Preparation method of lutetium [177Lu] oxygen octreotide precursor compound

Abstract

The invention provides a preparation method of a lutetium [177Lu] oxygen octreotide precursor compound, which comprises the following steps: sequentially carrying out coupling reaction on Fmoc-Thr (tBu)-OH, Fmoc-Cys (Trt)-OH, Fmoc-Thr (tBu)-OH, Fmoc-D-Trp (tBu)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Cys (Trt)-OH and DOTA-(tBu) 3-CO2H on Wang resin through a solid-phase synthesis method to obtain DOTA-(tBu) 3-D-Phe-Cys (Trt)-D-Trp (Boc)-Lys (Boc)-Thr (tBu)-C performing oxidation reaction to remove Trt protecting groups from two Cys and form disulfide bonds between the two Cys for cyclization, and performing cracking reaction to remove Wang resin and protecting groups, thereby obtaining the lutetium [177Lu] oxygen octreotide precursor compound as shown in the formula I. The lutetium [177Lu] oxygen octreotide precursor compound is shown in the formula I. The invention further discloses a preparation method of the lutetium [177Lu] oxygen octreotide compound. According to the preparation method, the purity of the prepared lutetium [177Lu] oxyoctreotide precursor compound can be remarkably improved, the impurity content is reduced, the product yield is improved, the subsequent purification process is simplified, and a foundation is laid for large-scale industrial production and clinical popularization and application of lutetium [177Lu] oxyoctreotide.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, the present invention relates to a lutetium [ 177 The invention discloses a preparation method and application of a Lu]oxyoctreotide precursor compound. Background Art

[0002] In 2018, the U.S. FDA approved the lutetium [ 177 Lu]oxoctreotide (Lutathera) is now available for the treatment of SSTR-positive gastroenteropancreatic neuroendocrine tumors (GEP-NETs) that have progressed after standard therapy. 177 As the first radioactive drug approved for radioligand therapy (RLT), Lu]oxyoctreotide specifically binds to the somatostatin receptor on the surface of tumor cells, reaches the tumor cells and releases radionuclides to produce radiation to kill tumor cells. Recent clinical trial results also show that its clinical application range is expected to be further expanded.

[0003] Lutetium 177 Lu] oxyoctreotide is a radionuclide [ 177 Lu] is chelated with a precursor compound (MS011) shown in the following formula I as a chelating agent, wherein the precursor compound is composed of a polypeptide composed of 8 amino acids connected to a chelating agent DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), wherein the octadentate ligand formed by the four nitrogen atoms on the dodecane heterocycle plus the four acetic acid groups connected to the nitrogen atoms provides electrons to bind to the lutetium [ 177 Lu] atoms form a stable chelate.

[0004] The industrial production of radionuclide-coupled drugs places high demands on the yield and product purity of the precursor compound preparation method. Impurities will seriously affect the subsequent radionuclide labeling process. 177 The preparation methods of lutetium [Lu] oxoctreotide precursor compounds generally have the problems of low yield and low purity, which is difficult to achieve 177 The efficient and large-scale production of Lu]-oxoctreotide API to meet clinical drug demand poses a major challenge. 177 The preparation process of Lu]oxyoctreotide precursor compounds has become one of the technical problems to be solved urgently in the pharmaceutical field. Summary of the invention

[0005] In view of this, the present invention aims to provide a lutetium [ 177 Lu] oxyoctreotide precursor compound preparation method, the preparation method can significantly improve the prepared lutetium [ 177 Lu] oxyoctreotide precursor compound purity, reduce impurity content, improve product yield, simplify the subsequent purification process, for Lutetium [ 177 This laid a foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide.

[0006] The above object of the present invention is achieved through the following technical solutions:

[0007] A lutetium represented by formula I 177 The invention discloses a method for preparing a precursor compound of Lu] oxygen octreotide, which comprises using a solid phase synthesis method to synthesize 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 is sequentially coupled on Wang resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin, and then an oxidation reaction is performed to remove the Trt protecting group of the two Cys and form a disulfide bond between them to form a cyclization, and then a cleavage reaction is performed to remove the Wang resin and the protecting group to obtain the lutetium shown in formula I [ 177 Lu] oxyoctreotide precursor compound,

[0008]

[0009] According to some embodiments of the present invention, the preparation method comprises the following steps:

[0010] (1) in an organic solvent containing a condensation reaction reagent, subjecting Fmoc-Thr(tBu)-OH to a coupling reaction on a Wang resin to obtain Fmoc-Thr(tBu)-Wang resin;

[0011] (2) in an organic solvent containing a condensation reaction reagent, 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 subjected to coupling reaction on Fmoc-Thr(tBu)-Wang resin to obtain Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin;

[0012] (3) in an organic solvent containing a condensation reaction reagent, subjecting DOTA-(tBu)3-CO2H to a coupling reaction on Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin; and

[0013] (4) performing an oxidation reaction in an organic solvent containing an oxidant, so that the two Lys in DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin are freed from the Trt protecting group and form a disulfide bond with each other to thereby cyclize;

[0014] (5) In a mixed solution containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), triisopropylsilane (Tis) and purified water, a cleavage reaction is performed to remove the Wang resin and the protecting group to obtain the lutetium [ 177 Lu] oxoctreotide 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.

[0015] According to some embodiments of the present invention, in step (1), the Wang resin is swollen in an organic solvent before performing the coupling reaction.

[0016] According to some embodiments of the present invention, 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.

[0017] According to some embodiments of the present invention, in step (1), the condensation reaction reagent comprises one of the following combinations: benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylethylamine (DIEA); O-benzotriazole-N,N,N',N'-tetramethyluronium 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-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. Among them, the molar equivalent ratio of the Fmoc-Thr(tBu)-OH, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:1:1:2; the molar equivalent ratio of the Fmoc-Thr(tBu)-OH, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 4-dimethylaminopyridine can be 1:1:1:2:0.1; the molar equivalent ratio of the Fmoc-Thr(tBu)-OH, 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine can be 1:1:1:0.1.

[0018] According to some embodiments of the present invention, in step (1), the degree of substitution of the Fmoc-Thr(tBu)-Wang resin is 0.40 to 0.70 mmol / g.

[0019] According to some embodiments of the present invention, in step (1), the method further comprises capping the obtained Fmoc-Thr(tBu)-Wang resin in an organic solvent comprising a capping agent and an organic base; preferably, the capping agent is acetic anhydride (Ac2O); wherein the organic base may be N,N-diisopropylethylamine and / or triethylamine, preferably N,N-diisopropylethylamine; preferably, the capping is performed at 20 to 30°C for 15 to 20 hours.

