Method for preparing lutetium[ 177lu]dotatate and precursor compound thereof

Abstract

Provided is a method for preparing lutetium[177Lu]dotatate and a precursor compound thereof, comprising performing coupling reactions on Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH, and DOTA-(tBu)3-CO2H successively on a Wang resin by means of a solid-phase synthesis method to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin, performing an oxidation reaction to remove Trt protecting groups from two Cys residues and form a disulfide bond therebetween, thus achieving cyclization, and then performing a cleavage reaction to remove the Wang resin and protecting groups to obtain a lutetium[177Lu]dotatate precursor compound represented by formula I. The preparation method can significantly improve the purity of the prepared lutetium[177Lu]dotatate precursor compound, reduce the impurity content, improve the product yield, and simplify the subsequent purification process.

Description

A type of lutetium 177 Preparation methods of Lu]oxooctreotide and its precursor compounds Technical Field

[0001] This application belongs to the field of pharmaceutical technology. Specifically, this application relates to a lutetium [ 177 Preparation method of Lu]oxooctreotide and its precursor compounds. Background Technology

[0002] In 2018, the U.S. FDA approved lutetium from Novartis subsidiary Advanced Accelerator Applications of France. 177 Lutathera, an oxooctreotide, has been marketed for the treatment of SSTR-positive gastrointestinal pancreatic neuroendocrine tumors (GEP-NETs) that have progressed after standard therapy. 177 Lu]Octrete, the first approved radiopharmaceutical for radioligand therapy (RLT), reaches tumor cells by specifically binding to the growth hormone-inhibiting receptor on the surface of tumor cells and releasing radionuclides to generate radiation that kills tumor cells. Recent clinical trial results also indicate that its clinical application scope may be further expanded.

[0003] lute 177 Lu]Oxyoctreotide is obtained through radionuclide [ 177 Lu was prepared by chelating with a precursor compound (MS011) of Formula I as a chelating agent, which is composed of a polypeptide linked to the chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) consisting of eight amino acids. The octardate ligand formed by the four nitrogen atoms on the dodecane heterocycle plus the four acetic acid groups attached to the nitrogen atoms provides electrons, thereby chelating with the centrally located lutetium [ 177 Lu atoms form stable chelates.

[0004] The industrial production of radionuclide-conjugated drugs places high demands on the yield and product purity of precursor compound preparation methods, as impurities can severely affect the subsequent radionuclide labeling process. Existing lutetium [ 177 The preparation methods for Lu]oxooctreotide precursor compounds generally suffer from low yield and low purity, which hinders the realization of lutetium [ 177 The efficient and large-scale production of lutetium oxyoctreotide raw material poses a significant challenge to meet clinical needs. Therefore, improving lutetium oxyoctreotide... 177 The preparation process of Lu]oxooctreotide precursor compounds has become one of the technical problems that urgently need to be solved in the pharmaceutical field. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a lutetium [ 177 Preparation method of Lu]O-octreotide precursor compound, a lutetium [ 177 A method for preparing lutetium oxorubicin (LXO) is described, which can significantly improve the yield of LXO. 177 The purity of lutetium oxooctreotide precursor compounds is improved, impurity content is reduced, product yield is increased, and subsequent purification processes are simplified, thus providing a basis for the purification of lutetium oxooctreotide precursor compounds. 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxooctreotide.

[0006] The above-mentioned objective of this application is achieved through the following technical solution:

[0007] A type of lutetium shown in Formula I [ 177 A method for preparing Lu]oxooctreotide precursor compounds, comprising solid-phase synthesis of Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH, and DOTA-(tBu). 3-CO2H undergoes a sequential coupling reaction on the king resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-king resin. Further oxidation causes the two Cys groups to lose their Trt protecting groups and form disulfide bonds with each other, thus cyclizing. Finally, a cleavage reaction removes the king resin and protecting groups, yielding lutetium as shown in Formula I. 177 Lu]O-octreotide precursor compound.

[0008] According to some embodiments of this application, the preparation method includes the following steps:

[0009] (1) In an organic solvent containing condensation reaction reagents, Fmoc-Thr(tBu)-OH is coupled onto the king resin to obtain Fmoc-Thr(tBu)-king resin.

[0010] (2) In an organic solvent containing condensation reaction reagents, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-D-Phe-OH are sequentially coupled onto Fmoc-Thr(tBu)-King resin to obtain Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin;

[0011] (3) In an organic solvent containing condensation reaction reagents, DOTA-(tBu)3-CO2H is coupled onto Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin; and

[0012] (4) An oxidation reaction is carried out in an organic solvent containing an oxidant, so that the two Lys in the DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin lose the Trt protecting group and form a disulfide bond with each other, thereby cyclizing;

[0013] (5) The resin and protecting group are removed by pyrolysis in a mixture containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan) (DODT), triisopropylsilane (Tis), and purified water to obtain lutetium [Formula I]. 177 Lu]O-octreotide precursor compound, wherein the cleavage reaction is carried out at 5–10 °C for 1 hour, and then at 26–30 °C for 3 hours.

[0014] According to some embodiments of this application, in step (1), the resin is swollen in an organic solvent before the coupling reaction is carried out.

[0015] According to some embodiments of this application, in step (1), the coupling reaction is carried out at 25-30°C for 2 hours; preferably, the Fmoc-Thr(tBu)-OH is first activated at 0-10°C, preferably 5°C for 3-5 minutes, and then the coupling reaction is carried out at 25-30°C for 2 hours.

[0016] According to some embodiments of this application, in step (1), the condensation reaction reagent comprises one of the following combinations: benzotriazole-1-yl-oxytripyrrolylphosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIEA); O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 4-dimethylaminopyridine (DMAP); or 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide (DIC), and 4-dimethylaminopyridine. Preferably, the condensation reaction reagent comprises benzotriazole-1-yl-oxytripyrrolylphosphine hexafluorophosphate, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine. The molar equivalent ratio of Fmoc-Thr(tBu)-OH, benzotriazole-1-yl-oxytripyrrolylphosphine hexafluorophosphate, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine can be 1:1:1:2; the molar equivalent ratio of Fmoc-Thr(tBu)-OH, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and 4-dimethylaminopyridine can be 1:1:1:2:0.1; and the molar equivalent ratio of Fmoc-Thr(tBu)-OH, 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide, and 4-dimethylaminopyridine can be 1:1:1:0.1.

[0017] According to some embodiments of this application, in step (1), the degree of substitution of the Fmoc-Thr(tBu)-King resin is 0.40 to 0.70 mmol / g.

[0018] According to some embodiments of this application, step (1) further includes end-capping the obtained Fmoc-Thr(tBu)-King resin in an organic solvent containing an end-capping agent and an organic base; preferably, the end-capping agent is acetic anhydride (Ac2O); wherein, the organic base can be N,N-diisopropylethylamine and / or triethylamine, preferably N,N-diisopropylethylamine; preferably, the end-capping is performed at 20-30°C for 15-20 hours.

[0019] According to some embodiments of this application, in step (2), the coupling reaction is carried out at 18-32°C, preferably 20-30°C, for 2-3 hours; preferably, the Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH are first activated at 0-10°C, preferably 5°C, for 5-10 minutes, and then the coupling reaction is carried out at 18-32°C, preferably 20-30°C, for 2-3 hours.

