Continuous liquid phase preparation method of first peptide chain of semeglutide

Abstract

The invention provides a continuous liquid phase preparation method of a first peptide chain of semeglutide. The invention provides a method for preparing a first peptide chain of semeglutide through liquid phase synthesis. The method comprises the steps of first esterification and amide condensation reaction, first deprotection reaction, second esterification and amide condensation reaction, second deprotection reaction and third esterification and amide condensation reaction.

Description

Technical Field

[0001] This invention belongs to the field of flow chemistry, and more specifically, this invention relates to a continuous liquid-phase preparation method for the first peptide chain of smegglutinin. Background Technology

[0002] Sermaglutide, developed by Novo Nordisk, is a novel hypoglycemic and weight-loss drug. Its injectable formulation was first approved by the U.S. Food and Drug Administration (FDA) on December 5, 2017. This drug belongs to the class of glucagon-like peptide-1 receptor agonists. By mimicking the body's own GLP-1 hormone, it promotes insulin secretion and inhibits glucagon release+ in a glucose concentration-dependent manner, significantly delaying gastric emptying and reducing appetite, thereby effectively lowering blood sugar and controlling weight. As one of the core drugs in modern comprehensive management programs for type 2 diabetes, sermaglutide has reshaped the treatment landscape of metabolic diseases due to its potent hypoglycemic effect, significant weight loss, and clear cardiovascular benefits. With the rapid growth in global demand for this drug, continuous optimization of its manufacturing process, increased production capacity, and exploration of optimal application strategies for different indications are of paramount clinical and economic value in further improving drug accessibility, meeting the treatment needs of a wider range of patients. The CAS number for smegglutinin is 910463-68-2, and its sequence is His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Octadec anedioic-Glu-PEG2-PEG2)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0003]

[0004] Traditional methods for synthesizing the first peptide chain of semaglutide, Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OBn, primarily involve solid-phase synthesis, as illustrated in Chinese patent applications CN106928343A, CN101133082A, CN106478806A, and CN106749613A. These methods employ the Fmoc strategy for stepwise solid-phase coupling to synthesize the first peptide chain of semaglutide. The main process mentioned in Chinese patent application CN106749613A involves solid-phase fragment condensation, which results in a stepwise coupling of amino acids into a cycle. However, this method is incomplete, the solid-phase support is limited by substitution values, leading to a low overall yield. Furthermore, it produces numerous impurities, making purification difficult and scale-up challenging, wasting solvents, and generating large amounts of waste liquid.

[0005] Traditional batch synthesis of short peptides has insurmountable drawbacks, including: 1) Extremely cumbersome and time-consuming operation: Purification relies on sequential steps, typically using crystallization, precipitation, or column chromatography, which is time-consuming and requires heavy manual operation. 2) Low yield and high loss: Each step of separation and purification is accompanied by unavoidable product loss, which accumulates significantly with peptide chain elongation, making it unsuitable for synthesizing the first peptide chain Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OBn. 3) Huge solvent consumption, resulting in significant environmental and cost pressures: The extensive use of organic solvents imposes cost and environmental burdens. 4) Difficulty in synthesizing complex or modified peptides: Many protecting groups and reaction conditions may be difficult to orthogonalize in the solution phase, limiting complex modifications.

[0006] In summary, the current production process of the first peptide chain of smegglutinin suffers from poor environmental affinity, long synthesis cycle, low purity and yield, and high production cost, which is not conducive to large-scale production. Therefore, it is urgent to solve this problem in this field. Summary of the Invention

[0007] The purpose of this invention is to provide a continuous liquid-phase preparation method for the first peptide chain of smegglutinin.

[0008] In a first aspect, the present invention provides a method for preparing the first peptide chain of smegglutinin using liquid-phase synthesis, the method comprising the following steps: (1) First esterification and amide condensation reaction: Using Fmoc-Glu(OtBu)-OH as raw material, the first pre-activated reaction solution was synthesized by esterification reaction, and H-Gly-OBz was added. HCl was used to perform an amide condensation reaction to synthesize intermediate compound 2. (2) First deprotection reaction: Using compound 2 synthesized in (1) as raw material, intermediate product compound 3 is obtained through deprotection reaction; (3) Second esterification and amide condensation reaction: Using Fmoc-Aib-OH as raw material, the second pre-activated reaction solution is synthesized by esterification reaction, and the compound 3 obtained in (2) is added to carry out amide condensation reaction to synthesize intermediate product compound 4; (4) Second deprotection reaction: Using compound 4 synthesized in (3) as a starting material, intermediate compound 5 is obtained through a deprotection reaction; and (5) Third esterification and amide condensation reaction: Using Fmoc-His(Trt)-OH as raw material, the third pre-activated reaction solution is synthesized by esterification reaction, and compound 5 obtained in (4) is added to carry out amide condensation reaction to synthesize the first peptide chain of smegglutinin.

[0009] In one or more embodiments, step (1) includes: (1A) Using Fmoc-Glu(OtBu)-OH as raw material, Fmoc-Glu(OtBu)-OH, alkali, and a first organic solvent are mixed to obtain feed solution A. (1B) The condensing agent is mixed with the second organic solvent to obtain feed solution B. (1C) The feed solution A obtained in (1A) is mixed with the feed solution B obtained in (1B), and the mixture is then subjected to an esterification reaction to synthesize the first pre-activated reaction solution. (1D) H-Gly-OBz HCl and alkali are mixed to obtain feed solution C. (1E) The first pre-activated reaction solution obtained in (1C) is mixed with feed solution C, and intermediate product compound 2 is synthesized by amide condensation reaction.

[0010] In one or more embodiments, step (1) further includes one or more of the following conditions: Steps (1A) to (1E) are performed consecutively; and / or, The condensing agent can be any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA, N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine; and / or, The first organic solvent, the second organic solvent, and the third organic solvent are each independently selected from any one or more of dichloromethane, ethyl acetate, acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, and 1,4-dioxane, preferably any one or more of N,N-dimethylformamide, tetrahydrofuran, and toluene, more preferably tetrahydrofuran, and even more preferably, the first organic solvent, the second organic solvent, and the third organic solvent are all tetrahydrofuran; and / or, In step (1A), the concentration of Fmoc-Glu(OtBu)-OH in feed solution A is 0.01~2.0 mol / L, preferably 0.05~0.5 mol / L or 0.05~0.15 mol / L; and / or, In step (1A), the molar ratio of Fmoc-Glu(OtBu)-OH to alkali in feed solution A is 1:1 to 4:1, preferably 1:1 to 2:1; and / or, In step (1B), the concentration of the condensing agent in the feed solution B is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (1C), after feed liquid A and feed liquid B are mixed, the molar ratio of Fmoc-Glu(OtBu)-OH to the condensing agent is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (1C) is carried out in the first microreactor; and / or, The reaction temperature of step (1C) is 10~100℃, preferably 20~70℃, more preferably 30±1℃; and / or, In step (1C), feed liquid A and feed liquid B are pumped into the first microreactor via a first injection pump; and / or, In step (1C), the injection flow rates of feed solution A and feed solution B are 0.02~0.04 mL / min; and / or, In step (1C), the residence time of feed liquid A and feed liquid B is 1~20 min, preferably 5~10 min; and / or, In the feed liquid C of step (1D), H-Gly-OBz The concentration of HCl is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L, 0.1~0.2 mol / L, or 0.13~0.15 mol / L; and / or, The step (1E) is carried out in the second microreactor; and / or, The reaction temperature of step (1E) is 10~100℃, preferably 20~70℃, more preferably 30±1℃; and / or, In step (1E), after the first pre-activated reaction solution and feed solution C are mixed, Fmoc-Glu(OtBu)-OH and H-Gly-OBz The molar ratio of HCl is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, In step (1E), the first pre-activated reaction solution and the feed solution C are pumped into the second microreactor via a second injection pump; and / or, In step (1E), the injection flow rate of the first pre-activated reaction solution is 0.03~0.05 mL / min; and / or, In step (1E), the injection flow rate of the feed solution C is 0.015~0.03 mL / min; and / or, In step (1E), the residence time of the feed liquid C is 1~20 min, for example 5~10 min.

[0011] In one or more embodiments, step (2) includes: (2A) The intermediate product compound 2 obtained in step (1) is mixed with a fourth organic solvent to obtain feed liquid D. (2B) The reagent that has removed the Fmoc protecting group is mixed with the fifth organic solvent to obtain feed solution E. (2C) The feed liquid D in (2A) is mixed with the feed liquid E in (2B), and after deprotection reaction, intermediate product compound 3 is obtained.

[0012] In one or more embodiments, step (2) further includes one or more of the following conditions: Steps (2A) to (2C) are performed consecutively; and / or, The reagent used to remove the Fmoc protecting group is DBU; and / or, The fourth and fifth organic solvents are each independently selected from one or more of methanol, toluene, N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, preferably one or more of methanol, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, more preferably acetonitrile, and even more preferably both the fourth and fifth organic solvents are acetonitrile; and / or, In step (2A), the concentration of compound 2 in the feed solution D is 0.01~0.5 mol / L, preferably 0.05~0.3 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (2B), the concentration of the reagent for removing the Fmoc protecting group in the feed solution E is 0.01~0.5 mol / L, preferably 0.05~0.2 mol / L or 0.05~0.15 mol / L; and / or, In step (2C), after feed solution D and feed solution E are mixed, the molar ratio of compound 2 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1.1 to 1:1.5; and / or, The step (2C) is carried out in a third microreactor; and / or, The reaction temperature in step (2C) is 25~70℃, preferably 40~60℃; and / or, In step (2C), the feed solution D and feed solution E are pumped into the third microreactor via a third injection pump; and / or, In step (2C), the injection flow rates of feed solution D and feed solution E are 0.025~0.055 mL / min; and / or, In step (2C), the residence time of feed liquid D and feed liquid E is 1~20min, preferably 4~10min.

[0013] In one or more embodiments, step (3) includes: (3A) Using Fmoc-Aib-OH as raw material, Fmoc-Aib-OH, alkali, and a sixth organic solvent are mixed to obtain feed liquid F. (3B) The condensing agent is mixed with the seventh organic solvent to obtain feed liquid G. (3C) The feed solution F obtained in (3A) is mixed with the feed solution G obtained in (3B), and the second pre-activated reaction solution is synthesized by esterification. (3D) The intermediate product compound 3 obtained in step (2) is mixed with the eighth organic solvent to obtain feed solution H. (3E) The second pre-activated reaction solution obtained in (3C) is mixed with feed solution H and the intermediate product compound 4 is synthesized by amide condensation reaction.

[0014] In one or more embodiments, step (3) further includes one or more of the following conditions: Steps (3A) through (3E) are performed consecutively; and / or, The condensing agent is any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA (N,N-diisopropylethylamine), N-methylmorpholine, pyridine, Na₂CO₃, NaHCO₃, K₂CO₃, KHCO₃, diethylamine, and triethylamine, preferably triethylamine; and / or, The sixth, seventh, and eighth organic solvents are each independently selected from any one or more of N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, acetonitrile, 1,4-dioxane, dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, preferably any one or more of dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, more preferably dichloromethane or ethyl acetate, and even more preferably, the sixth, seventh, and eighth organic solvents are all dichloromethane or ethyl acetate; and / or, In step (3A), the concentration of Fmoc-Aib-OH in the feed solution F is 0.05~0.20 mol / L, preferably 0.05~0.15 mol / L; and / or, In step (3A), the molar ratio of Fmoc-Aib-OH to alkali in the feed solution F is 1:1 to 4:1, preferably 1:1 to 2:1; and / or, In step (3B), the concentration of the condensing agent in the feed liquid G is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (3C), after feed liquid F and feed liquid G are mixed, the molar ratio of Fmoc-Aib-OH to condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (3C) is carried out in the fourth microreactor; and / or, The reaction temperature of step (3C) is 30~80℃, preferably 40~70℃, more preferably 45~60℃; and / or, In step (3C), the feed liquid F and feed liquid G are pumped into the fourth microreactor via a fourth injection pump; and / or, In step (3C), the injection flow rates of feed solution F and feed solution G are 0.02~0.08 mL / min, preferably 0.05~0.06 mL / min; and / or, In step (3C), the residence time of feed liquid F and feed liquid G is 1~20 min, for example, 5±1 min; and / or, In step (3D), the concentration of compound 3 in the feed solution H is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L, or 0.04~0.06 mol / L; and / or, In step (3E), after the second pre-activated reaction solution and the feed solution H are mixed, the molar ratio of Fmoc-Aib-OH to compound 3 is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (3E) is carried out in the fifth microreactor; and / or, The reaction temperature of step (3E) is 30~80℃, preferably 40~70℃, more preferably 45~60℃; and / or, In step (3E), the second pre-activated reaction solution and the feed solution H are pumped into the fifth microreactor via the fifth injection pump; and / or, In step (3E), the injection flow rate of the second pre-activated reaction solution and the feed solution H is 0.02~0.08 mL / min, preferably 0.05~0.06 mL / min; and / or, In step (3E), the residence time of the second pre-activated reaction solution and the feed solution H is 1~20 min, preferably 5±1 min.

[0015] In one or more embodiments, step (4) includes: (4A) The intermediate product compound 4 obtained in step (3) is mixed with the ninth organic solvent to obtain feed liquid I. (4B) The reagent that removes the Fmoc protecting group is mixed with the tenth organic solvent to obtain feed solution J. (4C) The feed liquid I in (4A) is mixed with the feed liquid J in (4B), and after deprotection reaction, intermediate product compound 5 is obtained.

