A method for preparing polypeptide heterodimer

Abstract

The present application discloses a method for preparing a polypeptide heterodimer, comprising: preparing a compound of formula (II) or its salt using a compound of formula (I) or its salt; preparing a compound of formula (IV) or its salt using a compound of formula (III) or its salt; preparing a compound of formula (V) or its salt using a compound of formula (IV) or its salt; preparing a compound of formula (VI) or its salt using a compound of formula (V) or its salt; preparing a compound of formula (VII) or its salt using a compound of formula (VI) or its salt; preparing a compound of formula (VIII) or its salt using a compound of formula (VII) or its salt; preparing a compound of formula (IX) or its salt using a compound of formula (VIII) or its salt; and preparing a compound of formula (X) or its salt using a compound of formula (II) or its salt and a compound of formula (IX) or its salt.

Description

Technical Field

[0001] The present application relates to the field of polypeptide synthesis, and more specifically to a method for preparing polypeptide heterodimers. Background Art

[0002] As a non-invasive technique for visualizing, characterizing, and quantifying molecular biological processes in vivo, molecular imaging has gained importance in a wide range of diagnostic and therapeutic applications. An important issue in molecular imaging is the development of specific imaging tracers with high affinity and specificity, low nonspecific uptake, adequate retention, and effective permeability. Over the past few decades, a great deal of research has been conducted on radiolabeled peptides for diagnostic and therapeutic applications due to their favorable properties, including extraordinary tissue permeability, rapid clearance, low immunogenicity, ease of synthesis, and low cost.

[0003] However, peptides also have some major disadvantages that cannot be ignored, such as relatively low tumor affinity and short retention time. To overcome these shortcomings, a variety of approaches have been developed, among which heterodimeric peptides have become a very promising targeting strategy. Generally, heterodimeric peptides are composed of two covalently linked peptides that can bind to two different receptors simultaneously. Compared with monomeric peptides, bispecific heterodimeric peptides have a variety of superior properties, including enhanced efficacy, affinity, and relatively long blood circulation time, which ensures their application in imaging and therapy. Summary of the invention

[0004] In some embodiments, the present application provides a method for preparing a polypeptide heterodimer, wherein the polypeptide heterodimer is a compound of formula (X) or a salt thereof.

[0005] In some embodiments, a method for preparing a compound is provided, comprising: preparing a compound of formula (I) or a salt thereof; preparing a compound of formula (II) or a salt thereof from a compound of formula (I) or a salt thereof; preparing a compound of formula (III) or a salt thereof; preparing a compound of formula (IV) or a salt thereof from a compound of formula (III) or a salt thereof; preparing a compound of formula (V) or a salt thereof from a compound of formula (IV) or a salt thereof; preparing a compound of formula (VI) or a salt thereof from a compound of formula (V) or a salt thereof; preparing a compound of formula (VII) or a salt thereof from a compound of formula (VI) or a salt thereof; preparing a compound of formula (VIII) or a salt thereof from a compound of formula (VII) or a salt thereof; preparing a compound of formula (IX) or a salt thereof from a compound of formula (VIII) or a salt thereof; and preparing a compound of formula (X) or a salt thereof from a compound of formula (II) or a salt thereof and a compound of formula (IX) or a salt thereof.

[0006]

[0007]

[0008] In some embodiments, the compound of formula (II) or its salt is prepared from the compound of formula (I) or its salt, comprising contacting the compound of formula (I) or its salt with BCN-PEG4-NHS in acetonitrile at a pH of 7-8. In some embodiments, the compound of formula (IV) or its salt is prepared from the compound of formula (III) or its salt, comprising contacting the compound of formula (III) or its salt with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt in dichloromethane. In some embodiments, the compound of formula (V) or its salt is prepared from the compound of formula (IV) or its salt, comprising contacting the compound of formula (IV) or its salt with hydrazine hydrate in N,N-dimethylformamide. In some embodiments, the compound of formula (VI) or its salt is prepared from the compound of formula (V) or its salt, comprising contacting the compound of formula (V) or its salt with Fmoc-PEG4-propionic acid, PyBop and N,N-diisopropylethylamine in N,N-dimethylformamide. In some embodiments, the compound of formula (VII) or its salt is prepared from the compound of formula (VI) or its salt, comprising contacting the compound of formula (VI) or its salt with piperidine in N,N-dimethylformamide. In some embodiments, the compound of formula (VIII) or its salt is prepared from the compound of formula (VII) or its salt, comprising contacting the compound of formula (VII) or its salt with NOTA(tBu)2-N3 and PyBop in N,N-dimethylformamide. In some embodiments, the compound of formula (IX) or its salt is prepared from the compound of formula (VIII) or its salt, comprising contacting the compound of formula (VIII) or its salt, water and triisopropylsilane in trifluoroacetic acid.

[0009] In some embodiments, the compound of formula (III) or its salt is contacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt by reacting 90.3±10% by weight of the compound of formula (III) or its salt with 21.0±10% by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt at room temperature for 24 hours. In some embodiments, the compound of formula (IV) or its salt is contacted with hydrazine hydrate by reacting 80.2±10% by weight of the compound of formula (IV) or its salt with 8±10% by weight of hydrazine hydrate at 10-30°C for 5 hours. In some embodiments, the compound of formula (V) or its salt is contacted with Fmoc-PEG4-propionic acid, PyBop and N,N-diisopropylethylamine by mixing 64.7±10% by weight of the compound of formula (V) or its salt with 32.0±10% by weight of Fmoc-PEG4-propionic acid and 34.2±10% by weight of PyBop and adding 17.02±10% by weight of N,N-diisopropylethylamine dropwise for 2 hours at 10-30° C. In some embodiments, the compound of formula (VI) or its salt is contacted with piperidine by reacting 84.1±10% by weight of the compound of formula (VI) or its salt with 41.88±10% by weight of piperidine at 10-30° C. for 2 hours. In some embodiments, the compound of formula (VII) or its salt is contacted with NOTA (tBu) 2-N3 and PyBop by reacting 60.0 ± 10% by weight of the compound of formula (VII) or its salt with 25.2 ± 10% by weight of NOTA (tBu) 2-N3 and 27.3 ± 10% by weight of PyBop at 10-30°C for 2 hours. In some embodiments, the compound of formula (VIII) or its salt, water and triisopropylsilane are contacted by reacting 66.0 ± 10% by weight of the compound of formula (VIII) or its salt, 16.5 ± 10% by weight of water and 12.75 ± 10% by weight of triisopropylsilane at room temperature for 2-3 hours.