[0020] According to some embodiments of this application, in step (2), the condensation reaction reagent comprises one of the following combinations: 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide; O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole hexafluorophosphate-1-yl-oxytripyrrolylphosphine and N,N'-diisopropylcarbodiimide. Preferably, the condensation reaction reagent comprises: 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide. Wherein, the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide can be 1:1.05:1.05; the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc- The molar equivalent ratio of Cys(Trt)-OH or Fmoc-D-Phe-OH, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:1:1:2; the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, benzotriazole-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate and N,N'-diisopropylcarbodiimide can be 1:1:2.

[0021] According to some embodiments of this application, in step (2), the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH to Fmoc-Thr(tBu)-King resin is independently 2 to 4:1, preferably 3:1.

[0022] According to some embodiments of this application, step (2) further includes extending the reaction by 0.5 to 2 hours after the coupling reaction product is detected negative by Kaiser.

[0023] According to some embodiments of this application, step (2) further includes, before carrying out the coupling reaction, removing the Fmoc protecting groups introduced by Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Trt)-OH respectively in an organic solvent containing a deprotecting agent; wherein, the deprotecting agent may be piperidine (PIP), or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene, preferably piperidine; preferably, the step of removing the Fmoc protecting groups is carried out twice at 20-30°C, each time for 15-30 minutes, preferably 20 minutes.

[0024] According to some embodiments of this application, in step (3), the coupling reaction is carried out at 20-30°C for 2-3 hours; preferably, the DOTA-(tBu)3-CO2H is first activated at 0-10°C for 5-10 minutes, and then the coupling reaction is carried out at 20-30°C for 2-3 hours.

[0025] According to some embodiments of this application, in step (3), the condensation reaction reagent comprises one of the following combinations: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine; O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole hexafluorophosphate-1-yl-oxytripyrrolylphosphine, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. Preferably, the condensation reaction reagent comprises benzotriazole hexafluorophosphate-1-yl-oxytripyrrolylphosphine, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. The molar equivalent ratio of DOTA-(tBu)3-CO2H, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine can be 1:0.98:1:2; the molar equivalent ratio of DOTA-(tBu)3-CO2H, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:0.98:1:2; and the molar equivalent ratio of DOTA-(tBu)3-CO2H, benzotriazole hexafluorophosphate-1-yl-oxytripyrrolidinephosphide, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:1:1:2.

[0026] According to some embodiments of this application, step (3) further includes a step of removing the Fmoc protecting group introduced by Fmoc-D-Phe-OH in an organic solvent containing a deprotecting agent before carrying out the coupling reaction; wherein the deprotecting agent may be piperidine (PIP), or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene, preferably piperidine; preferably, the step of removing the Fmoc protecting group is carried out twice at 20-30°C, each time for 15-30 minutes, preferably 20 minutes.

[0027] According to some embodiments of this application, in step (4), the oxidation reaction is carried out at 27-32°C for 25-55 minutes, preferably 35-45 minutes.

[0028] According to some embodiments of this application, in step (4), the oxidant is iodine; preferably, the molar equivalent ratio of iodine to Fmoc-Thr(tBu)-King resin is 3 to 5:1, more preferably 4:1. Preferably, the concentration of iodine in the reaction system is 0.15 to 0.25 mol / L, more preferably 0.2 mol / L.

[0029] According to some embodiments of this application, step (4) further includes washing the obtained cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-King resin in an organic solvent containing a reducing agent after the oxidation reaction; wherein the reducing agent may be vitamin C and / or sodium sulfite, preferably vitamin C; preferably, the washing is performed twice at 20-30°C, each time for 10 minutes.

[0030] According to some embodiments of this application, in step (5), the volume ratio of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)diethylthiol, triisopropylsilane and purified water is 80-90:4-10:3-5:3-5, preferably 90:4:3:3.

[0031] According to some embodiments of this application, in step (5), the ratio of the mixture containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)diethylthiol (DODT), triisopropylsilane (Tis) and purified water to the cyclized resin obtained in step (4) is 10-30 ml: 1 g, preferably 30 ml: 1 g.

[0032] According to some embodiments of this application, step (5) further includes filtering the product obtained from the pyrolysis reaction and allowing the filtrate to settle in methyl tert-butyl ether (MTBE) at a temperature ≤20°C for 15–20 hours. Preferably, the volume ratio of methyl tert-butyl ether to the mixture containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)diethylthiol (DODT), triisopropylsilane (Tis), and purified water is 10–12:1.

[0033] According to some preferred embodiments of this application, in steps (1) to (5), the organic solvent is independently N,N-dimethylformamide (DMF) and / or N-methyl-2-pyrrolidone, preferably N,N-dimethylformamide.

[0034] In addition, this application also provides a lutetium [ 177 A method for preparing lutetium oxorlene according to Formula I of this application. 177 The method for preparing the Lu]oxooctreotide precursor compound yielded lutetium of formula I. 177 Lu]Oxyoctreotide precursor compound and lutetium[ 177 Lu] chelation.

[0035] This application has at least the following beneficial effects:

[0036] This application provides an optimized lutetium [ 177 The method for preparing the Lu]oxooctreotide precursor compound reduces the content of individual impurities in the crude precursor compound and improves the purity of the precursor compound product, resulting in a purity ≥90.0%, which is more conducive to subsequent purification processes, thereby achieving an individual impurity content ≤0.10% by weight, which is beneficial for lutetium [ 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide. Attached Figure Description

[0037] The embodiments of this application are described in detail below with reference to the accompanying drawings, wherein:

[0038] Figure 1 shows the lutetium [provided in this application] 177 Flowchart of the preparation method of Lu]oxooctreotide precursor compound;

[0039] Figure 2 shows the lutetium prepared in Example 1. 177 NMR spectrum of MS011-A, a precursor compound of Lu-oxy-octreotide;

[0040] Figure 3 shows the lutetium prepared in Example 1. 177 LC-MS spectrum of Lu]oxooctreotide precursor compound MS011-A;

[0041] Figure 4 shows the lutetium prepared in Example 1. 177 Infrared spectrum of MS011-A, a precursor compound of octreotide [Lu];

[0042] Figures 5-7 show the lutetium [prepared according to Example 1]. 177 HPLC chromatogram of crude solution of Lu]oxooctreotide precursor compound MS011. Detailed Implementation

[0043] The present application will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present application and are not intended to limit the scope of the present application.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the reagents used in the following examples are commercially available products.

[0045] Abbreviation Table

[0046] Example 1: Lutetium [ 177 Preparation of Lu]Oxyoctreotide precursor compounds

[0047] 1. Lutetium 177 Synthetic route of Lu]O-octreotide precursor compound (MS011)

[0048] Key materials: Wang Resin, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-D-Phe-OH,DOTA-(tBu)3-CO2H

[0049] Reagents: PyBOP, HOBt, DIEA, Ac2O, DIC, TFA, DODT, Tis, water, piperidine, iodine, vitamin C

[0050] Solvents: DMF, MeOH, DCM, MTBE, ACN

[0051] 2. Synthesis process of MS011

[0052] 2.1 Coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0053] Using blank Wang Resin (Xi'an Lanxiao Technology Co., Ltd., crosslinking degree of 1%, mesh size of 100-200) with a substitution degree of 1.0-1.2 mmol / g, under nitrogen protection, 300±1g of Wang Resin (1eq) and 3.0±0.1LDMF (10vol, v / w) were added sequentially to the synthesis vessel R-2105. The mixture was stirred under nitrogen bubbling for 30-35 minutes, then stirring was stopped and the solvent in the vessel was emptied.