[0016] In one or more embodiments, step (4) further includes one or more of the following conditions: Steps (4A) to (4C) are performed consecutively; and / or, The reagent used to remove the Fmoc protecting group is DBU; and / or, The ninth and tenth organic solvents are each independently selected from one or more of tetrahydrofuran, methanol, toluene, N,N-dimethylformamide, ethyl acetate, 1,4-dioxane, acetonitrile, and dichloromethane, preferably one or more of tetrahydrofuran, methanol, dichloromethane, and acetonitrile, more preferably acetonitrile, and even more preferably both the ninth and tenth organic solvents are acetonitrile; and / or, In step (4A), the concentration of compound 4 in feed liquid I is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (4B), the concentration of the reagent for removing the Fmoc protecting group (e.g., DBU) in the feed solution J is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (4C), after feed liquid I and feed liquid J are mixed, the molar ratio of compound 4 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1.1 to 1:1.5; and / or, The step (4C) is carried out in the sixth microreactor; and / or, The reaction temperature of step (4C) is 25~50℃, preferably 25~40℃; and / or, In step (4C), feed liquid I and feed liquid J are pumped into the sixth microreactor via the sixth injection pump; and / or, In step (4C), the injection flow rates of feed solution I and feed solution J are 0.11~0.22 mL / min; and / or, In step (4C), the residence time of feed liquid I and feed liquid J is 1~10 min, preferably 1~5 min, and more preferably 1~4 min.

[0017] In one or more embodiments, step (5) includes: (5A) Using Fmoc-His(Trt)-OH as raw material, Fmoc-His(Trt)-OH, alkali, and the eleventh organic solvent are mixed to obtain feed liquid K. (5B) The condensing agent is mixed with the twelfth organic solvent to obtain feed liquid L. (5C) The feed solution K obtained in (5A) is mixed with the feed solution L obtained in (5B), and the mixture is then synthesized into a third pre-activated reaction solution via esterification. (5D) The intermediate compound 5 obtained in step (4) is mixed with the thirteenth organic solvent to obtain feed liquid M. (5E) The third pre-activated reaction solution obtained in (5C) is mixed with the feed solution M and synthesized as the first peptide chain of smegglutinin by amide condensation reaction.

[0018] In one or more embodiments, step (5) further includes one or more of the following conditions: Steps (5A) to (5E) are performed consecutively; and / or, The condensing agent is any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA, N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine; and / or, The eleventh, twelfth, and thirteenth organic solvents are each independently selected from any one or more of 1,4-dioxane, N,N-dimethylformamide, ethyl acetate, toluene, acetonitrile, dichloromethane, and tetrahydrofuran, preferably any one or more of dichloromethane and tetrahydrofuran, more preferably dichloromethane, and even more preferably all of the eleventh, twelfth, and thirteenth organic solvents are dichloromethane; and / or, In step (5A), the concentration of Fmoc-His(Trt)-OH in the feed solution K is 0.01~1.0 mol / L, preferably 0.05~0.5 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (5A), the molar ratio of Fmoc-His(Trt)-OH to alkali (e.g., triethylamine) in the feed liquid K is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, In step (5B), the concentration of the condensing agent (e.g., HATU) in the feed liquid L is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (5C), after mixing feed liquid K and feed liquid L, the molar ratio of Fmoc-His(Trt)-OH to condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (5C) is carried out in the seventh microreactor; and / or, The reaction temperature in step (5C) is 30~80℃, preferably 40~70℃, more preferably 55~70℃; and / or, In step (5C), the feed liquid K and feed liquid L are pumped into the seventh microreactor via the seventh injection pump; and / or, In step (5C), the injection flow rates of feed solution K and feed solution L are 0.03~0.3 mL / min, preferably 0.03-0.25 mL / min, more preferably 0.11~0.22 mL / min; and / or, In step (5C), the residence time of the feed liquid K and the feed liquid L is 1~20 min, preferably 5~20 min, for example 5~10 min; and / or, In step (5D), the concentration of compound 5 in the feed solution M is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.01~0.1 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L, or 0.04~0.06 mol / L; and / or, In step (5E), after the third pre-activated reaction solution and the feed solution M are mixed, the molar ratio of Fmoc-His(Trt)-OH to compound 5 is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (5E) is carried out in the eighth microreactor; and / or, The reaction temperature of step (5E) is 30~80℃, preferably 40~70℃, more preferably 55~70℃; and / or, In step (5E), the third pre-activation reaction solution and the feed solution M are pumped into the eighth microreactor via the eighth injection pump; and / or, In step (5E), the injection flow rate of the third pre-activated reaction solution and the feed solution M is 0.1~0.5 mL / min, preferably 0.22~0.44 mL / min; and / or, In step (5E), the residence time of the third pre-activated reaction solution and the feed solution M is 1~20 min, preferably 5~20 min, and more preferably 5~10 min.

[0019] In one or more embodiments, any two, three, four, or all five adjacent steps of steps (1) to (5) are performed consecutively in series; preferably, all five steps of steps (1) to (5) are performed consecutively; more preferably, steps (1A) to (1E), steps (2A) to (2C), steps (3A) to (3E), steps (4A) to (4C), and steps (5A) to (5E) are performed consecutively.

[0020] A second aspect of the present invention provides a method for preparing smegglutinin intermediate compound 2, compound 3, compound 4 or compound 5 using liquid-phase synthesis, wherein: The method for preparing smegglutinin intermediate compound 2 includes step (1) as described in any embodiment of the present invention. The method for preparing smegglutinin intermediate compound 3 includes steps (1) and (2) as described in any embodiment of the present invention, preferably steps (1) and (2) are performed consecutively; The method for preparing smegglutinin intermediate compound 4 includes steps (1), (2), and (3) as described in any embodiment of the present invention, preferably steps (1), (2), and (3) are performed sequentially; The method for preparing the intermediate compound 5 of smegglutinin includes steps (1), (2), (3) and (4) as described in any embodiment of the present invention, preferably steps (1), (2), (3) and (4) are performed sequentially.

[0021] A third aspect of the present invention provides a continuous synthesis apparatus, the apparatus comprising: a first microreactor, a second microreactor, a third microreactor, a fourth microreactor, a fifth microreactor, a sixth microreactor, a seventh microreactor, and an eighth microreactor in sequential fluid communication; a first injection pump in fluid communication with the first microreactor; a second injection pump in fluid communication with the second microreactor; a third injection pump in fluid communication with the third microreactor; a fourth injection pump in fluid communication with the fourth microreactor; a fifth injection pump in fluid communication with the fifth microreactor; a sixth injection pump in fluid communication with the sixth microreactor; a seventh injection pump in fluid communication with the seventh microreactor; an eighth injection pump in fluid communication with the eighth microreactor; a connecting pipe connecting each microreactor and the injection pump; and control components for operating the apparatus to implement the method for preparing the first peptide chain of smegglutinin by liquid-phase synthesis according to any embodiment of the present invention, or the method for preparing intermediate product compound 2, compound 3, compound 4, or compound 5 of smegglutinin by liquid-phase synthesis according to any embodiment of the present invention.

[0022] In one or more embodiments, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth injection pumps are syringe pumps, peristaltic pumps, or horizontal flow pumps, more preferably syringe pumps.

[0023] In one or more embodiments, the first microreactor, second microreactor, third microreactor, fourth microreactor, fifth microreactor, sixth microreactor, seventh microreactor and / or eighth microreactor is a capillary microreactor, a fixed-bed microreactor, a glass chip microreactor or a stainless steel and silicon carbide microchannel reactor, more preferably a capillary microreactor.

[0024] A fourth aspect of the invention provides the use of the continuous synthesis apparatus described in any embodiment of the invention in the continuous liquid-phase synthesis for the preparation of the first peptide chain of smegglutinin, or in the continuous liquid-phase synthesis for the preparation of intermediate product compounds 2, 3, 4 or 5 of smegglutinin.

[0025] In one or more embodiments, the method for preparing the first peptide chain of semaglutide by continuous liquid-phase synthesis is as described in any embodiment of the present invention, and / or, the method for preparing intermediate compound 2, compound 3, compound 4 or compound 5 of semaglutide by continuous liquid-phase synthesis is as described in any embodiment of the present invention.

[0026] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0027] Figure 1Fmoc-Glu(OtBu)-OH (compound 1) and H-Gly-OBz A schematic diagram of the synthesis of the first peptide chain of smegglutinin using HCl (compound 6) as a starting material.

[0028] Figure 2 A schematic diagram of the continuous synthesis apparatus of the present invention, wherein A represents the first injection pump, B represents the second injection pump, C represents the third injection pump, D represents the fourth injection pump, E represents the fifth injection pump, F represents the sixth injection pump, G represents the seventh injection pump, N represents the first microreactor, O represents the second microreactor, P represents the third microreactor, Q represents the fourth microreactor, R represents the fifth microreactor, S represents the sixth microreactor, T represents the seventh microreactor, U represents the eighth microreactor, and N represents the first peptide chain of smegglutinin.

[0029] Figure 3 The synthetic dipeptide (compound 2) was prepared using the method of Example 7, and its LCMS purity was 100%.

[0030] Figure 4 The first deprotected product (compound 3) was prepared and synthesized using the method of Example 18, and its LCMS purity was 100%.

[0031] Figure 5 The synthetic tripeptide (compound 4) was prepared using the method described in Example 36, and its LCMS purity was 91.78%.

[0032] Figure 6 The second deprotected product (compound 5) was prepared and synthesized using the method of Example 46, and its LCMS purity was 98.55%.

[0033] Figure 7 The first peptide chain (tetrapeptide) of smegglutinin was prepared and synthesized using the method of Example 64, and its LCMS purity was 91.75%. Detailed Implementation

[0034] Through in-depth research, the inventors have discovered a method for preparing the first peptide chain of semaglutide (Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OBn) via liquid-phase synthesis. The inventors innovatively employ an acid-base salt formation method for post-treatment, which easily yields high-purity products and improves yield. Compared to solid-phase synthesis, this liquid-phase synthesis method significantly shortens the reaction time and avoids the use of expensive resins and packed columns, thus reducing costs. Furthermore, this invention optimizes the batch liquid-phase synthesis method into a continuous liquid-phase synthesis method. Using the continuous liquid-phase synthesis method, the target product, the first peptide chain of semaglutide, can be obtained with an overall yield of over 85.5% and a total residence time of no more than 18 minutes, representing a significant improvement over the batch synthesis route's overall yield of 29.4% and total reaction time of 13.3 hours. This invention also provides a continuous synthesis apparatus suitable for the continuous liquid-phase synthesis of the first peptide chain of semaglutide.

[0035] the term

[0036] In this invention, " / " represents "or".

[0037] In this invention, the terms "above", "below", "higher than", and "lower than" all include the stated number. For example, "above 85.5%" includes the endpoint value of 85.5%, and also includes values ​​greater than 85.5%.

[0038] In this invention, the numerical ranges mentioned all include their endpoint values.

[0039] The term "semaglutide first peptide chain" refers to the tetrapeptide structure Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-OBn in semaglutide, as shown in Formula I: (Formula I).

[0040] The term "intermediate product" refers to a chemical substance generated during the synthesis process that is not the final target product and typically requires further processing or reaction to obtain the target product. In this invention, the target product is the first peptide chain of smegraglutide, and the intermediate products generated during the synthesis of the first peptide chain of smegraglutide include: compounds 2, 3, 4, and 5. Compound 2, Compound 3, Compound 4, Compound 5.

[0041] In this paper, the term "intermittent" refers to the fact that each operation in the synthesis of the target product (smegglutinin first peptide chain) is performed separately, with each operation having a clear start and end, rather than being continuous.

[0042] The term "continuous" herein refers to at least two or more operations in the synthesis of the target product (semaglutide first peptide chain) or intermediate products (compounds 2, 3, 4, and 5) being performed continuously and without interruption. In some embodiments, at least two or more operations in the steps of synthesizing intermediate product compound 2, compound 3, compound 4, compound 5 or the target product semaglutide first peptide chain are continuous. In some embodiments, the steps of synthesizing intermediate product compound 2, compound 3, compound 4, compound 5 and / or synthesizing the target product semaglutide first peptide chain are continuous.

[0043] The term "step" in this document refers to a reaction process, such as an amide condensation reaction being a step, or a deprotection reaction being a step. It should be understood that a step may involve multiple operations; for example, adding reactants is one operation, and stirring is another.

[0044] The term "yield" refers to the ratio of the actual mass of the product obtained from the synthesis of the intermediate or target product to the theoretical mass of the target product, usually expressed as a percentage. The term "overall yield" refers to the yield of Fmoc-Glu(OtBu)-OH and H-Gly-OBz. Using HCl as a starting material, the overall yield of the target product, semaglutide, first peptide chain, was synthesized. The overall yield is typically the product of the yields of intermediate products in each step of the synthesis process. For example, in this invention, the overall yield of the target product, semaglutide, first peptide chain, is calculated as: yield of compound 2 × yield of compound 3 × yield of compound 4 × yield of compound 4 × yield of semaglutide, first peptide chain.