[0020] According to some embodiments of the present invention, 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 is 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.

[0021] According to some embodiments of the present invention, 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'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate 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'-tetramethyluronium 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-oxytripyrrolidinophosphorus hexafluorophosphate and N,N'-diisopropylcarbodiimide can be 1:1:2.

[0022] According to some embodiments of the present invention, 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)-Wang resin is independently 2 to 4:1, preferably 3:1.

[0023] According to some embodiments of the present invention, in step (2), the step further includes extending the reaction for 0.5 to 2 hours after the coupling reaction product is negative by Kaiser detection.

[0024] According to some embodiments of the present invention, in step (2), it also includes the steps of 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 in an organic solvent containing a deprotecting agent before carrying out the coupling reaction; wherein the deprotecting agent can 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.

[0025] According to some embodiments of the present invention, 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.

[0026] According to some embodiments of the present invention, in step (3), the condensation reaction reagent comprises one of the following combinations: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine; O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. Preferably, the condensation reaction reagent comprises benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine. Among them, the molar equivalent ratio of the DOTA-(tBu)3-CO2H, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine can be 1:0.98:1:2; the molar equivalent ratio of the DOTA-(tBu)3-CO2H, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:0.98:1:2; the molar equivalent ratio of the DOTA-(tBu)3-CO2H, benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine can be 1:1:1:2.

[0027] According to some embodiments of the present invention, in step (3), it also 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 can 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.

[0028] According to some embodiments of the present invention, in step (4), the oxidation reaction is carried out at 27-32° C. for 25-55 minutes, preferably 35-45 minutes.

[0029] According to some embodiments of the present invention, in step (4), the oxidant is iodine; preferably, the molar equivalent ratio of iodine to Fmoc-Thr(tBu)-Wang resin is 3 to 5: 1, preferably 4: 1. Preferably, the concentration of iodine in the reaction system is 0.15 to 0.25 mol / L, preferably 0.2 mol / L.

[0030] According to some embodiments of the present invention, in step (4), the process further comprises, after the oxidation reaction, washing the obtained cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-Wang resin in an organic solvent containing a reducing agent; wherein the reducing agent may be vitamin C and / or sodium sulfite, preferably vitamin C; preferably, the washing is carried out twice at 20-30°C, each time for 10 minutes.

[0031] According to some embodiments of the present invention, in step (5), the volume ratio of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanethiol), triisopropylsilane and purified water is 80-90:4-10:3-5:3-5, preferably 90:4:3:3.

[0032] According to some embodiments of the present invention, in step (5), the ratio of the mixed solution comprising trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (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.

[0033] According to some embodiments of the present invention, in step (5), the product obtained from the cleavage reaction is filtered, and the filtrate is allowed to settle in methyl tert-butyl ether (MTBE) at a temperature of ≤20°C for 15 to 20 hours. Preferably, the volume ratio of methyl tert-butyl ether to the mixed solution comprising trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethanedithiol) (DODT), triisopropylsilane (Tis) and purified water is 10 to 12:1.

[0034] According to some preferred embodiments of the present invention, in steps (1) to (5), the organic solvent is independently N,N-dimethylformamide (DMF) and / or N-methyl-2-pyrrolidone, preferably N,N-dimethylformamide.

[0035] In addition, the present invention also provides a lutetium [ 177 Lu] oxyoctreotide preparation method, comprising the steps of: 177 The lutetium [Lu] obtained by the preparation method of the oxoctreotide precursor compound of formula I 177 Lu] oxyoctreotide precursor compound and lutetium [ 177 Lu] chelation.

[0036] The present invention has at least the following beneficial effects:

[0037] The present invention provides an optimized lutetium [ 177 The invention discloses a method for preparing a precursor compound of lutetium [Lu], which reduces the content of a single impurity in the crude product of the precursor compound and improves the purity of the precursor compound product, so that the product purity is ≥90.0%, which is more conducive to the subsequent purification process, thereby achieving a single impurity content of ≤0.10% by weight, and is a precursor compound of lutetium [Lu], which reduces the content of a single impurity in the crude product of the precursor compound and improves the purity of the precursor compound product, so that the product purity is ≥90.0%, which is more conducive to the subsequent purification process, 177 This laid the foundation for the large-scale industrial production and clinical application of Lu]oxyoctreotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0039] Figure 1 The lutetium provided by the present invention [ 177 Process flow chart of the preparation method of Lu]oxyoctreotide precursor compound.

[0040] Figure 2 The lutetium prepared in Example 1 [ 177 NMR spectrum of Lu]oxyoctreotide precursor compound MS011-A.

[0041] Figure 3 The lutetium prepared in Example 1 [ 177 LC-MS spectrum of Lu]-oxyoctreotide precursor compound MS011-A.

[0042] Figure 4 The lutetium prepared in Example 1 [ 177 Infrared spectrum of Lu]oxyoctreotide precursor compound MS011-A.

[0043] Figure 5-7 is the lutetium prepared according to Example 1 [ 177 HPLC spectrum of crude solution of Lu]-oxyoctreotide precursor compound MS011. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0045] 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 unless otherwise specified.

[0046] Abbreviations

[0047]

[0048]

[0049] Example 1 Lutetium177 Preparation of Lu]-oxyoctreotide precursor compound

[0050] 1. Lutetium 177 Synthesis route of Lu]oxyoctreotide precursor compound (MS011)

[0051] Key materials: Wang Resin, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH,

[0052] Fmoc-D-Trp(Boc)-OH,Fmoc-Tyr(tBu)-OH,Fmoc-D-Phe-OH,DOTA-(tBu)3-CO2H

[0053] Reagents: PyBOP, HOBt, DIEA, Ac2O, DIC, TFA, DODT, Tis, water, piperidine, iodine, vitamin C

[0054] Solvents: DMF, MeOH, DCM, MTBE, ACN

[0055] 2. Synthesis process of MS011

[0056] 2.1 Coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0057] Using blank Wang resin (Wang Resin, Xi'an Lanxiao Technology Co., Ltd., cross-linking degree of 1%, mesh size of 100-200) with a substitution degree of 1.0-1.2 mmol / g, 300±1 g of Wang resin (1 eq) and 3.0±0.1 L of DMF (10 vol, v / w) were added to the synthesis tank R-2105 in sequence under nitrogen protection, and nitrogen bubbling was stirred for 30-35 minutes. The stirring was stopped and the solvent in the kettle was emptied.

