplan has the following peptide sequence:

[0004] Ac-Lys 1 -Val 2 -Glu 3 -Arg 4 -Phe 5 -Asp 6 -N-Me-Asp 7 -Tle 8 -Tyr 9 -AzaTrp 10 -Glu 11 -Tyr 12 -Pro 13 -Chg 14 -Lys 15 (PEG24-γ-Glu-Pal)-OH, which forms a lactam bridge between Lys at position 1 and Asp at position 6; its molecular formula is: C 172 H 278 N 24 O 55 , the specific structural formula is as follows:

[0005]

[0006] The current preparation method of Zilucoplan is mainly solid-phase stepwise coupling synthesis. However, due to the complexity of the Zilucoplan structure, the traditional synthesis method has disadvantages such as difficult coupling and long coupling time, and it is easy to cause the loss of PEG24, resulting in low yield and purity.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] In order to solve any one or more of the above defects existing in the synthesis of Zilucoplan, the present invention provides a new method for preparing Zilucoplan.

[0009] In one aspect, the present invention provides a method for preparing Zilucoplan, comprising the following steps: coupling Fmoc-protected amino acid compounds to a resin in sequence from the C-terminus to the N-terminus according to the peptide sequence of Zilucoplan;

[0010] In the coupling, the first Fmoc-protected amino acid compound used is Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH.

[0011] In a specific embodiment of the present invention, the Fmoc-protected amino acid compounds used in the sequential coupling are: Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH, Fmoc-Chg-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-L-7-AzaTrp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tle-OH, Fmoc-N-Me-Asp(otBu)-OH, Fmoc-Asp(R1)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Val-OH and Fmoc-Lys(R2)-OH;

[0012] Wherein R1 is OAll, and R2 is selected from at least one of Alloc and Dde.

[0013] In a specific embodiment of the present invention, the preparation method comprises:

[0014] (a) coupling the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH with the resin to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin;

[0015] (b) coupling the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin with Fmoc-protected amino acid compounds in the order of amino acids from the C-terminus to the N-terminus of the Zilucoplan main chain, and then capping the terminal Fmoc to obtain a polypeptide resin I with a Lys side chain protection at position 1 and an Asp side chain protection at position 6;

[0016] (c) removing the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in the polypeptide resin I, and cyclizing to form a lactam bridge to obtain polypeptide resin II; and cleaving the polypeptide resin II to obtain the Zilucoplan.

[0017] In a specific embodiment of the present invention, the resin comprises at least one of CTC resin and Wang resin. Further, the substitution degree of the resin is 0.4-1.2 mmol / g.

[0018] In a specific embodiment of the present invention, the substitution degree of the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is 0.20 to 0.85 mmol / g.

[0019] In a specific embodiment of the present invention, in step (a), the coupling method comprises: using a solvent to swell the resin, adding the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH and DIEA to react, and then adding MeOH to seal.

[0020] In a specific embodiment of the present invention, in step (a), the coupling time is 2 to 3 hours. Furthermore, the coupling temperature is 20 to 30°C.

[0021] In a specific embodiment of the present invention, in step (a), the molar ratio of the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH to the resin is (1.5-2.5):1.

[0022] In a specific embodiment of the present invention, in step (b), each coupling step includes a deprotection reaction and a coupling reaction; the coupling reaction time is 1.5 to 2.5 hours. Furthermore, the coupling reaction temperature is 20 to 30°C.

[0023] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid compound to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-3):1.

[0024] In a specific embodiment of the present invention, when coupling Fmoc-L-7-AzaTrp-OH, the molar ratio of the Fmoc-L-7-AzaTrp-OH to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-2.5):1.

[0025] In a specific embodiment of the present invention, the coupling reaction is carried out under the action of a condensing agent; the condensing agent includes at least one of Oxyma / DIC, HOBT / DIC, PyBop / DIEA and HBTU / NMM. Further, in the coupling reaction, the solvent used includes at least one of DMF and NMP.

[0026] In a specific embodiment of the present invention, in the coupling reaction, the Fmoc-protected amino acid and the condensing agent are pre-activated at 0 to 5° C. Further, the activation treatment time is 5 to 10 minutes.

[0027] In a specific embodiment of the present invention, the deprotection reaction is carried out in a deprotection reagent; the temperature of the deprotection reaction is 20-30°C.

[0028] In a specific embodiment of the present invention, the deprotection reagent comprises an aprotic organic solvent containing an organic base, the organic base comprises piperidine, and the aprotic organic solvent comprises at least one of DCM, DMF and NMP. Further, in the deprotection reagent, the volume fraction of the organic base is 15% to 25%.

[0029] In a specific embodiment of the present invention, in step (b), the end-capping comprises: removing the terminal Fmoc with a deprotecting agent, and performing terminal acetylation with DIEA and acetic anhydride as blocking agents.

[0030] In a specific embodiment of the present invention, in step (c), a mixed removing agent is used to remove the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in the polypeptide resin I; the mixed removing agent includes palladium tetratriphenylphosphine, phenylsilane and NMM. Furthermore, the solvent for the removal reaction includes DCM.

[0031] In a specific embodiment of the present invention, in step (c), the method for cyclization to form a lactam bridge comprises: under the action of PyBop and DIEA, the Lys side chain at position 1 that has been deprotected reacts with the Asp side chain at position 6 that has been deprotected to form a lactam ring.