[0045] The term "purity" refers to the proportion of the synthesized intermediate or target product to the total product content, usually expressed as a percentage. Generally, total product content = intermediate / target product content + impurity content. Purity can be determined using methods such as LCMS; the ratio of the peak area of ​​the intermediate / target product to the total peak area represents the purity of the intermediate / target product.

[0046] The term "Fmoc-Glu(OtBu)-OH" refers to fluorenemethyloxycarbonyl-L-glutamic acid-5-tert-butyl ester.

[0047] The term "H-Gly-OBz" "HCl" refers to glycine benzyl ester hydrochloride.

[0048] The term "HATU" refers to O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, also known as 1-hydroxy-7-azabenzotriazol.

[0049] The term “DBU” refers to 1,8-diazabicyclo[5.4.0]undec-7-ene.

[0050] The term "Fmoc-Aib-OH" refers to N-fluorenylmethoxycarbonyl-2-aminoisobutyric acid.

[0051] The term "Fmoc-His(Trt)-OH" refers to 9-fluorenylmethoxycarbonyl-N-triphenylmethyl-L-histidine.

[0052] Method for preparing the first peptide chain of smegglutinin

[0053] This invention provides a method for preparing the first peptide chain of smegglutinin using liquid-phase synthesis (preferably continuous liquid-phase synthesis), such as... Figure 1 As shown, the reaction involves sequentially performing a first esterification and amide condensation reaction, a first deprotection reaction, a second esterification and amide condensation reaction, a second deprotection reaction, and a third esterification and amide condensation reaction on the raw material Fmoc-Glu(OtBu)-OH to obtain the crude first peptide chain of smegglutinin.

[0054] This invention provides a method for preparing the first peptide chain of smegglutinin using liquid-phase synthesis (preferably continuous liquid-phase synthesis), the method comprising the following steps: (1) First esterification and amide condensation reaction: Using Fmoc-Glu(OtBu)-OH as raw material, the first pre-activated reaction solution was synthesized by esterification reaction, and H-Gly-OBz was added. HCl was used to perform an amide condensation reaction to synthesize intermediate compound 2. (2) First deprotection reaction: Using compound 2 synthesized in (1) as raw material, intermediate product compound 3 is obtained through deprotection reaction; (3) Second esterification and amide condensation reaction: Using Fmoc-Aib-OH as raw material, the second pre-activated reaction solution is synthesized by esterification reaction, and the compound 3 obtained in (2) is added to carry out amide condensation reaction to synthesize intermediate product compound 4; (4) Second deprotection reaction: Using compound 4 synthesized in (3) as a starting material, intermediate compound 5 is obtained through a deprotection reaction; and (5) Third esterification and amide condensation reaction: Using Fmoc-His(Trt)-OH as raw material, the third pre-activated reaction solution is synthesized by esterification reaction, and compound 5 obtained in (4) is added to carry out amide condensation reaction to synthesize the first peptide chain of smegglutinin.

[0055] In this invention, step (1) includes: (1A) Using Fmoc-Glu(OtBu)-OH as raw material, Fmoc-Glu(OtBu)-OH, alkali, and a first organic solvent are mixed to obtain feed solution A. (1B) The condensing agent is mixed with the second organic solvent to obtain feed solution B. (1C) The feed solution A obtained in (1A) is mixed with the feed solution B obtained in (1B), and the mixture is then subjected to an esterification reaction to synthesize the first pre-activated reaction solution. (1D) H-Gly-OBz HCl and alkali are mixed to obtain feed solution C. (1E) The first pre-activated reaction solution obtained in (1C) is mixed with feed solution C, and intermediate product compound 2 is synthesized by amide condensation reaction.

[0056] In some implementations, steps (1A) through (1E) are performed sequentially.

[0057] In some embodiments, the condensing agent may be any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU.

[0058] In some embodiments, the base may be any one or more of DIEA (N,N-diisopropylethylamine), N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine.

[0059] In some embodiments, the first organic solvent, the second organic solvent, and the third organic solvent may each be independently selected from any one or more of dichloromethane, ethyl acetate, acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, and 1,4-dioxane, preferably any one or more of N,N-dimethylformamide, tetrahydrofuran, and toluene, and more preferably tetrahydrofuran. In some embodiments, the first organic solvent, the second organic solvent, and the third organic solvent may be the same, for example, all of them may be tetrahydrofuran.

[0060] In some embodiments, the concentration of Fmoc-Glu(OtBu)-OH in the feed solution A of step (1A) is 0.01~2.0 mol / L, preferably 0.05~0.5 mol / L or 0.05~0.15 mol / L, for example 0.1±0.01 mol / L. In some embodiments, the molar ratio of Fmoc-Glu(OtBu)-OH to alkali (e.g., triethylamine) in the feed solution A of step (1A) is 1:1~4:1, preferably 1:1~2:1, for example 2:1.

[0061] In some embodiments, the concentration of condensing agent (e.g., HATU) in the feed liquid B of step (1B) is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L, for example 0.2±0.01 mol / L.

[0062] In some embodiments, in step (1C), after mixing feed solution A and feed solution B, the molar ratio of Fmoc-Glu(OtBu)-OH to the condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:2. In some embodiments, step (1C) is carried out in a first microreactor. In some embodiments, the reaction temperature of step (1C) is 10 to 100°C, preferably 20 to 70°C, for example, 30 ± 1°C. In some embodiments, feed solution A and feed solution B are pumped into the first microreactor by a first injection pump. In some specific embodiments, the injection flow rate of feed solution A and feed solution B is 0.02 to 0.04 mL / min, and / or the residence time is 1 to 20 min, for example, 5 to 10 min.

[0063] In some embodiments, the feed liquid C in step (1D) contains H-Gly-OBz The concentration of HCl is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L, 0.1~0.2 mol / L or 0.13~0.15 mol / L, for example 0.139±0.01 mol / L.

[0064] In some embodiments, step (1E) is performed in a second microreactor. In some embodiments, the first pre-activated reaction solution and feed solution C are mixed to react Fmoc-Glu(OtBu)-OH and H-Gly-OBz. The molar ratio of HCl is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:1. In some embodiments, the reaction temperature of step (1E) is 10 to 100°C, preferably 20 to 70°C, for example, 30 ± 1°C. In some embodiments, the first pre-activated reaction solution and the feed solution C are pumped into the second microreactor via a second injection pump. In some specific embodiments, the injection flow rate of the first pre-activated reaction solution is 0.03 to 0.05 mL / min, and / or the injection flow rate of the feed solution C is 0.015 to 0.03 mL / min, and / or the residence time is 1 to 20 min, for example, 5 to 10 min.

[0065] In this invention, step (2) includes: (2A) The intermediate product compound 2 obtained in step (1) is mixed with a fourth organic solvent to obtain feed liquid D. (2B) The reagent that has removed the Fmoc protecting group is mixed with the fifth organic solvent to obtain feed solution E. (2C) The feed liquid D in (2A) is mixed with the feed liquid E in (2B), and after deprotection reaction, intermediate product compound 3 is obtained.

[0066] In some implementations, steps (2A) through (2C) are performed sequentially.

[0067] In some embodiments, the reagent for removing the Fmoc protecting group can be any one or more of DBU, piperidine, triethylamine, 4-methylpiperidine, morpholine, and 1-methylpiperazole, preferably DBU.

[0068] In some embodiments, the fourth and fifth organic solvents may each be independently selected from one or more of methanol, toluene, N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, preferably one or more of methanol, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, and more preferably acetonitrile. In some embodiments, the fourth and fifth organic solvents may be the same, for example, both being acetonitrile or methanol.

[0069] In some embodiments, the concentration of compound 2 in the feed liquid D of step (2A) is 0.01~0.5 mol / L, preferably 0.05~0.3 mol / L, 0.05~0.2 mol / L or 0.05~0.15 mol / L, for example 0.1±0.01 mol / L.

[0070] In some embodiments, the concentration of the reagent for removing the Fmoc protecting group (e.g., DBU) in the feed solution E of step (2B) is 0.01~0.5 mol / L, preferably 0.05~0.2 mol / L or 0.05~0.15 mol / L, for example 0.1±0.01 mol / L.

[0071] In some embodiments, in step (2C), after feed liquid D and feed liquid E are mixed, the molar ratio of compound 2 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1 to 1:1.5, for example, 1:1.

[0072] In some embodiments, step (2C) is carried out in a third microreactor. In some embodiments, the reaction temperature of step (2C) is 25–70°C, preferably 40–60°C. In some embodiments, feed solutions D and E are pumped into the third microreactor via a third injection pump. In some specific embodiments, the injection flow rate of feed solutions D and E is 0.025–0.055 mL / min, and / or the residence time is 1–20 min, for example, 4–10 min.

[0073] In this invention, step (3) includes: (3A) Using Fmoc-Aib-OH as raw material, Fmoc-Aib-OH, alkali, and a sixth organic solvent are mixed to obtain feed liquid F. (3B) The condensing agent is mixed with the seventh organic solvent to obtain feed liquid G. (3C) The feed solution F obtained in (3A) is mixed with the feed solution G obtained in (3B), and the second pre-activated reaction solution is synthesized by esterification. (3D) The intermediate product compound 3 obtained in step (2) is mixed with the eighth organic solvent to obtain feed solution H. (3E) The second pre-activated reaction solution obtained in (3C) is mixed with feed solution H and the intermediate product compound 4 is synthesized by amide condensation reaction.

[0074] In some implementations, steps (3A) through (3E) are performed sequentially.

[0075] In some embodiments, the condensing agent may be any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU.

[0076] In some embodiments, the base may be any one or more of DIEA (N,N-diisopropylethylamine), N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine.

[0077] In some embodiments, the sixth, seventh, and eighth organic solvents may each be independently selected from one or more of N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, acetonitrile, 1,4-dioxane, dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, preferably one or more of dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, and more preferably dichloromethane or ethyl acetate. In some embodiments, the sixth, seventh, and eighth organic solvents may be the same, for example, all of them may be dichloromethane or ethyl acetate.

[0078] In some embodiments, the concentration of Fmoc-Aib-OH in the feed solution F of step (3A) is 0.05~0.20 mol / L, preferably 0.05~0.15 mol / L, for example 0.1±0.01 mol / L. In some embodiments, the molar ratio of Fmoc-Aib-OH to alkali (e.g., triethylamine) in the feed solution F of step (3A) is 1:1~4:1, preferably 1:1~2:1, for example 2:1.

[0079] In some embodiments, the concentration of the condensing agent (e.g., HATU) in the feed liquid G of step (3B) is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L, for example 0.2±0.01 mol / L.

[0080] In some embodiments, in step (3C), after mixing feed solution F and feed solution G, the molar ratio of Fmoc-Aib-OH to the condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:2. In some embodiments, step (3C) is carried out in a fourth microreactor. In some embodiments, the reaction temperature of step (3C) is 30 to 80°C, preferably 40 to 70°C, for example, 45 to 60°C, more specifically, for example, 60 ± 1°C. In some embodiments, feed solution F and feed solution G are pumped into the fourth microreactor via a fourth injection pump. In some specific embodiments, the injection flow rate of feed solution F and feed solution G is 0.02 to 0.08 mL / min, preferably 0.05 to 0.06 mL / min, for example, 0.055 ± 0.001 mL / min, and / or, the residence time is 1 to 20 min, for example, 5 ± 1 min.

[0081] In some embodiments, the concentration of compound 3 in the feed solution H of step (3D) is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L or 0.04~0.06 mol / L, for example 0.05±0.01 mol / L.

[0082] In some embodiments, in step (3E), after mixing the second pre-activated reaction solution and the feed solution H, the molar ratio of Fmoc-Aib-OH to compound 3 is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:1. In some embodiments, step (3E) is carried out in a fifth microreactor. In some embodiments, the reaction temperature of step (3E) is 30 to 80°C, preferably 40 to 70°C, for example, 45 to 60°C, more specifically, for example, 60 ± 1°C. In some embodiments, the second pre-activated reaction solution and the feed solution H are pumped into the fifth microreactor by a fifth injection pump. In some specific embodiments, the injection flow rate of the second pre-activated reaction solution and the feed solution H is 0.02 to 0.08 mL / min, preferably 0.05 to 0.06 mL / min, for example, 0.055 ± 0.001 mL / min, and / or, the residence time is 1 to 20 min, for example, 5 ± 1 min.

[0083] In this invention, step (4) includes: (4A) The intermediate product compound 4 obtained in step (3) is mixed with the ninth organic solvent to obtain feed liquid I. (4B) The reagent that removes the Fmoc protecting group is mixed with the tenth organic solvent to obtain feed solution J. (4C) The feed liquid I in (4A) is mixed with the feed liquid J in (4B), and after deprotection reaction, intermediate product compound 5 is obtained.

[0084] In some implementations, steps (4A) to (4C) are performed sequentially.

[0085] In some embodiments, the reagent for removing the Fmoc protecting group can be any one or more of DBU, piperidine, triethylamine, 4-methylpiperidine, morpholine, and 1-methylpiperazole, preferably DBU.

[0086] In some embodiments, the ninth and tenth organic solvents may each be independently selected from one or more of tetrahydrofuran, methanol, toluene, N,N-dimethylformamide, ethyl acetate, 1,4-dioxane, acetonitrile, and dichloromethane, preferably one or more of tetrahydrofuran, methanol, dichloromethane, and acetonitrile, and more preferably acetonitrile. In some embodiments, the ninth and tenth organic solvents may be the same, for example, both being acetonitrile.