[0058] Add 2.1±0.1 L DMF (7 vol, v / w) into synthesis tank R-2105, stir with nitrogen bubbling for 3-5 minutes, drain the solvent; repeat this step once more.

[0059] 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 to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0060] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 366.0±1 g DIEA (8.0 eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 3-5 minutes.

[0061] The solution in the activation tank R-0304 is added dropwise to the synthesis tank R-2105, and the activation tank R-0304 is eluted with 0.20±0.01Kg DMF, and the elution liquid is transferred to the synthesis tank R-2105.

[0062] The temperature inside the synthesis tank R-2105 was controlled at 25-30°C and the reaction was carried out for 2 hours. After the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0063] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.1±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.1±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0064] Under nitrogen protection, 0.85±0.1 Kg DMF, 722.8±1 g Ac2O (20 eq), and 91.5±1 g DIEA (2.0 eq) were added to the activation tank R-0304 in sequence and stirred for 5 to 10 minutes.

[0065] Under nitrogen protection, transfer the solution in activation tank R-0304 to synthesis tank R-2105, control the temperature in synthesis tank R-2105 at 20-30°C, and keep the reaction for 15-20 hours; after the insulation is completed, drain the solvent in synthesis tank R-2105.

[0066] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.1±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.1±0.1L DMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0067] Add 2.78±0.1Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.55±0.1Kg MTBE (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.78±0.1Kg DCM (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.55±0.1Kg MTBE (7vol, v / w), stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent, and repeat this step 3 times.

[0068] Take out the Fmoc-Thr(tBu)-Wang Resin from the synthesis tank R-2105, transfer it to the vacuum drying oven D-2101, control the jacket temperature at 30-35°C, vacuum degree ≤-0.08MPa, dry for 10 hours, turn the material over every 3-4 hours, weigh it from the 10th hour, weigh it every 2 hours, until the first process control test (In-Process Control 01, IPC01) loss on drying (LOD) ≤10%, unload the material, put it in a double-layer LDPE bag, weigh it, and label it. Take samples for central control and test the degree of substitution (degree of substitution Sub = 0.4-0.7mmol / g). During the test, the material is placed in a refrigerator at 2-8°C.

[0069] In the above operation steps of 2.1, the material feeding amount is calculated based on Wang resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0070] In the following operation steps 2.2 to 2.10, the material feeding amount is calculated based on Fmoc-Thr(tBu)-Wang Resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0071] 2.2 Fmoc-Cys(Trt)-OH coupling

[0072] Under nitrogen protection, 320±10 g Fmoc-Thr(tBu)-Wang Resin (1 eq) and 3.2±0.1 L DMF (10 vol, v / w) were added to the synthesis tank R-2105 in sequence, and the mixture was stirred with nitrogen bubbling for 30 to 35 minutes. The stirring was stopped and the solvent in the kettle was emptied.

[0073] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, drain the solvent; repeat this step once more.

[0074] Under nitrogen protection, add 6.98±0.1Kg PIP and 30.56±0.1Kg DMF to the buffer tank R-2107 in sequence, stir evenly, and set aside (a total of 8 times of use).

[0075] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0076] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0077] Under nitrogen protection, 307.5±1 g Fmoc-Cys(Trt)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.76±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0078] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0079] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0080] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0081] 2.3 Fmoc-Thr(tBu)-OH coupling

[0082] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30°C, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0083] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0084] Under nitrogen protection, 208.7±1 g Fmoc-Thr(tBu)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.56±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0085] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0086] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0087] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0088] 2.4 Fmoc-Lys(Boc)-OH coupling

[0089] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30°C, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0090] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0091] Under nitrogen protection, add 246±1g Fmoc-Lys(Boc)-OH (3.0eq), 74.5±1g HOBT (3.15eq) and 0.56±0.1Kg DMF into activation tank R-0304 in sequence, stir to dissolve, and cool to 5±5°C.

[0092] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0093] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0094] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once. Add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0095] 2.5Fmoc-D-Trp(Boc)-OH coupling

[0096] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0097] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0098] Under nitrogen protection, 276.5±1 g Fmoc-D-Trp(Boc)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0099] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0100] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0101] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0102] 2.6Fmoc-Tyr(tBu)-OH coupling

[0103] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0104] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0105] Under nitrogen protection, 241.3±1 g Fmoc-Tyr(tBu)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 Kg DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0106] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0107] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0108] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0109] 2.7 Fmoc-Cys(Trt)-OH coupling

[0110] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0111] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0112] Under nitrogen protection, 307.5±1 g Fmoc-Cys(Trt)-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 L DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0113] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0114] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0115] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0116] 2.8 Fmoc-D-Phe-OH coupling

[0117] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0118] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0119] Under nitrogen protection, 203.4±1 g Fmoc-D-Phe-OH (3.0 eq), 74.5±1 g HOBT (3.15 eq), and 0.62±0.1 L DMF were added to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0120] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 69.6±1g DIC (3.15eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0121] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0122] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0123] 2.9DOTA-(tBu)3-CO2H coupling

[0124] Release about 2.08±0.1Kg (6.5vol, w / w) from buffer tank R-2107 into PP barrel, control the temperature inside synthesis tank R-2105 at 20-30℃, add the solution in PP barrel into synthesis tank R-2105, stir with nitrogen bubbling for 20 minutes, drain the solvent in synthesis tank R-2105; repeat this step once more.

[0125] Add 2.24±0.1L DMF (7vol, v / w) into synthesis tank R-2105, stir and wash with nitrogen bubbling for 3-5 minutes, and drain the solvent; repeat this step 5 times (take samples for Kaiser test, the resin solution should be blue).

[0126] 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 to the activation tank R-0304 in sequence, stirred to dissolve, and cooled to 5±5°C.

[0127] The temperature inside the activation tank R-0304 was controlled at 0-10°C, and 90.5±1 g DIEA (4.0 eq) was added dropwise to the activation tank R-0304. After the addition was completed, the mixture was stirred for 5-10 minutes.

[0128] The solution in the activation tank R-0304 was added dropwise to the synthesis tank R-2105, and the activation tank R-0304 was eluted with 0.20±0.1Kg DMF, and the elution liquid was transferred to the synthesis tank R-2105; after the addition was completed, the temperature in the synthesis tank R-2105 was controlled at 20-30°C, nitrogen was bubbled, and the mixture was kept warm and stirred for 2-3 hours (samples were taken for Kaiser test, and the resin and solution were colorless and transparent; ninhydrin was used to test every 1 hour until negative); after the reaction was completed, the solvent in the synthesis tank R-2105 was emptied.