[0054] Add 2.1 ± 0.1 LDMF (7 vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3 to 5 minutes, and then purge the solvent; repeat this step once more.

[0055] Under nitrogen protection, 562.8±1g Fmoc-Thr(tBu)-OH (4.0eq), 736.9±1g PyBOP (4.0eq), 191.3±1g HOBT (4.0eq), and 0.82±0.01Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0056] Control the internal temperature of activation tank R-0304 to 0~10℃, add 366.0±1g DIEA (8.0eq) dropwise to activation tank R-0304, and stir for 3~5 minutes after the addition is completed.

[0057] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.01Kg DMF. The rinsing solution was then transferred into the synthesis tank R-2105.

[0058] The internal temperature of the synthesis tank R-2105 was controlled at 25-30℃, and the reaction was maintained at this temperature for 2 hours. After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0059] Add 6.0±0.1 LDMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.1±0.1 LDMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.1±0.1 LDMF (7 vol, v / w) to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0060] Under nitrogen protection, add 0.85±0.1Kg DMF, 722.8±1g Ac2O (20eq), and 91.5±1g DIEA (2.0eq) sequentially to the activation tank R-0304, and stir for 5-10 minutes.

[0061] Under nitrogen protection, the solution in the activation tank R-0304 is transferred to the synthesis tank R-2105. The internal temperature of the synthesis tank R-2105 is controlled at 20-30℃, and the reaction is maintained for 15-20 hours. After the heat preservation is completed, the solvent in the synthesis tank R-2105 is emptied.

[0062] Add 6.0±0.1 LDMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.1±0.1 LDMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.1±0.1 LDMF (7 vol, v / w) to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0063] Add 2.78±0.1 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 1.55±0.1 kg MTBE (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 2.78±0.1 kg DCM (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent; add 1.55±0.1 kg MTBE (7 vol, v / w), bubble and stir under nitrogen for 3–5 minutes, and drain the solvent. Repeat this step 3 times.

[0064] Remove Fmoc-Thr(tBu)-WangResin from synthesis tank R-2105 and transfer it to vacuum drying oven D-2101. Control the jacket temperature at 30-35℃ and the vacuum degree at ≤-0.08MPa. Dry for 10 hours, turning the material over every 3-4 hours. Weigh the material starting from the 10th hour, then weigh it every 2 hours until the first process control test (In-Process Control 01, IPC01) shows a loss on drying (LOD) ≤10%. Discharge the material, pack it into double-layer LDPE bags, weigh it, and label it. Take samples for intermediate control and test the degree of substitution (Substituent = 0.4-0.7 mmol / g). During the testing period, refrigerate the material at 2-8℃.

[0065] In the above operation steps 2.1, the material feeding amount is calculated based on the resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0066] In the following operating steps 2.2 to 2.10, the material feed rate is calculated based on Fmoc-Thr(tBu)-Wang Resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0067] 2.2Fmoc-Cys(Trt)-OH Coupling

[0068] Under nitrogen protection, 320±10g Fmoc-Thr(tBu)-Wang Resin(1eq) and 3.2±0.1LDMF(10vol, v / w) were added sequentially to the synthesis vessel R-2105. The mixture was stirred under nitrogen bubbling for 30-35 minutes, then the stirring was stopped and the solvent in the vessel was emptied.

[0069] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step once.

[0070] Under nitrogen protection, add 6.98±0.1Kg PIP and 30.56±0.1Kg DMF to buffer tank R-2107 in sequence, stir well, and set aside for use (total amount for 8 uses).

[0071] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0072] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0073] Under nitrogen protection, 307.5±1g Fmoc-Cys(Trt)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.76±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0074] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0075] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0076] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0077] 2.3Fmoc-Thr(tBu)-OH Coupling

[0078] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Stir with nitrogen bubbling for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0079] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0080] Under nitrogen protection, 208.7±1g Fmoc-Thr(tBu)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.56±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0081] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0082] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0083] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0084] 2,4Fmoc-Lys(Boc)-OH coupling

[0085] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Stir with nitrogen bubbling for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0086] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0087] Under nitrogen protection, 246±1g Fmoc-Lys(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.56±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0088] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0089] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0090] Add 6.0±0.1 L DMF to the activation tank R-0304 as a spray, transfer the eluent to the synthesis tank R-2105, and wash with nitrogen bubbling and stirring for 3–5 minutes, then drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 as a spray, transfer the eluent to the synthesis tank R-2105, and wash with nitrogen bubbling and stirring for 3–5 minutes, then drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 as a spray, wash with nitrogen bubbling and stirring for 3–5 minutes, then drain the solvent. Repeat this step twice.

[0091] 2,5Fmoc-D-Trp(Boc)-OH coupling

[0092] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0093] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0094] Under nitrogen protection, 276.5±1g Fmoc-D-Trp(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0095] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0096] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0097] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0098] 2.6Fmoc-Tyr(tBu)-OH coupling

[0099] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0100] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0101] Under nitrogen protection, 241.3±1g Fmoc-Tyr(tBu)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0102] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0103] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0104] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0105] 2.7Fmoc-Cys(Trt)-OH Coupling

[0106] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0107] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0108] Under nitrogen protection, 307.5±1g Fmoc-Cys(Trt)-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1LDMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0109] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0110] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0111] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0112] 2,8Fmoc-D-Phe-OH coupling

[0113] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0114] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0115] Under nitrogen protection, 203.4±1g Fmoc-D-Phe-OH (3.0eq), 74.5±1g HOBT (3.15eq), and 0.62±0.1LDMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0116] Control the internal temperature of the activation vessel R-0304 to 0-10℃, add 69.6±1g DIC (3.15eq) dropwise to the activation vessel R-0304, and stir for 5-10 minutes after the addition is completed.

[0117] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0118] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0119] 2.9DOTA-(tBu)3-CO2H Coupling

[0120] Release approximately 2.08 ± 0.1 kg (6.5 vol, w / w) from buffer tank R-2107 into PP tank. Control the internal temperature of synthesis tank R-2105 at 20–30 °C. Add the solution from PP tank into synthesis tank R-2105. Bubble and stir with nitrogen for 20 minutes. Empty the solvent from synthesis tank R-2105. Repeat this step once more.

[0121] Add 2.24±0.1LDMF (7vol, v / w) to the synthesis vessel R-2105, bubble and stir with nitrogen for 3-5 minutes, and drain the solvent; repeat this step 5 times (take a sample for Kaiser test, the resin solution should be blue).

[0122] Under nitrogen protection, 200.5±1g DOTA-(tBu)3-CO2H (2.0eq), 47.3±1g HOBT (2.0eq), 182.1±1g PyBOP (2.0eq), and 0.62±0.1Kg DMF were added sequentially to the activation vessel R-0304, stirred to dissolve, and then cooled to 5±5℃.

[0123] Control the internal temperature of activation tank R-0304 to 0~10℃, add 90.5±1g DIEA (4.0eq) dropwise to activation tank R-0304, and stir for 5~10 minutes after the addition is completed.

[0124] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was rinsed with 0.20±0.1Kg DMF. The rinsing solution was then transferred to the synthesis tank R-2105. After the addition was completed, the temperature inside the synthesis tank R-2105 was controlled at 20-30℃, nitrogen was bubbled, and the mixture was stirred for 2-3 hours (a sample was taken for Kaiser analysis; the resin and solution were colorless and transparent; ninhydrin was used for testing every hour until a negative result was obtained). After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0125] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0126] 2.10 Solid-phase cyclization

[0127] Under nitrogen protection, 3.3±0.1Kg DMF and 177.7±1g iodine (4.0eq) were added sequentially to the activation vessel R-0304 and stirred until homogeneous.

