A kind of synthetic method of NH2-PEG4-RK(Dde)-OH

Abstract

The invention discloses a method for synthesizing NH2-PEG4-RK(Dde)-OH, wherein NH2-PEG4-RK(Dde)-OH is synthesized by a segmented synthesis method, wherein the segmented synthesis method is a two-stage method or a three-stage method; the two-stage method uses 5-Glu as a carbon terminal site, and NH2-PEG4-RK(Dde)-OH is synthesized by two synthetic fragments: a fragment Wang-(9-6)-NH2 and a fragment Fmoc-(1-5)-OH; the three-stage method uses 3-Asn and 6-Leu as two carbon terminal sites, and NH2-PEG4-RK(Dde)-OH is synthesized by three synthetic fragments: a fragment Wang-(9-7)-NH2, a fragment Fmoc-(4-6)-OH and a fragment Fmoc-(1-3)-OH; and the synthetic fragments are coupled by a condensing agent. The method has a simple process, high product purity and high yield, and is suitable for industrial production of NH2-PEG4-RK(Dde)-OH.

Description

Technical Field

[0001] The invention relates to the technical field of organic synthesis, and in particular to a method for synthesizing NH2-PEG4-RK(Dde)-OH. Background Art

[0002] Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor and a member of the epidermal growth factor receptor (EGFR) family. HER2 amplification and overexpression are closely related to the high invasiveness, high recurrence and high mortality of tumors. It is highly expressed to varying degrees in a variety of malignant tumors (such as breast cancer, gastric cancer, lung cancer, etc.) and is an important target for molecular imaging diagnosis and targeted therapy (monoclonal antibodies, antibody-drug conjugates, etc.). The carcinogenic mechanism of HER2 mainly includes: promoting tumor cell proliferation, increasing tumor cell invasiveness, inhibiting tumor cell apoptosis and promoting tumor angiogenesis and lymphangiogenesis, thereby providing favorable conditions for tumor growth and metastasis.

[0003] At present, commonly used HER2 targeting ligands include antibodies, small molecule peptides, or proteins that specifically bind to receptors. In cancer treatment, peptides can be used as targeting ligands to specifically deliver cytotoxic drugs to the tumor vasculature, tumor microenvironment, or cancer cells. On the other hand, peptides can also be delivered intracellularly to target cancer-specific upregulated transcription factors, oncogenes, or enzymes. In diagnostic imaging, short peptides have good penetration and can penetrate deep into solid tumors. They have short metabolic cycles and can be quickly cleared from the circulation, and have the advantages of low toxicity. Through the efficient peptide screening method of in situ single-cell sequencing on microarrays, two new octapeptides YLFFVFER (H6) and KLRLEWNR (H10) have attracted people's attention. The probes of H6 and H10 also showed high specificity and affinity in HER2-positive tumor tissues. The KD of H6 and H10 were 6.70×10 -8 mol / L and 3.04×10 -8 mmol / L. The plasma half-life of H6 is 17 minutes, and that of H10 is 13 minutes. Due to the shorter half-life of H10, H10 may be cleared from the blood faster than H6 and have lower toxicity.

[0004] The structure of KLRLEWNR (H10) is: lysine-leucine-arginine-leucine-glutamic acid-tryptophan-asparagine-arginine, and the structural formula is as follows:

[0005]

[0006] The existing KLRLEWNR (H10) modification method is: structurally modifying the H10 peptide chain, retaining the integrity of the peptide chain, exchanging the C and N ends, and coupling a PEG long chain with an amino group at the end to the arginine, which can not only increase the water solubility of the entire peptide chain, but also enable the targeted compound to be coupled with anticancer drugs as an intermediate to achieve precise treatment of tumors. The structure of the modified compound is as follows:

[0007]

[0008] The structural formula of the compound is: NH2-PEG4-Arg-Asn-Trp-Glu-Leu-Arg-Leu-Lys(Dde)-OH; English name: N 2 -(1-amino-3,6,9,12-tetraoxapentadecan-15-oyl)-L-arginyl-L-asparaginyl-L-tryptophyl-L-glutamyl-L-leucyl-L-arginyl-L-leucyl-N 6 -[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine; Chinese name: N 2 -(1-amino-3,6,9,12-tetrahydropentadecan-15-oxo)-L-arginyl-L-asparaginyl-L-tryptophanyl-L-glutamyl-L-leucine-L-arginyl-L-leucine-N 6 -[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine; abbreviation: NH2-PEG4-RK(Dde)-OH.

[0009] The crude NH2-PEG4-RK(Dde)-OH obtained by conventional solid phase synthesis and connection method has low purity and many impurities. Some impurities have similar structures to the main components, which leads to similar chromatographic behavior to the main components, thus increasing the difficulty of separation and requiring multiple HPLC preparation and purification. In addition, NH2-PEG4-RK(Dde)-OH contains Asn residues. During the multiple preparation and purification processes, Asn was found to be hydrolyzed to form Asp. However, after a series of process improvements, such as increasing the feed ratio of protected amino acids, changing the type of condensing agent, and reducing the degree of substitution of resins, the purity of the crude NH2-PEG4-RK(Dde)-OH has not been significantly improved. Summary of the invention

[0010] The purpose of the present invention is to provide a synthesis method for improving the purity of the product in view of the difficulty in synthesizing the above-mentioned NH2-PEG4-RK(Dde)-OH.

[0011] To achieve the above purpose, the technical solution provided by the present invention is:

[0012] A method for synthesizing NH2-PEG4-RK(Dde)-OH, wherein the NH2-PEG4-RK(Dde)-OH is synthesized by a segmented synthesis method, wherein the segmented synthesis method is a two-stage method or a three-stage method; the two-stage method uses 5-Glu as a carbon terminal site, and the NH2-PEG4-RK(Dde)-OH is synthesized through two synthetic segments: a Wang-(9-6)-NH2 segment and a Fmoc-(1-5)-OH segment;

[0013] The three-stage method uses 3-Asn and 6-Leu as two carbon terminal sites, respectively, so that NH2-PEG4-RK(Dde)-OH is synthesized through three synthetic fragments: fragment Wang-(9-7)-NH2, fragment Fmoc-(4-6)-OH, and fragment Fmoc-(1-3)-OH;

[0014] The synthetic fragments are coupled via a condensing agent.

[0015] In the two-stage method, the reactants are fragment Wang-(9-6)-NH2, fragment Fmoc-(1-5)-OH, HATU, HOAt and DIPEA, preferably, the reaction molar ratio is fragment 1: 1.5: 2.0: 2.0: 4.0; in the three-stage method, the reactants are fragment Wang-(9-7)-NH2, fragment Fmoc-(4-6)-OH, fragment Fmoc-(1-3)-OH, HATU, HOAt, DIPEA, preferably, the reaction molar ratio is 1: 1.5: 1.5: 2.0: 2.0: 4.0; after the reaction, the product is eluted with Wang resin lysis buffer, preferably, the Wang resin lysis buffer is TFA: TIPS: H2O = 95: 2.5: 2.5, v / v, mixed evenly, and frozen to 5±5°C; the crude product obtained by the reaction is refined by HPLC.

[0016] The synthesis method of the fragment Wang-(9-6)-NH2 is to react Wang-Lys(Dde) with a protected amino acid, HBTU and DIPEA, preferably, the reaction molar ratio is 1.0:3.0:3.0:3.0.

[0017] The synthesis method of Wang-Lys(Dde) is to react Wang resin with Fmoc-Lys(Dde)-OH, HOBt, DIC and DMAP. Preferably, the reaction molar ratio is 1.0:2.5:2.5:2.5:0.05.

[0018] The synthesis method of the fragment Fmoc-(1-5)-OH is to react CTC-Glu(OtBu) with protected amino acid, HBTU and DIPEA, preferably, the reaction molar ratio is 1.0:3.0:3.0:3.0; after the reaction, the product is eluted with CTC resin lysis solution, preferably, the CTC resin lysis solution is TFA:DCM=1:99, v / v, mixed evenly, and frozen to 5±5°C.

[0019] The synthesis method of CTC-Glu(OtBu) is to react CTC resin with Fmoc-Glu(OtBu)-OH and DIPEA, preferably, the reaction molar ratio is 1.0:2.0:4.0.