[0129] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0130] 2.10 Solid phase cyclization

[0131] Under nitrogen protection, add 3.3±0.1 Kg DMF and 177.7±1 g iodine (4.0 eq) into the activation tank R-0304 in sequence and stir evenly.

[0132] Add the solution in the activation tank R-0304 dropwise into the synthesis tank R-2105, control the temperature in the synthesis tank R-2105 at 27-32°C, keep the reaction warm for 40±5 minutes, and drain the solvent in the synthesis tank R-2105.

[0133] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0134] Under nitrogen protection, 184.9±1 g vitamin C (3.0 eq), 0.99±0.05 kg methanol and 5.9±0.1 kg DMF were added to the activation tank R-0304 in sequence and stirred to dissolve.

[0135] Control the temperature inside the synthesis tank R-2105 at 20-30°C, add half of the solution (about 2.24±0.05L) in the activation tank R-0304 into the synthesis tank R-2105, keep warm and stir for 10 minutes, and drain the solvent in the synthesis tank R-2105.

[0136] Control the temperature inside the synthesis tank R-2105 at 20-30°C, add the remaining solution (about 2.24±0.05L) in the activation tank R-0304 into the synthesis tank R-2105, keep warm and stir for 10 minutes, and drain the solvent in the synthesis tank R-2105.

[0137] Add 6.0±0.1L DMF to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.24±0.1LDMF (7vol, v / w) to the activation tank R-0304 for spraying, transfer the eluent to the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 2.24±0.1LDMF (7vol, v / w) to the synthesis tank R-2105 for spraying, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step twice.

[0138] Add 2.98±0.2Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 1.66±0.1Kg MTBE (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; add 2.98±0.2Kg DCM (7vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once; add 1.66±0.1Kg MTBE (70vol, v / w) into the synthesis tank R-2105, stir and wash with nitrogen bubbling for 3 to 5 minutes, and drain the solvent; repeat this step once.

[0139] Take out the peptide resin in the synthesis tank R-2105, transfer it to the vacuum drying oven D-2101, control the jacket temperature at 30-35°C, vacuum degree ≤-0.08MPa, dry for 10 hours, turn the material every 3-4 hours, weigh it from the 10th hour, weigh it every 2 hours until LOD ≤ 10%, unload it, put it into a double-layer LDPE bag, weigh it, and label it. The material is placed in a refrigerator at 2-8°C.

[0140] 2.11 Peptide resin cleavage

[0141] Under nitrogen protection, add 153±1 kg MTBE (300 vol, v / w) into the settling tank, cool to -20 to -10°C, start stirring at a speed of 100 rpm.

[0142] Under nitrogen protection, 27.5±0.1Kg TFA, 930±1g DODT, 480±1g Tis, and 0.62±0.05Kg purified water (volume ratio is 90:4:3:3) were added to the cracking kettle in sequence, the internal temperature was reduced to -10~-5℃, and the rotation speed was 100 rpm.

[0143] Under nitrogen protection, add 690±1g of peptide resin into the cleavage kettle, control the internal temperature at -10~0℃, and rinse the feed port with a small amount of cleavage solution.

[0144] Within 20 minutes, the temperature in the cracking reactor was raised to 5-10°C and maintained at 5-10°C for 1 hour with stirring.

[0145] Within 30 minutes, the temperature in the cracking reactor was raised to 26-30°C and maintained at 26-30°C for 3 hours with stirring.

[0146] Filter, transfer the filtrate into the settling tank R-2101 (control the temperature of the settling tank not exceeding 20°C), wash the resin in the cracking kettle with TFA 3 times, stir for 3 to 5 minutes each time, use 0.22±0.02Kg TFA for each washing, and transfer the washing liquid into the settling tank together.

[0147] The sedimentation tank is stirred for 2 to 4 hours and allowed to settle for 15 to 20 hours.

[0148] The material in the settling tank was transferred to a filter with a screen (three-in-one M-2101) for filtration and washed with MTBE, using 1.5±0.1 Kg MTBE (2.94 vol, v / w) for each washing; each washing lasted 10 to 30 minutes; and the washing operation was repeated 3 times.

[0149] The filter cake in the three-in-one was dried under reduced pressure for 12 hours, stirred every 1 to 2 hours, the jacket temperature was 25 to 35°C, and the vacuum degree was ≤-0.085MPa. After drying, the material in the three-in-one was transferred to the tray drying oven D-2101, and continued to be dried under reduced pressure, weighed every 2 hours, until LOD ≤ 10%.

[0150] After drying, the material is transferred into a double-layer LDPE bag and thermoformed; then put into an aluminum foil bag and thermoformed; a desiccant is added between the LDPE bag and the aluminum foil bag, and the bag is weighed, labeled, and stored at -20±5°C to obtain the crude MS011 (MS011-A).

[0151] In the operation step 2.11, the material feeding amount is calculated based on the peptide resin; the stirring speed under nitrogen bubbling is set to 100 rpm.

[0152] The NMR spectrum, LC-MS spectrum and IR spectrum of the synthesized product are shown in Figure 2-4 .

[0153] Lutetium [ 177 HPLC spectrum of crude solution of Lu]-oxyoctreotide precursor compound MS011 Figure 5-7 .

[0154]

[0155] Sample storage conditions: room temperature. The purity test method of MS011 crude solution is as follows:

[0156]

[0157] Example 2 Screening of resin substitution degree

[0158] Three substitution degrees of Fmoc-Thr(tBu)-Wang resin were used for peptide chain coupling (coupling was performed using an automatic synthesis instrument; 4.0eqFmoc-AA / HOBt / DIC, reaction at room temperature for 3.0h; removal of Fmoc: 10min+10min; then 3eqAA / DIEA / HBTU was used to manually re-add DOTA-(tBu)3-CO2H, reaction for 2.5h; DMF washing, Kaiser detection was colorless; 4.0eq iodine was added for cyclization reaction for 40min and then cleaved: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio) 20V, reaction at 5-10°C for 1.0h, and then reaction at 25-30°C for 3.0h; 10V MTBE sedimentation gave crude products MS011-20240126-E0005-C01, MS011-20240126-E0005-C02 and MS011-20240126-E0005-C03 respectively.