[0128] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105. The temperature inside the synthesis tank R-2105 was controlled at 27-32℃, and the reaction was maintained at this temperature for 40±5 minutes. The solvent in the synthesis tank R-2105 was then drained.

[0129] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0130] Under nitrogen protection, 184.9±1g vitamin C (3.0eq), 0.99±0.05Kg methanol, and 5.9±0.1Kg DMF were added sequentially to the activation vessel R-0304 and stirred until dissolved.

[0131] Control the internal temperature of synthesis tank R-2105 at 20-30℃, add half of the solution (about 2.24±0.05L) from activation tank R-0304 into synthesis tank R-2105, keep warm and stir for 10 minutes, and then drain the solvent from synthesis tank R-2105.

[0132] Control the internal temperature of synthesis tank R-2105 at 20-30℃, add the remaining solution (approximately 2.24±0.05L) in activation tank R-0304 to synthesis tank R-2105, keep warm and stir for 10 minutes, and then drain the solvent from synthesis tank R-2105.

[0133] Add 6.0±0.1 L DMF to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Add 2.24±0.1 L DMF (7 vol, v / w) to the activation tank R-0304 and spray. Transfer the eluent to the synthesis tank R-2105 and wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step once. Add 2.24±0.1 L DMF (7 vol, v / w) to the synthesis tank R-2105 and spray. Wash with nitrogen bubbling and stirring for 3-5 minutes. Drain the solvent. Repeat this step twice.

[0134] Add 2.98±0.2 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Add 1.66±0.1 kg MTBE (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Add 2.98±0.2 kg DCM (7 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Repeat this step once. Add 1.66±0.1 kg MTBE (70 vol, v / w) to synthesis vessel R-2105, bubble and agitate under nitrogen for 3–5 minutes, then drain the solvent. Repeat this step once.

[0135] Remove the peptide resin from synthesis tank R-2105 and transfer it to vacuum drying oven D-2101. Control the jacket temperature at 30-35℃ and the vacuum degree at ≤-0.08MPa. Dry for 10 hours, turning the material over every 3-4 hours. Starting from the 10th hour, weigh the material every 2 hours until LOD ≤10%. Discharge the material, pack it into double-layer LDPE bags, weigh it, and label it. Refrigerate the material at 2-8℃.

[0136] 2.11 Peptide Resin Cleavage

[0137] Under nitrogen protection, add 153±1Kg MTBE (300vol, v / w) to the settling tank, cool to -20~-10℃, start stirring, and rotate at 100 rpm.

[0138] Under nitrogen protection, 27.5±0.1Kg TFA, 930±1g DODT, 480±1g Tis, and 0.62±0.05Kg purified water (volume ratio of 90:4:3:3) were added sequentially to the pyrolysis vessel. The internal temperature was lowered to -10 to -5℃, and the rotation speed was 100 rpm.

[0139] Under nitrogen protection, 690±1g of peptide resin was added to the pyrolysis vessel, the internal temperature was controlled at -10 to 0℃, and the feed port was rinsed with a small amount of pyrolysis solution.

[0140] Within 20 minutes, the temperature inside the pyrolysis vessel is raised to 5-10°C and maintained at 5-10°C with stirring for 1 hour.

[0141] Within 30 minutes, the temperature inside the pyrolysis reactor is raised to 26–30°C and maintained at 26–30°C with stirring for 3 hours.

[0142] Filter the solution and transfer the filtrate to the settling tank R-2101 (control the temperature of the settling tank to not exceed 20℃). Wash the resin in the pyrolysis vessel with TFA three times, stirring for 3 to 5 minutes each time. Use 0.22±0.02Kg of TFA for each wash and transfer the washing solution to the settling tank together.

[0143] Stir in the settling tank for 2-4 hours, then let it stand for 15-20 hours to settle.

[0144] Transfer the material in the settling tank into a filter with a screen (3-in-1 M-2101) for filtration, and wash with MTBE, using 1.5±0.1Kg MTBE (2.94vol, v / w) each time; each wash lasts 10 to 30 minutes; repeat the washing operation 3 times.

[0145] The filter cake from the three-in-one process is dried under reduced pressure for 12 hours, with stirring every 1 to 2 hours. The jacket temperature is 25 to 35°C, and the vacuum degree is ≤-0.085MPa. After drying, the material from the three-in-one process is transferred to a tray drying oven D-2101 and dried under reduced pressure. The material is weighed every 2 hours until the LOD is ≤10%.

[0146] Remove from the oven; transfer the material into a double-layer LDPE bag and thermoplasticize; then pack it into an aluminum foil bag and thermoplasticize; add a desiccant between the LDPE and aluminum foil bags, weigh, label, and store at -20±5℃ to obtain MS011 crude product (MS011-A).

[0147] In the operation step 2.11, the material feed amount is calculated based on peptide resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0148] The nuclear magnetic resonance spectrum, LC-MS spectrum and infrared spectrum of the synthesized product are shown in Figures 2-4.

[0149] lutetium prepared according to Example 1 177 Figure 5-7 shows the HPLC chromatogram of the crude solution of Lu]oxooctreotide precursor compound MS011.

[0150] Sample storage conditions: room temperature. The purity test method for MS011 crude solution is as follows:

[0151] Example 2: Screening of Resin Substitution Degree

[0152] Peptide chain coupling was performed using Fmoc-Thr(tBu)-King resins with three different degrees of substitution (coupling was performed using an automated synthesis apparatus; 4.0 eq Fmoc-AA / HOBt / DIC, reaction at room temperature for 3.0 h; Fmoc removal: 10 min + 10 min; then 3 eq Fmoc-Thr(tBu)-King resins were used). AA / DIEA / HBTU was manually re-added with DOTA-(tBu)3-CO2H and reacted for 2.5 h; DMF washing was performed, and the product was colorless according to Kaiser analysis; 4.0 eq of iodine was added for cyclization reaction for 40 min, followed by pyrolysis: TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (volume ratio) 20V, reacted at 5-10℃ for 1.0 h, and then at 25-30℃ for 3.0 h; 10V MTBE precipitation yielded crude products MS011-20240126-E0005-C01, MS011-20240126-E0005-C02, and MS011-20240126-E0005-C03, respectively.

[0153] Based on the experimental results (see Table 1), the purity and yield of the crude peptides obtained by coupling with the three degrees of substitution were consistent. Therefore, the degree of substitution of Fmoc-Thr(tBu)-Wang resin ranges from 0.40 to 0.70 mmol / g.

[0154] Table 1

[0155] Example 3: Screening of DOTA-(tBu)3-CO2H Coupling Reagents

[0156] The experiment revealed that the coupling reaction using the DOTA-(tBu)3-OH / HOBT / DIC system may generate an impurity lacking DOTA (Des-DOTA) (4.0 eq, fully protected cleavage after 3.3 h of coupling (synthesized using 2-CTC Resin). Based on the fully protected cleavage (using 2-CTC... Based on the retention time estimation from Resin synthesis, the DOTA-deficient impurity content (RT) of 11.276 was approximately 0.33% (peak area percentage) (no molecular weight of the DOTA-deficient impurity was found in LC-MS). Therefore, the DOTA coupling process was optimized. Three coupling systems were screened: DOTA-(tBu)3-OH / HBTU / HOBT / DIEA, DOTA-(tBu)3-OH / PyBOP / HOBT / DIEA, and DOTA-(tBu)3-OH / HATU / HOAT / DIEA. The yields of DOTA-(tBu)3-OH coupling with these three condensing agents were basically consistent, and no DOTA-deficient peptides were found in any of them. The crude peptide purity obtained by coupling using the DOTA-(tBu)3-OH / HATU / HOAT / DIEA system was slightly lower than that of the other two.