[0020] The synthesis method of the fragment Wang-(9-7)-NH2 is to react Wang-Lys(Dde) with a protected amino acid, HBTU and DIPEA, preferably, the reaction molar ratio is 1.0:3.0:3.0:3.0.

[0021] The synthesis method of the fragment Fmoc-(4-6)-OH is to react CTC-Leu with a protected amino acid, HBTU, and DIPEA, preferably, the reaction molar ratio is 1.0:3.0:3.0:3.0; after the reaction, the product is eluted with a CTC resin lysis solution, preferably, the CTC resin lysis solution is TFA:DCM=1:99, v / v, mixed evenly, and frozen to 5±5°C.

[0022] The synthesis method of CTC-Leu is to react CTC resin with Fmoc-Leu-OH and DIPEA, preferably, the reaction molar ratio is 1.0:2.0:4.0.

[0023] The synthesis method of the fragment Fmoc-(1-3)-OH is to react CTC-Asn(Trt) with a protected amino acid, HBTU and DIPEA, preferably, the reaction molar ratio is 1.0:3.0:3.0:3.0; after the reaction, the product is eluted with a CTC resin lysis solution, preferably, the CTC resin lysis solution is TFA:DCM=1:99, v / v, mixed evenly, and frozen to 5±5°C.

[0024] The synthesis method of CTC-Asn(Trt) is to react CTC resin with Fmoc-Asn(Trt)-OH and DIPEA, preferably, the reaction molar ratio is 1.0:2.0:4.0.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention provides a segmented synthesis method of NH2-PEG4-RK(Dde)-OH, which can solve the problems that difficult sequences are easily formed when preparing NH2-PEG4-RK(Dde)-OH by solid phase synthesis, missing peptides and misconnected peptides are easily formed in the process of coupling one by one, resulting in poor purity of crude products, more structurally similar impurities, making preparation and purification difficult, and affecting product purity and yield.

[0027] The invention studies a variety of segmented synthesis methods of NH2-PEG4-RK(Dde)-OH, and obtains a segmented synthesis method most suitable for the synthesis of NH2-PEG4-RK(Dde)-OH after investigation; the method has a simple process, and the product purity and yield are high, and is suitable for the industrial production of NH2-PEG4-RK(Dde)-OH. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The segmented synthesis site of NH2-PEG4-RK(Dde)-OH of the present application. DETAILED DESCRIPTION

[0029] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0030] The test methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the art unless otherwise specified.

[0031] The abbreviations and notes involved in the present invention are shown in Table 1:

[0032] Table 1 Abbreviations

[0033]

[0034] The protected amino acid in the present application is selected from: Fmoc-Lys(Dde)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, FmocAsn(Trt)-OH, and Fmoc-PEG4-OH.

[0035] In order to solve the problems that difficult sequences are easily formed when preparing NH2-PEG4-RK(Dde)-OH by solid phase synthesis, missing peptides and misconnected peptides are easily formed in the coupling process one by one, the crude product has poor purity, many structurally similar impurities, and the preparation and purification are difficult, which affects the purity and yield of the product, the present application proposes a segmented synthesis method of NH2-PEG4-RK(Dde)-OH.

[0036] The solid phase synthesis one-by-one connection method, the improved solid phase synthesis one-by-one connection method and the segmented synthesis method of the present invention are introduced below:

[0037] (I) Preparation of NH2-PEG4-RK(Dde)-OH by solid phase synthesis and ligation:

[0038] First, CTC-Lys(Dde) resin was prepared, and Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Trp(Boc)-OH, FmocAsn(Trt)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-PEG4-OH were coupled respectively to obtain: CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin, which was cleaved by 95% TFA to obtain crude NH2-PEG4-RK(Dde)-OH, which was prepared and purified by HPLC, and freeze-dried to obtain refined NH2-PEG4-RK(Dde)-OH.

[0039] The process route for the full solid phase synthesis of NH2-PEG4-RK(Dde)-OH is:

[0040]

[0041] During the experiment, it was found that the purity of the crude NH2-PEG4-RK(Dde)-OH obtained by conventional solid phase synthesis and connection method was 60-70%, with many impurities. Some impurities may have structural similarities with the main component, which makes their chromatographic behavior similar to the main component, thereby increasing the difficulty of separation. It is necessary to undergo multiple HPLC preparation and purification to obtain a refined product with a purity of >95% and a single impurity of <3.0%. In addition, the polypeptide contains Asn residues. During the multiple preparation and purification processes, it was found that Asn was hydrolyzed to form Asp. The specific experimental process of preparing NH2-PEG4-RK(Dde)-OH by solid phase synthesis and connection method is shown in Comparative Example 1.

[0042] (II) Improved solid phase synthesis and one-by-one connection method for preparing NH2-PEG4-RK(Dde)-OH:

[0043] With regard to the problems in the above conventional solid phase synthesis method for preparing NH2-PEG4-RK(Dde)-OH by connecting one by one, the following process conditions were used for improvement and optimization:

[0044] (1) Increase the feed ratio of protected amino acid and change the feed ratio of CTC-Lys(Dde) resin (substitution degree: 0.70 mmol / g): protected amino acid: HBTU: DIPEA from 1:3:3:3 to 1:5:5:5;

[0045] (2) Changing the type of condensing agent, such as using HOBt-PyBOP-DIPEA or HATU-HOAt-DIPEA as condensing agent instead of the original HBTU-DIPEA condensing agent;

[0046] (3) Reduce the degree of substitution of the resin, such as using CTC-Lys(Dde) (substitution degree 0.4-0.5 mmol / g).

[0047] According to the above improvement plan, a series of comparative tests were carried out. The test results showed that the purity of the crude NH2-PEG4-RK(Dde)-OH was less than 80%, the purity of the refined product after HPLC preparation was greater than 95%, and the total yield was 40-50%.

[0048] It can be seen that with the change of process parameters, the purity of the crude NH2-PEG4-RK(Dde)-OH cannot be significantly improved, and the purity of the crude product is still less than 80%. This may be related to the Dde group in CTC-Lys(Dde). The Dde group can be selectively removed under the condition of 2% hydrazine hydrate, but it may not be stable under certain conditions, especially in an alkaline environment. The Dde group may react, causing its protective effect to weaken or disappear.

[0049] The piperidine used in removing the Fmoc group and the DIPEA used in coupling the protected amino acid are both organic bases. The instability of the Dde group will inevitably bring about a series of by-products. Six amino acids with protective groups need to be coupled to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2. During the coupling process, the amino acid with the protective group has strong hydrophobicity, and the reactivity of the carbon end may be affected. During the coupling process, a "difficult sequence" may be formed. In the coupling process one by one, missing peptides and misconnected peptides are easily formed. The specific experimental process of preparing NH2-PEG4-RK(Dde)-OH by the improved solid phase synthesis one by one connection method is shown in Comparative Examples 2-5.

[0050] (III) This application studies various segmented synthesis methods of NH2-PEG4-RK(Dde)-OH, see Table 2, and selects relatively stable Leu, Glu, and Asn in the 9-peptide chain as the carbon terminal sites, such as Figure 1 shown.

[0051] Table 2 Segmentation sites

[0052]

[0053] This application investigated five segmentation methods, A, B, C, D, and E, as shown in Table 3, and found that the segmentation method for synthesizing NH2-PEG4-RK(Dde)-OH is a very effective method for improving the purity of the product.

[0054] By testing the products obtained by different segmentation methods, it was found that the following segmentation methods are better: using 5-Glu as a carbon-terminal site to form a two-stage method, the synthesis of which is composed of the fragment Wang-(9-6)-NH2 and the fragment Fmoc-(1-5)-OH; using 3-Asn and 6-Leu as the two carbon-terminal sites respectively, a three-stage method is formed, and the synthesis of which is composed of the fragment Wang-(9-7)-NH2, the fragment Fmoc-(4-6)-OH and the fragment Fmoc-(1-3)-OH.