[0087] In some embodiments, the concentration of compound 4 in the feed liquid I of step (4A) is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L or 0.05~0.15 mol / L, for example 0.1±0.01 mol / L.

[0088] In some embodiments, the concentration of the reagent for removing the Fmoc protecting group (e.g., DBU) in the feed liquid J of step (4B) is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L or 0.05~0.15 mol / L, for example 0.1±0.01 mol / L.

[0089] In some embodiments, in step (4C), after feed liquid I and feed liquid J are mixed, the molar ratio of compound 4 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1 to 1:1.5, for example 1:1.

[0090] In some embodiments, step (4C) is carried out in a sixth microreactor. In some embodiments, the reaction temperature of step (4C) is 25-50°C, preferably 25-40°C. In some embodiments, feed solution I and feed solution J are pumped into the sixth microreactor via a sixth injection pump. In some specific embodiments, the injection flow rate of feed solution I and feed solution J is 0.11-0.22 mL / min, and / or the residence time is 1-10 min, preferably 1-5 min, for example 1-4 min.

[0091] In this invention, step (5) includes: (5A) Using Fmoc-His(Trt)-OH as raw material, Fmoc-His(Trt)-OH, alkali, and the eleventh organic solvent are mixed to obtain feed liquid K. (5B) The condensing agent is mixed with the twelfth organic solvent to obtain feed liquid L. (5C) The feed solution K obtained in (5A) is mixed with the feed solution L obtained in (5B), and the mixture is then synthesized into a third pre-activated reaction solution via esterification. (5D) The intermediate compound 5 obtained in step (4) is mixed with the thirteenth organic solvent to obtain feed liquid M. (5E) The third pre-activated reaction solution obtained in (5C) is mixed with the feed solution M and synthesized as the first peptide chain of smegglutinin by amide condensation reaction.

[0092] In some implementations, steps (5A) through (5E) are performed sequentially.

[0093] In some embodiments, the condensing agent may be any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU.

[0094] In some embodiments, the base may be any one or more of DIEA (N,N-diisopropylethylamine), N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine.

[0095] In some embodiments, the eleventh, twelfth, and thirteenth organic solvents may each be independently selected from any one or more of 1,4-dioxane, N,N-dimethylformamide, ethyl acetate, toluene, acetonitrile, dichloromethane, and tetrahydrofuran, preferably any one or more of dichloromethane and tetrahydrofuran, and more preferably dichloromethane. In some embodiments, the eleventh, twelfth, and thirteenth organic solvents may be the same, for example, all of them may be dichloromethane.

[0096] In some embodiments, the concentration of Fmoc-His(Trt)-OH in the feed solution K of step (5A) is 0.01~1.0 mol / L, preferably 0.05~0.5 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L, for example, 0.1±0.01 mol / L. In some embodiments, the molar ratio of Fmoc-His(Trt)-OH to alkali (e.g., triethylamine) in the feed solution K of step (5A) is 1:1~4:1, preferably 1:1~2:1, for example, 2:1.

[0097] In some embodiments, the concentration of the condensing agent (e.g., HATU) in the feed liquid L of step (5B) is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L, for example 0.2±0.01 mol / L.

[0098] In some embodiments, in step (5C), after mixing feed liquid K and feed liquid L, the molar ratio of Fmoc-His(Trt)-OH to the condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:2. In some embodiments, step (5C) is carried out in a seventh microreactor. In some embodiments, the reaction temperature of step (5C) is 30 to 80°C, preferably 40 to 70°C, for example, 55 to 70°C, more specifically, for example, 70 ± 1°C. In some embodiments, feed liquid K and feed liquid L are pumped into the seventh microreactor via a seventh injection pump. In some specific embodiments, the injection flow rates of feed liquid K and feed liquid L are 0.03~0.3 mL / min, preferably 0.03-0.25 mL / min, for example 0.11~0.22 mL / min, more specifically for example 0.22±0.01 mL / min, and / or the residence time is 1~20 min, preferably 5~20 min, for example 5~10 min.

[0099] In some embodiments, the concentration of compound 5 in the feed liquid M of step (5D) is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.01~0.1 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L or 0.04~0.06 mol / L, for example 0.05±0.01 mol / L.

[0100] In some embodiments, in step (5E), after the third pre-activated reaction solution and the feed solution M are mixed, the molar ratio of Fmoc-His(Trt)-OH to compound 5 is 1:1 to 1:10, preferably 1:1 to 1:4, for example, 1:1. In some embodiments, step (5E) is carried out in an eighth microreactor. In some embodiments, the reaction temperature of step (5E) is 30 to 80°C, preferably 40 to 70°C, for example, 55 to 70°C, more specifically, for example, 70 ± 1°C. In some embodiments, the third pre-activated reaction solution and the feed solution M are pumped into the eighth microreactor via an eighth injection pump. In some specific embodiments, the injection flow rate of the third pre-activated reaction solution and the feed solution M is 0.1~0.5 mL / min, preferably 0.22~0.44 mL / min, for example 0.44±0.01 mL / min, and / or the residence time is 1~20 min, preferably 5~20 min, for example 5~10 min.

[0101] In some implementations, any two, three, four, or all five adjacent steps in steps (1) to (5) are performed in series.

[0102] In some implementations, all five steps (1) to (5) are performed consecutively. In some implementations, steps (1A) to (1E), steps (2A) to (2C), steps (3A) to (3E), steps (4A) to (4C), and steps (5A) to (5E) are performed consecutively.

[0103] Method for preparing smegglutinin intermediate

[0104] The present invention also provides a method for preparing smegglutinin intermediate compound 2, the method comprising step (1) as described in any embodiment of the present invention. In some embodiments, step (1) comprises steps (1A) to (1E) as described in any embodiment of the present invention. In some embodiments, steps (1A) to (1E) are performed sequentially.

[0105] The present invention also provides a method for preparing smegglutinin intermediate compound 3, the method comprising steps (1) and (2) as described in any embodiment of the present invention. In some embodiments, step (1) comprises steps (1A) to (1E) as described in any embodiment of the present invention, and step (2) comprises steps (2A) to (2C) as described in any embodiment of the present invention. In some embodiments, steps (1A) to (1E) are performed sequentially, and / or steps (2A) to (2C) are performed sequentially. In some embodiments, steps (1) to (2) are performed sequentially, for example, steps (1A) to (1E) and steps (2A) to (2C) are performed sequentially.

[0106] The present invention also provides a method for preparing smegglutinin intermediate compound 4, the method comprising steps (1), (2), and (3) as described in any embodiment of the present invention. In some embodiments, step (1) comprises steps (1A) to (1E) as described in any embodiment of the present invention, step (2) comprises steps (2A) to (2C) as described in any embodiment of the present invention, and step (3) comprises steps (3A) to (3E) as described in any embodiment of the present invention. In some embodiments, steps (1A) to (1E) are performed sequentially, and / or steps (2A) to (2C) are performed sequentially, and / or steps (3A) to (3E) are performed sequentially. In some embodiments, steps (1) to (3) are performed sequentially, for example, steps (1A) to (1E), steps (2A) to (2C), and steps (3A) to (3E) are performed sequentially.

[0107] The present invention also provides a method for preparing smegglutinin intermediate compound 5, the method comprising steps (1), (2), (3), and (4) as described in any embodiment of the present invention. In some embodiments, step (1) comprises steps (1A) to (1E) as described in any embodiment of the present invention, step (2) comprises steps (2A) to (2C) as described in any embodiment of the present invention, step (3) comprises steps (3A) to (3E) as described in any embodiment of the present invention, and step (4) comprises steps (4A) to (4C) as described in any embodiment of the present invention. In some embodiments, steps (1A) to (1E) are performed sequentially, and / or steps (2A) to (2C) are performed sequentially, and / or steps (3A) to (3E) are performed sequentially, and / or steps (4A) to (4C) are performed sequentially. In some implementations, steps (1) to (4) are performed sequentially, for example, steps (1A) to (1E), steps (2A) to (2C), steps (3A) to (3E), and steps (4A) to (4C) are performed sequentially.

[0108] Continuous synthesis unit

[0109] The present invention also provides a continuous synthesis apparatus, comprising: a first microreactor, a second microreactor, a third microreactor, a fourth microreactor, a fifth microreactor, a sixth microreactor, a seventh microreactor, and an eighth microreactor in sequential fluid communication; a first injection pump in fluid communication with the first microreactor; a second injection pump in fluid communication with the second microreactor; a third injection pump in fluid communication with the third microreactor; a fourth injection pump in fluid communication with the fourth microreactor; a fifth injection pump in fluid communication with the fifth microreactor; a sixth injection pump in fluid communication with the sixth microreactor; a seventh injection pump in fluid communication with the seventh microreactor; an eighth injection pump in fluid communication with the eighth microreactor; connecting pipes connecting each microreactor and the injection pumps; and control components for operating the apparatus to perform the liquid-phase synthesis of the first peptide chain of smegglutinin.

[0110] In this invention, the continuous synthesis apparatus can be as follows: Figure 2The continuous synthesis apparatus is shown. In some embodiments, the first injection pump is used to deliver feed liquid A and feed liquid B into the first microreactor, and the second injection pump is used to deliver feed liquid C into the second microreactor to complete step (1). In some embodiments, the third injection pump is used to mix feed liquid D and feed liquid E and deliver them into the third microreactor to complete step (2). In some embodiments, the fourth injection pump is used to mix feed liquid F and feed liquid G and deliver them into the fourth microreactor, and the fifth injection pump is used to deliver feed liquid H into the fifth microreactor to complete step (3). In some embodiments, the sixth injection pump is used to mix feed liquid I and feed liquid J and deliver them into the sixth microreactor to complete step (4). In some embodiments, the seventh injection pump is used to mix feed liquid K and feed liquid L and deliver them into the seventh microreactor, and the eighth injection pump is used to deliver feed liquid M into the eighth microreactor to complete step (5).

[0111] In this invention, the injection pumps (including the first, second, third, fourth, fifth, sixth, seventh, and eighth injection pumps) can be, for example, but not limited to, syringe pumps, peristaltic pumps, horizontal flow pumps, etc. In some embodiments, the injection pumps (including the first, second, third, fourth, fifth, sixth, seventh, and eighth injection pumps) are syringe pumps.

[0112] In this invention, the microreactors (including the first microreactor, second microreactor, third microreactor, fourth microreactor, fifth microreactor, sixth microreactor, seventh microreactor, and eighth microreactor) can be, for example, but not limited to, capillary microreactors, fixed-bed microreactors, glass chip microreactors, stainless steel and silicon carbide microchannel reactors, etc. In some embodiments, the microreactors (including the first microreactor, second microreactor, third microreactor, fourth microreactor, fifth microreactor, sixth microreactor, seventh microreactor, and eighth microreactor) are capillary microreactors, such as capillary microreactors made of PTFE.

[0113] In some embodiments, the liquid holding volume of the first microreactor can be 0.2-3.0 mL. In some embodiments, the liquid holding volume of the second microreactor can be 0.5-2.0 mL. In some embodiments, the liquid holding volume of the third microreactor can be 0.1-4.0 mL. In some embodiments, the liquid holding volume of the fourth microreactor can be 0.2-2.0 mL. In some embodiments, the liquid holding volume of the fifth microreactor can be 0.4-2.5 mL. In some embodiments, the liquid holding volume of the sixth microreactor can be 0.2-2.0 mL. In some embodiments, the liquid holding volume of the seventh microreactor can be 0.5-3.5 mL. In some embodiments, the liquid holding volume of the eighth microreactor can be 1.0-4.0 mL.

[0114] application

[0115] This invention provides the application of the continuous synthesis apparatus described in any embodiment of the invention in the continuous liquid-phase synthesis of the first peptide chain of smegglutinin.

[0116] The present invention also provides the application of the continuous synthesis apparatus described in any embodiment of the present invention in the continuous liquid-phase synthesis of smegglutinin intermediates compound 2, compound 3, compound 4 or compound 5.

[0117] The advantages of this invention include: (1) Compared with the existing solid-phase synthesis method for preparing the first peptide chain of smegraglutide, the present invention adopts a liquid-phase synthesis method, which linearly connects the various steps in the preparation of the first peptide chain of smegraglutide, greatly shortening the reaction time. At the same time, it avoids the use of expensive resins and packed columns in solid-phase synthesis, thus reducing costs. The liquid-phase synthesis method of the present invention uses an innovative acid-base salt formation method for post-processing, which can easily obtain high-purity products and improve yield.

[0118] (2) The present invention further optimizes the batch liquid phase synthesis method into a continuous liquid phase synthesis method, and performs esterification, amide condensation and deprotection reactions in a continuous manner, which strengthens the mixing process and improves selectivity. It can obtain the target product smegglutinin first peptide chain with an overall yield of more than 85.5% and a total residence time of no more than 18 minutes. Compared with the batch synthesis route, the overall yield is 29.4% and the total reaction time is 13.3h, which is a significant improvement.