[0032] In a specific embodiment of the present invention, the cleavage treatment is carried out in a cleavage reagent; the cleavage reagent includes an aqueous solution containing TFA and TIS. Further, in the cleavage reagent, the volume fraction of TFA is 85% to 95%, and the volume fraction of TIS is 4% to 6%.

[0033] In a specific embodiment of the present invention, the process further comprises: collecting the solution after the cracking treatment, precipitating the solution in a poor solvent, and collecting the solid; the poor solvent comprises MTBE.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] (1) The present invention provides a novel method for preparing Zilucoplan, which significantly improves the yield and purity of Zilucoplan;

[0036] (2) In the preparation method of the present invention, the time of each coupling reaction is shortened, and the coupling time of the PEG24 site is significantly shortened, thereby greatly improving the production efficiency;

[0037] (3) In the preparation method of the present invention, the amount of Fmoc-PEG24-PA material with high traditional cost is reduced, which significantly reduces the material cost. DETAILED DESCRIPTION

[0038] 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.

[0039] The Chinese meanings of some English abbreviations involved in the present invention are shown in Table 1.

[0040] Table 1 Meaning of English abbreviations

[0041]

[0042] The existing synthesis method of Zilucoplan usually adopts a solid phase step-by-step coupling synthesis method based on the peptide sequence of Zilucoplan. Specifically, it includes: coupling Fmoc-Lys(Dde)-OH with wang resin, and completing the synthesis of the main chain by step-by-step coupling according to the peptide sequence of Zilucoplan, and then performing cyclization between specific side chains; after the cyclization is completed, the Dde protecting group on the Lys side chain is removed, and then PEG24 and other subsequent amino acids are gradually coupled. However, this synthesis method has a long time for each coupling step and low efficiency; and it requires excessive use of Fmoc-PEG24-PA, which is expensive and has high production costs; coupling is difficult, the coupling time is long, and PEG24 is prone to loss, which increases the difficulty of downstream purification and further increases production costs.

[0043] The present invention prepares the 15-position Lys of Zilucoplan and its side chain amino acid as starting materials, couples them to a resin, and then synthesizes the main chain of Zilucoplan by step-by-step coupling. On the one hand, the use of new starting materials can effectively avoid difficult coupling sites and reduce coupling steps; on the other hand, by improving the swelling property of the resin, the difficulty of subsequent coupling is reduced, the coupling time is shortened, and the reaction efficiency is greatly improved.

[0044] Based on this, the present invention provides a method for preparing Zilucoplan, comprising the following steps: coupling Fmoc-protected amino acid compounds to a resin in sequence from the C-terminus to the N-terminus according to the peptide sequence of Zilucoplan;

[0045] In the coupling, the first Fmoc-protected amino acid compound used was Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH.

[0046] Among them, the molecular formula of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH is C 97 H 170 N4O 33 , the specific structural formula is as follows:

[0047]

[0048] The present invention uses Lys at position 15 of Zilucoplan and its side chain amino acid as starting materials, adopts unconventional coupling sites to combine with resin, and can more effectively avoid the lack of PEG24, Glu and hexadecanoic acid; at the same time, the reaction step with Fmoc-PEG24-PA is omitted, on the one hand, the high-cost raw material Fmoc-PEG24-PA is avoided, and on the other hand, the reaction process between the polypeptide resin and Fmoc-PEG24-PA is avoided. Since the molecular weight of Fmoc-PEG24-PA is relatively large, the conventional coupling time with the polypeptide resin is relatively long. The synthetic route of the present invention eliminates this reaction process and significantly shortens the coupling time.

[0049] Furthermore, the present invention has found through research that when the resin is coupled with the starting material Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH of the present invention, the obtained Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin has an extended spatial structure, can be effectively swelled, reduces the possibility of resin polycondensation, greatly improves the difficulty of subsequent amino acid coupling, can not only improve the reaction efficiency, but also help to improve the purity of the product.

[0050] The starting material Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH of the present invention can be prepared in-house according to the structure provided by the present invention, but is not limited thereto.

[0051] The present invention provides a method for preparing Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH, but the specific preparation method is not limited thereto. The preparation method of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH may include the following steps:

[0052] (A) Compound 1 and Compound 2 are subjected to a first condensation reaction in a solvent under the action of a first condensation agent, and then reduced by hydrogenation using palladium carbon as a catalyst to obtain an intermediate;

[0053] (B) The intermediate and compound 3 are subjected to a second condensation reaction in a solvent under the action of a second condensation agent, and then reduced by hydrogenation using palladium carbon as a catalyst to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH;

[0054] Among them, the structural formulas of compound 1, compound 2 and compound 3 are as follows:

[0055]

[0056] Furthermore, the molar ratio of compound 1 to compound 2 is 1:1, and the molar ratio of the intermediate to compound 3 is 1:1.

[0057] Among them, compound 2 can be prepared by reacting Fmoc-PEG24-CH2CH2COOH (N-fluorenylmethoxycarbonyl-tetracosylethylene glycol-carboxylic acid) and CbZ-Cl (benzyl chloroformate) in a molar ratio of 1:1.2 in a solvent (such as acetonitrile) under the action of a base (such as diethylamine) at room temperature.

[0058] Compound 3 can be prepared by condensation reaction of L-glutamic acid-α-benzyl ester and palmitic acid in a solvent (such as DMF) under the action of a condensation agent (DIC / oxyma), wherein the molar ratio of DIC, oxyma, L-glutamic acid-α-benzyl ester and palmitic acid is 1.2﹕1.2﹕1﹕1.2.