[0055] That is, segmentation method B and segmentation method D have advantages; among them, segmentation method B, the method of disconnecting at the 5-position Glu has a higher product purity, and the 4+5 two-stage method is simpler, and the purity of the obtained crude product is >90%; at the same time, the crude products obtained by different segmentation methods are purified, and the purity of the purified refined product is >99.0%, the single impurity is <0.5%, and its total yield is >75%. This method has low production cost, simple process and easy operation, and is most suitable for the industrial production of NH2-PEG4-RK(Dde)-OH.

[0056] Table 3 Segmentation method

[0057]

[0058] The specific experimental process of the segmented synthesis method of NH2-PEG4-RK(Dde)-OH of the present application is shown in Examples 1 to 5.

[0059] The present invention is further described in detail below in conjunction with specific embodiments and comparative examples:

[0060] Comparative Example 1 Preparation of NH2-PEG4-RK(Dde)-OH by solid phase synthesis and ligation

[0061] The material molar ratio of CTC-Lys(Dde) resin (substitution degree: 0.70 mmol / g), protected amino acid, HBTU, and DIPEA is 1:3:3:3.

[0062] Preparation of intermediate CTC-Lys(Dde), feed amounts are shown in Table 4:

[0063] Table 4 Preparation and Feeding Amount of CTC-Lys(Dde) in Comparative Example 1

[0064]

[0065] Process:

[0066] Washing and swelling: Weigh CTC resin, add it to the peptide solid phase synthesis tube, measure N,N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the uniform dispersion of the resin in the solvent. Generally, the amount of solvent is 4.0-6.0eq of the amount of resin, the same below), wash 3 times (N2 bubbling, 1 min each time, and remove after washing); Add DMF to the peptide solid phase synthesis tube, bubble N2 for 60 min, and drain the liquid after sufficient swelling;

[0067] Coupling: Based on the molar weight of CTC resin N0, weigh Fmoc-Lys(Dde)-OH, take an appropriate amount of DMF, add them to a beaker, stir to dissolve, and add them to the peptide solid phase synthesis tube; weigh DIPEA and add it to the peptide solid phase synthesis tube. The coupling reaction lasts 2.0~3.0h, the reaction liquid is removed, DMF is added, and washed 3 times (N2 bubbling, 1min each time, and the liquid is drained after completion);

[0068] Blocking: Add an appropriate amount of a mixture of dichloromethane (DCM): methanol: DIPEA = 3:1:1 to the peptide solid phase synthesis tube, and block the reaction twice (N2 bubbling, 10 min each time, and drain the liquid after completion);

[0069] Contraction: Add appropriate amount of methanol to the peptide solid phase synthesis tube and contract it 3 times (N2 bubbling, the time is 5min, 5min, 10min respectively, and the liquid is drained after the end);

[0070] Drying: Dry with nitrogen flow for 4.0~5.0h until the resin particles are no longer sticky and are in the form of quicksand;

[0071] CTC-Lys(Dde) resin was obtained, and the substitution degree of CTC-Lys(Dde) resin was detected to be 0.70 mmol / g.

[0072] Preparation of crude NH2-PEG4-RK(Dde)-OH, feed amounts are shown in Table 5:

[0073] Table 5 Comparative Example 1 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0074]

[0075] Process:

[0076] Washing and swelling: Weigh CTC-Lys (Dde) resin, add it to the peptide solid phase synthesis tube, weigh N, N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the uniform dispersion of the resin in the solvent. Generally, the amount of solvent is 4.0-6.0eq of the amount of resin, the same below), wash twice (N2 bubbling, 1 min each time, and remove after washing); Add DMF to the peptide solid phase synthesis tube, bubble N2 for 30 min, and drain the liquid after sufficient swelling;

[0077] Coupling Fmoc-Leu-OH:

[0078] Removal of Fmoc group and color development: Add 20% piperidine solution to the peptide solid phase synthesis tube, remove Fmoc twice (N2 bubbling, time is 5min and 7min respectively, and the liquid is drained after completion), wash with DMF 6 times (N2 bubbling, 1min each time, and drain after washing); Kaiser method (take a small amount of resin, wash with DMF, add 3-5 drops of ninhydrin solution, heat at 120℃ for 3min; observe whether color is developed), detect the color change of resin; activation, coupling, color development, washing: take the synthesis scale N0 as the base number, weigh the materials according to a certain feed ratio: Fmoc-Leu-OH (3.0eq), HBTU (3.0eq); add Into a beaker, measure an appropriate amount of DMF, stir to dissolve, add DIPEA (3.0eq), activate the reaction for 3 minutes, and then add it to the peptide solid phase synthesis tube; room temperature 20-35 ° C, coupling reaction 0.5~1.0 hour; Kaiser method (take a small amount of resin, after DMF washing, add 3-5 drops of ninhydrin solution, heat at 120 ° C for 3 minutes; observe whether color is developed to determine whether the reaction is complete; if color is developed, continue coupling for 0.5 hours); drain the reaction solution and wash with DMF 3 times (N2 bubbling, 1 minute each time, and drain after washing).

[0079] According to the dosage in the feed list, the above operation was repeated to couple the following 6 amino acid fragments and Fmoc-PEG4-propionic acid fragment: coupling Fmoc-Arg(Pbf)-OH; coupling Fmoc-Leu-OH; coupling Fmoc-Glu(OtBu)-OH; coupling Fmoc-Trp(Boc)-OH; coupling Fmoc-Asn(Trt)-OH; coupling Fmoc-Arg(Pbf)-OH; coupling Fmoc-PEG4-propionic acid;

[0080] Removal of Fmoc group and color development: the same operation as before;

[0081] Contraction: Add appropriate amount of methanol to the peptide solid phase synthesis tube and contract it 3 times (N2 bubbling, the time is 5min, 5min, 10min respectively, and the liquid is drained after the end);

[0082] Drying: Drying with nitrogen flow for 4.0-5.0 h until the resin particles are no longer sticky and are in the form of quicksand, to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin;

[0083] Lysis: Lysis solution preparation: Prepare lysis solution (TFA:TIPS:H2O=95:2.5:2.5v / v) at a ratio of 10ml / 1g resin, mix well, and freeze to 5±5℃;

[0084] Pre-freeze ether: prepare the required ether according to the ratio of 70ml / 1g resin, freeze it in a freezer for more than 2 hours, and set aside; add CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin to the reaction bottle, slowly add the prepared pre-cooled lysis solution under stirring, stir well, and perform lysis reaction at room temperature 20-30℃ for 2.0~3.0h r; filter and collect the filtrate; wash the filter cake once with a small amount of lysis solution; combine the filtrate; add frozen ether (4-5 ml ether / 1 ml lysis solution) to the reaction bottle, slowly add the filtrate under stirring to precipitate the product, stir for 20 minutes, filter, and add appropriate amount of frozen ether to fully wash more than 3 times; the obtained product is vacuum dried at room temperature 20-30°C for 6.0-7.0h to obtain crude NH2-PEG4-RK(Dde)-OH, with a crude product yield of 112.27% and a crude product purity of 63.775%.

[0085] Preparation of NH2-PEG4-RK(Dde)-OH refined product, feed amount see Table 6:

[0086] Table 6 Comparative Example 1 Preparation of NH2-PEG4-RK(Dde)-OH Refined Product Feeding Amount

[0087]

[0088] Purification method: Chromatographic column: DEVOTE Dasiogel C18 250mm*100mm, 8μm; Mobile phase: Mobile phase A: 0.1% TFA aqueous solution; Mobile phase B: acetonitrile; Detection wavelength: 220nm; Flow rate: 150ml / min; Purification gradient see Table 7:

[0089] Table 7 Comparative Example 1 Purification Gradient

[0090]