[0159] According to the experimental results (see Table 1), the purity and yield of the crude peptide obtained by coupling with three degrees of substitution are consistent. Therefore, the degree of substitution of Fmoc-Thr(tBu)-Wang resin ranges from 0.40 to 0.70 mmol / g.

[0160] Table 1

[0161]

[0162] Example 3 Screening of DOTA-(tBu)3-CO2H Coupling Reagents

[0163] The experiment found that the coupling reaction using the DOTA-(tBu)3-OH / HOBT / DIC system may produce impurities with missing DOTA (Des-DOTA) (4.0 eq, fully protected cleavage after 3.3 h of coupling (synthesized using 2-CTC Resin). According to the fully protected cleavage (synthesized using 2-CTC Resin synthesis) retention time inferred that the impurity content of DOTA missing with RT of 11.276 is about 0.33% (peak area percentage) (the molecular weight of the impurity missing DOTA was not found in LC-MS). To this end, the DOTA coupling process was optimized, and three coupling systems were experimentally screened, namely DOTA-(tBu)3-OH / HBTU / HOBT / DIEA, DOTA-(tBu)3-OH / PyBOP / HOBT / DIEA and DOTA-(tBu)3-OH / HATU / HOAT / DIEA coupling systems. The yields of DOTA-(tBu)3-OH coupled with these three condensing agents were basically the same, and no peptides missing DOTA were found. The purity of the crude peptide obtained by coupling with the DOTA-(tBu)3-OH / HATU / HOAT / DIEA system was slightly lower than that of the other two.

[0164] Considering the comprehensive 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.

[0165] Table 2

[0166]

[0167] Example 4 Screening of coupling condensation agents for the first amino acid Fmoc-Thr(tBu)-OH

[0168] Wang Resin (1.18 mmol / g) was taken and the first Fmoc-Thr(tBu)-OH (3.0 eq) was coupled using three condensation systems, namely HOBT / DIC / DMAP, HBTU / HOBT / DMAP / DIEA, and PyBOP / HOBT / DIEA coupling systems. The reaction temperature was 27-30°C and nitrogen bubbling was used 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, respectively.

[0169] The experimental results (see Table 3) show that the HOBT / DIC / DMAP system is less effective than the HBTU / HOBT / DMAP / DIEA system when three condensation systems are used to couple the first Fmoc-Thr(tBu)-OH. However, the use of DMAP in the coupling process of Fmoc-Thr(tBu)-OH may lead to the risk of racemization during the coupling of Fmoc-Thr(tBu)-OH. Therefore, the PyBOP / HOBT / DIEA system is used to couple the first Fmoc-Thr(tBu)-OH.

[0170] Table 3

[0171]

[0172] Example 5 Screening of key process parameters for coupling of the first amino acid Fmoc-Thr(tBu)-OH

[0173] The experimental results (see Table 4) show that the reaction temperature has a great influence on the uploading rate of the first Fmoc-Thr(tBu)-OH. Considering the uploading rate, the Fmoc-Thr(tBu)-OH / PyBOP / HOBT / DIEA=4 / 4 / 4 / 8 system was selected for coupling, the reaction temperature was 25°C-30°C, and the reaction time was 2h.

[0174] Table 4

[0175]

[0176]

[0177] Example 6 Screening of the Ratio of Other Amino Acids in Peptide Resin Synthesis

[0178] The reaction temperature was controlled at 25-30°C, and the reaction was extended for 1 hour after the Kaiser test showed that the reaction was colorless. 4.0 eq of iodine was used for the cyclization reaction. The cleavage conditions were: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio) 30V, and the reaction was carried out at 5-10°C for 1 hour, then the temperature was slowly raised to 25-30°C and the reaction was carried out for 3.0 hours.

[0179] The amino acid feed ratio screening experiment obtained the following products: MS011-20240126-E0017-01, MS011-20240126-E0017-02 and MS011-20240126-E0017-03.

[0180] The experimental results (see Table 5) show that the yield and purity of the crude peptide product synthesized using 2eq, 3eq and 4eq (molar equivalent of Fmoc-AA-OH relative to Fmoc-Thr(tBu)-Wang Resin) amino acid feed amounts are basically consistent. In order to make the synthesis process more stable, 3eqAA is used for the development of subsequent processes.

[0181] Table 5

[0182]

[0183]

[0184] Fmoc-AA-OH represents an Fmoc-protected amino acid except Fmoc-Thr(tBu)-OH (the same below).

[0185] Example 7 Screening of coupling temperature for other amino acids in peptide resin synthesis

[0186] Take MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-Wang 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, the reaction was extended for 1 h after the Kaiser test showed colorless, the peptide resin was cyclized with 4.0 eq iodine, and after cleavage, MS011-20240126-E0020-01, MS011-20240126-E0020-02 and MS011-20240126-E0020-03 were obtained respectively.

[0187] The effects of coupling temperatures of 18-22°C, 23-27°C and 28-32°C on the purity of the crude peptide were investigated. According to the experimental results (see Table 6), the purity of the crude product obtained at a reaction temperature of 18-22°C was 82.13%, the purity of the crude product obtained at a reaction temperature of 23-27°C was 82.77%, and the purity of the crude product obtained at a reaction temperature of 28-32°C was 84.32%. The purity of the crude products in the three reaction temperature ranges was consistent, ranging from 82% to 84%, and the impurity spectra were consistent, and the temperature range for solid phase coupling of MS011 was determined to be 20-30°C.

[0188] Table 6

[0189]

[0190]

[0191] Example 8 Screening of coupling time of other amino acids in peptide resin synthesis

[0192] Take MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-Wang 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°C, the reaction is colorless after Kaiser test, and the reaction is extended for 0.5h, 1.0h and 2.0h. The peptide resin is cyclized with 4.0eq iodine, and after cleavage, MS011-20240126-E0030-01, MS011-20240126-E0030-02 and MS011-20240126-E0030-03 are obtained respectively.

[0193] The experimental results (see Table 7) show that when the reaction time is extended for 0.5 h, 1 h and 2 h after the Kaiser test is negative, the purity and yield of the obtained crude peptide are basically the same, and the impurity spectrum is also basically the same.