[0010] In some embodiments, the preparation of the compound of formula (I) or its salts comprises: contacting a first resin with Fmoc-amino acid, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a condensed first resin; contacting the condensed first resin with piperidine to obtain a deprotected first resin; contacting the deprotected first resin with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or its salts; and contacting the linear peptide of the compound of formula (I) or its salts with DMSO to obtain the compound of formula (I) or its salts. In some embodiments, the preparation of the compound of formula (III) or its salts comprises: contacting a second resin with Fmoc-amino acid, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a condensed second resin; contacting the condensed second resin with piperidine to obtain a deprotected second resin; and contacting the deprotected second resin with trifluoroacetic acid to obtain the compound of formula (III) or its salts.

[0011] In some embodiments, preparing the compound of formula (I) or its salt comprises: contacting a first resin with Fmoc-Cys(Trt)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a first resin after a first condensation; contacting the first resin after the first condensation with piperidine to obtain a first resin after a first deprotection; contacting the first resin after the first deprotection with Fmoc-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a second first resin after a condensation; contacting the first resin after the second condensation with piperidine to obtain a second first resin after a deprotection; contacting the first resin after the second deprotection with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a third first resin after a condensation; contacting the first resin after the third condensation with piperidine to obtain a third first resin after a deprotection; contacting the first resin after the third deprotection with Fmoc-Asn(Trt)-OH , 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a first resin after a fourth condensation; the first resin after the fourth condensation is contacted with piperidine to obtain a first resin after a fourth deprotection; the first resin after the fourth deprotection is contacted with Fmoc-Cys(Trt)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a first resin after a fifth condensation; the first resin after the fifth condensation is contacted with piperidine to obtain a first resin after a fifth deprotection; The first resin after the fifth deprotection is contacted with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the first resin after the sixth condensation; the first resin after the sixth condensation is contacted with piperidine to obtain the first resin after the sixth deprotection; the first resin after the sixth deprotection is contacted with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or its salt; and the linear peptide of the compound of formula (I) or its salt is contacted with DMSO to obtain the compound of formula (I) or its salt.In some embodiments, the preparation of the compound of formula (III) or its salts includes: contacting the second resin with Fmoc-Gly-OH, N,N-diisopropylethylamine, and then contacting with methanol to obtain a second resin after a first condensation; contacting the second resin after the first condensation with piperidine to obtain a second resin after a first deprotection; contacting the second resin after the first deprotection with Fmoc-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a second resin after a second condensation; contacting the second resin after the second condensation with piperidine to obtain a second resin after a second deprotection; contacting the second resin after the second deprotection with Fmoc-Lys(ivDde)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a third condensation. The second resin after the third condensation is contacted with piperidine to obtain the third deprotected second resin; the third deprotected second resin is contacted with Fmoc-D-Tyr(tBu)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fourth condensed second resin; the fourth condensed second resin is contacted with piperidine to obtain the fourth deprotected second resin; the fourth deprotected second resin is contacted with Fmoc-Asp(OtBu)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fifth condensed second resin; the fifth condensed second resin is contacted with piperidine to obtain the fifth deprotected second resin; and the fifth deprotected second resin is contacted with trifluoroacetic acid to obtain a compound of formula (III) or a salt thereof. In some embodiments, the first resin is a Rink Amide AM resin. In some embodiments, the second resin is a dichlorotrityl chloride resin. In some embodiments, the first resin is an unprotected or deprotected Rink Amide AM resin. In some embodiments, the second resin is an unprotected or deprotected dichlorotrityl chloride resin.

[0012] In some embodiments, the preparation of the compound of formula (X) or its salts further comprises: subjecting the crude product of the compound of formula (X) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 95% 0.3% acetic acid aqueous solution, 5% acetonitrile, 5 minutes: 88% 0.3% acetic acid aqueous solution, 12% acetonitrile, 85 minutes: 68% 0.3% acetic acid aqueous solution, 32% acetonitrile, and 100 minutes: 50% 0.3% acetic acid aqueous solution, 50% acetonitrile. In some embodiments, the preparation of the compound of formula (I) or its salts further comprises liquid chromatography purification. In some embodiments, the preparation of the compound of formula (I) or its salts further comprises: subjecting the crude product of the compound of formula (I) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 98% 0.05% trifluoroacetic acid aqueous solution, 2% acetonitrile, 60 minutes: 78% 0.05% trifluoroacetic acid aqueous solution, 22% acetonitrile, and 100 minutes: 100% acetonitrile. In some embodiments, the preparation of the compound of formula (II) or its salts further comprises subjecting the crude product of the compound of formula (II) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 95% of 0.1% trifluoroacetic acid aqueous solution, 5% of acetonitrile, 5 minutes: 85% of 0.1% trifluoroacetic acid aqueous solution, 15% of acetonitrile, and 75 minutes: 50% of 0.1% trifluoroacetic acid aqueous solution, 75% of acetonitrile. In some embodiments, the preparation of the compound of formula (IX) or its salts further comprises subjecting the crude product of the compound of formula (IX) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 95% of 0.1% trifluoroacetic acid aqueous solution, 5% of acetonitrile, 5 minutes: 90% of 0.1% trifluoroacetic acid aqueous solution, 15% of acetonitrile, and 60 minutes: 70% of 0.1% trifluoroacetic acid aqueous solution, 50% of acetonitrile. In some embodiments, the preparation of the compound of formula (X) or its salts further includes subjecting the trifluoroacetate salt of the compound of formula (X) to liquid chromatography gradient elution salt conversion, including: using 100 mmol / L ammonium acetate aqueous solution as the mobile phase for 30 minutes; using 0.1% acetic acid aqueous solution as the mobile phase for 20 minutes; and using 50% 0.1% acetic acid aqueous solution and 50% acetonitrile as the mobile phase.

[0013] In some embodiments, the present application provides the use of a compound of formula (I) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (II) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (III) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (IV) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (V) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (VI) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (VII) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the present application provides the use of a compound of formula (VIII) or its salt. In some embodiments, the present application provides the use of a compound of formula (IX) or its salt in the preparation of a compound of formula (X) or its salt. In some embodiments, the preparation of the compound of formula (X) is prepared by the preparation method disclosed in the present application.

[0014] In some embodiments, the present application provides the use of the preparation method disclosed in the present application in preparing radiolabeled compounds for diagnosis and treatment.