[0059] Furthermore, the solvent used in step (A) and step (B) may be DMF. The amount of the solvent used can be adjusted routinely to dissolve the reactants and ensure that the reaction proceeds stably.

[0060] Furthermore, the temperature of the first condensation reaction is 25° C., and the time of the first condensation reaction is 2 hours; the temperature of the second condensation reaction is 25° C., and the time of the second condensation reaction is 2 hours.

[0061] Furthermore, the first condensing agent and the second condensing agent are both DIC and oxyma; in the first condensing agent, the molar ratio of DIC, oxyma and compound 1 is 1:1:1; in the second condensing agent, the molar ratio of DIC, oxyma and the intermediate is 1:1:1.

[0062] Furthermore, the amount of palladium on carbon used in step (A) and step (B) is 0.5 eq; the conditions for hydrogenation reduction include: stirring under a hydrogen environment, and the pressure is 101 KPa to 202 KPa, such as 202 KPa.

[0063] Furthermore, in step (B), after hydrogenation reduction, MTBE sedimentation and centrifugation washing are performed twice, and the mixture is dried in a vacuum drying oven and then treated to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH.

[0064] The product was characterized by mass spectrometry (MS) and confirmed to be the target product. Mass spectrometry (MS) (m / z): calculated value C 97 H 170 N4O 33 , 1919.17; test value 1919.34.

[0065] In a specific embodiment of the present invention, the Fmoc-protected amino acid compounds used in the sequential coupling are: Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH, Fmoc-Chg-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-L-7-AzaTrp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tle-OH, Fmoc-N-Me-Asp(otBu)-OH, Fmoc-Asp(R1)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Val-OH and Fmoc-Lys(R2)-OH;

[0066] Wherein R1 is at least one of OAll, and R2 is at least one selected from Alloc and Dde.

[0067] According to the main chain sequence of Zilucoplan, the amino acid compounds protected by Fmoc are sequentially coupled to complete the synthesis of the main chain of Zilucoplan. A protecting group different from Fmoc is set on the side chain of Fmoc-Asp(R1)-OH and Fmoc-Lys(R2)-OH. After the main chain is completed, R1 and R2 are removed, and then subsequent cyclization is performed.

[0068] In a specific embodiment of the present invention, the preparation method comprises:

[0069] (a) coupling Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH with resin to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin;

[0070] (b) Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is coupled with Fmoc-protected amino acid compounds in the order of amino acids from the C-terminus to the N-terminus of the Zilucoplan main chain, and then the terminal Fmoc is capped to obtain polypeptide resin I with Lys side chain protection at position 1 and Asp side chain protection at position 6;

[0071] (c) removing the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in polypeptide resin I, and cyclizing to form a lactam bridge to obtain polypeptide resin II; and cleaving polypeptide resin II to obtain Zilucoplan.

[0072] Among them, polypeptide resin I is specifically Ac-Lys(Alloc)-Val-Glu(OtBu)- Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin.

[0073] In a specific embodiment of the present invention, the resin includes at least one of CTC resin and Wang Resin. Further, the substitution degree of the resin is 0.4-1.2 mmol / g.

[0074] In some embodiments, the degree of substitution of the resin may be 0.4 mmol / g, 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, 0.9 mmol / g, 1 mmol / g, 1.1 mmol / g, 1.2 mmol / g, or a range consisting of any two thereof.

[0075] In a specific embodiment of the present invention, the substitution degree of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is 0.20-0.85 mmol / g.

[0076] In some embodiments, the degree of substitution of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin can be 0.2 mmol / g, 0.3 mmol / g, 0.4 mmol / g, 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g, 0.8 mmol / g, 0.85 mmol / g, or a range consisting of any two thereof.

[0077] In a specific embodiment of the present invention, in step (a), the coupling method comprises: using a solvent to swell the resin, adding Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH and DIEA to react, and then adding MeOH to seal.

[0078] In a specific embodiment of the present invention, in step (a), the coupling time is 2 to 3 hours. Furthermore, the coupling temperature is 20 to 30°C.

[0079] The present invention uses a new starting material and adopts an unconventional coupling site to bind to the resin, which effectively shortens the time of a single coupling of PEG24 and significantly improves production efficiency. In some embodiments, the coupling time can be 2h, 2.5h, 3h, etc., but is not limited thereto. It is understood that the reaction time of each step of the present invention can be further extended on the basis of the limited range, and the scheme of extending the reaction time is also within the scope of protection of the present invention.

[0080] In a specific embodiment of the present invention, in step (a), the molar ratio of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH to the resin is (1.5-2.5):1.

[0081] By using the starting material of the present invention, when coupling with the resin, the amount of raw materials can be reduced and the cost can be reduced while ensuring or even improving the yield. In some embodiments, the molar ratio of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH to the resin can be 1.5:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.5:1 or a range of any two of them.

[0082] In actual operation, in step (a), the resin can be mixed with DCM for 20 to 40 minutes, and then Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH is added, and then DIEA is added, and the mixture is mixed and reacted at 20 to 30°C for 2 to 3 hours, and then MeOH is added, and the mixture is mixed and reacted for 20 to 40 minutes, and then filtered and washed with DCM.

[0083] In some embodiments, in step (a), the amount of DCM used to swell the resin can be adjusted according to actual needs. For example, the ratio of DCM to resin can be (8-12) mL: 1 mmol, that is, 8-12 mL of DCM solvent is used for every 1 mmol of resin, but it is not limited to this.