[0091] Process: Dissolution and filtration of crude NH2-PEG4-RK(Dde)-OH: Dissolve crude NH2-PEG4-RK(Dde)-OH with 400ml diluent (90% mobile phase A-10% mobile phase B) by ultrasonic dissolution; Place a 0.22µm microporous glass fiber filter membrane in the funnel, fully wet it, add NH2-PEG4-RK(Dde)-OH crude solution, connect a vacuum pump, open the control valve, filter, and replace the filter paper according to the actual situation; After the last filtration of the crude NH2-PEG4-RK(Dde)-OH solution, add 100ml of diluent to the filter. The solution was filtered and the filtrate was combined with the crude NH2-PEG4-RK(Dde)-OH solution; loading: the crude NH2-PEG4-RK(Dde)-OH solution was loaded; elution: linear gradient elution was performed according to the purification gradient; sample collection: the target peak was collected in segments (the retention time of NH2-PEG4-RK(Dde)-OH was about 46 minutes) and the preparation chromatogram was saved; merging: the samples with purity less than 95.0% were combined into repurified samples; concentration: the sample fractions with purity greater than 95.0% were combined and concentrated; freeze drying: the combined target peptide solution was divided into freeze drying trays, and the thickness of each tray was 1.0~1.5 cm; put the product into the box, lock the box door, quickly freeze the product to below -45℃ and keep it warm for 2 hours, turn on the vacuum pump to evacuate the front box, the vacuum degree is <50Pa, and heat and sublimate according to the following curve: set the heat transfer oil to 0℃ in 2 hours and keep it for more than 12 hours; set the heat transfer oil to 25℃ in 2 hours and keep it for more than 6 hours; release the vacuum, open the vacuum leak valve to release the vacuum in the box to normal pressure; sampling and packaging: release the vacuum, open the vacuum leak valve to release the vacuum in the box to normal pressure; open the box door and quickly take out the freeze-dried plate from the box; immediately take samples according to the sampling operation procedures after taking out of the box; transfer the drug into a low-density ethylene bottle for drug packaging, calculate the yield of the refined product CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH: 42.06%, label it, seal it, and store it in the freezer; the purity of the refined product is 94.680%.

[0092] Comparative Example 2 Preparation of NH2-PEG4-RK(Dde)-OH by Improved Solid Phase Synthesis and One-by-One Connection Method

[0093] The material molar ratio of CTC-Lys(Dde) resin (substitution degree: 0.70 mmol / g), protected amino acid, HBTU, and DIPEA is 1:5:5:5.

[0094] (1) The preparation of the intermediate CTC-Lys(Dde) was the same as that in Comparative Example 1.

[0095] (2) Preparation of crude NH2-PEG4-RK(Dde)-OH The reaction was the same as in Comparative Example 1, and the feed amounts were shown in Table 8:

[0096] Table 8 Comparative Example 2 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0097]

[0098] The process is roughly the same as that of Comparative Example 1. Due to the increase in the feed amount of the amino acid fragment, the amount of solvent is appropriately increased, wherein activation and coupling: based on the synthesis scale N0, the materials are weighed according to a certain feed ratio: Fmoc-Leu-OH (5.0eq), HBTU (5.0eq); added to a beaker, an appropriate amount of DMF is measured, stirred to dissolve, DIPEA (5.0eq) is added, and the activation reaction is performed for 3 minutes before adding to the polypeptide solid phase synthesis tube; the room temperature is 20-35°C, the coupling reaction is performed for 0.5-1.0 hours to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin; NH2-PEG4-RK(Dde)-OH crude product is obtained by cleavage, the crude product yield is 110.90%, and the crude product purity is 66.751%.

[0099] (3) Preparation of refined NH2-PEG4-RK(Dde)-OH

[0100] The purification conditions were the same as those in Comparative Example 1, and the process was roughly the same as that in Comparative Example 1. The total yield (CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH refined product) was calculated to be 43.75%. The product was labeled, sealed, and stored in a frozen state. The purity of the refined product was 95.244%.

[0101] Comparative Example 3 Preparation of NH2-PEG4-RK(Dde)-OH by Improved Solid Phase Synthesis and One-by-One Connection Method

[0102] The material molar ratio of CTC-Lys(Dde) resin (substitution degree: 0.70 mmol / g), protected amino acid, HOBT, PyBop, and DIPEA is 1:1.5:1.5:1.4:2.8.

[0103] (1) The preparation of the intermediate CTC-Lys(Dde) was the same as that in Comparative Example 1.

[0104] (2) Preparation of crude NH2-PEG4-RK(Dde)-OH The reaction was the same as in Comparative Example 1, and the feed amounts were shown in Table 9:

[0105] Table 9 Comparative Example 3 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0106]

[0107] The process is roughly the same as that of Comparative Example 1, wherein activation and coupling: based on the synthesis scale N0, weigh the materials according to a certain feed ratio: Fmoc-Leu-OH (1.50eq), HOBT (1.50eq), PyBop (1.40eq); add them to a beaker, measure an appropriate amount of DMF, stir to dissolve, add DIPEA (2.80eq), activate the reaction for 3 minutes, and then add it to the polypeptide solid phase synthesis tube; at room temperature of 20-35°C, couple the reaction for 0.5-1.0 hour to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin; cleave to obtain a crude NH2-PEG4-RK(Dde)-OH product, with a crude product yield of 113.96% and a crude product purity of 68.527%.

[0108] (3) Preparation of refined NH2-PEG4-RK(Dde)-OH

[0109] The purification conditions were the same as those in Comparative Example 1, and the process was roughly the same as that in Comparative Example 1. The total yield (CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH refined product) was calculated to be 45.58%. The mixture was labeled, sealed, and stored in a freezer. The purity of the refined product was 95.443%.

[0110] Comparative Example 4 Preparation of NH2-PEG4-RK(Dde)-OH by Improved Solid Phase Synthesis and One-by-One Connection Method

[0111] (1) The preparation of the intermediate CTC-Lys(Dde) was the same as that in Comparative Example 1.

[0112] (2) Preparation of crude NH2-PEG4-RK(Dde)-OH The reaction was the same as in Comparative Example 1, and the feed amounts were shown in Table 10:

[0113] Table 10 Comparative Example 4 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0114]

[0115] The process is roughly the same as that of Comparative Example 1, wherein activation and coupling are performed: based on the synthesis scale N0, the materials are weighed according to a certain feed ratio: Fmoc-Leu-OH (3.00eq), HATU (3.00eq), and HOAT (3.60eq); added to a beaker, an appropriate amount of DMF is measured, stirred to dissolve, DIPEA (6.00eq) is added, and the activation reaction is performed for 3 minutes before adding to the polypeptide solid phase synthesis tube; the room temperature is 20-35°C, and the coupling reaction is performed for 0.5-1.0 hours to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin; and the crude product NH2-PEG4-RK(Dde)-OH is obtained, with a crude product yield of 112.88% and a crude product purity of 70.138%.

[0116] (3) Preparation of refined NH2-PEG4-RK(Dde)-OH

[0117] The purification conditions were the same as those in Comparative Example 1, and the process was roughly the same as that in Comparative Example 1. The total yield (CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH refined product) was calculated to be 47.57%. The product was labeled, sealed, and stored in a freezer. The purity of the refined product was 95.477%.

[0118] Comparative Example 5: Preparation of NH2-PEG4-RK(Dde)-OH by Improved Solid Phase Synthesis and Connection

[0119] The material molar ratio of CTC-Lys(Dde) resin (substitution degree, 0.43 mmol / g), protected amino acid, HBTU, and DIPEA is 1:3:3:3.

[0120] The preparation reaction of the intermediate CTC-Lys (Dde) is the same as that of Comparative Example 1, and the feed amounts are shown in Table 11:

[0121] Table 11 Comparative Example 5 Preparation of Intermediate CTC-Lys(Dde) Feeding Amount

[0122]

[0123] The process is substantially the same as that of Comparative Example 1; CTC-Lys (Dde) resin is obtained, and the substitution degree of CTC-Lys (Dde) resin is detected to be 0.43 mmol / g.

[0124] The preparation reaction of the crude NH2-PEG4-RK(Dde)-OH product is the same as that of Comparative Example 1, and the feed amounts are shown in Table 12:

[0125] Table 12 Comparative Example 5 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0126]

[0127] The process is roughly the same as that of Comparative Example 1; activation and coupling: taking the synthesis scale N0 as the base number, weigh the materials according to a certain feed ratio: Fmoc-Leu-OH (3.00eq), HBTU (3.00eq); add them to a beaker, measure an appropriate amount of DMF, stir to dissolve, add DIPEA (3.00eq), activate the reaction for 3 minutes, and then add it to the polypeptide solid phase synthesis tube; at room temperature of 20-35°C, couple the reaction for 0.5-1.0 hour to obtain CTC-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin; obtain the crude product NH2-PEG4-RK(Dde)-OH, the crude product yield is 113.34%, and the crude product purity is 75.911%.