[0119] (3) This invention provides a continuous synthesis apparatus suitable for the continuous liquid-phase synthesis of the first peptide chain of smegglutinin. The continuous process achieved using a microreactor is simple to operate, reacts rapidly, effectively simplifies experimental procedures, reduces waste generation, effectively improves amide condensation reactions, enhances the selectivity of peptide synthesis, reduces the amount of organic solvent used, shortens reaction time, and effectively prevents the spillage of triethylamine, dichloromethane, etc., which can cause irritating odors and harm to humans and the environment. Simultaneously, this continuous synthesis apparatus allows for online purification of intermediates after each reaction step, fundamentally eliminating impurity accumulation. Precise control of temperature, mixing, and residence time within the microchannels significantly improves reaction efficiency and selectivity. The entire process can be fully automated and continuous, employing a "scale-up" mode, eliminating the need for traditional process scale-up and significantly shortening the development cycle. Furthermore, the system is closed, reagents are recyclable, and solvent consumption is greatly reduced, combining high efficiency, environmental friendliness, and scalable industrial potential.

[0120] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Based on attempts in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0121] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. All reagents and raw materials used in this invention are commercially available. All reagents used in the experiments are AR grade.

[0122] The continuous synthesis apparatus of the present invention is based on Figure 1 The reaction route for the synthesis of the first peptide chain of smegglutinin shown is constructed as illustrated in the schematic diagram of the continuous synthesis apparatus. Figure 2 As shown, A to G are all syringe pumps used for sample injection; N to U are all microreactors, serving as the reaction apparatus for the synthesis reaction. In this continuous synthesis apparatus, all connecting pipes are made of PTFE, and all microreactors are capillary microreactors made of PTFE.

[0123] Unless otherwise stated, percentages and portions are by weight.

[0124] Example

[0125] Example 1: First Continuous Esterification and Amide Condensation

[0126] With Fmoc-Glu(OtBu)-OH (compound 1) and H-Gly-OBz Using HCl (compound 6) as a starting material, compound 2, also known as a dipeptide, is generated through a first continuous esterification and amide condensation reaction. The reaction route is as follows: .

[0127] Take 0.85 g of Fmoc-Glu(OtBu)-OH (compound 1) and 0.0505 g of triethylamine, add dichloromethane as a solvent, and bring the volume to 20 mL. This is prepared as feed solution A, with a compound 1 concentration of 0.1 mol / L and a molar ratio of compound 1 to triethylamine of 2:1. Take 1.52 g of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea (HATU) hexafluorophosphate, add dichloromethane as a solvent, and bring the volume to 20 mL. This is prepared as feed solution B, with a HATU concentration of 0.2 mol / L. Feed solutions A and B are pumped into the first microreactor N by the first injection pump A, wherein the molar ratio of compound 1 to HATU is 1:2. The flow rates of both feed solutions A and B are 0.02 mL / min, the residence time is 10 min, and the temperature of the first microreactor N is 30℃, to carry out a continuous esterification reaction (also known as a pre-activation reaction), yielding a pre-activated reaction solution. H-Gly-OBz is then used. 0.42 g of HCl (compound 6) and 0.0505 g of triethylamine were added, and dichloromethane was added as a solvent to bring the volume to 15 mL, which was then used as feed solution C. The concentration of compound 6 in feed solution C was 0.139 mol / L. The pre-activated reaction solution and feed solution C were pumped into the second microreactor O by the second injection pump B, wherein the molar ratio of compound 1 to compound 6 was 1:1, the flow rate of the pre-activated reaction solution was 0.04 mL / min, the flow rate of feed solution C was 0.015 mL / min, the residence time was 10 min, and the temperature of the second microreactor O was 30 °C, to carry out a continuous amide condensation reaction to obtain reaction solution D. Reaction solution D was purified to obtain the amide condensation product compound 2, whose structure is as follows: .

[0128] HPLC analysis showed that the yield of compound 2 was 68%.

[0129] Example 2: First Continuous Esterification and Amide Condensation Reaction

[0130] In this embodiment, the solvent in feed solutions A, B, and C was replaced with ethyl acetate, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 24%.

[0131] Example 3: First Continuous Esterification and Amide Condensation Reaction

[0132] In this embodiment, the solvent in feed solutions A, B, and C was replaced with acetonitrile, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 49%.

[0133] Example 4: First Continuous Esterification and Amide Condensation Reaction

[0134] In this embodiment, the solvent in feed solutions A, B, and C was replaced with N,N-dimethylformamide, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 75%.

[0135] Example 5: First Continuous Esterification and Amide Condensation Reaction

[0136] In this embodiment, the solvent in feed solutions A, B, and C was replaced with toluene, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 60%.

[0137] Example 6: First Continuous Esterification and Amide Condensation Reaction

[0138] In this embodiment, the solvent in feed solutions A, B, and C was replaced with 1,4-dioxane, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 42%.

[0139] Example 7: First Continuous Esterification and Amide Condensation Reaction

[0140] In this embodiment, the solvent in feed solutions A, B, and C was replaced with tetrahydrofuran, and the other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 99%. Its LCMS purity was as follows: Figure 3 As shown, the LCMS purity of the obtained compound 2 is 100%.

[0141] Example 8: First Continuous Esterification and Amide Condensation Reaction

[0142] In this embodiment, the solvents in feed solutions A, B, and C were replaced with tetrahydrofuran. The flow rates of feed solutions A and B, pumped into the first microreactor N by the first injection pump A, were both set to 0.04 mL / min, with a residence time of 5 min. The flow rates of the pre-activated reaction solution and feed solution C, pumped into the second microreactor O by the second injection pump B, were both set to 0.03 mL / min, with a residence time of 5 min. All other conditions were the same as in Example 1. HPLC analysis showed that the yield of compound 2 was 87%.

[0143] Example 9: First Continuous Esterification and Amide Condensation Reaction

[0144] In this embodiment, the solvent in feed solutions A, B, and C was replaced with N,N-dimethylformamide, and the other conditions were the same as in Example 8. HPLC analysis showed that the yield of compound 2 was 64%.

[0145] Based on the results of Examples 1 to 9, the reaction conditions of Example 7 were used, and the solvents in feed solutions A, B, and C were all tetrahydrofuran. The first continuous esterification and amide condensation reaction produced compound 2 with the best effect. Therefore, in subsequent examples, the reaction conditions of Example 7 were used to synthesize compound 2.

[0146] Example 10, First Continuous Deprotection Reaction

[0147] Compound 2 was synthesized using the reaction conditions of Example 7. Compound 3 was synthesized using Compound 2 as a reactant via the following reaction route: .

[0148] Take 1.14 g of compound 2, add methanol as the solvent, and bring the volume to 20 mL. This is prepared as feed solution D, with a compound 2 concentration of 0.1 mol / L. Take 0.304 g of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), add methanol as the solvent, and bring the volume to 20 mL. This is prepared as feed solution E, with a DBU concentration of 0.1 mol / L. Pump feed solutions D and E into the third microreactor P using the third injection pump C. The molar ratio of compound 2 to DBU is 1:1. The flow rates of both feed solutions D and E are 0.025 mL / min, the residence time is 10 min, and the temperature of the third microreactor P is 60 °C. A continuous deprotection reaction is carried out to obtain reaction solution H. Purify reaction solution H by acid-base salt formation to obtain the first deprotected product, compound 3, with the following structure: .

[0149] HPLC analysis showed that the yield of compound 3 was 83%.

[0150] Example 11, First Continuous Deprotection Reaction

[0151] In this embodiment, the solvent in feed solutions D and E was replaced with toluene, and the other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 68%.

[0152] Example 12, First Continuous Deprotection Reaction

[0153] In this embodiment, the solvent in feed solutions D and E was replaced with N,N-dimethylformamide, and the other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 69%.

[0154] Example 13, First Continuous Deprotection Reaction

[0155] In this embodiment, the solvent in feed solutions D and E was replaced with 1,3-dimethyl-2-imidazolinone, and the other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 54%.

[0156] Example 14, First Continuous Deprotection Reaction

[0157] In this embodiment, the solvent in feed solutions D and E was replaced with ethyl acetate, and the other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 82%.

[0158] Example 15, First Continuous Deprotection Reaction

[0159] In this embodiment, the solvent in feed solutions D and E was replaced with dichloromethane. The flow rate of feed solutions D and E pumped into the third microreactor P by the third injection pump C was set to 0.025 mL / min, and the residence time was 10 min. The other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 85%.

[0160] Example 16, First Continuous Deprotection Reaction

[0161] In this embodiment, the solvent in feed solutions D and E was replaced with tetrahydrofuran. The flow rate of feed solutions D and E pumped into the third microreactor P by the third injection pump C was set to 0.055 mL / min, and the residence time was 4 min. The other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 90%.

[0162] Example 17, First Continuous Deprotection Reaction

[0163] In this embodiment, the solvent in feed solutions D and E was replaced with dichloromethane, and the other conditions were the same as in Example 16. HPLC analysis showed that the yield of compound 3 was 93%.

[0164] Example 18, First Continuous Deprotection Reaction

[0165] In this embodiment, the solvent in feed solutions D and E was replaced with acetonitrile, and the other conditions were the same as in Example 16. HPLC analysis showed that the yield of compound 3 was 100%, and the LCMS purity of compound 3 was 100%. Figure 4 As shown.

[0166] Example 19, First Continuous Deprotection Reaction

[0167] In this embodiment, the solvent in feed solutions D and E was replaced with methanol, and the other conditions were the same as in Example 16. HPLC analysis showed that the yield of compound 3 was 99%.

[0168] Example 20, First Continuous Deprotection Reaction

[0169] In this embodiment, the solvent in feed solutions D and E was replaced with tetrahydrofuran. The flow rate of feed solutions D and E pumped into the third microreactor P by the third injection pump C was set to 0.055 mL / min, the residence time was 4 min, the microreactor temperature was 40 °C, and the other conditions were the same as in Example 10. HPLC analysis showed that the yield of compound 3 was 83%.

[0170] Example 21, First Continuous Deprotection Reaction

[0171] In this embodiment, the solvent in feed solutions D and E was replaced with acetonitrile, and the other conditions were the same as in Example 20. HPLC analysis showed that the yield of compound 3 was 90%.

[0172] Based on the results of Examples 10 to 21, the reaction conditions of Example 18, in which the solvents in feed solutions D and E are both acetonitrile, resulted in the best effect in generating compound 3 in the first continuous deprotection reaction. Therefore, in subsequent examples, the reaction conditions of Example 18 were used to synthesize compound 3.

[0173] Example 22: Second Continuous Esterification and Amide Condensation Reaction

[0174] Compound 3 was synthesized using the reaction conditions of Example 18. Using compound 3, Fmoc-Aib-OH (i.e., compound 7), and triethylamine as reactants, the tripeptide product compound 4 was synthesized via the following reaction route: .

[0175] Take 0.65 g of Fmoc-Aib-OH (compound 7) and 0.101 g of triethylamine, add N,N-dimethylformamide as a solvent, and bring the volume to 20 mL. This is prepared as feed solution F, with a compound 7 concentration of 0.1 mol / L and a molar ratio of compound 7 to triethylamine of 2:1. Take 1.52 g of HATU, add N,N-dimethylformamide as a solvent, and bring the volume to 20 mL. This is prepared as feed solution G, with a HATU concentration of 0.2 mol / L. Feed solutions F and G were pumped into the fourth microreactor Q via the fourth injection pump D, with the molar ratio of compound 7 to HATU being 1:2. The flow rates of both feed solutions F and G were 0.055 mL / min, the residence time was 5 min, and the temperature of the fourth microreactor Q was 30 °C. A continuous esterification reaction (also known as a pre-activation reaction) was carried out to obtain a pre-activated reaction solution. 0.70 g of the first deprotected product (compound 3 obtained using the method in Example 18) was taken and N,N-dimethylformamide was added as a solvent to bring the volume to 40 mL, which was then used as feed solution H. The concentration of compound 3 in feed solution H was 0.05 mol / L. The pre-activated reaction solution and feed solution H are pumped into the fifth microreactor R by the fifth injection pump E, wherein the molar ratio of compound 7 to compound 3 is 1:1, the flow rate of the pre-activated reaction solution and feed solution H is 0.11 mL / min, the residence time is 5 min, the temperature of the fifth microreactor R is 30℃, and a continuous amide condensation reaction is carried out to obtain reaction solution I.

[0176] The reaction solution I was purified to obtain the amide condensation product compound 4, whose structure is as follows: .

[0177] HPLC analysis showed that the yield of compound 4 was 77%.

[0178] Example 23: Second Continuous Esterification and Amide Condensation Reaction

[0179] In this embodiment, the solvent in feed solutions F, G, and H was replaced with 1,3-dimethyl-2-imidazolinone, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 53%.

[0180] Example 24: Second Continuous Esterification and Amide Condensation Reaction

[0181] In this embodiment, the solvent in feed solutions F, G, and H was replaced with acetonitrile, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 68%.

[0182] Example 25: Second Continuous Esterification and Amide Condensation Reaction

[0183] In this embodiment, the solvent in feed solutions F, G, and H was replaced with 1,4-dioxane, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 40%.

[0184] Example 26: Second Continuous Esterification and Amide Condensation Reaction

[0185] In this embodiment, the solvent in feed solutions F, G, and H was replaced with dichloromethane, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 82%.

[0186] Example 27: Second Continuous Esterification and Amide Condensation Reaction

[0187] In this embodiment, the solvent in feed solutions F, G, and H was replaced with ethyl acetate, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 82%.

[0188] Example 28: Second Continuous Esterification and Amide Condensation Reaction

[0189] In this embodiment, the solvent in feed solutions F, G, and H was replaced with toluene, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 73%.

[0190] Example 29: Second Continuous Esterification and Amide Condensation Reaction

[0191] In this embodiment, the solvent in feed solutions F, G, and H was replaced with tetrahydrofuran, and the other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 75%.