[0157] Considering the overall purity, yield (see Table 2) and cost, the DOTA-(tBu)3-OH / PyBOP / HOBT / DIEA system was used for DOTA-(tBu)3-CO2H coupling, and the remaining amino acids were coupled using the Fmoc-AA / HOBT / DIC system.

[0158] Table 2

[0159] Example 4: Screening of coupling condensers for the first amino acid Fmoc-Thr(tBu)-OH

[0160] WangResin (1.18 mmol / g) was used to couple the first-terminal Fmoc-Thr(tBu)-OH (3.0 eq) using three condensation systems: HOBT / DIC / DMAP, HBTU / HOBT / DMAP / DIEA, and PyBOP / HOBT / DIEA coupling systems. The reaction temperature was 27-30℃, and the reaction was carried out under nitrogen bubbling for 3.0 h to obtain peptide resins Fmoc-Thr(tBu)-Wang Resin: MS011-20240126-E0001-04, MS011-20240126-E0005-B02, and MS011-20240126-E005-D01.

[0161] The experimental results (see Table 3) show that the HOBT / DIC / DMAP system performed poorly in coupling the first Fmoc-Thr(tBu)-OH using three condensation systems, while the HBTU / HOBT / DMAP / DIEA system performed better than the PyBOP / HOBT / DIEA system. However, the use of DMAP in the coupling of Fmoc-Thr(tBu)-OH may lead to racemization during the coupling process. Considering all factors, the PyBOP / HOBT / DIEA system was chosen for coupling the first Fmoc-Thr(tBu)-OH.

[0162] Table 3

[0163] Example 5: Screening of key process parameters for the coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0164] The experimental results (see Table 4) show that the reaction temperature has a significant impact on the loading rate of the first Fmoc-Thr(tBu)-OH. Considering the loading rate, the Fmoc-Thr(tBu)-OH / PyBOP / HOBT / DIEA = 4 / 4 / 4 / 8 coupling system was selected, with a reaction temperature of 25℃-30℃ and a reaction time of 2h.

[0165] Table 4

[0166] Example 6: Screening of the proportion of other amino acids in peptide resin synthesis

[0167] The reaction temperature was controlled at 25-30℃, and the reaction was extended by 1 hour after Kaiser test showed that it was colorless. The cyclization reaction was carried out with 4.0 eq of iodine. The pyrolysis conditions were: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio) 30V. The reaction was carried out at 5-10℃ for 1 hour, and then the temperature was slowly increased to 25-30℃ for 3.0 hours.

[0168] The following products were obtained from the amino acid feeding ratio screening experiment: MS011-20240126-E0017-01, MS011-20240126-E0017-02 and MS011-20240126-E0017-03.

[0169] The experimental results (see Table 5) show that the yield and purity of crude peptide products synthesized using 2eq, 3eq, and 4eq (molar equivalents of Fmoc-AA-OH to Fmoc-Thr(tBu)-Wang Resin) amino acid feed amounts are basically consistent. In order to make the synthesis process more stable, 3eqAA was used for the development of subsequent processes.

[0170] Table 5

[0171] Fmoc-AA-OH represents Fmoc-protected amino acids other than Fmoc-Thr(tBu)-OH (the same applies below).

[0172] Example 7: Screening of Coupling Temperatures for Other Amino Acids in Peptide Resin Synthesis

[0173] Take MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-King resin prepared according to Example 1, 5.00 g, 3.2 mmol) and Fmoc-AA-OH (3.0 eq), AA:HOBt:DIC = 1:1.05:1.05. After the reaction was colorless as detected by Kaiser test, the reaction time was extended by 1 h. The peptide resin was cyclized with 4.0 eq of iodine and cleaved to obtain MS011-20240126-E0020-01, MS011-20240126-E0020-02 and MS011-20240126-E0020-03, respectively.

[0174] The effects of coupling temperatures of 18-22℃, 23-27℃, and 28-32℃ on the purity of crude peptides were investigated. Based on the experimental results (see Table 6), the purity of the crude product obtained at the reaction temperature of 18-22℃ was 82.13%, the purity of the crude product obtained at the reaction temperature of 23-27℃ was 82.77%, and the purity of the crude product obtained at the reaction temperature of 28-32℃ was 84.32%. The purity of the crude product was consistent across the three reaction temperature ranges, ranging from 82% to 84%, and the impurity profiles were consistent. Therefore, the temperature range for solid-phase coupling of MS011 was determined to be 20-30℃.

[0175] Table 6

[0176] Example 8: Screening of Coupling Time of Other Amino Acids in Peptide Resin Synthesis

[0177] Take MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-King resin prepared according to Example 1, 5.00 g, 3.2 mmol) and Fmoc-AA-OH (3.0 eq), AA:HOBt:DIC = 1:1.05:1.05, control the reaction temperature at 20-30℃, and extend the reaction for 0.5 h, 1.0 h and 2.0 h after the reaction is colorless as detected by Kaiser test. Cyclate the peptide resin with 4.0 eq of iodine, and after cleavage, obtain MS011-20240126-E0030-01, MS011-20240126-E0030-02 and MS011-20240126-E0030-03 respectively.

[0178] The experimental results (see Table 7) show that after Kaiser test was negative, the purity and yield of the crude peptides obtained by extending the reaction time by 0.5 h, 1 h and 2 h were basically the same, and the impurity profiles were also basically the same.

[0179] Table 7

[0180] Example 9: Screening of deprotection time for remaining amino acids during peptide resin synthesis

[0181] The effect of Fmoc removal time after solid-phase coupling on the purity of crude peptides was investigated. 5.00 g (3.2 mmol) of MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-King resin prepared according to Example 1) and Fmoc-AA-OH (3.0 eq) were used, with an AA:HOBt:DIC ratio of 1:1.05:1.05. The reaction temperature was controlled at 20-30 °C. The reaction was colorless as detected by the Kaiser test. The peptide resin was cyclized with 4.0 eq of iodine, and after cleavage, MS011-20240126-E0030-A01, MS011-20240126-E0030-A02, and MS011-20240126-E0030-A03 were obtained.

[0182] The experimental results (see Table 8) show that the Fmoc removal reaction times of 15+15 min, 20+20 min, and 30+30 min have basically the same purity and impurity distribution, and the synthesis yield and pyrolysis yield are basically the same. The Fmoc removal time is tentatively set at 20 min+20 min.

[0183] Table 8

[0184] Example 10: Screening of Iodine Dosage in Peptide Resin Synthesis

[0185] Take 5.00 g (1.34 mmol) of peptide resin MS011-20240126-E0029-01 (DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin prepared according to Example 1) and iodine (concentration 0.2 mol / L), the reaction temperature is 20-30℃, and the reaction time is 40 min; Pyrolysis conditions: TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (volume ratio), pyrolysis liquid volume 30V, maintained at 5-10℃ for 1h, slowly restored to room temperature for 30min, maintained at 23-27℃ for 3.0h, MBTE sedimentation volume 10V, yielding crude products MS011-20240126-E0036-A01, MS011-20240126-E0036-A02 and MS011-20240126-E0036-A03, respectively.