[0128] (3) Preparation of refined NH2-PEG4-RK(Dde)-OH

[0129] The purification conditions were the same as those in Comparative Example 1, and the process was roughly the same as that in Comparative Example 1. The total yield (CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH refined product) was calculated to be 51.24%. The product was labeled, sealed, and stored in a frozen state. The purity of the refined product was 95.867%.

[0130] The results of the solid phase synthesis of NH2-PEG4-RK(Dde)-OH in Comparative Examples 1-5 are summarized in Table 13:

[0131] Table 13 Results of the solid phase synthesis of NH2-PEG4-RK(Dde)-OH in Comparative Examples 1-5

[0132]

[0133] Wherein, resin: CTC-Lys(Dde) resin; AA: protected amino acid.

[0134] It can be seen from the above table that reducing the substitution degree of CTC-Lys(Dde) resin can improve the purity of the crude NH2-PEG4-RK(Dde)-OH, but the purity of the crude product is still less than 80%, and reducing the substitution degree will inevitably increase the production cost.

[0135] Example 1 Segmental Method A: Preparation of NH2-PEG4-RK(Dde)-OH

[0136] Process route:

[0137] Preparation of Wang-(9-7)-NH2:

[0138]

[0139] Preparation of Fmoc-(1-6)-OH:

[0140]

[0141] Preparation of NH2-PEG4-RK(Dde)-OH:

[0142]

[0143] Preparation of Wang-(9-7)-NH2

[0144] Preparation of intermediate Wang-Lys (Dde), feed amounts are shown in Table 14:

[0145] Table 14 Example 1 Preparation of intermediate Wang-Lys (Dde) Feeding amount

[0146]

[0147] Process:

[0148] Washing and swelling: Weigh Wang resin and add it to a peptide solid phase synthesis tube. Measure N,N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the uniform dispersion of the resin in the solvent. Generally, the amount of solvent is 4.0-6.0eq of the resin, the same below). Wash 3 times (N2 bubbling, 1 min each time, and remove after washing); Add DMF to the peptide solid phase synthesis tube, bubble with N2 for 60 min, and drain the liquid after sufficient swelling; Coupling: Based on the molar amount of Wang resin N0, weigh Fmoc-Lys(Dde)-OH, HOBt, and DMAP, measure an appropriate amount of DMF, add it to a beaker, weigh DIC, add it to a beaker under stirring, activate for 10 min, develop and clarify, and add it to the peptide solid phase synthesis tube. Into a tube; coupling reaction for 7.0~8.0h, the reaction solution was removed, DMF was added, and washed 3 times (N2 bubbling, 1min each time, and the liquid was drained after the end); blocking: an appropriate amount of pyridine: acetic anhydride = 1:1 mixture was added to the polypeptide solid phase synthesis tube, and the blocking reaction was repeated twice (N2 bubbling, 30min each time, and the liquid was drained after the end); shrinking: an appropriate amount of methanol was added to the polypeptide solid phase synthesis tube, and the tube was shrunk 3 times (N2 bubbling, the time was 5min, 5min, and 10min respectively, and the liquid was drained after the end); drying: drying with nitrogen flow for 4.0~5.0h until the resin particles were no longer sticky and were in the form of quicksand; Wang-Lys (Dde) resin was obtained, and the substitution degree of Wang-Lys (Dde) resin was detected to be 0.67mmol / g.

[0149] (1.2) Preparation of Wang-(9-7)-NH2, feed amounts are shown in Table 15:

[0150] Table 15 Example 1 Preparation of Wang-(9-7)-NH2 Feeding Amount

[0151]

[0152] Process:

[0153] Washing and swelling: Weigh Wang-Lys (Dde) resin, add it to the peptide solid phase synthesis tube, weigh N,N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the uniform dispersion of the resin in the solvent. Generally, the amount of solvent is 4.0-6.0eq of the amount of resin, the same below), wash twice (N2 bubbling, 1 min each time, and remove after washing); Add DMF to the peptide solid phase synthesis tube, bubble N2 for 30 min, and drain the liquid after sufficient swelling;

[0154] Coupling Fmoc-Leu-OH: Removal of Fmoc group, color development: Add 20% piperidine solution to the peptide solid phase synthesis tube, remove Fmoc twice (N2 bubbling, time is 5min and 7min respectively, and the liquid is drained after completion), wash with DMF 6 times (N2 bubbling, 1min each time, and drain after washing); Kaiser method (take a small amount of resin, wash with DMF, add 3-5 drops of ninhydrin solution, heat at 120℃ for 3min; observe whether color is developed), detect the color change of resin; activation, coupling, color development, washing: take the synthesis scale N0 as the base number, weigh the materials according to a certain feed ratio: Fmoc-Leu-OH (3.0eq), HBTU (3.0eq); add Into a beaker, take an appropriate amount of DMF, stir to dissolve, add DIPEA (3.0eq), activate the reaction for 3 minutes, then add it to the peptide solid phase synthesis tube; room temperature 20-35 ° C, coupling reaction 0.5~1.0 hour; Kaiser method (take a small amount of resin, after DMF washing, add 3-5 drops of ninhydrin solution, heat at 120 ° C for 3 minutes; observe whether color is developed to determine whether the reaction is complete. If color is developed, continue coupling for 0.5 hours); drain the reaction solution, wash with DMF 3 times (N2 bubbling, 1 minute each time, and drain after washing);

[0155] Repeat the above operation to couple Fmoc-Arg(Pbf)-OH according to the dosage in the material list;

[0156] Removal of Fmoc group and color development: the operation is the same as before; shrinkage: add an appropriate amount of methanol to the polypeptide solid phase synthesis tube and shrink it 3 times (N2 bubbling, the time is 5min, 5min, 10min respectively, and the liquid is drained after the end); drying: nitrogen flow blowing drying for 4.0~5.0h, until the resin particles are no longer sticky and are in the form of quicksand, and Wang-Lys(Dde)-Leu-Arg(Pbf)-NH2 resin is obtained.

[0157] (2) Preparation of Fmoc-(1-6)-OH

[0158] (2.1) Preparation of intermediate CTC-Leu, feed amounts are shown in Table 16:

[0159] Table 16 Example 1 Preparation of intermediate CTC-Leu Feeding amount

[0160]

[0161] Process: The preparation of CTC-Lys (Dde) is roughly the same as that in Comparative Example 1, wherein coupling: based on the molar amount of CTC resin N0, weigh Fmoc-Leu-OH, measure an appropriate amount of DMF, add them to a beaker, stir to dissolve, and then add them to a polypeptide solid phase synthesis tube; weigh DIPEA and add it to a polypeptide solid phase synthesis tube; couple the reaction for 2.0 to 3.0 hours, remove the reaction solution, add DMF, and wash 3 times (N2 bubbling, 1 minute each time, and drain the liquid after the end); obtain CTC-Leu resin, and the substitution degree of CTC-Leu resin is detected to be 0.78 mmol / g.