[0192] Example 30: Second Continuous Esterification and Amide Condensation Reaction

[0193] In this embodiment, the temperature of both the fourth microreactor Q and the fifth microreactor R was set to 35°C, and the other conditions were the same as in Example 29. HPLC analysis showed that the yield of compound 4 was 80%.

[0194] Example 31: Second Continuous Esterification and Amide Condensation Reaction

[0195] In this embodiment, the temperature of both the fourth microreactor Q and the fifth microreactor R was set to 40°C, and the other conditions were the same as in Example 29. HPLC analysis showed that the yield of compound 4 was 82%.

[0196] Example 32: Second Continuous Esterification and Amide Condensation Reaction

[0197] In this embodiment, the temperature of both the fourth microreactor Q and the fifth microreactor R was set to 45°C, and the other conditions were the same as in Example 29. HPLC analysis showed that the yield of compound 4 was 87%.

[0198] Example 33: Second Continuous Esterification and Amide Condensation Reaction

[0199] In this embodiment, the solvent in feed solutions F, G, and H was replaced with dichloromethane, and the temperatures of the fourth microreactor Q and the fifth microreactor R were both set to 50°C. All other conditions were the same as in Example 22. HPLC analysis showed that the yield of compound 4 was 91%.

[0200] Example 34: Second Continuous Esterification and Amide Condensation Reaction

[0201] In this embodiment, the solvent in feed solutions F, G, and H was replaced with N,N-dimethylformamide, and the other conditions were the same as in Example 33. HPLC analysis showed that the yield of compound 4 was 89%.

[0202] Example 35: Second Continuous Esterification and Amide Condensation Reaction

[0203] In this embodiment, the temperature of both the fourth microreactor Q and the fifth microreactor R was set to 60°C, and the other conditions were the same as in Example 34. HPLC analysis showed that the yield of compound 4 was 91%.

[0204] Example 36: Second Continuous Esterification and Amide Condensation Reaction

[0205] In this embodiment, the temperature of both the fourth microreactor Q and the fifth microreactor R was set to 60°C, and the other conditions were the same as in Example 33. HPLC analysis showed that the yield of compound 4 was 94.2%, and the LCMS purity of compound 4 was 91.78%. Figure 5 As shown.

[0206] Based on the results of Examples 22 to 36, it can be seen that the synthesis of compound 4 via the second continuous esterification and amide condensation reaction was most effective when using the reaction conditions of Example 36, with dichloromethane as the solvent in feed solutions F, G, and H, and the temperatures of the fourth microreactor Q and the fifth microreactor R both at 60°C. Therefore, in subsequent examples, the reaction conditions of Example 36 were used to synthesize compound 4.

[0207] Example 37, Second Continuous Deprotection Reaction

[0208] In this embodiment, compound 4 was synthesized using the reaction conditions of Example 36. Compound 5 was synthesized using compound 4 as a reactant via the following reaction route: .

[0209] Take 1.31 g of the tripeptide product (compound 4 obtained by the method in Example 36), add tetrahydrofuran, and bring the volume to 20 mL. This is prepared as feed solution I, with a concentration of compound 4 of 0.1 mol / L. Take 0.304 g of DBU, add tetrahydrofuran as a solvent, and bring the volume to 20 mL. This is prepared as feed solution J, with a concentration of DBU of 0.1 mol / L. Pump feed solutions I and J into the sixth microreactor S using the sixth injection pump F. The molar ratio of compound 4 to DBU is 1:1. The flow rates of both feed solutions I and J are 0.11 mL / min, the residence time is 10 min, and the temperature of the sixth microreactor S is 25 °C. A continuous deprotection reaction is carried out to obtain reaction solution M. Purify reaction solution K by acid-base salt formation to obtain the second deprotected product compound 5, whose structure is as follows: .

[0210] HPLC analysis showed that the yield of compound 5 was 90%.

[0211] Example 38, Second Continuous Deprotection Reaction

[0212] In this embodiment, the solvent for feed solution I and feed solution J was changed to methanol, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 87%.

[0213] Example 39, Second Continuous Deprotection Reaction

[0214] In this embodiment, the solvent for feed solution I and feed solution J was changed to toluene, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 66%.

[0215] Example 40, Second Continuous Deprotection Reaction

[0216] In this embodiment, the solvent for feed solution I and feed solution J was changed to N,N-dimethylformamide, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 59%.

[0217] Example 41, Second Continuous Deprotection Reaction

[0218] In this embodiment, the solvent for feed solution I and feed solution J was changed to ethyl acetate, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 57%.

[0219] Example 42, Second Continuous Deprotection Reaction

[0220] In this embodiment, the solvent for feed solution I and feed solution J was changed to 1,4-dioxane, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 68%.

[0221] Example 43, Second Continuous Deprotection Reaction

[0222] In this embodiment, the solvent for feed solution I and feed solution J was replaced with acetonitrile, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 90%.

[0223] Example 44, Second Continuous Deprotection Reaction

[0224] In this embodiment, the solvent for feed liquid I and feed liquid J was changed to dichloromethane, the temperature of the sixth microreactor S was set to 60°C, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 81%.

[0225] Example 45, Second Continuous Deprotection Reaction

[0226] In this embodiment, the flow rates of feed liquid I and feed liquid J pumped into the sixth microreactor S by the sixth injection pump F were both set to 0.22 mL / min, the residence time was 4 min, and the other conditions were the same as in Example 44. HPLC analysis showed that the yield of compound 5 was 98%.

[0227] Example 46, Second Continuous Deprotection Reaction

[0228] In this embodiment, the solvent for feed solution I and feed solution J was replaced with acetonitrile, and the other conditions were the same as in Example 45. HPLC analysis showed that the yield of compound 5 was 100%, and the LCMS purity of compound 5 was 98.55%. Figure 6 As shown.

[0229] Example 47, Second Continuous Deprotection Reaction

[0230] In this embodiment, the temperature of the sixth microreactor S was set to 40°C, and the other conditions were the same as in Example 46. HPLC analysis showed that the yield of compound 5 was 85%.

[0231] Example 48, Second Continuous Deprotection Reaction

[0232] In this embodiment, the solvent for feed solution I and feed solution J was replaced with dichloromethane, and the other conditions were the same as in Example 46. HPLC analysis showed that the yield of compound 5 was 81%.

[0233] Example 49, Second Continuous Deprotection Reaction

[0234] In this embodiment, the solvent for feed solution I and feed solution J was replaced with acetonitrile. The flow rate of feed solution I and feed solution J pumped into the sixth microreactor S by the sixth injection pump F was set to 0.22 mL / min, the residence time was 1 min, the temperature of the sixth microreactor S was 60 °C, and the other conditions were the same as in Example 37. HPLC analysis showed that the yield of compound 5 was 80%.

[0235] Example 50, Second Continuous Deprotection Reaction

[0236] In this embodiment, the temperature of the sixth microreactor S was set to 40°C, and the other conditions were the same as in Example 49. HPLC analysis showed that the yield of compound 5 was 71%.

[0237] Based on the results of Examples 37 to 50, it can be seen that the second continuous deprotection reaction for synthesizing compound 5 was most effective when using the reaction conditions of Example 46, with acetonitrile as the solvent for feed liquids I and J, and the temperature of the sixth microreactor S set to 60°C. Therefore, in subsequent examples, the reaction conditions of Example 46 were used to synthesize compound 5.

[0238] Example 51: Third Continuous Esterification and Amide Condensation Reaction

[0239] In this embodiment, compound 5 was synthesized using the reaction conditions of Example 46. Compound 5, Fmoc-His(Trt)-OH (compound 8), and triethylamine were used as reactants to synthesize the first peptide chain of smegglutinin, i.e., the tetrapeptide, via the following reaction route: .

[0240] Take 1.24 g of Fmoc-His(Trt)-OH (compound 8) and 0.101 g of triethylamine, add 1,4-dioxane as a solvent, and dilute to 20 mL to prepare feed solution K. The concentration of compound 8 in feed solution K is 0.1 mol / L, and the molar ratio of compound 8 to triethylamine is 2:1. Take 1.52 g of HATU, add 1,4-dioxane as a solvent, and dilute to 20 mL to prepare feed solution L to prepare feed solution L. The concentration of HATU in feed solution L is 0.2 mol / L. Feed solutions F and K were pumped into the seventh microreactor T by the seventh injection pump G, wherein the molar ratio of compound 8 to HATU was 1:2. The flow rates of both feed solutions F and K were 0.11 mL / min, the residence time was 20 min, and the temperature of the seventh microreactor T was 30 °C, for continuous esterification reaction (i.e., pre-activation reaction), to obtain a pre-activated reaction solution. 0.87 g of the second deprotected product (compound 5 obtained using the method of Example 45) was taken, and 1,4-dioxane was added as a solvent to bring the volume to 40 mL, which was then used as feed solution M. The concentration of compound 5 in feed solution M was 0.05 mol / L. The pre-activated reaction solution and feed solution H are pumped into the eighth microreactor U by the eighth injection pump H, wherein the molar ratio of compound 8 to compound 5 is 1:1, the flow rate of the pre-activated reaction solution and feed solution H is 0.22 mL / min, the residence time is 20 min, the temperature of the eighth microreactor U is 30 °C, and a continuous amide condensation reaction is carried out to obtain reaction solution N.

[0241] The reaction solution N was purified to obtain the first peptide chain of the amide condensation product compound smegraglutide, the structure of which is as follows: .

[0242] HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 35%.

[0243] Example 52: Third Continuous Esterification and Amide Condensation Reaction

[0244] In this embodiment, the solvents for feed solutions K, L, and M were replaced with N,N-dimethylformamide, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 46%.

[0245] Example 53: Third Continuous Esterification and Amide Condensation Reaction

[0246] In this embodiment, the solvents for feed solutions K, L, and M were replaced with ethyl acetate, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 37%.

[0247] Example 54: Third Continuous Esterification and Amide Condensation Reaction

[0248] In this embodiment, the solvents for feed solutions K, L, and M were replaced with toluene, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 40%.

[0249] Example 55: Third Continuous Esterification and Amide Condensation Reaction

[0250] In this embodiment, the solvents for feed solutions K, L, and M were replaced with acetonitrile, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 51%.

[0251] Example 56: Third Continuous Esterification and Amide Condensation Reaction

[0252] In this embodiment, the solvents for feed solutions K, L, and M were replaced with dichloromethane, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 57%.

[0253] Example 57: Third Continuous Esterification and Amide Condensation Reaction

[0254] In this embodiment, the solvents for feed solutions K, L, and M were replaced with tetrahydrofuran, and the other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 53%.

[0255] Example 58: Third Continuous Esterification and Amide Condensation Reaction

[0256] In this embodiment, feed solutions F and K are pumped into the seventh microreactor T by the seventh syringe pump G at a flow rate of 0.22 mL / min and a residence time of 10 min. The pre-activated reaction solution and feed solution H are pumped into the eighth microreactor U by the eighth syringe pump H at a flow rate of 0.44 mL / min and a residence time of 10 min. All other conditions are the same as in Example 57. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 54%.

[0257] Example 59: Third Continuous Esterification and Amide Condensation Reaction

[0258] In this embodiment, the solvents for feed solutions K, L, and M were replaced with dichloromethane, and the other conditions were the same as in Example 58. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 62%.

[0259] Example 60: Third Continuous Esterification and Amide Condensation Reaction

[0260] In this embodiment, the temperature of the seventh microreactor T and the eighth microreactor U was set to 55°C, and the other conditions were the same as in Example 59. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 83%.

[0261] Example 61: Third Continuous Esterification and Amide Condensation Reaction

[0262] In this embodiment, the solvents for feed solutions K, L, and M were replaced with tetrahydrofuran. The flow rates of feed solutions F and K, pumped into the seventh microreactor T by the seventh injection pump G, were both set to 0.22 mL / min, with a residence time of 10 min. The temperature of the seventh microreactor T was 55°C. The flow rates of the pre-activated reaction solution and feed solution M, pumped into the eighth microreactor U by the eighth injection pump H, were both set to 0.44 mL / min, with a residence time of 10 min. The temperature of the eighth microreactor U was 55°C. All other conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 80%.

[0263] Example 62: Third Continuous Esterification and Amide Condensation Reaction

[0264] In this embodiment, the temperature of the seventh microreactor T and the eighth microreactor U was set to 65°C, and the other conditions were the same as in Example 61. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 87%.

[0265] Example 63: Third Continuous Esterification and Amide Condensation Reaction

[0266] In this embodiment, the solvents for feed solutions K, L, and M were replaced with dichloromethane, and the other conditions were the same as in Example 61. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 89%.

[0267] Example 64: Third Continuous Esterification and Amide Condensation Reaction

[0268] In this embodiment, the temperature of the seventh microreactor T and the eighth microreactor U was set to 70°C, and the other conditions were the same as in Example 63. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 91%, and the LCMS purity of the first peptide chain of smegglutinin was 91.75%. Figure 7 As shown.

[0269] Example 65: Third Continuous Esterification and Amide Condensation Reaction

[0270] In this embodiment, the solvents for feed solutions K, L, and M were replaced with dichloromethane. The flow rates of feed solutions F and K, pumped into the seventh microreactor T by the seventh injection pump G, were both set to 0.22 mL / min, with a residence time of 5 min. The temperature of the seventh microreactor T was 70°C. The flow rates of the pre-activated reaction solution and feed solution M, pumped into the eighth microreactor U by the eighth injection pump H, were both set to 0.44 mL / min, with a residence time of 5 min. The temperature of the eighth microreactor U was 70°C. The remaining conditions were the same as in Example 51. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 84%.