[0015] The preparation method of the present application provides a high-yield and effective preparation method. In addition, the selected purification method also provides a high-yield and high-purity purification method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The following is a reaction flow chart for preparing NGR (compound of formula (I)) in some embodiments of the present application.

[0017] Figure 2 In some embodiments of the present application, the reaction is performed to obtain NGR-PEG4-BCN (compound of formula (II)).

[0018] Figure 3 The following is a reaction flow chart for preparing FP-(ivDde)-RGD (compound of formula (III)) in some embodiments of the present application.

[0019] Figure 4 The following is a reaction flow chart for preparing N3-NOTA-PEG4-c-RGD (compound of formula (IX)) in some embodiments of the present application.

[0020] Figure 5In some embodiments of the present application, the reaction obtains NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X)). DETAILED DESCRIPTION

[0021] In order to further explain the technical means and effects adopted by the present application to achieve the predetermined purpose, the specific implementation methods, structures, features and effects of the present application are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0022] Example 1

[0023] (I) Preparation of NGR

[0024] Please refer to Figure 1 , is a reaction flow chart for preparing NGR (compound of formula (I)) in some embodiments of the present application.

[0025] 1. Resin pretreatment

[0026] (1) Swelling and washing: Weigh 334.5 g of Fmoc-Rink Amide AM resin into a reactor, add dichloromethane as solvent, and stir for 30 minutes. Drain the liquid in the reactor. Wash twice with N,N-dimethylformamide (DMF) and drain the liquid.

[0027] (2) Deprotection of Fmoc: Add 20% piperidine / DMF solution to the reaction kettle, stir for 30 minutes, then drain the liquid. Wash with DMF 6 times, and drain the liquid.

[0028] 2. First to sixth C-terminal amino acid coupling

[0029] (1) Condensation: Weigh an appropriate amount of Fmoc-amino acid and 1-hydroxybenzotriazole in a 5L reaction bottle or beaker, add DMF and stir to disperse, cool to -5-5°C, add 90mL of N,N′-diisopropylcarbodiimide (DIC) under stirring to activate for 5-10min. Add to the reaction kettle containing the product of the previous step (pretreated resin or peptide resin), control the temperature at 20-30°C and stir to react for 4h. Stop stirring after the reaction is complete and drain the liquid. Wash with DMF 6 times and drain the liquid.

[0030] (2) Deprotection of Fmoc: Add 20% piperidine / DMF solution to the reaction kettle, stir at 20-30°C for 30 minutes, then drain the liquid. Wash with DMF 6 times and drain the liquid.

[0031] The above condensation and Fmoc protection removal cycle is carried out six times (the first to sixth condensation). The Fmoc-amino acid added in the first to sixth condensation can be Fmoc-Cys(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, and finally H-Gly-Cys(Trt)-Asn(Trt)-Gly-Arg(Pbf)-Cys(Trt)-Rink Amide AM resin is obtained.

[0032] Specifically, in the first condensation, 351.4 g of Fmoc-Cys(Trt)-OH and 82.1 g of 1-hydroxybenzotriazole can be weighed into a reaction bottle or beaker to react and condense Cys; in the second condensation, 44.1 g of Fmoc-Arg(Pbf)-OH and 9.2 g of 1-hydroxybenzotriazole can be weighed into a reaction bottle or beaker to react.

[0033] After the above condensation and deprotection steps, the reaction can be detected by ninhydrin test to check whether it is complete; after the reaction is complete, the next step can be carried out. Take out a small amount of product in a test tube, add 2 drops of detection reagent A (80g phenol dissolved in 100mL anhydrous ethanol), detection reagent B (5g ninhydrin added to 100mL anhydrous ethanol), and detection reagent C (pyridine), heat at 100℃ for 5min, and take out for observation. After the condensation reaction, if the resin is colorless, proceed to the next step; after the deprotection reaction, if the resin is colored, proceed to the next step.

[0034] (3) Obtaining peptide resin: After the above cycle is completed for condensation and deprotection, add dichloromethane to the reactor to wash the resin three times and drain the liquid. Wash with MeOH three times and drain the liquid. Pump the resin in the reactor until it is half dry, take it out, and air-dry it at room temperature until constant weight is obtained. 755.5 g of peptide resin (H-Gly-Cys(Trt)-Asn(Trt)-Gly-Arg(Pbf)-Cys(Trt)-RinkAmide AM resin) is obtained.

[0035] 3. Cleavage to synthesize NGR linear crude peptide (H-Gly-Cys-Asn-Gly-Arg-Cys-NH2)

[0036] Weigh 300.0g of peptide resin and place it in a 5L reaction bottle. Add the cleavage reagent (trifluoroacetic acid: 1,2-ethanedithiol: water: TIPS = 90:2.5:5:2.5) at 10ml / g of peptide resin and stir for 3h at 20-30℃. Filter, add the filtrate to 8 times about 24L of methyl tert-butyl ether (methyl tert-butyl ether is placed at -20±5℃ for 2-4h for standby use) and stir for about 0.5-1h. Filter, wash the filter cake with methyl tert-butyl ether (2L), filter to dryness and dry at room temperature for 15-24h to obtain a loose off-white solid powder, namely NGR linear crude peptide, 99.20g, purity: 70.3%.

[0037] 4. Purification of NGR Linear Crude Peptide

[0038] Purification is performed by collecting a purified solution through liquid chromatography.

[0039] Equipment: Chengdu Glider Preparative Liquid Chromatograph (Model: GL6000-300mL / min). Chromatographic column: ID 100mm*450mm; Filler: Unsil-10-100C18 UItra 1200g; Packing column length: 250mm, Product No.: GL20190215001A; Flow rate: 200mL / min; Wavelength: 210nm

[0040] Sample pre-injection treatment: NGR linear crude peptide was dissolved by stirring in water and then filtered using a 0.45 μm filter membrane as the sample solution. The sample solution concentration was 0.04 g / mL.

[0041] The column was balanced with 2% phase B until the baseline was stable, and the balance lasted for 15 minutes. The sample was loaded using a C pump at a flow rate of 200 mL / min. After loading, pure water was added to rinse the injection line for 2 minutes. After loading, a gradient elution was performed: 0 minutes: 98% phase A, 2% phase B, 60 minutes: 78% phase A, 22% phase B, 80 minutes: 0% phase A, 100% phase B. Phase A is a 0.05% trifluoroacetic acid aqueous solution (which can be obtained by measuring 0.5 mL of trifluoroacetic acid and adding it to 1 L of water), and phase B is acetonitrile.