[0084] In some embodiments, in step (a), the molar ratio of DIEA to Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH is (1.5-2.5):1, such as 1.5:1, 1.8:1, 2:1, 2.2:1, 2.5:1 or a range consisting of any two of them.

[0085] In some embodiments, in step (a), the number of washing times using DCM can be 4 times, 5 times, 6 times, etc.

[0086] In a specific embodiment of the present invention, in step (b), each coupling step includes a deprotection reaction and a coupling reaction; the coupling reaction time is 1.5 to 2.5 hours. Furthermore, the coupling reaction temperature is 20 to 30°C.

[0087] Since the entire spatial structure of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin has good elasticity, the condensation of the resin can be reduced, and the efficiency of the coupling reaction can be significantly improved. In some embodiments, in step (b), in each coupling step, the coupling reaction time can be 1.5h, 1.8h, 2h, 2.2h, 2.5h or a range consisting of any two thereof.

[0088] In a specific embodiment of the present invention, in the coupling reaction, the molar ratio of the Fmoc-protected amino acid compound to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-3):1.

[0089] In some embodiments, the molar ratio of the Fmoc-protected amino acid compound to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin can be 2:1, 2.2:1, 2.5:1, 2.8:1, 3:1 or a range consisting of any two of them.

[0090] In a specific embodiment of the present invention, when coupling Fmoc-L-7-AzaTrp-OH, the molar ratio of Fmoc-L-7-AzaTrp-OH to Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-2.5):1.

[0091] The material cost of Fmoc-L-7-AzaTrp-OH is relatively high. In the preparation method of the present invention, while reducing the amount of Fmoc-L-7-AzaTrp-OH, a yield significantly higher than that of the traditional route can be achieved, which significantly reduces the cost. In some embodiments, the molar ratio of Fmoc-L-7-AzaTrp-OH to Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin can be 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1 or a range consisting of any two of them.

[0092] In a specific embodiment of the present invention, the coupling reaction is carried out under the action of a condensing agent; the condensing agent includes at least one of Oxyma / DIC, HOBT / DIC, PyBop / DIEA and HBTU / NMM. Further, in the coupling reaction, the solvent used includes at least one of DMF and NMP.

[0093] In some embodiments, the molar ratio of Oxyma, DIC and Fmoc-protected amino acid compounds is (1-1.1):(1-1.1):1, such as 1:1:1.

[0094] In a specific embodiment of the present invention, in the coupling reaction, the Fmoc-protected amino acid compound and the condensation agent are pre-activated at 0 to 5° C. Further, the activation time is 5 to 10 minutes.

[0095] In a specific embodiment of the present invention, the deprotection reaction is carried out in a deprotection reagent; the temperature of the deprotection reaction is 20-30°C.

[0096] In a specific embodiment of the present invention, the deprotection reagent includes an aprotic organic solvent containing an organic base, the organic base includes piperidine, and the aprotic organic solvent includes at least one of DCM, DMF and NMP. Further, in the deprotection reagent, the volume fraction of the organic base is 15% to 25%.

[0097] In actual operation, in each coupling of step (b), the step of deprotection reaction may include: placing the resin in a deprotection reagent, mixing at 20-30°C for 3-7 minutes, and washing with a solvent; then adding the deprotection reagent, mixing at 20-30°C for 8-12 minutes, and washing with a solvent. The solvent used for washing may include DMF. In each coupling of step (b), the step of coupling reaction may include: mixing the Fmoc-protected amino acid compound and Oxyma in a solvent, then adding DIC at 0-5°C, activating for 5-10 minutes, and obtaining an activation solution; adding the activation solution to the resin after the deprotection reaction, reacting at 20-30°C for 1.5-2.5 hours, and washing with a solvent.

[0098] In some embodiments, in the deprotection reaction, the ratio of the deprotection reagent to the resin is (25-35) mL: 1 mmol; in the deprotection reaction, the solvent used for washing includes but is not limited to DMF.

[0099] In some embodiments, in the coupling reaction, the ratio of the solvent to the Fmoc-protected amino acid compound is (3-3.5) mL: 1 mmol; in the coupling reaction, the solvent used for washing includes but is not limited to DMF.

[0100] In a specific embodiment of the present invention, in step (a) and step (b), the progress of the coupling reaction is monitored by ninhydrin detection. According to the ninhydrin detection results, when the coupling time of each Fmoc-protected amino acid compound in the present invention is as follows, it is more helpful to shorten the reaction time and improve the yield and purity. For example, the coupling time of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH is 3h, the coupling time of Fmoc-Chg-OH is 1.5h, the coupling time of Fmoc-Pro-OH is 1.5h, the coupling time of Fmoc-Tyr(tBu)-OH is 1.5h, the coupling time of Fmoc-Glu(otBu)-OH is 1.5h, the coupling time of Fmoc-L-7-AzaTrp-OH is 1.5h, the coupling time of Fmoc-Tyr(tBu)-OH is 1.5h, and the coupling time of Fmoc-Chg-OH is 1.5h. The coupling time for Fmoc-Tle-OH is 1.5h, the coupling time for Fmoc-N-Me-Asp(otBu)-OH is 1.5h, the coupling time for Fmoc-Asp(R1)-OH is 1.5h, the coupling time for Fmoc-Phe-OH is 1.5h, the coupling time for Fmoc-Arg(Pbf)-OH is 1.5h, the coupling time for Fmoc-Glu(otBu)-OH is 1.5h, the coupling time for Fmoc-Val-OH is 1.5h and the coupling time for Fmoc-Lys(R2)-OH is 1.5h.