[0271] Example 66: A series of first continuous esterification and amide condensation reactions and a first continuous deprotection reaction.

[0272] In this embodiment, compound 2 was synthesized by tandemly performing the first continuous esterification and amide condensation reaction and the first continuous deprotection reaction using the method of Example 7. Then, compound 3 was synthesized from compound 2 using the method of Example 18. HPLC analysis showed that the yield of compound 3 was 99%.

[0273] Example 67: A series of first continuous deprotection reactions and second continuous esterification and amide condensation reactions.

[0274] In this embodiment, the first continuous esterification and amide condensation reaction, the first continuous deprotection reaction, and the second continuous esterification and amide condensation reaction were cascaded together to synthesize compound 2 using the method of Example 7. Using compound 2 as a starting material, compound 3 was synthesized using the method of Example 18. Using compound 3 and Fmoc-Aib-OH (compound 7) as starting materials, compound 4 was synthesized using the method of Example 36. HPLC analysis showed that the yield of compound 4 was 93%.

[0275] Example 68: Second continuous esterification and amide condensation reaction and second deprotection reaction in series

[0276] In this embodiment, compound 2 was synthesized by cascading a first continuous esterification and amide condensation reaction, a first continuous deprotection reaction, and a second continuous esterification and amide condensation reaction, followed by a second deprotection reaction, using the method of Example 7. Compound 3 was then synthesized from compound 2 using the method of Example 18. Compound 4 was then synthesized from compound 3 and Fmoc-Aib-OH (compound 7) using the method of Example 36. Finally, compound 5 was synthesized from compound 4 using the method of Example 45. HPLC analysis showed that the yield of compound 5 was 94%.

[0277] Example 69: A second continuous deprotection reaction and a third continuous esterification and amide condensation reaction in series.

[0278] In this embodiment, the first continuous esterification and amide condensation reaction, the first continuous deprotection reaction, the second continuous esterification and amide condensation reaction, the second deprotection reaction, and the third continuous esterification and amide condensation reaction were cascaded together to synthesize compound 2 using the method of Example 7. Using compound 2 as a starting material, compound 3 was synthesized using the method of Example 18. Then, using compound 3 and Fmoc-Aib-OH (compound 7) as starting materials, compound 4 was synthesized using the method of Example 36. Next, using compound 4 as a starting material, compound 5 was synthesized using the method of Example 45. Finally, using compound 5 and Fmoc-His(Trt)-OH as starting materials, the first peptide chain (tetrapeptide) of smegglutinin was synthesized using the method of Example 62. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 91%.

[0279] Example 70: Comparison of intermittent synthesis method and continuous synthesis method

[0280] While exploring methods for synthesizing the first peptide chain of smegraglutide, the inventors discovered that the batch synthesis method had a longer overall reaction time but a lower overall yield. In contrast, the continuous synthesis method could link the various steps in the synthesis route of the first peptide chain of smegraglutide together, thereby significantly shortening the overall reaction time and increasing the overall yield.

[0281] The formula for calculating the total yield is: Total yield = Yield of compound 2 × Yield of compound 3 × Yield of compound 4 × Yield of compound 4 × Yield of the first peptide chain of smegglutinin.

[0282] The sequential synthesis method comprises the following steps in series: compound 2 is synthesized using the method of Example 7; compound 3 is synthesized using the method of Example 18; compound 4 is synthesized using the method of Example 36; compound 5 is synthesized using the method of Example 45; and the first peptide chain of smegglutinin is synthesized using the method of Example 62. This sequential synthesis method utilizes... Figure 2 The synthesis was carried out using the continuous synthesis apparatus shown. HPLC analysis revealed that the yields of compound 2 were 99%, compound 3 was 100%, compound 4 was 94.2%, compound 5 was 100%, and the yield of the first peptide chain of semaglutide was 91.7%. The overall yield of the first peptide chain of semaglutide was calculated to be 85.5% (99% × 100% × 94.2% × 100% × 91.7%). The LCMS purity of the first peptide chain of semaglutide was 91.75%, and the total reaction time was 28 min.

[0283] The intermittent synthesis method is carried out using the following steps: Intermittent first esterification and amide condensation reaction: Take 0.85 g of Fmoc-Glu(OtBu)-OH (compound 1), 1.52 g of HATU and 0.101 g of triethylamine, add 20 mL of tetrahydrofuran, and stir the reaction in an ice bath for 1 h to carry out the first esterification reaction (i.e., pre-activation reaction). Add H-Gly-OBz. 0.42 g of HCl (compound 6) was added and stirred at 30 °C for 2 h to carry out the first amide condensation reaction, yielding the dipeptide product compound 2. HPLC analysis showed that the yield of compound 2 was 85%, and its LCMS purity was 88%.

[0284] Intermittent first deprotection reaction: 1.14 g of dipeptide product (compound 2) was taken and 20 mL of acetonitrile was added. The mixture was stirred until dissolved, and 0.304 g of DBU was added dropwise. The mixture was stirred at 25 °C for 40 min to carry out the first deprotection reaction, yielding the first deprotected product, compound 3. HPLC analysis showed that the yield of compound 3 was 99%, and its LCMS purity was 99%.

[0285] Intermittent second esterification and amide condensation reaction: 0.65 g of Fmoc-Aib-OH (compound 7), 1.52 g of HATU, and 0.101 g of triethylamine were added to 20 mL of dichloromethane. The mixture was stirred in an ice bath for 1 h to carry out the second esterification reaction (i.e., pre-activation reaction). Then, 0.70 g of the first deprotected product (compound 3) was added, and the mixture was stirred at 30 °C for another 3 h to carry out the second amide condensation reaction, yielding the tripeptide product compound 4. HPLC analysis showed that the yield of compound 4 was 59.2%, and its LCMS purity was 48%.

[0286] Intermittent second deprotection reaction: 1.31 g of the tripeptide product (compound 4) was added to 20 mL of acetonitrile and stirred until dissolved. Then, 0.304 g of DBU was added dropwise. The mixture was stirred at 25 °C for 40 min to carry out the second deprotection reaction, yielding the second deprotected product, compound 5. HPLC analysis showed that the yield of compound 5 was 99%, and its LCMS purity was 98%.

[0287] Intermittent third esterification and amide condensation reaction: 1.24 g of Fmoc-His(Trt)-OH (compound 8), 1.52 g of HATU, and 0.101 g of triethylamine were added to 20 mL of dichloromethane. The mixture was stirred in an ice bath for 1 h to carry out the third pre-activation reaction. Then, 0.70 g of the second deprotected product (compound 3) was added, and the mixture was stirred at 30 °C for another 4 h to carry out the third amide condensation reaction, yielding the first peptide chain of smegglutinin. HPLC analysis showed that the yield of the first peptide chain of smegglutinin was 59.6%, and its LCMS purity was 44%.

[0288] The total yield of the batch synthesis method was calculated to be 29.4% (85%×99%×59.2%×99%×59.6%).

[0289] The results showed that the continuous synthesis method could chain together the various steps in the synthetic route of semaglutide's first peptide chain, thus significantly shortening the total reaction time from 13.3 h (3 h + 0.67 h + 4 h + 0.67 h + 5 h) in the batch synthesis method to 28 min (5 min + 4 min + 5 min + 4 min + 10 min). Furthermore, the continuous synthesis method achieved a higher yield of semaglutide's first peptide chain (85.5%), which was superior to the batch method (29.4%).

[0290] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. A method for preparing the first peptide chain of smegglutinin using liquid-phase synthesis, the method comprising the following steps: (1) First esterification and amide condensation reaction: Using Fmoc-Glu(OtBu)-OH as raw material, the first pre-activated reaction solution was synthesized by esterification reaction, and H-Gly-OBz was added. HCl was used to perform an amide condensation reaction to synthesize intermediate compound 2. (2) First deprotection reaction: Using compound 2 synthesized in (1) as raw material, intermediate product compound 3 is obtained through deprotection reaction; (3) Second esterification and amide condensation reaction: Using Fmoc-Aib-OH as raw material, the second pre-activated reaction solution is synthesized by esterification reaction, and the compound 3 obtained in (2) is added to carry out amide condensation reaction to synthesize intermediate product compound 4; (4) Second deprotection reaction: Using compound 4 synthesized in (3) as a starting material, intermediate compound 5 is obtained through a deprotection reaction; and (5) Third esterification and amide condensation reaction: Using Fmoc-His(Trt)-OH as raw material, the third pre-activated reaction solution is synthesized by esterification reaction, and compound 5 obtained in (4) is added to carry out amide condensation reaction to synthesize the first peptide chain of smegglutinin.

2. The method as described in claim 1, characterized in that, Step (1) includes: (1A) Using Fmoc-Glu(OtBu)-OH as raw material, Fmoc-Glu(OtBu)-OH, alkali, and a first organic solvent are mixed to obtain feed solution A. (1B) The condensing agent is mixed with the second organic solvent to obtain feed solution B. (1C) The feed solution A obtained in (1A) is mixed with the feed solution B obtained in (1B), and the mixture is then subjected to an esterification reaction to synthesize the first pre-activated reaction solution. (1D) H-Gly-OBz HCl and alkali are mixed to obtain feed solution C. (1E) The first pre-activated reaction solution obtained in (1C) is mixed with feed solution C, and intermediate product compound 2 is synthesized by amide condensation reaction; Preferably, step (1) further includes one or more of the following conditions: Steps (1A) to (1E) are performed consecutively; and / or, The condensing agent is any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA, N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine; and / or, The first organic solvent, the second organic solvent, and the third organic solvent are each independently selected from any one or more of dichloromethane, ethyl acetate, acetonitrile, N,N-dimethylformamide, toluene, tetrahydrofuran, and 1,4-dioxane, preferably any one or more of N,N-dimethylformamide, tetrahydrofuran, and toluene, more preferably tetrahydrofuran, and even more preferably, the first organic solvent, the second organic solvent, and the third organic solvent are all tetrahydrofuran; and / or, In step (1A), the concentration of Fmoc-Glu(OtBu)-OH in feed solution A is 0.01~2.0 mol / L, preferably 0.05~0.5 mol / L or 0.05~0.15 mol / L; and / or, In step (1A), the molar ratio of Fmoc-Glu(OtBu)-OH to alkali in feed solution A is 1:1 to 4:1, preferably 1:1 to 2:1; and / or, In step (1B), the concentration of the condensing agent in the feed liquid B is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (1C), after feed liquid A and feed liquid B are mixed, the molar ratio of Fmoc-Glu(OtBu)-OH to the condensing agent is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (1C) is carried out in the first microreactor; and / or, The reaction temperature of step (1C) is 10~100℃, preferably 20~70℃, more preferably 30±1℃; and / or, In step (1C), feed liquid A and feed liquid B are pumped into the first microreactor via a first injection pump; and / or, In step (1C), the injection flow rates of feed solution A and feed solution B are 0.02~0.04 mL / min; and / or, In step (1C), the residence time of feed liquid A and feed liquid B is 1~20 min, preferably 5~10 min; and / or, In the feed liquid C of step (1D), H-Gly-OBz The concentration of HCl is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L, 0.1~0.2 mol / L, or 0.13~0.15 mol / L; and / or, The step (1E) is carried out in the second microreactor; and / or, The reaction temperature of step (1E) is 10~100℃, preferably 20~70℃, more preferably 30±1℃; and / or, In step (1E), after the first pre-activated reaction solution and feed solution C are mixed, Fmoc-Glu(OtBu)-OH and H-Gly-OBz The molar ratio of HCl is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, In step (1E), the first pre-activated reaction solution and the feed solution C are pumped into the second microreactor via a second injection pump; and / or, In step (1E), the injection flow rate of the first pre-activated reaction solution is 0.03~0.05 mL / min; and / or, In step (1E), the injection flow rate of the feed solution C is 0.015~0.03 mL / min; and / or, In step (1E), the residence time of the feed liquid C is 1~20 min, for example 5~10 min.

3. The method as described in claim 1, characterized in that, Step (2) includes: (2A) The intermediate product compound 2 obtained in step (1) is mixed with a fourth organic solvent to obtain feed liquid D. (2B) The reagent that has removed the Fmoc protecting group is mixed with the fifth organic solvent to obtain feed solution E. (2C) The feed liquid D in (2A) is mixed with the feed liquid E in (2B), and after deprotection reaction, intermediate product compound 3 is obtained; Preferably, step (2) further includes one or more of the following conditions: Steps (2A) to (2C) are performed consecutively; and / or, The reagent used to remove the Fmoc protecting group is DBU; and / or, The fourth and fifth organic solvents are each independently selected from one or more of methanol, toluene, N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, preferably one or more of methanol, ethyl acetate, dichloromethane, tetrahydrofuran, and acetonitrile, more preferably acetonitrile, and even more preferably both the fourth and fifth organic solvents are acetonitrile; and / or, In step (2A), the concentration of compound 2 in the feed solution D is 0.01~0.5 mol / L, preferably 0.05~0.3 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (2B), the concentration of the reagent for removing the Fmoc protecting group in the feed solution E is 0.01~0.5 mol / L, preferably 0.05~0.2 mol / L or 0.05~0.15 mol / L; and / or, In step (2C), after feed solution D and feed solution E are mixed, the molar ratio of compound 2 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1.1 to 1:1.5; and / or, The step (2C) is carried out in a third microreactor; and / or, The reaction temperature in step (2C) is 25~70℃, preferably 40~60℃; and / or, In step (2C), the feed liquid D and feed liquid E are pumped into the third microreactor via a third injection pump; and / or, In step (2C), the injection flow rates of feed solution D and feed solution E are 0.025~0.055 mL / min; and / or, In step (2C), the residence time of feed liquid D and feed liquid E is 1~20 min, preferably 4~10 min.