[0042] A total of 5 injections were made, with approximately 20 g of sample loaded into each injection.

[0043] The target peak was collected in sections, and the fractions were collected by HPLC detection, and the purified solution with a purity of ≥98.0% was collected to obtain 6.34 L of NGR line peptide purified solution (containing 31.9 g of NGR line peptide), with a purity of 99.0% and a yield of 33.2%.

[0044] 5. Form a disulfide bond to obtain NGR (compound of formula (I))

[0045] Add 1.27 L of DMSO to the purified NGR peptide solution and react for 36–72 hours under stirring conditions.

[0046] A sample was taken for HPLC to confirm whether the reaction was complete. After the reaction was complete, 27.54 g of the compound of formula (I) was obtained by calibration.

[0047] Purity: 92.9%; Yield: 86.6%. Mass spectrum 606.2 (theoretical value 605.2).

[0048] (II) Preparation of NGR-PEG4-BCN

[0049] Please refer to Figure 2 , is a reaction formula for reacting NGR with BCN-PEG4-NHS to obtain NGR-PEG4-BCN (compound of formula (II)) in some embodiments of the present application.

[0050] 1. Synthesis of NGR-PEG4-BCN (compound of formula (II))

[0051] (1) Add NGR (compound of formula (I)) and acetonitrile (3.58 L) under stirring;

[0052] (2) Use NaHCO3 to adjust the reaction solution pH to 7–8;

[0053] (3) Bicyclo[6.1.0]nonyne-PEG4-N-hydroxysuccinimide (BCN-PEG4-NHS) (25.7 g) was weighed, diluted and dissolved in acetonitrile, and then added dropwise to the reaction solution. The system was colorless and clear, and the reaction was stirred at room temperature;

[0054] (4) After 5 hours of reaction, samples were taken and sent to HPLC for testing. Purity of reaction solution: 82.4%

[0055] 2. Purification of NGR-PEG4-BCN (compound of formula (II))

[0056] Purification is performed by collecting a purified solution through liquid chromatography.

[0057] Equipment: Chengdu Glider Preparative Liquid Chromatograph (Model: GL6000-300mL / min). Chromatographic column: ID 100mm*450mm; Filler: Unsil-10-100C18 UItra 1200g; Packing column length: 250mm, Product No.: GL20190215001A; Flow rate: 200mL / min; Wavelength: 210nm

[0058] Sample pre-injection treatment: Concentrate about 10.8 L of reaction solution to 7.2 L at 35°C, filter with a 0.45 μm filter membrane, and load the sample in 4 batches, 1.8 L each time (containing about 12.0 g of NGR-PEG4-BCN).

[0059] The column was balanced with 2% phase B until the baseline was stable, and the balance lasted for 15 minutes. The sample was loaded using a C pump at a flow rate of 200 mL / min. A total of 5 injections were made, with each injection loading about 20 g. After the loading was completed, pure water was added to rinse the injection line for 2 minutes. Then a gradient elution was run: 0 minutes: 95% phase A, 5% phase B, 5 minutes: 85% phase A, 15% phase B, 75 minutes: 50% phase A, 50% phase B. Phase A is a 0.1% trifluoroacetic acid aqueous solution (which can be obtained by measuring 1 mL of trifluoroacetic acid and adding it to 1 L of water), and phase B is acetonitrile.

[0060] (4) The target peak was collected in sections, and the fractions were collected by HPLC detection. The purified solution with a purity of ≥97.0% was collected for use. A total of 5.2 L of purified solution was obtained. After the content was calibrated with a reference substance, it was found to contain 26.0 g of INT2. Purity: 97.8%; Yield: 59.0%; Mass spectrum: 1028.5 (theoretical value: 1028.4).

[0061] (III) Preparation of FP-(ivDde)-RGD

[0062] Please refer to Figure 3 , is a reaction flow chart for preparing FP-(ivDde)-RGD (compound of formula (III)) in some embodiments of the present application.

[0063] 1. Resin pretreatment

[0064] Weigh 285.9 g of dichlorotrityl chloride resin, add it into a reaction kettle, add dichloromethane as a solvent, stir for 30 minutes, and drain the liquid in the reaction kettle.

[0065] 2. First C-terminal amino acid coupling

[0066] (1) Condensation: Weigh 269.9 g of Fmoc-Gly-OH into a 5 L reaction bottle, add dichloromethane and stir to disperse the raw materials, add to the reactor containing the pretreated resin, add 315 mL of N,N-diisopropylethylamine, control the temperature at 20-30 °C and stir to react for 4 hours, add 0.2 L of methanol to seal the resin, react for 0.5 hours, discharge the reaction liquid, wash with DMF 6 times, and discharge the liquid to obtain Fmoc-Gly-CTC resin.

[0067] (2) Deprotection of Fmoc: Add 20% piperidine / DMF solution to the reaction kettle containing the product of the previous step (pretreated resin or peptide resin), stir at 20-30° for 30 minutes, and then drain the liquid. Wash with DMF 6 times and drain the liquid.

[0068] 3. Second to fifth C-terminal amino acid coupling

[0069] (1) Condensation: Weigh an appropriate amount of Fmoc-amino acid and 1-hydroxybenzotriazole in a 5L reaction bottle or beaker, add DMF and stir to disperse, cool to -5-5°C, add 90mL of DIC to activate for 5-10min while stirring. Add to the reaction kettle containing the product of the previous step (pretreated resin or peptide resin), control the temperature at 20-30°C and stir to react for 4h. Stop stirring after the reaction is complete and drain the liquid. Wash with DMF 6 times and drain the liquid.

[0070] (2) Deprotection of Fmoc: Add 20% piperidine / DMF solution to the reaction kettle, stir at 20-30° for 30 minutes, then drain the liquid. Wash with DMF 6 times and drain the liquid.

[0071] The above-mentioned condensation and Fmoc deprotection cycle is carried out four times (second to fifth condensation), wherein the Fmoc-amino acid added in the second to fifth condensation can be Fmoc-Arg(Pbf)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-D-Tyr(tBu)-OH, and Fmoc-Asp(OtBu)-OH, respectively, to finally obtain Fmoc-Asp(OtBu)-D-Tyr(tBu)-Lys(ivDde)-Arg(Pbf)-Gly-CTC resin.

[0072] Specifically, in the second condensation, 333.8 g of Fmoc-Arg(Pbf)-OH and 69.7 g of 1-hydroxybenzotriazole can be weighed into a reaction bottle or beaker for reaction.