[0162] (2.2) Preparation of Fmoc-(1-6)-OH, feed amounts are shown in Table 17:

[0163] Table 17 Example 1 Preparation of Fmoc-(1-6)-OH Feed Amount

[0164]

[0165] Process: washing and swelling: weigh CTC-Leu resin, add it to the peptide solid phase synthesis tube, weigh N,N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the resin being evenly dispersed in the solvent. In general, the amount of solvent is 4.0-6.0eq of the resin, the same below), wash twice (N2 bubbling, 1 min each time, and remove after washing); add DMF to the peptide solid phase synthesis tube, bubble N2 for 30 min, and drain the liquid after sufficient swelling; coupling Fmoc-Glu(OtBu)-OH: remove Fmoc group, color development: add 20% piperidine solution to the peptide solid phase synthesis tube, remove Fmoc 2 times (N2 bubbling, time is 5min, 7min respectively, after finishing, drain the liquid), wash with DMF 6 times (N2 bubbling, 1min each time, drain after washing); Kaiser method (take a small amount of resin, after DMF washing, add 3-5 drops of ninhydrin solution, heat at 120℃ for 3min; observe whether color is developed), detect the color change of resin; activation, coupling, color development, washing, based on the synthesis scale N0, weigh the materials according to a certain feed ratio: Fmoc-Glu(OtBu)-OH (3.0eq), HBTU (3.0eq) ; Add to a beaker, take an appropriate amount of DMF, stir to dissolve, add DIPEA (3.0eq), activate the reaction for 3 minutes, and then add to the peptide solid phase synthesis tube; room temperature 20-35 ° C, coupling reaction 0.5~1.0 hour; Kaiser method (take a small amount of resin, after DMF washing, add 3-5 drops of ninhydrin solution, heat at 120 ° C for 3 minutes; observe whether color is developed to determine whether the reaction is complete. If color is developed, continue coupling for 0.5 hours); drain the reaction solution and wash with DMF 3 times (N2 bubbling, 1 minute each time, and drain after washing);

[0166] According to the dosage in the material list, repeat the above operation to couple the following three amino acid fragments and Fmoc-PEG4-propionic acid fragment: coupling Fmoc-Trp(Boc)-OH; coupling Fmoc-Asn(Trt)-OH; coupling Fmoc-Arg(Pbf)-OH; coupling Fmoc-PEG4-propionic acid.

[0167] Contraction: Add appropriate amount of methanol to the peptide solid phase synthesis tube and contract it 3 times (N2 bubbling, the time is 5min, 5min, 10min respectively, and the liquid is drained after the end); Drying: Dry with nitrogen flow for 4.0~5.0h until the resin particles are not sticky and are in the form of quicksand, and CTC-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH-Fmoc resin is obtained; Cleavage; Preparation of cleavage solution: Prepare cleavage solution at a ratio of 10ml / 1g resin: (TFA: D CM=1:99v / v), mix well, and freeze to 5±5℃; pre-freeze ether: prepare the required ether according to the ratio of 10ml / 1g resin, freeze it in a freezer for more than 2 hours, and set aside; add CTC-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH-Fmoc resin to the reaction bottle, slowly add the prepared pre-cooled lysis solution under stirring, stir well, and react at room temperature 20-30℃ for 2.0~3.0hr; filter and collect the filtrate. Wash the filter cake once with a small amount of lysis solution. Combine the filtrate; the filtrate is concentrated to dryness under reduced pressure in a water bath at <50℃, add frozen ether, precipitate the product, stir for 20min, filter, and add appropriate amount of frozen ether to fully wash more than 3 times; the obtained product is vacuum dried at room temperature 20-30℃ for 6.0~7.0h to obtain the product Fmoc-(1-6)-OH, with a yield of 100.21%. The purity of the product is 81.824%.

[0168] Preparation of crude NH2-PEG4-RK(Dde)-OH, feed amounts are shown in Table 18:

[0169] Table 18 Example 1 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0170]

[0171] Process:

[0172] Washing and swelling: weigh Wang-Lys(Dde)-Leu-Arg(Pbf)-NH2 resin, add it to the peptide solid phase synthesis tube, weigh N,N-dimethylformamide (DMF) and add it to the peptide solid phase synthesis tube (the amount of solvent is based on the uniform dispersion of the resin in the solvent. Generally, the amount of solvent is 4.0-6.0eq of the amount of resin, the same below), wash twice (N2 bubbling, 1 min each time, and remove after washing); add DMF to the peptide solid phase synthesis tube, N2 bubble for 30 min, and drain the liquid after sufficient swelling; coupling Fmoc-(1-6)-OH: remove Fmoc group, color development: add 20% piperidine solution to the peptide solid phase synthesis tube, remove Fmoc 2 times (N2 bubbling, time is 5min and 7min respectively, and the liquid is drained after the end), washed with DMF 6 times (N2 bubbling, 1min each time, and drained after washing); Kaiser method (take a small amount of resin, wash with DMF, add 3-5 drops of ninhydrin solution, heat at 120℃ for 3min; observe whether color is developed), detect the color change of resin; activation, coupling, color development, washing: based on the synthesis scale N0, weigh the materials according to a certain feed ratio: Fmoc-(1-6)-OH (1.5eq), HATU (2.0eq) HAOt (2.0eq); add to a beaker, take an appropriate amount of DMF, stir to dissolve, add DIPEA (2.0eq), activate the reaction for 3 minutes, and then add to the peptide solid phase synthesis tube; room temperature 20-35℃, coupling reaction 0.5~1.0 hour; Kaiser method (take a small amount of resin, wash with DMF, add 3-5 drops of ninhydrin solution, heat at 120℃ for 3 minutes; observe whether color is developed to determine whether the reaction is complete.If color is developed, continue coupling for 0.5 hours); drain the reaction solution and wash with DMF 3 times (N2 bubbling, 1 minute each time, drain after washing); remove Fmoc group and develop color: the operation is the same as before; shrink: add appropriate amount of methanol to the peptide solid phase synthesis tube, shrink 3 times (N2 bubbling, the time is 5 minutes, 5 minutes, 10 minutes respectively, and drain the liquid after the end); dry: dry with nitrogen flow for 4.0~5.0 hours until the resin particles are not sticky and are in the form of quicksand, and obtain Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin; cleavage: preparation of cleavage solution :Prepare the lysis solution (TFA:TIPS:H2O=95:2.5:2.5v / v) at a ratio of 10ml / 1g resin, mix well, and freeze to 5±5℃; Prefreeze ether: Prepare the required ether at a ratio of 70ml / 1g resin, freeze it in a freezer for more than 2 hours, and set aside; Add Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-Asn(Trt)-Arg(Pbf)-PEG4-NH2 resin to the reaction bottle, slowly add the prepared pre-cooled lysis solution under stirring, stir well, and react at room temperature 20-30℃ for 2.0~3.0hr; Filter and collect the filtrate. Wash the filter cake once with a small amount of lysis solution. Combine the filtrates; add ice-cold ether (4-5 ml ether / 1 ml lysate) to the reaction flask, slowly add the filtrate under stirring to precipitate the product, stir for 20 min, filter, and wash thoroughly with appropriate amount of ice-cold ether for more than 3 times; the obtained product is vacuum dried at room temperature 20-30 ° C for 6.0-7.0 h to obtain crude NH2-PEG4-RK(Dde)-OH, with a crude product yield of 103.88% and a crude product purity of 80.896%.

[0173] Preparation of NH2-PEG4-RK(Dde)-OH refined product, feed amount see Table 19:

[0174] Table 19 Example 1 Preparation of NH2-PEG4-RK(Dde)-OH Refined Product Feeding Amount

[0175]

[0176] The purification conditions are the same as those of Comparative Example 1; process: dissolution and filtration of crude NH2-PEG4-RK(Dde)-OH: ultrasonically dissolve the crude NH2-PEG4-RK(Dde)-OH with 200 ml of diluent (90% mobile phase A-10% mobile phase B); place a 0.22 µm microporous glass fiber filter membrane in the funnel, fully wet it, add the crude NH2-PEG4-RK(Dde)-OH solution, connect the vacuum pump, open the control valve, filter, and replace the filter paper according to the actual situation. After the last filtration of the crude NH2-PEG4-RK(Dde)-OH solution, add 100 ml of diluent to the filter and filter, and combine the filtrate with the crude NH2-PEG4-RK(Dde)-OH solution; Loading: Load the crude NH2-PEG4-RK(Dde)-OH solution; Elution: Perform linear gradient elution according to the purification gradient; Collecting: Collect the target peak in segments (RK retention time is about 46 minutes), and save the preparation chromatogram; Merge: Combine samples with a purity of less than 97.0% into a repurified sample; Concentrate: Combine and concentrate the sample fractions with a purity greater than 98.0%; Freeze-dry: Dispense the combined target peptide solution into freeze-drying trays, with a thickness of 1.0~1.5 cm; product enters the box, locks the box door, quickly freezes the product to below -45℃ and keeps it warm for 2 hours, turns on the vacuum pump to evacuate the front box, the vacuum degree is <50Pa, and sublimates according to the following curve: set the heat transfer oil to 0℃ in 2 hours and keep it for more than 12 hours; set the heat transfer oil to 25℃ in 2 hours and keep it for more than 6 hours; release the vacuum, open the vacuum leak valve to release the vacuum in the box to normal pressure. Sampling and packaging: release the vacuum, open the vacuum leak valve to release the vacuum in the box to normal pressure. Open the box door and quickly take out the freeze-dried plate from the box. Immediately take samples according to the sampling operation procedures after leaving the box. Transfer the drug to a low-density ethylene bottle for drug packaging, the total yield (Wang-Lys(Dde)-Leu-Arg(Pbf) resin→NH2-PEG4-RK(Dde)-OH refined product): 62.37%, label it, seal it, and store it in the freezer. The purity of the refined product is; 98.040%.