[0101] In a specific embodiment of the present invention, in step (b), the end-capping comprises: removing the terminal Fmoc with a deprotecting agent, and performing terminal acetylation with DIEA and acetic anhydride as blocking agents.

[0102] In the end-capping of step (b), a deprotection reagent is first used to remove the terminal Fmoc. This step can be performed with reference to the deprotection reaction in each coupling. The terminal acetylation can specifically include: mixing the blocking reagent with DMF to obtain an end-capping solution, adding the end-capping solution to the resin from which the terminal Fmoc has been removed, mixing at 20 to 30° C. for 1 to 1.5 hours, and washing to obtain Ac-Lys(Alloc)-Val-Glu(OtBu)- Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin.

[0103] In some embodiments, in the capping reagent, the volume ratio of DIEA to acetic anhydride is 10:6; the corresponding capping solution is prepared by mixing DIEA, acetic anhydride and DMF in a volume ratio of 10:6:84.

[0104] In a specific embodiment of the present invention, in step (c), a mixed removing agent is used to remove the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in the polypeptide resin I; the mixed removing agent includes palladium tetratriphenylphosphine, phenylsilane and NMM. Furthermore, the solvent for the removal reaction includes DCM.

[0105] In actual operation, in the mixed removing agent, the molar ratio of tetrakistriphenylphosphine palladium, phenylsilane and NMM is 0.1:24:24; the molar ratio of tetrakistriphenylphosphine palladium to resin is 0.3:1; the ratio of DCM usage to resin usage is (25-35) mL:1 mmol.

[0106] In a specific embodiment of the present invention, in step (c), the method for cyclization to form a lactam bridge comprises: under the action of PyBop and DIEA, the Lys side chain at position 1 that has been deprotected reacts with the Asp side chain at position 6 that has been deprotected to form a lactam ring.

[0107] In a specific embodiment of the present invention, the molar ratio of PyBop to DIEA is 1:1.2.

[0108] In actual operation, the cyclization to generate a lactam bridge may include: adding PyBop to a reaction container, adding DMF to dissolve, and then adding DIEA to obtain a mixed solution; adding the mixed solution to the resin with the deprotected group, mixing at 20-30° C. for 1 hour, and washing.

[0109] In a specific embodiment of the present invention, the lysis treatment is carried out in a lysis reagent; the lysis reagent includes an aqueous solution containing TFA and TIS. Further, in the lysis reagent, the volume fraction of TFA is 85% to 95%, and the volume fraction of TIS is 4% to 6%.

[0110] For example, in different embodiments, in the lysis reagent, the volume fraction of TFA can be 85%, 88%, 90%, 92%, 95% or a range consisting of any two thereof; the volume fraction of TIS can be 4%, 4.5%, 5%, 5.5%, 6% or a range consisting of any two thereof.

[0111] In a specific embodiment of the present invention, the reaction temperature of the pyrolysis treatment is 20 to 30° C., and the time of the pyrolysis treatment is 2 to 2.5 hours.

[0112] In a specific embodiment of the present invention, the process further comprises: collecting the solution after the cracking treatment, precipitating in a poor solvent, and collecting the solid; the poor solvent comprises MTBE.

[0113] In some embodiments, the method of the present invention may further include: purifying and freeze-drying the crude solid product precipitated from the poor solvent. Specific purification methods include but are not limited to HPLC separation and purification.

[0114] Example 1

[0115] This embodiment provides a method for preparing Zilucoplan, comprising the following steps:

[0116] (1) 10.20 g (10 mmol) of CTC resin with a substitution degree of 0.98 mmol / g was added to 100 mL of dichloromethane. After mixing for 30 min, 38.41 g (20 mmol) of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH was added, and then 7 mL (40 mmol) of DIEA was added. After mixing and reacting at 25 °C for 3 h, 100 mL of MeOH was added. After continuing to mix and react for 30 min, the mixture was filtered and the resin was washed with DCM for 5 times, 200 mL each time, to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin. The substitution degree was 0.81 mmol / g according to UV detection, and the total amount was 7.5 mmol.

[0117] (2) Take 4.05 g (5 mmol) of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin, add 150 mL of deprotection reagent (the deprotection reagent is a DMF solution with a volume fraction of 20%), mix at 25°C for 5 min, filter, add 150 mL of DMF, mix and wash for 3 min, and filter; add 150 mL of the above deprotection reagent, mix at 25°C for 10 min, filter, add 150 mL of DMF, mix and wash for 3 min, filter, and repeat washing with DMF 6 times to obtain a deprotected resin.

[0118] Add 5.69g (15mmol) of Fmoc-Chg-OH, 2.13g (15mmol) of Oxyma, and 50mL of DMF to the reaction vessel, and add 2.32ml (15mmol) of DIC to activate for 10min at 0-5℃. Slowly add the activation solution to the deprotected resin and mix at 25℃ for 1.5h. After the reaction is completed and the Kaiser Test is negative, filter, add 150mL of DMF, mix and wash for 3min, and filter. Repeat washing with DMF for 6 times to obtain Fmoc-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin.

[0119] According to the above coupling method, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-L-7-AzaTrp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tle-OH, Fmoc-N-Me-Asp(otBu)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Alloc)-OH were coupled in sequence to obtain Fmoc-Lys(Alloc)-Val-Glu(OtBu)- Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin.

[0120] In each specific coupling step, the coupling equivalent and coupling time of each Fmoc-protected amino acid compound are shown in Table 2.