[0073] After the above condensation and deprotection steps, the reaction can be detected by ninhydrin test to check whether it is complete; after the reaction is complete, the next step can be carried out. Take out a small amount of product in a test tube, add 2 drops of detection reagent A (80g phenol dissolved in 100mL anhydrous ethanol), detection reagent B (5g ninhydrin added to 100mL anhydrous ethanol), and detection reagent C (pyridine), heat at 100℃ for 5min, and take out for observation. After the condensation reaction, if the resin is colorless, proceed to the next step; after the deprotection reaction, if the resin is colored, proceed to the next step.

[0074] (3) Obtaining peptide resin: After the above cycle is completed to complete condensation and deprotection, add dichloromethane to the reactor to wash the resin three times and drain the liquid. Wash with MeOH three times and drain the liquid. Pump the resin in the reactor until it is half dry, then take it out and air-dry it at room temperature until constant weight is collected. 476.0 g of peptide resin (H-Asp(OtBu)-D-Tyr(tBu)-Lys(ivDde)-Arg(Pbf)-Gly-CTC resin) is obtained.

[0075] 4. Cleavage to synthesize FP-(ivDde)-RGD(H-Asp(OtBu)-D-Tyr(tBu)-Lys(ivDde)-Arg (Pbf)-Gly-OH)

[0076] (1) Add 4000 mL of dichloromethane and 20 mL of trifluoroacetic acid to the reaction flask under stirring;

[0077] (2) Add 400.0 g of peptide resin under stirring and react at room temperature for 3 hours. After the reaction is completed, filter out the resin using a sand core funnel, wash the resin with dichloromethane, and combine the filtrate;

[0078] (3) N,N-diisopropylethylamine was added dropwise to adjust the pH to 7-8, and the mixture was concentrated under reduced pressure at 35-40°C until no fraction flowed out;

[0079] (4) Add 1200 mL of ethyl acetate and stir to dissolve, wash with 1000 mL of saturated brine and separate the liquids, add 1000 mL of saturated brine to the organic phase again, wash and separate the liquids, add 120.0 g of anhydrous sodium sulfate to the organic phase and dry for 1 hour, concentrate the organic phase at 35-40°C until no fraction flows out, add 800 mL of ethyl acetate to dissolve, add dropwise to 8000 mL of n-heptane, stir and crystallize for 10-20 hours;

[0080] (5) Filter, rinse the filter cake three times with 400 mL of n-heptane, continue to drain for 5-8 hours, and then vacuum dry at 35°C to constant weight to obtain 181.7 g of off-white solid. Purity: 91.6%, yield: 59.7%, mass spectrum 1027.6 (theoretical value 1027.6).

[0081] (IV) Preparation of N3-NOTA-PEG4-c-RGD

[0082] Please refer to Figure 4 , is a reaction flow chart for preparing N3-NOTA-PEG4-c-RGD (compound of formula (IX)) using FP-(ivDde)-RGD (compound of formula (III)) in some embodiments of the present application.

[0083] Synthesis of FP-(ivDde)-c-RGD (compound of formula (IV))

[0084] (1) Add dichloromethane and 90.3 g of FP-(ivDde)-RGD (compound of formula (III)) into a reaction flask, stir to dissolve, add 21.0 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) and react at room temperature for 24 h;

[0085] (2) Monitor the reaction by TLC or HPLC. After the reaction is completed, the system shrinks until no fraction flows out. Dissolve the concentrate in 450 mL of DMF and add it dropwise to pure water to precipitate a white solid. Continue stirring and crystallizing for 15–20 h.

[0086] (3) Filter, rinse the filter cake three times with purified water, and dry it in a forced air drying oven at 40° C. for 20 h to obtain 85.2 g of an off-white solid with a purity of 85.9%, a yield of 96.7%, and a mass spectrum of 1189.6 (theoretical value is 1189.6).

[0087] Synthesis of FP-c-RGD (compound of formula (V))

[0088] (1) Add 400 mL of DMF and 8 mL of hydrazine hydrate into a reaction flask under stirring;

[0089] (2) Add 80.2 g of FP-(ivDde)-c-RGD (compound of formula (IV)) to the reaction bottle, stir to dissolve, and react at 10–30 °C for 5 h;

[0090] (3) HPLC monitoring of the reaction, after completion of the reaction, the reaction solution was added dropwise to methyl tert-butyl ether / n-heptane (7:3 / v:v) (8400 mL) for crystallization for 5 h;

[0091] (4) Filter, wash the filter cake three times with n-heptane and then drain, put the filter cake into a forced air drying oven and dry it at 35° C. for 15-24 h to obtain 65.3 g of off-white solid. Purity: 85.3%, yield: 99.2%, mass spectrum 983.5 (theoretical value: 983.5).

[0092] Synthesis of Fmoc-NH-PEG4-FP-c-RGD (compound of formula (VI))

[0093] (1) Add DMF (325 mL) to a reaction flask, then add 64.7 g of FP-c-RGD (compound of formula (V)), 32.0 g of Fmoc-PEG4-propionic acid (PA), and 34.2 g of PyBop, and stir to dissolve;

[0094] (2) After adding N,N-diisopropylethylamine (23 mL) dropwise, the reaction was stirred at 10–30 °C for 2 h;

[0095] (3) HPLC or TLC monitoring of the reaction. After the reaction is completed, the reaction solution is added dropwise to methyl tert-butyl ether (9750 mL) and stirred for crystallization for 2–5 h;

[0096] (4) Filter, wash the filter cake with methyl tert-butyl ether for 6 times and then drain, put the filter cake into a forced air drying oven at 35° C. and dry it for 15-24 hours, and then collect the material to obtain 84.7 g of off-white solid powder. Purity: 83.0%, yield: 88.3%, mass spectrum 1453.7 (theoretical value is 1452.7).

[0097] Synthesis of NH2-PEG4-FP-c-RGD (compound of formula (VII))

[0098] (1) Add 201.6 mL of DMF and 50.4 mL of piperidine to a reaction flask, add 84.1 g of Fmoc-NH-PEG4-FP-c-RGD to the reaction flask under stirring, stir to dissolve, and react at 10–30 °C for 2 h;

[0099] (2) Monitor the reaction solution by TLC or HPLC. After the reaction is completed, add the reaction solution dropwise into 7560 mL of methyl tert-butyl ether. After a white solid precipitates, continue stirring for 1–2 h.