[0186] The experimental results (see Table 9) show that solid-phase cyclization of peptide resin with iodine dosages of 3 eq, 4 eq, and 5 eq all resulted in the conversion of linear peptides into cyclic peptides. The reaction was complete, no linear peptide impurities were found, and the purity and yield remained basically consistent. Finally, the optimal iodine cyclization reaction equivalent was determined to be 4.0 eq.

[0187] Table 9

[0188] Example 11 Screening of cyclization reaction time in peptide resin synthesis

[0189] Take 5.00 g (1.34 mmol) of peptide resin MS011-20240126-E0029-01 (DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin prepared according to Example 1) and iodine (4.0 eq, concentration: 0.2 mol / L), at a temperature of 25-30℃, and a reaction time of 25 min and 40 min. min and 45 min; pyrolysis conditions: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio), pyrolysis liquid volume 30V, maintained at 5-10℃ for 1h, slowly restored to room temperature for 30min, maintained at 23-27℃ for 3.0h, MBTE sedimentation volume 10V, yielding MS011-20240126-E0036-F01, MS011-20240126-E0036-F02 and MS011-20240126-E0036-F03.

[0190] The experimental results (see Table 10) show that when the iodine solid-phase cyclization reaction time is 25 min, 40 min and 55 min, the linear peptides are all converted into cyclic peptides, the reaction is complete, no linear peptide impurities are found, and the purity and yield are basically consistent. The optimal iodine cyclization time is determined to be 40 min.

[0191] Table 10

[0192] Example 12: Screening of cyclization reaction temperature in peptide resin synthesis

[0193] Take 5.00 g (1.34 mmol) of peptide resin MS011-20240126-E0029-01 (DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin prepared according to Example 1) and iodine (4.0 eq, concentration: 0.2 mol / L), and react for 40 min; cleavage Solution conditions: TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (volume ratio), lysis buffer volume 30V, maintained at 5-10℃ for 1h, slowly restored to room temperature for 30min, maintained at 23-27℃ for 3.0h, MBTE sedimentation volume 10V, yielding MS011-20240126-E0036-C01, MS011-20240126-E0036-C02 and MS011-20240126-E0036-C03.

[0194] The experimental results (see Table 11) show that linear peptide impurities were generated at solid-phase cyclization temperatures of 18-23℃ and 23-27℃, with impurities of 44% and 21.6%, respectively. However, no obvious linear peptide impurities were found at cyclization temperatures of 27-32℃. Therefore, the solid-phase iodine cyclization temperature was determined to be 27-32℃.

[0195] Table 11

[0196] Example 13: Screening of iodine concentration in the cyclization reaction during peptide resin synthesis

[0197] Take 5.00 g (1.34 mmol) of peptide resin MS011-20240126-E0029-01 (DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin prepared according to Example 1) and iodine (4.0 eq, concentrations: 0.15 mol / L, 0.20 mol / L and 0.25 mol / L), and heat at 28-30°C. The reaction was carried out at 0℃ for 40 min. The pyrolysis conditions were: TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (volume ratio), pyrolysis liquid volume 30V, maintained at 5-10℃ for 1 h, slowly restored to room temperature for 30 min, maintained at 23-27℃ for 3.0 h, MBTE sedimentation volume 10V, yielding MS011-20240126-E0036-F04, MS011-20240126-E0036-F05 and MS011-20240126-E0036-F06 respectively.

[0198] The experimental results (see Table 12) show that when the concentration of iodine in the peptide resin cyclization reaction is 0.15 mol / L, 0.2 mol / L and 0.25 mol / L, all linear peptides can be oxidized into rings, and the purity and impurities of the crude product are consistent. The preferred iodine oxidation concentration is 0.2 mol / L.

[0199] Table 12

[0200] Example 14 Screening of lysis buffer formulations

[0201] The lysis conditions were screened using MS011-20240126-E0003-B01 peptide resin (37.71 g of cyclized peptide resin prepared according to Example 1, with a peptide resin synthesis yield of 116%). The reaction temperature was 25-30℃, and the reaction time was 3 h. The effect of different lysis buffers on the purity of the crude peptide was investigated.

[0202] Table 13

[0203] Based on the lysis reaction solution formulation in Table 13 and the purity of the obtained crude peptide, it can be seen that DODT or DTT needs to be added to the formulation to control lysis byproducts. However, adding DTT to the lysis solution formulation causes the product to be reduced to a linear peptide. Taking all factors into consideration, the lysis solution formulation is set as TFA / DODT / Tis / H2O=90 / 4 / 3 / 3(v / v).

[0204] Example 15 Screening of Pyrolysis Reaction Temperature

[0205] 1. Initial screening

[0206] The lysis conditions were screened using MS011-20240126-E0003-B01 peptide resin (37.71 g of cyclized peptide resin prepared according to Example 1, with a peptide resin synthesis yield of 116%). The lysis buffer formulation was TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (v / v), and the lysis buffer volume was 20 V. The effect of different reaction temperatures on the purity of the crude peptide was investigated.

[0207] Table 14

[0208] Table 14 shows that by controlling the pyrolysis process at different reaction temperatures, it was found that reacting at low temperatures of 5±5℃ and 10±5℃ for 1 hour, and then heating to 20-30℃ for 3 hours, resulted in crude products with slightly higher purity than those obtained by reacting at low temperatures of 0±5℃ for 1 hour. Considering all factors, it is recommended to control the low-temperature pyrolysis temperature at 5-10℃ and then heat to 20-30℃ for 3 hours.

[0209] 2. Refine the screening process

[0210] The lysis conditions were screened using MS011-20240126-E0050-02 peptide resin (235.80 g of cyclized peptide resin prepared according to Example 1, yield: 120.39%). The lysis buffer formulation was TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (v / v), 30V. The effect of different lysis reaction temperatures on the purity of the crude peptide was investigated.

[0211] Table 15

[0212] Table 15 shows that the purity of the crude product obtained by pyrolysis at 25±0.5℃ is 73.5%, the purity of the crude product obtained by pyrolysis at 32±0.5℃ is 73.1%, and the purity and impurity distribution of the crude products obtained by pyrolysis at 27±0.5℃ and 29±0.5℃ are basically the same, the pyrolysis yield is basically the same, and the purity is about 78%. Taking all factors into consideration, the reaction temperature is set at 26-30℃.

[0213] Example 16: Screening of Pyrolysis Reaction Volume

[0214] The lysis conditions were screened using MS011-20240126-E0003-B01 peptide resin (37.71 g of the cyclized peptide resin prepared according to Example 1, with a peptide resin synthesis yield of 116%). The lysis buffer formulation was TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (v / v), and the reaction was carried out at 5-10℃ for 1 h and 27-30℃ for 3 h. The effect of different lysis buffer volumes on the purity of the crude peptide was investigated.

[0215] Table 16

[0216] Table 16 shows the screening results for lysis volumes of 10V, 15V, 20V, and 30V. The crude peptide purities were 69.39%, 73.51%, 75.84%, and 77.57%, respectively. Increasing the lysis reaction volume can improve the purity of the crude lysate, but an excessively large lysis volume will lead to a larger crystallization volume. Therefore, the lysis volume was set at 30V, and the effect of larger lysis volumes on the quality of the crude product was no longer investigated.