[0121] (3) Add 150 mL of a deprotecting agent (a 20% by volume piperidine solution in DMF) to the resin obtained in step (2), mix at 25° C. for 5 min, filter with suction, add 150 mL of DMF, mix and wash for 3 min, and filter with suction; then add 150 mL of the above deprotecting agent, mix at 25° C. for 10 min, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF for 6 times to obtain a deprotected resin.

[0122] Add end-capping solution (prepared by mixing 15 mL acetic anhydride, 9 mL DIEA and 126 mL DMF) to the deprotected resin and mix at 25°C for 1 hour. After the reaction is completed and the Kaiser Test is negative, filter with suction, add 150 mL DMF, mix and wash for 3 minutes, filter with suction; repeat washing with DMF for 6 times to obtain Ac-Lys(Alloc)-Val-Glu(OtBu)-Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin.

[0123] (4) Add 150 mL of a mixed removing agent solution (the mixed removing agent solution is a DCM solution containing 0.1 eq of tetrakistriphenylphosphine palladium, 12 eq of phenylsilane and 12 eq of NMM) to the resin prepared in step (3), mix at 25°C for 2 h, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF 8 times to obtain a deprotected resin.

[0124] Add 7.8 g of PyBop and 50 mL of DMF to a reaction container, then add 7 mL of DIEA and mix well to obtain a mixed solution; add the mixed solution to the deprotected resin obtained above, mix at 25°C for 0.5 h, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF 6 times.

[0125] (5) Add the peptide resin obtained in step (4) to 150 mL of a cleavage reagent (TFA / TIS / H2O=90﹕5﹕5 (volume ratio)), stir the reaction at 25°C for 2 h, filter, wash the filter cake twice with TFA, and combine the filtrates. Slowly pour the combined filtrates into frozen MTBE (≤-10°C), stir for 30 min to fully precipitate the solid, filter, wash the filter residue three times with MTBE, and vacuum dry the solid (25°C, 8 h) to obtain 17.89 g of crude Zilucoplan (MW: 3559.97, yield 100.53%) with an HPLC purity of 41.09%.

[0126] Table 2 Information of each step

[0127]

[0128] Note: The test method for resin thickness is: the experiment uses a glass reactor with an inner diameter of 30mm / outer diameter of 55mm / length of 120mm. After each step of reaction, the thickness of the resin in the reactor is measured after it is evenly spread. The subsequent test method for resin thickness is the same.

[0129] Example 2

[0130] This example is prepared with reference to the method of Example 1, with the only difference being that in step (1), the amount of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH used is different.

[0131] In this example, the amount of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH used is 15 mmol.

[0132] The yield of the corresponding crude Zilucoplan was 97.34%, and the HPLC purity was 39.84%.

[0133] Example 3

[0134] This example is prepared with reference to the method of Example 1, with the only difference being that in step (1), the amount of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH used is different.

[0135] In this example, the amount of Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH used is 25 mmol.

[0136] The yield of the corresponding crude Zilucoplan was 100.84%, and the HPLC purity was 42.17%.

[0137] Example 4

[0138] This example is based on the preparation method of Example 1, with the only difference being that in step (1), the mixing reaction time at 25°C is different.

[0139] In this embodiment, the mixture was mixed and reacted at 25° C. for 2 h.

[0140] The yield of the corresponding crude Zilucoplan was 100.34%, and the HPLC purity was 41.05%.

[0141] Example 5

[0142] This example is based on the preparation method of Example 1, with the only difference being that in step (1), the mixing reaction time at 25°C is different.

[0143] In this embodiment, the mixture was mixed and reacted at 25° C. for 4 h.

[0144] The yield of the corresponding crude Zilucoplan was 100.55%, and the HPLC purity was 40.79%.

[0145] Example 6

[0146] This example refers to the preparation method of Example 1, with the only difference being that in step (2), the coupling time of each Fmoc-protected amino acid compound is 2 h.

[0147] The yield of the corresponding crude Zilucoplan was 99.74%, and the HPLC purity was 40.21%.

[0148] Example 7

[0149] This example is based on the preparation method of Example 1, with the only difference being that in step (2), the coupling time of each Fmoc-protected amino acid compound is 2.5 h.

[0150] The yield of the corresponding crude Zilucoplan was 99.51%, and the HPLC purity was 40.02%.

[0151] Example 8

[0152] This example refers to the preparation method of Example 1, with the only difference being that in step (2), the amount of each Fmoc-protected amino acid compound used is 2 eq.

[0153] The yield of the corresponding crude Zilucoplan was 96.54%, and the HPLC purity was 37.81%.

[0154] Example 9

[0155] This example is based on the preparation method of Example 1, with the only difference being that in step (2), the amount of Fmoc-L-7-AzaTrp-OH used is 3 eq.

[0156] The yield of the corresponding crude Zilucoplan was 102.14%, and the HPLC purity was 42.51%.

[0157] Comparative Example 1

[0158] Comparative Example 1 provides a method for preparing Zilucoplan, comprising the following steps:

[0159] (1) Add 10.20 g (10 mmol) of CTC resin with a substitution degree of 0.98 mmol / g into 100 mL of dichloromethane. After mixing for 30 min, add 30 mmol of Fmoc-Lys(Dde)-OH and 7 mL (40 mmol) of DIEA. After mixing and reacting at 25 °C for 3 h, add 100 mL of MeOH. After continuing to mix and react for 30 min, filter and wash the resin with DCM 5 times, 200 mL each time, to obtain Fmoc-Lys(Dde)-CTC resin. The substitution degree was 0.38 mmol / g by UV detection, for a total of 6.9 mmol.