[0177] Example 2 Segmental Method B: Preparation of NH2-PEG4-RK(Dde)-OH

[0178] Process route:

[0179] Preparation of Wang-(9-6)-NH2:

[0180]

[0181] Preparation of Fmoc-(1-5)-OH:

[0182]

[0183] Preparation of NH2-PEG4-RK(Dde)-OH:

[0184]

[0185] (1) Preparation of Wang-(9-6)-NH2

[0186] (1.1) Preparation of the intermediate Wang-Lys(Dde)-OH, same as in Example 1:

[0187] (1.2) Preparation of Wang-(9-6)-NH2, feed amounts are shown in Table 20:

[0188] Table 20 Example 2 Preparation of Wang-(9-6)-NH2 Feeding Amount

[0189]

[0190] The process is roughly the same as that of preparing Wang-(9-7)-NH2 in Example 1 to obtain Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-NH2 resin.

[0191] (2) Preparation of Fmoc-(1-5)-OH

[0192] The preparation method is roughly the same as that of Fmoc-(1-6)-OH in Example 1.

[0193] (2.1) Preparation of CTC-Glu(OtBu), feed amounts are shown in Table 21:

[0194] Table 21 Example 2 Preparation of CTC-Glu (OtBu) Feeding Amount

[0195]

[0196] CTC-Glu(OtBu) resin was obtained, and the substitution degree of CTC-Glu(OtBu) resin was detected to be 0.60 mmol / g.

[0197] (2.2) Preparation of Fmoc-(1-5)-OH, feed amounts see Table 22:

[0198] Table 22 Example 2 Preparation of Fmoc-(1-5)-OH Feed Amount

[0199]

[0200] The product Fmoc-(1-5)-OH was obtained with a yield of 100.66% and a product purity of 92.969%.

[0201] (3) Preparation of crude NH2-PEG4-RK(Dde)-OH

[0202] The preparation method is roughly the same as that of Example 1 for preparing NH2-PEG4-RK(Dde)-OH, and the feed amounts are shown in Table 23:

[0203] Table 23 Example 2 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0204]

[0205] The crude product NH2-PEG4-RK(Dde)-OH was obtained with a crude product yield of 101.10% and a crude product purity of 91.266%.

[0206] (4) Preparation of refined NH2-PEG4-RK(Dde)-OH. The purification conditions were the same as those in Comparative Example 1, and the process was substantially the same as that in Comparative Example 1, wherein the crude NH2-PEG4-RK(Dde)-OH was dissolved in a diluent, filtered, and then loaded onto a sample. Elution was performed according to a purification gradient, and the target peak was collected. The receiving solutions with a purity greater than 98.0% were combined and concentrated under reduced pressure, and then packaged into freeze-drying plates. The samples were freeze-dried according to the freeze-drying curve. The samples were weighed after freeze-drying, and the total yield (Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu resin→NH2-PEG4-RK(Dde)-OH refined product) was 76.81%. The samples were labeled, sealed, and stored frozen. The purity of the refined product was 99.602%.

[0207] Example 3 Segmental Method C: Preparation of NH2-PEG4-RK(Dde)-OH

[0208] Process route:

[0209] Preparation of Wang-(9-4)-NH2:

[0210]

[0211] Preparation of Fmoc-(1-3)-OH:

[0212]

[0213] Preparation of NH2-PEG4-RK(Dde)-OH:

[0214]

[0215] (1) Preparation of Wang-(9-4)-NH2:

[0216] The preparation method is roughly the same as the method for preparing Wang-(9-7)-NH2 in Example 1, and the feed amounts are shown in Table 24:

[0217] Table 24 Example 3 Preparation of Wang-(9-4)-NH2 Feeding Amount

[0218]

[0219] Obtain Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-NH2 resin.

[0220] (2) Preparation of Fmoc-(1-3)-OH

[0221] The preparation method is roughly the same as that of Example 1 for preparing Fmoc-(1-6)-OH.

[0222] (2.1) Preparation of CTC-Asn(Trt), feed amounts are shown in Table 25:

[0223] Table 25 Example 3 Preparation of CTC-Asn (Trt) Feeding Amount

[0224]

[0225] CTC-Asn(Trt) resin was obtained, and the substitution degree of CTC-Asn(Trt) resin was detected to be 0.58 mmol / g.

[0226] (2.2) Preparation of Fmoc-(1-3)-OH, feed amounts see Table 26:

[0227] Table 26 Example 3 Preparation of Fmoc-(1-3)-OH Feed Amount

[0228]

[0229] The product Fmoc-(1-3)-OH was obtained in an amount of 22.08 g, with a yield of 101.32% and a product purity of 97.971%.

[0230] (3) Preparation of crude NH2-PEG4-RK(Dde)-OH

[0231] The preparation method is roughly the same as that of Example 1 for preparing NH2-PEG4-RK(Dde)-OH, and the feed amounts are shown in Table 27:

[0232] Table 27 Example 3 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0233]

[0234] The crude product NH2-PEG4-RK(Dde)-OH was obtained with a crude product yield of 105.18% and a crude product purity of 75.171%.

[0235] Preparation of NH2-PEG4-RK(Dde)-OH refined product, feed amount see Table 28:

[0236] Table 28 Example 3 Preparation of NH2-PEG4-RK(Dde)-OH Refined Product Feeding Amount

[0237]

[0238] The purification conditions are the same as those of Comparative Example 1, and the process is roughly the same as that of Comparative Example 1, wherein the crude NH2-PEG4-RK(Dde)-OH is dissolved in a diluent, filtered, and loaded, eluted according to the purification gradient, and the target peak is collected. The receiving solution with a purity greater than 98.0% is combined and concentrated under reduced pressure, and then divided into freeze-drying plates, and freeze-dried according to the freeze-drying curve. Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-Glu(OtBu)-Trp(Boc)-NH2 resin→NH2-PEG4-RK(Dde)-OH The total yield of the refined product is 41.78%, and the label is affixed, sealed, and frozen for storage. The purity of the refined product is 97.676%.

[0239] Example 4 Segmental Method D: Preparation of NH2-PEG4-RK(Dde)-OH

[0240] Process route:

[0241] Preparation of Wang-(9-7)-NH2: the same as the preparation of Wang-(9-7)-NH2 in Example 1

[0242] Preparation of Fmoc-(4-6)-OH:

[0243]

[0244] Preparation of Fmoc-(1-3)-OH: the same as the preparation of Fmoc-(1-3)-OH in Example 3

[0245] Preparation of NH2-PEG4-RK(Dde)-OH:

[0246]

[0247] (1) Preparation of Wang-(9-7)-NH2

[0248] The preparation method is shown in the preparation of Wang-(9-7)-NH2 in Example 1.

[0249] (2) Preparation of Fmoc-(4-6)-OH

[0250] The preparation method is roughly the same as that of Fmoc-(1-6)-OH in Example 1, and the feed amounts are shown in Table 29:

[0251] Table 29 Example 4 Preparation of Fmoc-(4-6)-OH Feed Amount

[0252]

[0253] The product Fmoc-(4-6)-OH was obtained with a yield of 102.57% and a product purity of 95.888%.

[0254] (3) Preparation of Fmoc-(1-3)-OH

[0255] The preparation method is shown in the preparation of Fmoc-(1-3)-OH in Example 3.

[0256] (4) Preparation of crude NH2-PEG4-RK(Dde)-OH

[0257] The preparation method is roughly the same as that of NH2-PEG4-RK(Dde)-OH in Example 1, and the feed amounts are shown in Table 30:

[0258] Table 30 Example 4 Preparation of NH2-PEG4-RK(Dde)-OH Crude Product Feeding Amount

[0259]

[0260] The crude product NH2-PEG4-RK(Dde)-OH was obtained with a crude product yield of 102.55% and a crude product purity of 92.793%.