[0194] Table 7

[0195]

[0196]

[0197] Example 9 Screening of Deprotection Time of Other Amino Acids in Peptide Resin Synthesis

[0198] The effect of Fmoc removal time after solid phase coupling on the purity of crude peptide was investigated. Take MS011-20240126-E0012-A01 peptide resin (Fmoc-Thr(tBu)-Wang 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°C, the reaction is colorless as detected by Kaiser test, the peptide resin is cyclized with 4.0 eq iodine, and MS011-20240126-E0030-A01, MS011-20240126-E0030-A02 and MS011-20240126-E0030-A03 are obtained after cleavage.

[0199] The experimental results (see Table 8) show that the removal of Fmoc reaction time is 15+15min, 20+20min, and 30+30min, the purity and impurity distribution are basically the same, the synthesis yield and the cracking yield are basically the same, and the removal of Fmoc time is tentatively set at 20min+20min.

[0200] Table 8

[0201]

[0202] Example 10 Screening of iodine dosage in peptide resin synthesis

[0203] Take 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)-Wang resin prepared according to Example 1, 5.00 g, 1.34 mmol) and iodine (concentration 0.2 mol / L), the reaction temperature is 20-30°C, and the reaction time is 40 min; Cracking conditions: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio), lysate volume 30V, maintained at 5-10°C for 1h, slowly restored to room temperature for 30min, maintained at 23-27°C for 3.0h, MBTE sedimentation volume 10V, and crude products MS011-20240126-E0036-A01, MS011-20240126-E0036-A02 and MS011-20240126-E0036-A03 were obtained respectively.

[0204] The experimental results (see Table 9) show that the solid phase cyclization of the peptide resin using iodine dosages of 3eq, 4eq and 5eq all converted the linear peptide into a cyclic peptide. The reaction was complete, no linear peptide impurities were found, and the purity and yield remained basically consistent. Finally, the preferred iodine cyclization reaction equivalent was determined to be 4.0eq.

[0205] Table 9

[0206]

[0207] Example 11 Screening of Cyclization Reaction Time in Peptide Resin Synthesis

[0208] Take 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)-Wang resin prepared according to Example 1, 5.00 g, 1.34 mmol) and iodine (4.0 eq, concentration: 0.2 mol / L), temperature 25-30°C, reaction time: 25 min, 40 min and 45min; lysis conditions: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio), lysis solution volume 30V, maintained at 5-10°C for 1h, slowly restored to room temperature for 30min, maintained at 23-27°C for 3.0h, MBTE sedimentation volume 10V, to obtain MS011-20240126-E0036-F01, MS011-20240126-E0036-F02 and MS011-20240126-E0036-F03.

[0209] The experimental results (see Table 10) show that when the iodine solid phase cyclization reaction time is 25min, 40min and 55min, the linear peptides are converted into cyclic peptides, the reaction is complete, no linear peptide impurities are found, the purity and yield are basically consistent, and the optimal iodine cyclization time is determined to be 40min.

[0210] Table 10

[0211]

[0212] Example 12 Screening of Cyclization Reaction Temperature in Peptide Resin Synthesis

[0213] 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)-Wang resin prepared according to Example 1, 5.00 g, 1.34 mmol) and iodine (4.0 eq, concentration: 0.2 mol / L) were reacted for 40 min; Decomposition conditions: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio), lysis solution volume 30V, maintained at 5-10°C for 1h, slowly restored to room temperature for 30min, maintained at 23-27°C for 3.0h, MBTE sedimentation volume 10V, to obtain MS011-20240126-E0036-C01, MS011-20240126-E0036-C02 and MS011-20240126-E0036-C03.

[0214] The experimental results (see Table 11) show that iodine solid phase cyclization temperatures of 18-23°C and 23-27°C both produce linear peptide impurities at 44% and 21.6%, respectively, while no obvious linear peptide impurities were found under the cyclization conditions of 27-32°C. Therefore, the temperature of solid phase iodine cyclization was determined to be 27-32°C.

[0215] Table 11

[0216]

[0217] Example 13 Screening of iodine concentration for cyclization reaction in peptide resin synthesis

[0218] 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)-Wang resin prepared according to Example 1, 5.00 g, 1.34 mmol) and iodine (4.0 eq, concentration: 0.15 mol / L, 0.20 mol / L and 0.25 mol / L) were added and the temperature was 28-3 0℃, reaction time 40min; cleavage conditions: TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (volume ratio), cleavage solution 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, and obtained MS011-20240126-E0036-F04, MS011-20240126-E0036-F05 and MS011-20240126-E0036-F06 respectively.

[0219] The experimental results (see Table 12) show that when the iodine concentration 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.

[0220] Table 12

[0221]

[0222]

[0223] Example 14 Lysis solution formulation screening

[0224] MS011-20240126-E0003-B01 peptide resin (37.71 g of cyclized peptide resin prepared according to Example 1, peptide resin synthesis yield of 116%) was used for cleavage condition screening, reaction temperature 25-30°C, reaction time 3h, and the effect of different cleavage solutions on the purity of crude peptide was investigated:

[0225] Table 13

[0226]

[0227]

[0228] Combining the cleavage reaction solution formula 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 formula to control the cleavage by-products, and the addition of DTT to the cleavage solution formula causes the product to be reduced to a linear peptide. Taking all factors into consideration, the cleavage solution formula is determined to be TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v).

[0229] Example 15 Cracking reaction temperature screening

[0230] 1. Preliminary screening

[0231] MS011-20240126-E0003-B01 peptide resin (37.71 g of cyclized peptide resin prepared according to Example 1, peptide resin synthesis yield 116%) was used for cleavage condition screening, the cleavage solution formula was TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v), the cleavage solution dosage was 20V, and the effect of different reaction temperatures on the purity of the crude peptide was investigated:

[0232] Table 14

[0233]

[0234] Table 14 shows that by controlling the cracking process at different reaction temperatures, it was found that the purity of the crude product obtained by reacting at low temperatures of 5±5°C and 10±5°C for 1 hour and then heating to 20-30°C for 3 hours was slightly higher than that of the crude product obtained by reacting at low temperatures of 0±5°C for 1 hour. Taking all factors into consideration, the low-temperature cracking temperature was controlled at 5-10°C and then heated to 20-30°C for 3 hours.

[0235] 2. Refine the screening

[0236] MS011-20240126-E0050-02 peptide resin (235.80 g of cyclized peptide resin prepared according to Example 1, yield: 120.39%) was used for cleavage condition screening. The cleavage solution formula was TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v), 30 V, and the effect of different cleavage reaction temperatures on the purity of the crude peptide was investigated:

[0237] Table 15

[0238]

[0239] Table 15 shows that the purity of the crude product obtained by cleavage at 25±0.5℃ is 73.5%, the purity of the crude product obtained by cleavage at 32±0.5℃ is 73.1%, the purity and impurity distribution of the crude products obtained by cleavage at 27±0.5℃ and 29±0.5℃ are basically the same, the cleavage yield is basically the same, and the purity is about 78%. Taking all factors into consideration, the reaction temperature is set at 26-30℃.