[0100] (3) Filter, wash the filter cake three times with methyl tert-butyl ether and then drain, place the filter cake in a forced air drying oven at 35° C. and dry for 15–24 h to obtain 61.2 g of off-white solid powder. Purity: 85.4%, yield: 86.1%, mass spectrum 1231.7 (theoretical value: 1230.7).

[0101] Synthesis of N3-NOTA(tBu)2-PEG4-FP-c-RGD (compound of formula (VIII))

[0102] (1) Add 300 mL of DMF to a reaction flask, then add 60.0 g of NH2-PEG4-FP-c-RGD, 25.2 g of NOTA(tBu)2-N3, and 27.3 g of PyBop, and stir to dissolve.

[0103] (2) Add 19.2 mL of N,N-diisopropylethylamine dropwise and stir at 10–30 °C for 5 h;

[0104] (3) HPLC monitoring of the reaction. After the reaction is completed, the reaction solution is added dropwise to 9000 mL of methyl tert-butyl ether and stirred for crystallization for 2–5 h;

[0105] (4) Filter, wash the filter cake three times with 300 mL of methyl tert-butyl ether, drain and place in a forced air drying oven at 35° C. for 15–24 h to obtain 67.5 g of off-white solid powder. Purity: 78.8%, yield: 81.6%, mass spectrum 1697.9 (theoretical value: 1696.9).

[0106] Synthesis of N3-NOTA-PEG4-c-RGD (compound of formula (IX))

[0107] (1) Under stirring conditions, add 627 mL of trifluoroacetic acid, 16.5 mL of water, and 16.5 mL of triisopropylsilane into a reaction flask;

[0108] (2) Add 66.0 g of N3-NOTA(tBu)2-PEG4-FP-c-RGD and stir at room temperature for 2–3 h;

[0109] (3) After the reaction is completed, the reaction solution is added dropwise to 6600 mL of methyl tert-butyl ether for crystallization for 2–5 h;

[0110] (4) Filter, wash the filter cake three times with methyl tert-butyl ether and then drain, dry the filter cake at room temperature for 15-24 hours, and collect 48.9 g of off-white solid powder.

[0111] Purity: 80.4%, yield: 102.9%.

[0112] Purification of N3-NOTA-PEG4-c-RGD (compound of formula (IX))

[0113] Purification is performed by collecting a purified solution through liquid chromatography.

[0114] Equipment: Chengdu GLE Preparative Liquid Chromatograph (Model: GL6000-300mL / min)

[0115] Preparative chromatographic column: ID 100mm*450mm; Filler: Unsil-10-100 C18 UItra 1200g; Packing column length: 250mm; Product number: GL20190215001A; Flow rate: 200mL / min; Wavelength: 210nm

[0116] Sample pretreatment: 45.2 g of N3-NOTA-PEG4-c-RGD (compound of formula (IX)) was dissolved in 2250 mL of water and filtered through a 0.45 μm filter membrane before injection.

[0117] The column was equilibrated with 5% phase B until the baseline was stable, and the equilibration lasted for 15 minutes. The sample was loaded using a C pump at a flow rate of 200 mL / min. A total of 5 injections were made, with each injection loading about 9.0 g. After the loading was completed, a gradient elution was performed: 0 minutes: 95% phase A, 5% phase B, 5 minutes: 90% phase A, 10% phase B, 60 minutes: 70% phase A, 30% phase B. Phase A is a 0.1% trifluoroacetic acid aqueous solution (which can be obtained by measuring 1 mL of trifluoroacetic acid and adding it to 1 L of water), and phase B is acetonitrile.

[0118] The target peak was collected in sections, and HPLC was performed to detect the purified solution with a purity of 98.0% or more and a single impurity of less than 0.5%. Finally, 11.70 L of purified solution was obtained, which was calibrated to contain 16.86 g of N3-NOTA-PEG4-c-RGD (compound of formula (IX)). Purity: 99.0%, yield: 37.3%, mass spectrum 1220.6 (theoretical value is 1220.6).

[0119] (V) Preparation of NGR-PEG4-click-NOTA-PEG4-c-RGD

[0120] Please refer to Figure 5, is a reaction formula for obtaining NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X)) by reacting NGR-PEG4-BCN (compound of formula (II)) with N3-NOTA-PEG4-c-RGD (compound of formula (IX)) in some embodiments of the present application.

[0121] Synthesis of NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X))

[0122] (1) 16.00 g of a trifluoroacetic acid / acetonitrile mixture of NGR-PEG4-BCN (compound of formula (II)) (HPLC gradient elution purification solution of compound of formula (II)) and 16.20 g of N3-NOTA-PEG4-c-RGD (compound of formula (IX)) were mixed and reacted at 10-30°C for 15-20 h.

[0123] (2) The reaction was monitored by HPLC and the final product was purified after filtration through a 0.45 μm filter membrane.

[0124] Purification of NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X))

[0125] Purification is performed by collecting a purified solution through liquid chromatography.

[0126] Equipment: Chengdu GLE Preparative Liquid Chromatograph (Model: GL6000-300mL / min)

[0127] Preparative chromatographic column: ID 100mm*450mm; Filler: Unsil-10-100 C18 UItra 1200g; Packing column length: 250mm; Product number: GL20190215001A; Flow rate: 200mL / min; Wavelength: 210nm

[0128] The chromatographic column was equilibrated with 5% phase B until the baseline was stable, and the equilibration lasted for 15 minutes. The sample was loaded using a C pump at a loading flow rate of 200 mL / min. A total of 4 injections were made, with each injection loading 2.9 L (containing approximately 8.0 g of NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X))). After the loading was completed, pure water was added to rinse the injection line for 2 minutes, and then a gradient elution was run: 0 minutes: 95% phase A, 5% phase B, 5 minutes: 88% phase A, 12% phase B, 85 minutes: 68% phase A, 32% phase B, 100 minutes: 50% phase A, 50% phase B. Phase A is a 0.3% acetic acid aqueous solution (which can be obtained by measuring 3 mL of glacial acetic acid and adding it to 1 L of water), and phase B is acetonitrile.

[0129] The target fractions were collected in sections and sent to HPLC for testing. The purity of the collected and combined fractions was greater than or equal to 98%, and a total of 8.20 L of qualified purified solution was obtained. Purity: 99.8%.

[0130] NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X)) salt transfer

[0131] The salt transfer was performed by liquid chromatography.