[0160] (2) Take 5 mmol of Fmoc-Lys(Dde)-CTC resin, add 150 mL of deprotection reagent (the deprotection reagent is a DMF solution with a volume fraction of 20%), mix at 25°C for 5 minutes, filter, add 150 mL of DMF, mix and wash for 3 minutes, and filter; then add 150 mL of the above deprotection reagent, mix at 25°C for 10 minutes, filter, add 150 mL of DMF, mix and wash for 3 minutes, filter, and repeat washing with DMF for 6 times to obtain a deprotected resin.

[0161] Add 5.69g (15mmol) of Fmoc-Chg-OH, 2.13g (15mmol) of Oxyma, and 50mL of DMF to the reaction vessel, and add 2.32ml (15mmol) of DIC to activate for 10min at 0-5℃. Slowly add the activation solution to the deprotected resin and mix at 25℃ for 2h. After the reaction is completed and the Kaiser Test is negative, filter, add 150mL of DMF, mix and wash for 3min, and filter. Repeat the DMF washing for 6 times to obtain Fmoc-Chg-Lys(Dde)-CTC resin.

[0162] According to the above coupling method, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-L-7-AzaTrp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tle-OH, Fmoc-N-Me-Asp(otBu)-OH, Fmoc-Asp(OAll)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Val-OH and Fmoc-Lys(Alloc)-OH were coupled in sequence to obtain Fmoc-Lys(Alloc)-Val-Glu(OtBu)- Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(Dde)-CTC resin.

[0163] In each specific coupling step, the coupling equivalent and coupling time of each Fmoc-protected amino acid compound are shown in Table 3.

[0164] (3) Add 150 mL of a deprotecting agent (a 20% by volume piperidine solution in DMF) to the resin obtained in step (2), mix at 25° C. for 5 min, filter with suction, add 150 mL of DMF, mix and wash for 3 min, and filter with suction; then add 150 mL of the above deprotecting agent, mix at 25° C. for 10 min, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF for 6 times to obtain a deprotected resin.

[0165] Add end-capping solution (prepared by mixing 15 mL acetic anhydride, 9 mL DIEA and 126 mL DMF) to the deprotected resin and mix at 25°C for 1 hour. After the reaction is completed and the Kaiser Test is negative, filter with suction, add 150 mL DMF, mix and wash for 3 minutes, filter with suction; repeat washing with DMF for 6 times to obtain Ac-Lys(Alloc)-Val-Glu(OtBu)-Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(Dde)-CTC resin.

[0166] (4) Add 150 mL of a mixed removing agent solution (the mixed removing agent solution is a DCM solution containing 0.1 eq of tetrakistriphenylphosphine palladium, 12 eq of phenylsilane and 12 eq of NMM) to the resin prepared in step (3), mix at 25°C for 2 h, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF 8 times to obtain a deprotected resin.

[0167] Add 7.8 g of PyBop and 50 mL of DMF to a reaction container, then add 7 mL of DIEA and mix well to obtain a mixed solution; add the mixed solution to the deprotected resin obtained above, mix at 25°C for 0.5 h, filter with suction, add 150 mL of DMF, mix and wash for 3 min, filter with suction, and repeat washing with DMF 6 times.

[0168] (5) The polypeptide resin obtained in step (4) was added to 150 mL of 2% hydrazine hydrate / DMF solution to remove the Dde protecting group. After mixing at 25°C for 60 min, the mixture was filtered with suction. 150 mL of DMF was added, and the mixture was mixed and washed for 3 min, and then filtered with suction. Then 150 mL of the above deprotection reagent (2% hydrazine hydrate / DMF solution) was added, and the mixture was mixed at 25°C for 10 min, and then filtered with suction. 150 mL of DMF was added, and the mixture was mixed and washed for 3 min, and then filtered with suction. The mixture was washed with DMF for 6 times to obtain a deprotected resin.

[0169] Add 20.52g (15mmol) of Fmoc-PEG24-PA, 15.90g (15mmol) of Oxyma, and 50mL of DMF to the reaction vessel, and add 2.32mL (15mmol) of DIC to activate for 10min at 0-5℃. Slowly add the activation solution to the deprotected resin and mix at 25℃ for 8h. After the reaction is completed and the Kaiser Test is negative, filter, add 150mL of DMF, mix and wash for 3min, and filter. Repeat washing with DMF for 6 times to obtain Ac-Lys(Alloc)-Val-Glu(OtBu)-Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(Fmoc-PEG24)-CTC resin.

[0170] According to the above coupling method, Fmoc-γ-Glu(OtBu)-OH and palmitic acid were coupled in sequence to obtain Ac-Lys(Alloc)-Val-Glu(OtBu)-Arg(Pbf)-Phe-Asp(OAll)-N-Me-Asp(OtBu)-Tle-Tyr(tBu)-AzaTrp-Glu(OtBu)-Tyr(tBu)-Pro-Chg-Lys(PEG24-γ-Glu(OtBu)-Pal)-CTC resin.