[0240] Example 16 Cleavage Reaction Volume Screening

[0241] MS011-20240126-E0003-B01 peptide resin (37.71 g of cyclized peptide resin prepared according to Example 1, peptide resin synthesis yield 116%) was used for cleavage condition screening, the cleavage solution formula was TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v), 5-10°C for 1h, 27-30°C for 3h. The effect of different cleavage solution volumes on the purity of crude peptides was investigated:

[0242] Table 16

[0243]

[0244]

[0245] Table 16 shows the screening results of 10V, 15V, 20V, and 30V cleavage volumes. The crude peptide purities are 69.39%, 73.51%, 75.84%, and 77.57%, respectively. Increasing the cleavage reaction volume can improve the purity of the cleavage crude product, but too large a cleavage volume will result in a larger crystallization volume. Therefore, the cleavage volume was set at 30V, and the effect of a larger volume of cleavage solution on the quality of the crude product was no longer studied.

[0246] Example 17 Screening of the dosage of methyl tert-ether as a cracking and settling agent

[0247] MS011-20240126-E0024-03 peptide resin (11.33 g of cyclized peptide resin prepared according to Example 1, yield: 124%) was used for cleavage condition screening. The cleavage solution formula was TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v), 30 V; cleavage at 5-10°C for 1 h and then slowly heated up: cleavage at 23-27°C for 3 h, and the effects of different amounts of methyl tert-ether on the purity and yield of the crude peptide were investigated:

[0248] Table 17

[0249]

[0250] 8V, 10V, and 15V indicate that the volume of methyl tert-butyl ether is 8, 10, and 15 times the volume of the cleavage reaction solution, respectively.

[0251] Table 17 shows the results of using 8V, 10V, and 15V methyl tert-butyl ether to settle the cleavage reaction liquid and crystallize. The yield of the 8V methyl tert-butyl ether sedimentation product is 62.5%, the yield of the 10V sedimentation product is 72.1%, and the yield of the 15V sedimentation product is 76.2%. The 10V methyl tert-butyl ether and 15V methyl tert-butyl ether sedimentation yields are significantly higher than those of 8V. Considering comprehensively, the 10-12V methyl tert-butyl ether of the cleavage reaction liquid is selected for sedimentation and crystallization.

[0252] Example 18 Cleavage Reaction Time Screening

[0253] MS011-20240126-E0039-02 peptide resin (226.03 g of cyclized peptide resin prepared according to Example 1; synthesis yield: 114.10%) was used for cleavage condition screening. The cleavage solution formula was TFA / DODT / Tis / H2O=90 / 4 / 3 / 3 (v / v), 30V, 5-10°C for 1h, and the effect of different reaction times of slowly heating to 26-30°C on the purity of the crude peptide was investigated:

[0254] Table 18

[0255]

[0256] Table 18 shows the results of the peptide resin being cleaved at 5-10°C for 1 hour and then slowly heated up, and then cleaved at 26-30°C for 2 hours, 3 hours, and 4 hours, respectively. The purity of the crude peptide cleaved at 26-30°C for 3 and 4 hours was basically the same, and was higher than the purity of the crude product cleaved for 2 hours. Considering all factors, the cleavage time was selected to be 3 hours.

[0257] Comparative Example 1: Preparation of Lutetium using 2-CTC resin 177 Lu]Oxyoctreotide precursor compound

[0258] 1. Solid Phase Synthesis of Peptide Resins

[0259] The synthetic route is the same as in Example 1, and the resin used is 2-CTC resin. The experimental results are as follows:

[0260]

[0261]

[0262] in conclusion:

[0263] (1) The substitution degree of peptide resin of MS011-20240126-E0001-01 was low, and the equivalent of Fmoc-Thr(tBu)-OH was small (0.55 eq); MS011-2024026-E0001-03 was changed to 0.76 eq, and sub=0.56 mmol / g, 41.17 g, synthesis scale: 23.05 mmol;

[0264] (2) The peptide resin MS011-20240126-E0001-036 obtained was 88.44 g (actual weight gain: 38.44 g), theoretical weight: 85.86 g (theoretical weight gain: 55.86 g), yield: 68.81%, while the yield of Wang resin can generally reach 100%.

[0265] (3) The cleavage effect is poor. HPLC shows that the yield of full protection cleavage is 87.93%, while the yield using Wang resin can generally reach 90-100%.

[0266] (4) When coupled with DOTA, approximately 0.3% of the peptide was deleted.

[0267] 2.2-CTC peptide resin cleavage

[0268] The cleavage route is the same as in Example 1, and the resin used is 2-CTC resin. The experimental results are as follows:

[0269]

[0270] in conclusion:

[0271] Some of the impurities with MS+56 are the result of the sulfhydryl group in Cys combining with the butyl carbonium cation produced by the cleavage of tBu, which cannot be removed by subsequent secondary cleavage.

[0272] 3.2-CTC peptide resin solid phase iodine cyclization

[0273]

[0274]

[0275] Conclusion: Compared with Wang resin, 2-CTC resin has a lower yield in peptide resin synthesis and is not suitable for lutetium [ 177 Preparation of Lu]-oxyoctreotide precursor compound

[0276] The above are only several exemplary embodiments of the present invention, and do not limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the above disclosed technical content to make some changes or modifications are equivalent to equivalent implementation cases, and all belong to the scope of the technical solution of the present invention.