[0132] Preparative chromatographic column: ID 100mm*450mm; filler: Unsil-10-100 C18 UItra 1200g; packed column length: 250mm; product number: GL20190215001A; flow rate: 200mL / min; wavelength: 210nm.

[0133] Sample pretreatment: 8.2 L of NGR-PEG4-click-NOTA-PEG4-c-RGD (compound of formula (X)) purified solution was concentrated under reduced pressure at 30°C to 6.85 L and then loaded onto the sample.

[0134] Mobile phase: Phase A1: 0.1% acetic acid aqueous solution, Phase A2: 100 mmol / L ammonium acetate aqueous solution, Phase B: acetonitrile.

[0135] The column was equilibrated with 5% B phase and 95% A1 phase for 15 minutes. The sample was loaded using a C pump at a flow rate of 200 mL / min. After loading, the A2 phase was used as the mobile phase for 30 minutes to transfer the salt, the A1 phase was used as the mobile phase for 20 minutes to flush, and finally 50% A1 phase and 50% B phase were used as the mobile phase until the main peak was received and then stopped for elution.

[0136] Collect all target peaks to obtain 3.2 L of salt conversion solution. After concentrated under reduced pressure at 30°C, load the sample and freeze-dry to obtain 23.0 g of off-white fluffy solid powder. Purity: 99.7%, yield: 78.0%, mass spectrum 2250.1 (theoretical value 2249.1).

[0137] Example 2

[0138] The synthesis of compounds of formula (I) to (X) is the same as Example 1, except that phase A is a 0.3% trifluoroacetic acid aqueous solution when purifying the compound of formula (X).

[0139] The target fractions were collected in sections and sent to HPLC for testing. The purity of the collected and combined fractions was greater than or equal to 98%, and the purity of the purified liquid was 99.3%.

[0140] It can be seen that the purified solution obtained in Example 1 has a higher purity.

[0141] The above-mentioned implementation modes are only preferred implementation modes of the present application and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present application shall fall within the scope of protection required by the present application.

Claims

1. A method for preparing a compound, characterized in that: include: Preparation of compounds of formula (I) or their salts; Prepare the compound of formula (II) or its salt using the compound of formula (I) or its salt; preparing a compound of formula (III) or a salt thereof; Preparing a compound of formula (IV) or a salt thereof from a compound of formula (III) or a salt thereof; Prepare the compound of formula (V) or its salt using the compound of formula (IV) or its salt; Using a compound of formula (V) or a salt thereof to prepare a compound of formula (VI) or a salt thereof; Preparing a compound of formula (VII) or a salt thereof from a compound of formula (VI) or a salt thereof; Prepare the compound of formula (VIII) or its salt using the compound of formula (VII) or its salt; Prepare the compound of formula (IX) or its salt using the compound of formula (VIII) or its salt; as well as Prepare the compound of formula (X) or its salt using the compound of formula (II) or its salt and the compound of formula (IX) or its salt, 2. The preparation method according to claim 1, characterized in that The method of preparing the compound of formula (X) or its salt from the compound of formula (II) or its salt and the compound of formula (IX) or its salt comprises: In a trifluoroacetic acid / acetonitrile mixture, the compound of formula (II) or its salt and the compound of formula (IX) or its salt are reacted at 10-30° C. for 15-20 hours.

3. The preparation method according to claim 1, characterized in that: The method of preparing the compound of formula (II) or its salt from the compound of formula (I) or its salt comprises: contacting the compound of formula (I) or a salt thereof with BCN-PEG4-NHS in acetonitrile at a pH of 7-8; and / or The method of preparing the compound of formula (IV) or its salt from the compound of formula (III) or its salt comprises: contacting the compound of formula (III) or its salt with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt in dichloromethane; and / or The method of preparing the compound of formula (V) or its salt from the compound of formula (IV) or its salt comprises: In N,N-dimethylformamide, contacting the compound of formula (IV) or its salt with hydrazine hydrate; and / or The method of preparing the compound of formula (VI) or its salt from the compound of formula (V) or its salt comprises: In N,N-dimethylformamide, the compound of formula (V) or its salt is contacted with Fmoc-PEG4-propionic acid, PyBop and N,N-diisopropylethylamine; and / or The method of preparing the compound of formula (VII) or its salt from the compound of formula (VI) or its salt comprises: In N,N-dimethylformamide, the compound of formula (VI) or its salt is contacted with piperidine; and / or The method of preparing the compound of formula (VIII) or its salt from the compound of formula (VII) or its salt comprises: In N,N-dimethylformamide, the compound of formula (VII) or its salt is contacted with NOTA(tBu)2-N3 and PyBop; and / or The method of preparing the compound of formula (IX) or its salt from the compound of formula (VIII) or its salt comprises: The compound of formula (VIII) or its salt, water and triisopropylsilane are contacted in trifluoroacetic acid.

4. The preparation method according to claim 3, characterized in that: The contacting of the compound of formula (III) or its salt with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt comprises reacting 90.3±10% by weight of the compound of formula (III) or its salt with 21.0±10% by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or its salt at room temperature for 24 hours; and / or The contacting of the compound of formula (IV) or its salt with hydrazine hydrate comprises reacting 80.2±10% by weight of the compound of formula (IV) or its salt with 8±10% by weight of hydrazine hydrate at 10-30° C. for 5 hours; and / or The contacting of the compound of formula (V) or its salt with Fmoc-PEG4-propionic acid, PyBop and N,N-diisopropylethylamine comprises mixing 64.7±10% by weight of the compound of formula (V) or its salt with 32.0±10% by weight of Fmoc-PEG4-propionic acid and 34.2±10% by weight of PyBop and adding dropwise 17.02±10% by weight of N,N-diisopropylethylamine to react at 10-30°C for 2 hours; and / or The contacting of the compound of formula (VI) or its salt with piperidine comprises reacting 84.1±10% by weight of the compound of formula (VI) or its salt with 41.88±10% by weight of piperidine at 10-30° C. for 2 hours; and / or The contacting of the compound of formula (VII) or its salt with NOTA(tBu)2-N3 and PyBop comprises reacting 60.0±10% by weight of the compound of formula (VII) or its salt with 25.2±10% by weight of NOTA(tBu)2-N3 and 27.3±10% by weight of PyBop at 10-30°C for 2 hours; and / or The contacting of the compound of formula (VIII) or its salt, water and triisopropylsilane is to react 66.0±10% by weight of the compound of formula (VIII) or its salt, 16.5±10% by weight of water and 12.75±10% by weight of triisopropylsilane at room temperature for 2-3 hours.