4. The method as described in claim 1, characterized in that, Step (3) includes: (3A) Using Fmoc-Aib-OH as raw material, Fmoc-Aib-OH, alkali, and a sixth organic solvent are mixed to obtain feed solution F. (3B) The condensing agent is mixed with the seventh organic solvent to obtain feed liquid G. (3C) The feed solution F obtained in (3A) is mixed with the feed solution G obtained in (3B), and the mixture is then synthesized into a second pre-activated reaction solution via esterification. (3D) The intermediate product compound 3 obtained in step (2) is mixed with the eighth organic solvent to obtain feed solution H. (3E) The second pre-activated reaction solution obtained in (3C) is mixed with feed solution H, and intermediate product compound 4 is synthesized by amide condensation reaction; Preferably, step (3) further includes one or more of the following conditions: Steps (3A) through (3E) are performed consecutively; and / or, The condensing agent is any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA (N,N-diisopropylethylamine), N-methylmorpholine, pyridine, Na₂CO₃, NaHCO₃, K₂CO₃, KHCO₃, diethylamine, and triethylamine, preferably triethylamine; and / or, The sixth, seventh, and eighth organic solvents are each independently selected from any one or more of N,N-dimethylformamide, 1,3-dimethyl-2-imidazolinone, acetonitrile, 1,4-dioxane, dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, preferably any one or more of dichloromethane, ethyl acetate, toluene, and tetrahydrofuran, more preferably dichloromethane or ethyl acetate, and even more preferably, the sixth, seventh, and eighth organic solvents are all dichloromethane or ethyl acetate; and / or, In step (3A), the concentration of Fmoc-Aib-OH in the feed solution F is 0.05~0.20 mol / L, preferably 0.05~0.15 mol / L; and / or, In step (3A), the molar ratio of Fmoc-Aib-OH to alkali in the feed solution F is 1:1 to 4:1, preferably 1:1 to 2:1; and / or, In step (3B), the concentration of the condensing agent in the feed liquid G is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (3C), after feed liquid F and feed liquid G are mixed, the molar ratio of Fmoc-Aib-OH to condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (3C) is carried out in the fourth microreactor; and / or, The reaction temperature of step (3C) is 30~80℃, preferably 40~70℃, more preferably 45~60℃; and / or, In step (3C), the feed liquid F and feed liquid G are pumped into the fourth microreactor via a fourth injection pump; and / or, In step (3C), the injection flow rates of feed solution F and feed solution G are 0.02~0.08 mL / min, preferably 0.05~0.06 mL / min; and / or, In step (3C), the residence time of feed liquid F and feed liquid G is 1~20 min, for example, 5±1 min; and / or, In step (3D), the concentration of compound 3 in the feed solution H is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L, or 0.04~0.06 mol / L; and / or, In step (3E), after the second pre-activated reaction solution and the feed solution H are mixed, the molar ratio of Fmoc-Aib-OH to compound 3 is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (3E) is carried out in the fifth microreactor; and / or, The reaction temperature of step (3E) is 30~80℃, preferably 40~70℃, more preferably 45~60℃; and / or, In step (3E), the second pre-activated reaction solution and the feed solution H are pumped into the fifth microreactor via the fifth injection pump; and / or, In step (3E), the injection flow rate of the second pre-activated reaction solution and the feed solution H is 0.02~0.08 mL / min, preferably 0.05~0.06 mL / min; and / or, In step (3E), the residence time of the second pre-activated reaction solution and the feed solution H is 1~20 min, preferably 5±1 min.

5. The method as described in claim 1, characterized in that, Step (4) includes: (4A) The intermediate product compound 4 obtained in step (3) is mixed with the ninth organic solvent to obtain feed liquid I. (4B) The reagent that removes the Fmoc protecting group is mixed with the tenth organic solvent to obtain feed solution J. (4C) The feed liquid I in (4A) is mixed with the feed liquid J in (4B), and after deprotection reaction, intermediate product compound 5 is obtained; Preferably, step (4) further includes one or more of the following conditions: Steps (4A) to (4C) are performed consecutively; and / or, The reagent used to remove the Fmoc protecting group is DBU; and / or, The ninth and tenth organic solvents are each independently selected from one or more of tetrahydrofuran, methanol, toluene, N,N-dimethylformamide, ethyl acetate, 1,4-dioxane, acetonitrile, and dichloromethane, preferably one or more of tetrahydrofuran, methanol, dichloromethane, and acetonitrile, more preferably acetonitrile, and even more preferably both the ninth and tenth organic solvents are acetonitrile; and / or, In step (4A), the concentration of compound 4 in feed liquid I is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (4B), the concentration of the reagent for removing the Fmoc protecting group (e.g., DBU) in the feed solution J is 0.01~0.5 mol / L, preferably 0.05~0.4 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (4C), after feed liquid I and feed liquid J are mixed, the molar ratio of compound 4 to the reagent for removing the Fmoc protecting group (e.g., DBU) is 1:1 to 1:2, preferably 1:1.1 to 1:1.5; and / or, The step (4C) is carried out in the sixth microreactor; and / or, The reaction temperature of step (4C) is 25~50℃, preferably 25~40℃; and / or, In step (4C), feed liquid I and feed liquid J are pumped into the sixth microreactor via the sixth injection pump; and / or, In step (4C), the injection flow rates of feed solution I and feed solution J are 0.11~0.22 mL / min; and / or, In step (4C), the residence time of feed liquid I and feed liquid J is 1~10 min, preferably 1~5 min, and more preferably 1~4 min.

6. The method as described in claim 1, characterized in that, Step (5) includes: (5A) Using Fmoc-His(Trt)-OH as raw material, Fmoc-His(Trt)-OH, alkali, and the eleventh organic solvent are mixed to obtain feed liquid K. (5B) The condensing agent is mixed with the twelfth organic solvent to obtain feed liquid L. (5C) The feed solution K obtained in (5A) is mixed with the feed solution L obtained in (5B), and the mixture is then synthesized into a third pre-activated reaction solution via esterification. (5D) The intermediate compound 5 obtained in step (4) is mixed with the thirteenth organic solvent to obtain feed liquid M. (5E) The third pre-activated reaction solution obtained in (5C) is mixed with feed solution M and synthesized as the first peptide chain of smegglutinin by amide condensation reaction. Preferably, step (5) further includes one or more of the following conditions: Steps (5A) to (5E) are performed consecutively; and / or, The condensing agent is any one or more of HATU, HBTU, TBTU, and PyBOP, preferably HATU; and / or, The base is any one or more selected from DIEA, N-methylmorpholine, pyridine, Na2CO3, NaHCO3, K2CO3, KHCO3, diethylamine, and triethylamine, preferably triethylamine; and / or, The eleventh, twelfth, and thirteenth organic solvents are each independently selected from any one or more of 1,4-dioxane, N,N-dimethylformamide, ethyl acetate, toluene, acetonitrile, dichloromethane, and tetrahydrofuran, preferably any one or more of dichloromethane and tetrahydrofuran, more preferably dichloromethane, and even more preferably all of the eleventh, twelfth, and thirteenth organic solvents are dichloromethane; and / or, In step (5A), the concentration of Fmoc-His(Trt)-OH in the feed solution K is 0.01~1.0 mol / L, preferably 0.05~0.5 mol / L, 0.05~0.2 mol / L, or 0.05~0.15 mol / L; and / or, In step (5A), the molar ratio of Fmoc-His(Trt)-OH to alkali (e.g., triethylamine) in the feed liquid K is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, In step (5B), the concentration of the condensing agent (e.g., HATU) in the feed liquid L is 0.1~2.0 mol / L, preferably 0.1~1.0 mol / L or 0.1~0.3 mol / L; and / or, In step (5C), after mixing feed liquid K and feed liquid L, the molar ratio of Fmoc-His(Trt)-OH to condensing agent (e.g., HATU) is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (5C) is carried out in the seventh microreactor; and / or, The reaction temperature in step (5C) is 30~80℃, preferably 40~70℃, more preferably 55~70℃; and / or, In step (5C), the feed liquid K and feed liquid L are pumped into the seventh microreactor via the seventh injection pump; and / or, In step (5C), the injection flow rates of feed solution K and feed solution L are 0.03~0.3 mL / min, preferably 0.03-0.25 mL / min, more preferably 0.11~0.22 mL / min; and / or, In step (5C), the residence time of the feed liquid K and the feed liquid L is 1~20 min, preferably 5~20 min, for example 5~10 min; and / or, In step (5D), the concentration of compound 5 in the feed solution M is 0.01~2.0 mol / L, preferably 0.01~1.0 mol / L, 0.01~0.1 mol / L, 0.02~0.08 mol / L, 0.03~0.07 mol / L, or 0.04~0.06 mol / L; and / or, In step (5E), after the third pre-activated reaction solution and the feed solution M are mixed, the molar ratio of Fmoc-His(Trt)-OH to compound 5 is 1:1 to 1:10, preferably 1:1 to 1:4; and / or, The step (5E) is carried out in the eighth microreactor; and / or, The reaction temperature of step (5E) is 30~80℃, preferably 40~70℃, more preferably 55~70℃; and / or, In step (5E), the third pre-activation reaction solution and the feed solution M are pumped into the eighth microreactor via the eighth injection pump; and / or, In step (5E), the injection flow rate of the third pre-activated reaction solution and the feed solution M is 0.1~0.5 mL / min, preferably 0.22~0.44 mL / min; and / or, In step (5E), the residence time of the third pre-activated reaction solution and the feed solution M is 1~20 min, preferably 5~20 min, and more preferably 5~10 min.

7. The method according to any one of claims 1-6, characterized in that, Any two, three, four, or all five adjacent steps in steps (1) to (5) can be performed consecutively in a series. Preferably, all five steps (1) to (5) are performed consecutively; More preferably, steps (1A) to (1E), steps (2A) to (2C), steps (3A) to (3E), steps (4A) to (4C), and steps (5A) to (5E) are performed consecutively.

8. A method for preparing smegglutinin intermediates compound 2, compound 3, compound 4, or compound 5 using liquid-phase synthesis, characterized in that: The method for preparing smegglutinin intermediate compound 2 includes step (1) as described in claim 1 or 2. The method for preparing smegglutinin intermediate compound 3 includes steps (1) and (2) as described in any one of claims 1-3, preferably steps (1) and (2) are performed sequentially; The method for preparing the intermediate compound 4 of smegglutinin includes steps (1), (2) and (3) as described in any one of claims 1-4, preferably steps (1), (2) and (3) are performed sequentially; The method for preparing smegglutinin intermediate compound 5 includes steps (1), (2), (3) and (4) as described in any one of claims 1-5, preferably steps (1), (2), (3) and (4) are performed sequentially.

9. A continuous synthesis apparatus, characterized in that, The continuous synthesis apparatus comprises: a first microreactor, a second microreactor, a third microreactor, a fourth microreactor, a fifth microreactor, a sixth microreactor, a seventh microreactor, and an eighth microreactor that are in sequential fluid communication; a first injection pump in fluid communication with the first microreactor; a second injection pump in fluid communication with the second microreactor; a third injection pump in fluid communication with the third microreactor; a fourth injection pump in fluid communication with the fourth microreactor; a fifth injection pump in fluid communication with the fifth microreactor; a sixth injection pump in fluid communication with the sixth microreactor; a seventh injection pump in fluid communication with the seventh microreactor; an eighth injection pump in fluid communication with the eighth microreactor; a connecting pipe connecting each microreactor and the injection pump; and control components for operating the apparatus to implement the method for preparing the first peptide chain of smegglutinin by liquid-phase synthesis according to any one of claims 1-7, or the method for preparing intermediate product compound 2, compound 3, compound 4, or compound 5 of smegglutinin by liquid-phase synthesis according to claim 8. Preferably, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth injection pumps are syringe pumps, peristaltic pumps, or horizontal flow pumps, more preferably syringe pumps; and / or, Preferably, the first microreactor, second microreactor, third microreactor, fourth microreactor, fifth microreactor, sixth microreactor, seventh microreactor and / or eighth microreactor are capillary microreactors, fixed-bed microreactors, glass chip microreactors or stainless steel and silicon carbide microchannel reactors, more preferably capillary microreactors.

10. The application of the continuous synthesis apparatus according to claim 9 in the continuous liquid phase synthesis for the preparation of the first peptide chain of smegglutinin, or in the continuous liquid phase synthesis for the preparation of intermediate product compounds 2, 3, 4 or 5 of smegglutinin; Preferably, the method for preparing the first peptide chain of semaglutide by continuous liquid-phase synthesis is as described in any one of claims 1-7, and / or the method for preparing intermediate compound 2, compound 3, compound 4 or compound 5 of semaglutide by continuous liquid-phase synthesis is as described in claim 8.

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