Claims

1. A lutetium represented by formula I [ 177 The invention discloses a method for preparing a precursor compound of Lu] oxygen octreotide, which comprises using a solid phase synthesis method to synthesize 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 is sequentially coupled on Wang resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin, and then an oxidation reaction is performed to remove the Trt protecting group of the two Cys and form a disulfide bond between them to form a cyclization, and then a cleavage reaction is performed to remove the Wang resin and the protecting group to obtain the lutetium shown in formula I [ 177 Lu] oxyoctreotide precursor compound, 2. The preparation method according to claim 1, comprising the following steps: (1) in an organic solvent containing a condensation reaction reagent, subjecting Fmoc-Thr(tBu)-OH to a coupling reaction on a Wang resin to obtain Fmoc-Thr(tBu)-Wang resin; (2) in an organic solvent containing a condensation reaction reagent, 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 subjected to coupling reaction on Fmoc-Thr(tBu)-Wang resin to obtain Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin; (3) in an organic solvent containing a condensation reaction reagent, subjecting DOTA-(tBu)3-CO2H to a coupling reaction on Fmoc-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin to obtain DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin; and (4) performing an oxidation reaction in an organic solvent containing an oxidant, so that the two Lys in DOTA-(tBu)3-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-Wang resin are freed from the Trt protecting group and form a disulfide bond with each other to thereby cyclize; (5) In a mixed solution containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (DODT), triisopropylsilane (Tis) and purified water, a cleavage reaction is performed to remove the Wang resin and the protecting group to obtain the lutetium [ 177 Lu] oxoctreotide 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.

3. The preparation method according to claim 2, wherein In step (1), the Wang resin is swollen in an organic solvent before carrying out the coupling reaction; Preferably, in step (1), the coupling reaction is carried out at 25-30°C for 2 hours; further 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; Preferably, in step (1), the condensation reaction reagent comprises one of the following combinations: benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate (PyBOP), 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylethylamine (DIEA); O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole, N,N-diisopropylethylamine and 4-dimethylaminopyridine (DMAP); or 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide (DIC) and 4-dimethylaminopyridine; Further preferably, in step (1), the condensation reaction reagent comprises benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Further preferably, in step (1), the molar equivalent ratio of Fmoc-Thr(tBu)-OH, benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:1:1:2; the molar equivalent ratio of Fmoc-Thr(tBu)-OH, O-benzotriazole-N,N,N',N'-tetramethyluronium 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, 1-hydroxybenzotriazole, N,N'-diisopropylcarbodiimide and 4-dimethylaminopyridine is 1:1:1:0.1; Preferably, in step (1), the degree of substitution of the Fmoc-Thr(tBu)-Wang resin is 0.40 to 0.70 mmol / g; Preferably, in step (1), the method further comprises capping the obtained Fmoc-Thr(tBu)-Wang resin in an organic solvent comprising a capping agent and an organic base; further preferably, the capping agent is acetic anhydride (Ac2O); further preferably, the organic base is N,N-diisopropylethylamine and / or triethylamine, more preferably N,N-diisopropylethylamine; further preferably, the capping is performed at 20 to 30°C for 15 to 20 hours.

4. The preparation method according to claim 2, wherein 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 is 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 25-30°C for 2-3 hours; Preferably, 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'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate and N,N'-diisopropylcarbodiimide; Further preferably, in step (2), the condensation reaction reagent comprises 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide; Further preferably, 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, 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide is 1:1.05:1.

05. H, Fmoc-Cys(Trt)-OH or Fmoc-D-Phe-OH, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine in a molar equivalent ratio of 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-oxytripyrrolidinophosphorus hexafluorophosphate and N,N'-diisopropylcarbodiimide is 1:1:2; Preferably, 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)-Wang resin is independently 2 to 4:1, preferably 3:1; Preferably, in step (2), the method further comprises extending the reaction for 0.5 to 2 hours after the coupling reaction product is negative by Kaiser test; Preferably, in step (2), it also includes the step of removing the Fmoc protecting group 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 in an organic solvent containing a deprotecting agent before carrying out the coupling reaction; further preferably, the deprotecting agent is piperidine (PIP), or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene (DBU), more preferably piperidine; further 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.

5. The preparation method according to claim 2, wherein: 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; Preferably, in step (3), the condensation reaction reagent comprises one of the following combinations: 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 1-hydroxy-7-azobenzotriazole (HOAt) and N,N-diisopropylethylamine; O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; or benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Further preferably, in step (3), the condensation reaction reagent comprises benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine; Further preferably, in step (3), the molar equivalent ratio of DOTA-(tBu)3-CO2H, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium 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'-tetramethyluronium 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-oxytripyrrolidinophosphorus hexafluorophosphate, 1-hydroxybenzotriazole and N,N-diisopropylethylamine is 1:1:1:2; Preferably, in step (3), it also 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; further preferably, the deprotecting agent is piperidine (PIP), or piperidine and 1,8-diazabicyclo[5.4.0-7]undec-7-ene, more preferably piperidine; further 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.

6. The preparation method according to claim 2, wherein: In step (4), the oxidation reaction is carried out at 27-32° C. for 25-55 minutes, preferably 35-45 minutes; Preferably, in step (4), the oxidant is iodine; further preferably, the molar equivalent ratio of iodine to Fmoc-Thr(tBu)-Wang resin is 3 to 5:1, preferably 4:1; further preferably, the concentration of iodine in the reaction system is 0.15 to 0.25 mol / L, preferably 0.2 mol / L; Preferably, in step (4), the method further comprises washing the obtained cyclized DOTA-(tBu)3-D-Phe-Cys-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys-Thr(tBu)-Wang resin in an organic solvent containing a reducing agent after the oxidation reaction; further preferably, the reducing agent is vitamin C and / or sodium sulfite, preferably vitamin C; further preferably, the washing is carried out twice at 20-30°C, each time for 10 minutes.

7. The preparation method according to claim 2, wherein: In step (5), the volume ratio of trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethyl mercaptan), triisopropylsilane and purified water is 80-90:4-10:3-5:3-5, preferably 90:4:3:3; Preferably, in step (5), the ratio of the mixed solution containing trifluoroacetic acid (TFA), 2,2'-(1,2-ethylenedioxy)bis(ethylenethiol) (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. Preferably, in step (5), the process further comprises filtering the product obtained from the cleavage reaction and allowing the filtrate to settle in methyl tert-butyl ether (MTBE) at a temperature of ≤20°C for 15 to 20 hours; more preferably, the volume ratio of methyl tert-butyl ether to the mixed solution comprising trifluoroacetic acid, 2,2'-(1,2-ethylenedioxy)bis(ethanediol), triisopropylsilane and purified water is 10 to 12:

1.

8. The preparation method according to any one of claims 1 to 7, wherein In steps (1) to (5), the organic solvent is N,N-dimethylformamide (DMF) and / or N-methyl-2-pyrrolidone, preferably N,N-dimethylformamide.

9. A lutetium 177 A method for preparing Lu] oxyoctreotide, comprising treating the lutetium [ 177 Lu] oxyoctreotide precursor compound and lutetium [ 177 Lu] chelation.

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