5. The preparation method according to claim 1, characterized in that: The preparation of the compound of formula (I) or its salts comprises: contacting the first resin with protected amino acid, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a condensed first resin; contacting the condensed first resin with piperidine to obtain a deprotected first resin; contacting the deprotected first resin with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or a salt thereof; and contacting a linear peptide of the compound of formula (I) or a salt thereof with DMSO to obtain the compound of formula (I) or a salt thereof; and / or The preparation of the compound of formula (III) or its salts comprises: contacting the second resin with protected amino acid, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a condensed second resin; contacting the condensed second resin with piperidine to obtain a deprotected second resin; and The deprotected second resin is contacted with trifluoroacetic acid to obtain the compound of formula (III) or its salts.

6. The preparation method according to claim 5, characterized in that: The preparation of the compound of formula (I) or its salts comprises: contacting the first resin with Fmoc-Cys(Trt)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a first condensed first resin; contacting the first resin after the first condensation with piperidine to obtain the first resin after the first deprotection; contacting the first deprotected first resin with Fmoc-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the second condensed first resin; contacting the first resin after the second condensation with piperidine to obtain the first resin after the second deprotection; contacting the second deprotected first resin with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the third condensed first resin; contacting the first resin after the third condensation with piperidine to obtain the first resin after the third deprotection; Contacting the third deprotected first resin with Fmoc-Asn(Trt)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fourth condensed first resin; contacting the first resin after the fourth condensation with piperidine to obtain the first resin after the fourth deprotection; contacting the fourth deprotected first resin with Fmoc-Cys(Trt)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fifth condensed first resin; contacting the first resin after the fifth condensation with piperidine to obtain the first resin after the fifth deprotection; contacting the fifth deprotected first resin with Fmoc-Gly-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the sixth condensed first resin; contacting the first resin after the sixth condensation with piperidine to obtain the first resin after the sixth deprotection; contacting the first resin after the sixth deprotection with trifluoroacetic acid to obtain a linear peptide of the compound of formula (I) or a salt thereof; and contacting a linear peptide of the compound of formula (I) or a salt thereof with DMSO to obtain the compound of formula (I) or a salt thereof; and / or The preparation of the compound of formula (III) or its salts comprises: The second resin is contacted with Fmoc-Gly-OH and N,N-diisopropylethylamine, and then with methanol to obtain a second resin after the first condensation; contacting the second resin after the first condensation with piperidine to obtain the second resin after the first deprotection; contacting the first deprotected second resin with Fmoc-Arg(Pbf)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a second condensed second resin; contacting the second condensed second resin with piperidine to obtain the second deprotected second resin; contacting the second deprotected second resin with Fmoc-Lys(ivDde)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain a third condensed second resin; contacting the second resin after the third condensation with piperidine to obtain the second resin after the third deprotection; Contacting the third deprotected second resin with Fmoc-D-Tyr(tBu)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fourth condensed second resin; contacting the second resin after the fourth condensation with piperidine to obtain the second resin after the fourth deprotection; contacting the fourth deprotected second resin with Fmoc-Asp(OtBu)-OH, 1-hydroxybenzotriazole and N,N′-diisopropylcarbodiimide to obtain the fifth condensed second resin; contacting the fifth condensed second resin with piperidine to obtain the fifth deprotected second resin; and contacting the second resin after the fifth deprotection with trifluoroacetic acid to obtain the compound of formula (III) or its salt; and / or The first resin is Rink Amide AM resin, and / or The second resin is dichlorotrityl chloride resin.

7. The preparation method according to claim 1, characterized in that: The method for preparing the compound of formula (X) or its salts further comprises: The crude product of the compound of formula (X) or its salt is subjected to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 0.3% acetic acid in water 95%, acetonitrile 5%, 5 minutes: 0.3% acetic acid in water 88%, acetonitrile 12%, 85 minutes: 0.3% acetic acid in water 68%, acetonitrile 32%, and 100 minutes: 0.3% acetic acid in water 50%, acetonitrile 50%.

8. The preparation method according to claim 1, characterized in that: The method for preparing the compound of formula (I) or its salts further comprises subjecting the crude compound of formula (I) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 0.05% trifluoroacetic acid aqueous solution 98%, acetonitrile 2%, 60 minutes: 0.05% trifluoroacetic acid in water 78%, acetonitrile 22%, and 80 min: acetonitrile 100%; and / or The preparation of the compound of formula (II) or its salts also includes subjecting the crude product of the compound of formula (II) or its salts to liquid chromatography gradient elution, wherein the mobile phase includes: 0 minutes: 0.1% trifluoroacetic acid aqueous solution 95%, acetonitrile 5%, 5 minutes: 0.1% trifluoroacetic acid in water 85%, acetonitrile 15%, and 75 minutes: 0.1% trifluoroacetic acid aqueous solution 50%, acetonitrile 75%; and / or The method for preparing the compound of formula (IX) or its salts further comprises subjecting the crude compound of formula (IX) or its salts to liquid chromatography gradient elution, wherein the mobile phase comprises: 0 minutes: 0.1% trifluoroacetic acid aqueous solution 95%, acetonitrile 5%, 5 minutes: 0.1% trifluoroacetic acid in water 90%, acetonitrile 15%, and 60 minutes: 0.1% trifluoroacetic acid aqueous solution 70%, acetonitrile 50%; and / or The preparation of the compound of formula (X) or its salts also includes subjecting the trifluoroacetate of the compound of formula (X) to liquid chromatography gradient elution and salt conversion, including: A 100 mmol / L ammonium acetate aqueous solution was used as the mobile phase for 30 min; 0.1% acetic acid in water was used as the mobile phase for 20 minutes; and The mobile phase was 50% 0.1% acetic acid in water and 50% acetonitrile.

9. Use of a compound of formula (I) or a salt thereof, a compound of formula (II) or a salt thereof, a compound of formula (III) or a salt thereof, a compound of formula (IV) or a salt thereof, a compound of formula (V) or a salt thereof, a compound of formula (VI) or a salt thereof, a compound of formula (VII) or a salt thereof, a compound of formula (VIII) or a salt thereof, and / or a compound of formula (IX) or a salt thereof in the preparation of a compound of formula (X) or a salt thereof by the preparation method according to any one of claims 1 to 8.

10. Use of the preparation method according to any one of claims 1 to 8 in the preparation of radiolabeled compounds for diagnosis and treatment.

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