[0261] Preparation of NH2-PEG4-RK(Dde)-OH refined product, feed amount see Table 31:

[0262] Table 31 Example 4 Preparation of NH2-PEG4-RK(Dde)-OH Refined Product Feeding Amount

[0263]

[0264] The purification conditions are the same as those of Comparative Example 1, and the process is roughly the same as that of Comparative Example 1, wherein the crude NH2-PEG4-RK(Dde)-OH is dissolved in a diluent, filtered, and loaded, eluted according to the purification gradient, and the target peak is collected. The receiving solution with a purity greater than 98.0% is combined and concentrated under reduced pressure, and then divided into freeze-drying trays, freeze-dried according to the freeze-drying curve, labeled, sealed, and frozen for storage. Wang-Lys(Dde)-Leu-Arg(Pbf)-NH2 resin → NH2-PEG4-RK(Dde)-OH The total yield of the refined product is 77.17%, which is labeled, sealed, and frozen for storage. The purity of the refined product is 99.186%.

[0265] Example 5 Segmental Method E: Preparation of NH2-PEG4-RK(Dde)-OH

[0266] Process route

[0267] Preparation of Wang-(9-6)-NH2: Same as the preparation of Wang-(9-6)-NH2 in Example 2

[0268] Preparation of Fmoc-(4-5)-OH

[0269]

[0270] Preparation of Fmoc-(1-3)-OH: the same as the preparation of Fmoc-(1-3)-OH in Example 3

[0271] Preparation of NH2-PEG4-RK(Dde)-OH

[0272]

[0273] (1) Preparation of Wang-(9-6)-NH2

[0274] The preparation method is shown in Example 2 for the preparation of Wang-(9-6)-NH2.

[0275] (2) Preparation of Fmoc-(4-5)-OH

[0276] The preparation method is roughly the same as that of Fmoc-(1-5)-OH in Example 2, and the feed amounts are shown in Table 32:

[0277] Table 32 Example 5 Preparation of Fmoc-(4-5)-OH Feed Amount

[0278]

[0279] The product Fmoc-(4-5)-OH was obtained with a yield of 100.92% and a product purity of 98.556%.

[0280] (3) Preparation of Fmoc-(1-3)-OH

[0281] The preparation method is shown in Example 3 for the preparation of Fmoc-(1-3)-OH

[0282] (4) Preparation of crude NH2-PEG4-RK(Dde)-OH

[0283] The preparation method is roughly the same as that of NH2-PEG4-RK(Dde)-OH in Example 1, and the feed amounts are shown in Table 33:

[0284] Table 33 Example 5 Preparation of NH2-PEG4-RK(Dde)-OH crude product Feeding amount

[0285]

[0286] The crude product NH2-PEG4-RK(Dde)-OH was obtained with a crude yield of 102.00% and a crude purity of 85.159%.

[0287] (5) Preparation of refined NH2-PEG4-RK(Dde)-OH

[0288] The purification conditions are the same as those of Comparative Example 1, and the process is roughly the same as that of Comparative Example 1, wherein the crude NH2-PEG4-RK(Dde)-OH is dissolved in a diluent, filtered, and loaded, eluted according to the purification gradient, and the target peak is collected. The receiving solution with a purity greater than 98.0% is combined and concentrated under reduced pressure, and then divided into freeze-drying plates, and freeze-dried according to the freeze-drying curve. The yield of the refined product of CTC-Lys(Dde)→NH2-PEG4-RK(Dde)-OH is 70.15%, and the product is labeled, sealed, and frozen for storage. The purity of the refined product is 98.313%.

[0289] Example 1-5 The results of the segmented synthesis of NH2-PEG4-RK(Dde)-OH are shown in Table 34:

[0290] Table 34 Summary of the results of the segmented synthesis of NH2-PEG4-RK(Dde)-OH in Examples 1-5

[0291]

[0292] In the synthesis of NH2-PEG4-RK(Dde)-OH, since the amino acids at positions 2, 3, 4, and 5 carry consecutive protecting groups, it is easy to form a difficult sequence. In the process of coupling one by one, it is easy to form missing peptides and misconnected peptides, which leads to an increase in impurities in the crude NH2-PEG4-RK(Dde)-OH.

[0293] From the above table, it can be seen that the segmented method for synthesizing NH2-PEG4-RK(Dde)-OH can overcome difficult sequences and is a very effective method for improving the purity of the product. Through the detection of crude products obtained by different segmented methods, it was found that the two-stage method B of 4+5 and the three-stage method D of 3+3+3 have significant advantages, and the method is relatively easy to operate, and the purity of the obtained crude product is >90%. Purification of the crude products obtained by different segmented methods shows that the purity of the refined products after purification by segmented methods B and D is >99.0%, and the total yield is also >75%. This method has low production cost, simple process and easy operation, and is suitable for industrial production.

[0294] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for synthesizing NH2-PEG4-RK(Dde)-OH, characterized in that: NH2-PEG4-RK(Dde)-OH is synthesized by a segmented synthesis method, wherein the segmented synthesis method is a two-stage method or a three-stage method; The two-stage method uses Glu as a carbon-terminal site to synthesize NH2-PEG4-RK(Dde)-OH through two synthetic fragments: the fragment Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-NH2 and the fragment Fmoc-NH-PEG4-Arg(Pbf)-Asn(Trt)-Trp(Boc)-Glu(OtBu)-OH; The three-stage method uses Asn and Leu as two carbon-terminal sites, respectively, so that NH2-PEG4-RK(Dde)-OH is synthesized through three synthetic fragments: fragment Wang-Lys(Dde)-Leu-Arg(Pbf)-NH2, fragment Fmoc-Trp(Boc)-Glu(OtBu)-Leu-OH and fragment Fmoc-NH-PEG4-Arg(Pbf)-Asn(Trt)-OH; The synthetic fragments are coupled via a condensing agent.

2. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 1, characterized in that: The synthesis method of the fragment Wang-Lys(Dde)-Leu-Arg(Pbf)-Leu-NH2 is to react Wang-Lys(Dde) with protected amino acid, HBTU and DIPEA.

3. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 2, characterized in that: The Wang-Lys(Dde) synthesis method comprises reacting Wang resin with Fmoc-Lys(Dde)-OH, HOBt, DIC and DMAP.

4. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 1, characterized in that: The synthesis method of the fragment Fmoc-NH-PEG4-Arg(Pbf)-Asn(Trt)-Trp(Boc)-Glu(OtBu)-OH is to react CTC-Glu(OtBu) with protected amino acid, HBTU and DIPEA; after the reaction, CTC resin lysis solution is used to elute to obtain the product.

5. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 4, characterized in that: The synthesis method of CTC-Glu(OtBu) is to react CTC resin with Fmoc-Glu(OtBu)-OH and DIPEA.

6. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 1, characterized in that: The synthesis method of the fragment Wang-Lys(Dde)-Leu-Arg(Pbf)-NH2 is to react Wang-Lys(Dde) with protected amino acid, HBTU and DIPEA.

7. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 1, characterized in that: The synthesis method of the fragment Fmoc-Trp(Boc)-Glu(OtBu)-Leu-OH is to react CTC-Leu with protected amino acid, HBTU and DIPEA; after the reaction, the product is eluted with CTC resin lysis solution.

8. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 7, characterized in that: The synthesis method of CTC-Leu is to react CTC resin with Fmoc-Leu-OH and DIPEA.

9. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 1, characterized in that: The synthesis method of the fragment Fmoc-NH-PEG4-Arg(Pbf)-Asn(Trt)-OH is to react CTC-Asn(Trt) with protected amino acid, HBTU and DIPEA; after the reaction, CTC resin lysis solution is used to elute to obtain the product.

10. The method for synthesizing NH2-PEG4-RK(Dde)-OH according to claim 9, characterized in that: The synthesis method of CTC-Asn(Trt) is to react CTC resin with Fmoc-Asn(Trt)-OH and DIPEA.

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