[0171] (6) Add the peptide resin obtained in step (5) to 200 mL of cleavage reagent (TFA / TIS / H2O=90﹕5﹕5 (volume ratio)), stir the reaction at 25°C for 2 h, filter, wash the filter cake twice with TFA, and combine the filtrates. Slowly pour the combined filtrates into frozen MTBE (≤-10°C), stir for 30 min to fully precipitate the solid, filter, wash the filter residue three times with MTBE, and vacuum dry the solid (25°C, 8 h). The crude yield of Zilucoplan is 91.13%, and the HPLC purity is 24.43%.

[0172] Table 3 Information of each step

[0173]

[0174] Comparative Example 2

[0175] Comparative Example 2 refers to the preparation method of Comparative Example 1, except that in step (2), the coupling time of each Fmoc-protected amino acid compound is 1.5 h.

[0176] The yield of the corresponding crude Zilucoplan was 94.71%, and the HPLC purity was 22.84%.

[0177] The preparation method of Zilucoplan of the present invention not only shortens the reaction time and improves the production effect, but also significantly improves the yield and purity of Zilucoplan.

[0178] 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 Zilucoplan, characterized in that: The method comprises the following steps: coupling Fmoc-protected amino acid compounds to the resin from the C-terminus to the N-terminus in sequence according to the peptide sequence of Zilucoplan; in the sequential coupling, the Fmoc-protected amino acid compounds used are: Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH, Fmoc-Chg-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(otBu)-OH, Fmoc-L- 7-AzaTrp-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Tle-OH, Fmoc-N-Me-Asp(otBu)-OH, Fmoc-Asp(R1)-OH, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(otBu)-OH, Fmoc-Val-OH and Fmoc-Lys(R2)-OH; wherein R1 is OAll, and R2 is selected from at least one of Alloc and Dde; In the coupling, the first Fmoc-protected amino acid compound used is Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH, and the structural formula is as follows: ； The preparation method comprises: (a) coupling the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH with the resin, wherein the resin is selected from at least one of CTC resin and Wang resin, and the degree of substitution of the resin is 0.4 to 1.2 mmol / g, to obtain Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin; (b) coupling the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin with Fmoc-protected amino acid compounds in the order of amino acids from the C-terminus to the N-terminus of the Zilucoplan main chain, and then capping the terminal Fmoc to obtain a polypeptide resin I with a Lys side chain protection at position 1 and an Asp side chain protection at position 6; (c) removing the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in the polypeptide resin I, and cyclizing to form a lactam bridge to obtain polypeptide resin II; and cleaving the polypeptide resin II to obtain the Zilucoplan; In step (b), each coupling step includes a deprotection reaction and a coupling reaction; the coupling reaction time is 1.5 to 2.5 hours.

2. The method for preparing Zilucoplan according to claim 1, characterized in that: The substitution degree of the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is 0.2-0.85 mmol / g.

3. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (a), the coupling method comprises: using a solvent to swell the resin, adding the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH and DIEA to react, and then adding MeOH to seal.

4. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (a), the coupling time is 2 to 3 hours; the coupling temperature is 20 to 30°C.

5. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (a), the molar ratio of the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-OH to the resin is (1.5-2.5):

1.

6. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (b), in each coupling step, the coupling reaction temperature is 20-30°C.

7. The method for preparing Zilucoplan according to claim 1, characterized in that: In the coupling reaction, the molar ratio of the Fmoc-protected amino acid compound to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-3):

1.

8. The method for preparing Zilucoplan according to claim 1, characterized in that: When coupling Fmoc-L-7-AzaTrp-OH, the molar ratio of the Fmoc-L-7-AzaTrp-OH to the Fmoc-Lys(PEG24-γ-Glu(OtBu)-Pal)-resin is (2-2.5):

1.

9. The method for preparing Zilucoplan according to claim 1, characterized in that: The coupling reaction is carried out under the action of a condensing agent; the condensing agent includes at least one of Oxyma / DIC, HOBT / DIC, PyBop / DIEA and HBTU / NMM.

10. The method for preparing Zilucoplan according to claim 9, characterized in that: In the coupling reaction, the Fmoc-protected amino acid compound and the condensing agent are activated at 0-5°C in advance.

11. The method for preparing Zilucoplan according to claim 1, characterized in that: The deprotection reaction is carried out in a deprotection reagent; the temperature of the deprotection reaction is 20-30°C.

12. The method for preparing Zilucoplan according to claim 11, characterized in that: The deprotection reagent comprises an aprotic organic solvent containing an organic base, the organic base comprises piperidine, and the aprotic organic solvent comprises at least one of DCM, DMF and NMP.

13. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (b), the end-capping comprises: removing the terminal Fmoc using a deprotecting agent, and performing terminal acetylation using DIEA and acetic anhydride as blocking agents.

14. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (c), a mixed removing agent is used to remove the protecting groups on the side chain of Lys at position 1 and the side chain of Asp at position 6 in the polypeptide resin I; the mixed removing agent includes tetrakistriphenylphosphine palladium, phenylsilane and NMM.

15. The method for preparing Zilucoplan according to claim 1, characterized in that: In step (c), the method for cyclization to form a lactam bridge comprises: under the action of PyBop and DIEA, the Lys side chain at position 1 which has been deprotected reacts with the Asp side chain at position 6 which has been deprotected to form a lactam ring.

16. The method for preparing Zilucoplan according to claim 1, characterized in that: The cleavage treatment is carried out in a cleavage reagent; the cleavage reagent includes an aqueous solution containing TFA and TIS.

17. The method for preparing Zilucoplan according to claim 1, characterized in that: Also includes: The solution after the cracking treatment is collected, precipitated in a poor solvent, and the solid is collected; the poor solvent includes MTBE.

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