[0217] Example 17 Screening of the dosage of methyl tert-ether as a cracking settling agent

[0218] The lysis conditions were screened using MS011-20240126-E0024-03 peptide resin (11.33 g of cyclized peptide resin prepared according to Example 1, yield: 124%). The lysis buffer formulation was TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (v / v), 30V; lysis was carried out at 5-10℃ for 1 h, followed by slow heating: lysis was carried out at 23-27℃ for 3 h. The effect of different amounts of methyl ether on the purity and yield of the crude peptide was investigated.

[0219] Table 17

[0220] 8V, 10V, and 15V indicate that the volume of methyl tert-butyl ether is 8, 10, and 15 times the volume of the pyrolysis reaction solution, respectively.

[0221] Table 17 shows the results of sedimentation crystallization of the pyrolysis reaction solution using 8V, 10V, and 15V methyl tert-butyl ether. The yield of the sedimentation product with 8V methyl tert-butyl ether was 62.5%, with 10V it was 72.1%, and with 15V it was 76.2%. The sedimentation yields of 10V and 15V methyl tert-butyl ether were significantly higher than those with 8V. Considering all factors, 10-12V methyl tert-butyl ether from the pyrolysis reaction solution was selected for sedimentation crystallization.

[0222] Example 18 Screening of Pyrolysis Reaction Time

[0223] The lysis conditions were screened using MS011-20240126-E0039-02 peptide resin (226.03 g of cyclized peptide resin prepared according to Example 1; synthesis yield: 114.10%). The lysis buffer formulation was TFA / DODT / Tis / H2O = 90 / 4 / 3 / 3 (v / v), lysis was performed at 30V, 5-10℃ for 1 h, and the effect of different reaction times with slow heating to 26-30℃ on the purity of the crude peptide was investigated.

[0224] Table 18

[0225] Table 18 shows the results of lysis of peptides at 5–10℃ for 1 h followed by slow heating, and then lysis at 26–30℃ for 2 h, 3 h, and 4 h, respectively. The purity of the crude peptides lysed at 26–30℃ for 3 h and 4 h was basically the same, and higher than that of the crude peptides lysed at 2 h. Considering all factors, the lysis time of 3 h was selected.

[0226] Comparative Example 1 uses 2-CTC resin to prepare lutetium [ 177 Lu]Oxyoctreotide precursor compound

[0227] 1. Solid-phase synthesis of peptide resins

[0228] The synthesis route is the same as in Example 1, but 2-CTC resin is used. The experimental results are as follows:

[0229] in conclusion:

[0230] (1) The substitution degree value of peptide resin in MS011-20240126-E0001-01 was too low, and the feed equivalent of Fmoc-Thr(tBu)-OH was too small (0.55eq); MS011-2024026-E0001-03 was changed to 0.76eq, and the sub was measured to be 0.56mmol / g, 41.17g, and the synthesis scale was 23.05mmol;

[0231] (2) The peptide resin MS011-20240126-E0001-0368.44g (actual weight gain 38.44g) was obtained, with a theoretical weight of 85.86g (theoretical weight gain 55.86g) and a yield of 68.81%. The yield of using Wang resin can generally reach 100%.

[0232] (3) The pyrolysis effect is poor. HPLC shows that the yield of fully protected pyrolysis is 87.93%, while the yield of using royal resin can generally reach 90-100%.

[0233] (4) When DOTA is coupled, approximately 0.3% of the peptide is missing.

[0234] 2,2-CTC peptide resin cleavage

[0235] The pyrolysis route was the same as in Example 1, and 2-CTC resin was used. The experimental results are as follows:

[0236] in conclusion:

[0237] Some of the impurities in MS+56 are thiol groups in Cys combined with allyl carbocations generated from the cleavage of tBu, which cannot be removed by subsequent secondary cleavage.

[0238] 3.2-CTC peptide resin solid-phase iodine cyclization

[0239] Conclusion: Compared with king resin, 2-CTC resin has a lower yield in peptide resin synthesis and is not suitable for lutetium [ 177 Preparation of Lu]Oxyoctreotide precursor compounds

[0240] The above description is merely a few exemplary embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any modifications or variations made by those skilled in the art using the disclosed technical content without departing from the scope of the technical solution of this application are equivalent to equivalent implementations and fall within the scope of the technical solution of this application.

Claims

1. A lutetium [Lu] octreotide precursor compound of formula I ###0001### I 177 A process for the preparation of a lutetium [Lu] octreotide precursor compound of formula I, characterized in that, Includes the following steps: By solid-phase synthesis, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH and DOTA-(tBu)3-CO2H were sequentially coupled onto a king resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-king resin; The DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin is oxidized to remove the Trt protecting group from the two Cys groups and form disulfide bonds with each other, thereby cyclizing them. The obtained cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-royal resin was subjected to a pyrolysis reaction to remove the royal resin and protecting group, yielding lutetium as shown in Formula I. 177 Lu]O-octreotide precursor compound.

2. The production method according to claim 1, characterized by, Includes the following steps: (1) In an organic solvent containing condensation reaction reagents, Fmoc-Thr(tBu)-OH is coupled onto the king resin to obtain Fmoc-Thr(tBu)-king resin. (2) In an organic solvent containing condensation reaction reagents, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH and Fmoc-D-Phe-OH are sequentially coupled onto the Fmoc-Thr(tBu)-King resin to obtain Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin; (3) In an organic solvent containing condensation reaction reagents, DOTA-(tBu)3-CO2H is coupled onto the Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin; (4) An oxidation reaction is carried out in an organic solvent containing an oxidant, so that the two Lys in the DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-King resin lose the Trt protecting group and form a disulfide bond with each other, thereby cyclizing; (5) The cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-King resin obtained in step (4) is subjected to a pyrolysis reaction to remove the King resin and protecting group, in a mixture containing trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanedioxide), triisopropylsilane and purified water, to obtain lutetium […] as shown in Formula I. 177 The cleavage reaction of the Lu]oxooctreotide precursor compound was carried out at 5–10 °C for 1 hour, followed by 3 hours at 26–30 °C.

3. The preparation method according to claim 2, characterized in that, In step (1), the resin is swollen in an organic solvent before the coupling reaction is carried out.

4. The preparation method according to claim 2, characterized in that, In step (1), the coupling reaction is carried out at 25–30°C for 2 hours.

5. The preparation method according to claim 4, characterized in that, In step (1), the Fmoc-Thr(tBu)-OH is first activated at 0-10℃ for 3-5 minutes, and then coupled at 25-30℃ for 2 hours.

6. The preparation method according to claim 5, characterized in that, In step (1), the Fmoc-Thr(tBu)-OH is first activated at 5°C for 3 to 5 minutes.

7. The preparation method according to claim 2, characterized in that, In step (1), the condensation reaction reagent comprises one of the following combinations: Combination 1: Benzotriazole-1-yl-oxytripyrrolidinyl phosphate hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Combination 2: O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 4-dimethylaminopyridine; Combination 3: 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine.

8. The preparation method according to claim 7, characterized in that, In step (1), the molar equivalent ratio of Fmoc-Thr(tBu)-OH to the condensation reaction reagents benzotriazine-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:1:1:2; The molar equivalent ratio of Fmoc-Thr(tBu)-OH to the condensation reaction reagents of O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 4-dimethylaminopyridine is 1:1:1:2:0.1; The molar equivalent ratio of Fmoc-Thr(tBu)-OH to the condensation reaction reagents 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine is 1:1:1:0.

1.

9. The preparation method according to claim 2, characterized in that, In step (1), the degree of substitution of the Fmoc-Thr(tBu)-King resin is 0.40 to 0.70 mmol / g.

10. The method of claim 2, wherein, In step (1), the obtained Fmoc-Thr(tBu)-King resin is further end-capped in an organic solvent containing an end-capping agent and an organic base.

11. The method of claim 10, wherein, The capping agent is acetic anhydride; the organic base is N,N-diisopropylethylamine and / or triethylamine; the capping is performed at 20–30°C for 15–20 hours.

12. The method of claim 2, wherein, In step (2), the coupling reaction is carried out at 18-32°C for 2-3 hours.

13. The method of claim 12, wherein, In step (2), the coupling reaction is carried out at 20–30°C for 2–3 hours.

14. [Amended according to Rule 91 on 12.01.2026] The production method according to claim 12, characterized in that, In step (2), the Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH are first activated at 0-10℃ for 5-10 minutes, and then coupled at 18-32℃ for 2-3 hours.

15. The preparation method according to claim 13, characterized in that, In step (2), the Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH are first activated at 5°C for 5 to 10 minutes, and then coupled at 25 to 30°C for 2 to 3 hours.

16. The method of claim 2, wherein, In step (2), the condensation reaction reagent comprises one of the following combinations: Combination 1: 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide; Combination 2: O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Combination 3: benzotriazole hexafluorophosphate-1-yl-oxytripyrrolylphosphine and N,N'-diisopropylcarbodiimide.

17. [Amended according to Rule 91 on 12.01.2026] The production method according to claim 16, characterized in that, In step (2), the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide is 1:1.05:1.05; The molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:1:1:2; The molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, benzotriazol-1-yl-oxytripyrrolidinylphosphine hexafluorophosphate and N,N'-diisopropylcarbodiimide is 1:1:

2.

18. The method of claim 2, wherein, In step (2), the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH to Fmoc-Thr(tBu)-King resin is independently 2 to 4:

1.

19. [Amended according to Rule 91 12.01.2026] The method of manufacture according to claim 18, characterized in that, In step (2), the molar equivalent ratio of Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH to Fmoc-Thr(tBu)-King resin is 3:

1.

20. [R 91 CORRECTED 12.01.2026] The method of making of claim 2, wherein, Step (2) further includes extending the reaction by 0.5 to 2 hours after the Kaiser test for the coupling reaction product is negative.

21. The method of claim 2, wherein, Step (2) further includes removing the Fmoc protecting groups introduced by Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH and Fmoc-Cys(Trt)-OH respectively in an organic solvent containing a deprotecting agent before carrying out the coupling reaction.

22. The method of claim 21, wherein, The deprotecting agent is piperidine, or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene.

23. The method of manufacturing according to claim 21 or 22, wherein, The step of removing the Fmoc protecting group is performed twice at 20-30°C, each time for 15-30 minutes.

24. The method of claim 23, wherein, The step of removing the Fmoc protecting group is performed twice at 20-30°C, each time for 20 minutes.

25. The method of claim 2, wherein, In step (3), the coupling reaction is carried out at 20-30°C for 2-3 hours.

26. The method of claim 26, wherein, In step (3), the DOTA-(tBu)3-CO2H is first activated at 0-10℃ for 5-10 minutes, and then coupled at 20-30℃ for 2-3 hours.

27. The method of claim 2, wherein, In step (3), the condensation reaction reagent comprises one of the following combinations: Combination 1: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine; Combination 2: O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Combination 3: benzotriazole hexafluorophosphate-1-yl-oxytripyrrolidinephosphide, 1-hydroxybenzotriazole and N,N-diisopropylethylamine.

28. The preparation method according to claim 27, characterized in that, In step (3), the molar equivalent ratio of DOTA-(tBu)3-CO2H, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine is 1:0.98:1:2; The molar equivalent ratio of DOTA-(tBu)3-CO2H, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:0.98:1:2; The molar equivalent ratio of DOTA-(tBu)3-CO2H, benzotriazole-1-yl-oxytripyrrolidinephosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:1:1:

2.

29. The method of claim 2, wherein, Step (3) further includes a step of removing the Fmoc protecting group introduced by Fmoc-D-Phe-OH in an organic solvent containing a deprotecting agent before carrying out the coupling reaction.

30. The preparation method according to claim 29, characterized in that, The deprotecting agent is piperidine, or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene.

31. The method of manufacturing according to claim 29 or 30, wherein, The step of removing the Fmoc protecting group is performed twice at 20-30°C, each time for 15-30 minutes.

32. The method of claim 31, wherein, The step of removing the Fmoc protecting group is performed twice at 20-30°C, each time for 20 minutes.

33. The method of claim 2, wherein, In step (4), the oxidation reaction is carried out at 27–32°C for 25–55 minutes.

34. [Amended according to Rule 91, Corrected 12.01.2026] The method of manufacture according to claim 33, characterized in that, In step (4), the oxidation reaction is carried out at 27–32°C for 35–45 minutes.

35. The method of claim 2, wherein, In step (4), the oxidant is iodine.

36. The method of claim 35, wherein, The molar equivalent ratio of iodine to Fmoc-Thr(tBu)-royal resin is 3 to 5:

1.

37. The method of claim 36, wherein the method is performed in a single step. The molar equivalent ratio of iodine to Fmoc-Thr(tBu)-royal resin is 4:

1.

38. The method of claim 35, wherein the method is performed in a single step. The concentration of iodine in the reaction system is 0.15–0.25 mol / L.

39. The method of claim 38, wherein, The concentration of iodine in the reaction system is 0.2 mol / L.

40. The method of claim 2, wherein, Step (4) further includes washing the obtained cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-King resin in an organic solvent containing a reducing agent after the oxidation reaction.

41. The method of claim 40, wherein the method is carried out at a temperature of about 20°C to about 30°C. The reducing agent is vitamin C and / or sodium sulfite.

42. The method of claim 40 or 41, wherein, The washing process is performed twice at 20-30°C, each time for 10 minutes.

43. The method of claim 2, wherein, In step (5), the volume ratio of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)diethylthiol, triisopropylsilane and purified water is 80-90:4-10:3-5:3-5.

44. The method of claim 43, wherein the method is performed in a single step. The volume ratio of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanethiol), triisopropylsilane, and purified water is 90:4:3:

3.

45. The method of claim 2, wherein the method is carried out at a temperature of about 20°C to about 30°C. In step (5), the ratio of the mixture containing trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)diethylthiol, triisopropylsilane and purified water to the cyclized royal resin obtained in step (4) is 10-30 mL: 1 g.

46. The method of claim 2, wherein, Step (5) further includes filtering the product obtained from the pyrolysis reaction and allowing the filtrate to settle in methyl tert-butyl ether at a temperature of ≤20°C for 15 to 20 hours.

47. The method of claim 46, wherein the method is carried out at a temperature of about 20°C to about 30°C. The volume ratio of the methyl tert-butyl ether to the mixture comprising trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanethiol), triisopropylsilane and purified water is 10 to 12:

1.

48. The method of manufacturing according to claim 1 or 2, wherein, In steps (1) to (5), the organic solvent is N,N-dimethylformamide and / or N-methyl-2-pyrrolidone.

49. A lutetium [Lu] octreotide preparation method characterized by, 177 Lu] octreotide preparation method characterized by, a lutetium [Lu] octreotide precursor compound obtainable by the process according to any one of claims 1 to 48. 177 chelated with a lutetium [Lu] octreotide precursor compound. 177 Lu] chelated.

Images